Cell proliferation has been shown to have multiple functions in development and pattern formation, including roles in growth, morphogenesis, and gene expression. Previously, we determined that the earliest known morphological event downstream of the male sex determining gene, Sry, is the induction of proliferation. In this study, we used proliferation inhibitors to block cell division during early gonad development, at stages before the XY gonad has committed to the testis pathway. Using the expression of sex-specific genes and the formation of testis morphology as markers of testis determination, we found that proliferation within a specific 8-h window was critical for the establishment of the male pathway and the formation of the testis. Inhibition of proliferation before or after this critical period led to smaller gonads, but did not block testis formation. The critical period of proliferation coincides with the initiation of Sry expression and is essential for the differentiation of Sertoli cells, suggesting that proliferation is a vital component of the initiation of the male pathway by Sry. We believe these studies suggest that proliferation is involved not only in the elaboration of organ pattern, but also in the choice between patterns (male and female) in the bipotential gonad
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