The human FLT1 regulatory element directs vascular expression and modulates angiogenesis pathways in vitro and in vivo
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This paper aims to identify enhancers controlling human cardiovascular development. The authors identified a set of highly conserved regulatory elements bioinformatically and tested one of these in vivo, in medaka, and in vitro, in hESC differentiated into endothelial cells. They find deletion of this enhancer had no significant effect on the expression of FLT1 mRNA in the human cell line or on the formation of tube formation. However, deletion of the orthologous enhancer in medaka showed phenotypic effects in blood clot formation. Together, the data provide insights into the function of an enhancer controlling FLT1. This manuscript will be of interest to scientists interested in gene regulation, vascular biology and developmental biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
There is growing evidence that mutations in non-coding cis -regulatory elements (CREs) disrupt proper development. However, little is known about human CREs that are crucial for cardiovascular development. To address this, we bioinformatically identified cardiovascular CREs based on the occupancy of the CRE by the homeodomain protein NKX2-5 and cardiac chromatin histone modifications. This search defined a highly conserved CRE within the FLT1 locus termed enFLT1 . We show that the human enFLT1 is an enhancer capable of driving reporter transgene expression in vivo throughout the developing cardiovascular system of medaka. Deletion of the human enFLT1 enhancer ( ΔenFLT1 ) triggered molecular perturbations in extracellular matrix organisation and blood vessel morphogenesis in vitro in endothelial cells derived from human embryonic stem cells and vascular defects in vivo in medaka. These findings highlight the crucial role of the human FLT1 enhancer and its function as a regulator and buffer of transcriptional regulation in cardiovascular development.
Article activity feed
-
Evaluation Summary:
This paper aims to identify enhancers controlling human cardiovascular development. The authors identified a set of highly conserved regulatory elements bioinformatically and tested one of these in vivo, in medaka, and in vitro, in hESC differentiated into endothelial cells. They find deletion of this enhancer had no significant effect on the expression of FLT1 mRNA in the human cell line or on the formation of tube formation. However, deletion of the orthologous enhancer in medaka showed phenotypic effects in blood clot formation. Together, the data provide insights into the function of an enhancer controlling FLT1. This manuscript will be of interest to scientists interested in gene regulation, vascular biology and developmental biology.
(This preprint has been reviewed by eLife. We include the public reviews from …
Evaluation Summary:
This paper aims to identify enhancers controlling human cardiovascular development. The authors identified a set of highly conserved regulatory elements bioinformatically and tested one of these in vivo, in medaka, and in vitro, in hESC differentiated into endothelial cells. They find deletion of this enhancer had no significant effect on the expression of FLT1 mRNA in the human cell line or on the formation of tube formation. However, deletion of the orthologous enhancer in medaka showed phenotypic effects in blood clot formation. Together, the data provide insights into the function of an enhancer controlling FLT1. This manuscript will be of interest to scientists interested in gene regulation, vascular biology and developmental biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
This paper aims to identify enhancers controlling human cardiovascular development. The authors identified a set of highly conserved regulatory elements bioinformatically and tested one of these in vivo, in medaka, and in vitro, in hESC differentiated into endothelial cells. They find deletion of this enhancer had no significant effect on the expression of FLT1 mRNA in the human cell line or on the formation of tube formation. However, deletion of the orthologous enhancer in medaka showed phenotypic effects in blood clot formation. This paper is of interest to researchers in cardiovascular genetics. The data provides insights into the function of an enhancer controlling FLT1. The paper demonstrates the utility of transgenic assays of enhancer function using medeka but the major limitation is that it is …
Reviewer #1 (Public Review):
This paper aims to identify enhancers controlling human cardiovascular development. The authors identified a set of highly conserved regulatory elements bioinformatically and tested one of these in vivo, in medaka, and in vitro, in hESC differentiated into endothelial cells. They find deletion of this enhancer had no significant effect on the expression of FLT1 mRNA in the human cell line or on the formation of tube formation. However, deletion of the orthologous enhancer in medaka showed phenotypic effects in blood clot formation. This paper is of interest to researchers in cardiovascular genetics. The data provides insights into the function of an enhancer controlling FLT1. The paper demonstrates the utility of transgenic assays of enhancer function using medeka but the major limitation is that it is comprised mostly of negative results, and the key claim that this enhancer in humans has a crucial role in regulating cardiovascular development is not justified by the data.
-
Reviewer #2 (Public Review):
In their manuscript, Stolper et al identified and characterized a conserved enhancer element located in the intron of FLT1, a gene known for its role in vasculogenesis. They successfully demonstrated that the human enhancer sequence (enFLT1) was able to drive robust cardiovascular gene expression in vivo in medaka. However, deletion of enFLT1 in vitro did not significantly alter the expression of FLT1 nor affected angiogenesis, although it induced differential expression of many genes related to cardiovascular development. Finally, they showed that deletion of the medaka endogenous enflt1 enhancer by CRISPR/Cas9 caused an increase in blood clot formation in the yolk of F0 injected embryos. This study employed rigorous analyses following a logical thought process, to narrow down enhancer candidates and …
Reviewer #2 (Public Review):
In their manuscript, Stolper et al identified and characterized a conserved enhancer element located in the intron of FLT1, a gene known for its role in vasculogenesis. They successfully demonstrated that the human enhancer sequence (enFLT1) was able to drive robust cardiovascular gene expression in vivo in medaka. However, deletion of enFLT1 in vitro did not significantly alter the expression of FLT1 nor affected angiogenesis, although it induced differential expression of many genes related to cardiovascular development. Finally, they showed that deletion of the medaka endogenous enflt1 enhancer by CRISPR/Cas9 caused an increase in blood clot formation in the yolk of F0 injected embryos. This study employed rigorous analyses following a logical thought process, to narrow down enhancer candidates and identify the chosen target. The experimental validations were well designed and executed. Unfortunately, many of the results fall short in demonstrating a direct regulation of the claimed target gene (FLT1) by enFLT1 and the function of the endogenous enhancer in vivo.
-
Reviewer #3 (Public Review):
This manuscript from Stolper and colleagues describes a highly conserved transcriptional enhancer element within the FLT1 locus that is bound by Nkx2-5 in human pluripotent stem cell-derived cardiomyocytes and is proposed to regulate FLT1 expression. Stolper and co further demonstrate that a region encompassing 358 bases of the human FLT1 enhancer can drive reporter gene expression in the cardiovascular and neural systems of medaka. This is a strength of the study, though the assessment of reporter gene expression should be substantially expanded. The team next deleted a region encompassing 635 bases of the FLT1 enhancer in human embryonic stem cells, differentiated these cells into endothelial cells and investigated the impact of enhancer deletion on endothelial cell gene expression and tube forming …
Reviewer #3 (Public Review):
This manuscript from Stolper and colleagues describes a highly conserved transcriptional enhancer element within the FLT1 locus that is bound by Nkx2-5 in human pluripotent stem cell-derived cardiomyocytes and is proposed to regulate FLT1 expression. Stolper and co further demonstrate that a region encompassing 358 bases of the human FLT1 enhancer can drive reporter gene expression in the cardiovascular and neural systems of medaka. This is a strength of the study, though the assessment of reporter gene expression should be substantially expanded. The team next deleted a region encompassing 635 bases of the FLT1 enhancer in human embryonic stem cells, differentiated these cells into endothelial cells and investigated the impact of enhancer deletion on endothelial cell gene expression and tube forming ability. Intriguingly, while expression levels of FLT1 were not significantly altered in differentiated endothelial cells, the expression of a small number of genes was changed, suggesting that the enhancer might regulate genes other than Flt1. Enhancer deletion revealed that there was not a major impact on the ability of endothelial cells differentiated from embryonic stem cells to form tubes in angiogenesis assays, while the ability of cells in which exon 1 of Flt1 was deleted to form tubes was elevated, consistent with established roles of Flt1 in regulating endothelial cell proliferation. These data suggest that at least in this model, the Flt1 enhancer studied does not play a major role in regulating Flt1 expression. The greatest strength of the study is the finding that deletion of the enhancer in medaka resulted in an increased proportion of embryos exhibiting blood clots, suggestive of cardiovascular dysfunction. This observation deserves more extensive investigation; a detailed analysis of cardiac and vascular development in enhancer mutant fish should be undertaken. I expect an expanded study to be of significant and broad ranging interest to the vascular and developmental biology communities.
-
