DETECTION OF PRIMARY AND MATURE TRANSCRIPTS OF CALCITONIN-GENE-RELATED PEPTIDE GENES IN RAT PARAFOLLICULAR CELLS BY LIGHT, FLUORESCENCE ANDCONFOCAL MICROSCOPY

Alternative splicing of primary transcripts from the calcitonia/alpha calcitonin gene-related peptide (alpha CGRP) gene result in mature mRN As encoding either calcitonin or alpha CGRP. We have produced sequence -specific, synthetic, biotinylated oligodeoxynucleotide probes that re cognize calcitonin (exon 4), and alpha CGRP (exon 6) sequences as well as sequences common to both splice variants (exon 3) of this gene. Pr obes to exons 4 and 3 revealed strong cytoplasmic signals in rat paraf ollicular cells. In addition, a punctate nuclear signal was obtained w ith these probes. The alpha CGRP-specific (exon 6) probe resulted in w eak cytoplasmic labelling of parafollicular cells, but produced a punc tate nuclear labelling similar to that seen with the exon 4 and 3 prob es. RNase digestion removed all the cytoplasmic and nuclear signals ob tained with all probes. Hybridization with a thyroglobulin-specific pr obe failed to label parafollicular cells. A control (human enterovirus ) probe yielded negative results, while a probe to rat somatostatin pr oduced cytoplasmic labelling of a small subpopulation of parafollicula r cells, Finally, a probe specific for beta CGRP mRNA labelled most, i f not all, parafollicular cells. Fluorescent alkaline phosphatase deve lopment of in situ hybridizations could be combined with indirect immu nofluorescence for CGRP. Analysis by fluorescence and confocal microsc opy revealed that CGRP immunoreactive cells contained calcitonin, alph a CGRP and beta CGRP hybridization signals. Our results demonstrate th at all three genes may be simultaneously expressed by thyroid parafoll icular cells and show that synthetic biotinylated oligonucleotide prob es can be used for highly precise localizations of primary transcripts in the nuclei of these cells. The punctate distribution of the nuclea r hybridizations may be correlated to the locations of spliceosomes an d/or nuclear transport routes.