Estrutura e funcións celulares

Titulación: Grao en Matemáticas -- Materia: Bioloxía BásicaA célula é a unidade básica da vida. Todos e cada un dos organismos están formados por unha ou máis células. Todas estas células comparten características esenciais que se conservaron ao longo da evolución. Por exemplo, todas as células conteñen material xenético que dirixe todas as actividades celulares e se transmite ás células fillas. Todas están rodeadas por unha membrana plasmática que define a identidade da célula, xa que separa o seu contido interno (o citoplasma) do medio externo. Ademais, todas as células eucariotas conteñen varios orgánulos rodeados de membrana, que proporcionan compartimentos especializados con distintas funcións. Estas células posúen ademais unha rede de filamentos de proteínas que proporcionan un armazón que determina a forma celular, a posición dos orgánulos e os movementos da célula no seu conxunto. Polo tanto, comprender a estrutura celular (como é) e como funcionan os seus compoñentes resulta fundamental para descifrar o comportamento celular. Así, poderemos coñecer como o conxunto das actividades da célula se realiza de forma controlada, eficaz e herdable. Nesta unidade faise un estudo da membrana plasmática, dos distintos orgánulos da célula e do citoesqueleto. Estudarase a importancia biolóxica destas estruturas a nivel xeral e as súas funcións na comunicación entre a célula e o seu medio, a síntese, procesamento e transporte de proteínas e lípidos, os distintos tipos de movemento celular e a obtención de enerxía. Analizaremos como a regulación das actividades da célula permite acadar un equilibrio entre crecemento, maduración, división e morte celular.Tamén estudaremos como a alteración dos mecanismos de regulación celular deriva no desenvolvemento do cancro.Universidade de Santiago de Compostela. Servizo de Normalización Lingüístic

Identification of Radial Glia Progenitors in the Developing and Adult Retina of Sharks

Neural stem cells give rise to transient progenitors termed neuroepithelial cells (NECs) and radial glial cells (RGCs). RGCs represent the major source of neurons, glia and adult stem cells in several regions of the central nervous system (CNS). RGCs are mostly transient in mammals, but they are widely maintained in the adult CNS of fishes, where they continue to be morphologically similar to RGCs in the mammalian brain and fulfill similar roles as progenitors and guide for migrating neurons. The retina of fishes offers an exceptional model to approach the study of adult neurogenesis because of the presence of constitutive proliferation from the ciliary marginal zone (CMZ), containing NECs, and from adult glial cells with radial morphology (the Müller glia). However, the cellular hierarchies and precise contribution of different types of progenitors to adult neurogenesis remain unsolved. We have analyzed the transition from NECs to RGCs and RGC differentiation in the retina of the cartilaginous fish Scyliorhinus canicula, which offers a particularly good spatial and temporal frame to investigate this process. We have characterized progenitor and adult RGCs by immunohistochemical detection of glial markers as glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). We have compared the emergence and localization of glial markers with that of proliferating cell nuclear antigen (PCNA, a proliferation maker) and Doublecortin (DCX, which increases at early stages of neuronal differentiation). During retinal development, GFAP-immunoreactive NECs located in the most peripheral CMZ (CMZp) codistribute with DCX-immunonegative cells. GFAP-immunoreactive RGCs and Müller cells are located in successive more central parts of the retina and codistribute with DCX- and DCX/GS-immunoreactive cells, respectively. The same types of progenitors are found in juveniles, suggesting that the contribution of the CMZ to adult neurogenesis implies a transition through the radial glia (RG) stateThis work was supported by the Spanish Dirección General de Investigación Científica y Técnica-FEDER (BFU2010-15816 and BFU2014-58631-P), the Xunta de Galicia (10PXIB200051PR, IN845B-2010/159 and CN 2012/237)S

Use of vivo-morpholinos for gene knockdown in the postnatal shark retina

Work in the catshark Scyliorhinus canicula has shown that the evolutionary origin of postnatal neurogenesis in vertebrates is earlier than previously thought. Thus, the catshark can serve as a model of interest to understand postnatal neurogenic processes and their evolution in vertebrates. One of the best characterized neurogenic niches of the catshark CNS is found in the peripheral region of the retina. Unfortunately, the lack of genetic tools in sharks limits the possibilities to deepen in the study of genes involved in the neurogenic process. Here, we report a method for gene knockdown in the juvenile catshark retina based on the use of Vivo-Morpholinos. To establish the method, we designed Vivo-Morpholinos against the proliferation marker PCNA. We first evaluated the possible toxicity of 3 different intraocular administration regimes. After this optimization step, we show that a single intraocular injection of the PCNA Vivo-Morpholino decreases the expression of PCNA in the peripheral retina, which leads to reduced mitotic activity in this region. This method will help in deciphering the role of other genes potentially involved in postnatal neurogenesis in this animal modelGrant PID 2020-115121 GB-I00 funded by MCIN/AEI/10.13039/501100011033 to A. Barreiro-Iglesias. Grant ED 431C 2021/18 funded by Xunta de Galicia to E. CandalS

Expression of radial glial markers (GFAP, BLBP and GS) during telencephalic development in the catshark (Scyliorhinus canicula)

Radial glial cells (RGCs) are the first cell populations of glial nature to appear during brain ontogeny. They act as primary progenitor (stem) cells as well as a scaffold for neuronal migration. The proliferative capacity of these cells, both in development and in adulthood, has been subject of interest during past decades. In contrast with mammals where RGCs are restricted to specific ventricular areas in the adult brain, RGCs are the predominant glial element in fishes. However, developmental studies on the RGCs of cartilaginous fishes are scant. We have studied the expression patterns of RGCs markers including glial fibrillary acidic protein (GFAP), brain lipid binding protein (BLBP), and glutamine synthase (GS) in the telencephalic hemispheres of catshark (Scyliorhinus canicula) from early embryos to post-hatch juveniles. GFAP, BLBP and GS are first detected, respectively, in early, intermediate and late embryos. Expression of these glial markers was observed in cells with radial glia morphology lining the telencephalic ventricles, as well as in their radial processes and endfeet at the pial surface and their expression continue in ependymal cells (or tanycytes) in early juveniles. In addition, BLBP- and GS-immunoreactive cells morphologically resembling oligodendrocytes were observed. In late embryos, most of the GFAP- and BLBP-positive RGCs also coexpress GS and show proliferative activity. Our results indicate the existence of different proliferating subpopulations of RGCs in the embryonic ventricular zone of catshark. Further investigations are needed to determine whether these proliferative RGCs could act as neurogenic and/or gliogenic precursors.This work was supported by the Spanish Ministerio de Economía y Competitividad-FEDER (BFU2014-5863-1P); Xunta de Galicia-FEDER, Grant number: Rede Galega INBIOEST ED341D R2016/032S

Mitral cell development in the olfactory bulb of sharks: evidences of a conserved pattern of glutamatergic neurogenesis

In mammals, the development of the olfactory bulb (OB) relies in part on the expression of transcription factors involved in the specifications/differentiation of glutamatergic cells. In a previous study from our group, a high molecular similarity was reported between mammals and cartilaginous fishes regarding the neurogenic mechanisms underlying the development of glutamatergic cells in the telencephalon. However, information about the transcriptional program operating in the development of the glutamatergic system (mainly represented by mitral cells) in the OB is lacking in the catshark Scyliorhinus canicula, a cartilaginous fish. Using immunohistochemistry and in situ hybridization techniques, we have found that, previously to the appearance of the olfactory primordium (OP), proliferating cells expressing Pax6 with molecular hallmarks of progenitor radial glia were located in the ventrolateral pallial ventricular zone. Later in development, when the OP is recognizable, a stream of Pax6-positive cells were observed between the ventricular zone and the OP, where transcription factors involved in mitral cell development in mammals (ScTbr2, ScNeuroD, Tbr1) are expressed. Later in development, these transcription factors became expressed in a layered-like structure where ScVglut1, a marker of mitral cells, is also present. Our data suggest that the transcriptional program related with the specification/differentiation of glutamatergic cells in the telencephalon has been conserved throughout the evolution of vertebrates. These results, in combination with previous studies concerning GABAergic neurogenesis in sharks, have evidenced that the OB of mammals and sharks shares similarities in the timing and molecular programs of development.This work was supported by the Spanish Ministerio de Economía y Competitividad-FEDER (BFU2014-5863-1P and BFU2017-8986-1P) and CNRS Université Pierre et Marie Curie Grant No. ANR-16-CE13-0013-02S

Developmental genoarchitectonics as a key tool to interpret the mature anatomy of the chondrichthyan hypothalamus according to the prosomeric model

The hypothalamus is a key vertebrate brain region involved in survival and physiological functions. Understanding hypothalamic organization and evolution is important to deciphering many aspects of vertebrate biology. Recent comparative studies based on gene expression patterns have proposed the existence of hypothalamic histogenetic domains (paraventricular, TPa/PPa; subparaventricular, TSPa/PSPa; tuberal, Tu/RTu; perimamillary, PM/PRM; and mamillary, MM/RM), revealing conserved evolutionary trends. To shed light on the functional relevance of these histogenetic domains, this work aims to interpret the location of developed cell groups according to the prosomeric model in the hypothalamus of the catshark Scyliorhinus canicula, a representative of Chondrichthyans (the sister group of Osteichthyes, at the base of the gnathostome lineage). To this end, we review in detail the expression patterns of ScOtp, ScDlx2, and ScPitx2, as well as Pax6-immunoreactivity in embryos at stage 32, when the morphology of the adult catshark hypothalamus is already organized. We also propose homologies with mammals when possible. This study provides a comprehensive tool to better understand previous and novel data on hypothalamic development and evolutionThis work was supported by Ministerio de Economía, Industria y Competitividad – Agencia Estatal de Investigación (grant No. BFU2017-89861-P) partially financed by the European Social Fund, and by Xunta de Galicia (grant No. ED431C 2021/18)S

Prosomeric organization of the hypothalamus in an elasmobranch, the catshark Scyliorhinus canicula

The hypothalamus has been a central topic in neuroanatomy because of its important physiological functions, but its mature organization remains elusive. Deciphering its embryonic and adult organization is crucial in an evolutionary approach of the organization of the vertebrate forebrain. Here we studied the molecular organization of the hypothalamus and neighboring telencephalic domains in a cartilaginous fish, the catshark, Scyliorhinus canicula, focusing on ScFoxg1a, ScShh, ScNkx2.1, ScDlx2/5, ScOtp, and ScTbr1 expression profiles and on the identification αacetylated-tubulin-immunoreactive (ir), TH-ir, 5-HT-ir, and GFAP-ir structures by means of immunohistochemistry. Analysis of the results within the updated prosomeric model framework support the existence of alar and basal histogenetic compartments in the hypothalamus similar to those described in the mouse, suggesting the ancestrality of these subdivisions in jawed vertebrates. These data provide new insights into hypothalamic organization in cartilaginous fishes and highlight the generality of key features of the prosomeric model in jawed vertebrates.This work was supported by grants from the Spanish Dirección General de Investigación-FEDER (BFU2010- 15816), the Xunta de Galicia (10PXIB200051PR, CN 2012/237), European Community-Research Infrastructure Action under the FP7 “Capacities” Specific Programme (ASSEMBLE 227799), the Région Centre, Région Bretagne (EVOVERT grant number 049755; PEPTISAN project), National Research Agency (grant ANR-09-BLAN-026201), CNRS, Université d’Orléans and Université Pierre et Marie Curie. GNSD would like to thank Spanish SEPE for its funding supportS

Pluronic®/casein micelles for ophthalmic delivery of resveratrol: in vitro, ex vivo, and in vivo tests

Ocular health may strongly benefit from the supply of antioxidant agents that counteract free radicals and reactive oxygen species responsible for long-term eye diseases. Additionally, natural antioxidants like resveratrol can inhibit bacteria growth and restore natural microbiota. However, their use is hindered by limited solubility, fast degradation, and low ocular permeability. This work aimed to overcome these limitations by preparing single and mixed micelles of Pluronic® F127 and casein that serve as resveratrol nanocarriers. Single and mixed (0.1 % casein) micelles (0.0 to −17.0 mV; 2.4 to 32.7 nm) increased 50-fold resveratrol solubility, remained stable for one month at 4 °C, withstood fast dilution, underwent sol-to-gel transitions in the 23.9–27.1 °C range, and exhibited potent antioxidant properties. All formulations successfully passed the HET-CAM assay but showed Pluronic®-casein dose-dependent toxicity in the zebrafish embryo model. Resveratrol-loaded single and mixed micelles (10–15 mM Pluronic® F127) displayed antimicrobial activity against S. aureus and P. aeruginosa. The micelles favored resveratrol accumulation in cornea and sclera, but mixed micelles showed larger lag times and provided lower amount of resveratrol permeated through sclera. In vivo (rabbit) tests confirmed the safety of resveratrol-loaded single micelles and their capability to supply resveratrol to anterior and posterior eye segmentsThe work was supported by MCIN/AEI/10.13039/501100011033 [PID 2020-113881RB-I00 to A.C. and C.A.-L., and PID2020-115121GB-I00 to L.S. and A.B.-I.], Spain, Xunta de Galicia [ED431C 2020/17], and FEDER. M. Vivero-Lopez acknowledges Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria) for a predoctoral research fellowship [ED481A-2019/120]S

Pluronic®/casein micelles for ophthalmic delivery of resveratrol: In vitro, ex vivo, and in vivo tests

Received 10 July 2022; Received in revised form 3 October 2022; Accepted 7 October 2022Ocular health may strongly benefit from the supply of antioxidant agents that counteract free radicals and reactive oxygen species responsible for long-term eye diseases. Additionally, natural antioxidants like resveratrol can inhibit bacteria growth and restore natural microbiota. However, their use is hindered by limited solubility, fast degradation, and low ocular permeability. This work aimed to overcome these limitations by preparing single and mixed micelles of Pluronic® F127 and casein that serve as resveratrol nanocarriers. Single and mixed (0.1 % casein) micelles (0.0 to −17.0 mV; 2.4 to 32.7 nm) increased 50-fold resveratrol solubility, remained stable for one month at 4 °C, withstood fast dilution, underwent sol-to-gel transitions in the 23.9–27.1 °C range, and exhibited potent antioxidant properties. All formulations successfully passed the HET-CAM assay but showed Pluronic®-casein dose-dependent toxicity in the zebrafish embryo model. Resveratrol-loaded single and mixed micelles (10–15 mM Pluronic® F127) displayed antimicrobial activity against S. aureus and P. aeruginosa. The micelles favored resveratrol accumulation in cornea and sclera, but mixed micelles showed larger lag times and provided lower amount of resveratrol permeated through sclera. In vivo (rabbit) tests confirmed the safety of resveratrol-loaded single micelles and their capability to supply resveratrol to anterior and posterior eye segments.Depto. de Optometría y VisiónFac. de Óptica y OptometríaTRUEFondos FEDER - European Union’s Horizon 2020Ministerio de Ciencia e Innovación de España - MCIN/AEI/10.13039/ 501100011033Xunta de Galicia (España)pu

Study of the glial cytoarchitecture of the developing olfactory bulb of a shark using immunochemical markers of radial glia

During development of the olfactory bulb (OB), glial cells play key roles in axonal guiding/targeting, glomerular formation and synaptic plasticity. Studies in mammals have shown that radial glial cells and peripheral olfactory glia (olfactory ensheathing cells, OECs) are involved in the development of the OB. Most studies about the OB glia were carried out in mammals, but data are lacking in most non-mammalian vertebrates. In the present work, we studied the development of the OB glial system in the cartilaginous fish Scyliorhinus canicula (catshark) using antibodies against glial markers, such as glial fibrillary acidic protein (GFAP), brain lipid-binding protein (BLBP), and glutamine synthase (GS). These glial markers were expressed in cells with radial morphology lining the OB ventricle of embryos and this expression continues in ependymal cells (tanycytes) in early juveniles. Astrocyte-like cells were also observed in the granular layer and surrounding glomeruli. Numerous GS-positive cells were present in the primary olfactory pathway of embryos. In the developmental stages analysed, the olfactory nerve layer and the glomerular layer were the regions with higher GFAP, BLBP and GS immuno-reactivity. In addition, numerous BLBP-expressing cells (a marker of mammalian OECs) showing proliferative activity were present in the olfactory nerve layer. Our findings suggest that glial cells of peripheral and central origin coexist in the OB of catshark embryos and early juveniles. These results open the path for future studies about the differential roles of glial cells in the catshark OB during embryonic development and in adulthoodOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by the Spanish Ministerio de Economía y Competitividad-FEDER (BFU2014-5863-1P) and Ministerio de Ciencia e Innovacion-FEDER (BFU2017-8986-1P) and Xunta de Galicia (to EC, grant number: ED431C 2021/18)S