Tcf7l1 is expressed in a mutually exclusive manner with markers of specified NC and is required for proper forebrain development. (A-D) Expression of Tcf7l1 mRNA, the neuroectoderm marker Sox2, early NC marker Tfap2α, Sox9 mRNA as a marker of specified NC, and the Wnt reporter BAT-gal in the NPB and the ANF (encircled) of the mouse embryo between 1 and 5 somite pairs (s). Note the overlap of Tcf7l1 transcripts with Tfap2α protein, and of Sox9 mRNA with BAT-gal signal. Arrowheads (C) indicate Sox9 transcripts in the neural plate. (E) Mapping of recombination delivered by AP2α-Cre using Rosa26EYFPfl/+ reporter mice at various stages (E7.0-E10.5). EYFP was detected either directly or using anti-GFP antibody. Two different optical sections from the frontal view of the 5 s embryo are displayed (open arrowheads). The recombination occurred along the entire NPB (arrowheads, 10 s stage). Overlapping staining of GFP and Sox10 confirms recombination in the whole cranial NC population at the 10 s stage. (F,G) In situ hybridization shows the loss of Tcf7l1 mRNA expression in AP2α-Cre;Tcf7l1fl/fl mutants at 5 s stage. Neurectoderm is broader in the region of the prosencephalic-mesencephalic boundary (solid bar), whereas the anterior tip of the ANF is narrower (dashed bar) in the mutants. (H) Control embryo (left) and typical examples of ‘strong' (middle) and ‘mild' (right) AP2α-Cre;Tcf7l1fl/fl phenotype. Arrow, severe reduction of the telencephalic tissue and exencephaly in the strong phenotype; arrowhead, reduced telencephalon and bilateral anopthalmia in the mild phenotype. (I) Quantification of strong and mild phenotype incidence between E10.5 and E13.5. ANF, anterior neural fold; NPB, neural plate border; NNE, non-neural ectoderm; PM, prosencephalic-mesencephalic boundary. Embryos are shown from a lateral view unless stated otherwise. Nuclei were stained with Hoechst. See also Fig. S1.
Tcf7l1 deletion induces loss of ANF specifiers without affecting posterior brain structures. (A-B′) In situ hybridization shows unchanged expression of Fgf8 and Gbx2 mRNA in the midbrain-hindbrain boundary (MHB) (arrowheads). Lateral views. (C-C″) Tcf7l2 mRNA (arrowheads) in the posterior prosencephalon of control and AP2α-Cre;Tcf7l1fl/fl mouse embryos displaying mild or strong phenotype at E9.0. Lateral views. (D-G′) Tcf7l1 deletion results in marked reduction of Six3 and Sox2 mRNA expression at 7-8 s stage, Foxg1 at 11-12 s stage and of Fgf8 at E9.5 in the ANF of AP2α-Cre;Tcf7l1fl/fl embryos. Frontal views. Arrowheads (G′) indicate the expansion of Sox2− cells along the anterior lateral border of the ANF in the AP2α-Cre;Tcf7l1fl/fl mutants.
The NC population expands anteriorly in the absence of Tcf7l1. (A) Whole-mount immunohistochemical staining of Sox1 and Sox9 at 5 s stage. Lateral views. Higher magnification (bottom panels, black and white) revealed reduction of Sox1 staining in the ANF (dashed line) of AP2α-Cre;Tcf7l1fl/fl mutants. Nuclei were stained with Hoechst. (B) Frontal views of Sox1 and Sox9 immunofluorescence as in A. Higher magnification documents the expansion (lower panels, dashed line) of Sox9+ cells along the ANF border in AP2α-Cre;Tcf7l1fl/fl mutants. (C) Ectopic expression of the NC marker Foxd3 along the ANF of AP2α-Cre;Tcf7l1fl/fl mouse embryos at 10 s stage. Lateral (top) and frontal (bottom) views. (D) Sox10 mRNA is less abundant in Tcf7l1 conditional mutants at the 6 s stage but becomes ectopically expressed later at E9.5 (bottom panels, arrowhead) in the anterior head of AP2α-Cre;Tcf7l1fl/fl mutants. Lateral views. (E) Immunostaining of Sox10 and Tfap2α illustrating the reduced number of Sox10+ cells (arrows). The Sox10+ population was found to be arrested in the abnormally wide zone of the ANF, which expresses ectopic Tfap2α (bars) in the AP2α-Cre;Tcf7l1fl/fl embryos at 8 s stage. Lateral views (top three) and frontal view (bottom). (F) Expression of the NC inducer Pax3 is strongly increased along the ANF of the mutants when compared with the control embryos at 9 s stage. Lateral (top) and frontal (bottom) views. V, trigeminal ganglion. See also Fig. S2.
Aberrant activation of Wnt signaling in AP2α-Cre;Tcf7l1fl/fl and AP2α-Cre;β-catEx3fl/+ mutants. (A) In situ hybridization shows upregulation of the Wnt target Sp5 along the NPB of the ANF in AP2α-Cre;Tcf7l1fl/fl mutants at 6 s stage (arrowheads). Side (top) and frontal (bottom) views. (B) Increase in expression of the Wnt ligand Wnt1 along the NPB of the ANF in AP2α-Cre;Tcf7l1fl/fl mutants at 5-6 s and E9.0 stage (arrowheads). Frontal views. (C-E) BAT-gal signal (detected with β-galactosidase antibody; arrow) and Sox9 immunostaining (arrowheads) expand anteriorly in AP2α-Cre;Tcf7l1fl/fl;BAT-gal+/− mutants at the 8 s stage (D). Both BAT-gal activation and ectopic Sox9 expansion are weaker in the neural ectoderm of AP2α-Cre;β-catEx3fl/+;BAT-gal+/− mouse embryos at the same stage (E). Again, Sox9 immunofluorescence reflected the BAT-gal expansion in AP2α-Cre;β-catEx3fl/+;BAT-gal+/− mutants (E, arrowhead). Green line demarcates the border between the neural and non-neural ectoderm. Note that BAT-gal signal was clearly activated in the most anterior non-neural ectoderm in the frontal view AP2α-Cre;β-catEx3fl/+;BAT-gal+/−. Side (top) and frontal (middle) views and higher magnification of the frontal views (boxed regions, black and white, bottom). (F) Transverse sections (as illustrated) of AP2α-Cre;Tcf7l1fl/fl and control embryos at 4-5 s stage stained for N-cadherin and Sox9. Tcf7l1 conditional mutants showed no difference in the onset of delamination (arrows) at this stage. (G-I′) Transverse sections immunostained for N-cadherin and Sox9 at E9.5. The Sox9+ population is increased and N-cadherin staining is decreased in AP2α-Cre;Tcf7l1fl/fl and AP2α-Cre;β-catEx3fl/+ mutants compared with controls. Tcf7l1 conditional mutants with the strong phenotype display aberrant Sox9+ cells in the hindbrain prior to delamination (H, arrowheads) and N-cadherin expression is weaker in the anterior forebrain (H′, arrow) and in the dorsal hindbrain (H, arrows). Similarly, AP2α-Cre;β-catEx3fl/+ embryos exhibit aberrant Sox9+ cells in the hindbrain (I, arrowheads), accompanied by substantial reduction in N-cadherin (I,I′, arrows). (J-L) Lef1 immunostaining of coronal sections of control (J), AP2α-Cre;Tcf7l1fl/fl (K) and AP2α-Cre;β-catEx3fl/+ (L) embryos at E9.5. D, dorsal; V, ventral; fb, forebrain; t, tail. See also Fig. S3.
The constitutive repressor dnTCF4 is able to rescue the ANF phenotype in AP2α-Cre;Tcf7l1fl/fl mutants. (A) Construction of the Rosa26dnTCF4fl/+ and Rosa26ctβcat-Tcf7l1fl/+ mouse strains used in the rescue experiments. SA, splice acceptor; pA, polyadenylation signal. (B) Expression of Sox10 transcripts was either unaffected or only mildly affected in AP2α-Cre;Tcf7l1fl/fl;Rosa26dnTCF4fl/+ compound mutants, confirming the rescue capability of dnTCF4. (C) AP2α-Cre;Tcf7l1fl/fl;Rosa26ctβcat−Tcf7l1fl/+ compound mutants displayed no rescue, as confirmed by Sox10in situ hybridization. Asterisk indicates an artifact caused during preparation. (D) Quantification of strong and mild phenotype incidence and rescue capability of dnTCF4 and ctβcat-Tcf7l1, scored at E10.5. *P=0.0382, **P=0.0016, Fisher's exact test; n.s., not significant. n, number of embryos analyzed. See also Fig. S4.