Phospholipase D1 is required for angiogenesis of intersegmental blood vessels in zebrafish.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid and choline. Studies in cultured cells and Drosophila melanogaster have implicated PLD in the regulation of many cellular functions, including intracellular vesicle trafficking, cell proliferation and differentiation. However, the function of PLD in vertebrate development has not been explored. Here we report cloning and characterization of a zebrafish PLD1 (pld1) homolog. Like mammalian PLDs, zebrafish Pld1 contains two conservative HKD motifs. Maternally contributed pld1 transcripts are uniformly distributed in early embryo. Localized expression of pld1 is observed in the notochord during early segmentation, in the somites during later segmentation and in the liver at the larval stages. Studies in intact and cell-free preparations demonstrate evolutionary conservation of regulation. Inhibition of Pld1 expression using antisense morpholino oligonucleotides (MO) interfering with the translation or splicing of pld1 impaired intersegmental vessel (ISV) development. Incubating embryos with 1-butanol, which diverts production of phosphatidic acid to a phosphatidylalcohol, caused similar ISV defects. To determine where Pld1 is required for ISV development we performed transplantation experiments. Analyses of the mosaic Pld1 deficient embryos showed partial suppression of ISV defects in the segments containing transplanted wild-type notochord cells but not in the ones containing wild-type somitic cells. These results provide the first evidence that function of Pld1 in the developing notochord is essential for vascular development in vertebrates.

Essential role for fibrillin-2 in zebrafish notochord and vascular morphogenesis.

Recent studies demonstrate that lysyl oxidase cuproenzymes are critical for zebrafish notochord formation, but the molecular mechanisms of copper-dependent notochord morphogenesis are incompletely understood. We, therefore, conducted a forward genetic screen for zebrafish mutants that exhibit notochord sensitivity to lysyl oxidase inhibition, yielding a mutant with defects in notochord and vascular morphogenesis, puff daddygw1 (pfdgw1). Meiotic mapping and cloning reveal that the pfdgw1 phenotype results from disruption of the gene encoding the extracellular matrix protein fibrillin-2, and the spatiotemporal expression of fibrillin-2 is consistent with the pfdgw1 phenotype. Furthermore, each aspect of the pfdgw1 phenotype is recapitulated by morpholino knockdown of fibrillin-2. Taken together, the data reveal a genetic interaction between fibrillin-2 and the lysyl oxidases in notochord formation and demonstrate the importance of fibrillin-2 in specific early developmental processes in zebrafish.

Caveolin-1alpha and -1beta perform nonredundant roles in early vertebrate development.

Caveolins are integral membrane proteins that localize predominantly to lipid rafts, where they oligomerize to form flask-shaped organelles termed caveolae and play important roles in membrane trafficking, signal transduction, and other cellular processes. To investigate potential roles for caveolin-1 (cav-1) in development, cav-1alpha and -1beta cDNAs were functionally characterized in the zebrafish. Cav-1alpha and -1beta mRNAs exhibited overlapping but distinct expression patterns throughout embryogenesis. Targeted depletion of either Cav-1 isoform, using antisense morpholino oligomers, resulted in a substantial loss of caveolae and dramatic neural, eye, and somite defects by 12 hours after fertilization, the time at which mRNA levels of both isoforms substantially increased in wild-type animals. Morphant phenotypes were rescued by injection of homotypic (cav-1alpha/cav-1alpha) but not heterotypic (cav-1alpha/cav-1beta) zebrafish and human cav-1 cRNAs, revealing nonredundant and evolutionarily conserved functions for the individual Cav-1 isoforms. Mutation of a known Cav-1 phosphorylation site unique to Cav-1alpha (Y14-->F) resulted in a failure to rescue the cav-1alpha morphant phenotype, verifying an essential role for Cav-1alpha specifically and implicating this residue in early developmental functions. Cav-1alpha and -1beta morphants also exhibited disruption in the actin cytoskeleton. These results support important and previously unanticipated roles for the Caveolin-1 isoforms in vertebrate organogenesis.

Essential and overlapping roles for laminin alpha chains in notochord and blood vessel formation.

Laminins are major constituents of basement membranes and have wide ranging functions during development and in the adult. They are a family of heterotrimeric molecules created through association of an alpha, beta and gamma chain. We previously reported that two zebrafish loci, grumpy (gup) and sleepy (sly), encode laminin beta1 and gamma1, which are important both for notochord differentiation and for proper intersegmental blood vessel (ISV) formation. In this study we show that bashful (bal) encodes laminin alpha1 (lama1). Although the strongest allele, bal(m190), is fully penetrant, when compared to gup or sly mutant embryos, bal mutants are not as severely affected, as only anterior notochord fails to differentiate and ISVs are unaffected. This suggests that other alpha chains, and hence other isoforms, act redundantly to laminin 1 in posterior notochord and ISV development. We identified cDNA sequences for lama2, lama4 and lama5 and disrupted the expression of each alone or in mutant embryos also lacking laminin alpha1. When expression of laminin alpha4 and laminin alpha1 are simultaneously disrupted, notochord differentiation and ISVs are as severely affected as sly or gup mutants. Moreover, live imaging of transgenic embryos expressing enhanced green fluorescent protein in forming ISVs reveals that the vascular defects in these embryos are due to an inability of ISV sprouts to migrate correctly along the intersegmental, normally laminin-rich regions.

Angiogenesis within the developing mouse neural tube is dependent on sonic hedgehog signaling: possible roles of motor neurons.

Embryonic morphogenesis of vascular and nervous systems is tightly coordinated, and recent studies revealed that some neurogenetic factors such as Sonic hedgehog (Shh) also exhibit angiogenetic potential. Vascularization within the developing mouse neural tube depends on vessel sprouting from the surrounding vascular plexus. Previous studies implicated possible roles of VEGF/Flk-1 and Angiopoietin-1(Ang-1)/Tie-2 signaling as candidate molecules functioning in this process. Examining gene expressions of these factors at embryonic day (E) 9.5 and 10.5, we unexpectedly found that both VEGF and Ang-1 were expressed in the motor neurons in the ventral neural tube. The motor neurons were indeed located in the close vicinity of the infiltrating vessels, suggesting involvement of motor neurons in the sprouting. To substantiate this possibility, we inhibited induction of the motor neurons in the cultured mouse embryos by cyclopamine, a Shh signaling blocker. The vessel sprouting was dramatically impaired by inhibition of Shh signaling, together with nearly complete loss of the motor neurons. Expression of Ang-1, but not VEGF, within the neural tube was remarkably reduced in the cyclopamine treated embryos. These results suggest that the neural tube angiogenesis is dependent on Shh signaling, and mediated, at least in part, by the Ang-1 positive motor neurons.

The architecture of internal blood vessels in human fetal vertebral bodies.

The internal vascular system of vertebral bodies was investigated in 17-24 wk human fetuses by acrylic dye injection and by corrosion casting/scanning electron microscopy. The regions of intervertebral spaces did not contain blood vessels. The radial metaphyseal vessels were at the stage of centripetal ingrowth into the vertebral body cartilage and their terminal, blindly ending segments had a form of cuff-like capillary plexuses. The anterolateral equatorial arteries communicating with the vessels of the ossification centre were only rarely found. The centre was usually supplied by 2 posterior (nutrient) arteries which branched into an arcade-like array of arterioles equipped with occasional sphincters and giving origin to a dense network of peripherally located capillaries. Numerous blind capillary buds formed the advancing border of the ossification centre. The veins usually accompanied the arteries. In the ossification centre the venous compartment consisted of sinuses drained by larger posterior veins. In the 17 wk fetus, an axial avascular area was observed in the place of notochord localisation, indicating the formation of a ring-shaped ossification centre around the notochord remnants at earlier stages of fetal development.