Supplementary Material for: Conservation of Ovary-Specific Genes, Foxl2, Aromatase, and Rspo1, in the Common Indian Garden Lizard, Calotes versicolor, That Lacks Chromosomal or Temperature-Dependent Sex Determination

Foxl2,Rspo1, and Aromatase are genes important in the ovary developmental pathway in mammals and birds. Here, we show their presence in the lizard, Calotes versicolor, which is known to lack a chromosomal as well as a temperature-dependent mode of sex determination and has an indeterminate, bipotential gonad throughout embryonic development. The expression of the 3 genes, as well as that of CvSox9 and Wnt4 - the known testis and ovary pathway genes - was studied by RT-PCR and whole tissue RNA in situ hybridization (WRISH) on the developing mesonephros gonadal complex (MGC). The expression of all 3 genes was initiated in the gonad shortly after its evagination from the mesonephros (day 5 onwards). CvFoxl2 generally was expressed in those MGCs in which CvSox9 was either not expressed or lowly expressed and vice versa. On the other hand, CvArom was expressed rather sporadically and randomly, showing no association with CvFoxl2, CvRspo1, or CvSox9, though in later stages WRISH preparations showed its coincidence with CvWnt4. CvRspo1 was expressed in almost all embryos right from day 5. Immunofluorescence localization of Rspo1 and Foxl2 proteins showed their presence in the gonads from day 10 onwards, and by day 25 it was primarily confined to the cortex but away from the coelomic epithelium of the gonadal cortex. Apparently both proteins were localized in the pregranulosa cells, Rspo1 in the cytoplasm and Foxl2 in the nucleus. Thus, it is clear that both CvFoxl2 and CvRspo1 are active in ovary formation, but whether they are expressed in the same or different cells is unknown. Though the transcription pattern of CvArom remains circumspect for its role in differentiation of the ovary, earlier evidence on aromatase inhibitor-induced reversal to the male sex indicates its importance in ovary function.