Long chain fatty acids (LCFA) constitute a particular class of lipids whose developmental roles are coming under increasing scrutiny. These LCFAs not only have important cell specific functions but their dysregulation also forms the underlying cause of several lipid based disorders. Fatty acids are elongated in vivo by multi-enzymatic machinery in a stepwise manner. The first step of this process is carried out by a family of enzymes, conserved from yeast to humans, known as Elongases (Elos). It has been suggested that Elos work independently to elongate a fatty acid to a distinct length and perform distinct functions. Work in different species has implicated Elos in different biological roles such as barrier function, fertility, behavior and lipid homeostasis, but our understanding of the precise roles of these elongases in fatty acid elongation and how it relates to their physiological roles is not very well understood. To better understand the functions of Elos, I have characterized all Drosophila elongases for their developmental roles. In this thesis, I have shown that Drosophila elongases that are more conserved across species have more important functions (eg viability) as compared to the less conserved elongases. More specifically, I have discovered an elongases, sit still (sits), disruption of which in the nervous system results in striking locomotor and behavior defects, as well as decreased lifespan. I have also characterized another elongase, baldspot, which is important for imparting the epidermal barrier function in Drosophila, a conserved function of elongases across species. By elucidating the in vivo functions of these two Drosophila elongases, this work provides insight into the developmental roles of Elos and their links to diseases such as psoriasis, icthyosis, macular degeneration, Adrenoleukodystrophy (ALD) and Multiple Sclerosis (MS).

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The mammalian skin provides a necessary barrier between the organism and the environment, defending against loss of water and solutes, preventing the invasion of pathogens as well as protecting against chemical and physical assault. Claudin (Cldn)-based Tight Junctions (TJs) are the main functional part of the skin barrier. In particular, Cldn6 through its cytoplasmic tail has been shown to be important for barrier function. In other to further investigate the role of the Cldn6 tail in TJ-function, we developed Cldn6 mouse mutants carrying varying truncations of the Cldn6 tail. Both of these mice present with epidermal differentiation perturbations and delayed barrier function that is repaired later in life. These studies support the importance of the tail portion of the Cldn molecules in epidermal differentiation and barrier function. In addition, both of these mouse models are useful for the study of barrier function in preterm infants and in aging, with the hope of developing novel therapeutics for the alleviation of barrier dysfunction. Cdx is a family of homeodomain (HD) transcription factors (TFs) essential for many key developmental processes. In particular, Cdx2 is important for the establishment and maintenance of posterior identity in the developing endoderm. In spite of this, only a few Cdx targets in the developing endoderm have been discovered. In addition, the interplay between Cdx and its targets within the endoderm is poorly understood. In this study, we show that the forkhead box transcription factor, Foxa2 is a Cdx2 target. We also show that Foxa2 and Cdx2 physically and genetically interact to regulate a subset of genes that are implicated in endodermal development. These studies help to further our understanding of endoderm biology with the goal of developing new strategies to diagnose and treat diseases associated with defective endoderm development.

Epigenetic regulatory mechanisms play important roles in the control of lineage-specific differentiation during development. However, mechanisms that regulate higher-order chromatin remodelling and transcription of keratinocyte-specific genes that are clustered in the genome into three distinct loci (Keratin type I/II loci and Epidermal Differentiation Complex (EDC)) during differentiation of the epidermis are poorly understood. By using 3D-Fluorescent In Situ Hybridization (FISH), we determined that in the epidermal keratinocytes, the KtyII and EDC loci are located closely to each other in the nuclear compartment enriched by the nuclear speckles. However, in KtyII locus knockout mice, EDC locus moved away from the KtyII locus flanking regions and nuclear speckles towards the nuclear periphery, which is associated with marked changes in gene expression described previously. Chromatin architectural protein Ctcf has previously been implicated in the control of long-range enhancer-promoter contacts and inter-chromosomal interactions. Ctcf is broadly expressed in the skin including epidermal keratinocytes and hair follicles. Conditional Keratin 14-driven Ctcf ablation in mice results in the increase of the epidermal thickness, proliferation, alterations of the epidermal barrier and the development of epidermal pro-inflammatory response. Epidermal barrier defects in Krt14CreER/Ctcf fl/fl mice are associated with marked changes in gene expression in the EDC and KtyII loci, which become topologically segregated in the nucleus upon Ctcf ablation. Therefore, these data suggest that Ctcf serves as critical determinant regulating higher-order chromatin organization in lineage-specific gene loci in epidermal keratinocytes, which is required for the proper control of gene expression, maintenance of the epidermal barrier and its function.

La barrière épidermique du stratum corneum (SC) est doublée par une barrière secondaire des jonctions serrées (JS) qui influent sur la formation de barrière principale. Dans mes travaux, je me suis concentré sur l'étude de la présence et l'évolution des éléments jonctionnelles composants ces deux barrières ; les cornéodesmosomes au niveau du SC et les JSs au niveau de la granuleuse. En plus, je me suis intéressé à l'implication des glycanes dans la fonction barrière épidermique. Ces travaux ont été réalisés soit dans un contexte physiologique soit par la modulation de la barrière épidermique par des facteurs intrinsèques et extrinsèques. Nos résultats confirment que les JSs jouent un rôle subalterne par rapport à la barrière du SC et montrent que les glycanes persistent à la surface des cornéocytes humains. La composition et la répartition utlrastructurale des glycanes évoluent à travers les assises du SC jusqu'à la desquamation d'une manière concordante avec la répartition des cornéodesmosomes. Certaines modifications intrinsèques naturelles lors du vieillissement ou pathologiques notamment l'état pelliculaire et la dermatite atopique, ont permis d'appréhender le rôle de ces composants dans la cohésion du SC et la prestance d'une barrière fonctionnelle. Les modifications extrinsèques de la barrière par l'application de solvants, d'excipients ou de formulations perméabilisantes montrent l'importance de l'organisation utlrastructurale des composants jonctionnelles et non jonctionnelles du SC dans le maintien d'une barrière efficace / The stratum corneum (SC) barrier is doubled by the secondary barrier of tight junctions which influences the formation of the main barrier. In my work, I focused on the study of the junctional elements composing those two barriers; corneodesmosomes in the SC and the tight junction at the granular layer level. In addition, I got interested in the involvement of glycans in the epidermal barrier function. This work was carried out either in skin physiological conditions or by the modulation of the epidermal barrier by intrinsic or extrinsic factors. Our results confirm that tight junctions play a subordinate role compared to the SC barrier and that glycans remain present at the surface of human corneocytes. The composition and the ultrastructure distribution of glycans evolve from the SC compactum to the SC disjunctum, towards desquamation in a comparable manner to the repartition of corneodesmosomes. Natural intrinsic changes during aging and pathological changes, including dandruff and atopic dermatitis, helped us to understand the role of those components in the cohesion of the SC and the conservation of functional barrier. Extrinsic modulation of the barrier by the application of solvents, excipients or topical formulations shows the importance of the ultrastructural organization of junctional and non-junctional SC components in maintaining an effective barrier

Epigenetic regulatory mechanisms play important roles in the control of lineage-specific differentiation during development. However, mechanisms that regulate higher-order chromatin remodelling and transcription of keratinocyte-specific genes that are clustered in the genome into three distinct loci (Keratin type I/II loci and Epidermal Differentiation Complex (EDC) during differentiation of the epidermis are poorly understood. By using 3D-Fluorescent In Situ Hybridization (FISH), we determined that in the epidermal keratinocytes, the KtyII and EDC loci are located closely to each other in the nuclear compartment enriched by the nuclear speckles. However, in KtyII locus knockout mice, EDC locus moved away from the KtyII locus flanking regions and nuclear speckles towards the nuclear periphery, which is associated with marked changes in gene expression described previously. Chromatin architectural protein Ctcf has previously been implicated in the control of long-range enhancer-promoter contacts and inter-chromosomal interactions. Ctcf is broadly expressed in the skin including epidermal keratinocytes and hair follicles. Conditional Keratin 14-driven Ctcf ablation in mice results in the increase of the epidermal thickness, proliferation, alterations of the epidermal barrier and the development of epidermal pro-inflammatory response. Epidermal barrier defects in Krt14CreER/Ctcf fl/fl mice are associated with marked changes in gene expression in the EDC and KtyII loci, which become topologically segregated in the nucleus upon Ctcf ablation. Therefore, these data suggest that Ctcf serves as critical determinant regulating higher-order chromatin organization in lineage-specific gene loci in epidermal keratinocytes, which is required for the proper control of gene expression, maintenance of the epidermal barrier and its function.

Yes / Background and Objective Visible light has beneficial effects on cutaneous wound healing, but the role of potential photoreceptors in human skin is unknown. In addition, inconsistency in the parameters of blue and red light‐based therapies for skin conditions makes interpretation difficult. Red light can activate cytochrome c oxidase and has been proposed as a wound healing therapy. UV‐blue light can activate Opsin 1‐SW, Opsin 2, Opsin 3, Opsin 4, and Opsin 5 receptors, triggering biological responses, but their role in human skin physiology is unclear. Materials and Methods Localization of Opsins was analyzed in situ in human skin derived from face and abdomen by immunohistochemistry. An ex vivo human skin wound healing model was established and expression of Opsins confirmed by immunohistochemistry. The rate of wound closure was quantitated after irradiation with blue and red light and mRNA was extracted from the regenerating epithelial tongue by laser micro‐dissection to detect changes in Opsin 3 (OPN3) expression. Retention of the expression of Opsins in primary cultures of human epidermal keratinocytes and dermal fibroblasts was confirmed by qRT‐PCR and immunocytochemistry. Modulation of metabolic activity by visible light was studied. Furthermore, migration in a scratch‐wound assay, DNA synthesis and differentiation of epidermal keratinocytes was established following irradiation with blue light. A role for OPN3 in keratinocytes was investigated by gene silencing. Results Opsin receptors (OPN1‐SW, 3 and 5) were similarly localized in the epidermis of human facial and abdominal skin in situ. Corresponding expression was confirmed in the regenerating epithelial tongue of ex vivo wounds after 2 days in culture, and irradiation with blue light stimulated wound closure, with a corresponding increase in OPN3 expression. Expression of Opsins was retained in primary cultures of epidermal keratinocytes and dermal fibroblasts. Both blue and red light stimulated the metabolic activity of cultured keratinocytes. Low levels of blue light reduced DNA synthesis and stimulated differentiation of keratinocytes. While low levels of blue light did not alter keratinocyte migration in a scratch wound assay, higher levels inhibited migration. Gene silencing of OPN3 in keratinocytes was effective (87% reduction). The rate of DNA synthesis in OPN3 knockdown keratinocytes did not change following irradiation with blue light, however, the level of differentiation was decreased. Conclusions Opsins are expressed in the epidermis and dermis of human skin and in the newly regenerating epidermis following wounding. An increase in OPN3 expression in the epithelial tongue may be a potential mechanism for the stimulation of wound closure by blue light. Since keratinocytes and fibroblasts retain their expression of Opsins in culture, they provide a good model to investigate the mechanism of blue light in wound healing responses. Knockdown of OPN3 led to a reduction in early differentiation of keratinocytes following irradiation with blue light, suggesting OPN3 is required for restoration of the barrier function. Understanding the function and relationship of different photoreceptors and their response to specific light parameters will lead to the development of reliable light‐based therapies for cutaneous wound healing. / European Commission 7th Framework Programme for Research and Technical Development - Marie Curie Innovative Training Networks (ITN), Grant agreement no.: 607886

Skin is the largest organ in the body that protects the organism from environmental, chemical and physical traumas of each passing day. The protective skin epidermal permeability barrier (EPB) is formed within the exterior layers of the epidermis, which are regularly sloughed off and repopulated by movement of inner cells. The epidermal permeability barrier is established during in utero development and maintained through lifetime. Impaired epidermal barrier formation is one of the major features of several dermatoses such as psoriasis and atopic dermatitis. Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (Ctip2), also known as Bcl11b, is a C���H��� zinc finger protein expressed in many organs and tissues. It has been shown to regulate the development of thymocyte, tooth and corticospinal motor neurons. Ctip2 is highly expressed in mouse epidermis during skin organogenesis and in adulthood. It is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Germline (Ctip2- null mice) and selective ablation of Ctip2 in mouse epidermis (Ctip2[superscript ep-/-] mice) leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation and terminal differentiation as well as altered lipid distribution during embryogenesis. Sphingolipids account for ~50% of total skin lipids by weight and are crucial components of epidermal barrier. We have recently identified Ctip2 as a key regulator of skin lipid metabolism. Germline deletion of Ctip2 in mouse embryos leads to altered lipid composition in the developing mouse epidermis by modulating the expression levels of key enzymes involved in lipid metabolism (bio-synthesis and catabolism). We also demonstrated that Ctip2 is recruited to the promoter regions of several genes involved in the ceramide and sphingomyelin biosynthesis pathways and could directly regulate their expression. Thus, we have identified Ctip2 as a key regulator of several lipid metabolizing genes and hence epidermal sphingolipid biosynthesis during skin development. To study the role of Ctip2 in adult skin homeostasis, we have utilized Ctip2[superscript ep-/-] mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. We showed that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as high infiltration of T lymphocytes and immune cells. We have also observed increased expression of Th2-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression is significantly upregulated in the mutant epidermis as early as postnatal day 1 and Ctip2 was recruited to the promoter region of the TSLP gene in mouse epidermal keratinocytes. The results suggest that upregulation of TSLP expression in the Ctip2[superscript ep-/-] epidermis could be due to a derepression of gene transcription in absence of Ctip2. Thus, our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult skin, as well as a non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines in adult mouse skin. Present results establish an initiating role of epidermal TSLP in AD pathogenesis via a novel repressive regulatory mechanism mediated by Ctip2 in mouse epidermal keratinocytes. Altogether, our study indicates that Ctip2 could be involved in a diverse range of biological events in skin including barrier formation, maintenance and epidermal homeostasis. Ctip2 appears to be a master regulator in skin barrier functions by directly regulating the transcription of a subset of genes involved in lipid metabolism and inflammatory responses. / Graduation date: 2013

Le mécanisme qui sous-tend la réponse inflammatoire en cas de défaut de la barrière épidermique reste à élucider. Dans cette étude, nous montrons qu’en cas de rupture de la barrière formée par le stractum corneum épidermal, une réponse inflammatoire mixte de type 17 et 2 est induite. Nous décrivons ici une régulation réciproque entre les axes cytokiniques IL-23/IL-17/IL-22 et TSLP/IL-4 qui conditionne l’apparition du phénotype inflammatoire au niveau cutané. Par ailleurs, nous démontrons également que la flore bactérienne présente à la surface de la peau est engagée dans l’induction de l’IL-23 et de la réponse de type 17 alors que le PAR2 stimule, quant à lui, l’expression de TSLP et à la réponse de type 2. Nos résultats montrent donc la complexité et l’hétérogénéité des réponses inflammatoires en conditions de rupture de la barrière cutanée et ont des implications au niveau des thérapies pour les maladies inflammatoires de la peau. / Dysfunction of the epidermal barrier has been recognized as a critical factor in the development of skin inflammation; yet, the mechanism underlying the inflammatory responses triggered by epidermal defects remains still elusive. Here, by employing mice with corneodesmosin (CDSN) gene ablated in keratinocytes, we show that upon the breakdown of the epidermal barrier, type 17 and type 2 inflammatory responses are co-induced in the skin. Furthermore, we delineate a counter-regulation between IL-23/IL-17/IL-22 and TSLP/IL-4 cytokine axes, which shapes the outcome of the inflammatory phenotype in skin. Moreover, we show that the bacteria skin flora are engaged in the induction of IL-23 and the type 17 response, whereas the protease activation receptor PAR2 mediates TSLP expression and the type 2 response. Our results shed light on the complexity and heterogeneity of inflammatory responses in barrier-defective skin, and have implications for treating skin inflammatory diseases.