Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 May 17.
Published in final edited form as: Dev Cell. 2011 May 17;20(5):689–699. doi: 10.1016/j.devcel.2011.04.010

β-catenin-dependent FGF signaling sustains cell survival in the anterior embryonic head by countering Smad4

Hunki Paek 2,3, Jee-Yeon Hwang 1, R Suzanne Zukin 1, Jean M Hébert 1,2,*
PMCID: PMC3099549  NIHMSID: NIHMS293135  PMID: 21571225

SUMMARY

Growing evidence suggests that FGFs secreted from embryonic signaling centers are key mediators of cell survival. However, the mechanisms regulating FGF-dependent cell survival remain obscure. At the rostral end of the embryo, for example, ablation of FGF signaling leads to the rapid death of the precursor cells that form the anterior head, including the telencephalon. Here we outline a core genetic circuit that regulates survival in the embryonic mouse head: WNT signaling through β-catenin directly maintains FGF expression and requires FGF function in vivo to oppose pro-apoptotic TGFβ signaling through SMAD4. Moreover, these antagonistic pathways converge on the transcriptional regulation of apoptosis, and genes such as Cdkn1a, suggesting a mechanism for how signaling centers in the embryonic head regulate cell survival.

INTRODUCTION

Signaling centers, small groups of cells at the edge of tissues that secrete inductive factors, play key roles in the development of many organs across species. In cases where fibroblast growth factors (FGFs) mediate the activity of a signaling center, for example in the emerging mid-hindbrain, limbs, and branchial arches, ablation of FGF signaling can result in massive cell death and loss of the tissue (Chi et al., 2003; Mariani et al., 2008; Trumpp et al., 1999). The requirement for FGF signaling in certain early tissues indicates that the survival of their precursor cells is not a default state, but needs to be actively maintained. It is thus critical to decipher what factors regulate levels of FGF signaling and what factors act downstream of FGFs to maintain cell survival in these tissues.

Here we focus on the anterior-most region of the embryo, which also depends on FGF signaling for cell survival (Paek et al., 2009). The anterior end of the embryo forms the rostral craniofacial tissues and the telencephalon, precursor to the cerebral hemispheres. This part of the embryo is induced by a signaling center located at the ridge between the anterior most neuroectoderm and underlying ectoderm. This ridge is required to maintain the survival of anterior neural precursor cells and it expresses several FGF ligands, including Fgf8 (Wilson and Houart, 2004; Hébert and Fishell, 2008). At least in zebrafish, this ridge also expresses the Winglessint (WNT) antagonist tlc which like the ridge induces telencephalic character, perhaps by regulating FGF8, which itself can rescue telencephalic character in the absence of a ridge (Houart et al., 2002; Houart et al., 1998; Shimamura and Rubenstein, 1997). However, knocking out Fgf8 in the ridge does not lead to loss of the telencephalon and surrounding craniofacial tissue, probably due to compensation by the other Fgf genes. Consistent with this possibility, ablating FGF signaling in the anterior embryo by deleting the three expressed FGF receptor genes results in massive cell death and a loss of the telencephalon and surrounding craniofacial tissue (Paek et al., 2009).

Early development of the anterior embryo also involves other secreted signals that are either mitogenic, such as WNTs, or cytostatic and potentially apoptotic, such as members of the TGFβ superfamily (Wilson and Houart, 2004). Although these pathways are essential for patterning telencephalic neuroectoderm, their roles, if any, in regulating cell survival are unknown. Moreover, how they might interact with FGFs in this process is also unclear. In this study, we show that β-catenin-mediated WNT signaling is indirectly required for early survival of telencephalic precursor cells by directly promoting FGF expression and function. Moreover, the cell death observed with loss of β-catenin and FGF activity is rescued by deleting Smad4, a mediator of TGFβ signaling. Cdkn1a (p21Cip1) is a gene that is regulated by SMAD signaling and that promotes cell cycle arrest and apoptosis in other cell types (Gartel and Tyner, 2002; Seoane et al., 2004). Interestingly, expression of Cdkn1a correlates with cell death in the different mutants analyzed here. Moreover, we find that Cdkn1a expression is regulated in part by β-catenin mediated suppression of SMAD binding to a cis activating element upstream of Cdkn1a. These results provide a core circuit for how precursor cells in the anterior embryonic head integrate and respond to multiple pro-survival and apoptotic signals.

RESULTS

β-catenin-mediated WNT signaling is required for cell survival in the anterior embryo

In vertebrates, a lack of canonical WNT signaling in the anterior neural plate is necessary for telencephalon induction (Wilson and Houart, 2004). Consistent with this view, no WNT signaling can be detected at the time of telencephalon induction in the anterior neural plate of mouse embryos that carry a transgenic reporter gene for canonical WNT signaling, Tcf-lacZ (Mohamed et al., 2004), and are stained with X-gal at the 7 somite stage (Supp. Fig. 1A). However, Tcf-lacZ expression is detected in telencephalic tissue at the 10 somite stage subsequent to induction of the telencephalic marker Foxg1 and Tcf-lacZ expression increases until at least the 16–18 somite stage throughout the telencephalon with strongest staining in the presumptive dorsal regions (Fig. 1A, Supp. Fig. 1A,B). In embryos that do not carry Tcf-lacZ, no X-gal staining is detected (Supp. Fig. 1A). The timing of Tcf-lacZ expression coincides with the onset of WNT ligand expression in the telencephalon and suggests that although WNT signaling initially inhibits telencephalon induction, it may shortly thereafter play a role in the normal development of this tissue (Lee et al., 2000).

Figure 1. β-catenin-mediated WNT signaling is required for cell survival in the anterior embryo.

Figure 1

Open in a new tab

(A) X-gal staining of 16 somite stage Tcf-lacZ mouse embryo. Tcf-lacZ expression is detected throughout the telencephalon with strongest staining in the presumptive dorsal regions (n=3/3). Scale bar: 500 μm.

(B) The telencephalic hemispheres are apparent in E10.5 controls but not Ctnnb1cko/cko mutants in which all anterior tissue is truncated (arrowhead). Scale bar: 500 μm.

(C) Serial horizontal sections from 16–18 somite stage control and Ctnnb1cko/cko mutant embryos (anterior up). The areas between the arrows are Foxg1-expressing telencephalic precursors, as determined by in situ hybridization of serial sections (data not shown). TUNEL and activated Caspase-3 immunostaining revealed little staining (red) outside of the midline in controls (n=3), whereas in mutants (n=4) 20–30% of telencephalic precursor cells were positive (***: p < 0.0001 for TUNEL and p = 0.0002 for Casp3, mean±SEM). Hoechst counterstain (blue). Scale bar: 100 μm.

(D) qRT-PCR analysis of the Tcf-lacZ transcript from 16–18 somite stage telencephalons from control (n=5) and Ctnnb1cko/cko mutant (n=5) embryos. Ctnnb1cko/cko mutants had a 58% decrease in lacZ RNA compared with controls (p = 0.0028, mean±SEM). Primers to Gapdh and Hprt1 were used to normalize levels of RNA.

To address the role of WNT signaling in the anterior neural plate subsequent to telencephalon induction, we deleted from anterior precursor cells at the ~13 somite stage the Ctnnb1 gene encoding β-catenin, a central intracellular mediator of canonical WNT signaling. Mice carrying a conditional null allele of Ctnnb1 in which exons 2 through 6 are floxed (Ctnnb1cko; Brault et al., 2001) were crossed to mice carrying the Foxg1Cre allele with which efficient recombination of floxed alleles occurs reproducibly between the 10 and 16 somite stages in anterior head precursor cells (Hébert and McConnell, 2000; Paek et al., 2009; Storm et al., 2006). In all experiments described here, Foxg1Cre-carrying heterozygous littermates served as negative controls. Mutants were recovered at mendelian ratios until E12.5 and at least some made it to birth but died soon afterward presumably due to a failure to nurse. Mutants underwent normal induction of the telencephalic marker Foxg1 (Supp. Fig. 1B; Supp. Fig. 2A), but at E10.5 when the bilateral hemispheres of the telencephalon are apparent in controls, no telencephalic structure could be identified in the mutants (Fig. 1B). The loss of telencephalic tissue was completely penetrant and essentially invariable in its severity (n = 55/55 mutants).

Cell death and proliferation were examined as potential causes for the loss of telencephalic tissue. TUNEL and activated Caspase-3 immunostaining revealed that 20–30% of telencephalic precursor cells were undergoing cell death at once in 16–18 somite stage mutants whereas little cell death could be detected in controls except in the midline where it normally occurs (Fig. 1C; Supp. Fig. 2A). Cell death is also observed in the ectoderm and mesoderm surrounding the telencephalon in mutants accounting for the truncation of all anterior tissues. Despite the relatively high percentage of dying cells, the number of telencephalic cells positive for the mitotic marker phospho-histone H3 (p-HH3) and the general proliferative marker Ki67 are unaffected at the 16–18 somite stage (Supp. Fig. 2B and data not shown). The number of P-HH3+ telencephalic cells is also unaffected 4–5 hours earlier at the 13 somite stage just prior to a detectable increase in cell death (Supp. Fig. 2D). This suggests that proliferation may be unaffected, although changes in some cell cycle parameters, which can not be readily measured due to the short time frame between recombination of Ctnnb1 and cell death, are still possible. A lack of change in the numbers of P-HH3+ cells is also consistent with the possibility that cells are becoming post-mitotic immediately before or as they die. Nevertheless, the truncation and loss of anterior tissue is due to cell death.

As well as a central role in canonical WNT signaling, β-Catenin also plays an important role in cell adhesion (Nelson and Nusse, 2004). Hence it is possible that the cell death observed in the Ctnnb1cko/cko mutant is due to the dissociation of cells from the neuroepithelium as cell adhesion becomes impaired, as previously suggested (Junghans et al., 2005). However, a crucial role for canonical WNT signaling in maintaining cell survival is apparent from the experiments presented here. First, at the 16–18 somite stage, although telencephalic cells are dying in large numbers, they have not yet dissociated from the neuroepithelium, which maintains a normal apical structure based on immunostaining for PAR3 and ZO1, two tight junction markers (Fig. 2B–D). Strikingly, immunostaining for β-catenin itself reveals that this protein is still present at the apical surface in mutants, similar to controls, suggesting that at this subcellular location it is more stable than in the cytosol or nucleus where it normally mediates WNT signaling and where levels are difficult to detect with this antibody in either controls or mutants (Fig. 2A–A″).

Figure 2. Cell adhesion is maintained at a time when precursor cells are dying.

Figure 2

Open in a new tab

(A) Anti-β-catenin immunostaining prominently labels the apical surface of telencephalic neuroepithelial cells (arrowheads in A″) in 17 somite stage control (n=3/3) and Ctnnb1cko/cko mutant (n=3/3) embryos. A″ is a higher magnification of the boxed area in A′, which is a higher magnification of the boxed area in A. Scale bar: 100 μm.

(B–C) Likewise, immunostaining for ZO-1 and PAR3 is indistinguishable in serial sections in control (n=3/3) and mutant (n=3/3) embryos. Scale bar: 50 μm.

(D) Enlarged views of (C) showing only blue Hoechst staining. Shrunken nuclei with intense staining can be observed throughout the telencephalic neuroepithelium of mutants only (arrows), indicative of apoptosis.

Second, a decrease in canonical WNT signaling in the early telencephalon of the Foxg1Cre;Ctnnb1cko/cko mutants was confirmed by assessing levels of the Tcf-lacZ transcript, which should only be produced when a nuclear TCF/LEF/β-catenin complex binds the Tcf regulatory elements and activates expression of lacZ. Real time quantitative RT-PCR (qRT-PCR) on telencephalic tissue dissected from 16–18 somite stage embryos revealed that mutants had a ~58% decrease in lacZ RNA compared with controls (Fig. 1D), consistent with a loss of canonical signaling. Conversely, conditional expression of a stabilized β-catenin allele, Ctnnb1Δex3 in which exon 3 that encodes the residues that trigger protein degradation is floxed (Harada et al., 1999), led to a 5 fold increase in lacZ RNA levels (Supp. Fig. 2C). Therefore, Tcf-lacZ RNA levels reflect levels of β-catenin activity in the canonical WNT pathway, which mediates gene transcription. Note that the β-galactosidase protein expressed from the Tcf-lacZ reporter in the Ctnnb1cko/cko mutants is too stable to report real time decreases in canonical WNT signaling with either X-gal or anti-β-gal staining within the relevant time frame (less than 12 hours between the initiation of recombination at 10 somites and the loss of tissue at 18 somites) (data not shown).

Third, the cell death phenotype observed in Ctnnb1cko/cko mutant embryos could be recapitulated using WNT inhibitors in cultures of telencephalic explants. Whole telencephalic tissue was dissected from 16–18 somite stage Tcf-lacZ embryos and cultured with or without the WNT ligand inhibitors DKK1 or sFRP1. Application of either inhibitor resulted in between 30–40% of TUNEL-positive cells after 48 hours, a greater than 10-fold increase compared to control explants (Fig. 3A,B). This increase in cell death coincided with a ~60% decrease in levels of lacZ mRNA, suggesting that a substantial decrease in WNT signaling is responsible for cell death (Fig. 3C). Together these results indicate that a reduction of canonical WNT signaling causes cell death in telencephalic precursors. Moreover, the experiments presented in the sections below reveal that β-catenin promotes cell survival by transcriptionally activating and repressing pro-survival and apoptotic genes, respectively, rather than maintaining cell adhesion.

Figure 3. Inhibition of WNT activity causes telencephalic cell death.

Figure 3

Open in a new tab

(A) TUNEL staining of vehicle-treated (control), DKK1-treated (1μg/ml) and sFRP1-treated (1μg/ml) telencephalic explants after 48h culture. Scale bar: 100 μm.

(B) Application of both DKK1 (n=4) and sFRP1 (n=3) resulted in 30–40% of cells dying, a greater than 10-fold increase compared to control explants (n=3). Quantification of cell death was obtained by counting all cells in all sections of at least three explants of each type, excluding cells within 25 μm from the edges of the explants (compared with control: p = 0.0021 for DKK1 and 0.0002 for sFRP1, mean±SEM).

(C) qRT-PCR analysis of the Tcf-lacZ transcript from vehicle-treated control (n=5) and DKK1-treated explants (n=4) reveals a 60% decrease in levels of lacZ RNA (**: p< 0.01, mean±SEM).

β-catenin is required to promote FGF signaling

The phenotype of the Foxg1Cre;Ctnnb1cko/cko mutants, loss of the telencephalon and surrounding craniofacial structures due to rapid cell death (Fig. 1B,C), bears a striking similarity to the phenotype obtained by conditionally deleting three FGF receptor genes using the same Foxg1Cre driver (Paek et al., 2009). The similarity in phenotypes suggests a possible regulatory interaction between β-catenin and FGF signaling in the early telencephalon. Abolishing FGF signaling was previously shown not to affect expression of WNT ligand genes in the dorsal telencephalic midline (Paek et al., 2009). However, the converse, that β-catenin regulates expression of FGF ligand genes, remained possible.

Several FGF ligand genes, including Fgf8, Fgf17, and Fgf18, are expressed at the anterior tip of the early telencephalon (Maruoka et al., 1998). By RNA in situ hybridization on whole 16–18 somite stage embryos, expression of Fgf8 at the anterior tip of the embryo was greatly decreased or absent in Ctnnb1cko/cko mutants (Fig. 4A). Conversely, expression became wide spread in the telencephalic neuroepithelium of Ctnnb1+/Δex3 mutants, most dramatically as observed in E10.0 sections (Fig. 4B). Even later in development, conditional expression of Ctnnb1Δex3 using a dorsal telencephalic Cre driver, Emx1Cre (Gorski et al., 2002), resulted in ectopic cortical Fgf8 expression (data not shown).

Figure 4. β-catenin directly promotes FGF signaling.

Figure 4

Open in a new tab

(A) Whole mount RNA in situ hybridization of 16–18 somite stage embryos shows a decrease in Fgf8 expression (purple) at the anterior tip of the embryo (arrows) in Ctnnb1cko/cko mutants. Scale bar: 500 μm.

(B) RNA in situ hybridization of control and Ctnnb1+/Δex3 embryos on coronal and sagittal sections using a radioactive Fgf8 probe. Fgf8 expression (white) is wide spread in the telencephalic neuroepithelium (arrowhead) of Ctnnb1+/Δex3 mutants compared to the restricted midline expression in controls. t: telencephalon. Scale bar: 500 μm.

(C) qRT-PCR analyses indicate that not only is Fgf gene expression reduced with loss of β-catenin/WNT signaling, but also expression of Spry1, a downstream marker for signaling. RNA levels for Fgf8, Fgf17, and Fgf18 are significantly decreased (~50%) in both the Ctnnb1cko/cko telencephalon (left; n=4 each for control and mutant) and cultured telencephalic explants treated with DKK1 after 24 and 48 hours (right; n=5 each for control and mutant); p values are between 0.0006 and 0.08, and between 0.008 and 0.03, respectively, mean±SEM. The RNA level of Spry1 is also reduced by 55% in Ctnnb1cko/cko mutants (p = 0.002, mean±SEM; mutant: n=4, control: n=3). Conversely, levels of Fgf and Spry1 expression are increased by 150 – 260% in Ctnnb1+/Δex3 mutants (p between 0.01 and 0.001, mean±SEM).

(D) μChIP assay with β-catenin antibody followed by qPCR for control and Tcf-lef binding site regions of Fgf8 (left) and Fgf18 (right) using telencephalons from wildtype 16–18 somite stage embryos (difference compared with Gapdh: p=0.03 and 0.004, respectively, mean±SEM). Telencephalons from 20 and 30 embryos were used to perform 4 and 6 independent μChIP experiments for Fgf8 and Fgf18, respectively (5 embryos per assay).

(E) Luciferase activity was measured in dissociated 16–18 somite stage telencephalic cells from control (n=6) and Ctnnb1+/Δex3 (n=3) embryos. Cells were transfected with Fgf18 reporter constructs after 16 hours in culture. Activity was 3.2 fold higher in Ctnnb1+/Δex3 cells with the wildtype construct than either control cells with the wildtype construct or Ctnnb1+/Δex3 cells with the mutated construct (p=0.03, mean±SEM).

Importantly, at the 16–18 somite stage when β-catenin is required for cell survival (Fig. 1), RNA levels for Fgf8, Fgf17, and Fgf18 measured using qRT-PCR are significantly decreased in the Ctnnb1cko/cko telencephalon (Fig. 4C; ~50% decrease in all cases; note that differences observed by qRT-PCR are likely underestimates due to mispriming). Similar drops in Fgf8, Fgf17, and Fgf18 expression are observed in cultured telencephalic explants treated with DKK1, confirming that reduction in expression is due to loss of WNT signaling (Fig. 4C). Fgf gene expression is decreased in these explants after 48 hours (when cell death is easily detected, Fig. 3, 4C) and even after only 24 hours (a time point that precedes a detectable increase in cell death; Fig. 4C and data not shown), consistent with a causal relation between loss of FGF signaling and cell death. Moreover, the RNA level of Spry1, a gene whose transcription is induced by FGFs (Mason, 2007), is also reduced after 48 hours by 55% and 41% in Ctnnb1cko/cko mutants and DKK1-treated explants, respectively, indicating that β-catenin-mediated WNT signaling is required not only for FGF expression, but also activity (Fig. 4C, 5D). Note that the decrease in the levels of transcripts is not a result of having less viable cells since it can occur prior to detectable cell death, since amounts are normalized to Gapdh and Hprt levels, and since the expression of at least one other gene is upregulated (see below). Conversely, RNA levels for the Fgf genes are increased between 1.5 and 2.6 fold in the Ctnnb1+/Δex3 mutants (Fig. 4C). The in vivo mutant analysis together with the use of DKK1 in culture suggest that β-catenin-mediated WNT signaling regulates Fgf activity in the early telencephalon.

Figure 5. FGFs mediate β-catenin dependent cell survival.

Figure 5

Open in a new tab

(A) Whole E10.5 embryos showing the presence of anterior tissue (at), albeit malformed, in the Ctnnb1+/Δex3 mutant and a truncation of anterior tissue in both the Foxg1Cre;Fgfr1cko/cko;Fgfr2cko/cko;Fgfr3−/− triple and Foxg1Cre;Ctnnb1+/Δex3;Fgfr1cko/cko;Fgfr2cko/cko;Fgfr3−/− mutants. d: diencephalon; h: hindbrain; m: midbrain; t: telencephalon. Scale bar: 500 μm.

(B) TUNEL analysis indicates that loss of the telencephalon in the Ctnnb1+/Δex3;Fgfr triple mutant is due to cell death at the 16–18 somite stage (23% TUNEL+ telencephalic precursor cells; p < 0.0001, mean±SEM; n=2 each for control and mutant) Arrows: telencephalic neuroepithelium. Scale bar: 100 μm.

(C) Application of FGF8 (50 ng/ml) to DKK1 treated explants for 48 hours leads to a substantial rescue of cell death (p < 0.0001, mean±SEM; control: n=3, mutant: n=2). Scale bar: 100 μm.

(D) qRT-PCR reveals a 41% decrease of Spry1 RNA in explants treated with DKK1 alone (n=5) compared with vehicle-treated controls (n=8) and a restoration of levels to 88% of control in DKK1 plus FGF8 treated explants (n=4) (***: p < 0.0001, *: p < 0.05, mean±SEM).

In the presence of WNTs, β-catenin is stabilized and translocates to the nucleus where it associates with the TCF/LEF transcriptional complex to regulate the expression of WNT-responsive genes (Logan and Nusse, 2004). Therefore β-catenin may directly regulate transcription of FGF genes. Using the consensus TCF/LEF binding site to scan the genomic regions surrounding Fgf8, a potential site was identified in the 5′ region (3120 base pairs from the ATG start codon; note that no TCF/LEF binding sites were identified in sequences surrounding Fgf8 that were previously suggested to act as enhancers; Beermann et al., 2006). In addition, there are three potential TCF/LEF binding sites upstream of Fgf18 (467 to 882 base pairs from the ATG start site; Reinhold and Naski, 2007). To determine whether β-catenin is directly associated with these sites in telencephalic cells in vivo, micro-chromatin immunoprecipitation (μChIP) followed by qPCR was performed using telencephalons isolated from wildtype 16–18 somite stage embryos. Immunoprecipitation with an anti-β-catenin antibody led to 4.5 and 21 fold enrichment (over non-specific IgG) in DNA containing the TCF/LEF binding sites from Fgf8 and Fgf18, respectively (Fig. 4D). In contrast, sequences from the Gapdh promoter or other sequences surrounding the Fgf8 and Fgf18 loci were not enriched. Thus β-catenin is specifically associated with TCF/LEF binding sites upstream of both Fgf8 and Fgf18.

Whether binding of β-catenin to the TCF/LEF sites regulates transcription of Fgf18 was addressed with a reporter assay in which luciferase expression was driven by the Fgf18 upstream sequences containing either wildtype or mutated TCF/LEF binding sites, as described (Reinhold and Naski, 2007). Dissociated telencephalic cells from 16–18 somite stage control or Ctnnb1+/Δex3 embryos were transfected with the constructs and cultured for 16 hours. Luciferase activity with the wildtype TCF/LEF binding sites was more than 3 times higher in Ctnnb1+/Δex3 cells than control cells, whereas no increase was detected with the mutated construct (Fig. 4E). This suggests that β-catenin-mediated WNT signaling directly regulates levels of Fgf gene transcription in the early telencephalon. In addition, β-catenin mediated cell survival in the neuroepithelium is likely autonomous to this cell layer since FGF genes are directly promoted by β-catenin and specifically upregulated in the neuroepithelium and not the surrounding tissues of Ctnnb1+/Δex3 mutants (Fig. 4B). This leaves open the possibility that cells in the surrounding mesoderm and ectoderm die due indirectly to the death of neuroepithelial cells.

FGFs mediate β-catenin-dependent cell survival

Given that the Ctnnb1cko/cko and Fgfr triple mutant phenotypes are similar and that β-catenin regulates Fgf gene transcription (Fig. 1, 3, 4; Paek et al., 2009), it is possible that cells die in the Ctnnb1cko/cko mutants due indirectly to a loss of FGF signaling. This hypothesis was tested by combining the conditional gain-of-function Ctnnb1+/Δex3 allele with all three Fgfr loss-of-function alleles to determine whether stabilized β-catenin, tantamount to constitutive WNT signaling, was sufficient to rescue the cell death that is due to loss of FGF signaling. The Ctnnb1+/Δex3 mutation on its own exhibits disorganization of the anterior embryo, but without detectable cell loss (Fig. 4B, 5A). As with Fgfr triple mutants, the Foxg1Cre;Ctnnb1+/Δex3;Fgfr1cko/cko;Fgfr2cko/cko;Fgfr3−/− quadruple mutants at E10.5 exhibited a severe truncation of the telencephalon and surrounding tissues (Fig. 5A; also similar to the Ctnnb1cko/cko mutants, Fig. 1B). Although not a focus of this study, the optic vesicles are enlarged in the quadruple mutants similar to the Ctnnb1+/Δex3 mutants. As with the Ctnnb1cko/cko and Fgfr triple mutants, loss of the telencephalon in the quadruple mutants was due to extensive cell death (23% of telencephalic cells were TUNEL+ at the 16–18 somite stage; Fig. 5B). Therefore stabilized β-catenin and constitutive WNT signaling are not sufficient to rescue the survival of cells that lack FGF signaling.

These results do not exclude the possibility that FGFs in a parallel manner are also dependent on β-catenin-mediated WNT signaling to promote survival. This possibility was tested using exogenous FGFs in an attempt to rescue the cell death observed in telencephalic explants treated with the WNT antagonist DKK1. As above, DKK1 treatment for 48 hours increased the fraction of TUNEL+ cells to 26% compared with 2% in control explants (Fig. 3A,B, 5C). However, in explants treated with both DKK1 and FGF8, cell death was significantly rescued (only 5% TUNEL+ cells). Consistent with FGF signaling regulating survival, the level of Spry1 RNA measured by qRT-PCR is decreased 41% in DKK1 treated explants compared to controls and the level is restored to 88% of controls in explants treated with both DKK1 and FGF8 (Fig. 5D). Therefore, although WNT signaling is dependent on FGF signaling to promote cell survival, FGF signaling does not require WNT signaling.

SMAD4 mediates cell death in Ctnnb1cko/cko mutants

Members of the TGFβ family of secreted factors are known to promote programmed cell death during development. For example, BMPs are necessary and sufficient to cause cell death in telencephalic precursor cells and TGFβ is required for the normal death of ciliary, dorsal root, and spinal motor neurons (Fernandes et al., 2007; Furuta et al., 1997; Krieglstein et al., 2000; Ohkubo et al., 2002). Notably, in the limb bud, loss of Bmpr1a upregulates FGF expression which inhibits interdigital cell death leading to webbed-feet (Pajni-Underwood et al., 2007). Therefore we reasoned that the widespread cell death in the Ctnnb1cko/cko and Fgfr triple mutants might be due to unchecked TGFβ signaling.

To test this hypothesis, a floxed allele of Smad4 (Yang et al., 2002), which encodes an intracellular mediator of canonical TGFβ signaling, was conditionally disrupted in Ctnnb1cko/cko mutants. In Foxg1Cre;Smad4cko/cko;Ctnnb1cko/cko double mutants at the 16–18 somite stage, TUNEL staining in the telencephalic neuroepithelium was reduced to control levels (~1% TUNEL+ cells) compared with 23% in Ctnnb1cko/cko single mutants (Fig. 1C, 6). Loss of Smad4 on its own had no obvious phenotype, with levels of cell death in these mutants similar to wildtype controls (data not shown). Together these data indicate that Smad4 is essential for mediating the death observed in the Ctnnb1cko/cko mutants and likely the Fgfr triple mutants. Loss of Smad4 does not, on the other hand, rescue the delamination of telencephalic precursors that is due to the β-catenin at adherens junctions that occurs by the 25–30 somite stage (data not shown).

Figure 6. Smad4 mediates cell death in Ctnnb1cko/cko mutants.

Figure 6

Open in a new tab

Horizontal sections from 16–18 somite stage control, Ctnnb1cko/cko, Smad4cko/cko;Ctnnb1cko/cko, and Bmpr1acko/cko;Bmpr1b−/−;Ctnnb1cko/cko mutants. In Smad4cko/cko;Ctnnb1cko/cko double mutants the percentage of TUNEL+ cells in the telencephalic neuroepithelium (arrows) was reduced to control levels (~1%, p=0.76; n=3) compared with 23% in Ctnnb1cko/cko mutants (p<0.0001). However, cell death remains high in Bmpr1acko/cko;Bmpr1b−/−;Ctnnb1cko/cko mutants (n=4). Scale bar: 100 μm.

SMAD4 can potentially mediate signaling of many ligands in the TGFβ superfamily, including BMPs. To test whether BMP signaling promotes cell death in the β-catenin mutants, we examined the anterior end of embryos carrying mutant alleles of Ctnnb1 as well as Bmpr1a and Bmpr1b, the only two type I BMP receptor genes expressed in the early telencephalic neuroepithelium. Previous studies indicated that deletion of Bmpr1a using the Foxg1Cre driver occurs between the 10 and 13 somite stages (Fernandes et al., 2007). Foxg1Cre;Bmpr1acko/cko;Bmpr1b/−;Ctnnb1cko/cko mutants at the 16–18 somite stage exhibited similar high levels of cell death as the Ctnnb1cko/cko single mutants (Fig. 6), indicating that ligands other than BMPs are likely to promote cell death in these mutants.

Other members of the type I family of TGFβ receptors, ALK4, ALK5, and/or ALK7, were tested for their role in promoting cell death when WNT signaling is disrupted. Cell death was examined in 16–18 somite stage telencephalic explants treated with DKK1 with or without the ALK4/5/7 specific inhibitor SB-431542 (Inman et al., 2002). After 48 hours of culture, TUNEL staining was performed on vehicle-treated (control), DKK1-treated, and DKK1 plus SB-431542-treated explants. Control and DKK1 plus SB-431542-treated explants were not significantly different with both having ~3% TUNEL+ cells (control: 3.4±0.4%, DKK1+SB-431542: 3.0±0.3%, mean±SEM), which was significantly different than the 33.9±6.0% in DKK1-treated explants (p < 0.001). This suggest that ALK4, 5, or 7 may be the receptors in vivo that signal through SMAD4 to mediate cell death in the absence of WNT and FGF signaling. Be that as it may, TGFβ1, a potential ligand for these receptors, does not increase cell death when added on its own to cultured telencephalic explants (Supp. Fig. 3), suggesting that although signaling through SMAD4 is necessary for death, it is not sufficient and requires in addition loss of pro-survival factors such as FGFs or WNTs.

Cdkn1a expression correlates with cell death

The survival of the cells that form the telencephalon and surrounding tissues is not a default state, but appears to be tightly regulated by pro-survival (FGFs and WNTs) and pro-apoptotic signals (TGFβs) in their environment. p21Cip1 is able to promote cell death (Gartel and Tyner, 2002) and the transcription of its gene, Cdkn1a, is at least in part regulated by a balance of activation by the SMAD-FOXO complex and repression by the MYC- MIZ complex (Seoane et al., 2002; Seoane et al., 2004).

To determine whether Cdkn1a expression reflects the survival state of anterior cells in the different mutants above, anti-p21Cip1 immunostaining was performed on sections of 16–18 somite stage embryos. In controls and Smad4cko/cko mutants, in which hardly any cells are dying, few p21Cip1+ cells were observed in the telencephalic neuroepithelium (~2%; Fig. 7A,B). In contrast, 26 and 35% of cells were p21Cip1+ in Ctnnb1cko/cko and Ctnnb1+/Δex3;Fgfr1cko/cko;Fgfr2cko/cko;Fgfr3−/− mutants (Fig. 7A,B), in which there is extensive cell death. Moreover, in the Smad4cko/cko;Ctnnb1cko/cko mutants in which cells are rescued, but not in the Bmpr1acko/cko;Bmpr1b−/−;Ctnnb1cko/cko mutants in which cell death still occurs, the number of p21Cip1+ cells almost returned to control levels (~5%; Fig. 7A,B and data not shown). Hence p21Cip1 expression correlates strongly with levels of cell death in the early telencephalon of the different mutants examined.

Figure 7. Cdkn1a expression is repressed by WNTs and FGFs and promoted by TGFβ/SMAD4.

Figure 7

Open in a new tab

(A) Horizontal sections from 16–18 somite stage control, Ctnnb1cko/cko, and Smad4cko/cko;Ctnnb1cko/cko mutants immunostained for p21Cip1 (dashed boxed areas in the top row are shown enlarged in the bottom row). Scale bar: 100 μm.

(B) In controls, few p21Cip1+ cells were observed in the telencephalic neuroepithelium, whereas the number is increased in both Ctnnb1cko/cko (n=4) and Ctnnb1+/Δex3;Fgfr1cko/cko;Fgfr2cko/cko;Fgfr3−/− (n=2) mutants. In Smad4cko/cko;Ctnnb1cko/cko mutants (n=3), the number of p21Cip1+ cells returns close to control levels (*** p < 0.0001, mean±SEM).

(C) Compared to controls (n=8), DKK1-treated explants (n=4) show an increase in Cdkn1a RNA levels by qRT-PCR. The addition of FGF8 (n=4) or SB-431542 (n=5) to DKK1-treated explants leads to a significant decrease in levels of Cdkn1a RNA compared with DKK1 alone (*: p < 0.05, **: p < 0.01, mean±SEM).

(D) A μChIP assay using SMAD4 and phospho-SMAD2 antibodies reveals an increased association of these proteins to the SBE sites upstream of Cdkn1a in 16–18 somite stage telencephalons from Ctnnb1cko/cko embryos compared with controls (p < 0.01, mean±SEM). 30 telencephalons of each genotype were used for 3 independent μChIP assays for each antibody (5 telencephalons per assay).

(E) Model for how pro- (green) and anti-apoptotic (red) factors regulate cell survival; the FOXO/SMAD complex promotes Cdkn1a expression whereas the MYC/MIZ complex represses it (see Discussion for details; adapted from Seoane et al., 2004).

The changes in p21Cip1 expression observed in the different mutants were corroborated using cultured explants for which relative levels of Cdkn1a RNA were compared by qRT-PCR. Compared to vehicle treated controls, DKK1 treatment led to a 43% increase in Cdkn1a RNA levels (Figure 7C), consistent with the increase in cell death observed in these explants (Fig. 3A,B). Moreover, addition of FGF8 or SB-431542 to DKK1-treated explants, which both rescued cell death (Fig. 5C and see text above), led to a significant decrease in levels of Cdkn1a RNA compared with DKK1 alone (45 and 81% decreases, respectively; Fig. 7C). Taken together, the levels of Cdkn1a expression observed in vivo and in vitro further support the conclusion that β-catenin-mediated FGF activity promotes cell survival and that in the absence of this activity cell death is driven by SMAD4.

Cdkn1a expression not only provides a marker for cell death, but also provides a focal point for understanding the mechanisms by which a cell can integrate antagonistic signals. In cultured neural cell lines, activation of SMAD2, 3, and 4 in response to TGFβ can promote Cdkn1a transcription in two ways. First, SMADs bind to Smad Binding Elements (SBEs) upstream of Cdkn1a as part of a SMAD/FOXO complex that promotes transcription, and second SMAD activation suppresses Myc expression, which represses Cdkn1a expression (Fig. 7E; Chen et al., 2002; Seoane et al., 2004). Since β-catenin and FGFs promote Myc expression in other cell types (Cavalieri and Goldfarb, 1987; He et al., 1998; Medici et al., 2008), increased Cdkn1a expression in Ctnnb1cko/cko mutants is likely due at least in part to reduced Myc expression. Indeed, Myc RNA is significantly decreased in Ctnnb1cko/cko mutants compared with controls when measured by qRT-PCR (30% decrease; control: 1.00±0.07, mutant: 0.70±0.07, p = 0.036, mean±SEM, Supp. Fig. 4). In contrast, Foxo1, Foxo3a, and Foxo4 RNA levels appear unaffected in these mutants (Supp. Fig. 4).

However, a change in Myc RNA levels does not exclude the possibility that loss of Ctnnb1 also promotes Cdkn1a expression through increased SMAD activity. To address this possibility, μChIP assays were performed on 16–18 somite stage telencephalic tissue using anti-SMAD4 and anti-phospho-Smad2/3 antibodies to determine the levels of SMADs bound to the SBEs upstream of Cdkn1a in control and Ctnnb1cko/cko mutants. Cdkn1a sequences were significantly enriched by 1.6 and 1.3 fold in Ctnnb1cko/cko mutants compared with controls for SMAD4 and phopho-SMAD2/3, respectively, indicating increased binding of these factors to the Cdkn1a SBEs in the mutant (Fig. 7D). Increased binding of SMAD4 is not likely due to increased levels of SMAD4 protein, since these appear unaffected in Ctnnb1cko/cko telencephalons or telencephalic explants treated with DKK1 or FGF8 (Supp. Fig. 4C,D). Hence β-catenin may repress Cdkn1a expression both by repressing SMAD activity and promoting Myc expression.

DISCUSSION

Our results reveal how extracellular signals regulate the survival of precursor cells in the anterior head of the embryo. In response to WNTs, β-catenin activates FGF expression and function, which is essential for cell survival. In maintaining cell survival, FGFs are epistatic to WNT signaling since β-catenin is entirely dependent on FGF function to maintain survival whereas FGF8 can promote cell survival in the absence of WNT signaling. Moreover, without WNT signaling, the apoptotic activity of SMAD4-mediated TGFβ signaling goes unchecked leading to cell death.

Regulation of FGFs and cell survival

WNTs are not the only factors that promote FGF ligand expression in the early anterior embryo. SHH is also essential for maintaining expression of FGF genes (Ohkubo et al., 2002; Rash and Grove, 2007). In Shh−/− mutants, telencephalic precursors and surrounding tissues undergo higher than normal rates of cell death and the anterior head is severely reduced in size, consistent with the loss of FGFs in these mutants (Chiang et al., 1996; Fernandes et al., 2007; Rash and Grove, 2007). Moreover, both gain- and loss-of-function studies demonstrate that SHH in generating ventral telencephalic cells acts genetically upstream of FGFs; namely, SHH indirectly activates FGFs by inhibiting GLI3-repressor activity (Hébert and Fishell, 2008). Thus WNTs and SHH are each independently required to promote FGF expression and function, without which most or all telencephalic precursor cells die.

WNTs are predominantly expressed in the dorsomedial telencephalon whereas SHH is restricted to a ventromedial domain, both of which flank the anterior-medial domain of FGF expression (Monuki and Walsh, 2001). Here, β-Catenin is shown to directly bind and transcriptionally activate FGF genes. In the wildtype telencephalon, despite widespread TCF-lacZ expression throughout the telencephalon (Fig. 1 and Supp. Fig. 1), only cells in the anterior part express FGFs. On the other hand, with increased β-Catenin activity (using the Ctnnb1Δex3 mutants), Fgf8 expression is upregulated throughout the neuroepithelium (Fig. 4B), suggesting that normal levels of the β-Catenin/TCF complex are insufficient to promote expression of FGF genes throughout the neuroepithelium, but perhaps sufficient to prime their promoters. This also suggests that another factor, such as SHH signaling, may be required in combination with WNT signaling to induce FGF expression, potentially explaining why only anterior medial cells normally express FGFs. Whether GLI3, acting downstream of SHH, also directly regulates FGF gene expression remains unclear. In any case, it is interesting to speculate that FGF function is regulated by both dorsal and ventral cues to coordinate the survival and expansion of the broad dorsal and ventral areas of the early telencephalon.

Regulation of Cdkn1a

Cdkn1a expression in the telencephalon correlates with cell death (Fig. 7). Therefore, whether or not Cdkn1a expression itself induces apoptosis, understanding how Cdkn1a expression integrates the same signaling pathways that regulate apoptosis may provide a paradigm for understanding how cell survival is controlled in the nascent telencephalon. FGFs are likely to repress Cdkn1a expression in two ways. The first is by activating Foxg1, whose expression depends on FGFs (Paek et al., 2009; Shimamura and Rubenstein, 1997). Importantly, FOXG1 binds the FOXO/SMAD complex and inhibits it from activating Cdkn1a expression (Seoane et al., 2004). Consistent with this mechanism, although loss of β-catenin activity does not seem to affect expression levels of FOXO or SMAD4 (Supp. Fig. 4), the binding of SMADs to the SBEs of Cdkn1a is increased in the Ctnnb1cko/cko (β-catenin) mutant, in which FGF activity is lost (Fig. 7D). This increased binding at the SBE sites is therefore likely due at least in part to reduced inhibition by FOXG1.

Second, FGFs are likely to inhibit Cdkn1a expression by promoting MYC expression and activity. MYC, acting in a complex with MIZ, directly represses Cdkn1a (Chen et al., 2002; Seoane et al., 2002). In Ctnnb1cko/cko mutant telencephalons, in which FGF expression is lost, Myc RNA is decreased. In addition, MYC protein is stabilized and activated by mitogen signaling through the Erk and Akt pathways (Albihn et al.; Dai et al., 2006; Lutterbach and Hann, 1994; Sears et al., 2000), which are activated by FGFs. Therefore repression of Cdkn1a by WNTs and FGFs occurs indirectly in at least two ways, through FOXG1 and through MYC (Fig. 7E). On the other hand, activation of Cdkn1a expression by SMAD4 is likely to be direct given that a FOXO/SMAD complex can directly bind sequences upstream of Cdkn1a and activate its transcription (Seoane et al., 2004). In sum, this study provides a framework for understanding how key factors regulate the survival of the precursor cells that form the anterior head of the embryo.

EXPERIMENTAL PROCEDURES

Generation of mutant embryos

The crosses used in this study are listed in a table in the Supplemental Data along with references for the alleles not already listed in the text.

Explant cultures

The telencephalon (both sides including the ventral midline) was dissected from 16–18 somite stage embryos. Isolated telencephalic tissue was cultured on a micropore floating filter (Falcon) on DMEM (Gibco) supplemented with 10% FBS and streptomycin/penicillin in a 5% CO2 incubator at 37°C for 48 hours (unless otherwise specified). DKK1 or sFRP (both 1μg/ml, R&D), FGF8b (50ng/ml, R&D), WNT3A (100 ng/ml, R&D), TGFβ1(12.5 ng/ml, R&D), or SB-431542 (2μM, Tocris) were added to the media immediately after placing the explants on the filters.

RNA is situ hybridization

35S-labeled and digoxygenin (DIG)-labeled RNA in situ hybridizations were carried out as described previously (Paek et al., 2009).

Cell proliferation and TUNEL assays

Horizontal sections of 16–18 somite stage embryos were used for either phospho-histone H3 (P-HH3) staining , as previously described (Storm et al., 2006), or for TUNEL analysis according to the manufacturer’s specifications (Roche). Hoechst 33342 was used as a counterstain. The fraction of P-HH3+ or TUNEL+ cells was determined by counting the number of these cells in a segment of Foxg1 expressing telencephalic neuroepithelium divided by the total number of cells (Hoechst+) in that segment. Segments excluded a 90° wedge at the most anterior tip, where cell death normally occurs even in controls, and extended to the start of the optic stalk. At least three segments are counted in each case and there positions along the dorsal-ventral axis are matched between controls and mutants. For explants, the tissue was isolated after 48 hours of culture, frozen in OCT, sectioned and assayed for TUNEL. The fraction of TUNEL+ cells was determined by counting the number of these cells within 25 μm from the periphery (which often exhibited high levels of death regardless of treatment due presumably to dissection and manipulation) divided by the total number of cells (Hoechst+). At least three segments from each of three separate samples are counted for each treatment condition. Statistical analysis was performed with the unpaired two-tailed student’s t test.

Immunofluorescence

Immunostaining was performed as previously described (Kang et al., 2009) on 16–18 somite stage embryos. The primary antibodies used are: rabbit anti-SMAD4 (1:150, Millipore), rabbit anti-PH3 (1:200, Millipore), mouse anti-PAR3 (1:4, Hybridoma Bank), rabbit anti-cleaved Caspase 3 (1:200, cell signal Technology), rabbit anti-β-catenin (1:100, Upstate), rabbit anti-p21 (1:25, abcam), mouse anti-β-galactosidase (1:1000, Hybridoma Bank), mouse anti-ZO1(1:4, Hybridoma Bank) and mouse anti-PAR-3 (1:2, Hybridoma Bank). Cell counts were quantified and compared using the unpaired two-tailed student’s t test.

Real Time Quantitative PCR

Total RNA was prepared from individual 16–18 somite stage telencephalons or cultured telencephalic explants using PureLink RNA Mini Kit (Invitrogen) following the manufacturer’s instructions. 0.2 μg of total RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen). Real time PCR was carried out in triplicate with a SYBR Green PCR mixture (Applied Biosystems) in an ABI PRISM 7300 real-time PCR system (Applied Biosystems). After normalization with Gapdh and Hprt1, relative RNA levels in samples were calculated by the comparative threshold cycle (Ct) method. The amplification efficiency was not calculated (and assumed to be 100%) due to limited quantities of cDNA. The sequences of primers for qRT-PCR are listed in a table in the Supplemental Data. Statistical comparisons were done using the unpaired two-tailed student’s t test.

Micro ChIP assay

Micro ChIP (μChIP) assays were carried out on 16–18 somite stage telencephalons (5 per assay) as described previously (Dahl and Collas, 2008). Normal mouse IgG was used as a control for each cross-linked extract to normalize for non-specific antibody binding. 5 μg of antibodies were used for immunoprecipitation; rabbit anti-β-catenin (Millipore), anti-SMAD4 (Santa Cruz), and anti-phospho-SMAD2 (Millipore). Levels of associated DNA fragments were assessed by qPCR and quantified using the unpaired two-tailed student’s t test. Primer sequences are provided in a table in the Supplemental Data.

Luciferase Reporter assays

Plasmids comprised of the wildtype or mutant mouse Fgf18 promoter have been previously described (Reinhold and Naski, 2007). 16–18 somite stage telencephalic cells were dissociated in 0.5% trypsin-EDTA at 37°C for 5min, placed in 96-well plates (103 cells per well), and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 16 hours, luciferase activity was analyzed using the Dual luciferase Assay System (Promega). Statistical analysis was done using the unpaired two-tailed student’s t test.

Supplementary Material

01

Acknowledgments

We are grateful to Nick Baker for suggestions and input on the manuscript; Rolf Kemler, Daniel Dufort, Stewart Anderson, Makoto Mark Taketo, Harald von Boehmer, and Chuxia Deng for mice; and Michael Naski for Fgf18 reporter plasmids. J.M.H. was supported by NIH (MH083804), the Feinberg, the Sinsheimer, and the McDonnell Foundations.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Albihn A, Johnsen JI, Henriksson MA. MYC in oncogenesis and as a target for cancer therapies. Adv Cancer Res. 107:163–224. doi: 10.1016/S0065-230X(10)07006-5. [DOI] [PubMed] [Google Scholar]
  2. Beermann F, Kaloulis K, Hofmann D, Murisier F, Bucher P, Trumpp A. Identification of evolutionarily conserved regulatory elements in the mouse Fgf8 locus. Genesis. 2006;44:1–6. doi: 10.1002/gene.20177. [DOI] [PubMed] [Google Scholar]
  3. Brault V, Moore R, Kutsch S, Ishibashi M, Rowitch DH, McMahon AP, Sommer L, Boussadia O, Kemler R. Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development. 2001;128:1253–1264. doi: 10.1242/dev.128.8.1253. [DOI] [PubMed] [Google Scholar]
  4. Cavalieri F, Goldfarb M. Growth factor-deprived BALB/c 3T3 murine fibroblasts can enter the S phase after induction of c-myc gene expression. Mol Cell Biol. 1987;7:3554–3560. doi: 10.1128/mcb.7.10.3554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen CR, Kang Y, Siegel PM, Massague J. E2F4/5 and p107 as Smad cofactors linking the TGFbeta receptor to c-myc repression. Cell. 2002;110:19–32. doi: 10.1016/s0092-8674(02)00801-2. [DOI] [PubMed] [Google Scholar]
  6. Chi CL, Martinez S, Wurst W, Martin GR. The isthmic organizer signal FGF8 is required for cell survival in the prospective midbrain and cerebellum. Development. 2003;130:2633–2644. doi: 10.1242/dev.00487. [DOI] [PubMed] [Google Scholar]
  7. Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, Beachy PA. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature. 1996;383:407–413. doi: 10.1038/383407a0. [DOI] [PubMed] [Google Scholar]
  8. Dahl JA, Collas P. A rapid micro chromatin immunoprecipitation assay (microChIP) Nat Protoc. 2008;3:1032–1045. doi: 10.1038/nprot.2008.68. [DOI] [PubMed] [Google Scholar]
  9. Dai MS, Jin Y, Gallegos JR, Lu H. Balance of Yin and Yang: ubiquitylation-mediated regulation of p53 and c-Myc. Neoplasia. 2006;8:630–644. doi: 10.1593/neo.06334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fernandes M, Gutin G, Alcorn H, McConnell SK, Hébert JM. Mutations in the BMP pathway in mice support the existence of two molecular classes of holoprosencephaly. Development. 2007;134:3789–3794. doi: 10.1242/dev.004325. [DOI] [PubMed] [Google Scholar]
  11. Furuta Y, Piston DW, Hogan BL. Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development. Development. 1997;124:2203–2212. doi: 10.1242/dev.124.11.2203. [DOI] [PubMed] [Google Scholar]
  12. Gartel AL, Tyner AL. The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther. 2002;1:639–649. [PubMed] [Google Scholar]
  13. Gorski JA, Talley T, Qiu M, Puelles L, Rubenstein JL, Jones KR. Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. J Neurosci. 2002;22:6309–14. doi: 10.1523/JNEUROSCI.22-15-06309.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harada N, Tamai Y, Ishikawa T, Sauer B, Takaku K, Oshima M, Taketo MM. Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. Embo J. 1999;18:5931–5942. doi: 10.1093/emboj/18.21.5931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW. Identification of c-MYC as a target of the APC pathway. Science. 1998;281:1509–1512. doi: 10.1126/science.281.5382.1509. [DOI] [PubMed] [Google Scholar]
  16. Hébert JM, Fishell G. The genetics of early telencephalon patterning: some assembly required. Nat Rev Neurosci. 2008;9:678–685. doi: 10.1038/nrn2463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hébert JM, McConnell SK. Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures. Dev Biol. 2000;222:296–306. doi: 10.1006/dbio.2000.9732. [DOI] [PubMed] [Google Scholar]
  18. Houart C, Caneparo L, Heisenberg C, Barth K, Take-Uchi M, Wilson S. Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling. Neuron. 2002;35:255–265. doi: 10.1016/s0896-6273(02)00751-1. [DOI] [PubMed] [Google Scholar]
  19. Houart C, Westerfield M, Wilson SW. A small population of anterior cells patterns the forebrain during zebrafish gastrulation. Nature. 1998;391:788–792. doi: 10.1038/35853. [DOI] [PubMed] [Google Scholar]
  20. Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2002;62:65–74. doi: 10.1124/mol.62.1.65. [DOI] [PubMed] [Google Scholar]
  21. Junghans D, Hack I, Frotscher M, Taylor V, Kemler R. Beta-catenin-mediated cell-adhesion is vital for embryonic forebrain development. Dev Dyn. 2005;233:528–539. doi: 10.1002/dvdy.20365. [DOI] [PubMed] [Google Scholar]
  22. Kang W, Wong LC, Shi SH, Hébert JM. The transition from radial glial to intermediate progenitor cell is inhibited by FGF signaling during corticogenesis. J Neurosci. 2009;29:14571–14580. doi: 10.1523/JNEUROSCI.3844-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Krieglstein K, Richter S, Farkas L, Schuster N, Dunker N, Oppenheim RW, Unsicker K. Reduction of endogenous transforming growth factors beta prevents ontogenetic neuron death. Nat Neurosci. 2000;3:1085–1090. doi: 10.1038/80598. [DOI] [PubMed] [Google Scholar]
  24. Lee SM, Tole S, Grove E, McMahon AP. A local Wnt-3a signal is required for development of the mammalian hippocampus. Development. 2000;127:457–467. doi: 10.1242/dev.127.3.457. [DOI] [PubMed] [Google Scholar]
  25. Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 2004;20:781–810. doi: 10.1146/annurev.cellbio.20.010403.113126. [DOI] [PubMed] [Google Scholar]
  26. Lutterbach B, Hann SR. Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis. Mol Cell Biol. 1994;14:5510–5522. doi: 10.1128/mcb.14.8.5510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mariani FV, Ahn CP, Martin GR. Genetic evidence that FGFs have an instructive role in limb proximal-distal patterning. Nature. 2008;453:401–405. doi: 10.1038/nature06876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Maruoka Y, Ohbayashi N, Hoshikawa M, Itoh N, Hogan BL, Furuta Y. Comparison of the expression of three highly related genes, Fgf8, Fgf17 and Fgf18, in the mouse embryo. Mech Dev. 1998;74:175–177. doi: 10.1016/s0925-4773(98)00061-6. [DOI] [PubMed] [Google Scholar]
  29. Mason I. Initiation to end point: the multiple roles of fibroblast growth factors in neural development. Nat Rev Neurosci. 2007;8:583–596. doi: 10.1038/nrn2189. [DOI] [PubMed] [Google Scholar]
  30. Medici D, Razzaque MS, Deluca S, Rector TL, Hou B, Kang K, Goetz R, Mohammadi M, Kuro OM, Olsen BR, et al. FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis. J Cell Biol. 2008;182:459–465. doi: 10.1083/jcb.200803024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mohamed OA, Clarke HJ, Dufort D. Beta-catenin signaling marks the prospective site of primitive streak formation in the mouse embryo. Dev Dyn. 2004;231:416–424. doi: 10.1002/dvdy.20135. [DOI] [PubMed] [Google Scholar]
  32. Monuki ES, Walsh CA. Mechanisms of cerebral cortical patterning in mice and humans. Nat Neurosci. 2001;4:1199–1206. doi: 10.1038/nn752. [DOI] [PubMed] [Google Scholar]
  33. Nelson WJ, Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways. Science. 2004;303:1483–1487. doi: 10.1126/science.1094291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ohkubo Y, Chiang C, Rubenstein JL. Coordinate regulation and synergistic actions of BMP4, SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles. Neuroscience. 2002;111:1–17. doi: 10.1016/s0306-4522(01)00616-9. [DOI] [PubMed] [Google Scholar]
  35. Paek H, Gutin G, Hébert JM. FGF signaling is strictly required to maintain early telencephalic precursor cell survival. Development. 2009;136:2457–2465. doi: 10.1242/dev.032656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pajni-Underwood S, Wilson CP, Elder C, Mishina Y, Lewandoski M. BMP signals control limb bud interdigital programmed cell death by regulating FGF signaling. Development. 2007;134:2359–2368. doi: 10.1242/dev.001677. [DOI] [PubMed] [Google Scholar]
  37. Rash BG, Grove EA. Patterning the dorsal telencephalon: a role for sonic hedgehog? J Neurosci. 2007;27:11595–11603. doi: 10.1523/JNEUROSCI.3204-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reinhold MI, Naski MC. Direct interactions of Runx2 and canonical Wnt signaling induce FGF18. J Biol Chem. 2007;282:3653–3663. doi: 10.1074/jbc.M608995200. [DOI] [PubMed] [Google Scholar]
  39. Sears R, Nuckolls F, Haura E, Taya Y, Tamai K, Nevins JR. Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev. 2000;14:2501–2514. doi: 10.1101/gad.836800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Seoane J, Le HV, Massague J. Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature. 2002;419:729–734. doi: 10.1038/nature01119. [DOI] [PubMed] [Google Scholar]
  41. Seoane J, Le HV, Shen L, Anderson SA, Massague J. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell. 2004;117:211–223. doi: 10.1016/s0092-8674(04)00298-3. [DOI] [PubMed] [Google Scholar]
  42. Shimamura K, Rubenstein JL. Inductive interactions direct early regionalization of the mouse forebrain. Development. 1997;124:2709–2718. doi: 10.1242/dev.124.14.2709. [DOI] [PubMed] [Google Scholar]
  43. Storm EE, Garel S, Borello U, Hébert JM, Martinez S, McConnell SK, Martin GR, Rubenstein JL. Dose-dependent functions of Fgf8 in regulating telencephalic patterning centers. Development. 2006;133:1831–1844. doi: 10.1242/dev.02324. [DOI] [PubMed] [Google Scholar]
  44. Trumpp A, Depew MJ, Rubenstein JL, Bishop JM, Martin GR. Cre-mediated gene inactivation demonstrates that FGF8 is required for cell survival and patterning of the first branchial arch. Genes Dev. 1999;13:3136–3148. doi: 10.1101/gad.13.23.3136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wilson SW, Houart C. Early steps in the development of the forebrain. Dev Cell. 2004;6:167–181. doi: 10.1016/s1534-5807(04)00027-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yang X, Li C, Herrera PL, Deng CX. Generation of Smad4/Dpc4 conditional knockout mice. Genesis. 2002;32:80–1. doi: 10.1002/gene.10029. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS293135-supplement-01.pdf (297.4KB, pdf)

RESOURCES