Grant: Discovery Grants Program - Individual

We have previously explored the evolutionary history of the deubiquitinating enzymes. We determined that the genes encoding USP4 and USP15 were generated by an ancient whole genome duplication event (and are therefore ohnologs), whereas USP11 arose from a small segmental duplication of USP4 and then rapidly diverged in sequence. To determine the level of functional redundancy we crossed mice with inactivating mutations of Usp4 and Usp15 and found that while mice were viable when null for either gene no progeny could be generated that were null for both. This suggests that there are essential protein substrates that can be deubiquitinated by either, but in the absence of both such substrates are ubiquitinated and degraded by the proteasome. We found that the Wnt/beta catenin pathway is deficient in compound null cells, and have established that LGR5 (a modulator of Wnt signaling) is absent in the liver of midgestation embryos of that genotype. This would explain the failure of hematopoiesis that we believe is the cause of embryonic lethality. To accelerate the research we established a collaboration to generate zebrafish in which the orthologs of Usp4 and Usp15 have been deleted and are currently performing genetic crosses to determine if our mouse findings are recapitulated in this species. A failure of hematopoiesis would be much easier to study in the transparent zebrafish embryo. Single cell RNAseq will be performed on blood cells from fish of various genotypes to determine which cell types and molecular pathways are deficient. Our next objective will be an analysis of lgr genes in teleost fish and their potential role in hematopoiesis. Given that Lgr5 is an essential gene in mouse we were surprised to discover that lgr5 does not exist in zebrafish, but that the ohnolog genes lgr4 and lgr6 were present in the zebrafish genome. A graduate student will be recruited to establish the evolutionary history of the teleost lgr genes and determine temporal relationships to evolution of the deubiquitinating enzyme genes. I propose to use in situ hybridization to study gene expression in the zebrafish to determine if lgr4 and/or lgr6 play any role in hematopoiesis. In parallel lgr genes will be studied in medaka, a fish species with all three lgr ohnologs. The lgr genes of both zebrafish and medaka will be deleted using CRISPR/Cas9 methodology and phenotypes will be analyzed with particular attention to hematopoiesis. Collectively these experiments will provide insight into the evolution and subfunctionalization of duplicated genes, the understanding of which is our long term objective.