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.

RIPK3 cleavage is dispensable for necroptosis inhibition but restricts NLRP3 inflammasome activation.

Caspase-8 activity is required to inhibit necroptosis during embryogenesis in mice. In vitro studies have suggested that caspase-8 directly cleaves RIPK1, CYLD and the key necroptotic effector kinase RIPK3 to repress necroptosis. However, recent studies have shown that mice expressing uncleavable RIPK1 die during embryogenesis due to excessive apoptosis, while uncleavable CYLD mice are viable. Therefore, these results raise important questions about the role of RIPK3 cleavage. To evaluate the physiological significance of RIPK3 cleavage, we generated Ripk3D333A/D333A mice harbouring a point mutation in the conserved caspase-8 cleavage site. These mice are viable, demonstrating that RIPK3 cleavage is not essential for blocking necroptosis during development. Furthermore, unlike RIPK1 cleavage-resistant cells, Ripk3D333A/D333A cells were not significantly more sensitive to necroptotic stimuli. Instead, we found that the cleavage of RIPK3 by caspase-8 restricts NLRP3 inflammasome activation-dependent pyroptosis and IL-1ß secretion when Inhibitors of APoptosis (IAP) are limited. These results demonstrate that caspase-8 does not inhibit necroptosis by directly cleaving RIPK3 and further underscore a role for RIPK3 in regulating the NLRP3 inflammasome.

Hyaluronic acid turnover controls the severity of cerebral cavernous malformations in bioengineered human micro-vessels.

Cerebral cavernous malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system upon loss of the CCM multimeric protein complex. The endothelial cells within CCM lesions are characterized by overactive MEKK3 kinase and KLF2/4 transcription factor signaling, leading to pathological changes such as increased endothelial cell spreading and reduced junctional integrity. Concomitant to aberrant endothelial cell signaling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the central nervous system. Here, we adapted a three-dimensional microfluidic system to examine CCM1 deficient human micro-vessels in distinctive extracellular matrices. We validate that pathological hallmarks are maintained in this model. We further show that key genes responsible for homeostasis of hyaluronic acid, a major extracellular matrix component of the central nervous system, are dysregulated in CCM. Supplementing the matrix in our model with distinct forms of hyaluronic acid inhibits pathological cell spreading and rescues barrier function. Hyaluronic acid acts by dampening cell-matrix adhesion signaling in CCM, either downstream or in parallel of KLF2/4. This study provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signaling impact vascular malformations.

Canthin-6-One Inhibits Developmental and Tumour-Associated Angiogenesis in Zebrafish.

Tumour-associated angiogenesis play key roles in tumour growth and cancer metastasis. Consequently, several anti-angiogenic drugs such as sunitinib and axitinib have been approved for use as anti-cancer therapies. However, the majority of these drugs target the vascular endothelial growth factor A (VEGFA)/VEGF receptor 2 (VEGFR2) pathway and have shown mixed outcome, largely due to development of resistances and increased tumour aggressiveness. In this study, we used the zebrafish model to screen for novel anti-angiogenic molecules from a library of compounds derived from natural products. From this, we identified canthin-6-one, an indole alkaloid, which inhibited zebrafish intersegmental vessel (ISV) and sub-intestinal vessel development. Further characterisation revealed that treatment of canthin-6-one reduced ISV endothelial cell number and inhibited proliferation of human umbilical vein endothelial cells (HUVECs), suggesting that canthin-6-one inhibits endothelial cell proliferation. Of note, canthin-6-one did not inhibit VEGFA-induced phosphorylation of VEGFR2 in HUVECs and downstream phosphorylation of extracellular signal-regulated kinase (Erk) in leading ISV endothelial cells in zebrafish, suggesting that canthin-6-one inhibits angiogenesis independent of the VEGFA/VEGFR2 pathway. Importantly, we found that canthin-6-one impairs tumour-associated angiogenesis in a zebrafish B16F10 melanoma cell xenograft model and synergises with VEGFR inhibitor sunitinib malate to inhibit developmental angiogenesis. In summary, we showed that canthin-6-one exhibits anti-angiogenic properties in both developmental and pathological contexts in zebrafish, independent of the VEGFA/VEGFR2 pathway and demonstrate that canthin-6-one may hold value for further development as a novel anti-angiogenic drug.

HOPX-associated molecular programs control cardiomyocyte cell states underpinning cardiac structure and function.

Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function.

Vasculature is getting Hip(po): Hippo signaling in vascular development and disease.

The Hippo signaling pathway regulates developmental organ growth, regeneration, and cell fate decisions. Although the role of the Hippo pathway, and its transcriptional effectors YAP and TAZ, has been well documented in many cell types and species, only recently have the roles for this pathway come to light in vascular development and disease. Experiments in mice, zebrafish, and in vitro have uncovered roles for the Hippo pathway, YAP, and TAZ in vasculogenesis, angiogenesis, and lymphangiogenesis. In addition, the Hippo pathway has been implicated in vascular cancers and cardiovascular diseases, thus identifying it as a potential therapeutic target for the treatment of these conditions. However, despite recent advances, Hippo's role in the vasculature is still underappreciated compared with its role in epithelial tissues. In this review, we appraise our current understanding of the Hippo pathway in blood and lymphatic vessel development and highlight the current knowledge gaps and opportunities for further research.

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.

ahctf1 and kras mutations combine to amplify oncogenic stress and restrict liver overgrowth in a zebrafish model of hepatocellular carcinoma.

The nucleoporin (NUP) ELYS, encoded by AHCTF1, is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using both larval and adult zebrafish models of hepatocellular carcinoma (HCC), in which the expression of an inducible mutant kras transgene (krasG12V) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly decreases liver volume, while non-hyperplastic tissues are unaffected. We demonstrate that in the context of cancer, ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly, and chromosome segregation, leading to DNA damage and activation of a Tp53-dependent transcriptional programme that induces cell death and cell cycle arrest. Heterozygous expression of both ahctf1 and ranbp2 (encoding a second nucleoporin), or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocks krasG12V-driven hepatocyte hyperplasia. Gene expression analysis of patient samples in the liver hepatocellular carcinoma (LIHC) dataset in The Cancer Genome Atlas shows that high expression of one or more of the transcripts encoding the 10 components of the NUP107-160 subcomplex, which includes AHCTF1, is positively correlated with worse overall survival. These results provide a strong and feasible rationale for the development of novel cancer therapeutics that target ELYS function and suggest potential avenues for effective combinatorial treatments.

Plexin D1 negatively regulates zebrafish lymphatic development.

Lymphangiogenesis is a dynamic process that involves the directed migration of lymphatic endothelial cells (LECs) to form lymphatic vessels. The molecular mechanisms that underpin lymphatic vessel patterning are not fully elucidated and, to date, no global regulator of lymphatic vessel guidance is known. In this study, we identify the transmembrane cell signalling receptor Plexin D1 (Plxnd1) as a negative regulator of both lymphatic vessel guidance and lymphangiogenesis in zebrafish. plxnd1 is expressed in developing lymphatics and is required for the guidance of both the trunk and facial lymphatic networks. Loss of plxnd1 is associated with misguided intersegmental lymphatic vessel growth and aberrant facial lymphatic branches. Lymphatic guidance in the trunk is mediated, at least in part, by the Plxnd1 ligands, Semaphorin 3AA and Semaphorin 3C. Finally, we show that Plxnd1 normally antagonises Vegfr/Erk signalling to ensure the correct number of facial LECs and that loss of plxnd1 results in facial lymphatic hyperplasia. As a global negative regulator of lymphatic vessel development, the Sema/Plxnd1 signalling pathway is a potential therapeutic target for treating diseases associated with dysregulated lymphatic growth.

Dynamically regulated focal adhesions coordinate endothelial cell remodelling in developing vasculature.

The assembly of a mature vascular network involves coordinated endothelial cell (EC) shape changes, including the process of EC elongation. How EC elongation is dynamically regulated in vivo is not fully understood. Here, we have generated a zebrafish mutant that is deficient for the integrin adaptor protein Talin 1 (Tln1). Using a new focal adhesion (FA) marker line expressing endothelial Vinculinb-eGFP, we demonstrate that EC FAs function dynamically and are lost in our tln1 mutants, allowing us to uncouple the primary roles of FAs in EC morphogenesis from the secondary effects that occur due to systemic vessel failure or loss of blood flow. Tln1 loss led to compromised F-actin rearrangements, perturbed EC elongation and disrupted cell-cell junction linearisation in vessel remodelling. Finally, chemical induction of actin polymerisation restored actin dynamics and EC elongation during vascular morphogenesis. Together, we identify that FAs are essential for EC elongation and junction linearisation in flow-pressured vessels and that they influence actin polymerisation in cellular morphogenesis. These observations can explain the severely compromised vessel beds and vascular leakage observed in mutant models that lack integrin signalling. This article has an associated 'The people behind the papers' interview.

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.

Blood vessel occlusion by Cryptococcus neoformans is a mechanism for haemorrhagic dissemination of infection.

Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis.

Pkd1 and Wnt5a genetically interact to control lymphatic vascular morphogenesis in mice.

BACKGROUND: Lymphatic vascular development is regulated by well-characterized signaling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity, and morphogenesis. Canonical and non-canonical WNT signaling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. RESULTS: Here we find that loss of non-canonical WNT signaling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1;Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1-/- mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a-/- mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signaling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. CONCLUSION: Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signaling during the formation of lymphatic vascular networks.

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.

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.

Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis.

The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair, and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation, and migration. Extracellular signal-regulated kinase (Erk) is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response, however, involves a Vegfr-dependent mechanism that initiates concomitantly with the wound inflammatory response. This work reveals a highly dynamic sequence of signalling events in regenerative angiogenesis and validates a new resource for the study of vascular Erk-signalling in real-time.

Network patterning, morphogenesis and growth in lymphatic vascular development.

The lymphatic vasculature is a vital component of the vertebrate vascular system that mediates tissue fluid homeostasis, lipid uptake and immune surveillance. The development of the lymphatic vasculature starts in the early vertebrate embryo, when a subset of blood vascular endothelial cells of the cardinal veins acquires lymphatic endothelial cell fate. These cells sprout from the veins, migrate, proliferate and organize to give rise to a highly structured and unique vascular network. Cellular cross-talk, cell-cell communication and the interpretation of signals from surrounding tissues are all essential for coordinating these processes. In this chapter, we highlight new findings and review research progress with a particular focus on LEC migration and guidance, expansion of the LEC lineage, network remodeling and morphogenesis of the lymphatic vasculature.

The zebrafish grime mutant uncovers an evolutionarily conserved role for Tmem161b in the control of cardiac rhythm.

The establishment of cardiac function in the developing embryo is essential to ensure blood flow and, therefore, growth and survival of the animal. The molecular mechanisms controlling normal cardiac rhythm remain to be fully elucidated. From a forward genetic screen, we identified a unique mutant, grime, that displayed a specific cardiac arrhythmia phenotype. We show that loss-of-function mutations in tmem161b are responsible for the phenotype, identifying Tmem161b as a regulator of cardiac rhythm in zebrafish. To examine the evolutionary conservation of this function, we generated knockout mice for Tmem161b. Tmem161b knockout mice are neonatal lethal and cardiomyocytes exhibit arrhythmic calcium oscillations. Mechanistically, we find that Tmem161b is expressed at the cell membrane of excitable cells and live imaging shows it is required for action potential repolarization in the developing heart. Electrophysiology on isolated cardiomyocytes demonstrates that Tmem161b is essential to inhibit Ca2+ and K+ currents in cardiomyocytes. Importantly, Tmem161b haploinsufficiency leads to cardiac rhythm phenotypes, implicating it as a candidate gene in heritable cardiac arrhythmia. Overall, these data describe Tmem161b as a highly conserved regulator of cardiac rhythm that functions to modulate ion channel activity in zebrafish and mice.