Functional Conservation of Clock Output Signaling between Flies and Intertidal Crabs

• Published in: Journal of biological rhythms 2011-12, Vol.26 (6), p.518-529
• Main Authors: Beckwith, Esteban J., Lelito, Katherine R., Hsu, Yun-Wei A., Medina, Billie M., Shafer, Orie, Ceriani, M. Fernanda, de la Iglesia, Horacio O.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Behavior, Animal - physiology; Biological clocks; Biological Clocks - physiology; Brachyura - anatomy & histology; Brachyura - physiology; Brain - anatomy & histology; Brain - metabolism; Cancer productus; Circadian Rhythm - physiology; Crustaceans; Drosophila melanogaster; Drosophila melanogaster - anatomy & histology; Drosophila melanogaster - physiology; Drosophila Proteins - classification; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Gene Duplication; Insects; Molecular Sequence Data; Neurons - metabolism; Neuropeptides - classification; Neuropeptides - genetics; Neuropeptides - metabolism; Peptides - classification; Peptides - genetics; Peptides - metabolism; Phenotype; Phylogeny; Sequence Alignment; Signal Transduction - physiology
• ISSN: 0748-7304; 1552-4531
• DOI: 10.1177/0748730411420242

Intertidal species have both circadian and circatidal clocks. Although the behavioral evidence for these oscillators is more than 5 decades old, virtually nothing is known about their molecular clockwork. Pigment-dispersing hormones (PDHs) were originally described in crustaceans. Their insect homologs, pigment-dispersing factors (PDFs), have a prominent role as clock output and synchronizing signals released from clock neurons. We show that gene duplication in crabs has led to two PDH genes (β-pdh-I and β-pdh-II). Phylogenetically, β-pdh-I is more closely related to insect pdf than to β-pdh-II, and we hypothesized that β-PDH-I may represent a canonical clock output signal. Accordingly, β-PDH-I expression in the brain of the intertidal crab Cancer productus is similar to that of PDF in Drosophila melanogaster, and neurons that express PDH-I also show CYCLE-like immunoreactivity. Using D. melanogaster pdf-null mutants (pdf01) as a heterologous system, we show that β-pdh-I is indistinguishable from pdf in its ability to rescue the mutant arrhythmic phenotype, but β-pdh-II fails to restore the wild-type phenotype. Application of the three peptides to explanted brains shows that PDF and β-PDH-I are equally effective in inducing the signal transduction cascade of the PDF receptor, but β-PDH-II fails to induce a normal cascade. Our results represent the first functional characterization of a putative molecular clock output in an intertidal species and may provide a critical step towards the characterization of molecular components of biological clocks in intertidal organisms.