Thiébaud P et al. (2026), Vestigial-like 4 Regulates Neurogenesis and Neu...

XB-ART-61742

J Dev Biol 2026 Feb 11;141:. doi: 10.3390/jdb14010008.

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Vestigial-like 4 Regulates Neurogenesis and Neural Crest Formation During Xenopus Development.

Thiébaud P, Simon E, Moisan F, Fedou S, Rezvani HR, Thézé N.

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VESTIGIAL-LIKE proteins constitute a family of evolutionarily conserved proteins that act as cofactors in regulating gene expression through their binding to TEAD transcription factors. Among the four members of this family in vertebrates, VESTIGIAL-LIKE 4 has emerged as a tumor suppressor that competes with YAP in binding TEADs, thus inhibiting the HIPPO pathway downstream of YAP. Nevertheless, very few studies have addressed its function during early vertebrate development. Here, we used gain- and loss-of-function strategies to investigate the role of vestigial-like 4 during Xenopus laevis development. Our data show that vestigial-like 4 is a key regulator of neurogenesis and neural crest formation. In embryos depleted of vestigial-like 4, neurogenesis is severely impaired, and neither neurog1 nor neurod1 is able to stimulate neurogenesis. Vestigial-like 4 is also required for neural crest formation through pax3 and sox9 regulation, and this property does not necessarily require its interaction with tead. Collectively, our findings demonstrate that vestigial-like 4 is an important regulator of neurogenesis and neural crest formation. Although vestigial-like 4 can bind to tead proteins in the embryo, its function does not depend solely on this interaction, suggesting a complex level of regulation with which vestigial-like 4 regulates early steps in development and differentiation.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: egr2 en2 hes1 neurod1 neurog1 pax3 pax6 snai2 sox17a sox2 sox9 tbxt tead1 tead2 tubb2b twist1 vgll1 vgll2 vgll3 vgll4 vgll4l zic1
GO keywords: regulation of neurogenesis [+]
???displayArticle.antibodies??? H3f3a Ab9 HA Ab9
???displayArticle.morpholinos??? hbg1 cMO1 tead1 MO1 tead4 MO1 vgll4 MO1 vgll4 MO2 vgll4 MO3 vgll4 MO4

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	Figure 1. Temporal and spatial expression of Xenopus laevis vgll4 genes. Vgll4.La, vgll4.Lb and vgll4.Sa mRNAs were analyzed by RT–PCR with specific primers. (A) RT–PCR analysis of embryos from stage 2 to stage 42. (B) RT–PCR analysis of dissected parts of stage 11 embryos. AC, animal cap; DM, dorsal mesoderm; Ed, endoderm; Emb, total stage 11 embryo; VM, ventral mesoderm. (C) RT–PCR analysis of adult tissues. Br, brain; He, heart; In, intestine; Li, liver; Sk, skeletal muscle; Ov, ovary; Sp, spleen; St, stomach; Te, testis. Ornithine decarboxylase (odc) gene expression was used as a control—Control without reverse transcription.
	Figure 2. Vgll4 knockdown alters neural differentiation. Embryos were injected into one blastomere of 2-cell-stage embryos with 10 ng of vgll4 MO and analyzed by in situ hybridization for gene expression, as indicated in the panel. mRNA encoding the lineage tracer ß-galactosidase was co-injected with the MOs to identify the injected side. The asterisk indicates the injected side. Scale bar, 500 µm. A dorsoanterior view of neurula embryos is shown. A ventral view of gastrula embryos is shown for tbxt and sox17a. Reduced gene expression is indicated by a white arrowhead. Results are shown in adjacent panels. The numbers on the top of each bar indicate the number of embryos analyzed from three independent experiments. [[ Abbreviations: MO = morpholino ]]
	Figure 3. Vgll4 is required for the proneural activities of neurogenin 1 and neurod1. (A) Embryos were injected into one cell of a two-cell stage embryo with 1 ng of neurogenin 1 (neurog1) or neurod1 mRNA and 20 ng of control MO or with 20 ng of vgll4 MO. The embryos were analyzed via in situ hybridization for tubb2b expression at the neurula stage. mRNA encoding the lineage tracer ß-galactosidase was co-injected with the MOs to identify the injected side. (B) Embryos were injected into one cell at the two-cell stage embryo with 20 ng of control MO or with 20 ng of vgll4 MO and analyzed at the neurula stage via in situ hybridization for neurog1 expression. A dorsoanterior view of neurula embryos is shown. The injected side is indicated by an asterisk. Scale bar, 500 µm. Reduced gene expression is indicated by a white arrowhead. The quantification of the results is shown in adjacent panels. The numbers on the top of each bar indicate the number of embryos analyzed in three independent experiments. [[ Abbreviations: MO = morpholino ]]
	Figure 4. Overexpression of vgll4 blocks neurogenesis. (A) Schematic of the vgll4 fusion constructs. The vgll4 sequence is in gray, with TDU domains in blue. The sizes of the proteins, amino acids and different domains are indicated. EnR, repressor domain; VP16, activator domain. (B) Embryos were injected into one cell at the two-cell stage with increasing concentrations of vgll4 mRNA as indicated and analyzed via in situ hybridization for neurog1 expression. (C) Embryos were injected into one cell of a two-cell stage embryo with a constitutively active (VP16) or repressive form (EnR) of vgll4 and analyzed via in situ hybridization for tubb2b and neurog1 expression. A dorsoanterior view of neurula embryos is shown. The injected side is indicated by an asterisk. Scale bar, 500 µm. Reduced gene expression is indicated by a white arrowhead. The quantification of the results is shown inside the panels. In (B,C), the phenotype is fully penetrant.
	Figure 5. Proliferation and apoptosis are unchanged in vgll4-depleted embryos. (A) Embryos were injected into one cell at the two-cell stage embryo with control MO or vgll4 MO and analyzed at the blastula stage by immunostaining to detect phosphohistone (pHH3). (B) Embryos were injected into one cell at the two-cell stage embryo with control MO or vgll4 MO and analyzed at the neurula stage by in situ hybridization for p27xic expression and by TUNEL assay. The dorsoanterior view is shown. The injected side is indicated by an asterisk. Scale bar, 500 um. Three independent experiments were analyzed and the quantification of the results is shown inside the panels. (C) Quantification of phosphohistone (pHH3). (D) Non-injected embryo analyzed by TUNEL staining. [[ Abbreviations: MO = morpholino ]]
	Figure 6. Vgll4 is required for neural crest formation and normal craniofacial development. (A) Embryos were injected into one cell at the two-cell stage embryo with vgll4 MO or control MO and analyzed at the early neurula stage via in situ hybridization for the expression of the indicated genes. (B) Embryos were injected into one cell at the two-cell stage embryo with vgll4 MO alone or with vgll4 mRNA (vgll4) and analyzed at the neurula stage via in situ hybridization for pax3 expression. Reduced gene expression is indicated by a white arrowhead and increased expression is indicated by a black arrowhead. Control MO or a mismatch MO were tested under the same conditions. A dorsoanterior view of the embryos is shown. The injected side is indicated by an asterisk. Scale bar, 500 µm. Three independent experiments were analyzed and the quantification of the results is shown inside the panels. (C) Embryos were injected into one cell of a two-cell-stage embryo with the vgll4 MO or control MO and analyzed at the tailbud stage via in situ hybridization for twist expression. A lateral view of the embryo is shown. The injected side is indicated by an asterisk. Scale bar, 500 µm. The quantification of the results is shown inside the panels. (D) Alcian blue staining of dissected craniofacial cartilage from vgll4 MO- or control MO-injected tadpoles at stage 45. The left panel is a diagram illustrating Meckel’s (M), palatoquadrate (Q), ceratohyal (C) and gill (G) cartilage. The black lines indicate the midline. [[ Abbreviations: MO = morpholino ]]
	Figure 7. Vgll4 overexpression stimulates neural crest development. (A) Embryos were injected into one cell at the two-cell stage embryo with vgll4 mRNA as indicated and analyzed at the early neurula stage via in situ hybridization for pax3 expression. β-gal mRNA (lacZ) was injected as a control. (B) Embryos were injected into one cell at the two-cell stage embryo with vgll4-VP16 or vgll4-EnR mRNA and analyzed at the neurula stage via in situ hybridization for pax3 and hes4 expression. Increased or decreased gene expression is indicated by a black or white arrowhead, respectively. A dorsoanterior view of the embryos is shown. The injected side is indicated by an asterisk. Scale bar, 500 µm. Three independent experiments were analyzed and the quantification of the results is shown inside the panels. [[ Abbreviations: MO = morpholino ]]
	Figure 8. TEAD morphant embryos phenocopy vgll4 morphants. (A) Embryos were injected into one cell of a two-cell-stage embryo with tead MO or TEA-EnR mRNA as indicated and analyzed at the early neurula stage via in situ hybridization for pax3 or tubb2 expression. Reduced gene expression is indicated by a white arrowhead. A dorsoanterior view of neurula embryos is shown. The injected side is indicated by an asterisk. Scale bar, 500 um. Three independent experiments were analyzed and the quantification of the results is shown inside the panels. (B) Embryos were injected into two-cell embryos at the two-cell stage with tead1-Myc alone or in combination with vgll4-HA mRNA as indicated and subjected to immunoprecipitation (IP) followed by immunoblotting (IB) with anti-Myc or anti-HA antibodies. [[ Abbreviations: MO = morpholino ]]
	Figure 9. Vgll4 can bypass this interaction to activate pax3. (A) Schematic of the vgll4 (V4) fusion constructs. The sizes of the proteins with deleted regions (Δ) are indicated. The C-terminal HA tag and TDU domains are shown in black and blue boxes, respectively. In the V4mutTDU construct, both TDU domains are mutated (X). In situ hybridization of pax3 expression in embryos injected with the different constructs is indicated on the right (+/−). (B) Embryos were injected into one cell at the two-cell stage embryo with the indicated vgll4 construct mRNA and analyzed at the early neurula stage via in situ hybridization for pax3 expression. A dorsoanterior view of neurula embryos is shown. The injected side is indicated by an asterisk. Scale bar, 500 µm. Increased pax3 expression is indicated by a black arrowhead. Three independent experiments were analyzed, and the quantification of the results is shown inside the panels.
	Figure 10. Vgll4 activity is both dependent and independent of YAP. (A) Embryos were injected into one cell at the two-cell stage embryo with vgll4 (V4), yap mRNA or vgll4 MO (V4MO) and analyzed at the early neurula stage via in situ hybridization for pax3 expression. A dorsoanterior view of neurula embryos is shown. The injected side is indicated by an asterisk. Scale bar, 500 µm. Enhanced gene expression is indicated by a black arrowhead. (B) Quantification of the results. The numbers on the top of each bar indicate the number of embryos analyzed from three independent experiments. (C) Embryos were injected into two cells at the two-cell stage with pGL284LUC and pRL-TK DNA with the indicated mRNAs. Animal cap explants were removed at the blastula stage, and their luciferase activity was tested. Relative firefly luciferase activity (RLU) was normalized to Renilla luciferase activity. The data are presented as the means +/- s.e.m.s from three independent experiments carried out in duplicate. * p < 0.05; Student’s t-test.
	Figure 11. Proposed model showing the role of vgll4 in neuronal differentiation and neural crest formation in Xenopus embryos. The model integrates gain- and loss-of-function data from the present study and data from the literature (see text). In the neural plate, vgll4 stimulates neurog1 expression upstream of neurod1 and tubb2. Vgll4 might also directly stimulate neurod1 expression. A possible interaction through hes1 stimulation is shown as a dashed line. Vgll4 stimulates pax3 expression during neural crest formation while inhibiting zic1 and hes4 (hairy2) expression. Sox9 and snai2 can be activated by vgll4 or as a consequence of pax3 stimulation by vgll4.
	Figure S1. The Vgll4 gene can produce two distinct mRNAs through alternative promoters. (A) Conserved syntenic regions between the human (HSA), mouse (MMU), Xenopus laevis (XLA) and Xenopus tropicalis (XTR) chromosome regions containing the VGLL4 locus. Gene symbols are according to HUGO. The chromosome number for each species is indicated, as are the two subgenomes of Xenopus laevis (XLAL and XLAS). Genes are represented as colored boxes, with the arrow indicating the orientation of the transcription unit. Boxes with the same color correspond to orthologous genes. The following versions of the genomes used for establishment are as follows: human, GRCH38; p7; mouse, GRCm38; and Xenopus laevis version 9.1. The drawing is not on scale to avoid complexity. (B) Deduced amino acid sequence comparison between the vgll4.La and vgll4.Lb proteins. Distinct N-terminal sequences are boxed in blue and TDU domains are boxed in yellow. Identical and conserved amino acids are indicated by an asterisk or a dot, respectively. (C) Structure of the Xenopus laevis vgll4.L gene and its corresponding mRNAs. Common exons are in grey boxes, the untranslated region is in white, and the intron is represented by a black line. The amino acid-deduced exon size is indicated under the exons.
	Figure S2. (A) Vgll4 translation-blocking morpholinos target vgll4.La and vgll4.Sa. The position of the MO with respect to the 5’ sequence of the mRNA is indicated by a red line. (B) Western blot analysis with anti-GFP using lysates from embryos injected with 50 or 200 pg of vgll4.La or vgll4.Sa. Sa mRNA alone or in combination with the indicated morpholino. Tubulin is shown as a loading control.
	Figure S3. Control morpholino effect on gene expression, and vgll4 morphant embryos. (A) Embryos were injected into one cell at the two-cell stage embryo with control MO and analysed by in situ hybridization for the expression of the indicated genes. A dorsoanterior view of the embryos is shown for tubb2, en2, egr2 and pax6, and a ventral view is shown for tbxt and sox17a. (B) Embryos were injected into one cell of a two-cell-stage embryo with vgll4 MO in the absence or presence of vgll4 mRNA and analyzed via in situ hybridization for tubb2 gene expression. The injected side is indicated by an asterisk. Reduced gene expression is indicated by a white arrowhead. Scale bar, 500 µm. Three independent experiments were analysed, and the quantification of the results is shown in the panels. (C) Embryos were injected into one cell of a two-cell-stage embryo with the control MO, or vgll4 MO, and analyzed by in situ hybridization for sox2 gene expression.