In most mammals, the expression of Sry initiates the development of testes and thusdetermines the male phenotype. However, despite the pivotal role of SRY, the molecular andcellular mechanisms underlying its regulation and mode of action remain elusive. This projectsought to address two of the key missing pieces of the Sry puzzle: how is Sry expressionregulated? What does SRY directly regulate?One of the limitations in the study of SRY over the last two decades, has been the lack ofmolecular tools, notably, an antibody that can detect and be used to visualise mouse SRY invivo. Consequently, my first goal was to generate an anti-mouse SRY antibody. I comparedthe specificity and effectiveness of this antibody with several commercially available antimouseSRY antibodies. The two commercially available antibodies cross-reacted with otherSOX proteins in immunofluorescence analyses of transfected cells, and one of those wasunable to detect SRY on Western blots. The antibody generated in this project was both avidand specific, and was able to detect endogenous SRY in developing Sertoli cells in embryonicmouse genital ridges.Next, I examined the regulation of Sry in vivo. In this part of the project I used a Wt1(+KTS)—/— mouse model to gain an understanding of the role of WT1 in the regulation ofSRY. These mice demonstrate complete male-to-female phenotypic sex reversal, and previousstudies have shown a significant reduction in the level of Sry transcript in the gonads,suggesting that WT1 may play a role in regulating Sry. I observed that SRY protein is presentin the gonads of mutant mice, with similar nuclear localization to that of wild type. However,the amount of SRY protein per cell is greatly reduced in the mutant mouse, and proliferationat and near the coelomic epithelium at 11.5 dpc is reduced to a level similar to that found infemales. Coincident with this, is a significant reduction in the number of SRY-positive cellswithin the mutant gonad. This reduction in somatic cells does not affect XX Wt1 (+KTS)—/—mice, since they do not exhibit a significant reduction in the number of SF1 positive cells.These results suggest that the observed XY mutant gonadal phenotype is caused by a malespecificregulation of somatic cell numbers acting through the regulation of SRY, and not ageneral bipotential proliferative effect of WT1. This in turn suggests that WT1 is important inthe direct regulatory cascade regulating Sry expression.Finally, I set out to identify genes that may be regulated by SRY, using chromatinimmunoprecipitation (ChIP) to positively identify direct targets. An anti-mouse SRY antibodyprecipitated a region 7.5 kb upstream of the transcriptional start site of the cerebellin 4precursor (Cbln4) gene, which encodes a secreted protein and is expressed in Sertoli cells ofthe developing gonad. Its expression profile mimics that of the male sex-determining geneSox9. SRY as well as SOX9, bound two SRY/SOX binding sites within the Cbln4 enhancerregion in vitro and in vivo. In transgenic XY mouse embryos with reduced Sox9 expression,Cbln4 expression was reduced also, and over-expression of Sox9 in XX mice wasaccompanied by an increase in the level of Cbln4 mRNA. Finally, ectopic up-regulation ofSRY in vivo resulted in an ectopic expression of Cbln4. Our findings suggest that both SRYand SOX9 contribute to the male-specific increase in Cbln4 expression in the developingtestis. Furthermore, we identify for the first time in vivo, a gene that is a direct target of SRY.In summary, I have generated the first anti-mouse SRY antibody and used it to demonstratethe direct role of WT1 in the molecular cascade of Sry regulation. Furthermore, I haveidentified a previously unknown, direct target gene of SRY, namely Cbln4.