Detection of lamprin mRNA in the anadromous sea lamprey using in situ hybridization

An optimal in situ hybridization protocol is described for the detection of gene expression of a structural protein unique to lampreys, lamprin, in the cartilages of prolarval, metamorphic and adult sea lamprey, Petromyzon marinus. A 156 bp antisense RNA probe labeled with (35)S-UTP was transcribed in vitro from a recombinant plasmid containing a cDNA 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 signal to noise ratio was achieved by fixing tissues 30 min in 4% paraformaldehyde and prehybridizing with a probe incorporating a nonradioactive S-UTP. Strong signals were visualized in all cartilaginous elements of the lamprey neurocranium; however, lamprin mRNA transcripts were not detected in branchial and pericardial cartilages suggesting differential expression of the lamprin gene. No signals were observed in tissue sections that had been treated with RNase A prior to hybridization or in sections hybridized with sense RNA probes. This technique has great potential for use in studies of the spatial and temporal distribution of cartilaginous components during developmental stages of lampreys.