During development, animals generate distinct cell populations with specific identities, functions, and morphologies. We mapped transcriptionally distinct populations across 489,686 cells from 62 stages during wild-type zebrafish embryogenesis and early larval development (3–120 h post-fertilization). Using these data, we identified the limited catalog of gene expression programs reused across multiple tissues and their cell-type-specific adaptations. We also determined the duration each transcriptional state is present during development and identify unexpected long-term cycling populations. Focused clustering and transcriptional trajectory analyses of non-skeletal muscle and endoderm identified transcriptional profiles and candidate transcriptional regulators of understudied cell types and subpopulations, including the pneumatic duct, individual intestinal smooth muscle layers, spatially distinct pericyte subpopulations, and recently discovered best4+ cells. To enable additional discoveries, we make this comprehensive transcriptional atlas of early zebrafish development available through our website, Daniocell. 

Single-cell analysis of shared signatures and transcriptional diversity during zebrafish development.

http://www.cell.com/article/S1534580722002878/pdf

KCNJ8/ABCC9-containing K-ATP channel modulates brain vascular smooth muscle development and neurovascular coupling.

Chlorogenic acid (CGA) is a highly abundant bioactive compound in green coffee beans. CGA has protective roles in various cardiovascular diseases, suggesting that it may affect endothelial cells lining the blood vessels. However, cellular mechanisms underlying its beneficial effects remained unclear. We therefore performed quantitative proteomics of CGA-treated human endothelial cells, followed by enrichment/pathway analyses, and functional validation using various assays. EA.hy926 cells were treated with 5 µM CGA for 24-h before nanoLC-LTQ-Orbitrap MS/MS analyses. Comparing with control cells, the CGA-treated cells had 185 differentially expressed proteins. Of these, up-regulations of podocalyxin-1 and lamin A/C were confirmed by immunoblotting. Enrichment analysis revealed that CGA mainly affected RNA-binding proteins involved in protein targeting to membrane and exosomal secretion. KEGG pathway analysis of proteins in RNA metabolic process/gene expression cluster revealed the involvement of Rap1 signaling, PI3K/AKT signaling and spliceosome, suggesting their potential roles in modulating tight junction (TJ) barrier and angiogenesis. Functional validation revealed significant increases in ZO-1 (a TJ-associated protein) expression and transendothelial electrical resistance (TEER) in the CGA-treated cells. Finally, CGA enhanced capillary-like endothelial tube formation and secretion of angiopoietin-2 (an angiogenic factor). These novel findings provide insights into cellular mechanisms underlying the beneficial effects of CGA on cardiovascular system via enhancement of endothelial TJ barrier function and angiogenesis. 

Chlorogenic acid enhances endothelial barrier function and promotes endothelial tube formation: A proteomics approach and functional validation.

During development, animals generate distinct cell populations with specific identities, functions, and morphologies. We mapped transcriptionally distinct populations across 489,686 cells from 62 stages during wild-type zebrafish embryogenesis and early larval development (3–120 hours post-fertilization). Using these data, we identified the limited catalog of gene expression programs reused across multiple tissues and their cell-type-specific adaptations. We also determined the duration each transcriptional state is present during development and suggest new long-term cycling populations. Focused analyses of non-skeletal muscle and the endoderm identified transcriptional profiles of understudied cell types and subpopulations, including the pneumatic duct, individual intestinal smooth muscle layers, spatially distinct pericyte subpopulations, and homologs of recently discovered human best4+ enterocytes. The transcriptional regulators of these populations remain unknown, so we reconstructed gene expression trajectories to suggest candidates. To enable additional discoveries, we make this comprehensive transcriptional atlas of early zebrafish development available through our website, Daniocell.

https://www.biorxiv.org/content/biorxiv/early/2023/04/15/2023.03.20.533545.full.pdf

Single-cell analysis of shared signatures and transcriptional diversity during zebrafish development

The vasculature consists of vessels of different sizes that are arranged in a hierarchical pattern. Two cell populations work in concert to establish this pattern during embryonic development and adopt it to changes in blood flow demand later in life: endothelial cells that line the inner surface of blood vessels, and adjacent vascular mural cells, including smooth muscle cells and pericytes. Despite recent progress in elucidating the signalling pathways controlling their crosstalk, much debate remains with regard to how mural cells influence endothelial cell biology and thereby contribute to the regulation of blood vessel formation and diameters. In this Review, I discuss mural cell functions and their interactions with endothelial cells, focusing on how these interactions ensure optimal blood flow patterns. Subsequently, I introduce the signalling pathways controlling mural cell development followed by an overview of mural cell ontogeny with an emphasis on the distinguishing features of mural cells located on different types of blood vessels. Ultimately, I explore therapeutic strategies involving mural cells to alleviate tissue ischemia and improve vascular efficiency in a variety of diseases.

Biology of vascular mural cells.

The vascular permeability of the endothelium is finely controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions. In the majority of normal adult tissues, endothelial cells in blood vessels maintain vascular permeability at a relatively low level, while in response to inflammation, they limit vascular barrier function to induce plasma leakage and extravasation of immune cells as a defense mechanism. Thus, the dynamic but also simultaneously tight regulation of vascular permeability by endothelial cells is responsible for maintaining homeostasis and, as such, impairments of its underlying mechanisms result in hyperpermeability, leading to the development and progression of various diseases including coronavirus disease 2019 (COVID-19), a newly emerging infectious disease. Recently, increasing numbers of studies have been unveiling the important role of Rap1, a small guanosine 5'-triphosphatase (GTPase) belonging to the Ras superfamily, in the regulation of vascular permeability. Rap1 enhances VE-cadherin-mediated endothelial cell-cell junctions to potentiate vascular barrier functions via dynamic reorganization of the actin cytoskeleton. Importantly, Rap1 signaling activation reportedly improves vascular barrier function in animal models of various diseases associated with vascular hyperpermeability, suggesting that Rap1 might be an ideal target for drugs intended to prevent vascular barrier dysfunction. Here, we describe recent progress in understanding the mechanisms by which Rap1 potentiates VE-cadherin-mediated endothelial cell-cell adhesions and vascular barrier function. We also discuss how alterations in Rap1 signaling are related to vascular barrier dysfunction in diseases such as acute pulmonary injury and malignancies. In addition, we examine the possibility of Rap1 signaling as a target of drugs for treating diseases associated with vascular hyperpermeability.

Rap1 Small GTPase Regulates Vascular Endothelial-Cadherin-Mediated Endothelial Cell-Cell Junctions and Vascular Permeability.

Recruitment of mural cells to the vascular wall is essential for forming the vasculature as well as maintaining proper vascular functions. In recent years, zebrafish genetic tools for mural cell biology have improved substantially. Fluorescently labeled zebrafish mural cell reporter lines enable us to study, with higher spatiotemporal resolution than ever, the processes of mural cell development from their progenitors. Furthermore, recent phenotypic analysis of platelet-derived growth factor beta mutant zebrafish revealed well-conserved organotypic mural cell development and functions in vertebrates with the unique features of zebrafish. However, comprehensive reviews of zebrafish mural cells are lacking. Therefore, herein, we highlight recent advances in zebrafish mural cell tools. We also summarize the fundamental features of zebrafish mural cell development, especially at early stages, and functions.

Zebrafish Vascular Mural Cell Biology: Recent Advances, Development, and Functions.

Summary Lateral plate mesoderm (LPM) cells differentiate into various cell types including endothelial and hematopoietic cells. In zebrafish embryos, LPM cells migrate toward the midline along the ventral surfaces of somites during which their cell fate specification depends upon efficient integrin-mediated cell adhesion and migration. Herein, we present a protocol for analysis of integrin-mediated cell adhesion of LPM cells isolated from zebrafish embryos. This allows the study of the molecular mechanisms underlying integrin activation required for LPM cell fate specification. For complete details on the use and execution of this protocol, please refer to Rho et al. (2019) .

Protocol for analysis of integrin-mediated cell adhesion of lateral plate mesoderm cells isolated from zebrafish embryos.

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Rap1 Small GTPase Regulates Vascular Endothelial-Cadherin-Mediated Endothelial Cell-Cell Junctions and Vascular Permeability.

Zebrafish Vascular Mural Cell Biology: Recent Advances, Development, and Functions.

Protocol for analysis of integrin-mediated cell adhesion of lateral plate mesoderm cells isolated from zebrafish embryos.