BIOLOGICAL OVERVIEW

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP) plays a key role in the initiation of the ecdysis motor program. Drosophila bearing targeted ablations of CCAP neurons (Ccap-KO animals) have been used to investigate the role of Ccap in the execution and circadian regulation of ecdysis. Ccap-KO animals show specific defects at ecdysis, yet the severity and nature of the defects vary at different developmental stages. The majority of Ccap-KO animals die at the pupal stage from the failure of pupal ecdysis (pupation), whereas larval ecdysis and adult eclosion behaviors show only subtle defects. Interestingly, the most severe failure seen at eclosion appears to be in a function required for abdominal inflation, which could be cardioactive in nature. Although Ccap-KO populations exhibit circadian eclosion rhythms, the daily distribution of eclosion events (i.e., gating) is abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of Ccap as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila (Park, 2003).

Ecdysis is a complex yet stereotyped behavior whose timing must be precisely coordinated with the molting cycle such that it is turned on only when the old cuticle is sufficiently resorbed that it can successfully be shed. In addition, the timing of some ecdyses, typically that to the adult (adult ecdysis or eclosion), can be under the control of the circadian clock. While molting (the production of the new cuticle) is regulated by the ecdysteroid class of steroid hormones, the timing as well as the execution of ecdysis behavior is controlled by a number of neuropeptides: Eclosion hormone (EH), Ecdysis triggering hormone (ETH, and associated Pre-ecdysis triggering hormone, PETH: see Drosophila ETH), and Crustacean cardioactive peptide (Ccap) (reviewed by Ewer, 2002). Of these, Ccap is believed to be the neuropeptide that turns on the ecdysis motor program. In addition to a role in the execution of ecdysis, strong circumstantial evidence suggests that Ccap may be one of the factors that regulate the circadian timing of adult ecdysis (eclosion). For example, the Lark RNA-binding protein has been implicated in the circadian control of Drosophila eclosion (Newby, 1993), and it is localized (McNeil, 1998; Zhang, 2000) preferentially in the cytoplasm of CCAP neurons (Park, 2003 and references therein).

Although this model for the hormonal control of ecdysis is consistent with most of the available data, a number of observations suggest that the control of this behavior occurs via a more complicated mechanism. For instance, adult ecdysis still occurs in Drosophila lacking EH neurons (McNabb, 1997). Likewise, although the genetic deletion of the gene encoding ETH causes most animals to die at the first larval ecdysis (Park, 2002a), many of these animals still display ecdysis-like behavior at the end of this molt (Park, 2003).

The complex phenotypes of these variants also raises the possibility that the role of Ccap in the control of ecdysis may not be as simple as currently proposed. This study has used Drosophila to investigate the roles of Ccap in the control and circadian regulation of ecdysis. Genetic ablation of the Ccap neurons causes defects at ecdysis. However, the type of defects observed at the ecdyses to different developmental stages as well as the severity of these defects suggest that the role of Ccap in the control of ecdysis varies during postembryonic development. In addition, although populations of flies lacking Ccap neurons exhibit a circadian rhythmicity of eclosion, the daily timing of eclosion events is abnormal in these animals, implying a modulatory role for Ccap in the circadian control of this behavior (Park, 2003).

Strong circumstantial evidence implicates CCAP in the control of ecdysis behavior. In the moth Manduca, in vitro experiments using isolated abdominal central nervous systems suggest that CCAP is required for turning off the pre-ecdysis motor program (Gammie, 1997b) and turning on that for ecdysis (Gammie, 1997b; Zitnan, 2000). The phenotypes caused by ablating Ccap neurons in Drosophila are, overall, consistent with this model. Indeed, both larval and pupal pre-ecdysis are longer than normal in KO animals, in agreement with an inhibitory action of Ccap on the expression of this preparatory behavior. In addition, the lack of Ccap neurons causes the complete failure of pupal ecdysis (pupation), strongly suggesting that the Ccap neuropeptide is essential for turning on ecdysis behavior (Park, 2003).

Although Ccap neurons are essential for pupation, the ecdysis motor program of both larval and adult KO animals appears qualitatively normal, implicating additional mechanisms in the control of these behaviors. Ccap may play a minor role at these times or, alternatively, other neuropeptides may compensate for the loss of Ccap. Irrespective of the exact mechanism, the results of this study strongly suggest that other pathways, independent or compensatory, exist, which control the expression of these motor programs. To date the only gene that is known to be essential for ecdysis is the ETH gene, and flies carrying the null ETH alleles die at the first larval ecdysis. However, the ETH peptides are believed to act upstream of Ccap (reviewed by Ewer, 2002), and in Drosophila, ETH is released before Ccap at larval and pupal ecdysis, consistent with this hypothesis. Thus, it is unlikely that the ETH peptides act in parallel with Ccap or can compensate for it absence. In addition, the findings that the lack of Ccap does not cause larval lethality argue against a simple linear pathway in which the essential function of ETH is to cause release of Ccap leading to the initiation of the ecdysis motor program. EH is also believed to act upstream of Ccap (reviewed by Ewer, 2002). However, the exact role of EH in the control of ecdysis is currently unclear, since EH KO animals are usually able to ecdyse, although their behavior is somewhat disorganized. An examination of the ecdysis of animals lacking both EH and Ccap neurons compared with that of Ccap-KO and EH KO animals will reveal the extent to which EH can compensate for the lack of Ccap, and vice versa (Park, 2003).

In addition to compensatory mechanisms, other mechanisms may contribute to the varying importance of Ccap at different ecdyses. For instance, subsets of Ccap neurons may participate at some ecdyses but not at others. In the abdominal CNS of the Manduca for example, 2 pairs of CCAP-immunoreactive neurons up-regulate the second messenger cGMP at larval ecdysis, whereas only one pair does so at pupal and adult ecdysis (Ewer, 1997). Since this cGMP response likely increases the excitability of the Ccap neurons [it is known to do so for the thoracic set (Gammie, 1997a)], this change in the pattern of cGMP expression could change the relative participation of the different Ccap neurons at each ecdysis. It is not known if this sort of mechanism applies to Drosophila, since no cGMP response is detected in Ccap neurons at any ecdysis in this species (Ewer, 1996; Baker, 1999). Nevertheless, the differential activation of a subset of peptidergic neurons at different times in development could provide a mechanism for modifying the extent of the participation of these neurons in different behavioral or developmental contexts. Alternatively, the role of Ccap may change during postembryonic development because of changes in the expression of Ccap receptors. Although the Ccap receptor has not been conclusively identified (but see Park, 2002b), the completion of the Drosophila genome sequence and its subsequent analyses (Brody, 2000; Hewes, 2001) have produced a list of potential candidates (Park, 2003).

The most dramatic feature of KO animals at adult eclosion is not in the expression of the ecdysis motor program itself, but a function that may be cardioactive in nature. It may be that the Ccap neurons are important for increasing hemolymph pressure, and CCAP is known to be a cardioactive peptide in insects (see Dircksen, 1998) including Drosophila (Nichols, 1999), and to be released at eclosion in Manduca. Alternatively, the defect may be in fluid homeostasis. In crabs, for instance, the shedding of the old carapace is preceded by a massive release of hyperglycemic hormone (HH) which causes a swelling of the body via an anti-diuretic mechanism (Chung, 1999). CCAP is also released at this time (Phlippen, 2000) and could regulate HH release. Regardless of the bases for the defects observed in eclosing KO animals, their phenotype suggests that maintaining a high internal body pressure is critical for adult eclosion, and implicates the Ccap neurons in this process (Park, 2003).

Features of lark gene expression in the Ccap neurons, as well as the potential for synaptic contact between Ccap and clock neurons suggests that Ccap may play a role in mediating the circadian control of adult eclosion. Although the rhythmic eclosion profile of Ccap-KO populations shows that Ccap neurons are not essential for the circadian gating of eclosion, the distribution of eclosion events in this population indicates that these neurons modulate the gating process. This modulation may occur via a direct connection with clock neurons or other peptidergic neurons (e.g., those expressing PDF), and the anatomy of Ccap neurons in the brain is consistent with this hypothesis. The robust circadian rhythmicity of Ccap-KO populations indicates that there are multiple (and potentially redundant) cellular pathways mediating the output of the clock (Park, 2003).

Several lines of evidence suggest that Ccap neurons mediate the effects of light on eclosion, indirectly via the EH neurons. In Manduca, strong circumstantial evidence suggests that CCAP acts downstream of EH (reviewed by Ewer, 2002). In Drosophila, Ccap release occurs after EH release at larval ecdysis (A. C. Clark, M. del Campo, and J. Ewer, unpublished data cited in Park, 2003), suggesting that the same relationship may exist in the fly. Importantly, EH KO and Ccap-KO animals both show an altered response to the light-on signal, and recent evidence suggests that light can cause a premature release of EH (S. McNabb and J. W. Truman, personal communication to Park, 2003). Thus, it is possible that certain Ccap neurons mediate the light-on response that is channeled through the EH neurons (Park, 2003).

GENE STRUCTURE

Analysis of the 5' regulatory region of Ccap shows that it is devoid of a canonical TATA box; however, the region immediately upstream of transcription start does contain a putative arthropod initiator element and a downstream promoter element, as potential core regulatory elements. A potential TATA box is found 367 bp 5' of this putative arthropod initiator element and preliminary results suggest that an additional stage-specific transcript may be initiated from this upstream location (Park, 2003).

PROTEIN STRUCTURE

Amino Acids- 151 (NCBI sequence)

Structural Domains

The 701-nucleotide-long Drosophila Ccap cDNA encodes a 155 amino acid precursor. Conceptual translation of this precursor indicates that several peptides may be derived from the Ccap gene via post-translational processing. The N-terminal 23 amino acid residues are characteristic of a signal peptide. In addition, the presence of three consensus endoproteolytic cleavage sites (double or triple basic amino acids) suggests that four peptides could be produced from this precursor. The amino acid sequence of one of these is identical to that of Ccap, which is, so far, 100% invariant among a number of crustacean and insect species (reviewed by Dircksen, 1998). The presence of the consensus modification site (GRKR) suggests that Ccap is likely amidated at the C terminus in this fly (see Kolhekar, 1997) as it is in other arthropods. The other 3 putative peptides are called here Ccap-associated peptides (CCAP-AP) I, II and III. Comparisons between the conceptually translated products of Drosophila Ccap and those of other sequenced Ccap genes shows that, of the associated peptides, only CCAP-AP III exhibits any significant homology among CCAP precursors (see Loi, 2001). The Drosophila Ccap gene includes three exons separated by two introns, 208 and 53 nucleotides long. The second intron occurs within the sequence encoding the Ccap peptide, as is also seen (Loi, 2001) in the Manduca CCAP gene (Park, 2003).

date revised: 22 June 2003

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