Ex vivo cultures combined with vivo-morpholino induced gene knockdown provide a system to assess the role of WT1 and GATA4 during gonad differentiation

Abstract

Gonad morphogenesis relies on the correct spatiotemporal expression of a number of genes that together fulfill the differentiation of the bipotential gonad into testes or ovaries. As such, the transcription factors WT1 and GATA4 are pivotal for proper gonadal development. Here we address the contributions of GATA4 and WT1 to the sex differentiation phase in testes and ovaries. We applied an ex vivo technique for cultivating gonads in hanging droplets of media that were supplemented with vivo-morpholinos to knockdown WT1 and GATA4 either alone or in combination at the same developmental stage. We show that WT1 is equally important for both, the initial establishment and the maintenance of the sex-specific gene expression signature in testes and ovaries. We further identified Foxl2 as a novel putative downstream target gene of WT1. Moreover, knockdown of WT1 reduced mRNA levels of several molecular components of the hedgehog signaling pathway in XY gonads, whereas Gata4 vivo-morpholino treatment increased transcripts of Dhh and Ptch1 in embryonic testes. The data suggest that for its proper function, WT1 relies on the correct expression of the GATA4 protein. Furthermore, GATA4 down-regulates several ovarian promoting genes in testes, such as Ctnnb1, Fst, and Bmp2, suggesting that this repression is required for maintaining the male phenotype. In conclusion, this study provides novel insights into the role of WT1 and GATA4 during the sex differentiation phase and represents an approach that can be applied to assess other proteins with as yet unknown functions during gonadal development.

Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors

“…Direct testicular injection of a buffered salt solution can be used to introduce shRNA into germ cells, albeit with low transfection efficiency (<5%) (Ho et al 2017). Early gonadal development can be disrupted by cultivating organs in hanging droplets of media supplemented with vivo -morpholinos (Rudigier et al 2017). Cre can be targeted to mouse Leydig cells by germline transmission of transgenes, such as Sf1 -Cre (Bingham et al 2006), CYP11A1 -iCre (Wu et al 2007), and Cyp17a1 -iCre (Bridges et al 2008).…”

Evidence that downregulation of Wilms’ tumor 1 (WT1) is involved in cortical stromal cell differentiation into theca cells in adult bovine ovaries

“…Interestingly, WT1 plays important roles in maintaining adrenal‐gonadal primordium identity and in regulating gonad and granulosa cell differentiation (Bandiera et al, ; Gao et al, ). Furthermore, WT1 has important functions during sex determination and fetal testes development (Hammes et al, ; Rudigier, Dame, Scholz, & Kirschner, ; Wen, Wang, Tang, Cheng, & Liu, ). The cortical stroma had higher WT1 expression than that in the theca layer.…”

“…These differences may also be explained by , 2014). Furthermore, WT1 has important functions during sex determination and fetal testes development (Hammes et al, 2001;Rudigier, Dame, Scholz, & Kirschner, 2017;Wen, Wang, Tang, Cheng, & Liu, 2016). The cortical stroma had higher WT1 expression than that in the theca layer.…”

GATA4/6 regulate DHH transcription in rat adrenocortical autografts

“…Four patients who underwent bilateral adrenalectomy and ACTH replacement were able to withdraw from glucocorticoid replacement immediately after adrenal autotransplantation 8 . Dexamethasone-induced adrenal atrophy in mice was restored with daily ACTH stimulation 9 . ACTH stimulation after autotransplantation preserves autografts and may involve an unidentified pathway which promotes adrenal cortical regeneration and recovers endocrine function.…”

“…The HH signalling pathway may participate in adrenocortical autograft regeneration as well as adrenal cortex development. The regulation of Dhh transcription during gonadal development involves transcription factors such as Wt1, Gata4, Gata6, Sox9 and Sry 9,10 . In this study, we examined whether they affect Dhh transcription in adrenocortical autografts.…”