Pascale Leroy

The gene product of the recently cloned mouse obese gene (ob) is important in regulating adipose tissue mass. ob RNA is expressed specifically by mouse adipocytes in vivo in each of several different fat cell depots, including brown fat. ob RNA is also expressed in cultured 3T3-442A preadipocyte cells that have been induced to differentiate. Mice with lesions of the hypothalamus, as well as mice mutant at the db locus, express a 20-fold higher level of ob RNA in adipose tissue. These data suggest that both the db gene and the hypothalamus are downstream of the ob gene in the pathway that regulates adipose tissue mass and are consistent with previous experiments suggesting that the db locus encodes the ob receptor. In db/db and lesioned mice, quantitative differences in expression level of ob RNA correlated with adipocyte lipid content. The molecules that regulate expression level of the ob gene in adipocytes probably are important in determining body weight, as are the molecules that mediate the effects of ob at its site of action.

ABSTRACT Embryonic stem cells, derived from the inner cell mass of murine blastocysts, can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes, such as peroxisome proliferator-activated receptors γ and δ and CCAAT/enhancer binding protein β, during differentiation of retinoic acidtreated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes.

A molecular-marker linkage map of hexaploid wheat (Triticum aestivum L. em. Thell) provides a framework for integration with teh classical genetic map and a record of the chromosomal rearrangements involved in the evolution of this crop species. We have constructed restriction fragment length polymorphism (RFLP) maps of the A-, B-, and D-genome chromosomes of homoeologous groups 4, 5, and 7 of wheat using 114 F7 lines from a synthetic X cultivated wheat cross and clones from 10 DNA libraries. Chromosomal breakpoints for known ancestral reciprocal translocations involving these chromosomes and for a known pericentric inversion on chromosome 4A were localized by linkage and aneuploid analysis. Known genes mapped include the major vernalization genes Vrn1 and Vrn3 on chromosome arms 5AL and 5DL, the red-coleoptile gene Rc1 on 7AS, and presumptively the leaf-rust (Puccinia recondita f.sp. tritici) resistance gene Lr34 on 7DS and the kernel-hardness gene Ha on 5DS. RFLP markers previously obtained for powdery-mildew (Blumeria graminis f.sp. tritici) resistance genes Pm2 and Pm1 were localized on chromosome arms 5DS and 7AL.

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.