Unlike other neuronal counterparts, primary synaptic proteins are not known to be involved in vascular physiology. Here, we demonstrate that neurexins and neuroligins, which constitute large and complex families of fundamental players in synaptic activity, are produced and processed by endothelial and vascular smooth muscle cells throughout the vasculature. Moreover, they are dynamically regulated during vessel remodeling and form endogenous complexes in large vessels as well as in the brain. We used the chicken chorioallantoic membrane as a system to pursue functional studies and demonstrate that a monoclonal recombinant antibody against ␤-neurexin inhibits angiogenesis, whereas exogenous neuroligin has a role in promoting angiogenesis. Finally, as an insight into the mechanism of action of ␤-neurexin, we show that the anti-␤-neurexin antibody influences vessel tone in isolated chicken arteries. Our finding strongly supports the idea that even the most complex and plastic events taking place in the nervous system (i.e., synaptic activity) share molecular cues with the vascular system. angiogenesis ͉ vessel tone ͉ cell-to-cell adhesion ͉ nervous-vascular parallels ͉ synapses

Recently, it has been shown that targeted mutagenesis using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to generate knockout zebrafish lines for analysis of their function and/or developing disease models. A number of different methods have been developed for the design and assembly of gene-specific ZFNs and TALENs, making them easily available to most zebrafish researchers. Regardless of the choice of targeting nuclease, the process of generating mutant fish is similar. It is a time-consuming and multi-step process that can benefit significantly from development of efficient high throughput methods. In this study, we used ZFNs assembled through either the CompoZr (Sigma-Aldrich) or the CoDA (context-dependent assembly) platforms to generate mutant zebrafish for nine genes. We report our improved high throughput methods for 1) evaluation of ZFNs activity by somatic lesion analysis using colony PCR, eliminating the need for plasmid DNA extractions from a large number of clones, and 2) a sensitive founder screening strategy using fluorescent PCR with PIG-tailed primers that eliminates the stutter bands and accurately identifies even single nucleotide insertions and deletions. Using these protocols, we have generated multiple mutant alleles for seven genes, five of which were targeted with CompoZr ZFNs and two with CoDA ZFNs. Our data also revealed that at least five-fold higher mRNA dose was required to achieve mutagenesis with CoDA ZFNs than with CompoZr ZFNs, and their somatic lesion frequency was lower (<5%) when compared to CopmoZr ZFNs (9–98%). This work provides high throughput protocols for efficient generation of zebrafish mutants using ZFNs and TALENs.

Integrin alpha5beta1 is among the proteins overexpressed on tumor vessels and is a potential target for diagnostics and therapeutics. Here, we mapped the distribution of alpha5beta1 integrin in three murine tumor models and identified sites of expression that are rapidly accessible to intravascular antibodies. When examined by conventional immunohistochemistry, alpha5beta1 integrin expression was strong on most blood vessels in RIP-Tag2 transgenic mouse tumors, adenomatous polyposis coli (apc) mouse adenomas, and implanted MCa-IV mammary carcinomas. Expression increased during malignant progression in RIP-Tag2 mice. However, immunoreactivity was also strong in normal pancreatic ducts, intestinal smooth muscle, and several other sites. To determine which sites of expression were rapidly accessible from the bloodstream, we intravenously injected anti-alpha5beta1 integrin antibody and 10 minutes to 24 hours later examined the amount and distribution of labeling. The injected antibody strongly labeled tumor vessels at all time points but did not label most normal blood vessels or gain access to pancreatic ducts or intestinal smooth muscle. Intense vascular labeling by anti-alpha5beta1 integrin antibody co-localized with the uniform CD31 immunoreactivity of tumor vessels and contrasted sharply with the patchy accumulation of nonspecific IgG at sites of leakage. This strategy of injecting antibodies revealed the uniform overexpression and rapid accessibility of alpha5beta1 integrin on tumor vessels and may prove useful in assessing other potential therapeutic targets in cancer.

Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retroviral insertion or CRISPR/Cas9 approaches, and developed a high-throughput screening pipeline for analyzing hair cell development and regeneration. We screened 254 gene mutations and identified 7 genes specifically affecting hair cell regeneration. These hair cell regeneration genes fell into distinct and somewhat surprising functional categories. By examining the regeneration of caudal fin and liver, we found these hair cell regeneration genes often also affected other types of tissue regeneration. Therefore, our results demonstrate guided screening is an effective approach to discover regeneration candidates, and hair cell regeneration is associated with other tissue regeneration.

Rissone et al. demonstrate that adenylate kinase AK2, an enzyme mutated in reticular dysgenesis (RD) in humans, prevents oxidative stress during hematopoiesis. Using a zebrafish model, as well as induced pluripotent stem cells derived from an RD patient, they find that AK2 deficiency affects hematopoietic stem and progenitor development with increased oxidative stress. Antioxidant treatment rescues the hematopoietic phenotypes.

The scientific interest in the family of the so-called nervous vascular parallels has been growing steadily for the past 15 years, either by addition of new members to the group or, lately, by deepening the analysis of established concepts and mediators. Proteins governing both neurons and vascular cells are known to be involved in events such as cell fate determination and migration/guidance but not in the last and apparently most complex step of nervous system development, the formation and maturation of synapses. Hence, the recent addition to this family of the specific synaptic proteins, Neurexin and Neuroligin, is a double innovation. The two proteins, which were thought to be "simple" adhesive links between the pre- and post-synaptic sides of chemical synapses, are in fact extremely complex and modulate the most subtle synaptic activities. We will discuss the relevant data and the intriguing challenge of transferring synaptic activities to vascular functions.

Neurexins constitute a family of proteins originally identified as synaptic transmembrane receptors for a spider venom toxin. In mammals, the 3 known Neurexin genes present 2 alternative promoters that drive the synthesis of a long (alpha) and a short (beta) form and contain different sites of alternative splicing (AS) that can give rise to thousands of different transcripts. To date, very little is known about the significance of this variability, except for the modulation of binding to some of the Neurexin ligands. Although orthologs of Neurexins have been isolated in invertebrates, these genes have been studied mostly in mammals. With the aim of investigating their functions in lower vertebrates, we chose Danio rerio as a model because of its increasing importance in comparative biology. We have isolated 6 zebrafish homologous genes, which are highly conserved at the structural level and display a similar regulation of AS, despite about 450 Myr separating the human and zebrafish species. Our data indicate a strong selective pressure at the exonic level and on the intronic borders, in particular on the regulative intronic sequences that flank the exons subject to AS. Such a selective pressure could help conserve the regulation and consequently the function of these genes along the vertebrates evolutive tree. AS analysis during development shows that all genes are expressed and finely regulated since the earliest stages of development, but mark an increase after the 24-h stage that corresponds to the beginning of synaptogenesis. Moreover, we found that specific isoforms of a zebrafish Neurexin gene (nrxn1a) are expressed in the adult testis and in the earliest stages of development, before the beginning of zygotic transcription, indicating a potential delivery of paternal RNA to the embryo. Our analysis suggests the existence of possible new functions for Neurexins, serving as the basis for novel approaches to the functional studies of this complex neuronal protein family and more in general to the understanding of the AS mechanism in low vertebrates.

Neuroligins constitute a family of transmembrane proteins localized at the postsynaptic side of both excitatory and inhibitory synapses of the central nervous system. They are involved in synaptic function and maturation and recent studies have linked mutations in specific human Neuroligins to mental retardation and autism. We isolated the human Neuroligin homologs in Danio rerio. Next, we studied their gene structures and we reconstructed the evolution of the Neuroligin genes across vertebrate phyla. Using reverse-transcriptase polymerase chain reaction, we analyzed the expression and alternative splicing pattern of each gene during zebrafish embryonic development and in different adult organs. By in situ hybridization, we analyzed the temporal and spatial expression pattern during embryonic development and larval stages and we found that zebrafish Neuroligins are expressed throughout the nervous system. Globally, our results indicate that, during evolution, specific subfunctionalization events occurred within paralogous members of this gene family in zebrafish. Developmental Dynamics 239:688-702,