In vertebrates, the otic placode forms inner ear and epibranchial placodes produce sensory ganglia within branchial clefts. Fibroblast growth factor (FGF) family of protein ligands from the surrounding tissues are responsible for otic and epibranchial placode induction. Members of pax2/5/8 family of transcription factors function as mediators during otic induction. To understand the temporal and spatial requirements of Fgf and their interaction with pax2/8 for otic induction, we used heat shock inducible transgenic lines of zebrafish to misexpress fgf3/8 and pax2a/8 under the control of hsp70 promoter. Loss of function studies were done to examine the functions of pax2/8 genes in regulating otic and epibranchial development. We show that global transient activation of hs:fgf3 or hs:fgf8 at mid-late gastrula stages (7-8 hpf) severely impairs otic induction, in part by disrupting formation of the principal signaling centers in the hindbrain. Additionally, mosaic studies show that high-level misexpression blocks otic fate cell-autonomously, whereas low to moderate levels promote otic development. At later stages high-level Fgf misexpression, both globally and locally does not inhibit otic fate, but rather causes a dramatic expansion of endogenous otic domains. Misexpression of hs:pax2a or hs:pax8 also expands endogenous otic domains but is not sufficient to bypass the requirement for Fgf signaling. Co-misexpression of Fgf with pax2a or pax8 leads to production of ectopic otic tissue in a broad range of cranial ectoderm. These data show that changes in timing, distribution and level of Fgf signaling and its downstream effectors influences otic induction. We show that otic and epibranchial placodes are induced at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces otic expression of pax8 and sox3, which cooperate synergistically to establish otic fate. Subsequently, pax8 along with pax2a/pax2b downregulate foxi1 expression in otic cells, which is necessary for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Blocking functions of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These results support the model whereby the otic placode forms first and induces epibranchial placodes through pax2/8-dependent Fgf24 signaling.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

En els darrers anys s'ha produït un gran avenç en l'enteniment dels mecanismes implicats en la inducció de la placoda òtica. Tanmateix, poc es coneix encara de com s'estableix un domini amb competència neural i un altre no-neural i aquest ha estat l'objectiu d'aquesta tesi doctoral. Hem analitzat els mecanismes moleculars rellevants per la regionalització primarenca de la placoda òtica i hem explorat el paper de Sox3 i Lmx1b en l'establiment i manteniment d'un territori competent neural, emprant l'embrió de pollet com organisme model. Els resultats mostren que el gen Sox3, inicialment expressat en un territori extens, es regionalitza en un domini òtic i epibranquial proneural. La sobreexpressió de Sox3 a estadis preòtics, indueix la generació de precursors neuronals que expressen Sox2 i Delta1, però aquests no aconsegueixen progressar a estadis de major diferenciació. A la vegada, Sox3 és capaç de inhibir la expression de Lmx1b, un gen expressat en el domini no-neural, suggerint que el seu patró final depèn de l'activitat neurogènica de la oïda interna. Finalment, presento evidències que la senyalització mitjançada per BMP té un paper primerenc en l'establiment de l'expressió de Lmx1b en el territory òtic, però que ni l'activitat de BMP ni l'expressió de Lmx1b influencien el procés de determinació neural. En conclusió, els nostres resultats posen de relleu nova informacióour dels mecanismes moleculars que governen els primers passos de la competencia neural i regionalizació de la placoda òtica en un territori neural i un no-neural. / During the last years, a great progress has been made in understanding the mechanisms involved in otic induction but the mechanism behind otic patterning into neural and non-neural domains is still an open question and the major aim of this work was to address this question. We have analyzed the molecular mechanisms underling the early regionalization of the otic placode, and explored the role of Sox3 and Lmx1b in the establishment and maintenance of a neural competent domain in the otic placode by using the chick as a model system. The results show that Sox3 expression initially expressed in a broad domain gets regionalized in otic/epibranchial proneural domain. Overexpression of Sox3 at preotic stages can induce ectopic neuronal precursor cells expressing Sox2 and Delta1 but does not allow the ectopically developed neuronal precursor cells for further differentiation. Sox3, besides providing neural competence to the proneural domain, regulates the posterior non-neural gene Lmx1b suggesting that its final expression pattern depends on the neural activity. Finally, I present evidence that BMP signaling has an early role in inducing Lmx1b expression in the otic field but that neither BMP activity nor Lmx1b expression influence neural commitment. Taken together, our results provide new information and shed light on the molecular mechanisms that underlie the first steps of the neural competence and otic patterning in proneural and non-neural domain.

Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming. First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.

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