Injury response checkpoint and developmental timing in insects

Abstract

In insects, localized tissue injury often leads to global (organism-wide) delays in development and retarded metamorphosis. In Drosophila, for example, injuries to the larval imaginal discs can retard pupariation and prolong metamorphosis. Injuries induced by treatments such as radiation, mechanical damage and induction of localized cell death can trigger similar delays. In most cases, the duration of the developmental delay appears to be correlated with the extent of damage, but the effect is also sensitive to the developmental stage of the treated animal. The proximate cause of the delays is likely a disruption of the ecdysone signaling pathway, but the intermediate steps leading from tissue injury and/or regeneration to that disruption remain unknown. Here, we review the evidence for injury-induced developmental delays, and for a checkpoint or checkpoints associated with the temporal progression of development and the on-going efforts to define the mechanisms involved.

Keywords:

• Drosophila Development
• Developmental Timing
• Ecdysone
• PTTH
• Injury
• Regeneration

Abbreviations

PTTH	=	prothoracicotropic hormone
E	=	ecdysone
20E	=	20-hydroxyecdysone
DILP8	=	Drosophila Insulin-Like Peptide 8

Developmental Delays are Triggered by a Variety of Injuries

In insects, various types of tissue damage can trigger a systemic injury response which results in prolonged larval and/or pupal stages. For example, in Galleria, small incisions to the integument prolong larval instars by approximately one day.¹ In Blattella and Periplaneta, amputation of appendages results in developmental retardation.^2-5 In Drosophila, exposure to microwave frequency electromagnetic fields (EMF) delays the onset of pupariation.⁶ Finally, in Drosophila, Ephestia and Galleria, damage to imaginal discs via irradiation or induction of cell death clones results in prolonged larval instars.^4,7-17 These delays appear to be systemic as when one wing imaginal disc is damaged, growth slows or stops in the second wing disc.^4,9-11 In most cases, development appears to arrest until the damaged tissue is healed, whereupon development commences and the emerging adult appears morphologically normal.^9,13,15,17

The extent of the injury-induced developmental delay is correlated with two factors: (1) the amount of damaged tissue present and (2) the developmental stage of the animal at the time injury is sustained.^{2,3,6,8,10,11,14,16,21} There is a positive correlation between the amount of amputated or damaged tissue and the length of the molting delay.^{2,3,6,8,10,16,18,19} For example, in Periplaneta and Blattella, molting is significantly delayed when a single limb is autotomized (self-amputated as an escape mechanism) and autotomy of a second limb causes a measurable extra delay when done simultaneously with the first autotomy.^2–5,19 In Drosophila, small lesions induced by temperature-sensitive cell death clones in the wing discs do not produce a measurable delay.¹⁰ However, increasing the amount of lethal tissue delays both pupariation and eclosion.¹⁰ There also appear to be dose-dependent effects of X-irradiation, ethyl methanesulfonate (EMS) treatment, and electromagnetic field (EMF) exposure on pupariation times, supporting the notion that the amount of damaged tissue dictates, at least in part, the length of the molting delay.^6,8,14,22

The duration of injury-induced developmental delays is affected not only by the amount of tissue damage present but also by the developmental stage during which the damage occurs. Damage induced by X-irradiation has different effects on developmental timing depending on the age of the animal at the time of treatment.^8,14 Larvae irradiated early (48–84 hours after hatching) pupariate late while those irradiated closer to the normal pupariation time often pupariate precociously.^8,14 Delays induced by other means also show a stage-dependent response; lesions induced by targeted cell death in imaginal discs only appear to retard pupariation when induced at or before a certain critical stage in development – around the beginning of the third larval instar.^10,14,17 Interestingly, damage induced via irradiation to second instar Drosophila larvae does not delay the subsequent molt but does prolong the third larval stage suggesting the presence of an injury response developmental checkpoint in the last larval instar.¹⁴ The injury response checkpoint senses damaged tissue and responds by activating a series of events that inhibit developmental transitions. This checkpoint appears to be analogous to cell cycle checkpoints which sense incomplete or inaccurate cell cycle events and respond by inhibiting cell cycle transitions.²³

In Drosophila, we know of several critical “moments of passage” that occur during the third larval stage. These include critical weight (CW), minimum viable weight (MVW), and the mid-third instar transition (MIT). Critical weight is defined as the minimum size at which starvation no longer delays the onset of metamorphosis while minimum viable weight refers to the minimum weight required to survive the next developmental transition without additional feeding.^24,25 In Drosophila, CW and MVW occur early in the third larval instar.²⁶ The mid-third instar transition (MIT) is a developmental landmark that occurs during the middle of the third larval instar and is characterized by widespread changes in gene expression.²⁷ Tissue damage is associated with delayed developmental progression when induced before or after both CW and MVW has been achieved suggesting that the injury response checkpoint does not coincide with either CW or MVW.^14,15 Tissue damage induced via temperature-sensitive cell death induction in wing discs no longer triggers delayed development after animals progress past the MIT.¹⁵ Together these studies suggest the presence of an injury response checkpoint that delays developmental transitions in response to tissue damage sustained prior to attainment of the MIT.

Regenerating Tissue May Activate the Injury Response Checkpoint

Damaged imaginal tissues and amputated appendages in many insects have the ability to regenerate through a process involving localized cell proliferation.²⁸ Regeneration of certain tissues is associated with molting delays in insects and such an influence on the molt cycle has been associated with regeneration of imaginal discs and amputated appendages.^11,13,29-37 Experiments in Ephestia indicate that the presence of regenerating imaginal tissue is sufficient to induce systemic developmental delays. For example, Rahn³⁵ implanted fragments of wing imaginal discs into the body cavities of Ephestia larvae. The implanted fragment regenerated a complete disc and during this process, further growth of host larval structures and pupation were delayed.³⁵ Similarly, Dewes³⁶ transplanted male genital imaginal disc fragments into host Ephestia larvae of various ages. Regeneration of the disc fragments was associated with delayed onset of pupation, and, in general, the greater the amount of disc regeneration that occurred, the greater the pupation delay.³⁶ No delay in development was observed when the disc fragment failed to regenerate.³⁶

In Drosophila, the larval stage during which damage is inflicted influences the amount of regeneration that occurs. Imaginal wing disc damage induced in young larvae, before the MIT, results in almost complete regeneration of wings, delayed development, and adults that are morphologically normal.^13,15 Damage induced in older larvae, after the MIT, results in almost no tissue regeneration, no developmental retardation, and wingless adults.^13,15 These studies suggest that the injury response checkpoint may signify a change in the ability of imaginal tissues to regenerate. A similar ‘regeneration critical’ period has been observed in other organisms including cockroaches and assassin bugs (Rhodnius prolixus).^2,21

Simpson¹⁰ proposed a direct relationship between growth of imaginal discs and the timing of pupariation. She suggested that discs that have not completed a certain amount of growth produce a signal that inhibits pupariation. In fact, there is abundant evidence that links proliferating imaginal cells with a delay in pupariation.^{11,12,29,30,32,34,38} For example, the mutations lethal(2)giant discs (l(2)gd) and lethal(1)discs large-1 (l(1)d.lg-1) each result in extensive overgrowth of all imaginal discs and display a third larval instar which is extended by up to 9 days and often a failure to pupariate.^32,34 In addition to imaginal discs, regeneration of amputated appendages has also been shown to be associated with a developmental delay in some insects. For example, O’Farrell and Stock^3,19 investigated the regeneration of appendages and its effect on molting in the larvae of Blattella germanica and Periplaneta americana. They demonstrated that removal of an appendage postponed the subsequent molt until the wound had healed and regeneration of the appendage was complete. They inferred that regenerating tissue triggers a systemic inhibition of development. These studies suggest that it may be localized cell proliferation in regenerating tissue, rather than signals produced by damaged tissue, that leads to inhibition of developmental progression. However, the developmental delay must depend on more than just dividing cells since even extensive hypertrophy of the haematopoietic system in l(1)Tum¹ mutant larvae does not result in delayed pupariation.³⁹

The Injury Response Checkpoint Modulates Ecdysone Signaling

Ecdysteroids control the timing of developmental transitions including molts for growth (larval-larval molts), and for metamorphosis (larval-pupal and pupal-adult molts).^40-42 Pulses of ecdysteroids trigger a variety of developmental and reproductive processes including, but not limited to, embryonic organ development, imaginal disc morphogenesis, glue protein secretion and polytene chromosome puffing, cuticle tanning, degeneration of larval tissues during metamorphosis, proliferation of histoblasts during metamorphosis and control of oogenesis in adult females.^40,43-56 The production of ecdysteroids by the prothoracic gland cells of the ring gland is stimulated by prothoracicotropic hormone (PTTH), a cerebral neuropeptide (Fig. 1A).^57-62

Figure 1. A Proposed Model for the Injury Response Checkpoint. (A) Synthesis of Ecdysone (E) by the prothoracic cells of the larval Ring Gland is stimulated by PTTH. ^57–62 PTTH release stimulates production of E which is released into the hemolymph. ⁶⁰ Ecdysone is converted to its active form 20-hydroxyecdysone (20E) by enzymes expressed in target tissues where 20E triggers a series of downstream events leading to developmental progression.⁸⁷ (B) Tissue damage and regenerating tissues lead to activation of JNK signaling and increased expression of dilp8. ^22,69 DILP8 disrupts ecdysteroid production by decreasing expression of ecdysteroidogenic enzymes including dib and phm and possibly delaying the PTTH peak. ^22,69 Tissue damage also influences ptth expression via a retinoid-dependent pathway which is separate from the DILP8 pathway. ^14,22 Wing imaginal disc damage is associated with decreased expression of the ecdysone receptor and several ecdysone response genes, however it is unclear whether or not this is a direct effect of the injury response signals or a by-product of reduced ecdysteroid production. ¹⁵ Increased expression of ecdysone oxidase, which encodes an enzyme

Since developmental progression is under ecdysteroid control it is not surprising that developmental perturbations elicited by injury and regeneration also have an endocrine basis and appear to be due to a disruption in the production of ecdysteroids (Fig. 1B).^{1,2,4,7,8,14,15,21,38} In the kissing bug, Rhodnius prolixus, leg amputation results in a temporary decrease in the hemolymph ecdysteroid titer. Abolishing nervous system activity with the neurotoxin, tetrodotoxin, delays the recovery of the ecdysteroid titer.²¹ Recovery of the ecdysteroid titer closely parallels the recovery of electrical activity in the corpora cardiaca (CC) region of the ring gland which receives PTTH from axons of neurosecretory cells.^21,63 This suggests that the decrease in hemolymph ecdysteroids is due to decreased stimulation of ecdysteroidogenesis by PTTH. In Drosophila, developmental delays induced by X-irradiation are associated with a decrease in ptth expression and the delays can be suppressed via ectopic feeding of 20-hydroxyecdysone (20E) or by disrupting components of the retinoid biosynthesis pathway suggesting a role for retinoids in inhibiting PTTH production or release following tissue damage (Fig. 1).¹⁴

Additional studies in Drosophila have demonstrated that the decrease in the hemolymph ecdysteroid titer is due to additional factors, not simply a decrease in ptth expression. Damage to wing imaginal discs also results in decreased expression of many genes encoding ecdysteroidogenic enzymes including neverland (nvd), spookier (spok), disembodied (dib) and phantom (phm) (Fig. 1).^14,15,22 Genes encoding transcription factors, including molting defecting (mld) and without children (woc), which are also required for ecdysone synthesis also show reduced expression following wing disc damage.^15,64,65 Another factor which contributes to the decline in circulating ecdysone is ecdysone oxidase, an enzyme which inactivates ecdysone by converting it to 3-dehydroecdysone.⁶⁶ Expression of ecdysone oxidase (Eo) is increased following localized cell death in the wing disc (Fig. 1).¹⁵ These studies suggest that not only is there reduced synthesis of ecdysone in the ring gland, but circulating ecdysone may also be degraded to lower the hemolymph ecdysteroid titer. It is possible that the decreased expression of ecdysteroidogenic enzymes and ecdysone oxidase is a by-product of decreased ptth expression; however this possibility has not been fully explored.

In Drosophila larvae carrying lethal mutations including dlg [lethal (1)discs large], lgd [lethal (2)giant discs], dco [lethal (3)discs overgrown], c43 [lethal (3)c43] and fat [lethal (2)fat], all of which are characterized by imaginal disc overgrowth and developmental delays, the ecdysteroid surge that normally occurs at pupariation is absent.³⁸ No accumulation of unusual metabolites or conjugates has been detected in imaginal disc overgrowth mutants indicating that the reduced titer of ecdysteroids is primarily due to reduced ecdysone production by the ring gland rather than increased metabolic inactivation.³⁸ Implantation of wild type ring glands can suppress the pupariation delay observed in larvae homozygous for the imaginal disc overgrowth mutation lgl (lethal (2)giant larvae), indicating that the pupariation delay is caused by failure of ecdysone production or release.^39,67 Together these studies indicate that injured or regenerating tissues produce a signal that inhibits ecdysteroid production or secretion by the larval ring gland, and that this injury-response pathway involves regulation of the neuropeptide PTTH.

An Insulin-like Signal Mediates the Systemic Injury Response

The signal which triggers delayed development following injury appears to originate from either damaged tissues or regenerating/proliferating tissues. Studies have suggested that x-rays, which induce chromosome aberrations in dividing cells, cause the death of cells which in turn release a diffusible morphogenetic substance which results in developmental delays.^12,68 It was recently shown in Drosophila that the signal produced by damaged and regenerating imaginal tissues is an insulin-like peptide (DILP8), which is stimulated by c-jun-terminal kinase (JNK) signaling and is capable of disrupting the timing of developmental progression through inhibition of ecdysteroid production (Fig. 1).^22,69

Increased expression of dilp8 leads to pupariation delays and retards the expression of disembodied (dib) and phantom (phm), two genes required for ecdysone synthesis in the ring gland (Fig. 1).^22,69 These delays can be rescued by feeding larvae 20E which is consistent with the idea that injury and regeneration induced developmental disruptions have an endocrine basis.^22,69 However, there are conflicting reports regarding the mechanism by which DILP8 influences ecdysteroid production. Colombani⁶⁹ demonstrated that ectopic expression of dilp8 in wing imaginal discs retards the onset of ptth expression in the ring gland by as much as 36 hours. In contrast, Garelli²² demonstrated that dilp8 overexpression had only a minimal effect on ptth expression and suggested that a second signal may act on PTTH-secreting neurons to contribute to the decrease in ecdysteroid synthesis following tissue damage. The idea that multiple pathways inhibit development following injury is supported by the fact that cell death-induced wing disc damage inhibits PTTH synthesis and the resulting developmental delay can be enhanced by the consumption of β-carotene in the diet.^14,22 The effects of DILP8 on development appear to be independent of retinoids (Fig. 1).²²

Of all the insulin-like peptides tested, dilp8 is the only one that has been shown to be differentially expressed in imaginal disc tumors as well as in imaginal tissues that have been damaged through overexpression of the proapoptotic gene reaper (rpr) or EMS treatment.^11,22 Mutations in other insulin/insulin-like growth factor 1 (IGF1) signaling pathway components, are often associated with delays in pupariation and a decrease in body and organ size.^70–72 However, Garelli and colleagues²² demonstrated that IGF1 signaling was largely unaffected in larvae overexpressing dilp8 suggesting that DILP8 may function through a novel insulin-like signaling pathway to regulate ecdysone synthesis.

Remaining Questions and Future Perspectives

The mechanism by which damaged or regenerating tissues trigger developmental delays associated with a reduction in the hemolymph ecdysteroid titer remain poorly understood. As shown in the proposed injury-response checkpoint model (Fig. 1), the injury-response checkpoint clearly involves humoral signals including insulin-like peptides and retinoids which appear to act directly on the ring gland or on PTTH-secreting neurons.^14,22,69 However, the checkpoint also appears to involve signaling through the central nervous system (CNS), at least in some organisms. For example, developmental delays induced by amputation of appendages in Periplaneta and Blattella require intact nerve connections from the amputated limb to the CNS.² In contrast, delays observed following injury to wing imaginal discs in Galleria are not suppressed by either transaction of the nerve cord or brain implantation suggesting that the delays are triggered by a humoral signal.¹

It is currently unclear whether both larval and imaginal tissues are capable of triggering developmental delays upon injury. Several studies have found that pupariation can occur in the absence of imaginal discs and that larval tissues contain all of the necessary components to initiate metamorphosis.^10,73,74 It has been clearly shown that proliferating or damaged imaginal tissues can produce signals which retard development.^{12,29,31,32,34} The effects of larval tissue injuries on developmental timing are less well characterized, however Mala and colleagues¹ have demonstrated that incisions to larval integument can trigger a slight developmental delay in Galleria.

Many genetic mutations have been identified which, like physical damage, are capable of triggering a developmental delay or arrest.⁷⁵ For example, flies bearing mutations in known cell cycle regulators display prolonged larval stages and delays in adult eclosion.^76–78 Mutations that appear to specifically disrupt the development of imaginal discs also display a systemic retardation of development.^79,80 In fact there are numerous mutations – too many to discuss at length in this review – that are associated with developmental delays and/or arrest at various stages. It is possible that a number of these mutations activate the injury response checkpoint; however it is unknown how common this may be.

It is known that injury in insects triggers developmental delays associated with a reduction in the hemolymph ecdysteroid titer. Similarly, in vertebrates, severe injury, chronic disease and infection can each lead to developmental delays – most notably disruptions in gamete development and delayed onset of puberty.^81–84 As in insects, injury-induced developmental delays are often associated with changes in steroid hormone titers.^81,85 The exact mechanism underlying injury- and infection-induced developmental delays in vertebrates remains poorly understood but may involve signaling via cytokines released from either damaged/infected tissues or cells of the immune system.^83,84,86 Understanding how injured or infected tissues trigger changes in endocrine function is critical to understanding and treating various disease states and analysis of the injury-induced molting checkpoint in insects promises to provide valuable insights into the interactions between injured tissues and endocrine centers.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.