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

Apr 12, 2007Journal of biochemistry

Separate brain locations of daily rhythm and FMRFamide signals in houseflies and fruit flies

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Abstract

cDNA cloning of an fmrf mRNA from the housefly Musca domestica was achieved.

FMRFamide-like peptides and PDF were previously suggested to coexist in the same cells in insect brains.
Double-labelled in situ hybridization showed that the fmrf and pdf mRNAs are expressed in distinctly different cells in housefly brains.
Similar findings were confirmed in the fruit fly Drosophila melanogaster, indicating separate expression of these neuropeptides.
The results suggest that FMRFamide and PDF peptides are unlikely to be released from the same neurons.

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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 beta-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.

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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

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