Spatial, a gene expressed in thymic stromal cells, depends on three-dimensional thymus organization for its expression

Abstract

Although the importance of thymic stroma in thymopoiesis has been recognized, the underlying molecular details regarding stromal cell biology remain obscure. To study this area, we have cloned genes expressed in thymic stromal cells. Spatial is alternatively spliced to generate two mRNAs in thymus and lymph node (LN) but it is not expressed in the spleen. In mouse embryos, the short form begins expression at day 10 while the long form is not detected until day 12. Both mRNAs encode proline rich proteins and their closest homology is to homeobox and POU domain transcription factors. Spatial is not expressed in thymocytes, but it is expressed in 2-deoxyguanosine-treated day 14 fetal thymic organ culture (FTOC) and in reaggregated FTOC. These data suggest that a normal three-dimensional organization of stromal cells is required for Spatial expression. An antiserum raised against a C-terminal peptide detected proteins of 38 and 32 kDa in Western blots of total thymus proteins. In frozen thymus sections, subcapsular epithelial cells were stained with the anti-Spatial antiserum. Paracortical subcapsular cells of unknown function were also stained in the LN. Both forms of Spatial fused to the green fluorescent protein (GFP) localize to the nucleus in transfected cells.