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. 2015 Apr;35(8):1449-61.
doi: 10.1128/MCB.00765-14. Epub 2015 Feb 17.

YAP regulates the expression of Hoxa1 and Hoxc13 in mouse and human oral and skin epithelial tissues

Ming Liu  1 , Shuangyun Zhao  2 , Qingjie Lin  3 , Xiu-Ping Wang  4
Affiliations

YAP regulates the expression of Hoxa1 and Hoxc13 in mouse and human oral and skin epithelial tissues

Ming Liu et al. Mol Cell Biol. 2015 Apr.

Abstract

Yes-associated protein (YAP) is a Hippo signaling transcriptional coactivator that plays pivotal roles in stem cell proliferation, organ size control, and tumor development. The downstream targets of YAP have been shown to be highly context dependent. In this study, we used the embryonic mouse tooth germ as a tool to search for the downstream targets of YAP in ectoderm-derived tissues. Yap deficiency in the dental epithelium resulted in a small tooth germ with reduced epithelial cell proliferation. We compared the gene expression profiles of embryonic day 14.5 (E14.5) Yap conditional knockout and YAP transgenic mouse tooth germs using transcriptome sequencing (RNA-Seq) and further confirmed the differentially expressed genes using real-time PCR and in situ hybridization. We found that YAP regulates the expression of Hoxa1 and Hoxc13 in oral and dental epithelial tissues as well as in the epidermis of skin during embryonic and adult stages. Sphere formation assay suggested that Hoxa1 and Hoxc13 are functionally involved in YAP-regulated epithelial progenitor cell proliferation, and chromatin immunoprecipitation (ChIP) assay implies that YAP may regulate Hoxa1 and Hoxc13 expression through TEAD transcription factors. These results provide mechanistic insights into abnormal YAP activities in mice and humans.

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Figures

FIG 1
FIG 1
Yap deficiency in the dental epithelium leads to development of a small tooth germ with reduced cell proliferation. (A and B) E13.5 Yap CKO tooth germ exhibited budding similar to that seen in wild types. (C and D) At E14.5, wild-type tooth developed into a cap stage with inner and outer dental epithelia and a compact cluster of enamel knot cells at the tip of the enamel organ. Condensed dental mesenchymal cells were immediately underneath the enamel organ. The E14.5 Yap CKO tooth was smaller than that in wild-type mice but still exhibited histodifferentiation of the enamel organ and condensed dental mesenchyme. (E to H) At the E16.5 and E18.5 bell stage of tooth development, Yap CKO tooth germs were smaller than the control tooth germs. However, they still exhibited a typical pattern of histodifferentiation, with outer and inner dental epithelia as well as star-shaped stellate reticulum cells in the center of the enamel organ. (I and J) In situ hybridization of E14.5 mice showed that Yap transcripts were downregulated in Yap CKO dental epithelium compared with that in wild-type tooth. (K and L) Confocal images of YAP immunofluorescence staining showed reduced YAP protein expression in the dental epithelium of Yap CKO tooth compared to that in wild-type tooth. (M and N) EdU incorporation assay revealed profound cell proliferation in the enamel organ and dental mesenchyme of E14.5 tooth germ, with the enamel knot area at the tip of the enamel organ devoid of proliferating cells. Dental epithelial cell proliferation was greatly reduced in the E14.5 Yap CKO tooth. (O and P) Apoptotic cells were detected within the enamel knot of wild-type and Yap CKO tooth germs in similar patterns. Dashed lines indicate the boundary between the dental epithelium and mesenchyme of tooth germs. Scale bars, 100 μm.
FIG 2
FIG 2
Yap deficiency in the dental epithelium does not affect the expression of some key signaling and adhesion molecules. (A to L) At E14.5, the levels of expression of Shh, Fgf4, Sox9, Notch1, Fgf3, and Wnt3a in wild-type and Yap CKO tooth germs were similar. Shh, Fgf3, Fgf4, and Wnt3a were expressed in the enamel knot area at the tip of developing tooth germs. (M and N) Confocal images of E-cadherin immunofluorescence staining showed similar expression levels of E-cadherin in wild-type and Yap CKO tooth germs. (O and P) Confocal images of P-cadherin immunofluorescence staining showed that P-cadherin was highly expressed in the inner and outer dental epithelia and enamel knot cells of the enamel organ. The expression of P-cadherin in the Yap CKO tooth was similar to that in wild-type tooth. Dashed lines indicate the boundary between dental epithelium and mesenchyme. Scale bars, 100 μm.
FIG 3
FIG 3
Hoxa1 and Hoxc13 transcripts show changes concurrent with those of Yap in E14.5 tooth germs. (A) The expression levels of Hoxa1 and Hoxc13 were significantly decreased and Yap transcript levels were reduced in E14.5 Yap CKO tooth germs compared to those in wild types. (B) The expression levels of Hoxa1 and Hoxc13 were significantly increased and Yap expression levels were elevated in the E14.5 YAP Tg tooth germs compared to those in wild-type mice. (C to K) During E14.5 cap-stage tooth development, Yap is expressed in both dental epithelial and mesenchymal tissues, with intense expression in the inner and outer dental epithelia. Hoxa1 and Hoxc13 showed intense expression in the outer dental epithelium and enamel knot area. Yap, Hoxa1, and Hoxc13 mRNA transcripts were all significantly downregulated in the dental epithelial cells of Yap CKO mice, whereas they were significantly increased in the YAP Tg tooth germs. In panels A and B, data represent the experimental means of the results from three biologically different samples ± standard errors of the means (SEM). *, P < 0.05; **, P < 0.01. Dashed lines indicate the boundaries between dental epithelium and mesenchyme. Scale bars, 100 μm.
FIG 4
FIG 4
Hoxa1 and Hoxc13 transcripts exhibit changes concurrent with those of Yap in the epidermis of embryonic and adult mouse skin. (A to I) Yap, Hoxa1, and Hoxc13 transcripts were downregulated in E14.5 Yap CKO mouse epidermis, whereas they were significantly increased in the YAP Tg mouse skin. (J to O) Yap mRNA transcripts were upregulated in the epidermis of 10-week-old YAP Tg mouse, which was given Dox for 7 days to induce YAP transgene expression. After Dox treatment, the epidermal thickness of YAP Tg mouse was significantly increased, and Hoxa1 and Hoxc13 mRNA expression levels were also elevated. Dashed lines indicate the boundaries between epidermis and dermis. Scale bars, 100 μm.
FIG 5
FIG 5
YAP regulates HOXA1 and HOXC13 expression in human keratinocytes. (A) YAP transcript levels were significantly reduced in HaCaT cells after 24, 48, and 72 h of YAP siRNA treatment. (B) The relative expression levels of HOXA1 were significantly downregulated in HaCaT cells after 24 h and 48 h of YAP siRNA treatment. (C) The relative expression levels of HOXC13 were significantly downregulated in HaCaT cells after 24 h and 48 h of YAP siRNA treatment. (D) The expression levels of YAP were significantly increased in the HaCaT-iYAPS127A cells at 6 h, 24 h, and 48 h after Dox administration. (E) The relative expression levels of HOXA1 were significantly upregulated in HaCaT-iYAPS127A cells at 6 h, 24 h, and 48 h after Dox administration. (F) The relative expression levels of human HOXC13 were significantly upregulated in the HaCaT-iYAPS127A cells at 6 h after Dox administration. (G) Sphere formation assay indicated that the number of spheres formed from single HaCaT-iYAPS127A cells treated with Dox was greatly reduced in YAP siRNA-treated cells compared to those treated with scrambled (Scrbl) control siRNAs (P < 0.05). Either HOXA1 or HOXC13 siRNA treatment alone or a combination of HOXA1 siRNA treatment and HOXC13 siRNA treatment significantly decreased the number of spheres formed from single HaCaT-iYAPS127A cells treated with Dox (P < 0.01). *, P < 0.05; **, P < 0.01. n = 3.
FIG 6
FIG 6
Endogenous YAP binds to the Hoxa1 and Hoxc13 promoter/enhancer regions containing TEAD binding elements. (A and B) Schematic representation of the sequence and location of putative TEAD binding sites in mouse Hoxa1 and Hoxc13 promoter/enhancer regions. One putative TEAD binding sequence (CAGCATCT) is at −4586 to −4578 in mouse Hoxa1 (A), and two putative TEAD binding sequences (TTGTATTT) are at −1950 to −1942 and at −1761 to −1753 in mouse Hoxc13 (B). Arrow pairs indicate the locations of the PCR primers. (C and D) ChIP of endogenous YAP binding to a TEAD binding element within the Hoxa1 and Hoxc13 promoter/enhancer regions in E14.5 YAP Tg mouse skin. A 310-bp PCR product containing CAGCATCT in the mouse Hoxa1 gene was amplified from isolated chromatin DNA pulled down through YAP antibody. (D) A 359-bp PCR product containing TTGTATTT in the mouse Hoxc13 gene was amplified from isolated chromatin DNA pulled down through YAP antibody. The input chromatin is shown as a positive control for the ChIP. (E) A schematic representation of the regulation of Hoxa1 and Hoxc13 by YAP through TEAD transcription factors. ON, transcription of Hoxa1 and Hoxc13 is active when the YAP-TEAD complex is recruited into the promoter/enhancer regions of Hoxa1 and Hoxc13 genes.
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