INT-1-RELATED GENES IN XENOPUS EMBRYOS

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Inappropriate expression of proto-oncogenes may be causally related to a variety of cancers and to birth defects. This has led to considerable interest in elucidating the normal pattern of expression and functions of proto-oncogenes which are expressed during development of vertebrate embryos. The proto-oncogene Int-1 has a particularly interesting pattern of expression-it is expressed in a transient manner in the dorsal midline of the neural tube of vertebrate embryos, and may be involved in signalling between cells. The hypothesis that vertebrate embryos express additional genes related to int-1 has not been widely studied. In the proposed project, we shall test this hypothesis by completion of the following specific aims: 1. cDNAs related to int-1 will be cloned by PCR approaches from Xenopus embryo mRNA, using oligonucleotide primers complementary to conserved regions of int-1 and IRP from several species. That these techniques are appropriate is evident from our recent cloning of at least four unique Xenopus cDNAs homologous to int-1 by this approach. 2. To determine whether transcripts complementary to the int-1-related cDNAs are regulated in a temporal manner during development. To determine whether the expression of the transcripts related to the int-1-related cDNAs is restricted to specific regions of embryos, Xenopus embryos will be microdissected, RNA extracted, and RNA blot analyses undertaken. 3. The least conserved region of the int-1-related proteins will be expressed in E. coli following ligation of the cDNAs in frame in the expression vector pRIT2T. Polyclonal antisera will be prepared in rabbits. Whole mount immunocytochemistry of Xenopus embryos will be used to determine the spatial distribution of the int-1-related proteins. 5. mRNA transcribed from nearly full-length int-1-related cDNAs will be microinjected into fertilized Xenopus eggs to deregulate normal expression. Analysis of resulting embryonic phenotypes will provide insight into the normal functions of the int-1-related proteins. 6. Subtractive cDNA probes will be used to clone mRNAs changing in response to expression of int-1-related proteins. 7. Several lines of investigation will pursue cellular receptors for int-1 and int-1-related proteins, as well as second messenger systems involved in signal transduction. Collectively these specific aims will provide new information on the expression and functions of potential proto-oncogenes important cell signalling and communication in vertebrate embryos, as well as human cancers and birth defects.