Targeted Disruption of Lats1 and Lats2 in Mice Impairs Testis Development and Alters Somatic Cell Fate

Abstract

Hippo signaling plays an essential role in the development of numerous tissues. Although it was previously shown that the transcriptional effectors of Hippo signaling Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) can fine-tune the regulation of sex differentiation genes in the testes, the role of Hippo signaling in testis development remains largely unknown. To further explore the role of Hippo signaling in the testes, 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 the somatic cells of the testes using an Nr5a1-cre strain (Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre). We report here that early stages of testis somatic cell differentiation were not affected in this model but progressive testis cord dysgenesis was observed starting at gestational day e14.5. Testis cord dysgenesis was further associated with the loss of polarity of the Sertoli cells and the loss of SOX9 expression but not WT1. In parallel with testis cord dysgenesis, a loss of steroidogenic gene expression associated with the appearance of myofibroblast-like cells in the interstitial space was also observed in mutant animals. Furthermore, the loss of YAP phosphorylation, the accumulation of nuclear TAZ (and YAP) in both the Sertoli and interstitial cell populations, and an increase in their transcriptional co-regulatory activity in the testes suggest that the observed phenotype could be attributed at least in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper differentiation of testis somatic cells.

Keywords: Hippo signaling; Lats1/2; Sertoli cells; fetal Leydig cells; transgenic mouse model.

Figure 1

Testes of Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice are larger than controls and are histomorphologically abnormal. (A) Photographs of testes from 2-week-old Lats1^flox/flox;Lats2^flox/flox (control) and Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice. Ruler graduations are in millimeters. (B,C) Photomicrographs comparing the testes of 2-week-old (B) Lats1^flox/flox;Lats2^flox/flox and (C) Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice. (D,E) Photomicrographs illustrating the remaining seminiferous tubules (D) and interstitium (E) in Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice at higher magnification. Arrowhead = cells with a large nucleus and pale abundant cytoplasm. Red arrow = cell with an elongated large nucleus with an abundant cytoplasm. Black arrow = spindle-shaped cells. Asterisk = pyknotic cell. Dashes = delimitation of the seminiferous tubules. Scale bar in C is valid for B and scale bar in E is valid for D. Hematoxylin and eosin stain.

Figure 2

Progressive testicular dysgenesis in the Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice. Photomicrographs comparing testis histology of Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre with that of Lats1^flox/flox;Lats2^flox/flox controls during development. Dashed lines = testis cords dysgenesis, black arrow = germ cells, red arrow = interstitial spindle-shaped cells, arrowhead = spindle-shaped cells in the testis cords. Scale bar (lower right) is valid for all images. Hematoxylin and eosin stain.

Figure 3

Efficiency of Lats1 and Lats2 knockdown in the testes of Lats1^flox/flox;Lats2^flox/flox;Nr5a1-cre mice. (A–C) RT-qPCR analysis of Lats1 and Lats2 mRNA levels in the testes of (A) e14.5, (B) e17.5, and (C) 1 dpp 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 < 0.0001). (D) Immunohistochemical analysis of phospho-YAP expression in the testes of mice of the indicated genotypes. Scale bar (lower right) is valid for all images. Arrowhead = cells with inefficient recombination. Arrow = cells with efficient recombination.

Figure 4

YAP/TAZ activity increases in the testes of Lats1^flox/flox;Lats2^flox/flox;Nr5a1^cre/+ mice. (A–E) Immunohistochemical analysis of YAP expression in testes of e17.5 mice of the indicated genotypes. (F–J) Immunohistochemical analysis of TAZ expression in testes of e17.5 mice of the indicated genotypes. Arrow = YAP expression in non-recombined Sertoli cells. Scale bar in (G) is valid for (A,B,F). Scale bar in (J) is valid for (C–E,H,I). (K,L) RT-qPCR analysis of YAP/TAZ downstream targets mRNA levels in the testes of e17.5 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 < 0.0001).

Figure 5

Loss of Lats1 and Lats2 affects the polarity of the Sertoli cells. (A) Immunohistochemical analysis of GJA1 expression in testes of mice of the indicated genotypes. Scale bar (lower right) is valid for all images. (B) RT-qPCR analysis of Ocln and Pard6b mRNA levels in the testes of 1 dpp 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 < 0.01; **** p < 0.0001).

Figure 6

Loss of Lats1 and Lats2 affects expression of key Sertoli cell markers. (A) Immunohistochemical analysis of WT1 expression in testes of mice of the indicated genotypes. (B) Immunohistochemical analysis of SOX9 expression in testes of mice of the indicated genotypes. Arrow = immunopositive cells that lost their polarity. Arrowhead = immunopositive cells with normal polarity. Scale bar (lower right) is valid for all images. (C) RT-qPCR analysis of Sertoli cell markers in testes of 1dpp 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 7

Loss of Lats1 and Lats2 affects FLCs. (A) Immunohistochemical analysis of CYP17A1 expression in testes of mice of the indicated genotypes. Scale bar (lower right) is valid for all images. (B,C) RT-qPCR analysis of steroidogenesis (B) and progenitor FLC markers (C) in testes of 1dpp 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 < 0.01; **** p < 0.0001).

Figure 8

Loss of Lats1 and Lats2 leads to fibrosis in the testis interstitial tissue. (A,B) Immunohistochemical analysis of VIM expression in testes of e17.5 mice of the indicated genotypes. (C,D) Immunohistochemical analysis of α-SMA expression in testes of 1dpp mice of the indicated genotypes. (E) RT-qPCR analysis of myofibroblast markers in testes of 1dpp 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 < 0.01; *** p < 0.001). (F,G) Masson’s trichrome staining of 2-week-old mice of the indicated phenotype. Scale bars in the right panels are valid for the left panels.