Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / Title from title screen of research.pdf file (viewed on December 22, 2006). The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2006" Includes bibliographical references.

Osteopetrosis is a metabolic bone disease in mammals characterized by a generalized skeletal sclerosis caused by reduced bone resorption. This reduced bone resorption is manifested in afflicted animals by abnormal bone shape, reduced or absent marrow cavities, extramedullary hemopoiesis, abnormal mineral homeostasis and absent or delayed tooth eruption. The available osteopetrotic animal mutations have been a constant source of fruitful investigations concerning the systemic regulation of osteoclastogenesis and bone metabolism. Tooth eruption, on the other hand, is a localized manifestation of the timely activation of bone resorption and bone formation on opposite sides of an erupting tooth. Its rate-limiting step is the speed of bone resorption to form the eruption pathway. In this dissertation, we used two osteopetrotic rat mutations, toothless (tl) and microphthalmia blanc (mib), to investigate the abnormal development of osteoclasts and tooth eruption in mutant rats with an emphasis on the role of systemic and local factors. The significant contributions to this work are listed below. 1. In the toothless rat, a mutation lacking erupted dentition due to severely reduced bone resorption, colony-stimulating factor-1 (CSF-1) promoted tooth eruption but this was delayed compared to normal rats. Eruption was accompanied by changes in the populations of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells in the dental follicle and TRAP+ osteoclasts on adjacent alveolar bone surfaces. These cell populations were dramatically increased in treated mutants compared to untreated tl rats, but the timing of their appearance was delayed compared to normal littermates. This lag in the appearance of osteoclasts and their precursors corresponded to the delay in eruption of first molars in treated tl rats. 2. CSF-1 also accelerated the eruption of molars in normal rats. CSF-1 increased the number of TRAP+ mononuclear cells in the dental follicle and TRAP+ osteoclasts on adjacent alveolar bone surfaces, but had no effect on the timing of their appearance in normal rats. 3. Our data revealed a differential effect on tooth eruption of the growth factors CSF-1 and epidermal growth factor (EGF). CSF-1 accelerated eruption of molars in normal rats, but had no effect on incisor eruption. On the other hand, EGF accelerated incisor eruption; but did not affect molar eruption in normal rats. 4. We have described the mechanism for the transient, mild form of osteopetrosis inherited by mib rats. Mutant animals possess a typical sclerosis at birth, which diminished--but was not resolved--during the first postnatal month. These characteristics are caused by early reductions in osteoclast number and function which improve to normal levels by 4 weeks. Osteoclast numbers were severely reduced in mib rats between birth and 2 weeks, but improved to near normal levels by 4 weeks. Neonatal abnormalities in osteoclast function included reduced staining for the functional enzymes TRAP and TrATPase, decreased levels of mRNA for both TrATPase and CAll, and inability to form a well-developed ruffled border. None of these defects were apparent after the first postnatal month. 5. Finally, we have shown that the dental abnormalities caused by the mild, transient form of osteopetrosis in mib rats are limited to incisor defects and delayed eruption of all teeth. Histologic and radiographic examination of mutant incisors revealed that, contrary to the situation in normal rats, the apex of the incisors of mib rats failed to extend past the first molar region to the third molar. The incisor apex of newborn mib rats was misshaped due to ankylosis of incisor matrices with alveolar bone. This ankylosis was temporary, being resolved by the third postnatal day. The delayed eruption of incisors in mib rats and abnormal shape and occlusion of these teeth in older animals is a consequence of the temporary ankylosis in newborn rats.

No description available.

A Síndrome de Marfan (SMF) é a enfermidade hereditária mais comum dentre as que afetam o sistema conjuntivo, causada por mutações da glicoproteína fibrilina-1, o principal componente estrutural das microfibrilas elásticas da matriz extracelular. As manifestações fenotípicas da SMF são sistêmicas e acometem tipicamente os sistemas ocular, esquelético e cardiovascular, este uma importante causa de morbi-mortalidade. Entretanto, não está claro como a mutação induz a doença. Estudos anteriores sugerem anomalias morfológicas do retículo endoplasmático (RE) ou retenção intracelular da fibrilina-1 nos estágios avançados da SMF. Entretanto, a contribuição do enovelamento da fibrilina-1 mutada e do estresse do RE na fisiopatologia celular da SMF não é conhecida. Proteínas mal-enoveladas podem levar à retenção intracelular e/ou aumento da degradação através da via de degradação associada ao RE (ERAD), além da indução da resposta a proteínas mal-enoveladas (UPR), ambas com potencial contribuição à fisiopatologia de doenças, incluindo a SMF. Assim, estudamos em fibroblastos embrionários isolados de camundongos (MEFs) com SMF se a fibrilina-1 mutada é reconhecida pelo controle de qualidade do RE pelo seu mal- enovelamento e induz estresse do RE por sua retenção intracelular. Demonstramos que a mutação na fibrilina-1 per se não promoveu chaperonas marcadoras de UPR ou geração de oxidantes. Além disso, não levou a uma maior sensibilização das células à indução exógena de estresse do RE, nem promoveu maior morte celular após inibição do proteassoma. Além disso, não foi observada retenção intracelular da fibrilina-1 nas células SMF, e mesmo após inibição da via secretora ou indução de estresse do RE, a inibição da secreção da fibrilina-1 foi similar nos MEFs SMF e wild-type (WT). A dissulfeto isomerase proteica (PDI), uma importante chaperona redox do RE, interage com fibrilina-1, e seu silenciamento levou a um aumento na secreção da fibrilina-1 pelos MEFs WT, mas não SMF. Além disso, o silenciamento da PDI promoveu a desorganização da matriz extracelular depositada de fibrilina-1 nos MEFs WT, enquanto nos MEFs SMF, a desorganização basal da matriz não foi adicionalmente alterada. Em paralelo, investigações in vivo mostraram que o estresse do RE não é induzido em camundongos SMF com 1 ou 3 meses de idade, apesar de manifestações fenotípicas evidentes. Entretanto, concomitante à progressão da doença, detectamos a ocorrência de estresse do RE nas aortas ascendentes dos camundongos aos 6 meses. Esta detecção foi exclusiva desta região da aorta e não ocorreu em outros órgãos afetados ou não afetados pela SMF. Assim, a manifestação do fenótipo clássico da SMF não requer uma perda da homeostase do RE diretamente induzida pela fibrilina-1 mutada. Ao contrário, esta é capaz de evadir mecanismos de controle de qualidade mediados pela PDI, sendo secretada normalmente. Assim, esta evasão do controle de qualidade pela PDI é uma condição permissiva essencial para o fenótipo da SMF. Por outro lado, o estresse do RE é uma característica evolutiva do aneurisma da aorta ascendente na SMF concomitante ao agravamento do fenótipo neste tecido / Marfan syndrome (MFS) is the most common connective tissue hereditary disease, caused by mutations in the glycoprotein fibrillin-1, the main structural component of extracellular matrix elastic microfibrils. MFS phenotypic manifestations are systemic and typically involve the ocular, skeletal and cardiovascular systems, the latter a major cause of morbidity/mortality. However, how gene mutation induxes disease is yet unclear. Previous studies suggest endoplasmic reticulum (ER) morphological abnormalities or fibrillin-1 intracellular retention in advanced MFS stages. However, the contribution of mutated fibrillin-1 folding and ER stress to MFS cellular pathophysiology is unknown. Un/misfolded proteins may associate with their intracellular retention and/or increased degradation through ER-associated degradation (ERAD), in addition to inducing the unfolded protein response (UPR), both sharing potential contributions to disease pathophysiology, including MFS. Thus, we studied in embryonic fibroblasts (MEFs) isolated from WT and MFS mice, if mutated fibrillin-1 can be recognized by ER quality control as a misfolded protein, able to induce ER stress due to its intracellular retention. We showed that fibrillin-1 mutation by itself did not promote UPR chaperone markers or oxidant generation. Moreover, it did not sensitize cells to exogenous ER stress nor affected cell survival curves after proteasome inhibition. Furthermore, no intracellular retention of fibrillin-1 was observed in MFS cells, and even after secretory pathway inhibition or ER stress induction, fibrillin-1 secretion inhibition was similar in MFS and wild-type (WT) MEFs. Protein disulfide isomerase (PDI), an important ER redox chaperone, interacts with fibrillin-1 and its silencing induced an increased fibrillin-1 secretion in WT, but not MFS MEFs. Besides, PDI silencing promoted fibrillin-1 extracellular matrix disorganization in WT MEFs, whereas in MFS MEFs, the basal matrix disorganization was not further modified. Parallel in vivo evaluations demonstrated that ER stress is also not induced in 1 and 3 month-old mice MFS, despite evident phenotypical manifestations. However, concomitant to accelerated disease progression at 6 months, ER stress was detectable in ascendant aorta, but not in other disease-affected or unaffected organs. Thus, classic MFS phenotype manifestations do not require loss of ER homeostasis directly induced by mutated fibrillin-1. Contrarily, the latter can evade a PDI-mediated quality control mechanism to be normally secreted. Therefore, evading such PDI-mediated quality control is an essential permissive condition for enabling the MFS phenotype. On the other hand, ER stress is an evolutive feature of MFS ascendant aorta aneurysm concomitant to phenotype progression in this tissue

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Inorganic Phosphate (Pi) is one of the key nutrients required by all living organisms on earth. This nutrient is of vital importance to higher plants but it is not readily available for uptake from the soil, implying constant stress on plants. During photosynthetic dark and light reactions, phosphate is a prerequisite for all reactions to occur and to ensure plant survival. This statement implies that a careful homeostatic control of this nutrient is necessary in order to maintain a balanced carbon flow in all sub-cellular plant compartments. Phosphate limitation is a threat to plant survival and one way of addressing this nutritional hurdle is by feeding plants with fertilizer. This method of crop development and general plant maintenance by humans has a devastating effect on the environment, as phosphate causes eutrophication and various other consequences which are detrimental to animal life. Plants, however, are naturally equipped with Pi transporters which are activated conditionally depending on the external Pi availability. These transporters are present in most sub-cellular compartments and some of them have been identified and characterised, while others remain to be a prediction. If these transporters are characterised accordingly it might eventually mean that the use of fertilizers may no longer be necessary. In order to contribute to successful Pi-efficient crop development, a clearer understanding of P-dynamics in the soil and its recycling ability inside the plant itself is necessary. During this study it was attempted to characterise a putative high affinity Pi transporter, PHT1;5, from Arabidopsis thaliana via a Escherichia coli and yeast heterologous expression system and its Km value predicted in order to verify/hypothesise whether it is a high or low affinity transporter. This transporter is expressed in leaves and could be a promising tool for future carbon partitioning studies during phosphate limitation. / AFRIKAANSE OPSOMMING: Anorganiese fosfaat (Pi) word beskou as een van die belangrikste nutriente benodig vir alle lewe op aarde. Dit vervul ‘n hoof rol in talle noodsaaklike prosesse in hoër plante en is veral ‘n voorvereiste vir fotosintetiese reaksies om plaas te vind. In ‘n plant se natuurlike omgewing is anorganiese fosfaat nie geredelik bekskikbaar in grond nie en dus word daar vermoed dat plante onder konstante fosfaat stres gevind word. Omdat fosfaat so ‘n belangrike rol speel tydens fotosintese is dit noodsaaklik dat daar ‘n balans op sellulêre vlak gehandhaaf word, veral wat die verspreiding van koolhidrate tussen die verskillende kompartemente van die sel betref. Plante se oorlewing word bedreig deur ‘n tekort aan fosfaat in die omgewing en die enigste onmiddelike oplossing daarvoor is deur die toediening van bemestingstowwe. Hierdie metode van landery ontwikkeling en algemene instandhouding van plante deur die mensdom het ’n baie negatiewe effek op die omgewing. ‘n Oormaat fosfaat lei tot eutrifikasie en het verkeie ander negatiewe nagevolge wat dodelik is vir die dierelewe. Plante beskik ook oor natuurlike interne fosfaat transporters om hierdie tekort te oorkom. Hierdie transporters word op grond van eksterne fosfaat beskikbaarheid ge-aktiveer of ge-deaktifeer. Die transporters is teenwoordig in meeste sub-sellulêre kompartemente en sommige is al ge-identifiseer en gekarakteriseer, terwyl ander slegs ‘n voorspelling bly. Gedurende hierdie studie was ‘n poging aangewend om ‘n anorganiese fosfaat transporter van Arabidopsis thaliana, PHT1;5, te karakteriseer met behulp van mikro-organismes soos Escherichia coli en gis. Die poging het ingesluit om ‘n Km waarde vir hierdie transporter te voorspel en sodoende ‘n hipotese daar te stel van of dit hoë of lae affiniteit het vir fosfaat. Die transporter word groot en deels aangetref in blare en kan dus dien as ‘n belowende apparaat vir toekomstige koolhidraat uitruiling studies gedurende fosfaat tekort.

A Síndrome de Marfan (SMF) é a enfermidade hereditária mais comum dentre as que afetam o sistema conjuntivo, causada por mutações da glicoproteína fibrilina-1, o principal componente estrutural das microfibrilas elásticas da matriz extracelular. As manifestações fenotípicas da SMF são sistêmicas e acometem tipicamente os sistemas ocular, esquelético e cardiovascular, este uma importante causa de morbi-mortalidade. Entretanto, não está claro como a mutação induz a doença. Estudos anteriores sugerem anomalias morfológicas do retículo endoplasmático (RE) ou retenção intracelular da fibrilina-1 nos estágios avançados da SMF. Entretanto, a contribuição do enovelamento da fibrilina-1 mutada e do estresse do RE na fisiopatologia celular da SMF não é conhecida. Proteínas mal-enoveladas podem levar à retenção intracelular e/ou aumento da degradação através da via de degradação associada ao RE (ERAD), além da indução da resposta a proteínas mal-enoveladas (UPR), ambas com potencial contribuição à fisiopatologia de doenças, incluindo a SMF. Assim, estudamos em fibroblastos embrionários isolados de camundongos (MEFs) com SMF se a fibrilina-1 mutada é reconhecida pelo controle de qualidade do RE pelo seu mal- enovelamento e induz estresse do RE por sua retenção intracelular. Demonstramos que a mutação na fibrilina-1 per se não promoveu chaperonas marcadoras de UPR ou geração de oxidantes. Além disso, não levou a uma maior sensibilização das células à indução exógena de estresse do RE, nem promoveu maior morte celular após inibição do proteassoma. Além disso, não foi observada retenção intracelular da fibrilina-1 nas células SMF, e mesmo após inibição da via secretora ou indução de estresse do RE, a inibição da secreção da fibrilina-1 foi similar nos MEFs SMF e wild-type (WT). A dissulfeto isomerase proteica (PDI), uma importante chaperona redox do RE, interage com fibrilina-1, e seu silenciamento levou a um aumento na secreção da fibrilina-1 pelos MEFs WT, mas não SMF. Além disso, o silenciamento da PDI promoveu a desorganização da matriz extracelular depositada de fibrilina-1 nos MEFs WT, enquanto nos MEFs SMF, a desorganização basal da matriz não foi adicionalmente alterada. Em paralelo, investigações in vivo mostraram que o estresse do RE não é induzido em camundongos SMF com 1 ou 3 meses de idade, apesar de manifestações fenotípicas evidentes. Entretanto, concomitante à progressão da doença, detectamos a ocorrência de estresse do RE nas aortas ascendentes dos camundongos aos 6 meses. Esta detecção foi exclusiva desta região da aorta e não ocorreu em outros órgãos afetados ou não afetados pela SMF. Assim, a manifestação do fenótipo clássico da SMF não requer uma perda da homeostase do RE diretamente induzida pela fibrilina-1 mutada. Ao contrário, esta é capaz de evadir mecanismos de controle de qualidade mediados pela PDI, sendo secretada normalmente. Assim, esta evasão do controle de qualidade pela PDI é uma condição permissiva essencial para o fenótipo da SMF. Por outro lado, o estresse do RE é uma característica evolutiva do aneurisma da aorta ascendente na SMF concomitante ao agravamento do fenótipo neste tecido / Marfan syndrome (MFS) is the most common connective tissue hereditary disease, caused by mutations in the glycoprotein fibrillin-1, the main structural component of extracellular matrix elastic microfibrils. MFS phenotypic manifestations are systemic and typically involve the ocular, skeletal and cardiovascular systems, the latter a major cause of morbidity/mortality. However, how gene mutation induxes disease is yet unclear. Previous studies suggest endoplasmic reticulum (ER) morphological abnormalities or fibrillin-1 intracellular retention in advanced MFS stages. However, the contribution of mutated fibrillin-1 folding and ER stress to MFS cellular pathophysiology is unknown. Un/misfolded proteins may associate with their intracellular retention and/or increased degradation through ER-associated degradation (ERAD), in addition to inducing the unfolded protein response (UPR), both sharing potential contributions to disease pathophysiology, including MFS. Thus, we studied in embryonic fibroblasts (MEFs) isolated from WT and MFS mice, if mutated fibrillin-1 can be recognized by ER quality control as a misfolded protein, able to induce ER stress due to its intracellular retention. We showed that fibrillin-1 mutation by itself did not promote UPR chaperone markers or oxidant generation. Moreover, it did not sensitize cells to exogenous ER stress nor affected cell survival curves after proteasome inhibition. Furthermore, no intracellular retention of fibrillin-1 was observed in MFS cells, and even after secretory pathway inhibition or ER stress induction, fibrillin-1 secretion inhibition was similar in MFS and wild-type (WT) MEFs. Protein disulfide isomerase (PDI), an important ER redox chaperone, interacts with fibrillin-1 and its silencing induced an increased fibrillin-1 secretion in WT, but not MFS MEFs. Besides, PDI silencing promoted fibrillin-1 extracellular matrix disorganization in WT MEFs, whereas in MFS MEFs, the basal matrix disorganization was not further modified. Parallel in vivo evaluations demonstrated that ER stress is also not induced in 1 and 3 month-old mice MFS, despite evident phenotypical manifestations. However, concomitant to accelerated disease progression at 6 months, ER stress was detectable in ascendant aorta, but not in other disease-affected or unaffected organs. Thus, classic MFS phenotype manifestations do not require loss of ER homeostasis directly induced by mutated fibrillin-1. Contrarily, the latter can evade a PDI-mediated quality control mechanism to be normally secreted. Therefore, evading such PDI-mediated quality control is an essential permissive condition for enabling the MFS phenotype. On the other hand, ER stress is an evolutive feature of MFS ascendant aorta aneurysm concomitant to phenotype progression in this tissue

Introdução: As células-tronco apresentam capacidade de proliferação, autorrenovação, potencial de diferenciação e já foram descritas na hipófise estando envolvidas na renovação celular e regulação homeostática, porém pouco se sabe sobre o seu perfil de expressão nos quadros de hipopituitarismo. Dentre os marcadores de células-tronco descritos previamente na hipófise, destacam-se os genes Sox2, Nanog, Nestina, Cd44 e Oct4. Outro marcador, o gene Nr2e1 (Tlx), encontrado em células-tronco neuronais, apresenta-se elevado durante a embriogênese e na vida adulta no cérebro de camundongos, mas, até o momento, não foi caracterizado na hipófise. Objetivo: Analisar a imunolocalização do SOX2 e o padrão de expressão de marcadores de células-tronco/progenitoras, fatores de transcrição precoce, marcadores de apoptose e proliferação celular na hipófise de três linhagens de camundongos com hipopituitarismo de causa genética por alteração em fatores precoces de diferenciação glandular, as linhagens Ames (Prop1) e Snell (Pou1f1), e por fator tardio de conjugação dos hormônios glicoproteicos, a linhagem alfaGSU, nocaute do gene Cga. Material e Métodos: Foram coletadas hipófises nos tempos P0 (ao nascimento), P7 (final da primeira onda de crescimento glandular), 4 semanas (4S-período da puberdade) e 8 semanas (8S-vida adulta). Nas três linhagens de animais, realizou-se imuno-histoquímica com SOX2 e RT-qPCR com os marcadores de células-tronco/progenitoras Sox2, Nanog, Nestina, Cd44, Oct4 e Nr2e1, fatores de transcrição precoces (Hesx1, Hes1 e Otx2), fator de proliferação celular (Ki67), fatores de diferenciação celular (S100beta e Sox9) e marcadores de apoptose (Caspases 3 e 7). A quantificação relativa dos genes-alvo nos animais mutantes teve como calibrador os seus respectivos selvagens. Resultados: A imunolocalização do SOX2 foi observada na zona que circunda a fenda de Rathke (camada marginal) e em nichos difusos pela glândula nas três linhagens estudadas. Na linhagem alfaGSU, evidenciou-se uma redução de Nanog, Nr2e1, Oct4, e Hesx1 em 4S e de Nestina em 8S. Na linhagem Snell, observou-se aumento na expressão de Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e aumento de Sox2, Cd44, Hesx1, Otx2 e Sox9 em 8S, associado à redução de Ki67 em ambos os períodos. Na linhagem Ames, evidenciou-se aumento de Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e 8S. O gene Nr2e1 esteve hiperexpresso em todos os tempos. Houve redução do Ki67 em 4S. As caspases 3 e 7 não se apresentaram alteradas em nenhuma linhagem e/ou tempo. Discussão e conclusão: O padrão de imunolocalização de SOX2 encontrado nas três linhagens estudadas foi semelhante ao descrito em animais sem hipopituitarismo. A evidência da presença do Nr2e1 o coloca como um novo marcador de células-tronco/progenitoras na hipófise. A expressão elevada dos marcadores de células-tronco/progenitoras nas linhagens Ames e Snell sugere que a ausência dos fatores de transcrição precoces não permitiria que a célula tronco/progenitora iniciasse o processo de diferenciação celular, enquanto o oposto ocorreria na linhagem alfaGSU. Adicionalmente, estes achados justificam a hipoplasia hipofisária observada em animais com defeitos em fatores de transcrição expressos no início da diferenciação hipofisária, nos quais o acúmulo de células-tronco pode ser um indicador da indiferenciação hipofisária / Introduction: The role of stem cells, with their capacity for proliferation, self-renewal, and differentiation, has already been described in the cell turnover and homeostatic regulation of the pituitary gland. However, little is known about the expression profiles of these markers in hypopituitarism. Among the stem cell markers previously described in the pituitary include the genes for Sox2, Nanog, nestin, CD44 and Oct4. Another gene marker, Nr2e1 (Tlx), found in neural stem cells, is highly expressed during embryogenesis and adulthood, but so far has not been characterized in the pituitary. Objective: To analyze the immunohistochemical profile of SOX2, as well as the pattern of expression of various markers of stem/progenitor cells, early transcription factors, apoptosis factors and cell proliferation in three pituitary strains of mice with a genetic cause of hypopituitarism. Strains studied with hypopituitarism due to changes in factors of precocious glandular differentiation, include the Ames (Prop1) and Snell (Pou1f1) lineages; hypopituitarism due to the delayed conjugation of glycoprotein hormones include the alfaGSU strain, which is caused by the knockout of the Cga gene. Material and Methods: We collected pituitaries at four time points including P0 (birth), P7 (considered the end of the first wave of growth glandular), 4 weeks (4S - puberty period) and 8 weeks (8S - adulthood). All three strains were subjected to immunohistochemical analysis of SOX2 and RT-qPCR of markers of stem/progenitor cells Sox2, Nanog, Nestin, Cd44, Oct4 and Nr2e1, early transcription factors (Hesx1, Otx2 and Hes1), cell proliferation (Ki67), cell differentiation factors (S100beta and Sox9) and apoptosis (caspases 3 and 7) markers. Relative quantification of target genes in mutant animals was normalized to their respective wild type littermate. Results: The immunolocalization of SOX2 was observed in the area surrounding the Rathke cleft (marginal layer), as well as in diffuse niches throughout the gland in all three strains studied. The alfaGSU strain showed a reduction of Nanog, Nr2e1, Oct4 and Hesx1 at 4S, and Nestin at 8S. The Snell mice exhibited an increase of expression in Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta and Sox9 in at 4S and increased Sox2, Cd44, Hesx1, Otx2 and Sox9 at 8S, associated with the reduction of Ki67 in both periods. The Ames strain showed an increase of Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta and Sox9 at 4S and 8S; the gene Nr2e1 was over expressed at all times; and there was reduction in Ki67 at 4S. Caspases 3 and 7 had not changed in any strain, at any time. Discussion and Conclusion: The pattern of immunolocalization of SOX2 found in the three strains studied was similar to that described in animals without hypopituitarism. The presence of Nr2e1 in our study suggests it as a new marker of stem/progenitor cells in the pituitary. The high expression of markers of stem/progenitor cells in the Ames and Snell strains suggests that the absence of early transcription factors Prop1 and Pou1f1 do not allow the stem/ progenitors cells to start the process of cell differentiation, while the opposite occurs in the alfaGSU lineage. Additionally, these findings explain the pituitary hypoplasia observed in animals with defects in early transcription factors, as indicated by the accumulation of stem cells in the Snell and Ames lineages, preventing the initiation of pituitary differentiation

Introdução: As células-tronco apresentam capacidade de proliferação, autorrenovação, potencial de diferenciação e já foram descritas na hipófise estando envolvidas na renovação celular e regulação homeostática, porém pouco se sabe sobre o seu perfil de expressão nos quadros de hipopituitarismo. Dentre os marcadores de células-tronco descritos previamente na hipófise, destacam-se os genes Sox2, Nanog, Nestina, Cd44 e Oct4. Outro marcador, o gene Nr2e1 (Tlx), encontrado em células-tronco neuronais, apresenta-se elevado durante a embriogênese e na vida adulta no cérebro de camundongos, mas, até o momento, não foi caracterizado na hipófise. Objetivo: Analisar a imunolocalização do SOX2 e o padrão de expressão de marcadores de células-tronco/progenitoras, fatores de transcrição precoce, marcadores de apoptose e proliferação celular na hipófise de três linhagens de camundongos com hipopituitarismo de causa genética por alteração em fatores precoces de diferenciação glandular, as linhagens Ames (Prop1) e Snell (Pou1f1), e por fator tardio de conjugação dos hormônios glicoproteicos, a linhagem alfaGSU, nocaute do gene Cga. Material e Métodos: Foram coletadas hipófises nos tempos P0 (ao nascimento), P7 (final da primeira onda de crescimento glandular), 4 semanas (4S-período da puberdade) e 8 semanas (8S-vida adulta). Nas três linhagens de animais, realizou-se imuno-histoquímica com SOX2 e RT-qPCR com os marcadores de células-tronco/progenitoras Sox2, Nanog, Nestina, Cd44, Oct4 e Nr2e1, fatores de transcrição precoces (Hesx1, Hes1 e Otx2), fator de proliferação celular (Ki67), fatores de diferenciação celular (S100beta e Sox9) e marcadores de apoptose (Caspases 3 e 7). A quantificação relativa dos genes-alvo nos animais mutantes teve como calibrador os seus respectivos selvagens. Resultados: A imunolocalização do SOX2 foi observada na zona que circunda a fenda de Rathke (camada marginal) e em nichos difusos pela glândula nas três linhagens estudadas. Na linhagem alfaGSU, evidenciou-se uma redução de Nanog, Nr2e1, Oct4, e Hesx1 em 4S e de Nestina em 8S. Na linhagem Snell, observou-se aumento na expressão de Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e aumento de Sox2, Cd44, Hesx1, Otx2 e Sox9 em 8S, associado à redução de Ki67 em ambos os períodos. Na linhagem Ames, evidenciou-se aumento de Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e 8S. O gene Nr2e1 esteve hiperexpresso em todos os tempos. Houve redução do Ki67 em 4S. As caspases 3 e 7 não se apresentaram alteradas em nenhuma linhagem e/ou tempo. Discussão e conclusão: O padrão de imunolocalização de SOX2 encontrado nas três linhagens estudadas foi semelhante ao descrito em animais sem hipopituitarismo. A evidência da presença do Nr2e1 o coloca como um novo marcador de células-tronco/progenitoras na hipófise. A expressão elevada dos marcadores de células-tronco/progenitoras nas linhagens Ames e Snell sugere que a ausência dos fatores de transcrição precoces não permitiria que a célula tronco/progenitora iniciasse o processo de diferenciação celular, enquanto o oposto ocorreria na linhagem alfaGSU. Adicionalmente, estes achados justificam a hipoplasia hipofisária observada em animais com defeitos em fatores de transcrição expressos no início da diferenciação hipofisária, nos quais o acúmulo de células-tronco pode ser um indicador da indiferenciação hipofisária / Introduction: The role of stem cells, with their capacity for proliferation, self-renewal, and differentiation, has already been described in the cell turnover and homeostatic regulation of the pituitary gland. However, little is known about the expression profiles of these markers in hypopituitarism. Among the stem cell markers previously described in the pituitary include the genes for Sox2, Nanog, nestin, CD44 and Oct4. Another gene marker, Nr2e1 (Tlx), found in neural stem cells, is highly expressed during embryogenesis and adulthood, but so far has not been characterized in the pituitary. Objective: To analyze the immunohistochemical profile of SOX2, as well as the pattern of expression of various markers of stem/progenitor cells, early transcription factors, apoptosis factors and cell proliferation in three pituitary strains of mice with a genetic cause of hypopituitarism. Strains studied with hypopituitarism due to changes in factors of precocious glandular differentiation, include the Ames (Prop1) and Snell (Pou1f1) lineages; hypopituitarism due to the delayed conjugation of glycoprotein hormones include the alfaGSU strain, which is caused by the knockout of the Cga gene. Material and Methods: We collected pituitaries at four time points including P0 (birth), P7 (considered the end of the first wave of growth glandular), 4 weeks (4S - puberty period) and 8 weeks (8S - adulthood). All three strains were subjected to immunohistochemical analysis of SOX2 and RT-qPCR of markers of stem/progenitor cells Sox2, Nanog, Nestin, Cd44, Oct4 and Nr2e1, early transcription factors (Hesx1, Otx2 and Hes1), cell proliferation (Ki67), cell differentiation factors (S100beta and Sox9) and apoptosis (caspases 3 and 7) markers. Relative quantification of target genes in mutant animals was normalized to their respective wild type littermate. Results: The immunolocalization of SOX2 was observed in the area surrounding the Rathke cleft (marginal layer), as well as in diffuse niches throughout the gland in all three strains studied. The alfaGSU strain showed a reduction of Nanog, Nr2e1, Oct4 and Hesx1 at 4S, and Nestin at 8S. The Snell mice exhibited an increase of expression in Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta and Sox9 in at 4S and increased Sox2, Cd44, Hesx1, Otx2 and Sox9 at 8S, associated with the reduction of Ki67 in both periods. The Ames strain showed an increase of Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta and Sox9 at 4S and 8S; the gene Nr2e1 was over expressed at all times; and there was reduction in Ki67 at 4S. Caspases 3 and 7 had not changed in any strain, at any time. Discussion and Conclusion: The pattern of immunolocalization of SOX2 found in the three strains studied was similar to that described in animals without hypopituitarism. The presence of Nr2e1 in our study suggests it as a new marker of stem/progenitor cells in the pituitary. The high expression of markers of stem/progenitor cells in the Ames and Snell strains suggests that the absence of early transcription factors Prop1 and Pou1f1 do not allow the stem/ progenitors cells to start the process of cell differentiation, while the opposite occurs in the alfaGSU lineage. Additionally, these findings explain the pituitary hypoplasia observed in animals with defects in early transcription factors, as indicated by the accumulation of stem cells in the Snell and Ames lineages, preventing the initiation of pituitary differentiation