Kazunori Sumida

Structural isoforms of the circadian neuropeptide PDF expressed in the optic lobes of the cricket Gryllus bimaculatus: Immunocytochemical evidence from specific monoclonal antibodies

Pigment‐dispersing factor (PDF) is an 18‐mer peptide that acts as a principal neurotransmitter of the insect circadian clock. Our previous study, utilizing anti‐Uca β‐PDH polyclonal antibody (pAb) to immunolabel the optic lobe of the cricket Gryllus bimaculatus, suggested the existence of an alternative PDF‐like peptide in the outer cells of the first neuropile, or lamina (La), which were much less immunoreactive than the inner cells of the second neuropile, the medulla (Me). To obtain structural information about such a PDF‐like peptide, we prepared 10 anti‐Gryllus PDF monoclonal (mAb) and pAb antibodies and analyzed their detailed epitope specificities. The PDFMe and PDFLa inner cells and their axonal projections were clearly immunoreactive to all these antibodies, revealing the widespread immunocytochemical organization of the PDF system in the optic lobe, as seen previously with anti‐Uca β‐PDH pAb and anti‐Gryllus PDF mAb, the epitope structures of which were also clarified in this study. The lamina outer cells, which we found lacked a target pdf mRNA, displayed specific immunoreactivities, indicating that the cells contain a distinct PDF‐like peptide possessing both N‐ and C‐terminal structures. These cells were not immunolabeled by some other monoclonal antibodies, however, implying that the PDFLa outer cells have a PDF isoform peptide devoid of Asn at positions 6 and 16. This isoform was also identified in a varicose arborization in the lamina. These results suggest not only the structure of the peptide, but also the possibility of additional functions of this novel PDF isoform. J. Comp. Neurol. 499:404–421, 2006.

A Circadian Neuropeptide PDF in the Honeybee, Apis mellifera: cDNA Cloning and Expression of mRNA

Pigment-dispersing factor (PDF) is a pacemaker hormone regulating the locomotor rhythm in insects. In the present study, we cloned the cDNAs encoding the Apis PDF precursor protein, and found that there are at least seven different pdf mRNAs yielded by an alternative splicing site and five alternative polyadenylation sites in the 5′UTR and 3′UTR regions. The amino acid sequence of Apis PDF peptide has a characteristic novel amino acid residue, aspargine (Asn), at position 17. Quantitative real-time PCR of total and 5′UTR insertion-type pdf mRNAs revealed, for the first time, that the expression levels change in a circadian manner with a distinct trough at the beginning of night in LD conditions, and at the subjective night under DD conditions. In contrast, the expression level of 5′UTR deletion-type pdf mRNAs was about half of that of the insertion type, and the expression profile failed to show a circadian rhythm. As the expression profile of the total pdf mRNA exhibited a circadian rhythm, transcription regulated at the promoter region was supposed to be controlled by some of the clock components. Whole mount in situ hybridization revealed that 14 lateral neurons at the frontal margin of the optic lobe express these mRNA isoforms. PDF expressing cells examined with a newly produced antibody raised against Apis PDF were also found to have a dense supply of axon terminals in the optic lobes and the central brain.

Molecular cloning and circadian expression profile of insect neuropeptide PDF in black blowfly, Phormia regina

Pigment-dispersing factor PDF is an 18-amino acid insect neuropeptide that mediates a circadian rhythmicity in locomotor activity. PDF is coded in a precursor protein together with another neuropeptide named PDF-associated peptide, PAP. PDF is highly conserved among insects, whereas the homology of PAPs is very low with considerably varied amino acid sequences. Since such dissimilarity has suggested that the function of PAP peptide is not associated with that of PDF, we have attempted to analyze the sequences of PDF precursor proteins among a series of species of insects and hypothesized that PDF precursors are classified into at least three different classes:Drosophila-Musca, Meimuna-Romalea, andGryllus. In order to exemplify this hypothesis, we here describe the molecular cloning of thepdf-gene of the black blowflyPhormia regina and anin silico screening for thepdf-gene in the genome databank of the mosquitoAnopheles gambie, both species belonging to the Diptera. It was found that deduced amino acid sequences of PDF peptides are almost completely conserved among all Dipterans and also the amino acid sequences of PAPs are considerably highly preserved (55–82% similarity) among the species of Diptera. The results confirmed the validity of grouping the PDF precursor proteins.In situ hybridization was carried out in fly brains to identify the precise locations ofpdf-expressing cells and to examine any daily cycling ofpdf mRNA. Intense signals forpdf mRNA were identified in the medulla, but not in the pars lateralis where PDF neurons were strongly immunostained by the antibody raised against PDF peptide. Hybridization was also performed for the brain samples at two hour intervals throughout the day. Although very intense hybridization signals were observed at ZT8 even in some neurites, no prominent rhythmicity ofpdf mRNA expression was observed.