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. 2016 Feb 1;143(3):504-15.
doi: 10.1242/dev.126920. Epub 2015 Dec 30.

Yap and Taz play a crucial role in neural crest-derived craniofacial development

Jun Wang  1 , Yang Xiao  2 , Chih-Wei Hsu  3 , Idaliz M Martinez-Traverso  4 , Min Zhang  3 , Yan Bai  3 , Mamoru Ishii  5 , Robert E Maxson  5 , Eric N Olson  6 , Mary E Dickinson  7 , Joshua D Wythe  8 , James F Martin  9
Affiliations

Yap and Taz play a crucial role in neural crest-derived craniofacial development

Jun Wang et al. Development. .

Abstract

The role of the Hippo signaling pathway in cranial neural crest (CNC) development is poorly understood. We used the Wnt1(Cre) and Wnt1(Cre2SOR) drivers to conditionally ablate both Yap and Taz in the CNC of mice. When using either Cre driver, Yap and Taz deficiency in the CNC resulted in enlarged, hemorrhaging branchial arch blood vessels and hydrocephalus. However, Wnt1(Cre2SOR) mutants had an open cranial neural tube phenotype that was not evident in Wnt1(Cre) mutants. In O9-1 CNC cells, the loss of Yap impaired smooth muscle cell differentiation. RNA-sequencing data indicated that Yap and Taz regulate genes encoding Fox transcription factors, specifically Foxc1. Proliferation was reduced in the branchial arch mesenchyme of Yap and Taz CNC conditional knockout (CKO) embryos. Moreover, Yap and Taz CKO embryos had cerebellar aplasia similar to Dandy-Walker spectrum malformations observed in human patients and mouse embryos with mutations in Foxc1. In embryos and O9-1 cells deficient for Yap and Taz, Foxc1 expression was significantly reduced. Analysis of Foxc1 regulatory regions revealed a conserved recognition element for the Yap and Taz DNA binding co-factor Tead. ChIP-PCR experiments supported the conclusion that Foxc1 is directly regulated by the Yap-Tead complex. Our findings uncover important roles for Yap and Taz in CNC diversification and development.

Keywords: Cranial neural crest; Craniofacial development; Yap and Taz.

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Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Efficient deletion of Yap and Taz in CNC-derived cells. Hippo signaling activity, indicated by the level of phosphorylated Yap (pYAP) in control embryos (A,B) and in Wnt1Cre; Yapf/f; Tazf/f embryos (C,D). pYAP levels were reduced in CNC-derived cells (white arrows) but not in non-CNC derived cells (red arrows). Hippo-active cells (green) were stained with pYAP; nuclei (blue) were stained with DAPI.
Fig. 2.
Fig. 2.
Vascular defects, hemorrhage and neural tube defects following Yap and Taz inactivation using the Wnt1Cre2SOR Cre driver. Both Wnt1Cre2SOR; Yap; Taz dCKO (A-D) and Wnt1Cre2SOR; Yapf/f; Tazf/+ (M-P) embryos show lethality at E10.5 with severe vascular defects, hemorrhage and defects in neural tube closure, whereas control embryos (I-L) and Wnt1Cre2SOR; Yapf/+; Tazf/f embryos (E-H) show no obvious defects. (Ma-Pa) Zoom images of branchial arch vessel defects, hemorrhage and neural tube defects of Wnt1Cre2SOR; Yapf/f; Tazf/+ embryos. Three-dimensional imaging using optical projection tomography microscopy indicates that the neural tube of control embryos closed normally (Q), whereas Wnt1Cre2SOR; Yap; Taz dCKO embryos have a defect in neural tube closure (R).
Fig. 3.
Fig. 3.
Histological analysis showing the disruption of mandibular structure in Yap and Taz-deficient embryos. Coronal sections stained with hematoxylin and eosin showing that, in contrast to control embryos (A-D), both Wnt1Cre; Yap; Taz dCKO (E-L) and Wnt1Cre; Yapf/f; Tazf/+ (M-P) embryos have disorganized, sparse mesenchyme (black arrows) and enlarged vessels (red stars) in the mandible. Boxed areas are shown at higher magnification in panels to the right, as labeled.
Fig. 4.
Fig. 4.
Severe vessel defects caused by deletion of Yap and Taz. Whole-mount CD31 immunofluorescence staining in a control (A) and a Wnt1Cre; Yap; Taz dCKO mutant (B) reveals vessel defects and endothelial-lined hemangiomas in the forebrain and mandible in Wnt1Cre; Yap; Taz dCKO mutants (B). (C) The boxed area in B at a higher magnification, focusing on the endothelial-lined hemangiomas in the branchial arch in the Yap; Taz dCKO embryo. Whereas control embryos have normal vessel development in brain (D-F), Wnt1Cre; Yap; Taz dCKO mutants have vessel regression and disorganization (different regions are labeled 1-4) (G-I). Endothelial cells are stained with CD31 antibody (green), smooth muscle cells are stained with SMA antibody (red) and auto fluorescence is blue.
Fig. 5.
Fig. 5.
The regulation of multiple signals by the Hippo pathway in CNC-derived cells. RNA-Seq analysis was performed by using mandibular tissues from E10.5 control embryos and Wnt1Cre Taz; Yap dCKO mutants. (A) RNA-Seq analysis indicates that Yap and Taz expression levels (indicated by reads) decreased in Wnt1Cre; Taz; Yap dCKO embryos compared with control embryos. (B) Gene ontology analysis shows genes that are upregulated in Wnt1Cre; Taz; Yap dCKO embryos compared with control embryos, which includes genes that regulate adherens junctions, vasoconstriction and the cytoskeleton. (C) Genes that are downregulated in Wnt1Cre; Taz; Yap dCKO embryos compared with control embryos, which includes genes that regulate cell proliferation and vasculogenesis and that negatively regulate canonical Wnt. Numbers in bars in B and C indicate gene number of each gene ontology term. (D) Heat map of RNA-Seq data shows that, compared with controls, Wnt1Cre; Taz; Yap dCKO mutants downregulate expression of Foxc1 and upregulate expression of Foxe1, Prox1, Pdgfb and Jak-Stat genes, including Jak3, Ptk2b, Stat3, Stat5a and Stat5b.
Fig. 6.
Fig. 6.
Regulation of proliferation and apoptosis in CNC cells by the Hippo pathway. (A-J) Compared with control embryos, Wnt1Cre; Yap; Taz dCKO embryos have a significantly diminished percentage of pHH3-positive proliferating cells in the mandible at E9.5 and E10.5. Proliferating cells are stained with pHH3 antibody (green), smooth muscle cells with SMA antibody (red) and nuclei with DAPI (blue). *P<0.05. (K-N) Compared with O9-1 cells transfected with control (con) siRNA, O9-1 cells transfected with Yap and Taz siRNA have significantly reduced proliferation, and O9-1 cells transfected with Lats1 and Lats2 siRNA have significantly increased proliferation (*P<0.05). Proliferating cells are stained with pHH3 antibody (green), nuclei with DAPI (red). Arrows indicate proliferating cells. (O-S) Compared with control embryos, Wnt1Cre; Yap; Taz dCKO embryos have a significantly increased percentage of apoptotic cells in the mandible at E9.5 (*P<0.05). Apoptotic cells are stained with TUNEL (green), nuclei with DAPI (blue). All error bars represent s.e.m. Arrows indicate apoptotic cells.
Fig. 7.
Fig. 7.
Yap is required for smooth muscle differentiation. (A) Strategy for establishing a Yap-knockout (KO) O9-1 cell line by using CRISPR/Cas9 system. Specifically, exon 3 was deleted from Yap. (B) Western blot data shows diminished SMA and pYap expression in Yap KO O9-1 cells compared with wild-type (wt) O9-1 cells. (C) Under differentiation conditions, most wild-type O9-1 cells give rise to smooth muscle cells. (D) Yap KO O9-1 cells did not give rise to smooth muscle cells. Arrows indicate SMA-positive cells. Smooth muscle cells are stained with SMA antibody (red), nuclei with DAPI (blue). (E) Cell counting data show that the percentage of SMA-positive cells is significantly reduced in Yap KO O9-1 cells compared with wild-type O9-1 cells (*P<0.01). Error bars represent s.e.m.
Fig. 8.
Fig. 8.
Regulation of Foxc1 by Yap and Taz. Immunohistochemical staining of Foxc1 in sagittal sections of the mandible show that the majority of cells in control embryos express Foxc1 (A-Bb), whereas the expression of Foxc1 is diminished in Wnt1Cre; Yap; Taz dCKO embryos (C-Db). Boxed areas are shown at higher magnification in panels to the right, as labeled. (E) Cell counting data show that the percentage of cells positive for Foxc1 expression in the mandible is significantly reduced in Wnt1Cre; Yap; Taz dCKO embryos compared with control embryos (*P<0.01). (F-H) Immunohistochemical staining of Foxc1 in O9-1 cells shows significantly decreased Foxc1 expression in Yap knockout (KO); Taz knockdown (KD) O9-1 cells compared with wild-type O9-1 cells (*P<0.05). Arrows indicate Foxc1-positive cells. (I) Western blot analysis of Foxc1 in O9-1 cells shows decreased Foxc1 expression in response to decreased Yap and Taz expression level. (J) Conserved Tead binding site located in the upstream region of FOXC1. Peaks in ATAC-seq data (accession no. GSE70751) indicate chromatin accessibility (Prescott et al., 2015). (K) In vivo real-time PCR using ChIP DNA indicates that Foxc1 is bound by the Yap-Tead complex in embryonic facial tissue. P<0.05. All error bars represent s.e.m.
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