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Discovery and characterization of novel vascular and hematopoietic genes downstream of etsrp in zebrafish.

Gomez GA, Veldman MB, Zhao Y, Burgess S, Lin S - PLoS ONE (2009)

Bottom Line: Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel.Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development.The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The transcription factor Etsrp is required for vasculogenesis and primitive myelopoiesis in zebrafish. When ectopically expressed, etsrp is sufficient to induce the expression of many vascular and myeloid genes in zebrafish. The mammalian homolog of etsrp, ER71/Etv2, is also essential for vascular and hematopoietic development. To identify genes downstream of etsrp, gain-of-function experiments were performed for etsrp in zebrafish embryos followed by transcription profile analysis by microarray. Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel. Regulation of these genes by etsrp was confirmed by ectopic induction in etsrp overexpressing embryos and decreased expression in etsrp deficient embryos. Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development. The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development.

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Hapln1b and sh3gl3 are mediators of vascular development.(A–F) Flk1:gfp transgenic embryos injected with 2 ng of a translation blocking morpholino (MO) targeting hapln1b arrests angiogenic sprouting of the intersomitic vessels (asterisks in panels B, D, F; compare to their counterparts in A, C, and E respectively), resulting in delayed and improper dorsal longitudinal anastomotic vessel formation (arrow in D). Furthermore, the caudal vascular plexus is dilated relative to wild-type controls at 28 hours post fertilization (hpf) (compare the identical sized bracket in panels E and F). (G and H) Injection of 3 ng of translation blocking MO targeting sh3gl3 results in a thinner dorsal aorta (arrow in H) relative to wild type controls (G) at 72 hpf. Abbreviations: dlav, dorsal longitudinal anastomotic vessels; isv, intersomitic vessels; da, dorsal aorta; and pcv, posterior cardinal vein.
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pone-0004994-g006: Hapln1b and sh3gl3 are mediators of vascular development.(A–F) Flk1:gfp transgenic embryos injected with 2 ng of a translation blocking morpholino (MO) targeting hapln1b arrests angiogenic sprouting of the intersomitic vessels (asterisks in panels B, D, F; compare to their counterparts in A, C, and E respectively), resulting in delayed and improper dorsal longitudinal anastomotic vessel formation (arrow in D). Furthermore, the caudal vascular plexus is dilated relative to wild-type controls at 28 hours post fertilization (hpf) (compare the identical sized bracket in panels E and F). (G and H) Injection of 3 ng of translation blocking MO targeting sh3gl3 results in a thinner dorsal aorta (arrow in H) relative to wild type controls (G) at 72 hpf. Abbreviations: dlav, dorsal longitudinal anastomotic vessels; isv, intersomitic vessels; da, dorsal aorta; and pcv, posterior cardinal vein.

Mentions: To determine if the genes highlighted here might play a functional role in vascular development we used antisense morpholino (MO) oligos to knockdown hapln1b and sh3gl3 in flk:gfp transgenic fish. Hapln1b knockdown using 2 ng of morpholino results in the arrested angiogenic sprouting of many intersomitic vessels (asterisks in Figure 6B, 6D and 6F), preventing them from forming stereotypical dorsolateral anastamosing vessels (dlavs) by 48 hpf (arrow in Figure 6D). Furthermore, the vascular remodeling that occurs at the caudal vascular plexus around 28–30 hpf is severely defective in hapln1b morphants (compare bracketed region in Figure 6E and 6F). These defects were observed in 87 out of 92 (92.6%) embryos injected with hapln1b-MO while standard control MO (5 ng) injected embryos never exhibited this phenotype (0 out of 70 embryos). Sh3gl3 morphants (3 ng) had a less severe phenotype than hapln1b morphants. Sh3gl3-MO treated embryos had relatively normal intersomitic vessel development, but displayed reduced circulation as visualized by red blood cell movement in the axial vasculature when compared to controls (data not shown). A closer examination of the axial vessels at 72 hpf revealed a significant reduction in the diameter of the dorsal aorta with a concomitant increase in the diameter of the posterior cardinal vein in morphants (arrow in Figure 6H compared to 6G). This constriction of the dorsal aorta was likely the cause of the observed decrease in circulation. 61 of 83 (73.5%) embryos injected with sh3gl3-MO displayed the shrunken dorsal aorta/reduced circulation phenotype while none of the standard control MO injected (5 ng, 0 out of 70 embryos) or uninjected control embryos did. The demonstration that hapln1b or sh3gl3 knockdown results in distinct vascular phenotypes suggest that other genes identified in this gene expression screen will be functionally important and should be studied in further detail.


Discovery and characterization of novel vascular and hematopoietic genes downstream of etsrp in zebrafish.

Gomez GA, Veldman MB, Zhao Y, Burgess S, Lin S - PLoS ONE (2009)

Hapln1b and sh3gl3 are mediators of vascular development.(A–F) Flk1:gfp transgenic embryos injected with 2 ng of a translation blocking morpholino (MO) targeting hapln1b arrests angiogenic sprouting of the intersomitic vessels (asterisks in panels B, D, F; compare to their counterparts in A, C, and E respectively), resulting in delayed and improper dorsal longitudinal anastomotic vessel formation (arrow in D). Furthermore, the caudal vascular plexus is dilated relative to wild-type controls at 28 hours post fertilization (hpf) (compare the identical sized bracket in panels E and F). (G and H) Injection of 3 ng of translation blocking MO targeting sh3gl3 results in a thinner dorsal aorta (arrow in H) relative to wild type controls (G) at 72 hpf. Abbreviations: dlav, dorsal longitudinal anastomotic vessels; isv, intersomitic vessels; da, dorsal aorta; and pcv, posterior cardinal vein.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654924&req=5

pone-0004994-g006: Hapln1b and sh3gl3 are mediators of vascular development.(A–F) Flk1:gfp transgenic embryos injected with 2 ng of a translation blocking morpholino (MO) targeting hapln1b arrests angiogenic sprouting of the intersomitic vessels (asterisks in panels B, D, F; compare to their counterparts in A, C, and E respectively), resulting in delayed and improper dorsal longitudinal anastomotic vessel formation (arrow in D). Furthermore, the caudal vascular plexus is dilated relative to wild-type controls at 28 hours post fertilization (hpf) (compare the identical sized bracket in panels E and F). (G and H) Injection of 3 ng of translation blocking MO targeting sh3gl3 results in a thinner dorsal aorta (arrow in H) relative to wild type controls (G) at 72 hpf. Abbreviations: dlav, dorsal longitudinal anastomotic vessels; isv, intersomitic vessels; da, dorsal aorta; and pcv, posterior cardinal vein.
Mentions: To determine if the genes highlighted here might play a functional role in vascular development we used antisense morpholino (MO) oligos to knockdown hapln1b and sh3gl3 in flk:gfp transgenic fish. Hapln1b knockdown using 2 ng of morpholino results in the arrested angiogenic sprouting of many intersomitic vessels (asterisks in Figure 6B, 6D and 6F), preventing them from forming stereotypical dorsolateral anastamosing vessels (dlavs) by 48 hpf (arrow in Figure 6D). Furthermore, the vascular remodeling that occurs at the caudal vascular plexus around 28–30 hpf is severely defective in hapln1b morphants (compare bracketed region in Figure 6E and 6F). These defects were observed in 87 out of 92 (92.6%) embryos injected with hapln1b-MO while standard control MO (5 ng) injected embryos never exhibited this phenotype (0 out of 70 embryos). Sh3gl3 morphants (3 ng) had a less severe phenotype than hapln1b morphants. Sh3gl3-MO treated embryos had relatively normal intersomitic vessel development, but displayed reduced circulation as visualized by red blood cell movement in the axial vasculature when compared to controls (data not shown). A closer examination of the axial vessels at 72 hpf revealed a significant reduction in the diameter of the dorsal aorta with a concomitant increase in the diameter of the posterior cardinal vein in morphants (arrow in Figure 6H compared to 6G). This constriction of the dorsal aorta was likely the cause of the observed decrease in circulation. 61 of 83 (73.5%) embryos injected with sh3gl3-MO displayed the shrunken dorsal aorta/reduced circulation phenotype while none of the standard control MO injected (5 ng, 0 out of 70 embryos) or uninjected control embryos did. The demonstration that hapln1b or sh3gl3 knockdown results in distinct vascular phenotypes suggest that other genes identified in this gene expression screen will be functionally important and should be studied in further detail.

Bottom Line: Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel.Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development.The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The transcription factor Etsrp is required for vasculogenesis and primitive myelopoiesis in zebrafish. When ectopically expressed, etsrp is sufficient to induce the expression of many vascular and myeloid genes in zebrafish. The mammalian homolog of etsrp, ER71/Etv2, is also essential for vascular and hematopoietic development. To identify genes downstream of etsrp, gain-of-function experiments were performed for etsrp in zebrafish embryos followed by transcription profile analysis by microarray. Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel. Regulation of these genes by etsrp was confirmed by ectopic induction in etsrp overexpressing embryos and decreased expression in etsrp deficient embryos. Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development. The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development.

Show MeSH
Related in: MedlinePlus