Genetic analysis of ecdysis behavior in Drosophila reveals partially overlapping functions of two unrelated neuropeptides

Abstract

Ecdysis behavior allows insects to shed their old exoskeleton at the end of every molt. It is controlled by a suite of interacting hormones and neuropeptides, and has served as a useful behavior for understanding how bioactive peptides regulate CNS function. Previous findings suggest that crustacean cardioactive peptide (CCAP) activates the ecdysis motor program; the hormone bursicon is believed to then act downstream of CCAP to inflate, pigment, and harden the exoskeleton of the next stage. However, the exact roles of these signaling molecules in regulating ecdysis remain unclear. Here we use a genetic approach to investigate the functions of CCAP and bursicon in Drosophila ecdysis. We show that null mutants in CCAP express no apparent defects in ecdysis and postecdysis, producing normal adults. By contrast, a substantial fraction of flies genetically null for one of the two subunits of bursicon [encoded by the partner of bursicon gene (pburs)] show severe defects in ecdysis, with escaper adults exhibiting the expected failures in wing expansion and exoskeleton pigmentation and hardening. Furthermore, flies lacking both CCAP and bursicon show much more severe defects at ecdysis than do animals null for either neuropeptide alone. Our results show that the functions thought to be subserved by CCAP are partially effected by bursicon, and that bursicon plays an important and heretofore undescribed role in ecdysis behavior itself. These findings have important implications for understanding the regulation of this vital insect behavior and the mechanisms by which hormones and neuropeptides control the physiology and behavior of animals.

Figure 1.

A–H, Isolation of null mutants in CCAP (A–D) and pburs (E–H) genes. A, Schematic of lesion in CCAP gene caused by the imprecise excision of the EY mobile P-element; F and R: PCR primer pair used for diagnostic PCR, which showed a larger product in excision line (B, CCAP^exc) than in controls (B, w) due to retention of some mobile element sequences. C, D, In situ CNS expression of ccap RNA. Prominent expression in CCAP neurons (D) was absent in the CNS of homozygous excision flies (C). E, Schematic of lesion in pburs. Insertions d02171 (Ex1) and e02061 (Ex2), both of which contain FRT sites, were used to create a FLP-induced genetic deletion that exclusively removed the pburs gene. Diagnostic PCR product 2 was absent in homozygous excision flies, whereas DNA distal to Ex1 (PCR product 1) and proximal to Ex2 (PCR product 3) appeared intact in the resulting hybrid element. G, H, In situ CNS expression of pburs RNA. Prominent expression in pburs neurons of controls (H) was absent in the CNS of homozygous excision flies (G). See Table 1 for PCR primer sequences; for E, PCR products 1, 2, and 3 were amplified using primer pairs: pburs_F1 + X1, pburs_F2 + pburs_R1, and X2 + pburs_R2, respectively.

Figure 2.

Pupal ecdysis behavior sequence in the absence of CCAP. A–C, Duration of pupal preecdysis (A), ecdysis (B), and entire ecdysis sequence (preecdysis plus ecdysis) (C) in flies lacking CCAP neurons versus flies mutant for CCAP and controls. Animals bearing targeted ablations of CCAP neurons [expressing cell death gene, reaper, under the control of CCAP-GAL4; (rpr, column 1)] express a weak pullback behavior and then fail to ecdyse (Park et al., 2003). By contrast, controls [flies expressing LacZ under the control of CCAP-GAL4; (LacZ, column 2)] and flies hemizygous for CCAP (columns 3 and 4) express both preecdysis and ecdysis behavior. Although there are differences in the duration of the ecdysial phases among these latter genotypes, these differences do not correlate with the CCAP genotype. Times are averages ± SEM; N = 10–12 per group. # and hatching of column 1 indicate that preecdysis ended with weak pullback behavior. Different letters above columns indicate significantly different timing (p < 0.05). Hemizygous CCAP mutant animals were heterozygous for CCAP excision (CCAP^exc) and two different genetic deletions that include CCAP, Df1 [Df(3)23D1] and Df2 [Df(3)B+L³⁸]; see Materials and Methods for more details.

Figure 3.

Role of CCAP and pburs in the completion of pupal ecdysis behavior. Top, Success of ecdysis behavior, indicated as the proportion of animals that completed ecdysis within the indicated time intervals, in animals lacking CCAP neurons (CCAP KO group), the CCAP neuropeptide (CCAP⁻ group), the PBURS neurohormone (pburs⁻ group), and both CCAP and PBURS (CCAP⁻ + pburs⁻ group). Genotypes are coded by the combinations of black squares within each table column, and are defined below. Animals lacking PBURS showed severe defects at ecdysis (columns 7–10), which were rescued by a P{pburs} transgene (column 11). Although flies mutant for CCAP completed ecdysis within the normal time (columns 3 and 4), removing CCAP function in animals lacking PBURS greatly potentiated the defects expressed by pburs mutants (columns 13–15 vs columns 7–10). The defects of these CCAP + pburs double mutants were similar to those expressed by flies bearing targeted ablations of CCAP neurons (column 1; cf. Park et al., 2003). Defects expressed by double mutants were fully rescued by P{CCAP} + P{pburs} transgenes (column 16), and were rescued to levels comparable to those of pburs mutants by a P{CCAP} transgene (column 17; columns 7–10 vs 17; p > 0.05). Genotypes, abbreviated in the leftmost column, are as follows: CCAP^exc and pburs^exc correspond, respectively, to null CCAP and pburs alleles produced in this study; Df(3)B+L38 and Df(3)23D1 are genetic deletions that include the CCAP gene; Df(2)6036, Df(2)135, Df(2)110, and Df(2)A217 are genetic deletions that include the pburs gene; PB{L} and PB{R} represent the two mobile elements flanking pburs that were used to create the pburs excision; P{CCAP} and P{pburs} represent transgenes bearing CCAP and pburs rescue constructs, respectively; CCAP⁺ and pburs⁺ represent chromosomes bearing the endogenous, wild-type alleles of the CCAP and pburs genes, respectively. See Materials and Methods for further details. N > 10 animals per group.

Figure 4.

Role of CCAP and pburs in the regulation of head eversion and wing expansion. Morphology of the pharate adult and adult is summarized as the proportion of animals within the indicated categories. Categories and genotypes are displayed as described in Figure 3. The terminal phenotypes expressed by the different genotypes were consistent with the behavioral defects shown in Figure 3; slight differences are likely due to the much greater number of animals examined (N > 50 animals per genotype). Examples of animals in these groups are shown in Figure 6: 6A, normal adult; 6B, unexpanded wings; 6C, no head; 6D, >50% head.

Figure 5.

Role of CCAP and pburs in the regulation of the pharate adult and adult phenotypes: wing (left) and leg (right) length. Groups and genotypes are displayed as described in Figure 3. N > 30 animals per genotype. Different letters above columns indicate significantly different categories (p < 0.05).

Figure 6.

Terminal morphology of flies lacking CCAP or PBURS function. A, Flies mutant for CCAP expressed normal morphology and tanning. B, By contrast, adult flies mutant for pburs failed to inflate their wings and showed abnormal tanning (as evidenced, e.g., by matte exoskeleton). C, D, Examples of pharate adults mutant for pburs that showed extreme (C) and more mild (D) morphological defects caused by abnormal pupal ecdysis, based on the proportion of the head that was everted and the length of the wings and legs (black arrowheads and white arrows, respectively). E, Pharate control fly, showing normal head eversion, and normal wing and leg extension.

Figure 7.

Temporal pattern of adult emergence under LD regime in flies lacking CCAP function. Histogram represents the average percentage (±SEM) of flies that emerged within a 1 h time window. Black and white bars represent dark and light periods of the photoperiod, respectively. A–D, Flies homozygous (A) and hemizygous (B) for CCAP^exc showed a profile of eclosion similar to that of controls (C) and of homozygous CCAP^exc rescued with a wild-type CCAP transgene (D), including a similar surge of emergence 0–3 h after lights-on. Each panel represents the profile for each population, averaged across different days and for four independent experiments, each including two populations per genotype.

Figure 8.

Spatial pattern of expression of bursicon subunits in the ventral CNS of pharate pupae. A, Pattern of BURS-IR (brown) and pburs in situ RNA expression (blue), showing that pburs is expressed in a subset of four pairs of BURS-IR neurons. B–D, Summary of pattern of BURS-IR (B), and burs (C) and pburs in situ RNA expression (D). Circles represent the complement of CCAP neurons in the brain, SEG, and ventral nervous system. Filled circles, strong staining; +, weak staining. For each stain, 8–10 preparations were scored.

Figure 9.

Bursicon subunits are released at pupal ecdysis. Aa, Ab, Ba, Bb, Pattern of BURS-IR (a) and PBURS-IR (b) before (A) and after (B) pupal ecdysis. Note that prominent immunoreactivity in lateral axon (arrows in Aa and Ab, for BURS-IR and PBURS-IR, respectively) is not visible after ecdysis (Ba and Bb, for BURS-IR and PBURS-IR, respectively). C, Quantitation of immunoreactivity in lateral axon before (pre) and after (post) ecdysis for BURS-IR and PBURS-IR in wild-type (+/+) animals, and for BURS-IR in pburs hemizygous animals [pburs^exc/Df(2)Exel6036], showing that bursicon subunits are secreted at pupal ecdysis, and that BURS is released at this time even in the absence of PBURS. Asterisks indicate statistically significant change in immunoreactivity (p < 0.05).

Figure 10.

A, pupal is pburs (A) pu¹/pburs^exc fly (top) showing partially expanded wings and abnormal tanning (e.g., matte exoskeleton). These defects are rescued in pu¹/pburs^exc fly bearing a P{pburs} transgene (bottom) (black arrow points to reflection indicative of properly sclerotized exoskeleton). B, C, Summary of morphological defects expressed by pu¹ hemizygotes, demonstrating allelism with pburs. Wing expansion defects (B, column 1) as well as incomplete wing (C, wing, column 1) and leg (C, leg, column 1) extension are rescued by P{pburs} transgene. Defects expressed by pu¹/pburs^exc animals (B, C, column 1) are less severe than those expressed by pburs hemizygotes, (B, C, column 3). Genotypes are displayed as described in Figure 3. In C, different letters above wing and leg columns indicate significantly different categories (p < 0.05).