Gli2 and Gli3 Play Distinct Roles in the Dorsoventral Patterning of the Mouse Hindbrain ⁎ Mélanie Lebel A,B,1, Rong Mo A, Kenji Shimamura C, Chi-Chung Hui A,B

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Developmental Biology 302 (2007) 345–355 www.elsevier.com/locate/ydbio

Gli2 and Gli3 play distinct roles in the dorsoventral patterning of the mouse hindbrain ⁎ Mélanie Lebel a,b,1, Rong Mo a, Kenji Shimamura c, Chi-chung Hui a,b,

a Program in Developmental Biology, The Hospital for Sick Children, Room 13-314, Toronto Medical Discovery Tower, 101 College Street, Ontario, Canada M5G 1L7 b Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada M5S 1A8 c Division of Morphology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan Received for publication 29 March 2006; revised 1 August 2006; accepted 2 August 2006 Available online 9 August 2006

Abstract

Sonic Hedgehog (Shh) signaling plays a critical role during dorsoventral (DV) patterning of the developing neural tube by modulating the expression of neural patterning genes. Overlapping activator functions of Gli2 and Gli3 have been shown to be required for motoneuron development and correct neural patterning in the ventral spinal cord. However, the role of Gli2 and Gli3 in ventral hindbrain development is unclear. In this paper, we have examined DV patterning of the hindbrain of Shh−/−, Gli2−/− and Gli3−/− embryos, and found that the respective role of Gli2 and Gli3 is not only different between the hindbrain and spinal cord, but also at distinct rostrocaudal levels of the hindbrain. Remarkably, the anterior hindbrain of Gli2−/− embryos displays ventral patterning defects as severe as those observed in Shh−/− embryos suggesting that, unlike in the spinal cord and posterior hindbrain, Gli3 cannot compensate for the loss of Gli2 activator function in Shh-dependent ventral patterning of the anterior hindbrain. Loss of Gli3 also results in a distinct patterning defect in the anterior hindbrain, including dorsal expansion of Nkx6.1 expression. Furthermore, we demonstrate that ventral patterning of rhombomere 4 is less affected by loss of Gli2 function revealing a different requirement for Gli proteins in this rhombomere. Taken together, these observations indicate that Gli2 and Gli3 perform rhombomere-specific function during DV patterning of the hindbrain. © 2006 Elsevier Inc. All rights reserved.

Keywords: Sonic hedgehog; Gli transcription factors; Neural tube; Hindbrain; Dorsoventral patterning; Rhombomere; Mouse

Introduction Pax6 and Irx3, are negatively regulated by Shh and their expression is excluded from the ventral parts of the neural tube. During development of the vertebrate nervous system, On the other hand, the class II patterning genes, including neural progenitors are specified according to their positions Nkx6.1 and Olig2, are positively regulated by Shh and are along the antero-posterior (AP) and dorsoventral (DV) axes of expressed in the ventral region of the neural tube. It was also the neural tube. The DV axis of the neural tube is established by suggested that pairs of class I and class II genes repress each dorsally derived signals, such as Bone Morphogenetic Proteins other, leading to sharp expression borders. The profile of (BMPs), and ventrally derived signals, including Sonic hedge- expression of these patterning genes forms a combinatorial hog (Shh). In the ventral neural tube, Shh signal modulates the code that leads to the specification of distinct progenitor expression of several patterning genes (Figs. 1A, B). The domains within the ventral neural tube. For example, Pax6+, current model suggests that the class I patterning genes, such as Nkx6.1+, Olig2+ and Irx3− neural progenitors will become motoneurons while Pax6+, Nkx6.1+, Olig2− and Irx3+ ⁎ Corresponding author. Program in Developmental Biology, The Hospital for progenitor cells will form V2 interneurons (reviewed in Wilson Sick Children, Room 13-314, Toronto Medical Discovery Tower, 101 College and Maden, 2005). Consistent with this model, Shh−/− neural Street, Toronto, Ontario, Canada M5G 1L7. Fax: +416 813 5252. E-mail address: [email protected] (C. Hui). tube showed no expression of class II patterning genes, which 1 Current address: MRC-National Institute for Medical Research, The results in a complete absence of motoneurons as well as V2 and Ridgeway, Mill Hill, London, NW7 1AA, U.K. V3 interneurons (Chiang et al., 1996).

In Drosophila, Hedgehog (Hh) signal is transduced by a most V3 interneurons, two structures requiring the highest Shh single bi-functional Gli transcription factor, Cubitus interruptus response for their specification (Ding et al., 1998; Matise et al., (Ci) (Méthot and Basler, 2001). In the mouse, Hh signaling is 1998). Gli3−/− mice do not exhibit defect in the ventral spinal mediated by the combinatorial activity of three Gli transcription cord but its repressor function is required for the correct factors, Gli1, Gli2 and Gli3. Biochemical studies have shown specification of the ventro-lateral progenitor domains (p0, p1 as that Gli2 and Gli3 each contain both activation and repression well as progenitors of dl4 to dl6 dorsal interneurons) (Persson et domains, although Gli2 is a stronger activator than repressor al., 2002; reviewed in Jacob and Briscoe, 2003). Mutant analysis while Gli3 is a stronger repressor than activator (Sasaki et al., of compound mutant mice has revealed a functional redundancy 1999). In contrast, Gli1 contains only activation domains and between Gli2 and Gli3 in the patterning of the ventral spinal appears to be exclusively a transcriptional activator (Dai et al., cord. Overlapping functions of Gli2 and Gli3 are required for 1999; Ruiz i Altaba, 1999). Genetic analysis of Gli mutant mice the correct organization of the ventral spinal cord as well as the showed that Gli1 does not play a primary role in Shh signaling formation of motoneurons (Motoyama et al., 2003; Bai et al., during development (Park et al., 2000). Gli2 plays an activator 2004; Lei et al., 2004). Furthermore, the knock in of Gli3 into the role in the ventral spinal cord; Gli2−/− mice lack a floor plate and Gli2 locus produced a substantial, though incomplete, rescue of the Gli2−/− phenotype (Bai et al., 2004). Together, these experiments established that Gli3 could function as an activator in ventral neural tube patterning when Gli2 function is eliminated. On the other hand, removal of Gli3 rescues the specification of several ventral neural cell types in Shh−/− mice, indicating that a critical function of Shh is to suppress Gli3 repressor function in the ventral neural tube. In Shh−/− mice, the repressor form of Gli3 is not restricted anymore to the dorsal neural tube and is present in the ventral neural tube, which suppresses the formation of ventral cell types (Litingtung and Chiang, 2000; Wijgerde et al., 2002). Shh signal is required for the patterning of the ventral neural tube at all rostrocaudal levels, from forebrain to spinal cord (Ericson et al., 1995a,b). Genes involved in the patterning of ventral spinal cord, such as Nkx2.2, Nkx6.1 and Pax6, are known to play equally important functions in the patterning of the ventral hindbrain. In the hindbrain, the p3 progenitor domain gives rise to several types of neurons, among them visceral and branchial motoneurons as well as serotonergic neurons. Nkx2.2 is required for the specification of these neurons (Briscoe et al., 1999; Pattyn et al., 2003a,b, 2004) as well as the specification of V3 interneurons in the spinal cord (Briscoe et al., 1999). Similarly, Nkx6.1, Pax6 and Olig2 are involved in the specification of somatic motoneurons in the hindbrain (Ericson et al., 1997; Osumi et al., 1997; Pattyn et al., 2003a; Takahashi and Osumi, 2002) and in the spinal cord (Ericson et al., 1997; Lu et al., 2002; Sander et al., 2000; Takebayashi et al., 2002; Zhou and Anderson,

Fig. 1. The expression of patterning genes is controlled by Shh through Gli2 and Gli3 transcription factors. (A) Expression of class I (Pax7, Dbx2, Irx3, Pax6 and Dbx1) and class II (Nkx6.1, Olig2, Nkx2.2 and Nkx6.2) patterning genes in the neural tube forms a combinatorial code leading to the specification of progenitor domains in the ventral neural tube. pMN, progenitor domain of motoneurons; p0 to p3: progenitor domains of V0 to V3 ventral interneurons. (B) Shh modulates the expression of class II genes by either directly activating the expression of class II genes in the ventral neural tube trough Gli2 activator (Gli2A) and Gli3 activator (Gli3A) function or by inhibiting the repressor function of Gli3 and Gli2 (Gli3R, Gli2R) in the ventral neural tube, thereby allowing the expression of class II genes. Mutual repression between class I and class II genes limits the expression of class I genes to the medial part of the neural tube. (C) Patterning genes and terminal markers examined in this study. Neural progenitors located in the ventricular zone express different combination of patterning genes, as illustrated at the left. After the progenitors exit the cell cycle, they migrate to the mantle zone and express specific terminal marker, as illustrated at the right. Olig2 and Phox2b play specific role in specification of the somatic and visceral motoneurons in the ventral spinal cord and hindbrain. M. Lebel et al. / Developmental Biology 302 (2007) 345–355 347

2002). Furthermore, other genes involved in the patterning of Vector) were used to amplify the signal. Signal was visualized using VIP substrate ventral spinal cord, including Nkx6.2, Dbx1, Dbx2 and Irx3,are kit (Vector) and Red substrate kit for alkaline phosphatase (Vector). also expressed in a similar pattern in the hindbrain (Takahashi and Osumi, 2002). These observations suggest that similar regulation Results of these patterning genes by Shh is involved in the specification of ventral progenitors in the spinal cord and hindbrain. Motoneurons are specified through different mechanisms in the It is generally thought that the respective functions of Gli2 spinal cord and in the hindbrain and Gli3 in Shh signaling are conserved along the entire neural tube. However, studies of Ptc1;Gli mutant mice re- It was previously proposed that motoneurons were specified vealed that the mechanism may be different in the hindbrain. in the ventral neural tube by a mechanism involving Shh and Ptc1−/−;Gli2−/− mice exhibit severe ventralization of the Gli3 (Litingtung and Chiang, 2000). Gli3, whose expression is hindbrain, but not the spinal cord. This suggests that although modulated by Shh signal, restricts the expression of the class II Gli2 plays a critical role in Shh signal transduction in both the gene Olig2 to the motoneuron progenitor domain. Olig2 also spinal cord and the hindbrain, Gli3 can function as a strong restricts the expression of the class I gene Irx3 to the interneuron activator in the hindbrain and fulfill most of Gli2 activator progenitor domains dorsal to the motoneuron progenitor domain function when the pathway is overtly activated (Motoyama et (Litingtung and Chiang, 2000; Briscoe et al., 2000). To al., 2003). In this study, we examined the functions of Gli2 determine whether this model also describes the specification and Gli3 in the DV patterning of the hindbrain. Strikingly, we of motoneuron progenitor domain in the ventral hindbrain, we showed that Gli2−/− and Gli3−/− hindbrains exhibit distinct examined the development of motoneurons in single and ventral patterning defects than Gli2−/− and Gli3−/− spinal compound mutants of Gli2, Gli3 and Shh. The expression of cords. Our results revealed that Gli2 and Gli3 play distinct Olig2 and Irx3 as well as Isl1/2 was examined at different levels roles in Shh signal transduction in the developing hindbrain of the spinal cord as well as in rhombomere (r) 5. While Olig2 through differential modulation of patterning gene expression labels the motoneuron progenitor domain, Isl1/2 antibody marks in a rhombomere-specific manner. all subtypes of motoneurons (Varela-Echavarria et al., 1996). Irx3 is expressed in the V2 interneuron progenitor domain and Materials and methods extends dorsally in the neural tube (Briscoe et al., 2000)(Fig. 1C). No major differences were found between the expression of Embryos harvesting and histology these genes at the forelimb, trunk and hindlimb levels, thus results for only one level (hindlimb level) were shown. We chose −/− −/− −/− Shh , Gli2 and Gli3 mutant mice have been described previously to examine r5 because it is the most anterior location in the (Chiang et al., 1996; Ding et al., 1998; Matise et al., 1998; Hui and Joyner, 1993) mouse neural tube where Olig2 expression (in somatic and maintained in a CD-1 background. Mutant embryos were generated by heterozygote intercrosses, and were fixed in 4% PFA in PBS, overnight at 4°C motoneurons) is found (Takahashi and Osumi, 2002). In WT with agitation. Embryos harvested for whole-mount in situ hybridization spinal cord, the Olig2 protein was restricted to a broad stripe in − − − − (WISH) were dehydrated and kept in 100% methanol at −20°C for a maximum the ventral neural tube, but was absent from Shh / and Gli2 / ; of few weeks. For sectioning, embryos were dehydrated through a series of Gli3−/− embryos (Figs. 2A, B, D). Interestingly, our observa- μ ethanol and xylene washes, and then embedded in paraffin wax. 7 m sections tions contrast with previous reports of Olig2 expression in the were prepared using microtome and then dried at 57°C overnight. Gli2−/−;Gli3−/− neural tube (Bai et al., 2004; Lei et al., 2004). In situ hybridization The cause of this discrepancy remains unclear but mutant mice used in this study are maintained in a different genetic Plasmids for generating the riboprobes were: Shh (Echelard et al., 1993), background. Consistent with the current model, Olig2 expres- Gli1, Gli2, Gli3 (Hui et al., 1994), Dbx1 (Shoji et al., 1996), Nkx6.1 (Cai et al., sion was expanded ventrally in Gli2−/− spinal cords and was 2000), Nkx2.2 (Hartigan and Rubenstein, 1996), Olig2 (Takebayashi et al., rescued in Shh−/−;Gli3−/− spinal cords (Figs. 2C, E). Irx3 2000), Pax2 (Dressler et al., 1990), Ngn2 (Fode et al., 1998), Hoxb1 (Popperl et al., 1995), Hoxd4 (Folberg et al., 1997), Phox2b (Pattyn et al., 1997) and Isl1 expression domain was located immediately dorsal to the Olig2 (Gong et al., 1995). Paraffin sections or whole-mount embryos were subjected to domain and expanded well into the dorsal neural tube (Fig. 2K). in situ hybridization with digoxigenin-dUTP labeled riboprobes as described Irx3 expression in the spinal cord was complementary to Olig2 previously (Ding et al., 1998). For flat mounts, WISH was performed first, then expression in all mutants examined. It was expanded ventrally the hindbrain was dissected out in glycerol and flatten open under a coverslip. and spanned the midline in Shh−/− and Gli2−/−;Gli3−/− embryos For each marker, a minimum of three embryos of each genotype was analyzed. −/− −/− −/− Similar results were obtained for all embryos analyzed and a representative (Figs. 2L, N), while Gli2 and Shh ;Gli3 embryos embryo is shown. displayed normal Irx3 expression in their ventral neural tube (Figs. 2M, O). Isl1/2 staining revealed that motoneurons are − − − − − − Immunohistochemistry absent in Shh / and Gli2 / ;Gli3 / mutant spinal cord s(Figs. 2V, X). Motoneurons were detected in the ventral spinal cord of Primary antibodies used were: Mouse Nkx2.2 (Developmental Research Gli2−/− and Shh−/−;Gli3−/− mice, although they were shifted Hybridoma Bank), Rabbit Olig2 (kindly provided by M. Nakafuku; Takebayashi et ventrally and reduced in number in some embryos (Figs. 2W, Y). al., 2000), Rabbit Irx3 (Kobayashi et al., 2002) and Mouse Isl1/2 (Developmental Research Hybridoma Bank). Secondary antibodies were: anti-mouse-biotin The hindbrain expression of Olig2, Irx3 and Isl1/2 in WT, −/− −/− −/− (Vector) and anti-rabbit-biotin (Vector). Vectastain ABC-HRP standard kit (for Shh and Gli2 ;Gli3 embryos was similar to those Nkx2.2 and Isl1/2) and Vectastain ABC-AP standard kit (for Olig2) (both kits from observed in the spinal cord (Figs. 2F, G, I, P, Q, S, Z, AA, CC). 348 M. Lebel et al. / Developmental Biology 302 (2007) 345–355

Fig. 2. Motoneurons are specified through different mechanisms in the spinal cord and in the hindbrain. Immunostaining of transverse sections with specific antibodies was performed to detect the expression of Olig2 (A–J), Irx3 (K–T) and Isl1/2 (U–DD) in E10.5 embryos. Both the spinal cord and the hindbrain (r5) were examined in WT, Shh−/−, Gli2−/−, Gli2−/−;Gli3−/− and Shh−/−;Gli3−/− embryos as indicated.

However, the phenotype observed in Gli2−/− and Shh−/−;Gli3−/− cord and in the hindbrain has different requirement for Gli2 and hindbrains was clearly different from that in the spinal cord. No or Gli3. In the spinal cord, Olig2 expression appeared to be re- very few Olig2-positive cells and Isl1/2-positive cells could be pressed by Gli3 in the absence of Shh signal, and was restored detected in these mutants (Figs. 2H, J, BB, DD). Irx3 expression when Gli3 was removed in the Shh−/− background. Some Gli was ventrally expanded and spanned the midline in the hindbrain activity is nevertheless required for Olig2 expression, as it was not of both Gli2−/− and Shh−/−;Gli3−/− embryos (Figs. 2R, T). These expressed in Gli2−/−;Gli3−/− neural tubes. However, Olig2 was results indicate that the specification of motoneurons in the spinal expressed in Gli2−/− spinal cords, suggesting that Gli2 and Gli3

Fig. 3. Expression of neural patterning genes requires both Gli activator and repressor functions. Immunostaining (A–J) and RNA in situ hybridization (K–DD) were performed on transverse sections using specific antibodies/RNA probes to reveal the expression of Nkx2.2 (A–J), Nkx6.1 (K–T) and Dbx1 (U–DD) in E10.5 embryos. Both the spinal cord and hindbrain (r5) were examined in WT, Shh−/−, Gli2−/−, Gli2−/−;Gli3−/− and Shh−/−;Gli3−/− embryos, as indicated. M. Lebel et al. / Developmental Biology 302 (2007) 345–355 349 have redundant function in the ventral spinal cord. A different and hindbrain. In both mutants, expression of Dbx1 and Nkx6.1 scenario is observed in the hindbrain, where Olig2 expression was normal in the spinal cord (Figs. 3M, O, W, Y). In contrast, could not be detected in Shh−/−, Gli2−/−;Gli3−/−, Shh−/−;Gli3−/− similar to Gli2−/−;Gli3−/− embryos, the expression domain of or Gli2−/− embryos indicating that both Shh signal and Gli2 both Nkx6.1 and Dbx1 was slightly reduced in size and shifted function are essential for the expression of Olig2 in the hindbrain ventrally in r5 of Gli2−/− and Shh−/−;Gli3−/− embryos (Figs. and that Gli3 cannot compensate for the loss of Gli2. 3R, T, BB, DD compared to Figs. 3P, S, Z, CC). Together, these observations further suggest a stronger contribution for Gli2 The expression of patterning genes is regulated by different and Shh signal in the ventral patterning of r5 than in the spinal mechanisms requiring both Gli2 and Gli3 cord.

To investigate whether the expression of other patterning genes The expression of patterning genes is regulated by distinct is modulated by Gli2 and Gli3 through different mechanisms in mechanisms at different levels of the hindbrain the spinal cord and hindbrain, the expression of class II patterning genes (Nkx2.2 and Nkx6.1) and class I patterning gene (Dbx1), To better visualize the hindbrain phenotype of Gli2−/− and which encompasses nearly the entire dorsoventral axis of the Gli3−/− embryos and to extend our observation to other neural tube (Fig. 1C), was examined in WT and various mutant rhombomeres, we performed flat mount (FM) RNA in situ embryos. Nkx2.2 is expressed in the ventral most region of the hybridization to detect the expression of patterning genes. This neural tube and is essential for the specification of V3 technique was also used to confirm that Shh and Gli genes were interneurons (Briscoe et al., 1999) as well as other ventral neurons in the hindbrain (Varela-Echavarria et al., 1996). Nkx2.2 expression could not be detected in either the spinal cord or r5 of Shh−/−, Gli2−/−, Gli2−/−;Gli3−/− and Shh−/−;Gli3−/− embryos (Figs. 3A–J). Thus, unlike Olig2, Nkx2.2 expression requires Shh signal and Gli2 function in both r5 and the spinal cord. In WT spinal cord and hindbrain, the domain of Nkx6.1 expression encompasses the progenitor domains of V3 and V2 interneurons as well as motoneurons (Briscoe et al., 2000; Sander et al., 2000). Typical of a class II gene, Nkx6.1 expression was absent in Shh−/− spinal cords and hindbrains (Figs. 3K, L, P, Q). In the absence of Gli3, Shh signal is nevertheless dispensable for Nkx6.1 expression since expression of Nkx6.1 was restored in Shh−/−;Gli3−/− embryos (Figs. 3O, T). Nkx6.1 expression could be found in the neural tube of Gli2−/−;Gli3−/− embryos, although its domain was reduced (Figs. 3N, S). We reasoned that this reduction results from the loss of more ventral progenitor cells (that also express Nkx6.1)inGli2−/−;Gli3−/− embryos. Nkx6.1 expression was also highly disorganized in some mutants (see Supplementary Fig. 1), suggesting that although Gli function is dispensable for the expression of Nkx6.1,itis required for the correct positioning of Nkx6.1 expression domain in the ventral neural tube. Dbx1 is a class I patterning gene, which is repressed by Shh signaling in the ventral neural tube (Briscoe et al., 2000; Pierani et al., 1999, 2001). Its expression is ventrally shifted in the Shh−/− spinal cord, while it is absent from the hindbrain and the anterior spinal cord (Figs. 3U, V, Z, AA) (see below). This discrepancy likely originates from a partial rescue by Indian hedgehog (Ihh), which is secreted from the primitive gut (Wijgerde et al., 2002). Dbx1 expression is present in the spinal cord and r5 of all other mutants examined; Gli2−/−, Gli2−/−;Gli3−/− and Shh−/−;Gli3−/− (Figs. 3W–Y, BB –DD). As for Nkx6.1, Dbx1 expression nevertheless appears slightly more ventral and disorganized in Fig. 4. Expression of neural patterning genes is regulated by distinct mechanisms the spinal cord of some Gli2−/−;Gli3−/− embryos (Supplementary at different levels of the hindbrain. Whole-mount in situ hybridization of E10.5 – −/− – Fig. 1). WT (A C) and Shh embryos (D F) was performed using specific RNA probes for Nkx2.2 (A, D), Nkx6.1 (B, E) and Dbx1 (C, F). Arrow indicates Despite the fact that Nkx6.1 and Dbx1 are expressed in both −/− −/− −/− −/− −/− rhombomere 7 (r7). Flat mounts of E10.5 WT (G, K, O), Shh (H, L, P), Gli2 spinal cord and r5 of Gli2 and Shh ;Gli3 embryos, (I, M, Q) and Gli3−/− (J, N, R) hybridized with specific probes for Nkx2.2 (G–J), subtle differences could be identified between the spinal cord Nkx6.1 (K–N) and Dbx1 (O–R). r1–r7, rhombomere 1–rhombomere 7. 350 M. Lebel et al. / Developmental Biology 302 (2007) 345–355 expressed uniformly at all AP levels of the hindbrain and that detected by immunohistochemistry in the ventral hindbrain of Gli2 and Gli3 expression was not altered in the hindbrain of WT and Gli3−/− embryos (Supplementary Figs. 3A, D). There Gli3−/− and Gli2−/− mice, respectively (Supplementary Fig. 2 was no difference in Dbx1 expression between the different and data not shown). rhombomeres of Gli3−/− hindbrains (Fig. 4R). In contrast, the We first performed WISH on WT and Shh−/− E10.5 expression of Nkx6.1 was different in Gli3−/− hindbrains embryos using Nkx2.2, Nkx6.1 and Dbx1 RNA probes. As when compared with WT. In Gli3−/− embryos, Nkx6.1 expected, no expression of Nkx2.2 could be detected in Shh−/− expression was stronger at r1, r2 and r3, as well as dorsally embryos while Dbx1 expression could be detected only in the expanded at r1, while it is expressed at normal levels in the posterior spinal cord, along the primitive gut (Figs. 4A, D, C, other rhombomeres (Fig. 4N). F, G, H, O, P). Strikingly, Nkx6.1 expression could be In summary, the misexpression of these patterning genes in detected in the posterior hindbrain of Shh−/− embryos, while it Gli2−/− and Gli3−/− hindbrains can be generalized as follow. was absent from all other locations along the neural tube (Figs. First, the Gli2−/− hindbrain shows a more severe phenotype 4B, E). FM analysis in Shh−/− embryos identified Nkx6.1 in the anterior hindbrain than in the posterior hindbrain. expression at r7 (Fig. 4L). However, Nkx6.1 expression could Second, Nkx6.1 expression is elevated in the anterior hind- be detected only in the mantle layer of the neural tube, where brain of Gli3−/− embryos when compared to the posterior post-mitotic cells lie, but not in the ventricular zone, where hindbrain and spinal cord. Third, the expression of Nkx6.1 neural progenitors are located and where Nkx6.1 is also and Nkx2.2 is less affected in Gli2−/− r4 than in the neighbor expressed in WT embryos (Supplementary Figs. 3P, Q). Aside rhombomeres. for the r7 expression of Nkx6.1, our results indicate that Shh To further characterize the hindbrain defects, we examined signal is required for the expression of Nkx2.2, Nkx6.1 and the expression of Ngn2, a pro-neural gene expressed in ventral Dbx1 in the hindbrain. Another rhombomere-specific pheno- interneurons of the hindbrain (ventral domain) and in a subset of type was observed in Gli2−/− hindbrains, where Nkx2.2 dorsal interneurons (dorsal domain) (Davenne et al., 1999), and expression is selectively retained in r4, in addition of traces Pax2, a neuronal marker expressed in dl6, V0 and V1 of expression at r7 (Figs. 4G, I). interneurons (ventral domain) and in dl4 interneurons (dorsal There was also a striking difference between the expression domain) (Dressler et al., 1990)(Fig. 1C). In Shh−/− embryos, of Nkx6.1 and Dbx1 at different levels of Gli2−/− hindbrains. the ventral expression domain of Pax2 and Ngn2 was absent at Both genes were expressed normally at r7 and in the spinal cord, all levels of the spinal cord and hindbrain, except for the r7 but in the hindbrain segments anterior to r7, their expression expression of Pax2, which is found in the mantle layer of the domain showed a gradual ventral shift. In the case of Nkx6.1, neural tube similar to Nkx6.1 expression (Figs. 5A–D, E–F, I–J very little or no expression could be detected in r1, r2 and r3 of and Supplementary Figs. 3L, Q). Since Pax2 and Nkx6.1 Gli2−/− embryos (Figs. 4K, M). Dbx1 expression spanned the expression is restricted to post-mitotic cells at r7 of Shh−/− midline at r1 and r2, although its expression was always present embryos, we reasoned that their expression in this location is in Gli2−/− hindbrains (Figs. 4O, Q). It is also interesting to note regulated independently of Shh signaling. The dorsal expres- that Nkx6.1 expression was less affected in r4 than in the sion domain of Pax2 and Ngn2 was shifted ventrally along the neighboring rhombomeres (Fig. 4M). hindbrain midline of Shh−/− embryos. In Gli2−/− hindbrains, the Nkx2.2 expression was normal in Gli3−/− hindbrains, ventral expression domain of both Pax2 and Ngn2 showed a although the AP difference in signal intensity appeared gradual ventral shift along the AP axis of the neural tube (Figs. slightly more accentuated in Gli3−/− embryos than in WT 5E, G, I, K). Pax2 expression spanned the midline at r1 and its embryos. However, similar amount of Nkx2.2 protein was ventral expression was generally weaker. Ngn2 expression

Fig. 5. Expression of neurogenic genes and neural differentiation markers has distinct requirement for Gli2 and Gli3 at different levels of the hindbrain. Whole-mount in situ hybridization of E10.5 WT (A–B) and Shh−/− (C–D) embryos was performed using specific RNA probes for Pax2 (A, C) and Ngn2 (B, D). Flat mounts of E10.5 WT (E, I), Shh−/− (F, J), Gli2−/− (G, K) and Gli3−/− (H, L) hindbrains hybridized with specific probes for Pax2 (E–H) and Ngn2 (I–L). Note that, in WT embryos, the dorsal expression domain of Ngn2 is substantially broader in r6–r7 than in r3–r5 and, in Fig. 5I, the sample was truncated on the sides. M. Lebel et al. / Developmental Biology 302 (2007) 345–355 351 spanned the midline of r2 and r3 and could not be detected in the anterior part of r2 and at r1. However, Ngn2 expression was less affected in r4 than in the surrounding rhombomeres. Finally, the ventral expression domain of both Ngn2 and Pax2 was affected in the anterior hindbrain of Gli3−/− embryos (Figs. 5H, L). Their expression level was increased at r1, r2 and r3 as well as expanded dorsally at r1. Pax2 expression could also be detected at the midline of r4. Together, these results indicate that the expression of Pax2 and Ngn2 is controlled by similar mechanisms as the expression of Dbx1 and Nkx6.1 at a given location of the hindbrain. For these four genes, Gli2 appears to play a more important role in their expression in the anterior hindbrain.

All subtypes of motoneurons are differentially regulated along the AP axis of the hindbrain

Finally, we examined how the development of hindbrain motoneurons was affected by loss of Gli2 or Gli3 function by monitoring the expression of Olig2, Phox2b and Isl1 using FM in situ hybridization. Olig2 is normally expressed in the progenitors of somatic motoneurons at r5 and r7 as well as in the spinal cord. Around E10.5, Olig2 is expressed along the entire hindbrain and spinal cord, in what will become the oligodendrocyte progenitor domain (Mizuguchi et al., 2001; Novitch et al., 2001; Sun et al., 2001). Phox2b is expressed in the progenitors of visceral and branchial motoneurons in the entire hindbrain while Isl1 is a terminal marker expressed in all subtypes of motoneurons (Fig. 1C). In E10.5 WT embryos, the expression of Olig2 was mostly restricted to r5, r7 and to the spinal cord but lower levels of expression could already be detected in the oligodendrocyte precursors at r4 and r6. In Gli2−/− hindbrains, Olig2 expression was slightly more ventral at r7 but was eliminated in the anterior part of r5 (Fig. 6B). Fig. 6. All subtypes of motoneurons are differentially regulated along the antero-posterior axis of the hindbrain. Flat mounts of E10.5 WT (A, D, G), The ventral expression domain of Phox2b (which marks the Gli2−/− (B, E, H) and Gli3−/− (C, F, I) hindbrains hybridized with specific progenitors of visceral and branchial motoneurons) was also probes for Olig2 (A–C), Phox2b (D–F) and Isl1 (G–I). Red arrows indicate − − shifted ventrally in the posterior hindbrain of Gli2 / embryos the anterior end of Phox2b expression in the ventral neural tube. Flat mounts of − − − − − − and was lost in r5 as well (red arrow in Fig. 6E). In contrast, E10.5 WT (J, N), Gli2 / (K, O) Gli3 / (L, P) and Shh / (M, Q) hindbrains – – Phox2b expression was not reduced in r4. Similarly, Isl1 hybridized with specific probes for Hoxb1 (J M) and Hoxd4 (N Q). expression is ventrally shifted in the posterior hindbrain of Gli2−/− embryos and strongly reduced in r5 (Fig. 6H). Like the It was previously shown that the subtypes of motoneurons expression of Phox2b, Isl1 expression was not diminished in present in each rhombomere are strongly influenced by the r4. No Isl1 or Phox2b expression could be detected in r3, expression of Hox genes (Jungbluth et al., 1999; Bell et al., while only a very faint signal could be detected at the midline 1999). To test whether the loss of motoneurons in Gli2−/− of r2. These results indicate that motoneuron development was hindbrains results from Hox gene misexpression, we examined not severely disturbed in the Gli2−/− spinal cord and r7, but the expression of Hoxb1 and Hoxd4 in the hindbrain of E9.5 that motoneuron progenitors were lost in more anterior (data not shown) and E10.5 WT, Gli2−/−, Gli3−/− and Shh−/− rhombomeres. All motoneurons were affected in Gli2−/− embryos. Hoxb1, which plays a major role in the specification embryos, with no subtype specificity. It is also interesting of r4 and the neurons within (Gaufo et al., 2000; Studer et al., that the size reduction of the motoneuron progenitor field 1996; Goddard et al., 1996), was expressed in r4 of WT appears to form a gradient along the antero-posterior axis of embryos as well as in all the mutants examined (Figs. 6J–M). the hindbrain; extensive defects are observed in the anterior Hoxd4 marks the entire spinal cord as well as r7 and is hindbrain while the posterior hindbrain is less affected, with therefore a good indicator of the integrity of the r6/r7 barrier in the exception of r4. In contrast, the expression of Olig2, the developing hindbrain (Folberg et al., 1997). As expected, Phox2b and Isl1 was not significantly affected in the Gli3−/− Hoxd4 was expressed in the spinal cord and r7 of WT and all hindbrain, except for a slight increase of Isl1/2 expression mutants examined, with its characteristic sharp border between domain in r4 (Figs. 6C, F, I). r6 and r7 (Figs. 6N–Q). These observations suggest that the 352 M. Lebel et al. / Developmental Biology 302 (2007) 345–355 hindbrain phenotype of Gli2−/− and Gli3−/− embryos was not cord, Shh-dependent Gli2 activator function is indispensable caused by a remodeling of rhombomere identity, but likely due for these processes in r5. In the Gli2−/− spinal cord, only the to lack of rhombomere-specific function of Gli2 and Gli3 most ventral cell types (floor plate and V3 interneurons) are during hindbrain DV patterning. missing (Ding et al., 1998; Matise et al., 1998). This is a relatively mild phenotype, as Gli2 is the major transcriptional Discussion activator of the Shh pathway and Gli activator function is thought to be required for the expression of all class II genes in In this study, we examined the specific role of Gli2 and Gli3 the ventral neural tube. Analysis of compound mutant mice in patterning of the ventral hindbrain. First, we demonstrated indicates that Gli3 can compensate for the loss of Gli2 and that Gli2−/− embryos exhibit a more severe ventral patterning fulfill some of its functions in ventral neural patterning defect in the hindbrain than those observed in the spinal cord. (Motoyama et al., 2003; Bai et al., 2004; Lei et al., 2004). Thus, unlike in the spinal cord, Gli2 performs some unique We show here that the expression of neural patterning genes is functions in the hindbrain that cannot be substituted for by Gli3. severely altered in the anterior hindbrain of Gli2−/− embryos. Second, both Gli2−/− and Gli3−/− embryos display rhombo- Similar to those observed in Shh−/− embryos, the expression of mere-specific ventral patterning defects (summarized in Fig. 7). all class II patterning genes is lost in the anterior hindbrain of In the anterior hindbrain of Gli2−/− embryos, the expression of Gli2−/− embryos suggesting that, unlike in the spinal cord, Nkx2.2 and Nkx6.1 is completely lost. In contrast, Gli3−/− Gli3 cannot compensate for the loss of Gli2 function. embryos show normal Nkx2.2 expression but expanded Nkx6.1 Interestingly, Gli2 appears to act as the sole Gli activator in expression in the anterior hindbrain. Among all rhombomeres, this region. Consistent with this hypothesis, the anterior ventral patterning of r4 appears to be less affected by loss of hindbrain (r1–r3) of Gli2−/−;Gli3−/− embryos show similar Gli2 function. Together, these observations unveiled a differ- ventral defects as those observed in Gli2−/− embryos ential role for Gli2 and Gli3 in Shh-dependent ventral patterning (Supplementary Figs. 3P–S, X–Y). In contrast, the posterior at different AP levels along the hindbrain. hindbrain (r5–r7) of Gli2−/−;Gli3−/− embryos display a more The analysis of Gli2−/− and Shh−/−;Gli3−/− embryos severe ventral phenotype than that of Gli2−/− embryos uncovered a major difference in Shh-dependent ventral indicating that, as in the spinal cord, Gli2 and Gli3 do share patterning between the hindbrain (r5) and spinal cord; although overlapping activator functions in the posterior hindbrain. inactivation of Gli3 repressor alone by Shh is sufficient for It was previously shown that Gli3 modulates the expression Olig2 expression and motoneuron differentiation in the spinal of a subset of patterning genes in the medial neural tube

Fig. 7. Summary of neural patterning and differentiation genes in E10.5 Gli2−/− and Gli3−/− hindbrains. Expression of class II genes that are normally expressed in the ventral most region of the neural tube, such as Nkx2.2 (A), is lost at all levels of Gli2−/− hindbrains with the exception of r4, while its expression domain remains normal in Gli3−/− hindbrains. The motoneuron domain, as identified by the expression of the terminal marker Isl1 (B) as well as those of Olig2 and Phox2b (not depicted), is slightly reduced in the spinal cord of Gli2−/− embryos but is further reduced in r5 and r6, and is absent from r1, r2 and r3. In contrast, like in the spinal cord, the motoneuron domain is only slightly affected in r4. No significant change in this domain could be observed in Gli3−/− hindbrains. (C) Patterning genes whose expression extends to the ventro-medial part of the neural tube, such as Nkx6.1, also have a slightly reduced expression domain in the Gli2−/− spinal cord. Similar phenotype was observed in the Ngn2 and Pax2 ventral expression domain. A further reduction of their expression domain is observed in the Gli2−/− hindbrains (r6, r5 and r3), and no significant expression remains in r1 and r2 in Gli2−/− embryos. In contrast, the expression of these genes was minimally affected in Gli2−/− r4. In Gli3−/− hindbrains, the expression domain of these genes was expanded dorsally and their expression level was increased. (D) Medially expressed class I patterning genes (such as Dbx1) are expressed normally in the spinal cord of Gli2−/− embryos, but their expression is shifted ventrally in r3 to r6, the more severe phenotype being observed in r3. In r1 and r2, the expression of these genes is restricted at the midline of the neural tube. In the Gli3−/− neural tube, Dbx1 expression was slightly expanded dorsally (as reported previously), but no significant difference could be detected between its expression in the spinal cord and in the different rhombomeres. M. Lebel et al. / Developmental Biology 302 (2007) 345–355 353

(Persson et al., 2002). However, the phenotype observed in the between these species. Study of zebrafish Gli mutants and anterior hindbrain of Gli3−/− embryos is noticeably different, as morphants revealed that Gli1 acts as an activator whereas loss of Gli3 function affects the expression of other patterning Gli2 and Gli3 function as bi-functional effectors of hedgehog and differentiation genes, including Nkx6.1, Pax2 and Ngn2. signaling during neural tube development (Vanderlaan et al., For example, Nkx6.1 expression is expanded in the anterior 2005; Tyurina et al., 2005). The removal of Gli1 in zebrafish hindbrain, but is unaffected in the posterior hindbrain and spinal results in the elimination of Nk2.2 expression (which marks cord of Gli3−/− embryos. In contrast, Dbx1 expression is the entire floor plate) in both the spinal cord and hindbrain. In expanded dorsally in the Gli3−/− spinal cord (Persson et al., contrast, motoneurons are absent from the hindbrain but are 2002), but is not further altered in the Gli3−/− anterior hindbrain intact in the spinal cord of these mutants (Karlstrom et al., (this study). The expression of Fgf8 is up-regulated in the mid/ 2003; Chandrasekhar et al., 1999). In you-too (yot) mutants, hindbrain boundary of Gli3−/− embryos (Aoto et al., 2002) and which encode a dominant repressor form of Gli2, the it has been recently shown that this mid/hindbrain boundary expression of Nk2.2 is eliminated in the entire spinal cord expression of Fgf8 is mediated by Shh-dependent inhibition of and hindbrain with the exception of r4. Formation of Gli3 repressor activity (Blaess et al., 2006). It is possible that motoneurons also appears to be less affected in yot r4 than elevated Fgf8 signals emanating from the mid/hindbrain at other levels of the spinal cord, as determined by the boundary of Gli3−/− embryos could modulate the expression expression of the terminal marker Isl1 (Vanderlaan et al., of neural patterning genes in the anterior hindbrain. Consistent 2005). These observations indicate that the differential with this hypothesis, Pax2 expression, which is known to be requirement for Gli activities in the hindbrain is not restricted modulated by Fgf8 during chick development (Shamim et al., to the mouse but extends to other species. However, none of 1999), is up-regulated in the Gli3−/− anterior hindbrain. In the zebrafish Gli mutants or morphants analyzed so far addition to the Fgf signals from the mid/hindbrain boundary, displays a stronger phenotype in the anterior hindbrain than in other signaling pathways might also be involved in modifying the posterior hindbrain comparable to the hindbrain pheno- Gli functions along the AP axis of the developing neural tube. type of Gli2−/− and Gli3−/− mice. It remains to be established One possible candidate is retinoic acid, which is a posterior whether there is also a rhombomere-specific role for Gli signal generated by the spinal cord and the somites, at the time genes in the control of neural patterning genes in the when DV neural patterning occurs (Maden et al., 1998; Solomin zebrafish. et al., 1998; Colbert et al., 1995). Development of the nervous system requires coordination Another interesting observation from this study is that of growth and patterning along the AP and DV axes. Our ventral neural tube development is less affected by Gli2 study here shows that Gli2 and Gli3 perform distinct functions inactivation in r4. For all ventral patterning and differentiation in Shh-dependent ventral patterning at different AP levels of genes examined (Nkx2.2, Nkx6.1, Ngn2, Isl1, Phox2b and the hindbrain. As discussed above, the activities of Gli2 and Olig2), the phenotype of Gli2−/− embryos is significantly less Gli3 are probably modified by local signaling pathways that severe in r4 than in the neighbor rhombomeres. While some of control AP patterning of the developing hindbrain. A recent these genes have a slightly stronger expression in WT r4, the report has demonstrated that Gli2 and Gli3 play differential difference in expression observed in Gli2−/− r4 is far greater roles in DV patterning of the mid/hindbrain boundary (Blaess than that observed in WT hindbrains. Gli3 is likely responsible et al., 2006). Complementary to our data, their results for the expression of these genes in Gli2−/− r4 as Nkx2.2 and suggested that Shh signaling along the DV axis regulates the Isl1/2 expression is completely lost in the r4 of Gli2−/−;Gli3−/− mid/hindbrain boundary expression of Fgf8,whichis embryos (Supplementary Figs. 3E, J). We speculate that Gli3 important for AP patterning of the midbrain and hindbrain. interacts with an r4-specific factor to activate the expression of Together, these observations illustrate the importance of Gli2 class II genes in this rhombomere. The presence of DV and Gli3 in the cross-regulation of AP and DV patterning of patterning defects in the r4 of Hoxb1−/− mice (Gaufo et al., the developing hindbrain. 2000) and the observation that Gli3 and Hoxd proteins functionally interact during limb development (Chen et al., Acknowledgments 2004) raise an intriguing possibility that Hoxb1 could modify Gli3 function in r4. Interestingly, Gli3 influences the expression This research was funded by operating grants from the of neural differentiation genes in r4 without modulating the Canadian Institutes of Health Research (CIHR) to C.H. M.L. expression of patterning genes (such as Olig2, Nkx6.1 and was supported by a Canada Graduate Scholarship Doctoral Dbx1). The expression of Isl1/2 and Pax2 is significantly Award. We thank M. Nakafuku for providing the Olig2 expanded in Gli3−/− r4 revealing a novel role for Gli3 in ventral antibody, S. Cordes, M. Featherstone, and J. Rossant for in neural tube development. Whether this r4-specific “ventral” situ probes, and P. Barnfield, B.G. Bruneau and H.O. Cheung function of Gli3 involves an interaction with Hoxb1 awaits for critical reading of the manuscript. further investigation. A different requirement for Gli function in r4 was also Appendix A. Supplementary data reported in the zebrafish. Like the mouse, zebrafish possesses multiple Gli genes (at least four, see Ke et al., 2005) but the Supplementary data associated with this article can be found, respective functions of individual Gli genes are divergent in the online version, at doi:10.1016/j.ydbio.2006.08.005. 354 M. Lebel et al. / Developmental Biology 302 (2007) 345–355

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