TISSUE-SPECIFIC AND AGE-SPECIFIC EXPRESSION PATTERNS OF ALTERNATIVELYSPLICED AGRIN MESSENGER-RNA TRANSCRIPTS IN EMBRYONIC RAT SUGGEST NOVEL DEVELOPMENTAL ROLES

Agrin is an extracellular matrix protein that mediates the nerve-induc ed clustering of nicotinic acetylcholine receptors on target muscle ce lls, and thus plays a key role in development of the neuromuscular syn apse. Alternative exon usage within the rat agrin gene predicts numero us protein isoforms, which differ by the inclusion or exclusion of sma rt inserts at three sites in the C-terminal half of the molecule; the insert status at two of these sites, termed Y and Z, profoundly influe nces the acetylcholine receptor clustering activity. We have examined the cellular expression patterns of agrin messenger RNA transcripts du ring rat embryogenesis by in situ hybridization with isoform-specific probes. Six 36-mer oligonucleotide probes were designed to distinguish between mRNA isoforms at either the Y site: the encoded protein conta ins either no insert (Y0) or a 4-amino acid insert (Y4), or the Z site : the encoded protein contains either no insert or one of 8 (Z8), 11 ( Z11), or 19 (Z19) amino acids. Strikingly different expression pattern s were observed for the individual Y- and Z-site encoding messages. Wh ile optional exon usage predicts the possibility of eight different ag rin isoforms at the two splice sites, we detected only four isoforms i n vivo: Y4Z0, Y0Z0, Y4Z8, and Y4Z19. The Y4Z0 transcript, which compri sed the majority of the agrin expressed, was localized exclusively to nervous tissue and exhibited a distribution profile suggestive of a po tential role in neurogenesis and/or neural differentiation. From embry onic day 13 to birth, Y4Z0 was found in mitotic ventricular zones, spi nal, cranial, and sympathetic ganglia, and diffusely throughout the br ain. In contrast, Y0Z0 was not expressed in neurons, but specifically labeled capillary endothelial cells within the developing nervous syst em. Y4Z8 and Y4Z19, the forms most active in acetylcholine receptor ag gregation, were expressed at low levers only in spinal and brainstem m otor neurons; Z19 expression declined from embryonic day 15 to adultho od, whereas Z8 expression increased slightly during this period. Trans cripts encoding the Z11 insert could not be detected. These data sugge st potential novel biological roles for agrin beyond that originally p roposed in synapse formation.