Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.

Chromatin remodeling and histone modifications facilitate access of transcription factors to DNA by promoting the unwinding and destabilization of histone-DNA interactions. We present DPF3, a new epigenetic key factor for heart and muscle development characterized by a double PHD finger. DPF3 is associated with the BAF chromatin remodeling complex and binds methylated and acetylated lysine residues of histone 3 and 4. Thus, DPF3 may represent the first plant homeodomains that bind acetylated lysines, a feature previously only shown for the bromodomain. During development Dpf3 is expressed in the heart and somites of mouse, chicken, and zebrafish. Morpholino knockdown of dpf3 in zebrafish leads to incomplete cardiac looping and severely reduced ventricular contractility, with disassembled muscular fibers caused by transcriptional deregulation of structural and regulatory proteins. Promoter analysis identified Dpf3 as a novel downstream target of Mef2a. Taken together, DPF3 adds a further layer of complexity to the BAF complex by representing a tissue-specific anchor between histone acetylations as well as methylations and chromatin remodeling. Furthermore, this shows that plant homeodomain proteins play a yet unexplored role in recruiting chromatin remodeling complexes to acetylated histones.[Keywords: Heart and skeletal muscle development and function; PHD finger; BAF chromatin remodeling complex; SMARCD3-BAF60; acetylated and methylated histones; Mef2] Supplemental material is available at http://www.genesdev.org.

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.

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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...

The differentiation of HSCs into myeloid lineages requires the transcription factor PU.1. Whereas PU.1-dependent induction of myeloid-specific target genes has been intensively studied, negative regulation of stem cell or alternate lineage programs remains incompletely characterized. To test for such negative regulatory events, we searched for PU.1-controlled microRNAs (miRs) by expression profiling using a PU.1-inducible myeloid progenitor cell line model. We provide evidence that PU.1 directly controls expression of at least 4 of these miRs (miR-146a, miR-342, miR-338, and miR-155) through temporally dynamic occupation of binding sites within regulatory chromatin regions adjacent to their genomic coding loci. Ectopic expression of the most robustly induced PU.1 target miR, miR-146a, directed the selective differentiation of HSCs into functional peritoneal macrophages in mouse transplantation assays. In agreement with this observation, disruption of Dicer expression or specific antagonization of miR-146a function inhibited the formation of macrophages during early zebrafish (Danio rerio) development. In the present study, we describe a PU.1-orchestrated miR program that mediates key functions of PU.1 during myeloid differentiation.

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...

Organ morphogenesis requires cellular shape changes and tissue rearrangements that occur in a precisely timed manner. Here, we show that zebrafish heart and soul (Has)/protein kinase C iota (PRKCi) is required tissue-autonomously within the myocardium for normal heart morphogenesis and that this function depends on its catalytic activity. In addition, we demonstrate that nagie oko (Nok) is the functional homolog of mammalian protein associated with Lin-seven 1 (Pals1)/MAGUK p55 subfamily member 5 (Mpp5), and we dissect its earlier and later functions during myocardial morphogenesis. Has/PRKCi and Nok/Mpp5 are required early for the polarized epithelial organization and coherence of myocardial cells during heart cone formation. Zygotic nok/mpp5 mutants have later myocardial defects, including an incomplete heart tube elongation corresponding with a failure of myocardial cells to correctly expand in size. Furthermore, we show that nok/mpp5 acts within myocardial cells during heart tube elongation.Together, these results demonstrate that cardiac morphogenesis depends on the polarized organization and coherence of the myocardium, and that the expansion of myocardial cell size contributes to the transformation of the heart cone into an elongated tube.KEY WORDS: Organ morphogenesis, prkci, Myocardium, heart and soul, nagie oko, mpp5, pals1, Zebrafish Development 133,[107][108][109][110][111][112][113][114][115]