Vascular development begins when mesodermal cells differentiate into endothelial cells, which then form primitive vessels. It has been hypothesized that endothelial-specific gene expression may be regulated combinatorially, but the transcriptional mechanisms governing specificity in vascular gene expression remain incompletely understood. Here, we identify a 44 bp transcriptional enhancer that is sufficient to direct expression specifically and exclusively to the developing vascular endothelium. This enhancer is regulated by a composite cis-acting element, the FOX:ETS motif, which is bound and synergistically activated by Forkhead and Ets transcription factors. We demonstrate that coexpression of the Forkhead protein FoxC2 and the Ets protein Etv2 induces ectopic expression of vascular genes in Xenopus embryos, and that combinatorial knockdown of the orthologous genes in zebrafish embryos disrupts vascular development. Finally, we show that FOX:ETS motifs are present in many known endothelial-specific enhancers and that this motif is an efficient predictor of endothelial enhancers in the human genome.

Cell, Volume 135, Issue 6, 1053-1064, 12 December 2008

The lymphatic system plays a key role in tissue fluid regulation and tumour metastasis, and lymphatic defects underlie many pathological states including lymphoedema, lymphangiectasia, lymphangioma and lymphatic dysplasia1, 2, 3. However, the origins of the lymphatic system in the embryo, and the mechanisms that direct growth of the network of lymphatic vessels, remain unclear. Lymphatic vessels are thought to arise from endothelial precursor cells budding from the cardinal vein under the influence of the lymphatic hallmark gene Prox1 (prospero homeobox 1; ref. 4). Defects in the transcription factor gene SOX18 (SRY (sex determining region Y) box 18) cause lymphatic dysfunction in the human syndrome hypotrichosis-lymphoedema-telangiectasia5, suggesting that Sox18 may also play a role in lymphatic development or function. Here we use molecular, cellular and genetic assays in mice to show that Sox18 acts as a molecular switch to induce differentiation of lymphatic endothelial cells. Sox18 is expressed in a subset of cardinal vein cells that later co-express Prox1 and migrate to form lymphatic vessels. Sox18 directly activates Prox1 transcription by binding to its proximal promoter. Overexpression of Sox18 in blood vascular endothelial cells induces them to express Prox1 and other lymphatic endothelial markers, while Sox18-null embryos show a complete blockade of lymphatic endothelial cell differentiation from the cardinal vein. Our findings demonstrate a critical role for Sox18 in developmental lymphangiogenesis, and suggest new avenues to investigate for therapeutic management of human lymphangiopathies.

Nature 456, 643-647 (4 December 2008) | doi:10.1038/nature07391; Received 21 December 2007

Serum response factor (SRF) is a transcription factor that controls the expression of cytoskeletal proteins and immediate early genes in different cell types. Here, we found that SRF expression is restricted to endothelial cells (ECs) of small vessels such as capillaries in the mouse embryo. EC-specific Srf deletion led to aneurysms and hemorrhages from 11.5 days of mouse development (E11.5) and lethality at E14.5. Mutant embryos presented a reduced capillary density and defects in EC migration, with fewer numbers of filopodia in tip cells and ECs showing defects in actin polymerization and intercellular junctions. We show that SRF is essential for the expression of VE-cadherin and β-actin in ECs both in vivo and in vitro. Moreover, knockdown of SRF in ECs impaired VEGF- and FGF-induced in vitro angiogenesis. Taken together, our results demonstrate that SRF plays an important role in sprouting angiogenesis and small vessel integrity in the mouse embryo.

Developmental Cell, Vol 15, 448-461, 16 September 2008

Purhonen et al. (1) have refuted the data published in >50 reports (2, 3), neglecting to quote key articles or utilize relevant models, and have drawn unsubstantiated conclusions about the contribution of endothelial progenitor cells (EPCs) to tumor angiogenesis that are not supported by their nonquantitative data and superficially executed experiments. Their study (1) is flawed in experimental design and data interpretation. For example, they do not cite their own publication demonstrating the existence of VEGFR2+ EPCs (4) and neglect mentioning clinical validation (5, 6) and acknowledging mouse genetic models (2, 3), which provide convincing evidence for functional incorporation of EPCs into neovessels. Every figure lacks stereoconfocal-microscopic quantification of vessels that are presented as poorly defined longitudinal–linear streaks. Plasma VEGF-A levels were not measured in vivo in mice treated with VEGF-A, questioning their low level of VEGFR2+ EPC detection (3). Indeed, their FACS analysis is inaccurate because of (i) unconvincing CD31/VE-cadherin/VEGFR2 expression detected on MS-1 endothelium used as positive control and (ii) failure to show long-term marrow engraftment of donor-derived hematopoietic and authentic VEGFR2+LacZ+ colony-forming EPCs. APCmin mice develop only obstructive adenomas, rather than adenocarcinomas; therefore, it is an inappropriate model to study EPC incorporation, as Spring et al. (7) (not quoted) demonstrate that EPCs do not contribute to adenomas but contribute only to carcinomas/metastatic tumors. In the parabiotic model, wild-type EPCs compete with GPF+ EPCs, which underestimates EPC recruitment. Finally, study of 6-month-old VEGF-A-loaded Matrigel plugs in mice is impossible because Matrigel plugs are degraded within 2 months, particularly when VEGF-A by itself does not induce neoangiogenesis. No quantification of patent vessels in Matrigel plugs was provided. This article fails to disprove the established role of EPCs in supporting neoangiogenesis in certain tumors (3, 5) and metastatic transition (2)

PNAS 2008 105:E54; published ahead of print August 20, 2008, doi:10.1073/pnas.0804876105