Indiana University-Purdue University Indianapolis (IUPUI) / The maternal liver exhibits robust adaptations to pregnancy to accommodate the metabolic needs of developing and growing placenta and fetus by largely unknown mechanisms. We found that achaete-scute homolog 1 (Ascl1), a basic helix-loop-helix transcription factor essential for neuronal development, is highly activated in maternal hepatocytes during the second half of gestation in mice. Our aim is to investigate whether and how Ascl1 plays a pregnancy-dependent role. We deleted the Ascl1 gene in the maternal liver using three independent mouse models from mid-gestation until term and identified multiple Ascl1-dependent phenotypes. When Ascl1 was deficient in maternal hepatocytes, maternal livers exhibited aberrant hepatocyte histology, fat accumulation, increased hepatocyte cell cycle, and enlarged size, accompanied by reduced albumin production and elevated levels of free fatty acids, ALT, and AST in the maternal blood, indicating maternal liver dysfunction. In the same situation, maternal spleen and pancreas displayed marked enlargement without an overt structural change; the placenta exhibited striking overgrowth with increased ALP production; and the cecal microbiome showed alterations in the relative abundance of several bacterial subpopulations. Moreover, litters born from maternal hepatic Ascl1 null mutated dam experienced abnormal postnatal growth after weaning. RNA-seq analysis revealed Ascl1-regulated genes in the maternal liver associated with Ascl1-dependent phenotypes. Of particular interest, we found that, in maternal hepatocytes, Ascl1 loss-of-function caused the activation of paternally imprinted gene insulin-like growth factor 2 (Igf2) encoding a major placental and fetal growth factor. IGF2 is also a known mitogen for hepatocytes and several hematopoietic lineages. Thus, IGF2 is a potential inducer of Ascl1-dependent phenotypes including placental overgrowth and maternal organ enlargement. Our studies revealed Ascl1 as a novel regulator of maternal liver physiology during pregnancy. Ascl1 activation in maternal hepatocytes is essential for normal placental growth and appropriate maternal organ adaptations, ensuring the health of both the mother and the fetus.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumour in adults, as well as the most common. The current standard therapy is maximal safe resection, followed by radiotherapy in combination with the alkylating agent, temozolomide. Despite this multi-model treatment approach, median survival is just 14.6 months and new therapies are urgently needed. Glioblastoma stem cells (GSCs) are a highly tumourigenic subpopulation of GBM cells believed to promote therapeutic resistance along with angiogenesis and metastasis. Cancer stem cells share crucial characteristics with normal stem cells such as their ability to self- renew, maintain proliferation and differentiate multi-potently. One strategy to target GSCs is to force them to differentiate into post-mitotic cells, as this would cause them to lose their long-term repopulation potential and would therefore limit tumour growth. In this study, I investigated if the transcription factor ASCL1 could drive neuronal differentiation in GSCs. ASCL1 is a key regulator of neurogenesis in the developing CNS and is sufficient to reprogramme fibroblasts, astrocytes and induced pluripotent cells into neurons. However, ASCL1 also promotes proliferation, and in GSCs, it has been shown to drive tumourigenesis by upregulating Wnt signaling. Given its role in two opposing functions, ASCL1 is tightly regulated by multi-site phosphorylation on serine-proline resides. It is phosphorylated when driving proliferation in cycling cells and is un(der)phosphorylated when activating the transcription of its downstream targets involved in differentiation. I found that endogenous ASCL1 was phosphorylated in GSC lines and hypothesized that dephosphorylating ASCL1 may drive differentiation. I tested this by overexpressing a phosphomutant form of ASCL1, and found that it drove cell cycle exit through the downregulation of Cyclin D2, CDK4 and CDK6. However, ASCL1 did not drive overt differentiation which suggests GSCs may not respond to differentiation cues.

No description available.

Ce projet vise à fournir une nouvelle compréhension moléculaire du programme de transcription impliqué dans la différenciation des cellules souches neurales en oligodendrocytes myélinisant. La logique de ce travail repose sur des études antérieures ayant montré le rôle des facteurs de transcription bHLH Olig2 et Ascl1, opérant en synergie dans la spécification des OPCs, les cellules progénitrices d‘oligodendrocytes . L‘objectif central de ce travail était de comprendre au niveau génomique et transcriptomique les mécanismes par lesquels Ascl1 et Olig2 agissent pour spécifier les OPCs. Nous avons suivi une stratégie utilisant l'analyse du transcriptome et des profils de fixation des facteurs de transcription par immuno- précipitation de la chromatine. Nous avons pu identifier les cibles directes de Ascl1 et Olig2 dans les OPC et lors de la différenciation des oligodendrocytes. Nous avons également identifié de nouveaux marqueurs spécifiques des différents stades des lignées oligodendrocyte et nous sommes concentrés sur Chd7 et Tns3, deux gènes régulé par Ascl1 etOlig2 et enrichis dans la lignée oligodendrogliale à deux stades intéressants, la phase de spécification précoce et la transition entre la migration et la différenciation des oligodendrocytes, respectivement. De plus, nous avons porté notre attention sur le rôle spécifique des oligodendrocyte dans la synthèse de la créatine et son rôle possible de support métabolique dans la synthèse de myéline et de support axonal. Nous avons également initié une approche de repositionnement toxicogénomique pour identifier de nouvelles molécules à tester dans le cadre de maladie demyélinisantesLa plupart des traitements disponibles pour traiter les maladies démyélinisantes sont basées sur une approche immune modulatrice et anti-inflammatoire. A ce jour, aucun n'est en mesure de promouvoir directement la réparation de la myéline de manière efficace. Nous espérons que les gènes dont l'expression est régulée dans les lésions de démyélinisation identifiés lors de cette étude permettront de mieux comprendre le mécanisme de remyelinisation et le développement de nouvelles stratégies dans les maladies démyélinisantes telles que la sclérose en plaques ou dans les leucodystrophies. / Our project aims to provide a new molecular understanding of the transcription program involved in neural stem cells differentiation into oligodendrocytes. The rational of this work relies on previous studies demonstrating that the bHLH transcription factors Olig2 and Ascl1 work in synergy to specify OPCs, the oligodendrocyte progenitor cells. One central goal of this work was to understand at a genomic and transcriptomic level, how Ascl1 and Olig2 work together to specify OPCs. We followed a strategy using genome-wide transcriptome analysis and chromatin immuno-precipitation to characterize Ascl1 and Olig2 directly regulated genes in OPCs and during oligodendrocyte differentiation.We identified new specific markers of different stage of the neural lineages and new important genes correlated to OPCs differentiation. We focused on Chd7 and Tns3, two genes which expressions are driven by Ascl1 and Olig2 and enriched in the oligodendroglial lineage at two interesting stage, the early specification stage and the transition between migrating and differentiating oligodendrocytes, respectively. Moreover, we identified the myelinating oligodendrocyte as the cell in charge of the creatine synthesis in the brain and potentially driving axonal metabolic support. We also used an approach a toxicogenomic and drug repositioning approach to identify new molecules known to modify OPCs and myelin genes but untested in the context of demyelinating diseases. As currently, most of the available treatments for demyelinating diseases are based on immuno-modulatory and anti-inflammatory drugs but none are able to directly promote myelin repair, we expect that these identified genes involved in oligodendrogenesis and whose expression are regulated in demyelinated lesions will allow the development of new therapeutic strategies promoting an efficient remyelination in demyelinating diseases such as Multiple sclerosis or leukodystrophies.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23552号 / 生博第463号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 今吉 格, 教授 見学 美根子, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

<p>Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the <i>MEN1</i> and <i>VHL</i> tumour suppressor genes, is still elusive. The protein product of the <i>MEN1</i> gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling.</p><p>Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous <i>Men1</i> mice. For comparison, normal and <i>MEN1</i> non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between <i>Men1</i> expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in <i>Men1</i> mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and <i>Men1</i> mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of <i>Men1</i> and wt mice were observed.</p><p>In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression.</p><p>Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.</p>

No description available.

No description available.

No description available.

Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the MEN1 and VHL tumour suppressor genes, is still elusive. The protein product of the MEN1 gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling. Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous Men1 mice. For comparison, normal and MEN1 non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between Men1 expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in Men1 mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and Men1 mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of Men1 and wt mice were observed. In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression. Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.