RESUMO
Prime editing efficiency is modest in cells that are quiescent or slowly proliferating where intracellular dNTP levels are tightly regulated. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. We report that prime editing efficiency in primary cells and in vivo is increased by mutations that enhance the enzymatic properties of MMLV-reverse transcriptase and can be further complemented by targeting SAMHD1 for degradation.
RESUMO
The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.
Assuntos
Integrases , Peixe-Zebra , Camundongos , Animais , Camundongos Knockout , Peixe-Zebra/genética , Alelos , Integrases/genética , Técnicas de Inativação de GenesRESUMO
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 â¼13 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.
Assuntos
Temperatura Baixa , Edição de Genes , Peixe-Zebra , Animais , Humanos , Sítios de Ligação , Resposta ao Choque Frio , Sistemas CRISPR-Cas , Mamíferos , Ribonucleoproteínas , Peixe-Zebra/genéticaRESUMO
Single cell ATAC-seq (scATAC-seq) has become the most widely used method for profiling open chromatin landscape of heterogeneous cell populations at a single-cell resolution. Although numerous software tools and pipelines have been developed, an easy-to-use, scalable, reproducible, and comprehensive pipeline for scATAC-seq data analyses is still lacking. To fill this gap, we developed scATACpipe, a Nextflow pipeline, for performing comprehensive analyses of scATAC-seq data including extensive quality assessment, preprocessing, dimension reduction, clustering, peak calling, differential accessibility inference, integration with scRNA-seq data, transcription factor activity and footprinting analysis, co-accessibility inference, and cell trajectory prediction. scATACpipe enables users to perform the end-to-end analysis of scATAC-seq data with three sub-workflow options for preprocessing that leverage 10x Genomics Cell Ranger ATAC software, the ultra-fast Chromap procedures, and a set of custom scripts implementing current best practices for scATAC-seq data preprocessing. The pipeline extends the R package ArchR for downstream analysis with added support to any eukaryotic species with an annotated reference genome. Importantly, scATACpipe generates an all-in-one HTML report for the entire analysis and outputs cluster-specific BAM, BED, and BigWig files for visualization in a genome browser. scATACpipe eliminates the need for users to chain different tools together and facilitates reproducible and comprehensive analyses of scATAC-seq data from raw reads to various biological insights with minimal changes of configuration settings for different computing environments or species. By applying it to public datasets, we illustrated the utility, flexibility, versatility, and reliability of our pipeline, and demonstrated that our scATACpipe outperforms other workflows.
Assuntos
Genoma , Peixe-Zebra , Animais , Genoma/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
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 ß (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.
Assuntos
Miócitos de Músculo Liso , Pericitos , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Células Endoteliais/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericitos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Células Endoteliais , Fator C de Crescimento do Endotélio Vascular , Animais , Artérias , Sinais (Psicologia) , Células Endoteliais/fisiologia , Fator C de Crescimento do Endotélio Vascular/fisiologia , Peixe-ZebraRESUMO
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 Pdgfrß, 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.
Assuntos
Vasos Coronários/crescimento & desenvolvimento , Vasos Coronários/metabolismo , Coração/fisiologia , Organogênese/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Regeneração/fisiologia , Animais , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Miócitos de Músculo Liso/metabolismo , Pericárdio/metabolismo , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologiaRESUMO
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, and comparison with bulk RNA-seq from wild-type and pdgfrb mutant larvae further refined a pericyte gene set. 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, and 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.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericitos/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mutação , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
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.
Assuntos
Vasos Linfáticos , Peixe-Zebra , Animais , Células Endoteliais , Homeostase , Camundongos , Morfogênese , Peixe-Zebra/genéticaRESUMO
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.
Assuntos
Caenorhabditis elegans/genética , Reparo do DNA , DNA de Cadeia Simples/genética , DNA/genética , Edição de Genes/métodos , Camundongos/genética , Peixe-Zebra/genética , Animais , Células HEK293 , Humanos , Células K562RESUMO
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.
Assuntos
Músculo Liso Vascular/metabolismo , Pericitos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Diferenciação Celular , Vasos Coronários/metabolismo , Desenvolvimento Embrionário , Músculo Liso Vascular/embriologia , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Proteínas Proto-Oncogênicas c-sis/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Pontos de Checagem do Ciclo Celular , Células Endoteliais , Vasos Linfáticos , Neovascularização Fisiológica , Fator C de Crescimento do Endotélio Vascular , Veias , Proteínas de Peixe-Zebra , Animais , Animais Geneticamente Modificados , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fase G1 , Vasos Linfáticos/citologia , Sistema de Sinalização das MAP Quinases , Neovascularização Fisiológica/efeitos dos fármacos , Roscovitina/farmacologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Veias/citologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Adaptação Fisiológica , Movimento Celular , Meio Ambiente , Homeostase , Sistema da Linha Lateral/citologia , Peixe-Zebra/fisiologia , Animais , Biomarcadores/metabolismo , Contagem de Células , Fatores de Transcrição Forkhead/metabolismo , Brânquias/citologia , Células Ciliadas Auditivas/citologia , Concentração de Íons de Hidrogênio , Imageamento Tridimensional , Receptores Notch/metabolismo , Salinidade , Transdução de Sinais , Pele/citologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Anotação de Sequência Molecular/métodos , Transcriptoma , Peixe-Zebra/genética , Regiões 3' não Traduzidas , Animais , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Ontologia Genética , Genoma , Análise de Sequência de RNARESUMO
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.
Assuntos
Artérias/metabolismo , Fator II de Transcrição COUP/genética , Células Endoteliais/metabolismo , Elementos Facilitadores Genéticos/genética , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Veias/metabolismo , Artérias/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator II de Transcrição COUP/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina/métodos , Regulação da Expressão Gênica , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Veias/citologiaRESUMO
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.
Assuntos
Vasos Linfáticos/embriologia , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Embrião não Mamífero/metabolismo , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/ultraestrutura , Face/anatomia & histologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Imageamento Tridimensional , Larva/anatomia & histologia , Larva/metabolismo , Vasos Linfáticos/anatomia & histologia , Vasos Linfáticos/ultraestrutura , Camundongos , Morfogênese , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Vasos Linfáticos , Peixe-Zebra , Animais , Encéfalo , Células Endoteliais , Camundongos , RegeneraçãoRESUMO
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 pdgfrß 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.