Advances in genomic technologies over the past decade have yielded an unprecedented level of information about animal genomes. However, much of the information underlying these 2D models of cell and tissue function stills awaits experimental verification and further investigation in intact living organisms. Another factor limiting the application of genome wide approaches is the absence of functional characterization for ? 1/2 of all annotated genes. The targeted mutations generated by the International Knockout Mouse Consortium (IKMC), including KOMP, provide a revolutionary resource for functionally annotating the mammalian genome. We propose to characterize the phenotypes of mutations in the KOMP2-generated KO lines that result in develop- mental arrest or abnormal morphology at or prior to E9.5. We will functional define ~200 previously uncharacterized genes whose functions are essential during pre- and early post-implantation development. The investigators of this group have previously studied the phenotypes of ~200 embryonic lethal mouse mutants, leading to many surprising findings, including the novel cellular mechanisms that create the mammalian endoderm, the requirement for primary cilia in the Hedgehog signaling pathway and many more. Our specific goals are to (1) Characterize the functions of new genes essential for early embryonic development, by second tier phenotyping of ?200 lines lethal at or before e9.5. We will define the stage of arrest, analyze morphological abnormalities, assess proliferation and cell death. Only genes in which knock- outs have not been characterized will be studied, and those encoding uncharacterized proteins will have highest priority, making this an unbiased screen for novel essential genes, and providing the first evidence on bio- logical function of 200 essential genes. (2) Use our expertise to characterize at cellular resolution the roles of previously uncharacterized genes in preimplantation development, early embryonic morphogenesis and placental development. We will characterize novel regulators of pluripotency of early embryonic lineages, the gastrulation epithelial-mesenchymal transition, early mesoderm migration and ventral folding, which are reiteratively used morphogenetic programs essential for many aspects of human development. Characterization of cellular and developmental functions of regulators of placentation is crucial for fetal and child health.
Our specific Aims are:
Aim 1. - Establishing a Phenotyping Pipeline, which will initiate at three trans NIH KOMP2 production and phenotyping centers.
Aim 2 - Tier two phenotyping of ~200 mutations that cause lethality before E9.5.
Aim 3 - Tier 3 phenotyping of lethal mutations that affect development of the blastocyst, early post-implantation morphogenesis and placentation. The embryonic lethal KO mutations generated by KOMP and IKMC provide an unprecedented opportunity to expand and enrich the functional annotation of the mammalian genome. The embryonic lethal genes thus identified will not only be indispensable for early mouse development, but also critical for multiple aspects of human development and tissue homeostasis.
The critical stage of human embryonic development occurs over the first 6 weeks of pregnancy when human embryo is intractable for examination and very often the pregnancy has yet to be acknowledged. Several structural birth defects likely arise from impairment of these critical morphogenetic processes in the early embryo. This proposal investigates early embryonic lethal KOMP2 KO alleles which will provide models for a range of congenital and structural birth defects, infertility and spontaneous abortion. Thus the availabilit of early lethal mouse knockout lines brings focus to an understudied component of human health.