Brain and retina regionalization in sharksstudy based on the spatiotemporal expression pattern of Pax6 and other neurochemical markers

Resumo

Pax6 is a highly conserved transcription factor which has shown to be crucial for the normal development of the Central Nervous System (CNS), being involved in regionalization but also in regulating morphogenesis. The Pax6 expression pattern has been widely analyzed in the developing CNS of many vertebrates, however studies in elasmobranchs are lacking despite their key phylogenetic position as sister group to osteichthyans which makes them suitable for obtaining knowledge about the evolution of CNS development. The aims of this thesis were: to study the spatiotemporal Pax6 expression in the developing and postembryonic shark brain, to comparatively analyse the distribution of Pax6 and that of different neurochemical markers such as glutamic acid decarboxylase (GAD, the gammaaminobutyric acid synthesizing enzyme), calretinin (CR), tyrosine hydroxylase (TH; the ratelimiting enzyme of the catecholamine synthesis) and the signalling molecule Shh in order to obtain more accurate information on shark brain regionalization, and to study proliferation and differentiation patterns in the developing cerebellar body in relation to Pax6. Moreover, we aimed to study the spatiotemporal distribution of Pax6 during retinogenesis and in the mature retina, as well as the Pax6 patterns in relation to proliferation [proliferating cell nuclear antigen (PCNA)] and differentiation markers [GAD, CR, TH, serotonin (5-HT), and CERN922-opsin]. We have analyzed embryos and juvenile specimens of two species representatives of elasmobranch fish: Scyliorhinus canicula & Haploblepharus fuscus, by means of immunohistochemical and in situ hybridization techniques. The spatiotemporal progression of the Pax6 expression (protein and mRNA) in the developing brain of sharks studied revealed many similarities to those in other vertebrates, which supports the existence of an evolutionary conserved pattern. The recognition of this pattern makes the Pax6 distribution in the developing brain one of the most useful tools for comparative studies, especially in the search for homologies. The distribution pattern of Pax6 cells in sharks, especially when compared with the distribution of markers as TH, GAD, CR and Shh, highlights the presence of longitudinal and transverse boundaries, such as the alar-basal, diencephalic-mesencephalic and pallial-subpallial boundaries, and provides accurate regional information about prosomeric domains. In sharks, likewise in other vertebrates, Pax6 represents a good marker of dorsal telencephalic territories and of the pallial-subpallial border. Interestingly, proliferation and differentiation patterns revealed in sharks during the development of the cerebellar body with Pax6, PCNA, GAD/GABA, and CR have shown remarkable similarities with those of the cerebella of tetrapods, thus showing the importance of elasmobranch models to reconstruct the evolutionary developmental history of the vertebrate cerebellum. Concerning the retina, two distinct cell populations were observed regarding intensity of Pax6 immunostaining, These Pax6 populations were also observed in double immunolabelling with PCNA, interestingly PCNA was present in Pax6 weakly labelled cells. While strongly Pax6 immunoreactive cells were PCNA negative. The time course of Pax6 expression in relation to the patterns of cells proliferation and differentiation in the shark retina suggests a shift in the role of Pax6 over time. Thus, Pax6 may be involved in the maintenance of the proliferative stage in retinoblasts and in the regulation of the differentiation of particular cell types as layering progresses from the vitreal to the scleral retina. In addition, the persistence of Pax6 expression in cells of the adult retina of sharks indicates a possible role in the ageing eye. The developmental study of GABAergic markers in the developing retina of sharks showed that besides its role as neurotransmitter, GABA may have a role in regulating retinogenesis since GABAergic cells are observed before retinal layering was evident. GAD and Pax6 codistributed in the retina throughout development, and we have also found colocalization of Pax6 and GAD in postmitotic (PCNA negative) cells in amacrine and horizontal cells as they differentiate in the layered retina. Moreover, double and triple immunolabelling with differentiation markers such as CR, TH, 5-HT showed that Pax6 colocalize with CR in some cells of the GCL, with 5-HT and GAD in amacrine cells of the INLi and with GAD in cells of the HCL. Thus, a number of Pax6-ir cells are immunonegative for these markers, which indicates that the presence of Pax6 is not sufficient for directing cells towards one of these specific fates. Conversely, some postmitotic (PCNA-negative) cells lack Pax6, which indicates that Pax6 alone is dispensable for the acquisition and/or maintenance of CR-ir, 5-HT-ir and GAD-ir phenotypes. Overall, our results highlight the importance of studies on developing elasmobranchs as they contribute to infer the ancestral condition of brain organization in gnathostomes. Moreover, in this study we provide evidences that sharks are especially suitable to describe the dynamics of brain and retina development, as their lengthened growth and the large size of the embryonic brain allow disclosing fine details of developmental processes.