Antagonistic roles of Notch and p63 in controlling mammary epithelial cell fates.

The breast epithelium has two major compartments, luminal and basal cells, that are established and maintained by poorly understood mechanisms. The p53 homolog, p63, is required for the formation of mammary buds, but its function in the breast after birth is unknown. We show that in primary human breast epithelial cells, maintenance of basal cell characteristics depends on continued expression of the p63 isoform, DeltaNp63, which is expressed in the basal compartment. Forced expression of DeltaNp63 in purified luminal cells confers a basal phenotype. Notch signaling downmodulates DeltaNp63 expression and mimics DeltaNp63 depletion, whereas forced expression of DeltaNp63 partially counteracts the effects of Notch. Consistent with Notch activation specifying luminal cell fate in the mammary gland, Notch signaling activity is specifically detected in mice at sites of pubertal ductal morphogenesis where luminal cell fate is determined. Basal cells in which Notch signaling is active show decreased p63 expression. Both constitutive expression of DeltaNp63 and ablation of Notch signaling are incompatible with luminal cell fate. Thus, the balance between basal and luminal cell compartments of the breast is regulated by antagonistic functions of DeltaNp63 and Notch.Cell Death and Differentiation advance online publication, 9 April 2010; doi:10.1038/cdd.2010.37.

The role of LEKTI in fate determination of keratinocyte stem cells

SPINK5 (serine protease inhibitor Kazal-type 5) encodes the putative proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor). In skin, LEKTI expression is restricted to the stratum granulosum of the epidermis and the inner root sheath of hair follicles. Mutations that create premature termination codons in SPINK5 have been reported as the cause of Netherton syndrome (NS), a human autosomal recessive disorder characterized by congenital ichthyosis with defective cornification, a specific hair shaft defect known as trichorrexis invaginata or 'bamboo hair', and severe atopic manifestations, including atopic dermatitis and hayfever. Althought recombinant human LEKTI inhibits a battery of serine proteases including plasmin, trypsin, subtilisin A, cathepsin G, and elastase, the precise role of LEKTI in the physiopathology of NS remains unclear. Spink5−/− mice display a NS-like phenotype. Surprisingly, a psoriasis-like hyperplasia, basement membrane breakdown followed by evagination of spindle-shaped epidermal cells into the dermal compartment, and the presence of numerous sweat gland-like structures were also observed when the skin of Spink5−/− newborn mice, which die at birth, was transplanted onto the back of nude mice. Collectively, these observations suggest that LEKTI may play a role on cell proliferation and stem cell fate. Our current work aims at elucidating the mechanisms by which LEKTI impact these biological processes. Using keratinocyte stem cells obtained from NS patients, we have identified LEKTI as a regulator node in several signaling pathways involved in stem cell behavior.

Inactivation of Notch 1 in immature thymocytes does not perturb CD4 or CD8T cell development.

Notch proteins influence cell-fate decisions in many developing systems. Several gain-of-function studies have suggested a critical role for Notch 1 signaling in CD4-CD8 lineage commitment, maturation and survival in the thymus. However, we show here that tissue-specific inactivation of the gene encoding Notch 1 in immature (CD25+CD44-)T cell precursors does not affect subsequent thymocyte development. Neither steady-state numbers nor the rate of production of CD4+ and CD8+ mature thymocytes is perturbed in the absence of Notch 1. In addition, Notch 1-deficient thymocytes are normally sensitive to spontaneous or glucocorticoid-induced apoptosis. In contrast to earlier reports, these data formally exclude an essential role for Notch 1 in CD4-CD8 lineage commitment, maturation or survival.

Harnessing epithelial stem cell potency

Increase in potency of adult stem/progenitor cells holds great expectations for regenerative medicine; reprogramming is achieved by manipulating the genome or indirectly by manipulating the microenvironment. However, the genetic approach, which can result in lineage conversion up to ground pluripotent embryonic state, will certainly face strict regulatory constraints and consequently translation to the clinic may be difficult. Manipulating stem cell fate without altering the genome of adult stem cells is a promising alternative. My laboratory has demonstrated that non hairy squamous epithelia e.g. the cornea, the oral cavity, the oesophagus, the vagina, contain clonogenic stem cells that can respond to skin morphogenetic signals and form epidermis, cycling hair follicles and sebaceous glands. This capacity is maintained in serial transplantation, crosses primary germ line boundaries and is intrinsic to the stem cells, as cells which have never been exposed to cell culture behave in a similar fashion. Even more surprising, the thymus contains a population of clonogenic epithelial cells of endodermal origin that maintain a thymic identity in culture and have the capacity to incorporate into a thymic network, but can acquire the functionality of bona fide multipotent stem cells of the skin when exposed to proper developmental signals. Thymic epithelial cells exposed to a skin microenvironment exhibit a down-regulation or silencing of transcription factors important for thymic function. Hence, it is possible to reveal unsuspected potency and even to robustly reprogram stem cells by solely manipulating the microenvironment.

In vivo fate mapping with SCL regulatory elements identifies progenitors for primitive and definitive hematopoiesis in mice.

One of the principal issues facing biomedical research is to elucidate developmental pathways and to establish the fate of stem and progenitor cells in vivo. Hematopoiesis, the process of blood cell formation, provides a powerful experimental system for investigating this process. Here, we employ transcriptional regulatory elements from the stem cell leukemia (SCL) gene to selectively label primitive and definitive hematopoiesis. We report that SCL-labelled cells arising in the mid to late streak embryo give rise to primitive red blood cells but fail to contribute to the vascular system of the developing embryo. Restricting SCL-marking to different stages of foetal development, we identify a second population of multilineage progenitors, proficient in contributing to adult erythroid, myeloid and lymphoid cells. The distinct lineage-restricted potential of SCL-labelled early progenitors demonstrates that primitive erythroid cell fate specification is initiated during mid gastrulation. Our data also suggest that the transition from a hemangioblastic precursors with endothelial and blood forming potential to a committed hematopoietic progenitor must have occurred prior to SCL-marking of definitive multilineage blood precursors.

Mechanisms of spindle positioning during "Caenorhabditis elgans" asymmetric cell division

Résumé : Le positionnement correct du fuseau mitotique est crucial pour les divisions cellulaires asymétriques, car il gouverne le contrôle spatial de la division cellulaire et assure la ségrégation adéquate des déterminants cellulaires. Malgré leur importance, les mécanismes contrôlant le positionnement du fuseau mitotique sont encore mal compris. Chez l'embryon au stade une-cellule du nématode Caenorhabditis elegans, le fuseau mitotique est positionné de manière asymétrique durant l'anaphase grâce à l'action de générateurs de force situés au cortex cellulaire, et dont la nature était jusqu'alors indéterminée. Ces générateurs de force corticaux exercent une traction sur les microtubules astraux et sont dépendants de deux protéines Gα et de leurs protéines associées. Cette thèse traite de la nature de la machinerie responsable pour la génération des forces de tractions, ainsi que de son lien avec les protéines Gα et associées. Nous avons combiné des expériences de coupure par faisceau laser du fuseau mitotique avec le contrôle temporel de l'inactivation de gènes ou de l'exposition à des produits pharmacologiques. De cette manière, nous avons établi que la dynéine, un moteur se déplaçant vers l'extrémité négative des microtubules, ainsi que la dynamique des microtubules, sont toutes deux requises pour la génération efficace des forces de tractions. Nous avons démontré que les protéines Gα et leurs protéines associées GPR-1/2 et LIN-5 interagissent in vivo avec LIS-1, un composant du complexe de la dynéine. De plus, nous avons découvert que les protéines Gα, GPR-1/2 et LIN-5 promeuvent la présence du complexe de la dynéine au cortex cellulaire. Nos résultats suggèrent un mécanisme par lequel les protéines Gα permettent le recrutement cortical de GPR-1/2 et LIN-5, assurant ainsi la présence de la dynéine au cortex. Conjointement avec la dynamique des microtubules, ce mécanisme permet la génération des forces de tractions afin d'obtenir une division cellulaire correcte. Comme les mécanismes contrôlant le positionnement du fuseau mitotique et les divisions cellulaires asymétriques sont conservés au cours de l'évolution, nous espérons que les mécanismes élucidés par ce travail sont d'importance générale pour la génération de la diversité cellulaire durant le développement. De plus, ces mécanismes pourraient être applicables à d'autres divisions asymétriques, comme celle des cellules souches, dont le disfonctionnement peut entraîner la génération de cellules cancéreuses. Abstract : Proper spindle positioning is crucial for asymmetric cell division, because it controls spatial aspects of cell division and the correct inheritance of cell-fate determinants. However, the mechanisms governing spindle positioning remain incompletely understood. In the Caenorhabditis elegans one-cell stage embryo, the spindle becomes asymmetrically positioned during anaphase through the action of as-yet unidentified cortical force generators that pull on astral microtubules and that depend on two Gα proteins and associated proteins. This thesis addresses the nature of the force generation machinery and the link with the Gα and associated proteins. By performing spindle-severing experiments following temporally restricted gene inactivation and drug exposure, we established that microtubule dynamics and the minus-end directed motor dynein are both required for generating efficient pulling forces. We discovered that the Gα proteins and their associated proteins GPR-1/2 and LIN-5 interact in vivo with LIS-1, a component of the dynein complex. Moreover, we uncovered that LIN-5, GPR-1/2 and the Gα proteins promote the presence of the dynein complex at the cell cortex. Our findings suggest a mechanism by which the Gα proteins enable GPR-1/2 and LIN-5 recruitment to the cortex, thus ensuring the presence of cortical dynein. Together with microtubule dynamics, this allows pulling forces to be exerted and proper cell division to be achieved. Because the mechanisms of spindle positioning and asymmetric cell division are conserved across evolution, we expect the underlying mechanism uncovered here to be of broad significance for the generation of cell diversity during development. Moreover, this mechanism could be relevant for other asymmetric cell divisions, such as stem cell divisions, whose dysfunction may lead to the generation of cancer cells.

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.

PPARbeta/delta regulates paneth cell differentiation via controlling the hedgehog signaling pathway.

BACKGROUND & AIMS: All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. METHODS: To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice. RESULTS: We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. CONCLUSIONS: We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.

Merlin, a "Magic" Linker between Extracellular Cues and Intracellular Signaling Pathways that Regulate Cell Motility, Proliferation, and Survival.

Genetic alterations of neurofibromatosis type 2 (NF2) gene lead to the development of schwannomas, meningiomas, and ependymomas. Mutations of NF2 gene were also found in thyroid cancer, mesothelioma, and melanoma, suggesting that it functions as a tumor suppressor in a wide spectrum of cells. The product of NF2 gene is merlin (moesin-ezrin-radixin-like protein), a member of the Band 4.1 superfamily proteins. Merlin shares significant sequence homology with the ERM (Ezrin-Radixin-Moesin) family proteins and serves as a linker between transmembrane proteins and the actin-cytoskeleton. Merlin is a multifunctional protein and involved in integrating and regulating the extracellular cues and intracellular signaling pathways that control cell fate, shape, proliferation, survival, and motility. Recent studies showed that merlin regulates the cell-cell and cell-matrix adhesions and functions of the cell surface adhesion/extracellular matrix receptors including CD44 and that merlin and CD44 antagonize each other's function and work upstream of the mammalian Hippo signaling pathway. Furthermore, merlin plays important roles in stabilizing the contact inhibition of proliferation and in regulating activities of several receptor tyrosine kinases. Accumulating data also suggested an emerging role of merlin as a negative regulator of growth and progression of several non-NF2 associated cancer types. Together, these recent advances have improved our basic understanding about merlin function, its regulation, and the major signaling pathways regulated by merlin and provided the foundation for future translation of these findings into the clinic for patients bearing the cancers in which merlin function and/or its downstream signaling pathways are impaired or altered.

The ubiquitin-proteasome system in glioma cell cycle control.

A major determinant of cell fate is regulation of cell cycle. Tight regulation of this process is lost during the course of development and progression of various tumors. The ubiquitin-proteasome system (UPS) constitutes a universal protein degradation pathway, essential for the consistent recycling of a plethora of proteins with distinct structural and functional roles within the cell, including cell cycle regulation. High grade tumors, such as glioblastomas have an inherent potential of escaping cell cycle control mechanisms and are often refractory to conventional treatment. Here, we review the association of UPS with several UPS-targeted proteins and pathways involved in regulation of the cell cycle in malignant gliomas, and discuss the potential role of UPS inhibitors in reinstitution of cell cycle control.

RETINOBLASTOMA RELATED1 Regulates Asymmetric Cell Divisions in Arabidopsis.

Formative, also called asymmetric, cell divisions produce daughter cells with different identities. Like other divisions, formative divisions rely first of all on the cell cycle machinery with centrally acting cyclin-dependent kinases (CDKs) and their cyclin partners to control progression through the cell cycle. However, it is still largely obscure how developmental cues are translated at the cellular level to promote asymmetric divisions. Here, we show that formative divisions in the shoot and root of the flowering plant Arabidopsis thaliana are controlled by a common mechanism that relies on the activity level of the Cdk1 homolog CDKA;1, with medium levels being sufficient for symmetric divisions but high levels being required for formative divisions. We reveal that the function of CDKA;1 in asymmetric cell divisions operates through a transcriptional regulation system that is mediated by the Arabidopsis Retinoblastoma homolog RBR1. RBR1 regulates not only cell cycle genes, but also, independent of the cell cycle transcription factor E2F, genes required for formative divisions and cell fate acquisition, thus directly linking cell proliferation with differentiation. This mechanism allows the implementation of spatial information, in the form of high kinase activity, with intracellular gating of developmental decisions.

Simultaneous irradiation of fibroblasts and carcinoma cells repress the secretion of soluble factors able to stimulate carcinoma cell migration.

Stroma mediated wound healing signals may share similarities with the ones produced by tumor's microenvironment and their modulation may impact tumor response to the various anti-cancer treatments including radiation therapy. Therefore we conducted this study, to assess the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts irrespective of their RhoB status do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms which are TGF-β1 and MMP-mediated respectively. Lastly, we found that simultaneous irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-β and MMP secretion. This last result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response.

The role of Notch receptor signaling in T helper 17 cell differentiation

Antigenic recognition by naive CD4+ T cells induces their proliferation and differentiation into functionally distinct T helper (Th) cell. Each CD4+ Th cell subset expresses specific transcription factors and produces signature cytokines that coordinate immune responses against encountered pathogens. Among the factors influencing CD4+ Th cell differentiation, Notch signaling pathway has been reported to play a role in the differentiation and function of multiple CD4+Thcell subsets. Notch signaling is an evolutionarily conserved cell-to-cell signaling cascade involved in many cell fate decision processes. How Notch signaling modulates the differentiation of CD4+ Th cell subsets and whether Notch signaling alone is sufficient or not for the differentiation of CD4+ Th cells is still a matter of debate. Th17 cells are a distinct subset of CD4+ Th cells. They play a role in the control of extracellular bacterial and fungal infections and may lead to inflammatory and autoimmune diseases if not properly regulated. Th17 cells are defined by the expression of RAR-related orphan receptor (ROR)a and RORyT transcription factors and their secretion of IL-17A, IL-17F cytokines. The involvement of Notch signaling in Th17 cell differentiation has mostly been studied in vitro. However, neither the experimental conditions when Notch signaling might be involved in Th17 cell differentiation in vitro and in vivo nor the precise role of Notch in this process remain clear. To better define how Notch signaling impacts Th17 differentiation, we used mice with T cell specific ablation of Notchl and Notch2 (N1 N2ACD4Cre) or of Notch transcriptional repressor RBP- JK (RBP-J ACD4Cre). We show that impaired Notch signaling in T cells, when TCR activating signal were reduced, increased RORyT and IL-17 mRNA levels during in vitro Th17 cell differentiation. Following immunization with OVA in CFA, an adjuvant that induces mostly Th17 cell response, increased IL-17A mRNA and intracellular IL-17A levels were observed in draining lymph nodes of Notch-deficient CD4+T cells. Our data suggest that Notch limited Th17 cell differentiation. Despite high levels of IL-17 mRNA and intracellular IL-17 proteins observed in Notch-deficient T cells, their release of Th17 cytokines ex vivo was markedly decreased, indicating a role for Notch signaling. During the second part of this thesis, we observed that the impact of Notch on Th17 cell differentiation and effector functions was context-dependent using different in vivo experimental models, in which Th17 cells and IL-17A were reported to contribute in the disease development. Collectively, our data reveal that Notch signaling controls the fine-tuning of Th17 cell differentiation and effector functions by limiting their differentiation but promoting selectively cytokine release through Notch-dependent mechanisms that still need to be defined. -- Lors d'une réponse immunitaire et grâce à la reconnaissance antigénique, les lymphocytes CD4+ T naïfs prolifèrent, puis se différencient en CD4+ T auxiliaires ("T helper" ou Th) fonctionnellement distincts. Chaque sous-population de lymphocytes CD4+ T auxiliaires exprime des facteurs de transcription et des cytokines spécifiques qui coordonnent la réponse immunitaire contre les pathogènes rencontrés. Parmi les facteurs influençant la différenciation des lymphocytes CD4+ T auxiliaires, la voie de signalisation Notch a été identifiée comme ayant un rôle dans la différenciation et la fonction des différents sous-types de cellules CD4+ T auxiliaires. La voie de signalisation Notch est une voie évolutivement conservée, qui est impliquée dans la signalisation entre les cellules et dans de nombreux processus de décisions cellulaires. La manière dont la voie de signalisation Notch régule la différenciation des lymphocytes CD4+ T en sous-types de cellules CD4+ auxiliaires, mais également la question de savoir si la voie de signalisation Notch est capable ou non d'induire la différenciation des cellules CD4+T auxiliaires, restent à débattre. Les cellules T auxiliaires 17 (Th17) sont un sous-type distinct de cellules CD4+T. Elles jouent un rôle important dans la défense immunitaire contre des pathogènes tels que les bactéries extracellulaires et les champignons. Une dérégulation de la réponse des cellules Th17 peut conduire à des inflammations mais également à des maladies auto-immunes. Les cellules Th17 sont définies par l'expression de leurs facteurs de transcription RAR-related orphan receptor (ROR)a, RORyT et par la sécrétion de cytokines comme IL-17A, IL-17F. Le rôle de la voie de signalisation Notch dans la différenciation des cellules Th17 a principalement été démontré in vitro. Malgré tout, ni les conditions expérimentales dans lesquelles cette voie pourrait être impliquée dans la différenciation des cellules Th17 in vitro et in vivo, mais également ni la fonction exacte de Notch dans ces processus, ne sont des questions résolues. Afin de mieux définir comment la voie de signalisation Notch est impliquée dans la différenciation des cellules Th17, nous avons utilisé des souris avec une déficience spécifique dans les cellules T des récepteurs Notchl et Notch2 (N1N2ACD4Cre) ou du répresseur transcriptionnel de Notch RBP-JK (RBP-J ACD4Cre). Nous avons montré que lorsque la voie de signalisation Notch est déficiente, les niveaux d'ARN messager (ARNm) de RORyT et de IL-17A sont augmentés dans les cellules Th17 pendant la différenciation in vitro, en présence de niveaux réduits des signaux activant les cellules T CD4+. Une augmentation dans les niveaux d'ARNm de IL-17A et de IL-17A intracellulaire au niveau protéinique a été observée dans les cellules T CD4+ Notch déficientes, au niveau des ganglions drainants après immunisation avec l'OVA dans le CFA, un adjuvant induisant une réponse des cellules Th17. Nos résultats suggèrent que Notch pourrait réguler négativement l'expression de IL-17A au niveau transcriptionnel mais également protéinique. Malgré une augmentation de IL-17A au niveau de l'ARNm et protéinique dans les cellules CD4+ T Notch déficientes, paradoxalement la sécrétion de IL-17A mais également de cytokines associées aux fonctions effectrices des cellules Th17 sont profondément diminuées 6X vivo, suggérant un rôle de la voie de signalisation Notch dans ce processus. Dans la deuxième partie de ce travail de thèse, nous avons observé que l'impact de Notch dans la différenciation des cellules Th17 et dans leurs fonctions effectrices était dépendant du contexte dans d'autres modèles expérimentaux in vivo, où les cellules Th17 et l'IL-17A ont été identifiées comme ar-.riCociêSM dans le développement ds la pathologie. En résumé, nous avons montré que la voie de la signalisation Notch contrôle la régulation précise de la différenciation des cellules Th17 en limitant leur différenciation, mais en promouvant sélectivement leur relâchement en cytokines associés aux cellules Th17 par l'intermédiaire de mécanismes dépendant de Notch, qui restent toujours à déterminer. -- Lors d'une réponse immunitaire et grâce à la reconnaissance antigénique, les lymphocytes CD4+ T naïfs prolifèrent, puis se différencient en CD4+ T auxiliaires ("T helper" ou Th) fonctionnellement distincts. Chaque sous-population de lymphocytes T auxiliaires exprime des facteurs de transcription et des cytokines spécifiques qui coordonnent une réponse immunitaire contre différents pathogènes. Les mécanismes liés à la différenciation des lymphocytes CD4+ T auxiliaires sont complexes et régulés. Une mauvaise régulation de la différenciation des lymphocytes CD4+ T auxiliaires peut conduire à des maladies auto-immunes, mais également à des processus inflammatoires. Parmi les facteurs influençant la différenciation des lymphocytes T auxiliaires, la voie de signalisation Notch a été identifiée comme ayant un rôle dans la différenciation et la fonction des différents sous-types de cellules CD4+ T auxiliaires. La voie de signalisation Notch est une voie évolutivement conservée, qui est impliquée dans la signalisation entre les cellules, mais également dans de nombreux processus de décisions cellulaires. Quelle est l'implication de la voie de signalisation Notch dans la différenciation des lymphocytes CD4+ en sous-types de cellules CD4+T auxiliaires et comment cette voie agit dans ce processus, sont des questions débattues. Les cellules T auxiliaires 17 (Th17) sont une sous-population distincte de lymphocytes CD4+. Elles jouent un rôle important dans la défense immunitaire contre les bactéries extracellulaires et les champignons. Une dérégulation de la réponse des cellules Th17 a été associée à des maladies auto-immunes et à l'inflammation. Les cellules Th17 sont définies par l'expression du facteur de transcription RAR-related orphan receptor (ROR)yT et des cytokines comme IL-17A, IL-17F. Le rôle de la voie de signalisation Notch dans la différenciation des cellules Th17 a été principalement démontré dans des études expérimentales in vitro. Malgré tout, les conditions expérimentales exactes dans lesquelles la voie de signalisation de Notch pourrait être impliquée dans la différenciation des cellules Th17, mais également le rôle de Notch dans ce processus ne sont pas encore clairement élucidés. Afin de mieux définir comment la voie de signalisation Notch est impliquée dans la différenciation des cellules Th17, nous avons utilisé des souris avec une déficience spécifique dans les cellules T des récepteurs Notchl et Notch2 (N1 N2ACD4Cre) ou du répresseur transcriptionnel de Notch RBP-JK (RBP-JACD4CRE). Nous avons montré que lorsque la voie de signalisation Notch est déficiente, les niveaux d'ARN messager (ARNm) de RORyT et de IL-17 sont augmentés dans les cellules Th17 pendant leur différenciation in vitro. Cet effet de Notch sur la transcription apparaît être facultatif lorsque les conditions environnementales sont en excès in vitro. Après immunisation avec un adjuvant qui induit principalement une réponse des cellules Th17, nous avons observé que les niveaux de ARNm de IL-17A et aussi de IL-17A intracellulaire au niveau protéinique étaient augmentés dans les ganglions drainants dans les cellules CD4+ Notch déficientes. Ces résultats suggèrent que Notch pourrait réguler négativement l'expression de IL- 17 au niveau transcriptionnel mais également protéinique. Malgré des niveaux plus élevés de IL- 17 ARNm et aussi IL-17A intracellulaire dans les cellules T Notch déficientes, le relâchement en cytokines Th17 est profondément diminué indiquant un rôle de la voie de signalisation Notch dans ces processus de sécrétion. Dans la deuxième partie de cette thèse, nous avons observé que le rôle de Notch dans ia différenciation dss cellules Ti,17 et dans leurs fonctions effectrices était dépendant du contexte dans d'autres modèles expérimentaux, qui ont été rapportés comme une réponse induisant des cellules Th17. En résumé, nos données montrent que la voie de la signalisation Notch contrôle la régulation précise de la différenciation des cellules Th17 en limitant leur différenciation mais en promouvant sélectivement le relâchement en cytokines associées aux cellules Th17 par des mécanismes dépendant de Notch qui restent toujours à déterminer. Par conséquent, l'inhibition de la voie de signalisation Notch pourrait être utilisée dans des situations inflammatoires ou d'auto-immunité où la réponse des cellules Th17 est exacerbée.

Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress.

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.

Transcriptional response to cardiac injury in the zebrafish: systematic identification of genes with highly concordant activity across in vivo models.

BACKGROUND: Zebrafish is a clinically-relevant model of heart regeneration. Unlike mammals, it has a remarkable heart repair capacity after injury, and promises novel translational applications. Amputation and cryoinjury models are key research tools for understanding injury response and regeneration in vivo. An understanding of the transcriptional responses following injury is needed to identify key players of heart tissue repair, as well as potential targets for boosting this property in humans. RESULTS: We investigated amputation and cryoinjury in vivo models of heart damage in the zebrafish through unbiased, integrative analyses of independent molecular datasets. To detect genes with potential biological roles, we derived computational prediction models with microarray data from heart amputation experiments. We focused on a top-ranked set of genes highly activated in the early post-injury stage, whose activity was further verified in independent microarray datasets. Next, we performed independent validations of expression responses with qPCR in a cryoinjury model. Across in vivo models, the top candidates showed highly concordant responses at 1 and 3 days post-injury, which highlights the predictive power of our analysis strategies and the possible biological relevance of these genes. Top candidates are significantly involved in cell fate specification and differentiation, and include heart failure markers such as periostin, as well as potential new targets for heart regeneration. For example, ptgis and ca2 were overexpressed, while usp2a, a regulator of the p53 pathway, was down-regulated in our in vivo models. Interestingly, a high activity of ptgis and ca2 has been previously observed in failing hearts from rats and humans. CONCLUSIONS: We identified genes with potential critical roles in the response to cardiac damage in the zebrafish. Their transcriptional activities are reproducible in different in vivo models of cardiac injury.