RESUMEN
Nob3 is a major obesity quantitative trait locus (QTL) identified in an intercross of New Zealand Obese (NZO) mice with C57BL/6J (B6), and by introgression of its 38 Mbp peak region into B6 (B6.NZO-Nob3.38). B6.NZO-Nob3.38 mice carrying the NZO allele exhibited markedly increased body weight, fat mass, lean mass and a lower energy expenditure, than the corresponding B6 allele carriers. For positional cloning of the responsible obesity gene, five additional congenic lines (RCS) were generated and characterized, allowing to define a critical genomic interval comprising 43 genes. mRNA profiling and western blotting indicated that Ifi202b, a member of the Ifi200 family of interferon inducible transcriptional modulators, was expressed in NZO-allele carriers but was undetectable in tissues of homozygous B6-allele carriers due to a microdeletion, including the first exon and the 5'-flanking region of Ifi202b in B6. Transcriptome analysis of adipose tissue of RCS revealed a marked induction of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-Hsd1) expression in mice expressing Ifi202b. Furthermore, siRNA-mediated Ifi202b suppression in 3T3-L1 adipocytes resulted in a significant inhibition of 11ß-Hsd1 expression, whereas an adenoviral-mediated overexpression of Ifi202b increased 11ß-Hsd1 mRNA levels. Expression of human IFI orthologues was significantly increased in visceral adipose tissue of obese subjects. We suggest that the disruption of Ifi202b in B6 is responsible for the effects of the obesity QTL Nob3, and that Ifi202b modulates fat accumulation through expression of adipogenic genes such as 11ß-Hsd1.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Deleción Cromosómica , Cromosomas Humanos Par 1/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Obesidad/enzimología , Obesidad/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Región de Flanqueo 5'/genética , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Peso Corporal/genética , Exones/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Persona de Mediana Edad , Familia de Multigenes/genética , Sitios de Carácter Cuantitativo/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinación Genética/genética , Adulto JovenRESUMEN
Leaf hairs (trichomes) of Arabidopsis thaliana are a model system for studying cell development, differentiation and cell cycle regulation. To exploit this model system with ultimate spatial resolution we applied single cell sampling, thus avoiding the averaging effect induced by complex tissue mixtures. In particular, we analysed gene expression profiles of two selected stages of the developing trichome: trichome initial cells and mature trichomes, as well as pavement cells. Ten single cells per sample were collected by glass microcapillaries and used for the generation of radioactive probes for subsequent hybridization to nylon filters representing approximately 8000 genes of A. thaliana. Functional categorization of genes transcribed in trichome initials, mature trichomes and pavement cells demonstrated involvement of these surface cells in the stress response. In silico promoter analysis of genes preferentially expressed in trichome initials revealed enrichment in MYB-binding sites and presence of elements involved in hormonal, metal, sulphur response and cell cycle regulation. Three candidate genes preferentially expressed in trichome initials were selected for further analysis: At3g16980 (putative RNA polymerase II), At5g15230 (GASA4) and At4g27260 (GH3.5, WES1). Promoter:GUS studies confirmed expression of the putative RNA polymerase II and the gibberellin responsive GASA4 in trichome initials and partially in mature trichomes. Functional implication of the three selected candidates in trichome development and hence in cell cycle regulation in A. thaliana is discussed. We suggest that these genes are involved in differentiation and initiation of endocycling during trichome development.
Asunto(s)
Arabidopsis/genética , Perfilación de la Expresión Génica/métodos , Epidermis de la Planta/genética , Hojas de la Planta/genética , Arabidopsis/citología , Biología Computacional , Regulación de la Expresión Génica de las Plantas , Epidermis de la Planta/citología , Epidermis de la Planta/crecimiento & desarrollo , Hojas de la Planta/citología , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Every multicellular organism consists of numerous organs, tissues and specific cell types. To gain detailed knowledge about the morphogenesis of these complex structures, it is inevitable to advance biochemical analyses to ultimate spatial and temporal resolution since individual cell types contribute differently to the overall performance of living objects. Single cell sampling combined with systems biological approaches was recently applied to investigations of Arabidopsis thaliana trichomes (leaf hairs). These are single celled structures that provide ideal model systems to address various aspects of plant cell development and differentiation at the level of individual cells. A previously suggested function of trichomes in plant stress responses could thus be confirmed. Furthermore, trichome-specific "omics" data collected in several laboratories are mutually conclusive which demonstrates the applicability of systems biological approaches at the single cell level.