Academic journal article Genetics

The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles

Academic journal article Genetics

The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles

Article excerpt

HORMONES are major determinants of behavior, playing essential roles in mating, feeding, stress response, and other activities related to survival and reproduction. Identifying the neural circuits through which hormones act, however, has been complicated by the fact that many hormones directly enter the central nervous system and exert their effects at broadly dispersed sites. Establishing the "connectome" of hormonal action thus clearly requires tools different from those used to study synaptic connectivity in neural circuits. Pfaff and his colleagues pioneered the strategy of using sites of hormone binding as a guide to mapping behavioral circuits: By determining the principal sites of estrogen binding in female rat brains they elucidated the network underlying the rodent lordosis response (Pfaff and Keiner 1973; Pfaff et al. 1994). Receptor mapping has similarly provided key insights into the networks underlying other behaviors in both vertebrates and invertebrates, such as feeding (Wu et al. 2003; Scott et al. 2009), sleep and circadian rhythms (Marcus et al. 2001; Im and Taghert 2010), offspring care (Insel 1990), and pair bonding (Young et al. 1997). In general, however, the labor-intensive nature of receptor mapping and the complexity of most hormonally governed neural networks has made them difficult to fully unravel.

A comparatively tractable neuroendocrine network that has been extensively characterized in insects governs the shedding of the exoskeleton at the time of molting, a process called ecdysis (Zitnan and Adams 2012; White and Ewer 2014). This process is initiated by two related peptides (ETH1 and ETH2) encoded by the Ecdysis Triggering Hormone (ETH) gene. The ETH peptides activate a stereotyped sequence of motor programs that varies with species and developmental stage. The execution of the motor programs is accompanied by the successive release of several factors, including eclosion hormone (EH), crustacean cardioactive peptide (CCAP), and bursicon. In Drosophila, neurons that secrete these factors have been shown by calcium imaging to become sequentially active after exposure to ETH peptides-most potently by ETH1-suggesting that they directly coordinate motor programming. In addition, these neurons have been shown by in situ hybridization to express the A isoform of an identified ETH receptor (ETHRA; Kim et al. 2006a,b).

The ETHR gene encodes an alternatively spliced G-protein coupled receptor first identified in the Drosophila genome in 2001 (Hewes and Taghert 2001). Splicing of the gene into two receptor isoforms appears to be highly conserved in insects (Roller et al. 2010), and in Drosophila the ETHRA and ETHRB splice isoforms are known to differ both in their affinities for the ETH peptides (Iversen et al. 2002; Y. Park et al. 2003) and their expression patterns within the nervous system at the pupal stage as determined by in situ hybridization (Kim et al. 2006b). The in vivo functions of ETHR and its two splice variants have not been characterized in detail in any insect. In the red flour beetle, Tribolium, global RNAi knockdown of ETHRA, but not ETHRB, has been shown to produce ecdysis deficits (Arakane et al. 2008). More recently in Drosophila, targeted ETHRA overexpression and tandem knockdown of both splice isoforms have been used to demonstrate that ETHR expression levels influence the timing of execution of ecdysis motor programs (Kim et al. 2015). Manipulation of ETHR expression in neurons that express both CCAP and bursicon alters the onset of the ecdysis motor program, while duration of the pre-ecdysis motor program is influenced by ETHR expression levels in a second group of neurons that express the peptide leucokinin (Lk).

To more comprehensively investigate the function of both the ETHR gene and the neurons that express it in Drosophila, we here use the recently introduced Trojan exon method of genetic targeting (Diao et al. 2015). Our results demonstrate that neurons that express the ETHRA isoform are required for ecdysis at all developmental stages and that functionally distinct subsets regulate not only behavior, but also fluid balance at the time of the pupal molt. …

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