DEV024786 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Notch2 and Notch3 are expressed at very low levels in XX gonads. (A-F) Whole-mount in situ hybridization for Notch2 (A,B) and Notch3 (D,E) on XX gonads at 11.5 and 12.5 dpc (10×). (C,F) 12.5 dpc XX Notch2^LacZ and Notch3^LacZ gonads were stained for β-gal and sectioned (20×). (G,H) Notch2 and Notch3 are both expressed in the interstitium of XY gonads by 13.5 dpc. 13.5 dpc XY Notch2^LacZ (G) and Notch3^LacZ (H) gonads were stained for β-gal and then sectioned (100×). g, gonad; m, mesonephros; TC, testis cord; I, interstitium. Scale bars: 20 µm.

• Supplemental Figure S2 -

  Fig. S2. In Rosa^Notch; Sf1-cre gonads, Notch signaling is constitutively active in somatic cell precursors at 11.5 dpc, and in differentiated Sertoli and Leydig cells. (A-E) Immunofluorescent staining of PECAM1 (red, germ cells and vasculature) and NICD-GFP (green, active Notch) in gonads from Rosa^Notch; Sf1-cre mice, and in Rosa^Notch controls at 10.5 dpc (B). At 10.5 dpc, no GFP was detected above background in experimental versus controls without Sf1-Cre (A,B). At 11.5 dpc, GFP was enriched in nuclei of pre-Sertoli cells and other somatic cell (C). (D,E) After testis cords formed (broken lines), GFP expression was detected in Sertoli cells and some interstitial cells (Leydig progenitors). (F,G) Lower magnification views of Fig. 4C,D. Compared with wild type (F), Rosa^Notch; Sf1-cre gonads (G) showed loss of SF1 (green)-positive cells in the interstitium at 13.5 dpc. PECAM1 (red) labels vasculature and germ cells; SF1 labels both Sertoli cells around germ cells, and Leydig cells in the interstitium. Scale bars: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. The early progenitor cell marker LHX9 labels some interstitial cells at all stages. (A) 11.5 and (B) 13.5 dpc XY gonads stained by immunofluoresence for LHX9 (red, nucleus), PECAM1 (green, germ cell and vasculature) and DNA (blue, Syto13). LHX9-positive cells are detected in the coelomic epithelium and inside the gonad at both stages (arrows). Scale bars: 50 µm. g, gonad; m, mesonephros; TC, testis cord (broken lines); CE, coelomic epithelium.

• Supplemental Figure S4 -

  Fig. S4. Germ cell loss in Rosa^Notch; Sf1-cre gonad at postnatal day 1. (A,B) An increase in caspase 3-positive cells (red) inside testis cords (broken lines) suggests germ cells undergo apoptosis in the Rosa^Notch; Sf1-cre gonad (B), compared with wild type (A). (Syto13 stains DNA, blue.)