Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.

SUMMARYNeuroligins are postsynaptic cell adhesion proteins that bind specifically to presynaptic membrane proteins called neurexins. Mutations in human neuroligin genes are associated with autism spectrum disorders in some families. The nematode Caenorhabditis elegans has a single neuroligin gene (nlg-1), and approximately a sixth of C. elegans neurons, including some sensory neurons, interneurons and a subset of cholinergic motor neurons, express a neuroligin transcriptional reporter. Neuroligin-deficient mutants of C. elegans are viable, and they do not appear deficient in any major motor functions. However, neuroligin mutants are defective in a subset of sensory behaviors and sensory processing, and are hypersensitive to oxidative stress and mercury compounds; the behavioral deficits are strikingly similar to traits frequently associated with autism spectrum disorders. Our results suggest a possible link between genetic defects in synapse formation or function, and sensitivity to environmental factors in the development of autism spectrum disorders. RESEARCH ARTICLENLG-1 is 26-28% identical (45-47% similar) to the four human neuroligins and is most similar overall (28% identical, 47% similar) to human neuroligin 4 (supplementary material Fig. S1).Through a combination of cDNA sequencing and reverse transcription PCR (RT-PCR) analysis of transcripts, we documented several types of nlg-1 alternative splicing (Figs 1, 2). Exons 13 and 14 are variably present in nlg-1 transcripts; the skipping of these two exons occurs independently, and we have detected transcripts containing only exon 13, only exon 14, both exons, and neither exon. In addition, we have identified tandem alternative splice acceptor sites at the 5Ј-ends of exons 4 and 16, and tandem alternative splice donor sites at the 3Ј-end of exon 14 (Figs 1, 2). If these splicing events are independent, there could be as many as 24 distinct NLG-1 isoforms. nlg-1 is expressed in a subset of neurons and muscle cellsWe used a transgenic transcriptional reporter, with the nlg-1 promoter driving YFP expression (FRM77, Fig. 1), to examine the cellular expression of nlg-1. We found that nlg-1 is expressed in a subset of neurons in C. elegans adults, including ~20 cells in the ventral nerve cord and ~20 cells in the head (Fig. 3). We identified the nlg-1-expressing cells in the ventral nerve cord as the cholinergic VA and DA motor neurons (Fig. 3). We also identified the two AIY and two URB interneurons and the four URA motor neurons in the head, and the two PVD mechanosensory and two HSN motor neurons in the body, as nlg-1-expressing cells. Of these cells, the AIY interneurons are cholinergic (Altun-Gultekin et al., 2001), the PVD neurons are glutamatergic (Lee et al., 1999), and the HSN neurons release both serotonin and acetylcholine (ACh) (Desai et al., 1988;Duerr et al., 2001). Neurotransmitter assignments have not been reported for the remaining nlg-1-expressing neurons; however, they do not express GABAergic, dopaminergic, serotonergic or glutamatergic repo...

Objective We previously showed that endothelial epsin deficiency causes elevated VEGFR2 and enhanced VEGF signaling, resulting in aberrant tumor angiogenesis and tumor growth in adult mice. However, direct evidence demonstrating that endothelial epsins regulate angiogenesis specifically through VEGFR2 downregulation is still lacking. In addition, whether the lack of epsins causes abnormal angiogenesis during embryonic development remains unclear. Approach and Results A novel strain of endothelial epsin-deleted mice that are heterozygous for VEGFR2 (Epn1fl/fl; Epn2−/−; Flkfl/+; iCDH5 Cre mice) was created. Analysis of embryos at different developmental stages shows that deletion of epsins causes defective embryonic angiogenesis and retards embryo development. In vitro angiogenesis assays using isolated primary endothelial cells (EC) from Epn1fl/fl; Epn2−/−; iCDH5 Cre (EC-iDKO) and Epn1fl/fl; Epn2−/−; Flkfl/+; iCDH5 Cre (EC-iDKO-Flkfl/+) mice demonstrated that VEGFR2 reduction in epsin depleted cells is sufficient to restore normal VEGF signaling, EC proliferation, EC migration and EC network formation. These findings were complemented by in vivo wound healing, inflammatory angiogenesis, and tumor angiogenesis assays in which reduction of VEGFR2 was sufficient to rescue abnormal angiogenesis in endothelial epsin-deleted mice. Conclusions Our results provide the first genetic demonstration that epsins function specifically to downregulate VEGFR2 by mediating activated VEGFR2 internalization and degradation and that genetic reduction of VEGFR2 level protects against excessive angiogenesis caused by epsin loss. Our findings indicate epsins may be a potential therapeutic target in conditions where tightly regulated angiogenesis is crucial, such as in diabetic wound healing and tumors.

The Caenorhabditis elegans UNC-13 protein and its mammalian homologues are important for normal neurotransmitter release. We have identified a set of transcripts from the unc-13 locus in C. elegans resulting from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but that vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. We have analyzed the sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. We have also isolated a deletion allele that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products. INTRODUCTIONTransfer of information between neurons or between neurons and muscles requires the Ca 2ϩ -dependent release of neurotransmitters from synaptic vesicles. According to the SNARE hypothesis, specific synaptic vesicle proteins interact with specific plasma membrane proteins at docking sites at the active zone (Sö llner et al., 1993a,b). Interacting proteins include synaptobrevin (also known as VAMP) and synaptotagmin in the synaptic vesicle and syntaxin and SNAP-25 in the presynaptic membrane (Hayashi et al., 1993). Soluble factors, including N-ethylmaleimide-sensitive factor and ␣-and ␤-soluble N-ethylmaleimide-sensitive factor attachment proteins (Sö llner et al., 1993b), are involved in the formation and/or regulation of these proteins. Although many components of synaptic vesicle targeting and neurotransmitter release have been identified, the regulation of these processes cannot be completely explained.unc-13 was originally identified in Caenorhabditis elegans as a gene important for normal locomotion. Mutations in this gene result in severely uncoordinated movement, increased accumulation of the neurotransmitter acetylcholine, and resistance to acetylcholinesterase inhibitors (Brenner, 1974;Rand and Russell, 1985;Hosono et al., 1989;Nguyen et al., 1995). These phenotypes suggest a decrease in the synaptic release of acetylcholine, and other studies have indicated that unc-13 mutants are deficient in the release of most or all neurotransmitters (Miller et al., 1996).The unc-13 gene encodes a protein (UNC-13) containing C1 and C2 homology domains (Maruyama and Brenner, 1991). Mammalian homologues of UNC-13, Munc13-1, Munc13-2, and Munc13-3 in rat brain (Brose et al., 1995) and Hmunc13 in human kidney (Song et al., 1999), also contain one C1 and two or three C2 domains. C1 and C2 r...

Lymphatic valves prevent the backflow of the lymph fluid and ensure proper lymphatic drainage throughout the body. Local accumulation of lymphatic fluid in tissues, a condition called lymphedema, is common in individuals with malformed lymphatic valves. The vascular endothelial growth factor receptor 3 (VEGFR3) is required for the development of lymphatic vascular system. The abundance of VEGFR3 in collecting lymphatic trunks is high before valve formation and, except at valve regions, decreases after valve formation. We found that in mesenteric lymphatics, the abundance of epsin 1 and 2, which are ubiquitin-binding adaptor proteins involved in endocytosis, was low at early stages of development. After lymphatic valve formation, the initiation of steady shear flow was associated with an increase in the abundance of epsin 1 and 2 in collecting lymphatic trunks, but not in valve regions. Epsin 1 and 2 bound to VEGFR3 and mediated the internalization and degradation of VEGFR3, resulting in termination of VEGFR3 signaling. Mice with lymphatic endothelial cell-specific deficiency of epsin 1 and 2 had dilated lymphatic capillaries, abnormally high VEGFR3 abundance in collecting lymphatics, immature lymphatic valves, and defective lymph drainage. Deletion of a single Vegfr3 allele or pharmacological suppression of VEGFR3 signaling restored normal lymphatic valve development and lymph drainage in epsin-deficient mice. Our findings establish a critical role for epsins in the temporal and spatial regulation of VEGFR3 abundance and signaling in collecting lymphatic trunks during lymphatic valve formation.

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A r t i c l e A m e n d m e n t sDuring the assembly of Figure 10B, incorrect flow cytometry panels were inadvertently included for the Tg sm/p22phox + tempol sample. In addition, a different replicate is now provided for the Tg sm/p22phox sample in Figure 10A. The correct figure panels are below.The authors regret the errors.

We have cloned the cha-1 gene from Caenorhabditis elegans using the method of transposon tagging, cha-1 is the structural gene for ChAT, the enzyme that synthesizes ACh. Sequence analysis of cDNAs predicts a protein of 71.5 kDa; comparison of the deduced amino acid sequence with ChAT sequences from other species confirms that cha-1 encodes ChAT. Comparison of cDNA and genomic sequences reveals that transcription is from right to left on the genetic map, and that some of the transcripts may result from trans-splicing of the 22-base spliced leader SL 1. The cha-1 gene is organized into 11 exons. The first exon contains only untranslated sequences, and is followed by an extremely long intron. The coding sequence of the cha-1 transcript is disrupted by mutations in the cha-1 gene. We have determined the sites of four transposon insertions and the end-points of two deletions that lead to the cha-1 mutant phenotype; one of the deletions appears to eliminate gene function completely. Comparison of the Drosophila, rat, and C. elegans genes reveals conserved motifs and conserved intron sites.