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Summary The formation of new vessels requires a tight synchronization between proliferation, differentiation, and sprouting. However, how these processes are differentially activated, often by neighboring endothelial cells (ECs), remains unclear. Here, we identify cell cycle progression as a regulator of EC sprouting and differentiation. Using transgenic zebrafish illuminating cell cycle stages, we show that venous and lymphatic precursors sprout from the cardinal vein exclusively in G1 and reveal that cell-cycle arrest is induced in these ECs by overexpression of p53 and the cyclin-dependent kinase (CDK) inhibitors p27 and p21. We further demonstrate that, in vivo, forcing G1 cell-cycle arrest results in enhanced vascular sprouting. Mechanistically, we identify the mitogenic VEGFC/VEGFR3/ERK axis as a direct inducer of cell-cycle arrest in ECs and characterize the cascade of events that render “sprouting-competent” ECs. Overall, our results uncover a mechanism whereby mitogen-controlled cell-cycle arrest boosts sprouting, raising important questions about the use of cell cycle inhibitors in pathological angiogenesis and lymphangiogenesis., Graphical abstract, Highlights • Endothelial cells sprout from the PCV in G1 phase of the cell cycle • VEGFC/FLT4/ERK induce p53-, p21-, and p27-mediated cell-cycle arrest to enable sprouting • Forced G1 cell-cycle arrest results in ectopic sprouting of undifferentiated ECs, Jerafi-Vider et al. report a mechanism of endothelial cell sprouting, whereby the mitogenic VEGFC/FLT4/ERK signaling pathway induces p53-, p21-, and p27-mediated cell-cycle arrest, conferring angiogenic potential. These findings have important implications for the putative short-term effects of using cell cycle inhibitors in settings of pathological angiogenesis and lymphangiogenesis.

The emergence and growth of new vessels requires a tight synchronization between proliferation, differentiation and sprouting, traditionally thought to be controlled by mitogenic signals, especially of the VEGF family. However, how these cues are differentially transduced, by sometimes even neighboring endothelial cells (ECs), remains unclear. Here we identify cell-cycle progression as a new regulator of EC sprouting and differentiation. Using transgenic zebrafish illuminating cell-cycle stages, we show that venous and lymphatic precursors sprout from the Cardinal Vein (CV) exclusively in G0/G1, and reveal that cell-cycle arrest is induced in these ECs by p53 and the CDK-inhibitors p27 and p21. Moreover, we demonstrate that in vivo, chemical and genetic cell-cycle inhibition, results in massive vascular growth. Mechanistically, we identify the mitogenic VEGFC/VEGFR3/ERK axis as direct inducer of cell-cycle arrest in angiogenic ECs and characterize the cascade of events governing venous vs. lymphatic segregation and sprouting. Overall, our results uncover an unexpected mechanism whereby mitogen-controlled cell-cycle arrest boosts sprouting, raising important questions about the use of cell-cycle inhibitors in pathological angiogenesis and lymphangiogenesis.

In this Letter, affiliation number 1 was incorrectly listed as ‘Department of Biology, Technion – Israel Institute of Technion, Haifa 32000, Israel’ instead of ‘Department of Biology, Technion- Israel Institute of Technology, Haifa 32000, Israel’. The original Letter has not been corrected.

Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development--known as the phylotypic period--is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent 'mid-developmental transition' that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.

How cells acquire their fate is a fundamental question in developmental and regenerative biology. Multipotent progenitors undergo cell-fate restriction in response to cues from the microenvironment, the nature of which is poorly understood. In the case of the lymphatic system, venous cells from the cardinal vein are thought to generate lymphatic vessels through trans-differentiation. Here we show that in zebrafish, lymphatic progenitors arise from a previously uncharacterized niche of specialized angioblasts within the cardinal vein, which also generates arterial and venous fates. We further identify Wnt5b as a novel lymphatic inductive signal and show that it also promotes the ‘angioblast-to-lymphatic’ transition in human embryonic stem cells, suggesting that this process is evolutionarily conserved. Our results uncover a novel mechanism of lymphatic specification, and provide the first characterization of the lymphatic inductive niche. More broadly, our findings highlight the cardinal vein as a heterogeneous structure, analogous to the haematopoietic niche in the aortic floor.