The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development

The vertebrate embryonic vasculature develops from angioblasts, which are specified from mesodermal precursors and develop in close association with blood cells. The signals that regulate embryonic vasculogenesis and angiogenesis are incompletely understood. Here, we show that R-spondin 3 (Rspo3), a member of a novel family of secreted proteins in vertebrates that activate Wnt/beta-catenin signaling, plays a key role in these processes. In Xenopus embryos, morpholino antisense knockdown of Rspo3 induces vascular defects because Rspo3 is essential for regulating the balance between angioblast and blood cell specification. In mice, targeted disruption of Rspo3 leads to embryonic lethality caused by vascular defects. Specifically in the placenta, remodeling of the vascular plexus is impaired. In human endothelial cells, R-spondin signaling promotes proliferation and sprouting angiogenesis in vitro, indicating that Rspo3 can regulate endothelial cells directly. We show that vascular endothelial growth factor is an immediate early response gene and a mediator of R-spondin signaling. The results identify Rspo3 as a novel, evolutionarily conserved angiogenic factor in embryogenesis.     

Semaphorin signaling guides cranial neural crest cell migration in zebrafish

Cranial neural crest cells (NCCs) migrate into the pharyngeal arches in three primary streams separated by two cranial neural crest (NC)-free zones. Multiple tissues have been implicated in the guidance of cranial NCC migration; however, the signals provided by these tissues have remained elusive. We investigate the function of semaphorins (semas) and their receptors, neuropilins (nrps), in cranial NCC migration in zebrafish. We find that genes of the sema3F and sema3G class are expressed in the cranial NC-free zones, while nrp2a and nrp2b are expressed in the migrating NCCs. sema3F/3G expression is expanded homogeneously in the head periphery through which the cranial NCCs migrate in lzr/pbx4 mutants, in which the cranial NC streams are fused. Antisense morpholino knockdown of Sema3F/3G or Nrp2 suppresses the abnormal cranial NC phenotype of lzr/pbx4 mutants, demonstrating that aberrant Sema3F/3G-Nrp2 signaling is responsible for this phenotype and suggesting that repulsive Sema3F/3G-Npn2 signaling normally contributes to the guidance of migrating cranial NCCs. Furthermore, global over-expression of sema3Gb phenocopies the aberrant cranial NC phenotype of lzr/pbx4 mutants when endogenous Sema3 ligands are knocked down, consistent with a model in which the patterned expression of Sema3 ligands in the head periphery coordinates the migration of Nrp-expressing cranial NCCs.     

Prickle 1 regulates cell movements during gastrulation and neuronal migration in zebrafish

During vertebrate gastrulation, mesodermal and ectodermal cells undergo convergent extension, a process characterised by prominent cellular rearrangements in which polarised cells intercalate along the medio-lateral axis leading to elongation of the antero-posterior axis. Recently, it has become evident that a noncanonical Wnt/Frizzled (Fz)/Dishevelled (Dsh) signalling pathway, which is related to the planar-cell-polarity (PCP) pathway in flies, regulates convergent extension during vertebrate gastrulation. Here we isolate and functionally characterise a zebrafish homologue of Drosophila prickle (pk), a gene that is implicated in the regulation of PCP. Zebrafish pk1 is expressed maternally and in moving mesodermal precursors. Abrogation of Pk1 function by morpholino oligonucleotides leads to defective convergent extension movements, enhances the silberblick (slb)/wnt11 and pipetail (Ppt)/wnt5 phenotypes and suppresses the ability of Wnt11 to rescue the slb phenotype. Gain-of-function of Pk1 also inhibits convergent extension movements and enhances the slb phenotype, most likely caused by the ability of Pk1 to block the Fz7-dependent membrane localisation of Dsh by downregulating levels of Dsh protein. Furthermore, we show that pk1 interacts genetically with trilobite (tri)/strabismus to mediate the caudally directed migration of cranial motor neurons and convergent extension. These results indicate that, during zebrafish gastrulation Pk1 acts, in part, through interaction with the noncanonical Wnt11/Wnt5 pathway to regulate convergent extension cell movements, but is unlikely to simply be a linear component of this pathway. In addition, Pk1 interacts with Tri to mediate posterior migration of branchiomotor neurons, probably independent of the noncanonical Wnt pathway.     

Semaphorin 4D regulates gonadotropin hormone-releasing hormone-1 neuronal migration through PlexinB1-Met complex

In mammals, reproduction is dependent on specific neurons secreting the neuropeptide gonadotropin hormone–releasing hormone-1 (GnRH-1). These cells originate during embryonic development in the olfactory placode and migrate into the forebrain, where they become integral members of the hypothalamic–pituitary–gonadal axis. This migratory process is regulated by a wide range of guidance cues, which allow GnRH-1 cells to travel over long distances to reach their appropriate destinations. The Semaphorin4D (Sema4D) receptor, PlexinB1, is highly expressed in the developing olfactory placode, but its function in this context is still unknown. Here, we demonstrate that PlexinB1-deficient mice exhibit a migratory defect of GnRH-1 neurons, resulting in reduction of this cell population in the adult brain. Moreover, Sema4D promotes directional migration in GnRH-1 cells by coupling PlexinB1 with activation of the Met tyrosine kinase (hepatocyte growth factor receptor). This work identifies a function for PlexinB1 during brain development and provides evidence that Sema4D controls migration of GnRH-1 neurons.     

Chemokine signaling regulates sensory cell migration in zebrafish

Chemokines play an important role in the migration of a variety of cells during development. Recent investigations have begun to elucidate the importance of chemokine signaling within the developing nervous system. To better appreciate the neural function of chemokines in vivo, the role of signaling by SDF-1 through its CXCR4 receptor was analyzed in zebrafish. The SDF-1-CXCR4 expression pattern suggested that SDF-1-CXCR4 signaling was important for guiding migration by sensory cells known as the migrating primordium of the posterior lateral line. Ubiquitous induction of the ligand in transgenic embryos, antisense knockdown of the ligand or receptor, and a genetic receptor mutation all disrupted migration by the primordium. Furthermore, in embryos in which endogenous SDF-1 was knocked down, the primordium migrated towards exogenous sources of SDF-1. These data demonstrate that SDF-1 signaling mediated via CXCR4 functions as a chemoattractant for the migrating primordium and that chemokine signaling is both necessary and sufficient for directing primordium migration.     

Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development

Cranial neural crest cells migrate into the periocular region and later contribute to various ocular tissues including the cornea, ciliary body and iris. After reaching the eye, they initially pause before migrating over the lens to form the cornea. Interestingly, removal of the lens leads to premature invasion and abnormal differentiation of the cornea. In exploring the molecular mechanisms underlying this effect, we find that semaphorin3A (Sema3A) is expressed in the lens placode and epithelium continuously throughout eye development. Interestingly, neuropilin-1 (Npn-1) is expressed by periocular neural crest but down-regulated, in a manner independent of the lens, by the subpopulation that migrates into the eye and gives rise to the cornea endothelium and stroma. In contrast, Npn-1 expressing neural crest cells remain in the periocular region and contribute to the anterior uvea and ocular blood vessels. Introduction of a peptide that inhibits Sema3A/Npn-1 signaling results in premature entry of neural crest cells over the lens that phenocopies lens ablation. Furthermore, Sema3A inhibits periocular neural crest migration in vitro. Taken together, our data reveal a novel and essential role of Sema3A/Npn-1 signaling in coordinating periocular neural crest migration that is vital for proper ocular development.     

Tbl3 regulates cell cycle length during zebrafish development

The regulation of cell cycle rate is essential for the correct timing of proliferation and differentiation during development. Changes to cell cycle rate can have profound effects on the size, shape and cell types of a developing organ. We previously identified a zebrafish mutant ceylon (cey) that has a severe reduction in T cells and hematopoietic stem/progenitor cells (HSPCs). Here we find that the cey phenotype is due to absence of the gene transducin (beta)-like 3 (tbl3). The tbl3 homolog in yeast regulates the cell cycle by maintaining rRNA levels and preventing p53-induced cell death. Zebrafish tbl3 is maternally expressed, but later in development its expression is restricted to specific tissues. Tissues expressing tbl3 are severely reduced in cey mutants, including HSPCs, the retina, exocrine pancreas, intestine, and jaw cartilage. Specification of these tissues is normal, suggesting the reduced size is due to a reduced number of differentiated cells. Tbl3 MO injection into either wild-type or p53-/- mutant embryos phenocopies cey, indicating that loss of tbl3 causes specific defects in cey. Progression of both hematopoietic and retinal development is delayed beginning at 3 day post fertilization due to a slowing of the cell cycle. In contrast to yeast, reduction of Tbl3 causes a slowing of the cell cycle without a corresponding increase in p53 induced cell death. These data suggest that tbl3 plays a tissue-specific role regulating cell cycle rate during development.     

meis1 regulates the development of endothelial cells in zebrafish

The differentiation of endothelial cells is tightly connected with the formation of blood vessels during vertebrate development. The signaling pathways mediated by vascular endothelial growth factor (vegf) are required for these processes. Here we show that a proto-oncogene, meis1, plays important roles in the vascular development in zebrafish. Knockdown of meis1 by anti-sense meis1 morpholino (meis1 MO) led to the impairment of intersegmental vessel (ISV) formation. In meis1 morphants, the expression of an artery marker was reduced in dorsal aorta (DA), and the expression of vein markers was expanded in DA and posterior cardinal vein (PCV), suggesting the defects on artery development. Furthermore, the expression of vegf receptor, flk1, was significantly decreased in these embryos. Interestingly, flk1 MO-injected embryos exhibited similar defects as meis1 morphants. Thus, these results implicate that meis1 is a novel regulator involved in endothelial cell development, presumably affecting the vegf signaling pathway.     

Cranial vasculature in zebrafish forms by angioblast cluster-derived angiogenesis

Formation of embryonic vasculature involves vasculogenesis as endothelial cells differentiate and aggregate into vascular cords and angiogenesis which includes branching from the existing vessels. In the zebrafish which has emerged as an advantageous model to study vasculogenesis, cranial vasculature is thought to originate by a combination of vasculogenesis and angiogenesis, but how these processes are coordinated is not well understood. To determine how angioblasts assemble into cranial vasculature, we generated an etsrp:GFP transgenic line in which GFP reporter is expressed under the promoter control of an early regulator of vascular and myeloid development, etsrp/etv2. By utilizing time-lapse imaging we show that cranial vessels originate by angiogenesis from angioblast clusters, which themselves form by the mechanism of vasculogenesis. The two major pairs of bilateral clusters include the rostral organizing center (ROC) which gives rise to the most rostral cranial vessels and the midbrain organizing center (MOC) which gives rise to the posterior cranial vessels and to the myeloid and endocardial lineages. In Etsrp knockdown embryos initial cranial vasculogenesis proceeds normally but endothelial and myeloid progenitors fail to initiate differentiation, migration and angiogenesis. Such angioblast cluster-derived angiogenesis is likely to be involved during vasculature formation in other vertebrate systems as well.     

Fibronectin regulates epithelial organization during myocardial migration in zebrafish

Several genes have been implicated in heart tube formation, yet we know little about underlying cellular mechanisms. We analyzed the cellular architecture of the migrating myocardial precursors, and find that they form coherent epithelia that mature as they move medially. Mutant analyses indicate that the cardia bifida locus natter (nat) is required for the integrity of the myocardial epithelia. We positionally cloned nat and show that it encodes Fibronectin. During myocardial migration, Fibronectin is deposited at the midline between the endoderm and endocardial precursors, and laterally around the myocardial precursors. Further analyses show that Fibronectin deposition at the midline is required for the timely migration of myocardial precursors, but dispensable for the migration process itself. In the complete absence of Fibronectin, adherens junctions between myocardial precursors do not form properly, suggesting that cell-substratum interactions are required for epithelial organization. These data suggest that myocardial migration is dependent on epithelial integrity.     

Autotaxin regulates vascular development via multiple lysophosphatidic acid (LPA) receptors in zebrafish

Autotaxin (ATX) is a multifunctional ecto-type phosphodiesterase that converts lysophospholipids, such as lysophosphatidylcholine, to lysophosphatidic acid (LPA) by its lysophospholipase D activity. LPA is a lipid mediator with diverse biological functions, most of which are mediated by G protein-coupled receptors specific to LPA (LPA1-6). Recent studies on ATX knock-out mice revealed that ATX has an essential role in embryonic blood vessel formation. However, the underlying molecular mechanisms remain to be solved. A data base search revealed that ATX and LPA receptors are conserved in wide range of vertebrates from fishes to mammals. Here we analyzed zebrafish ATX (zATX) and LPA receptors both biochemically and functionally. zATX, like mammalian ATX, showed lysophospholipase D activity to produce LPA. In addition, all zebrafish LPA receptors except for LPA5a and LPA5b were found to respond to LPA. Knockdown of zATX in zebrafish embryos by injecting morpholino antisense oligonucleotides (MOs) specific to zATX caused abnormal blood vessel formation, which has not been observed in other morphant embryos or mutants with vascular defects reported previously. In ATX morphant embryos, the segmental arteries sprouted normally from the dorsal aorta but stalled in midcourse, resulting in aberrant vascular connection around the horizontal myoseptum. Similar vascular defects were not observed in embryos in which each single LPA receptor was attenuated by using MOs. Interestingly, similar vascular defects were observed when both LPA1 and LPA4 functions were attenuated by using MOs and/or a selective LPA receptor antagonist, Ki16425. These results demonstrate that the ATX-LPA-LPAR axis is a critical regulator of embryonic vascular development that is conserved in vertebrates.     

Heart-targeted overexpression of Nip3a in zebrafish embryos causes abnormal heart development and cardiac dysfunction

We transiently expressed a proapoptotic protein, Nip3a, by a heart-specific BMP4 promoter in zebrafish embryos and generated two variants of embryos with abnormal heart phenotypes (A and B). Embryos with phenotype A heart defects showed hypoplastic or elongated ventricles, elongated or enlarged atriums with no normal cardiac looping resulting a significant longer SV-BA distance, and bradycardia at 48 h post-fertilization (hpf). Embryos with phenotype B heart defects showed an enlarged fluid-filled pericardium, severe hypoplasia, non-contracting ventricles, and elongated or enlarged slowly beating atriums with no normal looping. Histological sections further revealed the absence of a proper atrioventricular boundary and no endocardial cells lining this region in both 48- and 72-hpf Nip3a-overexpressing embryos, implicating defective endocardial cushion formation. These phenotypes are reminiscent of atrioventricular canal defects in humans. In addition, induced apoptotic myocardium cells were clustered in the presumptive atrioventricular boundary as well as in the adjacent ventricle and atrium of 48- and 72-hpf Nip3a-overexpressing embryos. Nip3a expression was readily detected in 80% epiboly BMP4-Nip3a-injected embryos, and defects in heart development were observed in both the linear heart tube and subsequent chamber formation stages. These results showed that myocyte apoptosis is a universal pathogenic factor for congenital heart failure using zebrafish as a model organism.     

Semaphorin 3d promotes cell proliferation and neural crest cell development downstream of TCF in the zebrafish hindbrain

Neural crest cells (NCCs) are pluripotent migratory cells that are crucial to the development of the peripheral nervous system, pigment cells and craniofacial cartilage and bone. NCCs are specified within the dorsal ectoderm and undergo an epithelial to mesenchymal transition (EMT) in order to migrate to target destinations where they differentiate. Here we report a role for a member of the semaphorin family of cell guidance molecules in NCC development. Morpholino-mediated knockdown of Sema3d inhibits the proliferation of hindbrain neuroepithelial cells. In addition, Sema3d knockdown reduces markers of migratory NCCs and disrupts NCC-derived tissues. Similarly, expression of a dominant-repressor form of TCF (DeltaTCF) reduces hindbrain cell proliferation and leads to a disruption of migratory NCC markers. Moreover, expression of DeltaTCF downregulates sema3d RNA expression. Finally, Sema3d overexpression rescues reduced proliferation caused by DeltaTCF expression, suggesting that Sema3d lies downstream of Wnt/TCF signaling in the molecular pathway thought to control cell cycle in NCC precursors.     

Genetic analysis of melanophore development in zebrafish embryos

Vertebrate pigment cells are derived from neural crest, a tissue that also forms most of the peripheral nervous system and a variety of ectomesenchymal cell types. Formation of pigment cells from multipotential neural crest cells involves a number of common developmental processes. Pigment cells must be specified; their migration, proliferation, and survival must be controlled and they must differentiate to the final pigment cell type. We previously reported a large set of embryonic mutations that affect pigment cell development from neural crest (R. N. Kelsh et al., 1996, Development 123, 369-389). Based on distinctions in pigment cell appearance between mutants, we proposed hypotheses as to the process of pigment cell development affected by each mutation. Here we describe the cloning and expression of an early zebrafish melanoblast marker, dopachrome tautomerase. We used this marker to test predictions about melanoblast number and pattern in mutant embryos, including embryos homozygous for mutations in the colourless, sparse, touchdown, sunbleached, punkt, blurred, fade out, weiss, sandy, and albino genes. We showed that in homozygous mutants for all loci except colourless and sparse, melanoblast number and pattern are normal. colourless mutants have a pronounced decrease in melanoblast cell number from the earliest stages and also show poor melanoblast differentiation and migration. Although sparse mutants show normal numbers of melanoblasts initially, their number is reduced later. Furthermore, their distribution indicates a defect in melanoblast dispersal. These observations permit us to refine our model of the genetic control of melanophore development in zebrafish embryos.     

Endothelial Semaphorin 3fb regulates Vegf pathway-mediated angiogenic sprouting

Vessel growth integrates diverse extrinsic signals with intrinsic signaling cascades to coordinate cell migration and sprouting morphogenesis. The pro-angiogenic effects of Vascular Endothelial Growth Factor (VEGF) are carefully controlled during sprouting to generate an efficiently patterned vascular network. We identify crosstalk between VEGF signaling and that of the secreted ligand Semaphorin 3fb (Sema3fb), one of two zebrafish paralogs of mammalian Sema3F. The sema3fb gene is expressed by endothelial cells in actively sprouting vessels. Loss of sema3fb results in abnormally wide and stunted intersegmental vessel artery sprouts. Although the sprouts initiate at the correct developmental time, they have a reduced migration speed. These sprouts have persistent filopodia and abnormally spaced nuclei suggesting dysregulated control of actin assembly. sema3fb mutants show simultaneously higher expression of pro-angiogenic (VEGF receptor 2 (vegfr2) and delta-like 4 (dll4)) and anti-angiogenic (soluble VEGF receptor 1 (svegfr1)/ soluble Fms Related Receptor Tyrosine Kinase 1 (sflt1)) pathway components. We show increased phospho-ERK staining in migrating angioblasts, consistent with enhanced Vegf activity. Reducing Vegfr2 kinase activity in sema3fb mutants rescues angiogenic sprouting. Our data suggest that Sema3fb plays a critical role in promoting endothelial sprouting through modulating the VEGF signaling pathway, acting as an autocrine cue that modulates intrinsic growth factor signaling.     

The autism susceptibility gene met regulates zebrafish cerebellar development and facial motor neuron migration

During development, Met signaling regulates a range of cellular processes including growth, differentiation, survival and migration. The Met gene encodes a tyrosine kinase receptor, which is activated by Hgf (hepatocyte growth factor) ligand. Altered regulation of human MET expression has been implicated in autism. In mouse, Met signaling has been shown to regulate cerebellum development. Since abnormalities in cerebellar structure have been reported in some autistic patients, we have used the zebrafish to address the role of Met signaling during cerebellar development and thus further our understanding of the molecular basis of autism. We find that zebrafish met is expressed in the cerebellar primordium, later localizing to the ventricular zone (VZ), with the hgf1 and hgf2 ligand genes expressed in surrounding tissues. Morpholino knockdown of either Met or its Hgf ligands leads to a significant reduction in the size of the cerebellum, primarily as a consequence of reduced proliferation. Met signaling knockdown disrupts specification of VZ-derived cell types, and also reduces granule cell numbers, due to an early effect on cerebellar proliferation and/or as an indirect consequence of loss of signals from VZ-derived cells later in development. These patterning defects preclude analysis of cerebellar neuronal migration, but we have found that Met signaling is necessary for migration of hindbrain facial motor neurons. In summary, we have described roles for Met signaling in coordinating growth and cell type specification within the developing cerebellum, and in migration of hindbrain neurons. These functions may underlie the correlation between altered MET regulation and autism spectrum disorders.