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Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors, which depend on dynamic cell-cell interactions. Remodeling of endothelial cell-cell junctions promote morphogenetic cellular events while preserving vascular integrity. Here, we have analyzed the dynamics of endothelial cell-cell junctions during lumen formation in angiogenic sprouts. By live-imaging of the formation of intersegmental blood vessels in zebrafish, we demonstrate that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Formation and maintenance of these junctional fingers are positively regulated by blood pressure whereas inhibition of blood flow prevents their formation. Using fluorescent reporters, we show that the tension-sensor Vinculin localizes to junctional fingers. Furthermore, loss of vinculin function, in vinculin a and -b double knockouts, prevents junctional finger formation in angiogenic sprouts, whereas endothelial expression of a vinculin transgene is sufficient to restore junctional fingers. Taken together, our findings suggest a mechanism in which lumen expansion during angiogenesis leads to an increase in junctional tension, which triggers recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells may employ force-dependent junctional remodeling to react to changes in external forces to protect cell-cell contacts and to maintain vascular integrity during sprouting angiogenesis.

Loss-of-function variants in CCM1/KRIT1, CCM2, and CCM3/PDCD10 are associated with autosomal dominant cerebral cavernous malformations (CCMs). CRISPR/Cas9-mediated CCM3 inactivation in human endothelial cells (ECs) has been shown to induce profound defects in cell-cell interaction as well as actin cytoskeleton organization. We here show that CCM3 inactivation impairs fibronectin expression and consequently leads to reduced fibers in the extracellular matrix. Despite the complexity and high molecular weight of fibronectin fibrils, our in vitro model allowed us to reveal that fibronectin supplementation restored aberrant spheroid formation as well as altered EC morphology, and suppressed actin stress fiber formation. Yet, fibronectin replacement neither enhanced the stability of tube-like structures nor inhibited the survival advantage of CCM3(-/-) ECs. Importantly, CRISPR/Cas9-mediated introduction of biallelic loss-of-function variants into either CCM1 or CCM2 demonstrated that the impaired production of a functional fibronectin matrix is a common feature of CCM1-, CCM2-, and CCM3-deficient ECs.

Semaphorins regulate guidance during cell migration. In addition to repelling endothelial cells, Hamm et al. identify a novel mechanism by which Semaphorin3d/Neuropilin1 regulates collective endothelial cell migration through activating a kinase cascade, which regulates Actin network organization and cell–cell contacts., During cardiovascular development, tight spatiotemporal regulation of molecular cues is essential for controlling endothelial cell (EC) migration. Secreted class III Semaphorins play an important role in guidance of neuronal cell migration and were lately linked to regulating cardiovascular development. Recently, SEMA3D gene disruptions were associated with cardiovascular defects in patients; however, the mechanisms of action were not revealed. Here we show for the first time that Sema3d regulates collective EC migration in zebrafish through two separate mechanisms. Mesenchymal Sema3d guides outgrowth of the common cardinal vein via repulsion and signals through PlexinD1. Additionally, within the same ECs, we identified a novel function of autocrine Sema3d signaling in regulating Actin network organization and EC morphology. We show that this new function requires Sema3d signaling through Neuropilin1, which then regulates Actin network organization through RhoA upstream of Rock, stabilizing the EC sheet. Our findings are highly relevant for understanding EC migration and the mechanisms of collective migration in other contexts.

In this study, we explored the existence of a transcriptional network co-regulated by E2F7 and HIF1 alpha, as we show that expression of E2F7, like HIF1 alpha, is induced in hypoxia, and because of the previously reported ability of E2F7 to interact with HIF1 alpha. Our genome-wide analysis uncovers a transcriptional network that is directly controlled by HIF1 alpha and E2F7, and demonstrates both stimulatory and repressive functions of the HIF1 alpha -E2F7 complex. Among this network we reveal Neuropilin 1 (NRP1) as a HIF1 alpha-E2F7 repressed gene. By performing in vitro and in vivo reporter assays we demonstrate that the HIF1 alpha-E2F7 mediated NRP1 repression depends on a 41 base pairs 'E2F-binding site hub', providing a molecular mechanism for a previously unanticipated role for HIF1 alpha in transcriptional repression. To explore the biological significance of this regulation we performed in situ hybridizations and observed enhanced nrp1a expression in spinal motorneurons (MN) of zebrafish embryos, upon morpholino-inhibition of e2f7/8 or hif1 alpha. Consistent with the chemo-repellent role of nrp1a, morpholino-inhibition of e2f7/8 or hif1 alpha caused MN truncations, which was rescued in TALEN-induced nrp1a(hu10012) mutants, and phenocopied in e2f7/8 mutant zebrafish. Therefore, we conclude that repression of NRP1 by the HIF1 alpha-E2F7 complex regulates MN axon guidance in vivo.

During CNS development and adult neurogenesis, immature neurons travel from the germinal zones towards their final destination using cellular substrates for their migration. Classically, radial glia and neuronal axons have been shown to act as physical scaffolds to support neuroblast locomotion in processes known as gliophilic and neurophilic migration, respectively (Hatten, 1999; Marin and Rubenstein, 2003; Rakic, 2003). In adulthood, long distance neuronal migration occurs in a glial-independent manner since radial glia cells differentiate into astrocytes after birth. A series of studies highlight a novel mode of neuronal migration that uses blood vessels as scaffolds, the so-called vasophilic migration. This migration mode allows neuroblast navigation in physiological and also pathological conditions, such as neuronal precursor migration after ischemic stroke or cerebral invasion of glioma tumor cells. Here we review the current knowledge about how vessels pave the path for migrating neurons and how trophic factors derived by glio-vascular structures guide neuronal migration both during physiological as well as pathological processes.

The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

Cerebral cavernous malformations (CCMs) are prevalent slow-flow vascular lesions which harbour the risk to develop intracranial haemorrhages, focal neurological deficits, and epileptic seizures. Autosomal dominantly inherited CCMs were found to be associated with heterozygous inactivating mutations in 3 genes, CCM1(KRIT1), CCM2(MGC4607), and CCM3(PDCD10) in 1999, 2003 and 2005, respectively. Despite the availability of high-throughput sequencing techniques, no further CCM gene has been published since. Here, we report on the identification of an autosomal dominantly inherited frameshift mutation in a gene of thus far unknown function, FAM222B(C17orf63), through exome sequencing of CCM patients mutation-negative for CCM1-3. A yeast 2-hybrid screen revealed interactions of FAM222B with the tubulin cytoskeleton and STAMBP which is known to be associated with microcephaly-capillary malformation syndrome. However, a phenotype similar to existing models was not found, neither in fam222bb/fam222ba double mutant zebrafish generated by transcription activator-like effector nucleases nor in an in vitro sprouting assay using human umbilical vein endothelial cells transfected with siRNA against FAM222B. These observations led to the assumption that aberrant FAM222B is not involved in the formation of CCMs.

The Popeye domain containing (Popdc) genes encode a family of transmembrane proteins with an evolutionary conserved Popeye domain. These genes are abundantly expressed in striated muscle tissue, however their function is not well understood. In this study we have investigated the role of the popdc2 gene in zebrafish. Popdc2 transcripts were detected in the embryonic myocardium and transiently in the craniofacial and tail musculature. Morpholino oligonucleotide-mediated knockdown of popdc2 resulted in aberrant development of skeletal muscle and heart. Muscle segments in the trunk were irregularly shaped and craniofacial muscles were severely reduced or even missing. In the heart, pericardial edema was prevalent in the morphants and heart chambers were elongated and looping was abnormal. These pathologies in muscle and heart were alleviated after reducing the morpholino concentration. However the heart still was abnormal displaying cardiac arrhythmia at later stages of development. Optical recordings of cardiac contractility revealed irregular ventricular contractions with a 2:1, or 3:1 atrial/ventricular conduction ratio, which caused a significant reduction in heart frequency. Recordings of calcium transients with high spatiotemporal resolution using a transgenic calcium indicator line (Tg(cmlc2:gCaMP)s878) and SPIM microscopy confirmed the presence of a severe arrhythmia phenotype. Our results identify popdc2 as a gene important for striated muscle differentiation and cardiac morphogenesis. In addition it is required for the development of the cardiac conduction system.

Corrigendum to ‘‘The Popeye domain containing 2 (popdc2) gene in zebrafish is required for heart and skeletal muscle development’’ [Dev. Biol. 363 (2012) 438–450] Bettina C. Kirchmaier , Kar Lai Poon , Thorsten Schwerte , Jan Huisken , Christoph Winkler , Benno Jungblut , Didier Y. Stainier , Thomas Brand a,n a Celland Developmental Biology, University of Wurzburg, Biocenter, Am Hubland, 97074 Wurzburg, Germany b Harefield Heart Science Centre, National Heart & Lung Institute, Imperial College, London, Harefield UB9 6JH, UK c Institute of Zoology, University of Innsbruck, Technikerstr. 25, 6020 Innsbruck, Austria d Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA e Physiological Chemistry I, University of Wurzburg, Biocenter, Am Hubland, 97074 Wurzburg, Germany f Max Planck Institute for Heart and Lung Research, Parkstr. 1, 61231 Bad Nauheim, Germany

BACKGROUND: Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. METHODS AND RESULTS: Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2-associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. CONCLUSIONS: Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease. [KEYWORDS: Animals, Apoptosis/drug effects, Arterioles/pathology, Capillaries/pathology, Cell Hypoxia, Cells, Cultured/drug effects/metabolism, Collagen, Drug Combinations, Drug Evaluation, Preclinical, Endothelial Cells/ metabolism/pathology, GATA2 Transcription Factor/biosynthesis/genetics, Gene Expression Profiling, Heart Failure/etiology, Heme Oxygenase-1/biosynthesis/genetics, Laminin, Male, Mice, Mice, Inbred C57BL, MicroRNAs/antagonists & inhibitors/genetics/ physiology, Myocardial Infarc