Transcriptional inhibition of etv2 expression is essential for embryonic cardiac development.

E-twenty six variant 2 (Etv2) transcription factor participates in cardiac, vascular-endothelial and blood cell lineage specification decisions during embryonic development. Previous studies have identified genomic elements in the etv2 locus responsible for vascular endothelial cell specification. Using transgenic analysis in zebrafish, we report here an etv2 proximal promoter fragment that prevents transgene misexpression in myocardial progenitor cells. This inhibition of etv2 expression in the cardiac progenitor population is partly mediated by Scl and Nkx2.5, likely through direct binding to the etv2 promoter, and cis-regulatory elements located in the first and second introns. The results identify an etv2 cis-regulatory mechanism controlling cardiovascular fate choice implying that etv2 participates in a transcriptional network mediating developmental plasticity of endothelial progenitor cells during embryonic development.

Sox factors transcriptionally regulate ROBO4 gene expression in developing vasculature in zebrafish.

Despite their importance as members of the Roundabout (Robo) family in the control of axonal and vascular patterning, the transcriptional regulation of these genes is poorly understood. In this study, we show that members of the Sry-related high mobility box (Sox) transcription factor family as being transcriptional regulators of roundabout4 (robo4), a Robo gene family member that participates in sprouting angiogenesis in vivo, in zebrafish. Double whole mount in situ hybridization analysis in zebrafish embryos revealed co-localization of the vascular relevant Sox factors sox7 or sox18 mRNA with robo4 transcripts in developing intersomitic vessels. A 3-kb human ROBO4 promoter element was able to drive reporter expression in zebrafish to recapitulate the endogenous temporal intersomitic vessel expression pattern of robo4. EMSA analysis confirmed binding of Sox18 to a canonical Sox binding site (from -1170 bp to -1176 bp) in the ROBO4 promoter (3 kb), and mutation analysis indicated that this site was partially responsible for ROBO4 promoter activity in ECs. A combination of gain- and loss-of-function analysis identified Sox7 and Sox18 co-regulation of robo4 but not fli1a transcripts in zebrafish. Finally, Sox-mediated robo4 transcriptional regulation is conserved across evolution. These studies imply Sox-mediated transcriptional regulation of Robo4 in the developing embryonic vasculature.

Fli+ etsrp+ hemato-vascular progenitor cells proliferate at the lateral plate mesoderm during vasculogenesis in zebrafish.

BACKGROUND: Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we use ets-related protein-1 (etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp(+) cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli(+)etsrp(+) hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp(+) cell numbers at the LPM. CONCLUSIONS/SIGNIFICANCE: This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish.

Dusp-5 and Snrk-1 coordinately function during vascular development and disease.

Mitogen-activated protein kinases play an integral role in several cellular processes. To regulate mitogen-activated protein kinases, cells express members of a counteracting group of proteins called phosphatases. In this study, we have identified a specific role that one member of this family of phosphatases, dual-specific phosphatase-5 (Dusp-5) plays in vascular development in vivo. We have determined that dusp-5 is expressed in angioblasts and in established vasculature and that it counteracts the function of a serine threonine kinase, Snrk-1, which also plays a functional role in angioblast development. Together, Dusp-5 and Snrk-1 control angioblast populations in the lateral plate mesoderm with Dusp-5 functioning downstream of Snrk-1. Importantly, mutations in dusp-5 and snrk-1 have been identified in affected tissues of patients with vascular anomalies, implicating the Snrk-1-Dusp-5 signaling pathway in human disease.

Microinjecting recombinant rainbow trout Ea4-peptide of pro-IGF-I into zebrafish embryos causes abnormal development in heart, red blood cells, and vasculature.

E-peptides and mature insulin-like growth factors (IGFs) are produced from pre-pro-IGFs during post-translational processing and co-secreted into the circulation. Previously, we reported that introduction of a transgene encoding the secreted form of rainbow trout (rt) Ea4-peptide or human (h) Eb-peptide into newly fertilized eggs of medaka (Oryzias latipes) and zebrafish (Danio rerio) resulted in developmental defects in heart, red blood cells and vasculature. In addition to vasculature and red blood cell developmental defects, multiple phenocopies of heart developmental defects categorized by developmental arrest at cardiomyocyte, heart tube and heart looping stages were also observed. These results raise a question of whether rtEa4- or hEb-peptide exerts pleiotropic inhibitory effects on heart, vasculature and red blood cell development in fish embryos. To answer this question, various amounts of recombinant rtEa4-peptide were microinjected into zebrafish eggs at 1.5, 2.5 and 5.5 h post-fertilization (hpf). Although a dose-dependent developmental defect in heart, vasculature and red blood cells was observed in embryos microinjected with rtEa4-peptide at 1.5 and 2.5 hpf, the heart development in all of the microinjected embryos was arrested at the cardiomyocyte stage. Furthermore, the mRNA levels of Nkx2.5, GATA5, VEGF, GATA1 and GATA2 genes in defective embryos were significantly reduced by rtEa4-peptide. These results confirm our previous findings that rtEa4- or hEb-peptide exhibits pleiotropic effects in inhibiting heart, vasculature and red blood cell development in zebrafish embryos.

Trout Ea4- or human Eb-peptide of pro-IGF-I disrupts heart, red blood cell, and vasculature development in zebrafish embryos.

E-peptide of the pro-insulin-like growth factor (pro-IGF)-I is produced by proteolytic cleavage of the pro-hormone in post-translational processing. Introduction of a transgene encoding a secreted form of rtEa4- or hEb-peptide into newly fertilized zebrafish (Danio rerio) eggs by electroporation or microinjection resulted in embryos with abnormal cardiovascular features and reduced red blood cells and vasculature. Two different phenocopies of heart developmental defects were observed: (i) Group I embryos exhibited heart development arrested at the heart muscle stage and (ii) group II embryos exhibited heart development arrested at the heart tube stage. Both groups of embryos also exhibited reduction of red blood cells and vasculature. The mRNA levels of genes essential for heart development (GATA 5 and NKX2.5), hematopoiesis (GATA 1 and GATA 2), and vasculogenesis (VEGF) in normal and defective embryos were determined by quantitative real-time RT-PCR at 36 hr post-fertilization (hpf). Significant reduction of GATA 5, NKX2.5, GATA 1, GATA 2, and VEGF mRNA levels was observed in both groups of defective embryos. These results suggest that overexpression of rtEa4 or hEb transgene in zebrafish embryos disrupts heart development, hematopoiesis, and vasculogenesis by reducing the levels of GATA 5, NKX2.5, GATA 1, GATA 2, and VEGF mRNA.

Disruption of embryonic red blood cell development by Ea4-peptide of rainbow trout pro-IGF-I in medaka (Oryzias latipes).

E-peptides are proteolytic cleavage products of pre-pro-insulin-like growth factor (Pre-pro-IGF) I and II in posttranslational processing of the pre-pro-hormones. Rainbow trout (rt) Ea4- and human (h) Eb-peptides possess unique anti-tumor activities in established human and fish cancer cells. This paper reports on the effects of rtEa4- and hEb-peptides in fish embryos during early embryonic development. Introduction of either rtEa4 or hEb gene into newly fertilized medaka (Oryzias latipes) eggs by electroporation resulted in abnormal embryonic development of heart, red blood cells, and vasculature. Red blood cells in heart-defective embryos were stained with diaminofluorene (DAF) which revealed a drastic reduction of red blood cells in defective embryos, suggesting that, in addition to heart defects, embryonic red blood cell development may also be disrupted by the E-peptide. The temporal expression patterns of the transcription factor GATA-1 gene, an essential regulator of hematopoiesis during embryonic development, were determined by reverse transcription-polymerase chain reaction (RT-PCR). GATA-1 mRNA was detected at 24 hours postfertilization (hpf), then raised to a higher level at 30 hpf. It was maintained at this level until 100 hpf (>4 days). At 5 days postfertilization (dpf), the expression level of GATA- 1 gene decreased and remained at that level until hatching. The number of GATA-1 transcripts in transgenic embryos at 4 dpf was determined by quantitative real-time RT PCR. Whereas the GATA-1 mRNA in nontransgenic embryos at 4 dpf was 7.15 x 10(5)/3 microg of total RNA, that of the heart defective transgenic embryos at the same stage was 1.35 x10(5)/3 microg of total mRNA. These results suggest that rtEa4- or hEb-peptide may be responsible for disrupting embryonic hematopoiesis in medaka embryos by reducing the expression of the GATA-1 gene.