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.

In vitro and in vivo accumulation of the anticancer Ru complexes [RuII(cym)(HQ)Cl] and [RuII(cym)(PCA)Cl]Cl.

The cellular accumulation and the underlying mechanisms for the two ruthenium-based anticancer complexes [RuII(cym)(HQ)Cl] 1 (cym = η6-p-cymene, HQ = 8-hydroxyquinoline) and [RuII(cym)(PCA)Cl]Cl 2 (PCA = N-fluorophenyl-2-pyridinecarbothioamide) were investigated in HCT116 human colorectal carcinoma cells. The results showed that the cellular accumulation of both complexes increased over time and with higher concentrations, and that 2 accumulates in greater quantities in cells than 1. Inhibition studies of selected cellular accumulation mechanisms indicated that both 1 and 2 may be transported into the cells by both passive diffusion and active transporters, similar to cisplatin. Efflux experiments indicated that 1 and 2 are subjected to efflux through a mechanism that does not involve p-glycoprotein, as addition of verapamil did not make any difference. Exploring the influence of the Cu transporter by addition of CuCl2 resulted in a higher accumulation of 1 and 2 whilst the amount of Pt detected was slightly reduced when cells were treated with cisplatin. Complexes 1 and 2 were further explored in zebrafish where accumulation and distribution were determined with ICP-MS and LA-ICP-MS. The results correlated with the in vitro observations and zebrafish treated with 2 showed higher Ru contents than those treated with 1. The distribution studies suggested that both complexes mainly accumulated in the intestines of the zebrafish.

mafba is a downstream transcriptional effector of Vegfc signaling essential for embryonic lymphangiogenesis in zebrafish.

The lymphatic vasculature plays roles in tissue fluid balance, immune cell trafficking, fatty acid absorption, cancer metastasis, and cardiovascular disease. Lymphatic vessels form by lymphangiogenesis, the sprouting of new lymphatics from pre-existing vessels, in both development and disease contexts. The apical signaling pathway in lymphangiogenesis is the VEGFC/VEGFR3 pathway, yet how signaling controls cellular transcriptional output remains unknown. We used a forward genetic screen in zebrafish to identify the transcription factor mafba as essential for lymphatic vessel development. We found that mafba is required for the migration of lymphatic precursors after their initial sprouting from the posterior cardinal vein. mafba expression is enriched in sprouts emerging from veins, and we show that mafba functions cell-autonomously during lymphatic vessel development. Mechanistically, Vegfc signaling increases mafba expression to control downstream transcription, and this regulatory relationship is dependent on the activity of SoxF transcription factors, which are essential for mafba expression in venous endothelium. Here we identify an indispensable Vegfc-SoxF-Mafba pathway in lymphatic development.

Zebrafish facial lymphatics develop through sequential addition of venous and non-venous progenitors.

Lymphatic vessels are known to be derived from veins; however, recent lineage-tracing experiments propose that specific lymphatic networks may originate from both venous and non-venous sources. Despite this, direct evidence of a non-venous lymphatic progenitor is missing. Here, we show that the zebrafish facial lymphatic network is derived from three distinct progenitor populations that add sequentially to the developing facial lymphatic through a relay-like mechanism. We show that while two facial lymphatic progenitor populations are venous in origin, the third population, termed the ventral aorta lymphangioblast (VA-L), does not sprout from a vessel; instead, it arises from a migratory angioblast cell near the ventral aorta that initially lacks both venous and lymphatic markers, and contributes to the facial lymphatics and the hypobranchial artery. We propose that sequential addition of venous and non-venous progenitors allows the facial lymphatics to form in an area that is relatively devoid of veins. Overall, this study provides conclusive, live imaging-based evidence of a non-venous lymphatic progenitor and demonstrates that the origin and development of lymphatic vessels is context-dependent.

A Multitargeted Approach: Organorhodium Anticancer Agent Based on Vorinostat as a Potent Histone Deacetylase Inhibitor.

The combination of more than one bioactive moiety in a multitargeted anticancer agent may result in synergistic activity of its components. Using this concept, bioorganometallic compounds were designed to feature a metal center, a 2-pyridinecarbothioamide (PCA), and a hydroxamic acid, which is found in the anticancer drug vorinostat (SAHA). The organometallics showed inhibitory activity in the nanomolar range against histone deacetylases (HDACs) as the key target for SAHA. In particular, the Rh complex was a potent inhibitor of HDAC6 over HDAC1 and HDAC8. Whereas this complex was highly cytotoxic in human cancer cells, it showed low toxicity in hemolysis studies and zebrafish, demonstrating the role of the metal center. For this complex a slightly reduced expression of vascular endothelial growth factor receptor 2 (VEGFR2) was established, which was upregulated by SAHA. This finding indicates that the new organometallics display different modes of action than their bioactive components.

Vegfd can compensate for loss of Vegfc in zebrafish facial lymphatic sprouting.

Lymphangiogenesis is a dynamic process that involves the sprouting of lymphatic endothelial cells (LECs) from veins to form lymphatic vessels. Vegfr3 signalling, through its ligand Vegfc and the extracellular protein Ccbe1, is essential for the sprouting of LECs to form the trunk lymphatic network. In this study we determined whether Vegfr3, Vegfc and Ccbe1 are also required for development of the facial and intestinal lymphatic networks in the zebrafish embryo. Whereas Vegfr3 and Ccbe1 are required for the development of all lymphatic vessels, Vegfc is dispensable for facial lymphatic sprouting but not for the complete development of the facial lymphatic network. We show that zebrafish vegfd is expressed in the head, genetically interacts with ccbe1 and can rescue the lymphatic defects observed following the loss of vegfc. Finally, whereas knockdown of vegfd has no phenotype, double knockdown of both vegfc and vegfd is required to prevent facial lymphatic sprouting, suggesting that Vegfc is not essential for all lymphatic sprouting and that Vegfd can compensate for loss of Vegfc during lymphatic development in the zebrafish head.

Lymphatics, Cancer and Zebrafish.

Many solid tumors are known to metastasize through the lymphatic vasculature. This process is facilitated by the generation of new lymphatic vessels (tumor lymphangiogenesis) and also by the remodelling of existing lymphatics. Together these processes enable the spread of tumor cells to distant sites. Currently our understanding of tumor lymphangiogenesis has been informed from mouse tumor models and from studies of developmental lymphangiogenesis. Since the discovery of bona fide lymphatic vessels in zebrafish in 2006, zebrafish have become a well-established model of developmental lymphangiogenesis. The attributes that make zebrafish such an important model of blood vessel development-the ability to live image developing vessels, genetic tractability and the conserved nature of development-also make fish an attractive model of lymphatic vessel development. In particular, zebrafish have made important contributions to our understanding of the processes of lymphatic vessel sprouting from veins and the mechanisms by which lymphatic precursors remodel into mature vessels. To date, zebrafish have not been used to directly model tumor lymphangiogenesis. In this chapter we will summarise the contributions zebrafish have made to our understanding of lymphangiogenesis and investigate the possibilities of combining zebrafish transgenic cancer lines or tumor transplantation models with existing lymphatic reporter lines, which could provide valuable insights into the process of tumor-induced lymphangiogenesis. In addition the utility of using the zebrafish lymphatic model as a platform to screen and develop novel anti-lymphatic therapeutics will also be discussed.

lyve1 expression reveals novel lymphatic vessels and new mechanisms for lymphatic vessel development in zebrafish.

We have generated novel transgenic lines that brightly mark the lymphatic system of zebrafish using the lyve1 promoter. Facilitated by these new transgenic lines, we generated a map of zebrafish lymphatic development up to 15 days post-fertilisation and discovered three previously uncharacterised lymphatic vessel networks: the facial lymphatics, the lateral lymphatics and the intestinal lymphatics. We show that a facial lymphatic vessel, termed the lateral facial lymphatic, develops through a novel developmental mechanism, which initially involves vessel growth through a single vascular sprout followed by the recruitment of lymphangioblasts to the vascular tip. Unlike the lymphangioblasts that form the thoracic duct, the lymphangioblasts that contribute to the lateral facial lymphatic vessel originate from a number of different blood vessels. Our work highlights the additional complexity of lymphatic vessel development in the zebrafish that may increase its versatility as a model of lymphangiogenesis.

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.

Microtubule remodelling is required for the front-rear polarity switch during contact inhibition of locomotion.

When migrating mesenchymal cells collide, they exhibit a 'contact inhibition of locomotion' response that results in reversal of their front-rear polarity by extension of a new leading edge, which enables their migration away from the opposing contacted cell. The critical cytoskeletal rearrangements underpinning these mutual repulsion events are currently unknown. We found that during fibroblast cell-cell collisions, microtubules at the region of contact increase their frequency of catastrophe, their rates of shrinkage and growth, and concomitantly, a new microtubule array is established at a new leading edge. We show that Rho and ROCK activity is necessary for this repulsion response, and we observed increased microtubule stabilisation as a consequence of ROCK inhibition. Importantly, partial destabilisation of microtubules, by co-treatment with a low dose of nocodazole, restored microtubule dynamics to that of untreated cells and rescued contact inhibition of locomotion in ROCK-inhibited cells. Although there was an increase in microtubule growth or shrinkage rates in Y27632 cell-cell collisions, these failed to reach the same level of dynamicity compared with untreated collisions. Our data suggest that microtubule dynamics at contact sites must increase beyond a threshold for a cell to switch its front-rear polarity, and that microtubule stabilisation can lead to a failure of contact inhibition of locomotion.

Microinjection of ß-glucan Into Larval Zebrafish (Danio rerio) for the Assessment of a Trained-Like Immunity Phenotype.

The innate immune system can remember previous inflammatory insults, enabling long-term heightened responsiveness to secondary immune challenges in a process termed "trained immunity." Trained innate immune cells undergo metabolic and epigenetic remodelling and, upon a secondary challenge, provide enhanced protection with therapeutic potential. Trained immunity has largely been studied in innate immune cells in vitro or following ex vivo re-stimulation where the primary insult is typically injected into a mouse, adult zebrafish, or human. While highly informative, there is an opportunity to investigate trained immunity entirely in vivo within an unperturbed, intact whole organism. The exclusively innate immune response of larval zebrafish offers an attractive system to model trained immunity. Larval zebrafish have a functional innate immune system by 2 days post fertilisation (dpf) and are amenable to high-resolution, high-throughput analysis. This, combined with their optical transparency, conserved antibacterial responses, and availability of transgenic reporter lines, makes them an attractive alternative model to study trained immunity in vivo. We have devised a protocol where ß-glucan (one of the most widely used experimental triggers of trained immunity) is systemically delivered into larval zebrafish using microinjection to stimulate a trained-like phenotype. Following stimulation, larvae are assessed for changes in gene expression, which indicate the stimulatory effect of ß-glucan. This protocol describes a robust delivery method of one of the gold standard stimulators of trained immunity into a model organism that is highly amenable to several non-invasive downstream analyses. Key features • This protocol outlines the delivery of one of the most common experimental stimulators of trained immunity into larval zebrafish. • The protocol enables the assessment of a trained-like phenotype in vivo. • This protocol can be applied to transgenic or mutant zebrafish lines to investigate cells or genes of interest in response to ß-glucan stimulation.

Infection-experienced HSPCs protect against infections by generating neutrophils with enhanced mitochondrial bactericidal activity.

Hematopoietic stem and progenitor cells (HSPCs) respond to infection by proliferating and generating in-demand neutrophils through a process called emergency granulopoiesis (EG). Recently, infection-induced changes in HSPCs have also been shown to underpin the longevity of trained immunity, where they generate innate immune cells with enhanced responses to subsequent microbial threats. Using larval zebrafish to live image neutrophils and HSPCs, we show that infection-experienced HSPCs generate neutrophils with enhanced bactericidal functions. Transcriptomic analysis of EG neutrophils uncovered a previously unknown function for mitochondrial reactive oxygen species in elevating neutrophil bactericidal activity. We also reveal that driving expression of zebrafish C/EBPß within infection-naïve HSPCs is sufficient to generate neutrophils with similarly enhanced bactericidal capacity. Our work suggests that this demand-adapted source of neutrophils contributes to trained immunity by providing enhanced protection toward subsequent infections. Manipulating demand-driven granulopoiesis may provide a therapeutic strategy to boost neutrophil function and treat infectious disease.

Platinum(terpyridine) complexes with N-heterocyclic carbene co-ligands: high antiproliferative activity and low toxicity in vivo.

Pt(terpyridine) complexes are well-known DNA intercalators. The introduction of an NHC co-ligand rendered such a complex highly antiproliferative in cancer cells compared to its chlorido derivative. Despite the high potency, zebrafish embryos tolerated the compound well, especially compared to cisplatin. DNA interaction studies support a mode of action related to intercalation.

Ephrin-B2 regulates endothelial cell morphology and motility independently of Eph-receptor binding.

The transmembrane protein ephrin-B2 regulates angiogenesis, i.e. the formation of new blood vessels through endothelial sprouting, proliferation and remodeling processes. In addition to essential roles in the embryonic vasculature, ephrin-B2 expression is upregulated in the adult at sites of neovascularization, such as tumors and wounds. Ephrins are known to bind Eph receptor family tyrosine kinases on neighboring cells and trigger bidirectional signal transduction downstream of both interacting molecules. Here we show that ephrin-B2 dynamically modulates the motility and cellular morphology of isolated endothelial cells. Even in the absence of Eph-receptor binding, ephrin-B2 stimulates repeated cycling between actomyosin-dependent cell contraction and spreading episodes, which requires the presence of the C-terminal PDZ motif. Our results show that ephrin-B2 is a potent regulator of endothelial cell behavior, and indicate that the control of cell migration and angiogenesis by ephrins might involve both receptor-dependent and receptor-independent activities.

DMXAA (Vadimezan, ASA404) is a multi-kinase inhibitor targeting VEGFR2 in particular.

The flavone acetic acid derivative DMXAA [5,6-dimethylXAA (xanthenone-4-acetic acid), Vadimezan, ASA404] is a drug that displayed vascular-disrupting activity and induced haemorrhagic necrosis and tumour regression in pre-clinical animal models. Both immune-mediated and non-immune-mediated effects contributed to the tumour regression. The vascular disruption was less in human tumours, with immune-mediated effects being less prominent, but nonetheless DMXAA showed promising effects in Phase II clinical trials in non-small-cell lung cancer. However, these effects were not replicated in Phase III clinical trials. It has been difficult to understand the differences between the pre-clinical findings and the later clinical trials as the molecular targets for the agent have never been clearly established. To investigate the mechanism of action, we sought to determine whether DMXAA might target protein kinases. We found that, at concentrations achieved in blood during clinical trials, DMXAA has inhibitory effects against several kinases, with most potent effects being on members of the VEGFR (vascular endothelial growth factor receptor) tyrosine kinase family. Some analogues of DMXAA were even more effective inhibitors of these kinases, in particular 2-MeXAA (2-methylXAA) and 6-MeXAA (6-methylXAA). The inhibitory effects were greatest against VEGFR2 and, consistent with this, we found that DMXAA, 2-MeXAA and 6-MeXAA were able to block angiogenesis in zebrafish embryos and also inhibit VEGFR2 signalling in HUVECs (human umbilical vein endothelial cells). Taken together, these results indicate that at least part of the effects of DMXAA are due to it acting as a multi-kinase inhibitor and that the anti-VEGFR activity in particular may contribute to the non-immune-mediated effects of DMXAA on the vasculature.

Towards a new model of trained immunity: Exposure to bacteria and ß-glucan protects larval zebrafish against subsequent infections.

Once thought to be a feature exclusive to lymphocyte-driven adaptive immunity, immune memory has also been shown to operate as part of the innate immune system following infection to provide an elevated host response to subsequent pathogenic challenge. This evolutionarily conserved process, termed 'trained immunity', enables cells of the innate immune system to 'remember' previous pathogen encounters and mount stronger responses to the same, or different, pathogens after returning to a non-activated state. Here we show that challenging larval zebrafish, that exclusively rely on innate immunity, with live or heat-killed Salmonella typhimurium provides protection to subsequent infection with either Salmonella typhimurium or Streptococcus iniae, that lasts for at least 12 days. We also show that larvae injected with ß-glucan, the well-known trigger of trained immunity, demonstrate enhanced survival to similar live bacterial infections, a phenotype supported by increased cxcl8 expression and neutrophil recruitment to the infection site. These results support the conservation of a trained immunity-like phenotype in larval zebrafish and provide a foundation to exploit the experimental attributes of larval zebrafish to further understand this form of immunological memory.

A New Transgenic Line for Rapid and Complete Neutrophil Ablation.

Zebrafish lines expressing nitroreductase (NTR) in specific cell compartments, which sensitizes those cells to metronidazole (MTZ)-mediated ablation, have proven extremely useful for studying tissue regeneration and investigating cell function. In contrast to many cells, neutrophils are comparatively resistant to the NTR/MTZ targeted ablation strategy. Recently, a rationally engineered variant of NTR (NTR 2.0) has been described that exhibits greatly improved MTZ-mediated ablation efficacy in zebrafish. We show that a transgenic line with neutrophil-restricted expression of NTR 2.0 demonstrates complete neutrophil ablation, with an MTZ dose 100-fold less than current treatment regimens, and with treatment durations as short as 5 h.

Uricase-Deficient Larval Zebrafish with Elevated Urate Levels Demonstrate Suppressed Acute Inflammatory Response to Monosodium Urate Crystals and Prolonged Crystal Persistence.

Gout is caused by elevated serum urate leading to the deposition of monosodium urate (MSU) crystals that can trigger episodes of acute inflammation. Humans are sensitive to developing gout because they lack a functional urate-metabolizing enzyme called uricase/urate oxidase (encoded by the UOX gene). A hallmark of long-standing disease is tophaceous gout, characterized by the formation of tissue-damaging granuloma-like structures ('tophi') composed of densely packed MSU crystals and immune cells. Little is known about how tophi form, largely due to the lack of suitable animal models in which the host response to MSU crystals can be studied in vivo long-term. We have previously described a larval zebrafish model of acute gouty inflammation where the host response to microinjected MSU crystals can be live imaged within an intact animal. Although useful for modeling acute inflammation, crystals are rapidly cleared following a robust innate immune response, precluding analysis at later stages. Here we describe a zebrafish uox null mutant that possesses elevated urate levels at larval stages. Uricase-deficient 'hyperuricemic' larvae exhibit a suppressed acute inflammatory response to MSU crystals and prolonged in vivo crystal persistence. Imaging of crystals at later stages reveals that they form granuloma-like structures dominated by macrophages. We believe that uox-/- larvae will provide a useful tool to explore the transition from acute gouty inflammation to tophus formation, one of the remaining mysteries of gout pathogenesis.

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.

Isolation of Neutrophils from Larval Zebrafish and Their Transplantation into Recipient Larvae for Functional Studies.

Live imaging of neutrophils within optically transparent larval zebrafish has proved a powerful technique to investigate how specific gene products control neutrophil function. To resolve whether a gene contributes to neutrophil function in a cell-autonomous manner necessitates a way to examine gene-deficient neutrophils in an otherwise wild type background. To this end, here we describe methods to harvest fluorescent neutrophils from larval donor zebrafish and transplant them into age-matched recipients. We show that transplanted neutrophils can survive in recipient larvae for at least 3 days providing ample opportunity for functional studies. Focusing on bactericidal activity, we show that transplanted neutrophils phagocytose and kill live bacteria with similar kinetics to nontransplanted neutrophils, indicating that the transplantation process does not influence these neutrophil effector functions. Following the methods described here to transplant neutrophils between gene-deficient and wild type larval zebrafish will enable investigations into whether a gene's contribution to neutrophil function is cell-autonomous.