Elongation of the mouse anteroposterior axis depends on stem cell-like axial progenitors including a neuromesodermal (NM) bi-fated population existing in the primitive streak and later in the tail bud. Fate mapping experiments have demonstrated these NM progenitors reside in precise locations of the embryo. At E8.5, these cells are found in the node-streak border (NSB) and anterior epiblast on either side of the primitive streak. At tail bud stages (E10.5-E13.5), these progenitors reside in the chordoneural hinge (CNH). The coexpression of the transcription factors T (brachyury) and Sox2 has been proposed as a good marker to identify NM progenitors in vertebrates. However, this cell signature has never been thoroughly assessed during mouse axis elongation. In this thesis, I performed T and Sox2 double immunofluorescent stainings on different stages of mouse embryos and reconstructed their expression domains in the 3D images to investigate the spatiotemporal dynamics of NM progenitors during axis elongation. The results show the transient existence of T+Sox2+ cells in the posterior progenitor zone, from the headfold stage (E8.0) to the end of axis elongation (E13.5, 65somites). Moreover, the number of T+Sox2+ cells increases between E8.5 and E9.5 but gradually declines afterwards. I then investigated the time points for initiation and loss of NM progenitors by performing a series of heterotopic grafting experiments. It has been previously shown that distal epiblast (Sox2+T- cells) at LS-EB stages (E7.5) are fated to become NSB cells in E8.5 embryos. However, when cells from the distal region of LS-EB stage embryos (E7.5) were grafted to E8.5 NSB, these cells contribute extensity to the notochord but not either neural tissues or paraxial mesoderm. This indicates that NM progenitors may be not yet specified before the onset of T and Sox2 coexpression, while the notochord progenitors are already specified at E7.5. The grafting experiments also show the loss of NM progenitors at E14.5 after the end of axis elongation, which coincides with the disappearance of T+Sox2+ cells in the tail. Collectively, these results indicate that T+Sox2+ cells may represent a distinct cell state that defines NM progenitors. Wnt/β-catenin signalling has been shown to play an important role in maintaining the posterior progenitor zone. However, due to the wide expression of β-catenin and the early lethality of β-catenin null embryos, the exact effect of losing β-catenin in NM progenitors is still unknown. In this study, I took advantage of the Cre-ERT2 system and grafting technique to conditionally delete β-catenin specifically in NM progenitors during ex vivo culture. The results show that Wnt/β-catenin signalling is required cell autonomously for initiating mesoderm fate choice in NM progenitors. In its absence, mesoderm fated NM progenitors convert their fate and differentiate to neural derivatives. Moreover, the interchangeability between neural and mesodermal fate only exists in NM progenitors, as the loss of β-catenin in mesoderm committed progenitors does not affect their fate choice. Using image analysis and quantification software, I also show that Wnt/β-catenin signalling is crucial for the expansion of T+Sox2+ NM progenitors during axis elongation. Due to difficult access and a limited number of NM progenitors in vivo, in vitro generated NM progenitors from pluripotent cells, such as epiblast stem cells (EpiSCs), can offer an insight into the maintenance and differentiation of NM progenitors. Since the in vivo potential of EpiSCs had never been successfully demonstrated before, I first grafted EpiSCs into postimplantation embryos and cultured them ex vivo for 24-48 hours to assess their cell integration. The results show that EpiSCs can integrate successfully in streak stage embryos (E6.5-E7.5), but not at early somite stages (E8.5), when the epiblast has lost its pluripotency. I then further investigated the in vivo potential of EpiSC derivatives. The results show that increasing Wnt signalling in EpiSCs inhibits their ability to generate anterior neural tissues in vivo, which is consistent with the previous in vitro data. Recently, it has been demonstrated that NM progenitors can be derived from EpiSCs. These in vitro derived NM progenitors can incorporate into E8.5 embryos and give rise to both neural and mesodermal derivatives. In this thesis, I show that these in vitro derived NM progenitors do not incorporate successfully in E7.5 embryos. Collectively, by combining grafting experiments with a chimeric embryo formation assay, I can identify the in vivo stage of the in vitro counterparts of the embryonic cell types.

Although human congenital cerebellar malformations are common, their molecular and developmental basis is still poorly understood. Recently, cilia-related gene deficiencies have been implicated in several congenital disorders that exhibit cerebellar abnormalities such as Joubert syndrome, Meckel-Gruber syndrome, Bardet-Biedl syndrome, and Orofaciodigital syndrome. The association of cilia gene mutations with these syndromes suggests that cilia may be important for cerebellar development, but the nature of cilia involvement has not been elucidated. To assess the importance of cilia-related proteins during cerebellar development, we studied the effects of CNS-specific inactivation of two mouse genes whose protein products are critical for cilia formation and maintenance, IFT88, (also known as polaris or Tg737), which encodes intraflagellar transport 88 homolog, and Kif3a, which encodes kinesin family member 3a. We showed that loss of either of these genes caused severe cerebellar hypoplasia and foliation abnormalities, primarily attributable to a failure of expansion of the neonatal granule cell progenitor population. In addition, granule cell progenitor proliferation was sensitive to partial loss of IFT function in a hypomorphic mutant of IFT88 (IFT88orpk), an effect that was modified by genetic background. IFT88 and Kif3a were not required for the specification and differentiation of most other cerebellar cell types, including Purkinje cells. Together, our observations constitute the first demonstration that cilia proteins are essential for normal cerebellar development and suggest that granule cell proliferation defects may be central to the cerebellar pathology in human cilia-related disorders.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13559号 / 論医博第2288号 / 新制||医||1067(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 髙折 晃史, 教授 西浦 博, 教授 遊佐 宏介 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24676号 / 農博第2559号 / 新制||農||1100(附属図書館) / 学位論文||R5||N5457(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 寺内 良平, 教授 髙野 義孝, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

Le muscle strié squelettique est un tissu fascinant qui permet d’assurer les fonctions essentielles à notre existence : se mouvoir, maintenir sa posture, se nourrir, communiquer ou tout simplement respirer. De nombreuses situations, engendrant principalement une hypoactivité, peuvent provoquer un déconditionnement musculaire caractérisé par une perte de masse et de force ainsi qu’un développement d’infiltrations graisseuses (IMAT), altérant ainsi la fonction musculaire. Le développement d’IMAT est également observé lorsque les processus de régénération musculaire sont altérés. Les fibro-adipogenic progenitors (FAPs) représentent la population de cellules souches principalement impliquée dans le développement d’IMAT. L’interaction entre FAPs et cellules satellites/immunitaires semble être un trio indispensable pour une régénération optimale, sans développement d’IMAT. Au regard de la littérature scientifique, une modulation du niveau d’activité contractile permet de faire varier le niveau d’expression de nombreuses cytokines impliquées dans la modulation des FAPs et donc dans l’apparition d’IMAT. Nos travaux ont contribué à l’accroissement des connaissances scientifiques relatives à la thématique des infiltrations graisseuses et à leurs exacerbations dans des situations d’hypoactivité ou de régénération musculaire. Nous avons montré que 3 jours d’hypoactivité chez l’homme, induite par le modèle novateur de dry immersion, suffisent à augmenter le contenu musculaire en IMAT. Dans un contexte de régénération musculaire, induite par le modèle glycérol chez la souris, nous avons démontré une inhibition de l’apparition des IMAT en diminuant les contraintes mécaniques appliquées au muscle lésé. Nous avons également précisé le rôle de l’axe TNFα/TGF-β1, et donc celui des processus inflammatoires nécessaires dans l’apoptose des FAPs afin de limiter le développement des IMAT dans ce modèle. Ces trois études ouvrent de nombreuses perspectives, afin i) de préciser le rôle des IMAT dans la dysfonction musculaire, ii) de définir les mécanismes de régulation qui contrôlent le développement et l’accumulation d’IMAT. / Skeletal muscle is a fascinating tissue that ensures core functions: moving, maintaining postures, feeding, communicating or just breathing. Many situations, associated with hypoactivity, are able to involve muscle deconditioning defined by a loss of mass and strength, as well as fat infiltration development (IMAT), altogether impairing muscle function. IMAT development occurs also with disrupted regeneration processes. Fibro-adipogenic progenitors (FAPs) appear as the main stem cell population involved in IMAT development. The interaction between FAPs and satellite/immune cells seems to be a crucial trio for an efficient regeneration, without IMAT development. According to the literature, the degree of contractile activity is able to affect the expression levels of different cytokines involved in FAPs fate, and therefore in IMAT accumulation. Our work contributed to increase scientific knowledge on muscle fatty infiltrations and their exacerbations in hypoactivity or regeneration situations. We showed that 3 days of hypoactivity in human, induced by the innovative model of dry immersion, are sufficient to promote an increase in IMAT content. In a context of muscle regeneration, induced by the mouse glycerol model, we highlighted an almost complete inhibition of IMAT accumulation by decreasing mechanical constraints applied to the injured muscle. We also investigated the role of the TNFα/TGF-β1 axis, and therefore the potential role of the inflammatory stage in FAPs apoptosis and inhibition of IMAT development. Our work open up new prospects 1°) clarifying the role of IMAT in muscle dysfunction, and 2°) defining the underlying mechanisms controlling IMAT development and accumulation.

Les ephrines et leurs récepteurs Eph constituent une famille multigénique de facteurs de guidage cellulaire et axonal. Ces facteurs jouent un rôle-clé dans l’établissement de cartes neurales topographiques, notamment au niveau des connexions thalamocorticales, réseau neuronal majeur du cerveau des mammifères. Notre projet visait initialement à étudier l’implication des ephrines corticales dans la génèse des connexions thalamocorticales par une approche de gain de fonction. Pour ce faire, nous avons généré des souris transgéniques présentant une expression ectopique spécifique de l’ephrine-A5 dans le cortex en développement, en utilisant une technique de transgénèse d’addition par chromosome artificiel de bactéries (BAC). De façon surprenante, l’analyse de ces souris nous a révélé que les ephrines, à côté de leurs rôles classiques de facteurs de guidage, influençaient la taille du cortex cérébral en régulant l’apoptose des progéniteurs neuronaux. En effet, nous avons pu montrer que l’expression ectopique du ligand ephrine-A5 par les progéniteurs corticaux exprimant son récepteur EphA7 résultait en une déplétion précoce en progéniteurs corticaux par apoptose, et une diminution subséquente de la taille du cortex. Cette vague apoptotique est observée en l’absence de toute altération détectable de la prolifération, la différenciation et la migration neurale dans le cortex. Nous avons étayé notre étude in vivo par des expériences in vitro, qui ont montré que l’ephrine-A5 recombinante était capable d’induire rapidement la mort des progéniteurs neuronaux dissociés. Nous avons également montré que cette mort cellulaire impliquait l’activation de la caspase-3, confirmant ainsi l’effet direct des ephrines et de leurs récepteurs sur une ou plusieurs cascades apoptotiques. Par contre, la stimulation des neurones post-mitotiques corticaux par l’ephrine-A5 est accompagnée d’une activation de la caspase-3 sans mort cellulaire apparente. La signalisation ephrine/Eph induirait donc l’activation de la caspase-3 dans différents types cellulaires, sans que celle-ci ne soit systématiquement le reflet d’une mort cellulaire programmée. Parallèlement, afin d’évaluer l’importance physiologique de cette voie pro-apoptotique dépendante des ephrines, nous avons étudié des souris présentant une perte de fonction du récepteur EphA7. L’analyse de ces mutants nous a permis de mettre en évidence une diminution de l’apoptose des progéniteurs corticaux, une augmentation de la taille du cortex, ainsi qu’une hypercroissance exencéphalique de tout le cerveau antérieur dans les cas les plus extrêmes. Ces observations indiquent donc que les ephrines sont nécessaires au contrôle de la mort cellulaire programmée des progéniteurs du cortex cérébral. Nous avons également observé le même phénotype exencéphalique dans des mutants déficients en ephrines-A2, -A3 et -A5, dont l’analyse préliminaire suggère également des défauts de processus apoptotiques. Nos diverses expériences, combinant une approche par gain et perte de fonction, à la fois in vivo et in vitro, ont ainsi permis de proposer un nouveau rôle des ephrines en marge de leur implication dans la guidance axonale, à savoir un rôle dans le contrôle de la taille cérébrale par induction de l’apoptose des progéniteurs corticaux. La mise en évidence de cette nouvelle voie de signalisation pro-apoptotique pourrait avoir des implications importantes dans d’autres aspects de la biologie du développement et des cellules souches, ainsi que dans l’oncogénèse.

Mast cell progenitors originate from the bone marrow and migrate to the lungs via the blood. During maturation, these cells acquire granules that contain a potent array of bronchoconstrictive mediators. The number of pulmonary mast cells is augmented in asthmatic patients and in mice with allergic airway inflammation, possibly contributing to airway hyperreactivity. An increase in mast cells is likely due to an increased recruitment of committed mast cell progenitors from the blood. However, until now a committed mast cell progenitor population has not been found in adult peripheral blood. We isolated Lin- c-kithi ST2+ integrin β7hi CD16/32hi progenitors from murine blood and showed that these cells were committed to the mast cell lineage. Based on the expression of FcεRI, these cells were less mature in Th1-prone C57BL/6 mice than in Th2-prone BALB/c mice. Asthma is associated with elevated levels of IgE. Upon exposure to allergens, IgE immune complexes are formed. In a mouse model of allergic airway inflammation, we showed that intranasal administration of IgE immune complexes to antigen-sensitized mice resulted in an increased number of mast cell progenitors compared with antigen administration alone. The increase in mast cell progenitors was independent of the low-affinity IgE receptor CD23. Rather, signaling through the common FcRγ-chain was required to enhance the number of lung mast cell progenitors. Signaling through FcεRI was likely responsible for the increase. However a role for FcγRIV could not be excluded. CD11c+ cells, such as dendritic cells, are important for antigen sensitization. In a mouse model of allergic airway inflammation, these cells are also important for the development of airway hyperreactivity, eosinophilia and Th2 cytokine production in response to antigen challenge. We showed that CD11c+ cells are critical for the recruitment of lung mast cell progenitors and the subsequent increase in mast cells. These CD11c+ cells were needed for the upregulation of endothelial vascular cell adhesion molecule-1 (VCAM-1), which is a prerequisite for the antigen-induced recruitment of lung mast cell progenitors.

Elongation of the vertebrate rostrocaudal axis depends on localised populations of axial progenitors. Previous work has demonstrated the presence of Neuromesodermal (NM) progenitors that behave as multipotent stem cells, which contribute to the neurectoderm and mesoderm throughout axis elongation. They have been localised to the Node-­‐Streak Border (NSB) in the primitive streak region, and the Chordoneural Hinge (CNH) in its descendant, the tail bud. At primitive streak stages, the Caudal Lateral Ectoderm (CLE) on either side of the primitive streak itself is also fated for neurectoderm and mesoderm. However, fate mapping studies in mouse and chick have suggested that these progenitors are more transitory than those in the NSB and CNH, leading to the idea that two different types of progenitor cell exist in the primitive streak region; long-­‐term (stem cell-­‐like) and transient progenitors. In this thesis, I have examined the potency of the CLE cells by heterotopically grafting them into the NSB. Anterior CLE cells are exquisitely sensitive to their position and differentiate predominantly as neurectoderm, mesoderm, or both, depending on their exact location in the NSB. Most significantly, their descendants are retained in the CNH, indicating that CLE cells show equal potential to NSB progenitors on transplantation to the border environment. The relationship between fate and potency within the streak stage embryo suggest a mechanism by which stem cells are maintained not only by their intrinsic stem cell program, but are also influenced by their location. Furthermore, I have investigated the expression of candidate markers of NM-­‐progenitors, and have found that the combined expression of Sox2 and T genes in the progenitor area coincides with observed NM-­‐potency, and could serve as a marker for this stem cell population. Over time, axial elongation comes to a halt and NM-­‐progenitors are thereafter not longer active. It is still unclear how exactly this process occurs and specifically whether axial elongation ceases because NM progenitors are eliminated. I have investigated the events occurring immediately before the end of axial elongation. Morphological and gene expression analysis shows that apoptosis reaches a peak only after the complete axis has been laid down, and is not dramatically elevated in the progenitors themselves before that. In order to test signalling pathways that lead to progenitor maintenance, I have developed an in vitro tail growth assay that recapitulates in vivo development, as measured by several morphometric criteria. I show that, even though FGF signalling is critical for most cells in the tail bud including NM-­‐progenitors, it is not sufficient for NM-­‐ progenitor maintenance. In contrast, exposing tail buds to elevated Wnt/β-­‐catenin signalling does prolong the lifetime of NM-­‐progenitors in the ageing tail bud, as judged by the presence of Sox2-­‐T double-­‐positive cells. In this regard we have found that the time of cessation coincides with the disappearance of Sox2-­‐T double-­‐positive cells, the disappearance of Wnt3a and concomitant neuralisation of the progenitor region. This data suggest an important governing role for Wnt signalling in both maintenance and fate decision of NM progenitors. Thus the disappearance of Wnt signalling in the tail bud over time could well be the main reason for triggering the halt of caudal elongation.

Human haematopoiesis resembles a complex hierarchy, however most intermediate stages are only poorly defined. Efforts to characterise human progenitors have been inconsistent and failed to integrate previous knowledge. Furthermore, characterisation of normal progenitors has important implications in acute myeloid leukaemia (AML) biology. We previously established that leukaemic stem cells (LSCs) resemble the immunophenotypic progenitor compartments more closely than the stem cell fraction. Therefore, I set out to characterise human stem and progenitor cells (HSCPs) on phenotypic, molecular and functional level to complete the picture of human haematopoiesis. I purified HSPCs based on their immunophenotype from adult bone marrow (BM) and umbilical cord blood (CB) to investigate steady state and neonatal haematopoiesis. To define differentiation potentials, HSPCs were subjected to functional in vitro assays on bulk and clonal level. Limit dilution assays were used to determine the frequency of cells with multiple differentiation potentials. RNA sequencing revealed underlying lineage priming and specific gene expression signatures. I successfully characterized the incompletely defined Lin^-CD34⁺CD38^-CD45RA⁺ fraction in BM and CB, containing a CD10^lo lymphoid-primed multipotent progenitor (LMPP) with T cell, B cell, NK cell, granulocytic and monocytic differentiation potential, and succeeded in placing it in the haematopoietic hierarchy with relation to similar lympho-myeloid progenitors defined by other groups. This research lays the foundation to characterise early human progenitors with a comprehensive toolkit on a phenotypic, molecular and functional level. Findings from this thesis might provide knowledge about potential targets in LSCs.