Matthew-Wood syndrome is a rare human birth defect condition defined by the phenotypic constellation of clinical anophthalmia, diaphragmatic hernia, pulmonary hypoplasia and cardiac defects. Matthew-Wood syndrome has a high mortality rate, with most patients dying due to respiratory insufficiency as a consequence of pulmonary hypoplasia, within the first year of life. Mutations within STRA6 are causative for Matthew-Wood syndrome. STRA6 acts as a retinol transporter for retinol bound to its physiological carrier RBP4 allowing regulated entry of retinol into the cell. A mammalian model for Matthew-Wood syndrome was not found within the literature; however a morpholino knockdown of stra6 in the zebrafish did show phenotypic features consistent with those observed in human patients. The desire to create a mammalian model of Matthew-Wood syndrome drove the work contained within this thesis. Stra6-/- mice do not represent a model for Matthew-Wood syndrome with homozygous animals being viable, found in the expected ratio and demonstrating none of the developmental abnormalities observed in human patients. Retinal defects, cataracts and persistent hyperplastic primary vitereous affect the microphthalmic eye of Stra6-/- offspring of Stra6-/- mothers fed a retinoid-free diet from plug to birth indicating that Stra6 is required for normal eye development under low-retinoid stress. The disparity in phenotype between human Matthew-Wood patients and Stra6-/- mice may be the result of functional redundancy in the mouse between Stra6 and its paralogue, Stra6.2. Stra6.2 is well conserved through evolution and is found in diverse species, including the basal eumetazoan Trichoplax. STRA6.2 has become split across its resident chromosome with an associated break in gene synteny, in humans and great apes, causing most of the gene to no longer be transcribed. However a small portion of the gene, representing the final transmembrane domain and the C-terminal intracellular tail of the protein, remains expressed in human. stra6.2 is required for normal development in the zebrafish with stra6.2 morphants being phenotypically distinguishable from control injected embryos from the 10-somite stage by a larger head-tail distance indicating an axial extension defect. stra6.2 morphants also display microphthalmia, jaw malformation, shortened and curved body axis and retinal lamination defects. stra6.2 was found to be required to prevent an excess of retinoic acid resulting in an upregulation of retinoic acid-dependent gene expression through an increase in RA synthesis by Raldh enzymes in morphants. Stra6.2-/- mice are viable and fertile and phenotypically normal, even under retinoid-stress, supporting the notion of functional redundancy. In compound knockouts, normal development and postnatal survival can be maintained by a single copy of Stra6 in Stra6+/-;Stra6.2-/- animals. Stra6.2 is less able to support normal development and survival with ~50% of Stra6-/-;Stra6.2+/- animals dying before weaning or showing reduced growth although the remaining animals are indistinguishable from their littermates. Stra6 and Stra6.2 are functionally redundant for development under normal dietary conditions in the mouse and a single copy of either is able to support development in at least 50% of animals. Stra6-/-;Stra6.2-/- mice were therefore hypothesised to be the logical mouse model of Matthew-Wood syndrome, however these mice die early in gestation between E7.5-E9.5. The early embryonic lethality in Stra6-/-;Stra6.2-/- mouse embryos compared to postnatal survival in human Matthew-Wood patients, to which they are the comparable genetic model, could be attributed to the shortened STRA6.2 remaining within the human genome. The equivalent portion of Stra6 has validated signalling motifs, which may still be active in STRA6.2, allowing development to proceed in human ‘STRA6-/-’ embryos.

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Les récepteurs tyrosine kinases sont un groupe de molécules clés de signalisation, qui ont 2 fonctions: la détection des stimuli de l'environnement extérieur des cellules et la transmission de ces signaux à l’intérieur des cellules. Dans les 20 dernières années, notre laboratoire a choisi d'étudier la fonction d’Ephb6 kinase, un récepteur tyrosine kinase fortement exprimé dans les lymphocytes T.Comme Efnb1 et Efnb2 sont tous des ligands pour Ephb6, nous avons ensuite procédé à étudier leur rôle dans la fonction des cellules T in vitro et in vivo. Des cellules T spécifiques mutants (KO) dans les gènes Efnb1 ou Efnb2 ainsi que les doubles mutants Efnb1/b2 (double KO) ont été générés, mais il n’y avait que les souris double KO qui ont démontré de la déficience dans le développement des thymocytes, fonction de Th1 et Th17, la signalisation du récepteur d’IL-6, et les réponses antivirales. Des preuves solides indiquent que la reconnaissance d’auto-antigène par les cellules T est un événement précoce dans la pathogenèse de la PR. Donc, nous avons postulé que les cellules T spécifique Efnb1 / b2 double KO chez la souris peuvent protéger les souris de l’arthrite induite par collagène (CIA), un modèle de souris de la PR humaine. Nous avons trouvé que Efnb1et Efnb2 dans les cellules T étaient essentielles pour la production d'anticorps pathogéniques et de la migration des lymphocytes T vers les pattes enflammées chez les souris CIA. Notre étude clinique suggère que l'expression de EFNB1 dans les cellules T pourrait être un paramètre utile pour surveiller l'activité de la maladie de RA et la réponse de traitement. Pour élucider les événements dans le programme d'activation des lymphocytes T, nous avons exploré par l'analyse des micropuces d'ADN pour identifier des molécules qui ont été exprimées de manière différente dans le WT par rapport aux cellules T Ephb6 KO dans le stade précoce de l’activation des cellules T. Environ 30 molécules étaient sur ou sous exprimées plus de 3 fois dans les cellules T WT par rapport aux cellules T KO pendant les 16 premières heures après stimulation par l'anti-CD3. Stra6 (stimulée par le gène de l'acide rétinoïque 6) et Armc5 (Armadillo répéter contenant 5) ont été parmi ceux qui ont été validées pour leur expression altérée. STRA6 est un récepteur de haute affinité pour le plasma rétinol-binding protéine (RBP) et un médiateur pour absorption cellulaire de vitamine A. Cellules T KO et WT étaient similaires en termes de prolifération et les réponses immunitaires anti-virales de virus de la chorioméningite lymphocytaire (LCMV). Ainsi, la sur-régulation de Stra6 est soit un événement parallèle qui ne soit pas essentiel pour le programme d'activation des lymphocytes T, ou il est très essentiel que la redondance existe, et sa suppression ne montre aucun effet apparent sur l'activation des cellules T. ARMC5 est une protéine intracellulaire contenant sept répétitions en tandem d’armadillo et un domaine BTB. Les fonctions du ARMC5 dans le système immunitaire ne sont pas encore connues. Nos résultats d'hybridation in situ ont montré une expression élevée de Armc5 dans le thymus, et une expression modérée dans les ganglions lymphatiques et la rate. Nous avons généré des souris KO Armc5. Fait interessant, les cellules T Armc5 KO présentaient de la prolifération diminuée et de la différenciation compromise vers Th1 et Th17 in vitro. Les souris KO étaient résistantes à l'induction expérimentale d’encéphalite auto-immune, et ont été compromises dans les réponses immunitaires anti-LCMV. En utilisant de la levure 2-hybride test, nous avons identifié 8 protéines ARMC5-associantes, qui sont connues pour les rôles dans l'activation de la cellule, le cycle cellulaire et l'apoptose. Une étude mécanique est en cours. Nos résultats montrent que Armc5 est essentiel dans la programme d'activation/de prolifération/de différenciation des lymphocytes T. Nos études ont augmenté nos connaissances sur EFNB1, EFNB2, STRA6 et ARMC5 en biologie des lymphocytes T et leur pertinence à des troubles immunitaires dans des modèles animaux ainsi que chez l'être humaine. / Receptor tyrosine kinases are a group of key signaling molecules, which have dual functions: sensing the environmental stimuli outside the cells and transmitting them into the cells. 20 years ago, our laboratory started to study the function of Ephb6 kinase, a receptor tyrosine kinase highly expressed in T lymphocytes. As both Efnb1 and Efnb2 are the ligands for Ephb6, we then proceeded to study their roles in T cell function in vitro and in vivo. T cellspecific Efnb1, Efnb2 single gene knockout (KO), as well as Efnb1/b2 double KO mice were generated, but only the double KO mice showed compromised thymocyte development, Th1 and Th17 function, IL-6 receptor signaling, and anti-virus responses. Strong evidence indicates that T cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Thus, we postulated that T cell-specific Efnb1/b2 double KO in mice may protect mice from collagen-induced arthritis (CIA), a mouse model for human RA. We found that Efnb1 and Efnb2 in T cells were essential for pathogenic antibody production and T cell migration to the inflamed paws in mice with CIA. Our clinical study suggests that the expression of EFNB1 in T cells might be a useful parameter for monitoring RA disease activity and treatment responses. Naïve T cells have the ability to expansion and differentiation into effector cells once they encounter foreign antigens, during which a large number of molecules are modulated. Some of these molecules play essential regulatory roles, while others exert house keeping functions and/or act as supporters to cope with increased or changed metabolic demands. To fully elucidate events in the T cell activation program, we undertook unbiased exploration with DNA microarray analysis to identify molecules that were differentially expressed in WT versus Ephb6 KO T cells in the early T-cell activation stage. About 30 molecules were up- or down-regulated more than three folds in WT T cells compared with KO T cells. Stra6 (stimulated by retinoic acid gene 6) and Armc5 (Armadillo repeat-containing 5) were among those that had been validated for their altered expression. We generated mice with these two genes deleted to study their roles in T cell function in vitro and in vivo. STRA6 is a high-affinity receptor for plasma retinol-binding protein (RBP) and mediates cellular vitamin A uptake. Stra6 KO mice manifest normal spleen and thymus in size, cellularity and lymphocyte subpopulations. KO and WT T cells were similar regarding proliferation, differentiation and anti-viral immune responses to lymphocytic choriomeningitis virus (LCMV). Thus, the up-regulation of Stra6 is either a parallel event which is not essential for the T cell activation program or it is so critical that heavy redundancy exists. ARMC5 is an intracellular protein containing seven tandem armadillo repeats and one BTB domain. Functions of ARMC5 in the immune system are not known previously. Our in situ hybridization results showed high expression of Armc5 in the thymus and moderate expression in the spleen and lymph nodes. A transient increase of Armc5 expression in T cells after TCR activation was found. To investigate its roles in T cell function, Armc5 KO mice were generated. The KO mice weighed 40% less than their WT counterparts. Lymphoid organs (the thymus, spleen and lymph nodes) of the KO mice appeared to be of normal size, weight, cellularity, and lymphocyte subpopulations. Intriguingly, Armc5 KO T cells presented decreased proliferation and compromised differentiation towards Th1 and Th17 in vitro. The KO mice were resistant to experimental autoimmune encephalitis induction and were compromised in anti-LCMV immune responses. Using yeast 2-hybrid assay, we have identified 8 ARMC5-assciating proteins, which have known functions in cell cycling and apoptosis. Further mechanistic study is underway. Our results reveal that Armc5 is vital in the T cell activation/proliferation /differentiation program. Our studies have augmented our knowledge about EFNB1, EFNB2, STRA6 and ARMC5 in T cell biology and their relevance to immune disorders in animal models as well as in humans.