Regionalized cadherin-7 expression by radial glia is regulated by Shh and Pax7 during chicken spinal cord development

Neuroscience 142 (2006) 1133–1143

REGIONALIZED CADHERIN-7 EXPRESSION BY RADIAL GLIA IS REGULATED BY Shh AND Pax7 DURING CHICKEN SPINAL CORD DEVELOPMENT J. LUO,* M. J. JU AND C. REDIES

located in the dorsal region of the basal plate and express Evx-1 and Engrailed-1, respectively (Ericson et al., 1996). The V2 and VMN neurons are located more ventrally and are marked by Lim3 expression (Sharma et al., 1998). The V3 neurons are derived from the region between the floor plate and the VMN neurons and express Nkx2.2 (Briscoe et al., 1999). The transcription factors expressed in the spinal cord are divided into two classes, Class I genes and Class II genes, based on how they are regulated by graded Shh signaling. The expression of Class I genes is repressed by Shh; examples of Class I genes are the homeodomain proteins of the Pax family. The expression of Class II genes is induced by Shh; examples are members of the Nkx-family of transcription factors. Following the establishment of distinct progenitor cell domains by Shh, the mutually repressive interaction between Class I and Class II genes acts to refine and maintain domain integrity (Briscoe and Ericson, 1999; Briscoe et al., 2000). Cadherin-7 (Cad7), a classic type II cadherin, is a member of the cadherin family of morphoregulatory adhesion molecules (Nakagawa and Takeichi, 1995, 1998) and is expressed in a distinct dorsal domain of the basal plate (Nakagawa and Takeichi, 1998; Ju et al., 2004). It is also expressed by subsets of radial glia domains, developing gray matter regions, fiber tracts and synapses of several regions and in functional systems of the embryonic CNS, for example in the cerebellum (Arndt and Redies, 1998), the forebrain (Yoon et al., 2000; Redies et al., 2001) and the visual system (Wöhrn et al., 1998, 1999; Heyers et al., 2004). Like other classic cadherins (Redies, 2000), Cad7 was shown to play a role in the guidance of neuronal migration (Luo et al., 2004) and in axonal pathfinding (Treubert-Zimmermann et al., 2002). In summary, Cad7 plays a role in the functional differentiation of specific regions of the CNS. In the present work, we asked whether Cad7 expression in the spinal cord is regulated by graded Shh signaling. Moreover, the dorsal border of the Cad7 domain precisely co-localizes with the ventral border of the Pax7 domain and defines the border between the alar and basal plate (Ju et al., 2004). We thus asked whether Cad7 expression can be repressed by Pax7. By in vivo electroporation, different concentrations of Shh or Pax7 plasmids were transferred into the embryonic spinal cord and hindbrain of the chicken in order to study their effect on Cad7 expression. Shh was ectopically expressed also in the cerebellum at early stages of development, to ask whether it can induce Cad7 expression in an

Institute of Anatomy I, Friedrich Schiller University, Teichgraben 7, D-07740 Jena, Germany

Abstract—During development, several genes that specify neuronal subtype identity are expressed in distinct dorsoventral domains of the spinal cord and hindbrain. Cadherin-7 (Cad7), a member of the cadherin family of adhesion molecules, is expressed by radial glia in a dorsal domain of the spinal cord basal plate in chicken. To study the regulation of the Cad7 gene, we ectopically expressed two known dorsoventral patterning genes, Shh and Pax7, in the caudal neural tube and in two brain regions at different stages of development by in vivo electroporation. Results showed that Shh regulated the expression of Cad7 by radial glia in a concentration-dependent manner. Shh induced or repressed the expression of Cad7, at low and high concentrations, respectively. Furthermore, Pax7 inhibited the expression of Cad7. These results are compatible with a role of Shh and Pax7 in regulating endogenous Cad7 expression during spinal cord and hindbrain development. Our data show, for the first time, that Shh can regulate the expression not only of other gene regulatory factors, but also of Cad7, a morphoregulatory molecule that plays a role in axon elongation and neural circuit formation. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: in vivo electroporation, gene pattern, gene regulation, sonic hedgehog, cell adhesion.

During the development of the spinal cord and hindbrain, distinct classes of neurons are derived from precise dorsoventral progenitor domains of the neural tube, which are marked by the expression of transcription factors (Tanabe and Jessell, 1996; Jessell, 2000). The neurons generated in the dorsal part of neural tube (in the alar plate) function mostly in the transduction of sensory information, whereas the ventrally derived neurons (in the basal plate) differentiate into interneurons and motor neurons. In the alar plate, bone morphogenetic protein secreted from the roof plate appears to direct the differentiation of distinct sensory interneurons (Lee and Jessell, 1999). In the basal plate, sonic hedgehog (Shh), which is produced by the notochord and floor plate, acts as a morphogen and determines neuronal subtype identity of interneurons and motor neurons at different concentrations (Ericson et al., 1996; Briscoe and Ericson, 1999). The V0 and V1 neurons are *Corresponding author. Tel: ⫹49-3641-938519; fax: ⫹49-3641-938512. E-mail address: [email protected] (J. Luo). Abbreviations: Cad7, cadherin-7; GFP, green fluorescent protein; Hh, hedgehog; Shh, sonic hedgehog; TBS, Tris-buffered saline.

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.07.038

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area that does not normally express this molecule during early development.

EXPERIMENTAL PROCEDURES Animals, plasmids and antibodies Fertilized eggs of White Leghorn chicken (Gallus domesticus) were obtained from a local farm and incubated in a forced-draft incubator at 37 °C. Chicken embryos were staged according to Hamburger and Hamilton (1951) (HH). Embryos were studied for normal endogenous expression of Cad7 or Gli genes at stages 20, 23, 25, and 28 (at least three embryos for each stage). Embryos were electroporated with Shh or Pax7 expression plasmids at stages 12, 21, and 23 [in different areas (spinal cord, hindbrain, and cerebellum) or with different concentrations of Shh (0.5 ␮g/␮l and 2.0 ␮g/␮l); at least six embryos for each stage]. The experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and similar institutional regulations. Studies were designed to minimize the number of embryos used and their suffering. The plasmid pCAGGS– green fluorescent protein (GFP), a eukaryotic expression vector carrying a CMV/chicken ␤-actin promoter, was a kind gift of Dr. H. Ogawa (National Institute of Basic Biology, Okazaki, Japan; Momose et al., 1999). Full-length Shh cDNA was derived from plasmid pBluescript-Shh (pHH2b, kind gift of Dr. C. J. Tabin, Department of Genetics, Harvard Medical School, Boston, MA, USA), full-length Pax7 from plasmid pBluescript-Pax7 (N2-4⫹C4-9, kind gift of Dr. H. Nakamura, Institute of Development, Tohoku University, Sendai, Japan; Matsunaga et al., 2001), and full-length Cad7 cDNA from plasmid pBluescriptCad7 (NK-2, kind gift of Drs. S. Nakagawa and M. Takeichi, Kyoto University, Kyoto, Japan; Nakagawa and Takeichi, 1998). The cDNAs were cloned into blunted EcoRI sites of the plasmid pCAGGS after removing the GFP insert, respectively. The resulting plasmids were amplified in the XL1-blue strain of E. coli (Stratagene, La Jolla, CA, USA) and purified using Qiagen columns (Qiagen, Hilden, Germany). For electroporation, plasmid was dissolved in Gey’s balanced salt solution (GBSS; Sigma, Munich, Germany). For immunostaining of sections, mouse monoclonal antibodies against Cad7 (CCD7-1, kind gift of Drs. S. Nakagawa and M. Takeichi; Nakagawa and Takeichi, 1998), Shh (5E1; Ericson et al., 1996), Nkx2.2 (74.5A5; Ericson et al., 1997), Lim3 (67.4E12; Ericson et al., 1997), and Pax7 (Ericson et al., 1996) were used. The 5E1, 74.5A5, 67.4E12 and Pax7 antibodies were obtained from the Developmental Studies Hybridoma Bank (DSHB) developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA, USA. As secondary antibodies, Cy3-labeled goat anti-mouse IgG antiserum (Dianova, Hamburg, Germany) was used. Dye Hoechst 33258 (Molecular Probes, Eugene, OR, USA) was used for staining of cell nuclei.

In vivo electroporation Electroporation of developing chicken embryos was performed in ovo (Nakamura and Funahashi, 2001) at stage 12, and ex ovo (Luo and Redies, 2005) at stages 21 and 23, as described previously. In brief, for plasmid injection, capillary glass pipettes were pulled on a microelectrode puller (PUL-100 Micropipette Puller; WPI, Berlin, Germany) from glass tubes of 1.0 mm diameter (TW100F-4, WPI) and used through a mouthpiece (A5177, Sigma). Solution containing pCAGGS-GFP plasmid (final concentration of 0.25 ␮g/␮l) was mixed with pCAGGS-Shh plasmid (0.5 ␮g/␮l or 2.0 ␮g/␮l) or pCAGGS-Pax7 plasmid (2.0 ␮g/␮l), respectively. Fast Green (Sigma) was added to the solution (0.1%) to visualize the injection site. After injection, the electrodes

for in ovo electroporation (CUY 610-P2.5, Nepa Gene, Chiba, Japan) or for ex ovo electroporation (CUY 661–3⫻7, Nepa Gene) were placed on both sides of the embryo. Immediately, electric pulses of 12 V for in ovo electroporation, and 18 V for ex ovo electroporation (60 ms pulse length, six pulses, 100 ms intervals in each case) were applied by an electroporator (CUY 21, Nepa Gene). Visualizing GFP expression in the embryos under a fluorescence stereomicroscope (MZFLIII, Leica, Bensheim, Germany) allowed monitoring of the success of electroporation one to three days after electroporation.

Immunohistochemistry Eighteen or 20 micrometer-thick cryostat sections were immunostained according to previously published procedures (Luo and Redies, 2004). Briefly, after postfixation in 4% formaldehyde in ice-cold Hepes-buffered salt solution (100 mM Hepes, 140 mM NaCl, 5 mM KCl, 5 mM glucose, 0.4 mM Na2HPO4, and 0.04 mM Phenol Red, supplemented with 1 mM CaCl2 and 1 mM MgCl2, pH7.4; all reagents from Merck, Darmstadt, Germany) for 30 min, the sections were washed with Tris-buffered saline (TBS, 150 mM NaCl, 500 mM Tris, pH 7.4, containing 1 mM CaCl2 and MgCl2) and preincubated with blocking solution (5% skimmed milk, 0.3% Triton X-100 in TBS) at room temperature for 60 min. Then, primary antibody was applied overnight at 4 °C, followed by washing and incubation with secondary antibodies at room temperature for 60 min. Finally, cell nuclei were stained with dye Hoechst 33258. Fluorescence was imaged under a fluorescence microscope (BX40, Olympus, Hamburg, Germany) equipped with a digital camera (DP70, Olympus).

In situ hybridization The in situ hybridization was performed according to the protocol of Redies et al. (1993). Briefly, digoxigenin-labeled antisense cRNA probes were transcribed using chicken cDNAs encoding Gli1, Gli2, Gli3 (in pBluescript vector, kind gift of Dr. C. J. Tabin; Marigo et al., 1996; Schweitzer et al., 2000) and Cad7 (Nk-2, Nakagawa and Takeichi, 1998) as templates, respectively. Cryostat sections of 18 or 20 ␮m thickness were fixed with 4% formaldehyde in PBS (13 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4; pH 7.4) and were pretreated with proteinase K and acetic anhydride. Sections were hybridized with cRNA probes at a concentration of about 1 ng/␮l overnight at 70 °C in hybridization solution (50% formamide, 3⫻ SSC, 1⫻ Denhardt’s solution, 250 ␮g/ml yeast RNA and 250 ␮g/ml salmon sperm DNA). After the sections were washed, alkaline phosphatase-coupled anti-digoxigenin Fab fragments were applied to bind to the cRNA probes. For visualization of the labeled mRNAs, the sections were incubated with a substrate mixture of nitroblue tetrazolium salt (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (BCIP) overnight at 4 °C. The sections were viewed and photographed under a microscope (BX40, Olympus) equipped with a digital camera (DP70, Olympus). Digitized images from immunohistochemistry and in situ hybridization were adjusted in contrast and brightness with the Photoshop software (Adobe Systems, Mountain View, CA, USA). Labeling of the figures was done with the Illustrator software (Adobe Systems).

Apoptosis analysis To detect apoptotic cells, cryostat sections of transfected and untransfected sides of the electroporated embryos were processed using the In Situ Cell Death Detection Kit/TMR Red (Roche, Mannheim, Germany), according to the manufacturer’s instructions.

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RESULTS Endogenous expression of Cad7 in spinal cord The endogenous expression of Cad7 protein was studied by immunohistochemistry in transverse sections of chicken embryonic spinal cord at cervical and thoracic levels. At stage 20, Cad7 was expressed in the dorsal part of the basal plate (bracket in Fig. 1A; Ju et al., 2004). This expression persisted in the ventricular zone at least until stage 28 (brackets in Fig. 1B–D). From stage 21–23, the expression of Cad7 in the mantel zone gradually extended ventrally (arrows in Fig. 1B–D; Ju et al., 2004). At the same time, additional expression of Cad7 appeared in the floor plate ependymal cells (arrows in Fig. 1E, G; lower small arrows in Fig. 1I, J) and in the ventral floor plate, where the neurites from interneurons cross to the contralateral site (arrowheads in Fig. 1E, G; Ju et al., 2004). Gradually, the expression of Cad7 in the floor plate became stronger (arrowheads in Fig. 1B–D). Note that in the floor plate, the expression of Cad7 (Fig. 1E, G) overlapped partially with Shh (Fig. 1F, H; compare the arrows and arrowheads). Furthermore, by in situ hybridization, Cad7 mRNA was demonstrated in cell bodies of the ventricular zone in the dorsal part of the basal plate (upper small arrows in Fig. 1I, J; Ju et al., 2004). These cell bodies (arrowheads in Fig. 1K–N) represent radial glial cells because they possess fibrous processes that extend radially from the ventricular zone to the pial surface (arrows in Fig. 1K–N). Moreover, at stage 24, Cad7 mRNA was expressed in cells of the mantel layer (arrowheads in Fig. 1I). At stage 28, Cad7-positive interneurons have appeared in the alar plate (upper arrowhead in Fig. 1J) and a cluster of Cad7-positive neurons was observed in the lateral part of the motor column (middle arrowhead in Fig. 1J). Cad7 transcript was also seen in the floor plate cells (lower small arrows in Fig. 1I, J). Technical considerations In this study, we used in vivo electroporation to study the regulation of Cad7 expression by Shh and Pax7. Two separate plasmids (e.g. one encoding the target gene, another encoding the marker gene) were co-transfected. As shown previously (Momose et al., 1999; Haas et al., 2001; Luo and Redies, 2005), this procedure can yield a high degree of co-expression of both genes in the electroporated cells (Fig. 1O–R). In the present work, we used GFP as a marker gene for tracing the cells expressing the target gene ectopically (Figs. 1 O–R, 2– 4). The effect of electroporation on cell death possibly induced by in vivo electroporation was also evaluated. Results from the TUNEL assay demonstrated that the number of apoptotic cells on the electroporated side was not different from that on the un-transfected side of the spinal cord (data not shown; see also Luo and Redies, 2005).

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Ectopic Shh concentration and expression of downstream targets In order to get an idea of how much Shh was expressed ectopically after electroporation in vivo, the expression of genes induced downstream of Shh (Nkx2.2, Lim3, Gli1, Gli2 and Gli3) was monitored. The expression of Nkx2.2 is induced in the V3 neurons adjacent to the floor plate by relatively high concentrations of Shh (Briscoe et al., 1999, 2000). In Shh knock-out mice, the expression of Nkx2.2 is deleted in spinal cord (Stamataki et al., 2005). We considered that a large number of cells expressing Nkx2.2 in or around the electroporated area indicated very high concentrations of Shh. Lim3 is expressed in the V2 and VMN domains and by motor neurons in the spinal cord (Thor et al., 1999; William et al., 2003). Moderately high concentrations of Shh induce the expression of Lim3 (Ericson et al., 1997). We considered that a large number of cells expressing Lim3 indicated moderately high concentrations of Shh. Other downstream targets of Shh signaling are the transcription factors of the Gli family (Ruiz I Altaba et al., 2002; Jacob and Briscoe, 2003). At stage 20, Gli1 is expressed in the entire basal plate (bracket in Fig. 1T), Gli2 in an intermediate region (bracket in Fig. 1U) and Gli3 in both dorsal basal plate and alar plate (bracket in Fig. 1V). The expression domains of the three Gli genes overlap within the ventricular domain positive for Cad7 (compare the brackets in Fig. 1S–V). Gli expression is not observed in the floor plate (Figs. 1–3). We considered that a large number of cells expressing Gli3 indicated low concentrations of Shh. Several factors affect the level of ectopic Shh expression by electroporation in vivo. First, different expression vectors result in different efficiencies of transcription. For example, the mammalian expression vector pECE (Ellis et al., 1986) that contains an early SV40 promotor directs significantly lower levels of expression than the vector pCAGGS that carries the CMV/chicken ␤-actin promoter (Momose et al., 1999; Stamataki et al., 2005). In the present study, the pCAAGS vector was used. Second, the level of ectopic Shh expression depends on the concentration of electroporated Shh plasmid. We used two different concentrations of Shh plasmid (0.5 ␮g/␮l or 2.0 ␮g/␮l) in this study. Third, the efficiency of electroporation depends on the electroporation device, the electrodes, and electroporation parameters. In this study, the electroporation parameters were kept as constant as possible once they were established for each stage. Fourth, because Shh is a diffusible morphogen, the concentration of ectopic Shh depends on the number of cells electroporated in a given region. Low levels of ectopic Shh induce Cad7 expression in the spinal cord To study the in vivo regulation of Cad7 expression by different concentrations of Shh, transverse sections of chicken embryonic spinal cord were analyzed at the cervical and thoracic level. Regions with low levels of ectopic

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Fig. 1. Endogenous expression of Cad7, Shh and Gli genes in adjacent sections during chicken spinal cord development and expression of two co-transfected genes. (A–D) Endogenous expression of Cad7 in the spinal cord at stage 20 (A), stage 23 (B), stage 25 (C), and stage 28 (D). The brackets indicate the Cad7-positive domains in the dorsal part of basal plate. The arrows point to Cad7 expression in the mantel zone of the ventral part of the basal plate. The arrowheads indicate Cad7 expression in the floor plate. (E–H) Endogenous expression of Cad7 (E, G) and Shh (F, H) in the floor plate at stage 23 (E, F) and stage 28 (G, H). The arrows point to the perikarya of ependymal floor plate cells and the arrowheads point to neurites crossing the floor plate. Note that the expression of Cad7 and Shh overlaps partially. Cell nuclei (blue) were stained with dye Hoechst 33258. (I, J) In situ hybridization results showing Cad7 transcript in the dorsal part of the basal plate (upper small arrows), in the mantel zone (arrowheads), and in the floor plate (lower small arrows) at stage 24 (I) and stage 28 (J). The lower arrowhead in J points to neurites crossing the floor plate. Spinal ganglia and Schwann cells (large arrows) also show Cad7 expression. (K–N) The Cad7-expressing cells located in the ventricular zone (arrowheads) extend their fibrous processes from ependymal to the pial surface in a radial direction (arrows). L and N show a magnification of the areas boxed in K and M, respectively. (O–R) One day after electroporating spinal cord with a mixture of Pax7 and GFP plasmids, almost all of the GFP-positive cells (green in O) co-express Pax7 (red, arrows in O, P; co-expression indicated by yellow color in Q, R). Ectopic expression of Pax7 (arrows) is stronger than endogenous Pax7 expression (arrowheads). R shows a magnification of the area boxed in Q. GFP (green) but not Pax7 is expressed in the fiber fascicles at the surface of the spinal cord (arrow in R). (S–V) Endogenous expression of Cad7 (S) and the transcription of Gli1 (T), Gli2 (U), and Gli3 (V) at stage 20. Note that the expression of all Gli genes overlaps within the Cad7 expression domain. Scale bar⫽50 ␮m in K; 100 ␮m in A for (A–D), in E for (E, F), in M, in O for (O–Q), in S for (S–V); 200 ␮m in G for (G–J).

Shh expression are found on the right side of the sections displayed in Fig. 2A–H. Low levels of ectopic Shh expres-

sion were indicated by the following findings: (a) a small number of cells expressing Nkx2.2 at the center of the

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transfected region (red, inset in Fig. 2A), (b) upregulation of Gli1, Gli2, and Gli3 expression (right brackets in Fig. 2F–H), and (c) relatively few GFP-positive cells in the transfected area. Under these conditions, the Cad7-expressing area is enlarged (red, compare both sides in Fig. 2C, E). No change in the size and strength of the endogenous Pax7 expression was observed in the alar plate (red, compare both sides in Fig. 2D). The spinal cord is enlarged on the transfected side (Fig. 2A–H). High levels of ectopic Shh expression down-regulate Cad7 expression in the spinal cord In Fig. 2I, more cells in the transfected region express GFP. The level of Shh in the transfected region (green area, arrowheads) was relatively high, as indicated by more cells that ectopically expressed Nkx2.2 (red, upper bracket in Fig. 2I) at the center of the transfected area, where endogenous expression of Cad7 was partially repressed (compare both sides in Fig. 2K). Instead, expression of Cad7 was induced to expand to the dorsal side of the transfected area (upper bracket in Fig. 2K). Furthermore, the ventral border of endogenous expression of Pax7 moved dorsally (arrow, compare both sides in Fig. 2L). In the region where Pax7 disappeared, ectopic expression of Cad7 was induced (compare upper bracket in Fig. 2K and arrow in Fig. 2L). On the transfected side, the region of Lim3 expression in the mantel zone is ventrally adjacent to (and partially overlaps with) the lower Cad7positive region (Fig. 2J). In the sections displayed in Fig. 2M–T, ectopic expression of Shh in the transfected area was even higher, as indicated by the higher numbers of Nkx2.2-positive cells in the transfected area (red, upper bracket in Fig. 2M) and a down-regulation of Gli2 and Gli3 expression on the transfected side, respectively (arrowheads in Fig. 2S,T). Note also that the transcription of Gli1 was shifted dorsally on the transfected side (compare brackets in Fig. 2R). The region between the floor plate and the transfected area contains a large number of Lim3-positive cells, suggesting that Shh levels are moderately high in this area also (red, bracket in Fig. 2N). As a result of the inferred high concentrations of Shh, Cad7 expression was completely repressed in the ventricular layer on the transfected side (arrow in Fig. 2O and arrowhead in Fig. 2Q). Even in the area of ventral Pax7 down-regulation (arrowheads in Fig. 2P), no ectopic expression of Cad7 was observed (arrowheads in Fig. 2O). The regulation of Cad7 expression by graded Shh signaling was also studied at older stages (e.g. stages 23–28). Shh plasmid was electroporated in the spinal cord at stage 23, when Cad7 was already expressed endogenously in the dorsal part of the basal plate (Fig. 1B; Ju et al., 2004). Compared with the earlier stages (Fig. 2), results from embryos killed at stage 28 showed a similar regulation of Cad7 by ectopic Shh expression (Fig. 3A–P). A low concentration of Shh (Fig. 3A–H) induced Cad7 at the margin of the electroporated area (upper bracket in Fig. 3A, E). Here, low Shh concentration was indicated by an up-regulation of Gli expression (Fig. 3F–H). With high Shh

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concentration (Fig. 3I–P), as indicated by a large number of Nkx2.2 positive-cells (Fig. 3J) and a down-regulation of Gli2 and Gli3 expression in the electroporated region (Fig. 3O, P), endogenous Cad7 expression was repressed (arrowheads in Fig. 3I, M). To check whether cell fate was changed at older stages after electroporation of Shh, immunostaining against Islet-1 and Pax7 was performed. Compared with the control side, the expression domains of Islet-1 and Pax7 on the electroporated side were altered (Fig. 3C, D, K, L). It is therefore possible that cell fate of spinal cord neurons is changed after Shh is ectopically expressed at older stages. In summary, our results demonstrate that Shh induced or repressed the expression of Cad7 in a concentrationdependent manner. High concentrations of Shh downregulated endogenous Cad7 protein expression even at a relatively late stage of development. Shh regulates Cad7 expression in hindbrain and cerebellum To study the effect of Shh signaling on Cad7 expression in the basal plate of the hindbrain, electroporation was performed at stage 12 and sections were analyzed one or two days later. In general, results were similar to those obtained in the basal plate of the spinal cord. Low concentrations of ectopic Shh adjacent to the electroporated region induced ectopic expression of Cad7 (compare brackets in Fig. 3Q), whereas high concentrations effectively down-regulated endogenous Cad7 expression at the center of the transfected area (green, arrowhead in Fig. 3R) as well as in the immediately adjacent regions (arrows in Fig. 3R) where Shh concentration was presumably high also (compare brackets in Fig. 3R). To test whether Shh signaling induces Cad7 expression in other brain regions, which do not express Cad7 endogenously at the time of the experiment, Shh plasmid was electroporated into the cerebellar anlage at stages 21 and 23, respectively. Results revealed that ectopic expression of Cad7 was induced by Shh at stage 26 (brackets in Fig. 3S–U), that is, at a time when no endogenous expression of Cad7 can be observed in the cerebellum (Arndt and Redies, 1998). Like in the spinal cord and hindbrain, the area of ectopic Cad7 expression was often located adjacent to the electroporated area, where Shh concentrations were presumably lower than in the center of electroporation (arrowheads in Fig. 3S–V). Pax7 down-regulates the expression of Cad7 in the spinal cord and hindbrain To study the effect of Pax7 on the expression of Cad7, Pax7 expression plasmid was electroporated into the spinal cord and hindbrain. Electroporation was carried out at stage 12, when endogenous Cad7 expression first begins to be seen in the spinal cord and hindbrain (Ju et al., 2004). Results were obtained one or two days after electroporation and showed that most electroporated cells co-expressed GFP and Pax7 (Fig. 1O–R). Ectopic Pax7 down-regulated the expression of endogenous Cad7, but only in those cells that were electroporated (arrows in Fig. 4A–C, I–K, see en-

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Fig. 2. Regulation of Cad7 expression (red in C, E, K, O, Q) by ectopically induced Shh in spinal cord at early stages of development. Electroporated cells are marked by GFP expression (green), immunostaining (brackets) by red color and in situ hybridization (brackets) by purple. Adjacent sections are shown in (A–D), (E–H), (I–L), (M–P), and (Q–T). Ectopic expression of Shh in the spinal cord at different concentrations is indicated by the expression of genes downstream of Shh signaling (Nkx2.2 in A, I, M; Lim3 in B, J, N; and Pax7 in D, L, P; Gli genes in F–H, R–T). Note that, on the

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largements in D, L). Furthermore, electroporation of Pax7 was also performed in the spinal cord at a later stage (stage 23; Fig. 4E–H), when the domain of Cad7 expression was already prominent (Fig. 1B; Ju et al., 2004). Again, results revealed a down-regulation of endogenous Cad7 protein in cells that expressed Pax7 ectopically, but not in neighboring cells (arrow in Fig. 4G, see enlargement in H). Together, our results demonstrate that Pax7 represses the expression of Cad7. We also investigated whether the forced expression of Cad7 had an effect on Pax7 expressed in the alar plate of the spinal cord. Electroporation was performed at stage 12 and results were studied one or two days later (stage 20 and 24). Ectopic expression of Cad7 in the alar plate did not have any effect on Pax7 expression in this area (data not shown).

DISCUSSION

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nervous system (Nakagawa and Takeichi, 1998) and in the CNS (Luo et al., 2004), suggesting the possibility that it might play a role also in guiding the radial migration of early spinal interneurons to the mantel zone. At a later stage, Cad7 immunostaining is especially strong at the position where the neurites of interneurons cross the Cad7-positive floor plate (arrowheads in Fig. 1D, G). It has been shown experimentally that Cad7 plays a role in axonal guidance and fasciculation (Treubert-Zimmermann et al., 2002). Whether Cad7 plays a role in guiding neurites across the floor plate remains to be studied. Finally, the differential expression of several classic cadherins by pools of motor neurons has been implicated in the sorting of neurons (Price et al., 2002). This role is also suggested by the expression of cadherins in (pro-)nuclei of other CNS regions (Gänzler and Redies, 1995; Yoon et al., 2000). In summary, Cad7 is a morphoregulatory molecule which may play multiple roles in the development and functional differentiation of the embryonic spinal cord.

Cad7 is a multifunctional morphoregulatory molecule expressed by different cell types in the spinal cord

Graded Shh signaling regulates Cad7 expression

In the basal plate of the spinal cord, Cad7 is expressed by radial glia during early development. This result is indicated by the presence of Cad7 mRNA-containing perikarya in the ventricular layer (upper small arrows in Fig. 1I, J), and Cad7-immunoreactive fibrous immunostaining extending from the ependymal to the pial surface in a radial direction in this region (arrows in Fig. 1K–N). A regionally restricted expression by radial glia has also been described for several other classic cadherins in the CNS (Redies, 2000). Cad7 expression by radial glia stops abruptly at the basal/alar plate boundary (Ju et al., 2004). The abrupt change of cadherin expression at embryonic domain boundaries may serve to stabilize the embryonic boundaries in the brain (Espeseth et al., 1998; Redies, 2000; Inoue et al., 2001). Apart from radial glia, there are three other Cad7positive cell populations in the embryonic spinal cord [interneurons (upper arrowheads in Fig. 1I, J), floor plate ependymal cells (lower small arrows in Fig. 1I, J) and motor neurons (middle arrowhead in Fig. 1J)]. The Cad7positive interneurons are possibly born in the Cad7-positive domain of the dorsal basal plate, beginning at stage 17 (Ericson et al., 1996; Pierani et al., 1999). Cad7 was shown to play a role in cell migration both in the peripheral

Previously, with the method of in vitro explant culture, the regulation of transcription factors by graded Shh signals has been studied (Ericson et al., 1996; Briscoe et al., 1999). In response to Shh, distinct classes of neurons, which express different homeodomain transcription factors, are generated at defined position of the neural tube (Ericson et al., 1996; Briscoe et al., 1999, 2000; Jessell, 2000). In the present study, we altered the gradient of Shh in vivo by ectopically expressing Shh by electroporation, in order to study its effect on Cad7 expression by radial glial cells. The expression of five genes (Nkx2.2, Lim3 and Gli genes), which are known to be induced by different concentrations of Shh, was monitored to estimate the concentrations of Shh at and around the site of electroporation (Figs. 2, 3). Low concentrations of Shh induced ectopic expression of Cad7 (Figs. 2A–H, 3A–H), whereas high concentrations of Shh down-regulated endogenous Cad7 expression in the dorsal basal plate of the spinal cord (Figs. 2I–T, 3I–P). An induction of Cad7 expression was also demonstrated in hindbrain and cerebellum (Fig. 3Q– V). Endogenously, Cad7 is expressed at a distance to the source of Shh in the floor plate (brackets in Fig. 1A–D). In the Cad7-positive dorsal region of the basal plate, Shh concentrations are lower than in the ventral area in be-

electroporated side (right side), the spinal cord is enlarged and curved laterally. (A–H) Electroporation at stage 12 with a plasmid concentration of 0.5 ␮g/␮l. Analysis was done at stage 20 (A–D) or 23 (E–H). Only a few cells express Nkx2.2 ectopically (A), suggesting low levels of Shh in the electroporated area (see text). The area of Cad7 expression is expanded (right brackets in C and E) and the transcription domains of Gli1, Gli2 and Gli3 are enlarged (compare brackets in F–H). The inset in A shows a magnification of the area boxed in A, but without GFP signal. (I–L) Electroporation at stage 12 with a plasmid concentration of 0.5 ␮g/␮l. Analysis was done at stage 20. The concentration of Shh in the electroporated area was presumably higher than that in (A–H) because more cells ectopically expressed Nkx2.2 (red, large bracket in I) and a few cells expressed Nkx2.2 also dorsally (arrows in I); moreover, the ventral border of the Pax7 domain was moved dorsally (compare the brackets in L), leaving a dorsal, Pax7-negative area (arrow in L). Arrowheads indicate the area of high Shh concentration. (M–T) Electroporation at stage 12 with a plasmid concentration of 2.0 ␮g/␮l. Analysis was done at stage 20 (Q–T) or 23 (M–P). Very high levels of Shh concentration were presumably reached because the ectopic expression of Nkx2.2 was very strong (red, upper right bracket in M) and ectopic expression of Lim3 was also strongly induced (red, compare brackets in N). The ventral endogenous expression of Pax7 was down-regulated (arrowhead in P). Endogenous expression of Cad7 in the dorsal part of basal plate was completely repressed (arrow in O and arrowhead in Q). Massive electroporation of Shh (green in Q) caused an upregulation and a dorsal shift of Gli1 transcription (compare brackets in R), whereas the transcription of Gli2 (S) and Gli3 (T) was down-regulated (arrowheads in S, T). Scale bar⫽100 ␮m in A for (A–D), in E for (E–H), in I for (I–L), in M for (M–P), in Q for (Q–T).

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Fig. 3. Regulation of Cad7 expression (red in A, E, I, M, Q–V) by ectopically induced Shh in spinal cord at later stages of development (A–P), in hindbrain (Q, R) and in cerebellum (S–V). Electroporated cells are marked by GFP expression (green), immunostaining (brackets) by red color and in situ hybridization (brackets) by purple. Adjacent sections are shown in (A–H) and (I–P). (A-P) Electroporation of spinal cord at stage 23 with a plasmid concentration of 2.0 ␮g/␮l. Analysis was done at stage 28. A–H and I–P represent two different experiments inducing ectopic expression of Shh in the spinal cord at different concentrations, as indicated by the expression of genes downstream of Shh signaling (Nkx2.2 in B, J; Islet-1 in C, K; Pax7 in D, L; and Gli genes in F–H, N–P). In the experiment depicted in A–H, ectopic expression of Cad7 was induced in a region ventrally adjacent to the electroporated area (upper bracket in A, E), where Shh concentrations were presumably low. In the experiment depicted in I–P, endogenous Cad7 was repressed by high concentrations of Shh (arrowheads in I, M). Arrowheads in A, E indicate the transfected regions. Arrowheads in O, P indicate regions where the transcription of Gli2 (O) and Gli3 (P) is down-regulated. Arrows in C, K point to Islet-1-positive cells. (Q, R) Electroporation of hindbrain at stage 12 with a plasmid concentration of 0.5 ␮g/␮l (Q) and 2.0 ␮g/␮l (R). Analysis was done at stage 20 (Q) and stage 23 (R), respectively. Arrowheads point to Shh-transfected areas. Ectopic expression of Cad7 was induced (compare brackets on both sides). Note that the expression of Cad7 is induced at a distance (arrows in R) from the center of the electroporated region (green, arrowhead in R). (S–V) Electroporation of cerebellar anlage at stage 21 (S–U) and stage 23 (V) with a plasmid concentration of 2.0 ␮g/␮l. Analysis was done at stage 26 (S–U) and stage 28 (V), respectively. Ectopic expression of Cad7 (red) is indicated by brackets. The arrowheads indicate the transfected areas and the arrows indicate endogenous Cad7 expression in the hindbrain (S–V) or cerebellum (upper arrow in V). The asterisk in S indicates an artifact (fold of the section). Scale bars⫽200 ␮m in A for (A–P), in Q for (Q, R), in S, T, in U for (U, V).

tween the floor plate and the Cad7-positive region. A similar Cad7-negative intervening area has been observed

between the zona limitans intrathalamica, a source of graded Shh signal in the developing diencephalon (Shi-

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Fig. 4. Ectopic expression of Pax7 (green) down-regulates the expression of Cad7 (red) in the spinal cord and hindbrain. The same sections are shown in (A–C), (E–G), and (I–K), respectively. C, G and K display merged images. The areas boxed in C, G and K are shown at a higher magnification in D, H and L, respectively. (A–D) Spinal cord was electroporated at stage 12 and analyzed at stage 23. (E–H) Spinal cord was electroporated at stage 23 and analyzed at stage 28. (I–L) Hindbrain was electroporated at stage 12 and analyzed at stage 23. In each case, expression of Cad7 was down-regulated in the cells that were GFP-positive (arrows). Arrowheads indicate areas of endogenous Cad7 expression on the non-transfected side. Scale bars⫽100 ␮m in A for (A–C, E–G); 200 ␮m in I for (I–K).

mamura et al., 1995) and bilateral stripes of Cad7 expression running parallel to the zona limitans (Yoon et al., 2000). Together, these results suggest that the regulation of Cad7 expression by graded Shh signals is a developmental phenomenon that is not restricted to the spinal cord and hindbrain, but is widespread in the CNS. A similar conclusion has been reached for gene regulatory factors that are regulated by Shh (Agarwala et al., 2001; Hashimoto-Torii et al., 2003; Kiecker and Lumsden, 2004). To our knowledge, however, the present study is the first to report such a Shh-dependent mechanism for a morphoregulatory adhesion molecule that is involved directly in the functional differentiation of neurons. In mediating the effect of Shh on Cad7 expression, Gli genes (Gli1, Gli2 and Gli3) play a central role. Gli genes act downstream of Shh signaling. Their roles have diverged during vertebrate evolution. In zebrafish, Gli1 is necessary for neural tube development and the loss of Gli1 results in significant defects in the initial signaling of hedgehog (Hh). Gli2 deficiency causes only minor Hh signaling defects (Karlstrom et al., 2003). By contrast, in mouse, Gli1 is expressed in the ventral neural tube and Gli1-deficient mice do not show any developmental defects (Park et al., 2000). In mouse, Gli2 is required for the initial response of cells to Shh signaling and contributes to the induction of ventral neural tube (for example, the floor plate, V3 and VMN domains). Loss of Gli2 results in severe

defects in the development of the ventral neural tube (Park et al., 2000; Bai et al., 2002). Gli3 acts as an inhibitor of Shh signaling and is required for the dorsal–ventral patterning of mouse spinal cord (Persson et al., 2002). In the spinal cord, Gli genes work in combination and in a context-dependent fashion (Dai et al., 1999; Ruiz I Altaba et al., 2002; Jacob and Briscoe, 2003). Furthermore, a gradient of Gli activity is sufficient to mimic the effect of the Shh gradient (Stamataki et al., 2005). In the present study, Gli genes were used as markers to monitor the concentration of Shh in vivo after electroporation (Figs. 2, 3). Results from in situ hybridization revealed that, when the expression of Cad7 was induced by Shh, all three Gli genes were up-regulated (Figs. 2F–H, 3F–H), indicating relatively low levels of Shh. When Cad7 was repressed by Shh, Gli1 expression was up-regulated, whereas Gli2 and Gli3 expression was down-regulated (Figs. 2R–T, 3N–P), indicating high Shh concentrations. These results and experimental findings (Ruiz I Altaba et al., 2002; Jacob and Briscoe, 2003) suggest that the effect of Shh on Cad7 expression might be mediated by Gli genes. It is therefore speculated that, in chicken spinal cord, the transcription factor Gli1 and Gli2 may be the major mediator of Shh signaling, but Gli2 is only induced at low levels of Shh signaling. The activation of Gli2 will sequentially activate Gli3 activity and both Gli2 and Gli3 act together to induce Cad7 expression in the dorsal part of

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basal plate. This hypothesis is supported by experimental work showing that Gli3A can induce the expression of Cad7 in spinal cord (Stamataki et al., 2005). This effect, however, is observed only for the expression of Cad7 by radial glia; Cad7 expression by motor neurons was unaffected by changes in Shh concentration (arrows in Fig. 3I, M). Also, the lateral parts of the floor plate, which express Cad7 from stage 21–23, are exposed to the highest concentrations of Shh and remain Cad7 positive (Fig. 1E–H; Ju et al., 2004). These results suggest that Cad7 expression is regulated by different mechanisms and/or at different times in the various cell types of the spinal cord. Finally, ectopic expression of Shh down-regulates Cad7 protein in radial glia at stages 23–28 (arrowheads in Fig. 3I, M) when Cad7 expression has already set in (Fig. 1B, Ju et al., 2004). This down-regulation indicates a high degree of plasticity in gene expression even at relatively late stages of development. Pax7 restricts the expression of Cad7 to the basal plate In the present study, we show that Pax7 can repress the expression of Cad7 by radial glia in the basal plate (Fig. 4). Interestingly, Pax7 can suppress Cad7 expression in spinal cord and hindbrain, but not vice versa. Therefore, Cad7 acts downstream of Pax7-mediated patterning, as would be expected for a morphoregulatory molecule. During brain development, transcription factors of the Pax gene family play a role in the establishment of regional cell adhesion properties also in other areas of the CNS. For example, Pax6 expression at the cortico-striatal boundary in the mouse brain coincides with a boundary of R-cadherin expression; this adhesive boundary is missing in Pax6-deficient mice (Stoykova et al., 1996). Pax6 was also shown to promote R-cadherin-dependent outgrowth of pioneer axons in the brain (Andrews and Mastick, 2003). Some members of the Ig superfamily of adhesion molecules, for example N-CAM and Ng-CAM, are also regulated by the Pax family (Chalepakis et al., 1994; Holst et al., 1994, 1997; Kallunki et al., 1995). Acknowledgments—This work was supported by a grant of the Deutsche Forschungsgemeinschaft to C.R. (Re 616/4-4). We thank Dr. H. Ogawa for his kind gift of pCAGGS-GFP plasmid, Dr. S. Nakagawa and Dr. M. Takeichi for Cad7 plasmid, Dr. C. J. Tabin for Shh and Gli plasmids, Dr. H. Nakamura for Pax7 plasmid, Ms. F. Krebs for synthesis of the Gli antisense probes, Mr. J. Lin for performing in situ hybridization, and Ms. S. Hän␤gen for technical assistance.

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(Accepted 17 July 2006) (Available online 14 September 2006)