Neuronal Remodeling During Metamorphosis Is Regulated by the alan shepard (shep) Gene in Drosophila melanogaster

Abstract

Peptidergic neurons are a group of neuronal cells that synthesize and secrete peptides to regulate a variety of biological processes. To identify genes controlling the development and function of peptidergic neurons, we conducted a screen of 545 splicetrap lines and identified 28 loci that drove expression in peptidergic neurons when crossed to a GFP reporter transgene. Among these lines, an insertion in the alan shepard (shep) gene drove expression specifically in most peptidergic neurons. shep transcripts and SHEP proteins were detected primarily and broadly in the central nervous system (CNS) in embryos, and this expression continued into the adult stage. Loss of shep resulted in late pupal lethality, reduced adult life span, wing expansion defects, uncoordinated adult locomotor activities, rejection of males by virgin females, and reduced neuropil area and reduced levels of multiple presynaptic markers throughout the adult CNS. Examination of the bursicon neurons in shep mutant pharate adults revealed smaller somata and fewer axonal branches and boutons, and all of these cellular phenotypes were fully rescued by expression of the most abundant wild-type shep isoform. In contrast to shep mutant animals at the pharate adult stage, shep mutant larvae displayed normal bursicon neuron morphologies. Similarly, shep mutant adults were uncoordinated and weak, while shep mutant larvae displayed largely, although not entirely, normal locomotor behavior. Thus, shep played an important role in the metamorphic development of many neurons. P EPTIDERGIC neurons produce small peptides, called neuropeptides, which are secreted within the nervous system to influence the activity of other neurons or into the blood to act on other tissues. Through these targets, neuropeptides regulate a wide range of processes, which include development, feeding, growth, aggression, reproduction, and learning and memory (McShane et al. 1992;Park et al. 2003;Luquet et al. 2005;Crown et al. 2007;Nephew et al. 2009;Slaidina et al. 2009;Goncalves et al. 2012).One of the first genes identified to play a specific role in the development of peptidergic neurons was dimmed (dimm), which encodes a basic helix-loop-helix transcription factor that is required for the differentiation of diverse peptidergic neurons (Hewes et al. 2003;Park et al. 2008;Hamanaka et al. 2010). DIMM is a key regulator of expression of the neuropeptide biosynthetic enzyme, peptidylglycine-a-hydroxylating monooxygenase (PHM) (Park et al. 2008), and it promotes the differentiation of neurosecretory properties in many neurons (Hamanaka et al. 2010). Both DIMM and PHM are expressed widely and specifically in peptidergic neurons (Acampora et al. 1999;Michaud et al. 1998;Jiang et al. 2000;Hewes et al. 2003). In fact, DIMM was first identified by virtue of its pattern of peptidergic neuron expression through an enhancer-trap screen (Hewes et al. 2003). Similar expression pattern-based strategies may be useful for identification of other factors critical for peptidergic neuron de...

Drosophila Shep and C. elegans SUP-26 are RNA-binding proteins that play diverse roles in nervous system development

“…For example, the development of synaptic boutons at the bursicon neuron NMJ appears normal in shep mutant larvae; however, bouton number and size are reduced in adult shep mutants (Chen et al 2014). Consistent with these findings, we find that bouton number of the RP3 motor neuron is unaffected in larvae expressing presynaptic shep RNAi.…”

“…shep was originally identified in a screen for defects in sensory-motor responsiveness to gravity, which was the first indication that it plays a role in nervous system development (Armstrong et al 2006). shep was also uncovered in a number of screens for nervous system expression within olfactory neurons (Tunstall et al 2012) and peptidergic neurons (Chen et al 2014). Moreover, shep encodes one of few mRNAs whose 3' UTR is substantially elongated within the Drosophila nervous system during neurogenesis, suggesting that shep undergoes differential posttranscriptional regulation within neurons (Hilgers et al 2011).…”

“…Moreover, shep encodes one of few mRNAs whose 3' UTR is substantially elongated within the Drosophila nervous system during neurogenesis, suggesting that shep undergoes differential posttranscriptional regulation within neurons (Hilgers et al 2011). Cellular and genetic analyses reveal that bursicon neurons of adult shep mutants exhibit a reduced neuropil and aberrant morphogenesis of axonal branches and boutons (Chen et al 2014). Many of these phenotypes are associated with inappropriate metamorphic development and suggest that shep mainly functions in later stages of neurogenesis.…”

“…The UAS-shepRNAi VDRC line has previously been shown to effectively knockdown Shep in the Drosophila nervous system (Chen et al 2014). Dendrite morphology was visualized by the concomitant expression of membranetethered GFP using the UAS-CD4:GFP transgene.…”

Regulatory Mechanisms of Metamorphic Neuronal Remodeling Revealed Through a Genome-Wide Modifier Screen in Drosophila melanogaster

“…Loss of shep leads to defective outgrowth of peptidergic bursicon neurons, developmental lethality, and behavioral defects, all of which are largely adult-specific (Chen et al 2014). Loss of shep also interferes with the development of nociceptive and proprioceptive neurons in the larval peripheral nervous system (Schachtner et al 2015).…”