Academic journal article Genetics

Neuronal Remodeling during Metamorphosis Is Regulated by the Alan Shepard (Shep) Gene in Drosophila Melanogaster

Academic journal article Genetics

Neuronal Remodeling during Metamorphosis Is Regulated by the Alan Shepard (Shep) Gene in Drosophila Melanogaster

Article excerpt

PEPTIDERGIC 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, neuro- peptides 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 specificrolein 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 neuro- peptide 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 crit- ical for peptidergic neuron development.

In this study, we sought to identify similar factors through a splice-trap screen for genes with peptidergic cell-speci fi c expression patterns. We identified 28 insertions with differ- ent patterns of peptidergic cell reporter gene expression, driven by P-element splice-trap insertions in specific loci. These insertions drove reporter expression in insulin-like peptide 2 (ILP2), crustacean cardioactive peptide (CCAP)/ bursicon, -RFamide, Furin 1, and leucokinin (LK) cells and often caused defects typical of disrupted neuropeptide sig- naling. Thus, all 28 of these genes are strong candidate regulators of peptidergic cell development or function.

We mapped one of the splice-trap insertions to an exon of the alan shepard (shep) gene, and we chose this insertion for further analysis because it displayed an expression pattern that was highly similar to PHM and DIMM. shep in situ hybridization and anti-SHEP immunostaining later revealed that both the shep mRNA and SHEP protein expression is enriched in most neurons, yet shep mutants displayed defects in adult eclosion and wing expansion that suggested specific disruptions in signaling by bursicon and other neu- ropeptides. Consistent with these behavioral phenotypes, the shep mutant bursicon neurons had smaller somata, fewer axon branches, and smaller and fewer neuroendo- crine boutons, and all of these phenotypes were rescued by expression of a wild-type shep cDNA. Interestingly, pan- neuronal RNA interference to shep produced smaller CNS neuropils and defects in general locomotor behaviors, such as flipping and climbing. Most of the locomotor phenotypes were restricted to the adult stage, and the effects of shep mutations on neuronal growth were restricted to pupal de- velopment. Thus, shep regulates metamorphic growth of the bursicon neurons, and it may also serve as a general regu- lator of neuronal growth during metamorphic remodeling.

Materials and Methods

Stocks

Drosophila melanogaster stocks and crosses were cultured on standard cornmeal-yeast-agar media at 25^.Weobtained splice-trap strains, deficiency strains, and Gal4 strains from the Bloomington Drosophila Stock Center. We used three shep deficiencies: Df(3L)ED210 (FBab0035327), Df(3L)Exel6104 (FBab0038124), and Df(3L)Exel6103 (FBab0038123). The Gal4 lines used were 386-Gal4 (FBti0020938) (Bantignies et al. 2000), en-Gal4 (Fox et al. 2010), D42-Gal4 (Yeh et al. 1995), elav-Gal4 (FBti0002575) (Shakiryanova et al. …

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