Zebrafish etv2 knock-in line labels vascular endothelial and blood progenitor cells.

BACKGROUND: ETS transcription factor Etv2/Etsrp is one of the earliest markers for vascular and hematopoietic progenitors and functions as a key regulator of hematovascular development in multiple vertebrates, including zebrafish. Therefore, transgenic etv2 reporter lines provide a valuable tool to study vasculogenesis and hematopoiesis. However, previously generated zebrafish reporter lines do not fully recapitulate the endogenous pattern of etv2 expression. RESULTS: Here we used CRISPR/Cas9-mediated homology-independent DNA repair approach to knock-in a Gal4 transcriptional activator into the zebrafish etv2 genomic locus, thus generating etv2 ci32Gt gene trap line. etv2 ci32Gt ; UAS:GFP embryos show GFP expression in vascular endothelial, myeloid and red blood cells. Because gal4 insertion interrupts the etv2 locus, homozygous etv2 ci32Gt embryos display defects in vasculogenesis and myelopoiesis, and enable visualizing etv2-deficient hematovascular progenitors in live embryos. Furthermore, we performed differential transcriptome analysis of sorted GFP-positive cells from heterozygous and homozygous etv2 ci32Gt embryos. Approximately 500 downregulated genes were identified in etv2 ci32Gt homozygous embryos, which include multiple genes expressed in vascular endothelial and myeloid cells. CONCLUSIONS: The etv2 ci32Gt gene trap line and the data sets of misregulated genes will be valuable resources to study hematopoietic and vascular development.

ETS transcription factor Etsrp / Etv2 is required for lymphangiogenesis and directly regulates vegfr3 / flt4 expression.

The molecular mechanisms initiating the formation of the lymphatic system, lymphangiogenesis, are still poorly understood. Here we have identified a novel role in lymphangiogenesis for an ETS transcription factor, Etv2/Etsrp, a known regulator of embryonic vascular development. Through the use of fully validated photoactivatable morpholinos we show that inducible Etv2 inhibition in zebrafish embryos at 1 day post-fertilization (dpf) results in significant inhibition of lymphangiogenesis, while development of blood vessels is unaffected. In Etv2-inhibited embryos and larvae, the number of lymphatic progenitors is greatly reduced, the major lymphatic vessel, the thoracic duct, is absent or severely fragmented, and lymphangiogenesis-associated marker expression, including lyve1b, prox1a, and vegfr3/flt4, is strongly downregulated. We also demonstrate that lymphatic progenitors in Etv2 deficient embryos fail to respond to Vegfc signaling. Chromatin immunoprecipitation and sequencing (ChIP-Seq) studies using differentiated mouse embryonic stem (ES) cells as well as luciferase reporter studies in the ES cells and in zebrafish embryos argue that Etv2 directly binds the promoter/enhancer regions of Vegfc receptor Vegfr3/Flt4 and lymphatic marker Lyve1, and promotes their expression. Together these data support a model where Etv2 initiates lymphangiogenesis by directly promoting the expression of flt4 within the posterior cardinal vein.

A Novel Role for the Adenovirus L4 Region 22K and 33K Proteins in Adeno-Associated Virus Production.

Despite decades of research in adeno-associated virus (AAV) and the role of adenovirus in production, the interplay of AAV and adenovirus is not fully understood. Specific regions of the adenoviral genome containing E1, E2a, E4 open reading frame (ORF), and VA RNA have been demonstrated as necessary for AAV production; however, incorporating these regions into either a producer cell line or subcloning into an Ad helper plasmid may lead to inclusion of neighboring adenoviral sequence or ORFs with unknown function. Because AAV is frequently used in gene therapies, removing excessive adenovirus sequences improves the Ad helper plasmid size and manufacturability, and may lead to safer vectors for patients. Furthermore, deepening our understanding of the helper virus genes required for recombinant AAV (rAAV) production has the potential to increase yields and manufacturability of rAAV for clinical and commercial applications. One region continuously included in various Ad helper plasmid iterations is the adenoviral E2a promoter region that appears to be necessary for E2a expression. Due to the compact nature of viral genomes, the E2a promoter region overlaps with the Hexon Assembly/100K protein and the L4 region. The L4 region, which contains the coding sequences for 22K and 33K proteins, had not been thought to be necessary for AAV production. Through molecular techniques, this study demonstrates that the adenoviral 22K protein is essential for rAAV production in HEK293 cells by triple transfection and that the 33K protein synergistically increases rAAV yield.

Single-cell transcriptomic analysis of vascular endothelial cells in zebrafish embryos.

Vascular endothelial cells exhibit substantial phenotypic and transcriptional heterogeneity which is established during early embryogenesis. However, the molecular mechanisms involved in establishing endothelial cell diversity are still not well understood. Zebrafish has emerged as an advantageous model to study vascular development. Despite its importance, the single-cell transcriptomic profile of vascular endothelial cells during zebrafish development is still missing. To address this, we applied single-cell RNA-sequencing (scRNA-seq) of vascular endothelial cells isolated from zebrafish embryos at the 24 hpf stage. Six distinct clusters or subclusters related to vascular endothelial cells were identified which include arterial, two venous, cranial, endocardial and endothelial progenitor cell subtypes. Furthermore, we validated our findings by characterizing novel markers for arterial, venous, and endocardial cells. We experimentally confirmed the presence of two transcriptionally different venous cell subtypes, demonstrating heterogeneity among venous endothelial cells at this early developmental stage. This dataset will be a valuable resource for future functional characterization of vascular endothelial cells and interrogation of molecular mechanisms involved in the establishment of their heterogeneity and cell-fate decisions.

Integration of vascular progenitors into functional blood vessels represents a distinct mechanism of vascular growth.

During embryogenesis, the initial vascular network forms by the process of vasculogenesis, or the specification of vascular progenitors de novo. In contrast, the majority of later-forming vessels arise by angiogenesis from the already established vasculature. Here, we show that new vascular progenitors in zebrafish embryos emerge from a distinct site along the yolk extension, or secondary vascular field (SVF), incorporate into the posterior cardinal vein, and contribute to subintestinal vasculature even after blood circulation has been initiated. We further demonstrate that SVF cells participate in vascular recovery after chemical ablation of vascular endothelial cells. Inducible inhibition of the function of vascular progenitor marker etv2/etsrp prevented SVF cell differentiation and resulted in the defective formation of subintestinal vasculature. Similar late-forming etv2+ progenitors were also observed in mouse embryos, suggesting that SVF cells are evolutionarily conserved. Our results characterize a distinct mechanism by which new vascular progenitors incorporate into established vasculature.

Single-cell transcriptomic analysis identifies the conversion of zebrafish Etv2-deficient vascular progenitors into skeletal muscle.

Cell fate decisions involved in vascular and hematopoietic embryonic development are still poorly understood. An ETS transcription factor Etv2 functions as an evolutionarily conserved master regulator of vasculogenesis. Here we report a single-cell transcriptomic analysis of hematovascular development in wild-type and etv2 mutant zebrafish embryos. Distinct transcriptional signatures of different types of hematopoietic and vascular progenitors are identified using an etv2ci32Gt gene trap line, in which the Gal4 transcriptional activator is integrated into the etv2 gene locus. We observe a cell population with a skeletal muscle signature in etv2-deficient embryos. We demonstrate that multiple etv2ci32Gt; UAS:GFP cells differentiate as skeletal muscle cells instead of contributing to vasculature in etv2-deficient embryos. Wnt and FGF signaling promote the differentiation of these putative multipotent etv2 progenitor cells into skeletal muscle cells. We conclude that etv2 actively represses muscle differentiation in vascular progenitors, thus restricting these cells to a vascular endothelial fate.