Submitted by Helmut Patrocinio (hell.kenn@gmail.com) on 2017-11-10T16:29:31Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Marcos_Romualdo_Costa_TESE.pdf: 3272089 bytes, checksum: aaf244c49f569e8c070d41f2727a24c6 (MD5) / Approved for entry into archive by Ismael Pereira (ismael@neuro.ufrn.br) on 2017-11-10T17:31:16Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Marcos_Romualdo_Costa_TESE.pdf: 3272089 bytes, checksum: aaf244c49f569e8c070d41f2727a24c6 (MD5) / Made available in DSpace on 2017-11-10T17:31:31Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Marcos_Romualdo_Costa_TESE.pdf: 3272089 bytes, checksum: aaf244c49f569e8c070d41f2727a24c6 (MD5) Previous issue date: 2006 / Ao longo do desenvolvimento, as c?lulas neuroepiteliais do telenc?falo dividemse originando progenitores respons?veis pela gera??o sequencial dos diferentes tipos de neur?nios, astr?citos e oligodendr?citos do c?rtex cerebral. At? o presente, os progenitores telencef?licos estariam localizados nas zonas ventricular (ZV) e subventricular (ZSV). Sua posi??o ao longo dos eixos dorsoventral e rostro-caudal ? relacionada com territ?rios g?nicos e tipos celulares espec?ficos. Desta forma, observa-se a gera??o de neur?nios corticais glutamat?rgicos ou GABA?rgicos na ZV e ZSV do telenc?falo dorsal e ventral de roedores, respectivamente. Neste trabalho investigamos o potencial proliferativo in vivo e in vitro da zona marginal (ZM), conhecida por possuir neur?nios migrat?rios e diferenciados durante a corticog?nese. Determinamos o fen?tipo de c?lulas proliferativas da ZM e atrav?s de an?lise clonal utilizando infec??o por retrovirus contendo o gene para GFP (prote?na flourescente verde) acompanhamos as linhagens derivadas destes progenitores in vitro. C?lulas proliferativas in vivo foram marcadas atrav?s da administra??o do BrdU (bromodeoxiuridina, marcador da fase S do ciclo celular), combinada a ensaios imunohistoqu?micos para a identifica??o deste ant?geno e da forma fosforilada da histona 3 (expressa no final da fase G2 e durante a fase M do ciclo celular). Identificamos c?lulas proliferativas na ZM de camundongos a partir do dia embrinon?rio 14 (E14 - logo ap?s a divis?o da pr?-placa quando a ZM se torna distingu?vel) e por toda a corticog?nese com um aumento na proporc?o de c?lulas proliferativas de ~tr?s vezes em E18. As c?lulas proliferativas na ZM n?o expressam Pax6 ou Tbr2, fatores transcricionais caracter?sticos dos precursores da ZV e ZSV respectivamente. Ao longo da corticog?nese, esta popula??o precursora apresenta um padr?o de express?o do fator transcricional Olig2 seguindo um gradiente l?tero-medial, de modo que no per?odo perinatal todas as c?lulas proliferativas na zona marginal expressam o gene olig2. A an?lise das linhagens clonais geradas a partir destes precursores revelou um elevado potencial gliog?nico (~70% de clones gliais puros) quando comparado a ZV /ZSV (3,3%). Al?m disso, a ZM apresentou um significativo potencial neurog?nico, originando cerca de 30% de clones contendo neur?nios. Mostramos que os clones gliais puros da ZM s?o significativamente maiores que os da ZV. Conclu?mos, portanto, que a ZM dorsal ? um nicho neurog?nico e gliog?nico no c?rtex cerebral em desenvolvimento apresentando c?lulas proliferativas in vivo e in vitro com caracter?sticas fenot?picas distintas dos progenitores da ZV e ZSV. Atrav?s de estudos de linhagem clonal in vitro, demonstramos diferentes comportamentos proliferativos e potenciais neuro-gliog?nicos das c?lulas isoladas da ZM e da ZV/ZSV, indicando a exist?ncia de um novo tipo de progenitor no telenc?falo. / During development, telencephalic neuroepithelial cells proliferate and give rise to progenitors, which are responsible for the sequential generation of different types of neurons, astrocytes and oligodendrocytes in the cerebral cortex. To date, telencephalic progenitors would be located in the ventricular (VZ) and subventricular (SVZ) zones. Their position along the rostro-caudal and dorsoventral axis is related to gene expression territories and the generation of specific cell types, such that dorsal telencephalic VZ/ZVZ generates glutamatergic neurons and ventral VZ/ZVZ GABAergic neurons. In this work we investigated the in vivo and in vitro proliferative potential of the marginal zone (MZ) described to harbor migrating and differentiating neurons during corticogenesis. We determined the phenotype of MZ proliferative cells and by clonal analysis with infection by GFP (green fluorescent protein) containing retroviruses we followed the lineages derived from the progenitors in vitro. Proliferative cells in vivo were labeled by BrdU (bromodeoxyuridine, S phase cell cycle marker) combined to immunohistochemistry for the identification of BrdU antigen and the phosphorylated form of H3 ?histone (expressed at the end of G2 and during M phase of the cell cycle). We identified proliferative cells in mice MZ from embryonic day (E)14 (just after preplate division when MZ becomes distinguishable) and through all corticogenesis with a three fold increase in E18. Proliferative cells in the MZ do not express Pax6 or Tbr2, transcriptional factors typical of VZ and SVZ precursors respectively. During corticogenesis, this precursor population displays a latero-medial gradient of expression of Olig2, such that perinatally, all proliferative cells in the MZ express Olig2. Clonal lineage analysis from these precursors revealed a high gliogenic potential (~70% pure glial clones) when compared to VZ/SVZ (2,3%). Furthermore, MZ displays neurogenic potential since 30% of all clones contained neurons identified by class III ?-tubulin immunolabeling. Here we show that pure glial clones in the MZ are significantly larger than those generated by VZ. Concluding, the dorsal MZ is a neurogenic and gliogenic niche in the developing cerebral cortex containing proliferative cells with distinct phenotypic characteristics from the VZ and SVZ. By clonal lineage analysis in vitro, we demonstrated different proliferative behaviors and neuro-gliogenic potential from cells isolated from the MZ and VZ/SVZ indicating a novel type of progenitor in the cerebral cortex.