INT-2 INFLUENCES THE DEVELOPMENT OF THE NODOSE GANGLION

The int-2 gene was first described as a common proviral integration si te in tumors induced by mouse mammary tumor virus (MMTV). During embry onic development int-2 is produced and released by cells in the rhombe ncephalon and diffuses to the ectoderm to induce formation of the otoc yst from the otic placode. int-2 also influences the development of th e vestibulocochlear ganglion that is derived from the otic placode. Du ring embryogenesis the otic and nodose placodes, primordia of the inne r ear and the nodose ganglia, respectively, are located adjacent to ea ch other in the embryonic ectoderm. The nodose ganglia provide sensory innervation to all of the viscera. Using Northern analysis we determi ned that a high level of int-2 is transcribed in stage 14 chick embryo s. This is the time when cells begin to migrate from the nodose placod es to form the nodose ganglia. Using human and mouse sequences to desi gn primers around the translation start site of the transcript, a part ial clone containing the translation start site of chick int-2 was obt ained by polymerase chain reaction amplification from chick genomic DN A and cloned. An antisense oligodeoxynucleotide was designed to the re gion of the translation start site, and in vitro and in vivo technique s were used to demonstrate that inhibition of int-2 translation using this antisense oligonucleotide causes delayed and abnormal development of the nodose placodes. For in vitro studies, explants of stage 12 ch ick embryos containing neural tube, adjacent surface ectoderm, and pha ryngeal endoderm were cultured with int-2 antisense oligonucleotide. F or the in vivo studies, pieces of resin impregnated with int-2 antisen se oligonucleotide were implanted into the neural tube of stage 12 chi ck embryos at the level of the otic and nodose placodes. We found that the development of the nodose placodes exposed to int-2 antisense oli godeoxynucleotide was delayed and abnormal. These differences were not seen in embryos or explants treated with similar concentrations of se nse or nonspecific oligomers. Western analysis and immunohistochemistr y showed that an int-2-immunoreactive protein was reduced in the phary ngeal region and nodose ganglia of the embryos exposed to int-2 antise nse oligodeoxynucleotide, whereas it was not affected in embryos treat ed with sense oligomer. We conclude that int-2 may be necessary for no rmal development of the nodose ganglia.