Determination of Endothelial Stalk versus Tip Cell Potential during Angiogenesis by H2.0-like Homeobox-1

SummaryTissue branching morphogenesis requires the hierarchical organization of sprouting cells into leading “tip” and trailing “stalk” cells [1, 2]. During new blood vessel branching (angiogenesis), endothelial tip cells (TCs) lead sprouting vessels, extend filopodia, and migrate in response to gradients of the secreted ligand, vascular endothelial growth factor (Vegf) [3]. In contrast, adjacent stalk cells (SCs) trail TCs, generate the trunk of new vessels, and critically maintain connectivity with parental vessels. Here, we establish that h2.0-like homeobox-1 (Hlx1) determines SC potential, which is critical for angiogenesis during zebrafish development. By combining a novel pharmacological strategy for the manipulation of angiogenic cell behavior in vivo with transcriptomic analyses of sprouting cells, we identify the uniquely sprouting-associated gene, hlx1. Expression of hlx1 is almost entirely restricted to sprouting endothelial cells and is excluded from adjacent nonangiogenic cells. Furthermore, Hlx1 knockdown reveals its essential role in angiogenesis. Importantly, mosaic analyses uncover a cell-autonomous role for Hlx1 in the maintenance of SC identity in sprouting vessels. Hence, Hlx1-mediated maintenance of SC potential regulates angiogenesis, a finding that may have novel implications for sprouting morphogenesis of other tissues

Cardiac contraction activates endocardial Notch signaling to modulate chamber maturation in zebrafish

Congenital heart disease often features structural abnormalities that emerge during development. Accumulating evidence indicates a crucial role for cardiac contraction and the resulting fluid forces in shaping the heart, yet the molecular basis of this function is largely unknown. Using the zebrafish as a model of early heart development, we investigated the role of cardiac contraction in chamber maturation, focusing on the formation of muscular protrusions called trabeculae. By genetic and pharmacological ablation of cardiac contraction, we showed that cardiac contraction is required for trabeculation through its role in regulating notch1b transcription in the ventricular endocardium. We also showed that Notch1 activation induces expression of ephrin b2a (efnb2a) and neuregulin 1 (nrg1) in the endocardium to promote trabeculation and that forced Notch activation in the absence of cardiac contraction rescues efnb2a and nrg1 expression. Using in vitro and in vivo systems, we showed that primary cilia are important mediators of fluid flow to stimulate Notch expression. Together, our findings describe an essential role for cardiac contraction-responsive transcriptional changes in endocardial cells to regulate cardiac chamber maturation

High-resolution imaging of cardiomyocyte behavior reveals two distinct steps in ventricular trabeculation

Over the course of development, the vertebrate heart undergoes a series of complex morphogenetic processes that transforms it from a simple myocardial epithelium to the complex 3D structure required for its function. One of these processes leads to the formation of trabeculae to optimize the internal structure of the ventricle for efficient conduction and contraction. Despite the important role of trabeculae in the development and physiology of the heart, little is known about their mechanism of formation. Using 3D time-lapse imaging of beating zebrafish hearts, we observed that the initiation of cardiac trabeculation can be divided into two processes. Before any myocardial cell bodies have entered the trabecular layer, cardiomyocytes extend protrusions that invade luminally along neighboring cell-cell junctions. These protrusions can interact within the trabecular layer to form new cell-cell contacts. Subsequently, cardiomyocytes constrict their abluminal surface, moving their cell bodies into the trabecular layer while elaborating more protrusions. We also examined the formation of these protrusions in trabeculation-deficient animals, including erbb2 mutants, tnnt2a morphants, which lack cardiac contractions and flow, and myh6 morphants, which lack atrial contraction and exhibit reduced flow. We found that, compared with cardiomyocytes in wild-type hearts, those in erbb2 mutants were less likely to form protrusions, those in tnnt2a morphants formed less stable protrusions, and those in myh6 morphants extended fewer protrusions per cell. Thus, through detailed 4D imaging of beating hearts, we have identified novel cellular behaviors underlying cardiac trabeculation

The Spinster Homolog, Two of Hearts, Is Required for Sphingosine 1-Phosphate Signaling in Zebrafish

SummaryThe bioactive lipid sphingosine 1-phosphate (S1P) and its G protein-coupled receptors play critical roles in cardiovascular, immunological, and neural development and function [1–6]. Despite its importance, many questions remain about S1P signaling, including how S1P, which is synthesized intracellularly, is released from cells. Mutations in the zebrafish gene encoding the S1P receptor Miles Apart (Mil)/S1P2 disrupt the formation of the primitive heart tube [5]. We find that mutations of another zebrafish locus, two of hearts (toh), cause phenotypes that are morphologically indistinguishable from those seen in mil/s1p2 mutants. Positional cloning of toh reveals that it encodes a member of the Spinster-like family of putative transmembrane transporters. The biological functions of these proteins are poorly understood, although phenotypes of the Drosophila spinster and zebrafish not really started mutants suggest that these proteins may play a role in lipid trafficking [7, 8]. Through gain- and loss-of-function analyses, we show that toh is required for signaling by S1P2. Further evidence indicates that Toh is involved in the trafficking or cellular release of S1P

Regulation of neurocoel morphogenesis by Pard6γb

AbstractThe Par3/Par6/aPKC protein complex plays a key role in the establishment and maintenance of apicobasal polarity, a cellular characteristic essential for tissue and organ morphogenesis, differentiation and homeostasis. During a forward genetic screen for liver and pancreas mutants, we identified a pard6γb mutant, representing the first known pard6 mutant in a vertebrate organism. pard6γb mutants exhibit defects in epithelial tissue development as well as multiple lumens in the neural tube. Analyses of the cells lining the neural tube cavity, or neurocoel, in wildtype and pard6γb mutant embryos show that lack of Pard6γb function leads to defects in mitotic spindle orientation during neurulation. We also found that the PB1 (aPKC-binding) and CRIB (Cdc-42-binding) domains and the KPLG amino acid sequence within the PDZ domain (Pals1-and Crumbs binding) are not required for Pard6γb localization but are essential for its function in neurocoel morphogenesis. Apical membranes are reduced, but not completely absent, in mutants lacking the zygotic, or both the maternal and zygotic, function of pard6γb, leading us to examine the localization and function of the three additional zebrafish Pard6 proteins. We found that Pard6α, but not Pard6β or Pard6γa, could partially rescue the pard6γbs441 mutant phenotypes. Altogether, these data indicate a previously unappreciated functional diversity and complexity within the vertebrate pard6 gene family

Radial glia regulate vascular patterning around the developing spinal cord

Vascular networks surrounding individual organs are important for their development, maintenance, and function; however, how these networks are assembled remains poorly understood. Here we show that CNS progenitors, referred to as radial glia, modulate vascular patterning around the spinal cord by acting as negative regulators. We found that radial glia ablation in zebrafish embryos leads to excessive sprouting of the trunk vessels around the spinal cord, and exclusively those of venous identity. Mechanistically, we determined that radial glia control this process via the Vegf decoy receptor sFlt1: sflt1 mutants exhibit the venous over-sprouting observed in radial glia-ablated larvae, and sFlt1 overexpression rescues it. Genetic mosaic analyses show that sFlt1 function in trunk endothelial cells can limit their over-sprouting. Together, our findings identify CNS-resident progenitors as critical angiogenic regulators that determine the precise patterning of the vasculature around the spinal cord, providing novel insights into vascular network formation around developing organs

Cse1l Is a Negative Regulator of CFTR-Dependent Fluid Secretion

Transport of chloride through the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates[1, 2]. Loss of CFTR function leads to cystic fibrosis (CF)[1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine and vas deferens. Conversely, un-controlled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera[5, 6] and other secretory diarrheas [7] as well as Polycystic Kidney Disease (PKD)[8–10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the un-controlled activation of the CFTR channel. Analyses in zebrafish embryos and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much needed model system to study CFTR regulation in vivo

Notch Signaling Functions as a Cell-Fate Switch between the Endothelial and Hematopoietic Lineages

Recent studies have begun to elucidate how the endothelial lineage is specified from the nascent mesoderm. However, the molecular mechanisms which regulate this process remain largely unknown. We hypothesized that Notch signaling might play an important role in specifying endothelial progenitors from the mesoderm, given that this pathway acts as a bipotential cell-fate switch on equipotent progenitor populations in other settings. We found that zebrafish embryos with decreased levels of Notch signaling exhibited a significant increase in the number of endothelial cells, whereas embryos with increased levels of Notch signaling displayed a reduced number of endothelial cells. Interestingly, there is a concomitant gain of endothelial cells and loss of erythrocytes in embryos with decreased Notch activity, without an effect on cell proliferation or apoptosis. Lineage-tracing analyses indicate that the ectopic endothelial cells in embryos with decreased Notch activity originate from mesodermal cells that normally produce erythrocyte progenitors. Taken together, our data suggest that Notch signaling negatively regulates the number of endothelial cells by limiting the number of endothelial progenitors within the mesoderm, probably functioning as a cell-fate switch between the endothelial and the hematopoietic lineages

Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signaling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumor formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppresses hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis