Lineage- and stage-specific gene expression in human hemopoietic cells

grantor: University of TorontoThis study describes the establishment of a system for mapping lineage- and stage-specific gene expression in human hemopoietic precursors, and demonstrates its ability to generate information relevant to mechanisms that control proliferation and differentiation during hemopoietic development. Two approaches were used to define patterns of gene expression at specific stages in hemopoietic differentiation. The first of these is global RT-PCR which amplifies all polyA\sp+ mRNA from a single cell. Hemopoietic precursors in different commitment states are phenotypically similar and cannot be separated from one another or purified to homogeneity using existing methods. The ability to study gene expression at the level of a single cell can provide an alternative approach to achieving homogeneity. The second strategy is sibling analysis, which identifies retrospectively the developmental potential of a cell subjected to PCR by analysis of the fate of its siblings. I was able to collect more than 200 cDNA samples from cells of different lineages and different stages of development. The set was then used to determine expression patterns of a wide panel of genes (RBTN2, CD34, fms, c-kit, HPK1, Bcl-2 family, p53, WAF1, RB, and WT) known to be involved in regulation of proliferation and differentiation of hemopoietic cells. (Abstract shortened by UMI.)M.Sc

Calcium-Dependent Adhesion Is Necessary for the Maintenance of Prosomeres

Cell adhesion has been suggested to function in the establishment and maintenance of the segmental organization of the central nervous system. Here we tested the role of different classes of adhesion molecules in prosencephalic segmentation. Specifically, we examined the ability of progenitors from different prosomeres to reintegrate and differentiate within various brain regions after selective maintenance or removal of different classes of calcium-dependent versus -independent surface molecules. This analysis implicates calcium-dependent adhesion molecules as central to the maintenance of prosomeres. Only conditions that spared calcium-dependent adhesion systems but ablated more general (calcium-independent) adhesion systems resulted in prosomere-specific integration after transplantation. Among the members of this class of adhesion molecules, R-cadherin shows a striking pattern of prosomeric expression during development. To test whether expression of this molecule was sufficient to direct progenitor integration to prosomeres expressing R-cadherin, we used a retroviral-mediated gain-of-function approach. We found that progenitors originally isolated from prosomere P2 (a region which does not express R-cadherin), when forced to express this molecule, can now integrate more readily into R-cadherin-expressing regions, such as the cortex, the ventral thalamus, and the hypothalamus. Nonetheless, our analysis suggests that while calcium-dependent molecules are able to direct prosomere-specific integration, they are not sufficient to induce progenitors to change their regional identity. While diencephalic progenitors from R-cadherin-expressing regions of prosomere 5 could integrate into R-cadherin-expressing regions of the cortex, they did not express the cortex-specific gene Emx1 or the telencephalic-specific gene Bf-1. Furthermore, diencephalic progenitors that integrate heterotopically into the cortex do not persist postnatally, whereas the same progenitors survive and differentiate when they integrate homotopically into the diencephalon. Together our results implicate calcium-dependent adhesion molecules as key mediators of prosomeric organization but suggest that they are not sufficient to bestow regional identities

Telencephalic progenitors maintain anteroposterior identities cell autonomously

Grafting experiments have demonstrated that determination of anteroposterior (AP) identity is an early step in neural patterning that precedes dorsoventral (DV) specification [1,2]. These studies used pieces of tissue, however, rather than individual cells to address this question. It thus remains unclear whether the maintenance of AP identity is a cell-autonomous property or a result of signaling between cells within the grafted tissue. Previously, we and others [3–5] have used transplants of dissociated brain cells to show that individual telencephalic precursor cells can adopt host-specific DV identities when they integrate within novel regions of the telencephalon. We have now undertaken a set of transplantations during the same mid-neurogenic period used in the previous studies to assess the ability of telencephalic progenitors to integrate and differentiate into more posterior regions of the neuraxis. We observed that telencephalic progenitors were capable of integrating and migrating within different AP levels of the central nervous system (CNS). Despite this, we found that telencephalic progenitors that integrated within the diencephalon and the mesencephalon continued to express a telencephalic marker until adulthood. We speculate that during neurogenesis individual progenitors are determined in terms of their AP but not their DV identity. Hence, AP identity is maintained cell autonomously within individual progenitors

Conditional Activation of Akt in Adult Skeletal Muscle Induces Rapid Hypertrophy

Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachexia, cancer, AIDS, prolonged bedrest, and diabetes. One strategy in the treatment of atrophy is to induce the pathways normally leading to skeletal muscle hypertrophy. The pathways that are sufficient to induce hypertrophy in skeletal muscle have been the subject of some controversy. We describe here the use of a novel method to produce a transgenic mouse in which a constitutively active form of Akt can be inducibly expressed in adult skeletal muscle and thereby demonstrate that acute activation of Akt is sufficient to induce rapid and significant skeletal muscle hypertrophy in vivo, accompanied by activation of the downstream Akt/p70S6 kinase protein synthesis pathway. Upon induction of Akt in skeletal muscle, there was also a significant decrease in adipose tissue. These findings suggest that pharmacologic approaches directed toward activating Akt will be useful in inducing skeletal muscle hypertrophy and that an increase in lean muscle mass is sufficient to decrease fat storage