Genomic imprinting provides an exception to the Mendelian rule of inheritance, as imprinted genes are preferentially expressed in a parent-of-origin specific manner. They play important roles in the development of embryonic and extra-embryonic lineages and postnatally in the maintenance of correct metabolic homeostasis as well as regulation of adult behaviour. The parental conflict theory predicts that maternally expressed genes act as growth suppressors, limiting the usage of maternal resources, and that paternally expressed genes function in an opposite manner to promote growth at the expense of maternal resources. Growth factor bound protein 10 (Grb10) is an imprinted gene encoding an intracellular adaptor protein that can interact with several receptor tyrosine kinases and downstream signalling molecules. Recently, our lab has identified Grb10 as a unique imprinted gene capable of influencing fetal growth, postnatal energy metabolism and adult behaviour depending on functions of each of the parental alleles in distinct tissues. Grb10 predominantly expressed from the maternal allele during embryogenesis affects fetal and placental growth along with postnatal glucose homeostasis, whereas paternal Grb10 expression within the CNS influences social behaviour. Delta-like homologue 1 is (Dlk1) a paternally expressed imprinted gene coding for a protein belonging to the Notch/Delta family that acts as a membrane-associated or a soluble protein known to regulate differentiation of various cell types, notably adipocytes. In vivo Dlk1 has been associated with perinatal survival, regulation of normal growth and development and maintenance of the correct course of adipogenesis. Here a hypothesis is proposed that Grb10, as a predominantly maternally expressed growth inhibitor and Dlk1, a paternally expressed growth promoter, act antagonistically in a common genetic pathway. To test this hypothesis, we have generated Grb10m/+/Dlk1+/p double knockout mice and performed a phenotypic characterisation in comparison with wild type as well as the respective single knockout animals. Results obtained from allometric and metabolic analyses, together with histological studies, reveal strong similarities between the phenotypes of Grb10m/+and Grb10m/+/Dlk1+/p knockout mice. We found that overgrowth of Grb10m/+/Dlk1+/p embryos and placentae resemble the phenotype seen in Grb10m/+ mutants and that tissue overgrowth most likely results from higher proliferation rates of Grb10m/+and Grb10m/+/Dlk1+/p cells. Furthermore, Grb10m/+and Grb10m/+/Dlk1+/p knockout mice each exhibit improved glucose clearance and share an unusual characteristic accumulation of lipid in neonatal liver. These results are consistent with the proposed hypothesis and indicate that the Dlk1 and Grb10 genes might be involved in the same genetic pathway. Moreover, the data suggest Dlk1 is an inhibitor of Grb10 which is in turn acting as a growth suppressor.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

L'empreinte génomique est un mécanisme épigénétique qui conduit à l'expression d'un seul des deux allèles parentaux pour une centaine de gènes autosomaux chez les mammifères. La majorité des gènes soumis à l'empreinte est regroupée en clusters et tous ces gènes sont sous le contrôle de séquences discrètes appelées ICR (Imprinting Control Region). Les ICRs sont marquées épigénétiquement par une méthylation d'ADN et des modifications des histones alléliques. La méthylation d'ADN au niveau de ces ICRs est un facteur clé de l'empreinte et va être établie dans les lignées germinales suivant le sexe de l'embryon. Après fécondation, le nouvel embryon portera les empreintes paternelles et maternelles, ces empreintes devront alors être maintenues pendant tout le développement et interprétés dans le but de conduire à l'expression allélique des gènes soumis à l'empreinte. Cependant, la méthylation d'ADN ne peut expliquer à elle seule tous les aspects de l'empreinte génomique. Ainsi, d'autres marques épigénétiques doivent agir dans le maintien et la lecture de ces empreintes. Nous avons mis en évidence dans un premier temps que le contrôle de l'expression allélique dans le cerveau de Grb10 repose sur la résolution d'un domaine bivalent allélique spécifiquement dans le cerveau. Ces résultats mettent en avant pour la première fois un domaine bivalent dans le contrôle de l'expression des gènes soumis à l'empreinte et propose un nouveau mécanisme dans l'expression tissu spécifique de ces gènes. D'autre part, bien que des études en cellules ES aient démontré un rôle de G9a dans le maintien des empreintes au cours du développement embryonnaire, nos données suggèrent que G9a ne serait pas essentielle a ce maintien dans un contexte in vivo. / Genomic imprinting is a developmental mechanism which leads to parent-of-origin-specific expression for about one hundred genes in mammals. Most of imprinted genes are clustered and all are under control of sequence of few kilobases called Imprinting Control Region or ICR. ICRs are epigenetically marked by allelic DNA methylation and histone modifications. DNA methylation on ICRs is a key factor which is established in germ cells according to the sex of the embryo. After fecundation, the new embryo will harbored both paternal and maternal imprints which have to be maintained during the development and read to lead to allelic expression of imprinted genes. However, allelic DNA methylation alone cannot explain every aspect of genomic imprinting. Thus, there should be other epigenetic marks which act in the maintaining and reading of the imprints.Our data first indicate that bivalent chromatin, in combination with neuronal factors, controls the paternal expression of Grb10 in brain, the bivalent domain being resolved upon neural commitment, during the developmental window in which paternal expression is activated. This finding highlights a novel mechanism to control tissue-specific imprinting. On an other hand, although previous studies in ES cells show a role for G9a in the maintaining of imprints during embryonic development, our data suggest that G9a would not be essential in an in vivo model.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to characterize the Grb10 and Grb14 mRNA and protein expression in COCs derived from follicles of different stages of development (1-3, 4-6, 6-8 and >8mm in diameter) and determinate the involvement of the estradiol in the Grb10 and/or Grb14 mRNA expression. Firstly, a pool of the 80 COCs from follicles at 3 to 8mm was used to demonstrate Grb14 and Grb10 mRNA expression in denuded oocytes and respective cumulus cells. The expression was detected in oocyte and cumulus cells. To characterize the experimental model, oocytes from different follicle size were submitted to nuclear maturation assessment and evaluated for competence to reach metaphase II. Maturation competence began in oocytes from follicles larger than 2mm in diameter (P<0,01) and increased throughout follicular development, since, oocytes from follicles of 1-3mm and 4-6mm had lower maturation rate than those from follicles of 6-8 and >8mm (P<0,05). In the first experiment, Grb10 and Grb14 mRNA expression was assessed by qRT-PCR in COCs from different follicle size. The expression of Grb10 mRNA in oocytes from 1-3 and 4-6mm follicles was higher (P<0,05) than those from 6-8 and >8mm follicles. However the Grb14 mRNA relative expression was higher in oocytes of group 1-3mm decreasing (P<0,05) as the follicle size increase. In addition, we performed the localization of the Grb10 protein by immunofluorescence analysis. Positive fluorescence signal for Grb10 was detected in all analyzed samples. To evaluate an hormonal influence on the Grb10 and Grb14 mRNA expression, oocytes from follicles at 3-8mm were cultured in the presence of follicular cells in medium supplemented with 17β-estradiol and/or fulvestrant (an estradiol antagonist). The mRNA expression did not show statistical difference between treatments groups (P>0,05). Based on this results, we conclude that Grb10 and Grb14 mRNA and Grb10 protein are expressed COCs in cattle. In addition, the differential expression observed in COCs from different follicle size suggest an involvement of the Grb10 and Grb14 genes in the bovine oocyte competence. / O objetivo do presente trabalho foi caracterizar as proteínas Grb10 e Grb14 em complexos cumulus-oócito (CCOs) de bovinos oriundos de folículos em diferentes fases de desenvolvimento e mostrar o envolvimento do estradiol na regulação da expressão de RNAm. Primeiramente, foram obtidos pool de CCOs de folículos de 3-8mm para verificar a expressão de RNAm para Grb10 e Grb14 no oócito desnudo e nas células do cumulus. Tanto o oócito quanto as células do cumulus expressaram RNAm para as proteínas em estudo. Com o intuito de caracterizar o modelo experimental utilizado, foi verificado que a competência à progressão meiótica se da em oócitos oriundos de folículos com diâmetro >2mm (P<0,01) e aumenta ao longo do desenvolvimento folicular, já que, oócitos oriundos de folículos de 1-3 e 4-6mm apresentam taxas de maturação inferiores aos oócitos oriundos de folículos de 6-8 e >8mm (P<0,05). O primeiro experimento foi delineado com o intuito de demonstrar uma expressão diferencial de mRNA para as proteínas Grb10 e Grb14 ao longo do desenvolvimento folicular. Para isso os grupos de CCOs (1-3, 4-6, 6-8 e >8mm) foram submetidos a extração RNA e transcrição reversa. A expressão relativa dos genes foi realizada por PCR em tempo real. Nesse experimento foi verificado que a expressão do gene Grb10 em CCOs esteve elevada nos grupos 1-3 e 4-6mm, diminuindo nos grupos 6-8 e >8mm (P<0,05). Já a expressão de mRNA de Grb14 esteve alta no grupo 1-3, diminuindo (P<0,05) conforme o aumento do tamanho folicular. Além disso, foi realizada a localização da proteína Grb10 pela técnica de imunofluorescência. A proteína Grb10 foi localizada tanto no oócito como no cumulus dos grupos de CCOs (1-3, 4-6, 6-8 e >8mm) porém a localização foi mais evidente em CCOs oriundos de folículos com diâmetro <6mm. Com o intuito de identificar uma influência hormonal na regulação da expressão de Grb10 e Grb14, foi realizado um terceiro experimento no qual CCOs oriundos de folículos de 3-8mm foram co-cultivados com metades foliculares em meio suplementado com estradiol-17β e/ou fulvestrant (antagonista do estradiol) durante 6h. A expressão de RNAm não apresentou diferença significativa entre os tratamentos (P>0,05). Com base nesses resultados pode-se concluir que há expressão de RNAm para Grb10 e Grb14 bem como localização da proteína Grb10 em CCOs de bovinos, e que a expressão diferencial de RNAm sugere um envolvimento desses genes na aquisição de competência oocitária ao longo do desenvolvimento folicular. Portanto novos estudos precisam ser feitos para entender um mecanismo de regulação na expressão de RNAm.

Imprinted genes are a subset of loci, positioned on autosomes and the X-chromosome, which are expressed monoallelically in a parent-of-origin specific manner. The influence of such genes on the regulation of embryonic growth and postnatal energy homeostasis is well established. The parental conflict hypothesis predicts that, in utero, paternally-expressed genes will promote maternal resource acquisition and thus growth, whereas maternally-expressed genes will oppose this action, restricting resource investment in a single brood in the interests of the lifetime reproductive success of the mother. Grb10 is an imprinted gene which encodes the cytoplasmic adaptor protein Growth factor receptor bound protein 10. In the majority of tissues, Grb10 is expressed from the maternally-derived chromosome. Consistent with conflict theory, transgenic mice inheriting a disrupted Grb10 allele through the maternal line (Grb10Δ2-4m/+) exhibit embryonic overgrowth, although the mechanisms and signalling pathways responsible for this effect are unclear. Grb10Δ2-4m/+ mice also demonstrate enhanced insulin signalling and improved whole body glucose clearance, consistent with the established role of imprinted genes in the regulation of postnatal metabolism. An integrated LacZ gene-trap in the Grb10Δ2-4 allele failed to fully recapitulate endogenous Grb10 expression, notably within the central nervous system. To address this issue, a second transgenic mouse line, Grb10KO, was created. This allele produced strong LacZ reporter expression in the central nervous system, but only when transmitted through the paternal line (Grb10KO+/p), establishing Grb10 as the only known imprinted gene with a reciprocal imprinting profile between the central nervous system and peripheral tissues. Grb10KO+/p mice exhibit a social dominance phenotype, suggesting distinct roles for maternally- and paternally-expressed Grb10, consistent with their respective sites of expression. The current study characterised the Grb10KO allele at the genetic level, and in doing so, revealed a phenotypic difference between Grb10KOm/p and Grb10Δ2-4m/p mice for which a possible explanation was provided. Importantly, with this knowledge, the current study elucidated the genetic and molecular basis for inconsistencies in reporter expression between the two transgenic lines, identifying a novel tissue-specific enhancer element at the locus. In addition to the central nervous system, this enhancer appeared to be active in the mammary epithelium, identifying a novel site of Grb10 expression, which was pregnancy-dependent and specifically from the maternally-inherited chromosome. Characterisation of the functional significance of expression in this tissue revealed that maternally-expressed Grb10 mediates a supply/demand system between lactating mother and suckling pup, acting as a supply promoter and demand suppressor. This role is inconsistent with conflict theory, but suggests the maintenance of the Grb10 imprint in the mammary epithelium might be associated with improved coadaptiveness between mother and offspring. Intriguingly, in utero, Grb10 is both a demand and supply suppressor. When considered together, these findings suggest a wider role for maternally-expressed Grb10 in the homeostatic control of growth and achievement of optimal fitness.

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