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Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal primer binding site (PBS) length for the pegRNA of ∼13 nucleotides depending on the sequence composition. However, optimal PBS length characterization has been based on prime editing outcomes using plasmid or lentiviral expression systems. In this study, we demonstrate that for prime editor (PE) ribonucleoprotein complexes, the auto-inhibitory interaction between the PBS and the spacer sequence affects pegRNA binding efficiency and target recognition. Destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency in multiple prime editing formats. In the case of end-protected pegRNAs, a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. Finally, we show that prime editor ribonucleoprotein complexes programmed with pegRNAs designed using these refined parameters efficiently correct disease-related genetic mutations in patient-derived fibroblasts and efficiently install precise edits in primary human T cells and zebrafish.

Pericytes reside in capillary beds where they share a basement membrane with endothelial cells and regulate their function. However, little is known about embryonic pericyte development, in part, due to lack of specific molecular markers and genetic tools. Here, we applied single cell RNA-sequencing (scRNA-seq) of platelet derived growth factor beta (pdgfrb)-positive cells to molecularly characterize pericytes in zebrafish larvae. scRNA-seq revealed zebrafish cells expressing mouse pericyte gene orthologs while comparison to bulk RNA-seq from wild type and pdgfrb mutant larvae further refined a pericyte geneset. Subsequent integration with mouse pericyte scRNA-seq profiles revealed a core set of conserved pericyte genes. Using transgenic reporter lines, we validated pericyte expression of two genes identified in our analysis: NDUFA4 mitochondrial complex associated like 2a (ndufa4l2a), and potassium voltage-gated channel, Isk-related family, member 4 (kcne4). Both reporter lines exhibited pericyte expression in multiple anatomical locations, while kcne4 was also detected in a subset of vascular smooth muscle cells. Thus, our integrated molecular analysis revealed a molecular profile for zebrafish pericytes and allowed us to develop new tools to observe these cells in vivo.SUMMARY STATEMENTAn integrated molecular analysis in zebrafish identifies new molecular markers for pericytes.

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA-repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

Lymphatic valves develop from pre-existing endothelial cells through a step-wise process involving complex changes in cell shape and orientation, along with extracellular matrix interactions, to form two intraluminal leaflets. Once formed, valves prevent back-flow within the lymphatic system to ensure drainage of interstitial fluid back into the circulatory system, thereby serving a critical role in maintaining fluid homeostasis. Despite the extensive anatomical characterization of lymphatic systems across numerous genus and species dating back several hundred years, valves were largely thought to be phylogenetically restricted to mammals. Accordingly, most insights into molecular and genetic mechanisms involved in lymphatic valve development have derived from mouse knockouts, as well as rare diseases in humans. However, we have recently used a combination of imaging and genetic analysis in the zebrafish to demonstrate that valves are a conserved feature of the teleost lymphatic system. Here, we provide a historical overview of comparative lymphatic valve anatomy together with recent efforts to define molecular pathways that contribute to lymphatic valve morphogenesis. Finally, we integrate our findings in zebrafish with previous work and highlight the benefits that this model provides for investigating lymphatic valve development.

The migration of lymphatic endothelial cells (LECs) is key for the development of the complex and vast lymphatic vascular network that pervades most tissues in an organism. In zebrafish, arterial intersegmental vessels together with chemokines have been shown to promote lymphatic cell migration from the horizontal myoseptum (HM). We observed that emergence of mural cells around the intersegmental arteries coincides with lymphatic departure from HM which raised the possibility that arterial mural cells promote LEC migration. Our live imaging and cell ablation experiments revealed that LECs migrate slower and fail to establish the lymphatic vascular network in the absence of arterial mural cells. We determined that mural cells are a source for the C-X-C motif chemokine 12 (Cxcl12a and Cxcl12b), vascular endothelial growth factor C (Vegfc) and collagen and calcium-binding EGF domain-containing protein 1 (Ccbe1). We showed that chemokine and growth factor signalling function cooperatively to induce robust LEC migration. Specifically, Vegfc-Vegfr3 signalling, but not chemokines, induces extracellular signal-regulated kinase (ERK) activation in LECs, and has an additional pro-survival role in LECs during the migration. Together, the identification of mural cells as a source for signals that guide LEC migration and survival will be important in the future design for rebuilding lymphatic vessels in disease contexts.

Type V CRISPR–Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.

Summary The formation of new vessels requires a tight synchronization between proliferation, differentiation, and sprouting. However, how these processes are differentially activated, often by neighboring endothelial cells (ECs), remains unclear. Here, we identify cell cycle progression as a regulator of EC sprouting and differentiation. Using transgenic zebrafish illuminating cell cycle stages, we show that venous and lymphatic precursors sprout from the cardinal vein exclusively in G1 and reveal that cell-cycle arrest is induced in these ECs by overexpression of p53 and the cyclin-dependent kinase (CDK) inhibitors p27 and p21. We further demonstrate that, in vivo, forcing G1 cell-cycle arrest results in enhanced vascular sprouting. Mechanistically, we identify the mitogenic VEGFC/VEGFR3/ERK axis as a direct inducer of cell-cycle arrest in ECs and characterize the cascade of events that render “sprouting-competent” ECs. Overall, our results uncover a mechanism whereby mitogen-controlled cell-cycle arrest boosts sprouting, raising important questions about the use of cell cycle inhibitors in pathological angiogenesis and lymphangiogenesis., Graphical abstract, Highlights • Endothelial cells sprout from the PCV in G1 phase of the cell cycle • VEGFC/FLT4/ERK induce p53-, p21-, and p27-mediated cell-cycle arrest to enable sprouting • Forced G1 cell-cycle arrest results in ectopic sprouting of undifferentiated ECs, Jerafi-Vider et al. report a mechanism of endothelial cell sprouting, whereby the mitogenic VEGFC/FLT4/ERK signaling pathway induces p53-, p21-, and p27-mediated cell-cycle arrest, conferring angiogenic potential. These findings have important implications for the putative short-term effects of using cell cycle inhibitors in settings of pathological angiogenesis and lymphangiogenesis.

Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these zebrafish mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects.

Endothelial cells emerge from the atrioventricular canal (AVC) to form nascent coronary blood vessels in the juvenile zebrafish heart. We found that pdgfrβ is first expressed in the epicardium around the AVC and later becomes localized mainly in the mural cells. pdgfrβ mutant fish display severe defects in mural cell recruitment and coronary vessel development. pdgfrβ+ mural cells are heterogeneous and those associated with coronary arteries also express cxcl12b. Mural cells positive for both pdgfrβ and cxcl12b transgenic reporters had elevated expression of smooth muscle cell genes. Interestingly, these mural cells were associated with coronary arteries even in the absence of Pdgfrβ, although smooth muscle gene expression was downregulated in these cells. We found that pdgfrβ expression dynamically changes in the epicardium derived cells, which we found to be a heterogeneous population. mdka was identified as a gene upregulated in subpopulations of pdgfrβ+ cells during heart regeneration. However, pdgfrβ but not mdka mutants showed defects in heart regeneration. Our results demonstrated that pdgfrβ+ cells and Pdgfrβ signaling are essential for coronary development and heart regeneration.SUMMARY STATEMENTHeterogeneous pdgfrβ positive cells are present in developing and regenerating zebrafish hearts and are required for development of mural cells and their association with the nascent coronary vessels during zebrafish heart development and regeneration.

Endothelial cells emerge from the atrioventricular canal to form coronary blood vessels in juvenile zebrafish hearts. We find that pdgfrb is first expressed in the epicardium around the atrioventricular canal and later becomes localized mainly in the mural cells. pdgfrb mutant fish show severe defects in mural cell recruitment and coronary vessel development. Single-cell RNA sequencing analyses identified pdgfrb+ cells as epicardium-derived cells (EPDCs) and mural cells. Mural cells associated with coronary arteries also express cxcl12b and smooth muscle cell markers. Interestingly, these mural cells remain associated with coronary arteries even in the absence of Pdgfrβ, although smooth muscle gene expression is downregulated. We find that pdgfrb expression dynamically changes in EPDCs of regenerating hearts. Differential gene expression analyses of pdgfrb+ EPDCs and mural cells suggest that they express genes that are important for regeneration after heart injuries. mdka was identified as a highly upregulated gene in pdgfrb+ cells during heart regeneration. However, pdgfrb but not mdka mutants show defects in heart regeneration after amputation. Our results demonstrate that heterogeneous pdgfrb+ cells are essential for coronary development and heart regeneration.

Vascular networks comprise endothelial cells and mural cells, which include pericytes and smooth muscle cells. To elucidate the mechanisms controlling mural cell recruitment during development and tissue regeneration, we studied zebrafish caudal fin arteries. Mural cells colonizing arteries proximal to the body wrapped around them, whereas those in more distal regions extended protrusions along the proximo-distal vascular axis. Both cell populations expressed platelet-derived growth factor receptor β (pdgfrb) and the smooth muscle cell marker myosin heavy chain 11a (myh11a). Most wrapping cells in proximal locations additionally expressed actin alpha2, smooth muscle (acta2). Loss of Pdgfrb signalling specifically decreased mural cell numbers at the vascular front. Using lineage tracing, we demonstrate that precursor cells located in periarterial regions and expressing Pgdfrb can give rise to mural cells. Studying tissue regeneration, we did not find evidence that newly formed mural cells were derived from pre-existing cells. Together, our findings reveal conserved roles for Pdgfrb signalling in development and regeneration, and suggest a limited capacity of mural cells to self-renew or contribute to other cell types during tissue regeneration.

Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle. To determine if this role was conserved in zebrafish, we analyzedpdgfbandpdgfrbmutant lines. Similar to mouse,pdgfbandpdgfrbmutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects,pdgfrbmutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells inpdgfrbmutants. Similar to mouse,pdgfrbmutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects.Summary statementGenetic analysis in zebrafish demonstrates the conserved role of Pdgfb/Pdgfrb signaling in pericyte and vascular smooth muscle cell formation during vascular development in vertebrates.

Vascular networks are comprised of endothelial cells and mural cells, which include pericytes and smooth muscle cells. It is well established that new endothelial cells are derived from pre-existing ones during the angiogenic phase of blood vessel growth. By contrast, mural cell ontogeny is less clear with an ongoing debate whether mural cells possess mesenchymal stem cell properties. To elucidate the mechanisms controlling mural cell recruitment during development and tissue regeneration, we studied the formation of zebrafish caudal fin arteries. Mural cells showed morphological heterogeneity: cells colonizing arteries proximal to the body wrapped around them, while those in more distal regions extended protrusions along the proximo-distal vascular axis. Despite these differences, both cell populations expressed platelet-derived growth factor receptor beta (Pdgfrb) and the smooth muscle cell marker myosin heavy chain 11a (Myh11a). Loss of Pdgfrb signalling during development or tissue regeneration resulted in a substantial decrease in mural cells at the vascular front, while those proximal to the body were less affected. Using lineage tracing, we demonstrate that precursor cells located in periarterial regions of the caudal fin and expressing Pgdfrb can give rise to mural cells, while in regeneration newly formed mural cells were not derived from pre-existing ones. Together, our findings reveal conserved roles for pdgfrb signalling in development and regeneration, while at the same time illustrating a limited capacity of mural cells to self-renew or contribute to other cell types during tissue regeneration.

The zebrafish is ideal for studying embryogenesis and is increasingly applied to model human disease. In these contexts, RNA-sequencing (RNA-seq) provides mechanistic insights by identifying transcriptome changes between experimental conditions. Application of RNA-seq relies on accurate transcript annotation for a genome of interest. Here, we find discrepancies in analysis from RNA-seq datasets quantified using Ensembl and RefSeq zebrafish annotations. These issues were due, in part, to variably annotated 3' untranslated regions and thousands of gene models missing from each annotation. Since these discrepancies could compromise downstream analyses and biological reproducibility, we built a more comprehensive zebrafish transcriptome annotation that addresses these deficiencies. Our annotation improves detection of cell type-specific genes in both bulk and single cell RNA-seq datasets, where it also improves resolution of cell clustering. Thus, we demonstrate that our new transcriptome annotation can outperform existing annotations, providing an important resource for zebrafish researchers.

The emergence and growth of new vessels requires a tight synchronization between proliferation, differentiation and sprouting, traditionally thought to be controlled by mitogenic signals, especially of the VEGF family. However, how these cues are differentially transduced, by sometimes even neighboring endothelial cells (ECs), remains unclear. Here we identify cell-cycle progression as a new regulator of EC sprouting and differentiation. Using transgenic zebrafish illuminating cell-cycle stages, we show that venous and lymphatic precursors sprout from the Cardinal Vein (CV) exclusively in G0/G1, and reveal that cell-cycle arrest is induced in these ECs by p53 and the CDK-inhibitors p27 and p21. Moreover, we demonstrate that in vivo, chemical and genetic cell-cycle inhibition, results in massive vascular growth. Mechanistically, we identify the mitogenic VEGFC/VEGFR3/ERK axis as direct inducer of cell-cycle arrest in angiogenic ECs and characterize the cascade of events governing venous vs. lymphatic segregation and sprouting. Overall, our results uncover an unexpected mechanism whereby mitogen-controlled cell-cycle arrest boosts sprouting, raising important questions about the use of cell-cycle inhibitors in pathological angiogenesis and lymphangiogenesis.

Lymphedema, the most common lymphatic anomaly, involves defective lymphatic valve development; yet the epigenetic modifiers underlying lymphatic valve morphogenesis remain elusive. Here, we showed that during mouse development, the histone-modifying enzyme histone deacetylase 3 (Hdac3) regulates the formation of both lymphovenous valves, which maintain the separation of the blood and lymphatic vascular systems, and the lymphatic valves. Endothelium-specific ablation of Hdac3 in mice led to blood-filled lymphatic vessels, edema, defective lymphovenous valve morphogenesis, improper lymphatic drainage, defective lymphatic valve maturation, and complete lethality. Hdac3-deficient lymphovenous valves and lymphatic vessels exhibited reduced expression of the transcription factor Gata2 and its target genes. In response to oscillatory shear stress, the transcription factors Tal1, Gata2, and Ets1/2 physically interacted with and recruited Hdac3 to the evolutionarily conserved E-box–GATA–ETS composite element of a Gata2 intragenic enhancer. In turn, Hdac3 recruited histone acetyltransferase Ep300 to form an enhanceosome complex that promoted Gata2 expression. Together, these results identify Hdac3 as a key epigenetic modifier that maintains blood-lymph separation and integrates both extrinsic forces and intrinsic cues to regulate lymphatic valve development.

Angiogenic sprouting needs to be tightly controlled. It has been suggested that the Notch ligand dll4 expressed in leading tip cells restricts angiogenesis by activating Notch signalling in trailing stalk cells. Here, we show using live imaging in zebrafish that activation of Notch signalling is rather required in tip cells. Notch activation initially triggers expression of the chemokine receptor cxcr4a. This allows for proper tip cell migration and connection to the pre-existing arterial circulation, ultimately establishing functional arterial-venous blood flow patterns. Subsequently, Notch signalling reduces cxcr4a expression, thereby preventing excessive blood vessel growth. Finally, we find that Notch signalling is dispensable for limiting blood vessel growth during venous plexus formation that does not generate arteries. Together, these findings link the role of Notch signalling in limiting angiogenesis to its role during artery formation and provide a framework for our understanding of the mechanisms underlying blood vessel network expansion and maturation.

Identification of tissue-specific and developmentally active enhancers provides insights into mechanisms that control gene expression during embryogenesis. However, robust detection of these regulatory elements remains challenging, especially in vertebrate genomes. Here, we apply fluorescent activated nuclei sorting (FANS) followed by Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) to identify developmentally active endothelial enhancers in the zebrafish genome. ATAC-Seq of nuclei from Tg(fli1a:egfp)y1 transgenic embryos revealed expected patterns of nucleosomal positioning at transcriptional start sites throughout the genome and association with active histone modifications. Comparison of ATAC-Seq from GFP-positive and -negative nuclei identified more than 5,000 open elements specific to endothelial cells. These elements flanked genes functionally important for vascular development and that displayed endothelial-specific gene expression. Importantly, a majority of tested elements drove endothelial gene expression in zebrafish embryos. Thus, FANS-assisted ATAC-Seq using transgenic zebrafish embryos provides a robust approach for genome-wide identification of active tissue-specific enhancer elements.

Rationale: Significant progress has revealed transcriptional inputs that underlie regulation of artery and vein endothelial cell fates. However, little is known concerning genome-wide regulation of this process. Therefore, such studies are warranted to address this gap. Objective: To identify and characterize artery- and vein-specific endothelial enhancers in the human genome, thereby gaining insights into mechanisms by which blood vessel identity is regulated. Methods and Results: Using chromatin immunoprecipitation and deep sequencing for markers of active chromatin in human arterial and venous endothelial cells, we identified several thousand artery- and vein-specific regulatory elements. Computational analysis revealed that NR2F2 (nuclear receptor subfamily 2, group F, member 2) sites were overrepresented in vein-specific enhancers, suggesting a direct role in promoting vein identity. Subsequent integration of chromatin immunoprecipitation and deep sequencing data sets with RNA sequencing revealed that NR2F2 regulated 3 distinct aspects related to arteriovenous identity. First, consistent with previous genetic observations, NR2F2 directly activated enhancer elements flanking cell cycle genes to drive their expression. Second, NR2F2 was essential to directly activate vein-specific enhancers and their associated genes. Our genomic approach further revealed that NR2F2 acts with ERG (ETS-related gene) at many of these sites to drive vein-specific gene expression. Finally, NR2F2 directly repressed only a small number of artery enhancers in venous cells to prevent their activation, including a distal element upstream of the artery-specific transcription factor, HEY2 (hes related family bHLH transcription factor with YRPW motif 2). In arterial endothelial cells, this enhancer was normally bound by ERG, which was also required for arterial HEY2 expression. By contrast, in venous endothelial cells, NR2F2 was bound to this site, together with ERG, and prevented its activation. Conclusions: By leveraging a genome-wide approach, we revealed mechanistic insights into how NR2F2 functions in multiple roles to maintain venous identity. Importantly, characterization of its role at a crucial artery enhancer upstream of HEY2 established a novel mechanism by which artery-specific expression can be achieved.

The zebrafish (Danio rerio) has emerged as a powerful model to study vertebrate development and disease. Its short generation time makes it amenable to genetic manipulation and analysis, and its small size and high fecundity make it especially well suited for large-scale forward genetic and chemical screens. Fast-developing zebrafish embryos are transparent, facilitating live imaging of a variety of developmental processes in wild-type and mutant animals. Published version

Vascular smooth muscle of the head derives from neural crest, but developmental mechanisms and early transcriptional drivers of the vSMC lineage are not well characterized. We find that in early development, the transcription factor foxc1b is expressed in mesenchymal cells that associate with the vascular endothelium. Using timelapse imaging, we observe that foxc1b expressing mesenchymal cells differentiate into acta2 expressing vascular mural cells. We show that in zebrafish, while foxc1b is co-expressed in acta2 positive smooth muscle cells that associate with large diameter vessels, it is not co-expressed in capillaries where pdgfrβ positive pericytes are located. In addition to being an early marker of the lineage, foxc1 is essential for vSMC differentiation; we find that foxc1 loss of function mutants have defective vSMC differentiation and that early genetic ablation of foxc1b or acta2 expressing populations blocks vSMC differentiation. Furthermore, foxc1 is expressed upstream of acta2 and is required for acta2 expression in vSMCs. Using RNA-Seq we determine an enriched intersectional gene expression profile using dual expression of foxc1b and acta2 to identify novel vSMC markers. Taken together, our data suggests that foxc1 is a marker of vSMCs and plays a critical functional role in promoting their differentiation.

Summary Mammalian inner ear and fish lateral line sensory hair cells (HCs) detect fluid motion to transduce environmental signals. Actively maintained ionic homeostasis of the mammalian inner ear endolymph is essential for HC function. In contrast, fish lateral line HCs are exposed to the fluctuating ionic composition of the aqueous environment. Using lineage labeling, in vivo time-lapse imaging and scRNA-seq, we discovered highly motile skin-derived cells that invade mature mechanosensory organs of the zebrafish lateral line and differentiate into Neuromast-associated (Nm) ionocytes. This invasion is adaptive as it is triggered by environmental fluctuations. Our discovery of Nm ionocytes challenges the notion of an entirely placodally derived lateral line and identifies Nm ionocytes as likely regulators of HC function possibly by modulating the ionic microenvironment. Nm ionocytes provide an experimentally accessible in vivo system to study cell invasion and migration, as well as the physiological adaptation of vertebrate organs to changing environmental conditions.

Despite the critical role of vascular mural cells (smooth muscle cells and pericytes) in supporting the endothelium of blood vessels, we know little of their early morphogenesis and differentiation.foxc1b:EGFPexpressing cells in zebrafish associate with the vascular endothelium (kdrl) and co-express a smooth muscle marker (acta2), but not a pericyte marker (pdgfrβ). The expression offoxc1bin early peri-endothelial mesenchymal cells allows us to follow the morphogenesis of mesenchyme intoacta2expressing vascular smooth muscle cells. We show that mural cells expressing different markers associate with vessels of different diameters, depending on their embryonic location and developmental timing, suggesting marker expression is predictive of functional differences. We identify gene expression signatures for an enriched vascular smooth muscle cell population (foxc1b + acta2)and all smooth muscle(acta2)using fluorescence-activated cell sorting and RNA-Seq. Finally, we demonstrate that progressive loss offoxc1a/foxc1bresults in decreased smooth muscle cell coverage. Together, our data highlight the early cellular dynamics and transcriptome profiles of smooth muscle cellsin vivo,usingfoxc1bas a unique tool to probe vascular smooth muscle cell differentiation.Summary StatementTracing the morphogenesis and transcriptome of early vascular smooth muscle cells using foxc1b

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

Recent studies of brain meningeal lymphatic endothelial cells in mouse and zebrafish have raised interest in their function. In this issue of Developmental Cell, Chen et al. (2019) reveal new developmental roles for these cells in facilitating tissue repair and guiding vascular re-growth in the central nervous system.

The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium. Studies in mice have suggested that GATA2 might function at early stages within hemogenic endothelium. Two orthologs of Gata2 exist in zebrafish: gata2a and gata2b. Here, we report that gata2b expression initiates during the convergence of PLM, becoming restricted to emerging HSCs. We observe Notch-dependent gata2b expression within the hemogenic subcompartment of the dorsal aorta that is in turn required to initiate runx1 expression. Our results indicate that Gata2b functions within hemogenic endothelium from an early stage, whereas Gata2a functions more broadly throughout the vascular system.

The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

The lymphatic system comprises a network of interconnected and blind-ended vessels whose central function is to maintain fluid homeostasis.1 Similar to the vessels of the circulatory system, the lymphatic system consists of a hierarchical network of small and large caliber vessels, which function together to allow fluid drainage. Lymphatic capillaries are lined by endothelial cells that are highly discontinuous, making them permeable to surrounding fluid to facilitate resorption. By contrast, larger lymphatic collecting ducts possess a thin smooth muscle layer and valves to promote unidirectional transport of fluid toward connection points with the circulatory system at the subclavian veins. In addition to fluid homeostasis, the lymphatic system and the constituent lymph nodes are a portal for lymphocytes, as well as antigen-presenting cells, while also functioning in lipid absorption in the digestive system.1 Given their wide-ranging importance in these normal physiological processes, it is not surprising that the lymphatic system is implicated in several pathological conditions. Indeed, inflammation is intimately associated with lymphatic function and is a central cause of lymphedema in a wide range of acquired and congenital diseases.1 Notably, congenital forms of lymphedema are associated with mutations in several genes important for lymphatic vessel formation, including those encoding the receptor tyrosine kinase FLT4 and its ligand, VEGFC.2 Articles, see p 1263 and 1276 The lymphatic system is also essential for cardiac homeostasis and has been increasingly implicated in related diseases. Importantly, lymphatic vessels can play a central role to facilitate repair of cardiac tissue after myocardial infarction.3 Indeed, injection of VEGFC after myocardial infarction can lead to sustained improvement in cardiac function, including increased ejection fraction, and reduction in both end-systolic volume and hypertrophy. In these cases, the presence of additional lymphatic vessels induced by VEGFC is thought to better resolve post-myocardial infarction edema …

The genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood, however less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report 17β-estradiol (E2) influences the production of runx1+ HSPCs in the AGM region by antagonizing VEGF-signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development (12–24hpf) significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity and specification of scl+ HE by decreasing the expression of VEGFAa and downstream arterial Notch-pathway components; heat-shock induction of VEGFAa/Notch rescued E2-mediated hemato-vascular defects. Conversely, repression of endogenous E2-activity increased somitic VEGF expression and vascular-target regulation, shifting the assignment of arterial/venous fate and HE localization; blocking E2-signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest yolk-derived estrogen sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral limits of VEGF regulation.

Summary The lymphatic system comprises blind-ended tubes that collect interstitial fluid and return it to the circulatory system. In mammals, unidirectional lymphatic flow is driven by muscle contraction working in conjunction with valves. Accordingly, defective lymphatic valve morphogenesis results in backflow leading to edema. In fish species, studies dating to the 18th century failed to identify lymphatic valves, a precedent that currently persists, raising the question of whether the zebrafish could be used to study the development of these structures. Here, we provide functional and morphological evidence of valves in the zebrafish lymphatic system. Electron microscopy revealed valve ultrastructure similar to mammals, while live imaging using transgenic lines identified the developmental origins of lymphatic valve progenitors. Zebrafish embryos bearing mutations in genes required for mammalian valve morphogenesis show defective lymphatic valve formation and edema. Together, our observations provide a foundation from which to further investigate lymphatic valve formation in zebrafish.

Gene editing in zebrafish has begun to reveal discordance between mutant phenotypes and those associated with knockdown via morpholino oligonucleotides (MOs). These studies suggest that MOs should not be used as a standalone tool and underscore the need for guidelines that require defined mutants to assess gene function in zebrafish.

etv2 is an endothelial-specific ETS transcription factor that is essential for vascular differentiation and morphogenesis in vertebrates. While recent data suggest that Etv2 is dynamically regulated during vascular development, little is known about the mechanisms involved in this process. Here, we find that etv2 transcript and protein expression are highly dynamic during zebrafish vascular development, with both apparent during early somitogenesis and subsequently down-regulated as development proceeds. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis stages, but not later, caused severe vascular defects, suggesting a specific role in early commitment of lateral mesoderm to the endothelial linage. Accordingly, Etv2-overexpressing cells showed an enhanced ability to commit to endothelial lineages in mosaic embryos. We further find that the etv2 3' untranslated region (UTR) is capable of repressing an endothelial autonomous transgene and contains binding sites for members of the let-7 family of microRNAs. Ectopic expression of let-7a could repress the etv2 3'UTR in sensor assays and was also able to block endogenous Etv2 protein expression, leading to concomitant reduction of endothelial genes. Finally, we observed that Etv2 protein levels persisted in maternal-zygotic dicer1 mutant embryos, suggesting that microRNAs contribute to its repression during vascular development. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is then down-regulated, in part through post-transcriptional repression by microRNAs, to allow normal vascular development.