The molecular mediators which orchestrate cell-cell inductive interactions in renal organogenesis are poorly understood. Toward this end, we have identified, cloned, and initially characterized a novel 25 kDa secreted glycoprotein, designated B6l, which is induced in response to treatment of endothelial cells with the proinflammatory cytokine tumor necrosis factor- alpha. We hypothesize that B61 functions as a novel cytokine in the inflammatory environment and in embryonic development. Several lines of evidence support this contention: 1) As an immediate-early gene, B6l is a member of a class of genes which play pivotal roles in orchestrating the initial response of a cell to a particular stimulus. 2) TNF and IL-1 induce a pleotropic proinflammatory phenotype in endothelial cells mediated by the induction of gene products that serve as important molecular intermediaries in the process of inflammation. Nearly all known TNF induced and secreted proteins derived from endothelium are cytokines or other mediators relevant to inflammation. c) Since inflammation and morphogenesis share common processes of growth, differentiation, and tissue pattern formation, it was reasoned that B6l might be expressed during embryonic development. Preliminary data demonstrates that B6l mRNA is differentially expressed in select tissues and at distinct times during mouse embryogenesis. d) B61 protein is distributed in the actively differentiating metanephrogenic mesenchyme of the neonatal kidney. The purpose of the proposed project is to establish that B6l plays a role in renal organogenesis and in embryogenesis in general. Both direct and indirect approaches to understanding B6l's function are proposed. Initially, the temporal and spatial distribution of B6l mRNA and protein expression during mouse embryogenesis will be characterized using the techniques of immunocytochemistry and in situ hybridization. Since the developing kidney provides a suitable model for studying the inductive cell-cell interactions which determine organogenesis, a well described renal organ culture model will be studied. The pattern of expression of both B61 and its putative receptor will be defined and correlated with morphology. Using recently developed anti-B61 antibodies and antisense oligonucleotides, experiments will be performed aimed at establishing that B61 has a functional role in morphogenesis. Finally, these latter experiments will be corroborated in an embryonic carcinoma model system of cell differentiation in which steady state B6l mRNA expression is induced during differentiation along a mesodermal differentiation pathway. To further explore the roll of B61 in modifying cell behavior, B61 will be either overexpressed in transfectants of undifferentiated embryonic """"""""stem cells"""""""" or its expression will be blocked by transfection of an antisense expression vector. These experiments will define the roll of B61 in mesodermal differentiation.
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