Targeted Disruption of Lats1 and Lats2 in Mice Impairs Adrenal Cortex Development and Alters Adrenocortical Cell Fate

Abstract

It has recently been shown that the loss of the Hippo signaling effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in adrenocortical steroidogenic cells impairs the postnatal maintenance of the adrenal gland. To further explore the role of Hippo signaling in mouse adrenocortical cells, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in steroidogenic cells using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that developing adrenocortical cells adopt characteristics of myofibroblasts in both male and female Lats1flox/flox;Lats2flox/flox;Nr5a1-cre mice, resulting in a loss of steroidogenic gene expression, adrenal failure and death by 2 to 3 weeks of age. A marked accumulation of YAP and TAZ in the nuclei of the myofibroblast-like cell population with an accompanying increase in the expression of their transcriptional target genes in the adrenal glands of Lats1flox/flox;Lats2flox/flox;Nr5a1-cre animals suggested that the myofibroblastic differentiation could be attributed in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper adrenocortical cell differentiation and suppresses their differentiation into myofibroblast-like cells.

Keywords: LATS1; LATS2; adrenal gland; adrenocortical cells; development.

Figure 1.

Adrenal glands of Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice are larger than controls and are histomorphologically abnormal. (A) Photographs of adrenal gland from 12 dpp Lats1^flox/flox;Lats2^flox/flox (control) and Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre males. Ruler graduations are in millimeters. (B) Adrenosomatic index (adrenal gland weight/corporal weight) comparing male Lats1^flox/flox;Lats2^flox/flox (n = 12) with Lats1^flox/flox; Lats2^flox/flox;Nr5a1-cre (n = 8) and female Lats1^flox/flox;Lats2^flox/flox (n = 10) with Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre (n = 8) mice. Data are expressed as means (columns) ± SEM (error bars). Asterisks = significantly different from control (** P < 0.01). (C, D) Photomicrographs comparing the adrenal glands of 12 dpp male (C)Lats1^flox/flox;Lats2^flox/flox and (D)Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice. (E, F) Immunofluorescence analysis of 20αHSD expression in adrenal glands from 12 dpp female (E)Lats1^flox/flox;Lats2^flox/flox and (F)Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice. (G, H) Immunohistochemical analysis of TH expression in adrenal glands from 12 dpp male (G)Lats1^flox/flox;Lats2^flox/flox and (H)Lats1^flox/flox;Lats2^flox/flox; Nr5a1-cre mice. Abbreviations: H&E, hematoxylin and eosin stain; TH, tyrosine hydroxylase. Arrow = cells with lipid droplets in their cytoplasm. Arrowhead = cells with a large nucleus and eosinophilic cytoplasm. Scale bar in D is valid for C, scale bar in F is valid for E and scale bar in G is valid for H.

Figure 2.

Efficiency of Lats1 and Lats2 knockdown in Lats1^flox/flox; Lats2^flox/flox;Nr5a1-cre mice. (A-C) RT-qPCR analysis of Lats1 and Lats2 mRNA levels in the adrenal glands of (A) e14.5, (B) e17.5 and (C) 1 dpp male mice of the indicated genotypes (n = 6 animals/genotype). All data were normalized to the housekeeping gene Rpl19 and are expressed as means (columns) ± SEM (error bars). Asterisks = significantly different from control (* P < 0.05; *** P < 0.001; **** P < 0.0001).

Figure 3.

Progressive appearance of spindle-shaped cells in the adrenal cortex of Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice. Photomicrographs comparing male adrenal gland histology of Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre with that of Lats1^flox/flox;Lats2^flox/flox controls at the indicated ages. Scale bar (lower right) is valid for all images. Hematoxylin and eosin stain.

Figure 4.

Reduction of CYP11B1 expression in the adrenal cortex of Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice during development. Immunohistochemical analysis of CYP11B1 expression in adrenal glands from e14.5, e15.5, e17.5 and 1 dpp male mice of the indicated genotypes. Scale bar (lower right) is valid for all images.

Figure 5.

Progressive reduction of STAR expression in the adrenal cortex of Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice during development. Immunohistochemical analysis of STAR expression in adrenal glands from e14.5, e15.5, e17.5 and 1 dpp male mice of the indicated genotypes. Scale bar (lower right) is valid for all images.

Figure 6.

Progressive increase of vimentin expression in the adrenal cortex of Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice during development. Immunohistochemical analysis of vimentin expression in adrenal glands from e14.5, e15.5, e17.5 and 1 dpp male mice of the indicated genotypes. Scale bar (lower right) is valid for all images.

Figure 7.

Progressive increase of α-SMA expression in a subpopulation of adrenocortical cells in Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice. Immunohistochemical analysis of α-SMA expression in adrenal glands from e14.5, e17.5, 1 dpp and 12 dpp male mice of the indicated genotypes. Scale bar (lower right) is valid for all images.

Figure 8.

Lats1 and Lats2 deletion causes a decrease in the phosphorylation of YAP and an increase in the expression of YAP and TAZ downstream target genes in Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice. (A, B) Immunohistochemical analysis of phospho-YAP expression in adrenal glands from e17.5 dpp male mice of the indicated genotypes. Scale bar in B is valid for all images. (C, D) RT-qPCR analysis of Hippo target genes mRNA levels in the adrenal glands of (C) e17.5 and (D) 1 dpp male mice of the indicated genotypes (n = 6 animals/genotype). All data were normalized to the housekeeping gene Rpl19 and are expressed as means (columns) ± SEM (error bars). Asterisks = significantly different from control (* P < 0.05; *** P < 0.001; **** P < 0.0001).