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