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First published online 16 October 2008
doi: 10.1242/dev.024786

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Notch signaling maintains Leydig progenitor cells in the mouse testis

¹ The Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
² The Department of Cellular Sociology, National Institute for Basic Biology, Okazaki, Japan.
³ Molecular and Integrative Physiology, University of Illinois at Urbana Champaign, IL 61801, USA.

View larger version (110K): [in this window] [in a new window]   Fig. 1. Dynamic expression of the Notch signaling pathway suggests a role in mouse gonad development. The coelomic domain of the gonad is upwards, anterior is leftwards and posterior is rightwards. Whole mount in situ hybridization of Notch2 (A,B), Notch3 (D,E), Hes5 (G,H) and Hes1 (J,K) were performed on 11.5 and 12.5 dpc XY whole gonads, and in some cases, stained gonads were sectioned (I,L). Whole-mount β-gal staining was performed on 12.5 dpc XY Notch2LacZ (C) and Notch3LacZ (F) gonads prior to sectioning. (M-R)XY Notch2LacZ gonads were stained with antibodies against β-gal (green) to detect Notch2, and PECAM1 (red, germ cells and vasculature) at 11.5, 12.0 and 13.5 dpc. Low (M-O) and high (P-R) magnification images are shown at each stage. Notch2 was detected throughout the XY gonad at 11.5 dpc (M,P), and localized to cells that are clustered around groups of germ cells at the beginning of testis cord development (N,Q). The Sertoli cell expression pattern was apparent at 12.5 dpc (C). At 13.5 dpc, β-gal expression was undetectable in Sertoli cells and shifted to interstitial cells in Notch2LacZ mice (O,R). Notch3 was not detected in XY gonads at 11.5 dpc (D). At 12.5 dpc, Notch3 was detected in the interstitium of the XY gonad (E,F). Hes5 and Hes1 were expressed in 11.5 dpc XY gonads (G,J). At 12.5 dpc, Hes5 was detected inside the cords (H,I), whereas Hes1 was detected in the interstitium (K,L). A,B,D,E,G,H,J,K were taken at 12x magnification; C,F were taken at 20x magnification; I,L,M-O were taken at 25x magnification; P-R were taken at 63x magnification. g, gonad; m, mesonephros; TC, testis cord; I, interstitium.

View larger version (74K): [in this window] [in a new window]   Fig. 2. Loss of Notch signaling results in an increase in fetal Leydig cells. The coelomic domain of the gonad is upwards, anterior is leftwards and posterior is rightwards. (A-E) 11.5 dpc mouse XY gonads were cultured in DMEM for 48 hours with DMSO (A,C) or the -secretase inhibitor DAPT (B,D). (A,B) Sertoli cells were stained with antibodies against SOX9 (red), and vasculature and germ cells, with antibodies against PECAM1 (green). (C,D) Leydig cells, detected with antibodies against 3β-HSD were significantly increased (138%; n=8 gonads; P<0.001) in DAPT-treated gonads (D,E), compared with the DMSO control (C,E). Numbers of Sertoli cells are not significantly affected (E; n=3 gonads; P=0.1792). (F-J) Deletion of the Notch downstream target gene Hes1 (Hes1-/-) resulted in normal numbers of SOX9-positive Sertoli cells (red, G,J; n=3 gonads; P=0.2138), but an increase of 3β-HSD-positive Leydig cells (I,J; 47%; n=3 gonads; P=0.039) and a loss of germ cells (PECAM1, green; white arrows in F,G). Data presented are mean±s.e.m. of cell numbers from at least three independent gonad samples. Scale bars: 50 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 3. Constitutively active Notch signaling in Sertoli cells does not change Sertoli cell fate. The coelomic domain of the gonad is upwards, anterior is leftwards and posterior is rightwards. (A-F) Immunofluorescent staining of PECAM1 (red) and SOX9 (green) on wild-type (A,C,E) and RosaNotch; Sf1-cre (B,D,F) mouse gonads at 11.5 (A,B), 12.5 (C,D) and 13.5 dpc (E,F). SOX9 appeared normal at all stages, although some mutant cords were smaller than wild type at 13.5 dpc (F). (G,H) Immunofluorescent staining of SOX9 (green, G) and GFP (green, a marker for NICD expression, H) on two adjacent sections of a 13.5 dpc mutant gonad showed that SOX9-positive Sertoli cells were also Notch active cells. Scale bars: 20 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 4. Constitutively active Notch signaling results in a loss of Leydig cells. The coelomic domain of the gonad is upwards, anterior is leftwards and posterior is rightwards. (A-D) Compared with wild type (A,C), RosaNotch; Sf1-cre mouse gonads (B,D) showed a loss of Leydig cells at 13.5 dpc based on immunofluorescent staining for 3β-HSD (red, A,B) or SF1 (red, C,D). PECAM1 (green) labels vasculature and germ cells in C and D. (E,F) In view of the low levels of activated caspase 3 (red), this loss was not due to cell death (PECAM1, green; Syto13 stains DNA, blue; n=3 gonads; P=0.7813). (G-J) LHX9 (red), which is expressed in interstitial somatic cells (G), shows an increase in RosaNotch; Sf1-cre gonads (H; n=3 gonads; P<0.01), whereas Leydig cell numbers are decreased based on 3β-HSD staining (I,J). Images shown are representative staining of at least three sections from three independent gonads. (K) A summary diagram of Notch/Hes1 results: loss of Notch function (LOF) leads to Leydig cell differentiation (red, oval cells), whereas gain of Notch function (GOF) maintains interstitial mesenchymal progenitor cells (purple, spindle shaped), and restricts their differentiation into Leydig cells. Scale bars: 50 µm.

View larger version (91K): [in this window] [in a new window]   Fig. 5. Constitutive Notch signaling restricts Leydig cell differentiation throughout fetal gonad development, but cannot change the fate of differentiated Leydig cells. The coelomic domain of the gonad is upwards, anterior is leftwards and posterior is rightwards. (A-D) H&E staining of mouse testis sections at P1, with (A,B) or without (C,D) immunostaining for 3β-HSD (black) in RosaNotch; Sf1-cre gonads (B,D) compared with gonads from mice carrying only one of the two transgenes (A,C). Spindle shaped cells increased in RosaNotch; Sf1-cre gonads (green arrows in D), whereas Eosin-staining Leydig cells (LC) declined. (E-H) Immunofluorescent staining for LHX9 (red, nuclear staining; cytoplasmic staining is nonspecific background) and DNA (Syto13, blue in E,F) or NICD (green in G,H) shows increased LHX9-positive cells in RosaNotch; Sf1-cre gonads, some of which were expressing Notch (white arrows in H, yellow staining cells). Broken lines outline testis cords. (I) RosaNotch; Hs-cre mice were heat-shocked at 13.5 dpc, after Leydig cell differentiation is initiated. At 15.5 dpc, many 3β-HSD-positive cells were detected (red), some also expressing NICD (green). Scale bars: 20 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Sertoli cell markers are not significantly different in RosaNotch; Sf1-cre gonads, but markers of differentiated Leydig cells are significantly downregulated. mRNA was isolated from a single pair of mouse gonads, and expression was compared by Q-RT-PCR between RosaNotch; Sf1-cre and wild-type gonads, normalized to Hprt. Sertoli cell-specific genes were similar (Sox9, Dhh, Pdgfa and Ptgds) or significantly downregulated (Amh) relative to normal expression levels (P=0.0534, 0.7942, 0.964, 0.4774 and 0.0323, respectively). Two differentiated Leydig cell genes, Scc and Cyp17a1, were significantly decreased in the mutant testis (P=0.0057, 0.00460). Interstitial markers, Arx, Pdgfra and Ptch1, were similar to wild type, while Lhx9 was significantly increased in RosaNotch; Sf1-cre gonads (P=0.0881, 0.2915, 0.5526 and 0.0212, respectively). NICD was significantly upregulated as expected. Data presented are mean±s.e.m. of Q-RT-PCR results from a total of four independent pairs of mutant and control gonad samples (each sample repeated at least three times for each gene).

View larger version (79K): [in this window] [in a new window]   Fig. 7. Testis cord formation and germ cell survival are affected by gain or loss of Notch signaling in gonadal somatic cells. The coelomic domain of the gonad is upwards, anterior is leftwards and posterior is rightwards. (A-F) Immunofluorescent staining for SOX9 (A,B,E,F) and Laminin (C,D, box inserts show 40x images of a confocal section) were used to evaluate mouse testis cord structure. At 13.5 dpc abnormal cord formation was detected in both, RosaNotch; Sf1-cre gonads (B,D) and Hes1-/- gonads (F). (G,H) At postnatal day (P) 1, fewer germ cells were detected inside testis cords based on Hematoxylin and Eosin staining (green arrows), although Sertoli cells appeared normal. (I,J) A significant decrease in the number of GCNA-positive cells (red; n=3, P<0.001) reflects germ cell loss in RosaNotch; Sf1-cre gonad at P1 (Syto13 stains DNA, blue). (K,L) C57BL/6 Hes1-/- gonads showed a significant germ cell loss at 13.5 dpc based on PECAM1 fluorescent immunostaining (P<0.001, n=3). Scale bars: 50 µm.

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