Spp (secreted phosphoprotein, osteopontin) is a gene that is expressed transiently in both neural and non-neural tissues during embryogenesis. In the nervous system, spp is observed almost exclusively in the auditory and vestibular ganglionic neurons, as well as in specific cell types of the inner ear. This pattern of expression and its regulation by growth factors suggest that spp may play an important role in development of the inner ear. At present, the function of the encoded protein is unknown, although much is known about its structure and distribution. It was originally thought to be involved exclusively in bone development, but since the protein has now been localized to tissues in other organs, such as the inner ear, ovary and the kidney, this belief is no longer tenable. By establishing transgenic mouse strains in which spp expression has been specifically altered we can investigate how expression of this gene affects the inner ear. In this way, we hope to learn more about the function of this protein and the molecular signals that control and direct inner ear ontogenesis. The murine gene has been extensively characterized and elements in the enhancer/promoter tat regulate expression have been identified. Vectors will be constructed in which these enhancer/promoter elements will be used to drive expression in transgenic mice of (1) RNA complementary to the normal mRNA (antisense RNA) or (2) a protein (diphtheria toxin) lethal to the cell. Mice carrying specific mutations in the spp gene will be produced by first engineering the mutation in embryonic stem cells, which are then incorporated into blastocysts, some of which give rise to mice with the mutation in the germ line. The consequences of alterations in spp expression in the inner ear will be examined both morphologically and physiologically. In parallel studies the properties of various inner ear cell types in cell culture will be studied with particular reference to the effects of SPP. Wild-type and mutant forms of SPP will be produced in an appropriate host and incorporated into extracellular matrices to assess their effect on various cell types. We believe that SPP conveys signals to cells and endows them with particular properties that are important developmentally. This approach will afford us the unique opportunity to manipulate the peripheral auditory and vestibular systems on a molecular level and to relate these data to known hereditary disorders that affect hearing and balance.