An interplay of growth factors and signaling pathways governs the development and maintenance of lymphatic vasculature, ensuring proper fluid homeostasis and immune function. Disruption of these regulatory mechanisms can lead to congenital lymphatic disorders and contribute to various pathological conditions. However, the mechanisms underlying the molecular regulation of these processes remain elusive. Here, we reveal a critical and previously unappreciated role for the signaling scaffold protein Shoc2 in lymphangiogenesis. We demonstrate that loss of Shoc2 results in near-complete loss of lymphatic vasculature in vivo and senescence of lymphatic endothelial cells in vitro. Mechanistically, Shoc2 is required for balancing signaling through the ERK1/2 pathway, and its loss results in increased mTORC1 signaling. This dysregulation impairs mitochondrial respiration and triggers an IRF/IFN-II response, ultimately leading to cellular senescence. Strikingly, expression of the Noonan Syndrome with Loose anagen Hair (NSLH)-causing Shoc2 variant S2G phenocopies the effects of Shoc2 loss. Together, these studies establish the critical role of Shoc2 in lymphangiogenesis and uncover a novel mechanistic link between Shoc2 signaling, mitochondrial function, innate immune response, and lymphatic development, with significant implications for Ras-pathway-related congenital disorders.

Vascular endothelial cells in vivo are exquisitely regulated by their local environment, which is disrupted or absent when using methods such as FACS sorting of cells isolated from animals or in vitro cell culture. Here, we profile the gene expression patterns of undisturbed endothelial cells in living animals using a novel “AngioTag” zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope tagged Rpl10a 60 S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. By performing TRAP-RNAseq on AngioTag animals, we demonstrate strong enrichment of endothelial-specific genes and have uncovered both novel endothelial genes and unique endothelial gene expression signatures for different adult organs. Finally, we generated a versatile “UAS: RiboTag” transgenic line to allow a wider array of different zebrafish cell and tissue types to be examined using TRAP-RNAseq methods. These new tools offer an unparalleled resource to study the molecular identity of cells in their normal in vivo context.

The pulmonary vasculature plays a critical role in gas exchange and in lung pathologies, but it is challenging to observe and experimentally manipulate deep within the lungs of living mammals. Unlike mammalian lungs, externally located zebrafish gills are readily accessible for high-resolution optical imaging and experimental manipulation, suggesting zebrafish might provide an excellent comparative vertebrate model for studying the development and function of gas-exchange organs and the gas-exchange blood vasculature. To characterize their resident cell populations, we performed single-cell RNA sequencing (scRNAseq) on adult zebrafish gills, revealing numerous cell types with transcriptional similarities to those found in mammalian lungs. We uncovered and characterized several different endothelial cell populations, including distinct clusters of arterial endothelial cells and lymphatic endothelial cells. The largest endothelial cell cluster closely resembles Cap2 or “Aerocyte” endothelial cells, a recently discovered unusual mammalian endothelial cell type found exclusively in lung alveoli. Zebrafish aerocytes localize to the analogous gas-exchange structures in fish, the highly vascularized gill lamellae. We use confocal and super-resolution imaging of transgenic and hybridization chain reaction-probed zebrafish, array tomography, and focused ion beam scanning electron microscopy to carry out a detailed and comprehensive characterization of gill aerocytes including 3-D ultrastructural reconstruction of one of these cells, showing that as in mammals these cells are closely associated with gas-exchange epithelia and that they possess unique properties that may help facilitate their gas-exchange function. Together, our findings help establish a new, experimentally accessible comparative vertebrate model for studying the gas-exchange blood vasculature.

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis.

ABSTRACTDynein cytoplasmic 1 light intermediate chain 1 (LIC1,DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified adync1li1zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis.In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling.In vivo, endothelial-specific expression of constitutively activeRab11aleads to excessive angiogenesis, similar to thedync1li1mutants. Increased angiogenesis is also evident in zebrafish harboring mutations inrilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis.

Salivary complement inhibitors occur in many of the blood feeding arthropod species responsible for transmission of pathogens. During feeding, these inhibitors prevent the production of proinflammatory anaphylatoxins, which may interfere with feeding, and limit formation of the membrane attack complex which could damage arthropod gut tissues. Salivary inhibitors are, in many cases, novel proteins which may be pharmaceutically useful or display unusual mechanisms that could be exploited pharmaceutically. Albicin is a potent inhibitor of the alternative pathway of complement from the saliva of the malaria transmitting mosquito, Anopheles albimanus. Here we describe the cryo-EM structure of albicin bound to C3bBb, the alternative C3 convertase, a proteolytic complex that is responsible for cleavage of C3 and amplification of the complement response. Albicin is shown to induce dimerization of C3bBb, in a manner similar to the bacterial inhibitor SCIN, to form an inactive complex unable to bind the substrate C3. Size exclusion chromatography and structures determined after 30 minutes of incubation of C3b, factor B (FB), factor D (FD) and albicin indicate that FBb dissociates from the inhibited dimeric complex leaving a C3b-albicin dimeric complex which apparently decays more slowly. Structural analysis using cryo-EM reveals that albicin, an inhibitor of the alternative pathway of complement from the saliva of a mosquito, binds the C3bBb complex through contacts with both chains of C3b as well as the protease factor B.

Inhibitors of complement and coagulation are present in the saliva of a variety of blood feeding arthropods that transmit parasitic and viral pathogens. Here we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of the serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all β-sheet fold containing two domains. A structure of the lufaxin-C3bB complex obtained by cryo-electron microscopy shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation where proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex the inhibitor binds both targets simultaneously and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.

Anti-angiogenic therapies have generated significant interest for their potential to combat tumor growth. However, tumor overproduction of pro-angiogenic ligands can overcome these therapies, hampering success of this approach. To circumvent this problem, we target the resynthesis of phosphoinositides consumed during intracellular transduction of pro-angiogenic signals in endothelial cells (EC), thus harnessing the tumor’s own production of excess stimulatory ligands to deplete adjacent ECs of the capacity to respond to these signals. Using zebrafish and human endothelial cells in vitro, we show ECs deficient in CDP-diacylglycerol synthase 2 are uniquely sensitive to increased vascular endothelial growth factor (VEGF) stimulation due to a reduced capacity to re-synthesize phosphoinositides, including phosphatidylinositol-(4,5)-bisphosphate (PIP2), resulting in VEGF-exacerbated defects in angiogenesis and angiogenic signaling. Using murine tumor allograft models, we show that systemic or EC specific suppression of phosphoinositide recycling results in reduced tumor growth and tumor angiogenesis. Our results suggest inhibition of phosphoinositide recycling provides a useful anti-angiogenic approach.

Rationale: The recent discovery of meningeal lymphatics in mammals is reshaping our understanding of fluid homeostasis and cellular waste management in the brain, but visualization and experimental analysis of these vessels is challenging in mammals. Although the optical clarity and experimental advantages of zebrafish have made this an essential model organism for studying lymphatic development, the existence of meningeal lymphatics has not yet been reported in this species. Objective: Examine the intracranial space of larval, juvenile, and adult zebrafish to determine whether and where intracranial lymphatic vessels are present. Methods and Results: Using high-resolution optical imaging of the meninges in living animals, we show that zebrafish possess a meningeal lymphatic network comparable to that found in mammals. We confirm that this network is separate from the blood vascular network and that it drains interstitial fluid from the brain. We document the developmental origins and growth of these vessels into a distinct network separated from the external lymphatics. Finally, we show that these vessels contain immune cells and perform live imaging of immune cell trafficking and transmigration in meningeal lymphatics. Conclusions: This discovery establishes the zebrafish as a important new model for experimental analysis of meningeal lymphatic development and opens up new avenues for probing meningeal lymphatic function in health and disease.

The preferential accumulation of vascular smooth muscle cells (vSMCs) on arteries versus veins during early development is a well-described phenomenon, but the molecular pathways underlying this polarization are not well understood. In zebrafish, the cxcr4a receptor (mammalian CXCR4) and its ligand cxcl12b (mammalian CXCL12) are both preferentially expressed on arteries at time points consistent with the arrival and differentiation of the first vSMCs during vascular development. We show that autocrine cxcl12b/cxcr4 activity leads to increased production of the vSMC chemoattractant ligand pdgfb by endothelial cells in vitro and increased expression of pdgfb by arteries of zebrafish and mice in vivo. Additionally, we demonstrate that expression of the blood flow-regulated transcription factor klf2a in primitive veins negatively regulates cxcr4/cxcl12 and pdgfb expression, restricting vSMC recruitment to the arterial vasculature. Together, this signalling axis leads to the differential acquisition of vSMCs at sites where klf2a expression is low and both cxcr4a and pdgfb are co-expressed, i.e. arteries during early development.

The post-transcriptional mechanisms contributing to molecular regulation of developmental lymphangiogenesis and lymphatic network assembly are not well understood. MicroRNAs are important post-transcriptional regulators during development. Here, we use high throughput small RNA sequencing to identify miR-204, a highly conserved microRNA dramatically enriched in lymphatic vs. blood endothelial cells in human and zebrafish. Suppressing miR-204 leads to loss of lymphatic vessels while endothelial overproduction of miR-204 accelerates lymphatic vessel formation, suggesting a critical positive role for this microRNA during developmental lymphangiogenesis. We also identify the NFATC1 transcription factor as a key miR-204 target in human and zebrafish, and show that NFATC1 suppression leads to lymphatic hyperplasia. The loss of lymphatics caused by miR-204 deficiency can be largely rescued by either endothelial autonomous expression of miR-204 or by suppression of NFATC1. Together, our results highlight a miR-204/NFATC1 molecular regulatory axis required for proper lymphatic development.

The preferential accumulation of vascular smooth muscle cells on arteries versus veins during early development is a well-described phenomenon, but the molecular pathways underlying this polarization are not well understood. During zebrafish embryogenesis the cxcr4a receptor (mammalian CXCR4) and its ligand cxcl12b (mammalian CXCL12) are both preferentially expressed on arteries at time points consistent with the arrival and differentiation of the first vascular smooth muscle cells (vSMCs). We show that autocrine cxcl12b/cxcr4 activity leads to increased production of the vSMC chemoattractant ligand pdgfb by endothelial cells in vitro and increased expression of pdgfb by arteries in vivo . Additionally, we demonstrate that expression of the well-characterized blood flow-regulated transcription factor klf2a in primitive veins negatively regulates cxcr4/cxcl12 and pdgfb expression, restricting vSMC recruitment to the arterial vasculature. Together, this signaling axis leads to the differential acquisition of smooth muscle cells at sites where klf2a expression is low and both cxcr4a and pdgfb are co-expressed, i.e. arteries during early development.

ABSTRACTVascular endothelial cellsin vivoare exquisitely regulated by their local environment, which is disrupted or absent when using methods such as FACS sorting of cells isolated from animals orin vitrocell culture. Here, we profile the gene expression patterns of undisturbed endothelial cells in living animals using a novel “AngioTag” zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specifickdrlpromoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. By performing TRAP-RNAseq on AngioTag animals, we demonstrate strong enrichment of endothelial specific genes and uncover novel endothelial genes and unique endothelial gene expression signatures for different adult organs. Finally, we generated a versatile “UAS:RiboTag” transgenic line to allow a wider array of different zebrafish cell and tissue types to be examined using TRAP-RNAseq methods. These new tools offer an unparalleled resource to study the molecular identity of cells in their normalin vivocontext.

Vascular endothelial cells are exquisitely regulated by their local environment, which is absent or disrupted when using methods such as FACS or cell culture to study native signaling pathways. Here, we profile the gene expression patterns of undisturbed endothelial cells in living animals using a novel ‘AngioTag’ zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specific promoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. By performing TRAP-RNAseq on AngioTag animals, we demonstrate strong enrichment of endothelial specific genes and uncover novel endothelially expressed genes. Additionally, we generated a ‘UAS:RiboTag’ transgenic line to allow for the study of a wider array of zebrafish cell and tissue types using TRAP-RNAseq methods. This new tool offers an unparalleled resource to study cause and effect relationships in the context of gene loss or gain of function .

Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

The lymphatic vascular system is a hierarchically organized complex network essential for tissue fluid homeostasis, immune trafficking and absorption of dietary fats in the human body. Despite its importance, the assembly of the lymphatic network is still not fully understood. The zebrafish is a powerful model organism that enables study of lymphatic vessel development using high-resolution imaging and sophisticated genetic and experimental manipulation. Although several studies have described early lymphatic development in the fish, lymphatic development at later stages has not been completely elucidated. In this study, we generated a new Tg(mrc1a:egfp)(y251) transgenic zebrafish that uses a mannose receptor, C type 1 (mrc1a) promoter to drive strong EGFP expression in lymphatic vessels at all stages of development and in adult zebrafish. We used this line to describe the assembly of the major vessels of the trunk lymphatic vascular network, including the later-developing collateral cardinal, spinal, superficial lateral and superficial intersegmental lymphatics. Our results show that major trunk lymphatic vessels are conserved in the zebrafish, and provide a thorough and complete description of trunk lymphatic vessel assembly.

Blood-feeding disease vectors mitigate the negative effects of hemostasis and inflammation through the binding of small-molecule agonists of these processes by salivary proteins. In this study, a lipocalin protein family member (LTBP1) from the saliva of Rhodnius prolixus, a vector of the pathogen Trypanosoma cruzi, is shown to sequester cysteinyl leukotrienes during feeding to inhibit immediate inflammatory responses. Calorimetric binding experiments showed that LTBP1 binds leukotrienes C-4 (LTC4), D-4 (LTD4), and E-4 (LTE4) but not biogenic amines, adenosine diphosphate, or other eicosanoid compounds. Crystal structures of ligand-free LTBP1 and its complexes with LTC4 and LTD4 reveal a conformational change during binding that brings Tyr114 into close contact with the ligand. LTC4 is cleaved in the complex, leaving free glutathione and a C-20 fatty acid. Chromatographic analysis of bound ligands showed only intact LTC4, suggesting that cleavage could be radiation-mediated.

The cerebral vasculature provides the massive blood supply that the brain needs to grow and survive. By acquiring distinctive cellular and molecular characteristics it becomes the Blood Brain Barrier (BBB), a selectively permeable and protective interface between the brain and the peripheral circulation that maintains the extra-cellular milieu permissive for neuronal activity. Accordingly, there is great interest in uncovering the mechanisms that modulate the formation and differentiation of the brain vasculature. By performing a forward genetic screen in zebrafish we isolated no food for thought (nfty72), a recessive late-lethal mutant that lacks most of the intra-cerebral Central Arteries (CtAs), but not other brain blood vessels. We found that the cerebral vascularization deficit of nfty72 is caused by an inactivating lesion in reck (reversion-inducing-cysteine-rich protein with Kazal motifs or ST15; Suppressor of Tumorigenicity 15 protein), which encodes a membrane-anchored tumor suppressor glycoprotein. Our findings highlight Reck as a novel and pivotal modulator of the canonical Wnt signaling pathway that acts in endothelial cells to enable intra-cerebral vascularization and proper expression of molecular markers associated with BBB formation. Additional studies with cultured endothelial cells suggest that, in other contexts, Reck impacts vascular biology via the Vascular Endothelial Growth Factor (VEGF) cascade. Together, our findings have broad implications for both vascular and cancer biology.

ZAP70 [zeta-chain (TCR)-associated protein kinase, 70-kDa], is required for T cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70(y442)) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70(y442) homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck: GFP(+) thymic T cells by 5 days postfertilization that persisted into adult stages. Morphological analysis, RNA sequencing, and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70(y442) mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70(y442) mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immune deficiency.