Detection of primary and mature transcripts of calcitonin-gene-related peptide genes in rat parafollicular cells by light, fluorescence and confocal microscopy.
Alternative splicing of primary transcripts from the calcitonin/alpha calcitonin gene-related peptide (alpha CGRP) gene result in mature mRNAs encoding either calcitonin or alpha CGRP. We have produced sequence-specific, synthetic, biotinylated oligodeoxynucleotide probes that recognize calcitonin (exon 4), and alpha CGRP (exon 6) sequences as well as sequences common to both splice variants (exon 3) of this gene. Probes to exons 4 and 3 revealed strong cytoplasmic signals in rat parafollicular cells. In addition, a punctate nuclear signal was obtained with these probes. The alpha CGRP-specific (exon 6) probe resulted in weak cytoplasmic labelling of parafollicular cells, but produced a punctate nuclear labelling similar to that seen with the exon 4 and 3 probes. RNase digestion removed all the cytoplasmic and nuclear signals obtained with all probes. Hybridization with a thyroglobulin-specific probe failed to label parafollicular cells. A control (human enterovirus) probe yielded negative results, while a probe to rat somatostatin produced cytoplasmic labelling of a small subpopulation of parafollicular cells. Finally, a probe specific for beta CGRP mRNA labelled most, if not all, parafollicular cells. Fluorescent alkaline phosphatase development of in situ hybridizations could be combined with indirect immunofluorescence for CGRP. Analysis by fluorescence and confocal microscopy revealed that CGRP immunoreactive cells contained calcitonin, alpha CGRP and beta CGRP hybridization signals. Our results demonstrate that all three genes may be simultaneously expressed by thyroid parafollicular cells and show that synthetic biotinylated oligonucleotide probes can be used for highly precise localizations of primary transcripts in the nuclei of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
van Lieshout
E M
EM
Department of Molecular Cell Biology, Statens Seruminstitut, Copenhagen, Denmark.
Hougaard
D M
DM
Larsson
L I
LI
D000818:Animals; D001710:Biotin; D015740:Calcitonin Gene-Related Peptide / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D005091:Exons / Q000502:physiology; D005260:Female; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D013961:Thyroid Gland / Q000378:metabolism*
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D001710:Biotin; D015740:Calcitonin Gene-Related Peptide