The goals of this project are to dissect the sex determination/differentiation pathway of mice by identifying new genes in this pathway, establishing how they influence the expression of each other, and by determining the functions of their products. Since the discovery of the testis-determining factor gene Sry, little has been learned about which other genes it regulates to activate testis differentiation or repress ovarian differentiation, and thus this pathway of differentiation still remains largely a mystery. We thus propose to search for new genes in this pathway by comparing mRNAs expressed in genital ridges from fetuses of normal XY males, normal XX females and XY fetuses that are destined to be sex reversed. The mouse strain C57BL/6JEi-Y-pos(B6Ypos) will be used to generate XY sex reversed fetuses. These mice carry the Y chromosome from Mus poschiavinus on the C57BL/6J(B6) genetic background and produce XY offspring that are either hermaphrodites or females (never normal males). Furthermore, rescue of the sex reversal phenotype with a Mus musculus Sry transgene has demonstrated that the Y-chromosome- associated determinant causing sex reversal is the testis-determining factor gene Sry. RNAs from genital ridges at 11.5 and 12.5 days post coitum (d.p.c.) from XX and XY B6 mice and from XX and XY B6YPOS mice will be compared using enhanced differential display (EDD). At this time, five candidate mRNAs have been identified. Messenger RNAs identified as being in the sex determination/differentiation pathway because they show differences in expression between male and female and XY sex reversed genital ridges will be pursued to establish their roles in the pathway. The function of some of the newly identified genes will be tested directly by introducing null alleles into mice using ES cell gene targeting technology and determining the effect on sex phenotype. Mice carrying null alleles of sex determination pathway genes will be used to determine how one gene in the pathway influences others by assaying RNA of homozygous null genital ridges for expression of other sex determination pathway mRNAs. It is likely that homozygous mutants of some of these genes will produce partial sex reversal phenotypes (as seen in some of the XY B6Ypos mice). In these cases mice will be bred that carry combinations of more than one sex determination null allele to establish the functional relationships of these genes in the pathway.
