Department of Molecular and Cellular Biology, The Biolabs, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
* Present address: Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
Author for correspondence (e-mail: amcmahon{at}mcb.harvard.edu)
Accepted 24 July 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Sonic hedgehog, Mouse, Growth, CNS
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dorsal signaling is initiated by the surface ectoderm at neural plate stages and continued by roof plate cells, which occupy the dorsal midline of the neural tube after neural tube closure. A number of TGFß family members are expressed in one or both of these two signaling regions. Several lines of evidence indicate that their individual or combinatorial actions specify distinct dorsal neural fates (reviewed by Lee and Jessell, 1999). The roof plate also expresses several members of the Wnt-family, including Wnt1 and Wnt3a (Parr et al., 1993
). Both, analysis of Wnt1/3a compound mutants, and the results of ectopic activation of Wnt signaling within the neural tube, suggest that these Wnt signals regulate cell proliferation (Dickinson et al., 1994
; Ikeya et al., 1997
; Lee et al., 2000
; Megason and McMahon, 2002
) (S. M. Lee, M. I., S. Megason, S. Takada. and A. P. M., unpublished). Thus, the roof plate coordinates growth and pattern by the production of two distinct classes of signal.
Accumulating evidence indicates that sonic hedgehog (Shh), a glycoprotein secreted by the notochord and floor plate, acts directly as a morphogen to specify distinct ventral cell identities (reviewed by Briscoe and Ericson, 1999; Jessell, 2000
). The ventral half of the spinal cord is missing in Shh mutants, while the dorsal half remains (Chiang et al., 1996
), consistent with notochord ablation experiments (Placzek et al., 1990
; Van Straaten and Heckking, 1991
; Yamada et al., 1991
). Shh may not be the exclusive ventralizing factor, for example, retinoid signaling is implicated in induction of v0 and v1 populations of ventral interneurons in the presumptive spinal cord (Pierani et al., 1999
). The notochord has also been shown to regulate cell proliferation in neural plate explants, consistent with the possibility that Shh secreted from this source acts as a mitogen (Van Straaten et al., 1989
; Placzek et al., 1993
). Indeed, ectopic expression of Shh in the dorsal neural tube (Rowitch et al., 1999
), or ectopic activation of the Shh pathway through the removal of patched 1 (Ptch1) activity, results in dramatic hyper-proliferative phenotypes (Goodrich et al., 1997
). Together these studies on the control of growth and pattern within presumptive spinal cord regions demonstrate that dorsal and ventral halves are largely regulated independently of one another.
The brain is considerably more complex. Several studies indicate that its organization is based upon an early segmental scaffold of repeating metameric units termed neuromeres. This is most obvious in the rhombomeres, the neuromeres of the hindbrain, where rhombomeric boundaries are barriers to cell mixing maintaining the clonal restriction of cell populations (reviewed by Lumsden and Krumlauf, 1996). In addition, appropriate expression of Hox genes and other regulatory factors within subsets of rhombomeres is critical for their patterning (reviewed by Krumlauf et al., 1993
; Wilkinson, 1993
). Whereas the midbrain is thought to arise from a single neuromere (reviewed by Lumsden and Krumlauf, 1996
), Puelles and colleagues have argued that the forebrain can be subdivided into six prosomeric units, three that generate the diencephalon excluding the hypothalamus (P1 to P3, caudal to rostral), and three that make up the telencephalon (Puelles and Rubenstein, 1993
; Rubenstein and Puelles, 1994
). Whether prosomeres exist in the same developmental and functional sense as rhombomeres is debatable; however, prosomere boundaries serve as a useful set of coordinates for the description of forebrain development.
As in presumptive spinal cord regions, signaling by Wnt, Hedgehog and TGFß-family members has been shown to regulate the growth and pattern of brain regions. For example, Shh is expressed in the ventral forebrain, midbrain and hindbrain and its ventralizing properties extend into these regions (reviewed by Briscoe and Ericson, 1999; Jessell, 2000
). Thus, Shh is a general ventralizing factor along the entire anteroposterior (AP) axis of the neural tube. How, then, does the same signal specify distinct cell types within different regions? Part of the answer appears to lie in the combinatorial action of Shh and other signaling factors, as well as intrinsic differences in the regional response to Shh that result from earlier patterning events (Dale et al., 1997
; Ye et al., 1998
). In addition to its role in cell fate specification, Shh has been proposed to act as a mitogen in the expansion of granule cell precursors in the external granule layer of the cerebellum, a relatively late event in CNS development (Dahmane and Ruiz-i-Altaba, 1999
; Wallace, 1999
; Wechsler-Reya and Scott, 1999
). Furthermore, misregulation of hedgehog signaling is implicated in the development of medullablastomas, a granule cell tumor (Vorechovsky et al., 1997
; Raffel et al., 1997
).
Although, Shh is expressed predominantly in the ventral neural tube at early stages, by 10.5 days post-coitum (dpc) there is a prominent dorsal extension at the zona limitans intrathalamica (ZLI), which lies at the boundary between P2 and P3 (Echelard et al., 1993; Shimamura et al., 1995
). This raises the possibility that Shh signaling in the diencephalon may play a broader role in its development. Consistent with this view, Shh mutants have a disproportionate reduction in the size of the diencephalon relative to the hindbrain region at 11.5 dpc (Chiang et al., 1996
).
We demonstrate that Shh signaling is critical for the proliferation and survival of neural precursors in the diencephalon and anterior midbrain, prior to the initiation of expression in the ZLI. Unlike other regions of the neural tube, Shh signaling is required for the normal development of both dorsal and ventral regions of the diencephalon and anterior midbrain, though analysis of the expression of Shh targets suggests that Shh does not signal directly within dorsal regions. Our data indicate a Shh-dependent signaling relay between ventral and dorsal regions that coordinates their growth. We suggest that fibroblast growth factor (FGF) and Wnt signaling may mediate these mitogenic and survival effects.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whole-mount RNA in situ hybridization
Whole-mount RNA in situ hybridization of embryos was performed as previously described (Parr et al., 1993). Digoxigenin probes were synthesized using the Digoxigenin RNA labeling Kit (Roche).
BrdU incorporation analysis
Dissected embryos were incubated in DMEM with BrdU for 30 minutes (S. Hayashi and A. P. M., unpublished) and processed for frozen sections. BrdU incorporation into newly replicated DNA was detected immunochemically as previously described (Dickinson et al., 1994) using anti-BrdU antibody (Pharmingen) and Alexa 568-conjugated anti-mouse IgG antibody (Molecular Probes). Labeled sections were counterstained with YoPro1 (Molecular Probes) and analyzed under the confocal microscope (Zeiss). The boundary between the telencephalon, diencephalon and midbrain were determined by morphological criteria and nuclei in each region were counted. Five wild-type and five Shh mutant embryos were examined and the statistical significance was calculated using Students t-test.
TUNEL assay
Frozen sections of embryos were treated with proteinase K and processed for TUNEL assay using ApopTag Red In Situ Apoptosis Detection Kit (Intergen Company).
Plasmid construction
The entire coding region of an Fgf15 cDNA (kindly provided by Dr Murre) was subcloned into the expression vector, pCIG (Megason and McMahon, 2002). This base vector contains a constitutive promoter, multiple cloning site and an internal ribosomal entry signal (IRES) that is followed by a cDNA encoding green fluorescent protein (GFP). pCIG-F15-transfected cells produce both FGF15 and GFP.
Explant culture and electroporation
Mouse embryonic brains were dissected between the 14- and 16-somite stages and placed in DNA solution (1 mg/ml). Electrodes were placed 4 mm apart at both sides of the explants, then rectangular pulses (22 V, 50 mseconds, three times) were given by a T820 electroporator and a BTX500 optimizer (BTX). The explants were cultured in collagen matrix with the medium which contained 50% DMEM (Gibco), 10% fetal bovine serum (Hyclone) and 40% rat serum (Harlan). Collagen gels were prepared as previously described (Tessier-Lavigne et al., 1987; Artinger and Bronner-Fraser, 1993
). After 40 hours, the explants were fixed in 4% paraformaldehyde and processed for whole-mount RNA in situ hybridization.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Pax6 expression provides a third useful landmark. Pax6 is expressed throughout the entire alar plate of the forebrain, the sharp posterior boundary demarcates the diencephalic-midbrain junction (Warren and Price, 1997; Mastick et al., 1997
; Gringley et al., 1997
) (Fig. 2E). Thus, the gap between Pax6 and En1 expression domains at mid-somite stages corresponds to the anterior midbrain. This region was also greatly reduced at the 21-somite stage in Shh mutants (Fig. 1I,J). Although the size of the En1 expression domain was not altered, there was a marked decrease in the size of the Pax6 expression domain in the forebrain, most likely reflecting the truncated development of P1 and P2 regions (Fig. 1I,J). Thus, the analysis of regional markers was consistent with the results of the morphological analysis: the dorsal parts of P1/2 and the anterior midbrain developed normally in Shh mutants until the 14-somite stage but shortly thereafter their growth was retarded.
|
Dbx1, which encodes a homeodomain protein, is expressed in the basal plate of P3 and the alar plates of both P1/2 and the entire midbrain. Dbx1 is also expressed in an intermediate zone where the sulcus limitans forms at hindbrain and spinal cord levels (Shoji et al., 1996) (Fig. 2K). Dbx1 expression was almost completely absent in Shh mutant brains at the 25-somite stage (Fig. 2L), while spinal cord expression was maintained (Pierani et al., 1999
) (data not shown). Expression was observed in the alar plate of Shh mutants at the 13-somite stage (data not shown). Thus, in contrast to the aforementioned markers, both dorsal and ventral expression of Dbx1 in the diencephalon and midbrain is dependent on Shh.
Pax7 encodes a paired-type homeodomain protein that is broadly expressed in the alar plate along most of the length of the early neural tube (Jostes et al., 1990). Pax7 extends into ventral regions of the presumptive spinal cord in the absence of Shh signaling (Litingtung and Chiang, 2000
; Briscoe et al., 2001
), consistent with the loss of ventral cell identities and dorsalization of the neural tube. In the forebrain and midbrain, Pax7 shows a similar ventral extension indicating that ventral cell fates were lost but dorsal cell fates were maintained in Shh mutants (data not shown). Thus, the observed loss of Dbx1 expression does not reflect a general failure in dorsal specification within these brain regions of Shh mutant embryos.
Cell proliferation is decreased in alar plates of the diencephalon and midbrain of Shh mutants
The failure of diencephalic/midbrain development could reflect altered proliferation of neural precursors within these primordia. To examine this possibility directly, we performed an analysis of BrdU incorporation at 15- to 16-somite stages (Fig. 3A,B). In wild-type embryos, the entire telencephalon, diencephalon and midbrain showed a BrdU incorporation rate of 60% using our labeling protocol (Fig. 3C-E,G). In Shh mutants, incorporation in the telencephalon was not significantly different from wild type (66.36±1.82%; Fig. 3F,G). By contrast, the diencephalon and midbrain showed significantly fewer S-phase cells (41.46±0.65% and 50.08±1.82%, respectively; Fig. 3F,G). Thus, a reduced rate of proliferation in diencephalic and midbrain precursors at 15- to 16-somite stages most probably contributes to the observed reduction in diencephalic and midbrain regions at later stages.
|
Wnt signaling is perturbed in P1 and P2 alar plates of Shh mutants
Recent studies indicate that Ccnd1 is a direct target of Wnt signaling. In the presence of a Wnt signal, ß-catenin forms a complex with TCF/LEF proteins, which are HMG-box containing transcription factors, and this complex activates transcription of target genes (Molenaar et al., 1996; Korinek et al., 1997
; Morin et al., 1997
), one of which is Ccnd1 (Shtutman et al., 1999
; Tetsu and McCormick, 1999
). Tcf4 is expressed in the ventral telencephalon, the alar plates of P1/2 and rhombomere 5 (r5) of the hindbrain at 8.5 dpc (Cho and Dressler, 1998
) (Fig. 4A,B). Wnt1 and Wnt3a are expressed in the roof plate of the diencephalon and in the absence of both signals there is a failure of diencephalic development (S. M. Lee, M. I., S. Megason, S. Takada. and A. P. M., unpublished). Thus, Tcf4 is well placed to respond to these Wnt signals.
|
Cell death is increased in alar plates of the diencephalon and midbrain of Shh mutants
The finding of ectopic cell death in the developing spinal cord and somites of Shh mutants suggests that Shh may promote the survival of certain cell types (Chiang et al., 1996; Borycki et al., 1999
; Litingtung and Chiang, 2000
). To determine whether cell death contributes to the diencephalic/midbrain phenotype, we performed a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay on Shh mutants. In wild-type brains, there were very few TUNEL-positive cells at the 15- and 17-somite stages (Fig. 5A) (data not shown). By contrast, a large number of TUNEL-positive cells were observed in Shh mutants (Fig. 5B) (data not shown), suggesting that ectopic cell death contributes to the dorsal brain phenotype of Shh mutant embryos.
|
Tcf4 and Bmp4 are indirect targets of Shh signaling
The altered dorsal expression of Tcf4 and Bmp4 together with the correlated changes in cell proliferation and cell death, raises the issue of whether Shh signals directly to dorsal regions of the diencephalon and midbrain at the 14- to 25-somite stages. To address this issue, we examined expression of Shh and Ptch. Ptch encodes the Shh receptor, its upregulation in response to Hedgehog signaling is a highly conserved transcriptional response that serves to limit the range of Hedgehog signaling (reviewed by Ingham and McMahon, 2001). As demonstrated in previous studies (Echelard et al., 1993
), Shh was expressed only in ventral regions at the 14-somite and earlier stages (Fig. 6A; data not shown). The first evidence of a dorsal expansion, at the presumptive ZLI, was not observed until the 24-somite stage (arrow in Fig. 6C; data not shown) (Shimamura et al., 1995
). Upregulation of Ptch was also confined to ventral regions corresponding approximately to the basal plate (Fig. 6B,D). Thus, the analysis of Shh expression and Shh target gene response indicates that Shh signaling is restricted to the basal plate; consequently, Tcf4 and Bmp4 are unlikely to be direct targets of Shh signaling.
|
In 12-somite stage wild-type embryos, Fgf15 was strongly expressed in the ventral and intermediate parts of P1/2 and the anterior midbrain just dorsal to the Shh expression domain (Fig. 7A,B). By the 14-somite stage, Fgf15 expression extended dorsally overlapping the Dbx1 and Tcf4 expression domains (Fig. 7D), dorsal expression of Fgf15 intensified by the 16-somite stage (Fig. 7F). Fgf15 was also strongly expressed just posterior to the midbrain/hindbrain isthmus (Fig. 7B,D,F). In Shh mutants, no Fgf15 expression was observed within the forebrain or midbrain between the 12- and 16-somite stages (arrowhead in Fig. 7C,E,G). By contrast, Fgf15 expression was detected caudal to the midbrain/hindbrain isthmus (arrow in Fig. 7C,E,G). Thus, Fgf15 expression is Shh dependent and, given its temporal and spatial expression, Fgf15 is well placed to participate in a signaling relay that connects ventral and dorsal regions of the diencephalon and midbrain.
|
Overexpression of Fgf15 expands the Tcf4 expression domain
These results suggest that Shh may regulate dorsal cell types in these regions through an Fgf15 relay. To test this model, we used electroporation of an Fgf15 expression construct into brain explants isolated from 14- to 16-somite stage mouse embryos to determine whether ectopic expression of Fgf15 would modulate Tcf4 expression. Electroporation was restricted to one side of the brain explant, providing an internal control for the ectopic expression of Fgf15 (Fig. 8A,B). Electroporation of a control vector expressing GFP into the diencephalic/midbrain region did not alter the Tcf4 expression domain (Fig. 8C), whereas overexpression of Fgf15 resulted in a robust expansion of the Tcf4 expression domain that was limited to the electroporated side of the explants (Fig. 8D). Furthermore, the hybridization signal was more intense on the electroporated side, suggesting that transcription of Tcf4 was also upregulated by Fgf15 (Fig. 8D). These results are consistent with the idea that Fgf15 regulates Tcf4 expression. Next, we examined whether expression of Fgf15 induces ectopic expression of Tcf4 by examining Bf1, Tcf4 and En1 expression. As described above, Bf1 is expressed in the telencephalic alar plates, Tcf4 in P1/2 alar plates and En1 in the middle/posterior midbrain. Thus, a gap between Bf1 and Tcf4 expression domains represents the P3 alar plate, and a gap between Tcf4 and En1 expression domains corresponds to the anterior midbrain. Comparable gaps were observed between these expression domains on the electroporated and control sides, suggesting that ectopic expression of Fgf15 leads to an expansion of the endogenous Tcf4 domain, rather than de novo activation of Fg15 in other brain regions (data not shown). Finally, to determine whether Fgf15 is sufficient to rescue Tcf4 expression we electroporated brain explants from Shh mutant embryos at the 10-somite stage. Although, GFP activity was visible within 8 hours post electroporation, we failed to observe any activation of Tcf4 or rescue of the diencephalic growth defect (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Shh signaling controls cell proliferation
Our morphological and marker analyses indicate that Shh is clearly essential for the dramatic growth of both dorsal and ventral regions of the diencephalic and anterior midbrain that occurs between the 14- and 25-somite stages. Furthermore, analysis of regional markers suggests that the primary patterning of these regions is not altered in Shh mutants, rather it is the subsequent failure of expansion of dorsal and ventral neural precursors that leads to a gross reduction in these brain regions. Consistent with this view, the diencephalon and anterior midbrain of Shh mutants showed a decreased BrdU incorporation rate at the 15- to 16-somite stages in comparison with adjacent telencephalic regions. In wild-type embryos, the anterior midbrain showed the highest level of Ccnd1 expression in the developing brain, while the diencephalon showed modest levels that correlate with the initiation of growth. The dramatic downregulation of Ccnd1 in the anterior midbrain and absence of activity in the diencephalic primordium of Shh mutants suggest that regulation of G1 cyclin activity is at least one mechanism by which Shh regulates growth of these brain primordia.
Ectopic expression studies have demonstrated that Shh can have a mitogenic role in the developing CNS (Rowitch et al., 1999). In particular in the cerebellum there is good evidence that Purkinje cell-supplied SHH is the principal mitogen for proliferation of cerebellar granule cell precursors (Dahmane and Ruiz i Altaba, 1999
; Wallace, 1999
; Wechsler-Reya and Scott, 1999
). Moreover, several G1 cyclins, including Ccnd1, are transcriptional targets of this Shh-mediated mitogenic response (Kenney and Rowitch, 2000
). Thus, it is reasonable to postulate that Shh may play a relatively direct role in regulating neural precursor proliferation in the diencephalic and midbrain regions. However, whereas this might be true in the basal plate in ventral regions, it is unlikely to be true for dorsally located precursors in the alar plate. Although recent evidence indicates that Shh may act directly over a distance of up to 300 µm (Lewis et al., 2001
), analysis of Shh target gene expression in diencephalic and midbrain anlagen provides no evidence of active Shh signaling in the dorsal half of these brain primordia. Thus, the evidence is more consistent with a Shh-dependent signaling relay controlling proliferation in dorsal regions.
Shh regulates Wnt and FGF signaling
Two other families of signaling factors have been implicated in the expansion of CNS precursor populations at early neural plate/neural tube stages: the Wnt and FGF families. Wnt1 and Wnt3a, either alone or in combination, are both necessary and sufficient for the expansion of CNS precursors in several regions of the developing CNS (McMahon and Bradley, 1990; Thomas and Capecchi, 1990
; McMahon et al., 1992
; Dickinson et al., 1994
; Ikeya et al., 1997
; Megason and McMahon, 2002
). Interestingly, in the absence of both Wnt1 and Wnt3a activities, there is a broad deficiency in both the diencephalon and midbrain that appears to result from a growth defect at early somite stages (S. M. Lee, M. I., S. Megason, S. Takada. and A. P. M., unpublished). Thus, whereas development of the midbrain is Wnt1 dependent (McMahon and Bradley, 1990
; Thomas and Capecchi, 1990
), diencephalic development is co-regulated by Wnt1 and Wnt3a, both of which are most likely secreted by cells at the dorsal midline (Parr et al., 1993
). Furthermore, characterization of the canonical Wnt-signaling pathway mediated by the Wnt1/Wnt3a class of ligand indicates that a transcriptional complex between ß-catenin and LEF/TCF factors is responsible for the activation of Wnt targets, one such target appears to be Ccnd1 (Tetsu and McCormick, 1999
; Shtutman et al., 1999
).
One member of the Lef/Tcf family, Tcf4 is expressed specifically in the alar plates of P1/2 at the 14-somite stage in wild-type embryos. This timing in expression correlates with the first appearance of a phenotype in the dorsal diencephalic region of Shh mutants. Interestingly, our results indicate that upregulation of Tcf4 is itself dependent on Shh signaling. Thus, the loss of Tcf4 activity might downregulate the response to dorsal Wnt1/3a signals, thereby contributing to a deficiency in the proliferation of dorsal diencephalic precursors. However, the absence of Tcf4 expression cannot by itself explain the diencephalic phenotype, as Tcf4 mutants do not exhibit a brain phenotype (Korinek et al., 1998). A possible functional redundancy amongst Lef/Tcf members that are more broadly expressed in the neural tube at this time could be a complicating factor (Galceran et al., 1999
). Furthermore, Tcf4 expression is restricted to the diencephalon but a similar phenotype is observed in the anterior midbrain. Signaling between P1 and the anterior midbrain has been proposed as a possible regulatory mechanism; however, the nature of this signaling is unclear. Interestingly, we observe that Dbx1 expression is absent in both the dorsal diencephalon and anterior midbrain of Shh mutants, suggesting that Dbx1 may act in some way to co-ordinate development of these brain regions, although the exact activity of Dbx1 has not been determined.
A key issue in our study is the molecular link between Shh ventrally and Tcf4 dorsally. Our data suggest that FGF15 could be one factor. Fgf15 is expressed ventrally in the diencephalon and midbrain at the appropriate time adjacent to the Shh expression domain. Fgf15 expression is clearly Shh dependent, although determining whether this regulation is direct or indirect will require a detailed analysis of the Fgf15 cis-regulatory regions. Given that Fgf15 shows differential expression at distinct anteroposterior positions of the developing neural tube, it is apparent that other Shh-independent regulatory controls must govern its precise spatial expression. FGF/Shh interactions have been demonstrated in other aspects of embryonic development, notably in the limb and lung. Shh is required for the maintenance of expression of several Fgfs in the apical ectodermal ridge of the developing limb bud (Laufer et al., 1994; Niswander et al., 1994
; Yang and Niswander, 1995
; Zuniga et al., 1999
; Lewis et al., 2001
) (reviewed by Martin, 1998
; Caruccio et al., 1999
; Kraus et al., 2001
) and for localization of Fgf10 in the lung bud (Pepicelli et al., 1998
). In the former, this regulation is mediated indirectly through a signaling relay (Zuniga et al., 1999
); in the latter, it is not clear whether regulation is direct or indirect.
Our data indicate that overexpression of Fgf15 leads to an apparent upregulation and expansion of the endogenous Tcf4 expression domain in wild-type embryos, but is insufficient to activate Tcf4 or rescue the proliferative deficiency in the diencephalon of Shh mutants when brain explants are electroporated at the 10-somite stage, prior to a visible diencephalic phenotype. Thus, while basal activation of Tcf4 is not Shh/FGF15-dependent (Fig. 4E), FGF15 may mediate a Shh signaling relay to dorsal regions to upregulate Tcf4 expression: however, additional factors are likely to be required for a wild-type response. The nature of these additional factors remains to be determined. Alternatively, there may be a narrow time window of FGF15 responsiveness that is not mimicked in the current set of experiments. Given that there is a dramatic visible reduction in the size of the diencephalic region that occurs over a seven-somite (12 hour) interval (Fig. 1F,H), this remains a possibility.
The molecular mechanism by which FGF15 signals is currently unclear. Mouse FGF15 is reported to be the ortholog of human FGF19, although the amino acid identity between them is significantly less (51%) than that observed between most human and mouse FGF orthologs (more than 90%) (Nishimura et al., 1999). Human FGF19 has been reported to bind exclusively to FGF receptor 4 in vitro (Xie et al., 1999
). However, Fgfr4 is not expressed in the brain at early somite stages. By contrast, Fgfr2 and Fgfr3 both localize to the diencephalic and midbrain primordia and display altered expression in Shh mutants. Given the high divergence between the mouse FGF15 and human FGF19, one possibility is that mouse FGF15 has a distinct receptor specificity from its suggested human counterpart. Signaling through these spatially restricted receptors might also explain the spatially restricted response to ectopic Fgf15 expression that was observed on electroporation of Fgf15 into brain explants.
FGFs have been known for sometime to act as both mitogens (Reynolds and Weiss, 1996; Gritti et al., 1996
; Lee et al., 1997
) and survival factors (Desire et al., 1998
; Learish et al., 2000
) for specific types of neural stem/precursor cells. However, the role of FGF signaling in the developing neural tube has only recently been investigated. Fgf8 is expressed specifically at the midbrain/hindbrain junction and expression is essential for the expansion of midbrain precursors (Crossley and Martin, 1995
; Crossley et al., 1996
; Meyers et al., 1998
). Although it is not clear how this expansion is effected, it is remarkable that this is the same target population that requires a Wnt1 input, pointing to additional links between FGF and Wnt signaling in the growth of specific brain primordia. FGF signaling is directed through tyrosine kinase receptors that are thought to activate various pathways, including mitogen-activated protein (MAP) kinase (reviewed by Boilly et al., 2000
), protein kinase C (Logan and Logan, 1991
; Hurley et al., 1996
) and signal transducers and activators of transcription (Su et al., 1997
). As both Myc and Ccnd1 are activated by a MAP-kinase cascade (Lavoie et al., 1996
; Aziz et al., 1999
), FGF signaling may independently regulate the cell cycle in the diencephalic/midbrain region. In this case, FGF15 and Wnt1/3a signaling may act cooperatively to regulate cell proliferation through the regulation of Ccnd1 and presumably other factors. With respect to this possibility, Wnt1 and MEK1 have been demonstrated to act cooperatively to inhibit glycogen synthase kinase-3ß activity leading to the accumulation of cyclin D1 (Rimerman et al., 2000
).
If FGF15 acts exclusively as a mitogen, an FGF15-mediated expansion of Tcf4-expressing cells, rather than a transcriptional expansion of the Tcf4 expression domain, could explain the observed increase in Tcf4 expression in wild-type brain explants ectopically expressing Fgf15. However, this model could not account for the observed upregulation of Tcf4 expression levels within its normal diencephalic domain in response to FGF15. Furthermore, the loss of Fgf15 expression and downregulation of Fgfr3 expression in Shh mutants occurs concomitant with a failure to elevate Tcf4 expression to normal levels prior to a detectable diencephalic growth defect.
In addition to decreased cell proliferation in the diencephalon and midbrain, we also observed an increased rate of cell death. This observation could reflect a link between mitogen activity and cell survival, or an alternative mechanism by which Shh enhances survival of neural precursors. Cell death in the neural tube has been associated with BMP4 signaling (Trousse et al., 2001; Graham et al., 1994
; Golden et al., 1999
). We observed ectopic upregulation of Bmp4 in the dorsal diencephalon and midbrain of Shh mutants but only after the 15-somite stage. Thus, ectopic Bmp4 expression is too late to account for the initial increase in cell death in Shh mutants but could play a role at later stages. Interestingly, BMP4 has been shown to suppress Dbx1 expression in vitro (Pierani et al., 1999
), thus, an upregulation in Bmp4 expression could account for the downregulation in Dbx1 expression observed at the 25-somite stage.
In summary, our data provide evidence that the proliferative activity of neural precursors within dorsal regions of the diencephalon and anterior midbrain is regulated in response to a signaling relay governed by the ventral activity of Shh. This result contrasts with the ventrally restricted actions of Shh in more caudal areas of the developing CNS. Interestingly, Shh mutants display a marked reduction in the development of dorsal telencephalic regions at later stages (26-somite, 9.5 dpc) that correlates with the downregulation of Bf1, a factor known to regulate the expansion of telencephalic precursors (Xuan et al., 1995). Thus, other regions of the forebrain might also rely upon a Shh-regulated relay to co-ordinate their growth.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Artinger, K. B. and Bronner-Fraser, M. (1993). Delayed formation of the floor plate after ablation of the avian notochord. Neuron 11, 1147-1161.[Medline]
Avivi, A., Zimmer, Y., Yayon, A., Yarden, Y. and Givol, D. (1991). Flg-2, a new member of the family of fibroblast growth factor receptors. Oncogene 6, 1089-1092.[Medline]
Aziz, N., Cherwinski, H. and McMahon, M. (1999). Complementation of defective colony-stimulating factor 1 receptor signaling and mitogenesis by Raf and v-Src. Mol. Cell. Biol. 19, 1101-1115.
Boilly, B., Vercoutter-Edouart, A. S., Hondermarck, H., Nurcombe, V. and le Bourhis, X. (2000). FGF signals for cell proliferation and migration through different pathways. Cytokine Growth Factor Rev. 11, 295-302.[CrossRef][Medline]
Borycki, A. G., Brunk, B., Tajbakhsh, S., Buckingham, M., Chiang, C. and Emerson, C. P., Jr (1999). Sonic hedgehog controls epaxial muscle determination through Myf5 activation. Development 126, 4053-4063.
Briscoe, J. and Ericson, J. (1999). The specification of neuronal identity by graded sonic hedgehog signalling. Semin. Cell Dev. Biol. 10, 353-362.[CrossRef][Medline]
Briscoe, J., Chen, Y., Jessell, T. M. and Struhl, G. (2001). A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube. Mol. Cell 7, 1279-1291.[CrossRef][Medline]
Caruccio, N. C., Martinez-Lopez, A., Harris, M., Dvorak, L., Bitgood, J., Simandl, B. K. and Fallon, J. F. (1999). Constitutive activation of sonic hedgehog signaling in the chicken mutant talpid(2): Shh-independent outgrowth and polarizing activity. Dev. Biol. 212, 137-149.[CrossRef][Medline]
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407-413.[CrossRef][Medline]
Cho, E. A. and Dressler, G. R. (1998). TCF-4 binds beta-catenin and is expressed in distinct regions of the embryonic brain and limbs. Mech. Dev. 77, 9-18.[CrossRef][Medline]
Crossley, P. H. and Martin, G. R. (1995). The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121, 439-451.
Crossley, P. H., Martinez, S. and Martin, G. R. (1996). Midbrain development induced by FGF8 in the chick embryo. Nature 380, 66-68.[CrossRef][Medline]
Dahmane, N. and Ruiz-i-Altaba, A. (1999). Sonic hedgehog regulates the growth and patterning of the cerebellum. Development 126, 3089-3100.
Dale, J. K., Vesque, C., Lints, T. J., Sampath, T. K., Furley, A., Dodd, J. and Placzek, M. (1997). Cooperation of BMP7 and SHH in the induction of forebrain ventral midline cells by prechordal mesoderm. Cell 90, 257-269.[Medline]
Davis, C. A., Holmyard, D. P., Millen, K. J. and Joyner, A. L. (1991). Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum. Development 111, 287-298.[Abstract]
Davis, C. A. and Joyner, A. L. (1988). Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development. Genes Dev 2, 1736-1744.[Abstract]
Desire, L., Head, M. W., Fayein, N. A., Courtois, Y. and Jeanny, J. C. (1998). Suppression of fibroblast growth factor 2 expression by antisense oligonucleotides inhibits embryonic chick neural retina cell differentiation and survival in vivo. Dev. Dyn. 212, 63-74.[CrossRef][Medline]
Dickinson, M. E., Krumlauf, R. and McMahon, A. P. (1994). Evidence for a mitogenic effect of Wnt-1 in the developing mammalian central nervous system. Development 120, 1453-1471.
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75, 1417-1430.[Medline]
Furuta, Y., Piston, D. W. and Hogan, B. L. (1997). Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development. Development 124, 2203-2212.
Galceran, J., Farinas, I., Depew, M. J., Clevers, H. and Grosschedl, R. (1999). Wnt3a/-like phenotype and limb deficiency in Lef1/Tcf1/ mice. Genes Dev. 13, 709-717.
Golden, J. A., Bracilovic, A., McFadden, K. A., Beesley, J. S., Rubenstein, J. L. and Grinspan, J. B. (1999). Ectopic bone morphogenetic proteins 5 and 4 in the chicken forebrain lead to cyclopia and holoprosencephaly. Proc. Natl. Acad. Sci. USA 96, 2439-2444.
Goodrich, L. V., Milenkovic, L., Higgins, K. M. and Scott, M. P. (1997). Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277, 1109-1113.
Graham, A., Francis-West, P., Brickell, P. and Lumsden, A. (1994). The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest. Nature 372, 684-686.[CrossRef][Medline]
Grindley, J. C., Hargett, L. K., Hill, R. E., Ross, A. and Hogan, B. L. (1997). Disruption of PAX6 function in mice homozygous for the Pax6Sey-1Neu mutation produces abnormalities in the early development and regionalization of the diencephalon. Mech. Dev. 64, 111-126.[CrossRef][Medline]
Gritti, A., Parati, E. A., Cova, L., Frolichsthal, P., Galli, R., Wanke, E., Faravelli, L., Morassutti, D. J., Roisen, F., Nickel, D. D. and Vescovi, A. L. (1996). Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J. Neurosci. 16, 1091-1100.[Abstract]
Hurley, M. M., Marcello, K., Abreu, C. and Kessler, M. (1996). Signal transduction by basic fibroblast growth factor in rat osteoblastic Py1a cells. J. Bone Miner. Res. 11, 1256-1263.[Medline]
Ikeya, M., Lee, S. M., Johnson, J. E., McMahon, A. P. and Takada, S. (1997). Wnt signalling required for expansion of neural crest and CNS progenitors. Nature 389, 966-970.[CrossRef][Medline]
Ingham, P. W. and McMahon, A. P. (2001). Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 15, 3059-3087.
Inoue, T., Nakamura, S. and Osumi, N. (2000). Fate mapping of the mouse prosencephalic neural plate. Dev. Biol. 219, 373-383.[CrossRef][Medline]
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20-29.[CrossRef][Medline]
Jostes, B., Walther, C. and Gruss, P. (1990). The murine paired box gene, Pax7, is expressed specifically during the development of the nervous and muscular system. Mech. Dev. 33, 27-37[CrossRef][Medline]
Kenney, A. M. and Rowitch, D. H. (2000). Sonic hedgehog promotes G(1) cyclin expression and sustained cell cycle progression in mammalian neuronal precursors. Mol. Cell Biol. 20, 9055-9067.
Korinek, V., Barker, N., Morin, P. J., van Wichen, D., de Weger, R., Kinzler, K. W., Vogelstein, B. and Clevers, H. (1997). Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC/ colon carcinoma. Science 275, 1784-1787.
Korinek, V., Barker, N., Moerer, P., van Donselaar, E., Huls, G., Peters, P. J. and Clevers, H. (1998). Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat. Genet. 19, 379-383.[CrossRef][Medline]
Kraus, P., Fraidenraich, D. and Loomis, C. A. (2001). Some distal limb structures develop in mice lacking Sonic hedgehog signaling. Mech. Dev. 100, 45-58.[CrossRef][Medline]
Krumlauf, R., Marshall, H., Studer, M., Nonchev, S., Sham, M. H. and Lumsden, A. (1993). Hox homeobox genes and regionalisation of the nervous system. J. Neurobiol. 24, 1328-1340.[Medline]
Laufer, E., Nelson, C. E., Johnson, R. L., Morgan, B. A. and Tabin, C. (1994). Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell 79, 993-1003.[Medline]
Lavoie, J. N., LAllemain, G., Brunet, A., Muller, R. and Pouyssegur, J. (1996). Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J. Biol. Chem. 271, 20608-20616.
Learish, R. D., Bruss, M. D. and Haak-Frendscho, M. (2000). Inhibition of mitogen-activated protein kinase kinase blocks proliferation of neural progenitor cells. Dev. Brain Res. 122, 97-109.[Medline]
Lee, K. J. and Jessell, T. M. (1999). The specification of dorsal cell fates in the vertebrate central nervous system. Annu. Rev. Neurosci. 22, 261-294.[CrossRef][Medline]
Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997). Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain. Development 124, 959-969.
Lee, S. M., Tole, S., Grove, E. and McMahon, A. P. (2000). A local Wnt-3a signal is required for development of the mammalian hippocampus. Development 127, 457-467.
Lewis, P. M., Dunn, M. P., McMahon, J. A., Logan, M., Martin, J. F., St-Jacques, B. and McMahon, A. P. (2001). Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1. Cell 105, 599-612.[CrossRef][Medline]
Litingtung, Y. and Chiang, C. (2000). Specification of ventral neuron types is mediated by an antagonistic interaction between Shh and Gli3. Nat. Neurosci. 3, 979-985.[CrossRef][Medline]
Logan, A. and Logan, S. D. (1991). Studies on the mechanisms of signalling and inhibition by pertussis toxin of fibroblast growth factor-stimulated mitogenesis in Balb/c 3T3 cells. Cell. Signal. 3, 215-223.[CrossRef][Medline]
Lumsden, A. and Krumlauf, R. (1996). Patterning the vertebrate neuraxis. Science 274, 1109-1115.
Mansukhani, A., DellEra, P., Moscatelli, D., Kornbluth, S., Hanafusa, H. and Basilico, C. (1992). Characterization of the murine BEK fibroblast growth factor (FGF) receptor: activation by three members of the FGF family and requirement for heparin. Proc. Natl. Acad. Sci. USA 89, 3305-3309.[Abstract]
Martin, G. R. (1998). The roles of FGFs in the early development of vertebrate limbs. Genes Dev. 12, 1571-1586.
Mastick, G. S., Davis, N. M., Andrew, G. L. and Easter, S. S., Jr (1997). Pax-6 functions in boundary formation and axon guidance in the embryonic mouse forebrain. Development 124, 1985-1997.
McMahon, A. P. and Bradley, A. (1990). The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62, 1073-1085.[Medline]
McMahon, A. P., Joyner, A. L., Bradley, A. and McMahon, J. A. (1992). The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell 69, 581-595.[Medline]
McWhirter, J. R., Goulding, M., Weiner, J. A., Chun, J. and Murre, C. (1997). A novel fibroblast growth factor gene expressed in the developing nervous system is a downstream target of the chimeric homeodomain oncoprotein E2A-Pbx1. Development 124, 3221-3232.
Megason, S. and McMahon, A. P. (2002). A mitogen gradient of dorsal midline Wnts organizes growth in the CNS. Development 129, 2087-2098.
Meyers, E. N., Lewandoski, M. and Martin, G. R. (1998). An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombinations. Nat. Genet. 18, 136-141.[CrossRef][Medline]
Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destree, O. and Clevers, H. (1996). XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86, 391-399.[Medline]
Morin, P. J., Sparks, A. B., Korinek, V., Barker, N., Clevers, H., Vogelstein, B. and Kinzler, K. W. (1997). Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275, 1787-1790.
Nishimura, T., Utsunomiya, Y., Hoshikawa, M., Ohuchi, H. and Itoh, N. (1999). Structure and expression of a novel human FGF, FGF-19, expressed in the fetal brain. Biochim Biophys Acta 1444, 148-151.[Medline]
Niswander, L., Jeffrey, S., Martin, G. R. and Tickle, C. (1994). A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371, 609-612.[CrossRef][Medline]
Ornitz, D. M. and Itoh, N. (2001). Fibroblast growth factors. Genome Biol. 2, 3005.1-3005.12.
Parr, B. A., Shea, M. J., Vassileva, G. and McMahon, A. P. (1993). Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds. Development 119, 247-261.
Pepicelli, C. V., Lewis, P. M. and McMahon, A. P. (1998). Sonic hedgehog regulates branching morphogenesis in the mammalian lung. Curr. Biol. 8, 1083-1086.[Medline]
Pierani, A., Brenner-Morton, S., Chiang, C. and Jessell, T. M. (1999). A sonic hedgehog-independent, retinoid-activated pathway of neurogenesis in the ventral spinal cord. Cell 97, 903-915.[Medline]
Placzek, M., Tessier-Lavigne, M., Yamada, T., Jessell, T. and Dodd, J. (1990). Mesodermal control of neural cell identity: floor plate induction by notochord. Science 250, 985-988.[Medline]
Placzek, M., Jessell, T. and Dodd, J. (1993). Induction of floor plate differentiation by contact-dependent, homeogenetic signals. Development 117, 205-218.
Puelles, L. and Rubenstein, J. L. (1993). Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosci. 16, 472-479.[CrossRef][Medline]
Raffel, C., Jenkins, R. B., Frederick, L., Hebrink, D., Alderete, B., Fults, D. W. and James, C. D. (1997). Sporadic medulloblastomas contain PTCH mutations. Cancer Res. 57, 842-845.[Abstract]
Reid, H. H., Wilks, A. F. and Bernard, O. (1990). Two forms of the basic fibroblast growth factor receptor-like mRNA are expressed in the developing mouse brain. Proc. Natl. Acad. Sci. USA 87, 1596-1600.[Abstract]
Reynolds, B. A. and Weiss, S. (1996). Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev. Biol. 175, 1-13.[CrossRef][Medline]
Rimerman, R. A., Gellert-Randleman, A. and Diehl, J. A. (2000). Wnt1 and MEK1 cooperate to promote cyclin D1 accumulation and cellular transformation. J. Biol. Chem. 275, 14736-14742.
Rowitch, D. H., St-Jacques, B., Lee, S. M., Flax, J. D., Snyder, E. Y. and McMahon, A. P. (1999). Sonic hedgehog regulates proliferation and inhibits differentiation of CNS precursor cells. J. Neurosci. 19, 8954-8965.
Rubenstein, J. L. and Puelles, L. (1994). Homeobox gene expression during development of the vertebrate brain. Curr. Top. Dev. Biol. 29, 1-63.[Medline]
Sherr, C. J. and Roberts, J. M. (1999). CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13, 1501-1512.
Shimamura, K., Hartigan, D. J., Martinez, S., Puelles, L. and Rubenstein, J. L. (1995). Longitudinal organization of the anterior neural plate and neural tube. Development 121, 3923-3933.
Shoji, H., Ito, T., Wakamatsu, Y., Hayasaka, N., Ohsaki, K., Oyanagi, M., Kominami, R., Kondoh, H. and Takahashi, N. (1996). Regionalized expression of the Dbx family homeobox genes in the embryonic CNS of the mouse. Mech. Dev. 56, 25-39.[CrossRef][Medline]
Shtutman, M., Zhurinsky, J., Simcha, I., Albanese, C., DAmico, M., Pestell, R. and Ben-Zeev, A. (1999). The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc. Natl. Acad. Sci. USA 96, 5522-5527.
Stark, K. L., McMahon, J. A. and McMahon, A. P. (1991). FGFR-4, a new member of the fibroblast growth factor receptor family, expressed in the definitive endoderm and skeletal muscle lineages of the mouse. Development 113, 641-651.[Abstract]
St-Jacques, B., Dassule, H. R., Karavanova, I., Botchkarev, V. A., Li, J., Danielian, P. S., McMahon, J. A., Lewis, P. M., Paus, R. and McMahon, A. P. (1998). Sonic hedgehog signaling is essential for hair development. Curr. Biol. 8, 1058-1068.[Medline]
Su, W. C., Kitagawa, M., Xue, N., Xie, B., Garofalo, S., Cho, J., Deng, C., Horton, W. A. and Fu, X. Y. (1997). Activation of Stat1 by mutant fibroblast growth-factor receptor in thanatophoric dysplasia type II dwarfism. Nature 386, 288-292.[CrossRef][Medline]
Tanabe, Y. and Jessell, T. M. (1996). Diversity and pattern in the developing spinal cord. Science 274, 1115-1123.
Tessier-Lavigne, M., Placzek, M., Lumsden, A. G. S., Dodd, J. and Jessell, T. M. (1987). Chemotropic guidance of developing axons in the mammalian central nervous system. Nature 336, 775-778.[CrossRef]
Tetsu, O. and McCormick, F. (1999). Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398, 422-426.[CrossRef][Medline]
Thomas, K. R. and Capecchi, M. R. (1990). Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature 346, 847-850.[CrossRef][Medline]
Trousse, F., Esteve, P. and Bovolenta, P. (2001). Bmp4 mediates apoptotic cell death in the developing chick eye. J. Neurosci. 21, 1292-1301.
Van Straaten, H. W., Hekking, J. W., Beursgens, J. P., Terwindt-Rouwenhorst, E. and Drukker, J. (1989). Effect of the notochord on proliferation and differentiation in the neural tube of the chick embryo. Development 107, 793-803.[Abstract]
Van Straaten, H. W. and Heckking, J. W. (1991). Development of the floo plate, neurons and axonal outgrowth pattern in the early spinal cord of the notochord deficient chick embryo. Anat. Embryol. 184, 55-63.[Medline]
Vorechovsky, I., Tingby, O., Hartman, M., Stromberg, B., Nister, M., Collins, V. P. and Toftgard, R. (1997). Somatic mutations in the human homologue of Drosophila patched in primitive neuroectodermal tumours. Oncogene 15, 361-366.[CrossRef][Medline]
Wallace, V. A. (1999). Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum. Curr. Biol. 9, 445-448.[CrossRef][Medline]
Walshe, J. and Mason, I. (2000). Expression of FGFR1, FGFR2 and FGFR3 during early neural development in the chick embryo. Mech. Dev. 90, 103-110.[CrossRef][Medline]
Warren, N. and Price, D. J. (1997). Roles of Pax-6 in murine diencephalic development. Development 124, 1573-1582.
Wechsler-Reya, R. J. and Scott, M. P. (1999). Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 22, 103-114.[Medline]
Wilkinson, D. G. (1993). Molecular mechanisms of segmental patterning in the vertebrate hindbrain and neural crest. BioEssays 15, 499-505.[Medline]
Xie, M. H., Holcomb, I., Deuel, B., Dowd, P., Huang, A., Vagts, A., Foster, J., Liang, J., Brush, J., Gu, Q. et al. (1999). FGF-19, a novel fibroblast growth factor with unique specificity for FGFR4. Cytokine 11, 729-735.[CrossRef][Medline]
Xuan, S., Baptista, C. A., Balas, G., Tao, W., Soares, V. C. and Lai, E. (1995). Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres. Neuron 14, 1141-1152.[Medline]
Yamada, T., Placzek, M., Tanaka, H., Dodd, J. and Jessell, T. M. (1991). Control of cell pattern in the developing nervous system: polarizing activity of the floor plate and notochord. Cell 64, 635-647.[Medline]
Yang, Y. and Niswander, L. (1995). Interaction between the signaling molecules WNT7a and SHH during vertebrate limb development: dorsal signals regulate anteroposterior patterning. Cell 80, 939-947.[Medline]
Ye, W., Shimamura, K., Rubenstein, J. L., Hynes, M. A. and Rosenthal, A. (1998). FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 93, 755-766.[Medline]
Zuniga, A., Haramis, A. P., McMahon, A. P. and Zeller, R. (1999). Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds. Nature 401, 598-602.[CrossRef][Medline]