Alcohol-derived DNA crosslinks are repaired by two distinct mechanisms.

Acetaldehyde is a highly reactive, DNA-damaging metabolite that is produced upon alcohol consumption1. Impaired detoxification of acetaldehyde is common in the Asian population, and is associated with alcohol-related cancers1,2. Cells are protected against acetaldehyde-induced damage by DNA crosslink repair, which when impaired causes Fanconi anaemia (FA), a disease resulting in failure to produce blood cells and a predisposition to cancer3,4. The combined inactivation of acetaldehyde detoxification and the FA pathway induces mutation, accelerates malignancies and causes the rapid attrition of blood stem cells5-7. However, the nature of the DNA damage induced by acetaldehyde and how this is repaired remains a key question. Here we generate acetaldehyde-induced DNA interstrand crosslinks and determine their repair mechanism in Xenopus egg extracts. We find that two replication-coupled pathways repair these lesions. The first is the FA pathway, which operates using excision-analogous to the mechanism used to repair the interstrand crosslinks caused by the chemotherapeutic agent cisplatin. However, the repair of acetaldehyde-induced crosslinks results in increased mutation frequency and an altered mutational spectrum compared with the repair of cisplatin-induced crosslinks. The second repair mechanism requires replication fork convergence, but does not involve DNA incisions-instead the acetaldehyde crosslink itself is broken. The Y-family DNA polymerase REV1 completes repair of the crosslink, culminating in a distinct mutational spectrum. These results define the repair pathways of DNA interstrand crosslinks caused by an endogenous and alcohol-derived metabolite, and identify an excision-independent mechanism.

T lymphocyte-dependent and -independent regulation of Cxcl8 expression in zebrafish intestines.

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 (<3 wk of age) and presence (>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.

Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium.

BACKGROUND & AIMS: The contribution of genetic factors to the pathogenesis of inflammatory bowel disease (IBD) has been established by twin, targeted-sequencing, and genome-wide association studies. These studies identified many risk loci, and research is underway to identify causal variants. These studies have focused mainly on protein-coding genes. We investigated other functional elements in the human genome, such as regulatory regions. METHODS: Using acetylated histone 3 lysine 27 chromatin immunoprecipitation and sequencing, we identified tens of thousands of potential regulatory regions that are active in intestinal epithelium (primary intestinal crypts and cultured organoids) isolated from resected material and from biopsies collected during ileo-colonoscopies and immune cells (monocytes, macrophages, CD34(+), CD4(+), and CD8(+)). We correlated these regions with susceptibility loci for IBD. RESULTS: We have generated acetylated histone 3 lysine 27 profiles from primary intestinal epithelium and cultured organoids, which we have made publically available. We found that 45 of 163 single nucleotide polymorphisms (SNPs) associated with IBD overlap specifically with active regulatory elements. In addition, by taking strong linkage disequilibrium into account, another 47 IBD-associated SNPs colocalized with active regulatory elements through other SNPs in their vicinity. Altogether, 92 of 163 IBD-associated SNPs correlated with distinct active regulatory elements-a frequency 2.5- to 3.5-fold greater than that expected from random sampling. The variations in these SNPs often create or disrupt known binding motifs; they might affect the binding of transcriptional regulators to alter expression of regulated genes. CONCLUSIONS: In addition to variants in protein coding genes, variants in noncoding DNA regulatory regions that are active in intestinal epithelium and immune cells are potentially involved in the pathogenesis of IBD.

Vegfc/Flt4 signalling is suppressed by Dll4 in developing zebrafish intersegmental arteries.

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-of-function 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.

Deficiency in macrophage-stimulating protein results in spontaneous intestinal inflammation and increased susceptibility toward epithelial damage in zebrafish.

Several genome-wide association studies have identified the genes encoding for macrophage-stimulating protein (MSP) and its receptor RON (Recepteur d'Origine Nantais) as possible susceptibility factors in inflammatory bowel disease. While it has been shown that the MSP-RON signaling pathway is involved in tissue injury responses, current mouse models for MSP and RON deficiency have not clearly demonstrated a role of MSP-RON signaling in the context of intestinal inflammation. In this study, we report that the recently identified zebrafish Msp mutant (msp(t34230)) develops spontaneous intestinal inflammation over time. From 14 to 28 weeks postfertilization Msp-deficient zebrafish show intestinal eosinophilia, increased intestinal expression of inflammatory marker mmp9, and activation of intestinal goblet cells. Moreover, these Msp mutant zebrafish are more susceptible toward ethanol-induced epithelial damage, which resulted in increased infiltration and proliferation of immune cells within the lamina propria and prolonged intestinal proinflammatory cytokine responses in some mutant fish. In light of the recent development of many tools to visualize, monitor, and genetically modify zebrafish, these Msp-deficient zebrafish will enable in-depth in vivo analysis of epithelial and macrophage-specific MSP-RON signaling in the context of intestinal inflammation.

T lymphocytes control microbial composition by regulating the abundance of Vibrio in the zebrafish gut.

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.

Zebrafish prox1b mutants develop a lymphatic vasculature, and prox1b does not specifically mark lymphatic endothelial cells.

BACKGROUND: The 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 FINDINGS: To 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. CONCLUSION: We conclude that Prox1b activity is not essential for embryonic lymphatic development in zebrafish.

Ccbe1 is required for embryonic lymphangiogenesis and venous sprouting.

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.

Mutations in CCBE1 cause generalized lymph vessel dysplasia in humans.

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.