Targeted Disruption of YAP and TAZ Impairs the Maintenance of the Adrenal Cortex

Abstract

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are functionally redundant transcriptional regulators that are downstream effectors of the Hippo signaling pathway. They act as major regulators of stem cell maintenance, cell growth, and differentiation. To characterize their roles in the adrenal cortex, we generated a mouse model in which Yap and Taz were conditionally deleted in steroidogenic cells (Yapflox/flox;Tazflox/flox;Nr5a1cre/+). Male Yapflox/flox;Tazflox/flox;Nr5a1cre/+ mice were characterized by an age-dependent degeneration of the adrenal cortex associated with an increase in apoptosis and a progressive reduction in the expression levels of steroidogenic genes. Evaluation of the expression levels of stem and progenitor cell population markers in the adrenal glands of Yapflox/flox;Tazflox/flox;Nr5a1cre/+ mice also showed the downregulation of sonic hedgehog (Shh), a marker of the subcapsular progenitor cell population. Gross degenerative changes were not observed in the adrenal glands of Yapflox/flox;Tazflox/flox;Nr5a1cre/+ females, although steroidogenic capacity and Shh expression were reduced, suggesting that mechanisms of adrenocortical maintenance are sex specific. These results define a crucial role for YAP and TAZ in the maintenance of the postnatal adrenal cortex.

Figure 1.

Localization of YAP and TAZ in the adrenal gland of mice. Immunohistochemical analysis of (A) YAP and (B) TAZ expression in adrenal glands from 10-week-old male mice. Immunohistochemical analysis of (C) YAP and (D) TAZ at higher magnification to illustrate expression in the capsule. Immunohistochemical analysis of (E) YAP and (F) TAZ expression in adrenal glands from a 10-week-old virgin female.

Figure 2.

Yap and Taz knockdown efficiency in male Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (A) PCR genotype analyses for Yap and Taz performed on adrenal glands from 10-week-old males of the indicated genotypes. Bands corresponding to the floxed and Cre-recombined alleles are indicated. (B) qRT-PCR analysis of Yap and Taz mRNA levels in the adrenal glands of 10-week-old males of the indicated genotypes (n = 6 animals per genotype). All data were normalized to the housekeeping gene Rpl19 and are expressed as means (columns) ± standard error of the mean (error bars). Asterisks indicate significantly different from control (**P < 0.01; ****P < 0.0001). (C, D) Immunohistochemical analysis of YAP expression in adrenal glands from 10-week-old male (C) Yap^flox/flox;Taz^flox/flox and (D) Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (E, F) Immunohistochemical analysis of TAZ in adrenal glands from 10-week-old male (E) Yap^flox/flox;Taz^flox/flox and (F) Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. Scale bar in (F) is valid for all images.

Figure 3.

YAP and TAZ are required for the maintenance of the adrenal cortex. (A−E) Photomicrographs comparing the adrenal glands of 20-week-old male (A) Yap^flox/flox;Taz^flox/flox, (B) Yap^flox/+;Taz^flox/+;Nr5a1^cre/+, (C) Yap^flox/+;Taz^flox/flox;Nr5a1^cre/+, (D) Yap^flox/flox;Taz^flox/+;Nr5a1^cre/+, and (E) Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (F) Higher magnification of the boxed area in photomicrograph (E). (G, H) Photomicrographs comparing the adrenal glands of 20-week-old male (G) Yap^flox/flox and (H) Yap^flox/flox;Nr5a1^cre/+ mice (arrows indicate multinuclear lipoid structures). Hematoxylin, phloxine, and saffron stain.

Figure 4.

Progressive adrenal cortex degeneration in male Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. Photomicrographs comparing adrenal gland histology of Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice with that of Yap^flox/flox;Taz^flox/flox controls at the indicated ages. Scale bar (lower right) is valid for all images. Hematoxylin, phloxine, and saffron stain.

Figure 5.

Zonation in Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (A−D) Immunohistochemical analysis of CTNNB1 expression in adrenal glands from 20-week-old male (A) Yap^flox/flox;Taz^flox/flox, (B) Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice, (C) 30-week-old male Yap^flox/flox;Taz^flox/flox, and (D) Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. Insets in (B) and (D) show the expression of CTNNB1 in the zG at higher magnification. Insert in (D) also shows the expression of CTNNB1 in hypertrophic cells of the foci in the zF of mutant animals. Scale bar in (D) is valid for all images.

Figure 6.

Adrenal gland degeneration in the Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ model is associated with apoptosis. (A, B) Immunohistochemical analysis of cleaved caspase-3 in adrenal glands from 20-week-old male (A) Yap^flox/flox;Taz^flox/flox and (B) Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. Insets in (B) show that cleaved caspase-3 expression was associated with the multinucleated vacuolar cells. Scale bar in (B) is valid for both images.

Figure 7.

Circulating hormone levels in male Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (A) Serum corticosterone levels from untreated and ACTH-treated 10-week-old male Yap^flox/flox;Taz^flox/flox and Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (B) Serum aldosterone levels from 20-week-old male Yap^flox/flox;Taz^flox/flox and Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (C) Plasma ACTH levels in 10-week-old male Yap^flox/flox;Taz^flox/flox and Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. Sample numbers analyzed varied by hormonal test and genotype. Values for Yap^flox/flox;Taz^flox/flox mice were basal corticosterone: n = 17; ACTH-induced corticosterone: n = 6; aldosterone: n = 12; and ACTH: n = 7. Values for Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice were basal corticosterone: n = 12; ACTH-induced corticosterone: n = 7; aldosterone: n = 9; and ACTH: n = 7. Data are expressed as mean (columns) ± standard error of the mean (error bars). Asterisks indicate significantly different from control (*P < 0.05).

Figure 8.

Lipid accumulation and expression of steroidogenic genes were altered in the adrenal glands of male Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (A, B) Oil Red O staining of adrenal glands of 10-week-old male (A) Yap^flox/flox;Taz^flox/flox and (B) Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (C−E) qRT-PCR analysis of genes involved in steroidogenesis in adrenal gland from (C) 2-week-old, (D) 10-week-old, and (E) 20-week-old male mice of the indicated genotypes (n = 6 animals per genotype). All data were normalized to the housekeeping gene Rpl19 and are expressed as mean (columns) ± standard error of the mean (error bars). Asterisks indicate significantly different from control (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 9.

Regeneration of the zF after dexamethasone treatment was impaired in male Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (A) Comparison of adrenal weights of male Yap^flox/flox;Taz^flox/flox and Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice 2 weeks after dexamethasone treatment and 2 weeks after dexamethasone treatment with a 2-week recovery period (n = 8 animals per genotype; vehicle = 0.05% ethanol). Data are expressed as means (columns) ± standard error of the mean (error bars) and are presented as percentage of vehicle controls. Asterisks indicate significantly different from control (**P < 0.01). (B−E) Photomicrographs comparing the adrenal glands of (B) Yap^flox/flox;Taz^flox/flox mice with those of (C) Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice 2 weeks after dexamethasone treatment, and adrenal glands of (D) Yap^flox/flox;Taz^flox/flox mice with those of (E) Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice 2 weeks after dexamethasone treatment with a 2-week recovery period. The line indicates the border between zG and the zF. The dashed line indicates the border between the zF and the medulla. Scale bar (lower right) is valid for all images. Hematoxylin, phloxine, and saffron stain. Dex, dexamethasone; Y;T, Yap^flox/flox;Taz^flox/flox; Y;T;N, Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+.

Figure 10.

Expression of genes involved in stem cell or progenitor cell maintenance in the adrenal glands of male Yap^flox/flox;Taz^flox/flox;Nr5a1^cre/+ mice. (A, B) qRT-PCR analysis of genes involved in stem cell or progenitor cell maintenance in adrenal glands from (A) newborn and (B) 10-week-old male mice of the indicated genotypes (n = 6 animals per genotype). All data were normalized to the housekeeping gene Rpl19 and are expressed as means (columns) ± standard error of the mean (error bars). Asterisks indicate significantly different from control (*P < 0.05; **P < 0.01).