Journal of Biochemistry Advance Access originally published online on April 10, 2007
Journal of Biochemistry 2007 141(6):867-877; doi:10.1093/jb/mvm094
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© 2007 The Japanese Biochemical Society.
Double-labelled in situ Hybridization Reveals the Lack of Co-localization of mRNAs for the Circadian Neuropeptide PDF and FMRFamide in Brains of the Flies Musca domestica and Drosophila melanogaster
1Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan; and 2Division of Biology, Faculty of Science, Fukuoka University, Fukuoka 814-0180, Japan
*To whom correspondence should be addressed. Tel/Fax: +81-92-642-2584, E-mail: shimoscc{at}mbox.nc.kyushu-u.ac.jp
Received January 9, 2007; Accepted March 31, 2007
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Abstract |
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Many lines of evidence have suggested that neuropeptides other than pigment-dispersing factor (PDF) are involved in regulating insect circadian rhythms, and FMRFamide-related peptides are additional candidates acting as such neuromodulators. Double-immunolabelling in insect brains with anti-crustacean ß-PDH and anti-FMRFamide antibodies had previously suggested that insect PDF and FMRFamide-like peptides may coexist in the same cells. However, it is critical for this kind of comparative investigations to use antibodies of proven specificity, to eliminate the possibility of both reciprocal cross-reactivity and the detection of unknown peptides. In the present study, we achieved the cDNA cloning of an fmrf mRNA from the housefly Musca domestica, for which co-localization of FMRFamide and PDF peptides was previously suggested. In order to examine the possible co-expression of this gene with the pdf gene, we carried out double-labelled in situ hybridization for simultaneous detection of both pdf and fmrf mRNAs in housefly, Musca brains. The results clearly indicated that they occur in distinctly different cells. This was also proven for the fruit fly Drosophila melanogaster by similar double-labelled in situ hybridization. The results thus revealed no reason to evoke the physiological release of FMRFamide and PDF peptides from the same neurons.
Key Words: circadian rhythm, double-labelled in situ hybridization, FMRFamides, neuropeptides, pigment-dispersing factor (PDF)
Abbreviations: AAP, Abridged Anchor Primer; AP, alkaline phosphatase; AUAP, Abridged Universal Anchor Primer; BCIP, 5-bromo-4-chloro-3-indolylphosphate; DCV, dense core vesicle; DIG, digoxigenin; FaRPs, FMRFamide-related peptides; FITC, fluorescein isothiocyanate; FMRFamide, the one-letter amino acid code denotes H–Phe–Met–Arg–Phe–NH2; HRP, horseradish peroxidase; NBT, 4-nitro-blue tetrazolium chloride; NTMT, a solution containing 100 mM NaCl, 100 mM Tris–HCl (pH 9.5), 50 mM MgCl2 and 1% Tween 20; PBS, phosphate buffered saline; PBST, PBS containing 0.1% Tween 20; PDF, pigment-dispersing factor; PDH, pigment-dispersing hormone; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription PCR; TBS, Tris-buffered saline; TBST, TBS containing 1% Tween 20