Ecdysis triggering hormone: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References
Gene name - Ecdysis triggering hormone

Synonyms -

Cytological map position - 60E5

Function - ligand

Keywords - molting, hormone

Symbol - ETH

FlyBase ID: FBgn0028738

Genetic map position - 3-

Classification - ecdysis-triggering hormone

Cellular location - secreted



NCBI links: Precomputed BLAST | Entrez Gene | UniGene
Recent literature
Kruger, E., Mena, W., Lahr, E. C., Johnson, E. C. and Ewer, J. (2015). Genetic analysis of Eclosion hormone action during Drosophila larval ecdysis. Development [Epub ahead of print]. PubMed ID: 26395475
Summary:
Insect growth is punctuated by molts, during which the animal produces a new exoskeleton. The molt culminates with ecdysis, an ordered sequence of behaviors that causes the old cuticle to be shed. This sequence is activated by Ecdysis Triggering Hormone (ETH), which acts on the CNS to activate neurons that produce neuropeptides implicated in ecdysis, including Eclosion hormone (EH), Crustacean Cardioactive Peptide (CCAP), and bursicon. Despite over 40 years of research on ecdysis, understanding of the precise roles of these neurohormones remains rudimentary. Of particular interest is EH, whose role beyond the well-accepted action of massively upregulating ETH release has remained elusive. This study reports on the isolation of an eh null mutant in Drosophila, and it's use to investigate the role of EH in larval ecdysis. Null mutant animals invariably died at around the time of ecdysis, revealing an essential role in its control. Unexpectedly, however, they failed to express the preparatory behavior of pre-ecdysis while directly expressing the motor program of ecdysis. In addition, although ETH release could not be detected in these animals, the lack of pre-ecdysis could not be rescued by injections of ETH, suggesting that EH is required within the CNS for ETH to trigger the normal ecdysial sequence. Using a genetically-encoded calcium probe it was shown that EH configures the response of the CNS to ETH. These findings show that EH plays an essential role in the Drosophila CNS in the control of ecdysis, in addition to its known role in the periphery of triggering ETH release.
Mena, W., Diegelmann, S., Wegener, C. and Ewer, J. (2016). Stereotyped responses of Drosophila peptidergic neuronal ensemble depend on downstream neuromodulators. Elife 5. PubMed ID: 27976997
Summary:
Neuropeptides play a key role in the regulation of behaviors and physiological responses including alertness, social recognition, and hunger, yet, their mechanism of action is poorly understood. This study focused on the endocrine control ecdysis behavior, which is used by arthropods to shed their cuticle at the end of every molt. Ecdysis is triggered by ETH (Ecdysis triggering hormone), and the response of peptidergic neurons that produce CCAP (crustacean cardioactive peptide), which are key targets of ETH and control the onset of ecdysis behavior, was shown to depend fundamentally on the actions of neuropeptides produced by other direct targets of ETH and released in a broad paracrine manner within the CNS; by autocrine influences from the CCAP neurons themselves; and by inhibitory actions mediated by GABA. These findings provide insights into how this critical insect behavior is controlled and general principles for understanding how neuropeptides organize neuronal activity and behaviors.
Meiselman, M., Lee, S. S., Tran, R. T., Dai, H., Ding, Y., Rivera-Perez, C., Wijesekera, T. P., Dauwalder, B., Noriega, F. G. and Adams, M. E. (2017). Endocrine network essential for reproductive success in Drosophila melanogaster. Proc Natl Acad Sci U S A 114(19): E3849-e3858. PubMed ID: 28439025
Summary:
Ecdysis-triggering hormone (ETH) was originally discovered and characterized as a molt termination signal in insects through its regulation of the ecdysis sequence. This study reports that ETH persists in adult Drosophila melanogaster, where it functions as an obligatory allatotropin to promote juvenile hormone (JH) production and reproduction. ETH signaling deficits lead to sharply reduced JH levels and consequent reductions of ovary size, egg production, and yolk deposition in mature oocytes. Expression of ETH and ETH receptor genes is in turn dependent on ecdysone (20E). Furthermore, 20E receptor knockdown specifically in Inka cells reduces fecundity. These findings indicate that the canonical developmental roles of 20E, ETH, and JH during juvenile stages are repurposed to function as an endocrine network essential for reproductive success.
Lee, S. S., Ding, Y., Karapetians, N., Rivera-Perez, C., Noriega, F. G. and Adams, M. E. (2017). Hormonal signaling cascade during an early-adult critical period required for courtship memory retention in Drosophila. Curr Biol 27(18): 2798-2809.e2793. PubMed ID: 28918947
Summary:
Formation and expression of memories are critical for context-dependent decision making. In Drosophila, a courting male rejected by a mated female subsequently courts less avidly when paired with a virgin female, a behavioral modification attributed to "courtship memory." This study shows the critical role of hormonal state for maintenance of courtship memory. Ecdysis-triggering hormone (ETH) is essential for courtship memory through regulation of juvenile hormone (JH) levels in adult males. Reduction of JH levels via silencing of ETH signaling genes impairs short-term courtship memory, a phenotype rescuable by the JH analog methoprene. JH-deficit-induced memory impairment involves rapid decay rather than failure of memory acquisition. A critical period governs memory performance during the first 3 days of adulthood. Using sex-peptide-expressing "pseudo-mated" trainers, it was found that robust courtship memory elicited in the absence of aversive chemical mating cues also is dependent on ETH-JH signaling. Finally, this study found that JH acts through dopaminergic neurons, and it is concluded that an ETH-JH-dopamine signaling cascade is required during a critical period for promotion of social-context-dependent memory.
BIOLOGICAL OVERVIEW

At the end of each developmental stage, insects perform a stereotypic behavioral sequence leading to ecdysis (molting or shedding) of the old cuticle. While ecdysis triggering hormone (ETH) is sufficient to trigger this sequence, it has remained unclear whether it is required. This study shows that deletion of ETH, the gene encoding ETH in Drosophila, leads to lethal behavioral and physiological deficits. Null mutants (eth-) fail to inflate the new respiratory system on schedule, do not perform the ecdysis behavioral sequence, and exhibit the phenotype buttoned-up, which is characterized by incomplete ecdysis and 98% mortality at the transition from first to second larval instar. Precisely timed injection of synthetic ETH1 (one of the peptides encoded by the ETH gene) restores all deficits and allows normal ecdysis to occur. These findings establish obligatory roles for ETH and its gene products in initiation and regulation of the ecdysis sequence. The ETH signaling system provides an opportunity for genetic analysis of a chemically coded physiological and behavioral sequence (Park, 2002a).

Insect development proceeds through a series of stages from egg to reproductive adult, each punctuated by ecdysis of the old cuticle. Initiation of the molt, which culminates in ecdysis, coincides with one or more bouts of ecdysteroid-induced gene expression appropriate to the next stage. The end of the molt is signaled by declining steroid levels, leading to ecdysis of the old exoskeleton surrounding the body and lining the respiratory system and gut (Park, 2002a).

Ecdysis is controlled by a genetic program specifying a precisely timed developmental sequence. Among the genes involved are those encoding peptide hormones that activate central pattern generators for pre-ecdysis and ecdysis behaviors. Ecdysis in Saturniid moths is triggered by a peptide factor from the brain, identified as eclosion hormone (EH: see Drosophila Eclosion hormone). EH causes secretion of ecdysis-triggering hormones (ETHs) from endocrine Inka cells (Zitnan, 1996; Kingan, 1997), which act on the CNS to trigger centrally patterned pre-ecdysis and ecdysis behaviors. It is well established that ETHs trigger ecdysis upon injection into moths and flies (Zitnan, 1996; Ewer, 1997; Baker, 1999; Park, 1999; Zitnan, 1999; Zitnan, 2000). These studies demonstrate the sufficiency of ETHs in triggering ecdysis, but do not prove their necessary involvement in the natural process (Park, 2002a).

During the hour preceding ecdysis of 1st instar Drosophila larvae, three main events occur: sclerotization of new mouthparts, tracheal dynamics and a defined behavioral sequence. Appearance of the new mouthparts, including mouthhooks and vertical plates, precedes ETH release, and no disruption of these events occurs in eth flies. However subsequent events are triggered by ETH release from a system of Inka cells homologous to those previously described in moths (Park, 2002a).

As direct gene products, many peptide signaling molecules provide a link between gene expression and behavior. Orchestration of the ecdysis behavioral sequence depends on coordinated induction and suppression of genes essential to its properly timed initiation. This includes induction of ETH gene expression in Inka cells and CNS sensitivity to ETH 1-2 days before ecdysis (Zitnan, 1999; Zitnan, 2000). The timing of ecdysis initiation is accomplished by ecdysteroid suppression of secretory competence in Inka cells until the animal is ready to perform the behavioral sequence and escape the old cuticle (Kingan, 2000). Once ecdysteroids drop to low levels during the hours preceding ecdysis, peptide hormones, including ETHs, eclosion hormone and Cardioacceleratory peptide, are released to initiate the behavioral sequence. Orchestration of ecdysis behavior therefore depends on coordinated expression of genes that are involved in peptide signaling (Park, 2002a and references therein).

Each of these peptides is capable of initiating ecdysis, but it has remained unclear to what extent they play obligatory roles. To test the hypothesis that ETH is required for ecdysis, genetic tools in Drosophila were used to delete its gene, ETH (Park, 1999). Genetic null Drosophila mutants carrying micro-deletions in the ETH locus are described. The consequences of this selective hormonal deficit are severe, and include failure both to inflate the new respiratory system and to perform the ecdysis behavioral sequence. Virtually all animals fail to survive the first ecdysis. These findings establish an obligatory role for eth and its gene products, and demonstrate how lack of a specific chemical signal results in a lethal behavioral deficit (Park, 2002a).

Larval ecdysis in Drosophila culminates a sequence of precisely timed morphological, physiological and behavioral events. These include appearance of new mouthparts, tracheal inflation, and pre-ecdysis and ecdysis behaviors. Park (2002) details a time line of these events, which occur during the 1 hour interval preceding ecdysis from 1st to the 2nd larval instar. Visible morphological changes become apparent ~1 hr prior to ecdysis, including the appearance of new mouth hooks and vertical plates. The first event is sclerotization of new mouth hooks adjacent to the old structures, referred to 'double mouth hooks'. About 30 minutes later, new vertical plates appear, leading to 'double vertical plates' (dVP). dVP was chosen as a reference point (time zero) to which all other events are related (Park, 2002a).

Before the appearance of dVP, the old tracheal lining or 'intima' in the main dorsal tracheal trunk separates from the new intima. About 10 minutes after the dVP stage the old intima collapse, becoming coiled in appearance. Tracheal collapse is followed immediately by inflation of the new trachea with air, which takes ~2-5 minutes. Before this time, the space between the old and new intima is filled with liquid, presumably molting fluid (Park, 2002a).

Pre-ecdysis behaviors commence upon completion of tracheal air filling. The first stage of pre-ecdysis consists of repetitive 'anteroposterior' contractions (A-P) beginning 15 minutes after dVP. This behavior is characterized by alternating telescopic contractions of 2-5 seconds duration and relaxations lasting 10-18 seconds. The second stage of pre-ecdysis beginning at 19 minutes involves rolling contractions called 'squeezing waves'. Squeezing waves (SW) are visible from the dorsal aspect and travel from posterior to anterior at 5-6 second intervals, ending with head retractions. During the A-P and SW behaviors, vigorous muscle contractions pull the mouthparts alternately in the posterior and anterior directions. It is believed that these movements may be crucial for later detachment of old vertical plates and mouthhooks from the new apparatus during subsequent ecdysis behavior (Park, 2002a).

Ecdysis behavior begins 25 minutes after dVP, with one or two forward head thrust movements, which detach old mouthparts and plant them in the substrate. The forward movement also coincides with shedding of old tracheal linings through segmental spiracular pits. Upon planting the old mouthparts onto the substrate, the forward thrust is followed by three to five vigorous backward thrusts to detach the old posterior spiracles. The behavior is interrupted by a 2-5 minute rest period, and is completed by a forward, lateral turning escape movement, freeing the animal from the old cuticle. Some variation in this pattern is observed, in which subunits of the ecdysis behavior are repeated or even entire recapitulations of the behavioral sequence are observed. The patterned behavior just described is also accompanied by some irregular behaviors prior to anterior-posterior contractions (A-P) with large variations between individuals and time of onset. These are (1) head swinging, (2) dorso-ventral contractions and (3) alternating anterior and posterior peristaltic squeezing (Park, 2002a).

Ecdysis from second to third instar follows a similar pattern. Even the time of behavioral onset is similar, with the exception that double mouth hooks appear at -104 minutes relative to dVP instead of -30 minutes as observed in the 1st to 2nd instar ecdysis (Park, 2002a).

The peptides Drosophila ETH1 and ETH2, which trigger adult ecdysis (Park, 1999), were injected into late first instar larvae to assess their ability to induce ecdysis at this stage of development. Injection of ETH1 (0.01 to 10 fmol) at the double mouth hooks stage induces the premature appearance of physiological and behavioral events accompanying ecdysis. The first response to peptide injection is premature tracheal collapse and inflation, with a latency of 3 minutes and 4 minutes, respectively, followed by pre-ecdysis and ecdysis behaviors. Weak anterior-posterior contractions were observed in 33% of injected animal. Strong squeezing waves occurred in all injected animals, with an average latency of 14±2 minutes. Ecdysis behavior consisting of forward thrust, backward thrust and turning escape movements appeared with a latency of 24±3 minutes (Park, 2002a).

The timing of Drosophila ETH1 injection is critical for successful ecdysis. All animals injected at the dVP stage underwent successful ecdysis. However injections performed earlier, for example at double mouth hooks, induced tracheal collapse and inflation, anterior-posterior contractions, squeezing waves and repeated bouts of ecdysis behavior that were unsuccessful in shedding the mouthparts, leading ultimately to death. These observations indicate that precise timing of events in the ecdysis sequence is critical for successful ecdysis (Park, 2002a).

ETH2 injections were less effective in eliciting tracheal dynamics and behaviors. At relatively high doses (>10 fmol), ETH2 induces tracheal collapse and inflation with a latency of 3 minutes and 4 minutes, respectively, and ecdysis behaviors with a latency of 34±5 minutes. Strikingly, ETH2 (10 fmol) elicits neither anterior-posterior contractions nor squeezing waves. Lower doses of ETH2 (1 fmol) induce tracheal collapse and inflation with latencies of 4 minutes and 5 minutes, but have no behavioral effects (Park, 2002a).

Inka cells of Drosophila express the gene ETH and contain the peptide ETH. Expression of ETH is evident in transformed Drosophila carrying the chimeric transgene eth3-egfp, where EGFP fluorescence and ETH-like immunoreactivity are colocalized in Inka cells. No other cells exhibit EGFP fluorescence, although some CNS neurons also show ETH-IR. The most intense EGFP fluorescence is observed as larvae approach the first ecdysis, whereas neither EGFP fluorescence nor ETH-IR is detected during embryonic development or early 1st instar. This, together with the observation that no hatching deficiency occurs in eth- mutants, indicates that ETHs are probably not required for early development, including the patterned behavior associated with egg hatching. EGFP fluorescence and ETH-IR are observed throughout the adult stage, suggesting possible mating or reproductive functions (Park, 2002a).

ETH triggers tracheal collapse and inflation. This conclusion is indicated by two observations. (1) EGFP fluorescence in Inka cells sharply declines just minutes before these events. Since the subcellular distribution of EGFP fluorescence and ETH-IR in Inka cells is identical, it is concluded that EGFP and processed ETHs are sorted into secretory granules of Inka cells (Klein, 1999), and that these peptides are co-released. (2) Injection of either Drosophila ETH1 or DrmETH2 into wild-type larvae or eth- mutants induces tracheal collapse and air inflation within minutes. These observations strongly implicate ETH in the control of tracheal dynamics before ecdysis (Park, 2002a).

It is notable that Inka cells and associated components of the epitracheal endocrine system are situated directly on tracheal tubes (Zitnan, 1996). While it has been recognized for some time that ETHs act directly on the CNS to elicit centrally patterned behaviors, this report documents for the first time functions for these peptides in respiratory physiology. What is the functional significance of Inka cell placement directly on tracheal tubes? It is speculated that their location in some way senses the readiness of the respiratory system to switch over from old to new trachea in preparation for pre-ecdysis and ecdysis behaviors. Whether the Inka cells indeed have such a sensory function remains to be demonstrated (Park, 2002a).

The mechanism of tracheal inflation is not known, but it is hypothesized that dissolved gas is liberated as a consequence of fluid movement out of the tracheal lumen. The hydrophobic surface of the cuticle lining the tracheal tube may then facilitate a reverse capillary force to complete air filling. This is the first indication that these processes are under hormonal control. Further work is needed to define the signaling steps involved in this process (Park, 2002a).

Upon completion of tracheal inflation, a behavioral sequence ensues consisting of pre-ecdysis and ecdysis behaviors. These behaviors and successful ecdysis are triggered upon injection of Drosophila ETH1, consistent with an earlier study showing that ETH injection induces premature eclosion behavior in the pharate adult (Park, 1999). It is striking that the same chemical signal initiates two quite different behaviors separated by two metamorphic molts (Park, 2002a).

Drosophila ETH1 is sufficient to evoke the ecdysis sequence in larval Drosophila, as was previously observed for adult Drosophila and for MasETH in M. sexta and BomETH in B. mori (Zitnan, 1996; Kingan, 1997; Park, 1999; Zitnan, 1999). The lethal phenotype observed in eth- deletion mutants provides confirmation that, in Drosophila, ETH is necessary for ecdysis. The same physiological and behavioral deficiencies occur in both eth25b and eth196 lines, where tracheal collapse and inflation are delayed for hours, and pre-ecdysis behaviors are absent. Ecdysis behavior is abnormal and occurs prematurely, soon after the dVP stage. These physiological and behavioral deficits contribute to the buttoned-up phenotype (Park, 2002a).

It is remarkable that tracheal dynamics and the behavioral sequence absent in eth- mutants can be completely restored by injection of Drosophila ETH1. Animals rescued by injection of Drosophila ETH1 shed the cuticle normally and develop through the second instar, although a higher mortality is observed in eth196 mutants. Since this line also has a significant deletion of the adjacent orc4 gene, it may suffer additional defects which may account for this. The deletion phenotype and its facile rescue by injection of Drosophila ETH1 provide the best evidence thus far that this blood-borne peptide is a necessary signal in the orchestration of key developmental events culminating in ecdysis. Interestingly, Drosophila ETH2 injection also rescues the buttoned-up phenotype, but without triggering pre-ecdysis behaviors. It therefore appears that the pre-ecdysis behaviors in Drosophila larvae are not crucial for completion of ecdysis (Park, 2002a).

An obvious physiological consequence of ETH deletion is failure of the respiratory system to inflate on schedule. This is consistent with the finding that ETH injection regulates collapse of old trachea and inflation of new trachea, and confirms that this peptide plays a vital signaling function for tracheal dynamics associated with ecdysis. Significantly, tracheal collapse and inflation are markedly delayed, but not eliminated in eth- larvae. Therefore ETH may act indirectly through downstream regulatory processes, which eventually succeed in mediating tracheal inflation. One candidate signal for tracheal inflation is Eclosion hormone. Adult Drosophila that lack functional eclosion hormone neurons fail to inflate the tracheal system properly upon adult eclosion and are reported to be insensitive to ETH (Park, 2002a).

The actual causes of lethality observed in eth- larvae could be a combination of factors, including respiratory and behavioral deficits. Lack of tracheal inflation and incomplete shedding of the old tracheal lining would obviously compromise respiratory functions. Failure to shed old mouthparts at the appropriate time is likely the main factor in production of the buttoned-up phenotype. This condition appears in null mutants, as well as in wild-type flies upon premature injection of Drosophila ETH1. In either case, the buttoned-up phenotype prevents complete ecdysis and further ingestion of food. All of these factors could be jointly involved in the lethality observed (Park, 2002a).

Of special significance is the fact that ETH deletion mutants engage in a premature ecdysis-like behavior. Although quite variable and different from authentic ecdysis, these sporadic bouts gradually became more similar to authentic ecdysis more than 2 hours after dVP. Animals are able to shed the old cuticle partially, albeit with buttoned-up mouthhooks. The early appearance of ecdysis-like behavior provides further evidence that ETH not only triggers ecdysis, but delays its onset until the appropriate time. Decapitation or removal of cephalic or thoracic ganglia accelerates ecdysis, supporting the hypothesis that ETH activates both excitatory and inhibitory centers in the CNS (Baker, 1999; Zitnan, 2000), with inhibition functioning to delay ecdysis until the appropriate time. In the absence of ETH, these inhibitory centers may not be triggered, leading to premature ecdysis or ecdysis-like behavior (Park, 2002a).

The ETH gene encodes three predicted amidated peptides: ETH1, ETH2 and ETH-AP. Two of these peptides, ETH1 and ETH2 have biological activity in both larval and adult stages (Park, 1999). In wild-type larvae, injection of Drosophila ETH1 elicits the entire sequence of ecdysis-related events, although for reasons not yet clear the A-P phase of pre-ecdysis is rather weak. Similarly, Drosophila ETH1 alone rescues the entire sequence in eth- mutants, including tracheal dynamics, anterior-posterior contractions (A-P), squeezing waves (SW) and ecdysis. ETH2 also elicits tracheal dynamics and ecdysis behavior, but only at higher doses. This peptide fails to trigger pre-ecdysis (A-P and SW). Lower doses of ETH2 (~1 fmol) induce only tracheal dynamics, without pre-ecdysis or ecdysis. With regard to relative potency, these findings are consistent with earlier work showing that Drosophila ETH1 is more active than DrmETH2 in triggering adult eclosion (Park, 1999). However, no functional differences were apparent in that study (Park, 2002a).

Given that Drosophila ETH1 is sufficient to trigger the entire sequence, the functional role of DrmETH2 is unclear. It might have been expected that DrmETH2 is involved in pre-ecdysis behavior, given that Drosophila ETH1 injection elicits only weak A-P contractions. However, DrmETH2 fails to elicit pre-ecdysis at all, yet elicits tracheal collapse and air filling at lower doses than are needed to generate ecdysis behavior. The higher potency of this peptide in eliciting tracheal collapse and air filling suggests that it may regulate tracheal dynamics in a way that has thus far escaped attention. It is also possible that because ETH is expressed in the adult stage, DrmETH2 plays some role in mating or reproduction. Further work is needed to resolve these issues (Park, 2002a).

In M. sexta, the ETH gene also encodes three peptides, PETH, ETH and ETH-AP (Zitnan, 1999). PETH induces only pre-ecdysis I, whereas ETH triggers pre-ecdysis II and ecdysis. It is also known that injection of ETH into naive animals elicits all three behavioral steps, including pre-ecdysis I, pre-ecdysis II and ecdysis (Zitnan, 1996). It would therefore be predicted that, if the ETH gene were deleted in M. sexta, ETH alone might be sufficient to rescue the missing behavioral sequence, leaving the role (necessity) of PETH similarly unclear. It might be possible to approach this issue using RNAi followed by injection of each peptide (Park, 2002a).

The phenotype of eth- null mutants results in 98%-100% lethality at the first larval ecdysis. This is striking in comparison with the phenotype of EH-cell knockout flies that show only ~50% lethality in larval stages, uncoordinated behaviors during adult eclosion and accumulated lethality of ~70%. Similarly, ablation of crustacean cardioactive peptide (CCAP: Gammie, 1997)-cells leads to no obvious defects during larval stages but major lethality at pupation. It has been proposed that ETH acts through central release of EH, because although wild-type flies show premature eclosion behavior upon injection of ETH, EH-cell knockout flies are insensitive to ETH. Furthermore, CCAP is seen as a downstream signal whose release is caused by EH (Park, 2002a).

If EH is an obligatory signal downstream of ETH why is the larval mortality of eth- animals as shown in this study so much higher than that of EH knockouts? Analysis of behavioral phenotypes in larval ecdysis of CCAP-cell knockout and EH-cell knockout flies may provide invaluable information to aid in the understanding of the roles of each peptide and signaling cascade for the ecdysis (Park, 2002a).

The cascade of peptides involved in regulation of insect ecdysis is growing. It is clear that ETH participates in a complex cascade that includes EH, and crustacean cardioactive peptide (CCAP) (Gammie, 1997). These molecules appear to be broadly conserved among Lepidoptera, Drosophila and other insects, and may be expandable to other arthropods such as crustaceans. A conceptual framework for the peptide signaling cascade regulating ecdysis has been provided by studies of Manduca sexta (Ewer, 1997; Gammie, 1999; Zitnan, 1999; Zitnan, 2000) and Drosophila. It is thought that a positive feedback pathway between peripheral secretion of EH from the CNS and ETH from Inka cells elevates circulating levels of both peptides (Ewer, 1997). Elevated levels of ETH acting on downstream targets within the CNS recruit sequential pre-ecdysis and ecdysis behaviors. Among these targets are CCAP-containing neurons (Gammie, 1997), which activate the central pattern generator for ecdysis behavior (Park, 2002a).

Ecdysis provides an excellent model system for analysis of a chemically coded behavioral sequence. The behavioral deficits associated with ETH deletion provide a particularly clear illustration of behavioral failure in the absence of the requisite signaling molecule. It is remarkable that complete rescue of the behavior occurs on simple injection of ETH. These findings indicate that the performance of innate, stereotypic behavioral sequences may depend upon achieving proper levels of peptides and other endogenous signals in the nervous system so as to activate and bias central pattern generators appropriate to developmental and sensory context. Other specific examples of how peptides may function as behavioral orchestrators in vivo include egg-laying, feeding and maternal behaviors (Park, 2002a).

Several developmental mutants of Drosophila display phenotypes defined by molting deficiencies. Many of these display 'double mouth hooks', suggesting a defect in an aspect of the ecdysis program. Many if not most of these phenotypes result from defective events upstream of ETH signaling, and it seems likely that they may result in ETH deficiency and the buttoned-up phenotype (Park, 2002a).

Mutations that lead to the double mouthhooks phenotype fall into three general categories: ecdysone synthesis and secretion, downstream transcriptional signaling and peptide processing enzymes. Ecdysone synthesis mutants include ecd (ecdysoneless), dare (defective in avoidance of repellents) and itpr (IP-3 receptor). Transcription factors regulated by ecdysone signaling include EcR-B, USP (ultraspiracle), ßFTZ-F1 (ftz transcription factor 1) and crc (cryptocephal). Finally, a mutant displaying the buttoned-up phenotype carrying defective peptide processing enzymes is amontillado, which encodes the enzyme prohormone convertase 2; these mutants are deficient in larval molting. Some mutants also are deficient in hatching behavior. Mutations of PHM (peptidylglycine alpha-hydroxylating monooxygenase), which is required for alpha-amidation at the C-terminal end, also generate a buttoned-up phenotype. It will be interesting to determine whether many if not most of the above mutations lead to ETH deficiency. The ETH deletion mutant and the ETH reporter fly line 2eth-egfp also provide opportunities to investigate roles for ecdysone in regulation of expression, processing and secretion of ETH, and its downstream pathways up to ecdysis behavior (Park, 2002a).

Rescheduling behavioral subunits of a fixed action pattern by genetic manipulation of peptidergic signaling

The ecdysis behavioral sequence in insects is a classic fixed action pattern (FAP) initiated by hormonal signaling. Ecdysis triggering hormones (ETHs) release the FAP through direct actions on the CNS. This study presents evidence implicating two groups of central ETH receptor (ETHR) neurons in scheduling the first two steps of the FAP: kinin (aka drosokinin, leucokinin) neurons regulate pre-ecdysis behavior and CAMB neurons (CCAP, AstCC, MIP, and Bursicon) initiate the switch to ecdysis behavior. Ablation of kinin neurons or altering levels of ETH receptor (ETHR) expression in these neurons modifies timing and intensity of pre-ecdysis behavior. Cell ablation or ETHR knockdown in CAMB neurons delays the switch to ecdysis, whereas overexpression of ETHR or expression of pertussis toxin in these neurons accelerates timing of the switch. Calcium dynamics in kinin neurons are temporally aligned with pre-ecdysis behavior, whereas activity of CAMB neurons coincides with the switch from pre-ecdysis to ecdysis behavior. Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior. These findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP (Kim, 2015).

Innate behaviors are stereotypic patterns of movement inherited from birth that require no prior experience for proper execution. Among such behaviors are fixed action patterns that, once initiated, run to completion independent of sensory inputs. Examples include courtship rituals, aggression displays, and ecdysis. Ecdysis represents a 'chemically-coded' behavioral sequence triggered by peptidergic ecdysis triggering hormones (ETH), which orchestrate a downstream peptidergic cascade leading to sequential activation of central pattern generators underlying patterned motor activity. The term FAP has fallen into disuse, since innate behaviors generally exhibit considerable plasticity. However the invariant nature of the ecdysis behavioral sequence makes it a clear example a classic FAP. In depth analysis of ecdysis behavior may provide a more thorough understanding of how hormones assemble and regulate behavioral circuitry in the brain, in particular circuits that operate sequentially (Kim, 2015).

ETHs are released by peripheral peptidergic Inka cells in response to declining levels of the steroid hormone 20-hydroxyecdysone. Presence of Inka cells in more than 40 species of arthropods, along with the sequence similarity of ETH peptides in diverse insect groups, suggests that ETH signaling is highly conserved in insects. Identification of the Ecdysis Triggering Hormone receptor (ETHR) gene in Drosophila melanogaster enabled elucidation of a complex downstream signaling cascade triggered by ETH. The ETHR gene encodes two functionally distinct subtypes of G protein coupled receptors (ETHR-A and -B) through alternative splicing. The presence of two ETH receptor subtypes has been observed in all insect species thus far examined. The two receptor subtypes show differences in ligand sensitivity and specificity and are expressed in separate populations of central neurons, suggesting that they have distinctive roles in ETH signaling (Kim, 2015).

A diversity of ETHR neurons in the moth Manduca sexta and fruitfly Drosophila melanogaster has been identified. One of the most striking properties of ETHR-A neurons is that they are virtually all peptidergic and conserved across insect orders. Groups of ETHR-A 'peptidergic ensembles' express a range of different neuropeptides, including kinins, diuretic hormone (DH), eclosion hormone (EH), FMRFamide, crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIPs), bursicon (burs and pburs), neuropeptide F (NPF), and short neuropeptide F (sNPF). It is hypothesized that released ETH acts directly on the CNS to activate these peptidergic ensembles for control of specific central pattern generator circuits that elicit stereotyped ecdysis behaviors. However evidence for direct actions of ETH on these target ensembles is yet to be reported (Kim, 2015).

Likely functions of certain ETHR-A peptidergic ensembles in Manduca have been inferred from pharmacological manipulations. For example, serially homologous L3/4 neurons of abdominal ganglia in Manduca express a cocktail of kinins and diuretic hormones; exposure of the isolated CNS to these peptides elicits a fictive pre-ecdysis I-like motor pattern. Similarly, the IN704 peptidergic ensemble that co-expresses CCAP and MIPs is implicated in initiation of ecdysis behavior, since co-application of these two peptides elicits fictive ecdysis behavior. Homologous peptidergic ensembles in Drosophila exhibit characteristic patterns and time courses of calcium mobilization indicative of electrical activity coincident with successive steps in the ecdysis FAP. Of particular interest are observations that bursicon, a hormone co-expressed in a subset of CCAP neurons, is required for ecdysis behavior (Kim, 2015).

This study tested hypotheses that two central peptidergic ensembles -- kinin neurons and a subset of CCAP neurons (CAMB) that co-express CCAP, Allatostatin CC, Myoinhibitory peptide, and Bursicon -- are direct targets of ETH and schedule pre-ecdysis and ecdysis behavior components of the ecdysis FAP, respectively in the fruit fly Drosophila. Manipulation of ETHR expression levels and signal transduction specifically in these ensembles influences scheduling of the FAP. Finally, possible mechanisms were examined underlying timing of the switch from pre-ecdysis to ecdysis behavior, and a model is proposed to explain mechanistically how these behaviors are sequentially activated (Kim, 2015).

The aims of this study were to implicate identified ETHR neuronal ensembles with specific steps in the ecdysis FAP. Through genetic manipulation of all known ETHR-A central neuron ensembles and subsets thereof, kinin and CAMB neurons were implicated in scheduling of pre-ecdysis and ecdysis, respectively. These findings confirm that these ensembles are targeted directly by ETH. A key observation is timing of calcium mobilization in kinin and CAMB ensembles following ETH application: kinin neurons mobilize calcium within minutes, while activity in CAMB neurons is delayed. CAMB neurons mobilize calcium only after kinin neuron activity ceases, some 10 minutes later. These temporal patterns of cellular activity correspond well with those of pre-ecdysis and ecdysis behaviors observed in vivo (Kim, 2015).

The findings demonstrate that the kinin peptide ensemble is necessary for proper scheduling of pre-ecdysis behavior, if not itself sufficient to elicit it. Kinin cell ablation abolishes pre-ecdysis behavior in a significant percentage (25%) of animals. The remaining 75% of individuals showed highly variable pre-ecdysis duration, ranging from 3-22 minutes, whereas duration of this behavior in control animals is tightly regulated at 9.1 ± 0.9 min. Furthermore, a 30% reduction in kinin receptor expression, caused by a piggyBac-element insertion into the promoter region of the gene, also disrupts fidelity of pre-ecdysis regulation; this phenotype is rescued by precise excision of the piggyBac insertion. Finally, RNA silencing of the kinin receptor in peripheral neurons using the Peb-Gal4 driver leads to reduced intensity of the behavior and greatly increases variability of pre-ecdysis duration. This is the first report demonstrating that kinin signaling affects scheduling of the ecdysis behavioral sequence via actions on peripheral neurons. Pan-neuronal silencing of kinin receptors also disrupted pre-ecdysis scheduling, but to a lesser extent (Kim, 2015).

Manipulation of ETHR expression levels in kinin neurons also alters scheduling of pre-ecdysis behavior significantly, confirming that these neurons are targeted directly by ETH and that they play an important role in pre-ecdysis regulation. It was reasoned that knockdown of receptor expression in these neurons would lead to a lower density of ETHR in the plasma membrane, thereby reducing sensitivity to ETH and delaying onset of pre-ecdysis. The experimental results demonstrated a reduction of pre-ecdysis duration. This reduction was attributed to a delay in pre-ecdysis onset, brought about by the need for higher ETH levels for neuronal activation. Since timing of the switch to ecdysis (controlled by CAMB neurons; see below) is unaffected, pre-ecdysis duration is shortened. In contrast, overexpression of ETHR in kinin neurons led to prolongation of pre-ecdysis duration. Following the same reasoning, this would result from premature kinin neuron activation attributable to higher sensitivity to rising ETH levels and overall longer pre-ecdysis (Kim, 2015).

Kinins were identified originally using bioassays for myotropic and diuretic functions and they have well-known actions on muscle and transport activity in epithelia. More recent studies demonstrated diverse functional roles for kinin signaling, including feeding, olfaction, and locomotory behavior. ETHR-A expression was previously demonstrated in kinin neurons of both Drosophila and Manduca, implicating them as direct targets of ETH. Imaging studies have shown that abdominal kinin neurons in fly larvae exhibit periodic calcium oscillations under normal conditions and are involved in turning behavior. These kinin neurons project to a terminal plexus in close association to kinin receptors, suggesting it functions as a site of peptide release. Interestingly, this same ensemble of kinin neurons in the pre-pupal preparation used in this study showed no such periodic activity, but instead exhibited synchronized calcium oscillations activity following exposure to ETH. This difference could be unique to the pharate stage (i.e., preceding ecdysis) animal, during which insects generally are unresponsive to external stimuli. In imaging studies, it was observed that ETH-induced calcium dynamics are initiated from the terminal plexus region and subsequently moving anteriorly to the cell bodies. These observations suggest that this plexus serves critical functions in both sending and receiving signals. Evidence presented in this study for regulation of pre-ecdysis behavior by kinin neurons demonstrates a new function for this peptide in Drosophila, which is reinforced by previous observations in Manduca that application of kinin causes a fictive pre-ecdysis motor pattern in the isolated CNS (Kim, 2015).

How do kinin neurons function in the promotion of pre-ecdysis behavior? While manipulation of kinin signaling clearly affects behavioral intensity and duration, attempts to initiating pre-ecdysis through temperature-dependent activation of kinin neurons expressing either TRPM8 and TRPA1 were unsuccessful. It is concluded that, while kinin functions as a modulatory influence necessary for proper scheduling of pre-ecdysis behavior, other as yet unidentified signals are necessary for behavioral initiation (Kim, 2015).

This study has demonstrated that CAMB neurons are both necessary and sufficient for the switch from pre-ecdysis to ecdysis behavior. This conclusion rests on results from a combination of experiments. First, CAMB cell ablation abolishes the switch to ecdysis, suggesting these neurons are necessary for the switch. Failure of ecdysis initiation is attributable to bursicon deficiency, since a previous report showed clearly that expression of the bursicon gene is required for initiation of pupal ecdysis. Calcium mobilization in CAMB neurons is delayed for ~10 min after onset of activity in kinin neurons, which fits well with the ~10 min delay before appearance of ecdysis behavior following onset of pre-ecdysis behavior observed in vivo. Altered levels of ETHR expression in CAMB neurons clearly affects timing of the switch to ecdysis behavior: receptor knockdown delays the switch, whereas overexpression accelerates it. In vitro experiments confirm that altered ETHR expression levels affect timing of calcium mobilization in CAMB neurons in register with changes in behavioral timing (Kim, 2015).

Finally, it was shown that activation of CAMB neurons through temperature-sensitive TRPM8 expression initiates ecdysis behavior in vivo. Thus, CAMB neurons are both necessary and sufficient for the switch to ecdysis behavior. However, activity in CAMB neurons alone does not result in robust ecdysis behavior. Expression of ecdysis behavior with parameters corresponding to that observed in wild-type flies requires activation of the entire CCAP ensemble (Kim, 2015).

It is interesting that, in all TRPM8 activation experiments, removal of the temperature stimulus led to re-capitulation of the entire ecdysis FAP. This might be explained by positive feedback influences on the Inka cell to release ETH, possibly via EH neurons. Alternatively, the ecdysis motor circuit may exert negative feedback on the pre-ecdysis circuit, which when removed, causes a post-inhibitory rebound leading to activation of the pre-ecdysis circuit and the entire FAP. Attempts to demonstrate such negative feedback here were inconclusive (Kim, 2015).

CAMB neurons express a combination of CCAP, Ast-CC, MIP, and bursicon. In Manduca, application of a CCAP/MIP cocktail is sufficient to elicit fictive ecdysis behavior. It would be parsimonious to extrapolate this result to Drosophila, since both of these peptides are found in CAMB neurons. Nevertheless, in Drosophila it is clear that bursicon is a key signaling molecule necessary for ecdysis initiation. It remains to be demonstrated precisely how absence of the bursicon gene blocks the switch to ecdysis. It will be interesting to elucidate possible functional roles of co-expressed peptides in CAMB neurons CCAP, Ast-CC, MIP in activation of the motor circuitry encoding the ecdysis motor pattern (Kim, 2015).

How is timing of the switch to ecdysis determined? Since both kinin and CAMB ensembles express ETHR, one would expect ETH to activate both ensembles simultaneously. Several previous studies provide evidence for the role of descending inhibition from cephalic and thoracic ganglia in setting the delay in the switch to ecdysis behavior. This study shows that expression of pertussis toxin in CAMB neurons accelerates the switch to ecdysis, consistent with disinhibition of Gαi/o input(s). It is hypothesized that a balance of excitatory and inhibitory inputs to the CAMB neurons contributes to the delay in their activity, excitatory input coming from ETH via Gαq signaling and Gαi/o from an as yet unidentified transmitter descending from cephalic and/or thoracic ganglia. The finding that RNAi-knockdown of MIP neurons lying outside the CCAP ensemble accelerates the switch to ecdysis behavior suggests one such possible inhibitory input (Kim, 2015).

It is possible, if not likely that ETH drives both inhibitory and excitatory inputs to CAMB neurons, with ETHR-B-expressing inhibitory inputs preceding excitatory input. Such a scenario follows from the fact that sensitivity of Drosophila ETHR-B to ETH was shown to be ~450-fold higher than that of ETHR-A (Park, 2003). Therefore, as ETH levels rise in the hemolymph, ETHR-B-expressing inhibitory neurons would be activated well before ETHR-A neurons. ETH would effectively inhibit CAMB neurons indirectly prior to direct excitation via ETHR-A activation (Kim, 2015).

Such a scenario pre-supposes that the EC50 values governing activation of ETH receptors determined previously from heterologous expression in mammalian CHO cells are valid in Drosophila neurons. Data presented in this study suggests this is so. The EC50 value for ETH1 against ETHR-A was found to be ~414 nM, while the EC50 for ETH2 was determined to be ~4.3 μM. A combination of ETH1 and ETH2, each at a concentration of 300 nM, was applied to the isolated CNS, and a pattern was obtained of calcium dynamics in kinin neurons lasting for ~10 min, which matches the duration of pre-ecdysis behavior under natural conditions. Furthermore, the switch to ecdysis behavior occurs ~10 min after initiation of calcium mobilization in kinin neurons, which corresponds to timing of the switch to ecdysis behavior in vivo. Doubling concentrations of the ETH1/ETH2 cocktail reduced the duration of calcium dynamics in kinin neurons to 5.5 min and accelerated the switch to ecdysis behavior. These results make it likely that the relative sensitivities of ETHR-B and ETHR-A are as established in Park (2005) and consequently activity in ETHR-B neurons would precede that of ETHR-A neurons (Kim, 2015).

This study has shown that altered levels of ETHR expression have significant consequences for timing of pre-ecdysis duration and timing of the ecdysis switch. These findings raise the possibility that scheduling of sequential steps in the ecdysis FAP may be a consequence of different sensitivities to the peptide ligand. In other words, delay in the switch to ecdysis could result from a lower density of ETHR in CAMB neurons, making them less sensitive to ETH. Possible differential sensitivity to ETH could be tested in variety of way, including assessing timing of responses to the ligand by acutely dissociated neurons and/or single cell PCR (Kim, 2015).

A mechanistic model is proposed to explain neural mechanisms underlying the Drosophila pupal ecdysis FAP (see A model depicting functional roles of kinin and CAMB neurons in scheduling of the ecdysis FAP.). Principle players in orchestration of pre-ecdysis and ecdysis behaviors are the kinin and CAMB ETHR ensembles, respectively. As ETH levels rise in the hemolymph, ETHR-B neurons are activated due to their high sensitivity (EC50 ~ 1 nM). These neurons release inhibitory signals acting through Gαi/o to inhibit CAMB neurons. As ETH levels rise further, kinin neurons receive direct excitatory input from ETH signaling via ETHR-A and Gαq to mobilize calcium from intracellular stores, leading to electrical activity in these neurons. ETH acts simultaneously on CAMB neurons, but inhibition from ETHR-B neurons descending from anterior ganglia prevents them from becoming active. As inhibition wanes, CAMB neurons become active, initiating the switch to ecdysis behavior (Kim, 2015).


GENE STRUCTURE

cDNA clone length - 735

Exons - 2

Bases in 3' UTR - 134


PROTEIN STRUCTURE

Amino Acids - 203

Structural Domains

Ecdysis-triggering hormones (ETH) initiate a defined behavioral sequence leading to shedding of the insect cuticle. eth, a gene encoding peptides with ETH-like structure and biological activity in Drosophila melanogaster, has been defined. The open reading frame contains three putative peptides based on canonical endopeptidase cleavage and amidation sites. Two of the predicted peptides (Drosophila ETH1 and DrmETH2) prepared by chemical synthesis induce premature eclosion upon injection into pharate adults. The promoter region of the gene contains a direct repeat ecdysteroid response element. Identification of ETH in Drosophila provides opportunities for genetic manipulation of endocrine and behavioral events underlying a stereotypic behavior (Park, 1999).


Ecdysis triggering hormone: Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

date revised: 26 April 2002

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