DEV019497 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Sequence of Sdmg1. Predicted transmembrane domains are underlined and the potential C-terminal dileucine targeting motif is highlighted in blue.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Phylogeny of DUF300 protein family members from selected model organisms. Scale bar indicates 0.1 substitutions per site. Uniprot or GenBank Accession Numbers are indicated. Multiple alignments and phylogenetic analysis were performed using CLUSTAL W (Thompson et al., 1994).

  Reference

  Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. In situ hybridisation for Sdmg1 in male and female embryonic gonads. Sdmg1 exhibits male-specific expression in the testis cords at 12.5 dpc and 13.5 dpc, and expression in 14.5 dpc male W^e/W^e gonads. In situ hybridisation was performed as described by Henrique et al. (Henrique et al., 1995). Scale bar: 0.2 mm.

  Reference

  Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J. and Ish-Horowicz, D. (1995). Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787-790.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Sdmg1 has a restricted expression profile in adult mouse tissues. (A) Northern blot showing expression of Sdmg1 RNA in testis (arrows). Methylene Blue staining of ribosomal RNA is shown as a loading control. (B) Western blot showing expression of Sdmg1 protein in testis (arrow). The anti-Sdmg1 band migrates at around 110 kDa. (C) Western blot showing an anti-Sdmg1 band can be detected at around 50 kDa after deglycosylation of testis extracts with PNGase. Northern and western blotting was performed as described by Adams and McLaren (Adams and McLaren, 2002). PNGase treatment of testis extracts was performed as recommended by the supplier (New England Biolabs). Abbreviations: Sk. Musc., skeletal muscle; PNGase, peptide:N-glycosylase F.

  Reference

  Adams, I. R. and McLaren, A. (2004). Identification and characterisation of mRif1: a mouse telomere-associated protein highly expressed in germ cells and embryo-derived pluripotent stem cells. Dev. Dyn. 229, 733-744.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. Retinoic acid or ketoconazole can induce Stra8 expression in cultured male gonads. (A,B) In situ hybridisation showing that Stra8 is normally expressed in 13.5 dpc female, but not male, gonads. (C,D) Sexually dimorphic expression of Stra8 is maintained in our culture system when 12.5 dpc male or female gonads are cultured for 2 days on agar blocks. (E,F) Treatment of 12.5 dpc male gonads with either 0.7 mM retinoic acid (RA) or 0.7 mM ketoconazole (ket) for 2 days in culture induces ectopic expression of Stra8 in our culture system, consistent with previous reports (Bowles et al., 2006; Koubova et al., 2006). In situ hybridisation was performed as described by Henrique et al. (Henrique et al., 1995). Scale bar: 0.1 mm.

  Additional reference

  Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J. and Ish-Horowicz, D. (1995). Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787-790.