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. 2022 Sep 16;7(1):bvac143.
doi: 10.1210/jendso/bvac143. eCollection 2022 Nov 17.

Effect of Inactivation of Mst1 and Mst2 in the Mouse Adrenal Cortex

Nour Abou Nader  1 , Étienne Blais  1 , Guillaume St-Jean  1 , Derek Boerboom  1 , Gustavo Zamberlam  1 , Alexandre Boyer  1
Affiliations

Effect of Inactivation of Mst1 and Mst2 in the Mouse Adrenal Cortex

Nour Abou Nader et al. J Endocr Soc. .

Abstract

Recent conditional knockout of core components of the Hippo signaling pathway in the adrenal gland of mice has demonstrated that this pathway must be tightly regulated to ensure proper development and maintenance of the adrenal cortex. We report herein that the most upstream kinases of the pathway, the mammalian STE20-like protein kinases 1 and 2 (MST1and MST2, respectively), are expressed in the mouse adrenal cortex with MST2 expression being restricted to the zona glomerulosa (zG). To further explore the role of Hippo signaling in adrenocortical cells, we conditionally deleted Mst1/2 in steroidogenic cells using an Nr5a1-cre strain (Mst1 flox/flox ; Mst2 flox/flox ; Nr5a1-cre). Our results show that the loss of MST1/2 leads to the premature and progressive accumulation of subcapsular GATA4+, WT1+ adrenal gonadal primordium (AGP)-like progenitor cells starting at 2 months of age without affecting aldosterone and corticosterone secretion. To help us understand this phenotype, microarray analyses were performed on adrenal glands from 2-month-old mutant and control mice. Gene expression analyses revealed that loss of Mst1/2 leads to the overexpression of known downstream target genes (Ajuba, Aqp1, Fn1, Ibsp, Igf1, Igfbp2, Mmp2, Thbs1) of the main effector of Hippo signaling, YAP; and underexpression of genes (Agtr1b, Ecgr4, Hsd3b6, Nr0b1, Tesc, Vsnl1) that are normally specifically expressed in the zG or overexpressed in the zG compared to the zona fasciculata (zF). Together, these results suggest that MST1/2 regulates Hippo signaling activity in the adrenal cortex and that these two kinases are also involved in the fine tuning of zG cell function or differentiation.

Keywords: MST1; MST2; adrenal gland; adrenocortical cells; transgenic mouse‌.

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Figures

Figure 1.
Figure 1.
Localization of MST1 and MST2 in the adrenal gland of mice. Immunohistochemical analysis of MST1 (A, B) and MST2 (C). (D) Expression in adrenal glands from 6-month-old virgin female (images B and D are increased magnifications of A and C, respectively). Scale bar in C is valid for A and scale bar in D is valid for B. Arrows, X-zone cells; Arrowheads, chromaffin cells; c, capsule; zF, zona fasciculata; zG, zona glomerulosa.
Figure 2.
Figure 2.
Recombination efficiency in Mst1flox/flox;Mst2flox/flox;Nr5a1-cre mice. (A, B) Immunohistochemical analysis of MST1 expression in adrenal glands from 6-month-old control (A) and mutant (B) virgin female. (C, D) Immunohistochemical analysis of MST2 expression in adrenal glands from 6-month-old control (C) and mutant (D) virgin female. Scale bar in (D) is valid for all images. Arrows, X-zone cells; arrowheads, chromaffin cells; cross, capsular cells. (E) RT-qPCR analyses of Lats1 and Lats2 mRNA levels in the adrenal glands of 4- and 6-month-old virgin female 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 < .0001).
Figure 3.
Figure 3.
Progressive appearance of spindle-shaped cells in the adrenal cortex of Mst1flox/flox;Mst2flox/flox;Nr5a1-cre mice. (A) Photomicrographs comparing female adrenal gland histology of Mst1flox/flox;Mst2flox/flox;Nr5a1-cre with that of Mst1flox/flox;Mst2flox/flox controls at the indicated ages. (B) Photomicrographs of the spindle-shaped cells observed in Mst1flox/flox;Mst2flox/flox;Nr5a1-cre mice at higher magnification. Scale bar (lower right) is valid for all images. Hematoxylin and eosin stain. Dashed line, delimitation of the spindle-shaped cell population. (C) Measure of the thickness of the zone formed by the spindle-shaped cells in 10-month-old control and mutant animals; 3 to 4 nonconsecutive sections of whole adrenal were measured per animal (n = 4). Data are expressed as means (columns) ± SEM (error bars). Asterisks, significantly different from control (**P < .01).
Figure 4.
Figure 4.
Spindle-shaped cells have characteristics of AGP-like progenitor cells. Immunohistochemical analysis of WT1 (A), GATA4 (B) and NR5A1 (C) expression in adrenal glands from a 2-month-old Mst1flox/flox;Mst2flox/flox;Nr5a1-cre mice virgin female. Scale bar in (C) is valid for all images.
Figure 5.
Figure 5.
Spindle-shaped cells affect DAB2+ cell positioning. Immunohistochemical analysis of DAB2 in adrenal glands from 2-month-old and 10-month-old Mst1flox/flox;Mst2flox/flox (A, B) and Mst1flox/flox;Mst2flox/flox;Nr5a1-cre (C, D) virgin females. Scale bar in D is valid for all images. Dashed lines, delimitation of DAB2+ cells.
Figure 6.
Figure 6.
Hippo signaling is inactive in the spindle-shaped cells of mutant animals. Immunohistochemical analysis of phospho-YAP in adrenal glands from 2-month-old and 10-month-old Mst1flox/flox;Mst2flox/flox (A, B) and Mst1flox/flox;Mst2flox/flox;Nr5a1-cre (C, D) virgin females. Scale bar in D is valid for all images. Dashed line, delimitation of the spindle-shaped cell population.
Figure 7.
Figure 7.
Effects of Mst1/2 ablation on gene expression. (A) Validation of the microarray data by RT-qPCR analysis (n = 6 for Mst1flox/flox;Mst2flox/flox and n = 6 for Mst1flox/flox;Mst2flox/flox;Nr5a1-cre 2-month-old virgin females). RT-qPCR data were normalized to the housekeeping gene Rpl19. All data are expressed as mean (columns) ± SEM (error bars). Asterisks indicate significant differences from controls (*P < .05; **P < .01; ***P < .001).
Figure 8.
Figure 8.
Network of genes with upregulated expression following the loss of Mst1/2 in the adrenal cortex. Gene network analysis of the upregulated genes in the adrenal cortex of Mst1flox/flox;Mst2flox/flox;Nr5a1-cre 2-month-old virgin females using the string database. Magenta, experimentally determined interactions; blue, interactions known from curated database; green, predicted interactions, gene neighborhood; violet, protein homology.
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