News and Publications

Molecular Regulation of Vascular System Development

L. Campagnolo, F. Kuhnert, M. Fitch, D. Lemons, H. Stuhlmann

To identify genes involved in the development of the mammalian circulatory system, we used retroviral gene-trap vectors 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.

AN EARLY ZINC FINGER GENE ESSENTIAL FOR NORMAL VASCULAR DEVELOPMENT

One gene we identified, Vezf1, has restricted expression in vascular structures in embryoid bodies and embryos derived from the embryonic stem cell clone. This gene encodes vascular endothelial zinc finger 1, a 56-kD protein that contains 6 putative zinc finger domains and a proline-rich region at its C terminus. Vezf1 is a member of a small subfamily of zinc finger genes that are highly conserved in higher vertebrate species.

During embryogenesis, Vezf1 is expressed early on in vascular endothelial cells and in mesenchymal cells. Because of this expression pattern, we hypothesized that Vezf1 plays an important role during vascular development. To test this hypothesis, we generated gain- and loss-of-function mutations in mouse embryonic stem cells and transgenic mice. We found that Vezf1 acts in a dosage-dependent manner on the growth, remodeling, and integrity of the vasculature. Vezf1^-/- embryos died between day 9.5 and day 16.5 of gestation. Mutant embryos had a variety of vascular abnormalities and hemorrhaging in the head and neck region. Interestingly, 20% of Vezf1^+/- embryos had haploinsufficiency (i.e., the protein produced by a single copy of the gene was not sufficient to ensure normal function) at the Vezf1 locus. Specifically, these embryos had extensive hypervascularization, hemorrhaging, and edema caused by abnormalities of lymphatic vessels budding from the jugular vein.

We also studied the effects of Vezf1 overexpression on development of the vascular system by generating mice with a tie2 promoter-Vezf1 cDNA transgene. Compared with control mice, transgenic mice had an increased number of small, abnormal vessels in the head and trunk region. Death due to massive hemorrhaging and additional CNS developmental defects occurred from day 12.5 of gestation to adulthood. It will be important to determine if forced expression of Vezf1 in the vascular endothelium can prevent the deaths that occur in the Vezf1^-/- embryos. We are addressing this possibility by generating Vezf1^-/- mice that also have the transgene.

We are examining the molecular pathways in which Vezf1 participates. The zinc finger domains are sufficient for DNA binding, and the proline-rich region contains a strong transcriptional transactivator. We are identifying target genes that are directly regulated by Vezf1 during vascular development. For this purpose, we are using RNA and expression profiles from wild-type and Vezf1^-/- embryos. We examined a panel of candidate genes whose in vivo expression overlaps expression of Vezf1 or whose deletion results in abnormalities similar to those that occur in Vezf^-/- embryos. These candidate genes include ligands and receptors of the vascular endothelial growth factor, angiopoietin, ephrin, and notch signaling pathways. However, Vezf1 does not appear to regulate expression of any of these genes. We are beginning to screen for novel target genes by using microarrays.

A NOVEL GENE EXPRESSED IN EARLY ENDOTHELIAL PROGENITOR CELLS AND DURING VASCULAR DEVELOPMENT

We identified a second gene, Zneu1, in our screen that has restricted expression in early endothelial progenitor cells and in the endothelium during vascular development. Interestingly, expression of Zneu1 precedes that of the early endothelial marker flk-1. Zneu1 encodes a 29-kD protein with 2 putative epidermal growth factor-like domains.

Because of its early onset and endothelial-restricted expression, we hypothesize that Zneu1 plays a crucial role in early processes of vascular development. We are testing this hypothesis by generating mice that lack the gene for Zneu1. In a second project, we will exploit expression of Zneu1 in endothelial progenitor cells to identify and distinguish vascular stem cells from differentiating embryonic stem cells and embryos. For this purpose, we marked the endogenous Zneu1 locus by inserting an autofluorescent reporter gene that is suitable for flow cytometry. In future experiments, we will isolate the marked progenitor cells and examine their potential to differentiate into different cell lineages in vitro and in vivo.

PUBLICATIONS

Sun, X., Lemons, D., Xiong, J.-W., Kuhnert, F., English, F., Licht, J., Stuhlmann, H. VEZF is an endothelial transcription factor that binds to GC/CT-rich sequences in the IL-3, flk-1 and flt-1 promoter. J. Biol. Chem., in press.