Use of hPSC-derived 3D organoids and mouse genetics to define the roles of YAP in the development of the esophagus

Abstract

Balanced progenitor activities are crucial for the development and maintenance of high turn-over organs such as the esophagus. However, the molecular mechanisms regulating these progenitor activities in the esophagus remain to be elucidated. Here, we demonstrated that Yap is required for the proliferation of esophageal progenitor cells (EPCs) in the developing murine esophagus. We found that Yap deficiency reduces EPC proliferation and stratification whereas persistent Yap activation increases cell proliferation and causes aberrant stratification of the developing esophagus. We further demonstrated that the role of YAP signaling is conserved in the developing human esophagus by utilizing 3D human pluripotent stem cell (hPSC)-derived esophageal organoid culture. Taken together, our studies combining loss/gain-of-function murine models and hPSC differentiation support a key role for YAP in the self-renewal of EPCs and stratification of the esophageal epithelium.

Keywords: 3D organoids; Esophagus; Hippo pathway; Human pluripotent stem cells; Progenitor cells; YAP.

Fig. 1.

Yap deletion examined at different developmental stages of the mouse esophagus. (A,B) Yap expression is incompletely ablated in the esophageal epithelium of Shh-Cre;Yap^loxp/loxp (Yap KO) mutants at E10.5 and E11.5 (n=3). Note the complete loss of Yap in the ventral esophageal epithelium (arrows). (C,D) Yap expression is completely ablated in the esophageal epithelium of Shh-Cre;Yap^loxp/loxp (Yap KO) mutants at E12.5 and E18.5. Note that expression of YAP is nuclear in EPCs at E10.5, and nuclear expression of Yap is localized to basally located EPCs at E11.5, E12.5 and E15.5. Apically oriented cells express cytoplasmic Yap starting at E11.5, E12.5 and E18.5. Note expression of both cytoplasmic and nuclear staining of YAP in wild type at E12.5 (arrows) and mostly nuclear in the basal cells (arrows) at E18.5 (n=3). Dotted line encircles the epithelium. d, dorsal; Epi, epithelium; es, esophagus; tr, trachea; v, ventral. Scale bars: 20 µm.

Fig. 2.

Yap deletion affects the development of the mouse esophagus. (A) Deletion of Yap reduces the size of the esophageal lumen and thickness of the epithelium in Shh-Cre;Yap^loxp/loxp (Yap KO) mutants at E12.5 (n=5). Black dotted line encircles the epithelium. (B) Expression of the progenitor markers p63 and Sox2 is maintained upon Yap deletion (n=3). Note the number of p63⁺ basal cells is significantly reduced at E12.5. (C,D) Yap deletion decreases the numbers of EPCs (p63⁺) and proliferating cells (pHH3⁺, arrows) at E12.5 (C) and E18.5 (D). White dotted line encircles the epithelium. Data represent mean+s.e.m. (n=5). *P<0.05, ***P<0.001 by unpaired, two-tailed Student's t-test. Scale bars: 20 µm.

Fig. 3.

Yap deletion reduces the number of epithelial layers and lumen size. (A,B) Yap deletion results in a flat and simplified esophageal epithelium with a reduction in the size of the esophageal lumen and epithelial thickness at E15.5 and E18.5 (n=5 for each). Epi, epithelium. Black dotted line indicates the border between the epithelium and mesenchyme. Data represent mean+s.e.m. (n=5). *P<0.05, **P<0.01, ***P<0.001 by unpaired, two-tailed Student's t-test. Scale bars: 20 µm.

Fig. 4.

Yap deletion reduces squamous stratification in the developing mouse esophagus. (A) Yap deletion leads to a simplified epithelium with a reduced number of Krt8⁺ cells at different stages of esophageal development. Note a few Krt8⁺ cells (arrows) in E18.5 mutant esophagus and the thickness of Krt8⁺ cells is reduced (n=3). (B,C) Yap deletion reduces squamous differentiation and only a few cells on the top layer of the epithelium (arrows) express Krt13 at E15.5 (B) and E18.5 (C) (n=5). The thickness of Krt13⁺ cells is reduced in Yap KO mutants. Note the apical nonspecific staining of Krt8 and Krt13 in p63⁺ basal cells of E18.5 mutant esophagus. Also note the normal shedding keratin in the middle of the esophageal lumen and that the thickness of Krt13⁺ cells is reduced at E15.5 and E18.5. Data represent mean+s.e.m. (n=5). *P<0.05, ***P<0.001 by unpaired, two-tailed Student's t-test. Scale bars: 20 µm.

Fig. 5.

Ectopic Yap promotes the proliferation and stratification of esophageal epithelium. (A) Yap overexpression increases the thickness of the esophageal epithelium in Shh-Cre;Yap^loxp/loxp;R26-Yap^5SA (Yap OE) compound mutants (n=4). Black dotted line indicates the border between epithelium and mesenchyme. (B) Thickness of the suprabasal cell layers (Krt8⁺) is increased in the esophagus of E18.5 Yap OE mutants (n=3). (C) Thickness of the basal cell layers (p63⁺ Krt5⁺) and differentiated cell layers (Krt13⁺) is increased in the esophagus of E18.5 Yap OE mutants (n=5). (D) Yap overexpression increases the number of p63⁺ basal cells and pHH3⁺ proliferating epithelial cells (arrows) at E18.5 (n=6). Note the proliferating parabasal cells in mutants as shown in the magnified view. Epi, epithelium. White dotted line encircles the epithelium. Data represent mean+s.e.m. (n=5). *P<0.05, ***P<0.001 by unpaired, two-tailed Student's t-test. Scale bars: 50 µm.

Fig. 6.

Yap crosstalks with Notch during the differentiation of EPCs in the developing mouse esophagus. (A) Yap deletion and overexpression reduces and increases expression of cleaved NICD1, a Notch signaling effector, respectively. Note NICD1 expression is enriched in the nuclei of suprabasal cells at E18.5 (n=3 for each). (B) Yap deletion reduces expression of Notch3 in the developing mouse esophagus at E18.5 (n=3). Note that Notch3 is relatively enriched in the nuclei of parabasal cells (arrows), in contrast to relative enrichment in the plasma membrane of basal cells (arrowheads). Epi, epithelium. White dotted line indicates the basement membrane. Scale bars: 40 µm.

Fig. 7.

YAP is required for the growth of organoids established with hPSC-derived EPCs. (A) Schematic of the experimental protocol describing YAP inhibition and knockdown in organoids established with human embryonic stem cell RUES2-derived EPCs. Note YAP is expressed in p63⁺ SOX2⁺ EPCs. (B) YAP inhibition with verteporfin (VP) reduces the numbers and size of esophageal organoids (n=3). (C) YAP inhibition with VP reduces the proliferation (Ki67⁺) of EPCs (n=6). (D) YAP inhibition reduces the stratification of hPSC-derived esophageal organoids. (E) YAP knockdown by siRNA reduces the numbers and size of esophageal organoids (n=3). (F) YAP knockdown reduces the proliferation (Ki67⁺) of EPCs (n=5). (G) YAP knockdown reduces the stratification of hPSC-derived esophageal organoids. Data represent mean+s.e.m. *P<0.05, ***P<0.001 by unpaired, two-tailed Student's t-test. Scale bars: 20 μm.