Pkd1 Regulates Lymphatic Vascular Morphogenesis during Development.

Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development

LOXL4 Is Induced by Transforming Growth Factor 1 through Smad and JunB/Fra2 and Contributes to Vascular Matrix Remodeling

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Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish

Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division

The interaction of heparan sulfate proteoglycans with endothelial transglutaminase-2 limits VEGF165-induced angiogenesis

International audienceSprouting angiogenesis is stimulated by vascular endothelial growth factor (VEGF165) that is localized in the extracellular matrix (ECM) and binds to heparan sulfate (HS)-bearing proteins known as heparan sulfate proteoglycans (HSPGs). VEGF165 presentation by HSPGs enhances VEGF receptor-2 (VEGFR2) signaling. We investigated the effect of TG2, which binds to HSPGs, on the interaction between VEGF165 and HS and angiogenesis. Mice with tg2 deficiency showed transiently enhanced retina vessel formation and increased vascularization of VEGF165-containing Matrigel implants. In addition, endothelial cells in which TG2 was knocked down exhibited enhanced VEGF165-induced sprouting and migration, which was associated with increased phosphorylation of VEGFR2 at Tyr(951) and its targets Src and Akt. TG2 knockdown did not affect the phosphorylation of VEGFR2 at Tyr(1175) or cell proliferation in response to VEGF165 and sprouting or signaling in response to VEGF121. Decreased phosphorylation of VEGFR2 at Tyr(951) was due to ECM-localized TG2, which reduced the binding of VEGF165 to endothelial ECM in a manner that required its ability to bind to HS but not its catalytic activity. Surface plasmon resonance assays demonstrated that TG2 impeded the interaction between VEGF165 and HS. These results show that TG2 controls the formation of VEGF165-HSPG complexes and suggest that this regulation could be pharmacologically targeted to modulate developmental and therapeutic angiogenesi

A blood capillary plexus-derived population of progenitor cells contributes to genesis of the dermal lymphatic vasculature during embryonic development

Despite the essential role of the lymphatic vasculature in tissue homeostasis and disease, knowledge of the organ-specific origins of lymphatic endothelial progenitor cells remains limited. The assumption that most murine embryonic lymphatic endothelial cells (LECs) are venous derived has recently been challenged. Here, we show that the embryonic dermal blood capillary plexus constitutes an additional, local source of LECs that contributes to the formation of the dermal lymphatic vascular network. We describe a novel mechanism whereby rare PROX1-positive endothelial cells exit the capillary plexus in a Ccbe1-dependent manner to establish discrete LEC clusters. As development proceeds, these clusters expand and further contribute to the growing lymphatic system. Lineage tracing and analyses of Gata2-deficient mice confirmed that these clusters are endothelial in origin. Furthermore, ectopic expression of Vegfc in the vasculature increased the number of PROX1-positive progenitors within the capillary bed. Our work reveals a novel source of lymphatic endothelial progenitors employed during construction of the dermal lymphatic vasculature and demonstrates that the blood vasculature is likely to remain an ongoing source of LECs during organogenesis, raising the question of whether a similar mechanism operates during pathological lymphangiogenesis.Cathy Pichol-Thievend, Kelly L. Betterman, Xiaolei Liu, Wanshu Ma, Renae Skoczylas, Emmanuelle Lesieur, Frank L. Bos, Dorte Schulte, Stefan Schulte-Merker, Benjamin M. Hogan, Guillermo Oliver, Natasha L. Harvey, and Mathias Francoi

SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development

Arterial specification and differentiation are influenced by a number of regulatory pathways, encompassing numerous growth factors, signaling molecules and transcription factors. While it is known that the Vegfa-Notch cascade plays a central role in this biological process, the transcriptional hierarchy controlling arterial specification has not been fully delineated. To elucidate the direct transcriptional regulators of Notch receptor expression in arterial endothelial cells, we used histone signatures, DNaseI hypersensitivity and ChIP-seq data to identify enhancers for the human NOTCH1 and zebrafish notch1b genes. These enhancers were able to direct arterial endothelial cell-restricted expression in transgenic models. Genetic disruption of SOXF binding sites clearly established a requirement for members of the SOXF group of transcription factors (SOX7,-17 and-18) to drive these enhancers activity in vivo. Further, endogenous deletion of the notch1b enhancer led to a significant augmentation of arterio-venous defects in notch-pathway deficient zebrafish. Loss of SoxF function revealed that these factors are necessary for the activity of NOTCH1 and notch1b enhancers, and for correct endogenous Notch1 gene transcription. These findings therefore position SOXF transcription factors directly upstream of Notch receptor expression during the acquisition of arterial identity in vertebrates

Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish

Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc-Vegfd-Ccbe1-Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.Neil I Bower, Katarzyna Koltowska, Cathy Pichol-Thievend, Isaac Virshup, Scott Paterson, Anne K Lagendijk, Weili Wang, Benjamin W Lindsey, Stephen J Bent, Sungmin Baek, Maria Rondon-Galeano, Daniel G Hurley, Naoki Mochizuki, Cas Simons, Mathias Francois, Christine A Wells, Jan Kaslin and Benjamin M Hoga