News and Publications

Molecular Regulation of Vascular System Development

To identify genes involved in the development of the mammalian circulatory system, we used a gene trap approach to perform a genetic screen in mouse embryonic stem cells and in embryos. Candidate genes were identified by their restricted expression of the reporter gene during in vitro differentiation of embryonic stem cells and in mouse embryos. We isolated 2 genes that are expressed in developing blood vessels.

An Early Zinc Finger Gene Essential For Normal Mouse Vascular Development

One of the genes, Vezf1, is expressed in endothelial cells and encodes a 56-kD nuclear transcription factor that contains 6 putative zinc finger domains of the Cys2/His2 type. We identified an internal nuclear localization signal, a DNA-binding domain, and a C-terminal transcriptional transactivation domain. Vezf1 is a member of a small subfamily of zinc finger genes that are highly conserved in higher vertebrate species.

The results of molecular genetic studies, including use of Vezf1-deficient and transgenic mice, suggest an essential role for Vezf1 function for proliferation, remodeling, and integrity of the developing vasculature. All embryos homozygous for Vezf1 and 20% of mutant embryos heterozygous for the gene had vascular and lymphatic endothelial abnormalities and hemorrhaging, and they died during midgestation. Preliminary findings indicated that the primary defect is endothelial in origin, because death of Vezf1-deficient embryos can be prevented by endothelial overexpression of Vezf1.

To explore the molecular pathways in which Vezf1 participates, we dissected the regulatory regions of the gene and identified potentially important factors that modulate its expression, among them Egr-1 and Ets factors. We are identifying target genes that are directly regulated by Vezf1. None of the potential candidate genes known to participate in vascular development, including those for ligands and receptors of the vascular endothelial growth factor, angiopoietin, ephrin, and notch signaling pathways, are directly regulated by Vezf1. Thus, Vezf1 may act through yet unknown molecular pathways. To uncover potential novel target genes involved in these pathways, we are performing differential screens and microarray analysis.

A Novel Gene Expressed In Early Endothelial Progenitor Cells and During Vascular Development

Expression of the second gene we identified, Egfl7, is restricted to early endothelial progenitor cells and the endothelium during vascular development. Interestingly, expression of Egfl7 precedes that of the early endothelial marker flk-1. With the exception of lung, where postnatal expression of the gene remains high, Egfl7 expression is downregulated in the quiescent vasculature in adults. However, expression is induced during vascular injury and endothelial regeneration.

Egfl7 encodes a 29-kD secreted protein with an N-terminal signal peptide and 2 internal epidermal growth factor-like domains. We will determine if EGFL7, the protein encoded by Egfl7, acts as a growth factor or a cytokine through direct binding of a yet unknown receptor or if it interacts with other proteins localized to the extracellular matrix of endothelial cells.

Because of its early onset and restricted expression, we hypothesize that Egfl7 plays a crucial role in early processes of vascular development. We are testing this hypothesis by generating mice that lack the gene or have reduced expression of the gene. In a second project, we are exploiting expression of Egfl7 in endothelial progenitor cells in order to identify and characterize vascular stem cells. For these studies, we marked the endogenous Egfl7 locus by adding an autofluorescent reporter gene. This approach will allow enrichment of potential progenitor cells by flow cytometry. In future experiments, we will isolate the marked progenitor populations and examine their potential for lineage differentiation in vitro and in vivo. Our studies in the mouse model will be complemented by using a human embryonic stem cell differentiation system.

Publications

Eto, K., Murphy, R., Kerrigan, S.W., Bertoni, A., Stuhlmann, H., Nakano, T., Leavitt, A.D., Shattil, S.J. Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. Proc. Natl. Acad. Sci. U. S. A. 99:12819, 2002.

Macara, I.G., Baldarelli, R., Field, C.M., Glotzer, M., Hayashi, Y., Hsu, S.C., Kennedy, M.B., Kinoshita, M., Longtine, M., Low, C., Maltais, L.J., McKenzie, L., Mitchison, T.J., Nishikawa, T., Noda, M., Petty, E.M., Peifer, M., Pringle, J.R., Robinson, P.J., Roth, D., Russell, S.E., Stuhlmann, H., Tanaka, M., Tanaka, T., Trimble, W.S., Ware, J., Zeleznik-Le, N.J., Zieger, B. Mammalian septins nomenclature. Mol. Biol. Cell 13:4111, 2002.

Stuhlmann, H. Gene trap vector screen for developmental genes in differentiating ES cells. Methods Enzymol. 365:386, 2003.

Hooper, J.D., Campagnolo, L., Goodarzi, G., Truong, T.N., Stuhlmann, H., Quigley, J.P. Mouse matriptase-2: identification, characterization and comparative mRNA expression analysis with mouse hepsin in adult and embryonic tissues. Biochem. J., in press.