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Tight junctions are required for the formation of tissue barriers and function as suppressors of signalling mechanisms that control gene expression and cell behaviour; however, little is known about the physiological and developmental importance of such signalling functions. Here, we demonstrate that depletion of MarvelD3, a transmembrane protein of tight junctions, disrupts neural crest formation and, consequently, development of neural crest-derived tissues during Xenopus embryogenesis. Using embryos and explant cultures combined with a small molecule inhibitor or mutant mRNAs, we show that MarvelD3 is required to attenuate JNK signalling during neural crest induction and that inhibition of JNK pathway activation is sufficient to rescue the phenotype induced by MarvelD3 depletion. Direct JNK stimulation disrupts neural crest development, supporting the importance of negative regulation of JNK. Our data identify the junctional protein MarvelD3 as an essential regulator of early vertebrate development and neural crest induction and, thereby, link tight junctions to the control and timing of JNK signalling during early development.

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor beta 1 integrin and occurs in the absence of blood flow. Downregulation of beta 1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a beta 1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.

The heartbeat and blood flow signal to endocardial cell progenitors through mechanosensitive proteins that modulate the genetic program controlling heart valve morphogenesis. To date, the mechanism by which mechanical forces coordinate tissue morphogenesis is poorly understood. Here we use high-resolution imaging to uncover the coordinated cell behaviours leading to heart valve formation. We find that heart valves originate from progenitors located in the ventricle and atrium that generate the valve leaflets through a coordinated set of endocardial tissue movements. Gene profiling analyses and live imaging reveal that this reorganization is dependent on extracellular matrix proteins, in particular on the expression of fibronectin1b. We show that blood flow and klf2a, a major endocardial flow-responsive gene, control these cell behaviours and fibronectin1b synthesis. Our results uncover a unique multicellular layering process leading to leaflet formation and demonstrate that endocardial mechanotransduction and valve morphogenesis are coupled via cellular rearrangements mediated by fibronectin synthesis., In the developing heart, blood flow transmits mechanical signals to progenitor cells that ultimately leads to valve formation. Here, the authors identify the origin of the valve progenitor cells and fibronectin1b as a transcriptional target of the mechanotransduced signals responsible for valve formation.

International audience; The pattern of blood flow has long been thought to play a significant role in vascular morphogenesis, yet the flow-sensing mechanism that is involved at early embryonic stages, when flow forces are low, remains unclear. It has been proposed that endothe-lial cells use primary cilia to sense flow, but this has never been tested in vivo. Here we show, by non-invasive, high-resolution imaging of live zebrafish embryos, that endothelial cilia progressively deflect at the onset of blood flow and that the deflection angle correlates with calcium levels in endothelial cells. We demonstrate that alterations in shear stress, ciliogenesis, or expression of the calcium channel PKD2 impair the endothelial calcium level and both increase and perturb vascular morpho-genesis. Altogether, these results demonstrate that endothelial cilia constitute a highly sensitive structure that permits the detection of low shear forces during vascular morphogenesis.