Evolutionary and functional relationships between Lgr proteins and ubiquitin-specific proteases

我们之前已经探索了去泛素化酶的进化史。我们确定编码USP4和USP15的基因是由一个古老的全基因组复制事件产生的（因此是同源的），而USP11是由USP4的一个小片段复制产生的，然后在序列上迅速分化。为了确定功能冗余的水平，我们将Usp4和Usp15失活突变的小鼠进行杂交，发现当其中一个基因缺失时，小鼠可以存活，但不可能产生两个基因都缺失的后代。这表明有必要的蛋白质底物可以被任何一种去泛素化，但在没有这两种底物的情况下，这些底物被蛋白酶体泛素化和降解。我们发现Wnt/ β - catenin通路在复合零细胞中是缺乏的，并且已经确定LGR5（一种Wnt信号的调节剂）在该基因型的妊娠中期胚胎的肝脏中缺失。这就解释了我们认为导致胚胎死亡的造血功能失败的原因。为了加速研究，我们建立了一项合作，以产生Usp4和Usp15的同源基因已被删除的斑马鱼，目前正在进行遗传杂交，以确定我们的小鼠发现是否在该物种中重现。在透明的斑马鱼胚胎中研究造血失败要容易得多。单细胞RNAseq将在不同基因型的鱼类血细胞上进行，以确定哪些细胞类型和分子途径存在缺陷。我们的下一个目标将是分析硬骨鱼中的lgr基因及其在造血中的潜在作用。考虑到Lgr5是小鼠必不可少的基因，我们惊讶地发现Lgr5在斑马鱼中不存在，而lgr4和lgr6却存在于斑马鱼基因组中。将招募一名研究生建立硬骨鱼lgr基因的进化史，并确定去泛素化酶基因进化的时间关系。我建议使用原位杂交技术来研究斑马鱼的基因表达，以确定lgr4和/或lgr6是否在造血中发挥作用。同时，将在具有所有三种lgr技术的medaka鱼中研究lgr基因。将使用CRISPR/Cas9方法删除斑马鱼和medaka的lgr基因，并对表型进行分析，特别关注造血。总的来说，这些实验将提供对复制基因的进化和亚功能化的深入了解，这是我们的长期目标。

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.