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Genetics. Feb 2008; 178(2): 883–901.
PMCID: PMC2248363

A Drosophila Gain-of-Function Screen for Candidate Genes Involved in Steroid-Dependent Neuroendocrine Cell Remodeling

Abstract

The normal functioning of neuroendocrine systems requires that many neuropeptidergic cells change, to alter transmitter identity and concentration, electrical properties, and cellular morphology in response to hormonal cues. During insect metamorphosis, a pulse of circulating steroids, ecdysteroids, governs the dramatic remodeling of larval neurons to serve adult-specific functions. To identify molecular mechanisms underlying metamorphic remodeling, we conducted a neuropeptidergic cell-targeted, gain-of-function genetic screen. We screened 6097 lines. Each line permitted Gal4-regulated transcription of flanking genes. A total of 58 lines, representing 51 loci, showed defects in neuropeptide-mediated developmental transitions (ecdysis or wing expansion) when crossed to the panneuropeptidergic Gal4 driver, 386Y-Gal4. In a secondary screen, we found 29 loci that produced wing expansion defects when crossed to a crustacean cardioactive peptide (CCAP)/bursicon neuron-specific Gal4 driver. At least 14 loci disrupted the formation or maintenance of adult-specific CCAP/bursicon cell projections during metamorphosis. These include components of the insulin and epidermal growth factor signaling pathways, an ecdysteroid-response gene, cabut, and an ubiquitin-specific protease gene, fat facets, with known functions in neuronal development. Several additional genes, including three micro-RNA loci and two factors related to signaling by Myb-like proto-oncogenes, have not previously been implicated in steroid signaling or neuronal remodeling.

NEURONS display extensive morphological and functional changes after terminal differentiation. The resulting changes in neuronal activity shape nervous system homeostasis, seasonal and developmentally staged behavior, learning, and responses to stress, injury, and disease (Burbach et al. 2001; Zitnanova et al. 2001; Thiery et al. 2002; Viau 2002; Karmarkar and Dan 2006; Romeo and McEwen 2006; Arancio and Chao 2007; Navarro et al. 2007). In recent years, exponential progress has been made toward understanding the molecular and cellular mechanisms underlying neuronal plasticity. However, the factors governing developmental remodeling in neuroendocrine systems remain poorly understood.

Metamorphosis of the insect nervous system involves extensive developmental reorganization. Differentiated larval neurons adopt one of two fates: programmed cell death or morphological remodeling (Truman 1992). The programmed cell death of many larval neurons occurs through autophagy or apoptosis (Weeks 2003; Choi et al. 2006). Neuronal remodeling involves the selective elimination of larval neurites and the outgrowth and elaboration of adult-specific projections (Levine and Truman 1982; Lee et al. 2000). These events support the transformation of the insect from a vermiform larva that is devoted to feeding into an active adult with well-developed legs, wings, and sensory organs and complex reproductive behavior. Molting and metamorphosis are coordinated by two families of hormones, the juvenile hormones and the ecdysteroids (Nijhout 1994). During metamorphosis, ecdysteroids act cell autonomously to control neuronal cell fates (Robinow et al. 1993; Lee et al. 2000; Brown et al. 2006) through evolutionarily conserved cellular mechanisms and signaling pathways (Draizen et al. 1999; Kinch et al. 2003; Watts et al. 2003; Choi et al. 2006; Hoopfer et al. 2006).

Successful molt completion requires precise timing of ecdysis behaviors, which lead to shedding of the old cuticle. This is controlled, in part, by declining ecdysteroid levels that act through a hierarchical cascade of neuropeptides and peptide hormones to trigger the behaviors (Ewer and Reynolds 2002). Intensive study over the past four decades has revealed many salient features of this hierarchy, although some components of the system remain to be identified. In the current model (Ewer and Reynolds 2002), one of the first steps is activation of the endocrine Inka cells, which are located peripherally on the tracheae. In Drosophila, the Inka cells produce two related peptide hormones, referred to collectively as ecdysis-triggering hormone (ETH). ETH stimulates the secretion of additional peptide hormones, including eclosion hormone (EH) and crustacean cardioactive peptide (CCAP). These and other neuropeptides contribute to the control of ecdysis behaviors (Clark et al. 2004; Kim et al. 2006). After ecdysis, some of the CCAP neurons are thought to secrete CCAP and additional neuropeptides to control postecdysis behaviors. These include bursicon, a heterodimeric neuropeptide hormone that controls wing expansion behaviors and cuticular sclerotization shortly after adult ecdysis.

The CCAP/bursicon neurons undergo substantial remodeling during the pupal stage (Luan et al. 2006). These changes likely underlie some of the differences following metamorphosis in the timing, pattern, and function of ecdysis and postecdysis behaviors. Many other neuropeptidergic cells with known or potential roles in the control of molting-related behaviors also undergo metamorphic remodeling (e.g., Riddiford et al. 1994; Schubiger et al. 1998). These changes are accessible for relatively high-throughput genetic screens. Although ecdysis and postecdysis behaviors are generally completed within a few minutes, the targeted ablation of the Drosophila CCAP/bursicon and EH neurons results in a variety of easily observable phenotypes that can be scored days later. These include failure to evert the adult head at pupal ecdysis and failure to expand the wings and sclerotize the adult cuticle (McNabb et al. 1997; Park et al. 2003). Similar phenotypes are produced through the targeted manipulation of cell signaling within these neurons (Cherbas et al. 2003; Park et al. 2003; Hodge et al. 2005; Luan et al. 2006).

Drosophila genetic mosaic methods provide powerful tools for the inhibition or stimulation of gene function in small numbers of neurons (down to the level of single cells) and at specific stages in development (Lee and Luo 1999; Mcguire et al. 2004). These tools allow the experimental manipulation of signaling pathways involved in metamorphic remodeling within the complex brain, where cells display the impact of these changes within the context of hormonal signals, metabolic and other microenvironmental cues, and other cellular interactions. Coupled with our detailed understanding of the neuroendocrine control of metamorphosis and molting behaviors, this system provides unique opportunities to perform unbiased genetic screens for novel regulators of neuronal remodeling. However, genes that regulate nerve cell remodeling during metamorphosis are likely to also regulate the embryonic or larval development of neurons (temporal pleiotropy) or the development of other tissues (spatial pleiotropy). If these other functions are essential to the survival of embryos or larvae, then they will preclude the observation of loss-of-function (LOF) mutant phenotypes involving the disruption of neuropeptidergic cell metamorphosis. Gain-of-function (GOF) screening may overcome these problems of spatial and temporal pleiotropy and can also reveal gene functions even when other genes have redundant functions (Rørth 1996; Rørth et al. 1998). As a proof of principle, GOF screens in several other well-studied Drosophila developmental models have identified many genes with previously confirmed roles (Rørth 1996; Rørth et al. 1998; Abdelilah-Seyfried et al. 2000; Kraut et al. 2001; Pena-Rangel et al. 2002; Tseng and Hariharan 2002; McGovern et al. 2003). GOF screens also have resulted in the identification of several novel and important developmental regulators, thus confirming the utility of this approach for gene discovery (Brennecke et al. 2003; Sherwood et al. 2004; Teleman et al. 2005, 2006).

Here, we performed a GOF screen to identify candidate regulators of ecdysteroid-dependent metamorphosis of neuropeptidergic cells. We first used the Gal4/upstream activating sequence (UAS) system to direct expression of several known cell-signaling regulatory molecules to three different populations of neuropeptidergic cells. These experiments established the feasibility of this approach by demonstrating our ability to detect ecdysis and wing expansion defects in the progeny. We then used this method to perform a systematic GOF screen of 6097 lines that provided neuropeptidergic cell-targeted activation of randomly selected loci in the Drosophila genome. These experiments revealed at least 14 loci with putative functions in the formation or maintenance of adult-specific neurite projections during metamorphosis. Our screen also revealed the existence of additional, as yet unidentified, neuropeptidergic cells with critical roles in the signaling hierarchy controlling ecdysis and wing expansion.

MATERIALS AND METHODS

Stocks:

Flies (Drosophila melanogaster) were cultured on standard cornmeal–yeast–agar media at 22°–25°, and test crosses were performed at 29° unless otherwise noted. EP lines with insertions on the second and third chromosomes (Rørth 1996; Rørth et al. 1998) were obtained from the Szeged Drosophila Stock Center. EP lines with insertions on the X chromosome and EY, WH, and XP lines (Bellen et al. 2004) with insertions on chromosomes X–4 were obtained from the Bloomington Drosophila Stock Center. The Gal4 drivers used were EH-Gal4 (w*; P{GAL4-Eh.2.4}C21; FBti0012534) (McNabb et al. 1997), c929-Gal4 (w*; P{GawB}crc929; FBti0004282) (O'Brien and Taghert 1998), 386Y-Gal4 (w*;; P{GAL4}386Y; FBti0020938) (Bantignies et al. 2000), and CCAP-Gal4 (y* w*; P{Ccap-GAL4.P}16; FBti0037998) (Park et al. 2003). Other lines were obtained from the Bloomington Drosophila Stock Center or were kindly provided by individual labs (supplemental Table 1 at http://www.genetics.org/supplemental/).

Insertion-site analysis:

Flanking DNA sequences for the target lines used in this screen were previously determined [EP and EY lines (Abdelilah-Seyfried et al. 2000; Bellen et al. 2004) and XP and WH lines (Thibault et al. 2004)]. For each target line obtained through the GOF screening, we used the University of California, Santa Cruz (Karolchik et al. 2003), and FlyBase (Grumbling et al. 2006) Genome Browsers and the BLASTN user service at the National Center for Biotechnology Information to identify transcripts near the insertion. We defined the putative target gene(s) as the first locus (loci) within 30 kb of the insertion site and in the same orientation (GOF) as the run-off transcript derived from the UAS sites in the target element. We also identified loci that were in reverse orientation, within 5 kb of the insertion, and located closer to the insertion than any transcripts in the forward orientation, as potential mediators of LOF phenotypes (cf. Abdelilah-Seyfried et al. 2000).

We used three criteria—phenocopy with UAS transgenes, induction of elevated immunostaining, or induction of in situ hybridization signals—to independently validate the identification of the misexpressed genes for selected GOF lines. The identifications of 9 loci have been validated, including 7 of the 17 loci included in our analysis of CCAP/bursicon cellular phenotypes (see supplemental Results and Discussion at http://www.genetics.org/supplemental/): cabut (cbt), fat facets (faf), forkhead box sub-group O (foxo), miR-310–miR-313, miR-276a, miR-279, Myb-interacting protein 120 (mip120), pointed (pnt), and split ends (spen).

Immunochemistry and quantification:

Immunostaining was performed on central nervous system (CNS) or whole-animal fillet preparations obtained from wandering larvae, prepupae at the indicated times after puparium formation (APF), staged pupae (Bainbridge and Bownes 1981), or adults at the indicated times after eclosion. After dissection in calcium-free saline [182 mm KCl, 46 mm NaCl, 2.3 mm MgCl2.6H2O, 10 mm 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris), pH 7.2], tissues were fixed for 1 hr at room temperature (RT) in either 4% paraformaldehyde (PFA) or 4% paraformaldehyde/7% picric acid (PFA/PA), and immunostaining was performed as described (Hewes et al. 2003, 2006). We used antisera directed against the following proteins: CCAP (1:4000, PFA/PA) (Park et al. 2003), Bursicon α-subunit (1:5000, PFA/PA) (Luan et al. 2006), green fluorescent protein (GFP) (1:500, PFA) (Invitrogen, Carlsbad, CA), Split ends (SPEN) (1:50, PFA) (Chen and Rebay 2000), and Myb-interacting protein 120 (MIP120) (1:1000, PFA) (Lewis et al. 2004). Confocal z-series projections were obtained using an Olympus (Center Valley, PA) Fluoview FV500 microscope. Many of the grayscale images were inverted in Adobe (San Jose, CA) Photoshop for better visualization of fine cellular processes. Images of external structures were obtained on an Olympus SZX12 stereomicroscope with a SPOT RT camera and software (Diagnostic Instruments, Sterling Heights, MI). Photomontages of the images were obtained using Adobe Photoshop. Test and control preparations, and preparations in each developmental time series, were stained and imaged in parallel.

For quantification of the extent of neurite pruning and outgrowth during metamorphosis and of bursicon secretion in adult animals, we used the threshold function in Adobe Photoshop (with the same threshold for all images) to convert the background to white and all remaining pixels (neurites and somata) to black. Somata and any obvious artifacts were manually cut from the image, and then we obtained a count of the black pixels. For time points during which pruning and outgrowth overlapped, the extent of each was obtained by manually cutting away portions of the arbor and then recounting the number of black pixels. We reported variances as standard errors (for means) or interquartile ranges (IQR, distance between the 75th percentile and the 25th percentile). Statistics were performed using NCSS-2001 software (NCSS, Kaysville, UT).

In situ hybridization with locked nucleic acid probes:

We performed in situ hybridization using a protocol modified from Li and Carthew (2005) with digoxigenin (DIG)-labeled locked nucleic acid (LNA) probes (Exiqon, Woburn, MA). The probes and the modified protocol are described in the supplemental Materials and Methods at http://www.genetics.org/supplemental/. We obtained near-infrared fluorescent images of the insoluble purple precipitate (McCauley and Bronner-Fraser 2006; Trinh et al. 2007), using a Zeiss Axio Imager Z1 system with an Hg arc lamp source, a 645- to 685-nm excitation band pass filter, and a 760-nm long pass emission filter. We also captured weak autofluorescence of the tissue with the fluorescein filters.

Expression patterns of the Gal4 drivers:

We used four Gal4 lines—EH-Gal4, CCAP-Gal4, c929-Gal4 [dimmed-Gal4 (Hewes et al. 2003)], and 386Y-Gal4—to direct GOF element expression to peptidergic cells. These lines were chosen to target different populations of cells, some of which are known to control key aspects of ecdysis and wing expansion behaviors. Reporter gene expression in the EH-Gal4 line is limited to just two CNS neurons, the ventromedial EH neurons (McNabb et al. 1997). The CCAP-Gal4 driver is expressed specifically in ~35 pairs of neurons in the brain and ventral nerve cord (VNC) that produce CCAP and bursicon (Park et al. 2003; Dewey et al. 2004; Luo et al. 2005). The c929-Gal4 driver is expressed in several peptidergic cell types, including 100–200 heterogeneous CNS neurons, intrinsic endocrine cells in the corpora cardiaca, the endocrine Inka cells, midgut cells, and peripheral nervous system (PNS) neurons. c929-Gal4 also drives transgene expression in scattered locations in other tissues, including fat body, epithelial cells, and salivary glands (Hewes et al. 2003). The 386Y-Gal4 element drives transgene expression in numerous CNS peptidergic neurons and in many peripheral endocrine cells, including the Inka cells (Bantignies et al. 2000; Taghert et al. 2001). This driver produced the largest number of Gal4-positive neurons and secretory cells of the three lines used for this screen. All four lines yield strong transgene expression in peptidergic cells beginning in late embryos or early larvae and continuing through the adult stage (McNabb et al. 1997; Bantignies et al. 2000; Taghert et al. 2001; Hewes et al. 2003; Park et al. 2003; Luan et al. 2006).

RESULTS

Overview of the GOF screen:

We conducted a modular GOF screen in which we misexpressed native Drosophila genes in neuropeptidergic cells and examined the effects on neuropeptide-mediated developmental transitions. The screen was performed in three phases. In phase I, we examined whether gene GOF in these Gal4 patterns could produce defects in molting-related behaviors by crossing EH-Gal4, c929-Gal4, and 386Y-Gal4 to a collection of UAS lines controlling expression of cell-signaling proteins. These included wild-type, dominant-negative, or constitutively active factors involved in cAMP signaling [dunce (dnc), Cyclic-AMP response element binding protein B at 17A (CrebB-17A), Jun-related antigen (Jra), kayak (kay)], Ca2+ signaling [Calmodulin (Cam)], ecdysteroid action [Ecdysone receptor (EcR)], endocytosis [shibire (shi)], and electrical excitability [Open rectifier K+ channel 1 (Ork1)] (supplemental Table 1). Each of the Gal4 drivers produced molting or metamorphosis defects in combination with multiple UAS lines (supplemental Table 2 at http://www.genetics.org/supplemental/). The mutant phenotypes (Figure 1) included (1) larval lethality, often associated with a failure to properly shed the larval mouthparts (Hewes et al. 2000), (2) retention of larval characteristics following pupariation, (3) pupal lethality associated with a failure to properly evert the adult head and to fully extend the legs and wings (head eversion defects) (Figure 1, C and D), (4) late pupal lethality associated with the completion of adult development [through stage P15(i) (Bainbridge and Bownes 1981)] and failure to eclose (pharate adult lethality), and (5) adults that displayed defective tanning of the cuticle and that partially or completely failed to expand their wings (Figure 1B). Similar phenotypes have previously been reported following expression of wild-type or dominant-negative constructs with all four of the above Gal4 drivers (McNabb et al. 1997; Bantignies et al. 2000; Cherbas et al. 2003; Clark et al. 2004; Dewey et al. 2004; Hodge et al. 2005; Kim et al. 2006; Luan et al. 2006).

Figure 1.
Head eversion and wing expansion phenotypes produced by gene GOF in peptidergic neurons. (A) Oregon R (wild-type) adult female. (B) Examples of y, w; CCAP-Gal4/EY(2)04392 females with normal wings (normal), partially expanded wings (PEW), and unexpanded ...

The behaviors leading to head eversion, adult eclosion, and adult wing expansion all occur during brief (<1 hr) periods (Baker and Truman 2002; Park et al. 2003). However, the disruption of any of these events leads to morphological defects that can be easily scored days later by visual examination of culture vials. Therefore, we used these three events to detect genetic interactions in phases II and III of the screen. In phase II, we crossed EH-Gal4, c929-Gal4, and 386Y-Gal4 to a collection of 1808 EP lines, each with an insertion on the second or third chromosome. We obtained 16 lines that displayed head eversion, adult eclosion, or wing expansion defects with the 386Y-Gal4 driver. In contrast, the c929-Gal4 driver yielded 2 lines (both of which also interacted with 386Y-Gal4), and the EH-Gal4 driver produced no hits in this phase of the screen (see supplemental Results and Discussion). Therefore, in phase III of the screen, we crossed 386Y-Gal4 only to a collection of 202 EP lines with insertions on the X chromosome and an additional set of 4087 EY, WH, and XP lines with insertions on the X and all three autosomes. With the exception of XP(3)d02595 (see below), lines that did not produce viable prepupae after two attempts were discarded and were not included in the above counts; we did not attempt to recover lines that may have produced mutant phenotypes that were restricted to larval ecdysis. In total (phases II and III combined), we obtained 57 of 6097 lines (0.93%, representing 50 independent loci) that produced defects in head eversion, adult eclosion, or wing expansion in the progeny when crossed to 386Y-Gal4. One additional line (and locus), XP(3)d02595, produced larval lethality associated with defects in larval ecdysis. Because each of these mutant phenotypes occurs naturally at low frequencies in wild-type stocks, we scored crosses as hits in the screen only when the defects occurred in at least 10% of the pupae or adults. These 57 lines, plus XP(3)d02595, were then crossed to two drivers, c929-Gal4 and CCAP-Gal4, that produce Gal4 expression in subsets of the 386Y-Gal4 pattern. Only 14% (8 of 58) of the lines produced phenotypes when crossed to c929-Gal4, while 59% (34 of 58) of the lines did so with CCAP-Gal4 (Table 1). Supplemental Table 3 at http://www.genetics.org/supplemental/ lists the lines obtained in phases II and III of the screen, the distance and orientation of each insertion with respect to the reference sequence landmark, the genes targeted by these insertions, and the mutant phenotypes observed with each Gal4 driver.

TABLE 1
Lines with head eversion, pharate adult lethal, or wing expansion phenotypes with each Gal4 driver

The frequency of gene hits obtained in the screen was dependent upon the class of response element used. The rank order of effectiveness in generating phenotypes was XP > EYEP > WH (Table 1). These differences likely reflect multiple factors, including the difference in insertion-site distributions obtained with PiggyBac (WH) vs. P (EP, EY, and XP) elements (Bellen et al. 2004) and the bidirectional (XP) vs. unidirectional (EP, EY, and WH) orientation of transcription off of the different drivers. However, the frequencies of hits obtained also may have been skewed by the preselection of lines deposited into the stock centers (Bellen et al. 2004; Thibault et al. 2004), and our results provide only a rough estimate of the relative effectiveness of these different elements in the screen.

Most of the insertions (52 of the 55 in regions with known transcripts) were located within +1.0 kb to −7.0 kb of a confirmed transcriptional start site (EST or cDNA) or intron splice acceptor site (Figure 2). Thus, in most cases, the affected transcripts appear to be located within 7 kb of the insertion. In three lines [EY(3)00681, EY(3)10546, and EY(3)13010], the GOF phenotype may result from transcription over longer distances (approaching 25 kb). We obtained indirect support of this hypothesis through identification of two insertions, EP(3)3523 and EY(3)13010, that are both upstream of the miR-276a locus. EY(3)13010 is located ~23 kb farther away (supplemental Table 3). Both insertions led to pharate adult lethality when crossed to 386Y-Gal4, but only EP(3)3523 produced wing expansion defects when crossed to CCAP-Gal4. Therefore, while both insertions may misexpress the same locus, the closer insertion appeared to produce a stronger gain-of-function phenotype, presumably through more efficient transcription of the nearer target. However, we cannot exclude the possibility that EY(3)13010 produced a similar phenotype through GOF of a second, unannotated gene.

Figure 2.
Scatter plot of target element insertion-site distances from the nearest promoter or exon. The distance for each target element to the 5′ end of the nearest promoter (black diamonds) or exon (magenta circles) was plotted on the y-axis. Negative ...

Metamorphosis of the CCAP/bursicon neurons:

As a basis for determining the cellular consequences of neuropeptidergic cell overexpression of the genes obtained in the screen, we first characterized the morphology of control CCAP/bursicon neurons in larvae and in pupae at various stages of metamorphosis. We labeled the cells with either mCD8::GFP, a membrane-linked fusion protein that provides excellent visualization of fine cellular processes (visualized directly or through anti-GFP immunostaining) (Lee and Luo 1999), or antisera recognizing CCAP and the bursicon α-subunit (hereafter referred to as bursicon). Consistent with prior observations (Dewey et al. 2004; Luo et al. 2005), in wandering third-instar larvae, most elements of the CCAP/bursicon cell pattern were visible following anti-bursicon immunostaining (Figure 3A). This generally produced a much stronger signal than anti-CCAP immunostaining (Figure 3B). However, the MP neurons and their processes in the brain and ventral nerve cord (MPA and MLT; Figure 3B) were not stained with the anti-bursicon antiserum, although they were strongly CCAP immunoreactive (cf. Dewey et al. 2004). With the exception of the brain DP neurons (which were weakly bursicon positive and CCAP negative), most of the CCAP- and bursicon-positive somata and neurites were also clearly visible after labeling with mCD8::GFP under control of the CCAP-Gal4 driver (Figure 3C). Some of the finer processes were more intensely labeled with the anti-neuropeptide antisera then with mCD8::GFP (e.g., MPA in Figure 3, B and C), presumably due to the concentration of secretory granules in portions of the arbor.

Figure 3.
Staining patterns and morphologies of the CCAP/bursicon neurons. (A) Anti-bursicon (BURS) and (B) anti-CCAP (CCAP) immunostaining in wandering third-instar larval CNS (CCAP-Gal4/+). Anti-bursicon immunostaining was also observed in a cluster of ...

During metamorphosis, the CCAP/bursicon cells underwent substantial remodeling, resulting in an adult pattern of neuritic projections that was markedly different from the larval pattern (Figure 3D) (Luan et al. 2006). To track the fate of individual larval neurons and portions of the neurite arbor, we examined anti-bursicon and anti-GFP immunostaining on CCAP-Gal4, UAS-mCD8::GFP animals at various stages during metamorphosis: at 3-hr intervals for the first 24 hr APF, at 6-hr intervals for the period between 24 and 60 hr APF, and at 72 hr APF (Figure 4 and supplemental Figure 1 at http://www.genetics.org/supplemental/). We could follow the neurons throughout metamorphosis—most of the larval CCAP/bursicon cells were retained—and we did not observe the development of new cells with this marker during the 72-hr period APF. We first detected the loss of bursicon/GFP immunoreactive neurites in the thoracic and abdominal ganglia at 3 hr APF. The peak amount of pruning occurred from 12 to 15 hr APF, after which the disappearance of larval neurites continued at a slower rate until ~30 hr APF. The first appearance of new, adult-specific projections occurred simultaneously with the latter stages of larval neurite pruning, and peak outgrowth occurred at 36–42 hr APF in the abdominal ganglia and at 48–54 hr APF in the thoracic ganglia. Outgrowth was essentially complete at 60 hr APF throughout the VNC.

Figure 4.
Remodeling of the CCAP/bursicon neurons during metamorphosis. (A) Anti-bursicon immunostaining of the ventral nerve cord (VNC) at 0, 12, 36, and 60 hr after puparium formation (APF) (n = 6–19). Additional time points are shown in supplemental ...

In addition to the pruning back of larval neurites and outgrowth of new, adult-specific neurites, there were three other notable changes in the CCAP/bursicon cell pattern in the CNS during metamorphosis. First, the somata in abdominal segments A1–A7 more than doubled in diameter (Figure 4) and later became multiangular in shape (not shown). Their orderly segmental arrangement was lost as they migrated or were pushed to new locations during the reorganization of the abdominal ganglia. Second, bursicon expression was turned off in 14 abdominal neurons. In larvae there was a pair of bursicon-immunoreactive neurons in each abdominal hemisegment. One cell in each pair was smaller and more weakly bursicon (and CCAP) immunoreactive. During the first 12–24 hr APF, these more weakly bursicon-positive neurons lost this epitope (Figure 4A and supplemental Figure 1), although they continued to express mCD8::GFP (Figure 4B). Third, a few CCAP/bursicon cells either died or turned off CCAP, bursicon, and CCAP-Gal4 expression during metamorphosis. The most prominent example of this class of cells was the MP neurons (Figure 3), which were strongly CCAP immunoreactive in larvae but undetectable shortly after head eversion (data not shown).

In larvae, many of the CCAP/bursicon neurons in the VNC had efferent projections. We observed five pairs of unbranched axons that were weakly bursicon and CCAP immunoreactive and that projected to larval muscles 12 and 13 (Hodge et al. 2005). There they terminated in a few higher-order branches with numerous strongly bursicon- and CCAP-immunoreactive type III endings (supplemental Figure 2 at http://www.genetics.org/supplemental/ and data not shown). Up to three additional pairs of axons projected out through terminal abdominal nerves. These projections did not terminate on the body wall muscles and did not branch, although strongly bursicon- and CCAP-immunoreactive varicosities were present near the distal tips (see below, Figure 6B).

Figure 6.
GOF of cbt prevented the outgrowth of adult-specific bursicon-immunoreactive neurites and the increase in diameter of the CCAP/bursicon cell somata during metamorphosis. (A–D) Anti-bursicon immunostaining in the CNS (A and C) and peripheral neurites ...

To determine the fate of the CCAP/bursicon efferents during metamorphosis, we performed anti-GFP immunostaining on CCAP-Gal4, UAS-mCD8::GFP animals (supplemental Figure 2). At 12–24 hr APF, the larval pattern of axons was still recognizable, although the peripheral terminals retracted back toward the CNS and lost most of the larval branches and boutons. We also observed axonal fragments that may have resulted from the severing of axons during pruning (see asterisk in supplemental Figure 2). By 30–36 hr APF, the larval efferents were gone in several preparations. At this time, we saw newly forming processes that were characterized by numerous small, branching fibers near the distal tips. By 42–48 hr APF, the adult pattern of tree-like projections and branches began to form, with eight efferents projecting out a short distance through the thick medial abdominal nerve trunk and three pairs of efferents projecting out of the two or three adjacent nerves [Ab1Nv, Ab2Nv, and AcNv3 (Demerec 1994)]. The peripheral projections also were thicker, were more varicose, and often terminated as a mass of multiple short, poorly organized, higher-order distal branches. At 54 hr APF, the peripheral projections displayed a nearly complete adult pattern, with efferents that ramified into further, higher-order branches as they extended farther posteriorly to form a tree-like arbor. The efferents closer to the abdominal midline extended a long distance posteriorly before branching into the terminal abdominal nerves and forming large, strongly immunoreactive varicosities. The lateral efferents branched and formed strongly immunoreactive varicosities much closer to their exit points from the CNS.

Disrupted metamorphosis of the CCAP/bursicon neurons:

Cell ablation experiments have shown that the CCAP/bursicon neurons are essential for head eversion behavior during pupal ecdysis as well as wing expansion and complete tanning of the adult cuticle after adult eclosion (Park et al. 2003). However, for 70% of the lines that produced mutant phenotypes when trans-heterozygous with CCAP-Gal4, wing expansion defects were the only defects observed (Table 1, supplemental Table 3). Overall, 88% of the lines (vs. 57 and 25% with 386Y-Gal4 and c929-Gal4, respectively) displayed wing expansion defects either alone or together with other mutant phenotypes. In contrast, only a third of the lines produced defects in pupal ecdysis when crossed to CCAP-Gal4. Thus, the GOF of many of these genes may have selectively disrupted the reorganization of the CCAP/bursicon neurons during metamorphosis (whereas the cell ablations of Park et al. 2003 disrupted both pupal and adult functions). To test this hypothesis, we examined anti-CCAP and anti-bursicon immunostaining and expression of mCD8::GFP (using direct fluorescence) in wandering third-instar larvae and in stage P14 pharate adults. To maximize our ability to detect cellular phenotypes, we focused the analysis on insertions representing the 16 loci that produced the strongest wing expansion defects when misexpressed with CCAP-Gal4. In addition, we analyzed the effect of directly driving the foxo gene, which produced stronger wing expansion defects than the original insertion [EY(3)11248] upstream of the foxo locus. We performed most of the crosses at 29° (in parallel with crosses to the wild-type stock, Oregon R, as controls).

The results of these experiments are described in supplemental Table 4 at http://www.genetics.org/supplemental/. The responses of the CCAP/bursicon cells to the GOF of these 17 genes fell into at least three distinct classes: (I) gross defects in axonal pathfinding that were evident in both the larval and the adult stages, (II) selective loss of adult-specific neurites, often associated with neuronal degeneration, and (III) defective bursicon secretion in adults without obvious defects in neuron morphology. Except for the first class, the GOF of these loci (in heterozygous animals) either had no effect on the larval morphology of the CCAP/bursicon neurons or resulted in very modest cellular defects, while the adult morphology was often profoundly disrupted. For any given line the changes in cellular morphology were not a good predictor of the external phenotypes observed (and vice versa). Nevertheless, these results were consistent overall with the trend toward greater disruption of adult wing expansion and tanning than of pupal head eversion behaviors.

In addition to the above insertion-specific effects, we observed a generalized response to transgene expression in the CCAP/bursicon neurons. For most insertions, we observed 30–50% lower expression of CCAP, bursicon, and mCD8::GFP in most somata, central neurites, and peripheral projections at both the wandering third instar and the P14 pharate adult stages (the VA neurons, and their dorsal efferent projections, were one notable exception). We also observed this generalized reduction in immunostaining of the CCAP/bursicon neurons when we crossed CCAP-Gal4 to UAS-GFP alone, even though this cross did not produce significant lethality or wing expansion/tanning defects. Therefore, this effect appeared to be a global cellular response to transgenic protein expression, and we did not examine it further. Instead, we focused our analysis on examples of the three specific classes of GOF phenotypes.

Class I—neurite pathfinding defects with EP(2)2003 (Ptr):

Of the 17 insertions that we examined, only one line, EP(2)2003 [inserted upstream of Patched-related (Ptr)], produced widespread, gross defects in the larval CCAP/bursicon neuron pattern of dendritic and axonal projections (supplemental Table 4). Six other lines [EP(2)2583, EP(2)2587, EP(3)3140, EP(3)3354, EP(3)3520, and EY(2)04392] produced subtle defects in small portions of the CNS arbor in larvae. However, in each of these cases, the larval projection pattern was mostly preserved.

With Ptr GOF, portions of the normal larval pattern of CNS projections, and most of the CCAP/bursicon somata, were present. However, some somata and neurites were missing. Only 30–50% of the normal efferents and ~75% of the bilateral pairs of somata in abdominal segments 1–7 (A1–A7) were visible. Many parts of the normal larval neuritic arbor were missing, often unilaterally [e.g., LB(A3) and LLT in Figure 5A′ and inset in Figure 5B′], or they were substantially scaled back in size and complexity [e.g., MA(A4) in Figure 5A′]. Ectopic neurites were also observed. Thus, the effects of Ptr GOF on the morphology of the CCAP/bursicon neurons were likely due to the disruption of neurite pathfinding. Similarly, Ptr produced striking defects in axonal and dendritic pathfinding during metamorphosis, leading to the bilaterally asymmetric loss of major portions of the normal neuritic arbor and the formation of ectopic neurites throughout the pharate adult CNS (supplemental Table 4, Figure 5C).

Figure 5.
GOF of Ptr produced neurite pathfinding defects in larval CCAP/bursicon neurons. In both larvae (A and B) and pharate adults (C), misexpression of Ptr (A′, B′, and C′) led to the loss of some somata and neurites (labels with dashed ...

Previous work has shown that these neurons are essential for successful completion of head eversion during ecdysis from the larval to the pupal stage (Park et al. 2003). Remarkably, despite the severe defects in larval CCAP/bursicon neuron morphology resulting from Ptr GOF, head eversion in heterozygous CCAP-Gal4, EP(2)2003 animals occurred normally (supplemental Table 3). These results show that heterozygous CCAP-Gal4, EP(2)2003 animals retained enough CCAP/bursicon, and enough larval CCAP/bursicon neuron connectivity, to allow normal functioning of the neuropeptidergic signaling hierarchy controlling pupal ecdysis behavior. In contrast, the adult function of the CCAP/bursicon neurons in controlling wing expansion was not preserved.

Class II—loss of adult-specific neurites:

Of the 17 lines examined for cellular changes following GOF with the CCAP-Gal4 driver, 14 displayed the loss of some or all adult-specific neurite projections. This group comprises EP(2)2237, EP(2)2583, EP(3)3140, EP(3)3520, EY(X)10575, EY(2)04392, EY(2)05304, EY(3)00559, EY(3)00146, XP(2)d07339, XP(3)d00809, XP(3)d02595, XP(3)d04253, and UAS-foxo, which was substituted for EY(3)11248 (supplemental Table 4). In each case, we detected only minor morphological changes in the CCAP/bursicon cells in larvae. These changes may have been physiologically significant, particularly when the gene dosage was increased (when more lines displayed head eversion defects). Nevertheless, the disruption of remodeling of the CCAP/bursicon neurons during metamorphosis (or the failure to retain adult projections) was the predominant phenotype for lines that produced defects in wing expansion or ecdysis when crossed to CCAP-Gal4.

We examined one insertion, EP(2)2237, to determine whether adult-specific neurites were lost through a pruning defect, an outgrowth defect, or atrophy. EP(2)2237 is located immediately 5′ of the cbt C2H2-type zinc-finger transcription factor, and overexpression of a cbt cDNA phenocopied EP(2)2237 expression in the eye disc and the wing disc (S. Munoz-Descalzo, personal communication). In wandering third-instar larvae, the morphology of the CCAP/bursicon neurons was largely unchanged following cbt GOF with EP(2)2237 (supplemental Table 4; Figure 6, A and B), except that we observed fewer and smaller boutons at the peripheral endings. In contrast, we observed severe defects at the P14 pharate adult stage (supplemental Table 4; Figure 6, C and D), including the loss of many central neurites and efferent projections, with the remaining efferents displaying substantially fewer/shorter branches and large varicosities (Figure 6D′). In addition, we observed the loss of 1–6 of the 14 abdominal bursicon neurons [the BAG cells (Luan et al. 2006)]. The remaining BAG somata were reduced in diameter and either intensely immunoreactive or only very weakly immunostained (Figure 6C′ and data not shown).

To determine whether these defects were due to either a failure of the CCAP/bursicon neurons to remodel or cellular atrophy after remodeling, we crossed CCAP-Gal4, UAS-mCD8::GFP flies to EP(2)2237 and performed anti-GFP immunostaining on whole-animal fillets and isolated CNSs at 18, 30, 42, 54, and 60 hr APF (Figure 6, E–H). In control CCAP-Gal4, UAS-mCD8::GFP/+ pupae at 18 hr, we observed numerous thick, anteriorly and posteriorly directed projections from the LB (Figure 6E) that appeared to set down the future location of an adult-specific axon tract located ~5 μm to each side of the CNS midline (Figure 6F). While these projections were also present following cbt GOF, they were shorter and less numerous (Figure 6E′). These differences persisted at later stages. In the controls at 30 hr, the central neurite projections were finer and much more numerous, and the abdominal arbor displayed a more adult-like pattern. At 42 hr, the neurite tracts were thicker and more varicose. By 54 hr, the adult abdominal longitudinal axon tracts were well formed, and there were many thin neurite projections extending into the neuropile (Figure 6F). In contrast, following cbt GOF, the new projections retained their thick, meandering, blunt-ended appearance throughout this period and were still individually discernible even at 54 hr (Figure 6F′). Although the adult abdominal longitudinal axon tracts were visible, much of the finer abdominal neuritic arbor failed to form.

cbt GOF also resulted in defects in formation of the peripheral arbor. At 18 hr (n = 7), the efferents in the control pupae remained long with accumulated material at the distal endings. The pattern was similar in CCAP-Gal4, UAS-mCD8::GFP/EP(2)2237 pupae, but the efferents were finer and had less distal end material. At 30 hr (n = 5), these differences were retained. However, there were fewer efferents visible in both genotypes, and many appeared to have pruned back to the CNS (Figure 6G). By 42 hr (n = 7), all of the larval efferents were gone, and new projections were visible extending from the CNS. In the control pupae, these projections began to acquire a more varicose appearance and terminated in a loosely associated network of finer branches, while the cbt GOF efferents were thinner and less varicose and terminated in fewer branches. At 54 hr (n = 7) and 60 hr (n = 3), the cbt-expressing efferents were short, with only a few small branches, while the control efferents formed a nearly complete adult arbor (Figure 6H). Thus, in both the CNS and the periphery, cbt GOF led to an arrest in the remodeling of the CCAP/bursicon neurons at an early stage of neurite outgrowth.

Among the 14 lines displaying the loss of some or all adult-specific neurites in pharate adults, there were differences in the phenotypes that suggest the presence of multiple different underlying mechanisms. In addition to EP(2)2237, six other insertions [EP(2)2583, EY(X)10575, EY(3)00559, EY(3)11248/UAS-foxo, XP(2)d07339, and XP(3)d02595] displayed the loss of many central neurites and efferents, which in all of the lines except XP(2)d07339 was accompanied by a reduced number of bursicon-immunoreactive BAG (and LSE) neuron somata. In some genotypes, a subset of the observed BAG somata was very small and weakly immunoreactive (Figure 7), and a few appeared to be fragmented. We observed a similar reduction in soma number by examining GFP fluorescence in pupae expressing UAS-mCD8::GFP in parallel with the gain-of-function transgenes (data not shown). While we cannot exclude the downregulation of bursicon, CCAP, and CCAP-Gal4 expression in the missing neurons as the cause of the apparent loss of cells, these results are consistent with cell death. Thus, neuronal degeneration often appeared to accompany, or follow, the loss of adult-specific neurites.

Figure 7.
GOF of klar resulted in specific loss of adult-specific bursicon-immunoreactive neurites and loss of six to eight CCAP/bursicon cell somata. (A and B) Anti-bursicon immunostaining in wandering third-instar (A) and P14 pharate adult (B) stage CNS from ...

A second group of five lines [EP(3)3140, EP(3)3520, EY(2)5304, XP(3)d00809, and XP(3)d04253] displayed similar losses of adult-specific neurites and loss of a few CCAP/bursicon neuron somata when crossed to CCAP-Gal4. The efferent projections displayed either failure or reduced capacity to form en passant varicosities and higher-order branches. EP(3)3140, for example, produced efferents lacking most branches and large varicosities and ending in large, strongly bursicon-immunoreactive club-shaped endings. Thus, the GOF of these loci appeared to disrupt the accumulation of secretory material at neuroendocrine release sites.

Two insertions produced other phenotypes (supplemental Table 4). EY(2)04392 displayed a loss of adult-specific neurites that was limited to the brain, subesophageal, and thoracic arbor in the CNS. EY(3)00146 displayed weak immunostaining or loss of many central neurites and efferents, but the number of bursicon-immunoreactive anterior BAG (equation M3) somata was increased from 8 to ~18. This was not accompanied by an increase in the number of somata labeled with UAS-mCD8::GFP (data not shown). In wild-type animals, the number of bursicon-immunoreactive abdominal neurons decreases from 28 to 14 during metamorphosis (compare the anti-bursicon staining to the CD8::GFP signal in Figure 4). Taken together, these results show that EY(3)00146 blocked the downregulation of bursicon expression in 14 abdominal neurons during metamorphosis.

Class III—defective bursicon secretion without gross changes in adult cell morphology:

Two insertions, EP(2)2587 and EP(3)3354, produced moderate to strong, highly penetrant wing expansion defects when crossed to CCAP-Gal4 without affecting the gross morphology of the CCAP/bursicon neurons in pharate adults (supplemental Table 4). Both EP(2)2587 and EP(3)3354 appear to drive the expression of micro-RNAs that all belong to the same family. EP(2)2587 [along with two other insertions obtained in the screen, EP(2)2586 and EP(2)2356] is inserted directly upstream of a cluster of four micro-RNAs, miR-310–miR-313 (supplemental Table 3). Of the four micro-RNAs in the miR-310–miR-313 cluster, miR-310 is the furthest downstream of the EP(2)2587. We confirmed the overexpression of this micro-RNA cluster in wandering-stage CCAP-Gal4/EP(2)2587 larvae by in situ hybridization with LNA probes to miR-310 (Figure 8A; n = 8), miR-312, and miR-313 (data not shown). In CCAP-Gal4/+ control CNS, we observed a weak stripe of expression of miR-310, miR-312, and miR-313 near the presumptive optic lobes (data not shown).

Figure 8.
GOF of the miR-310–miR-313 micro-RNA cluster during an early- to midmetamorphosis critical period prevented bursicon secretion in adults. (A) In situ hybridization (magenta) for miR-310 (antisense LNA probe) in a wandering third-instar larval ...

EP(3)3354 is inserted directly upstream of the putative transcription factor, jing interacting gene regulatory 1 (jigr1). There are also two nearby micro-RNAs. miR-92a is located ~9 kb downstream, in an intron of jigr1, and miR-92b is ~14 kb downstream, 1 kb beyond the 3′ end of the jigr1 gene. Interestingly, miR-92a, miR-92b, and miR-310–miR-313 are closely related (Aravin et al. 2003; Leaman et al. 2005) and share many of the same predicted target mRNAs (Enright et al. 2003; Stark et al. 2003; Rajewsky and Socci 2004; Grun et al. 2005). Thus, we hypothesize that EP(2)2587 and EP(3)3354 affect the CCAP/bursicon neurons through inhibition of common loci. Both insertions produced similar phenotypes, although the behavioral defects elicited with EP(2)2587 were stronger than with EP(3)3354.

The failure of EP(2)2587 and EP(3)3354 to affect the morphology of the CCAP/bursicon neurons led us to ask whether the secretion of bursicon was affected. In Drosophila, bursicon activity appears in the blood within the first 20 min after eclosion (Baker and Truman 2002; Luan et al. 2006). In control CCAP-Gal4/+ flies at 22°–23°, the median times until wing expansion were 23 min after eclosion (AE) in females (n = 26, IQR = 18) and 12 min AE in males (n = 23, IQR = 9). Although the sexual dimorphism in the time to wing expansion was statistically significant (P = 0.000048; Mann-Whitney U-test), all but one of the animals of either sex initiated wing expansion within 50 min AE (one female took 473 min). In the tobacco hornworm, Manduca sexta, the secretion of bursicon is accompanied by an ~80% depletion of the hormone from the neuroendocrine release sites in the transverse nerve (Truman 1973; Taghert and Truman 1982), and bursicon bioactivity in the hemolymph of both M. sexta and blowflies peaks within 1 hr of eclosion (Fraenkel and Hsiao 1965; Reynolds et al. 1979). Thus, we expected to be able to detect bursicon secretion through an immunofluorescence assay, as we and others have done previously to detect robust secretion of CCAP, EH, and ETH (Hewes and Truman 1991; Ewer et al. 1997; McNabb et al. 1997; Clark et al. 2004). We performed anti-bursicon immunostaining on fillet dissections of Drosophila adults staged at either 0 or 1 hr AE at 25°. We collected the flies without anesthesia to preclude anesthesia-induced behavioral changes. In control CCAP-Gal4/+ flies, we observed a 73% depletion in the intensity of anti-bursicon immunostaining over this period [Figure 8, B (top) and C]. In wild-type animals, there was a concomitant depletion of CCAP and PHM immunoreactivity at this time (data not shown). Taken together with the earlier bioassay data (Baker and Truman 2002), these results are consistent with the regulated neuroendocrine secretion of neuropeptides and other secretory granule contents from the peripheral projections of the CCAP/bursicon neurons within 1 hr AE.

When we examined CCAP-Gal4/EP(2)2587 flies, which at this temperature (25°) displayed 100% unexpanded wings, we observed two changes in the anti-bursicon immunostaining [Figure 8, B (bottom) and C]. First, the baseline levels of bursicon immunoreactivity were lower than those in the CCAP-Gal4/+ controls, consistent with the generalized 30–50% decrease in bursicon, CCAP, and mCD8::GFP levels in most lines expressing transgenes in the CCAP/bursicon neurons (see above). Second, there was no depletion of bursicon immunoreactivity during the 1 hr AE in CCAP-Gal4/EP(2)2587 flies. Thus, the GOF of miR-310–miR-313 in the CCAP/bursicon neurons inhibited bursicon secretion after eclosion. This inhibition may have resulted from direct disruption of the secretory apparatus in the CCAP/bursicon efferents. Alternatively, miR-310–miR-313 could have altered the electrical activity (or synaptic efficacy) of neurons within the CCAP/bursicon cell population that may provide synaptic input to the subset of neurons with neuroendocrine projections.

Critical window for the miR-310–miR-313 GOF phenotype:

EP(2)2587 expression did not perturb head eversion, even in homozygous CCAP-Gal4, EP(2)2587 animals. This result was in contrast to the apparent dosage-sensitive disruption of head eversion for a few other lines, including EP(2)2003, EP(2)2237, and EP(2)2583 (data not shown). Thus, the effects of miR-310–miR-313 GOF appeared to be strictly limited to adult-specific functions of the CCAP/bursicon neurons. This could have resulted from an acute block in the expression of one or more proteins required for exocytosis or for trans-synaptic activation of the CCAP/bursicon neurons. Alternatively, miR-310–miR-313 may have altered the earlier development of these cells. To test these hypotheses, we used the TARGET system (McGuire et al. 2003) to determine the critical period for the disruption of wing expansion by miR-310–miR-313 expression in neuropeptidergic cells. We generated a fly line containing 386Y-Gal4 and the tubulinP-Gal80ts transgene, which provides ubiquitous expression of a temperature-sensitive Gal80 protein (McGuire et al. 2003), and crossed these flies to EP(2)2587. Gal80ts binds to Gal4 at permissive temperatures (18°–22°) and represses Gal4 transcriptional activity. We observed normal wing expansion in ~100% of tubP-Gal80ts/EP(2)2587; 386Y-Gal4/+ animals raised throughout development. The inhibition by Gal80ts is released at restrictive temperatures (27.5°–30°), and when raised throughout development at 30°, 100% of tubP-Gal80ts/EP(2)2587; 386Y-Gal4/+ flies failed to inflate their wings.

To determine the critical period for miR-310-miR-313 expression, we then conducted temperature-shift experiments. We collected eggs for 24 hr at either 19° or 30°. Groups of animals then were shifted up or down to the other temperature at daily intervals until the adults emerged. After eclosion, we measured the percentage of adults displaying partially or completely uninflated wings (Figure 8D). When the temperature was shifted down (from 30° to 19°), miR-310–miR-313 was expressed early and then repressed after the temperature shift. If miR-310–miR-313 expression was repressed before a critical window opened, then animals would be able to expand their wings. Conversely, when the temperature was shifted up (from 19° to 30°), miR-310–miR-313 was expressed after the temperature shift. If this occurred after a critical window closed, then animals would be able to expand their wings. Therefore, we defined the opening and closing times for the critical window as the times at which the respective shift-down and shift-up curves each crossed the 50% mark for animals with uninflated wings. These results show that miR-310–miR-313 expression was effective in blocking wing expansion when the animals were at the restrictive temperature, 30°, between 6.5 and 8 days of age. The curve for timing the onset of the critical window (shift down) displayed a rapid exponential rise that sharply delineated the onset of sensitivity of the CCAP/bursicon cells to miR-310–miR-313 expression.

The time for the close of the critical window (shift up) was less well defined. In the control experiments by McGuire et al. (2004), the repression of control transcript expression after shifting to 19° was half-maximal at 15 hr and complete by 36 hr. A similar slow decline likely accounts for the greater variability and shallower curve for the shift-up tests in Figure 8D. When we examined the stage of development of tubP-Gal80ts/EP(2)2587; 386Y-Gal4/+ animals after 8.7 days at 19°, 71% of the animals were wandering-stage third-instar larvae, and the rest were feeding-stage third-instar larvae (n = 105). If the offset of miR-310–miR-313 occurred ~8–30 hr after the animals were shifted to 19°, then most of these animals would have been in the early stages of pupal development. Therefore, we conclude that the critical window for the effects of miR-310–miR-313 GOF on the ability of the CCAP/bursicon neurons to secrete bursicon coincides with the first half to middle third of metamorphosis.

DISCUSSION

Analysis of steroid-dependent neuroendocrine cell remodeling through GOF screening:

We screened 6097 randomly inserted GOF transposon insertions and identified 30 lines (29 unique loci) that produced wing expansion defects and 5 lines (3 loci) that produced pupal lethality (2 with head eversion or eclosion defects) when crossed to the CCAP-Gal4 driver. We also identified 19 loci that produced head eversion, eclosion, or wing expansion defects when expressed with the much broader neuropeptidergic cell driver, 386Y-Gal4, but not CCAP-Gal4. These results point to the existence of one or more undefined neuropeptidergic cell types in the 386Y-Gal4 pattern with important roles in the control of head eversion, eclosion, and wing expansion behaviors (supplemental Results and Discussion). Our phenotypic analysis of 17 of the loci that produced the most severe wing expansion defects with CCAP-Gal4 suggests functions for these genes in axonal pathfinding, development of competence for developmentally timed neuropeptide secretion, outgrowth of adult-specific neurite projections, neuropeptide expression, and neuronal maintenance. The other lines obtained in the screen revealed several additional genes with putative roles in neuroendocrine cell remodeling.

Combined with LOF data, GOF screens have proved to be an effective method for identification of genes involved in the development of many tissues (Rørth 1996; Rørth et al. 1998; Abdelilah-Seyfried et al. 2000; Kraut et al. 2001; Pena-Rangel et al. 2002; Tseng and Hariharan 2002; Brennecke et al. 2003; McGovern et al. 2003; Sherwood et al. 2004; Teleman et al. 2005, 2006). However, this method also poses special concerns that are not usually encountered with traditional LOF screens. For example, some genes produce phenotypes in cells in which they are not normally expressed. In many cases, these phenotypes are complementary to LOF phenotypes in other cells, and they are therefore useful for defining gene functions (e.g., Allan et al. 2003; Hewes et al. 2003, 2006). Alternatively, GOF phenotypes may arise from abnormal protein–protein interactions or through the activation of cellular homeostasis machinery, such as the unfolded protein response (Ryoo and Steller 2007). In future studies, it will be important to map the normal expression patterns of these genes and test for complementary LOF phenotypes to identify factors that (1) control remodeling of the CCAP/bursicon during metamorphosis and (2) regulate more general aspects of steroid-dependent neuronal plasticity. Nevertheless, our phenotypic analysis and other published studies provide several insights into their possible functions. Below, we describe a few of the putative associations among these genes.

Micro-RNAs:

GOF screens have proved to be effective for identifying functions of micro-RNAs, which are 20- to 22-nucleotide noncoding RNAs that assume diverse gene regulatory roles (Kloosterman and Plasterk 2006). Hundreds of micro-RNA genes have been identified through computational prediction and experimental verification (Berezikov et al. 2006), yet relatively few micro-RNA functions have been identified. In this study, we isolated 6 insertions, of a total of 58 (10%), that drove expression of three different micro-RNA loci. In other published GOF screens, these 6 insertions produced developmental phenotypes involving regulation of cell growth, differentiation, or apoptosis in the eye imaginal disc, adult dorsal thorax, and external sensory organs and motor axon guidance and synaptogenesis in embryos and larvae (Abdelilah-Seyfried et al. 2000; Kraut et al. 2001; Pena-Rangel et al. 2002; Tseng and Hariharan 2002; Muller et al. 2005). Other similar screens led to the discovery of micro-RNAs involved in tissue growth and energy homeostasis (Brennecke et al. 2003; Teleman et al. 2006). Thus, coupled with target prediction, LOF experiments, and mapping of micro-RNA expression patterns, GOF screening provides an additional tool for systematic examination of micro-RNA function.

The GOF phenotypes obtained with micro-RNAs in our screen likely result from the repression of unidentified mRNAs that are normally expressed in the CCAP/bursicon neurons. It will be of interest to determine the identity of these target mRNAs and whether the repression by the micro-RNAs occurs normally in other cells or in the CCAP/bursicon neurons at other stages during development. Through in situ hybridization of wandering third-instar larval CNS, we detected weak, heterogeneous expression of miR-279 in the VNC (data not shown) and of miR-310, miR-312, and miR-313 in the brain (see results). Therefore, these micro-RNAs may be involved in the developmental regulation of certain CNS neurons. In a previous study, miR-311 and miR-313 were shown to play significant roles in embryonic nervous system development (Leaman et al. 2005). Interestingly, the miR-310–miR-313 cluster and the closely related micro-RNAs, miR-92a and miR-92b were strongly expressed in embryos and then less abundant at later stages (Aravin et al. 2003; Sempere et al. 2003). For some of these micro-RNAs, significant downregulation occurred near the embryo–larval and larval–pupal transitions. Our observation of a critical period for miR-310–miR-313 action during early metamorphosis suggests that the upregulation of some of their target mRNAs was required for the CCAP/bursicon cells to acquire competence to secrete bursicon after eclosion.

Growth factor signaling:

Several of the genes identified in our screen are involved in signaling by growth factors. Two of the genes, foxo and Insulin-like receptor (InR), encode components of the insulin signaling pathway. In mammals, multiple forkhead O (FoxO) subfamily transcription factors play important roles in the regulation of neuronal apoptotic death in the context of low-level insulin signaling (Gilley et al. 2003; Barthelemy et al. 2004; Soriano et al. 2006; Biswas et al. 2007). In Drosophila, foxo is the only FoxO subfamily member in the genome, and it has been shown to mediate apoptosis in retinal neurons (Luo et al. 2007). Here, we found that foxo overexpression resulted in evident neurodegenerative phenotypes: reduced and fragmented neurites, reduced cell sizes, and loss of neuronal somata. Interestingly, overexpression of InR by EY(3)00681 in the 386Y-Gal4 pattern also produced pharate lethality. On the basis of these results, we are currently examining the role of insulin signaling in the regulation of neuropeptidergic cell remodeling during metamorphosis (T. Gu, T. Zhao and R. S. Hewes, unpublished observations).

In addition to insulin signaling, the epidermal growth factor receptor (EGFR) signaling pathway is a critical regulator of axon growth and guidance and neuronal cell survival (Dominguez et al. 1998; Doroquez and Rebay 2006). In this screen, we found two genes with known roles in EGFR signaling during neuronal development, the pnt ETS domain transcription factor and the spen nuclear corepressor (Chen and Rebay 2000; Kuang et al. 2000). Both pnt and spen elicited robust head eversion and wing expansion defects when expressed in the 386Y-Gal4 and CCAP-Gal4 patterns (supplemental Table 3), and spen overexpression resulted in the loss of most of the CCAP/bursicon neuron arbor and the disappearance of many abdominal CCAP/bursicon neuron somata (supplemental Table 4). Interestingly, reduced activity of pnt can suppress spen overexpression-mediated phenotypes (T. Zhao, T. Gu and R. S. Hewes, unpublished observations), which is consistent with a previous report that spen functions synergistically with pnt in regulation of the EGFR pathway (Chen and Rebay 2000). These results suggest that EGFR signaling controls aspects of neurite outgrowth and neuropeptidergic cell survival during metamorphosis. Alternatively, pnt and spen may each influence different signaling pathways that contribute to the development or maintenance of adult CCAP/bursicon projections. In the GOF screen, we also obtained spitz (spi), which encodes a ligand of the Drosophila EGFR. When expressed in the 386Y-Gal4 pattern, spi produced pharate adult lethality, presumably due to actions of this ligand on neighboring cells.

Myb proto-oncoprotein-like transcription factors:

Of the 14 genes that produced loss of adult-specific neurite projections, two appear to be involved in signaling by Myb-like protein complexes. The mip120 gene encodes one of five components of the Drosophila Myb complex (Beall et al. 2002), and stonewall (stwl) encodes a Myb-like transcription factor (Clark and McKearin 1996). In mammals, the apoptotic loss of postmitotic neurons following the loss of trophic support from nerve growth factor (NGF) requires the derepression of multiple transcriptional pathways. These include the FoxO pathway, the c-Jun and c-Jun N-terminal kinase pathway (JNK), and the cell cycle pathway, which involves phosphorylation of retinoblastoma (Rb) proteins as well as the derepression of Myb proteins (Brunet et al. 2001; Liu et al. 2004). All three of these pathways converge on Bim, a pro-apoptotic protein that is activated in response to NGF deprivation. Overexpression of either FOXO or Myb family proteins (including STWL) can result in apoptotic cell death (Liu and Greene 2001; Gilley et al. 2003; Brun et al. 2006). However, the apoptotic response to NGF under physiological conditions may require the combined derepression of all three of these pathways (Biswas et al. 2007). In this screen, foxo expression led to loss of CCAP/bursicon cell neurites and somata, while overexpression of mip120 and stwl both led to loss of neurites alone (supplemental Table 4). In the case of mip120, the GOF phenotype appears to have resulted from overexpression, rather than misexpression, since we found that MIP120 was ubiquitously expressed in the larval CNS (supplemental Results and Discussion and T. Gu, T. Zhao and R. S. Hewes, unpublished observations). Likewise, stwl was previously shown to be expressed ubiquitously in embryos (Tomancak et al. 2007). It will be of interest to determine whether these factors function together under physiological conditions to regulate nerve cell growth and survival in this system.

Ecdysteroid signaling:

Studies on a variety of cell types in Drosophila and M. sexta have shown that the metamorphic remodeling of larval neurons is mediated by ecdysteroids. These steroid actions are mediated largely cell autonomously (Williams and Truman 2005; Brown et al. 2006) through ecdysteroid receptors, which are heterodimers consisting of Ultraspiracle along with one of three isoforms of the ecdysteroid receptor (EcR), EcR-B1, -B2, or -A (Koelle et al. 1991; Talbot et al. 1993; Thomas et al. 1993; Yao et al. 1992, 1993). In Drosophila neuroendocrine cells as well as in other neuronal cell types, loss of the EcR-B isoforms leads to pruning defects. In contrast, EcR-A is thought to regulate the outgrowth of the adult-specific neurites (Truman et al. 1994; Truman 1996; Schubiger et al. 1998; Lee et al. 2000; Brown et al. 2006; Santos et al. 2006).

GOF of several of the loci identified in this screen (cbt, CG14438, faf, foxo, klar, mip120, miR-279, spen, stwl, and the genes under the control of EY(2)04392, XP(2)d07339, XP(3)d00809, XP(3)d02595, and XP(3)d04253) produced defects consistent with the disruption of ecdysteroid-dependent remodeling of the CCAP/bursicon neurons. For cbt, we observed pruning on schedule, but formation of the adult central and peripheral adult arbor was retarded, and outgrowth appeared to cease after completion of an initial phase (Figure 6). Interestingly, cbt encodes a C2H2 zinc-finger transcription factor (Munoz-Descalzo et al. 2005) that functions as a primary ecdysteroid response gene (Beckstead et al. 2005). Thus, EcR may directly regulate cbt expression, which in turn may regulate the expression of genes involved in controlling neuronal outgrowth.

Ubiquitin–proteasome system:

Finally, we identified several genes that have previously been shown to regulate axonal pathfinding or synapse formation in other cell types (cf. Kraut et al. 2001). One of these, faf, encodes a deubiquitinating protease that contributes to synaptic growth control at the neuromuscular junction (NMJ). The ubiquitin–proteosome system plays a crucial role in axon guidance and synaptogenesis in diverse species (Hegde 2004). In Drosophila, faf overexpression at the NMJ produces synapse overgrowth in third-instar larvae (Diantonio et al. 2001). Similarly, we found that faf overexpression produced modest overgrowth, including enlarged varicosities, of larval CCAP/bursicon neurites within the CNS. In contrast, during metamorphosis, faf overexpression produced degenerative phenotypes (supplemental Table 4), accompanied by loss of neurite arbor and CCAP/bursicon cell somata. Thus, the contribution of ubiquitin–proteasome system components to the development of CCAP/bursicon neurons, or in the balance of regulated proteolysis in different subcellular domains, may change as a function of developmental stage.

Taken together, these results provide several molecular clues into how the complex processes of neuronal differentiation, ecdysteroid-dependent regulation of neuronal remodeling, and neuronal growth and maintenance may be coordinated in a single cell type, the CCAP/bursicon cells. The challenge for the future will be to define the biological functions of the factors identified in this screen and their roles in neuronal remodeling.

Acknowledgments

We thank David McCauley for assistance with in situ imaging; Adam Diehl, Adrienne Emel, Kendal Hopkins, Brett Kirkconnell, Matthew Mote, Rahul Patel, Elizabeth Pearsall, Edward Spencer, and Ryan Wilkes for assistance with EP crosses, insertion mapping, or immunostaining; Bryce Jones, Kim Krittenbrink, David McCauley, Jeremiah Smith, and Audrey Kennedy for technical assistance; Frédéric Bantignies, Mariann Bienz, Gabrielle Boulianne, John Ewer, Sue McNabb, Paul Taghert, Marc Tatar, and the Szeged and Bloomington Drosophila stock centers for fly stocks; and Bing Zhang for comments on the manuscript. This work was supported by grants to R.H. from the National Science Foundation (IBN0344018 and EPSCoR 0132534), the Oklahoma Center for the Advancement of Science and Technology (HR03-048S), the Oklahoma Research Council/University of Oklahoma Vice President for Research, and the Oklahoma State Regents for Higher Education.

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