Supplemental Figure 1: Leydig cells are reduced at multiple stages in both male sterile mutants

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1 SUPPLEMENTAL FIGURE LEGENDS: Supplemental Figure 1: Leydig cells are reduced at multiple stages in both male sterile mutants (Sgpl1 -/- and Plekha1 -/- ). Using an antibody against CYP11a1 to label Leydig cells (red, examples indicated by arrows), the numbers of these cells is reduced before and during early adolescence (P7, P14 and P21) in both mutants. Sections are counterstained with DAPI (blue). Supplemental Figure 2: Uterine morphology is affected in female sterile mutants. Whole mounts show hypoplasic uteri in Sgpl1 -/-, Plekha1 -/- ;Pdgfrα +/-, Schip1 -/- ;Pdgfrα +/- and BC /- ;Pdgfrα +/- mice, while the uteri of Tiparp -/- mice appear enlarged. Histological sections indicate that the layers of the uterine wall (the endometrial stroma and myometrium) are present, but reduced. Sections are stained with hematoxylin and eosin. Supplemental Figure 3: Using CSPG4 as a marker (red), theca cells (arrows) are reduced in most female sterile mutants (Sgpl1 -/-, Plekha1 -/- ;Pdgfrα +/-, Schip1 -/- ;Pdgfrα +/- and BC /- ;Pdgfrα +/- ). Using ASMA as a marker (green), VSMCs are also reduced in throughout the ovaries of these mutants. Tiparp -/- ovaries appear to have normal numbers of both theca cells and VSMCs. The partially fertile Schip1 -/- ;Pdgfrα +/- and BC /- ;Pdgfrα +/- ovaries maintain small regions of CSPG4 positive cells in the stroma. Immunofluorescent sections are counterstained with DAPI (blue). Supplemental Figure 4: LH levels in sterile mutants are not decreased from wildtype. LH was measured in serum using an RIA assay. Error bars indicate SEM. Each bar represents an average of 6-10 mice, with the exception of Sgpl1 -/- and Plekha1 -/- ;Pdgfrα +/-. These mutants had decreased viability and fewer samples were measured (1-2 mice), thus error bars are not shown on these conditions.

2 Supplemental Figure 5: In Plekha1 -/- testes, both ASMA and Desmin (green) are reduced around vasculature in VSMCs (arrows) and around the testis cords, in areas associated with peritubular myoid cells. Sections are counterstained with DAPI (blue). Supplemental Figure 6: A model of the function of PDGF targets in the regulation of the male steroidogenic pathway. In the testis, Plekha1 represses Tiparp expression, allowing the expression of the male steroidogenic enzyme 17HSD3 and the synthesis of testosterone. In the ovary, Tiparp acts to repress this enzyme, preventing the synthesis of testosterone in the theca cell and allowing the synthesis of estrogen in the neighboring granulosa cell. Supplemental Figure 7: Pdgf receptors and ligands are expressed within the testis and ovary. A) In the testis, both receptors (Pdgfrα and Pdgfrβ) are expressed within the interstitial population (arrows) and to a lesser degree in the outer layers of the testis cords. In the ovary, both receptors (Pdgfrα and Pdgfrβ) are expressed with theca cells (arrows) surrounding follicles, as well as in some stroma populations. B) The ligands Pdgfa and Pdgfc are expressed in the testis cords (arrowheads) and interstitial populations. In the ovary, the ligand Pdgfa is expressed in stroma populations, with some weak detection in theca cells (arrow). Pdgfb is expressed in the granulosa of follicles (arrowhead) and the stroma. Pdgfc is broadly expressed in the ovary and found in granulosa, theca and stroma populations. Supplemental Figure 8: Conditional knockout of Pdgfrα leads to defects in the embryonic testis. A) Using Xgal staining, Sf1-Cre (blue) is detected in embryonic Leydig cells between the developing testis cords at E12.5. B, C) Sf1-Cre;Pdgfrα fl-/- testes exhibit impaired testis cord formation at both E13.5 and E14.5. D) Sf1-Cre;Pdgfrα fl-/- testes have reduced numbers of

3 steroidogenic cells (red), detected with an antibody against CYP11A1. Sections are counterstained with DAPI (blue) and the testis is indicated by brackets. Supplemental Figure 9: Schematic of the conditional allele of Pdgfrβ. A) Schematic of conditional allele. A) Map of the PDGFRβ locus, exons are indicated by blue boxes; bottom, red arrowheads indicate loxp sites, blue arrowheads indicate FRT sites flanking a PGK-neo cassette. DTA is a PGK-DTA cassette used for negative selection in ES cells. B) PCR of shows Neo is efficiently removed from the targeted allele in More-Cre + ;Pdgfrβ fl-/- mice. C) Western of Pdgfrβ immunoprecipitated from whole embryo extracts at E18.5 shows loss of PDGFRβ protein in More-Cre + ;Pdgfrβ fl-/- mice.

4 Supplemental Table 1: Sf1-cre + /Pdgfrα fl-/- /Pdgfrβ fl-/- mice have reduced viability after birth. +-/+-/+x --/+-/+- Actual wk 1 Expected wk 1 +-/+-/-- x +-/--/+- Actual E 18.5 Expected E /--/ % 3.1 % % 18.8 % -/--/ % 3.1 % % 6.3 % +/--/ % 6.3 % % 18.8 % -/--/ % 6.3 % % 6.3 % +/+-/ % 6.3 % % 18.8 % -/+-/ % 6.3 % % 6.3 % Total in litters 128 (18 litters) 65 (9 litters)

5 Supplemental Table 2: Primers used for genotyping and real time PCR. For genotyping mutants created with the gene trap array construct (Sgpl1, Plekha1, Tiparp, Schip and BC058969), the primers for the gene are listed in the indicated box, while the primer for splice acceptor region of the construct is indicated separately as GTA. Gene Genotyping PCR Real Time PCR Sgpl1 F: CGCTCAGAAGGCTCTGAGTCATGG na R: CCAAGTGTACCTGCTAAGTTCCAG Plekha1 F: TACTCAGATGAAAAGGCAGGAACC na R: GGATCTGGATTGCATCTCTAGCCC Tiparp F: TGTCAGATCCCTCCTTCGTGAGGC R: GTATAGTACCTAGCACTGTTCACC F: CGACTAATTGAAGAAGCCAACTCTCG R: CTTGGATGAAGTCCTGAGATGGATGC Schip1 F: TGACCATAGAAACTCCACAAGGG R: TACTATGAGGCTAGTAGAGAAGCC na BC F: CAGTATTCAACAGTCCAGTCTTGAG na R: CCTGGCTGTCCTGGAACTCACTCTG GTA R: CATCAAGGAAACCCTGGACTACTG na Pdgfrα and Pdgfrα fl R4: CCCTTGTGGTCATGCCAAAC R5: GCTTTTGCCTCCATTACACTGG R6:ACGAAGTTATTAGGTCCCTCGAC F: GAAACGATCGTGGTGACCTGTG R: TGACGGGCAGCACATTCATACT Pdgfrβ Pdgfrβ fl Cre B1:TGGACTCCGAGGACCTGTTCATT B2: AAAAGTACCAGTGAAACCTCGCTG B3: ATCAGCCTCGACTGTGCCTTCTAG F: GGAAAAGCAGGTTTGTGC R: TACCAGGAAGGCTTGGGAAG B: CCAGTTAGTCCACTTATGTTG F: TCCAATTTACTGACCGTACACCAA F: CATGTCTGAGACCCGGTACGTG R: GCAGCTTGAAGGAGAGCTGGAC R: CCTGATCCTGGCAATTTCGGCTA Cyp11a1 na F: TCCATTACCATCAGATGCAGAG R: GTCCACGATGTAAACTGACTCC Cyp17a1 na F: CTGGCCAGAGAAGTGCTCGTG R: TGCAGCTGCCAGGAGCTACTAC Cyp19a1 na F: TTCATGAGAGTCTGGATCAGTG R: CCACGCTTGCTGCCGAATCG Hsd17b1 na F: CTGCGTGGTTATGAGCAAGC R: CGCATTGCAGTCAAGAAGAGC Hsd17b3 na F: GACCACTGGAAGCTGTGTGAAGAT R: TCTCACCGGAAGTGCTCAGGAAAT Ubc na F: CGAGCCCAGTGTTACCACCAAG R: CACCCAAGAACAAGCACAAGGA na na

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