Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Single-cell analysis of lymphatic endothelial cell fate specification and differentiation during zebrafish development.

During development, the lymphatic vasculature forms as a second network derived chiefly from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is a key step in this process. Specification, differentiation and maintenance of LEC fate are all driven by the transcription factor Prox1, yet the downstream mechanisms remain to be elucidated. We here present a single-cell transcriptomic atlas of lymphangiogenesis in zebrafish, revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single-cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a LEC-VEC fate shift. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new downstream targets and regulatory regions of the genome controlled by Prox1 and presents evidence that Prox1 specifies LEC fate primarily by limiting blood vascular and haematopoietic fate. This extensive single-cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.

A Prox1 enhancer represses haematopoiesis in the lymphatic vasculature.

Transcriptional enhancer elements are responsible for orchestrating the temporal and spatial control over gene expression that is crucial for programming cell identity during development1-3. Here we describe a novel enhancer element that is important for regulating the expression of Prox1 in lymphatic endothelial cells. This evolutionarily conserved enhancer is bound by key lymphatic transcriptional regulators including GATA2, FOXC2, NFATC1 and PROX1. Genome editing of the enhancer to remove five nucleotides encompassing the GATA2-binding site resulted in perinatal death of homozygous mutant mice due to profound lymphatic vascular defects. Lymphatic endothelial cells in enhancer mutant mice exhibited reduced expression of genes characteristic of lymphatic endothelial cell identity and increased expression of genes characteristic of haemogenic endothelium, and acquired the capacity to generate haematopoietic cells. These data not only reveal a transcriptional enhancer element important for regulating Prox1 expression and lymphatic endothelial cell identity but also demonstrate that the lymphatic endothelium has haemogenic capacity, ordinarily repressed by Prox1.

Claudin5 protects the peripheral endothelial barrier in an organ and vessel-type-specific manner.

Dysfunctional and leaky blood vessels resulting from disruption of the endothelial cell (EC) barrier accompanies numerous diseases. The EC barrier is established through endothelial cell tight and adherens junctions. However, the expression pattern and precise contribution of different junctional proteins to the EC barrier is poorly understood. Here, we focus on organs with continuous endothelium to identify structural and functional in vivo characteristics of the EC barrier. Assembly of multiple single-cell RNAseq datasets into a single integrated database revealed the variability and commonalities of EC barrier patterning. Across tissues, Claudin5 exhibited diminishing expression along the arteriovenous axis, correlating with EC barrier integrity. Functional analysis identified tissue-specific differences in leakage properties and response to the leakage agonist histamine. Loss of Claudin5 enhanced histamine-induced leakage in an organotypic and vessel type-specific manner in an inducible, EC-specific, knock-out mouse. Mechanistically, Claudin5 loss left junction ultrastructure unaffected but altered its composition, with concomitant loss of zonula occludens-1 and upregulation of VE-Cadherin expression. These findings uncover the organ-specific organisation of the EC barrier and distinct importance of Claudin5 in different vascular beds, providing insights to modify EC barrier stability in a targeted, organ-specific manner.

mafba and mafbb differentially regulate lymphatic endothelial cell migration in topographically distinct manners.

Lymphangiogenesis, formation of lymphatic vessels from pre-existing vessels, is a dynamic process that requires cell migration. Regardless of location, migrating lymphatic endothelial cell (LEC) progenitors probe their surroundings to form the lymphatic network. Lymphatic-development regulation requires the transcription factor MAFB in different species. Zebrafish Mafba, expressed in LEC progenitors, is essential for their migration in the trunk. However, the transcriptional mechanism that orchestrates LEC migration in different lymphatic endothelial beds remains elusive. Here, we uncover topographically different requirements of the two paralogs, Mafba and Mafbb, for LEC migration. Both mafba and mafbb are necessary for facial lymphatic development, but mafbb is dispensable for trunk lymphatic development. On the molecular level, we demonstrate a regulatory network where Vegfc-Vegfd-SoxF-Mafba-Mafbb is essential in facial lymphangiogenesis. We identify that mafba and mafbb tune the directionality of LEC migration and vessel morphogenesis that is ultimately necessary for lymphatic function.

Epigenetic Regulation of Endothelial Cell Lineages During Zebrafish Development-New Insights From Technical Advances.

Epigenetic regulation is integral in orchestrating the spatiotemporal regulation of gene expression which underlies tissue development. The emergence of new tools to assess genome-wide epigenetic modifications has enabled significant advances in the field of vascular biology in zebrafish. Zebrafish represents a powerful model to investigate the activity of cis-regulatory elements in vivo by combining technologies such as ATAC-seq, ChIP-seq and CUT&Tag with the generation of transgenic lines and live imaging to validate the activity of these regulatory elements. Recently, this approach led to the identification and characterization of key enhancers of important vascular genes, such as gata2a, notch1b and dll4. In this review we will discuss how the latest technologies in epigenetics are being used in the zebrafish to determine chromatin states and assess the function of the cis-regulatory sequences that shape the zebrafish vascular network.

Proper migration of lymphatic endothelial cells requires survival and guidance cues from arterial mural cells.

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.

Real-time evaluation of glioblastoma growth in patient-specific zebrafish xenografts.

BACKGROUND: Patient-derived xenograft (PDX) models of glioblastoma (GBM) are a central tool for neuro-oncology research and drug development, enabling the detection of patient-specific differences in growth, and in vivo drug response. However, existing PDX models are not well suited for large-scale or automated studies. Thus, here, we investigate if a fast zebrafish-based PDX model, supported by longitudinal, AI-driven image analysis, can recapitulate key aspects of glioblastoma growth and enable case-comparative drug testing. METHODS: We engrafted 11 GFP-tagged patient-derived GBM IDH wild-type cell cultures (PDCs) into 1-day-old zebrafish embryos, and monitored fish with 96-well live microscopy and convolutional neural network analysis. Using light-sheet imaging of whole embryos, we analyzed further the invasive growth of tumor cells. RESULTS: Our pipeline enables automatic and robust longitudinal observation of tumor growth and survival of individual fish. The 11 PDCs expressed growth, invasion and survival heterogeneity, and tumor initiation correlated strongly with matched mouse PDX counterparts (Spearman R = 0.89, p < 0.001). Three PDCs showed a high degree of association between grafted tumor cells and host blood vessels, suggesting a perivascular invasion phenotype. In vivo evaluation of the drug marizomib, currently in clinical trials for GBM, showed an effect on fish survival corresponding to PDC in vitro and in vivo marizomib sensitivity. CONCLUSIONS: Zebrafish xenografts of GBM, monitored by AI methods in an automated process, present a scalable alternative to mouse xenograft models for the study of glioblastoma tumor initiation, growth, and invasion, applicable to patient-specific drug evaluation.

The RNA helicase Ddx21 controls Vegfc-driven developmental lymphangiogenesis by balancing endothelial cell ribosome biogenesis and p53 function.

The development of a functional vasculature requires the coordinated control of cell fate, lineage differentiation and network growth. Cellular proliferation is spatiotemporally regulated in developing vessels, but how this is orchestrated in different lineages is unknown. Here, using a zebrafish genetic screen for lymphatic-deficient mutants, we uncover a mutant for the RNA helicase Ddx21. Ddx21 cell-autonomously regulates lymphatic vessel development. An established regulator of ribosomal RNA synthesis and ribosome biogenesis, Ddx21 is enriched in sprouting venous endothelial cells in response to Vegfc-Flt4 signalling. Ddx21 function is essential for Vegfc-Flt4-driven endothelial cell proliferation. In the absence of Ddx21, endothelial cells show reduced ribosome biogenesis, p53 and p21 upregulation and cell cycle arrest that blocks lymphangiogenesis. Thus, Ddx21 coordinates the lymphatic endothelial cell response to Vegfc-Flt4 signalling by balancing ribosome biogenesis and p53 function. This mechanism may be targetable in diseases of excessive lymphangiogenesis such as cancer metastasis or lymphatic malformation.

Homeostatic maintenance of the lymphatic vasculature.

The lymphatic vasculature is emerging as a multifaceted regulator of tissue homeostasis and regeneration. Lymphatic vessels drain fluid, macromolecules, and immune cells from peripheral tissues to lymph nodes (LNs) and the systemic circulation. Their recently uncovered functions extend beyond drainage and include direct modulation of adaptive immunity and paracrine regulation of organ growth. The developmental mechanisms controlling lymphatic vessel growth have been described with increasing precision. It is less clear how the essential functional features of lymphatic vessels are established and maintained. We discuss the mechanisms that maintain lymphatic vessel integrity in adult tissues and control vessel repair and regeneration. This knowledge is crucial for understanding the pathological vessel changes that contribute to disease, and provides an opportunity for therapy development.

3,4-Difluorobenzocurcumin Inhibits Vegfc-Vegfr3-Erk Signalling to Block Developmental Lymphangiogenesis in Zebrafish.

Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing vasculature, plays critical roles in disease, including in cancer metastasis and chronic inflammation. Preclinical and recent clinical studies have now demonstrated therapeutic utility for several anti-lymphangiogenic agents, but optimal agents and efficacy in different settings remain to be determined. We tested the anti-lymphangiogenic property of 3,4-Difluorobenzocurcumin (CDF), which has previously been implicated as an anti-cancer agent, using zebrafish embryos and cultured vascular endothelial cells. We used transgenic zebrafish labelling the lymphatic system and found that CDF potently inhibits lymphangiogenesis during embryonic development. We also found that the parent compound, Curcumin, does not inhibit lymphangiogenesis. CDF blocked lymphatic and venous sprouting, and lymphatic migration in the head and trunk of the embryo. Mechanistically, CDF impaired VEGFC-VEGFR3-ERK signalling in vitro and in vivo. In an in vivo pathological model of Vegfc-overexpression, treatment with CDF rescued endothelial cell hyperplasia. CDF did not inhibit the kinase activity of VEGFR3 yet displayed more prolonged activity in vivo than previously reported kinase inhibitors. These findings warrant further assessment of CDF and its mode of action as a candidate for use in metastasis and diseases of aberrant lymphangiogenesis.

Vasohibin 1 selectively regulates secondary sprouting and lymphangiogenesis in the zebrafish trunk.

Previous studies have shown that Vasohibin 1 (Vash1) is stimulated by VEGFs in endothelial cells and that its overexpression interferes with angiogenesis in vivo Recently, Vash1 was found to mediate tubulin detyrosination, a post-translational modification that is implicated in many cell functions, such as cell division. Here, we used the zebrafish embryo to investigate the cellular and subcellular mechanisms of Vash1 on endothelial microtubules during formation of the trunk vasculature. We show that microtubules within venous-derived secondary sprouts are strongly and selectively detyrosinated in comparison with other endothelial cells, and that this difference is lost upon vash1 knockdown. Vash1 depletion in zebrafish specifically affected secondary sprouting from the posterior cardinal vein, increasing endothelial cell divisions and cell number in the sprouts. We show that altering secondary sprout numbers and structure upon Vash1 depletion leads to defective lymphatic vessel formation and ectopic lymphatic progenitor specification in the zebrafish trunk.

Localised Collagen2a1 secretion supports lymphatic endothelial cell migration in the zebrafish embryo.

The lymphatic vasculature develops primarily from pre-existing veins. A pool of lymphatic endothelial cells (LECs) first sprouts from cardinal veins followed by migration and proliferation to colonise embryonic tissues. Although much is known about the molecular regulation of LEC fate and sprouting during early lymphangiogenesis, we know far less about the instructive and permissive signals that support LEC migration through the embryo. Using a forward genetic screen, we identified mbtps1 and sec23a, components of the COP-II protein secretory pathway, as essential for developmental lymphangiogenesis. In both mutants, LECs initially depart the cardinal vein but then fail in their ongoing migration. A key cargo that failed to be secreted in both mutants was a type II collagen (Col2a1). Col2a1 is normally secreted by notochord sheath cells, alongside which LECs migrate. col2a1a mutants displayed defects in the migratory behaviour of LECs and failed lymphangiogenesis. These studies thus identify Col2a1 as a key cargo secreted by notochord sheath cells and required for the migration of LECs. These findings combine with our current understanding to suggest that successive cell-to-cell and cell-matrix interactions regulate the migration of LECs through the embryonic environment during development.

MAFB modulates the maturation of lymphatic vascular networks in mice.

BACKGROUND: Lymphatic vessels play key roles in tissue fluid homeostasis, immune cell trafficking and in diverse disease settings. Lymphangiogenesis requires lymphatic endothelial cell (LEC) differentiation, proliferation, migration, and co-ordinated network formation, yet the transcriptional regulators underpinning these processes remain to be fully understood. The transcription factor MAFB was recently identified as essential for lymphangiogenesis in zebrafish and in cultured human LECs. MAFB is activated in response to VEGFC-VEGFR3 signaling and acts as a downstream effector. However, it remains unclear if the role of MAFB in lymphatic development is conserved in the mammalian embryo. RESULTS: We generated a Mafb loss-of-function mouse using CRISPR/Cas9 gene editing. Mafb mutant mice presented with perinatal lethality associated with cyanosis. We identify a role for MAFB in modifying lymphatic network morphogenesis in the developing dermis, as well as developing and postnatal diaphragm. Furthermore, mutant vessels displayed excessive smooth muscle cell coverage, suggestive of a defect in the maturation of lymphatic networks. CONCLUSIONS: This work confirms a conserved role for MAFB in murine lymphatics that is subtle and modulatory and may suggest redundancy in MAF family transcription factors during lymphangiogenesis.

Yap1 promotes sprouting and proliferation of lymphatic progenitors downstream of Vegfc in the zebrafish trunk.

Lymphatic vascular development involves specification of lymphatic endothelial progenitors that subsequently undergo sprouting, proliferation and tissue growth to form a complex second vasculature. The Hippo pathway and effectors Yap and Taz control organ growth and regulate morphogenesis and cellular proliferation. Yap and Taz control angiogenesis but a role in lymphangiogenesis remains to be fully elucidated. Here we show that YAP displays dynamic changes in lymphatic progenitors and Yap1 is essential for lymphatic vascular development in zebrafish. Maternal and Zygotic (MZ) yap1 mutants show normal specification of lymphatic progenitors, abnormal cellular sprouting and reduced numbers of lymphatic progenitors emerging from the cardinal vein during lymphangiogenesis. Furthermore, Yap1 is indispensable for Vegfc-induced proliferation in a transgenic model of Vegfc overexpression. Paracrine Vegfc-signalling ultimately increases nuclear YAP in lymphatic progenitors to control lymphatic development. We thus identify a role for Yap in lymphangiogenesis, acting downstream of Vegfc to promote expansion of this vascular lineage.

The Alternative Splicing Regulator Nova2 Constrains Vascular Erk Signaling to Limit Specification of the Lymphatic Lineage.

The correct assignment of cell fate within fields of multipotent progenitors is essential for accurate tissue diversification. The first lymphatic vessels arise from pre-existing veins after venous endothelial cells become specified as lymphatic progenitors. Prox1 specifies lymphatic fate and labels these progenitors; however, the mechanisms restricting Prox1 expression and limiting the progenitor pool remain unknown. We identified a zebrafish mutant that displayed premature, expanded, and prolonged lymphatic specification. The gene responsible encodes the regulator of alternative splicing, Nova2. In zebrafish and human endothelial cells, Nova2 selectively regulates pre-mRNA splicing for components of signaling pathways and phosphoproteins. Nova2-deficient endothelial cells display increased Mapk/Erk signaling, and Prox1 expression is dynamically controlled by Erk signaling. We identify a mechanism whereby Nova2-regulated splicing constrains Erk signaling, thus limiting lymphatic progenitor cell specification. This identifies the capacity of a factor that tunes mRNA splicing to control assignment of cell fate during vascular differentiation.

Peri-arterial specification of vascular mural cells from naïve mesenchyme requires Notch signaling.

Mural cells (MCs) are essential for blood vessel stability and function; however, the mechanisms that regulate MC development remain incompletely understood, in particular those involved in MC specification. Here, we investigated the first steps of MC formation in zebrafish using transgenic reporters. Using pdgfrb and abcc9 reporters, we show that the onset of expression of abcc9, a pericyte marker in adult mice and zebrafish, occurs almost coincidentally with an increment in pdgfrb expression in peri-arterial mesenchymal cells, suggesting that these transcriptional changes mark the specification of MC lineage cells from naïve pdgfrblow mesenchymal cells. The emergence of peri-arterial pdgfrbhigh MCs required Notch signaling. We found that pdgfrb-positive cells express notch2 in addition to notch3, and although depletion of notch2 or notch3 failed to block MC emergence, embryos depleted of both notch2 and notch3 lost mesoderm- as well as neural crest-derived pdgfrbhigh MCs. Using reporters that read out Notch signaling and Notch2 receptor cleavage, we show that Notch activation in the mesenchyme precedes specification into pdgfrbhigh MCs. Taken together, these results show that Notch signaling is necessary for peri-arterial MC specification.

Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish.

Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc-Vegfd-Ccbe1-Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.