Incomplete formation of the enteric nervous system (ENS), known as Hirschsprung disease (HSCR), is a congenital ENS disorder which affect boys much more frequently than girls. Numerous genes have been associated with HSCR, including those involved in RET/GDNF and EDN3/EDNRB pathways. However, none of them can explain the male bias. Our laboratory instead found that p53-induced up-regulation of the RNA helicase-coding gene Ddx3y is the cause of the male bias in a HSCR mouse model called TashT. Intriguingly, over-expression of Ddx3y only effected male TashT animals, without any impact on females. This project will test the hypothesis that this is due to an obligate interaction between DDX3Y and another Y-linked protein called EIF2S3Y that also regulates the initiation of mRNA translation. This project builds on key BiFC data showing that both of these proteins directly interact, but not with their X-linked homologs DDX3X and EIF2S3X. To confirm the importance of DDX3Y-EIF2S3Y complex in causing male biased HSCR, we will generate mice overexpressing both Ddx3y and Eif2s3y. To better understand the role played by DDX3Y and EIF2S3Y (alone and in complex), we will also apply techniques such as split-TurboID and split-APEX2-seq to identify their binding partners and RNA targets, respectively. This study will clarify the reason behind male-biased HSCR, which will most likely help to understand other sex-based differences in other components of the nervous system.