DETECTION OF LAMPRIN MESSENGER-RNA IN THE ANADROMOUS SEA LAMPREY USING IN-SITU HYBRIDIZATION

An optimal in situ hybridization protocol is described for the detecti on of gene expression of a structural protein unique to lampreys, lamp rin, in the cartilages of prolarval, metamorphic and adult sea lamprey , Petromyzon marinus. A 156 bp antisense RNA probe labeled with S-35-U TP was transcribed in vitro from a recombinant plasmid containing a cD NA insert homologous to the largest (1.8 kb) of three known mRNAs for the lamprin gene and hybridized to 6 mu m paraffin sections. Optimal s ignal to noise ratio was achieved by fixing tissues 30 min in 4% paraf ormaldehyde and prehybridizing with a probe incorporating a nonradioac tive S-UTP. Strong signals were visualized in all cartilaginous elemen ts of the lamprey neurocranium; however, lamprin mRNA transcripts were not detected in branchial and pericardial cartilages suggesting diffe rential expression of the lamprin gene. No signals were observed in ti ssue sections that had been treated with RNase A prior to hybridizatio n or in sections hybridized with sense RNA probes. This technique has great potential for use In studies of the spatial and temporal distrib ution of cartilaginous components during developmental stages of lampr eys.