Novel Insights on Nitric Oxide Synthase and NO Signaling in Ascidian Metamorphosis.

Nitric oxide (NO) is a pivotal signaling molecule involved in a wide range of physiological and pathological processes. We investigated NOS/NO localization patterns during the different stages of larval development in the ascidia Ciona robusta and evidenced a specific and temporally controlled pattern. NOS/NO expression starts in the most anterior sensory structures of the early larva and progressively moves towards the caudal portion as larval development and metamorphosis proceeds. We here highlight the pattern of NOS/NO expression in the central and peripheral nervous system of Ciona larvae which precisely follows the progression of neural signals of the central pattern generator necessary for the control of the movements of the larva towards the substrate. This highly dynamic localization profile perfectly matches with the central role played by NO from the first phase of settlement induction to the next control of swimming behavior, adhesion to substrate and progressive tissue resorption and reorganization of metamorphosis itself.

Onecut Regulates Core Components of the Molecular Machinery for Neurotransmission in Photoreceptor Differentiation.

Photoreceptor cells (PRC) are neurons highly specialized for sensing light stimuli and have considerably diversified during evolution. The genetic mechanisms that underlie photoreceptor differentiation and accompanied the progressive increase in complexity and diversification of this sensory cell type are a matter of great interest in the field. A role of the homeodomain transcription factor Onecut (Oc) in photoreceptor cell formation is proposed throughout multicellular organisms. However, knowledge of the identity of the Oc downstream-acting factors that mediate specific tasks in the differentiation of the PRC remains limited. Here, we used transgenic perturbation of the Ciona robusta Oc protein to show its requirement for ciliary PRC differentiation. Then, transcriptome profiling between the trans-activation and trans-repression Oc phenotypes identified differentially expressed genes that are enriched in exocytosis, calcium homeostasis, and neurotransmission. Finally, comparison of RNA-Seq datasets in Ciona and mouse identifies a set of Oc downstream genes conserved between tunicates and vertebrates. The transcription factor Oc emerges as a key regulator of neurotransmission in retinal cell types.

Functional conserved non-coding elements among tunicates and chordates.

Non-coding regions with dozens to several hundred base pairs of extreme conservation have been found in all metazoan genomes. The distribution of these conserved non-coding elements (CNE) within and across genomes has suggested that many of them may have roles as transcriptional regulatory elements. A combination of bioinformatics and experimental approaches can be used to identify CNEs with regulatory activity in phylogenetically distant species. Nevertheless, the high divergent rate of genomic sequences of several organisms, such as tunicates, complicates the characterization of these conserved elements and very few examples really may prove their functional activity. We used a comparative approach to facilitate the identification of CNEs among distantly related or highly divergent species and experimentally demonstrated the functional significance of these novel CNEs. We first experimentally tested, in C. robusta and D. rerio transgenic embryos, the regulatory activity of conserved elements associated to genes involved in developmental control among different chordates (Homo sapiens and Danio rerio for vertebrates, Ciona robusta and Ciona savignyi for tunicates and Branchiostoma floridae for cephalochordates). Once demonstrated the cross-species functional conservation of these CNEs, the same gene loci were used as references to locate homologous regions and possible CNEs in available tunicate genomes. Comparison of tunicate-specific and chordate-specific CNEs revealed absence of conservation of the regulatory elements in spite of conservation of regulatory patterns, likely due to evolutionary specification of the respective developmental networks. This result highlights the importance of an integrative in-silico/in-vivo approach to CNEs investigation, encompassing both bioinformatics, essential for putative CNEs identification, and laboratory experiments, pivotal for the understanding of CNEs functionality.

Regulatory elements retained during chordate evolution: coming across tunicates.

Understanding the role of conserved noncoding elements (CNEs) throughout the genome is taking advantage of the improved efficiency of genome-sequencing techniques and bioinformatics tools. Tunicates diverged before the vertebrate whole genome duplications and, therefore, represent an optimal model system to study the evolution of complex regulatory networks. Here, we review the current knowledge on the characterization of CNEs during embryonic development, focusing on the evolutionary similarity and divergence between tunicates and other chordates. Many vertebrate specific CNEs that regulate developmental processes were identified based on high level of sequence conservation, but only few of them have been recognized in tunicates or other invertebrates because of genomic sequences divergence. We discuss recent studies demonstrating that a combination of different methodologies, based not only on high sequence identity, can collectively be used to identify CNEs with regulatory activity in phylogenetically distant species. Here, a low sequence constraints approach was successfully used to search orthologous chordate gene regions for cross-species conserved regulatory elements that control developmental genes.

New insights into protamine-like component organization in Mytilus galloprovincialis' sperm chromatin.

We have analyzed Mytilus galloprovincialis' sperm chromatin, which consists of three protamine-like proteins, PL-II, PL-III, and PL-IV, in addition to a residual amount of the four core histones. We have probed the structure of this sperm chromatin through digestion with micrococcal nuclease (MNase) in combination with salt fractionation. Furthermore, we used the electrophoretic mobility shift assay to define DNA-binding mode of PL-II and PL-III and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. Although in literature it is reported that M. galloprovincialis' sperm chromatin lacks nucleosomal organization, our results obtained by MNase digestion suggest the existence of a likely unusual organization, in which there would be a more accessible location of PL-II/PL-IV when compared with PL-III and core histones. So, we hypothesize that in M. galloprovincialis' sperm chromatin organization DNA is wrapped around a PL-III protein core and core histones and PL-II and PL-IV are bound to the flanking DNA regions (similarly to somatic histone H1). Furthermore, we propose that PL's K/R ratio affects their DNA-binding mode and self-association ability as reported previously for somatic and sperm H1 histones.