Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology

Language

English (en)

Date of Award

8-31-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Kenneth M Murphy

Abstract

Previous work has identified Mesp1 as an important regulator of the epithelial–mesenchymal transition: EMT) and of cardiovascular cell fate in differentiating embryonic stem cells: ESCs). To understand the molecular mechanisms underlying the actions of Mesp1, we sought to identify transcription targets of Mesp1. Mesp1 rapidly induced expression of PDGFRα in differentiating ESCs and directly bound to evolutionary conserved E-boxes within the PDGFRα promoter. This result suggested that PDGFRα could be a direct target of Mesp1. However, we found that PDGFRα was not sufficient for the induction of EMT in ESCs or the induction of Flk1+ mesoderm, but that it may play a role rather in the survival of Mesp1–induced mesodermal cells.

Although a clear role for Mesp1 in EMT and cardiovascular differentiation has been established, its function in hematopoietic development is still unclear. Previous lineage tracing demonstrated that Mesp1 activity labeled endothelial cells of embryonic dorsal aorta, which recently was shown to give rise to definitive hematopoietic progenitors. This suggested the potential that Mesp1 activity in endothelium might influence subsequent hematopoietic development. Although in vitro studies indicated that Mesp1 acted to suppress emergence of hematopoietic progenitors, we made the surprising observation in lineage tracing analysis of Mesp1 that all adult hematopoietic progenitors and mature lineages were efficiently labeled by Mesp1–Cre, and further that Mesp1 was necessary for hematopoietic differentiation of ESCs. In examining the downstream targets of Mesp1 in ESC–derived endothelial cells, we identified myeloid ecotropic viral integration site 1: Meis1).

Meis1 forms a heterodimer with Pbx1 that augments Hox-dependent gene expression. In addition, Meis1 has been associated with leukemogenesis and hematopoietic stem cell self-renewal. In examining potential roles of Meis1 in hematopoietic development, we identified two independent actions. One activity regulated cellular proliferation of early hematopoietic progenitors. The second activity was involved the fate choice between erythroid and megakaryocyte lineages. First, we found that endogenous Mesp1 indirectly induces Meis1 and Meis2 in endothelial cells derived from embryonic stem: ES) cells. Overexpression of Meis1 and Meis2 greatly enhanced the formation of hematopoietic colonies from ES cells, with the exception of erythroid colonies, by maintaining hematopoietic progenitor cells in a state of proliferation. Second, overexpression of Meis1 repressed the development of early erythroid progenitors, acting in vivo at the megakaryocyte–erythroid progenitor: MEP) stage to skew development away from erythroid generation and toward megakaryocyte development. This previously unrecognized action of Meis1 may explain the embryonic lethality observed in Meis1-/- mice that arises from failure of lymphatic–venous separation, and which can result as a consequence of defective platelet generation. These results show that Meis1 exerts two independent functions, with its role in proliferation of hematopoietic progenitors acting earlier in development from its influence on the fate choice at the MEP between megakaryocytic and erythroid development.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7B56GR0

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