Inactivation of Dicer1 in Steroidogenic factor 1-positive cells reveals tissue-specific requirement for Dicer1 in adrenal, testis, and ovary

Abstract

Background: The synthesis of microRNA (miRNA) is a multi-step process that requires the action of the ribonuclease Dicer1. Dicer1 is responsible for the final processing of miRNA and has been implicated in cellular processes such as proliferation, apoptosis, and differentiation. Mouse embryos lacking Dicer1 die in early embryogenesis. In this study, we investigated whether Dicer1 is required for development of adrenal, testis, and ovary in mouse embryos.

Results: To target Dicer1 deletion specifically in developing adrenals and gonads, we used Steroidogenic factor 1-cre (Sf1/Cre) line in which Cre recombinase is active in the progenitor cells of adrenals and gonads. Lack of Dicer1 in the SF1-positive cells did not affect formation and early differentiation of the adrenals and gonads. However, increasing numbers of apoptotic cells were first detected in the Dicer1 knockout adrenal cortex at 18.5 days post coitum (dpc), followed by apoptosis of somatic cells and germ cells in the testis at postnatal day 0. Affected adrenal and testes underwent complete degeneration 48 hrs after the onset of apoptosis. However, ovaries were not affected at least until postnatal day 5, when the animals died due to adrenal insufficiency.

Conclusions: Dicer1 is dispensable for formation and differentiation of fetal tissues derived from the SF1-positive adrenogonadal primordium. Dicer1 is essential for maintaining cell survival in adrenal and testis; however, development of the ovary from fetal stages to postnatal day 5 does not require the presence of Dicer1. Our results reveal a tissue-specific requirement of Dicer1 and microRNAs. Future research is needed to understand how the tissue-specific role of Dicer1 is established.

Figure 1

Effects of Dicer1 ablation on adrenal development. Adrenal glands from control (CT) or Sf1/Cre;Dicer1^loxP/loxP (KO) embryos (16.5 and 18.5 dpc), newborns (P0), and Day 5 neonates (P5) were collected for (A) gross morphological analysis, (B) immunofluorescence for SF1 (magenta), TH (green), and DAPI (blue), and (C) immunofluorescence for Ki-67 (green), HSD3b (magenta), and DAPI (blue). Higher magnification (2× of the original figure) of the proliferating cells is shown in the inlets. Arrow = adrenal; kd = kidney. Scale bars represent 250 μm.

Figure 2

Effects of Dicer1 ablation on adrenal apoptosis. TUNEL assay was performed on sections of adrenals at 14.5 dpc, 16.5 dpc, 18.5 dpc, P0, and P5. Green nuclear staining represents positive signals for fragmented DNA and blue staining was the DAPI nuclear counterstain. Immunofluorescence for CYP21 (magenta) was also performed on adrenal sections to label the adrenal cortex. Higher magnification (4× of the original figure) of the apoptotic cells is shown in the inlets. Scale bars represent 100 μm.

Figure 3

Effects of Dicer1 ablation on fetal testis development. Testes from control (CT) or Sf1/Cre;Dicer1^loxP/loxP (KO) embryos at 16.5 dpc and 18.5 dpc were collected for immunofluorescence for SOX9 (green) and DAPI counterstain (blue). Scale bars represent 100 μm.

Figure 4

Effects of Dicer1 ablation on neonatal testis development. Testes from control (WT) or Sf1/Cre;Dicer1^loxP/loxP (KO) newborns (P0), Day 2 (P2), and Day 5 (P5) neonates were collected for (A) Gross morphological analysis and (B) immunofluorescence for laminin (green) and DAPI counterstain (blue). Epi = epididymis; T = testis. Scale bars represent 250 μm.

Figure 5

Effects of Dicer1 ablation on differentiation of germ cells, Sertoli cells, and Leydig cells in the testis. Testes from control (WT) or Sf1/Cre;Dicer1^loxP/loxP (KO) newborns (P0), Day 2 (P2), and Day 5 (P5) neonates were collected for (A) immunofluorescence for germ cell nuclear marker TRA98 (magenta), Sertoli cell marker SOX9 (green) and DAPI counterstain (blue) and (B) immunofluorescence for Leydig cell marker HSD3b (magenta), proliferation marker Ki67 (green), and DAPI (blue). Higher magnification (2× of the original figure) of the proliferating cells for P0 is shown in the inlets. Scale bars represent 100 μm.

Figure 6

Effects of Dicer1 ablation on apoptosis in the testis. (A) Immunofluorescence for cleaved Caspase3 (CASP3) was performed on sections of testes at 18.5 dpc, P0, P2, and P5. Green nuclear staining represented positive signals for apoptotic cells and blue staining was the DAPI nuclear counterstain. Double staining of CASP3 (green) and TRA98 or laminin (magenta) was performed on testes from P0 (B and D) and P2 (C and E) KO testes. Dotted lines mark the testis cords. Arrowhead = apoptotic cells outside the testis cord. Arrow = apoptotic germ cells. Scale bars represent 100 μm.

Figure 7

Effects of Dicer1 ablation on ovary development. Ovaries from control (CT) or Sf1/Cre;Dicer1^loxP/loxP (KO) Day 5 neonate (P5) were collected for (A) immunofluorescence for Ki67 (green) and HSD3b (magenta), (B) immunofluorescence for TRA98 (green) and SF1 (magenta), (C) immunofluorescence for CASP3 (green) and HSD3b (magenta). All sections were counterstained with DAPI (blue). Scale bars represent 250 μm.