Pre-Sertoli Specific Gene Expression Profiling Reveals Differential Expression of Ppt1 and Brd3 Genes Within the Mouse Genital Ridge at the Time of Sex Determination1

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Abstract
In mammals, testis determination is initiated when the SRY gene is expressed in pre-Sertoli cells of the undifferentiated genital ridge. SRY directs the differentiation of these cells into Sertoli cells and initiates the testis differentiation pathway via currently ill-defined mechanisms. Because Sertoli cells are the first somatic cells to differentiate within the developing testis, it is likely that the signals for orchestrating testis determination are expressed within pre-Sertoli cells. We have previously generated a transgenic mouse line that expresses green fluorescent protein under the control of the pig SRY promoter, thus marking pre-Sertoli cells via fluorescence. We have now used suppression-subtractive hybridization (SSH) to construct a normalized cDNA library derived from fluorescence-activated cell sorting (FACS) purified pre-Sertoli cells taken from 12.0 to 12.5 days postcoitum (dpc) fetal transgenic mouse testes. A total of 35 candidate cDNAs for known genes were identified. Detection of Sf1, a gene known for its role in sex determination as well as Vanin-1, Vcp1, Sparc, and Aldh3a1, four genes previously identified in differential screens as gene overexpressed in developing testis compared with ovary, support the biological validity of our experimental model. Whole-mount in situ hybridization was performed on the 35 candidate genes for qualitative differential expression between male and female genital ridges; six were upregulated in the testis and one was upregulated in the ovary. The expression pattern of two genes, Ppt1 and Brd3, were examined in further detail. We conclude that combining transgenically marked fluorescent cell populations with differential expression screening is useful for cell expression profiling in developmental systems such as sex determination and differentiation.