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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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Early ectopic expression of transgenic flk1:gfp is induced by etsrp over-expression.(A) Injection of 75 pg of etsrp mRNA at one-cell stage results in the ectopic induction of flk1:gfp before the end of gastrulation. (B) No red fluorescence is observed in etsrp mRNA injected flk1:gfp transgenic embryos. (C) Injection of 500 pg of control H2B-RFP1 mRNA does not result in the early induction of flk1:gfp similar to uninjected embryos (not shown). (D) Uniform H2B-RFP1 expression in a H2B-RFP1 mRNA injected embryo. Panels A and C are composite images of light transmitted images merged with the green fluorescent channel, while panels B and D are composites of light transmitted images merged with the red fluorescent channel.
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pone-0004994-g001: Early ectopic expression of transgenic flk1:gfp is induced by etsrp over-expression.(A) Injection of 75 pg of etsrp mRNA at one-cell stage results in the ectopic induction of flk1:gfp before the end of gastrulation. (B) No red fluorescence is observed in etsrp mRNA injected flk1:gfp transgenic embryos. (C) Injection of 500 pg of control H2B-RFP1 mRNA does not result in the early induction of flk1:gfp similar to uninjected embryos (not shown). (D) Uniform H2B-RFP1 expression in a H2B-RFP1 mRNA injected embryo. Panels A and C are composite images of light transmitted images merged with the green fluorescent channel, while panels B and D are composites of light transmitted images merged with the red fluorescent channel.

Mentions: In order to identify novel blood or vascular related genes downstream of etsrp, the expression profiles of embryos ectopically expressing etsrp at late gastrulation stages, 80% epiboly to tail bud were compared to control embryos by microarray analysis. The flk1:gfp transgenic line was used to identify embryos successfully expressing etsrp since ectopic etsrp induces flk:gfp expression. Neither endogenous or transgenic flk1:gfp is normally expressed at 80% to tail-bud stage [20], [21], while early ectopic expression of flk1:gfp is evident in embryos injected with 75 pg of synthetic etsrp mRNA (Figure 1A). To ensure that the ectopic expression of flk1:gfp is induced by etsrp specifically, we injected up to 500 pg of synthetically transcribed mRNA encoding RFP tagged histone H2B, H2B-RFP1, as a control. Similar to uninjected controls, flk1:gfp expression is not induced in H2B-RFP1 injected embryos (Figure 1C and 1D).


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)

Early ectopic expression of transgenic flk1:gfp is induced by etsrp over-expression.(A) Injection of 75 pg of etsrp mRNA at one-cell stage results in the ectopic induction of flk1:gfp before the end of gastrulation. (B) No red fluorescence is observed in etsrp mRNA injected flk1:gfp transgenic embryos. (C) Injection of 500 pg of control H2B-RFP1 mRNA does not result in the early induction of flk1:gfp similar to uninjected embryos (not shown). (D) Uniform H2B-RFP1 expression in a H2B-RFP1 mRNA injected embryo. Panels A and C are composite images of light transmitted images merged with the green fluorescent channel, while panels B and D are composites of light transmitted images merged with the red fluorescent channel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654924&req=5

pone-0004994-g001: Early ectopic expression of transgenic flk1:gfp is induced by etsrp over-expression.(A) Injection of 75 pg of etsrp mRNA at one-cell stage results in the ectopic induction of flk1:gfp before the end of gastrulation. (B) No red fluorescence is observed in etsrp mRNA injected flk1:gfp transgenic embryos. (C) Injection of 500 pg of control H2B-RFP1 mRNA does not result in the early induction of flk1:gfp similar to uninjected embryos (not shown). (D) Uniform H2B-RFP1 expression in a H2B-RFP1 mRNA injected embryo. Panels A and C are composite images of light transmitted images merged with the green fluorescent channel, while panels B and D are composites of light transmitted images merged with the red fluorescent channel.
Mentions: In order to identify novel blood or vascular related genes downstream of etsrp, the expression profiles of embryos ectopically expressing etsrp at late gastrulation stages, 80% epiboly to tail bud were compared to control embryos by microarray analysis. The flk1:gfp transgenic line was used to identify embryos successfully expressing etsrp since ectopic etsrp induces flk:gfp expression. Neither endogenous or transgenic flk1:gfp is normally expressed at 80% to tail-bud stage [20], [21], while early ectopic expression of flk1:gfp is evident in embryos injected with 75 pg of synthetic etsrp mRNA (Figure 1A). To ensure that the ectopic expression of flk1:gfp is induced by etsrp specifically, we injected up to 500 pg of synthetically transcribed mRNA encoding RFP tagged histone H2B, H2B-RFP1, as a control. Similar to uninjected controls, flk1:gfp expression is not induced in H2B-RFP1 injected embryos (Figure 1C and 1D).

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