- Email: [email protected]
Primary cilia and forebrain development
MECHANISMS OF DEVELOPMENT
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/modo
Primary cilia and forebrain development Marc August Willaredt, Evangelia Tasouri, Kerry L. Tucker
*
Interdisciplinary Center for Neurosciences, Institute of Anatomy and Cell Biology, University of Heidelberg, 69120 Heidelberg, Germany
A R T I C L E I N F O
A B S T R A C T
Article history:
With a microtubule-based axoneme supporting its plasma membrane-ensheathed projec-
Available online 22 October 2012
tion from the basal body of almost all cell types in the human body, and present in only one copy per cell, the primary cilium can be considered an organelle sui generis. Although it was
Keywords: Primary cilia Cerebral cortex Forebrain Sonic hedgehog Gli3 Telencephalon
first observed and recorded in histological studies from the late 19th century, the tiny structure was essentially forgotten for many decades. In the past ten years, however, scientists have turned their eyes once again upon primary cilia and realized that they are very important for the development of almost all organs in the mammalian body, especially those dependent upon the signaling from members Hedgehog family, such as Indian and Sonic hedgehog. In this review, we outline the roles that primary cilia play in forebrain development, not just in the crucial transduction of Sonic hedgehog signaling, but also new results showing that cilia are important for cell cycle progression in proliferating neural precursors. We will focus upon cerebral cortex development but will also discuss the importance of cilia for the embryonic hippocampus, olfactory bulb, and diencephalon. Ó 2012 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Cilia are 1–3 lm membrane-bound, microtubule-based structures found on many cells in the vertebrate body (Davis et al., 2006). Cilia can be classified into two groups regarding their ultrastructure and function; both groups share at their base a centrosome-derived basal body that anchors the cilium within the cytoplasm. The axoneme in motile cilia is organized in a ‘9 + 2’ arrangement, where the double microtubule core in the middle is surrounded by nine microtubule doublets arranged in a circular pattern. Motility is achieved through the protein–protein interactions between the central core and the surrounding doublets. Non-motile cilia, also known as primary cilia, show a ‘9 + 0’ arrangement lacking the central microtubule core. Even though primary cilia have been considered to be completely non-motile, it has been reported that primary cilia at the embryonic node can generate a rhythmic movement, though not in the whiplike fashion of motile cilia (Nonaka et al., 1998). Both classes of cilia are
maintained by the transport of protein cargo along the axoneme mediated by a microtubule-based transport, namely intraflagellar transport (IFT) (Scholey and Anderson, 2006). IFT utilizes kinesin-II- and dynein-based motors to facilitate anterograde and retrograde transport, respectively. Interaction between protein cargo and the transport motors occurs through the IFT scaffolding proteins complexes IFTA and IFTB, which in turn are composed of IFTX proteins, in which X denotes the molecular weight of the given protein. Even though cilia in the past have been undervalued, recent studies of Hedgehog (Hh) signaling reveal striking evidence of the importance of cilia in signal transduction. Hh signaling has a critical role during development because of its implication in patterning events (Dessaud et al., 2008). There are three vertebrate ligands for Hh signaling, Sonic (Shh), Indian and Desert, that all bind to the specific receptor for Hh-family ligands Patched (Ptch1). In the absence of any ligand, Ptch1 prevents Smoothened (Smo), a G-coupled transmembrane protein, to associate with the membrane, thus
* Corresponding author. Address: Interdisciplinary Center for Neurosciences, Institute of Anatomy and Cell Biology, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany. Tel.: +49 6221 54 8687; fax: +49 6221 54 4952. E-mail address: [email protected] (K.L. Tucker). 0925-4773/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mod.2012.10.003
374
MECHANISMS OF DEVELOPMENT
repressing signal transduction. In the presence of a ligand, Ptch1 moves out of the cilium, thereby relieving the inhibition of Smo activity (Corbit et al., 2005; Rohatgi et al., 2007). Smo moves into the primary cilium (Corbit et al., 2005) and transduces the Hh signal through its effects upon the transcription factors Gli2 and Gli3, which also localize to primary cilia (Haycraft et al., 2005). Both transcription factors are activated upon association with cilia, while Gli3 is proteolytically converted from a full-length form to a shorter repressor form (Gli3R) in a cilia-dependent fashion. Dozens of recent papers have proven the dependence upon primary cilia for Shh signaling in the development of such disparate organs as the spinal cord, heart, mammary gland, kidneys, ovaries, and pancreas, while Indian hedgehog has been shown to signal through primary cilia in skeletal development (for a thorough review, see (Tasouri and Tucker, 2011)). More controversial has been the involvement of primary cilia in Wnt signaling. Although in vitro analysis indicated that primary cilia constrain canonical Wnt signaling (Corbit et al., 2008), the evidence from in vivo studies is far from clear. Comprehensive analysis of Wnt-dependent developmental processes in zebrafish and the mouse revealed no role for primary cilia (Huang and Schier, 2009; Ocbina et al., 2009), but perturbations in Wnt signaling have been recorded in the later development of many organ systems, including the forebrain, cerebellum, kidney, pancreas, mammary gland, and long bones (reviewed in (Tasouri and Tucker, 2011)). Whether changes in Wnt signaling reflect a secondary effect downstream of a Hh-based perturbation, or in fact indicate a direct role of primary cilia in later Wnt-dependent developmental processes is still unresolved. This review focuses upon the relevance of primary cilia for the development of the forebrain, with an emphasis upon the largest portion of the forebrain, the cerebral cortex. The following results are also summarized in Table 1.
2. Primary development
cilia
and
cerebral
cortex
Using a hypomorphic allele of the Ift88 gene, cobblestone, uncovered by ENU mutagenesis, Willaredt et al. (2008) were the first to examine the role that primary cilia have in cortical morphogenesis. They demonstrated that in cobblestone mutants both mRNA and protein levels of Ift88 are reduced by 70–80%. Cobblestone mutants not only displayed severe malformations of the hippocampus, choroid plexus and cortical hem, but additionally the dorsal–ventral boundary between the pallium and subpallium was not properly determined, although the dorsal and ventral domains were not shifted, as seen in other cilia mutants (Besse et al., 2011; Stottmann et al., 2009). Surprisingly, transmission and scanning electron microscopy presented in this study showed that primary cilia were still present and appeared intact at the ultrastructural level. These various phenotypes are comparable to the ones observed in the Gli3 deletion mutant Extra toes (XtJ) (Fotaki et al., 2006; Theil et al., 1999; Tole et al., 2000), which prompted the investigation of Gli3 proteolytic processing in cobblestone mutants. The authors observed an increase of the full-length form of Gli3. A number of phenotypes seen
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
in the cobblestone mutants, such as the formation of rosetteshaped heterotopias in the dorsal cortex or the defects in the determination of tissue of the dorsal telencephalon mirror ones observed in XtJ/XtJ embryos. Further analysis of Shh signaling targets, such as Ptch1 and Gli1, showed an increase in their expression in the cobblestone mutants (Willaredt et al., 2008). Examination of changes in Wnt signaling also revealed an upregulation of canonical Wnt signaling, as reflected in the increased and ectopic expression of Wnt7b, Wnt8b, and Axin2. Like Ift88, the protein encoded by the Ift172 gene is a member of the IFTB complex involved in anterograde ciliary traffic. Inactivation of this gene in the mouse resulted in phenotypes reminiscent of cobblestone, in that the mice displayed holoprosencephaly and exencephaly, but in addition the mutants suffered from craniofacial defects (Gorivodsky et al., 2009). Of great interest was their finding that nodal expression was eliminated in embryonic day 7.5 (E7.5) embryos, which is important for the subsequent induction of anterior mesendoderm that will go on to express Shh, Foxa2, and Gsc (Vincent et al., 2003). This may contribute to the forebrain phenotypes seen in other ciliary mutants. It is important to point out that the two aforementioned mutants (cobblestone, Ift172) were in IFTB complex proteins, which would imply that anterograde IFT would be compromised in these mutants. As seen directly below, one might not expect the same phenotype from a mutant in retrograde IFT. In contrast to Ift88 and Ift172, the protein Ift139, encoded by the gene Ttc21b, is a member of the IFTA complex that is involved in retrograde IFT. A mouse mutant for this gene showed a correspondingly different phenotype, in which IFT proteins and their cargo accumulated within the cilium (Stottmann et al., 2009; Tran et al., 2008). This resulted in an increase in Shh signaling, as evidenced by a pronounced ventralization of the forebrain and an upregulation of downstream components of the Shh signaling pathway, including Shh, Ptch1 and Foxa2. By crossing the Ttc21b mutant to a Shh mutant, the resulting phenotype was milder than the Ttc21b mutant alone, allowing the authors to conclude that from a genetic perspective, Ttc21b inhibits Shh signaling, presumably by promoting the exit of activated Smo from the cilium. With respect to Wnt signaling, the authors examined a Wnt-sensitive lacZ transgene and observed a downregulation, whereas betacatenin levels showed no change (Stottmann et al., 2009). Ftm encodes a protein that localizes to the basal body of cilia. The human ortholog of Ftm is called RPGRIP1L, and this gene is mutated in certain patients suffering from Joubert or Meckel syndrome, two well-characterized heritable ciliopathies (Arts et al., 2007; Delous et al., 2007). The mouse mutant for Ftm has been examined for its forebrain phenotype. In the forebrain of the targeted Ftm mutant (Vierkotten et al., 2007), a ventralization phenotype could be recorded, in that two standard markers identifying ventral forebrain, Dlx2 and Gsx2, were enlarged in their expression domains, while the expression domains of the dorsal cortical markers Ngn2 and Pax6 were diminished (Besse et al., 2011). However, this ventralization was limited to the most anterior part of the telencephalon. Unusually, expression of Shh and two downstream targets, Gli1 and Nkx2.1, were unaffected in the ventral forebrain, where they are normally to be found, in midgestation
MECHANISMS OF DEVELOPMENT
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
375
Table 1 – Summary of the proteins and gene alleles discussed in this review, with a short description of the forebrain phenotypes and references to the papers in which the findings and/or the particular mouse model are reported. Protein
Mutation
Phenotype
Ift88
cobblestone ENU-mutagenesis: Hypomorph
Willaredt et al. (2008) Malformation of hippocampus, choroid plexus, cortical hem. Rosette-shaped heterotopias in dorsal cortex. Pallial-subpallial boundary breaks down. Telencephalon– diencephalon boundary dissolved.
Ift172
Slb Targeted deletion: Full knockout
Holoprosencephaly & exencephaly. Midbrain-hindbrain boundary defects. Loss of nodal expression.
Ttc21b (Ift139) alien (aln) ENU-mutagenesis: Complete loss-of-function
References
Gorivodsky et al. (2009)
Forebrain ventralization. Tran et al. (2008); Stottmann et al. (2009) Forebrain reduced, midbrain expanded. Disorganized cortex.
Gli3
Extra toes (XtJ) 51.5-kb genomic deletion: Complete loss-of-function
Theil et al. (1999); Tole et al. (2000); Highly disorganized Fotaki et al. (2006) neocortex. Rosette-shaped heterotopias in dorsal cortex. Telencephalon– diencephalon boundary dissolved.
Ftm
Ftm Targeted deletion: Full knockout
Forebrain ventralization. Dorsal cortical markers diminished in anterior telencephalon. Shrunken, mushroom-like cilia. Olfactory bulb agenesis.
Besse et al. (2011); Vierkotten et al. (2007)
Gli3;Ftm
Gli3 targeted mutation: C-terminal deletion mutant; Ftm double mutant
Ventralized telencephalon phenotype rescued. No cilia at ventricle.
Besse et al. (2011); Bo¨se et al. (2002)
Kif3a
Kif3a targeted mutation: Normal cortical dorsal– Wilson et al. (2011); Marszalek et al. (1999) LoxP sites for inducible knockout ventral expression domains. No cilia at VZ in cortex. Larger cerebral cortex volumes. Rosette-shaped heterotopias.
Tctex-1
Tctex-1: In utero RNAi
Kif3a
Kif3a targeted mutation: Postnatal hippocampal LoxP sites for inducible knockout hypoplasia. Failure of GNP pool expansion and subsequent establishment of adult stem cells in dentate gyrus. Epistatic relationship between Smo & Kif3a.
Han et al. (2008)
Stumpy
Stumpy targeted mutation: Postnatal hippocampal LoxP sites for inducible knockout hypoplasia. Reduction in precursor proliferation.
Breunig et al. (2008); Town et al. (2008)
Tctex-1 knockdown: excessive neuronal differentiation, depletion of glial cells.
Li et al. (2011)
376
MECHANISMS OF DEVELOPMENT
Ftm mutants. Examination of primary cilia at the ventricle of the forebrain revealed first that the Ftm protein was absent in the mutant, as expected, and that the cilia exhibited a shrunken, mushroom-like dismorphology, although the basal bodies appeared to be of normal appearance and location at the apical membrane. Examination of tight junctions between proliferating precursors at the ventricle revealed no abnormalities, but further markers of polarity were not examined. The authors examined the ratio of full-length Gli3 to the shorter, proteolytically-processed isoform, and found that the ratio of the former to the latter increased, in keeping with results seen in both cobblestone mutants (Willaredt et al., 2008) and in many other organs in multiple ciliary mutants (Haycraft et al., 2005; Huangfu and Anderson, 2005; Liu et al., 2005; May et al., 2005; Tran et al., 2008). Most importantly, the ventralized telencephalon phenotype could be rescued by crossing the Ftm mutant to a knockout mutation of the Gli3 gene in which only the truncated isoform of Gli3 is expressed (Bo¨se et al., 2002). As this shorter isoform is thought to act as a transcriptional repressor, it shows that the major defect in the Ftm mutants is the absence of this isoform, and not the increased levels of full-length Gli3. Even more exciting was the finding that the Gli3;Ftm double mutants did not produce cilia at the ventricle (Besse et al., 2011), thereby proving that, in principle, defects attributable to the function of the primary cilium can be compensated for by expression of the relevant proteins lying downstream of the cilium in the signal transduction pathway. This may be of great relevance for therapeutic approaches to ciliopathies such as polycystic kidney disease. Although the studies described in the previous section were very useful in outlining the role of primary cilia with respect to Shh signaling, they all employed standard constitutive knockout/hypomorph approaches. Thus the relatively early contributions that Shh makes to development already at E7.0 could contribute to phenotypes analyzed much later. It would be preferable to affect ciliary activity later, and the next two studies have approached this problem by using either a conditional knockout (Wilson et al., 2011) or a siRNA-based (Li et al., 2011) approach. In both cases, the authors examined the relationship between primary cilia and cell cycle dynamics at the ventricular zone of the embryonic cortex. In the first case, the authors used a nestin::CRE deleter mouse line to specifically eliminate expression of Kif3a (Marszalek et al., 1999) in cortical progenitors at the beginning of the neurogenic program (Petersen et al., 2002). Kif3a is a motor protein primarily involved in anterograde IFT. At E12.5, cilia were lost from the ventricular zone of the cortex (Wilson et al., 2011). In distinct contrast to Gli3 mutants and the other reported ciliary mutants discussed above, the Kif3a mutants retained their normal dorsal– ventral cortical expression domains. However, mutant mice were born, showing larger cerebral cortex volumes and even rosette-shaped heterotopias identical in appearance to those seen in cobblestone (Willaredt et al., 2008) and Gli3 (Fotaki et al., 2006; Theil et al., 1999) mutants. As seen in the Ftm knockout mice, adherens junctions and apical-basal polarity of neuroepithelial precursors seemed normal in these mutants. The increase in cortical mass was not attributable to a decrease in apoptosis, which plays a very important role
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
in the development of the cortex (Kuan et al., 2000). In fact, levels of apoptosis in the Kif3a mutant were if anything increased by a factor of two. Examination of the rate of neurogenesis also showed an increase, which would in fact argue for smaller cortical volume as the precursor pool is more quickly diminished. The answer lay in cell cycle kinetics. A detailed examination showed that the cell cycle length in the precursors was shortened because of a shorter G1 phase, resulting in more cell divisions per embryonic day and ultimately an increased cortical volume. How to explain this change in G1? The authors looked at the usual suspect, Gli3. As is most often the case (Haycraft et al., 2005; Huangfu and Anderson, 2005; Liu et al., 2005; May et al., 2005; Tran et al., 2008; Willaredt et al., 2008), the authors observed an increase in the relative amounts of the full-length Gli3 isoform, at the expense of the processed form. This resulted in upregulation of some Shh targets such as Ptch1, Fgf15, and cyclinD1. Next, the authors examined a different Gli3 mouse mutant, one in which the domain where the Gli3 protein is proteolytically cleaved from the full-length to the short isoform is missing (Wang et al., 2007). They observed similar changes in cell cycle dynamics to that seen in the Kif3a mutant. Curiously, this mutant did not demonstrate the same level of overgrowth seen in the Kif3a mutant, so clearly there must be other functions that Kif3a is providing, potentially non-ciliary ones. Finally, the authors decided to examine the upregulation of Fgf15 more closely. Using in utero electroporation of E12.0 cortex with an Fgf15 overexpression construct, the authors could examine subsequent changes in cell cycle with standard BrdU labeling. Indeed, the portion of the cortex that was electroporated showed a shortened cell cycle length, showing that Fgf15 overexpression is sufficient to replicate the cell cycle phenotype seen in the Kif3a mutation. It was established decades ago that there is a connection between primary cilia and the cell cycle, in that cilia are normally present in the G0/G1 phase, and that they become reabsorbed into the cytoplasm when the cells reenter the cell cycle (Tucker et al., 1979a,b). A causal connection has lately been established, showing that cells can be pushed into S phase when the cilia reabsorption process is forced externally. To show this, one group (Li et al., 2011) employed a retinal pigmented epithelial cell line commonly used to examine ciliogenesis, RPE-1. In this cell line, serum withdrawal leads to cell cycle block and the formation of a primary cilium, while subsequent addition of serum causes ciliary reabsorption and entry into S phase. The authors investigated Tctex-1, a subunit of the light chain of dynein. Upon phosphorylation of residue Thr94, Tctex-1 dissociates from the dynein complex (Chuang et al., 2005). A knockdown of Tctex-1 using shRNA led to an inability of the cells to reenter S phase after serum withdrawal and treatment. The importance of Tctex-1 phosphorylation was examined in several further experiments. First, they showed that upon Thr94 phosphorylation, Tctex-1 localizes to the transition zone, a specialized structure between the basal body and the base of the ciliary axoneme. Second, the reentry block caused by Tctex-1 knockdown could be rescued by expression of a phosphomimetic mutant form of the protein. Third, exogenous introduction of this same phosphomimetic mutant form of Tctex-1 promoted both ciliary reabsorption and cell cycle reentry.
MECHANISMS OF DEVELOPMENT
To show the relevance of this protein for the embryonic cerebral cortex, the authors first demonstrated that radial glial cells express Tctex-1, and that the protein localizes to the transition zone of the ventricularly-oriented primary cilium of these cells. They used in utero electroporation to perform gain- and loss-of-function experiments. Tctex-1 knockdown resulted in excessive neuronal differentiation and a depletion of radial glial cells. In a complementary fashion, overexpression of the phosphomimetic version of Tctex-1 increased the mitotic index and shortened the length of G1 phase of the cell cycle of ventricular zone cells. Unfortunately, the authors did not directly correlate Tctex-1 knockdown/overexpression with the number and identity of cilia-bearing cells at the ventricular zone, but they argue that the results they obtain in vivo are reflective of the in vitro situation. All in all, the results from this (Li et al., 2011) and the previous (Wilson et al., 2011) study agree that, in cortical ventricular zone precursors, cilia restrain the G1 phase of the cell cycle. Similar results were found by manipulation of the centrosomal protein Nde1 in RPE-1 cells and in Zebrafish embryos (Kim et al., 2011). As discussed above, both the initial neuroepithelial cells that will generate the forebrain, as well as the radial glial population appearing later, display a prominent primary cilium that grows out of the apical membrane of the neuroepithelial cells and pokes out into the ventricle of the embryonic brain (Cohen and Meininger, 1987). These cilia are either lost or dysfunctional in the ciliary mutants that we have already discussed (Besse et al., 2011; Stottmann et al., 2009; Willaredt et al., 2008; Wilson et al., 2011). There is a second population of neuronal progenitors that derive from the radial glial cells, delaminating from the ventricular surface and migrating in the basal direction, hence their name ‘‘basal progenitors’’ (Haubensak et al., 2004). A recent study posed the question as to whether these cells bear cilia, and if so, in which orientation they are facing. The results were surprising and very exciting (Wilsch-Brauninger et al., 2012). The authors depended upon the expression of the transcription factor Tbr2 to identify this population and found that, although these cells maintained adherens junctions with their fellow cells, their primary cilium was found to localize at the basolateral membrane, facing away from the ventricle. In keeping with the gradual increase in neurogenesis seen over time in the cortex, the number of cells bearing such cilia increased with embryonic age. Overexpression of the transcription factor insulinoma-associated 1, which promotes the production of basal progenitors from their apical progenitor mothers (Farkas et al., 2008), resulted in an increase in the number of cells with basolateral cilia. The switch of the milieu that the primary cilium is exposed to, from the ventricular fluid to the extracellular matrix within the developing cortex, could have profound implications for the specific signal transduction cascades that are orchestrated at the cilium.
3. Primary development
cilia
and
hippocampus
As discussed above, hippocampal determination does not depend upon primary cilia function, but its proper morphogenesis does. In the hypomorphic Ift88 allele cobblestone, at
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
377
E12.5 the nascent hippocampus displays a disordered architecture and reduction in the expression of both EphB1 and Lhx2 expression (Willaredt et al., 2008), the second of these two markers is known to be crucial for hippocampus development. A much more interesting and detailed analysis has been performed, however, postnatally. Granule neurons reside in the hippocampal dentate gyrus and have fundamental roles in processes such as learning and memory. Their precursors, granule neuron precursors (GNPs), reside in the dentate neuroepithelium of the hippocampus and represent one of the few loci of neurogenesis in the adult brain (Altman and Bayer, 1990). After birth, they migrate and proliferate into the developing dentate gyrus to finally settle in the subgranular zone (SGZ). In the last decade studies have shown that Shh signaling has a major role in the maintenance of adult neural stem cells in the SGZ (Lai et al., 2003; Machold et al., 2003). A recent study by Han et al. (2008) has shed much light on the mechanisms through which these neural progenitors expand and transform themselves into postnatal neural stem cells, by testing the function that primary cilia have in Shh-dependent signaling in the expansion of GNPs (Han et al., 2008). Conditional deletion mutants in the gene Kif3a, which encodes a protein involved in anterograde IFT, were used to show that loss of this particular protein leads to a defective Shh signaling in GNPs, which has as a consequence defective proliferation of the GNPs. Kif3a was conditionally deleted using a Cre recombinase under the control of the human GFAP promoter, which is expressed in radial glial precursors of both the cerebral cortex and the hippocampus. Examination of the earliest populations of GNPs in these Kif3a mutants showed that, although GNPs underwent specification normally, they failed to expand later on to generate radial astrocytes. The authors also used in their study mutant mice where Smo, a necessary component of Shh signaling, was genetically ablated. The postnatal GNP population in these mutants was found to be more severely affected than in the Kif3a mutants. Analysis of double mutants-lacking both Kif3a and Smo showed an improvement in this severe phenotype, more resembling the Kif3a mutants. This observation not only points out that Smo lies genetically upstream to Kif3a, but it also demonstrates its importance in the expansion of GNPs and their conversion to adult progenitors. Moreover, when constitutively-active Smo (SmoM2) was expressed in the Kif3a mutant background, it drove the expansion of dentate gyrus, but in a cilia-dependent fashion. The abnormalities observed in the dentate gyrus are specific to GNPs and their onset overlaps with that of Shh signaling in the dentate gyrus during development. Other ciliary mutants, such as Ift88 and Ftm, also exhibited a reduction in GNP expansion. Taken together, these findings advocate that cilia-mediated defects in Shh signaling are the cause of the phenotype seen in these Kif3a mutant mice (Han et al., 2008). In the same line with these findings was also the study of Breunig et al. (2008) (Breunig et al., 2008), where defects in the proliferation of dentate gyrus GNPs were also observed, however less severe, in conditional mutant mice for a gene (stumpy, (Town et al., 2008)) encoding a protein found at the primary cilium. Stumpy mutants, like the Kif3a mutants, also showed alterations in Shh signaling. Han et al. (2008) reported lack of induction of downstream genes, such as Ptch1 (Han
378
MECHANISMS OF DEVELOPMENT
et al., 2008). Furthermore, Breunig et al. (2008) had reported, in addition to the lack of induction of Gli1, also an increase in the level of full-length Gli3 in the Stumpy mutant (Breunig et al., 2008).
4. Primary development
cilia
and
olfactory
bulb
Only one publication has addressed the role of primary cilia in the development of the olfactory bulbs (Besse et al., 2011). Ftm mutant embryos exhibited apparent olfactory bulb agenesis (Delous et al., 2007). However, the pallium of Ftm mutants contained an ectopic group of Tbr2-positive cells that could be distinguished from the cortical progenitors, whose identity as mitral cells of the olfactory bulb could be determined by Tbx21 expression, a marker for this cell type. This ectopically-located olfactory bulb-like structure derives from a primordial anlage that is correctly specified at E13.5 in Ftm mutants, but somehow becomes mislocalized dorsally and laterally, where it fails to be contacted by olfactory neurons and subsequently to properly laminate. Normal olfactory bulb development could be restored by crossing the Ftm mutant to the Gli3 deletion mutant (Bo¨se et al., 2002) described above (Section 2).
5. Primary development
cilia
and
diencephalon
Very little is known about the role of primary cilia and the development of diencephalic structures including the thalamus, hypothalamus, subthalamus, and epithalamus. The only report to investigate this area of the forebrain showed that in cobblestone, a hypomorphic allele of Ift88, the boundary between the telencephalon and the diencephalon was dissolved (Willaredt et al., 2008). A free mixing of tissue between the areas could be observed, with a particularly high incidence of rosettes containing newborn neurons. A similar, though not identical, phenotype has been reported for the Gli3 deletion mutant XtJ (Fotaki et al., 2006). Determination of substructures in the diencephalon has not been reported for ciliary mutations, but the development of hypothalamic and thalamic nuclei would be expected to be affected in ciliary mutants, as differentiation of these tissues is dependent upon Shh and Gli3 activity (Alvarez-Bolado et al., 2012; Haddad-Tovolli et al., 2012). Surprisingly, despite several reports showing defects in eye development in ciliary mutants (e.g., coloboma in the cobblestone mutant (Willaredt et al., 2008)), no reports have yet been published outlining a role for cilia in early eye development, but surely these will appear soon.
6.
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
than the latter, although it remains to be seen why cilia promote proliferations in some contexts (such as postnatal dentate gyrus) but restrain it in others (such as embryonic cortex). With respect to the latter, much work needs to be done in order to determine the morphological organizing roles of the cilia. Third, there are hints that Wnt signaling in the forebrain may also be modulated by cilia, but the evidence for this is still fragmentary. The field needs a closer, in vivo examination of Wnt signaling components at the cilium and the basal body, as well as functional experiments testing modulation of up- and downstream components of Wnt signaling pathways in the context of ciliary mutations, for which latter now a wealth of models have been developed (Table 1). Many issues remain to be investigated with respect to forebrain development. What do cilia have to do with boundary maintenance between the pallium-subpallium and the telencephalon–diencephalon? What is the significance of the cortical rosettes seen in ciliary and Gli3 mutants? Agenesis of the corpus callosum is a feature found in certain ciliopathies (e.g., (Thauvin-Robinet et al., 2006)), and axonal pathfinding defects are found in the cobblestone mutant (Willaredt and Tucker, unpublished observations) and presumably in other ciliary mutants. How then are primary cilia involved in axonal or dendritic development? Of great interest is the question of whether later stages of cortical development are affected in ciliary mutants. The establishment of the six layers of the isocortex follows neurogenesis and involves both radial migration from the ventricular zone as well as tangential migration of interneurons from the subpallium. Although an anatomically distinct entity, migrating neural crest cells have been shown recently to both bear primary cilia (Willaredt and Tucker, unpublished data) as well as depend, at least partially, upon basal body proteins for migration (Tobin et al., 2008). It would not be illogical to hypothesize that either excitatory or inhibitory newborn neurons (or both), may employ primary cilia in their migratory paths. Of course, it will also be also fascinating to see if astrogliogenesis or oligodendrocyte development are also affected by primary cilia. With respect to all of these topics, what other signaling pathways could be taking place at the cilium or the basal body? Finally, studies are starting to be published in which the primary cilia borne by cortical and hippocampal neurons are shown to important for critical neuronal functions such as synaptogenesis (Kumamoto et al., 2012). With an ever-growing list of neurotransmitter receptors that are found to localize specifically to neuronal cilia (e.g., (Domire et al., 2011; Handel et al., 1999; Iwanaga et al., 2011)), surely we will start to see that primary cilia are indeed acting within the brain like ‘‘cellular antennas’’, as they have so often been described in other organ systems and organisms.
Conclusions and outlook Acknowledgements
A clear picture is beginning to emerge from the relatively small number of studies that have been published to date. First, primary cilia are essential for the transduction of Shh signaling in the development of the cerebral cortex and other portions of the forebrain. Second, they control not only the proliferation of neural precursors but also the morphogenesis of a variety of tissues. The former point is better understood
The authors were funded by the German Research Society (DFG SFB 488, Teilprojekt B9) and would like to thank Joachim Kirsch for generous scientific support. They apologize to those colleagues whose papers, for lack of space, have not been cited. K.L.T. is a member of the Excellence Cluster CellNetworks at the University of Heidelberg.
MECHANISMS OF DEVELOPMENT
R E F E R E N C E S
Altman, J., Bayer, S.A., 1990. Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. J. Comp. Neurol. 301, 365–381. Alvarez-Bolado, G., Paul, F.A., Blaess, S., 2012. Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions. Neural Dev. 7, 4. Arts, H.H., Doherty, D., van Beersum, S.E., Parisi, M.A., Letteboer, S.J., Gorden, N.T., Peters, T.A., Marker, T., Voesenek, K., Kartono, A., Ozyurek, H., Farin, F.M., Kroes, H.Y., Wolfrum, U., Brunner, H.G., Cremers, F.P., Glass, I.A., Knoers, N.V., Roepman, R., 2007. Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome. Nat. Genet. 39, 882–888. Besse, L., Neti, M., Anselme, I., Gerhardt, C., Ruther, U., Laclef, C., Schneider-Maunoury, S., 2011. Primary cilia control telencephalic patterning and morphogenesis via Gli3 proteolytic processing. Development 138, 2079–2088. Bo¨se, J., Grotewold, L., Ruther, U., 2002. Pallister–Hall syndrome phenotype in mice mutant for Gli3. Hum. Mol. Genet. 11, 1129– 1135. Breunig, J.J., Sarkisian, M.R., Arellano, J.I., Morozov, Y.M., Ayoub, A.E., Sojitra, S., Wang, B., Flavell, R.A., Rakic, P., Town, T., 2008. Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling. Proc. Natl. Acad. Sci. USA 105, 13127–13132. Chuang, J.Z., Yeh, T.Y., Bollati, F., Conde, C., Canavosio, F., Caceres, A., Sung, C.H., 2005. The dynein light chain Tctex-1 has a dynein-independent role in actin remodeling during neurite outgrowth. Dev. Cell 9, 75–86. Cohen, E., Meininger, V., 1987. Ultrastructural analysis of primary cilium in the embryonic nervous tissue of mouse. Int. J. Dev. Neurosci. 5, 43–51. Corbit, K.C., Aanstad, P., Singla, V., Norman, A.R., Stainier, D.Y., Reiter, J.F., 2005. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021. Corbit, K.C., Shyer, A.E., Dowdle, W.E., Gaulden, J., Singla, V., Chen, M.H., Chuang, P.T., Reiter, J.F., 2008. Kif3a constrains betacatenin-dependent Wnt signalling through dual ciliary and non-ciliary mechanisms. Nat. Cell Biol. 10, 70–76. Davis, E.E., Brueckner, M., Katsanis, N., 2006. The emerging complexity of the vertebrate cilium: new functional roles for an ancient organelle. Dev. Cell 11, 9–19. Delous, M., Baala, L., Salomon, R., Laclef, C., Vierkotten, J., Tory, K., Golzio, C., Lacoste, T., Besse, L., Ozilou, C., Moutkine, I., Hellman, N.E., Anselme, I., Silbermann, F., Vesque, C., Gerhardt, C., Rattenberry, E., Wolf, M.T., Gubler, M.C., Martinovic, J., Encha-Razavi, F., Boddaert, N., Gonzales, M., Macher, M.A., Nivet, H., Champion, G., Bertheleme, J.P., Niaudet, P., McDonald, F., Hildebrandt, F., Johnson, C.A., Vekemans, M., Antignac, C., Ruther, U., Schneider-Maunoury, S., Attie-Bitach, T., Saunier, S., 2007. The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome. Nat. Genet. 39, 875– 881. Dessaud, E., McMahon, A.P., Briscoe, J., 2008. Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogenregulated transcriptional network. Development 135, 2489– 2503. Domire, J.S., Green, J.A., Lee, K.G., Johnson, A.D., Askwith, C.C., Mykytyn, K., 2011. Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet–Biedl syndrome proteins. Cell Mol. Life Sci. 68, 2951–2960. Farkas, L.M., Haffner, C., Giger, T., Khaitovich, P., Nowick, K., Birchmeier, C., Paabo, S., Huttner, W.B., 2008. Insulinomaassociated 1 has a panneurogenic role and promotes the
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
379
generation and expansion of basal progenitors in the developing mouse neocortex. Neuron 60, 40–55. Fotaki, V., Yu, T., Zaki, P.A., Mason, J.O., Price, D.J., 2006. Abnormal positioning of diencephalic cell types in neocortical tissue in the dorsal telencephalon of mice lacking functional Gli3. J. Neurosci. 26, 9282–9292. Gorivodsky, M., Mukhopadhyay, M., Wilsch-Braeuninger, M., Phillips, M., Teufel, A., Kim, C., Malik, N., Huttner, W., Westphal, H., 2009. Intraflagellar transport protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain. Dev. Biol. 325, 24–32. Haddad-Tovolli, R., Heide, M., Zhou, X., Blaess, S., Alvarez-Bolado, G., 2012. Mouse thalamic differentiation: gli-dependent pattern and gli-independent prepattern. Front. Neurosci. 6, 27. Han, Y.G., Spassky, N., Romaguera-Ros, M., Garcia-Verdugo, J.M., Aguilar, A., Schneider-Maunoury, S., Alvarez-Buylla, A., 2008. Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells. Nat. Neurosci. 11, 277–284. Handel, M., Schulz, S., Stanarius, A., Schreff, M., ErdtmannVourliotis, M., Schmidt, H., Wolf, G., Hollt, V., 1999. Selective targeting of somatostatin receptor 3 to neuronal cilia. Neuroscience 89, 909–926. Haubensak, W., Attardo, A., Denk, W., Huttner, W.B., 2004. Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis. Proc. Natl. Acad. Sci. USA 101, 3196–3201. Haycraft, C.J., Banizs, B., Aydin-Son, Y., Zhang, Q., Michaud, E.J., Yoder, B.K., 2005. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet. 1, e53. Huang, P., Schier, A.F., 2009. Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia. Development 136, 3089–3098. Huangfu, D., Anderson, K.V., 2005. Cilia and Hedgehog responsiveness in the mouse. Proc. Natl. Acad. Sci. USA 102, 11325–11330. Iwanaga, T., Hozumi, Y., Takahashi-Iwanaga, H., 2011. Immunohistochemical demonstration of dopamine receptor D2R in the primary cilia of the mouse pituitary gland. Biomed. Res. 32, 225–235. Kim, S., Zaghloul, N.A., Bubenshchikova, E., Oh, E.C., Rankin, S., Katsanis, N., Obara, T., Tsiokas, L., 2011. Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry. Nat. Cell Biol. 13, 351–360. Kuan, C.Y., Roth, K.A., Flavell, R.A., Rakic, P., 2000. Mechanisms of programmed cell death in the developing brain. Trends Neurosci. 23, 291–297. Kumamoto, N., Gu, Y., Wang, J., Janoschka, S., Takemaru, K., Levine, J., Ge, S., 2012. A role for primary cilia in glutamatergic synaptic integration of adult-born neurons. Nat. Neurosci. 15 (399–405), S1. Lai, K., Kaspar, B.K., Gage, F.H., Schaffer, D.V., 2003. Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nat. Neurosci. 6, 21–27. Li, A., Saito, M., Chuang, J.Z., Tseng, Y.Y., Dedesma, C., Tomizawa, K., Kaitsuka, T., Sung, C.H., 2011. Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors. Nat. Cell Biol. 13, 402–411. Liu, A., Wang, B., Niswander, L.A., 2005. Mouse intraflagellar transport proteins regulate both the activator and repressor functions of Gli transcription factors. Development 132, 3103– 3111. Machold, R., Hayashi, S., Rutlin, M., Muzumdar, M.D., Nery, S., Corbin, J.G., Gritli-Linde, A., Dellovade, T., Porter, J.A., Rubin, L.L., Dudek, H., McMahon, A.P., Fishell, G., 2003. Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron 39, 937–950.
380
MECHANISMS OF DEVELOPMENT
Marszalek, J.R., Ruiz-Lozano, P., Roberts, E., Chien, K.R., Goldstein, L.S., 1999. Situs inversus and embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of kinesin-II. Proc. Natl. Acad. Sci. USA 96, 5043–5048. May, S.R., Ashique, A.M., Karlen, M., Wang, B., Shen, Y., Zarbalis, K., Reiter, J., Ericson, J., Peterson, A.S., 2005. Loss of the retrograde motor for IFT disrupts localization of Smo to cilia and prevents the expression of both activator and repressor functions of Gli. Dev. Biol. 287, 378–389. Nonaka, S., Tanaka, Y., Okada, Y., Takeda, S., Harada, A., Kanai, Y., Kido, M., Hirokawa, N., 1998. Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 95, 829–837. Ocbina, P.J., Tuson, M., Anderson, K.V., 2009. Primary cilia are not required for normal canonical Wnt signaling in the mouse embryo. PLoS One 4, e6839. Petersen, P.H., Zou, K., Hwang, J.K., Jan, Y.N., Zhong, W., 2002. Progenitor cell maintenance requires numb and numblike during mouse neurogenesis. Nature 419, 929–934. Rohatgi, R., Milenkovic, L., Scott, M.P., 2007. Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372– 376. Scholey, J.M., Anderson, K.V., 2006. Intraflagellar transport and cilium-based signaling. Cell 125, 439–442. Stottmann, R.W., Tran, P.V., Turbe-Doan, A., Beier, D.R., 2009. Ttc21b is required to restrict sonic hedgehog activity in the developing mouse forebrain. Dev. Biol. 335, 166–178. Tasouri, E., Tucker, K.L., 2011. Primary cilia and organogenesis: is Hedgehog the only sculptor? Cell Tissue Res. 345, 21–40. Thauvin-Robinet, C., Cossee, M., Cormier-Daire, V., Van Maldergem, L., Toutain, A., Alembik, Y., Bieth, E., Layet, V., Parent, P., David, A., Goldenberg, A., Mortier, G., Heron, D., Sagot, P., Bouvier, A.M., Huet, F., Cusin, V., Donzel, A., Devys, D., Teyssier, J.R., Faivre, L., 2006. Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oralfacial-digital syndrome type 1: a French and Belgian collaborative study. J. Med. Genet. 43, 54–61. Theil, T., Alvarez-Bolado, G., Walter, A., Ruther, U., 1999. Gli3 is required for Emx gene expression during dorsal telencephalon development. Development 126, 3561–3571. Tobin, J.L., Di Franco, M., Eichers, E., May-Simera, H., Garcia, M., Yan, J., Quinlan, R., Justice, M.J., Hennekam, R.C., Briscoe, J., Tada, M., Mayor, R., Burns, A.J., Lupski, J.R., Hammond, P., Beales, P.L., 2008. Inhibition of neural crest migration underlies
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
craniofacial dysmorphology and Hirschsprung’s disease in Bardet–Biedl syndrome. Proc. Natl. Acad. Sci. USA 105, 6714– 6719. Tole, S., Goudreau, G., Assimacopoulos, S., Grove, E.A., 2000. Emx2 is required for growth of the hippocampus but not for hippocampal field specification. J. Neurosci. 20, 2618–2625. Town, T., Breunig, J.J., Sarkisian, M.R., Spilianakis, C., Ayoub, A.E., Liu, X., Ferrandino, A.F., Gallagher, A.R., Li, M.O., Rakic, P., Flavell, R.A., 2008. The stumpy gene is required for mammalian ciliogenesis. Proc. Natl. Acad. Sci. USA 105, 2853– 2858. Tran, P.V., Haycraft, C.J., Besschetnova, T.Y., Turbe-Doan, A., Stottmann, R.W., Herron, B.J., Chesebro, A.L., Qiu, H., Scherz, P.J., Shah, J.V., Yoder, B.K., Beier, D.R., 2008. THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia. Nat. Genet. 40, 403–410. Tucker, R.W., Pardee, A.B., Fujiwara, K., 1979a. Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells. Cell 17, 527–535. Tucker, R.W., Scher, C.D., Stiles, C.D., 1979b. Centriole deciliation associated with the early response of 3T3 cells to growth factors but not to SV40. Cell 18, 1065–1072. Vierkotten, J., Dildrop, R., Peters, T., Wang, B., Ruther, U., 2007. Ftm is a novel basal body protein of cilia involved in Shh signalling. Development 134, 2569–2577. Vincent, S.D., Dunn, N.R., Hayashi, S., Norris, D.P., Robertson, E.J., 2003. Cell fate decisions within the mouse organizer are governed by graded nodal signals. Genes Dev. 17, 1646–1662. Wang, C., Pan, Y., Wang, B., 2007. A hypermorphic mouse Gli3 allele results in a polydactylous limb phenotype. Dev. Dyn. 236, 769–776. Willaredt, M.A., Hasenpusch-Theil, K., Gardner, H.A.R.G., Kitanovic, I., Hirschfeld-Warneken, V.C., Gojak, C.P., Gorgas, K., Bradford, C.L., Spatz, J., Wolfl, S., Theil, T., Tucker, K.L., 2008. A crucial role for primary cilia in cortical morphogenesis. J. Neurosci. 28, 12887–12900. Wilsch-Brauninger, M., Peters, J., Paridaen, J.T., Huttner, W.B., 2012. Basolateral rather than apical primary cilia on neuroepithelial cells committed to delamination. Development 139, 95–105. Wilson, S.L., Wilson, J.P., Wang, C., Wang, B., McConnell, S.K., 2011. Primary cilia and Gli3 activity regulate cerebral cortical size. Dev. Neurobiol.
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/modo
Primary cilia and forebrain development Marc August Willaredt, Evangelia Tasouri, Kerry L. Tucker
*
Interdisciplinary Center for Neurosciences, Institute of Anatomy and Cell Biology, University of Heidelberg, 69120 Heidelberg, Germany
A R T I C L E I N F O
A B S T R A C T
Article history:
With a microtubule-based axoneme supporting its plasma membrane-ensheathed projec-
Available online 22 October 2012
tion from the basal body of almost all cell types in the human body, and present in only one copy per cell, the primary cilium can be considered an organelle sui generis. Although it was
Keywords: Primary cilia Cerebral cortex Forebrain Sonic hedgehog Gli3 Telencephalon
first observed and recorded in histological studies from the late 19th century, the tiny structure was essentially forgotten for many decades. In the past ten years, however, scientists have turned their eyes once again upon primary cilia and realized that they are very important for the development of almost all organs in the mammalian body, especially those dependent upon the signaling from members Hedgehog family, such as Indian and Sonic hedgehog. In this review, we outline the roles that primary cilia play in forebrain development, not just in the crucial transduction of Sonic hedgehog signaling, but also new results showing that cilia are important for cell cycle progression in proliferating neural precursors. We will focus upon cerebral cortex development but will also discuss the importance of cilia for the embryonic hippocampus, olfactory bulb, and diencephalon. Ó 2012 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Cilia are 1–3 lm membrane-bound, microtubule-based structures found on many cells in the vertebrate body (Davis et al., 2006). Cilia can be classified into two groups regarding their ultrastructure and function; both groups share at their base a centrosome-derived basal body that anchors the cilium within the cytoplasm. The axoneme in motile cilia is organized in a ‘9 + 2’ arrangement, where the double microtubule core in the middle is surrounded by nine microtubule doublets arranged in a circular pattern. Motility is achieved through the protein–protein interactions between the central core and the surrounding doublets. Non-motile cilia, also known as primary cilia, show a ‘9 + 0’ arrangement lacking the central microtubule core. Even though primary cilia have been considered to be completely non-motile, it has been reported that primary cilia at the embryonic node can generate a rhythmic movement, though not in the whiplike fashion of motile cilia (Nonaka et al., 1998). Both classes of cilia are
maintained by the transport of protein cargo along the axoneme mediated by a microtubule-based transport, namely intraflagellar transport (IFT) (Scholey and Anderson, 2006). IFT utilizes kinesin-II- and dynein-based motors to facilitate anterograde and retrograde transport, respectively. Interaction between protein cargo and the transport motors occurs through the IFT scaffolding proteins complexes IFTA and IFTB, which in turn are composed of IFTX proteins, in which X denotes the molecular weight of the given protein. Even though cilia in the past have been undervalued, recent studies of Hedgehog (Hh) signaling reveal striking evidence of the importance of cilia in signal transduction. Hh signaling has a critical role during development because of its implication in patterning events (Dessaud et al., 2008). There are three vertebrate ligands for Hh signaling, Sonic (Shh), Indian and Desert, that all bind to the specific receptor for Hh-family ligands Patched (Ptch1). In the absence of any ligand, Ptch1 prevents Smoothened (Smo), a G-coupled transmembrane protein, to associate with the membrane, thus
* Corresponding author. Address: Interdisciplinary Center for Neurosciences, Institute of Anatomy and Cell Biology, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany. Tel.: +49 6221 54 8687; fax: +49 6221 54 4952. E-mail address: [email protected] (K.L. Tucker). 0925-4773/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mod.2012.10.003
374
MECHANISMS OF DEVELOPMENT
repressing signal transduction. In the presence of a ligand, Ptch1 moves out of the cilium, thereby relieving the inhibition of Smo activity (Corbit et al., 2005; Rohatgi et al., 2007). Smo moves into the primary cilium (Corbit et al., 2005) and transduces the Hh signal through its effects upon the transcription factors Gli2 and Gli3, which also localize to primary cilia (Haycraft et al., 2005). Both transcription factors are activated upon association with cilia, while Gli3 is proteolytically converted from a full-length form to a shorter repressor form (Gli3R) in a cilia-dependent fashion. Dozens of recent papers have proven the dependence upon primary cilia for Shh signaling in the development of such disparate organs as the spinal cord, heart, mammary gland, kidneys, ovaries, and pancreas, while Indian hedgehog has been shown to signal through primary cilia in skeletal development (for a thorough review, see (Tasouri and Tucker, 2011)). More controversial has been the involvement of primary cilia in Wnt signaling. Although in vitro analysis indicated that primary cilia constrain canonical Wnt signaling (Corbit et al., 2008), the evidence from in vivo studies is far from clear. Comprehensive analysis of Wnt-dependent developmental processes in zebrafish and the mouse revealed no role for primary cilia (Huang and Schier, 2009; Ocbina et al., 2009), but perturbations in Wnt signaling have been recorded in the later development of many organ systems, including the forebrain, cerebellum, kidney, pancreas, mammary gland, and long bones (reviewed in (Tasouri and Tucker, 2011)). Whether changes in Wnt signaling reflect a secondary effect downstream of a Hh-based perturbation, or in fact indicate a direct role of primary cilia in later Wnt-dependent developmental processes is still unresolved. This review focuses upon the relevance of primary cilia for the development of the forebrain, with an emphasis upon the largest portion of the forebrain, the cerebral cortex. The following results are also summarized in Table 1.
2. Primary development
cilia
and
cerebral
cortex
Using a hypomorphic allele of the Ift88 gene, cobblestone, uncovered by ENU mutagenesis, Willaredt et al. (2008) were the first to examine the role that primary cilia have in cortical morphogenesis. They demonstrated that in cobblestone mutants both mRNA and protein levels of Ift88 are reduced by 70–80%. Cobblestone mutants not only displayed severe malformations of the hippocampus, choroid plexus and cortical hem, but additionally the dorsal–ventral boundary between the pallium and subpallium was not properly determined, although the dorsal and ventral domains were not shifted, as seen in other cilia mutants (Besse et al., 2011; Stottmann et al., 2009). Surprisingly, transmission and scanning electron microscopy presented in this study showed that primary cilia were still present and appeared intact at the ultrastructural level. These various phenotypes are comparable to the ones observed in the Gli3 deletion mutant Extra toes (XtJ) (Fotaki et al., 2006; Theil et al., 1999; Tole et al., 2000), which prompted the investigation of Gli3 proteolytic processing in cobblestone mutants. The authors observed an increase of the full-length form of Gli3. A number of phenotypes seen
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
in the cobblestone mutants, such as the formation of rosetteshaped heterotopias in the dorsal cortex or the defects in the determination of tissue of the dorsal telencephalon mirror ones observed in XtJ/XtJ embryos. Further analysis of Shh signaling targets, such as Ptch1 and Gli1, showed an increase in their expression in the cobblestone mutants (Willaredt et al., 2008). Examination of changes in Wnt signaling also revealed an upregulation of canonical Wnt signaling, as reflected in the increased and ectopic expression of Wnt7b, Wnt8b, and Axin2. Like Ift88, the protein encoded by the Ift172 gene is a member of the IFTB complex involved in anterograde ciliary traffic. Inactivation of this gene in the mouse resulted in phenotypes reminiscent of cobblestone, in that the mice displayed holoprosencephaly and exencephaly, but in addition the mutants suffered from craniofacial defects (Gorivodsky et al., 2009). Of great interest was their finding that nodal expression was eliminated in embryonic day 7.5 (E7.5) embryos, which is important for the subsequent induction of anterior mesendoderm that will go on to express Shh, Foxa2, and Gsc (Vincent et al., 2003). This may contribute to the forebrain phenotypes seen in other ciliary mutants. It is important to point out that the two aforementioned mutants (cobblestone, Ift172) were in IFTB complex proteins, which would imply that anterograde IFT would be compromised in these mutants. As seen directly below, one might not expect the same phenotype from a mutant in retrograde IFT. In contrast to Ift88 and Ift172, the protein Ift139, encoded by the gene Ttc21b, is a member of the IFTA complex that is involved in retrograde IFT. A mouse mutant for this gene showed a correspondingly different phenotype, in which IFT proteins and their cargo accumulated within the cilium (Stottmann et al., 2009; Tran et al., 2008). This resulted in an increase in Shh signaling, as evidenced by a pronounced ventralization of the forebrain and an upregulation of downstream components of the Shh signaling pathway, including Shh, Ptch1 and Foxa2. By crossing the Ttc21b mutant to a Shh mutant, the resulting phenotype was milder than the Ttc21b mutant alone, allowing the authors to conclude that from a genetic perspective, Ttc21b inhibits Shh signaling, presumably by promoting the exit of activated Smo from the cilium. With respect to Wnt signaling, the authors examined a Wnt-sensitive lacZ transgene and observed a downregulation, whereas betacatenin levels showed no change (Stottmann et al., 2009). Ftm encodes a protein that localizes to the basal body of cilia. The human ortholog of Ftm is called RPGRIP1L, and this gene is mutated in certain patients suffering from Joubert or Meckel syndrome, two well-characterized heritable ciliopathies (Arts et al., 2007; Delous et al., 2007). The mouse mutant for Ftm has been examined for its forebrain phenotype. In the forebrain of the targeted Ftm mutant (Vierkotten et al., 2007), a ventralization phenotype could be recorded, in that two standard markers identifying ventral forebrain, Dlx2 and Gsx2, were enlarged in their expression domains, while the expression domains of the dorsal cortical markers Ngn2 and Pax6 were diminished (Besse et al., 2011). However, this ventralization was limited to the most anterior part of the telencephalon. Unusually, expression of Shh and two downstream targets, Gli1 and Nkx2.1, were unaffected in the ventral forebrain, where they are normally to be found, in midgestation
MECHANISMS OF DEVELOPMENT
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
375
Table 1 – Summary of the proteins and gene alleles discussed in this review, with a short description of the forebrain phenotypes and references to the papers in which the findings and/or the particular mouse model are reported. Protein
Mutation
Phenotype
Ift88
cobblestone ENU-mutagenesis: Hypomorph
Willaredt et al. (2008) Malformation of hippocampus, choroid plexus, cortical hem. Rosette-shaped heterotopias in dorsal cortex. Pallial-subpallial boundary breaks down. Telencephalon– diencephalon boundary dissolved.
Ift172
Slb Targeted deletion: Full knockout
Holoprosencephaly & exencephaly. Midbrain-hindbrain boundary defects. Loss of nodal expression.
Ttc21b (Ift139) alien (aln) ENU-mutagenesis: Complete loss-of-function
References
Gorivodsky et al. (2009)
Forebrain ventralization. Tran et al. (2008); Stottmann et al. (2009) Forebrain reduced, midbrain expanded. Disorganized cortex.
Gli3
Extra toes (XtJ) 51.5-kb genomic deletion: Complete loss-of-function
Theil et al. (1999); Tole et al. (2000); Highly disorganized Fotaki et al. (2006) neocortex. Rosette-shaped heterotopias in dorsal cortex. Telencephalon– diencephalon boundary dissolved.
Ftm
Ftm Targeted deletion: Full knockout
Forebrain ventralization. Dorsal cortical markers diminished in anterior telencephalon. Shrunken, mushroom-like cilia. Olfactory bulb agenesis.
Besse et al. (2011); Vierkotten et al. (2007)
Gli3;Ftm
Gli3 targeted mutation: C-terminal deletion mutant; Ftm double mutant
Ventralized telencephalon phenotype rescued. No cilia at ventricle.
Besse et al. (2011); Bo¨se et al. (2002)
Kif3a
Kif3a targeted mutation: Normal cortical dorsal– Wilson et al. (2011); Marszalek et al. (1999) LoxP sites for inducible knockout ventral expression domains. No cilia at VZ in cortex. Larger cerebral cortex volumes. Rosette-shaped heterotopias.
Tctex-1
Tctex-1: In utero RNAi
Kif3a
Kif3a targeted mutation: Postnatal hippocampal LoxP sites for inducible knockout hypoplasia. Failure of GNP pool expansion and subsequent establishment of adult stem cells in dentate gyrus. Epistatic relationship between Smo & Kif3a.
Han et al. (2008)
Stumpy
Stumpy targeted mutation: Postnatal hippocampal LoxP sites for inducible knockout hypoplasia. Reduction in precursor proliferation.
Breunig et al. (2008); Town et al. (2008)
Tctex-1 knockdown: excessive neuronal differentiation, depletion of glial cells.
Li et al. (2011)
376
MECHANISMS OF DEVELOPMENT
Ftm mutants. Examination of primary cilia at the ventricle of the forebrain revealed first that the Ftm protein was absent in the mutant, as expected, and that the cilia exhibited a shrunken, mushroom-like dismorphology, although the basal bodies appeared to be of normal appearance and location at the apical membrane. Examination of tight junctions between proliferating precursors at the ventricle revealed no abnormalities, but further markers of polarity were not examined. The authors examined the ratio of full-length Gli3 to the shorter, proteolytically-processed isoform, and found that the ratio of the former to the latter increased, in keeping with results seen in both cobblestone mutants (Willaredt et al., 2008) and in many other organs in multiple ciliary mutants (Haycraft et al., 2005; Huangfu and Anderson, 2005; Liu et al., 2005; May et al., 2005; Tran et al., 2008). Most importantly, the ventralized telencephalon phenotype could be rescued by crossing the Ftm mutant to a knockout mutation of the Gli3 gene in which only the truncated isoform of Gli3 is expressed (Bo¨se et al., 2002). As this shorter isoform is thought to act as a transcriptional repressor, it shows that the major defect in the Ftm mutants is the absence of this isoform, and not the increased levels of full-length Gli3. Even more exciting was the finding that the Gli3;Ftm double mutants did not produce cilia at the ventricle (Besse et al., 2011), thereby proving that, in principle, defects attributable to the function of the primary cilium can be compensated for by expression of the relevant proteins lying downstream of the cilium in the signal transduction pathway. This may be of great relevance for therapeutic approaches to ciliopathies such as polycystic kidney disease. Although the studies described in the previous section were very useful in outlining the role of primary cilia with respect to Shh signaling, they all employed standard constitutive knockout/hypomorph approaches. Thus the relatively early contributions that Shh makes to development already at E7.0 could contribute to phenotypes analyzed much later. It would be preferable to affect ciliary activity later, and the next two studies have approached this problem by using either a conditional knockout (Wilson et al., 2011) or a siRNA-based (Li et al., 2011) approach. In both cases, the authors examined the relationship between primary cilia and cell cycle dynamics at the ventricular zone of the embryonic cortex. In the first case, the authors used a nestin::CRE deleter mouse line to specifically eliminate expression of Kif3a (Marszalek et al., 1999) in cortical progenitors at the beginning of the neurogenic program (Petersen et al., 2002). Kif3a is a motor protein primarily involved in anterograde IFT. At E12.5, cilia were lost from the ventricular zone of the cortex (Wilson et al., 2011). In distinct contrast to Gli3 mutants and the other reported ciliary mutants discussed above, the Kif3a mutants retained their normal dorsal– ventral cortical expression domains. However, mutant mice were born, showing larger cerebral cortex volumes and even rosette-shaped heterotopias identical in appearance to those seen in cobblestone (Willaredt et al., 2008) and Gli3 (Fotaki et al., 2006; Theil et al., 1999) mutants. As seen in the Ftm knockout mice, adherens junctions and apical-basal polarity of neuroepithelial precursors seemed normal in these mutants. The increase in cortical mass was not attributable to a decrease in apoptosis, which plays a very important role
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
in the development of the cortex (Kuan et al., 2000). In fact, levels of apoptosis in the Kif3a mutant were if anything increased by a factor of two. Examination of the rate of neurogenesis also showed an increase, which would in fact argue for smaller cortical volume as the precursor pool is more quickly diminished. The answer lay in cell cycle kinetics. A detailed examination showed that the cell cycle length in the precursors was shortened because of a shorter G1 phase, resulting in more cell divisions per embryonic day and ultimately an increased cortical volume. How to explain this change in G1? The authors looked at the usual suspect, Gli3. As is most often the case (Haycraft et al., 2005; Huangfu and Anderson, 2005; Liu et al., 2005; May et al., 2005; Tran et al., 2008; Willaredt et al., 2008), the authors observed an increase in the relative amounts of the full-length Gli3 isoform, at the expense of the processed form. This resulted in upregulation of some Shh targets such as Ptch1, Fgf15, and cyclinD1. Next, the authors examined a different Gli3 mouse mutant, one in which the domain where the Gli3 protein is proteolytically cleaved from the full-length to the short isoform is missing (Wang et al., 2007). They observed similar changes in cell cycle dynamics to that seen in the Kif3a mutant. Curiously, this mutant did not demonstrate the same level of overgrowth seen in the Kif3a mutant, so clearly there must be other functions that Kif3a is providing, potentially non-ciliary ones. Finally, the authors decided to examine the upregulation of Fgf15 more closely. Using in utero electroporation of E12.0 cortex with an Fgf15 overexpression construct, the authors could examine subsequent changes in cell cycle with standard BrdU labeling. Indeed, the portion of the cortex that was electroporated showed a shortened cell cycle length, showing that Fgf15 overexpression is sufficient to replicate the cell cycle phenotype seen in the Kif3a mutation. It was established decades ago that there is a connection between primary cilia and the cell cycle, in that cilia are normally present in the G0/G1 phase, and that they become reabsorbed into the cytoplasm when the cells reenter the cell cycle (Tucker et al., 1979a,b). A causal connection has lately been established, showing that cells can be pushed into S phase when the cilia reabsorption process is forced externally. To show this, one group (Li et al., 2011) employed a retinal pigmented epithelial cell line commonly used to examine ciliogenesis, RPE-1. In this cell line, serum withdrawal leads to cell cycle block and the formation of a primary cilium, while subsequent addition of serum causes ciliary reabsorption and entry into S phase. The authors investigated Tctex-1, a subunit of the light chain of dynein. Upon phosphorylation of residue Thr94, Tctex-1 dissociates from the dynein complex (Chuang et al., 2005). A knockdown of Tctex-1 using shRNA led to an inability of the cells to reenter S phase after serum withdrawal and treatment. The importance of Tctex-1 phosphorylation was examined in several further experiments. First, they showed that upon Thr94 phosphorylation, Tctex-1 localizes to the transition zone, a specialized structure between the basal body and the base of the ciliary axoneme. Second, the reentry block caused by Tctex-1 knockdown could be rescued by expression of a phosphomimetic mutant form of the protein. Third, exogenous introduction of this same phosphomimetic mutant form of Tctex-1 promoted both ciliary reabsorption and cell cycle reentry.
MECHANISMS OF DEVELOPMENT
To show the relevance of this protein for the embryonic cerebral cortex, the authors first demonstrated that radial glial cells express Tctex-1, and that the protein localizes to the transition zone of the ventricularly-oriented primary cilium of these cells. They used in utero electroporation to perform gain- and loss-of-function experiments. Tctex-1 knockdown resulted in excessive neuronal differentiation and a depletion of radial glial cells. In a complementary fashion, overexpression of the phosphomimetic version of Tctex-1 increased the mitotic index and shortened the length of G1 phase of the cell cycle of ventricular zone cells. Unfortunately, the authors did not directly correlate Tctex-1 knockdown/overexpression with the number and identity of cilia-bearing cells at the ventricular zone, but they argue that the results they obtain in vivo are reflective of the in vitro situation. All in all, the results from this (Li et al., 2011) and the previous (Wilson et al., 2011) study agree that, in cortical ventricular zone precursors, cilia restrain the G1 phase of the cell cycle. Similar results were found by manipulation of the centrosomal protein Nde1 in RPE-1 cells and in Zebrafish embryos (Kim et al., 2011). As discussed above, both the initial neuroepithelial cells that will generate the forebrain, as well as the radial glial population appearing later, display a prominent primary cilium that grows out of the apical membrane of the neuroepithelial cells and pokes out into the ventricle of the embryonic brain (Cohen and Meininger, 1987). These cilia are either lost or dysfunctional in the ciliary mutants that we have already discussed (Besse et al., 2011; Stottmann et al., 2009; Willaredt et al., 2008; Wilson et al., 2011). There is a second population of neuronal progenitors that derive from the radial glial cells, delaminating from the ventricular surface and migrating in the basal direction, hence their name ‘‘basal progenitors’’ (Haubensak et al., 2004). A recent study posed the question as to whether these cells bear cilia, and if so, in which orientation they are facing. The results were surprising and very exciting (Wilsch-Brauninger et al., 2012). The authors depended upon the expression of the transcription factor Tbr2 to identify this population and found that, although these cells maintained adherens junctions with their fellow cells, their primary cilium was found to localize at the basolateral membrane, facing away from the ventricle. In keeping with the gradual increase in neurogenesis seen over time in the cortex, the number of cells bearing such cilia increased with embryonic age. Overexpression of the transcription factor insulinoma-associated 1, which promotes the production of basal progenitors from their apical progenitor mothers (Farkas et al., 2008), resulted in an increase in the number of cells with basolateral cilia. The switch of the milieu that the primary cilium is exposed to, from the ventricular fluid to the extracellular matrix within the developing cortex, could have profound implications for the specific signal transduction cascades that are orchestrated at the cilium.
3. Primary development
cilia
and
hippocampus
As discussed above, hippocampal determination does not depend upon primary cilia function, but its proper morphogenesis does. In the hypomorphic Ift88 allele cobblestone, at
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
377
E12.5 the nascent hippocampus displays a disordered architecture and reduction in the expression of both EphB1 and Lhx2 expression (Willaredt et al., 2008), the second of these two markers is known to be crucial for hippocampus development. A much more interesting and detailed analysis has been performed, however, postnatally. Granule neurons reside in the hippocampal dentate gyrus and have fundamental roles in processes such as learning and memory. Their precursors, granule neuron precursors (GNPs), reside in the dentate neuroepithelium of the hippocampus and represent one of the few loci of neurogenesis in the adult brain (Altman and Bayer, 1990). After birth, they migrate and proliferate into the developing dentate gyrus to finally settle in the subgranular zone (SGZ). In the last decade studies have shown that Shh signaling has a major role in the maintenance of adult neural stem cells in the SGZ (Lai et al., 2003; Machold et al., 2003). A recent study by Han et al. (2008) has shed much light on the mechanisms through which these neural progenitors expand and transform themselves into postnatal neural stem cells, by testing the function that primary cilia have in Shh-dependent signaling in the expansion of GNPs (Han et al., 2008). Conditional deletion mutants in the gene Kif3a, which encodes a protein involved in anterograde IFT, were used to show that loss of this particular protein leads to a defective Shh signaling in GNPs, which has as a consequence defective proliferation of the GNPs. Kif3a was conditionally deleted using a Cre recombinase under the control of the human GFAP promoter, which is expressed in radial glial precursors of both the cerebral cortex and the hippocampus. Examination of the earliest populations of GNPs in these Kif3a mutants showed that, although GNPs underwent specification normally, they failed to expand later on to generate radial astrocytes. The authors also used in their study mutant mice where Smo, a necessary component of Shh signaling, was genetically ablated. The postnatal GNP population in these mutants was found to be more severely affected than in the Kif3a mutants. Analysis of double mutants-lacking both Kif3a and Smo showed an improvement in this severe phenotype, more resembling the Kif3a mutants. This observation not only points out that Smo lies genetically upstream to Kif3a, but it also demonstrates its importance in the expansion of GNPs and their conversion to adult progenitors. Moreover, when constitutively-active Smo (SmoM2) was expressed in the Kif3a mutant background, it drove the expansion of dentate gyrus, but in a cilia-dependent fashion. The abnormalities observed in the dentate gyrus are specific to GNPs and their onset overlaps with that of Shh signaling in the dentate gyrus during development. Other ciliary mutants, such as Ift88 and Ftm, also exhibited a reduction in GNP expansion. Taken together, these findings advocate that cilia-mediated defects in Shh signaling are the cause of the phenotype seen in these Kif3a mutant mice (Han et al., 2008). In the same line with these findings was also the study of Breunig et al. (2008) (Breunig et al., 2008), where defects in the proliferation of dentate gyrus GNPs were also observed, however less severe, in conditional mutant mice for a gene (stumpy, (Town et al., 2008)) encoding a protein found at the primary cilium. Stumpy mutants, like the Kif3a mutants, also showed alterations in Shh signaling. Han et al. (2008) reported lack of induction of downstream genes, such as Ptch1 (Han
378
MECHANISMS OF DEVELOPMENT
et al., 2008). Furthermore, Breunig et al. (2008) had reported, in addition to the lack of induction of Gli1, also an increase in the level of full-length Gli3 in the Stumpy mutant (Breunig et al., 2008).
4. Primary development
cilia
and
olfactory
bulb
Only one publication has addressed the role of primary cilia in the development of the olfactory bulbs (Besse et al., 2011). Ftm mutant embryos exhibited apparent olfactory bulb agenesis (Delous et al., 2007). However, the pallium of Ftm mutants contained an ectopic group of Tbr2-positive cells that could be distinguished from the cortical progenitors, whose identity as mitral cells of the olfactory bulb could be determined by Tbx21 expression, a marker for this cell type. This ectopically-located olfactory bulb-like structure derives from a primordial anlage that is correctly specified at E13.5 in Ftm mutants, but somehow becomes mislocalized dorsally and laterally, where it fails to be contacted by olfactory neurons and subsequently to properly laminate. Normal olfactory bulb development could be restored by crossing the Ftm mutant to the Gli3 deletion mutant (Bo¨se et al., 2002) described above (Section 2).
5. Primary development
cilia
and
diencephalon
Very little is known about the role of primary cilia and the development of diencephalic structures including the thalamus, hypothalamus, subthalamus, and epithalamus. The only report to investigate this area of the forebrain showed that in cobblestone, a hypomorphic allele of Ift88, the boundary between the telencephalon and the diencephalon was dissolved (Willaredt et al., 2008). A free mixing of tissue between the areas could be observed, with a particularly high incidence of rosettes containing newborn neurons. A similar, though not identical, phenotype has been reported for the Gli3 deletion mutant XtJ (Fotaki et al., 2006). Determination of substructures in the diencephalon has not been reported for ciliary mutations, but the development of hypothalamic and thalamic nuclei would be expected to be affected in ciliary mutants, as differentiation of these tissues is dependent upon Shh and Gli3 activity (Alvarez-Bolado et al., 2012; Haddad-Tovolli et al., 2012). Surprisingly, despite several reports showing defects in eye development in ciliary mutants (e.g., coloboma in the cobblestone mutant (Willaredt et al., 2008)), no reports have yet been published outlining a role for cilia in early eye development, but surely these will appear soon.
6.
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
than the latter, although it remains to be seen why cilia promote proliferations in some contexts (such as postnatal dentate gyrus) but restrain it in others (such as embryonic cortex). With respect to the latter, much work needs to be done in order to determine the morphological organizing roles of the cilia. Third, there are hints that Wnt signaling in the forebrain may also be modulated by cilia, but the evidence for this is still fragmentary. The field needs a closer, in vivo examination of Wnt signaling components at the cilium and the basal body, as well as functional experiments testing modulation of up- and downstream components of Wnt signaling pathways in the context of ciliary mutations, for which latter now a wealth of models have been developed (Table 1). Many issues remain to be investigated with respect to forebrain development. What do cilia have to do with boundary maintenance between the pallium-subpallium and the telencephalon–diencephalon? What is the significance of the cortical rosettes seen in ciliary and Gli3 mutants? Agenesis of the corpus callosum is a feature found in certain ciliopathies (e.g., (Thauvin-Robinet et al., 2006)), and axonal pathfinding defects are found in the cobblestone mutant (Willaredt and Tucker, unpublished observations) and presumably in other ciliary mutants. How then are primary cilia involved in axonal or dendritic development? Of great interest is the question of whether later stages of cortical development are affected in ciliary mutants. The establishment of the six layers of the isocortex follows neurogenesis and involves both radial migration from the ventricular zone as well as tangential migration of interneurons from the subpallium. Although an anatomically distinct entity, migrating neural crest cells have been shown recently to both bear primary cilia (Willaredt and Tucker, unpublished data) as well as depend, at least partially, upon basal body proteins for migration (Tobin et al., 2008). It would not be illogical to hypothesize that either excitatory or inhibitory newborn neurons (or both), may employ primary cilia in their migratory paths. Of course, it will also be also fascinating to see if astrogliogenesis or oligodendrocyte development are also affected by primary cilia. With respect to all of these topics, what other signaling pathways could be taking place at the cilium or the basal body? Finally, studies are starting to be published in which the primary cilia borne by cortical and hippocampal neurons are shown to important for critical neuronal functions such as synaptogenesis (Kumamoto et al., 2012). With an ever-growing list of neurotransmitter receptors that are found to localize specifically to neuronal cilia (e.g., (Domire et al., 2011; Handel et al., 1999; Iwanaga et al., 2011)), surely we will start to see that primary cilia are indeed acting within the brain like ‘‘cellular antennas’’, as they have so often been described in other organ systems and organisms.
Conclusions and outlook Acknowledgements
A clear picture is beginning to emerge from the relatively small number of studies that have been published to date. First, primary cilia are essential for the transduction of Shh signaling in the development of the cerebral cortex and other portions of the forebrain. Second, they control not only the proliferation of neural precursors but also the morphogenesis of a variety of tissues. The former point is better understood
The authors were funded by the German Research Society (DFG SFB 488, Teilprojekt B9) and would like to thank Joachim Kirsch for generous scientific support. They apologize to those colleagues whose papers, for lack of space, have not been cited. K.L.T. is a member of the Excellence Cluster CellNetworks at the University of Heidelberg.
MECHANISMS OF DEVELOPMENT
R E F E R E N C E S
Altman, J., Bayer, S.A., 1990. Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. J. Comp. Neurol. 301, 365–381. Alvarez-Bolado, G., Paul, F.A., Blaess, S., 2012. Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions. Neural Dev. 7, 4. Arts, H.H., Doherty, D., van Beersum, S.E., Parisi, M.A., Letteboer, S.J., Gorden, N.T., Peters, T.A., Marker, T., Voesenek, K., Kartono, A., Ozyurek, H., Farin, F.M., Kroes, H.Y., Wolfrum, U., Brunner, H.G., Cremers, F.P., Glass, I.A., Knoers, N.V., Roepman, R., 2007. Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome. Nat. Genet. 39, 882–888. Besse, L., Neti, M., Anselme, I., Gerhardt, C., Ruther, U., Laclef, C., Schneider-Maunoury, S., 2011. Primary cilia control telencephalic patterning and morphogenesis via Gli3 proteolytic processing. Development 138, 2079–2088. Bo¨se, J., Grotewold, L., Ruther, U., 2002. Pallister–Hall syndrome phenotype in mice mutant for Gli3. Hum. Mol. Genet. 11, 1129– 1135. Breunig, J.J., Sarkisian, M.R., Arellano, J.I., Morozov, Y.M., Ayoub, A.E., Sojitra, S., Wang, B., Flavell, R.A., Rakic, P., Town, T., 2008. Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling. Proc. Natl. Acad. Sci. USA 105, 13127–13132. Chuang, J.Z., Yeh, T.Y., Bollati, F., Conde, C., Canavosio, F., Caceres, A., Sung, C.H., 2005. The dynein light chain Tctex-1 has a dynein-independent role in actin remodeling during neurite outgrowth. Dev. Cell 9, 75–86. Cohen, E., Meininger, V., 1987. Ultrastructural analysis of primary cilium in the embryonic nervous tissue of mouse. Int. J. Dev. Neurosci. 5, 43–51. Corbit, K.C., Aanstad, P., Singla, V., Norman, A.R., Stainier, D.Y., Reiter, J.F., 2005. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021. Corbit, K.C., Shyer, A.E., Dowdle, W.E., Gaulden, J., Singla, V., Chen, M.H., Chuang, P.T., Reiter, J.F., 2008. Kif3a constrains betacatenin-dependent Wnt signalling through dual ciliary and non-ciliary mechanisms. Nat. Cell Biol. 10, 70–76. Davis, E.E., Brueckner, M., Katsanis, N., 2006. The emerging complexity of the vertebrate cilium: new functional roles for an ancient organelle. Dev. Cell 11, 9–19. Delous, M., Baala, L., Salomon, R., Laclef, C., Vierkotten, J., Tory, K., Golzio, C., Lacoste, T., Besse, L., Ozilou, C., Moutkine, I., Hellman, N.E., Anselme, I., Silbermann, F., Vesque, C., Gerhardt, C., Rattenberry, E., Wolf, M.T., Gubler, M.C., Martinovic, J., Encha-Razavi, F., Boddaert, N., Gonzales, M., Macher, M.A., Nivet, H., Champion, G., Bertheleme, J.P., Niaudet, P., McDonald, F., Hildebrandt, F., Johnson, C.A., Vekemans, M., Antignac, C., Ruther, U., Schneider-Maunoury, S., Attie-Bitach, T., Saunier, S., 2007. The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome. Nat. Genet. 39, 875– 881. Dessaud, E., McMahon, A.P., Briscoe, J., 2008. Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogenregulated transcriptional network. Development 135, 2489– 2503. Domire, J.S., Green, J.A., Lee, K.G., Johnson, A.D., Askwith, C.C., Mykytyn, K., 2011. Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet–Biedl syndrome proteins. Cell Mol. Life Sci. 68, 2951–2960. Farkas, L.M., Haffner, C., Giger, T., Khaitovich, P., Nowick, K., Birchmeier, C., Paabo, S., Huttner, W.B., 2008. Insulinomaassociated 1 has a panneurogenic role and promotes the
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
379
generation and expansion of basal progenitors in the developing mouse neocortex. Neuron 60, 40–55. Fotaki, V., Yu, T., Zaki, P.A., Mason, J.O., Price, D.J., 2006. Abnormal positioning of diencephalic cell types in neocortical tissue in the dorsal telencephalon of mice lacking functional Gli3. J. Neurosci. 26, 9282–9292. Gorivodsky, M., Mukhopadhyay, M., Wilsch-Braeuninger, M., Phillips, M., Teufel, A., Kim, C., Malik, N., Huttner, W., Westphal, H., 2009. Intraflagellar transport protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain. Dev. Biol. 325, 24–32. Haddad-Tovolli, R., Heide, M., Zhou, X., Blaess, S., Alvarez-Bolado, G., 2012. Mouse thalamic differentiation: gli-dependent pattern and gli-independent prepattern. Front. Neurosci. 6, 27. Han, Y.G., Spassky, N., Romaguera-Ros, M., Garcia-Verdugo, J.M., Aguilar, A., Schneider-Maunoury, S., Alvarez-Buylla, A., 2008. Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells. Nat. Neurosci. 11, 277–284. Handel, M., Schulz, S., Stanarius, A., Schreff, M., ErdtmannVourliotis, M., Schmidt, H., Wolf, G., Hollt, V., 1999. Selective targeting of somatostatin receptor 3 to neuronal cilia. Neuroscience 89, 909–926. Haubensak, W., Attardo, A., Denk, W., Huttner, W.B., 2004. Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis. Proc. Natl. Acad. Sci. USA 101, 3196–3201. Haycraft, C.J., Banizs, B., Aydin-Son, Y., Zhang, Q., Michaud, E.J., Yoder, B.K., 2005. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet. 1, e53. Huang, P., Schier, A.F., 2009. Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia. Development 136, 3089–3098. Huangfu, D., Anderson, K.V., 2005. Cilia and Hedgehog responsiveness in the mouse. Proc. Natl. Acad. Sci. USA 102, 11325–11330. Iwanaga, T., Hozumi, Y., Takahashi-Iwanaga, H., 2011. Immunohistochemical demonstration of dopamine receptor D2R in the primary cilia of the mouse pituitary gland. Biomed. Res. 32, 225–235. Kim, S., Zaghloul, N.A., Bubenshchikova, E., Oh, E.C., Rankin, S., Katsanis, N., Obara, T., Tsiokas, L., 2011. Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry. Nat. Cell Biol. 13, 351–360. Kuan, C.Y., Roth, K.A., Flavell, R.A., Rakic, P., 2000. Mechanisms of programmed cell death in the developing brain. Trends Neurosci. 23, 291–297. Kumamoto, N., Gu, Y., Wang, J., Janoschka, S., Takemaru, K., Levine, J., Ge, S., 2012. A role for primary cilia in glutamatergic synaptic integration of adult-born neurons. Nat. Neurosci. 15 (399–405), S1. Lai, K., Kaspar, B.K., Gage, F.H., Schaffer, D.V., 2003. Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nat. Neurosci. 6, 21–27. Li, A., Saito, M., Chuang, J.Z., Tseng, Y.Y., Dedesma, C., Tomizawa, K., Kaitsuka, T., Sung, C.H., 2011. Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors. Nat. Cell Biol. 13, 402–411. Liu, A., Wang, B., Niswander, L.A., 2005. Mouse intraflagellar transport proteins regulate both the activator and repressor functions of Gli transcription factors. Development 132, 3103– 3111. Machold, R., Hayashi, S., Rutlin, M., Muzumdar, M.D., Nery, S., Corbin, J.G., Gritli-Linde, A., Dellovade, T., Porter, J.A., Rubin, L.L., Dudek, H., McMahon, A.P., Fishell, G., 2003. Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron 39, 937–950.
380
MECHANISMS OF DEVELOPMENT
Marszalek, J.R., Ruiz-Lozano, P., Roberts, E., Chien, K.R., Goldstein, L.S., 1999. Situs inversus and embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of kinesin-II. Proc. Natl. Acad. Sci. USA 96, 5043–5048. May, S.R., Ashique, A.M., Karlen, M., Wang, B., Shen, Y., Zarbalis, K., Reiter, J., Ericson, J., Peterson, A.S., 2005. Loss of the retrograde motor for IFT disrupts localization of Smo to cilia and prevents the expression of both activator and repressor functions of Gli. Dev. Biol. 287, 378–389. Nonaka, S., Tanaka, Y., Okada, Y., Takeda, S., Harada, A., Kanai, Y., Kido, M., Hirokawa, N., 1998. Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 95, 829–837. Ocbina, P.J., Tuson, M., Anderson, K.V., 2009. Primary cilia are not required for normal canonical Wnt signaling in the mouse embryo. PLoS One 4, e6839. Petersen, P.H., Zou, K., Hwang, J.K., Jan, Y.N., Zhong, W., 2002. Progenitor cell maintenance requires numb and numblike during mouse neurogenesis. Nature 419, 929–934. Rohatgi, R., Milenkovic, L., Scott, M.P., 2007. Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372– 376. Scholey, J.M., Anderson, K.V., 2006. Intraflagellar transport and cilium-based signaling. Cell 125, 439–442. Stottmann, R.W., Tran, P.V., Turbe-Doan, A., Beier, D.R., 2009. Ttc21b is required to restrict sonic hedgehog activity in the developing mouse forebrain. Dev. Biol. 335, 166–178. Tasouri, E., Tucker, K.L., 2011. Primary cilia and organogenesis: is Hedgehog the only sculptor? Cell Tissue Res. 345, 21–40. Thauvin-Robinet, C., Cossee, M., Cormier-Daire, V., Van Maldergem, L., Toutain, A., Alembik, Y., Bieth, E., Layet, V., Parent, P., David, A., Goldenberg, A., Mortier, G., Heron, D., Sagot, P., Bouvier, A.M., Huet, F., Cusin, V., Donzel, A., Devys, D., Teyssier, J.R., Faivre, L., 2006. Clinical, molecular, and genotype-phenotype correlation studies from 25 cases of oralfacial-digital syndrome type 1: a French and Belgian collaborative study. J. Med. Genet. 43, 54–61. Theil, T., Alvarez-Bolado, G., Walter, A., Ruther, U., 1999. Gli3 is required for Emx gene expression during dorsal telencephalon development. Development 126, 3561–3571. Tobin, J.L., Di Franco, M., Eichers, E., May-Simera, H., Garcia, M., Yan, J., Quinlan, R., Justice, M.J., Hennekam, R.C., Briscoe, J., Tada, M., Mayor, R., Burns, A.J., Lupski, J.R., Hammond, P., Beales, P.L., 2008. Inhibition of neural crest migration underlies
1 3 0 ( 2 0 1 3 ) 3 7 3 –3 8 0
craniofacial dysmorphology and Hirschsprung’s disease in Bardet–Biedl syndrome. Proc. Natl. Acad. Sci. USA 105, 6714– 6719. Tole, S., Goudreau, G., Assimacopoulos, S., Grove, E.A., 2000. Emx2 is required for growth of the hippocampus but not for hippocampal field specification. J. Neurosci. 20, 2618–2625. Town, T., Breunig, J.J., Sarkisian, M.R., Spilianakis, C., Ayoub, A.E., Liu, X., Ferrandino, A.F., Gallagher, A.R., Li, M.O., Rakic, P., Flavell, R.A., 2008. The stumpy gene is required for mammalian ciliogenesis. Proc. Natl. Acad. Sci. USA 105, 2853– 2858. Tran, P.V., Haycraft, C.J., Besschetnova, T.Y., Turbe-Doan, A., Stottmann, R.W., Herron, B.J., Chesebro, A.L., Qiu, H., Scherz, P.J., Shah, J.V., Yoder, B.K., Beier, D.R., 2008. THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia. Nat. Genet. 40, 403–410. Tucker, R.W., Pardee, A.B., Fujiwara, K., 1979a. Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells. Cell 17, 527–535. Tucker, R.W., Scher, C.D., Stiles, C.D., 1979b. Centriole deciliation associated with the early response of 3T3 cells to growth factors but not to SV40. Cell 18, 1065–1072. Vierkotten, J., Dildrop, R., Peters, T., Wang, B., Ruther, U., 2007. Ftm is a novel basal body protein of cilia involved in Shh signalling. Development 134, 2569–2577. Vincent, S.D., Dunn, N.R., Hayashi, S., Norris, D.P., Robertson, E.J., 2003. Cell fate decisions within the mouse organizer are governed by graded nodal signals. Genes Dev. 17, 1646–1662. Wang, C., Pan, Y., Wang, B., 2007. A hypermorphic mouse Gli3 allele results in a polydactylous limb phenotype. Dev. Dyn. 236, 769–776. Willaredt, M.A., Hasenpusch-Theil, K., Gardner, H.A.R.G., Kitanovic, I., Hirschfeld-Warneken, V.C., Gojak, C.P., Gorgas, K., Bradford, C.L., Spatz, J., Wolfl, S., Theil, T., Tucker, K.L., 2008. A crucial role for primary cilia in cortical morphogenesis. J. Neurosci. 28, 12887–12900. Wilsch-Brauninger, M., Peters, J., Paridaen, J.T., Huttner, W.B., 2012. Basolateral rather than apical primary cilia on neuroepithelial cells committed to delamination. Development 139, 95–105. Wilson, S.L., Wilson, J.P., Wang, C., Wang, B., McConnell, S.K., 2011. Primary cilia and Gli3 activity regulate cerebral cortical size. Dev. Neurobiol.