A sphingosine-1-phosphate receptor regulates cell migration during vertebrate heart development

Coordinated cell migration is essential in many fundamental biological processes including embryonic development, organogenesis, wound healing and the immune response. During organogenesis, groups of cells are directed to specific locations within the embryo. Here we show that the zebrafish miles apart (mil) mutation specifically affects the migration of the heart precursors to the midline. We found that mutant cells transplanted into a wild-type embryo migrate normally and that wild-type cells in a mutant embryo fail to migrate, suggesting that mil may be involved in generating an environment permissive for migration. We isolated mil by positional cloning and show that it encodes a member of the lysosphingolipid G-protein-coupled receptor family. We also show that sphingosine-1-phosphate is a ligand for Mil, and that it activates several downstream signalling events that are not activated by the mutant alleles. These data reveal a new role for lysosphingolipids in regulating cell migration during vertebrate development and provide the first molecular clues into the fusion of the bilateral heart primordia during organogenesis of the heart.     

The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation

Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.     

Fgf10 regulates hepatopancreatic ductal system patterning and differentiation

During organogenesis, the foregut endoderm gives rise to the many different cell types that comprise the hepatopancreatic system, including hepatic, pancreatic and gallbladder cells, as well as the epithelial cells of the hepatopancreatic ductal system that connects these organs together and with the intestine. However, the mechanisms responsible for demarcating ducts versus organs are poorly understood. Here, we show that Fgf10 signaling from the adjacent mesenchyme is responsible for refining the boundaries between the hepatopancreatic duct and organs. In zebrafish fgf10 mutants, the hepatopancreatic ductal epithelium is severely dysmorphic, and cells of the hepatopancreatic ductal system and adjacent intestine misdifferentiate toward hepatic and pancreatic fates. Furthermore, Fgf10 also functions to prevent the differentiation of the proximal pancreas and liver into hepatic and pancreatic cells, respectively. These data shed light onto how the multipotent cells of the foregut endoderm, and subsequently those of the hepatopancreatic duct, are directed toward different organ fates.     

A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula

It has been proposed that haematopoietic and endothelial cells share a common progenitor, termed the haemangioblast. This idea was initially conceived as a result of the observation that these two cell types develop in close proximity to each other within the embryo. Support for this hypothesis was provided by studies on single-cell-derived colonies that can produce both haematopoietic and endothelial cells in vitro. Although these data point towards the existence of a common progenitor for these two lineages, the presence of a bipotential progenitor cell has yet to be demonstrated in vivo. Through the construction of single-cell-resolution fate maps of the zebrafish late blastula and gastrula, we demonstrate that individual cells can give rise to both haematopoietic and endothelial cells. These bipotential progenitors arise along the entire extent of the ventral mesoderm and contribute solely to haematopoietic and endothelial cells. We also find that only a subset of haematopoietic and endothelial cells arise from haemangioblasts. The endothelial descendants of the haemangioblasts all clustered in a specific region of the axial vessels regardless of the location of their progenitors. Our results provide in vivo evidence supporting the existence of the haemangioblast and reveal distinct features of this cell population.     

Mesodermal Wnt2b signalling positively regulates liver specification

Endodermal organs such as the lung, liver and pancreas emerge at precise locations along the primitive gut tube. Although several signalling pathways have been implicated in liver formation, so far no single gene has been identified that exclusively regulates liver specification. In zebrafish, the onset of liver specification is marked by the localized endodermal expression of hhex and prox1 at 22 hours post fertilization. Here we used a screen for mutations affecting endodermal organ morphogenesis to identify a unique phenotype: prometheus (prt) mutants exhibit profound, though transient, defects in liver specification. Positional cloning reveals that prt encodes a previously unidentified Wnt2b homologue. prt/wnt2bb is expressed in restricted bilateral domains in the lateral plate mesoderm directly adjacent to the liver-forming endoderm. Mosaic analyses show the requirement for Prt/Wnt2bb in the lateral plate mesoderm, in agreement with the inductive properties of Wnt signalling. Taken together, these data reveal an unexpected positive role for Wnt signalling in liver specification, and indicate a possible common theme for the localized formation of endodermal organs along the gut tube.     

Birc2 (cIap1) regulates endothelial cell integrity and blood vessel homeostasis

Integrity of the blood vessel wall is essential for vascular homeostasis and organ function. A dynamic balance between endothelial cell survival and apoptosis contributes to this integrity during vascular development and pathological angiogenesis. The genetic and molecular mechanisms regulating these processes in vivo are still largely unknown. Here, we show that Birc2 (also known as cIap1) is essential for maintaining endothelial cell survival and blood vessel homeostasis during vascular development. Using a forward-genetic approach, we identified a zebrafish null mutant for birc2, which shows severe hemorrhage and vascular regression due to endothelial cell integrity defects and apoptosis. Using genetic and molecular approaches, we show that Birc2 positively regulates the formation of the TNF receptor complex I in endothelial cells, thereby promoting NF-kappaB activation and maintaining vessel integrity and stabilization. In the absence of Birc2, a caspase-8-dependent apoptotic program takes place that leads to vessel regression. Our findings identify Birc2 and TNF signaling components as critical regulators of vascular integrity and endothelial cell survival, thereby providing an additional target pathway for the control of angiogenesis and blood vessel homeostasis during embryogenesis, regeneration and tumorigenesis.     

Vertebrate Smoothened functions at the primary cilium

The unanticipated involvement of several intraflagellar transport proteins in the mammalian Hedgehog (Hh) pathway has hinted at a functional connection between cilia and Hh signal transduction. Here we show that mammalian Smoothened (Smo), a seven-transmembrane protein essential for Hh signalling, is expressed on the primary cilium. This ciliary expression is regulated by Hh pathway activity; Sonic hedgehog or activating mutations in Smo promote ciliary localization, whereas the Smo antagonist cyclopamine inhibits ciliary localization. The translocation of Smo to primary cilia depends upon a conserved hydrophobic and basic residue sequence homologous to a domain previously shown to be required for the ciliary localization of seven-transmembrane proteins in Caenorhabditis elegans. Mutation of this domain not only prevents ciliary localization but also eliminates Smo activity both in cultured cells and in zebrafish embryos. Thus, Hh-dependent translocation to cilia is essential for Smo activity, suggesting that Smo acts at the primary cilium.