The differentiation of rat lens cells is controlled by fibroblast growth factor (FGF). The morphogenetic signal sent by FGF must set in train a series of transcriptional changes, affecting regulatory as well as structural genes. Which regulatory genes are affected by FGF and, in turn, what molecular effects are exerted by the products of these regulatory genes, is unknown. As a first step towards the identification of the mechanism(s) by which FGF directly or indirectly redirects the flow of genetic information, the FGF dependent steps in the activation of two sets of structural genes, the beta- and gamma-crystallin genes, will be followed. These genes were chosen because their product marks the fully differentiated lens fibre cell, because their structure is known in detail and because knowledge of the sequences involved in the regulation of these genes has rapidly accumulated in the last few years. The process of activation of these genes as a function of FGF-directed differentiation will be assessed at three levels: the availability of the chromatin for transcription (as measured by demethylation of chromosomal DNA), the presence of transcription factors (as assayed by transfection experiments) and the accumulation of the transcript (as reflected by RNA levels). This approach has become experimentally possible with the development of a rat lens epithelial cell explant culture, which can be manipulated to yield pure populations of cells at intermediate stages of lens cell differentiation. The intermediate stage(s) at which critical events in the activation of the transcription of the beta- and gamma-crystallin genes occur will be the source material for the next set of experiments, aimed at the isolation of regulatory genes acting at the DNA level. The experimental approach is based upon the hypothesis that such critical stages of differentiation will be characterized by the appearance or disappearance of transacting factors, identifiable by their DNA binding domains. The analysis of the molecular course of events in lens differentiation triggered by fibroblast growth factor will not only provide a fundamental insight in the process of differentiation, it will also show to what extent abnormal growth factor levels can be a cause of lens dysfunction.
