Comparative Genome Analysis of the Neurexin Gene Family in Danio rerio: Insights into Their Functions and Evolution

Abstract

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.

Characterization of the neuroligin gene family expression and evolution in zebrafish

“…The identity and conservation values are higher than 70 and 80%, respectively, further supporting the identity of the cloned genes. As we previously observed comparing zebrafish and human Neurexin proteins (Rissone et al, 2007), also zebrafish Neuroligins are very similar to their human homologs, notwithstanding the evolutionary distance from the common ancestor (Aparicio et al, 2002). This high degree of sequence similarity can be explained by the presence of positive evolutionary pressure acting on both Neurexins and Neuroligins strongly suggesting a conservation of their functions across vertebrate evolution.…”

“…Figure 5B shows the different isoforms expressed during zebrafish embryonic development. As the Neurexin genes (Rissone et al, 2007), many nlgns (1, 2a, 2b, 3a, and 4b) are expressed since the earliest stages of development and they increase their expression from 24 hr post fertilization (hpf).…”

Nervous vascular parallels: axon guidance and beyond

“…Both gene families present a high degree of evolutionary conservation that can be explained by the action of a positive selective pressure which in turn suggests a conservation of their functions across vertebrate evolution (Rissone et al, 2007;Rissone et al, 2010) Neurexins, which are produced as a long (a) and a short (b) forms by each of their genes, have been widely studied because of their exceptionally extended alternative splicing (Rissone et al, 2007;Tabuchi and Sudhof, 2002) and have been indirectly localized at the pre-synaptic membrane (Sudhof, 2001). Thousands of different isoforms were predicted to be produced by the three mammalian neurexin genes, so that they were proposed as molecular codes for the specific reciprocal neuronal recognition (Missler and Sudhof, 1998).…”

The synaptic proteins neurexins and neuroligins are widely expressed in the vascular system and contribute to its functions

“…Neurexins, produced in long (␣) and short (␤) forms, have been widely studied because of their extended alternative splicing (1,3) and have been localized indirectly at the presynaptic membrane (4). Neuroligins are localized at the postsynaptic membrane (5) and interact with neurexins from the opposite side (in trans) of the synaptic cleft in a calciumdependent manner (6).…”