In situ RT-PCR detection of CYP1A mRNA in pharyngeal epithelium and chondroid cells from chemically untreated fish: involvement in vertebrate craniofacial skeletal development?
Knowledge about the expression sites of cytochrome P450 1A (CYP1A) mRNA is crucial for a better understanding of the physiological function of CYP1A. We investigated the cellular localization of CYP1A mRNA in chemically untreated fish by use of an in situ reverse transcription-polymerase chain reaction (IS RT-PCR) technique. The fathead minnow (Pimephales promelas) was formalin-fixed, and paraffin-embedded. Sections (5 microm) were treated with trypsinogen. Following reverse transcription of CYP1A mRNA, the cDNA was amplified in situ by PCR with specific primers. Detection of the amplicons was accomplished by a second PCR performed with digoxigenin-labeled dUTP. CYP1A mRNA expression was detected in cytoplasm of chondroid cells surrounding hyaline cartilage in gill arches. This result was consistent with that of immunohistochemical analysis with a CYP1A1-specific monoclonal antibody. CYP1A mRNA also was found in stratified squamous epithelium of the pharynx and gill arches, but no staining was detected in those cells by immunohistochemical analysis. The results indicate that IS RT-PCR is an effective/sensitive technique for localizing low level CYP1A expression. In addition, the sites where we identified expression of CYP1A are targets of retinoic acid, sonic hedgehog and Hox genes, suggesting that functional CYP1A in vertebrates could participate in craniofacial skeletal development through involvement in the retinoic acid signaling cascade.
Iwata
H
H
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA.
Stegeman
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019902:Chondrocytes / Q000378:metabolism*; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000502:physiology; D004076:Digoxigenin / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D005260:Female; D005399:Fishes / Q000196:embryology / Q000235:genetics*; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010614:Pharynx / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D004076:Digoxigenin