Supplemental Information. Nodal Signaling Regulates Germ Cell. Development and Establishment of Seminiferous. Cords in the Human Fetal Testis

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1 Cell Reports, Volume 25 Supplemental Information Nodal Signaling Regulates Germ Cell Development and Establishment of Seminiferous Cords in the Human Fetal Testis Anne Jørgensen, Joni Macdonald, John E. Nielsen, Karen R. Kilcoyne, Signe Perlman, Lene Lundvall, Lea Langhoff Thuesen, Kristine Juul Hare, Hanne Frederiksen, Anna-Maria Andersson, Niels E. Skakkebæk, Anders Juul, Richard M. Sharpe, Ewa Rajpert-De Meyts, and Rod T. Mitchell

2 Fig. S1 Supplementary Figure 1. Key factors of Nodal and Activin signaling pathways are expressed in human fetal gonads. Related to Figure 1. Expression of Nodal, Cripto, Lefty, ALK4, ALK5, ActRIIB, INHBA, and INHBB in fetal ovary and testis samples from 1 st trimester and testis samples from 2 nd trimester. Gene expression is normalized to RPS29 and expressed relative to fetal testis samples from GW 7 9, which were set to a value of 1. Values represent mean ± SEM and for each age-group N=5-11. Significant difference compared to fetal testis GW 7-9, *** p<0.001, ** p<0.01, * p<0.05. Note, no fetal ovary samples were available for analysis for GW and GW ND: not determined, GW: gestational week.

3 Fig. S2 Supplementary Figure 2. Effects of inhibiting Nodal and Activin signaling in 1 st trimester human fetal testis ex vivo cultures. Related to Figure 1. A) Effects of SB treatment (20 µm for 14 days) in ex vivo culture of 1st trimester fetal testis on expression of the germ cell marker VASA and the meiosis marker SCP3. Adult human testis sample was included as a positive control. B) Effects of SB treatment (20 µm for 14 days) in ex vivo culture of 1st trimester fetal testis on expression of the granulosa cell marker FOXL2 to examine possible Sertoli-to-granulosa cell trans-differentiation. Fetal ovary sample was included as positive control. Counterstaining with Mayer s haematoxylin, scale bar corresponds to 50 µm.

4 Fig. S3 Supplementary Figure 3. Effects of simultaneous inhibition of Nodal and Activin signaling in 1 st trimester human fetal testis ex vivo cultures on proliferation and apoptosis. Related to Figure 1. A) Effects on expression of markers of proliferation and apoptosis following SB treatment (20 µm), for 7 or 14 days in ex vivo culture of 1 st trimester fetal testis. Immunohistochemical staining for the proliferation marker BrdU (which was added to culture media for the last 6 hours of culture), the apoptosis markers cleaved PARP (cparp) and staining determining TUNEL-positive cells, was examined with N=3 for the 7-day culture period and N=9 for the 14-day culture period. Counterstaining with Mayer s haematoxylin, scale bar corresponds to 50 µm. B) Quantification of proliferating (BrdU + ) germ cells and apoptotic (cparp + ) cells per mm 2 after 14 days of culture. Values represent mean ± SEM, with N=9. Significant difference compared to vehicle controls, *** P<0.001, ** P<0.01.

5 Fig. S4 Supplementary Figure 4. Effects of inhibiting Nodal and Activin signaling in 2 nd trimester human fetal testis. Related to Figure 3. Multinucleated germ cells present in 2 nd trimester fetal testis treated with SB (20 µm, 6 days) followed by xenografting for 6 weeks. Higher magnification of haematoxylin eosin staining shown in Figure 3A, scale bar corresponds to 50 µm and 10 µm, respectively. Boxes indicate area containing multinucleated germ cells.

6 Fig. S5 Supplementary Figure 5. Effects of manipulating Activin signalling in 1 st trimester fetal testis ex vivo cultures. Related to Figure 4. Morphology and expression pattern of germ cell markers OCT4 (gonocyte) and MAGE-A4 (pre-spermatogonia), Sertoli cell marker (AMH), interstitial cell marker (COUP-TFII), and Leydig cell marker (CYP11A1) investigated in 1 st trimester fetal testis samples treated with recombinant Activin A and Follistatin. With N=12 fetuses for Activin A treatment and N=12 for Follistatin treatment. Counterstaining with Mayer s haematoxylin, scale bar corresponds to 50 µm.

7 Fig. S6 Supplementary Figure 6. Effects of manipulating Nodal signalling in 1 st trimester ex vivo cultures. Related to Figure 4 and 5. A) Effects on proliferation and apoptosis of Nodal and Lefty treatment for 2 weeks in ex vivo culture of 1 st trimester fetal testis. Immunohistochemical staining for the proliferation marker BrdU (which were added to culture media for the last 6 hours of culture) and the apoptosis marker cleaved PARP (cparp). Counterstaining with Mayer haematoxylin, scale bar corresponds to 50 µm. B) Quantification of proliferating (BrdU + ) germ cells and apoptotic (cparp + ) cells per mm 2. Values represent mean ± SEM, with N=7 (Nodal) and N=10 (Lefty).

8 Fig. S7 Supplementary Figure 7. Effects of manipulating Activin signalling in 1 st trimester ex vivo cultures. Related to Figure 4 and 5. A) Quantification of androgens produced in the fetal testis tissue ex vivo cultures and secreted to the media droplets. Media was collected every 48 hours throughout the 14-day culture period and was pooled for each individual tissue piece. Androgens were measured by LC-MS/MS and are shown as ratios compared to vehicle controls for each metabolite. Values represent mean ± SEM, with N=10 for each treatment. B) Effects on proliferation and apoptosis of Activin and Follistatin treatment for 2 weeks in ex vivo culture of 1 st trimester fetal testis. Immunohistochemical staining for the proliferation marker BrdU (which were added to culture media for the last 6 hours of culture) and the apoptosis marker cleaved PARP (cparp). Counterstaining with Mayer haematoxylin, scale bar corresponds to 50 µm. C) Quantification of proliferating (BrdU + ) germ cells and apoptotic (cparp + ) cells per mm 2. Values represent mean ± SEM, with N=10.

9 Table S1 Supplementary table 1. Antibody dilutions, retrieval buffer and details. Related to Figure 1-6. For all antibodies, antigen retrieval was conducted by microwaving or placing sections in a pressure cooker in indicated retrieval buffer. Citrate buffer: 10 mm, ph 6.0; TEG buffer: 10 mmtris, 0.5 mm EGTA, ph 9.0; Urea buffer: 5% w/v carbamide, ph 5.5. Antibody Dilution (formalin/bouin fixed tissue) Retrieval buffer Company Number OCT4 1:50 / 1:100 TEG Santa Cruz Sc-5279 NANOG 1:50 Citrate R&D Systems AF-1997 AP2γ 1:50 / 1:50 Urea Santa Cruz Sc SALL4 1:100 TEG Santa Cruz Sc LIN28 1:500 Citrate R&D Systems AF-3757 C-KIT 1:200 TEG Dako A4502 MAGE-A4 1:250 / 1:250 TEG Non-commercial Gift from Prof. Spagnoli AMH 1:400 / 1:600 Citrate Santa Cruz Sc-6886 SOX9 1:400 / 1:2000 Citrate Millipore AB5535 cparp 1:75 / 1:75 Citrate Cell Signaling 5625 BrdU 1:100 Citrate Dako M0744 COUP-TFII 1:50 / 1:50 Citrate Perseus Proteomics PP-H CYP11A1 1:250 /1:300 TEG Sigma HPA γh2ax 1:800 TEG Abcam Ab26350 SCP3 1:800 TEG Novus NB VASA 1:1200 Citrate Abcam Ab13840 FOXL2 1:75 Citrate Non-commercial Gift from Dr. Wilhelm DMRT1 1:400 / 1:800 Citrate Sigma HPA027850

10 Table S2 Supplementary table 2. LC-MS/MS validation parameters for androgens and corticosteroids in cell media from fetal testis cultures. Related to Figure 1 and 5. Limits of quantification (LOQ), range of calibration curves based on 10 standards and results of inter-day control materials (n=32) at low and high levels from 11 batches. Control Low Control high LOQ Range mean RSD recovery mean RSD recovery (nm) (nm) (nm) (%) (%) (nm) (%) (%) Estrone 1-sulfate LOQ Cortisone 0.19 LOQ Cortisol 1.9 LOQ dehydroepiandrosterone sulfate 19 LOQ Corticosterone 0.1 LOQ deoxycortisol LOQ Δ4-androstenedione LOQ Testosterone LOQ α-hydroxyprogesterone 0.1 LOQ Progesterone LOQ

11 Table S3 Supplementary table 3. Primer sequences. Related to Figure 1 and Figure S1. Gene Fwd primer 5-3 Rev primer 5-3 Amplicon size GenBank Accession no. Activin βa (INHBA) GAACTTATGGAGCAGACCTCGG TTGCCATCACACTCCAAGCC 608 bp NM_ Activin βb (INHBB) GCCAGGAGCGCGTTTCCGAAATC CCGCTCGCCCCGCTCAAACAAG 326 bp NM_ ActRIIB (ACVR2B) CAACTTCTGCAACGAACGCTT GCGCCCCCGAGCCTTGATCTC 283 bp NM_ ALK4 (ACVR1B) GGAGCGTCTTGTCTTTGGAG TGCAACAGGATCGACTTGAG 238 bp NM_ ALK5 (TGFBR1) GATGGGCTCTGCTTTGTCTC CAAGGCCAGGTGATGACTTT 214 bp NM_ ALK7 (ACVR1C) GACATGAAAACATCCTTGGT ACTTCTGGTCACAAACAACC 585 bp NM_ LEFTY GCCTCGACAGTGCATCGCCTC CAAGTAAACAATGACACATTGGGC 477 bp NM_ NODAL AGCATGGTTTTGGAGGTGAC CCTGCGAGAGGTTGGAGTAG 160 bp NM_ CRIPTO (TDGF1) TCCTTCTACGGACGGAACTG ATCACAGCCGGGTAGAAATG 153 bp NM_ RPS20 AACAAGCCGCAACGTAAAATC ACGATCCCACGTCTTAGAACC 166 bp NM_ RPS29 CGCTCTTGTCGTGTCTGTTCA CCTTCGCGTACTGACGGAAA 91 bp NM_001032