Characterization of the insect cuticle sclerotization hormone bursicon and bursicon-regulated genes in the house fly Musca domestica

Bursicon is a neurohormone that regulates cuticle sclerotization (tanning and hardening) and the wing expansion processes in insects. Bursicon was discovered over forty years ago from the blowfly Calliphora erythrocephala in a neck-ligated fly assay. However due to the difficulties in hormone protein purification and the lack of molecular techniques, the genes encoding bursicon were not identified until in the years of 2004-2005. Studies in Drosophila melanogaster have indicated that bursicon is actually a heterodimeric cystine knot family protein containing two subunits, bursicon alpha (bur alpha or burs) and bursicon beta (bur beta or pburs), which are encoded by two individual genes burs alpha and burs beta (or burs and pburs). Although bursicon genes have been cloned from several insect species or predicted from insect genomes, little is known about its mechanisms in the cuticle sclerotization and wing expansion processes, especially the signal transduction pathway(s) and the genes regulated by bursicon.;In the current study, the house fly, Musca domestica, was selected as the research model, because of its relatively larger body size for easy dissection and microscopic manipulation when compared to Drosophila, its distinguished cuticle sclerotization phenotype for easy detection, and a house fly bioassay system we developed in our laboratory for the detection of bursicon activity. The burs alpha and burs beta genes were cloned from M. domestica using 5' and 3' rapid amplification of cDNA ends (RACEs). The M. domestica burs alpha gene has an open reading frame (ORF) of 531 bps, encoding a 176 amino acid (a.a.) polypeptide with a predicted molecular weight of 19.5 kDa, while the burs beta gene has an ORF of 444 bps, encoding a 147 a.a. polypeptide with a predicted molecular weight of 17 kDa. The M. domestica burs alpha and burs beta both share 79% sequence identity with their Drosophila counterparts, and the identities with the bursicon sequences from other insect species range from 47% to 61%. A developmental study revealed that both M. domestica burs alpha and burs beta transcripts were present in larval and pupal stages, maximally expressed in pharate adults, and declined sharply after adult emergence, suggesting the release of the hormone at adult emergence. Recombinant Musca and Drosophila bursicon heterodimer (r-bursicon) was expressed in mammalian 293 cells and insect Highfive(TM) cells. R-bursicon expressed in both systems showed a strong bursicon activity in the neck-ligated house fly assay, and also showed cross species activities between Musca and Drosophila . Fluorescence in situ hybridization (FISH) studies of bursicon distribution in the central nervous system of M. domestica and D. melanogaster indicated that in M. domestica, both burs alpha and burs beta transcripts were expressed in a set of neurosecretory cells (NSCs) in the subeosophagael ganglia (SEG) and abdominal ganglia (AG) in larvae and pupae, but only in the fused thoracic-abdominal ganglia (TAG) in the adults. This is similar to the expression pattern as detected in D. melanogaster although some differences do exist.;By using r-bursicon with DNA microarray analysis, a series of genes were identified that are very likely involved in the bursicon-regulated cuticle sclerotization and wing expansion processes. Among these identified genes, two genes were cloned and sequenced for further study in the house fly M. domestica. One of them, mdSu(H), is a homolog of the D. melanogaster Suppressor of Hairless (Su(H) ) gene. Su(H) is a neurogenic transcriptional factor and has been demonstrated to be involved in regulating several insect neurogenesis processes and many other physiological events in insects. Real-time RT-PCR analysis indicated that the level of mdSu(H) transcript was up-regulated by ∼2.5-3 folds 1 h after r-bursicon injection, which correlated well with the cuticle sclerotization process observed in the r-bursicon injected neck-ligated flies. Developmental studies showed that in normal (non-ligated) flies, this gene was also highly expressed after ecdysis, indicating its participation in post-ecdysis events, possibly cuticle sclerotization and/or the wing expansion process. Su(H) is a component of the Notch signaling pathway. After analyzing our DNA microarray data, we found that 11 other genes, which are either directly involved in or associated with the Notch signaling pathway, were also up-regulated upon r-bursicon stimulation in the neck-ligated fly assay. This indicates that the Notch pathway might be an important signal transduction pathway involved in the bursicon-mediated sclerotization and wing expansion processes. A second gene of interest to us, mdPH, which is a homolog of D. melanogaster pleckstrin homology (PH) gene encoding a pleckstrin homology (PH) protein, was also cloned and sequenced in M. domestica. Analysis of the mdPH gene showed that it is down-regulated by ∼2-3 fold upon r-bursicon stimulation in the neck-ligated flies, starting from 40 minutes after injection. Developmental analysis of mdPH transcripts in the non-ligated house flies also showed that it is down-regulated after adult emergence. PH domains are present in a number of proteins. Many of their functions are related to signal transduction, e.g. in the G protein coupled receptor signaling pathway, which is now widely accepted as a downstream transduction pathway during bursicon-mediated cuticle sclerotization and wing expansion. In this pathway, PH has an inhibitory effect on G protein coupled receptor (GPCR) by activating G protein coupled receptor kinase-2 (GRK-2) and then phosphorylating the GPCR, thus repressing the activation of GPCR and blocking the transduction of signal. Rapid down-regulation of the repressor PH upon r-bursicon administration allows activation of GPCR and subsequent signal transduction, leading to cuticle sclerotization and wing expansion. This result indicates that PH is also very likely an important element involved in the bursicon signal transduction pathway.