Analysis of a zebrafish VEGF receptor mutant reveals specific disruption of angiogenesis

Blood vessels form either by the assembly and differentiation of mesodermal precursor cells (vasculogenesis) or by sprouting from preexisting vessels (angiogenesis). Endothelial-specific receptor tyrosine kinases and their ligands are known to be essential for these processes. Targeted disruption of vascular endothelial growth factor (VEGF) or its receptor kdr (flk1, VEGFR2) in mouse embryos results in a severe reduction of all blood vessels, while the complete loss of flt1 (VEGFR1) leads to an increased number of hemangioblasts and a disorganized vasculature. In a large-scale forward genetic screen, we identified two allelic zebrafish mutants in which the sprouting of blood vessels is specifically disrupted without affecting the assembly and differentiation of angioblasts. Molecular cloning revealed nonsense mutations in flk1. Analysis of mRNA expression in flk1 mutant embryos showed that flk1 expression was severely downregulated, while the expression of other genes (scl, gata1, and fli1) involved in vasculogenesis or hematopoiesis was unchanged. Overexpression of vegf(121+165) led to the formation of additional vessels only in sibling larvae, not in flk1 mutants. We demonstrate that flk1 is not required for proper vasculogenesis and hematopoiesis in zebrafish embryos. However, the disruption of flk1 impairs the formation or function of vessels generated by sprouting angiogenesis.     

K-ras/PI3K-Akt signaling is essential for zebrafish hematopoiesis and angiogenesis

The RAS small GTPases orchestrate multiple cellular processes. Studies on knock-out mice showed the essential and sufficient role of K-RAS, but not N-RAS and H-RAS in embryonic development. However, many physiological functions of K-RAS in vivo remain unclear. Using wild-type and fli1:GFP transgenic zebrafish, we showed that K-ras-knockdown resulted in specific hematopoietic and angiogenic defects, including the impaired expression of erythroid-specific gene gata1 and sse3-hemoglobin, reduced blood circulation and disorganized blood vessels. Expression of either K-rasC40 that links to phosphoinositide 3-kinase (PI3K) activation, or Akt2 that acts downstream of PI3K, could rescue both hematopoietic and angiogenic defects in the K-ras knockdown. Consistently, the functional rescue by k-ras mRNA was significantly suppressed by wortmannin, a PI3K-specific inhibitor. Our results provide direct evidence that PI3K-Akt plays a crucial role in mediating K-ras signaling during hematopoiesis and angiogenesis in vivo, thus offering new targets and alternative vertebrate model for studying these processes and their related diseases.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo

Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express c-myb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, transplantation, and in vivo lineage-tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of HSCs. EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate-mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.     

Distinct signalling pathways regulate sprouting angiogenesis from the dorsal aorta and the axial vein

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is critical to most physiological processes and many pathological conditions. During zebrafish development, angiogenesis expands the axial vessels into a complex vascular network that is necessary for efficient oxygen delivery. Although the dorsal aorta and the axial vein are spatially juxtaposed, the initial angiogenic sprouts from these vessels extend in opposite directions, indicating that distinct cues may regulate angiogenesis of the axial vessels. We found that angiogenic sprouts from the dorsal aorta are dependent on vascular endothelial growth factor A (Vegf-A) signalling, and do not respond to bone morphogenetic protein (Bmp) signals. In contrast, sprouts from the axial vein are regulated by Bmp signalling independently of Vegf-A signals, indicating that Bmp is a vein-specific angiogenic cue during early vascular development. Our results support a paradigm whereby different signals regulate distinct programmes of sprouting angiogenesis from the axial vein and dorsal aorta, and indicate that signalling heterogeneity contributes to the complexity of vascular networks.     

Cloning and expression of zebrafish genes encoding the heme synthesis enzymes uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO).

Heme is synthesized from glycine and succinyl CoA by eight heme synthesis enzymes. Although genetic defects in any of these enzymes are known to cause severe human blood diseases, their developmental expression in mammals is unknown. In this paper, we report two zebrafish heme synthesis enzymes, uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO) that are well conserved in comparison to their human counterparts. Both UROS and PPO formed pairs of bilateral stripes in the lateral plate mesoderm at the 15-somite stage. At 24 h post-fertilization (hpf), UROS and PPO were predominantly expressed in the intermediate cell mass (ICM) that is the major site of primitive hematopoiesis. The expression of UROS and PPO was drastically suppressed in the bloodless mutants cloche and vlad tepes/gata 1 from 15-somite to 24hpf stages, indicating that both cloche and vlad tepes/gata 1 are required for the induction and maintenance of UROS and PPO expression in the ICM.     

Vap (Vascular Associated Protein): a novel factor involved in erythropoiesis and angiogenesis

Both endothelial and erythroid cells are generated in the intermediate cell mass (ICM) during zebrafish embryogenesis, but the nature of the genes that contribute to the processes of erythrocyte maturation and blood vessel network formation is not fully understood. From our in situ-based screening, we have identified a novel factor, Vap (Vascular Associated Protein) that is predominantly expressed in the ICM, and subsequently enriched in endothelial cells. Vap expression in the ICM was drastically suppressed in the cloche mutant that has defects in both vasculogenesis and hematopoiesis, whereas Vap expression was not affected in the vlad tepes/gata1 mutant. Knockdown of Vap using anti-sense morpholinos (VAP-MO) not only resulted in decreased numbers of erythrocytes but also in the strong suppression of hemoglobin production. Further, we found that Vap knockdown caused the disorganization of the intersegmental vessels (ISVs), which show irregular branching. We propose that Vap plays an important role in the maturation of endothelial and erythroid cells in zebrafish.     

15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation

A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.     

Angiogenic network formation in the developing vertebrate trunk

We have used time-lapse multiphoton microscopy of living Tg(fli1:EGFP)y1 zebrafish embryos to examine how a patterned, functional network of angiogenic blood vessels is generated in the early vertebrate trunk. Angiogenic vascular sprouts emerge from the longitudinal trunk axial vessels (the dorsal aorta and posterior cardinal vein) in two spatially and temporally distinct steps. Dorsal aorta-derived sprouts form an initial primary network of vascular segments, followed by emergence of vein-derived secondary vascular sprouts that interact and interconnect dynamically with the primary network to initiate vascular flow. Using transgenic silent heart mutant embryos, we show that the gross anatomical patterning of this network of vessels does not require blood circulation. However, our results suggest that circulatory flow dynamics play an important role in helping to determine the pattern of interconnections between the primary network and secondary sprouts, and thus the final arterial or venous identity of the vessels in the functional network. We discuss a model to explain our results combining genetic programming of overall vascular architecture with hemodynamic determination of circulatory flow patterns.     

Angiomotin-like2 gene (amotl2) is required for migration and proliferation of endothelial cells during angiogenesis

Angiogenesis involves sprouting, migration, and proliferation of endothelial cells. The angiomotin-like2 gene (amotl2) has been found in blood vessels in zebrafish embryos, but its function in angiogenesis and underlying mechanisms remain unknown. In this study, we demonstrate that knockdown of amotl2 in zebrafish Tg(fli1:EGFP)(y1) and Tg(fli1:nEGFP)(y7) transgenic embryos impairs the intersegmental vessel growth and suppresses proliferation of endothelial cells. Transplantation experiments indicate that function of amotl2 in intersegmental vessel growth is cell-autonomous. AMOTL2 knockdown in cultured human umbilical vein endothelial cells also inhibits cell proliferation and migration and disrupts cell polarity, ultimately interrupting the formation of vascular tube-like structures. Amotl2 promotes MAPK/ERK activation via c-Src, which is dependent on phosphorylation of tyrosine residue at position 103 but independent of the C-terminal PDZ-binding domain. Taking together, our data indicate that Amotl2 plays a pivotal role in polarity, migration and proliferation of angiogenic endothelial cells.     

Homeotic selector genes control the patterning of seven-up expressing cells in the Drosophila dorsal vessel

The linear cardiac tube of Drosophila, the dorsal vessel, is an important model organ for the study of cardiac specification and patterning in vertebrates. In Drosophila, the Hox segmentation gene abdominal-A (abd-A) is required for the specification of a functionally distinct heart region at the posterior of the dorsal vessel, from which blood is pumped anteriorly through a tube termed the aorta. Since we have previously shown that the posterior part of the aorta is specified during embryogenesis to form the adult heart during metamorphosis, we determined if the embryonic aorta is also patterned by the function of Hox segmentation genes. Using gain- and loss-of-function experiments, we demonstrate that the three Hox genes expressed in the posterior aorta and heart are sufficient to confer heart or posterior aorta fate throughout the dorsal vessel. Additionally, we demonstrate that Ultrabithorax and abd-A, but not Antennapedia, function to control cell number in the dorsal vessel. These studies add robustness to the model that homeotic selector genes pattern the Drosophila dorsal vessel, and further extend our understanding of how the cardiac tube is patterned in animal models.