Development 127, 4837-4843 (2000) 4837 Printed in Great Britain © The Company of Biologists Limited 2000 DEV2615

FGF signalling controls the timing of Pax6 activation in the neural tube

Nicolas Bertrand, François Médevielle and Fabienne Pituello* Centre de Biologie du Développement, UMR 5547-CNRS-Université P. Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 04, France *Author for correspondence (e-mail: [email protected])

Accepted 30 August; published on WWW 24 October 2000

SUMMARY

We have recently demonstrated that Pax6 activation occurs in the neural plate. We propose that the progression of Pax6 in phase with somitogenesis in the spinal cord. Here we activation in the neural tube is controlled by the caudal show that the presomitic exerts an inhibitory regression of the anterior limit of FGF activity. Hence, as activity on Pax6 expression. This repressive effect is part of its posteriorising activity, FGF8 downregulation mediated by the FGF signalling pathway. The presomitic acts as a switch from early (posterior) to a later (anterior) mesoderm displays a decreasing caudorostral gradient of state of neural epithelial development. FGF8, and grafting FGF8-soaked beads at the level of the neural tube abolishes Pax6 activation. Conversely, when Key words: FGF, Pax6, Neural tube, Patterning, Vertebrate FGF signalling is disrupted, Pax6 is prematurely activated development, Chick

INTRODUCTION Grafting a caudally, at the level of the presomitic mesoderm, results in a premature activation of Pax6 expression In the developing vertebrate central nervous system, the in the neural plate (Pituello et al., 1999). Conversely, replacing regionalised expression of genes helps to define a piece of rostral presomitic mesoderm with another one from distinct neural progenitor domains along the a more caudal region delays the upregulation of Pax6 (Pituello anteroposterior and dorsoventral axes (Bang and Goulding, et al., 1999). These observations, plus the fact that grafting a 1996). This regionalisation is achieved throughout different somite-sized piece of membrane under the caudal neural plate signalling molecules acting along these two axes (Lumsden is not sufficient to upregulate Pax6 expression, suggest that the and Krumlauf, 1996; Tanabe and Jessell, 1996). In the chick newly formed could be the source of a signal spinal cord, molecules such as bone morphogenetic responsible for the activation of Pax6 expression in the adjacent (BMPs), (SHH) and retinoic acid (RA) neural tube (Pituello et al., 1999). However, this model cannot participate in setting up restricted dorsoventral expression explain why neural plates isolated at the level of the presomitic domains of transcription regulators belonging to the Pax, Msx, mesoderm and cultured for 24 hours in the absence of somitic Dbx and Nkx families (Briscoe and Ericson, 1999; Pierani et tissue display high levels of Pax6 expression (Pituello et al., al., 1999). Among them, Pax6, a paired-type homeobox 1995, 1999). An alternative explanation could therefore be that , is a key component in establishing neural an inhibitory signalling pathway represses Pax6 expression progenitor cell domains (Briscoe et al., 2000). Consequently, prior to somite formation. loss of Pax6 function leads to mis-specification (Ericson et al., In this paper, we show that the activation of Pax6 expression, 1997; Osumi et al., 1997) or delayed differentiation (Sun et al., in the neural tube, results from the relief of an inhibitory 1998) of certain populations of motoneurons and impeded activity mediated by the presomitic mesoderm. Removal of the differentiation of ventral interneurons in the hindbrain and presomitic mesoderm from its rostral part to Hensen’s node is spinal cord. sufficient to observe a premature expression of Pax6 in the It is now well established that the refinement of the Pax6 neural plate, while grafting a piece of presomitic mesoderm, at expression domain is under the control of graded SHH the level of the neural tube, leads to a downregulation of Pax6 signalling (Ericson et al., 1997). However, the molecular expression. This repressive activity is mediated by the FGF mechanisms responsible for the activation of Pax6 expression, signalling pathway. Ectopic application of a subset of FGFs, in the neural tube, remain unknown. including FGF8 present in the presomitic mesoderm, leads to We have recently demonstrated that Pax6 activation is a downregulation of Pax6 expression in the neural tube. triggered by the paraxial mesoderm (Pituello et al., 1999). In Moreover, inhibition of the FGFR1 signalling pathway is seven- to ten-somite stage chicken , Pax6 transcription sufficient to observe a premature activation of Pax6 in the is sharply upregulated in the neural tube region flanking the neural plate and to suppress the repressor effect of the last formed somite (Goulding et al., 1993; Li et al., 1994). presomitic mesoderm. Finally, we show that genes normally 4838 N. Bertrand, F. Médevielle and F. Pituello restricted to the caudal neural plate, such as Cash4 (Henrique Instead, removal of the non-neural ectoderm and neural fold et al., 1997) and Sax1 (Rangini et al., 1989; Spann et al., 1994), showed no effect on the timing of Pax6 activation (data not are reactivated or maintained in the neural tube following the shown). This is a strong indication that the presomitic inhibition of Pax6 expression by FGF8. mesoderm is the source of an inhibitory activity that maintains Pax6 in a transcriptionally repressed state. We have previously shown that replacing a small piece of the MATERIALS AND METHODS presomitic mesoderm with a somite-sized membrane is not sufficient, however, to upregulate Pax6 expression (Pituello Microsurgery experiments et al., 1999). One possible interpretation of this result is that Host and donor embryos at the seven- to ten-somite stage were a signalling molecule diffusing not only transversally, but prepared, operated and incubated as described elsewhere (Pituello et also locally along the rostrocaudal axis of the , is the al., 1999). source of such inhibitory activity. Graft of FGF-soaked beads FGF8, present in the presomitic mesoderm, is Heparin-coated acrylic beads were rinsed in PBS for 15 minutes and capable of inhibiting Pax6 expression incubated in FGF8 or FGF4 (50 µg/ml), or in FGF7 (250 µg/ml). After 2 hours on ice, the beads were rinsed in PBS and grafted. Control At the level of the caudal neural plate, signalling molecules beads were incubated in PBS. A somite (level +I or +IV) or a piece such as fibroblast growth factors (FGFs) are present in the of presomitic mesoderm (level –II) was replaced with a bead and the presomitic mesoderm (Shamim and Mason, 1999; Vogel et al., embryo reincubated in Tyrode’s solution for 2.5 to 3 hours at 38°C. 1996). These molecules have been shown to be involved in posterior neural tissue specification in the chick embryo (Muhr Inhibition of FGFR1 signalling pathway et al., 1999; Storey et al., 1998). Fgf8 transcription is restricted Embryos were incubated in Tyrode’s solution supplemented with µ µ to the caudal region of the embryo being very strong at the SU5402 (Calbiochem) (10 M or 25 M) for 15 minutes before level of the primitive streak and Hensen’s node and displaying FGF8-soaked beads or presomitic mesoderm were grafted, and then reincubated for 3 hours in Tyrode’s solution containing SU5402 (10 a decreasing caudo-rostral gradient in the presomitic µM or 25 µM). mesoderm (Fig. 2A). Furthermore, transcripts encoding the transmembrane 1 to FGFs (FGFR1) are detected in In situ hybridisation the caudal neural plate as well as in the neural tube (Fig. 3A) Pax6 and Pax3 transcripts were analysed using the antisense RNA probe (Walshe and Mason, 2000). Altogether, these data suggested prepared as described (Pituello et al., 1995). Chick Cash4 cDNA was a gift from D. Henrique, chick Fgf8 cDNA was a gift from S. Martinez and chick Sax1 cDNA was a gift from A. Fainsod. A FGFR1 702bp-cDNA fragment was generated by PCR using the following primers: upstream 5′ GGAGAACA- AATACGGGAGCA 3′; downstream 5′ GGCAGCTCATAC- TCGGAGAC 3′. These primers were defined using the FGFR1 cDNA sequence found in EMBL database (Accession Number, M24637). The whole-mount in situ procedure was performed according to Wilkinson (1992). Double in situ hybridisation experiments and vibratome sectioning were performed as previously described (Pituello et al., 1995).

RESULTS

The presomitic mesoderm exerts an inhibitory effect on Pax6 expression To test the possibility that Pax6 expression is repressed in the caudal part of the embryo, we microsurgically removed all tissues in contact with the neural plate except the notochord. Removal at the level of the presomitic mesoderm of the non-neural ectoderm, neural fold and paraxial and lateral mesoderms on one side of seven- to ten-somite stage embryo (Fig. 1A), resulted in a strong premature activation of Pax6 Fig. 1. The paraxial mesoderm is the source of an inhibitory activity on expression on the operated side (Fig. 1B,C). This result Pax6 expression. (a,d) Diagrams of experiments performed. (b,c,e,f) Pax6 in suggests that some type of inhibitory activity present situ hybridisation on 10-somite stage operated embryos. (a) Removal of all in the caudal neural plate is normally responsible for the structures in contact with the caudal neural plate, except the notochord, leads to a premature activation of Pax6 expression (b,c). (d) Complete repressing Pax6 expression in that region. We then deletion of the presomitic mesoderm is sufficient to obtain the same result proceeded to identify the tissue responsible for this (e,f). Black arrowheads indicate the position of the last formed somite (+I). inhibition. A premature upregulation of Pax6 The red arrowheads mark the limits of the presomitic mesoderm deletion. expression was observed in the caudal neural plate in ec, ectoderm; en, endoderm; lm, lateral mesoderm; pm, presomitic absence of the presomitic mesoderm (Fig. 1D-F). mesoderm; nc: notochord; np, neural plate,. FGF signalling controls Pax6 activation 4839

Fig. 2. FGF8 is able to block Pax6 activation and repress Pax6 expression in the neural tube. (a) Fgf8 in situ hybridisation. (b-g) Pax6 in situ hybridisation on 11-somite stage (b-d) and 12-somite stage chick embryos (e-g). (h,i) Pax3 in situ hybridisation. (a) Fgf8 transcripts are very abundant in the caudal region of a ten-somite stage chick embryo and present in the presomitic mesoderm. (b,c) Replacing a piece of presomitic mesoderm in position –II with a FGF8-soaked bead, results in a block of Pax6 activation at the level of the last formed somite. The effect extends rostrally to level +II and the contralateral side is affected too. (e,f) Graft of a FGF8-soaked bead in position +I, in place of a somite, results in the downregulation of Pax6 expression. Downregulation also occurs caudally and rostrally to the bead and the contralateral side can be affected (f). Control beads have no effect on Pax6 activation (d) or expression (g). Cross sections showing Pax3 expression in the neural tube adjacent to a FGF8-soaked bead (h) or a PBS-soaked bead (i). FGF8 neither switches off Pax3 nor modifies its dorsoventral patterning (h). Arrowheads indicate the position of the last formed somite. that FGFs could be involved in Pax6 inhibition by the the neural tube (Fig. 2H-I). Grafting control beads in the same presomitic mesoderm. In order to test this possibility, paraxial conditions had no effect on Pax6 expression (Fig. 2D,G). When mesoderm was replaced with beads soaked in FGF8 and similar experiments were performed with beads soaked in other grafted at different anteroposterior levels, either (–II, +I FGFs, we found that FGF4 was able to mimic the effects of (position of the last formed somite) or +IV). After a 2.5 hour FGF8, while FGF7 (KGF), which is known to interact incubation, a –II-grafted bead ended at the –I level. Despite the specifically with a different receptor, the FGFR2 (IIIb) (Ornitz formation of a new somite, Pax6 transcripts were not detected et al., 1996), has no effect on Pax6 expression (data not shown). rostrally to the bead (Fig. 2B,C), indicating that FGF8 is able Taken together, these data indicate that a specific subset of to block the activation of Pax6 expression. In most cases, this FGFs is able to inhibit Pax6 expression in the neural tube, and repression effect reaches the position +II. In cases in which a suggest a role for such factors in its repression in the caudal somite from position +I or +IV was replaced with an FGF8b- neural plate. soaked bead, Pax6 expression was strongly downregulated on the side of the grafted bead. Variable levels of downregulation Blocking FGF signalling transduction pathway is were also detected in the contralateral side in which the neural sufficient to abolish the inhibitory effect of the tube remained in contact with the somite (Fig. 2F and data not presomitic mesoderm shown). Again, the inhibitory effect of FGF8 expands further To further investigate whether FGF8 is responsible for the rostrally and caudally than the position of the bead. Pax3 expression was not switched off by the graft of an FGF8b- soaked bead in position +I neither was affected its ventral limit of expression in

Fig. 3. FGF8 is responsible for the repressor effect of the caudal presomitic mesoderm on Pax6 expression. (a) Fgfr1 is expressed all along the central nervous system in seven- to ten-somite stage chick embryo. The downregulation of Pax6 expression following the graft of a FGF8-soaked bead (b) or a piece of caudal presomitic mesoderm (d,f) in position +I is suppressed in presence of SU5402, an inhibitor of the FGFR1 signalling pathway (c,e,g). Compare the caudal limit (red arrowheads) of Pax6 expression in a control embryo (d) and in an embryo incubated in SU5402 (e). (d,e) Black arrowheads mark Hensen’s node level. (f,g) arrowheads show the limits of presomitic mesoderm graft. 4840 N. Bertrand, F. Médevielle and F. Pituello inhibition of Pax6 transcription by the presomitic mesoderm, we performed the same grafting experiments in a context where the FGF transduction pathway is inhibited within the neural tube. A good candidate for transducing FGF8 signalling is FGFR1, which is expressed along the entire anteroposterior axis of the neural tube (Fig. 3A). FGFR1 transduction can be blocked by SU5402, a kinase (PTK) inhibitor that blocks the autophosphorylation of the receptor by interacting directly with its catalytic domain (Mohammadi et al., 1997). Even if SU5402 is also a weak inhibitor of phosphorylation of the PDGF receptor and its effect has only been assayed against a subset of PTKs (including FGFR1) in the initial study (Mohammadi et al., 1997), this molecule remains a potent tool in blocking FGF-mediated effects on chick tissues (McCabe et al., 1999; Muhr et al., 1999; Streit et al., 2000). First, we tested the ability of SU5402 to block the inhibitory effect of FGF8 on Pax6 expression in our bead graft assay. We found that Pax6 inhibition could be prevented by the presence of the inhibitor (Fig. 3B,C). SU5402 was, then, a suitable tool to use to determine if FGFs are the molecular support of the inhibitory effect of the presomitic mesoderm. Grafts of caudal paraxial mesoderm (level −IV, −V) to position +I normally leads to a downregulation of Pax6 expression in the neural tube (Fig. 3D,F). In the presence of SU5402 (10 µM), this repression does not take place (n=8/8) (Fig. 3E,G), suggesting that FGF signalling is required to mediate the inhibitory effect of the presomitic mesoderm on Pax6 expression. In a fraction of these SU5402-treated embryos (n=4/8), a premature activation of Pax6 expression in the neural plate was observed Fig. 4. FGF8 maintains or reactivates anteriorly genes that are (Fig. 3D,E). The number of SU5402-treated embryos expressed in the caudal neural plate. (a) Double in situ experiment on displaying such a caudal activation of Pax6 expression was a nine-somite stage chick embryo, showing the roughly complementary expression domain of Pax6 (in blue) and Sax1 (in clearly enhanced (n=10/13) by a 2.5-fold increase of SU5402 − concentration. One hypothesis may be that SU5402 affects a red). Graft of a FGF8-soaked bead in position II in an eight-somite stage chick embryo results in a maintenance of Sax1 expression possible inhibitory effect of SHH on Pax6 expression in the above the bead (b) while Sax1 transcripts are absent above a control caudal neural plate. However, it has been shown that SU5402 bead (c). Arrowheads indicate the position of the last formed somite does not affect the response to SHH in vitro (Muhr et al., (+I). Graft of a FGF8-soaked bead in an eight-somite stage chick 1999). It is, thus, unlikely that the caudal activation of Pax6 embryo in position +I results in a reactivation of Cash4 expression in observed in presence of SU5402 is due to inhibition of SHH the neural tube (d) while PBS-soaked bead is ineffective (e). signalling. Moreover, the presence of the notochord does not prevent a premature activation of Pax6 after ablation of the presomitic mesoderm strongly suggesting that SHH is not mostly for Sax1 (Fig. 4A). This raised the possibility that the involved in the mechanisms that keep Pax6 expression off in repression effect of FGF8 on Pax6 expression could be the caudal neural plate. Therefore, even if we cannot totally associated with the maintenance and/or reactivation of Cash4 exclude that other pathways participate in the control of Pax6 and/or Sax1. When a bead soaked in FGF8 was grafted in expression, the observation that increasing concentrations of position +I, Cash4 but not Sax1 was strongly upregulated SU5402 lead to an increased relief of inhibition strongly within the domain of Pax6 repression (Fig. 4D). Therefore, the supports the theory that FGF signalling is a major component inhibition of Pax6 expression is associated with the reactivation of the regulatory network that controls Pax6 activation in the of at least one caudal gene. The absence of Sax1 response may neural tube. reflect the fact that cells have already lost the competence to express the gene. FGF8 bead grafts were then performed at Pax6 inhibition can be associated with the level –II. In such conditions, the rostral limit of Sax1 maintenance and/or reactivation of caudally expression was shifted anteriorly (Fig. 4B). Yet, it did not reach expressed genes the anterior limit of Pax6 inhibition (compare with Fig. 2C) but Transcription factors expressed in the caudal neural plate such it shows that delaying Pax6 activation correlates with the as Cash4 and Sax1 have been shown to respond to FGF maintenance of Sax1 expression. These data show that the signalling (Henrique et al., 1997; Storey et al., 1998). For downregulation of Pax6 and the upregulation of caudal example, Cash4 and Sax1 are induced by grafting FGF8 beads restricted transcription factors are both controlled via FGF8, in extra-embryonic epiblast of HH3-3+ host embryos. In seven- suggesting that this signalling pathway controls the switch to ten-somite stage embryos the expression patterns of these from early (caudal) neural plate state to later (rostral) neural two genes appeared roughly complementary to that of Pax6 tube state. FGF signalling controls Pax6 activation 4841

DISCUSSION expression while a membrane of the same size has no effect. Indeed, the question of the role of the somite with respect to Graded FGF signalling represses Pax6 expression in its ability to induce Pax6 expression remains to be addressed. the neural plate FGFs constitute a family of about 20 structurally and We have shown that FGF8 could repress Pax6 expression in functionally related growth factors (Szebenyi and Fallon, the neural tube and that the presomitic mesoderm was the 1999). At least some of them, such as FGF4 (Shamim and source of a FGF activity responsible for the absence of Pax6 Mason, 1999), have been reported to be expressed in the expression in the neural plate. Expression of Fgf8, in the posterior region of the embryo, including the presomitic presomitic mesoderm, follows a decreasing caudorostral mesoderm. Furthermore, we observed that FGF4 was capable gradient, the anterior limit of which does not reach the level of repressing Pax6 expression (this study) without inducing of the last formed pair of somites. In seven- to ten-somite FGF8 expression (N. B., unpublished). We cannot thus exclude stage chick embryo, the caudal limit of Pax6 expression, in that other FGFR1 ligands than FGF8 are involved in the the neural tube, is rather sharp and faces the last formed repressive effect of the presomitic mesoderm on Pax6 somites. An explanation for this may be that a graded expression in the caudal region of the embryo. distribution of FGF8 protein exists in the presomitic Among the four high-affinity FGF receptor genes present in mesoderm and that FGF8 protein is present nearby the newly chick, Fgfr1 is the only one expressed in the posterior neural formed somite. This hypothesis is supported by the plate at the stages we considered in our study (Marcelle et al., expression pattern of sprouty 2, an avian homologue of 1994; Walshe and Mason, 2000). Therefore, it is the best sprouty (Chambers et al., 2000; Minowada et al., candidate for receiving and transducing the FGFs inhibitory 1999). FGF8 can induce sprouty 2 transcription in neural activity acting in the caudal region of the embryo on Pax6 tissue (Chambers et al., 2000) and even if it is not strictly expression. In mouse, Fgfr1 is also present in the neural plate expressed in all tissues in which FGFR are activated, sprouty (Yamaguchi et al., 1992). Using targeted gene disruption, it has 2 expression constitutes a useful means with which to analyse previously been shown that FGFR1-null embryos were FGF action in chick embryo (Chambers and Mason, 2000; developmentally retarded and died during gastrulation (Deng Chambers et al., 2000). Interestingly, from early et al., 1994; Yamaguchi et al., 1994). More informative for somitogenesis, sprouty 2 is expressed into the unsegmented neural tube development are studies on chimeric embryos paraxial mesoderm and in the neural plate extending to the formed using FGFR1-deficient embryonic stem cells (Ciruna level of the last formed pair of somites (Chambers and et al., 1997) and on hypomorphic mutants (Partanen et al., Mason, 2000). These data indicate that FGF8 action, in the 1998; Xu et al., 1999). Such mutant embryos are reported to caudal region of the embryo, extends further away than the display an expansion of the posterior neural structures. For site of its expression and that it ends at the level of the newly example, disruption of the FGFR1 alpha isoform give rise to formed somite consistent with the caudal limit of Pax6 unclosed neural tube (spina bifida), which began at around the expression in the neural tube. Moreover, both expression of 10th somite and extended posteriorly (Xu et al., 1999). Pax6 and sprouty 2 suggest that FGF8 action is stopped Anterior neuroepithelium markers such as Otx2, Fgf8, En1 and abruptly at the level of the somites. Two mechanisms that are Krox20 do not seem affected, while the posterior marker Hoxb9 not exclusive can be proposed to explain this sudden displays higher intensity and broader expression consistent interruption of FGF8 signalling. In Drosophila, sprouty is with histological observations that excess amount of neural proposed to be an intracellular antagonist of the Ras/MAP epithelium is present in the posterior embryonic portion of kinase pathway and it antagonises both FGF and epidermal mutant embryos. These observations have led to the proposal (EGF) signalling (Casci et al., 1999; Reich et that FGF/FGFR1 signals may play a role in regulating al., 1999). In chick, it is proposed that sprouty 2 acts as an patterning during posterior neural tube development (Xu et al., antagonist of FGF signalling by participating in a cell- 1999). However it has to be considered that in such mutants, autonomous regulatory negative- loop. That multiple abnormalities are observed in posterior structures, regulatory loop could serve to regulate the patterning events suggesting that the effect on neural tube development may be dependent on this signalling and in particular on FGF8 secondary to these developmental defects. signalling (Chambers et al., 2000). This proposition is based on several facts: (1) there is a functional conservation Initiation of Pax6 expression occurs when between the Drosophila and the mammalian sprouty protein; expression of Cash4 and Sax1 ceases (2) the chick sprouty 2 gene is highly identical to its We have shown that FGF8-mediated repression of Pax6 mammalian counterparts; and (3) there is a close correlation expression in the neural tube can be associated with of FGF and sprouty 2 expression while sprouty 2 expression reactivation of Cash4 expression and maintenance of Sax1 pattern bears little resemblance to the distribution of EGF expression. Induction of the expression of these genes by FGF (Chambers and Mason, 2000; Chambers et al., 2000). Thus, ligands has already been reported (Henrique et al., 1997; we can speculate that sprouty 2 alone limits the range of Storey et al., 1998). In seven- to ten-somite stage chick action of FGF8 by the proposed regulatory negative-feedback embryo, Cash4 and Sax1 are expressed in the caudal neural loop and that this loop suddenly turns off FGF8 signalling. plate (Henrique et al., 1997; Rangini et al., 1989; Spann et al., The second mechanism could be based on another negative 1994), exhibiting a roughly complementary expression pattern regulation acting at the level of the somite. If we assume that to that of Pax6. These observations raise the possibility that this negative regulation can be triggered by the somite itself, the FGF8 repression effect on Pax6 expression could be the this second hypothesis could also explain how a somite consequence of Cash4 or Sax1 upregulation. However, a strict grafted close to the neural plate can induce a premature Pax6 correlation between Pax6 inhibition and Cash4 and/or Sax1 4842 N. Bertrand, F. Médevielle and F. Pituello expression is unlikely: Sax1 is not upregulated when Pax6 is steps of neurogenesis (Campos-Ortega, 1998; Skeath and inhibited in rostral neural tube; in later stages embryo, Cash4 Carroll, 1994). Proneural functions of Cash4 have not been expression ceases well before Pax6 activation; furthermore, addressed in chick but in Drosophila and Xenopus embryos, by reactivation of Cash4 expression in the neural tube by FGF8 is rescue and mRNA injection experiments, respectively only observed as long as the gene is expressed in the caudal (Henrique et al., 1997). Thus, it is proposed that Cash4 neural plate while Pax6 can still be inhibited (N. B., functions as a neural determination gene specifying the neural unpublished). Nevertheless, we cannot exclude the possibility precursors that will generate the avian posterior nervous that these genes exhibit redundant capability in repressing Pax6 system (Henrique et al., 1997). As previously mentioned, Pax6 transcription. The expression of one of them in the caudal expression is activated in all the neural progenitor cells of the neural plate will be then sufficient to prevent Pax6 activation. developing spinal cord after Cash4 expression ceases. One Another possibility is that Pax6 and Cash4/Sax1 are meaning of the negative regulation of Pax6 expression we have independently regulated by FGF signalling. identified could be that Pax6 must be activated at a precise time in the course of the neurogenesis execution. Thus, Pax6 could Pax6 expression is controlled by an anteroposterior be one of the factors that act sequentially in the implementation acting signal prior to be submitted to dorsoventral of the neural program. Accordingly, it remains to be refinement determined what the importance of the timing of Pax6 Several studies indicate that FGF signalling is involved in expression activation might be with respect to the development multiple patterning events along the anteroposterior axis of the of the spinal cord and neuronal specification. developing neural tube (Crossley et al., 1996; Irving and Mason, In conclusion, the data presented here reveal the regulatory 2000; Lee et al., 1997; Martinez et al., 1999; Pownall et al., network governing the timing of Pax6 activation in the neural 1998; Shamim et al., 1999; Shimamura and Rubenstein, 1997). tube. A graded FGF signalling present in the caudal part and Moreover, FGF signalling is thought to be one of the major in the presomitic mesodermal regions of the embryo is signalling system implicated in the induction and development responsible for maintaining the expression in the neural of the posterior central nervous system (CNS) (Henrique et al., groove of transcription regulators genes such as Cash4 and 1997; Muhr et al., 1999; Storey et al., 1998). Transcription Sax1, and for maintaining, in a transcriptionally repressed factors, such as Cash4, which are induced by posteriorising state, the transcription factors expressed in the neural tube signals including FGFs, have been proposed to define a such as Pax6. As the anteroposterior axis of the embryo posterior neural progenitor cell domain (Henrique et al., 1997; elongates, the gradient of FGF signalling moves posteriorly Storey et al., 1998). We suggest that Pax6 transcription, which allowing the limit of Pax6 expression to progress caudally results from the relief of FGF signalling, draws up the reflecting neural tube extension. As FGF signalling is boundaries of a new progenitor cell domain complementary and involved in multiple patterning events along the rostral to the posterior neural progenitor cell domain. The anteroposterior axis of the developing neural tube, it appears progression of Pax6 expression domain, in the neural tube, is that the activation of the expression of Pax6, a gene that is allowed by the fact that the gradient of FGF signalling moves involved in dorsoventral patterning of the neural tube is posteriorly with the posterior CNS progenitor domain as the controlled by an anteroposterior acting signal. anteroposterior axis of the embryo elongates. Interestingly in the FGFR1 alpha mutant, the primary defect is the failure of This paper is dedicated to Dr Anne-Marie Duprat who retired node regression (Xu et al., 1999) a process essential for embryo recently. The authors would like to thank Dr A. Fainsod, Dr D. elongation. The FGF signalling pathway may therefore control Henrique and Dr S. Martinez for the gift of Sax1, Cash4 and Fgf8 both the growth and patterning in vertebrate embryo, cDNAs, respectively. Many thanks to F. Foulquier for excellent technical assistance. We thank Dr Philippe Cochard, Dr Alain Vincent synchronising these events. Pax6 expression, the initiation of and Dr Guillermo Oliver for their comments on the manuscript. This which is controlled by anteroposterior signalling, will then be work was supported by the Centre National de la Recherche refined by the dorsoventral acting signals. 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