Lymphatic vessels have important roles in fluid homeostasis, fat absorption, inflammation and cancer metastasis and develop in a dynamic process (called lymphangiogenesis) involving budding, migration and proliferation of lymphangioblasts. Using a genetic screen in zebrafish we identify ccbe1 (collagen and calcium-binding EGF domain-1) as indispensible for embryonic lymphangiogenesis. Ccbe1 acts at the same stage of development as Vegfc and is required for lymphangioblast budding and angiogenic sprouting from venous endothelium.

The development of arteries, veins and lymphatics from pre-existing vessels are intimately linked processes controlled by a number of well-studied reiteratively acting signalling pathways. To delineate the mechanisms governing vessel formation in vivo, we performed a forward genetic screen in zebrafish and isolated the mutant expando. Molecular characterisation revealed a loss-offunction mutation in the highly conserved kinase insert region of flt4. Consistent with previous reports, flt4 mutants were deficient in lymphatic vascular development. Recent studies have demonstrated a role for Flt4 in blood vessels and showed that Dll4 limits angiogenic potential by limiting Flt4 function in developing blood vessels. We found that arterial angiogenesis proceeded normally, yet the dll4 loss-of-function arterial hyperbranching phenotype was rescued, in flt4 signalling mutants. Furthermore, we found that the Flt4 ligand Vegfc drives arterial hyperbranching in the absence of dll4. Upon knockdown of dll4, intersegmental arteries were sensitised to increased vegfc levels and the overexpression of dll4 inhibited Vegfc/Flt4-dependent angiogenesis events. Taken together, these data demonstrate that dll4 functions to suppress the ability of developing intersegmental arteries to respond to Vegfc-driven Flt4 signalling in zebrafish. We propose that this mechanism contributes to the differential response of developing arteries and veins to a constant source of Vegfc present in the embryo during angiogenesis.

Lymphedema, lymphangiectasias, mental retardation and unusual facial characteristics define the autosomal recessive Hennekam syndrome. Homozygosity mapping identified a critical chromosomal region containing CCBE1, the human ortholog of a gene essential for lymphangiogenesis in zebrafish. Homozygous and compound heterozygous mutations in seven subjects paired with functional analysis in a zebrafish model identify CCBE1 as one of few genes causing primary generalized lymph-vessel dysplasia in humans.

Acetaldehyde is a highly reactive, DNA damaging metabolite, produced upon alcohol consumption 1. Impaired acetaldehyde detoxification is common in the Asian population, and is associated with alcohol related cancers 1,2. Cellular protection against acetaldehyde-induced damage is provided by DNA crosslink repair; when impaired this causes Fanconi anaemia (FA), a disease resulting in failed blood production and cancer predisposition 3,4. Strikingly, combined inactivation of acetaldehyde detoxification and the FA pathway induces mutation, accelerates malignancies and causes the rapid attrition of blood stem cells 5-7. A key question concerns the nature of DNA damage caused by acetaldehyde, and how this is repaired. Here we generate acetaldehyde-induced DNA interstrand crosslinks (AA-ICLs) and determine their repair mechanism in Xenopus egg extract. We discover that two replication-coupled pathways repair these lesions. The first is the FA pathway, that operates using excision, analogous to the mechanism used for chemotherapeutic crosslinks caused by cisplatin. Yet, this AA-ICL repair results in elevated mutation frequency and altered mutational spectrum. The second repair modality requires replication fork convergence but unexpectedly does not involve DNA incisions, instead the acetaldehyde-crosslink itself is broken. The Y-family DNA polymerase REV1 completes repair, culminating in a distinct mutation spectrum. This work defines how DNA interstrand crosslinks caused by an endogenous and alcohol-derived metabolite are repaired, identifying an excision-independent mechanism. To study the repair of alcohol-induced DNA damage, we generated an acetaldehyde-crosslinked DNA substrate. Acetaldehyde reacts with guanine creating a crosslink precursor, N2-propanoguanine (PdG) (Fig. 1a) 8. In a 5'-CpG sequence, PdG reacts with the N2-amine of guanine on the opposite strand to create an interstrand acetaldehyde crosslink (AA-ICL). The crosslink exists in equilibrium between three states 9. We synthesized a site-specific native AANAT-ICL within an oligonucleotide duplex (Extended Data Fig. 1a, b, d, Supplementary Information Fig. 1). A control reaction of PdG with deoxyinosine (dIno), lacking an N2-amine, did not crosslink, confirming AANAT-ICL site-specificity (Extended Data Fig. 1c, for gel source data see Supplementary Information Fig. 2). AANAT-ICLs were stable at physiological pH and temperature (< 10% reversal after 72 h at 37 C) (Extended Data Fig. 1e). Elevated temperature (55 C) or acid did however reverse AANAT-ICL, consistent with Schiff base Top strand Unhooked Bottom strand Unhooked

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Abbreviations: wpf: weeks post fertilization.Dysbiosis of the intestinal microbial community is considered a risk factor for development of chronic intestinal inflammation as well as other diseases such as diabetes, obesity and even cancer. Study of the innate and adaptive immune pathways controlling bacterial colonization has however proven difficult in rodents, considering the extensive cross-talk between bacteria and innate and adaptive immunity. Here, we used the zebrafish to study innate and adaptive immune processes controlling the microbial community. Zebrafish lack a functional adaptive immune system in the first weeks of life, enabling study of the innate immune system in the absence of adaptive immunity. We show that in wild type zebrafish, the initial lack of adaptive immunity associates with overgrowth of Vibrio species (a group encompassing fish and human pathogens), which is overcome upon adaptive immune development. In Rag1-deficient zebrafish (lacking adaptive immunity) Vibrio abundance remains high, suggesting that adaptive immune processes indeed control Vibrio species. Using cell transfer experiments, we confirm that adoptive transfer of T lymphocytes, but not B lymphocytes into Rag1-deficient recipients suppresses outgrowth of Vibrio. In addition, ex vivo exposure of intestinal T lymphocytes to Rag1-deficient microbiota results in increased interferon-gamma expression by these T lymphocytes, compared to exposure to wild type microbiota. In conclusion, we show that T lymphocytes control microbial composition by effectively suppressing the outgrowth of Vibrio species in the zebrafish intestine.

BackgroundThe expression of the Prospero homeodomain transcription factor (Prox1) in a subset of cardinal venous cells specifies the lymphatic lineage in mice. Prox1 is also indispensible for the maintenance of lymphatic cell fate, and is therefore considered a master control gene for lymphangiogenesis in mammals. In zebrafish, there are two prox1 paralogues, the previously described prox1 (also known as prox1a) and the newly identified prox1b.Principal FindingsTo investigate the role of the prox1b gene in zebrafish lymphangiogenesis, we knocked-down prox1b and found that depletion of prox1b mRNA did not cause lymphatic defects. We also generated two different prox1b mutant alleles, and maternal-zygotic homozygous mutant embryos were viable and did not show any lymphatic defects. Furthermore, the expression of prox1b was not restricted to lymphatic vessels during zebrafish development.ConclusionWe conclude that Prox1b activity is not essential for embryonic lymphatic development in zebrafish.

CXCL8 is a potent neutrophil recruiting chemokine. CXCL8 is produced by several innate immune cells, including neutrophils, macrophages, as well as epithelial cells. Although previously considered only to be produced as a result of TLR signaling in these cells, recent reports show that T cell–derived cytokines also induce CXCL8 in epithelial cells. Likewise, we observed that T cell inhibition diminished intestinal production of functional mouse homologs of CXCL8 in the early phase of enterocolitis. In this study, we specifically investigated whether adaptive cells contribute to innate cxcl8 expression in the intestines. To this end, we used the zebrafish as our model system. Unlike murine models that lack CXCL8, zebrafish have two CXCL8 chemokines that are both elevated after an acute inflammatory stimulus and recruit neutrophils. Furthermore, zebrafish develop innate and adaptive immunity sequentially, enabling analysis of intestinal cxcl8 expression in the absence (&lt;3 wk of age) and presence (&gt;3 wk of age) of adaptive immunity. In this study, we show that intestinal cxcl8-l1 but not cxcl8-l2 expression is regulated by T lymphocytes under homeostatic conditions. In contrast, during intestinal inflammation especially, cxcl8-l1 expression is upregulated independent of T lymphocyte presence. Furthermore, we show that human CXCL8 is able to induce intestinal zebrafish neutrophil recruitment and cxcl8-l1 expression, demonstrating that zebrafish can be used as a model to study CXCL8 function and regulation. In conclusion, these data provide evidence that Cxcl8-l1 and Cxcl8-l2 are differentially regulated via T lymphocyte–dependent and –independent mechanisms during homeostasis and inflammation.