Intestinal homeostasis is governed by fate choices of stem cells residing in the intestinal crypt base. This must involve niche-specific co-ordination of cell division to guarantee that epithelial cells divide at the right time and place. These mechanisms operate to ensure precise control of the numbers of stem and differentiated cells. Little is known about how proliferative fate decisions are regulated in intestinal crypts. Both the placement of daughter cells within a particular niche, and their decision to enter and progress through the cell cycle, contribute. This thesis investigates the spatiotemporal control of cell division in intestinal crypts to understand the relationship between cell-cycle specific fate choices and intestinal homeostasis. Firstly, I describe a novel mode of asymmetric cell division within intestinal crypts. Using high resolution microscopy of intestinal organoids, I show that a subset of mitoses produce daughters that become displaced from one another after cytokinesis. This post-mitotic separation or the ‘positional asymmetry’ of daughter cells occurs in all cycling epithelial cells. These divisions may facilitate divergent fate of daughter cells and provides a general mechanism for stochastic niche exit. Post-mitotic separation is facilitated by interkinetic nuclear migration and selective tethering to the basement membrane during mitosis. Importantly, these mechanisms are altered in tissue carrying mutations in Adenomatous polyposis coli (Apc), highlighting its importance for normal tissue homeostasis. Secondly, I aimed to understand the dynamics of cell-cycle commitment in intestinal crypt compartments by investigating the DNA Replication Licensing System. The licensing system is a master regulator of proliferative fate in all cells in adult tissue. At its core is the regulated loading of the Mcm2-7 protein complex onto origins of replication exactly once per cell cycle. Engagement of the licensing system directly indicates commitment to proliferative cell fate. A technique to visualise licensing in intestinal crypts was developed. This revealed distinct proliferation zones in intestinal crypts. Mcm licensing was most prevalent in the lower transit-amplifying compartment, the zone enriched for early TA progenitors. Licensing is inhibited in terminally differentiated cells, and not detected in the transit-amplifying cells most proximal to the differentiated zone. Strikingly, the majority of ‘active’ intestinal stem cells were found in an unlicensed state. These data suggest that licensing decisions are delayed or inhibited until late G1 phase in intestinal stem cells and explains their longer cell-cycle. We postulate that this may provide a time window for niche cues to act, either stimulating cell-cycle entry or allowing retention in a ‘shallow’ G0 state. High resolution imaging of cell-cycle phases throughout the epithelium revealed remarkable cell-cycle co-ordination. This manifested in uninterrupted ‘ribbons’ of cells in similar cell-cycle states. This was due to lineage specific cell cycle co-ordination where adjacent daughter cells progress through the cell cycle at the same rate. These field effects are the result of co-ordinated cell-cycle progression between daughter cells. These observations were validated using living organoids expressing fluorescent ubiquitination-based cell cycle indicators (FUCCI). These ribbons were occasionally interrupted by cells in other cell cycle phases suggesting the separation of sisters by daughters from another lineage. This suggests that cell-cycle coordination can facilitate post-mitotic separation, and influence stochastic niche exit.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21992号 / 医博第4506号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 坂井 義治, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The intestinal epithelium represents one of the most actively renewing tissues in the body, and is widely used as a model system to study epithelial cell biology. ASPP2, a member of the ASPP (apoptosis stimulating protein of p53) protein family, has been shown to act as a regulator of epithelial cell polarity and tumour suppressor. This study investigated whether the dual function of ASPP2 is involved in the regulation of intestinal homeostasis and tumourigenesis, with a particular interest in the distinction between epithelial cell autonomous and non-autonomous mechanisms. Germline and intestinal epithelial cell-specific ASPP2 conditional knockout mice were employed in this study. Deficiency of ASPP2 in the intestinal epithelium resulted in delayed recovery from dextran sulfate sodium (DSS)-induced acute colitis, concurrent with a reduction in the expression of proinflammatory cytokines such as interleukin (IL)-1β and IL-6. Moreover, ASPP2-deficient mice showed increased susceptibility to Azoxymethane/DSS-induced colorectal tumourigenesis. While wild-type and ASPP2-deficient crypts showed similar incidence of tumour formation, the local immune microenvironment of ASPP2-deficient mice favoured tumour progression. The intestinal organoid culture was established to supplement in vivo experiments. The feasibility of the system was demonstrated with small intestinal organoids, in the context of proliferation, differentiation, and cell death. Using the established workflow, a colonic organoid-based tissue regeneration model was developed. The intrinsic susceptibility of organoids to DSS-induced cell death was not affected by the loss of ASPP2. However, ASPP2-deficient colonic organoids were less responsive to the pro-proliferative effects of IL-6, but were more sensitive to tumour necrosis factor-α-induced cell death in the presence of IL-22. In conclusion, this project undertook parallel examinations of animal models and organoids, demonstrating that a deficiency of ASPP2 in the intestinal epithelium results in dysregulated epithelial-immune cell interactions. This may partially explain the pathological conditions observed in ASPP2-deficient mice. Importantly, this study highlights the possibility of using organoids to investigate epithelial cell non-autonomous factors implicated in intestinal pathogenesis.

Les cellules souches adultes sont des cellules non différenciées, essentielles au le renouvellement constant de nos tissus. Elles produisent des cellules différenciées nécessaires au fonctionnement de nos organes, tout en maintenant un réservoir de cellules souches dans le tissu. Cet équilibre entre prolifération et différentiation cellulaire est crucial pour le maintien d’un état constant du tissu appelé homéostasie tissulaire. Entre identité « souche » et différenciation : Quels programmes génétiques contrôlent ces états ? Cette question suscite un intérêt majeur tant pour la recherche dans le domaine des cellules souches que pour les perspectives thérapeutiques qui en découlent. Dans cette optique, ce travail de thèse a permis de mettre en évidence un nouveau rôle du gène spen dans le contrôle des cellules souches intestinales chez Drosophila melanogaster. Une inactivation du gène spen est à l’origine d’une accumulation aberrante des cellules souches au sein de l’intestin de drosophile. La mise en place d’un protocole de purification par FACS des cellules souches, associé à un séquençage à grande échelle des ARN, a permis de mettre à jour les réseaux de gènes régulés par Spen dans les cellules souches. Ainsi, en combinant des techniques de génétique et d’analyses in vivo, ce travail montre que Spen est un facteur clé du processus de spécification des cellules souches intestinales et de la régulation de leur prolifération. Cette étude participe ainsi à la compréhension de la fonction moléculaire des protéines de la famille SPEN dans les cellules souches et les dérégulations à l’origine des pathologies auxquelles elles sont associées. / Adult stem cells are non-differentiated cells that maintain tissue homeostasis by supplying differentiated cells while at the same time self-renewing. How is this balance between stem cell state and differentiated state controlled? This question became one of the major interests of the Stem cell research and Translation, mostly due to the potential therapeutic perspectives that it gives. Regarding this effort, this thesis work describes a new function of a gene call split-ends/spen in adult stem cell regulation in Drosophila intestine. SPEN familly is composed by essential genes, which codes conserved proteins from Plants to Metazoa. They are involved in key cellular processes such as cell death, differentiation or proliferation, and are associated with various molecular functions controlling transcriptional and post-transcriptional gene expression. We found that a spen inactivation in Drosophila intestine leads to an abnormal increase in adult stem cells. In this work, by combining genetics tools and in vivo stem cell analysis methods, we could show that Spen works as a key factor of intestinal stem cell commitment and plays a role in their proliferation control. How does genetics programs control cellular identity? In order to investigate the molecular signature of intestinal stem cells and progenitor cells knockdowned for spen, we combined genetics, cell sorting and mRNA sequencing analysis to uncovered Spen target genes regulated in intestinal stem cells. Here, we provide a new function of spen in adult stem cell regulation, which may also shed light on its mode of action in other developmental and pathological contexts.

For more than 20 years, T-cell specific factor 4 (Tcf4) is the most intensively studied member of the conserved Tcf/Lymphoid enhancer-binding factor (Lef) family of transcription factors. Together with β-catenin coactivator, Tcf4 represents the prominent nuclear effector of canonical Wnt signaling in the intestinal epithelium. Regulation of Wnt-β-catenin signaling in intestinal stem cells is crucial for tissue homeostasis and tumor formation initiation. Up to date, several mouse models were generated to manipulate Tcf4 abundance or activity in vivo and dissect its function. Moreover, mutational screens and expression profiling of human colorectal tumors were carried out to disclose a contribution of TCF4 to tumor progression. However, subsequent studies brought conflicting results in relation to the potential of Tcf4 to activate or repress Wnt target genes and drive or inhibit cell proliferation. Here in this study, we analyze publicly available datasets for global expression of TCF4 and its paralogs in human tissues and colorectal cancer (CRC) samples. Notably, we present newly generated Tcf4flox5 mouse with a conditional Tcf4 allele that can be used to eliminate expression of Tcf4 from two alternative promoters of the gene. Using this mouse strain we documented that Tcf4 loss led to the demise of...

The work presented in this thesis provides insights into the Drosophila smooth septate junction complex Ssk-Mesh that regulates ISC proliferation and tissue homeostasis in addition to the well-known barrier function in the epithelial integrity. With CRISPR-generated tag knockin alleles of Ssk and Mesh, I characterized the intracellular expression pattern of Ssk and Mesh. Ssk and Mesh had low but detectable expression in punctate format in the cytoplasm of enteroblasts (EBs). The protein expression profile of Ssk and Mesh correlated with their ability to regulate the ISC proliferation even though the septate junctions in EBs had not fully formed. Along with further differentiation into mature enterocytes (ECs), Ssk and Mesh gradually localized to the epithelial apical domain, where they coordinated with other junction proteins, such as Tsp2A and Coracle, to form the septate junction. RNAi-conducted genetic assays and mutant clonal analyses by knockout mutant alleles of Ssk and mesh further revealed that Ssk and Mesh restricted the activity of the transcription coactivator Yki, which governs the production of the cytokine Upd3 along the EB-EC differentiation lineage in adult midgut. Loss of Ssk or Mesh activated Yki to elevate the upd3 expression and thereby to induce the robust ISC proliferation non-autonomously. Although the total number of EBs in midgut is much fewer than that of ECs, surprisingly, knockdown Ssk or mesh in EBs resulted in a comparable upd3 upregulation and ISC proliferation as knockdown their expression in ECs. Leaky midgut caused by knockdown of Ssk or mesh in ECs activated the stress-responding mechanisms to repair the damaged intestinal epithelium, and was eventually associated with death of animals. The reduction of Ssk and Mesh in EBs displayed much milder gut leakage and lower lethality further confirmed that Ssk and Mesh in the two distinct cell types had their own roles in governing ISC proliferation.

No description available.