Fezf2 promotes neuronal differentiation through localised activation of Wnt/β-catenin signalling during forebrain development

Abstract

Brain regionalisation, neuronal subtype diversification and circuit connectivity are crucial events in the establishment of higher cognitive functions. Here we report the requirement for the transcriptional repressor Fezf2 for proper differentiation of neural progenitor cells during the development of the Xenopus forebrain. Depletion of Fezf2 induces apoptosis in postmitotic neural progenitors, with concomitant reduction in forebrain size and neuronal differentiation. Mechanistically, we found that Fezf2 stimulates neuronal differentiation by promoting Wnt/β-catenin signalling in the developing forebrain. In addition, we show that Fezf2 promotes activation of Wnt/β-catenin signalling by repressing the expression of two negative regulators of Wnt signalling, namely lhx2 and lhx9. Our findings suggest that Fezf2 plays an essential role in controlling when and where neuronal differentiation occurs within the developing forebrain and that it does so by promoting local Wnt/β-catenin signalling via a double-repressor model.

Keywords: Fezf2; Forebrain development; Wnt signalling; Xenopus.

Fig. 1.

fezf2 knockdown leads to defects in forebrain neuronal differentiation. (A) Whole-mount in situ hybridisation for arx in control MO (20/20) or fezf2 MO (15/18) injected Xenopus embryos. Arrowhead indicates the forebrain. (B-J) One blastomere at the 2-cell stage was injected with fezf2 MO and embryos were sectioned at stage 30 transversely across the forebrain, and stained for Sox3 (B,C), MyT1 (E,F) or TUNEL (H,I). FITC staining identifies the injected side (B,E,H). Arrowheads indicate MyT1⁺ (E,F) or TUNEL⁺ (H,I) cells. (D,G,J) Statistical analysis of Sox3⁺ (n=4 embryos), MyT1⁺ (n=6 embryos) and TUNEL⁺ (n=4 embryos) cells. All control sides have been normalised to 100%. Error bars represent s.e.m. *P<0.05; ***P<0.001; ns, not significant. Scale bar: 25 µm.

Fig. 2.

fezf2 promotes Wnt/β-catenin signalling and induces neuronal differentiation through Wnt/β-catenin in vitro and in vivo. (A) fezf2 misexpression in early Xenopus embryos leads to strong dorsoanteriorisation (31/35 embryos examined showed the illustrated phenotype) compared with lacZ (β-gal) controls (39/39). (B) fezf2 misexpression enhances Smad2/3 phosphorylation and inhibits Smad1/5/8 phosphorylation as assessed in western blots. Blastula stage (st. 8) indicates the pre-activation state. Elongation factor 4E (elF4E) was used as a loading control. (C,D) qPCR shows that fezf2 promotes the expression of xnr3 (C) and sia (D) in early embryos (n=3 replicates). (E) TOPFlash assay shows that fezf2 promotes Wnt/β-catenin signalling (n=4 replicates). (F-H) fezf2 expression colocalises with active Wnt signalling in the forebrain. (F) The transgenic construct. (G) Dorsal and lateral views of stage 30 embryos; GFP signal for Wnt activity (green); Katushka signal for fezf2 expression (red); +bf, merged image with bright-field. (H) Knockdown of fezf2 reduces Wnt activity in the forebrain as assessed by expression of the 7LEF-dEGFP F1.1 Wnt reporter line. Arrowhead indicates the diencephalon. (I) The Wnt inhibitor ΔNTcf3 antagonises Fezf2-induced neuronal differentiation in mouse neuronal progenitors, as assessed by the induction of axonogenesis. (J) Statistics of I (n=4 replicates). (K,L) Electroporation experiments show that the Wnt inhibitor ΔNTcf3 antagonises Fezf2-induced neuronal differentiation in the tadpole forebrain. (K) Transverse sections of the forebrain area of stage 30 embryos electroporated correspondingly and stained for MyT1 (red), GFP (green) and with DAPI (blue). Left images, merge; right images, MyT1 alone. (L) Statistics of K (n=5 embryos). Control side is normalised to 100%. (M) qPCR analysis shows that the Wnt inhibitor ΔNTcf3 antagonises Fezf2-induced ngn1 expression in stage 20 animal cap explants (n=3 replicates). In all qPCR analyses, ribosomal protein L8 (rpl8) was used as an internal control. *P<0.05, **P<0.01, ***P<0.001. Error bars represent s.e.m. Scale bars: 100 µm in I; 50 µm in K.

Fig. 3.

The Fezf2-regulated endogenous level of Wnt/β-catenin signalling governs forebrain neurogenesis. (A) Different Fezf2 constructs. Different N-terminal domains (Eh1-repressor, VP16 activator or Eve repressor) are shown in different colours. The zinc-finger DNA-binding domain is shown in blue. (B) Western blot of gastrula stage Xenopus embryos injected with nuclear lacZ (control), eve-fezf2, VP16-fezf2 and wt-fezf2 and assayed for phosphorylated Smad1 or Smad2 and α-Tubulin (loading control). (C) pTransgenesis system transgenic constructs to assess the impact of Fezf2 and/or Wnt activities on forebrain development. (D) Expression of NβT-GFP (a-e, stage 40 embryo) and arx (a′-e′, stage 30 embryo) in the forebrain of transgenic embryos harbouring the transgenes shown in C. Inset (a) shows the fluorescence from Katushka (red). (E) Quantification of neural tissue growth phenotypes from D.

Fig. 4.

Fezf2 functions through interaction with members of Groucho family. (A) Tle1, Tle2, Tle4 and Aes constructs. Note that Aes lacks the protein-interaction WD domain. (B) Wild-type Fezf2 and ΔEh1-Fezf2 with a mutated Eh1 domain. (C) Immunoprecipitation of extracts from Xenopus embryos injected with different combinations of the indicated mRNAs, showing that Fezf2 interacts with Tle1, Tle2 and Tle4 (lanes 6, 8 and 12) but not Aes (lanes 14, 15). The Eh1 domain is required for the proper interaction between Fezf2 and Tle1/2/4 (lanes 6 and 7, 8 and 9, 12 and 13).

Fig. 5.

Fezf2 represses the activity of lhx2 and lhx9 in the forebrain. (A) ChIP-qPCR analysis of Fezf2 binding to the lhx2 promoter. Region 1 showed very high enrichment (n=3 replicates). (B,C) qPCR analysis of lhx2 and lhx9 expression in p3hGR-VP16-Fezf2-injected animal cap explants aged to stage 12 and treated with CHX alone or CHX+DEX (n=3 replicates). (D,E) qPCR analysis of lhx2 and lhx9 expression in neuralised animal cap explants aged to stage 20 (n=3 replicates). (F,G) In situ hybridisation analysis shows that mild knockdown of fezf2 leads to expansion of the lhx2 (F) and lhx9 (G) expression area. Arrowhead indicates the epithalamus; bracket indicates the ventral diencephalon. (H,I) In situ hybridisation analysis of arx (H) or ngn1 (I) in stage 28 morphants (lateral views). Arrowheads indicate arx or ngn1 expression. (J) qPCR analysis shows that lhx2 and lhx9 antagonise expression of the fezf2-induced Wnt-responsive gene xnr3 in stage 14 animal cap explants (n=3 replicates). (K) qPCR analysis shows lhx2 and lhx9 antagonise fezf2-induced ngn1 expression in stage 20 animal cap explants (n=3 replicates). In all qPCR analyses, rpl8 was used as internal control. Error bars represent s.e.m. *P<0.05, ***P<0.001; ns, not significant.

Fig. 6.

Mechanistic model of Fezf2 function in the forebrain. (A) In the presence of Fezf2. Fezf2 interacts with Groucho-family repressors and inhibits the expression of lhx2/lhx9. Consequently, β-catenin binds to the Tcf complex and Wnt signalling is activated, promoting the expression of neurogenin 1 and thus stimulating neuronal differentiation. (B) In the absence of Fezf2. Lhx2/Lhx9 inhibits Wnt signalling, resulting in the degradation of β-catenin. In the absence of β-catenin, the Tcf complex is maintained in a repressive state. This repressive Tcf complex inhibits neurogenin 1 expression, thus inhibiting neurogenesis. Progenitor cells that have exited the proliferation state cannot differentiate and thus enter apoptosis.