Abstract. The main intrinsic polypeptide (MIP) is the major protein present in the lens fiber cell membrane and is the product of a gene which, as far as is known, is expressed only in the lens. We have used in situ hybridization and immunofluorescence microscopy to characterize the expression of this gene during the course of development in the rat. At progressive stages of lens morphogenesis, we find that synthesis of the protein is closely tied to the accumulation of MIP mRNA in cells that are committed to terminal differentiation, first in the elongating presumptive primary lens fibers and later in the secondary fibers as they differentiate from the anterior epithelial cells. The transcripts accumulate in the basal cytoplasm of the primary fibers and in the cytoplasm which surrounds the cell nucleus in the secondary fibers. We have compared this pattern of expression with that of a gene for a cytoplasmic protein, [~-crystallin I3-A1/A3. In sharp contrast to the localized concentrations seen for the MIP mRNA, I~A1/A3 transcripts are relatively uniformly distributed throughout the cytoplasm. Neither MIP nor crystallin gene appears to be transcriptionally active in the undifferentiated epithelial cell, but transcripts from the I3-A1/A3 gene appear earlier in fiber cell differentiation than do those from the gene for MIP. THE developing eye lens is unusually well suited for the study of the mechanisms of gene expression during morphogenesis. The cells of the lens epithelium follow a single pathway of differentiation, from the earliest stages of lens development and throughout life, maturing to become fiber cells which form the mass of the lens. This differentiation of epithelium to fiber, both during embryonic development and in the mature lens, is characterized by the appearance and subsequent massive accumulation of the cytoplasmic crystallins (tz, 13, and ~, in mammalians [Harding and Crabbe, 1984]) and of an integral membrane protein known as the main intrinsic polypeptide (MIP) ~ (Alcala et al., 1975, Broekhuyse et al., 1976Waggoner and Maisel, 1978; Bloemendal, 1979;Vallon et al., 1985). The specific function of MIP in the lens fiber cell membrane is still an unsolved problem. In our previous work we have discussed the controversy surrounding the proposed role of MIP in the formation of the membrane channels for intercellular communication (i.e., gap junctions). The evidence we presented based on cDNA cloning and analysis of the deduced amino acid sequence showed that MIP has the structural characteristics that would be expected if it were a channel-forming protein. Reconstitution experiments support this view (Nik-

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