ZEBRAFISH AS MODEL SYSTEM TO STUDY THE ROLE OF THE GLIS3 TRANSCRIPTION FACTOR IN THE PATHOGENESIS OF CONGENITAL HYPOTHYROIDISM

Congenital Hypothyroidism (CH) is the most common congenital endocrine disease that can be broadly classified as failure of the gland to develop normally (dysgenesis) or inadequate thyroid hormone (TH) production from eutopic thyroid gland (dyshormonogenesis). Biochemically, CH patients are characterized by low circulating levels of T4 and T3 associated with an increased production of TSH due to the feedback mechanisms controlling the hypothalamic-pituitary-thyroid (HPT) axis. CH is considered a disease with a strong genetic component, but with a largely missing explanation for its heritability. At present, genetic variants in candidate genes explain only the 10% of CH cases.\u2028The extraordinary progresses in high-throughput screening technologies lead to the identification of several genes involved in the thyroid development and TH synthesis. Recently, the transcription factor GLI-Similar protein 3 (GLIS3) has emerged as a new candidate gene for CH, but its role in thyroid development and function remains largely unexplored. GLIS3 is a member of the Kruppel-like zinc-finger transcription factors that can acts as activator or repressor of gene expression. Homozygous and compound heterozygous GLIS3 variants have been associated with NDH syndrome, characterized by neonatal diabetes (T1D and T2D), CH and polycystic kidney. Additionally, several missense heterozygous GLIS3 variants have been also identified in cohorts of patients with isolated CH. Interestingly, heterozygous GLIS3 variants are associated with other mutations in genes involved in thyroid functioning, supporting the hypothesis of the oligogenic CH origin. In both NDH and isolated CH patients, the thyroid disease is extremely heterogeneous. In fact, most of the affected cases present variable thyroid dysgenesis (athyreosis, thyroid hypoplasia and ectopy), whereas dyshormonogenesis with eutopic in situ gland is also reported. Evidences from Glis3 knockout mice indicate a relevant role of Glis3 in TH biosynthesis and postnatal follicle proliferation, with a mechanism of action downstream of the TSH/TSHR signalling and thyroid cell proliferation. However, no significant thyroid developmental defects were observed in this particular mice model. Since GLIS3 mutations are variably associated with thyroid dysgenesis, the aim of this study is to gain insight on GLIS3 activity during the early steps of thyroid development, using zebrafish as a model system. In zebrafish (zf), we observed the expression of glis3 transcript from the early developmental stages onwards, with a particularly evident signal at 1 dpf (day post fertilization) in the pharyngeal endoderm, the embryonic tissue that will give rise to endocrine organs, like thyroid and pancreas, and in the pronephric ducts. In apparent contrast with mouse data, glis3 is absent in the differentiated thyrocytes of zebrafish embryos at 2-3 dpf. Transient knockdown zebrafish embryos (called glis3_MOs), obtained by the microinjection of specific glis3 morpholinos, revealed a reduced expression of the early thyroid markers nkx2.4, and pax2a, at 1dpf. The defective specification of the thyroid primordium at this early developmental stage was not associated with reduced proliferation or increased apoptosis of thyroid precursors, thus indicating that this phenotype was likely due to alterations in the commitment of endodermal cells toward the thyroid fate. Such defect resulted in a reduction in size of the differentiated thyroid precursors with a diminished expression of both tg and slc5a5, and later on in the number of functional thyroid follicles. At 5dpf, the stage in which the thyroid gland is functional and responds to the HPT-axis, decreased levels of T4 associated with tshba elevation were also seen in glis3_MOs. The specificity of the thyroid defects was confirmed by the rescue phenotype after co-injection of the wild-type zf-glis3 transcript (WT mRNA) and the glis3 morpholino. In contrast with morpholino knock-down, the overexpression of the glis3 mRNA leads to an increase of the number of the thyroid precursors at 1dpf, thus leading to the differentiation of a larger thyroid tissue and an elevation of T4 levels at 5 dpf. Taken together, our results demonstrate that glis3 acts at the endodermal level controlling the commitment of endocrine precursors, potentially representing a required factor for the early specification of thyroid primordium. Given the current knowledge, GLIS3 is reported to interact with a pivotal element within the Sonic hedgehog (Shh) pathway. Previous experiments in the mouse model indicated a potential role for such pathway in thyroid development, but the underlying mechanisms and the stage of Shh action in this context are still elusive. Our experiments revealed that the expression of shha (the zf homologous of human Shh) was significantly reduced in the pharyngeal endoderm of glis3_MOs. Consistently, the injection of a morpholino against shha abolished the expression of glis3 in the endodermal layer, as well as the treatment with Cyclopamine (a Shh-antagonist) caused a reduced or absent expression of glis3, thus confirming the co-involvement of shha and glis3 during the thyroid cell specification. Interestingly enough, the overexpression of glis3 mRNA failed to rescue the thyroid defects in the Cyclopamine-treated embryos, suggesting that glis3 would act as downstream effector of the Shh pathway. In conclusion, this is the first evidence of the possible interaction between Shh and Glis3 during the early specification of thyroid primordium. Since the mechanisms involved in such a delicate event are largely unknown, our data provide important insights as glis3 appears as an endoderm factor required for the commitment of endoderm cells toward the thyroid fate. These data shed new light into the molecular mechanisms potentially involved in CH pathogenesis

GLIS3 and Thyroid: A Pleiotropic Candidate Gene for Congenital Hypothyroidism

Variations in the transcription factor Gli-similar 3 (GLIS3) gene have been associated to variable congenital endocrine defects, including both morphogenetic and functional thyroid alterations. Evidence from Glis3 knockout mice indicates a relevant role for GLIS3 in thyroid hormone biosynthesis and postnatal thyroid gland growth, with a mechanism of action downstream of the TSH/TSHR interaction. However, the pathophysiological role of this transcription factor during the embryonic thyroid development remains unexplored. In this manuscript, we will provide an overview of the current knowledge on GLIS3 function during development. As a perspective, we will present preliminary evidence in the zebrafish model in support of a potential role for this pleiotropic transcription factor in the early stages of thyroid gland development

Effects of Arousal, Dominance and Their Interaction on Pleasure in a Cultural Environment

Being typically hedonic, arts and culture by definition provide pleasurable experiences that are consumed for their own sake. If pleasure acts as the key emotion in these experiences, at the same time other emotions might be elicited by the rich and holistic environment and interaction that takes place between the cultural product and the consumer. By adopting the common theoretical framework of the PAD model (Mehrabian & Russell, 1974), but innovating in the structural links among the three main affective dimensions—pleasure, arousal, and dominance—this study empirically tests whether arousal, dominance, and their interaction generate pleasure. Findings show that arousal and dominance affect pleasure positively and significantly, whereas their interaction affects it negatively. Pleasure leads to a direct and positive effect on satisfaction that mediates a positive effect on behavioral intention

Les differences de productivite des exploitations laitieres de plaine et de montagne : premiers resultats

*INRA Station d'Economie et Sociologie rurales INA Paris-Grignon 78850 THIVERVAL GRIGNON (FRA) Diffusion du document : INRA Station d'Economie et Sociologie rurales INA Paris-Grignon 78850 THIVERVAL GRIGNON (FRA)National audienceCette communication compare les resultats technico-economiques des exploitations laitieres specialisees de plaine et de montagne sur la base du RICA 1985. L'analyse porte sur : -la dimension des exploitations et les caracteristiques des systemes ; -les performances economiques comparees des moyens de production ; -les niveaux de capitaux, de revenu, d'endettement, de subventions. La comparaison des resultats moyens montre que a superficie et travail equivalents, l'exploitation laitiere de montagne obtient un niveau de productivite du travail inferieur de pres de moitie a celui de l'exploitation de plaine. D'importantes economies realisees sur les charges et des subventions plus elevees permettent de reduire les differences de RBE a 30 %. Au niveau regional la Franche-Comte obtient des resultats superieurs a ceux de la plaine alors qu'ils sont inferieurs de pres de moitie en Auvergne. La comparaison par classe de production laitiere montre que les systemes montagnards plus extensifs permettent d'obtenir de meilleurs revenus dans les exploitations inferieures a 100000 l

La politique de la montagne en Suisse : enseignements et limites pour la France

*INRA Station d'Economie et Sociologie rurales INA Paris-Grignon 78850 THIVERVAL GRIGNON (FRA) Diffusion du document : INRA Station d'Economie et Sociologie rurales INA Paris-Grignon 78850 THIVERVAL GRIGNON (FRA)National audienceLa politique agricole suisse fait partie integrante de la politique d'independance nationale et de neutralite de la Confederation. Elle a notamment pour objectif d'assurer l'autosuffisance alimentaire en cas de crise grave (l'auto-approvisionnement atteint 64 % en 1983), la protection et l'entretien du territoire, un peuplement suffisant des regions marginales. Sans une politique specifique des zones de montagne, qui couvrent 70 % du territoire et rassemblent 46 000 exploitations d'une dizaine d'hectares en moyenne, ces objectifs en matiere de production agricole et d'amenagement de l'espace ne pourraient etre atteints. Definie a partir de 1951, la politique de la montagne cherche a favoriser le maintien et le developpement des exploitations familiales et a garantir leur revenu. Elle repose sur le renforcement des aides aux investissements (credits a taux nul), des prix agricoles eleves mais non differencies (superieurs de 50 a 100 % aux prix CEE), des paiements directs a l'UGB (qui atteignent 2000 FF par vache) et a l'hectare en pente exploite. Contrairement au systeme europeen, c'est le consommateur suisse qui paie la plus large part du soutien des marches par le biais de prix alimentaires eleves. Malgre ces efforts, on constate que l'agriculture de montagne ne percoit pas une part plus importante du budget federal que l'agriculture de plaine en 1983 (37 % du budget pour 37 % des exploitations). Comme en France, c'est donc la politique des prix et des marches qui prime dans la formation du revenu agricole suisse. Mais la forte croissance des prix agricoles et l'augmentation des transferts, a cependant permis une amelioration du revenu agricole montagnard de 28 % en valeur reelle depuis 10 ans, alors qu'il tendait a stagner en France. D'autre part, de nombreuses mesures sectorielles prises en Suisse pour favoriser l'exploitation des terres marginales, pourraient etre adaptees en France afin d

Avantages compares des principales cultures dans les systemes de production agricole europeens (1979-1988)

*INRA Station d'Economie et Sociologie rurales INA Paris-Grignon 78850 THIVERVAL GRIGNON (FRA) Diffusion du document : INRA Station d'Economie et Sociologie rurales INA Paris-Grignon 78850 THIVERVAL GRIGNON (FRA)National audienceCette etude est une partie de celle, plus vaste, entreprise a la demande de la CEE sur les couts de production des principaux produits agricoles. Elle repose sur l'idee que la prise en consideration des marges des cultures a l'hectare permet de mieux comprendre les substitutions, orientations et specialisations en cours dans le secteur de grande culture europeen, brievement retracees a l'aide d'un bilan de l'evolution des superficies des principales cultures en France, Allemagne, Italie et Royaume-Uni. C'est pourquoi des marges pour diverses activites ble tendre, ble dur, orge, mais, oleagineux, betterave sucriere et pomme de terre ont ete estimees par voie econometrique pour chacun de ces pays a l'aide d'un modele base sur une regression lineaire multiple et sont comparees systematiquement entre elles : on s'interroge sur la mesure selon laquelle, au-dela des prix a la production et des rendements physiques, elles contribuent a une explication du comportement des agriculteurs en matiere de choix de leurs systemes de production. Outre des marges calculees a partir des observations du RICA europeen pour les annees 1979 a 1985, une extrapolation fournit les donnees pour la periode recente 1986 a 1988. Une simulation permet enfin de definir a quelles conditions, rendements-seuils et prix-seuils, le ble tendre, les oleagineux et le mais sont aujourd'hui substituables. L'aspect regional des comparaisons des avantages entre cultures n'est aborde qu'assez succinctement au niveau francais. Mais divers developpements de ces travaux sont en cours

In vivo Functional Consequences of Human THRA Variants Expressed in the Zebrafish

Background: Heterozygous mutations in the thyroid hormone receptor alpha (THRA) gene cause resistance to thyroid hormone alpha (RTH\u3b1), a disease characterized by variable manifestations reminiscent of untreated congenital hypothyroidism but a raised triiodothyronine/thyroxine ratio and normal thyrotropin levels. It was recently described that zebrafish embryos expressing a dominant negative (DN) form of thraa recapitulate the key features of RTH\u3b1, and that zebrafish and human receptors are functionally interchangeable. Methods: This study expressed several human thyroid hormone receptor alpha (hTR\u3b1) variants in zebrafish embryos and analyzed the resulting phenotypes. Results: All hTR\u3b1-injected embryos showed variable defects, including cerebral and cardiac edema likely caused by an aberrant looping during heart development, anemia, and an incomplete formation of the vascular network. Moreover, the hTR\u3b1-injected embryos presented severe defects of motorneurons and craniofacial development, thus affecting their autonomous feeding and swimming behaviors. Surprisingly, expression of all hTR\u3b1 mutants had no detectable effect on thyrotropin beta and thyrotropin-releasing hormone transcripts, indicating that their DN action is limited on the thyroid hormone reception beta 2 targets at the hypothalamic/pituitary level in vivo. As previously described in vitro, treatment with high triiodothyronine doses can efficiently revert the observed defects only in embryos injected with missense hTR\u3b1 variants. Conclusion: Injection of human THRA variants in zebrafish embryos causes tissue-specific defects recapitulating most of the RTH\u3b1 clinical and biochemical manifestations. The described manipulation of zebrafish embryos represents a novel in vivo model to screen the functional consequences of THRA variants and the rescue potential of new therapeutic compounds

L'étude de l'organisation du travail dans les systèmes d'activités complexes. Points de vue de l'intérêt et les limites de la méthode Bilan Travail

National audienc

Genetics and management of congenital hypothyroidism

Several evidences support a relevant genetic origin for Congenital Hypothyroidism (CH), however familial forms are uncommon. CH can be due to morphogenetic or functional defects and several genes have been originally associated either with thyroid dysgenesis or dyshormonogenesis, with a highly variable expressivity and a frequently incomplete penetrance of the genetic defects. The phenotype-driven genetic analyses rarely yielded positive results in more than 10% of cases, thus raising doubts on the genetic origin of CH. However, more recent unsupervised approaches with systematic Next Generation Sequencing (NGS) analysis revealed the existence of hypomorphic alleles of these candidate genes whose combination can explain a significant portion of CH cases. The co-segregation studies of the hypothyroid phenotype with multiple gene variants in pedigrees confirmed the potential oligogenic origin of CH, which finally represents a suitable explanation for the frequent sporadic occurrence of this disease