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 β1 integrin and occurs in the absence of blood flow. Downregulation of β1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a β1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.

Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes. Although it is well established that members of the Claudin family of transmembrane tight junction proteins determine paracellular tightness within epithelial/endothelial barrier systems, functional evidence for their role in the morphogenesis of lumenized organs has been scarce. Here, we identify Claudin5a as a core component of an early cerebral-ventricular barrier system that is required for ventricular lumen expansion in the zebrafish embryonic brain before the establishment of the embryonic blood-brain barrier. Loss of Claudin5a or expression of a tight junction-opening Claudin5a mutant reduces brain ventricular volume expansion without disrupting the polarized organization of the neuroepithelium. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. Genetic analyses show that the apical neuroepithelial localization of Claudin5a depends on epithelial cell polarity and provide evidence for concerted activities between Claudin5a and Na B rain morphogenesis in the zebrafish embryo involves a wellstudied ventricular lumen expansion process which occurs at early stages of development between 17 and 21 h after fertilization (hpf) (1). Genetic analyses showed that the osmoregulatory ion pump ATPase, Na + /K + transporting, alpha 1 polypeptide (Atp1a1) is critically important for lumen expansion which suggests a role in the generation of hydrostatic pressure (1). However, the embryonic cerebral barrier expected to maintain luminal fluids and ions within the brain ventricles remains unknown. It is known that the bloodbrain barrier forms 2 days after brain ventricle expansion (2). Also, the choroid plexus, which develops from the ependymal layer lining the ventral floor of the cerebral ventricle, is not functional at these stages and forms the blood-cerebrospinal fluid barrier only at later stages and in the adult fish (3, 4). To elucidate the nature of the cerebral-ventricular barrier system required for initial lumen expansion, we focused our attention on the neuroepithelium lining the cerebral cavities. We hypothesized that Claudins (Cldn) may contribute to the cerebral-ventricular barrier based on their established roles in the regulation of tight junction (TJ) barriers (5, 6). Claudins are characterized as either barrier-or pore-forming. In a recent study, Bagnat and colleagues demonstrated an involvement of the pore-forming Cldn15 in gut lumen expansion in the zebrafish embryo (7). Moreover, the C-terminal half of Clostridium perfringens enterotoxin (C-CPE), a polypeptide with inhibitory activity to several barrier-forming Claudins including Cldn3, Cldn4, and Cldn6, affected murine blastocoel cavity expansion, which is another lumen expansion process that requires hydrostatic pressure (8). Together, these studies implied an important function of Claudins in brain ventricular lumen expansion.Her...

Abstract-Many vertebrate organs are derived from monolayered epithelia that undergo morphogenesis to acquire their shape. Whereas asymmetric left/right gene expression within the zebrafish heart field has been well documented, little is known about the tissue movements and cellular changes underlying early cardiac morphogenesis. Here, we demonstrate that asymmetric involution of the myocardium of the right-posterior heart field generates the ventral floor, whereas the noninvoluting left heart field gives rise to the dorsal roof of the primary heart tube. During heart tube formation, asymmetric left/right gene expression within the myocardium correlates with asymmetric tissue morphogenesis. Disruption of left/right gene expression causes randomized myocardial tissue involution. Time-lapse analysis combined with genetic analyses reveals that motility of the myocardial epithelium is a tissue migration process. Our results demonstrate that asymmetric morphogenetic movements of the 2 bilateral myocardial cell populations generate different dorsoventral regions of the zebrafish heart tube. Failure to generate a heart tube does not affect the acquisition of atrial versus ventricular cardiac cell shapes. Therefore, establishment of basic cardiac cell shapes precedes cardiac function. Together, these results provide the framework for the integration of single cell behaviors during the formation of the vertebrate primary heart tube. (Circ Res. 2008;102:e12-e19.)Key Words: heart tube Ⅲ cell polarity Ⅲ protein kinase C iota Ⅲ left-right asymmetry Ⅲ southpaw Ⅲ nagie oko H eart development in vertebrates involves the fusion of 2 myocardial progenitor fields at the embryonic midline. These heart fields derive from the left and right lateral plate mesoderm (LPM). 1 In zebrafish, the fusion of the 2 heart fields forms the heart cone, a central flat disc that is subsequently transformed into the primary heart tube that generates a 2-chambered heart with an anterior atrium and a posterior ventricle, which initiates circulation at 24 hours post fertilization (hpf). [2][3][4] Morphogenetic processes and tissue dynamics required for heart cone-to-tube transition are not well understood. Previous studies have described the asymmetric leftward movement of the primary heart tube (after 24 hpf), followed by the looping of the heart at 36 hpf, processes that depend on the left/right (L/R) signaling pathway and transform the linear heart tube into a looped heart with distinct bean-shaped heart chambers. 5 A key player in the hierarchy of the L/R signaling pathway is the nodal-related gene southpaw (spaw), which also affects the correct expression of downstream genes including pitx2, lefty1, and lefty2. 6 Combinatorial gene expression patterns of L/R signaling genes have been described within the heart cone. 7 However, whether this L/R asymmetric gene expression is underlying asymmetric cell behaviors is currently unknown.Myocardial precursor cells acquire a polarized epithelial morphology, which is a prerequisite for normal heart developme...

Signaling by Nodal and Bmp is essential for cardiac laterality. How activities of these pathways translate into left-right asymmetric organ morphogenesis is largely unknown. We show that, in zebrafish, Nodal locally reduces Bmp activity on the left side of the cardiac field. This effect is mediated by the extracellular matrix enzyme Hyaluronan synthase 2, expression of which is induced by Nodal. Unilateral reduction of Bmp signaling results in lower expression of nonmuscle myosin II and higher cell motility on the left, driving asymmetric displacement of the entire cardiac field. In silico modeling shows that left-right differences in cell motility are sufficient to induce a robust, directional migration of cardiac tissue. Thus, the mechanism underlying the formation of cardiac left-right asymmetry involves Nodal modulating an antimotogenic Bmp activity.

Endosomal membrane traffic serves crucial roles in cell physiology, signaling, and development. Sorting between endosomes and the trans-Golgi network (TGN) is regulated among other factors by the adaptor AP-1, an essential component of multicellular organisms. Membrane recruitment of AP-1 requires phosphatidylinositol 4-phosphate [PI(4)P], though the precise mechanisms and PI4 kinase isozyme (or isozymes) involved in generation of this PI(4)P pool remain unclear. The Wnt pathway is a major developmental signaling cascade and depends on endosomal sorting in Wnt-sending cells. Whether TGN/endosomal sorting modulates signaling downstream of Frizzled (Fz) receptors in Wnt-receiving cells is unknown. Here, we identify PI4-kinase type 2β (PI4K2β) as a regulator of TGN/endosomal sorting and Wnt signaling. PI4K2β and AP-1 interact directly and are required for efficient sorting between endosomes and the TGN. Zebrafish embryos depleted of PI4K2β or AP-1 lack pectoral fins due to defective Wnt signaling. Rescue experiments demonstrate requirements for PI4K2β-AP-1 complex formation and PI4K2β-mediated PI(4)P synthesis. Furthermore, PI4K2β binds to the Fz-associated component Dishevelled (Dvl) and regulates endosomal recycling of Fz receptors and Wnt target gene expression. These data reveal an evolutionarily conserved role for PI4K2β and AP-1 in coupling phosphoinositide metabolism to AP-1-mediated sorting and Wnt signaling.

Retinal lamination is known to depend on cell polarity and localized signaling. In vertebrates, atypical Protein Kinase C proteins, aPKCλ/ι and aPKCζ, are essential for apical-basal cell polarity. However, it is not known to what extent functional redundancy has precluded a comprehensive functional characterization of aPKC signaling during vertebrate retinogenesis. Here, we show that aPKCs λ and ζ are functionally redundant for multiple aspects of retinogenesis including mitotic division location and orientation, cell-type positioning, and retinal pigment epithelial (RPE) and photoreceptor cell morphogenesis. Genetic mosaic analyses demonstrate a cell-autonomous requirement of aPKCs for RPE and photoreceptor development, and a cell-non-autonomous function that is intrinsic to the neural retina for cell-type positioning. Our observations uncover a previously unappreciated involvement of aPKCζ during zebrafish retinogenesis and suggest that aPKC signaling primes the retinal environment for appropriate cell migration of post-mitotic progenitor cells, but is not essential for correct cell-type specification.

Endothelial cells respond to different levels of fluid shear stress through adaptations of their mechanosensitivity. Currently, we lack a good understanding of how this contributes to sculpting of the cardiovascular system. Cerebral cavernous malformation (CCM) is an inherited vascular disease that occurs when a second somatic mutation causes a loss of CCM1/KRIT1, CCM2, or CCM3 proteins. Here, we demonstrate that zebrafish Krit1 regulates the formation of cardiac valves. Expression of heg1, which encodes a binding partner of Krit1, is positively regulated by blood-flow. In turn, Heg1 stabilizes levels of Krit1 protein, and both Heg1 and Krit1 dampen expression levels of klf2a, a major mechanosensitive gene. Conversely, loss of Krit1 results in increased expression of klf2a and notch1b throughout the endocardium and prevents cardiac valve leaflet formation. Hence, the correct balance of blood-flow-dependent induction and Krit1 protein-mediated repression of klf2a and notch1b ultimately shapes cardiac valve leaflet morphology.

Background and Purpose— Cerebral cavernous malformations (CCMs) are vascular malformations of the brain that lead to cerebral hemorrhages. A pharmacological treatment is needed especially for patients with nonoperable deep-seated lesions. We and others obtained CCM mouse models that were useful for mechanistic studies and rapid trials testing the preventive effects of candidate drugs. The shortened lifespan of acute mouse models hampered evaluation of compounds that would not only prevent lesion appearance but also cure preexisting lesions. Indirubin-3′-monoxime previously demonstrated its efficacy to reverse the cardiac phenotype of ccm2 m201 zebrafish mutants and to prevent lesion development in an acute CCM2 mouse model. In the present article, we developed and characterized a novel chronic CCM2 mouse model and evaluated the curative therapeutic effect of indirubin-3′-monoxime after CCM lesion development. Methods— The chronic mouse model was obtained by a postnatal induction of brain-endothelial-cell-specific ablation of the Ccm2 gene using the inducible Slco1c1 -CreER T2 mouse line. Results— We obtained a fully penetrant novel CCM chronic mouse model without any obvious off-target phenotypes and compatible with long-term survival. By 3 months of age, CCM lesions ranging in size from small isolated lesions to multiple caverns developed throughout the brain. Lesion burden was quantified in animals from 1 week to 5 months of age. Clear signs of intracerebral hemorrhages were noticed in brain-endothelial-cell-specific ablation of the Ccm2 gene. In contrast with its preventive effect in the acute CCM2 mouse model, a 20 mg/kg indirubin-3′-monoxime treatment for 3 weeks in 3-month old animals neither had any beneficial effect on the lesion burden nor alleviated cerebral hemorrhages. Conclusions— The brain-endothelial-cell-specific ablation of the Ccm2 gene chronic model is a strongly improved disease model for the CCM community whose challenge today is to decipher which candidate drugs might have a curative effect on patients’ preexisting lesions. Visual Overview— An online visual overview is available for this article.