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Date of Award

9-2012

Document Type

Campus Access

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Animal Biotechnology and Biomedical Sciences

First Advisor

Jesse Mager

Second Advisor

Kimberly D. Tremblay

Third Advisor

James Chambers

Subject Categories

Developmental Biology | Genetics

Abstract

One of the many ways that the ubiquitously expressed Polycomb Group protein, Yin-Yang1 (Yy1 ), is believed to regulate gene expression is through direct binding to DNA elements found in promoters or enhancers of target loci. Additionally, YY1 contains diverse domains that enable a plethora of protein-protein interactions, including association with the Oct4/Sox2 pluripotency complex and the Polycomb Group silencing complexes. To elucidate the in vivo role of YY1 during gastrulation, Yy1 was deleted in the epiblast of mouse embryos using Sox2-Cre.Yy1 conditional knockout (cKO) embryos initiate gastrulation, but the primitive streak fails to extend anteriorly. Migration through the streak is severely impaired, and streak descendants fail to downregulate E-Cadherin resulting in an aberrant accumulation of streak cells. Intriguingly, we find an accumulation of Nodal and a concomitant reduction of Nodal antagonists suggesting that YY1 is normally required for proper Nodalregulation. We have observed that definitive endoderm is specified but fails to properly delaminate into the outer layer and mutant embryos also fail to accumulate any axial midline structure. Although anterior neuroectoderm is clearly specified, mesoderm specification is severely restricted. Our results reveal critical requirements of YY1 in several important developmental processes, including epithelial to mesenchymal transition (EMT), Nodal regulation and PRC2 mediated H3K27Me3 of the inactive X-chromosome.

Despite the localization of Oct4 and Sox2 transcripts in the neuroectoderm of the Yy1 epiblast cKO and the presence of stable transcripts of both genes in the Yy1 RNAi knock down blastocyst, both proteins are void in these models and Oct4 protein is absent in the peri-implantion Yy1 KO mouse. We believe YY1 is required for stabilization of the Oct4/Sox2 pluripotency complex in vivo. We have identified two endogenous forms of YY1 and we believe these posttranslational modifications of YY1 permit the protein to perform the diverse activities it performs in vivo . For example, in addition to the roles in transcriptional regulation and protein complex stabilization, we have also observed a role in YY1 in epigenetic regulation in vivo, including deposition of histone 3 lysine 27 trimethylation (H3K27Me3) on the inactive X-chromosome in female embryos and a role in imprinted gene expression of the Dlk1/Dio3 locus.

Detailed analysis of the peri-implantation lethal Yy1 KO mouse in utero revealed unexpected novel developmental events. A large scale follow up examination of wildtype implantation primarily through analysis of immunohistochemical data and gene expression profiling at the cellular level. We analyzed expression patterns of important developmental genes including Oct4, Sox2, Nanog, Cdx2, Gata6 and Sox17, as well as markers of epithelial biogenesis including ZO1, E-Cadherin and Laminin. Interestingly we identified consistent variances in cell populations within the ICM as well as likely primitive endoderm progenitors that produce Laminin and first appear at the periphery of the ICM. We also identified a novel upregulation of Sox17 specifically at the site of implantation. With these data we compose a staging diagram of peri-implantation embryonic and maternal changes during the elusive window of development.

These results are the first to elucidate the role of YY1 during gastrulation and peri- implantation, providing potential in vivo targets of YY1 and highlighting the diversity of function of YY1 in the early embryo. Additionally we have been able to advance molecular knowledge of peri-implantation development, in order to provide a platform from which to analyze other peri-implantation lethal KO mice, as well as to aid biomedical understanding of implantation and implantation failure in mammals.

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