High commitment of embryonic keratinocytes to terminal differentiation through a Notch1-caspase 3 regulatory mechanism.

Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

The human epidermal differentiation complex: cornified envelope precursors, S100 proteins and the 'fused genes' family.

  The skin is essential for survival and protects our body against biological attacks, physical stress, chemical injury, water loss, ultraviolet radiation and immunological impairment. The epidermal barrier constitutes the primordial frontline of this defense established during terminal differentiation. During this complex process proliferating basal keratinocytes become suprabasally mitotically inactive and move through four epidermal layers (basal, spinous, granular and layer, stratum corneum) constantly adapting to the needs of the respective cell layer. As a result, squamous keratinocytes contain polymerized keratin intermediate filament bundles and a water-retaining matrix surrounded by the cross-linked cornified cell envelope (CE) with ceramide lipids attached on the outer surface. These cells are concomitantly insulated by intercellular lipid lamellae and hold together by corneodesmosmes. Many proteins essential for epidermal differentiation are encoded by genes clustered on chromosomal human region 1q21. These genes constitute the 'epidermal differentiation complex' (EDC), which is divided on the basis of common gene and protein structures, in three gene families: (i) CE precursors, (ii) S100A and (iii) S100 fused genes. EDC protein expression is regulated in a gene and tissue-specific manner by a pool of transcription factors. Among them, Klf4, Grhl3 and Arnt are essential, and their deletion in mice is lethal. The importance of the EDC is further reflected by human diseases: FLG mutations are the strongest risk factor for atopic dermatitis (AD) and for AD-associated asthma, and faulty CE formation caused by TG1 deficiency causes life-threatening lamellar ichthyosis. Here, we review the EDC genes and the progress in this field.

The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-mum thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.

BCL9 is an essential component of canonical Wnt signaling that mediates the differentiation of myogenic progenitors during muscle regeneration.

Muscle stem cells and their progeny play a fundamental role in the regeneration of adult skeletal muscle. We have previously shown that activation of the canonical Wnt/beta-catenin signaling pathway in adult myogenic progenitors is required for their transition from rapidly dividing transient amplifying cells to more differentiated progenitors. Whereas Wnt signaling in Drosophila is dependent on the presence of the co-regulator Legless, previous studies of the mammalian ortholog of Legless, BCL9 (and its homolog, BCL9-2), have not revealed an essential role of these proteins in Wnt signaling in specific tissues during development. Using Cre-lox technology to delete BCL9 and BCL9-2 in the myogenic lineage in vivo and RNAi technology to knockdown the protein levels in vitro, we show that BCL9 is required for activation of the Wnt/beta-catenin cascade in adult mammalian myogenic progenitors. We observed that the nuclear localization of beta-catenin and downstream TCF/LEF-mediated transcription, which are normally observed in myogenic progenitors upon addition of exogenous Wnt and during muscle regeneration, were abrogated when BCL9/9-2 levels were reduced. Furthermore, reductions of BCL9/9-2 inhibited the promotion of myogenic differentiation by Wnt and the normal regenerative response of skeletal muscle. These results suggest a critical role of BCL9/9-2 in the Wnt-mediated regulation of adult, as opposed to embryonic, myogenic progenitors.

Genetic differentiation of common shrew Sorex araneus populations among different alpine valleys revealed by microsatellites

Geographical barriers may affect the genetic structure of populations by reducing gene exchanges among them. In Switzerland, the common shrew Sorer araneus Linnaeus, 1758 is mostly confined to mountainous areas because of a competing sister species, Millet's shrew S. coronatus Millet, 1828, which occupies most of the Swiss lowlands. The structure of common shrew populations found in different alpine valleys may therefore be affected by the topography. Using microsatellites, genetic structuring of seven shrew populations is investigated among four different valleys of, the Swiss Alps. Using the exact G-test, significant genetic structuring is detected between several valleys. Isolation by distance does not fully explain our results. It appears that high mountain ridges (> 2400 m) can significantly reduce gene flow. F- and R-statistics are estimated and compared to the exact G-tests results. Mantel tests show that F-ST, unlike R-ST, is significantly correlated with differentiation. F-ST remains however low even at high differentiation levels, while R-ST has a high variance. We discuss how these results may have wider implications with regards the interpretation of microsatellite data. Finally, a new microsatellite locus, L99, appears to discriminate S. araneus of the Vaud and Cordon races from both S. araneus Valais and S. coronatus.

Identification of a pheromone regulating caste differentiation in termites.

The hallmark of social insects is their caste system: reproduction is primarily monopolized by queens, whereas workers specialize in the other tasks required for colony growth and survival. Pheromones produced by reining queens have long been believed to be the prime factor inhibiting the differentiation of new reproductive individuals. However, there has been very little progress in the chemical identification of such inhibitory pheromones. Here we report the identification of a volatile inhibitory pheromone produced by female neotenics (secondary queens) that acts directly on target individuals to suppress the differentiation of new female neotenics and identify n-butyl-n-butyrate and 2-methyl-1-butanol as the active components of the inhibitory pheromone. An artificial pheromone blend consisting of these two compounds had a strong inhibitory effect similar to live neotenics. Surprisingly, the same two volatiles are also emitted by eggs, playing a role both as an attractant to workers and an inhibitor of reproductive differentiation. This dual production of an inhibitory pheromone by female reproductives and eggs probably reflects the recruitment of an attractant pheromone as an inhibitory pheromone and may provide a mechanism ensuring honest signaling of reproductive status with a tight coupling between fertility and inhibitory power. Identification of a volatile pheromone regulating caste differentiation in a termite provides insights into the functioning of social insect colonies and opens important avenues for elucidating the developmental pathways leading to reproductive and nonreproductive castes.

Differentiation associated regulation of microRNA expression in vivo in human CD8+ T cell subsets.

BACKGROUND: The differentiation of CD8+ T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown. METHODS: In order to explore the potential implication of microRNAs in CD8+ T cell differentiation in humans, microRNA expression profiles were analysed using microarrays and quantitative PCR in several human CD8+ T cell subsets defining the major steps of the T cell differentiation pathway. RESULTS: We found expression of a limited set of microRNAs, including the miR-17~92 cluster. Moreover, we reveal the existence of differentiation-associated regulation of specific microRNAs. When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease. CONCLUSIONS: This study establishes for the first time in a large panel of individuals the existence of differentiation associated regulation of microRNA expression in human CD8+ T lymphocytes in vivo, which is likely to impact on specific cellular functions.

A dynamic model of keratinocyte stem cell renewal and differentiation: role of the p21WAF1/Cip1 and Notch1 signaling pathways.

We present here a dynamic model of functional equilibrium between keratinocyte stem cells, transit amplifying populations and cells that are reversibly versus irreversibly committed to differentiation. According to this model, the size of keratinocyte stem cell populations can be controlled at multiple levels, including relative late steps in the sequence of events leading to terminal differentiation and by the influences of a heterogeneous extra-cellular environment. We discuss how work in our laboratory, on the interconnection between the cyclin/CDK inhibitor p21WAF1/Cip1 and the Notch1 signaling pathways, provides strong support to this dynamic model of stem cell versus committed and/or differentiated keratinocyte populations.

Experimental cutaneous Leishmaniasis: a powerful model to study in vivo the mechanisms underlying genetic differences in Th subset differentiation.

The murine model of infection with Leishmania major has allowed the demonstration in vivo of the importance CD4+ T cell subsets, distinguishable by the pattern of cytokines they produce, on the outcome of infectious diseases. Genetically determined resistance and susceptibility to infection with this parasite are the result of the development of Th1 and Th2 response, respectively. In this short paper, we present some results obtained in our group pertaining to the analysis of the mechanisms, operational during the early phase of this infection, responsible for the maturation of these functionally distinct CD4+ responses.

Sensitive gene expression profiling of human T cell subsets reveals parallel post-thymic differentiation for CD4+ and CD8+ lineages.

The differentiation of CD4(+) or CD8(+) T cells following priming of naive cells is central in the establishment of the immune response against pathogens or tumors. However, our understanding of this complex process and the significance of the multiple subsets of differentiation remains controversial. Gene expression profiling has opened new directions of investigation in immunobiology. Nonetheless, the need for substantial amount of biological material often limits its application range. In this study, we have developed procedures to perform microarray analysis on amplified cDNA from low numbers of cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4(+) or CD8(+) T cell differentiation. Surprisingly, our data revealed that while CD4(+) and CD8(+) T cell gene expression programs diverge at early stages of differentiation, they become increasingly similar as cells reach a late differentiation stage. This suggests that functional heterogeneity between Ag experienced CD4(+) and CD8(+) T cells is more likely to be located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes.

High quantitative and no molecular differentiation of a freshwater snail (Galba truncatula) between temporary and permanent water habitats.

We investigate the variation in quantitative and molecular traits in the freshwater snail Galba truncatula, from permanent and temporary water habitats. Using a common garden experiment, we measured 20 quantitative traits and molecular variation using seven microsatellites in 17 populations belonging to these two habitats. We estimated trait means in each habitat. We also estimated the distributions of overall genetic quantitative variation (QST), and of molecular variation (FST), within and between habitats. Overall, we observed a lack of association between molecular and quantitative variance. Among habitats, we found QST>FST, an indication of selection for different optima. Individuals from temporary water habitat matured older, at a larger size and were less fecund than individuals from permanent water habitat. We discuss these findings in the light of several theories for life-history traits evolution.

Notch1 is required for neuronal and glial differentiation in the cerebellum.

The mechanisms that guide progenitor cell fate and differentiation in the vertebrate central nervous system (CNS) are poorly understood. Gain-of-function experiments suggest that Notch signaling is involved in the early stages of mammalian neurogenesis. On the basis of the expression of Notch1 by putative progenitor cells of the vertebrate CNS, we have addressed directly the role of Notch1 in the development of the mammalian brain. Using conditional gene ablation, we show that loss of Notch1 results in premature onset of neurogenesis by neuroepithelial cells of the midbrain-hindbrain region of the neural tube. Notch1-deficient cells do not complete differentiation but are eliminated by apoptosis, resulting in a reduced number of neurons in the adult cerebellum. We have also analyzed the effects of Notch1 ablation on gliogenesis in vivo. Our results show that Notch1 is required for both neuron and glia formation and modulates the onset of neurogenesis within the cerebellar neuroepithelium.

The role of Notch receptors in T helper differentiation

After encountering antigens, naïve CD4+ Τ cells can differentiate into various effector Τ helper (Th) cell subsets, including CD4+ Thi, Th2, Thi7, regulatory Τ cells and the recently described follicular Τ helper cells (TFH cells). To date, most of the studies used either gain-of-function approaches that do not reflect the physiological Notch signaling intensity or loss-of-function models that block the entire Notch pathway. The contribution of single Notch receptors during Th differentiation occurring upon infection has not been investigated yet. In the present thesis, we wanted to assess the individual role of Notchi and Notch2 in Th differentiation, by using mice with Τ cell-specific deletion of Notchi, Notch2 or both (NiN2/iCD4Cre) in different models of infection/immunization.¦In the first part, we characterized the role of Notchi and Notch2 in Thi differentiation. We used experimental infection with the protozoan parasite Leishmania major, known to induce a protective Thi immune response in mice on the C57BL/6 background. Mice deficient for both Notchi and Notch2 developed unhealing lesions and were unable to control the parasite burden in their footpad. A profound defect in IFNy secretion by CD4+ Τ cells was shown to be responsible for the susceptibility of these mice. Although CD4+ Τ cells did not secrete IFNy following L. major infection, they exhibited higher IFNymRNA expression as well as higher frequency of CD4+IFNy+Τ cells in dLN. Altogether, these data indicate that Notch is dispensable for the differentiation of Thi cells expressing IFNy but controls, directly or not, the secretion of IFNy, allowing the development of a fully functional Thi immune response.¦In the second part of this thesis, we determined whether Notch is involved in differentiation of follicular Τ helper (TFH) cells. Using different models of immunization (NP-CGG, Schistosoma mansoni eggs) or infection (Leishmania mexicana), we showed that NiN2ACD4Cre mice were unable to generate TFH cells, displayed impaired germinal center (GC) formation as well as a profound defect in high affinity specific-antibodies secretion. We demonstrated an essential and previously unknown role of Notch in TFH cell development, the consequent GC formation and high affinity antibodies secretion, although the mechanisms by which Notch affects TFH development remain to be clearly demonstrated.¦-¦Lors d'une réponse immune, les lymphocytes Τ CD4+ se différencient en différentes sous- populations de lymphocytes Τ auxiliaires (T helper ou Th en anglais) incluant les populations de cellules Thi, Th2, Thn.7, Τ régulatrices ou Τ folliculaires. De nombreuses études ont montré un rôle de la voie de signalisation Notch dans la différentiation des lymphocytes Τ auxiliaires, bien que les résultats soient controversés. A ce jour, la majorité de ces études sont basées sur des modèles de gain de fonction qui ne reflètent pas le niveau physiologique du signal ou des modèles de perte de fonction pour lesquels toute la voie de signalisation est bloquée. De ce fait, nous avons voulu établir le rôle individuel de Notchi et Notch2 dans la réponse immune de type Thi et dans la différentiation des lymphocytes Τ auxiliaires folliculaires avec l'aide de souris déficientes pour Notchi, Notch2 ou les 2 (NiN2ACD4Cre) à la surface de leurs cellules T.¦Dans la première partie de cette thèse, nous avons analysé le rôle de Notch dans la différentiation de type Thi suite à infection avec le parasite Leishmania major, connu pour induire une forte réponse Thi dans des souris de souche C57BL/6. Les souris déficientes pour Notchi et Notch2 développent une importante lésion et sont incapables de contrôler la prolifération du parasite au site d'infection. Le profond défaut de la sécrétion d'IFNy par les cellules Τ des ganglions drainants est probablement responsable de la susceptibilité de ces souris à L. major. Bien que les cellules Τ ne sécrètent pas d'IFNy, nous avons observé des niveaux plus importants d'expression au niveau de l'ARN messager, et une proportion plus élevée de cellules positives pour CD4 et IFNy. Ces résultats indiquent que Notch est nécessaire pour la sécrétion d'IFNy mais pas pour la différentiation de cellules compétentes pour l'IFNy.¦Dans un second temps, nous avons voulu déterminer si Notch est impliqué dans la différentiation des cellules Τ folliculaires. En utilisant divers modèles d'immunisation (avec NP-CGG ou des oeufs de Schistosoma mansoni) ou d'infection (avec L. mexicana), nous avons montré que les souris NlN2ACD4Cre sont incapables de générer des cellules Τ folliculaires. En conséquence, la formation des centres germinatifs et la sécrétion d'anticorps de haute affinité sont profondément affectés. Nous avons démontré dans cette seconde partie un rôle crucial et inconnu à ce jour de Notch dans la différentiation des cellules Τ et en conséquence dans la formation des centres germinatifs et la sécrétion des anticorps de haute affinité, bien que les mécanismes par lesquels Notch contrôle cette différentiation restent à identifier.¦-¦Lors d'une réponse immune, les lymphocytes Τ CD// se différencient en différentes sous- populations de lymphocytes Τ auxiliaires de types Thi, Th2, Thi7, régulatrices ou folliculaires, définies selon la sécrétion de cytokines spécifiques. Le rôle de ces sous-populations dans le contrôle de diverses infections ou leur association avec de nombreuses maladies rend la compréhension des mécanismes de différentiation de ces cellules particulièrement importante. De nombreux facteurs sont impliqués dans ce processus, tels que la présence de diverses cytokines dans l'environnement, la nature de l'antigène ou encore la force de la stimulation. Par ailleurs, de nombreuses études ont montré un rôle de la voie de signalisation Notch dans la différentiation des lymphocytes T, bien que les résultats soient controversés. Dans cette thèse, nous avons voulu évaluer le rôle individuel des récepteurs Notch dans la différentiation des cellules Τ auxiliaires de type Thi et folliculaires à l'aide de souris dont les récepteurs Notch sont spécifiquement absents à la surface des lymphocytes T.¦Dans la première partie, nous avons utilisé le modèle d'infection au parasite Leishmania major, connu pour induire une forte réponse protectrice de type Thi dans la majorité des souches de souris. Suite à l'infection, les souris déficientes pour les récepteurs Notch sont incapables de contrôler la prolifération du parasite et développent une importante lésion au site d'infection. Cette susceptibilité est due à l'incapacité des cellules Τ auxiliaires à sécréter une cytokine spécifique des cellules de type Thi et nécessaire à l'éradication du parasite, l'IFNy. Ces résultats indiquent que les récepteurs Notch sont indispensables au développement d'une réponse Thi fonctionnelle, permettant la guérison suite à l'infection avec L. major.¦Dans la deuxième partie de cette thèse, nous avons voulu déterminer si Notch est impliqué dans la différentiation des lymphocytes Τ folliculaires. Ces cellules ont la particularité d'aider les lymphocytes Β à former des centres germinatifs au sein desquels les lymphocytes Β prolifèrent et sécrètent des anticorps, un processus nécessaire à la protection contre les pathogènes. Actuellement, l'efficacité de la majorité des vaccins repose sur la sécrétion d'anticorps par les lymphocytes B, aidés par les cellules Τ folliculaires. En raison du rôle important de ces cellules dans l'éradication des pathogènes et lors d'un processus de vaccination, il est important de connaître les facteurs et les mécanismes permettant la différentiation de ces cellules. Dans cette étude, nous montrons que la formation des cellules Τ folliculaires dépend de la voie de signalisation Notch, impliquant un rôle essentiel de cette molécule dans l'induction de la sécrétion d'anticorps par les lymphocytes B.

Phylogenetics of Olea (Oleaceae) based on plastid and nuclear ribosomal DNA sequences: tertiary climatic shifts and lineage differentiation times.

BACKGROUND AND AIMS: The genus Olea (Oleaceae) includes approx. 40 taxa of evergreen shrubs and trees classified in three subgenera, Olea, Paniculatae and Tetrapilus, the first of which has two sections (Olea and Ligustroides). Olive trees (the O. europaea complex) have been the subject of intensive research, whereas little is known about the phylogenetic relationships among the other species. To clarify the biogeographical history of this group, a molecular analysis of Olea and related genera of Oleaceae is thus necessary. METHODS: A phylogeny was built of Olea and related genera based on sequences of the nuclear ribosomal internal transcribed spacer-1 and four plastid regions. Lineage divergence and the evolution of abaxial peltate scales, the latter character linked to drought adaptation, were dated using a Bayesian method. KEY RESULTS: Olea is polyphyletic, with O. ambrensis and subgenus Tetrapilus not sharing a most recent common ancestor with the main Olea clade. Partial incongruence between nuclear and plastid phylogenetic reconstructions suggests a reticulation process in the evolution of subgenus Olea. Estimates of divergence times for major groups of Olea during the Tertiary were obtained. CONCLUSIONS: This study indicates the necessity of revising current taxonomic boundaries in Olea. The results also suggest that main lines of evolution were promoted by major Tertiary climatic shifts: (1) the split between subgenera Olea and Paniculatae appears to have taken place at the Miocene-Oligocene boundary; (2) the separation of sections Ligustroides and Olea may have occurred during the Early Miocene following the Mi-1 glaciation; and (3) the diversification within these sections (and the origin of dense abaxial indumentum in section Olea) was concomitant with the aridification of Africa in the Late Miocene.