- Email: [email protected]
Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure
Accepted Manuscript Title: Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure Author: Ezequiel J. Tolosa Mart´ın Fernandez-Zapico Natalia L. Battiato Roberto A. Rovasio PII: DOI: Reference:
S0171-9335(16)30006-1 http://dx.doi.org/doi:10.1016/j.ejcb.2016.02.003 EJCB 50868
To appear in: Received date: Revised date: Accepted date:
20-6-2015 23-1-2016 17-2-2016
Please cite this article as: Tolosa, E.J., Fernandez-Zapico, M., Battiato, N.L., Rovasio, R.A.,Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure, European Journal of Cell Biology (2016), http://dx.doi.org/10.1016/j.ejcb.2016.02.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ed pt ce Ac
Page 1 of 53
Highlights
Tolosa et al.,
Ac
ce pt
ed
M
an
us
cr
Cephalic NCCs migrate chemotactically to a concentration gradient of Shh morphogen. Expression of Shh at optic vesicle and Ptch-Smo on NCCs integrate the guide system. Molecular and pharmacological blocking inhibits oriented migration of cephalic NCC. Shh-dependent chemotaxis of NCCs was altered by in vitro-in vivo ethanol exposure. Results support a guiding activity for Shh in addition to its other canonic functions.
ip t
Page 2 of 53
1
Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure
ip t
Ezequiel J. Tolosa a,b, Martín Fernandez-Zapico b,
Center for Cellular and Molecular Biology, CEBICEM-IIBYT (UNC-CONICET)
an
a
us
cr
Natalia L. Battiato a and Roberto A. Rovasio a
National University of Córdoba, Av. Vélez Sarsfield 1611, (5016) Córdoba, Argentina. Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
Ac ce pt e
d
M
b
Corresponding author:
Roberto A. Rovasio, CEBICEM (FCEFN, UNC), IIBYT (UNC-CONICET) Av. Vélez Sarsfield 1611, (5016) Córdoba, Argentina. Email: [email protected]
Page 3 of 53
2
Abstract Our aim was to understand the involvement of Sonic hedgehog (Shh) morphogen in the
potential disorders of this guiding mechanism after ethanol exposure.
ip t
oriented distribution of neural crest cells (NCCs) toward the optic vesicle and to look for
In vitro directional analysis showed the chemotactic response of NCCs up Shh gradients
cr
and to notochord co-cultures (Shh source) or to their conditioned medium, a response
inhibited by anti-Shh antibody, receptor inhibitor cyclopamine and anti-Smo morpholino
us
(MO). Expression of the Ptch-Smo receptor complex on in vitro NCCs was also shown. In whole embryos, the expression of Shh mRNA and protein was seen in the ocular region, and
an
of Ptch, Smo and Gli/Sufu system on cephalic NCCs. Anti-Smo MO or Ptch-mutated plasmid (Ptch1∆loop2) impaired cephalic NCC migration/distribution, with fewer cells invading the
M
optic region and with higher cell density at the homolateral mesencephalic level. Beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic signal) supported the role of Shh as an in vivo guide molecule for cephalic NCCs.
d
Ethanol exposure perturbed in vitro and in vivo NCC migration. Early stage embryos
Ac ce pt e
treated with ethanol, in a model reproducing Fetal Alcohol Syndrome, showed later disruptions of craniofacial development associated with abnormal in situ expression of Shh morphogen.
The results show the Shh/Ptch/Smo-dependent migration of NCCs toward the optic vesicle, with the support of specific inactivation with genetic and pharmacological tools. They also help to understand mechanisms of accurate distribution of embryonic cells and of their perturbation by a commonly consumed teratogen, and demonstrate, in addition to its other known developmental functions, a new biological activity of cellular guidance for Shh.
Key words Cell guiding; Cell migration; Chemotaxis; Chick embryo; Ethanol; Neural crest cells; Sonic hedgehog
Page 4 of 53
3
Introduction Neural crest cells (NCCs) segregate from the closing neural tube, migrate along several pathways and colonize defined sites, giving rise to a variety of derivatives. The conserved distribution pattern of NCCs along vertebrate embryos and their morphogenetic behavior are modulated by a balance between genetic signals and those of the extracellular milieu (Bronner and Le Douarin, 2012; Le Douarin and Kalcheim, 1999; Rovasio et al., 1983). However, these
ip t
factors do not fully explain their directional migration.
Studies from our laboratory on different experimental models helped to re-discover
cr
chemotaxis as a significant modulating factor of directional cell response (Guidobaldi et al., 2012; Jaurena, 2011; Rovasio et al., 2012; Tolosa et al., 2012; Zanin et al., 2013). In this
us
communication at a distance, the cell recognizes an exogenous concentration gradient of diffusible molecules released by a “target” field and orients its locomotion as a function of the
an
slope vector (Rovasio et al., 2012). Chemotaxis was proven in bacteria (Hazelbauer, 2012), amoebas (García and Parent, 2008), leukocytes (Afonso et al., 2012), neurons (Paratcha et al., 2006) and axonal growth cones (Mortimer et al., 2008). We have shown chemotaxis of
M
mammal sperm toward the ovular region (Giojalas and Rovasio, 1998), with progesterone signals as the attractant (Guidobaldi et al., 2012). Curiously, the embryonic cell, paradigmatic
d
of accurate, directional motility, has been less studied with a chemotactic approach, except in
Ac ce pt e
a few documented systems in Drosophila (Ricardo and Lehmann, 2009), zebrafish (Breau et al., 2012), frog (Mayor and Theveneau, 2013), mouse (Kubota and Ito, 2000) and avian embryos (McLennan et al., 2012). Recently, we presented direct evidence of Stem Cell Factor (SCF) eliciting chemotactic migration of NCCs toward the skin (Rovasio et al., 2012), as well as the CXCR4-dependent chemotactic behavior of cephalic NCCs up to the chemokine Stromal cell-Derived Factor-1 (SDF-1) expressed in the optic field (Jaurena, 2011), and TrC/p75-dependent cephalic NCC migration up an in vitro and in vivo NT-3 concentration gradient released in the ocular region (Zanin et al., 2013). In our study model of the colonization pattern of NCCs to the optic vesicle region, it should be noted that NCCs start emigrating from mesencephalic levels in a narrow temporal window of 32 to 36 h (stages 8-10 HH) when the neural folds are closing, and then there is no emigration from this segment of the neural tube (Newgreen and Erickson, 1986). After detaching, mesencephalic NCCs migrate to both lateral sides under the ectoderm, toward the future facial region (Le Douarin and Kalcheim, 1999). Then, a subpopulation of ciliary NCCs detaches from the principal mass, changes its direction by about 90° and heads toward the
Page 5 of 53
4
optic vesicle stalk (Lee et al., 2003) (see Fig. 3 B, arrows), where the parasympathetic ciliary ganglion of the eye will form. It is noteworthy that there is no topographic, micro-anatomic or any other explanation to account for the clear directional change of ciliary NCCs to the optic vesicle field. The notion of Sonic hedgehog (Shh) as a chemotactic guide for NCC distribution arose from the association between the perturbed expression of Shh with cephalic anomalies
ip t
(Ahlgren et al., 2002; Marcucio et al., 2005; Paganelli et al., 2010) and enteric agangliosis (Tobin et al., 2008). Extracellular protein Shh is a highly conserved paracrine factor
cr
displaying significant activity in communication at a distance in a broad sense (Etheridge et al., 2010), and in the context of the concept of morphogen as a concentration-dependent
us
modulator of spatial cell patterns (Gilbert, 2013). Besides the well-known functions of Shh during early embryo development, there is evidence of Shh involvement in the migratory
an
behavior of fibroblasts (Bijlsma et al., 2007), axonal cone growth (Gore et al., 2008), olfactory bulb neuroblasts (Angot et al, 2008), primordial germ cells (Deshpande et al., 2001),
Marcucio et al., 2005; Tolosa et al., 2012).
M
enteric NCCs (Fu et al., 2004), and craniofacial NCCs (Ahlgren et al., 2002; Hu et al., 2015;
On the other hand, the relevant attributes of NCCs and the putative role of Shh as a guide
d
molecule suggested considering this embryonic model within a pathogenic context. Thus, the
Ac ce pt e
neurocristopathies family (Bolande, 1974) includes tumoral diseases, genetic-based anomalies and epigenetic determinants such as Retinoic Acid Syndrome (Salvarezza and Rovasio, 1997) or Fetal Alcohol Syndrome (FAS) (Jaurena et al., 2011; Rovasio and Battiato, 1995, 2002), all with serious social impact. Taking into account that several FAS anomalies imply regions colonized by NCCs in the craniofacial field (Le Douarin et al., 2007), and that prenatal effects of ethanol exposure include a low migratory capacity and perturbed NCC distribution (Chen et al., 2000; Jaurena et al., 2011; Rovasio and Battiato, 1995, 2002), it is reasonable to think that epigenetic perturbation of directional migration of this cell population may be an important etiopathogenic component of FAS. It is known that accurate cell distribution in the embryo relies on precise cell migration, which is disrupted in cortical cerebellar neuroblasts after ethanol exposure (Kumada et al., 2010). Also, the involvement of Shh expression in craniofacial morphogenesis is well known (Hu et al., 2015; Tapadia et al., 2005; Tobin et al., 2008) following abnormal cephalic development after perturbation of Shh expression by ethanol exposure (Ahlgren et al., 2002), or treatments with cyclopamine (Chen et al., 2002), or anti-Shh antibodies (Marcucio et al., 2005). These data encouraged us to explore the association between signals started with the Shh ligand, the oriented migration of cephalic
Page 6 of 53
5
NCCs and their potential lability to ethanol action in the final localization of this cell population. In this report on NCC cultures and directional analysis, we show the chemotactic response of NCCs up Shh extracellular gradients or in notochord co-cultures (Shh source) or their conditioned medium, and their inhibition by anti-Shh antibody, the receptor inhibitor cyclopamine and the anti-Smo MO. In whole embryos, we demonstrate the expression of Shh
ip t
mRNA and protein in the ocular region of the embryo and of Ptch-Smo receptor complex in in vitro and in vivo cephalic NCCs. Also, the MO blockade of the Shh-Ptch-Smo system
cr
impaired in vivo migration/distribution of cephalic NCCs. The insertion of beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic source) provided additional evidence on
us
the Shh function as an in vivo guidance molecule for the orientation of cephalic NCCs. Moreover, we showed that a teratogenic concentration of ethanol exposure perturbed the Shh-
an
dependent chemotaxis of in vitro NCCs, and that early-exposed embryos in a model reproducing FAS show a disruption of craniofacial development at later stages, associated with abnormal in situ expression of Shh morphogen.
M
These in vitro and in vivo results strongly suggest that: (1) the chemotactic mechanism is an essential element in the spatiotemporal orientation of NCCs toward specific regions of the
d
embryo; (2) mesencephalic NCCs migrate toward the optic vesicle guided by a chemotactic
Ac ce pt e
mechanism in response to Shh concentration gradients in a Ptch/Smo-dependent manner; (3) the accuracy of this chemotactic mechanism is perturbed by ethanol, a commonly consumed teratogen. This helps to understand a fundamental mechanism for the distribution of embryonic cells and their epigenetic perturbation, and also amplifies the functional scope of the morphogen Shh, involving it in new biological activities for the colonization of a specific region by NCCs, in addition to its other well-known developmental functions.
Materials and Methods
(Note: For full details, see Supplemental material)
In vitro experiments Culture of cephalic neural crest cells Cultures of mesencephalic NCCs were made from Gallus gallus (Cobb line) chick embryos stages 10-11 HH (Hamburger and Hamilton, 1951) as detailed (Rovasio et al., 2012), incubated with N2 defined medium (Barnes and Sato, 1980) plus 10% fetal calf serum (FCS) (Sigma Chem Co, St Louis, MO) during 20 h at 37 ±0.2 °C in 5% CO2 in air.
Page 7 of 53
6
Notochord and optic vesicle conditioned medium Notochord explants, as Shh sources (Martí et al., 1995), were obtained from the trunk level of stages 10-11 HH chick embryos by microdissection and collagenase digestion, cultured in N2 medium for 48 h and the conditioned medium (CM) recovered and used accordingly. Some lots were concentrated in a Centricon column (Amicon Inc., Beverly, MA, USA), obtaining fractions of ≤10,000 and >10,000 Daltons of molecules. Similarly, CM was
ip t
obtained from optic vesicle cultures (Jaurena, 2011). Video-microscopy and chemotaxis assay
cr
A real-time approach was applied using a chemotaxis chamber, computerized videomicroscopy and software based on objective directional criteria (Rovasio et al., 2012). NCC
us
cultures were perfused with a concentration gradient of Shh (1845-SH; R&D Systems, Minneapolis, MN, USA) at a concentration indicated in the Results section, or co-cultured
an
with notochord or optic vesicle explants, or their CM. Control cultures and inhibition assays were performed with only N2 medium in the chamber well, or control CM, or Shh preincubated with neutralizing anti-Shh antibody (5E1, Develop. Studies Hybrid. Bank)
M
(Marcucio et al., 2005), or heat-inactivated Shh, or the inhibitor cyclopamine (Chen et al., 2002), or culturing NCCs transfected with anti-Smo or anti-Shh MOs. Morphometric
d
parameters were cell area, perimeter and shape factor (4π x area)/perimeter2; dynamic
Ac ce pt e
parameters were curvilinear velocity (total distance traveled by the cell per unit time) and linearity (nondimensional quotient between linear distance and curvilinear distance); and chemotactic parameters were evaluated applying algorithms developed in our laboratory (Rovasio et al., 2012). Since several chemotactic parameters based on cell number and trajectory show equivalent results, we present only representative data, displaying other parameters only for particular points or to better visualize oriented cell migration. The attractant concentration refers to the gradient source, and all the experiments (and graphs of results) were carried out with the right-side well of the chemotaxis chamber as the source of putative attractant (Rovasio et al., 2012). After video assays and in-between sampling, NCCs were submitted to proliferation and viability testing. Ethanol exposure Experiments were also performed in the presence of 100 µM ethanol in both wells of the chemotaxis chamber as detailed in the Results section. At the end of each experiment, ethanol concentration was checked in both chamber compartments by head-space gas chromatography as explained (Jaurena et al., 2011).
Page 8 of 53
7
Immunolabeling of NCC cultures The purity of NCC culture was assessed with the HNK1 monoclonal antibody (Sigma Chem Co). Patched receptor and Smoothened signal transduction protein were labeled with specific antibodies (SC-6149 and SC-13943, Santa Cruz Biotech. Inc., CA, USA), then incubated with rhodamine- or fluoresceine isothiocyanate-conjugated secondary antibodies, and controlled by replacing antibodies with PBS or heat-inactivated antibodies.
ip t
Determination of trophic parameters
Cell survival was analyzed by the Live/Dead Kit (Molecular Probes, Eugene, OR)
cr
according to manufacturer instructions, using NCCs pre-incubated with sodium azide as positive control. Cell proliferation was assessed after incorporation of 5-bromo-2′-
us
deoxyuridine (BrdU) as detailed (Jaurena et al., 2011). Images of the same microscopic fields comprising almost all the surface of NCC outgrowth were obtained with phase contrast and
an
fluorescence optics, and submitted to analyses with Image-J software (NIH, Bethesda, MA, USA), according to previous descriptions (Jaurena et al., 2011; Rovasio et al., 2012). NCC in vitro transfection with anti-Smo and anti-Shh morpholinos
M
Prior to evaluation of chemotaxis, morpholino (MO) transfection of NCC cultures was performed with the Endo-Porter® tool (Gene Tools, LLC Philomath, USA), according to
d
manufacturer instructions, using 1 µM lisamine (sulforhodamine)-labeled MO against: 1) Smo
Ac ce pt e
mRNA (5’-AAAGCAGCAGCTCACCATGCTCCAT-3’ sulforhodamine) (Smo-MO); 2) Shh mRNA (5´-TGTCAACAGCAGCATTTCGACCATT-3’ sulforhodamine) (Shh-MO), or 3) standard control (5'-CCTCTTACCTCA GTTACAATTTATA-3' sulforhodamine) (Ctrl-MO), all of them added with 5 µl/ml Endo-Porter® in culture medium plus 10% serum, followed by 24 h incubation. The medium was then replaced with defined medium and the NCCs used for different assays.
Whole chick embryo experiments Obtaining and processing
Distribution of NCC markers and bioactive molecules was assessed by whole-mount in situ hybridization and immunocytochemistry on stage 10-11 HH chick embryos incubated in ovo as detailed elsewhere (Rovasio et al., 2012; Tolosa et al., 2012; Zanin et al., 2013), or cultivated in a shell-less culture system as described (Battiato et al., 1995; Jaurena et al., 2011; Rovasio and Battiato, 2002; Tolosa et al., 2012). After PBS-washing, the embryos were fixed with 4% paraformaldehyde in PBS for 4 h at 4 °C. For embryos submitted to in situ hybridization, 0.1% diethylpyrocarbonate (DEPC) (Sigma Chem Co) was added to all solutions (Tolosa et al., 2012), dehydrated and maintained in 100% methanol at -20 °C until
Page 9 of 53
8
use, keeping the embryos in PBS for immunolabeling. Embryo morphology was evaluated using a conventional series (Hamburger and Hamilton, 1951) and no differences were observed in results between in ovo or shell-less culture groups. In situ hybridization Whole mount in situ hybridization was performed as described (Tolosa et al., 2012). Probes were synthesized from plasmids containing partial cDNA of chick Shh (Franco et al.,
ip t
1999), chick Ptch1 (Marigo et al., 1996a), chick Sufu (Pearse et al., 1999), human Gli1 and chick Gli2 (Marigo et al., 1996b) and chick Gli 3 (Schweitzer et al., 2000) and a plasmid
cr
containing the full-length cDNA of chick Slug (Del Barrio and Nieto, 2004). Sense control
and antisense riboprobes were made from the same vector and were developed according to
us
conventional methods (Sambrook et al., 1989; Tolosa et al., 2012). Immunolabeling
an
Function-blocking 5E1 anti-Shh antibody (Sigma Chem Co), neural crest marker HNK1 monoclonal antibody (Sigma Chem Co), and FITC-labeled secondary antibodies were used. With the whole embryo in PBS, the cephalic ectoderm was pierced with a tungsten
M
microneedle to allow antibody penetration, followed by the conventional immunolabeling method, with 8 to 24 h incubation with antibodies, depending on the embryo age.
d
After in situ hybridization or immunolabeling, whole mount embryos were assessed for
Ac ce pt e
distribution of NCCs on the basis of Slug and HNK1 label density at optic vesicle and mesencephalic levels as previously described (Rovasio et al., 2012; Zanin et al., 2013). Regional tissue extraction and western blot From stage 10 HH chick embryos of control or ethanol treated groups (see below), cell masses corresponding to (1) migratory mesencephalic NCCs, (2) optic vesicles, and (3) neural tube and remainder of cephalic region, were dissected and processed with the conventional method to obtain the total proteins (Sambrook et al., 1989). Samples were submitted to electrophoresis in 12-15% SDS-PAGE, transferred and immunolabeled with anti-Shh 5E1 (Sigma Chem Co) and anti-tubulin (10D8) (SC-53646, Santa Cruz Biotech. Inc., CA, USA) antibodies, then with peroxidase-labeled secondary antibody (Sigma Chem Co) and developed with the diaminobenzidine technique or by means of chemoluminescence after incubation in a solution of luminol, coumaric acid and hydrogen peroxide (10 vol), and then recorded on a photographic plate (Medical X Ray Film, Agfa, Argentina). Morpholino microinjection and electroporation Stage 8-9 HH chick embryos were submitted to morpholino (MO) microinjection with Endo-Porter®, or to microinjection and electroporation according to conventional techniques
Page 10 of 53
9
(Nakamura et al., 2004). Microinjections were made into the neural groove, delivering 0.2-0.5 µl of 1 µM MO against Smo mRNA (5’-AAAGCAGCAGCTCACCATGCTCCAT-3’ sulforhodamine B), or the standard control MO (5'-CCTCTTACCTCAGTTACAATTTATA3' carboxyfluorescein (Gene Tools, Philomath, OR, USA). A specificity control of Smo MO was performed by means of co-injection along the MO with a solution containing full length mouse Smoothened cDNA cloned in pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) and GFP
ip t
cDNA cloned in pEGFP-N1 (Addgene, Cambridge, MA, USA) at a 1/10 dilution. Another lot of embryos were delivered with a DNA plasmid encoding for mutated sequence of the Ptch
cr
receptor (Ptc∆loop2) and green fluorescent protein (GFP) (Briscoe et al., 2001). Microelectrodes were placed parallel to the mesencephalic segment and a current was released as 5 pulses of
us
squared wave of 20-25 V and cycles of 50 ms on/100 ms off, from an ELP-5 electroporator (LIADE, FCEFN, Universidad Nacional de Córdoba, Argentina) (Chesini et al., 2011), and
an
the embryos were reincubated for 15-18 h. Transfections with Endo-Porter® were made delivering 0.5-1.0 µl of 1 µM MO plus 5 µl/ml of Endo-Porter® solution (Gene Tools, LLC Philomath, USA) and reincubated without electroporation. The location of MO as well as the
M
distribution and density of NCCs were determined by Slug in situ hybridization or immunolabeling with HNK1 antibody, in function of the label density in different regions of
d
the cephalic end as indicated in the Results section, after applying conventional methods with
Ac ce pt e
the Image-J software (NIH, Bethesda, MA, USA). Microbead implants
Blocking of the signal chain started by Shh and migration/distribution of cephalic NCCs exposed to an ectopic source of Shh were evaluated. In ex ovo culture stage 8-9 HH embryos, a cut of 200 µm was made in the ectoderm parallel to the edge of the mesencephalic segment and implanted with 2.5 µM cyclopamine embedded beads (AG 1-X®, 75-180 µm, Bio-Rad Lab, Hercules, CA USA), or with 10 µg/ml Shh (Affi-Gel Blue®, 75-150 µm, Bio-Rad Lab), according to the manufacturer instructions. Ethanol exposure
In ovo and ex ovo whole embryos were exposed to 100 mM ethanol as detailed in the Results section (Battiato et al., 1995; Jaurena et al., 2011; Rovasio and Battiato, 2002). Other fertile eggs were injected in the yolk with 100 µl of 25% ethanol in PBS, reincubated until 3-5 days of development (Jaurena et al., 2011; Rovasio and Battiato, 2002), and submitted to morphologic studies and in situ hybridization to look for expression of Shh in the craniofacial region.
Page 11 of 53
10
Statistics After a minimal sample test, no fewer than 60 cells and a mean of 700 cell contours and centroids were studied in each experimental condition repeated in triplicate. Means comparisons were made with Student’s t-test and nonparametric Mann-Whitney tests, and analysis of proportions with the z-test with Yates correction, or previous transformation to the
ip t
corresponding arc-sine values and then one way ANOVA, setting significance at p <0.05.
cr
Results
Cephalic NCCs respond with chemotaxis to in vitro Shh concentration gradients
us
This report shows in vitro chemotaxis of NCCs up concentration gradients of morphogen Sonic hedgehog (Shh) using an objective method and strict directional criteria (Rovasio et al.,
an
2012). Mesencephalic NCCs exhibited a chemotactic index up Shh at initial concentrations of 5 and 10 µg/ml, with a clearly significant bias (Fig. 1 A, C). The same chemotactic result was obtained when notochord explants were used as a Shh source, or with concentrated
M
notochord-conditioned medium (CM) (Fig. 1 A). Equivalent data were also seen when NCCs confronted optic vesicle explants (not shown), confirming our previous results (Jaurena et al.,
d
2011). In contrast, at 1 µg/ml Shh, oriented migration was not significantly different to
Ac ce pt e
controls, and when migratory response was evaluated at an initial concentration of 0.2 µg/ml Shh, there was a repulsive-type of cell displacement, as well as when non-concentrated notochord CM was used (Fig. 1 A). Chemotaxis assays using notochord explants as a Shh source in presence of 2.5 µM cyclopamine, or with 10 µg/ml Shh pre-treated with functionblocking 5E1 anti-Shh antibody, or 10 µg/ml Shh on NCCs transfected with 1 µM antiSmoothened morpholino (MO), also showed repulsive migratory behavior of NCCs (Fig. 1 A). The latter comes from blocking the synthesis/expression of the transmembrane protein Smo responsible for triggering the signal chain when the Ptch receptor recognizes the Shh ligand. NCCs transfected with control MO and exposed to 10 µg/ml Shh gradient exhibited a chemotactic behavior equivalent to that of non-transfected NCCs exposed to the same Shh concentration (Fig. 1 A). When NCCs were transfected with anti-Shh MO to block any slight (non-detected) endogen expression of Shh, and exposed to 10 µg/ml Shh gradient, the chemotactic response was similar to those of the control MO or 10 µg/ml Shh alone (Fig.1 A). At the end of the chemotaxis assays, NCC morphology of MO-treated cultures was normal, with its usual nuclear incorporation and a diffuse cytosolic label (Fig. 1 E). As a
Page 12 of 53
11
delivery control of MO, preliminary experiments were carried out treating NCCs with an excess of MO (10 µM), which allowed us to confirm MO fluorescence within large vesicles and diffusely in the cytoplasm (Fig. 1 F), a sign of good cell incorporation [http://www.genetools.com/customer_support]. Moreover, NCC chemotaxis diminished to control values when evaluated on gradients of 50 µg/ml Shh, as well as with chemotactic (10 µg/ml) or repulsive (0.2 µg/ml) concentrations
ip t
of Shh in the presence of 2.5 µM cyclopamine or heat inactivated 10 µg/ml Shh (Fig. 1 A). Other control conditions were applied by using NCCs exposed to gradients of preimmune
results to other nonstimulating control conditions (Fig. 1 A).
cr
IgG, concentrated defined medium, or cyclopamine alone, showing equivalent negative
us
The real-time cell tracks clearly show the migration of NCCs up the Shh-containing gradient, changing from a centrifugal migration equidistant from the origin, as observed in
an
control experiments (Fig. 1 B), to displacement progressively approaching the Shh source (Fig. 1 C). This Shh-induced bias of oriented NCC distribution also emerged from other directional parameters such as the percentage of oriented cells, indicating that a significant
M
proportion exhibits chemotaxis up the Shh gradient (Fig. 2).
The data also showed that, after exposure to an ethanol concentration sufficient to induce
d
FAS, the chemotactic index and the proportion of oriented NCCs as well as the repulsive cell
Ac ce pt e
migration diminished significantly up the Shh gradient (Figs. 1 A and 2). This loss of Shhdependent chemotaxis was also clearly seen in the real-time cell trajectories after ethanol treatment of NCCs (Fig. 1 D).
Bearing in mind that ethanol-exposed NCCs showed a slight but significant expression of Shh (see Fig. 3 C), chemotaxis determination was also carried out after transfection with antiShh MO. The results exhibit a western blot identical to control NCCs (not shown) and no difference in chemotaxis behavior compared with the control MO, as well as a significant difference after exposure to 10 µg/ml Shh plus ethanol treatment (Fig. 1). The literature on cell motility indicates that chemotaxis (cell orientation) is not necessarily or directly associated with chemokinesis (cell speed). In this report, there were no significant differences in the curvilinear velocity of cells when cephalic NCCs were exposed to most of the experimental conditions described, regardless of the orientation pattern exhibited, except for a significant speed reduction when the cells were exposed to 10 µg/ml Shh pre-treated with function-blocking 5E1 anti-Shh antibody, or after exposure to 0.2 µg/ml (but not to 10 µg/ml) Shh plus ethanol, compared to control (Table 1). Linearity, a good estimator of the straightness of a migratory cell (where the unit represents a rectilinear
Page 13 of 53
12
trajectory), was significantly higher when NCCs were confronted with Shh at 5 or 10 µg/ml in the source of the gradient (Table 1). Inversely, the effect was reversed to a “convoluted” trajectory after NCC exposure to function-blocking 5E1 anti-Shh antibody, as well as to Shh 10 µg/ml plus ethanol (Table 1). The dramatic effect of ethanol alone on these dynamic parameters was also confirmed (Table 1) (Rovasio and Battiato, 2002). On the other hand, the cell health parameters showed differences in experiments
ip t
involving Shh- and/or ethanol-exposure. Thus, the cytotoxic index of NCC after confronting gradients of initial concentrations of 0.2 µg/ml Shh or Shh pretreated with the function-
cr
blocking 5E1 anti-Shh antibody, or 0.2 µg/ml Shh plus ethanol, was not different from the
control (Table 1). Moreover, the proportion of cell death after exposure to 5 or 10 µg/ml Shh
us
was lower than in the control, and leveled to control values when NCCs were exposed to 10 µg/ml Shh plus ethanol (Table 1), suggesting a trophic effect of Shh. As expected, exposure to
an
only ethanol had a greater cytotoxic effect on NCCs (Table 1) (Jaurena et al., 2011). The proliferation of NCCs was stimulated when exposed to attracting chemotaxis (5 or 10 µg/ml Shh), but was inhibited by the 5E1 anti-Shh antibody, leveling to control (Table 1).
M
These data, added to diminution of cell proliferation after treatment with 0.2 µg/ml Shh plus ethanol, and their leveling to control value when exposed to 10 µg/ml Shh plus ethanol (Table
d
1), also indicate a morphogen trophic effect. In the same way, exposure to ethanol without
Ac ce pt e
Shh induces a significant reduction of the proliferative capacity of NCCs (Table 1) (Jaurena et al., 2011).
Expression of Shh ligand, receptor system and signal chain in NCC cultures and whole embryos
Since in vitro cephalic NCCs respond with chemotaxis up the Shh concentration gradient, a more physiological view requires examining this cell population within the whole embryo to find evidence of elements that may explain this directional cell migration. Expression of the Shh ligand in the target field Whole embryo mRNA in situ hybridization, protein immunolabeling and tissue western blotting show the expression of Shh in the optic vesicle region of the embryo at the time NCCs migrate into it (Fig. 3). For the Shh mRNA in situ hybridization, we used early whole embryos as positive control, alongside its later stages corresponding to NCC migration toward the optic vesicle (Fig. 3 A, B). In a detailed sequential timing study, we observed that the initial expression of Shh at the Hensen’s node and notochord extends to the cephalic end, enlarges and expands laterally at the future infundibulum level at stage 11 HH (Fig. 3 D), and
Page 14 of 53
13
then spreads to both sides of the optic vesicle stalk at stage 12 HH (Fig 3 E, H), finally diffusely invading the space lateral to the mesencephalic level of the neural tube at stages 1213 (Fig. 3 F, G, I). Clearly, Shh mRNA expression topologically coincides with the invasion site of NCCs into the retro-ocular region (Fig. 3 E, G, H), where the ciliary ganglion will be formed. After a long immunolabel exposure time, it was also possible to localize the Shh protein (Fig. 3 F, I) with a distribution coherent with results from mRNA in situ hidridization,
ip t
matching the migratory pathway and destination of ciliary NCCs (see also Fig. 3 B).
Even though these results do not reveal the cellular source of the Shh nor the mechanism
cr
of forming and stabilizing the Shh concentration gradient, the results from western blot of
regional cell populations showed a clear expression of Shh in optic vesicle tissues (Fig. 3 C),
us
strongly supporting the optic stalk wall as the origin of the craniocaudal Shh gradient (Fig. 3 F-I). There was also a slight but significant expression of Shh protein in NCCs after exposure
an
to ethanol (Fig. 3 C) (see below).
Expression of the Shh receptor system in in vitro-in vivo responding NCCs In the experimental conditions of in vitro chemotaxis assays, NCCs express the receptor
M
Ptch (Fig. 4 A) and the membrane-activating protein Smo (Fig. 4 B). This receptor complex distributes with a typical punctiform pattern, clearly observed in the focus plane of the cell
d
surface (Fig. 4 A, B, insets). In the whole embryo, there is a clear co-localization of this
Ac ce pt e
receptor system (Fig. 4 C, D) with the mesencephalic population of NCCs as visualized with HNK1 immunolabeling (see Figs. 3 A, B, 6 C and 7 C). Expression of the Gli-Sufu system
In situ hybridization revealed a weak, common expression of the Gli-Sufu system shared by stages 9-11 HH of chick embryos. Gli 1-2-3 transcription factors express in the neural tube wall (Fig. 5 A-D, black arrows), as well as in the NCCs migrating toward the optic vesicle region (Fig. 5 A, withe arrows), in the pre-migratory NCCs on the dorsal surface of the cephalic and trunk neural tube (Fig. 5 B, D, white arrows), and in the optic-mesencephalic NCCs (Fig. 5 C, D, demarcated area). A light, diffuse expression of Sufu was seen in the neural tube wall (Fig. 5 E, black arrows) and in the migratory mesencephalic NCCs (Fig. 5 E, demarcated area), compared with embryos after control riboprobes (Fig. 5 F).
Directional migration and whole embryo distribution of cephalic NCCs are perturbed after blocking the signal cascade triggered by Shh After the evidence of chemotaxis of in vitro NCCs responding to the Shh signal and the expression of the main putative actors involved in this cell behavior, a natural corollary was to
Page 15 of 53
14
test the result of functional blocking of the Shh-Ptch/Smo system on in vivo distribution of cephalic NCCs. It is important to remember that there is no explanation for the clear directional change of ciliary NCCs toward the optic vesicle field (see Introduction). Several types of blocking experiments were carried out on whole embryos, and the cell migration-distribution was then evaluated with NCC-markers Slug mRNA in situ hybridization or HNK1 monoclonal antibody. To interrupt the signal system triggered by the
ip t
Shh ligand, a double strategy was used: (1) blocking the Smoothened (Smo) protein by a specific MO delivered bilaterally (EndoPorter®) or unilaterally (electroporation), and (2)
cr
inducing overexpression of the Patched-1 (Ptch) receptor by a corresponding mutated DNA plasmid to prevent Shh recognition and binding. In both cases, stage 8-9 HH embryos were
us
microinjected into the closing neural tube with MO, plasmid or their respective controls. Transfection with anti-Smo morpholino
an
Lissamine (sulphorhodamine B)-labeled anti-Smo morpholino (MO) was supplied together with the Endo-Porter® molecule. After about 10 h reincubation, the label was clearly seen inside the in vivo NCCs migrating to the lateral mesencephalic region (Fig. 6 A,
M
arrowheads) with only some of them toward the optic vesicle (Fig. 6 A, arrows). After the end of reincubation until stage 12 HH, the embryos exhibited a perturbed distribution of NCCs
d
with a slight invasion of the stalk and dorsal area of the optical vesicle (Fig. 6 B, arrows),
Ac ce pt e
accumulation of NCCs in the middle mesencephalic region (Fig. 6 B, arrowheads), and delayed NCCs (Fig. 6 B, double arrow) compared to MO control embryos (Fig. 6 C) or uninjected embryos of the same age (see Figs. 3 B and 7 C). In the control group of embryos co-injected with the chick anti-Smo MO plus a full length mouse Smo cDNA plasmid, the results of NCC distribution did not differ from those of control embryos (not shown), indicating a chemotaxis behavior recovery.
An interesting observation suggested a degree of selectivity of anti-Smo MO in the cephalic Shh-NCCs system. Thus, in remote trunk and caudal segments, pre-migratory NCCs exposed to anti-Smo MO/Endo-Porter®, such as the mesencephalic NCCs, showed no perturbation of their normal migration/distribution pattern. These cells exhibited the typical temporary arrest of NCC migration at the neural tube-somite angle (Fig. 6 B, inset, arrows) and a migratory pathway over the cephalic half of each somite (Fig. 6 B, inset, arrowheads). In another group of experiments, the electroporation-induced unilateral delivery of the injected MO made it possible to take the opposite embryonic side as an internal control (Fig. 7 A-D), also providing direct evidence of the crucial participation of the Shh ligand in oriented migration of NCCs toward the optic vesicle. In situ anti-Smo MO induced an
Page 16 of 53
15
abnormal distribution pattern of NCCs, resulting in sub-colonization of the optic vesicle field (Fig. 7 E, encircled area), together with a higher concentration of NCCs at the same lateral mesencephalic level (Fig. 7 E, rectangular area). The quantitative fluorescence intensity determined in standardized areas enabled us to establish that, on the MO side, the distribution of NCCs in the optic vesicle or the mesencephalic regions was not equivalent to that of the control side, nor compared with the symmetric displacement of NCCs on control embryos
ip t
(Fig. 7 F), indicating that the correct route had been lost through the anti-Smo MO effect, leading to a bad distribution of the ciliary population of NCCs.
cr
The abnormal distribution of NCCs after blocking the Shh/Ptch-Smo system, taken
together with the previous in vitro and in vivo data of this report, support a failure of the
us
spatial orientation of a subpopulation of mesencephalic NCCs which, under normal conditions, would be guided by a chemotactic response to Shh gradients emerging from the
an
optic vesicle region. It is worth mentioning that the embryos treated with anti-Smo MO show a quite conserved general morphology, while the NCC distribution exhibited diverse degrees of abnormalities with a common denominator of a lower density of optic NCC population.
M
Some embryos of the anti-Smo MO-treated group showed a more pronounced abnormal NCC distribution, maintaining a lower NCC concentration at the optic vesicle of the
d
electroporated side and a heavy accumulation of NCCs in the mesencephalic segment, as well
Ac ce pt e
as in the dorsal area of the prosencephalic and mesencephalic neural tube (Fig. 7 G, H). Embryos alternatively injected with anti-Smo MO in the left instead of the right side, showed the same but inverted abnormal NCC distribution described above (not shown). This additional control was considered necessary because of the early functional relation of Shh with the bilateral symmetry of the embryo (Gilbert, 2013). Transfection with mutated Ptch DNA plasmid and electroporation Another strategy to evaluate the effect of Shh gradient on the migration-distribution of cephalic NCCs was to induce the expression of the GFP-conjugated Ptc∆loop2 mutated Ptch receptor, a successful method assayed in neuronal differentiation of Drosophila, mouse and chick embryos in Shh gradients (Briscoe et al., 2001). In the present report, the mutated Ptch receptor expressed in NCCs prevented the binding of the Shh ligand, thus inducing abnormal distribution of cephalic NCCs, with acceptable conservation of the embryo morphology (Fig. 8 A). Embryos carrying the mutated Ptch receptor show a significantly lower volume of NCCs invading the stalk and the bulk of the optic vesicle (Fig. 8 B, arrows), as well as a higher accumulation of NCCs at the same lateral level of the mesencephalon (Fig. 8 B, C,
Page 17 of 53
16
rectangular areas) and a bulk of arrested NCCs at the dorsal neural tube (Fig. 8 B, C, arrowheads). Insertion of Shh- and cyclopamine-embedded beads Shh-embedded beads were inserted in stage 8-9 HH embryos under the ectoderm covering the lateral mesencephalic level and after reincubation for 15-18 h, they showed a net influence of the Shh ligand compared to controls (Fig. 9 B-D). When PBS control beads were
ip t
placed near to ciliary subpopulation of NCCs, their distribution was not substantially affected (Fig. 9 A), but Shh-embedded beads in the vicinity of the cephalic NCC population
cr
significantly affected their normal distribution (Fig. 9 B). The Shh source implanted near the migratory pathway of ciliary NCCs induced low dispersion toward the homolateral optic
us
vesicle (Fig. 9 B, arrow), as well as cell accumulation around the Shh source (Fig. 9 B, white arrowhead). On embryos simultaneously implanted with control beads in the contralateral
an
side, NCC distribution was as normal on this side as in the non-treated embryos (Fig. 9 B, black arrowhead). Moreover, when Shh beads were implanted at a slightly more caudal level, a perturbed distribution of NCCs was seen on the same side (Fig. 9 C, arrow), as well as
M
accumulation of cells around the Shh source (Fig. 9 C, arrowhead and detail). These observations clearly show that a Shh gradient is important for recruiting a subpopulation of
d
NCCs, as long as it is expressed at a suitable stage and for enough time.
Ac ce pt e
To discard the idea that the asymmetric distribution of NCCs might be attributed to cell loss, fluorescence cell density was bilaterally determined in a significant number of embryos, and the values between both sides of the optic vesicle plus mesencephalic levels were found not to be different in embryos unilaterally implanted with Shh-beads (Fig. 9 D). The same bilateral equivalence of NCCs was also found in the optic vesicle plus mesencephalic regions of embryos injected with anti-Smo MO and electroporated (see Fig. 7 F). That is to say that the reduction of oriented migration toward the optic vesicle and the simultaneous increase of NCCs migrating toward the ectopic Shh source (or their permanence at the mesencephalic level on Smo MO-treated embryos) seems to respond to directional interference, leading to a loss of the normal route for the ciliary subpopulation of NCCs. In another experimental group, when cyclopamine-embedded beads were inserted in stage 8-9 HH of chick embryos as described above, after the reincubation period most NCCs accumulated in the vicinity of the bead. Even if a few pioneer ciliary NCCs have reached the optic vesicle (Fig. 9 E, arrow), those emigrating later from the neural tube show a clearly perturbed distribution, remaining arrested at the mesencephalic level in the vicinity of the cyclopamine-embedded beads (Fig. 9 E, arrowhead).
Page 18 of 53
17
Ethanol exposure perturbs Shh-dependent chemotaxis and later distribution of cephalic NCCs Previous data on the proposed Shh-induced cell guidance mechanism, as well as in vitro and in vivo ethanol perturbation of NCC migration and distribution, encourage research into the association between ethanol exposure, the morphology of NCC-derived cephalic fields
ip t
and Shh expression at later stages of development.
Ex ovo and in ovo chick embryos treated at stages 8-9 HH (early migration of cephalic
cr
NCCs) with a dose of ethanol sufficient to induce FAS and studied at stages 11-12 HH,
showed a rather conserved general morphology (Fig. 10 A, B). However, immunolabeling of
us
NCCs revealed altered migratory patterns and perturbed distribution of cells (Fig. 10 C, D). Notably, there were NCCs still in transit from the neural tube (Fig. 10 C, D, arrows), while
an
the controls of equivalent age showed no emigrating mesencephalic NCCs (see Figs. 3 B, 6 C and 7 C).
Other groups of control or ethanol-treated embryos were reincubated until 3-5 days of
M
development, showing a global development of craniofacial structures (Fig. 11 A, D). Embryos of the ethanol-treated group showed diverse degrees of facial underdevelopment,
d
with mainly a low growth of the frontal process and a delay of telencephalic vesicle
Ac ce pt e
separation (Fig. 11 D, arrow), premature medial fusion and poor development of nasal placodes (Fig. 11 D, arrowhead) as well as of the maxillary and mandibular processes (Fig. 11 C, double arrowheads).
In a parallel experimental group, submitted to Shh mRNA in situ hybridization, we observed the structures and anomalies described above and also significant changes in the craniofacial expression of Shh in territories colonized by cephalic NCCs (Fig. 11 B, C, E, F). In 3-4 day control embryos, Shh expression was seen by transparency in the cephalic neuroectoderm, ventral ectoderm of the frontonasal protuberance, pharyngeal endoderm of the primitive mouth, maxillo-mandibular processes, notochord and neural tube floor (Fig.11 B and the inset). At 5 days, control embryos showed Shh expression in the diencephalic-ocular (optical stalk) neuroectoderm (not shown), maxillary process, roof of the stomodeum, limb primordium, umbilical region, notochord and neural tube floor (Fig. 11 C). On the other hand, the ethanol-treated 3-4 day embryos showed conserved Shh expression in the mesencephalon, diencephalon, telencephalon, frontal protuberance (Fig. 11 E and the inset), but there was a significant reduction of Shh expression in the maxillo-mandibular processes (Fig. 11 E, arrows), nasal placode (Fig. 11 E, arrowheads), ventral stomodeal ectoderm (Fig. 11 E, *),
Page 19 of 53
18
notochord and neural tube floor (Fig. 11 E, enlarged detail), and the diencephalon-ocular connection (not shown). At 5 days, the ethanol-treated embryos maintained Shh expression in the umbilical region and limb primordium (Fig. 11 F, arrowheads), but showed significant low expression in the maxillary process (Fig. 11 F, Mx), roof of the stomodeum (Fig. 11 F, *), notochord and neural tube floor (Fig. 11 F, double arrow).
ip t
Discussion
There are decades of evidence about when and where the embryonic cell distributes, but
cr
knowledge is still limited about how and why it does so with precision toward a final
destination. The efficient motility of neural crest cells (NCCs) relies on a network of genetic-
us
environmental signals (Bronner and Le Douarin, 2012; Le Douarin and Kalcheim, 1999; Rovasio et al., 1983) and, adding directional signals to those elements, the modulation of cell
an
orientation becomes very complex.
Chemotaxis may be a central embryonic cell compass
M
In the last years, chemotaxis has been rediscovered and enabled deeper study of the directional cell regulation of the axonal growth cone (Gore et al., 2008), and of several
d
procaryotic (Hazelbauer, 2012) and eucaryotic cells (Giojalas and Rovasio, 1998; Guidobaldi
Ac ce pt e
et al., 2012; Insall and Andrew, 2007), including the NCCs (Mwizerwa et al., 2011; Rovasio et al., 2012; Tolosa et al., 2012; Toyofuku et al., 2008; Zanin et al., 2013). A key point in designing the present work was to make the spatiotemporal frame of in vitro experiments compatible with the in vivo biological model, focusing on the migration of mesencephalic NCCs that reach the optic vesicle in a time window of 30 to 35 hours (Newgreen and Erickson, 1986). This ciliary subpopulation of NCCs separates from the bulk of cells and turns 90° toward the optic vesicle (Lee et al., 2003), a directional change that has not yet been attributed to any known modulating factor. The present in vitro approach to the migration of in vivo cells to the optic primordium is supported by our previous work showing that “old” NCC cultures of >40 hours progressively lose chemotactic capacity (Zanin et al., 2013). An important and unsurprising result of the chemotactic activity of Shh on cephalic NCCs was its coherence with data of others showing that this morphogen seems to work, not as an individual determinant, but as part of a collectively concurring strategy to orient cell motility (Jaurena, 2011; Mayor and Theveneau, 2013; McLennan et al., 2012; Rovasio et al.,
Page 20 of 53
19
2012; Theveneau et al., 2013; Tolosa et al., 2012; Zanin et al., 2013). It would thus be not too simplistic to think that such an important function as the final location of a cell population may respond not to just one molecule, and that we should rather expect the confluence (redundance?) of several guiding factors. The evolutionary interpretation would be as compensation for possible failures of the directional distribution of embryonic cells, a redundant mechanism well-known in other patterns of cell distribution (Gilbert, 2013) and
ip t
neurotrophic neuronal differentiation (Le Douarin and Kalcheim, 1999), even though it is
poorly studied in guided embryonic cell migration. Other examples of cooperative guidance
cr
emerge from several versions of the trophic factor FGF (Kubota and Ito, 2000), the RGMa/Neogenin (Gessert et al., 2008), VEGF/Neurotrophin (McLennan et al., 2010) and
us
Semaphorin/Neuropilin (Schwarz et al., 2008) systems involved in NCC guidance, as well as other short and long range communication factors (Mayor and Theveneau, 2013; Theveneau
an
et al., 2013). In our laboratory, besides evidence of a chemotactic Stem cell factor/cKit system guiding the cephalic and trunk NCCs toward the skin (Rovasio et al., 2012), it was shown that the neurotrophin NT-3/TrkC-p75 (Zanin et al., 2013), chemokine SDF-1/CXCR4 (Jaurena,
M
2011) and morphogen Shh/Ptch-Smo systems (Tolosa et al., 2012, and this report) have
d
definite guidance functions for cephalic NCCs invading the optic vesicle field.
NCCs
Ac ce pt e
The morphogen Shh expresses at the optic vesicle and induces chemotaxis on cephalic
Present data from mRNA in situ hybridization and protein immunolabeling show the expression of Shh in the route and the destination of the ciliary subpopulation of cephalic NCCs. The Shh morphogen as a chemotactic factor was involved with Netrin in the orientation of commissural axons (Charron et al., 2003) and the migration of oligodendrocytes (Merchán et al., 2007). Our real-time results (Rovasio et al., 2012) clearly show that a subpopulation of mesencephalic NCCs respond with Ptch/Smo-dependent chemotaxis up concentration gradients of Shh, excluding other migratory guidance. The data came from experiments with purified Shh, as well as optic vesicle or notochord-conditioned media, and co-cultures with notochord explants, a known source of Shh (Martí et al., 1995). Further interruption of NCC chemotaxis up Shh gradients by a function-blocking antibody (Marcucio et al., 2005) or Smo-binding cyclopamine (Chen et al., 2002) or anti-Smo MO (Locker et al., 2006) provided additional validation for our in vitro results. Moreover, the low chemotactic response after perturbed expression/function of Shh did not result from cell death, because in vitro chemotaxis was determined in real-time motile (alive) cells and their trophic parameters
Page 21 of 53
20
were not different from control conditions. This also indicates that Shh-elicited chemotaxis may be a substantial mechanism for in vivo spatiotemporal orientation of NCCs to the optic vesicle. The diverse representations of oriented cell migration were coherent, all showing a bellshaped curve typical of chemotactic response (Gore et al., 2008; Guidobaldi et al., 2012; Rovasio et al., 2012; Tolosa et al., 2012; Zanin et al., 2013). This gradient-dependent
ip t
response indicates that, when the attractant concentration increases, the specific receptors remain totally occupied and are thus incapable of detecting changes coming from the
cr
extracellular milieu, and the chemotactic response falls. This self-regulation of chemotaxis may also be involved in the arrest of migration at the colonized site (source of the
us
concentration gradient), and may help to explain the less-understood mechanism of chemorepulsion (see below), since the “less attracted” cells arriving at the target site may be
an
redirected by other chemotactic systems toward a different field. This interesting “relay station” behavior is well known among social amoebas (García and Parent, 2008), leukocytes (Afonso et al., 2012), or metastatic cells (Roussos et al., 2011). This is a less explored
M
approach to the distribution of embryonic cells, except for some pioneering data showing that in the wild type Xenopus, the Ihh signal released by cranial NCCs is itself required to support
d
both autocrine and paracrine cell migration (Agüero et al., 2012). Our present results after
Ac ce pt e
blocking any expression of endogenous NCC-expressed Shh indicate that this is not the case for the chick NCCs-Shh-optic field system.
Cells go forward, but also go backward
Besides the bell-shaped chemotactic curve, a chemo-repulsion effect was observed in NCCs confronted with a concentration of Shh 0.2 µg/ml, as well as in the presence of cyclopamine, anti-Shh antibody or anti-Smo MO. This repulsive phenomenon has been more studied in leukocyte migration toward the tissues and back to the blood (Holmes et al., 2012), as well as in the directional axon growth cone when wiring the nervous system (Ferrario et al., 2012). However, chemorepulsion has scarcely been studied except in NCC migration in the trunk ventral pathway (Jia et al., 2005), the outflow tract of the heart (Toyofuku et al., 2008), enteric innervation (De Bellard et al., 2003), and melanoma metastatic expansion (Amatschek et al., 2011). In our laboratory, we have shown this concentration-dependent bi-phasic effect in trophic factor SCF (Rovasio et al., 2012), chemokine SDF-1 (Jaurena, 2011), and morphogen Shh (Tolosa et al., 2012, and this report) on cephalic NCCs; but no similar effect was observed with neurotrophic factor NT-3 under the same conditions and the same cell
Page 22 of 53
21
population (Zanin et al., 2013). Chemorepulsion remains unexplained today probably because there is little evidence for the notion that similar molecules drive the cell forward or backward, regulating the opposite displacements by means of small, but significant, changes in some factor(s) of the taxis-associated signal chains (Ferrario et al., 2012). It should also be remembered that the existence of repulsion could be masked by the study method, since the classical transfilter methods (Boyden, 1962) do not reveal the proportion of cells migrating
ip t
against the chemotactic gradient, nor the cell velocity/distance traveled. In this context, different to the reduction of NCC cell speed elicited by chemotactic trophic factor SCF
cr
(Rovasio et al., 2012), or chemokine SDF-1 (Jaurena, 2011), our present data indicate that
chemotactic concentrations of 10 µg/ml Shh do not induce changes of speed but an increase of
us
the straightness of NCCs, suggesting that the morphogen signal involves directional coordination, without essential participation in other parameters of the cell motility
an
mechanism.
Extracellular Shh ligand triggers chemotactic signal chains
M
The fact that a subpopulation of NCCs migrating in vitro and in vivo toward the optic vesicle expresses the Ptch/Smo receptor/activator system, indicates that they respond in a
d
selective manner to exogenous Shh ligand in in vitro assays or expressed in the optic vesicle
Ac ce pt e
field of the embryo, where the ciliary ganglion of the eye will form, mostly derived from this cell population (Le Douarin and Kalchein, 1999; Lee et al., 2003). In all of our experiments, we have seen that only a fraction of the NCCs respond to Shh, suggesting a molecular heterogeneity of its regulatory compass. Considering that NCCs invade several sites of the craniofacial region (Le Douarin, 2004; Le Douarin et al., 2007), it is clear that their receptor/signal chain complex is not homogeneous and not necessarily all NCCs respond simultaneously to directional stimulus of Shh or other molecules (Mayor and Theveneau, 2013; McLennan et al., 2012; Rovasio et al., 2012; Theveneau et al., 2013; Tolosa et al., 2012; Zanin et al., 2013).
Even though the spatiotemporal expression of Shh at the target site and its receptor on the migrating NCCs supports the proposed guide function, neither the mechanism of synthesis and release of Shh morphogen nor the factor(s) involved in gradient formation and stability can be inferred. We can only speculate about the consumption of the attractant molecule by NCCs or the modulation of attractant release at the source or its variant source-sink model (Yu et al., 2009).
Page 23 of 53
22
The significant reduction of in vitro and in vivo NCC migration up the Shh gradient after treatment with anti-Shh antibody, anti-Smo MO, Smo-blocking cyclopamine, or overexpression of mutated Ptch receptor, is clearly the result of losing the way toward the optic vesicle. This also explains the concurrent high population of NCCs at the mesencephalic level of the same side of the embryo, suggesting that the cells that could not orientate toward the optic vesicle distribute on the lateral regions. This evidence is also supported by the
ip t
recovery of the normal distribution of NCCs on embryos co-injected with chick anti-Smo MO plus a full-length mouse Smo cDNA plasmid. These results indicate that the Shh morphogen
cr
is important for guiding NCCs toward the ocular region, without disregarding other
concurrent molecules and mechanisms (Jaurena, 2011; Rovasio et al., 2012; Theveneau et al.,
us
2013; Tolosa et al., 2012; Zanin et al., 2013).
The above data may also explain the “abnormal expression levels of secreted guidance
an
signals from the eye along the dorsal surface of the budding eye vesicle, which deserve elucidation” in a report on perturbed migration of cephalic NCCs in the mutant chokh/rx3 of
(Langenberg et al., 2008).
M
zebrafish, lacking eye development without interference of the neural system development
The signal chain(s) activated by Shh > Ptch > Smo interactions is a major issue that is
d
almost unknown in the biological model we are studying, and we decided not to cover it here.
Ac ce pt e
Considering that the recognition of extracellular gradient and the directional change occur within minutes, it is reasonable to think that cell orientation occurs regardless of transcriptional activity, by connecting to a non-canonical pathway, excluding (at least initially) the transcriptional system Gli-Sufu. This was proposed as the activation by Shh of cytoskeletal-associated systems such as Ras/MEK-Erk1/2 (Chang et al., 2010), Tiam1/Rac1 (Sasaki et al., 2010), 5-Lipooxigenase/Rho (Bijlsma et al., 2007), PIP3/DOCK2/Rho (Nishikimi et al., 2009) and Gli3-Sufu (Zhang et al., 2005), with involvement of the Ptch receptor and with (or without) activation of Smo activating protein (Chang et al., 2010). On the other hand, at the final stages of chemotaxis, in a matter of hours, the involvement of canonical ways (Shh > Ptch-Smo > Gli >...> ADN) is reasonable, as a transcriptional phase associated to the final steps of oriented cell migration, distribution and arrest. It is currently accepted that both canonical and non-canonical signal pathways, in a sequential or combinatory manner and sometimes with redundance, modulate processes of cell motility, proliferation and differentiation (Ayers and Thérond, 2010; Bourikas et al., 2005; Jenkins, 2009; Okada et al., 2006; Tenzen et al., 2006). Under this rationale, although the expression of Gli-Sufu system was seen in the present work in NCCs and other cell populations of the
Page 24 of 53
23
embryo, it does not seem that their involvement may qualify as actors of early orientation of the cell. It may be imprudent to propose a “principal” factor to explain any biological phenomenon, but it is fair to think of conserved elements receiving convergent triggering signals from extracellular ligand(s), then being transduced to the cytoskeleton to start the shape changes and oriented cell motility. Detection of a chemotactic gradient is a conserved
ip t
strategy from prokaryote to eukaryote cells, and spatial detection of the gradient around the
cell and the transfer of the signals to intracellular chains is accepted for the latter, frequently
cr
working with the PI3-kinase /PIP3 system and Rho-GTPases cytoskeleton modulators. This general view, conserved in amoebas, leukocytes, embryonic cells, neurons and axons (von
us
Philipsborn and Bastmeyer, 2007), is a main focus of our present research on directional cell motility. As a first conclusion, our direct experimental evidence shows that the extracellular
an
Shh concentration gradient induces directional changes of cephalic NCCs with a dynamic response of chemotaxis, suggesting a general model for cell distribution toward the ocular
M
region (Fig. 12).
The Shh-dependent chemotaxis of cephalic NCCs is perturbed by ethanol exposure
d
The high capacity of NCCs to disperse along the embryo body and produce many
Ac ce pt e
derivatives gives them increased importance in the pathogeny of developmental and tumoral pathologies (Bolande, 1974; Le Douarin et al., 2007; Matera et al., 2008), such as in craniofacial phenotypes of Fetal Alcohol Syndrome (FAS) (Chen et al., 2000; Jaurena et al. 2011; Rovasio and Battiato, 1995, 2002).
We have previously shown the ethanol-induced irreversible damage of in vitro and in vivo migratory behavior of NCCs (Rovasio and Battiato, 1995, 2002), as well as the loss of chemotactic migration up chemokine SDF-1 gradients (Jaurena, 2011). In the present report, the arrest of normal chemotaxis up Shh gradients in the presence of a teratogenic concentration of ethanol matches other reports showing disregulation of Shh signals in chick and mouse embryos treated with ethanol (Yamada et al., 2005), also producing craniofacial anomalies attributable to NCC perturbation (Ahlgren et al., 2002). Moreover, western blot results showed that embryos exposed to ethanol express Shh in the NCCs, whereas the controls only express in the optic vesicle and the remainder of the neural tube. In physiological conditions, it is also known that endogenous Ihh signaling is continuously required for the normal migration of Xenopus cranial NCCs, suggesting that this Hedgehog molecule may act in both autocrine and paracrine fashion (Agüero et al., 2012).
Page 25 of 53
24
However, our present data did not enable us to establish whether the changes in Shh expression in NCCs are part of the ethanol-induced Shh perturbation (Yamada et al., 2005), or are a consequence of an induced relay mechanism by which some NCCs begin to release the morphogen, as has been found in amoebas (García and Parent, 2008), leukocytes (Afonso et al., 2012) and tumoral cells (Roussos et al., 2011). Our experiments of blocking the endogenous Shh by ethanol-exposed NCCs enable us to suggest that this Shh expression does
ip t
not change the perturbed chemotaxis observed in this experimental condition.
In addition to perturbation of NCC chemotaxis, ethanol exposure diminished the cell
cr
linearity in gradients of 10 µg/ml Shh. Moreover, while the cytotoxic effect increased as expected in NCC cultures treated with only ethanol (Jaurena et al., 2011; Rovasio and
us
Battiato, 2002), the presence of ethanol in Shh gradients led to a drop in the proportion of cell death equal to that in the control condition. A similar trophic behavior response was found
an
when the proliferation index was evaluated. The results as a whole support the idea that ethanol-induced development anomalies may be more associated with perturbed NCC distribution (Tapadia et al., 2005) than with cell death (Ahlgren et al., 2002). Matching our
M
previous data on the protection of ethanol-treated NCCs by co-treatment with neurotrophin-3 (Jaurena et al., 2011), the present data also open up new perspectives for therapeutic and/or
Ac ce pt e
and Battiato, 1995, 2002).
d
preventive tools applicable to ethanol-induced perturbations (Jaurena et al., 2011; Rovasio
Our data on later stage craniofacial morphogenesis after early ethanol exposure show great similarity with phenotypes of FAS and those induced after blocking the Shh signal chains (Ahlgren et al., 2002; Hu et al., 2015; Marcucio et al., 2005; Tapadia et al., 2005), and thus help to explain the pathogeny of this embryopathology. We frequently saw irregular expression of in situ Shh expression in these ethanol-treated abnormal embryos, mainly in the cephalic and notochord/neural tube areas. Curiously, in other regions, such as the central nervous system wall and the bud limbs, Shh expression was not different to that in equivalent control sites. Also, considering the context of embryonic development, it is clear that a brief period of abnormal NCC behavior could be sufficient to cause serious and irreversible consequences in later stages of development, such as we found in this experimental model (Rovasio and Battiato, 1995, 2002; Jaurena et al., 2011). Our results thus support the current agreement on the risk of ethanol exposure, even for a short period and/or at a low dose, during all the period of gestation and lactancy.
Conclusions
Page 26 of 53
25
This work enabled us to show that cephalic NCCs migrate in an oriented manner as a chemotactic response to an exogenous concentration gradient of the Shh morphogen. Whole embryo in situ Shh expression in the optic vesicle region at the time of colonization by cephalic NCCs, as well as in vitro and in vivo expression of the receptor Ptch and the activating protein Smo on cephalic NCCs, complete a guidance system to orient this cell population toward their target. In vitro and whole embryo molecular and pharmacological
ip t
blocking of this system inhibit the chemotactic migration of cephalic NCCs, preventing them finding or remaining on the correct route to colonize the optic field (see Fig. 12). The lack of
cr
a generalized response of NCCs suggests that the guide function of Shh is part of a more
complex mechanism of oriented migration, complemented with other guide molecules, as was
us
supported by our and other works. This contributes to the growing concept of the convergence or redundancy of molecular signals being responsible for the modulation of key aspects of cell
an
guidance in embryo development.
As a significant derivation of the above cues, another important result of this work was the selective perturbation of cephalic NCC chemotactic guidance after in vitro and in vivo
pathogeny of this embryopathology.
M
ethanol exposure at doses sufficient to induce FAS, which also helps to better understand the
d
These results, besides improving the understanding of a basic mechanism for precise
Ac ce pt e
embryonic cell distribution and their eventual perturbation by usual teratogen agents, also contribute a new guidance activity for the Shh morphogen, apart from its other important canonical functions.
Acknowledgments
We thank Mr Joss Heywood for critical reading of the manuscript. Plasmids with gene sequences to produce chick Shh probes were kindly donated by Dr. Andrés Carrasco (FM, UBA-CONICET, Argentina), and a plasmid containing the full-length cDNA of chick Slug was kindly provided by Dr. M. A. Nieto (Instituto Cajal, Madrid, Spain). Microbeads for implantation experiments were kindly provided by Dr. Manuel Aybar (Depto. Biología del Desarrollo, INSIBIO-CONICET-Universidad Nacional de Tucumán, Argentina). This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), the Agencia Nacional de Promoción Científica y Tecnológica (FONCYT), the Ministerio de Ciencia y Tecnología de la Provincia de Córdoba and the Secretaria de Ciencia y Tecnología de la Universidad Nacional de Córdoba (Argentina).
Page 27 of 53
26
References Afonso, P. V., Janka-Junttila, M., Lee, Y. J., McCann, C. P., Oliver, C. M., Aamer, K. A., Losert, W., Cicerone, M. T., Parent, C. A., 2012. LTB4 is a signal-relay molecule during neutrophil chemotaxis. Dev. Cell 22, 1079-1091. Agüero, T. H., Fernández, J. P., Vega López, G., Tríbulo, C., Aybar, M. J., 2012. Indian hedgehog signaling is required for proper formation, maintenance and migration of
ip t
Xenopus neural crest. Dev. Biol. 364, 99-113.
Ahlgren, S. C., Thakur, V., Bronner-Fraser, M., 2002. Sonic hedgehog rescues cranial neural
cr
crest from cell death induced by ethanol exposure. Proc. Natl. Acad. Sci. USA 99, 1047610481.
us
Amatschek, S., Lucas, R., Eger, A., Pflueger, M., Hundsberger, H., Knoll, C., Grosse-Kracht, S., Schuett, W., Koszik, F., Maurer, D., Wiesner, C., 2011. CXCL9 induces chemotaxis,
melanoma cells. Br. J. Cancer 104, 469-479.
an
chemorepulsion and endothelial barrier disruption through CXCR3-mediated activation of
Angot, E., Loulier, K., Nguyen-Ba-Charvet, K., T., Gadeau, A. P., Ruat, M., Traiffort, E.,
M
2008. Chemoattractive activity of Sonic Hedgehog in the adult subventricular zone modulates the number of neural precursors reaching the olfactory bulb. Stem Cells 26,
d
2311-2320.
Ac ce pt e
Ayers, K. L., Thérond, P. P., 2010. Evaluating Smoothened as a G-protein-coupled receptor for Hedgehog signaling. Trends Cell Biol. 20, 287-298. Barnes, D., Sato, G., 1980. Methods for growth of culture cell in serum-free medium. Anal. Biochem. 102, 255-270.
Battiato, N. L., Paglini, M. G., Salvarezza, S. B., Rovasio, R. A., 1996. Method for treatment and processing of whole chick embryos for autoradiography, immuno-cytochemistry and other techniques. Biotech. & Histochem. 71, 286-288. Bijlsma, M.F., Borensztajn, K. S., Roelink, H., Peppelenbosch, M. P., Spek, C. A., 2007. Sonic hedgehog induces transcription-independent cytoskeletal rearrangement and migration regulated by arachidonate metabolites. Cell Signal 19, 2596-2604. Bolande, R. P., 1974. The neurocristopathies: a unifying concept of disease arising in neural crest maldevelopment. Human Path. 5, 409-429. Bourikas, D., Pekarik, V., Baeriswyl, T., Grunditz, A., Sadhu, R., Nardó, M., Stoeckli, E. T., 2005. Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat. Neurosci. 8, 297-304.
Page 28 of 53
27
Boyden, S. V., 1962. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes. J. Exp. Med. 115, 453-466. Breau, M.A, Wilson, D., Wilkinson, D. G., Xu, D. G., 2012. Chemokine and Fgf signaling act as opposing guidance cues in formation of the lateral line primordium. Development 139, 2246–2253. Briscoe, J., Chen, Y., Jessell, T. M., Struhl, G., 2001. A Hedgehog-insensitive form of
ip t
Patched provides evidence for direct long-range morphogen activity of Sonic Hedgehog in the neural tube. Molec. Cell 7, 1279-1291.
cr
Briscoe, J., Ericson, J., 2001. Specification of neuronal fates in the ventral neural tube. Curr. Op. Neurobiol. 11, 43-49.
us
Bronner M. E., Le Douarin N. M., 2012. Development and evolution of the neural crest: An overview. Dev Biol 366:2-9.
an
Chang, H., Li, Q., Moraes, R. C., Lewis, M. T., Hamel T. A., 2010. Activation of Erk by sonic hedgehog independent of canonical hedgehog signaling. Int. J. Biochem. Cell Biol. 42, 1462-1471.
M
Charron, F., Stein, E., Jeong, J., McMahon, A. P., Tessier-Lavigne, M., 2003. The morphogen Sonic Hedgehog is an axonal chemoattractant that collaborates with Netrin-1 in midline
d
axon guidance. Cell 113, 11-23.
Ac ce pt e
Chen, J. K., Taipale, J., Cooper, M. K., Beachy, P. A., 2002. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Develop. 16, 2743-2748. Chen, S., Periasamy, A., Yang, B., Herman, B., Jacobson, K., Sulik, K, K., 2000. Differential sensitivity of mouse neural crest cells to ethanol-induced toxicity. Alcohol 20, 75-81. Chesini, E., Bigliani, J. C., Zanin, J. P., Rovasio, R. A., Taborda, R. A. M., 2011. Electroporator applying to research on molecular and developmental biology. XVIII Congress of Bioingeneering. SABI, pp. 1-8. Buenos Aires, Argentina. De Bellard, M. E., Rao, Y., Bronner-Fraser, M., 2003. Dual function of Slit2 in repulsion and enhanced migration of trunk, but not vagal, neural crest cells. J. Cell Biol. 162, 269-279. Del Barrio, M. G., Nieto, M. A., 2004. Relative expression of Slug, RhoB, and HNK-1 in the cranial neural crest of the early chicken embryo. Dev. Dyn. 229, 136–139. Deshpande, G., Swanhart, L., Chiang, P., Schedl, P., 2001. Hedgehog signaling in germ cell migration. Cell 106, 759-769. Etheridge, L. A., Crawford, T. Q., Zhang, S., Roelink, H., 2010. Evidence for a role of vertebrate Disp1 in long-range Shh signaling. Development 137, 133-140.
Page 29 of 53
28
Ferrario, J. E., Baskarana, P., Clark, C., Hendry, A., Lerner, O., Hintze, M., Allen, J., Chilton, J. K., Guthrie, S., 2012. Axon guidance in the developing ocular motor system and Duane retraction syndrome depends on Semaphorin signaling via alpha2-chimaerin. Proc. Natl. Acad. Sci. USA 109, 14669-14674. Franco, P. G., Paganelli, A. R., López, S. L., Carrasco, A. E., 1999. Functional association of retinoic acid and hedgehog signaling in Xenopus primary neurogenesis. Development 126,
ip t
4257-4265.
Fu, M., Lui, V. C. H., Sham, M. H., Pachnis, V., Tam, P. K. H., 2004. Sonic hedgehog
cr
regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut. J. Cell Biol. 166, 673-684.
us
García, G. L., Parent, C. A., 2008. Signal relay during chemotaxis. J. Microsc. 231, 529-534. Gessert, S., Maurus, D., Kuhl, M., 2008. Repulsive guidance molecule A (RGM A) and its
an
receptor neogenin during neural and neural crest cell development of Xenopus laevis. Biol. Cell 100, 659-673.
Gilbert, S. F., 2013. Developmental Biology. 10th ed. Sinauer Ass. Inc., Publ., Sunderland,
M
MA.
Giojalas, L. C., Rovasio, R. A., 1998. Mouse spermatozoa modify their motility parameters
Ac ce pt e
201-206.
d
and chemotactic response to factors from the oocyte microenvironment. Int. J. Androl. 21,
Gore, B. B., Wong, K. G., Tessier-Lavigne, M., 2008. Stem cell factor functions as an outgrowth-promoting factor to enable axon exit from the midline intermediate target. Neuron 57, 501-510.
Guidobaldi, H. A., Teves, M. E., Uñates, D. R., Giojalas, L. C., 2012. Sperm transport and retention at the fertilization site is orchestrated by a chemical guidance and oviduct movement. Reproduction 143, 587-596.
Hamburger, V., Hamilton, H. L., 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92.
Hazelbauer, G. L., 2012. Bacterial Chemotaxis: The Early Years of Molecular Studies. Annu Rev. Microbiol. 66, 285-303. Holmes, G. R., Anderson, S. R., Dixon, G., Robertson, A. L., Reyes-Aldasoro, C. C., Billings, S. A., Renshaw, S. A., Kadirkamanathan, V., 2012. Repelled from the wound, or randomly dispersed? Reverse migration behaviour of neutrophils characterized by dynamic modeling. J. R. Soc. Interface 9, 3229–3239.
Page 30 of 53
29
Hu, D., Young, N. M., Li, X., Xu, Y., Hallgrimsson, B., Marcucio, R. S., 2015. A dynamic Shh expression pattern, regulated by SHH and BMP signaling, coordinates fusion of primordia in the amniote face. Development 142, 567-574. Insall, R., Andrew, N., 2007. Chemotaxis in Dictyostelium: how to walk straight using parallel pathways. Curr. Op. Microbiol. 10, 578-581. Jaurena, M. B., Carri, N. G., Battiato, N. L., Rovasio, R. A., 2011. Trophic and proliferative
prevented by Neurotrophin 3. Neurotoxicol. Teratol. 33, 422-430.
ip t
perturbations of in vivo/in vitro cephalic neural crest cells after ethanol exposure are
cr
Jaurena, M.B., 2011. The chemokine Stromal cell-Derived Factor-1 participate in the oriented migration of neural crest cells in normal ontogenesis and exposed to ethanol. PhD Thesis
us
Dissertation. Biology Sciences School, pp. 78. National University of Cordoba, Argentina. Jenkins, D., 2009. Hedgehog signalling: emerging evidence for non-canonical pathways. Cell
an
Signal 21, 1023-1034.
Jia, L., Cheng, L., Raper, J., 2005. Slit/Robo signaling is necessary to confine early neural crest cells to the ventral migratory pathway in the trunk. Dev. Biol. 282, 411-421.
M
Kubota, Y., Ito, K., 2000. Chemotactic migration of mesencephalic neural crest cells in the mouse. Dev. Dyn. 217, 170-179.
d
Kumada, T., Komuro, Y., Li, Y., Wang Z, Littner Y, Komuro H., 2010. Inhibition of
Ac ce pt e
cerebellar granule cell turning by alcohol. Neurosci. 170, 1328-1344. Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
Langenberg, T., Kahana, A., Wszalek, J. A., Halloran, M. C., 2008. The eye organizes neural crest cell migration. Dev. Dyn. 237, 1645-1652. Le Douarin, N. M., Brito, J. M., Creuzet, S., 2007. Role of the neural crest in face and brain development. Brain Res. Rev. 55, 237-247. Le Douarin, N. M., Kalcheim, C., 1999. The Neural Crest. 2nd ed. Cambridge Univ. Press. Cambridge.
Le Douarin, N. M., 2004. The avian embryo as a model to study the development of the neural crest: a long and still ongoing story. Mech. Dev. 121, 1089-1102. Lee, V. M., Sechrist, J. W., Luetolf, S., Bronner-Fraser, M., 2003. Both neural crest and placode contribute to the ciliary ganglion and oculomotor nerve. Dev. Biol. 263, 176-190. Locker, M., Agathocleous, M., Amato, M. A., Parain, K., Harris, W. A., Perron, M., 2006. Hedgehog signaling and the retina: insights into the mechanisms controlling the proliferative properties of neural precursors. Genes Dev. 20, 3036–3048.
Page 31 of 53
30
Marcucio, R. S., Cordero, D. R., Hu, D., Helms, J. A., 2005. Molecular interactions coordinating the development of the forebrain and face. Dev. Biol. 284, 48-61. Marigo, V., Scott, M. P., Johnson, R. L., Goodrich, L. V., Tabin, C. J., 1996a. Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development 122, 1225-1233. Marigo, V., Johnson, R. L., Vortkamp, A., Tabin, C. J., 1996b. Sonic hedgehog differentially
ip t
regulates expression of GLI and GLI3 during limb development. Dev. Biol. 80, 273–283 Martí, E., Takada, R., Bumcrot, D. A., Sasaki, H., McMahon, A. P., 1995. Distribution of
cr
Sonic hedgehog peptides in the developing chick and mouse embryo. Development 121, 2537-2547.
us
Matera, I., Watkins-Chow, D. E., Loftus, S. K., Hou, L., Incao, A., Silver, D. L., Rivas, C., Elliott, E. C., Baxter, L. L., Pavan, W. J., 2008. A sensitized mutagenesis screen identifies
an
Gli3 as a modifier of Sox10 neurocristopathy. Hum. Mol. Genet. 17, 2118-2131. Mayor, R., Theveneau, E., 2013. The neural crest. Development 140, 2247–2251. McLennan, R., Teddy, J. M., Kasemeier-Kulesa, J. C., Romine, M. H., Kulesa, P. M., 2010.
Dev. Biol. 339, 114-125.
M
Vascular endothelial growth factor (VEGF) regulates cranial neural crest migration in vivo.
d
McLennan, R., Dyson, L., Prather, K. W., Morrison, J. A., Baker, R. B., Maini, P. K., Kulesa,
Ac ce pt e
P. M., 2012. Multiscale mechanisms of cell migration during development: theory and experiment. Development 139, 2935–2944. Merchán, P., Bribian, A., Sánchez-Camacho, C., Lezameta, M., Bovoleta, P., de Castro, F., 2007. Sonic hedgehog promotes the migration and proliferation of optic nerve oligodendrocyte precursors. Mol. Cell Neurosci. 36, 355-368. Mortimer, D., Fothergill, T., Pujic, Z., Richards, L. J., Goodhill, G. J., 2008. Growth cone chemotaxis. Trends Neurosci. 31, 90-98. Mwizerwa, O., Das, P., Nagy, N., Akbareian, S. E., Mably, J. D., Goldstein, A. M., 2011. Gdnf is mitogenic, neurotrophic, and chemoattractive to enteric neural crest cells in the embryonic colon. Dev. Dyn. 240, 1402-1411. Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., Funahashi, J., 2004. Gain- and loss-offunction in chick embryos by electroporation. Mech. Dev. 121, 1137-1143. Newgreen, D. F., Erickson, C. A., 1986. The migration of neural crest cells. Int. Rev. Cytol. 103, 89-145. Nishikimi, A., Fukuhara, H., Su, W., Hongu, T., Takasuga, S., Mihara, H., Cao, Q., Sanematsu, F., Kanai, M., Hasegawa, H., Tanaka, Y., Shibasaki, M., Kanaho, Y., Sasaki,
Page 32 of 53
31
T., Frohman, M. A., Fukui, Y., 2009. Sequential regulation of DOCK2 dynamics by two phospholipids during neutrophil chemotaxis. Science 324, 384-387. Okada, A., Charron, F., Morin, S., Shin, D. S., Wong, K., Fabre, P. J., Tessier-Lavigne, M., McConnell, S. K., 2006. Boc is a receptor for sonic hedgehog in the guidance of commissural axons. Nature 444, 369-373. Paganelli, A., Gnazzo, V., Acosta, H., López, S. L., Carrasco, A. E., 2010. Glyphosate-based
ip t
herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling. Chem. Res. Toxicol. 23, 1586-1595.
cr
Paratcha, G., Ibañez, C. F., Ledda, F., 2006. GDNF is a chemoattractant factor for neuronal precursor cells in the rostral migratory stream. Mol. Cell Neurosci. 31, 505-514.
us
Pearse II, R. V., Collier, L. S., Scott, M. P., Tabin, C. J., 1999. Vertebrate homologs of Drosophila suppressor of Fused interact with the Gli family of transcriptional regulators.
an
Dev Biol 212, 323-336.
Ricardo, S., Lehmann, R., 2009. An ABC transporter controls export of a Drosophila germ cell attractant. Science 13, 943-946.
Cancer 11, 573-587.
M
Roussos, E. T., Condeelis, J. S., Patsialou, A., 2011. Chemotaxis in cancer. Nature Rev.
d
Rovasio, R. A., Battiato, N. L., 1995. Role of early migratory neural crest cells in
Ac ce pt e
developmental anomalies induced by ethanol. Int. J. Dev. Biol. 39, 421-422. Rovasio, R. A., Battiato, N. L., 2002. Ethanol induces morphological and dynamic changes on in vivo and in vitro neural crest cells. Alcohol. Clin. Exp. Res. 26: 1286-1298. Rovasio, R. A., Delouvée, A., Yamada, K. M., Timpl, J. P., Thiery, J. P., 1983. Neural crest cell migration: requirements for exogenous fibronectin and high cell density. J. Cell Biol. 96, 462-473.
Rovasio, R. A., Faas, L., Battiato, N. L., 2012. Insights into Stem Cell Factor chemotactic guidance of neural crest cells revealed by a real-time directionality-based assay. Eur. J. Cell Biol. 91, 375-390.
Salvarezza, S. B., Rovasio, R. A., 1997. Exogenous retinoic acid decreases in vivo and in vitro proliferative activity during the early migratory stage of neural crest cells. Cell Prolifer. 30, 71-80. Sambrook, J., Fritsch, E. F., Maniatis, T., 1989. Molecular cloning – A laboratory manual. 2nd ed. Cold Spring Harbor Lab Press. New York.
Page 33 of 53
32
Sasaki, N., Kurisu, J., Kengaku, M., 2010. Sonic hedgehog signaling regulates actin cytoskeleton via Tiam1–Rac1 cascade during spine formation. Mol. Cell Neurosci. 45, 335-344. Schwarz, Q., Vieira, J. M., Howard, B., Eickholt, B. J., Ruhrberg, C., 2008. Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells. Development 135, 1605-1613.
ip t
Schweitzer, R., Vogan, K. J., Tabin, C. J., 2000. Similar expression and regulation of Gli2 and Gli3 in the chick limb bud. Mech. Dev. 98, 171-174.
cr
Tapadia, M. D., Cordero, D. R., Helms, J. A., 2005. It’s all in your head: new insights into craniofacial development and deformation. J. Anat. 207, 461-477.
us
Tenzen, T., Allen, B. L., Cole, F., Kang, J. S., Krauss, R. S., McMahon, A. P., 2006. The cell surface membrane proteins Cdo and Boc are components and targets of the Hedgehog
an
signaling pathway and feedback network in mice. Dev. Cell 10, 647-656. Theveneau, E., Steventon, B., Scarpa, E., García, S., Trepat, X., Streit, A., Mayor, R., 2013. Chase-and-run between adjacent cell populations promotes directional collective
M
migration. Nature Cell Biol. 15, 763-772.
Tobin, J. L., Di Franco, M., Eichers, E., May-Simera, H., García, M., Yan, J., Quinlan, R.,
d
Justice, M. J., Hennekam, R. C., Briscoe, J., Tada, M., Mayor, R., Burns, A. J., Lupski, J.
Ac ce pt e
R., Hammond, P., Beales, P. L., 2008. Inhibition of neural crest migration underlies craniofacial dysmorphology and Hirschsprung’s disease in Bardet–Biedl syndrome. Proc. Natl. Acad. Sci. USA 105, 6714-6719.
Tolosa, E. J., Jaurena, M. B., Zanin, J. P., Battiato, N. L., Rovasio, R. A., 2012. In situ hybridization of chemotactically bioactive molecules on cultured chick embryo. J. Histotech. 35, 114-129.
Toyofuku, T., Yoshida, J., Sugimoto, T., Yamamoto, M., Makino, N., Takamatsu, H., Takegahara, N., Suto, F., Hori, M., Fujisawa, H., Kumanogoh, A., Kikutani, H., 2008. Repulsive and attractive semaphorins cooperate to direct the navigation of cardiac neural crest cells. Dev. Biol. 321, 251-262. von Philipsborn, A., Bastmeyer, M., 2007. Mechanisms of gradient detection: a comparison of axon pathfinding with eukaryotic cell migration. Int. Rev. Cytol. 263, 1-62. Yamada, Y., Nagase, T., Nagase, M., Koshima, I., 2005. Gene expression changes of Sonic Hedgehog signaling cascade in a mouse embryonic model of Fetal Alcohol Syndrome. J. Craniof. Surg. 16, 1055-1061.
Page 34 of 53
33
Yu, S. R., Burkhardt, M., Nowak, M., Ries, J., Petrasek, Z., Scholpp, S., Schwille, P., Brand, M., 2009. Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules. Nature 461, 533-537. Zanin, J. P., Battiato, N. L., Rovasio, R. A., 2013. Neurotrophic factor NT-3 displays a noncanonical cell guidance signalling function for cephalic neural crest cells. Europ. J. Cell Biol. 92, 264-279.
ip t
Zhang, W., Zhao, Y., Tong, C., Wang, G., Wang, B., Jia, J., Jiang, J., 2005. Hedgehog-
Regulated Costal2-kinase complexes control phosphorylation and proteolytic processing of
Ac ce pt e
d
M
an
us
cr
cubitus interruptus. Dev. Cell 8, 267-278.
Page 35 of 53
34
Legends for figures Fig 1. A. Chemotactic index of NCCs confronted with Sonic hedgehog (Shh) gradients at different initial concentrations (from the right side), as well as with various control media and in the presence of ethanol. Attraction (black bars) and repulsion (gray bars) are shown. Ctrl: control. NE: notochord explants. NCCM: non-concentrated conditioned medium. CCM: concentrated conditioned medium. CDM: concentrated defined medium. Shh: µg/ml. Cyp: 2.5
ip t
µM cyclopamine. NE + Cyp: NE in presence of Cyp. Shh (µg/ml) + Cyp: Shh in presence of Cyp. Inac: inactivated Shh. 5E1: function blocking anti-Shh antibody. Ctrl IgG: preimmune
cr
control IgG. SmoMO: NCCs transfected with 1 µM chick anti-Smoothened morpholino.
ShhMO: NCCs transfected with 1 µM anti-Shh MO. Ctrl MO: NCCs transfected with 1 µM
us
control MO. Shh (µg/ml) + Etoh: Shh in presence of 100 mM ethanol. a: Difference versus Ctrl (p < 0.05). b: Difference versus NE (p < 0.05). c: Difference versus NCCM (p < 0.05). d:
an
Difference versus Shh 10 µg/ml (p < 0.05). B. Cell tracks of individual cells of a typical experiment in control condition. C. Tracks of NCCs migrating in a gradient of Shh 10 µg/ml in the source (right side). D. Migratory behavior of NCCs exposed to chemotactic gradient of
M
Shh in the presence of 100 mM ethanol. E. In vitro NCCs after Smo-MO transfection. F.
d
Delivery control of Smo-MO transfection (see details in the text).
Ac ce pt e
Fig. 2. Proportion of cells migrating in a 180° angle open toward (black bars) or against (gray bars) the Sonic hedgehog (Shh) source of the gradient at different initial concentrations (from the right side), as well as with several control media and in the presence of ethanol. The sum of repulsion plus attraction is not 100%, because the non-oriented population was not considered (see method details in Rovasio et al., 2012). Abbreviations as in Figure 1. a: Difference versus Ctrl (p < 0.05). b: Difference versus NE (p < 0.05). c: Difference versus NCCM (p < 0.05). d: Difference versus Shh 10 µg/ml (p < 0.05).
Fig. 3. Expression of the Shh ligand. A. Chick embryo of stage 12 HH showing main components. Prosencephalon (P), mesencephalon (M), rhombencephalon (R), optic vesicle (OV), neural crest cells (NCCs, demarcated area). Dark field. B. The same embryo showing HNK1-antibody immunolabeling of NCCs (demarcated area). The arrows indicate migration pathways of cephalic NCCs toward M and OV regions. C. Above: Immunoblot of tissues shows the expression of Sonic hedgehog (Shh) and tubulin (Tub = loading control) at neural cell crest population (NCCs), optic vesicle (OV) and the cephalic remaining (CR), in control
Page 36 of 53
35
(Ctrl) and ethanol-treated (Etoh) embryos. Below: Mean relative density graph corresponding to immunoblots. The Shh expression in control NCCs is significantly lower than in OV (p< 0.02), CR (p< 0.001) and Etoh-exposed NCCs, OV and CR (p< 0.05). D. Cephalic pole of chick embryo of stage 11 HH. Shh mRNA in situ hybridization (ISH) shows notochord expression with the cephalic expansion at prosencephalon level (arrow). E. Chick embryo of stage 12 HH shows ISH of Shh expression at the OV stalk (arrows). Broken line indicates
ip t
transversal section of H. F. Chick embryo of stage 12 HH shows immunolabel of Shh protein “detaching” from the OV stalk toward mesencephalic space (broken arrows). Horizontal
cr
broken line indicates transversal section of I. G. Posterolateral oblique view of a chick
embryo of stage 13 HH showing the detailed ISH Shh expression at the OV stalk (arrows). H.
us
Transversal section indicated in E, showing HNK1-labeled NCCs surrounding the OV stalk (arrows) and the ISH-Shh expression at the basal plate of forebrain and OV stalk
an
(arrowheads). I. Transversal sections indicated in F, showing at left side a control embryo (Ctrl) and at right side the immunolabeled Shh protein (arrows).
M
Fig. 4. A. Culture of mesencephalic neural crest cells (NCCs) immunolabeled for Patched (Ptch) receptor. B. Culture of NCCs immunolabeled for Smoothened (Smo) membrane
d
activating protein. In both enlarged details, note the punctiform pattern in the focus plane of
Ac ce pt e
cell surface. C. Cephalic pole of chick embryo of stage 10-11 HH showing in situ hybridization (ISH) expression of Ptch receptor on NCCs (demarcated area). D. Equivalent chick embryo after ISH of Smo protein in NCCs (demarcated area). OV: optic vesicle.
Fig. 5. A. In situ hybridization (ISH) for the transcription factor Gli1 in stage 11 HH chick embryo. B. ISH of Gli2 in stage 9 HH chick embryo. C. ISH of Gli3 in stage 10 HH chick embryo. D. ISH of Gli3 in stage 11 HH chick embryo. E. ISH of SUFU protein in stage 11 HH chick embryo. F. Representative embryo after a sense control riboprobe for Gli-SUFU system. Note a shared expression of Gli-SUFU at the neural tube wall (A-E, black arrows), the neural crest cells (NCCs) moving to optic vesicle stalk (A, white arrows), presumptive NCCs at the dorsal wall of cephalic and trunk neural tube (B, D, white arrows), and opticmesencephalic NCCs (C-E, demarcated area).
Fig. 6. A. Chick embryo at stage 10 HH, microinjected into the neural tube at stage 8 HH with anti-Smo morpholino (MO)/EndoPorter® and reincubated for 10 hours. The MO (red dots) is seen in the in vivo NCCs migrating toward mesencephalic level (arrowheads) and scarcely to
Page 37 of 53
36
the optic vesicle (OV) (arrows). B. The same embryo as A, after reincubation until stage 12 HH. The HNK1 labeled NCCs migrate toward mesencephalic segment (arrowheads), only a few to the OV (arrows, compare with C) and some others are still on the route (double arrow). Inset. Probable selectivity of the anti-Smo MO on cephalic NCCs/Shh-Ptch/Smo system. The HNK1-labeled NCCs of trunk level of anti-Smo MO transfected embryo (under the same influence as cephalic NCCs) show normal migratory behavior, with temporary arrest at the
ip t
neural tube-somite angle (arrows) and displacement along the cephalic half of each somite
(arrowheads) (see details in the text). C. HNK1-labeled stage 12 HH control embryo showing
cr
a normal NCC population filling mesencephalic and OV areas.
us
Fig. 7. A. Dark field, and B. Fluorescence microscopy of a stage 9 HH chick embryo just microinjected into the neural tube lumen with a lisamine-conjugated anti-Smo morpholino
an
(MO) and electroporated (+). C. and D. Control stage 11 HH chick embryo injected at stage 9 HH into the neural tube with a lisamine-conjugated control MO and electroporated (+). Note at C, the NCCs bilaterally distributed toward mesencephalic (M) and optic vesicle (OV)
M
regions (compare with Figs. 3 A, B and 6 C), and at D, the lateral location of the control MO. Broken vertical line indicates cranio-caudal axis. E. Chick embryo at stage 11 HH injected
d
into the neural tube with anti-Smo MO at stage 9 HH and electroporated (+). The HNK1
Ac ce pt e
immunolabeling shows a lower displacement of NCCs (arrow) towards the OV (circle) on the electroporated side (+) and a higher cell population in mesencephalic segment of the same side (rectangle). P: prosencephalon; M; mesencephalon; R: rhombencephalon. F. Graph of fluorescence intensity of NCCs in the OV and M regions (see E) of control (Ctrl) and antiSmo MO injected/electroporated embryos. Reduction of NCCs in the OV region and higher ell density at M segment are seen in the anti-Smo MO-treated group. Mean ±SE. a = p <0.05. G. and H. Embryos treated as explained for E show pronouced changes at OV (circle) and M (rectangle) levels, in line with those just described.
Fig. 8. A. Bright field, and B. HNK1-immunolabeled chick embryo at stage 12 HH, transfected at stage 8-9 HH with DNA plasmid corresponding to mutated Ptch receptor (Ptc∆loop2) and electroporated (+). C. Another embryo of the same group, both showing the perturbed dispersion of NCCs in the electroporated side (+): less cell density at the optic vesicle (OV, arrows, circles), cell accumulation at the mesencephalic level (rectangle), and arrested NCCs on the dorsal surface of the neural tube (arrowheads).
Page 38 of 53
37
Fig. 9. A. Chick embryo at stage 12 HH, implanted at stage 8-9 HH in right side region of mesencephalic level with beads embedded with PBS (white arrowhead). NCC dispersion is equivalent between both sides. B. Equivalent chick embryo implanted in the right side of the mesencephalic region with beads embedded with Shh (white arrowhead) and in the left side with PBS embedded beads (black arrowhead). Note NCCs stopped caudally to the optic vesicle (OV, arrow) and recruited around the ectopic source of Shh, with a normal dispersion
ip t
at the control left side. C. Similar chick embryo implanted in the right side of the caudal
mesencephalic region with beads embedded with Shh (arrowhead). In addition to the NCCs
cr
not reaching the OV (arrow), they accumulate around the ectopic source of Shh (arrowhead). The enlarged detail clearly shows the NCCs tightly around the bead. D. Density graph of
us
NCCs expressed as fluorescence intensity after immunolabeling with HNK1 antibody. Bilateral cell density corresponding to optic vesicle plus mesencephalic regions of control
an
embryos or unilaterally inserted with Shh embedded beads was not significantly different between both sides of the embryo. Mean ±SE. E. Chick embryo equivalent to A-C, implanted with cyclopamine-embedded beads in the right side of mesencephalic level (arrowhead).
M
There is a remarkable absence of a normal dispersion of NCCs toward the OV (arrow).
d
Fig. 10. A. Dark-field, B. Bright-field, C. and D. HNK1-fluorescence microscopy of chick
Ac ce pt e
embryos at stage 12 HH treated in ovo at stage 8-9 HH with 100 mM ethanol. Note the conserved normal gross morphology (A, B) coexisting with early disturbed dispersion of NCCs: deficient colonization of the OV region (C, D, compare with Figs. 3 B, 6 C and 7 C), as well as many cells still on the route (arrows). Broken line outlines the cephalic embryonic pole and the neural tube.
Fig. 11. A. and B. Craniofacial aspect of control chick embryos at stage 22-23 HH (3-4 days) treated in ovo at stage 8-9 HH with PBS. A. The global structure involves frontal process (F), telencephalic vesicles and eye primordium (O), nasal placodes (N), maxillary (Mx), mandibular (Mb) and hyoid (H) processes, and stomodeum (*). B. In situ hybridization for mRNA to Shh shows the expression by transparency in cephalic neuroectoderm, ventral ectoderm of frontonasal protuberance (arrowheads), pharyngeal endoderm of stomodeum (*), maxillo-mandibular processes (arrows), notochord and neural tube floor (detail on inset). C. A cephalo-thoracic view of control chick embryo at stage 26 HH (5 days) shows Shh expression at the maxillary process (Mx) and roof of the stomodeum (*), distal limbs and umbilical region (arrowheads), notochord and neural tube floor (arrows). D. and E. Craniofacial view of
Page 39 of 53
38
chick embryos of stage 22-23 HH (3-4 days) treated in ovo at stage 8-9 HH with ethanol. D. Note diverse degrees of facial underdevelopment, global low growth of the frontal process, delay of cephalic vesicle separation (arrow), poor development of nasal placodes with premature medial fusion (arrowhead), low development of maxillary (double black arrowheads) and mandibular (double white arrowheads) processes. E. After in situ hybridization for mRNA to Shh, structural anomalies and perturbed expression of Shh in
ip t
territories colonized by cephalic NCCs are seen to coexist. While the ethanol-treated embryo exhibits a conserved Shh expression in telencephalon and frontal protuberance, there is a
cr
significant reduction of Shh expression in the nasal placode (arrowheads), ventral stomodeal ectoderm (*), maxillo-mandibular processes (arrows), notochord and neural tube floor (detail
us
on inset). F. A cephalo-thoracic view of ethanol-treated chick embryo at stage 26 HH (5 days) shows reduction of Shh expression at the maxillary process and roof of the stomodeum (*),
umbilical region and distal limbs (arrowheads).
an
notochord and neural tube floor (arrows), with preservation of morphogen expression in the
M
Fig. 12. Simplified model of migration/distribution of mesencephalic NCCs to ocular region. (1) Neural tube emerging NCCs (dots) migrate dorso-laterally (horizontal arrow) occupying
d
the space between the neural tube and ectoderm. (2) Ciliary population of NCCs recognizes
Ac ce pt e
the concentration gradient (curved bands) of molecules released from the optic vesicle (OV) region, including Shh. (3) NCCs respond with orientated migration toward the optic area (curved arrow). (4) Reaching the attractant source, its higher relative concentration saturates the receptors and consequently stops the guide mechanism. (5) At this spatiotemporal intersection, some NCCs differentiate as neurons and glia of the ciliary ganglion. It is probable that ciliary NCC plasticity enables the recognition and response to other cooperating attractants (see text). P: prosencephalon; M: mesencephalon; R: rhombencephalon; S: somites.
Page 40 of 53
39
Table 1. Dynamic and viability parameters of in vitro NCCs after Shh/Ethanol exposure
Shh 10
30.00 ±1.5 vs Ctrl: NS
Shh 10 + 5E1
17.8 ±1.2 vs Ctrl: p <0.001 vs Shh 10: p <0.001
Shh 0.2 + Etoh
Etoh
27.90 ±4.5 vs Ctrl: NS vs Shh 10: NS
10.08 ±1.1
vs Ctrl: p <0.001 vs Shh 10: p <0.001 vs Shh 10 + Etoh: p <0.019
1
0.75 ±0.04
vs Ctrl: p <0.03
0.88 ±0.03
0.060 ±0.005
vs Ctrl: p <0.001
0.57 ±0.04 vs Ctrl: NS vs Shh 10: p <0.001
0.59 ±0.05
ip t
0.055 ±0.005
vs Ctrl: p <0.008
vs Ctrl: NS vs Shh 0.2: NS
0.44 ±0.02
vs Ctrl: p <0.001 vs Shh 10: p <0.001
0.23 ±0.03
Ac ce pt e
Shh 10 + Etoh
15.25 ±1.9 vs Ctrl: p <0.001 vs Shh 0.2: p <0.05
vs Ctrl: NS
vs Ctrl: p <0.001 vs Shh 10: p <0.001 vs Shh 10 + Etoh: p <0.001
0.102 ±0.006 0.125 ±0.019
cr
32.25 ±2.6 vs Ctrl: NS
vs Ctrl: NS
us
Shh 5
0.080 ±0.006 0.087 ±0.008
vs Ctrl: p<0.02
0.084 ±0.012
an
vs Ctrl: NS
Viability parameters Cytotoxic index Proliferation index 2 2 (n = 69) (n = 75)
vs Ctrl: NS vs Shh 10: NS
0.100 ±0.011
M
Control Shh 0.2
Dynamic parameters Curvilinear Linearity 1 velocity (n = 780) 1 (n = 780) 29.05 ±2.4 0.63 ±0.02 30.75 ±2.5 0.68 ±0.03
d
Experimental condition
vs Ctrl: NS vs Shh 0.2: NS
0.087 ±0.006
vs Ctrl: NS vs Shh 10: p <0.006
0.116 ±0.011 vs Ctrl: p <0.006 vs Shh 10: p <0.001 vs Shh 10 + Etoh: p <0.03
vs Ctrl: NS
0.126 ±0.010
vs Ctrl: p <0.05
0.130 ±0.0095 vs Ctrl: p <0.02
0.115 ±0.016 vs Ctrl: NS vs Shh 10: NS
0.062 ±0.017 vs Ctrl: p <0.03 vs Shh 0.2: p <0.02
0.108 ±0.017 vs Ctrl: NS vs Shh 10: NS
0.067 ±0.003 vs Ctrl: p <0.001 vs Shh 10: p <0.001 vs Shh 10 + Etoh: p <0.02
2
n : number of cell contours and centroids; n : number of cells; Shh: Sonic hedgehog (µg/ml); 5E1: function-blocking anti-Shh antibody; Etoh: ethanol 100 mM; NS: no significant difference.
Page 41 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 42 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 43 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 44 of 53
Ac ce p
te
d
M
an
us
cr
ip t
Figure Click here to download high resolution image
Page 45 of 53
Ac ce p
te
d
M
an
us
cr
ip t
Figure Click here to download high resolution image
Page 46 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 47 of 53
Ac ce p
te
d
M
an
us
cr
ip t
Figure Click here to download high resolution image
Page 48 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 49 of 53
Ac ce p
te
d
M
an
us
cr
ip t
Figure Click here to download high resolution image
Page 50 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 51 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 52 of 53
Ac ce p
te
d
M
an
us
cr
ip t
Figure Click here to download high resolution image
Page 53 of 53
S0171-9335(16)30006-1 http://dx.doi.org/doi:10.1016/j.ejcb.2016.02.003 EJCB 50868
To appear in: Received date: Revised date: Accepted date:
20-6-2015 23-1-2016 17-2-2016
Please cite this article as: Tolosa, E.J., Fernandez-Zapico, M., Battiato, N.L., Rovasio, R.A.,Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure, European Journal of Cell Biology (2016), http://dx.doi.org/10.1016/j.ejcb.2016.02.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ed pt ce Ac
Page 1 of 53
Highlights
Tolosa et al.,
Ac
ce pt
ed
M
an
us
cr
Cephalic NCCs migrate chemotactically to a concentration gradient of Shh morphogen. Expression of Shh at optic vesicle and Ptch-Smo on NCCs integrate the guide system. Molecular and pharmacological blocking inhibits oriented migration of cephalic NCC. Shh-dependent chemotaxis of NCCs was altered by in vitro-in vivo ethanol exposure. Results support a guiding activity for Shh in addition to its other canonic functions.
ip t
Page 2 of 53
1
Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure
ip t
Ezequiel J. Tolosa a,b, Martín Fernandez-Zapico b,
Center for Cellular and Molecular Biology, CEBICEM-IIBYT (UNC-CONICET)
an
a
us
cr
Natalia L. Battiato a and Roberto A. Rovasio a
National University of Córdoba, Av. Vélez Sarsfield 1611, (5016) Córdoba, Argentina. Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
Ac ce pt e
d
M
b
Corresponding author:
Roberto A. Rovasio, CEBICEM (FCEFN, UNC), IIBYT (UNC-CONICET) Av. Vélez Sarsfield 1611, (5016) Córdoba, Argentina. Email: [email protected]
Page 3 of 53
2
Abstract Our aim was to understand the involvement of Sonic hedgehog (Shh) morphogen in the
potential disorders of this guiding mechanism after ethanol exposure.
ip t
oriented distribution of neural crest cells (NCCs) toward the optic vesicle and to look for
In vitro directional analysis showed the chemotactic response of NCCs up Shh gradients
cr
and to notochord co-cultures (Shh source) or to their conditioned medium, a response
inhibited by anti-Shh antibody, receptor inhibitor cyclopamine and anti-Smo morpholino
us
(MO). Expression of the Ptch-Smo receptor complex on in vitro NCCs was also shown. In whole embryos, the expression of Shh mRNA and protein was seen in the ocular region, and
an
of Ptch, Smo and Gli/Sufu system on cephalic NCCs. Anti-Smo MO or Ptch-mutated plasmid (Ptch1∆loop2) impaired cephalic NCC migration/distribution, with fewer cells invading the
M
optic region and with higher cell density at the homolateral mesencephalic level. Beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic signal) supported the role of Shh as an in vivo guide molecule for cephalic NCCs.
d
Ethanol exposure perturbed in vitro and in vivo NCC migration. Early stage embryos
Ac ce pt e
treated with ethanol, in a model reproducing Fetal Alcohol Syndrome, showed later disruptions of craniofacial development associated with abnormal in situ expression of Shh morphogen.
The results show the Shh/Ptch/Smo-dependent migration of NCCs toward the optic vesicle, with the support of specific inactivation with genetic and pharmacological tools. They also help to understand mechanisms of accurate distribution of embryonic cells and of their perturbation by a commonly consumed teratogen, and demonstrate, in addition to its other known developmental functions, a new biological activity of cellular guidance for Shh.
Key words Cell guiding; Cell migration; Chemotaxis; Chick embryo; Ethanol; Neural crest cells; Sonic hedgehog
Page 4 of 53
3
Introduction Neural crest cells (NCCs) segregate from the closing neural tube, migrate along several pathways and colonize defined sites, giving rise to a variety of derivatives. The conserved distribution pattern of NCCs along vertebrate embryos and their morphogenetic behavior are modulated by a balance between genetic signals and those of the extracellular milieu (Bronner and Le Douarin, 2012; Le Douarin and Kalcheim, 1999; Rovasio et al., 1983). However, these
ip t
factors do not fully explain their directional migration.
Studies from our laboratory on different experimental models helped to re-discover
cr
chemotaxis as a significant modulating factor of directional cell response (Guidobaldi et al., 2012; Jaurena, 2011; Rovasio et al., 2012; Tolosa et al., 2012; Zanin et al., 2013). In this
us
communication at a distance, the cell recognizes an exogenous concentration gradient of diffusible molecules released by a “target” field and orients its locomotion as a function of the
an
slope vector (Rovasio et al., 2012). Chemotaxis was proven in bacteria (Hazelbauer, 2012), amoebas (García and Parent, 2008), leukocytes (Afonso et al., 2012), neurons (Paratcha et al., 2006) and axonal growth cones (Mortimer et al., 2008). We have shown chemotaxis of
M
mammal sperm toward the ovular region (Giojalas and Rovasio, 1998), with progesterone signals as the attractant (Guidobaldi et al., 2012). Curiously, the embryonic cell, paradigmatic
d
of accurate, directional motility, has been less studied with a chemotactic approach, except in
Ac ce pt e
a few documented systems in Drosophila (Ricardo and Lehmann, 2009), zebrafish (Breau et al., 2012), frog (Mayor and Theveneau, 2013), mouse (Kubota and Ito, 2000) and avian embryos (McLennan et al., 2012). Recently, we presented direct evidence of Stem Cell Factor (SCF) eliciting chemotactic migration of NCCs toward the skin (Rovasio et al., 2012), as well as the CXCR4-dependent chemotactic behavior of cephalic NCCs up to the chemokine Stromal cell-Derived Factor-1 (SDF-1) expressed in the optic field (Jaurena, 2011), and TrC/p75-dependent cephalic NCC migration up an in vitro and in vivo NT-3 concentration gradient released in the ocular region (Zanin et al., 2013). In our study model of the colonization pattern of NCCs to the optic vesicle region, it should be noted that NCCs start emigrating from mesencephalic levels in a narrow temporal window of 32 to 36 h (stages 8-10 HH) when the neural folds are closing, and then there is no emigration from this segment of the neural tube (Newgreen and Erickson, 1986). After detaching, mesencephalic NCCs migrate to both lateral sides under the ectoderm, toward the future facial region (Le Douarin and Kalcheim, 1999). Then, a subpopulation of ciliary NCCs detaches from the principal mass, changes its direction by about 90° and heads toward the
Page 5 of 53
4
optic vesicle stalk (Lee et al., 2003) (see Fig. 3 B, arrows), where the parasympathetic ciliary ganglion of the eye will form. It is noteworthy that there is no topographic, micro-anatomic or any other explanation to account for the clear directional change of ciliary NCCs to the optic vesicle field. The notion of Sonic hedgehog (Shh) as a chemotactic guide for NCC distribution arose from the association between the perturbed expression of Shh with cephalic anomalies
ip t
(Ahlgren et al., 2002; Marcucio et al., 2005; Paganelli et al., 2010) and enteric agangliosis (Tobin et al., 2008). Extracellular protein Shh is a highly conserved paracrine factor
cr
displaying significant activity in communication at a distance in a broad sense (Etheridge et al., 2010), and in the context of the concept of morphogen as a concentration-dependent
us
modulator of spatial cell patterns (Gilbert, 2013). Besides the well-known functions of Shh during early embryo development, there is evidence of Shh involvement in the migratory
an
behavior of fibroblasts (Bijlsma et al., 2007), axonal cone growth (Gore et al., 2008), olfactory bulb neuroblasts (Angot et al, 2008), primordial germ cells (Deshpande et al., 2001),
Marcucio et al., 2005; Tolosa et al., 2012).
M
enteric NCCs (Fu et al., 2004), and craniofacial NCCs (Ahlgren et al., 2002; Hu et al., 2015;
On the other hand, the relevant attributes of NCCs and the putative role of Shh as a guide
d
molecule suggested considering this embryonic model within a pathogenic context. Thus, the
Ac ce pt e
neurocristopathies family (Bolande, 1974) includes tumoral diseases, genetic-based anomalies and epigenetic determinants such as Retinoic Acid Syndrome (Salvarezza and Rovasio, 1997) or Fetal Alcohol Syndrome (FAS) (Jaurena et al., 2011; Rovasio and Battiato, 1995, 2002), all with serious social impact. Taking into account that several FAS anomalies imply regions colonized by NCCs in the craniofacial field (Le Douarin et al., 2007), and that prenatal effects of ethanol exposure include a low migratory capacity and perturbed NCC distribution (Chen et al., 2000; Jaurena et al., 2011; Rovasio and Battiato, 1995, 2002), it is reasonable to think that epigenetic perturbation of directional migration of this cell population may be an important etiopathogenic component of FAS. It is known that accurate cell distribution in the embryo relies on precise cell migration, which is disrupted in cortical cerebellar neuroblasts after ethanol exposure (Kumada et al., 2010). Also, the involvement of Shh expression in craniofacial morphogenesis is well known (Hu et al., 2015; Tapadia et al., 2005; Tobin et al., 2008) following abnormal cephalic development after perturbation of Shh expression by ethanol exposure (Ahlgren et al., 2002), or treatments with cyclopamine (Chen et al., 2002), or anti-Shh antibodies (Marcucio et al., 2005). These data encouraged us to explore the association between signals started with the Shh ligand, the oriented migration of cephalic
Page 6 of 53
5
NCCs and their potential lability to ethanol action in the final localization of this cell population. In this report on NCC cultures and directional analysis, we show the chemotactic response of NCCs up Shh extracellular gradients or in notochord co-cultures (Shh source) or their conditioned medium, and their inhibition by anti-Shh antibody, the receptor inhibitor cyclopamine and the anti-Smo MO. In whole embryos, we demonstrate the expression of Shh
ip t
mRNA and protein in the ocular region of the embryo and of Ptch-Smo receptor complex in in vitro and in vivo cephalic NCCs. Also, the MO blockade of the Shh-Ptch-Smo system
cr
impaired in vivo migration/distribution of cephalic NCCs. The insertion of beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic source) provided additional evidence on
us
the Shh function as an in vivo guidance molecule for the orientation of cephalic NCCs. Moreover, we showed that a teratogenic concentration of ethanol exposure perturbed the Shh-
an
dependent chemotaxis of in vitro NCCs, and that early-exposed embryos in a model reproducing FAS show a disruption of craniofacial development at later stages, associated with abnormal in situ expression of Shh morphogen.
M
These in vitro and in vivo results strongly suggest that: (1) the chemotactic mechanism is an essential element in the spatiotemporal orientation of NCCs toward specific regions of the
d
embryo; (2) mesencephalic NCCs migrate toward the optic vesicle guided by a chemotactic
Ac ce pt e
mechanism in response to Shh concentration gradients in a Ptch/Smo-dependent manner; (3) the accuracy of this chemotactic mechanism is perturbed by ethanol, a commonly consumed teratogen. This helps to understand a fundamental mechanism for the distribution of embryonic cells and their epigenetic perturbation, and also amplifies the functional scope of the morphogen Shh, involving it in new biological activities for the colonization of a specific region by NCCs, in addition to its other well-known developmental functions.
Materials and Methods
(Note: For full details, see Supplemental material)
In vitro experiments Culture of cephalic neural crest cells Cultures of mesencephalic NCCs were made from Gallus gallus (Cobb line) chick embryos stages 10-11 HH (Hamburger and Hamilton, 1951) as detailed (Rovasio et al., 2012), incubated with N2 defined medium (Barnes and Sato, 1980) plus 10% fetal calf serum (FCS) (Sigma Chem Co, St Louis, MO) during 20 h at 37 ±0.2 °C in 5% CO2 in air.
Page 7 of 53
6
Notochord and optic vesicle conditioned medium Notochord explants, as Shh sources (Martí et al., 1995), were obtained from the trunk level of stages 10-11 HH chick embryos by microdissection and collagenase digestion, cultured in N2 medium for 48 h and the conditioned medium (CM) recovered and used accordingly. Some lots were concentrated in a Centricon column (Amicon Inc., Beverly, MA, USA), obtaining fractions of ≤10,000 and >10,000 Daltons of molecules. Similarly, CM was
ip t
obtained from optic vesicle cultures (Jaurena, 2011). Video-microscopy and chemotaxis assay
cr
A real-time approach was applied using a chemotaxis chamber, computerized videomicroscopy and software based on objective directional criteria (Rovasio et al., 2012). NCC
us
cultures were perfused with a concentration gradient of Shh (1845-SH; R&D Systems, Minneapolis, MN, USA) at a concentration indicated in the Results section, or co-cultured
an
with notochord or optic vesicle explants, or their CM. Control cultures and inhibition assays were performed with only N2 medium in the chamber well, or control CM, or Shh preincubated with neutralizing anti-Shh antibody (5E1, Develop. Studies Hybrid. Bank)
M
(Marcucio et al., 2005), or heat-inactivated Shh, or the inhibitor cyclopamine (Chen et al., 2002), or culturing NCCs transfected with anti-Smo or anti-Shh MOs. Morphometric
d
parameters were cell area, perimeter and shape factor (4π x area)/perimeter2; dynamic
Ac ce pt e
parameters were curvilinear velocity (total distance traveled by the cell per unit time) and linearity (nondimensional quotient between linear distance and curvilinear distance); and chemotactic parameters were evaluated applying algorithms developed in our laboratory (Rovasio et al., 2012). Since several chemotactic parameters based on cell number and trajectory show equivalent results, we present only representative data, displaying other parameters only for particular points or to better visualize oriented cell migration. The attractant concentration refers to the gradient source, and all the experiments (and graphs of results) were carried out with the right-side well of the chemotaxis chamber as the source of putative attractant (Rovasio et al., 2012). After video assays and in-between sampling, NCCs were submitted to proliferation and viability testing. Ethanol exposure Experiments were also performed in the presence of 100 µM ethanol in both wells of the chemotaxis chamber as detailed in the Results section. At the end of each experiment, ethanol concentration was checked in both chamber compartments by head-space gas chromatography as explained (Jaurena et al., 2011).
Page 8 of 53
7
Immunolabeling of NCC cultures The purity of NCC culture was assessed with the HNK1 monoclonal antibody (Sigma Chem Co). Patched receptor and Smoothened signal transduction protein were labeled with specific antibodies (SC-6149 and SC-13943, Santa Cruz Biotech. Inc., CA, USA), then incubated with rhodamine- or fluoresceine isothiocyanate-conjugated secondary antibodies, and controlled by replacing antibodies with PBS or heat-inactivated antibodies.
ip t
Determination of trophic parameters
Cell survival was analyzed by the Live/Dead Kit (Molecular Probes, Eugene, OR)
cr
according to manufacturer instructions, using NCCs pre-incubated with sodium azide as positive control. Cell proliferation was assessed after incorporation of 5-bromo-2′-
us
deoxyuridine (BrdU) as detailed (Jaurena et al., 2011). Images of the same microscopic fields comprising almost all the surface of NCC outgrowth were obtained with phase contrast and
an
fluorescence optics, and submitted to analyses with Image-J software (NIH, Bethesda, MA, USA), according to previous descriptions (Jaurena et al., 2011; Rovasio et al., 2012). NCC in vitro transfection with anti-Smo and anti-Shh morpholinos
M
Prior to evaluation of chemotaxis, morpholino (MO) transfection of NCC cultures was performed with the Endo-Porter® tool (Gene Tools, LLC Philomath, USA), according to
d
manufacturer instructions, using 1 µM lisamine (sulforhodamine)-labeled MO against: 1) Smo
Ac ce pt e
mRNA (5’-AAAGCAGCAGCTCACCATGCTCCAT-3’ sulforhodamine) (Smo-MO); 2) Shh mRNA (5´-TGTCAACAGCAGCATTTCGACCATT-3’ sulforhodamine) (Shh-MO), or 3) standard control (5'-CCTCTTACCTCA GTTACAATTTATA-3' sulforhodamine) (Ctrl-MO), all of them added with 5 µl/ml Endo-Porter® in culture medium plus 10% serum, followed by 24 h incubation. The medium was then replaced with defined medium and the NCCs used for different assays.
Whole chick embryo experiments Obtaining and processing
Distribution of NCC markers and bioactive molecules was assessed by whole-mount in situ hybridization and immunocytochemistry on stage 10-11 HH chick embryos incubated in ovo as detailed elsewhere (Rovasio et al., 2012; Tolosa et al., 2012; Zanin et al., 2013), or cultivated in a shell-less culture system as described (Battiato et al., 1995; Jaurena et al., 2011; Rovasio and Battiato, 2002; Tolosa et al., 2012). After PBS-washing, the embryos were fixed with 4% paraformaldehyde in PBS for 4 h at 4 °C. For embryos submitted to in situ hybridization, 0.1% diethylpyrocarbonate (DEPC) (Sigma Chem Co) was added to all solutions (Tolosa et al., 2012), dehydrated and maintained in 100% methanol at -20 °C until
Page 9 of 53
8
use, keeping the embryos in PBS for immunolabeling. Embryo morphology was evaluated using a conventional series (Hamburger and Hamilton, 1951) and no differences were observed in results between in ovo or shell-less culture groups. In situ hybridization Whole mount in situ hybridization was performed as described (Tolosa et al., 2012). Probes were synthesized from plasmids containing partial cDNA of chick Shh (Franco et al.,
ip t
1999), chick Ptch1 (Marigo et al., 1996a), chick Sufu (Pearse et al., 1999), human Gli1 and chick Gli2 (Marigo et al., 1996b) and chick Gli 3 (Schweitzer et al., 2000) and a plasmid
cr
containing the full-length cDNA of chick Slug (Del Barrio and Nieto, 2004). Sense control
and antisense riboprobes were made from the same vector and were developed according to
us
conventional methods (Sambrook et al., 1989; Tolosa et al., 2012). Immunolabeling
an
Function-blocking 5E1 anti-Shh antibody (Sigma Chem Co), neural crest marker HNK1 monoclonal antibody (Sigma Chem Co), and FITC-labeled secondary antibodies were used. With the whole embryo in PBS, the cephalic ectoderm was pierced with a tungsten
M
microneedle to allow antibody penetration, followed by the conventional immunolabeling method, with 8 to 24 h incubation with antibodies, depending on the embryo age.
d
After in situ hybridization or immunolabeling, whole mount embryos were assessed for
Ac ce pt e
distribution of NCCs on the basis of Slug and HNK1 label density at optic vesicle and mesencephalic levels as previously described (Rovasio et al., 2012; Zanin et al., 2013). Regional tissue extraction and western blot From stage 10 HH chick embryos of control or ethanol treated groups (see below), cell masses corresponding to (1) migratory mesencephalic NCCs, (2) optic vesicles, and (3) neural tube and remainder of cephalic region, were dissected and processed with the conventional method to obtain the total proteins (Sambrook et al., 1989). Samples were submitted to electrophoresis in 12-15% SDS-PAGE, transferred and immunolabeled with anti-Shh 5E1 (Sigma Chem Co) and anti-tubulin (10D8) (SC-53646, Santa Cruz Biotech. Inc., CA, USA) antibodies, then with peroxidase-labeled secondary antibody (Sigma Chem Co) and developed with the diaminobenzidine technique or by means of chemoluminescence after incubation in a solution of luminol, coumaric acid and hydrogen peroxide (10 vol), and then recorded on a photographic plate (Medical X Ray Film, Agfa, Argentina). Morpholino microinjection and electroporation Stage 8-9 HH chick embryos were submitted to morpholino (MO) microinjection with Endo-Porter®, or to microinjection and electroporation according to conventional techniques
Page 10 of 53
9
(Nakamura et al., 2004). Microinjections were made into the neural groove, delivering 0.2-0.5 µl of 1 µM MO against Smo mRNA (5’-AAAGCAGCAGCTCACCATGCTCCAT-3’ sulforhodamine B), or the standard control MO (5'-CCTCTTACCTCAGTTACAATTTATA3' carboxyfluorescein (Gene Tools, Philomath, OR, USA). A specificity control of Smo MO was performed by means of co-injection along the MO with a solution containing full length mouse Smoothened cDNA cloned in pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) and GFP
ip t
cDNA cloned in pEGFP-N1 (Addgene, Cambridge, MA, USA) at a 1/10 dilution. Another lot of embryos were delivered with a DNA plasmid encoding for mutated sequence of the Ptch
cr
receptor (Ptc∆loop2) and green fluorescent protein (GFP) (Briscoe et al., 2001). Microelectrodes were placed parallel to the mesencephalic segment and a current was released as 5 pulses of
us
squared wave of 20-25 V and cycles of 50 ms on/100 ms off, from an ELP-5 electroporator (LIADE, FCEFN, Universidad Nacional de Córdoba, Argentina) (Chesini et al., 2011), and
an
the embryos were reincubated for 15-18 h. Transfections with Endo-Porter® were made delivering 0.5-1.0 µl of 1 µM MO plus 5 µl/ml of Endo-Porter® solution (Gene Tools, LLC Philomath, USA) and reincubated without electroporation. The location of MO as well as the
M
distribution and density of NCCs were determined by Slug in situ hybridization or immunolabeling with HNK1 antibody, in function of the label density in different regions of
d
the cephalic end as indicated in the Results section, after applying conventional methods with
Ac ce pt e
the Image-J software (NIH, Bethesda, MA, USA). Microbead implants
Blocking of the signal chain started by Shh and migration/distribution of cephalic NCCs exposed to an ectopic source of Shh were evaluated. In ex ovo culture stage 8-9 HH embryos, a cut of 200 µm was made in the ectoderm parallel to the edge of the mesencephalic segment and implanted with 2.5 µM cyclopamine embedded beads (AG 1-X®, 75-180 µm, Bio-Rad Lab, Hercules, CA USA), or with 10 µg/ml Shh (Affi-Gel Blue®, 75-150 µm, Bio-Rad Lab), according to the manufacturer instructions. Ethanol exposure
In ovo and ex ovo whole embryos were exposed to 100 mM ethanol as detailed in the Results section (Battiato et al., 1995; Jaurena et al., 2011; Rovasio and Battiato, 2002). Other fertile eggs were injected in the yolk with 100 µl of 25% ethanol in PBS, reincubated until 3-5 days of development (Jaurena et al., 2011; Rovasio and Battiato, 2002), and submitted to morphologic studies and in situ hybridization to look for expression of Shh in the craniofacial region.
Page 11 of 53
10
Statistics After a minimal sample test, no fewer than 60 cells and a mean of 700 cell contours and centroids were studied in each experimental condition repeated in triplicate. Means comparisons were made with Student’s t-test and nonparametric Mann-Whitney tests, and analysis of proportions with the z-test with Yates correction, or previous transformation to the
ip t
corresponding arc-sine values and then one way ANOVA, setting significance at p <0.05.
cr
Results
Cephalic NCCs respond with chemotaxis to in vitro Shh concentration gradients
us
This report shows in vitro chemotaxis of NCCs up concentration gradients of morphogen Sonic hedgehog (Shh) using an objective method and strict directional criteria (Rovasio et al.,
an
2012). Mesencephalic NCCs exhibited a chemotactic index up Shh at initial concentrations of 5 and 10 µg/ml, with a clearly significant bias (Fig. 1 A, C). The same chemotactic result was obtained when notochord explants were used as a Shh source, or with concentrated
M
notochord-conditioned medium (CM) (Fig. 1 A). Equivalent data were also seen when NCCs confronted optic vesicle explants (not shown), confirming our previous results (Jaurena et al.,
d
2011). In contrast, at 1 µg/ml Shh, oriented migration was not significantly different to
Ac ce pt e
controls, and when migratory response was evaluated at an initial concentration of 0.2 µg/ml Shh, there was a repulsive-type of cell displacement, as well as when non-concentrated notochord CM was used (Fig. 1 A). Chemotaxis assays using notochord explants as a Shh source in presence of 2.5 µM cyclopamine, or with 10 µg/ml Shh pre-treated with functionblocking 5E1 anti-Shh antibody, or 10 µg/ml Shh on NCCs transfected with 1 µM antiSmoothened morpholino (MO), also showed repulsive migratory behavior of NCCs (Fig. 1 A). The latter comes from blocking the synthesis/expression of the transmembrane protein Smo responsible for triggering the signal chain when the Ptch receptor recognizes the Shh ligand. NCCs transfected with control MO and exposed to 10 µg/ml Shh gradient exhibited a chemotactic behavior equivalent to that of non-transfected NCCs exposed to the same Shh concentration (Fig. 1 A). When NCCs were transfected with anti-Shh MO to block any slight (non-detected) endogen expression of Shh, and exposed to 10 µg/ml Shh gradient, the chemotactic response was similar to those of the control MO or 10 µg/ml Shh alone (Fig.1 A). At the end of the chemotaxis assays, NCC morphology of MO-treated cultures was normal, with its usual nuclear incorporation and a diffuse cytosolic label (Fig. 1 E). As a
Page 12 of 53
11
delivery control of MO, preliminary experiments were carried out treating NCCs with an excess of MO (10 µM), which allowed us to confirm MO fluorescence within large vesicles and diffusely in the cytoplasm (Fig. 1 F), a sign of good cell incorporation [http://www.genetools.com/customer_support]. Moreover, NCC chemotaxis diminished to control values when evaluated on gradients of 50 µg/ml Shh, as well as with chemotactic (10 µg/ml) or repulsive (0.2 µg/ml) concentrations
ip t
of Shh in the presence of 2.5 µM cyclopamine or heat inactivated 10 µg/ml Shh (Fig. 1 A). Other control conditions were applied by using NCCs exposed to gradients of preimmune
results to other nonstimulating control conditions (Fig. 1 A).
cr
IgG, concentrated defined medium, or cyclopamine alone, showing equivalent negative
us
The real-time cell tracks clearly show the migration of NCCs up the Shh-containing gradient, changing from a centrifugal migration equidistant from the origin, as observed in
an
control experiments (Fig. 1 B), to displacement progressively approaching the Shh source (Fig. 1 C). This Shh-induced bias of oriented NCC distribution also emerged from other directional parameters such as the percentage of oriented cells, indicating that a significant
M
proportion exhibits chemotaxis up the Shh gradient (Fig. 2).
The data also showed that, after exposure to an ethanol concentration sufficient to induce
d
FAS, the chemotactic index and the proportion of oriented NCCs as well as the repulsive cell
Ac ce pt e
migration diminished significantly up the Shh gradient (Figs. 1 A and 2). This loss of Shhdependent chemotaxis was also clearly seen in the real-time cell trajectories after ethanol treatment of NCCs (Fig. 1 D).
Bearing in mind that ethanol-exposed NCCs showed a slight but significant expression of Shh (see Fig. 3 C), chemotaxis determination was also carried out after transfection with antiShh MO. The results exhibit a western blot identical to control NCCs (not shown) and no difference in chemotaxis behavior compared with the control MO, as well as a significant difference after exposure to 10 µg/ml Shh plus ethanol treatment (Fig. 1). The literature on cell motility indicates that chemotaxis (cell orientation) is not necessarily or directly associated with chemokinesis (cell speed). In this report, there were no significant differences in the curvilinear velocity of cells when cephalic NCCs were exposed to most of the experimental conditions described, regardless of the orientation pattern exhibited, except for a significant speed reduction when the cells were exposed to 10 µg/ml Shh pre-treated with function-blocking 5E1 anti-Shh antibody, or after exposure to 0.2 µg/ml (but not to 10 µg/ml) Shh plus ethanol, compared to control (Table 1). Linearity, a good estimator of the straightness of a migratory cell (where the unit represents a rectilinear
Page 13 of 53
12
trajectory), was significantly higher when NCCs were confronted with Shh at 5 or 10 µg/ml in the source of the gradient (Table 1). Inversely, the effect was reversed to a “convoluted” trajectory after NCC exposure to function-blocking 5E1 anti-Shh antibody, as well as to Shh 10 µg/ml plus ethanol (Table 1). The dramatic effect of ethanol alone on these dynamic parameters was also confirmed (Table 1) (Rovasio and Battiato, 2002). On the other hand, the cell health parameters showed differences in experiments
ip t
involving Shh- and/or ethanol-exposure. Thus, the cytotoxic index of NCC after confronting gradients of initial concentrations of 0.2 µg/ml Shh or Shh pretreated with the function-
cr
blocking 5E1 anti-Shh antibody, or 0.2 µg/ml Shh plus ethanol, was not different from the
control (Table 1). Moreover, the proportion of cell death after exposure to 5 or 10 µg/ml Shh
us
was lower than in the control, and leveled to control values when NCCs were exposed to 10 µg/ml Shh plus ethanol (Table 1), suggesting a trophic effect of Shh. As expected, exposure to
an
only ethanol had a greater cytotoxic effect on NCCs (Table 1) (Jaurena et al., 2011). The proliferation of NCCs was stimulated when exposed to attracting chemotaxis (5 or 10 µg/ml Shh), but was inhibited by the 5E1 anti-Shh antibody, leveling to control (Table 1).
M
These data, added to diminution of cell proliferation after treatment with 0.2 µg/ml Shh plus ethanol, and their leveling to control value when exposed to 10 µg/ml Shh plus ethanol (Table
d
1), also indicate a morphogen trophic effect. In the same way, exposure to ethanol without
Ac ce pt e
Shh induces a significant reduction of the proliferative capacity of NCCs (Table 1) (Jaurena et al., 2011).
Expression of Shh ligand, receptor system and signal chain in NCC cultures and whole embryos
Since in vitro cephalic NCCs respond with chemotaxis up the Shh concentration gradient, a more physiological view requires examining this cell population within the whole embryo to find evidence of elements that may explain this directional cell migration. Expression of the Shh ligand in the target field Whole embryo mRNA in situ hybridization, protein immunolabeling and tissue western blotting show the expression of Shh in the optic vesicle region of the embryo at the time NCCs migrate into it (Fig. 3). For the Shh mRNA in situ hybridization, we used early whole embryos as positive control, alongside its later stages corresponding to NCC migration toward the optic vesicle (Fig. 3 A, B). In a detailed sequential timing study, we observed that the initial expression of Shh at the Hensen’s node and notochord extends to the cephalic end, enlarges and expands laterally at the future infundibulum level at stage 11 HH (Fig. 3 D), and
Page 14 of 53
13
then spreads to both sides of the optic vesicle stalk at stage 12 HH (Fig 3 E, H), finally diffusely invading the space lateral to the mesencephalic level of the neural tube at stages 1213 (Fig. 3 F, G, I). Clearly, Shh mRNA expression topologically coincides with the invasion site of NCCs into the retro-ocular region (Fig. 3 E, G, H), where the ciliary ganglion will be formed. After a long immunolabel exposure time, it was also possible to localize the Shh protein (Fig. 3 F, I) with a distribution coherent with results from mRNA in situ hidridization,
ip t
matching the migratory pathway and destination of ciliary NCCs (see also Fig. 3 B).
Even though these results do not reveal the cellular source of the Shh nor the mechanism
cr
of forming and stabilizing the Shh concentration gradient, the results from western blot of
regional cell populations showed a clear expression of Shh in optic vesicle tissues (Fig. 3 C),
us
strongly supporting the optic stalk wall as the origin of the craniocaudal Shh gradient (Fig. 3 F-I). There was also a slight but significant expression of Shh protein in NCCs after exposure
an
to ethanol (Fig. 3 C) (see below).
Expression of the Shh receptor system in in vitro-in vivo responding NCCs In the experimental conditions of in vitro chemotaxis assays, NCCs express the receptor
M
Ptch (Fig. 4 A) and the membrane-activating protein Smo (Fig. 4 B). This receptor complex distributes with a typical punctiform pattern, clearly observed in the focus plane of the cell
d
surface (Fig. 4 A, B, insets). In the whole embryo, there is a clear co-localization of this
Ac ce pt e
receptor system (Fig. 4 C, D) with the mesencephalic population of NCCs as visualized with HNK1 immunolabeling (see Figs. 3 A, B, 6 C and 7 C). Expression of the Gli-Sufu system
In situ hybridization revealed a weak, common expression of the Gli-Sufu system shared by stages 9-11 HH of chick embryos. Gli 1-2-3 transcription factors express in the neural tube wall (Fig. 5 A-D, black arrows), as well as in the NCCs migrating toward the optic vesicle region (Fig. 5 A, withe arrows), in the pre-migratory NCCs on the dorsal surface of the cephalic and trunk neural tube (Fig. 5 B, D, white arrows), and in the optic-mesencephalic NCCs (Fig. 5 C, D, demarcated area). A light, diffuse expression of Sufu was seen in the neural tube wall (Fig. 5 E, black arrows) and in the migratory mesencephalic NCCs (Fig. 5 E, demarcated area), compared with embryos after control riboprobes (Fig. 5 F).
Directional migration and whole embryo distribution of cephalic NCCs are perturbed after blocking the signal cascade triggered by Shh After the evidence of chemotaxis of in vitro NCCs responding to the Shh signal and the expression of the main putative actors involved in this cell behavior, a natural corollary was to
Page 15 of 53
14
test the result of functional blocking of the Shh-Ptch/Smo system on in vivo distribution of cephalic NCCs. It is important to remember that there is no explanation for the clear directional change of ciliary NCCs toward the optic vesicle field (see Introduction). Several types of blocking experiments were carried out on whole embryos, and the cell migration-distribution was then evaluated with NCC-markers Slug mRNA in situ hybridization or HNK1 monoclonal antibody. To interrupt the signal system triggered by the
ip t
Shh ligand, a double strategy was used: (1) blocking the Smoothened (Smo) protein by a specific MO delivered bilaterally (EndoPorter®) or unilaterally (electroporation), and (2)
cr
inducing overexpression of the Patched-1 (Ptch) receptor by a corresponding mutated DNA plasmid to prevent Shh recognition and binding. In both cases, stage 8-9 HH embryos were
us
microinjected into the closing neural tube with MO, plasmid or their respective controls. Transfection with anti-Smo morpholino
an
Lissamine (sulphorhodamine B)-labeled anti-Smo morpholino (MO) was supplied together with the Endo-Porter® molecule. After about 10 h reincubation, the label was clearly seen inside the in vivo NCCs migrating to the lateral mesencephalic region (Fig. 6 A,
M
arrowheads) with only some of them toward the optic vesicle (Fig. 6 A, arrows). After the end of reincubation until stage 12 HH, the embryos exhibited a perturbed distribution of NCCs
d
with a slight invasion of the stalk and dorsal area of the optical vesicle (Fig. 6 B, arrows),
Ac ce pt e
accumulation of NCCs in the middle mesencephalic region (Fig. 6 B, arrowheads), and delayed NCCs (Fig. 6 B, double arrow) compared to MO control embryos (Fig. 6 C) or uninjected embryos of the same age (see Figs. 3 B and 7 C). In the control group of embryos co-injected with the chick anti-Smo MO plus a full length mouse Smo cDNA plasmid, the results of NCC distribution did not differ from those of control embryos (not shown), indicating a chemotaxis behavior recovery.
An interesting observation suggested a degree of selectivity of anti-Smo MO in the cephalic Shh-NCCs system. Thus, in remote trunk and caudal segments, pre-migratory NCCs exposed to anti-Smo MO/Endo-Porter®, such as the mesencephalic NCCs, showed no perturbation of their normal migration/distribution pattern. These cells exhibited the typical temporary arrest of NCC migration at the neural tube-somite angle (Fig. 6 B, inset, arrows) and a migratory pathway over the cephalic half of each somite (Fig. 6 B, inset, arrowheads). In another group of experiments, the electroporation-induced unilateral delivery of the injected MO made it possible to take the opposite embryonic side as an internal control (Fig. 7 A-D), also providing direct evidence of the crucial participation of the Shh ligand in oriented migration of NCCs toward the optic vesicle. In situ anti-Smo MO induced an
Page 16 of 53
15
abnormal distribution pattern of NCCs, resulting in sub-colonization of the optic vesicle field (Fig. 7 E, encircled area), together with a higher concentration of NCCs at the same lateral mesencephalic level (Fig. 7 E, rectangular area). The quantitative fluorescence intensity determined in standardized areas enabled us to establish that, on the MO side, the distribution of NCCs in the optic vesicle or the mesencephalic regions was not equivalent to that of the control side, nor compared with the symmetric displacement of NCCs on control embryos
ip t
(Fig. 7 F), indicating that the correct route had been lost through the anti-Smo MO effect, leading to a bad distribution of the ciliary population of NCCs.
cr
The abnormal distribution of NCCs after blocking the Shh/Ptch-Smo system, taken
together with the previous in vitro and in vivo data of this report, support a failure of the
us
spatial orientation of a subpopulation of mesencephalic NCCs which, under normal conditions, would be guided by a chemotactic response to Shh gradients emerging from the
an
optic vesicle region. It is worth mentioning that the embryos treated with anti-Smo MO show a quite conserved general morphology, while the NCC distribution exhibited diverse degrees of abnormalities with a common denominator of a lower density of optic NCC population.
M
Some embryos of the anti-Smo MO-treated group showed a more pronounced abnormal NCC distribution, maintaining a lower NCC concentration at the optic vesicle of the
d
electroporated side and a heavy accumulation of NCCs in the mesencephalic segment, as well
Ac ce pt e
as in the dorsal area of the prosencephalic and mesencephalic neural tube (Fig. 7 G, H). Embryos alternatively injected with anti-Smo MO in the left instead of the right side, showed the same but inverted abnormal NCC distribution described above (not shown). This additional control was considered necessary because of the early functional relation of Shh with the bilateral symmetry of the embryo (Gilbert, 2013). Transfection with mutated Ptch DNA plasmid and electroporation Another strategy to evaluate the effect of Shh gradient on the migration-distribution of cephalic NCCs was to induce the expression of the GFP-conjugated Ptc∆loop2 mutated Ptch receptor, a successful method assayed in neuronal differentiation of Drosophila, mouse and chick embryos in Shh gradients (Briscoe et al., 2001). In the present report, the mutated Ptch receptor expressed in NCCs prevented the binding of the Shh ligand, thus inducing abnormal distribution of cephalic NCCs, with acceptable conservation of the embryo morphology (Fig. 8 A). Embryos carrying the mutated Ptch receptor show a significantly lower volume of NCCs invading the stalk and the bulk of the optic vesicle (Fig. 8 B, arrows), as well as a higher accumulation of NCCs at the same lateral level of the mesencephalon (Fig. 8 B, C,
Page 17 of 53
16
rectangular areas) and a bulk of arrested NCCs at the dorsal neural tube (Fig. 8 B, C, arrowheads). Insertion of Shh- and cyclopamine-embedded beads Shh-embedded beads were inserted in stage 8-9 HH embryos under the ectoderm covering the lateral mesencephalic level and after reincubation for 15-18 h, they showed a net influence of the Shh ligand compared to controls (Fig. 9 B-D). When PBS control beads were
ip t
placed near to ciliary subpopulation of NCCs, their distribution was not substantially affected (Fig. 9 A), but Shh-embedded beads in the vicinity of the cephalic NCC population
cr
significantly affected their normal distribution (Fig. 9 B). The Shh source implanted near the migratory pathway of ciliary NCCs induced low dispersion toward the homolateral optic
us
vesicle (Fig. 9 B, arrow), as well as cell accumulation around the Shh source (Fig. 9 B, white arrowhead). On embryos simultaneously implanted with control beads in the contralateral
an
side, NCC distribution was as normal on this side as in the non-treated embryos (Fig. 9 B, black arrowhead). Moreover, when Shh beads were implanted at a slightly more caudal level, a perturbed distribution of NCCs was seen on the same side (Fig. 9 C, arrow), as well as
M
accumulation of cells around the Shh source (Fig. 9 C, arrowhead and detail). These observations clearly show that a Shh gradient is important for recruiting a subpopulation of
d
NCCs, as long as it is expressed at a suitable stage and for enough time.
Ac ce pt e
To discard the idea that the asymmetric distribution of NCCs might be attributed to cell loss, fluorescence cell density was bilaterally determined in a significant number of embryos, and the values between both sides of the optic vesicle plus mesencephalic levels were found not to be different in embryos unilaterally implanted with Shh-beads (Fig. 9 D). The same bilateral equivalence of NCCs was also found in the optic vesicle plus mesencephalic regions of embryos injected with anti-Smo MO and electroporated (see Fig. 7 F). That is to say that the reduction of oriented migration toward the optic vesicle and the simultaneous increase of NCCs migrating toward the ectopic Shh source (or their permanence at the mesencephalic level on Smo MO-treated embryos) seems to respond to directional interference, leading to a loss of the normal route for the ciliary subpopulation of NCCs. In another experimental group, when cyclopamine-embedded beads were inserted in stage 8-9 HH of chick embryos as described above, after the reincubation period most NCCs accumulated in the vicinity of the bead. Even if a few pioneer ciliary NCCs have reached the optic vesicle (Fig. 9 E, arrow), those emigrating later from the neural tube show a clearly perturbed distribution, remaining arrested at the mesencephalic level in the vicinity of the cyclopamine-embedded beads (Fig. 9 E, arrowhead).
Page 18 of 53
17
Ethanol exposure perturbs Shh-dependent chemotaxis and later distribution of cephalic NCCs Previous data on the proposed Shh-induced cell guidance mechanism, as well as in vitro and in vivo ethanol perturbation of NCC migration and distribution, encourage research into the association between ethanol exposure, the morphology of NCC-derived cephalic fields
ip t
and Shh expression at later stages of development.
Ex ovo and in ovo chick embryos treated at stages 8-9 HH (early migration of cephalic
cr
NCCs) with a dose of ethanol sufficient to induce FAS and studied at stages 11-12 HH,
showed a rather conserved general morphology (Fig. 10 A, B). However, immunolabeling of
us
NCCs revealed altered migratory patterns and perturbed distribution of cells (Fig. 10 C, D). Notably, there were NCCs still in transit from the neural tube (Fig. 10 C, D, arrows), while
an
the controls of equivalent age showed no emigrating mesencephalic NCCs (see Figs. 3 B, 6 C and 7 C).
Other groups of control or ethanol-treated embryos were reincubated until 3-5 days of
M
development, showing a global development of craniofacial structures (Fig. 11 A, D). Embryos of the ethanol-treated group showed diverse degrees of facial underdevelopment,
d
with mainly a low growth of the frontal process and a delay of telencephalic vesicle
Ac ce pt e
separation (Fig. 11 D, arrow), premature medial fusion and poor development of nasal placodes (Fig. 11 D, arrowhead) as well as of the maxillary and mandibular processes (Fig. 11 C, double arrowheads).
In a parallel experimental group, submitted to Shh mRNA in situ hybridization, we observed the structures and anomalies described above and also significant changes in the craniofacial expression of Shh in territories colonized by cephalic NCCs (Fig. 11 B, C, E, F). In 3-4 day control embryos, Shh expression was seen by transparency in the cephalic neuroectoderm, ventral ectoderm of the frontonasal protuberance, pharyngeal endoderm of the primitive mouth, maxillo-mandibular processes, notochord and neural tube floor (Fig.11 B and the inset). At 5 days, control embryos showed Shh expression in the diencephalic-ocular (optical stalk) neuroectoderm (not shown), maxillary process, roof of the stomodeum, limb primordium, umbilical region, notochord and neural tube floor (Fig. 11 C). On the other hand, the ethanol-treated 3-4 day embryos showed conserved Shh expression in the mesencephalon, diencephalon, telencephalon, frontal protuberance (Fig. 11 E and the inset), but there was a significant reduction of Shh expression in the maxillo-mandibular processes (Fig. 11 E, arrows), nasal placode (Fig. 11 E, arrowheads), ventral stomodeal ectoderm (Fig. 11 E, *),
Page 19 of 53
18
notochord and neural tube floor (Fig. 11 E, enlarged detail), and the diencephalon-ocular connection (not shown). At 5 days, the ethanol-treated embryos maintained Shh expression in the umbilical region and limb primordium (Fig. 11 F, arrowheads), but showed significant low expression in the maxillary process (Fig. 11 F, Mx), roof of the stomodeum (Fig. 11 F, *), notochord and neural tube floor (Fig. 11 F, double arrow).
ip t
Discussion
There are decades of evidence about when and where the embryonic cell distributes, but
cr
knowledge is still limited about how and why it does so with precision toward a final
destination. The efficient motility of neural crest cells (NCCs) relies on a network of genetic-
us
environmental signals (Bronner and Le Douarin, 2012; Le Douarin and Kalcheim, 1999; Rovasio et al., 1983) and, adding directional signals to those elements, the modulation of cell
an
orientation becomes very complex.
Chemotaxis may be a central embryonic cell compass
M
In the last years, chemotaxis has been rediscovered and enabled deeper study of the directional cell regulation of the axonal growth cone (Gore et al., 2008), and of several
d
procaryotic (Hazelbauer, 2012) and eucaryotic cells (Giojalas and Rovasio, 1998; Guidobaldi
Ac ce pt e
et al., 2012; Insall and Andrew, 2007), including the NCCs (Mwizerwa et al., 2011; Rovasio et al., 2012; Tolosa et al., 2012; Toyofuku et al., 2008; Zanin et al., 2013). A key point in designing the present work was to make the spatiotemporal frame of in vitro experiments compatible with the in vivo biological model, focusing on the migration of mesencephalic NCCs that reach the optic vesicle in a time window of 30 to 35 hours (Newgreen and Erickson, 1986). This ciliary subpopulation of NCCs separates from the bulk of cells and turns 90° toward the optic vesicle (Lee et al., 2003), a directional change that has not yet been attributed to any known modulating factor. The present in vitro approach to the migration of in vivo cells to the optic primordium is supported by our previous work showing that “old” NCC cultures of >40 hours progressively lose chemotactic capacity (Zanin et al., 2013). An important and unsurprising result of the chemotactic activity of Shh on cephalic NCCs was its coherence with data of others showing that this morphogen seems to work, not as an individual determinant, but as part of a collectively concurring strategy to orient cell motility (Jaurena, 2011; Mayor and Theveneau, 2013; McLennan et al., 2012; Rovasio et al.,
Page 20 of 53
19
2012; Theveneau et al., 2013; Tolosa et al., 2012; Zanin et al., 2013). It would thus be not too simplistic to think that such an important function as the final location of a cell population may respond not to just one molecule, and that we should rather expect the confluence (redundance?) of several guiding factors. The evolutionary interpretation would be as compensation for possible failures of the directional distribution of embryonic cells, a redundant mechanism well-known in other patterns of cell distribution (Gilbert, 2013) and
ip t
neurotrophic neuronal differentiation (Le Douarin and Kalcheim, 1999), even though it is
poorly studied in guided embryonic cell migration. Other examples of cooperative guidance
cr
emerge from several versions of the trophic factor FGF (Kubota and Ito, 2000), the RGMa/Neogenin (Gessert et al., 2008), VEGF/Neurotrophin (McLennan et al., 2010) and
us
Semaphorin/Neuropilin (Schwarz et al., 2008) systems involved in NCC guidance, as well as other short and long range communication factors (Mayor and Theveneau, 2013; Theveneau
an
et al., 2013). In our laboratory, besides evidence of a chemotactic Stem cell factor/cKit system guiding the cephalic and trunk NCCs toward the skin (Rovasio et al., 2012), it was shown that the neurotrophin NT-3/TrkC-p75 (Zanin et al., 2013), chemokine SDF-1/CXCR4 (Jaurena,
M
2011) and morphogen Shh/Ptch-Smo systems (Tolosa et al., 2012, and this report) have
d
definite guidance functions for cephalic NCCs invading the optic vesicle field.
NCCs
Ac ce pt e
The morphogen Shh expresses at the optic vesicle and induces chemotaxis on cephalic
Present data from mRNA in situ hybridization and protein immunolabeling show the expression of Shh in the route and the destination of the ciliary subpopulation of cephalic NCCs. The Shh morphogen as a chemotactic factor was involved with Netrin in the orientation of commissural axons (Charron et al., 2003) and the migration of oligodendrocytes (Merchán et al., 2007). Our real-time results (Rovasio et al., 2012) clearly show that a subpopulation of mesencephalic NCCs respond with Ptch/Smo-dependent chemotaxis up concentration gradients of Shh, excluding other migratory guidance. The data came from experiments with purified Shh, as well as optic vesicle or notochord-conditioned media, and co-cultures with notochord explants, a known source of Shh (Martí et al., 1995). Further interruption of NCC chemotaxis up Shh gradients by a function-blocking antibody (Marcucio et al., 2005) or Smo-binding cyclopamine (Chen et al., 2002) or anti-Smo MO (Locker et al., 2006) provided additional validation for our in vitro results. Moreover, the low chemotactic response after perturbed expression/function of Shh did not result from cell death, because in vitro chemotaxis was determined in real-time motile (alive) cells and their trophic parameters
Page 21 of 53
20
were not different from control conditions. This also indicates that Shh-elicited chemotaxis may be a substantial mechanism for in vivo spatiotemporal orientation of NCCs to the optic vesicle. The diverse representations of oriented cell migration were coherent, all showing a bellshaped curve typical of chemotactic response (Gore et al., 2008; Guidobaldi et al., 2012; Rovasio et al., 2012; Tolosa et al., 2012; Zanin et al., 2013). This gradient-dependent
ip t
response indicates that, when the attractant concentration increases, the specific receptors remain totally occupied and are thus incapable of detecting changes coming from the
cr
extracellular milieu, and the chemotactic response falls. This self-regulation of chemotaxis may also be involved in the arrest of migration at the colonized site (source of the
us
concentration gradient), and may help to explain the less-understood mechanism of chemorepulsion (see below), since the “less attracted” cells arriving at the target site may be
an
redirected by other chemotactic systems toward a different field. This interesting “relay station” behavior is well known among social amoebas (García and Parent, 2008), leukocytes (Afonso et al., 2012), or metastatic cells (Roussos et al., 2011). This is a less explored
M
approach to the distribution of embryonic cells, except for some pioneering data showing that in the wild type Xenopus, the Ihh signal released by cranial NCCs is itself required to support
d
both autocrine and paracrine cell migration (Agüero et al., 2012). Our present results after
Ac ce pt e
blocking any expression of endogenous NCC-expressed Shh indicate that this is not the case for the chick NCCs-Shh-optic field system.
Cells go forward, but also go backward
Besides the bell-shaped chemotactic curve, a chemo-repulsion effect was observed in NCCs confronted with a concentration of Shh 0.2 µg/ml, as well as in the presence of cyclopamine, anti-Shh antibody or anti-Smo MO. This repulsive phenomenon has been more studied in leukocyte migration toward the tissues and back to the blood (Holmes et al., 2012), as well as in the directional axon growth cone when wiring the nervous system (Ferrario et al., 2012). However, chemorepulsion has scarcely been studied except in NCC migration in the trunk ventral pathway (Jia et al., 2005), the outflow tract of the heart (Toyofuku et al., 2008), enteric innervation (De Bellard et al., 2003), and melanoma metastatic expansion (Amatschek et al., 2011). In our laboratory, we have shown this concentration-dependent bi-phasic effect in trophic factor SCF (Rovasio et al., 2012), chemokine SDF-1 (Jaurena, 2011), and morphogen Shh (Tolosa et al., 2012, and this report) on cephalic NCCs; but no similar effect was observed with neurotrophic factor NT-3 under the same conditions and the same cell
Page 22 of 53
21
population (Zanin et al., 2013). Chemorepulsion remains unexplained today probably because there is little evidence for the notion that similar molecules drive the cell forward or backward, regulating the opposite displacements by means of small, but significant, changes in some factor(s) of the taxis-associated signal chains (Ferrario et al., 2012). It should also be remembered that the existence of repulsion could be masked by the study method, since the classical transfilter methods (Boyden, 1962) do not reveal the proportion of cells migrating
ip t
against the chemotactic gradient, nor the cell velocity/distance traveled. In this context, different to the reduction of NCC cell speed elicited by chemotactic trophic factor SCF
cr
(Rovasio et al., 2012), or chemokine SDF-1 (Jaurena, 2011), our present data indicate that
chemotactic concentrations of 10 µg/ml Shh do not induce changes of speed but an increase of
us
the straightness of NCCs, suggesting that the morphogen signal involves directional coordination, without essential participation in other parameters of the cell motility
an
mechanism.
Extracellular Shh ligand triggers chemotactic signal chains
M
The fact that a subpopulation of NCCs migrating in vitro and in vivo toward the optic vesicle expresses the Ptch/Smo receptor/activator system, indicates that they respond in a
d
selective manner to exogenous Shh ligand in in vitro assays or expressed in the optic vesicle
Ac ce pt e
field of the embryo, where the ciliary ganglion of the eye will form, mostly derived from this cell population (Le Douarin and Kalchein, 1999; Lee et al., 2003). In all of our experiments, we have seen that only a fraction of the NCCs respond to Shh, suggesting a molecular heterogeneity of its regulatory compass. Considering that NCCs invade several sites of the craniofacial region (Le Douarin, 2004; Le Douarin et al., 2007), it is clear that their receptor/signal chain complex is not homogeneous and not necessarily all NCCs respond simultaneously to directional stimulus of Shh or other molecules (Mayor and Theveneau, 2013; McLennan et al., 2012; Rovasio et al., 2012; Theveneau et al., 2013; Tolosa et al., 2012; Zanin et al., 2013).
Even though the spatiotemporal expression of Shh at the target site and its receptor on the migrating NCCs supports the proposed guide function, neither the mechanism of synthesis and release of Shh morphogen nor the factor(s) involved in gradient formation and stability can be inferred. We can only speculate about the consumption of the attractant molecule by NCCs or the modulation of attractant release at the source or its variant source-sink model (Yu et al., 2009).
Page 23 of 53
22
The significant reduction of in vitro and in vivo NCC migration up the Shh gradient after treatment with anti-Shh antibody, anti-Smo MO, Smo-blocking cyclopamine, or overexpression of mutated Ptch receptor, is clearly the result of losing the way toward the optic vesicle. This also explains the concurrent high population of NCCs at the mesencephalic level of the same side of the embryo, suggesting that the cells that could not orientate toward the optic vesicle distribute on the lateral regions. This evidence is also supported by the
ip t
recovery of the normal distribution of NCCs on embryos co-injected with chick anti-Smo MO plus a full-length mouse Smo cDNA plasmid. These results indicate that the Shh morphogen
cr
is important for guiding NCCs toward the ocular region, without disregarding other
concurrent molecules and mechanisms (Jaurena, 2011; Rovasio et al., 2012; Theveneau et al.,
us
2013; Tolosa et al., 2012; Zanin et al., 2013).
The above data may also explain the “abnormal expression levels of secreted guidance
an
signals from the eye along the dorsal surface of the budding eye vesicle, which deserve elucidation” in a report on perturbed migration of cephalic NCCs in the mutant chokh/rx3 of
(Langenberg et al., 2008).
M
zebrafish, lacking eye development without interference of the neural system development
The signal chain(s) activated by Shh > Ptch > Smo interactions is a major issue that is
d
almost unknown in the biological model we are studying, and we decided not to cover it here.
Ac ce pt e
Considering that the recognition of extracellular gradient and the directional change occur within minutes, it is reasonable to think that cell orientation occurs regardless of transcriptional activity, by connecting to a non-canonical pathway, excluding (at least initially) the transcriptional system Gli-Sufu. This was proposed as the activation by Shh of cytoskeletal-associated systems such as Ras/MEK-Erk1/2 (Chang et al., 2010), Tiam1/Rac1 (Sasaki et al., 2010), 5-Lipooxigenase/Rho (Bijlsma et al., 2007), PIP3/DOCK2/Rho (Nishikimi et al., 2009) and Gli3-Sufu (Zhang et al., 2005), with involvement of the Ptch receptor and with (or without) activation of Smo activating protein (Chang et al., 2010). On the other hand, at the final stages of chemotaxis, in a matter of hours, the involvement of canonical ways (Shh > Ptch-Smo > Gli >...> ADN) is reasonable, as a transcriptional phase associated to the final steps of oriented cell migration, distribution and arrest. It is currently accepted that both canonical and non-canonical signal pathways, in a sequential or combinatory manner and sometimes with redundance, modulate processes of cell motility, proliferation and differentiation (Ayers and Thérond, 2010; Bourikas et al., 2005; Jenkins, 2009; Okada et al., 2006; Tenzen et al., 2006). Under this rationale, although the expression of Gli-Sufu system was seen in the present work in NCCs and other cell populations of the
Page 24 of 53
23
embryo, it does not seem that their involvement may qualify as actors of early orientation of the cell. It may be imprudent to propose a “principal” factor to explain any biological phenomenon, but it is fair to think of conserved elements receiving convergent triggering signals from extracellular ligand(s), then being transduced to the cytoskeleton to start the shape changes and oriented cell motility. Detection of a chemotactic gradient is a conserved
ip t
strategy from prokaryote to eukaryote cells, and spatial detection of the gradient around the
cell and the transfer of the signals to intracellular chains is accepted for the latter, frequently
cr
working with the PI3-kinase /PIP3 system and Rho-GTPases cytoskeleton modulators. This general view, conserved in amoebas, leukocytes, embryonic cells, neurons and axons (von
us
Philipsborn and Bastmeyer, 2007), is a main focus of our present research on directional cell motility. As a first conclusion, our direct experimental evidence shows that the extracellular
an
Shh concentration gradient induces directional changes of cephalic NCCs with a dynamic response of chemotaxis, suggesting a general model for cell distribution toward the ocular
M
region (Fig. 12).
The Shh-dependent chemotaxis of cephalic NCCs is perturbed by ethanol exposure
d
The high capacity of NCCs to disperse along the embryo body and produce many
Ac ce pt e
derivatives gives them increased importance in the pathogeny of developmental and tumoral pathologies (Bolande, 1974; Le Douarin et al., 2007; Matera et al., 2008), such as in craniofacial phenotypes of Fetal Alcohol Syndrome (FAS) (Chen et al., 2000; Jaurena et al. 2011; Rovasio and Battiato, 1995, 2002).
We have previously shown the ethanol-induced irreversible damage of in vitro and in vivo migratory behavior of NCCs (Rovasio and Battiato, 1995, 2002), as well as the loss of chemotactic migration up chemokine SDF-1 gradients (Jaurena, 2011). In the present report, the arrest of normal chemotaxis up Shh gradients in the presence of a teratogenic concentration of ethanol matches other reports showing disregulation of Shh signals in chick and mouse embryos treated with ethanol (Yamada et al., 2005), also producing craniofacial anomalies attributable to NCC perturbation (Ahlgren et al., 2002). Moreover, western blot results showed that embryos exposed to ethanol express Shh in the NCCs, whereas the controls only express in the optic vesicle and the remainder of the neural tube. In physiological conditions, it is also known that endogenous Ihh signaling is continuously required for the normal migration of Xenopus cranial NCCs, suggesting that this Hedgehog molecule may act in both autocrine and paracrine fashion (Agüero et al., 2012).
Page 25 of 53
24
However, our present data did not enable us to establish whether the changes in Shh expression in NCCs are part of the ethanol-induced Shh perturbation (Yamada et al., 2005), or are a consequence of an induced relay mechanism by which some NCCs begin to release the morphogen, as has been found in amoebas (García and Parent, 2008), leukocytes (Afonso et al., 2012) and tumoral cells (Roussos et al., 2011). Our experiments of blocking the endogenous Shh by ethanol-exposed NCCs enable us to suggest that this Shh expression does
ip t
not change the perturbed chemotaxis observed in this experimental condition.
In addition to perturbation of NCC chemotaxis, ethanol exposure diminished the cell
cr
linearity in gradients of 10 µg/ml Shh. Moreover, while the cytotoxic effect increased as expected in NCC cultures treated with only ethanol (Jaurena et al., 2011; Rovasio and
us
Battiato, 2002), the presence of ethanol in Shh gradients led to a drop in the proportion of cell death equal to that in the control condition. A similar trophic behavior response was found
an
when the proliferation index was evaluated. The results as a whole support the idea that ethanol-induced development anomalies may be more associated with perturbed NCC distribution (Tapadia et al., 2005) than with cell death (Ahlgren et al., 2002). Matching our
M
previous data on the protection of ethanol-treated NCCs by co-treatment with neurotrophin-3 (Jaurena et al., 2011), the present data also open up new perspectives for therapeutic and/or
Ac ce pt e
and Battiato, 1995, 2002).
d
preventive tools applicable to ethanol-induced perturbations (Jaurena et al., 2011; Rovasio
Our data on later stage craniofacial morphogenesis after early ethanol exposure show great similarity with phenotypes of FAS and those induced after blocking the Shh signal chains (Ahlgren et al., 2002; Hu et al., 2015; Marcucio et al., 2005; Tapadia et al., 2005), and thus help to explain the pathogeny of this embryopathology. We frequently saw irregular expression of in situ Shh expression in these ethanol-treated abnormal embryos, mainly in the cephalic and notochord/neural tube areas. Curiously, in other regions, such as the central nervous system wall and the bud limbs, Shh expression was not different to that in equivalent control sites. Also, considering the context of embryonic development, it is clear that a brief period of abnormal NCC behavior could be sufficient to cause serious and irreversible consequences in later stages of development, such as we found in this experimental model (Rovasio and Battiato, 1995, 2002; Jaurena et al., 2011). Our results thus support the current agreement on the risk of ethanol exposure, even for a short period and/or at a low dose, during all the period of gestation and lactancy.
Conclusions
Page 26 of 53
25
This work enabled us to show that cephalic NCCs migrate in an oriented manner as a chemotactic response to an exogenous concentration gradient of the Shh morphogen. Whole embryo in situ Shh expression in the optic vesicle region at the time of colonization by cephalic NCCs, as well as in vitro and in vivo expression of the receptor Ptch and the activating protein Smo on cephalic NCCs, complete a guidance system to orient this cell population toward their target. In vitro and whole embryo molecular and pharmacological
ip t
blocking of this system inhibit the chemotactic migration of cephalic NCCs, preventing them finding or remaining on the correct route to colonize the optic field (see Fig. 12). The lack of
cr
a generalized response of NCCs suggests that the guide function of Shh is part of a more
complex mechanism of oriented migration, complemented with other guide molecules, as was
us
supported by our and other works. This contributes to the growing concept of the convergence or redundancy of molecular signals being responsible for the modulation of key aspects of cell
an
guidance in embryo development.
As a significant derivation of the above cues, another important result of this work was the selective perturbation of cephalic NCC chemotactic guidance after in vitro and in vivo
pathogeny of this embryopathology.
M
ethanol exposure at doses sufficient to induce FAS, which also helps to better understand the
d
These results, besides improving the understanding of a basic mechanism for precise
Ac ce pt e
embryonic cell distribution and their eventual perturbation by usual teratogen agents, also contribute a new guidance activity for the Shh morphogen, apart from its other important canonical functions.
Acknowledgments
We thank Mr Joss Heywood for critical reading of the manuscript. Plasmids with gene sequences to produce chick Shh probes were kindly donated by Dr. Andrés Carrasco (FM, UBA-CONICET, Argentina), and a plasmid containing the full-length cDNA of chick Slug was kindly provided by Dr. M. A. Nieto (Instituto Cajal, Madrid, Spain). Microbeads for implantation experiments were kindly provided by Dr. Manuel Aybar (Depto. Biología del Desarrollo, INSIBIO-CONICET-Universidad Nacional de Tucumán, Argentina). This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), the Agencia Nacional de Promoción Científica y Tecnológica (FONCYT), the Ministerio de Ciencia y Tecnología de la Provincia de Córdoba and the Secretaria de Ciencia y Tecnología de la Universidad Nacional de Córdoba (Argentina).
Page 27 of 53
26
References Afonso, P. V., Janka-Junttila, M., Lee, Y. J., McCann, C. P., Oliver, C. M., Aamer, K. A., Losert, W., Cicerone, M. T., Parent, C. A., 2012. LTB4 is a signal-relay molecule during neutrophil chemotaxis. Dev. Cell 22, 1079-1091. Agüero, T. H., Fernández, J. P., Vega López, G., Tríbulo, C., Aybar, M. J., 2012. Indian hedgehog signaling is required for proper formation, maintenance and migration of
ip t
Xenopus neural crest. Dev. Biol. 364, 99-113.
Ahlgren, S. C., Thakur, V., Bronner-Fraser, M., 2002. Sonic hedgehog rescues cranial neural
cr
crest from cell death induced by ethanol exposure. Proc. Natl. Acad. Sci. USA 99, 1047610481.
us
Amatschek, S., Lucas, R., Eger, A., Pflueger, M., Hundsberger, H., Knoll, C., Grosse-Kracht, S., Schuett, W., Koszik, F., Maurer, D., Wiesner, C., 2011. CXCL9 induces chemotaxis,
melanoma cells. Br. J. Cancer 104, 469-479.
an
chemorepulsion and endothelial barrier disruption through CXCR3-mediated activation of
Angot, E., Loulier, K., Nguyen-Ba-Charvet, K., T., Gadeau, A. P., Ruat, M., Traiffort, E.,
M
2008. Chemoattractive activity of Sonic Hedgehog in the adult subventricular zone modulates the number of neural precursors reaching the olfactory bulb. Stem Cells 26,
d
2311-2320.
Ac ce pt e
Ayers, K. L., Thérond, P. P., 2010. Evaluating Smoothened as a G-protein-coupled receptor for Hedgehog signaling. Trends Cell Biol. 20, 287-298. Barnes, D., Sato, G., 1980. Methods for growth of culture cell in serum-free medium. Anal. Biochem. 102, 255-270.
Battiato, N. L., Paglini, M. G., Salvarezza, S. B., Rovasio, R. A., 1996. Method for treatment and processing of whole chick embryos for autoradiography, immuno-cytochemistry and other techniques. Biotech. & Histochem. 71, 286-288. Bijlsma, M.F., Borensztajn, K. S., Roelink, H., Peppelenbosch, M. P., Spek, C. A., 2007. Sonic hedgehog induces transcription-independent cytoskeletal rearrangement and migration regulated by arachidonate metabolites. Cell Signal 19, 2596-2604. Bolande, R. P., 1974. The neurocristopathies: a unifying concept of disease arising in neural crest maldevelopment. Human Path. 5, 409-429. Bourikas, D., Pekarik, V., Baeriswyl, T., Grunditz, A., Sadhu, R., Nardó, M., Stoeckli, E. T., 2005. Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat. Neurosci. 8, 297-304.
Page 28 of 53
27
Boyden, S. V., 1962. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes. J. Exp. Med. 115, 453-466. Breau, M.A, Wilson, D., Wilkinson, D. G., Xu, D. G., 2012. Chemokine and Fgf signaling act as opposing guidance cues in formation of the lateral line primordium. Development 139, 2246–2253. Briscoe, J., Chen, Y., Jessell, T. M., Struhl, G., 2001. A Hedgehog-insensitive form of
ip t
Patched provides evidence for direct long-range morphogen activity of Sonic Hedgehog in the neural tube. Molec. Cell 7, 1279-1291.
cr
Briscoe, J., Ericson, J., 2001. Specification of neuronal fates in the ventral neural tube. Curr. Op. Neurobiol. 11, 43-49.
us
Bronner M. E., Le Douarin N. M., 2012. Development and evolution of the neural crest: An overview. Dev Biol 366:2-9.
an
Chang, H., Li, Q., Moraes, R. C., Lewis, M. T., Hamel T. A., 2010. Activation of Erk by sonic hedgehog independent of canonical hedgehog signaling. Int. J. Biochem. Cell Biol. 42, 1462-1471.
M
Charron, F., Stein, E., Jeong, J., McMahon, A. P., Tessier-Lavigne, M., 2003. The morphogen Sonic Hedgehog is an axonal chemoattractant that collaborates with Netrin-1 in midline
d
axon guidance. Cell 113, 11-23.
Ac ce pt e
Chen, J. K., Taipale, J., Cooper, M. K., Beachy, P. A., 2002. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Develop. 16, 2743-2748. Chen, S., Periasamy, A., Yang, B., Herman, B., Jacobson, K., Sulik, K, K., 2000. Differential sensitivity of mouse neural crest cells to ethanol-induced toxicity. Alcohol 20, 75-81. Chesini, E., Bigliani, J. C., Zanin, J. P., Rovasio, R. A., Taborda, R. A. M., 2011. Electroporator applying to research on molecular and developmental biology. XVIII Congress of Bioingeneering. SABI, pp. 1-8. Buenos Aires, Argentina. De Bellard, M. E., Rao, Y., Bronner-Fraser, M., 2003. Dual function of Slit2 in repulsion and enhanced migration of trunk, but not vagal, neural crest cells. J. Cell Biol. 162, 269-279. Del Barrio, M. G., Nieto, M. A., 2004. Relative expression of Slug, RhoB, and HNK-1 in the cranial neural crest of the early chicken embryo. Dev. Dyn. 229, 136–139. Deshpande, G., Swanhart, L., Chiang, P., Schedl, P., 2001. Hedgehog signaling in germ cell migration. Cell 106, 759-769. Etheridge, L. A., Crawford, T. Q., Zhang, S., Roelink, H., 2010. Evidence for a role of vertebrate Disp1 in long-range Shh signaling. Development 137, 133-140.
Page 29 of 53
28
Ferrario, J. E., Baskarana, P., Clark, C., Hendry, A., Lerner, O., Hintze, M., Allen, J., Chilton, J. K., Guthrie, S., 2012. Axon guidance in the developing ocular motor system and Duane retraction syndrome depends on Semaphorin signaling via alpha2-chimaerin. Proc. Natl. Acad. Sci. USA 109, 14669-14674. Franco, P. G., Paganelli, A. R., López, S. L., Carrasco, A. E., 1999. Functional association of retinoic acid and hedgehog signaling in Xenopus primary neurogenesis. Development 126,
ip t
4257-4265.
Fu, M., Lui, V. C. H., Sham, M. H., Pachnis, V., Tam, P. K. H., 2004. Sonic hedgehog
cr
regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut. J. Cell Biol. 166, 673-684.
us
García, G. L., Parent, C. A., 2008. Signal relay during chemotaxis. J. Microsc. 231, 529-534. Gessert, S., Maurus, D., Kuhl, M., 2008. Repulsive guidance molecule A (RGM A) and its
an
receptor neogenin during neural and neural crest cell development of Xenopus laevis. Biol. Cell 100, 659-673.
Gilbert, S. F., 2013. Developmental Biology. 10th ed. Sinauer Ass. Inc., Publ., Sunderland,
M
MA.
Giojalas, L. C., Rovasio, R. A., 1998. Mouse spermatozoa modify their motility parameters
Ac ce pt e
201-206.
d
and chemotactic response to factors from the oocyte microenvironment. Int. J. Androl. 21,
Gore, B. B., Wong, K. G., Tessier-Lavigne, M., 2008. Stem cell factor functions as an outgrowth-promoting factor to enable axon exit from the midline intermediate target. Neuron 57, 501-510.
Guidobaldi, H. A., Teves, M. E., Uñates, D. R., Giojalas, L. C., 2012. Sperm transport and retention at the fertilization site is orchestrated by a chemical guidance and oviduct movement. Reproduction 143, 587-596.
Hamburger, V., Hamilton, H. L., 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92.
Hazelbauer, G. L., 2012. Bacterial Chemotaxis: The Early Years of Molecular Studies. Annu Rev. Microbiol. 66, 285-303. Holmes, G. R., Anderson, S. R., Dixon, G., Robertson, A. L., Reyes-Aldasoro, C. C., Billings, S. A., Renshaw, S. A., Kadirkamanathan, V., 2012. Repelled from the wound, or randomly dispersed? Reverse migration behaviour of neutrophils characterized by dynamic modeling. J. R. Soc. Interface 9, 3229–3239.
Page 30 of 53
29
Hu, D., Young, N. M., Li, X., Xu, Y., Hallgrimsson, B., Marcucio, R. S., 2015. A dynamic Shh expression pattern, regulated by SHH and BMP signaling, coordinates fusion of primordia in the amniote face. Development 142, 567-574. Insall, R., Andrew, N., 2007. Chemotaxis in Dictyostelium: how to walk straight using parallel pathways. Curr. Op. Microbiol. 10, 578-581. Jaurena, M. B., Carri, N. G., Battiato, N. L., Rovasio, R. A., 2011. Trophic and proliferative
prevented by Neurotrophin 3. Neurotoxicol. Teratol. 33, 422-430.
ip t
perturbations of in vivo/in vitro cephalic neural crest cells after ethanol exposure are
cr
Jaurena, M.B., 2011. The chemokine Stromal cell-Derived Factor-1 participate in the oriented migration of neural crest cells in normal ontogenesis and exposed to ethanol. PhD Thesis
us
Dissertation. Biology Sciences School, pp. 78. National University of Cordoba, Argentina. Jenkins, D., 2009. Hedgehog signalling: emerging evidence for non-canonical pathways. Cell
an
Signal 21, 1023-1034.
Jia, L., Cheng, L., Raper, J., 2005. Slit/Robo signaling is necessary to confine early neural crest cells to the ventral migratory pathway in the trunk. Dev. Biol. 282, 411-421.
M
Kubota, Y., Ito, K., 2000. Chemotactic migration of mesencephalic neural crest cells in the mouse. Dev. Dyn. 217, 170-179.
d
Kumada, T., Komuro, Y., Li, Y., Wang Z, Littner Y, Komuro H., 2010. Inhibition of
Ac ce pt e
cerebellar granule cell turning by alcohol. Neurosci. 170, 1328-1344. Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
Langenberg, T., Kahana, A., Wszalek, J. A., Halloran, M. C., 2008. The eye organizes neural crest cell migration. Dev. Dyn. 237, 1645-1652. Le Douarin, N. M., Brito, J. M., Creuzet, S., 2007. Role of the neural crest in face and brain development. Brain Res. Rev. 55, 237-247. Le Douarin, N. M., Kalcheim, C., 1999. The Neural Crest. 2nd ed. Cambridge Univ. Press. Cambridge.
Le Douarin, N. M., 2004. The avian embryo as a model to study the development of the neural crest: a long and still ongoing story. Mech. Dev. 121, 1089-1102. Lee, V. M., Sechrist, J. W., Luetolf, S., Bronner-Fraser, M., 2003. Both neural crest and placode contribute to the ciliary ganglion and oculomotor nerve. Dev. Biol. 263, 176-190. Locker, M., Agathocleous, M., Amato, M. A., Parain, K., Harris, W. A., Perron, M., 2006. Hedgehog signaling and the retina: insights into the mechanisms controlling the proliferative properties of neural precursors. Genes Dev. 20, 3036–3048.
Page 31 of 53
30
Marcucio, R. S., Cordero, D. R., Hu, D., Helms, J. A., 2005. Molecular interactions coordinating the development of the forebrain and face. Dev. Biol. 284, 48-61. Marigo, V., Scott, M. P., Johnson, R. L., Goodrich, L. V., Tabin, C. J., 1996a. Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development 122, 1225-1233. Marigo, V., Johnson, R. L., Vortkamp, A., Tabin, C. J., 1996b. Sonic hedgehog differentially
ip t
regulates expression of GLI and GLI3 during limb development. Dev. Biol. 80, 273–283 Martí, E., Takada, R., Bumcrot, D. A., Sasaki, H., McMahon, A. P., 1995. Distribution of
cr
Sonic hedgehog peptides in the developing chick and mouse embryo. Development 121, 2537-2547.
us
Matera, I., Watkins-Chow, D. E., Loftus, S. K., Hou, L., Incao, A., Silver, D. L., Rivas, C., Elliott, E. C., Baxter, L. L., Pavan, W. J., 2008. A sensitized mutagenesis screen identifies
an
Gli3 as a modifier of Sox10 neurocristopathy. Hum. Mol. Genet. 17, 2118-2131. Mayor, R., Theveneau, E., 2013. The neural crest. Development 140, 2247–2251. McLennan, R., Teddy, J. M., Kasemeier-Kulesa, J. C., Romine, M. H., Kulesa, P. M., 2010.
Dev. Biol. 339, 114-125.
M
Vascular endothelial growth factor (VEGF) regulates cranial neural crest migration in vivo.
d
McLennan, R., Dyson, L., Prather, K. W., Morrison, J. A., Baker, R. B., Maini, P. K., Kulesa,
Ac ce pt e
P. M., 2012. Multiscale mechanisms of cell migration during development: theory and experiment. Development 139, 2935–2944. Merchán, P., Bribian, A., Sánchez-Camacho, C., Lezameta, M., Bovoleta, P., de Castro, F., 2007. Sonic hedgehog promotes the migration and proliferation of optic nerve oligodendrocyte precursors. Mol. Cell Neurosci. 36, 355-368. Mortimer, D., Fothergill, T., Pujic, Z., Richards, L. J., Goodhill, G. J., 2008. Growth cone chemotaxis. Trends Neurosci. 31, 90-98. Mwizerwa, O., Das, P., Nagy, N., Akbareian, S. E., Mably, J. D., Goldstein, A. M., 2011. Gdnf is mitogenic, neurotrophic, and chemoattractive to enteric neural crest cells in the embryonic colon. Dev. Dyn. 240, 1402-1411. Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., Funahashi, J., 2004. Gain- and loss-offunction in chick embryos by electroporation. Mech. Dev. 121, 1137-1143. Newgreen, D. F., Erickson, C. A., 1986. The migration of neural crest cells. Int. Rev. Cytol. 103, 89-145. Nishikimi, A., Fukuhara, H., Su, W., Hongu, T., Takasuga, S., Mihara, H., Cao, Q., Sanematsu, F., Kanai, M., Hasegawa, H., Tanaka, Y., Shibasaki, M., Kanaho, Y., Sasaki,
Page 32 of 53
31
T., Frohman, M. A., Fukui, Y., 2009. Sequential regulation of DOCK2 dynamics by two phospholipids during neutrophil chemotaxis. Science 324, 384-387. Okada, A., Charron, F., Morin, S., Shin, D. S., Wong, K., Fabre, P. J., Tessier-Lavigne, M., McConnell, S. K., 2006. Boc is a receptor for sonic hedgehog in the guidance of commissural axons. Nature 444, 369-373. Paganelli, A., Gnazzo, V., Acosta, H., López, S. L., Carrasco, A. E., 2010. Glyphosate-based
ip t
herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling. Chem. Res. Toxicol. 23, 1586-1595.
cr
Paratcha, G., Ibañez, C. F., Ledda, F., 2006. GDNF is a chemoattractant factor for neuronal precursor cells in the rostral migratory stream. Mol. Cell Neurosci. 31, 505-514.
us
Pearse II, R. V., Collier, L. S., Scott, M. P., Tabin, C. J., 1999. Vertebrate homologs of Drosophila suppressor of Fused interact with the Gli family of transcriptional regulators.
an
Dev Biol 212, 323-336.
Ricardo, S., Lehmann, R., 2009. An ABC transporter controls export of a Drosophila germ cell attractant. Science 13, 943-946.
Cancer 11, 573-587.
M
Roussos, E. T., Condeelis, J. S., Patsialou, A., 2011. Chemotaxis in cancer. Nature Rev.
d
Rovasio, R. A., Battiato, N. L., 1995. Role of early migratory neural crest cells in
Ac ce pt e
developmental anomalies induced by ethanol. Int. J. Dev. Biol. 39, 421-422. Rovasio, R. A., Battiato, N. L., 2002. Ethanol induces morphological and dynamic changes on in vivo and in vitro neural crest cells. Alcohol. Clin. Exp. Res. 26: 1286-1298. Rovasio, R. A., Delouvée, A., Yamada, K. M., Timpl, J. P., Thiery, J. P., 1983. Neural crest cell migration: requirements for exogenous fibronectin and high cell density. J. Cell Biol. 96, 462-473.
Rovasio, R. A., Faas, L., Battiato, N. L., 2012. Insights into Stem Cell Factor chemotactic guidance of neural crest cells revealed by a real-time directionality-based assay. Eur. J. Cell Biol. 91, 375-390.
Salvarezza, S. B., Rovasio, R. A., 1997. Exogenous retinoic acid decreases in vivo and in vitro proliferative activity during the early migratory stage of neural crest cells. Cell Prolifer. 30, 71-80. Sambrook, J., Fritsch, E. F., Maniatis, T., 1989. Molecular cloning – A laboratory manual. 2nd ed. Cold Spring Harbor Lab Press. New York.
Page 33 of 53
32
Sasaki, N., Kurisu, J., Kengaku, M., 2010. Sonic hedgehog signaling regulates actin cytoskeleton via Tiam1–Rac1 cascade during spine formation. Mol. Cell Neurosci. 45, 335-344. Schwarz, Q., Vieira, J. M., Howard, B., Eickholt, B. J., Ruhrberg, C., 2008. Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells. Development 135, 1605-1613.
ip t
Schweitzer, R., Vogan, K. J., Tabin, C. J., 2000. Similar expression and regulation of Gli2 and Gli3 in the chick limb bud. Mech. Dev. 98, 171-174.
cr
Tapadia, M. D., Cordero, D. R., Helms, J. A., 2005. It’s all in your head: new insights into craniofacial development and deformation. J. Anat. 207, 461-477.
us
Tenzen, T., Allen, B. L., Cole, F., Kang, J. S., Krauss, R. S., McMahon, A. P., 2006. The cell surface membrane proteins Cdo and Boc are components and targets of the Hedgehog
an
signaling pathway and feedback network in mice. Dev. Cell 10, 647-656. Theveneau, E., Steventon, B., Scarpa, E., García, S., Trepat, X., Streit, A., Mayor, R., 2013. Chase-and-run between adjacent cell populations promotes directional collective
M
migration. Nature Cell Biol. 15, 763-772.
Tobin, J. L., Di Franco, M., Eichers, E., May-Simera, H., García, M., Yan, J., Quinlan, R.,
d
Justice, M. J., Hennekam, R. C., Briscoe, J., Tada, M., Mayor, R., Burns, A. J., Lupski, J.
Ac ce pt e
R., Hammond, P., Beales, P. L., 2008. Inhibition of neural crest migration underlies craniofacial dysmorphology and Hirschsprung’s disease in Bardet–Biedl syndrome. Proc. Natl. Acad. Sci. USA 105, 6714-6719.
Tolosa, E. J., Jaurena, M. B., Zanin, J. P., Battiato, N. L., Rovasio, R. A., 2012. In situ hybridization of chemotactically bioactive molecules on cultured chick embryo. J. Histotech. 35, 114-129.
Toyofuku, T., Yoshida, J., Sugimoto, T., Yamamoto, M., Makino, N., Takamatsu, H., Takegahara, N., Suto, F., Hori, M., Fujisawa, H., Kumanogoh, A., Kikutani, H., 2008. Repulsive and attractive semaphorins cooperate to direct the navigation of cardiac neural crest cells. Dev. Biol. 321, 251-262. von Philipsborn, A., Bastmeyer, M., 2007. Mechanisms of gradient detection: a comparison of axon pathfinding with eukaryotic cell migration. Int. Rev. Cytol. 263, 1-62. Yamada, Y., Nagase, T., Nagase, M., Koshima, I., 2005. Gene expression changes of Sonic Hedgehog signaling cascade in a mouse embryonic model of Fetal Alcohol Syndrome. J. Craniof. Surg. 16, 1055-1061.
Page 34 of 53
33
Yu, S. R., Burkhardt, M., Nowak, M., Ries, J., Petrasek, Z., Scholpp, S., Schwille, P., Brand, M., 2009. Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules. Nature 461, 533-537. Zanin, J. P., Battiato, N. L., Rovasio, R. A., 2013. Neurotrophic factor NT-3 displays a noncanonical cell guidance signalling function for cephalic neural crest cells. Europ. J. Cell Biol. 92, 264-279.
ip t
Zhang, W., Zhao, Y., Tong, C., Wang, G., Wang, B., Jia, J., Jiang, J., 2005. Hedgehog-
Regulated Costal2-kinase complexes control phosphorylation and proteolytic processing of
Ac ce pt e
d
M
an
us
cr
cubitus interruptus. Dev. Cell 8, 267-278.
Page 35 of 53
34
Legends for figures Fig 1. A. Chemotactic index of NCCs confronted with Sonic hedgehog (Shh) gradients at different initial concentrations (from the right side), as well as with various control media and in the presence of ethanol. Attraction (black bars) and repulsion (gray bars) are shown. Ctrl: control. NE: notochord explants. NCCM: non-concentrated conditioned medium. CCM: concentrated conditioned medium. CDM: concentrated defined medium. Shh: µg/ml. Cyp: 2.5
ip t
µM cyclopamine. NE + Cyp: NE in presence of Cyp. Shh (µg/ml) + Cyp: Shh in presence of Cyp. Inac: inactivated Shh. 5E1: function blocking anti-Shh antibody. Ctrl IgG: preimmune
cr
control IgG. SmoMO: NCCs transfected with 1 µM chick anti-Smoothened morpholino.
ShhMO: NCCs transfected with 1 µM anti-Shh MO. Ctrl MO: NCCs transfected with 1 µM
us
control MO. Shh (µg/ml) + Etoh: Shh in presence of 100 mM ethanol. a: Difference versus Ctrl (p < 0.05). b: Difference versus NE (p < 0.05). c: Difference versus NCCM (p < 0.05). d:
an
Difference versus Shh 10 µg/ml (p < 0.05). B. Cell tracks of individual cells of a typical experiment in control condition. C. Tracks of NCCs migrating in a gradient of Shh 10 µg/ml in the source (right side). D. Migratory behavior of NCCs exposed to chemotactic gradient of
M
Shh in the presence of 100 mM ethanol. E. In vitro NCCs after Smo-MO transfection. F.
d
Delivery control of Smo-MO transfection (see details in the text).
Ac ce pt e
Fig. 2. Proportion of cells migrating in a 180° angle open toward (black bars) or against (gray bars) the Sonic hedgehog (Shh) source of the gradient at different initial concentrations (from the right side), as well as with several control media and in the presence of ethanol. The sum of repulsion plus attraction is not 100%, because the non-oriented population was not considered (see method details in Rovasio et al., 2012). Abbreviations as in Figure 1. a: Difference versus Ctrl (p < 0.05). b: Difference versus NE (p < 0.05). c: Difference versus NCCM (p < 0.05). d: Difference versus Shh 10 µg/ml (p < 0.05).
Fig. 3. Expression of the Shh ligand. A. Chick embryo of stage 12 HH showing main components. Prosencephalon (P), mesencephalon (M), rhombencephalon (R), optic vesicle (OV), neural crest cells (NCCs, demarcated area). Dark field. B. The same embryo showing HNK1-antibody immunolabeling of NCCs (demarcated area). The arrows indicate migration pathways of cephalic NCCs toward M and OV regions. C. Above: Immunoblot of tissues shows the expression of Sonic hedgehog (Shh) and tubulin (Tub = loading control) at neural cell crest population (NCCs), optic vesicle (OV) and the cephalic remaining (CR), in control
Page 36 of 53
35
(Ctrl) and ethanol-treated (Etoh) embryos. Below: Mean relative density graph corresponding to immunoblots. The Shh expression in control NCCs is significantly lower than in OV (p< 0.02), CR (p< 0.001) and Etoh-exposed NCCs, OV and CR (p< 0.05). D. Cephalic pole of chick embryo of stage 11 HH. Shh mRNA in situ hybridization (ISH) shows notochord expression with the cephalic expansion at prosencephalon level (arrow). E. Chick embryo of stage 12 HH shows ISH of Shh expression at the OV stalk (arrows). Broken line indicates
ip t
transversal section of H. F. Chick embryo of stage 12 HH shows immunolabel of Shh protein “detaching” from the OV stalk toward mesencephalic space (broken arrows). Horizontal
cr
broken line indicates transversal section of I. G. Posterolateral oblique view of a chick
embryo of stage 13 HH showing the detailed ISH Shh expression at the OV stalk (arrows). H.
us
Transversal section indicated in E, showing HNK1-labeled NCCs surrounding the OV stalk (arrows) and the ISH-Shh expression at the basal plate of forebrain and OV stalk
an
(arrowheads). I. Transversal sections indicated in F, showing at left side a control embryo (Ctrl) and at right side the immunolabeled Shh protein (arrows).
M
Fig. 4. A. Culture of mesencephalic neural crest cells (NCCs) immunolabeled for Patched (Ptch) receptor. B. Culture of NCCs immunolabeled for Smoothened (Smo) membrane
d
activating protein. In both enlarged details, note the punctiform pattern in the focus plane of
Ac ce pt e
cell surface. C. Cephalic pole of chick embryo of stage 10-11 HH showing in situ hybridization (ISH) expression of Ptch receptor on NCCs (demarcated area). D. Equivalent chick embryo after ISH of Smo protein in NCCs (demarcated area). OV: optic vesicle.
Fig. 5. A. In situ hybridization (ISH) for the transcription factor Gli1 in stage 11 HH chick embryo. B. ISH of Gli2 in stage 9 HH chick embryo. C. ISH of Gli3 in stage 10 HH chick embryo. D. ISH of Gli3 in stage 11 HH chick embryo. E. ISH of SUFU protein in stage 11 HH chick embryo. F. Representative embryo after a sense control riboprobe for Gli-SUFU system. Note a shared expression of Gli-SUFU at the neural tube wall (A-E, black arrows), the neural crest cells (NCCs) moving to optic vesicle stalk (A, white arrows), presumptive NCCs at the dorsal wall of cephalic and trunk neural tube (B, D, white arrows), and opticmesencephalic NCCs (C-E, demarcated area).
Fig. 6. A. Chick embryo at stage 10 HH, microinjected into the neural tube at stage 8 HH with anti-Smo morpholino (MO)/EndoPorter® and reincubated for 10 hours. The MO (red dots) is seen in the in vivo NCCs migrating toward mesencephalic level (arrowheads) and scarcely to
Page 37 of 53
36
the optic vesicle (OV) (arrows). B. The same embryo as A, after reincubation until stage 12 HH. The HNK1 labeled NCCs migrate toward mesencephalic segment (arrowheads), only a few to the OV (arrows, compare with C) and some others are still on the route (double arrow). Inset. Probable selectivity of the anti-Smo MO on cephalic NCCs/Shh-Ptch/Smo system. The HNK1-labeled NCCs of trunk level of anti-Smo MO transfected embryo (under the same influence as cephalic NCCs) show normal migratory behavior, with temporary arrest at the
ip t
neural tube-somite angle (arrows) and displacement along the cephalic half of each somite
(arrowheads) (see details in the text). C. HNK1-labeled stage 12 HH control embryo showing
cr
a normal NCC population filling mesencephalic and OV areas.
us
Fig. 7. A. Dark field, and B. Fluorescence microscopy of a stage 9 HH chick embryo just microinjected into the neural tube lumen with a lisamine-conjugated anti-Smo morpholino
an
(MO) and electroporated (+). C. and D. Control stage 11 HH chick embryo injected at stage 9 HH into the neural tube with a lisamine-conjugated control MO and electroporated (+). Note at C, the NCCs bilaterally distributed toward mesencephalic (M) and optic vesicle (OV)
M
regions (compare with Figs. 3 A, B and 6 C), and at D, the lateral location of the control MO. Broken vertical line indicates cranio-caudal axis. E. Chick embryo at stage 11 HH injected
d
into the neural tube with anti-Smo MO at stage 9 HH and electroporated (+). The HNK1
Ac ce pt e
immunolabeling shows a lower displacement of NCCs (arrow) towards the OV (circle) on the electroporated side (+) and a higher cell population in mesencephalic segment of the same side (rectangle). P: prosencephalon; M; mesencephalon; R: rhombencephalon. F. Graph of fluorescence intensity of NCCs in the OV and M regions (see E) of control (Ctrl) and antiSmo MO injected/electroporated embryos. Reduction of NCCs in the OV region and higher ell density at M segment are seen in the anti-Smo MO-treated group. Mean ±SE. a = p <0.05. G. and H. Embryos treated as explained for E show pronouced changes at OV (circle) and M (rectangle) levels, in line with those just described.
Fig. 8. A. Bright field, and B. HNK1-immunolabeled chick embryo at stage 12 HH, transfected at stage 8-9 HH with DNA plasmid corresponding to mutated Ptch receptor (Ptc∆loop2) and electroporated (+). C. Another embryo of the same group, both showing the perturbed dispersion of NCCs in the electroporated side (+): less cell density at the optic vesicle (OV, arrows, circles), cell accumulation at the mesencephalic level (rectangle), and arrested NCCs on the dorsal surface of the neural tube (arrowheads).
Page 38 of 53
37
Fig. 9. A. Chick embryo at stage 12 HH, implanted at stage 8-9 HH in right side region of mesencephalic level with beads embedded with PBS (white arrowhead). NCC dispersion is equivalent between both sides. B. Equivalent chick embryo implanted in the right side of the mesencephalic region with beads embedded with Shh (white arrowhead) and in the left side with PBS embedded beads (black arrowhead). Note NCCs stopped caudally to the optic vesicle (OV, arrow) and recruited around the ectopic source of Shh, with a normal dispersion
ip t
at the control left side. C. Similar chick embryo implanted in the right side of the caudal
mesencephalic region with beads embedded with Shh (arrowhead). In addition to the NCCs
cr
not reaching the OV (arrow), they accumulate around the ectopic source of Shh (arrowhead). The enlarged detail clearly shows the NCCs tightly around the bead. D. Density graph of
us
NCCs expressed as fluorescence intensity after immunolabeling with HNK1 antibody. Bilateral cell density corresponding to optic vesicle plus mesencephalic regions of control
an
embryos or unilaterally inserted with Shh embedded beads was not significantly different between both sides of the embryo. Mean ±SE. E. Chick embryo equivalent to A-C, implanted with cyclopamine-embedded beads in the right side of mesencephalic level (arrowhead).
M
There is a remarkable absence of a normal dispersion of NCCs toward the OV (arrow).
d
Fig. 10. A. Dark-field, B. Bright-field, C. and D. HNK1-fluorescence microscopy of chick
Ac ce pt e
embryos at stage 12 HH treated in ovo at stage 8-9 HH with 100 mM ethanol. Note the conserved normal gross morphology (A, B) coexisting with early disturbed dispersion of NCCs: deficient colonization of the OV region (C, D, compare with Figs. 3 B, 6 C and 7 C), as well as many cells still on the route (arrows). Broken line outlines the cephalic embryonic pole and the neural tube.
Fig. 11. A. and B. Craniofacial aspect of control chick embryos at stage 22-23 HH (3-4 days) treated in ovo at stage 8-9 HH with PBS. A. The global structure involves frontal process (F), telencephalic vesicles and eye primordium (O), nasal placodes (N), maxillary (Mx), mandibular (Mb) and hyoid (H) processes, and stomodeum (*). B. In situ hybridization for mRNA to Shh shows the expression by transparency in cephalic neuroectoderm, ventral ectoderm of frontonasal protuberance (arrowheads), pharyngeal endoderm of stomodeum (*), maxillo-mandibular processes (arrows), notochord and neural tube floor (detail on inset). C. A cephalo-thoracic view of control chick embryo at stage 26 HH (5 days) shows Shh expression at the maxillary process (Mx) and roof of the stomodeum (*), distal limbs and umbilical region (arrowheads), notochord and neural tube floor (arrows). D. and E. Craniofacial view of
Page 39 of 53
38
chick embryos of stage 22-23 HH (3-4 days) treated in ovo at stage 8-9 HH with ethanol. D. Note diverse degrees of facial underdevelopment, global low growth of the frontal process, delay of cephalic vesicle separation (arrow), poor development of nasal placodes with premature medial fusion (arrowhead), low development of maxillary (double black arrowheads) and mandibular (double white arrowheads) processes. E. After in situ hybridization for mRNA to Shh, structural anomalies and perturbed expression of Shh in
ip t
territories colonized by cephalic NCCs are seen to coexist. While the ethanol-treated embryo exhibits a conserved Shh expression in telencephalon and frontal protuberance, there is a
cr
significant reduction of Shh expression in the nasal placode (arrowheads), ventral stomodeal ectoderm (*), maxillo-mandibular processes (arrows), notochord and neural tube floor (detail
us
on inset). F. A cephalo-thoracic view of ethanol-treated chick embryo at stage 26 HH (5 days) shows reduction of Shh expression at the maxillary process and roof of the stomodeum (*),
umbilical region and distal limbs (arrowheads).
an
notochord and neural tube floor (arrows), with preservation of morphogen expression in the
M
Fig. 12. Simplified model of migration/distribution of mesencephalic NCCs to ocular region. (1) Neural tube emerging NCCs (dots) migrate dorso-laterally (horizontal arrow) occupying
d
the space between the neural tube and ectoderm. (2) Ciliary population of NCCs recognizes
Ac ce pt e
the concentration gradient (curved bands) of molecules released from the optic vesicle (OV) region, including Shh. (3) NCCs respond with orientated migration toward the optic area (curved arrow). (4) Reaching the attractant source, its higher relative concentration saturates the receptors and consequently stops the guide mechanism. (5) At this spatiotemporal intersection, some NCCs differentiate as neurons and glia of the ciliary ganglion. It is probable that ciliary NCC plasticity enables the recognition and response to other cooperating attractants (see text). P: prosencephalon; M: mesencephalon; R: rhombencephalon; S: somites.
Page 40 of 53
39
Table 1. Dynamic and viability parameters of in vitro NCCs after Shh/Ethanol exposure
Shh 10
30.00 ±1.5 vs Ctrl: NS
Shh 10 + 5E1
17.8 ±1.2 vs Ctrl: p <0.001 vs Shh 10: p <0.001
Shh 0.2 + Etoh
Etoh
27.90 ±4.5 vs Ctrl: NS vs Shh 10: NS
10.08 ±1.1
vs Ctrl: p <0.001 vs Shh 10: p <0.001 vs Shh 10 + Etoh: p <0.019
1
0.75 ±0.04
vs Ctrl: p <0.03
0.88 ±0.03
0.060 ±0.005
vs Ctrl: p <0.001
0.57 ±0.04 vs Ctrl: NS vs Shh 10: p <0.001
0.59 ±0.05
ip t
0.055 ±0.005
vs Ctrl: p <0.008
vs Ctrl: NS vs Shh 0.2: NS
0.44 ±0.02
vs Ctrl: p <0.001 vs Shh 10: p <0.001
0.23 ±0.03
Ac ce pt e
Shh 10 + Etoh
15.25 ±1.9 vs Ctrl: p <0.001 vs Shh 0.2: p <0.05
vs Ctrl: NS
vs Ctrl: p <0.001 vs Shh 10: p <0.001 vs Shh 10 + Etoh: p <0.001
0.102 ±0.006 0.125 ±0.019
cr
32.25 ±2.6 vs Ctrl: NS
vs Ctrl: NS
us
Shh 5
0.080 ±0.006 0.087 ±0.008
vs Ctrl: p<0.02
0.084 ±0.012
an
vs Ctrl: NS
Viability parameters Cytotoxic index Proliferation index 2 2 (n = 69) (n = 75)
vs Ctrl: NS vs Shh 10: NS
0.100 ±0.011
M
Control Shh 0.2
Dynamic parameters Curvilinear Linearity 1 velocity (n = 780) 1 (n = 780) 29.05 ±2.4 0.63 ±0.02 30.75 ±2.5 0.68 ±0.03
d
Experimental condition
vs Ctrl: NS vs Shh 0.2: NS
0.087 ±0.006
vs Ctrl: NS vs Shh 10: p <0.006
0.116 ±0.011 vs Ctrl: p <0.006 vs Shh 10: p <0.001 vs Shh 10 + Etoh: p <0.03
vs Ctrl: NS
0.126 ±0.010
vs Ctrl: p <0.05
0.130 ±0.0095 vs Ctrl: p <0.02
0.115 ±0.016 vs Ctrl: NS vs Shh 10: NS
0.062 ±0.017 vs Ctrl: p <0.03 vs Shh 0.2: p <0.02
0.108 ±0.017 vs Ctrl: NS vs Shh 10: NS
0.067 ±0.003 vs Ctrl: p <0.001 vs Shh 10: p <0.001 vs Shh 10 + Etoh: p <0.02
2
n : number of cell contours and centroids; n : number of cells; Shh: Sonic hedgehog (µg/ml); 5E1: function-blocking anti-Shh antibody; Etoh: ethanol 100 mM; NS: no significant difference.
Page 41 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 42 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 43 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 44 of 53
Ac ce p
te
d
M
an
us
cr
ip t
Figure Click here to download high resolution image
Page 45 of 53
Ac ce p
te
d
M
an
us
cr
ip t
Figure Click here to download high resolution image
Page 46 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 47 of 53
Ac ce p
te
d
M
an
us
cr
ip t
Figure Click here to download high resolution image
Page 48 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 49 of 53
Ac ce p
te
d
M
an
us
cr
ip t
Figure Click here to download high resolution image
Page 50 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 51 of 53
Ac
ce
pt
ed
M
an
us
cr
i
Figure Click here to download high resolution image
Page 52 of 53
Ac ce p
te
d
M
an
us
cr
ip t
Figure Click here to download high resolution image
Page 53 of 53