Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

1-1-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Kenneth Murphy

Abstract

During gastrulation, epiblast cells undergo an epithelial-to-mesenchymal transition: EMT) as they ingress through the primitive streak and form mesoderm. To better understand the molecular pathways of EMT during this developmental transition, we developed a model system utilizing mouse embryonic stem: ES) cells. We show that EMT occurs during ES cell differentiation and is dependent on the Wnt signaling pathway. We further show that the Wnt-dependent transcription factor Snail homolog 1: Snail) is expressed and capable of inducing EMT in differentiating ES cells. In addition to EMT, Snail accelerates differentiation, promotes mesoderm commitment, and restricts markers of primitive ectoderm and epiblast. Snail's impact on differentiation can be partly explained through its repression of ES cell-associated microRNAs, including the miR-200 family. The miR-200 family is down-regulated in a Wnt-dependent manner during ES cell differentiation. We find that maintenance of miR-200 expression prevents EMT and stalls differentiating ES cells at an epiblast-like stem cell: EpiSC) stage. Consistent with a role for Activin in EpiSC maintenance, we show that miR-200 requires Activin to efficiently maintain cells at the epiblast stage. Together, these findings demonstrate that Snail and miR-200 act in opposition to regulate EMT and exit from the EpiSC stage towards induction of germ layer fates. By modulating expression levels of Snail, Activin, and miR-200, we are able to control the timing of EMT and transition out of the EpiSC state. Beyond a role in gastrulation, Snail has also been demonstrated to be important in vasculogenesis. Snail-deficient mice display early vascular defects while Snail overexpressing tumors are associated with increased angiogenesis. We utilized our ES cell model as a means to better understand Snail's relationship with vasculogenesis. We find that unlike other types of mesoderm, Snail's induction of Flk1+ endothelial cells is cell-intrinsic and independent of Wnt, BMP, and Activin signaling pathways. Based on the transcriptional profile of Flk+ sorted cells, we hypothesize that Snail selectively induces endothelium in a subset of ES cells that resemble primitive endoderm. We further demonstrate that Snail's induction of endothelium requires the down-regulation of the miR-200 family, which directly target the 3'UTRs of Flk1 and Ets1.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7TD9VB2

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