Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by well-studied signaling and transcriptional mechanisms. Less well understood is the ongoing elaboration of vessels to form a network. This involves cell polarisation, coordinated migration, adhesion, mixing, regression and cell shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. We found a mutation in polycystic kidney disease 1a to be responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial sprouting of lymphatic precursors is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice also has no effect on sprouting of precursors 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.

How developing vascular networks acquire the right balance of arteries, veins and lymphatic vessels to efficiently supply and drain tissues is poorly understood. In zebrafish embryos, the robust and regular 50:50 global balance of intersegmental veins and arteries that form along the trunk prompts the intriguing question of how does the organism keep ‘count’? Previous studies have suggested that the ultimate fate of an intersegmental vessel (ISV) is determined by the identity of the approaching secondary sprout emerging from the posterior cardinal vein. Here, we show that the formation of a balanced trunk vasculature involves an early heterogeneity in endothelial cell behaviour and Notch signalling activity in the seemingly identical primary ISVs that is independent of secondary sprouting and flow. We show that Notch signalling mediates the local patterning of ISVs, and an adaptive flow-mediated mechanism subsequently fine-tunes the global balance of arteries and veins along the trunk. We propose that this dual mechanism provides the adaptability required to establish a balanced network of arteries, veins and lymphatic vessels.

Background: The interplay between Notch and Vegf signaling regulates angiogenesis in the embryo. Notch signaling limits the responsiveness of endothelial cells to Vegf to control sprouting. Despite the importance of this regulatory relationship, much remains to be understood about extrinsic factors that modulate the pathway. Results: During a forward genetic screen for novel regulators of lymphangiogenesis, we isolated a mutant with reduced lymphatic vessel development. This mutant also exhibited hyperbranching arteries, reminiscent of Notch pathway mutants. Positional cloning identified a missense mutation in the carbamoyl‐phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (cad) gene. Cad is essential for UDP biosynthesis, which is necessary for protein glycosylation and de novo biosynthesis of pyrimidine‐based nucleotides. Using a transgenic reporter of Notch activity, we demonstrate that Notch signaling is significantly reduced in cadhu10125 mutants. In this context, genetic epistasis showed that increased endothelial cell responsiveness to Vegfc/Vegfr3 signaling drives excessive artery branching. Conclusions: These findings suggest important posttranslational modifications requiring Cad as an unappreciated mechanism that regulates Notch/Vegf signaling during angiogenesis. Developmental Dynamics 244:1–9, 2015. © 2014 Wiley Periodicals, Inc.

Molecular mechanisms controlling the formation, stabilization and maintenance of blood vessel connections remain poorly defined. Here we identify blood flow and the large extracellular protein Svep1 as co-modulators of vessel anastomosis during developmental angiogenesis in zebrafish embryos. Both loss of Svep1 and blood flow reduction contribute to defective anastomosis of intersegmental vessels. The reduced formation and lumenisation of the dorsal longitudinal anastomotic vessel (DLAV) is associated with a compensatory increase in Vegfa/Vegfr pERK signalling, concomittant expansion of apelin-positive tip cells, but reduced expression of klf2. Experimentally, further increasing Vegfa/Vegfr signalling can rescue the DLAV formation and lumenisation defects, while its inhibition dramatically exacerbates the loss of connectivity. Mechanistically, our results suggest that flow and Svep1 co-regulate the stabilization of vascular connections, in part by modulating the Vegfa/Vegfr signalling pathway.