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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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Ectopic induction of microarray identified genes by etsrp overexpression.Ectopic gene expression was examined at 80% epiboly to tailbud stages in flk1:gfp transgenic embryos injected with 30 pg etsrp-mcherry DNA at the one cell stage. Embryos exhibiting both red and green fluorescence were selected for analysis. Representative control embryos are on the left and etsrp overexpressing (OE) embryos are on the right side of each panel. Represented genes: (A) krml2; (B) yrk; (C) lrrp33; (D) similar to hemicentin; (E) hapln1b; (F) sh3gl3; (G) rasgrp3; (H) similar to costimulatory protein; (I) tem8; (J) ldb2; (K) arhgap23; (L) est:AI721944; (M) fgd5; and (N) est:AW019729. Note that there is a low level of endogenous expression in the control embryos for the two EST's, L and N, at the polster (arrows). Ratios in bottom right hand corner in panels represent the number of embryos with ectopic induction of total embryos processed and scored in the injected groups; control embryos never displayed ectopic induction. All embryos are in lateral view, and those at tail bud stage are oriented with anterior to the left. Scale bar: 250 µm.
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pone-0004994-g002: Ectopic induction of microarray identified genes by etsrp overexpression.Ectopic gene expression was examined at 80% epiboly to tailbud stages in flk1:gfp transgenic embryos injected with 30 pg etsrp-mcherry DNA at the one cell stage. Embryos exhibiting both red and green fluorescence were selected for analysis. Representative control embryos are on the left and etsrp overexpressing (OE) embryos are on the right side of each panel. Represented genes: (A) krml2; (B) yrk; (C) lrrp33; (D) similar to hemicentin; (E) hapln1b; (F) sh3gl3; (G) rasgrp3; (H) similar to costimulatory protein; (I) tem8; (J) ldb2; (K) arhgap23; (L) est:AI721944; (M) fgd5; and (N) est:AW019729. Note that there is a low level of endogenous expression in the control embryos for the two EST's, L and N, at the polster (arrows). Ratios in bottom right hand corner in panels represent the number of embryos with ectopic induction of total embryos processed and scored in the injected groups; control embryos never displayed ectopic induction. All embryos are in lateral view, and those at tail bud stage are oriented with anterior to the left. Scale bar: 250 µm.

Mentions: To validate the results obtained with the etsrp OE microarray, WISH was performed on embryos at the same developmental stage as was done for the microarray, 80% epiboly to tailbud. To ensure that the OE samples examined were indeed ectopically expressing etsrp, embryos were injected with plasmid DNA encoding an Etsrp-mcherry fusion protein. This fusion protein was functionally active since it efficiently induced ectopic expression of flk:gfp when injected into transgenic embryos (data not shown). Embryos expressing Etsrp-mcherry and flk:gfp were selected to test for ectopic induction for 23 of the 31 genes identified in Table 2. 21 of 23 genes were clearly induced ectopically by etsrp OE. Figure 2 shows the ectopic induction of the 14 genes identified below to be relevant to blood and/or vascular expression. There was ubiquitous endogenous expression in 2/23 genes, arhgef9 and znf385, prohibiting any qualitative discernment of their changes in expression by etsrp OE (data not shown). Overall, this demonstrates that the microarray data is low in false positives and reliable.


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)

Ectopic induction of microarray identified genes by etsrp overexpression.Ectopic gene expression was examined at 80% epiboly to tailbud stages in flk1:gfp transgenic embryos injected with 30 pg etsrp-mcherry DNA at the one cell stage. Embryos exhibiting both red and green fluorescence were selected for analysis. Representative control embryos are on the left and etsrp overexpressing (OE) embryos are on the right side of each panel. Represented genes: (A) krml2; (B) yrk; (C) lrrp33; (D) similar to hemicentin; (E) hapln1b; (F) sh3gl3; (G) rasgrp3; (H) similar to costimulatory protein; (I) tem8; (J) ldb2; (K) arhgap23; (L) est:AI721944; (M) fgd5; and (N) est:AW019729. Note that there is a low level of endogenous expression in the control embryos for the two EST's, L and N, at the polster (arrows). Ratios in bottom right hand corner in panels represent the number of embryos with ectopic induction of total embryos processed and scored in the injected groups; control embryos never displayed ectopic induction. All embryos are in lateral view, and those at tail bud stage are oriented with anterior to the left. Scale bar: 250 µm.
© Copyright Policy
Related In: Results  -  Collection

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pone-0004994-g002: Ectopic induction of microarray identified genes by etsrp overexpression.Ectopic gene expression was examined at 80% epiboly to tailbud stages in flk1:gfp transgenic embryos injected with 30 pg etsrp-mcherry DNA at the one cell stage. Embryos exhibiting both red and green fluorescence were selected for analysis. Representative control embryos are on the left and etsrp overexpressing (OE) embryos are on the right side of each panel. Represented genes: (A) krml2; (B) yrk; (C) lrrp33; (D) similar to hemicentin; (E) hapln1b; (F) sh3gl3; (G) rasgrp3; (H) similar to costimulatory protein; (I) tem8; (J) ldb2; (K) arhgap23; (L) est:AI721944; (M) fgd5; and (N) est:AW019729. Note that there is a low level of endogenous expression in the control embryos for the two EST's, L and N, at the polster (arrows). Ratios in bottom right hand corner in panels represent the number of embryos with ectopic induction of total embryos processed and scored in the injected groups; control embryos never displayed ectopic induction. All embryos are in lateral view, and those at tail bud stage are oriented with anterior to the left. Scale bar: 250 µm.
Mentions: To validate the results obtained with the etsrp OE microarray, WISH was performed on embryos at the same developmental stage as was done for the microarray, 80% epiboly to tailbud. To ensure that the OE samples examined were indeed ectopically expressing etsrp, embryos were injected with plasmid DNA encoding an Etsrp-mcherry fusion protein. This fusion protein was functionally active since it efficiently induced ectopic expression of flk:gfp when injected into transgenic embryos (data not shown). Embryos expressing Etsrp-mcherry and flk:gfp were selected to test for ectopic induction for 23 of the 31 genes identified in Table 2. 21 of 23 genes were clearly induced ectopically by etsrp OE. Figure 2 shows the ectopic induction of the 14 genes identified below to be relevant to blood and/or vascular expression. There was ubiquitous endogenous expression in 2/23 genes, arhgef9 and znf385, prohibiting any qualitative discernment of their changes in expression by etsrp OE (data not shown). Overall, this demonstrates that the microarray data is low in false positives and reliable.

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