Biology subjects

Sumanas, S.

Publications and source records attributed to Sumanas, S..

7 recordsLinked to original sources

bioRxiv2026

Mapping the regulatory wiring of endothelial cell differentiation reveals a critical role for Hippo mechanosignaling

At each step of development, stem cells encounter a branching decision to differentiate into one type of cell or another. This decision is executed through the action of competing gene programs that specify different cellular identities. Consequently, creating a comprehensive map of the gene programs that drive each branching step of human cell differentiation remains a long-sought goal of developmental and stem cell biology. Here, we introduce a framework combining genome-wide CRISPR screens, single-cell readouts, and gene program inference to systematically discover and characterize regulators of cell differentiation. This framework includes a method for CRISPR perturbations paired with single-cell RNA-seq called circular capture Perturb-seq (CC-Perturb-seq), which improves gRNA detection from split-pool combinatorial indexing protocols and enables perturbing many candidate regulators to map their effects on gene programs. We applied this framework to map how human pluripotent stem cells differentiate into arterial endothelial cells (ECs), and identified 174 regulators that promote or inhibit EC differentiation. By tracking gene programs across three stages of differentiation, we resolved when and how regulators divert cells toward alternative fates. This map revealed an unexpected requirement for the Hippo mechanosignaling pathway in EC specification. During lateral mesoderm differentiation, Hippo mechanosignaling was required to activate ETV2, the master transcriptional regulator of endothelial identity. Inhibiting Hippo blocked ETV2 even in the presence of VEGF and diverted progenitors to a mesenchymal fate in vitro. Likewise, Hippo signaling was required for maximal etv2 expression in developing zebrafish embryos. Conversely, pharmacologically activating the Hippo pathway increased the purity of both arterial and venous EC differentiation in vitro. Our framework thus identifies novel regulators of endothelial cell differentiation, improves stem-cell differentiation protocols via manipulation of mechanosignaling, and provides a generalizable platform for mapping the regulatory wiring of human development.

genomics↗

bioRxiv2025

A human arteriovenous differentiation roadmap reveals vein developmental mechanisms and vascular effects of viruses

Extracellular signals and cell-fate trajectories during vein development remain elusive, despite trailblazing insights into artery development. Here we exploit human pluripotent stem cell differentiation and mouse embryology to present a model that answers longstanding questions: vein endothelial cell (EC) differentiation unfolds in two steps driven by opposing extracellular signals. First, VEGF differentiates mesoderm into "primed" ECs, newly-defined progenitors that co-express certain arterial (SOX17) and venous (APLNR) markers. Second, primed ECs execute vein differentiation upon VEGF/ERK inhibition; however, upon VEGF activation they can instead form artery ECs. The arteriovenous plasticity of primed ECs was supported by intersectional lineage tracing. Future venous genes including NR2F2 harbor poised chromatin in primed ECs, but are only transcribed upon VEGF/ERK inhibition. SOXF transcription factors, including SOX17, confer primed ECs with vein differentiation competence. Collectively, this two-step vein differentiation model--entailing primed EC intermediates and VEGF/ERK inhibition to trigger vein differentiation--has implications for VEGF-modulating therapies.

developmental biology↗

bioRxiv2025

Jam2 Signaling Functions Downstream of Hand2 To Initiate The Formation Of Organ-Specific Vascular Progenitors In Zebrafish

The mechanisms regulating the formation of organ-specific vasculature are still poorly understood. We have previously identified a population of late-forming endothelial progenitor cells in zebrafish embryos (termed secondary vascular field, SVF), which emerge from the lateral plate mesoderm after 24 hpf stage, when blood circulation has already been initiated. Here, we investigate the functional role of SVF cells and the molecular mechanisms that govern the emergence of these SVF cells and their contribution to vasculature. We found that the bHLH transcription factor Hand2 and junctional adhesion molecule Jam2b are expressed in the SVF-forming region and are required for the emergence of SVF cells in zebrafish embryos. Time-lapse imaging and jam2b:Cre-based lineage tracing showed that SVF cells serve as the major source of the intestinal vasculature, including the supraintestinal artery (SIA) and subintestinal vein (SIV), which are subsequently remodeled to provide blood flow to many internal organs. To analyze the functional role of jam2b and the related jam2a gene in vascular development, we generated double maternal-zygotic jam2a; jam2b mutants, which display a greatly reduced number of SVF cells and show defects in the development of the intestinal vasculature. Further analysis showed that hand2 functions in the SVF-forming region upstream of jam2b and is required to induce expression of the transcription factor etv2/etsrp, a known master regulator of vasculogenesis. In summary, our results identify new roles for Jam2 signaling and Hand2 function in the emergence of organ-specific vascular progenitors. The presence of similar progenitors in mammalian embryos suggests that this mechanism is evolutionarily conserved.

developmental biology↗

bioRxiv2024

Lineage labeling with zebrafish hand2 Cre and CreERT2 recombinase CRISPR knock-ins

BackgroundThe ability to generate endogenous Cre recombinase drivers using CRISPR- Cas9 knock-in technology allows lineage tracing, cell type specific gene studies, and in vivo validation of inferred developmental trajectories from phenotypic and gene expression analyses. This report describes endogenous zebrafish hand2 Cre and CreERT2 drivers generated with GeneWeld CRISPR-Cas9 precision targeted integration. Resultshand2-2A-cre and hand2-2A-creERT2 knock-ins crossed with ubiquitous loxP-based Switch reporters led to broad labeling in expected mesodermal and neural crest-derived lineages in cardiac, pectoral fins, pharyngeal arch, liver, intestine, and mesothelial tissues, as well as enteric neurons. Novel patterns of hand2 lineage tracing appeared in venous blood vessels. CreERT2 induction at 24 hours reveals late emerging hand2 progenitors in the 24 - 48 hour embryo contribute to the venous and intestinal vasculature. Induction in 3 dpf larva restricts hand2 lineage labeling to mesoderm-derived components of the branchial arches, heart, liver and enteric neurons. Conclusionshand2 progenitors from the lateral plate mesoderm and ectoderm contribute to numerous lineages in the developing embryo. Later emerging hand2 progenitors become restricted to a subset of lineages in the larva. The hand2 Cre and CreERT2 drivers establish critical new tools to investigate hand2 lineages in zebrafish embryogenesis and larval organogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/626907v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@12f58cborg.highwire.dtl.DTLVardef@16e95c8org.highwire.dtl.DTLVardef@12b8e9dorg.highwire.dtl.DTLVardef@a01848_HPS_FORMAT_FIGEXP M_FIG C_FIG Zebrafish hand2 Cre and CreERT2 drivers generated with GeneWeld CRISPR/Cas9 precision targeted integration label numerous lineages from the mesoderm and ectoderm. Temporal CreERT2 Tamoxifen regulated switching re{-} veals late emerging hand2 progenitors contribute to the embryonic venous vasculature and larval organs.

developmental biology↗

bioRxiv2023

SH2 domain protein E (SHE) and ABL signaling regulate blood vessel size

Blood vessels in different vascular beds vary in lumen diameter, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vascular lumen size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA lumen, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA lumen in she mutants correlated with an increased endothelial expression of claudin 5a and 5b (cldn5a / cldn5b), which encode proteins enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA lumen size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates lumen size during vascular tubulogenesis.

developmental biology↗

bioRxiv2021

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

During embryogenesis, the initial vascular network is thought to form by the process of vasculogenesis, or the specification of vascular progenitors de novo. After the initial blood circulation has been established, the majority of later-forming vessels are thought to arise by angiogenesis from the already established vasculature. Here we show that new vascular progenitors in zebrafish embryos contribute to functional vasculature even after blood circulation has been established. Based on the expression analysis of early vascular progenitor markers etv2 and tal1, we characterized a novel site of late vasculogenesis (termed secondary vascular field, SVF), located bilaterally along the yolk extension. Using time-lapse imaging of etv2 reporter lines, we show that SVF cells migrate and incorporate into functional blood vessels and contribute to the formation of the posterior cardinal vein and subintestinal vasculature, suggesting a novel mode of vascular growth. We further demonstrate that SVF cells participate in vascular recovery after chemical ablation of vascular endothelial cells. Inducible inhibition of etv2 function prevented SVF cell differentiation and resulted in the defective formation of subintestinal vasculature. In addition, we performed single-cell RNA-seq analysis to identify the transcriptional profile of SVF cells, which demonstrated similarities and differences between the transcriptomes of SVF cells and early vascular progenitors. Our results characterize a novel mechanism of how new vascular progenitors incorporate into established vasculature and revise our understanding of basic mechanisms that regulate vascular development.

developmental biology↗

bioRxiv2021

Single-cell transcriptome analysis of the zebrafish embryonic trunk

During embryonic development, cells differentiate into a variety of distinct cell types and subtypes with diverse transcriptional profiles. To date, transcriptomic signatures of different cell lineages that arise during development have been only partially characterized. Here we used single-cell RNA-seq to perform transcriptomic analysis of over 20,000 cells disaggregated from the trunk region of zebrafish embryos at the 30 hpf stage. Transcriptional signatures of 27 different cell types and subtypes were identified and annotated during this analysis. This dataset will be a useful resource for many researchers in the fields of developmental and cellular biology and facilitate the understanding of molecular mechanisms that regulate cell lineage choices during development.

developmental biology↗