Mutation studies in ascidians: a review.

Historically, mutations have had a significant impact on the study of developmental processes and phenotypic evolution. Lesions in DNA are created by artificial methods or detected by natural genetic variation. Random mutations are then ascribed to genetic change by direct sequencing or positional cloning. Tunicate species of the ascidian genus Ciona represent nearly fully realized model systems in which gene function can be investigated in depth. Additionally, tunicates are valuable organisms for the study of naturally occurring mutations due to the capability to exploit genetic variation down to the molecular level. Here, we summarize the available information about how mutations are studied in ascidians with examples of insights that have resulted from these applications. We also describe notions and methodologies that might be useful for the implementation of easy and tight procedures for mutations studies in Ciona.

Auto and cross regulatory elements control Onecut expression in the ascidian nervous system.

The expression pattern of Onecut genes in the central and peripheral nervous systems is highly conserved in invertebrates and vertebrates but the regulatory networks in which they are involved are still largely unknown. The presence of three gene copies in vertebrates has revealed the functional roles of the Onecut genes in liver, pancreas and some populations of motor neurons. Urochordates have only one Onecut gene and are the closest living relatives of vertebrates and thus represent a good model system to understand its regulatory network and involvement in nervous system formation. In order to define the Onecut genetic cascade, we extensively characterized the Onecut upstream cis-regulatory DNA in the ascidian Ciona intestinalis. Electroporation experiments using a 2.5kb genomic fragment and of a series of deletion constructs identified a small region of 262bp able to reproduce most of the Onecut expression profile during embryonic development. Further analyses, both bioinformatic and in vivo using transient transgenes, permitted the identification of transcription factors responsible for Onecut endogenous expression. We provide evidence that Neurogenin is a direct activator of Onecut and that an autoregulatory loop is responsible for the maintenance of its expression. Furthermore, for the first time we propose the existence of a direct connection among Neurogenin, Onecut and Rx transcription factors in photoreceptor cell formation.

Changes in Adhesion and the Expression of Adhesion Molecules in PBMCs after Aneurysmal Subarachnoid Hemorrhage: Relation to Cerebral Vasospasm.

Aneurysmal subarachnoid hemorrhage (aSAH) is a neurovascular disease produced by extravasation of blood to the subarachnoid space after rupture of the cerebral vessels. After bleeding, the immune response is activated. The role of peripheral blood mononuclear cells (PBMCs) in this response is a current subject of research. We have analysed the changes in PBMCs of patients with aSAH and their interaction with the endothelium, focusing on their adhesion and the expression of adhesion molecules. Using an in vitro adhesion assay, we observed that the adhesion of PBMCs of patients with aSAH is increased. Flow cytometry analysis shows that monocytes increased significantly in patients, especially in those who developed vasospasm (VSP). In aSAH patients, the expression of CD162, CD49d, CD62L and CD11a in T lymphocytes and of CD62L in monocytes increased. However, the expression of CD162, CD43, and CD11a decreased in monocytes. Furthermore, monocytes from patients who developed arteriographic VSP had lower expression of CD62L. In conclusion, our results confirm that after aSAH, monocyte count and adhesion of PBMCs increase, especially in patients with VSP, and that the expression of several adhesion molecules is altered. These observations can help predict VSP and to improve the treatment of this pathology.

Onecut is a direct neural-specific transcriptional activator of Rx in Ciona intestinalis.

Retinal homeobox (Rx) genes play a crucial and conserved role in the development of the anterior neural plate of metazoans. During chordate evolution, they have also acquired a novel function in the control of eye formation and neurogenesis. To characterize the Rx genetic cascade and shed light on the mechanisms that led to the acquisition of this new role in eye development, we studied Rx transcriptional regulation using the ascidian, Ciona intestinalis. Through deletion analysis of the Ci-Rx promoter, we have identified two distinct enhancer elements able to induce Ci-Rx specific expression in the anterior part of the CNS and in the photosensory organ at tailbud and larva stages. Bioinformatic analysis highlighted the presence of two Onecut binding sites contained in these enhancers, so we explored the role of this transcription factor in the regulation of Ci-Rx. By in situ hybridization, we first confirmed that these genes are co-expressed in the same cells. Through a series of in vivo and in vitro experiments, we then demonstrated that the two Onecut sites are responsible for enhancer activation in Ci-Rx endogenous territories. We also demonstrated in vivo that Onecut misexpression is able to induce ectopic activation of the Rx promoter. Finally, we demonstrated that Ci-Onecut is able to promote Ci-Rx expression in the sensory vesicle. Together, these results support the conclusion that in Ciona embryogenesis, Ci-Rx expression is under the control of the Onecut transcription factor and that this factor is necessary and sufficient to specifically activate Ci-Rx through two enhancer elements.

New insights into the evolution of metazoan tyrosinase gene family.

Tyrosinases, widely distributed among animals, plants and fungi, are involved in the biosynthesis of melanin, a pigment that has been exploited, in the course of evolution, to serve different functions. We conducted a deep evolutionary analysis of tyrosinase family amongst metazoa, thanks to the availability of new sequenced genomes, assessing that tyrosinases (tyr) represent a distinctive feature of all the organisms included in our study and, interestingly, they show an independent expansion in most of the analyzed phyla. Tyrosinase-related proteins (tyrp), which derive from tyr but show distinct key residues in the catalytic domain, constitute an invention of chordate lineage. In addition we here reported a detailed study of the expression territories of the ascidian Ciona intestinalis tyr and tyrps. Furthermore, we put efforts in the identification of the regulatory sequences responsible for their expression in pigment cell lineage. Collectively, the results reported here enlarge our knowledge about the tyrosinase gene family as valuable resource for understanding the genetic components involved in pigment cells evolution and development.