Gene trap analysis of germ cell signaling to Sertoli cells: NGF-TrkA mediated induction of Fra1 and Fos by post-meiotic germ cells

Analysis of complex signalisation networks involving distinct cell types is required to understand most developmental processes. Differentiation of ma le germ cells in adult mammals involves such a cross-talk between Sertoli c ells, the somatic component which supports and controls germinal differenti ation, and germ cells at their successive maturation stages. We developed a gene trapping strategy to identify genes, which, in Sertoli cells, are eit her up- or down-regulated by signals emitted by the germinal component. A l ibrary of similar to2,000 clones was constituted from colonies independentl y selected from the Sertoli line 15P-1 by growth in drug-containing medium after random integration of a promoter-less beta geo transgene (neo(r)-lacZ fusion), which will be expressed as a fusion transcript from a 'trapped' c ellular promoter, different in each clone. A first screen conducted on 700 events identified six clones in which beta -galactosidase activity was incr eased and one in which it was repressed upon addition of germ cells. The ta rgeted loci were identified by cloning and sequencing the genomic region 5' of the insert. One of them was identified as the gene encoding Fra1, a com ponent of the AP1 transcription regulatory complex. Accumulation of Fra1 mR NA was induced, both in 15P-1 and in freshly explanted Sertoli cells, by ad dition of either round spermatids or nerve growth factor (NGF). The effect of NGF was mediated by the TrkA receptor and the ERK1-ERK2 kinase kinase pa thway. Fos and Fra1 transcription were induced within the first hour after addition of the neurotrophin, but, unlike what is observed after serum indu ction in the same cells, a second wave of transcription of Fra1, but not of Fos, started 16 hours later and peaked at higher levels at about 20 hours. These results suggest that AP1 activation may be an important relay in the Sertoli-germ cell cross-talk, and validate the gene trapping approach as a tool for the identification of target genes in cell culture systems.