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Identification of vascular and hematopoietic genes downstream of etsrp by deep sequencing in zebrafish.

Gomez G, Lee JH, Veldman MB, Lu J, Xiao X, Lin S - PLoS ONE (2012)

Bottom Line: It is also required for hematopoiesis in zebrafish and mice.Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays.Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells.

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/Er71/Etv2 is a master control gene for vasculogenesis in all species studied to date. It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology.

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Analysis of gene expression in etsrp morphants.Flk1-gfp embryos were injected at the one cell stage with a mixture of translation blocking morpholinos for etsrp and analyzed by WISH at the 24hpf stage. Marked reduction is apparent in all genes examined in the axial vasculature, which includes the dorsal aorta and posterior cardinal vein, and is marked with a downward facing arrow in all images. The primitive myeloid cells stained in (L) myo1f wild-type controls are absent in their etsrp morphant counterparts. The staining in the axial trunk region of rgl2 in etsrp morphants marks primitive erythrocytes that are trapped due to lack of circulation. The expression of non-vascular structures is not affected otherwise in etsrp morphants. Embryos are positioned laterally with the anterior facing left. Abbreviations: cht, caudal hematopoietic tail region; cv, cranial vasculature; da, dorsal aorta. Scale bar: 250 µm.
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pone-0031658-g004: Analysis of gene expression in etsrp morphants.Flk1-gfp embryos were injected at the one cell stage with a mixture of translation blocking morpholinos for etsrp and analyzed by WISH at the 24hpf stage. Marked reduction is apparent in all genes examined in the axial vasculature, which includes the dorsal aorta and posterior cardinal vein, and is marked with a downward facing arrow in all images. The primitive myeloid cells stained in (L) myo1f wild-type controls are absent in their etsrp morphant counterparts. The staining in the axial trunk region of rgl2 in etsrp morphants marks primitive erythrocytes that are trapped due to lack of circulation. The expression of non-vascular structures is not affected otherwise in etsrp morphants. Embryos are positioned laterally with the anterior facing left. Abbreviations: cht, caudal hematopoietic tail region; cv, cranial vasculature; da, dorsal aorta. Scale bar: 250 µm.

Mentions: To determine whether the 50 selected ectopically induced genes are expressed in the developing embryonic zebrafish vasculature, embryos were processed for WISH at a developmental stage when most of the primitive vasculature has formed, 24-hour post fertilization (24hpf). Marked expression in the vasculature was noted for 39 of the 50 genes (Figure 3). To confirm that these genes are functionally downstream of etsrp in the vasculature, we examined their expression in etsrp morphants, in which case the expression in the axial vasculature is prominently reduced as demonstrated in Figure 4. Sparse expression in myeloid cells was only detected for one of these genes, myo1F (Figure 3L). Although there is some ubiquitous expression of some of these genes, the more pronounced expression in the vasculature is clearly demonstrated in higher magnification images of the trunk regions (Figure S1), where the axial vascular expression in morphants is clearly reduced in all genes. Of the 39 genes with vascular expression only fhl3 (Figure 3 AD′ and Figure S1AD′) and acsbg2 (Figure 3AL′ and Figure S1AL′) are preferentially expressed in the axial venous system at this stage.


Identification of vascular and hematopoietic genes downstream of etsrp by deep sequencing in zebrafish.

Gomez G, Lee JH, Veldman MB, Lu J, Xiao X, Lin S - PLoS ONE (2012)

Analysis of gene expression in etsrp morphants.Flk1-gfp embryos were injected at the one cell stage with a mixture of translation blocking morpholinos for etsrp and analyzed by WISH at the 24hpf stage. Marked reduction is apparent in all genes examined in the axial vasculature, which includes the dorsal aorta and posterior cardinal vein, and is marked with a downward facing arrow in all images. The primitive myeloid cells stained in (L) myo1f wild-type controls are absent in their etsrp morphant counterparts. The staining in the axial trunk region of rgl2 in etsrp morphants marks primitive erythrocytes that are trapped due to lack of circulation. The expression of non-vascular structures is not affected otherwise in etsrp morphants. Embryos are positioned laterally with the anterior facing left. Abbreviations: cht, caudal hematopoietic tail region; cv, cranial vasculature; da, dorsal aorta. Scale bar: 250 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3306315&req=5

pone-0031658-g004: Analysis of gene expression in etsrp morphants.Flk1-gfp embryos were injected at the one cell stage with a mixture of translation blocking morpholinos for etsrp and analyzed by WISH at the 24hpf stage. Marked reduction is apparent in all genes examined in the axial vasculature, which includes the dorsal aorta and posterior cardinal vein, and is marked with a downward facing arrow in all images. The primitive myeloid cells stained in (L) myo1f wild-type controls are absent in their etsrp morphant counterparts. The staining in the axial trunk region of rgl2 in etsrp morphants marks primitive erythrocytes that are trapped due to lack of circulation. The expression of non-vascular structures is not affected otherwise in etsrp morphants. Embryos are positioned laterally with the anterior facing left. Abbreviations: cht, caudal hematopoietic tail region; cv, cranial vasculature; da, dorsal aorta. Scale bar: 250 µm.
Mentions: To determine whether the 50 selected ectopically induced genes are expressed in the developing embryonic zebrafish vasculature, embryos were processed for WISH at a developmental stage when most of the primitive vasculature has formed, 24-hour post fertilization (24hpf). Marked expression in the vasculature was noted for 39 of the 50 genes (Figure 3). To confirm that these genes are functionally downstream of etsrp in the vasculature, we examined their expression in etsrp morphants, in which case the expression in the axial vasculature is prominently reduced as demonstrated in Figure 4. Sparse expression in myeloid cells was only detected for one of these genes, myo1F (Figure 3L). Although there is some ubiquitous expression of some of these genes, the more pronounced expression in the vasculature is clearly demonstrated in higher magnification images of the trunk regions (Figure S1), where the axial vascular expression in morphants is clearly reduced in all genes. Of the 39 genes with vascular expression only fhl3 (Figure 3 AD′ and Figure S1AD′) and acsbg2 (Figure 3AL′ and Figure S1AL′) are preferentially expressed in the axial venous system at this stage.

Bottom Line: It is also required for hematopoiesis in zebrafish and mice.Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays.Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells.

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/Er71/Etv2 is a master control gene for vasculogenesis in all species studied to date. It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology.

Show MeSH
Related in: MedlinePlus