InteractiveFly: GeneBrief

Prothoracicotropic hormone: Biological Overview | References

BIOLOGICAL OVERVIEW

In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. This report examines the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion and results in larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the overgrowth and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth-rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval (McBrayer, 2007).

The proper development of all multicellular organisms requires not only correct spatial control of cellular interactions, but also accurate timing of specific developmental programs of gene expression. These timed programs involve systemic signaling systems that respond to key nutritional and environmental cues to direct coordinated developmental responses throughout the animal. In humans, for example, passage from adolescence to adulthood is accompanied by rapid changes in growth and acquisition of sexual maturity. Likewise, in frogs a dramatic hormone-stimulated transformation remodels the immature, swimming tadpole into a sexually active, air-breathing adult. Perhaps the most dramatic examples, however, can be found in insects in which developmental transitions occur at regularly defined intervals. These transitions include molting, a process whereby the rigid exoskeleton is shed and resynthesized to accommodate increasing larval body size as a result of cell growth, and metamorphosis, a transformation in which the immature larva changes into a reproductively mature adult (McBrayer, 2007).

In most arthropods, the timing of molts and metamorphosis is coordinated by a rise in the titer of the steroid hormone 20-hydroxyecdysone (20E). In insects, the production and release of ecdysone in response to developmental cues is thought to be primarily regulated by a small, secreted peptide known as prothoracicotropic hormone (PTTH) (reviewed in Rybczynski, 2005). PTTH was originally purified from Bombyx mori brain extracts as a substance that could stimulate ecdysone production in the prothoracic glands (Kataoka, 1991). Active Bombyx PTTH is an 25 kDa disulfide-linked homodimer that is processed from a larger precursor protein. In Lepidoptera, it is produced primarily in a pair of bilateral neurosecretory cells that terminate in specialized neurohemal varicosities on the corpus allatum, a secretory gland of the neuroendocrine system. Once released from the corpus allatum into the hemolymph, PTTH targets the prothoracic gland, where it binds to an unknown receptor and triggers the production and release of ecdysone via one or more second-messenger pathways that include Ca2+, cAMP, and a MAP kinase cascade (McBrayer, 2007).

Understanding the regulation of PTTH production and release is key to deciphering the mechanisms that regulate developmental timing in insects. Studies in Lepidoptera and several other insect groups suggest that PTTH release is controlled by at least two components, weight gain and photoperiod. In some blood-sucking hemipteran species such as Rhodnius prolixus or the milkweed bug, Oncopeltus fasciatus, weight gain triggers PTTH release and molting. Since injection of air into the gut can also trigger a molt, distention of abdominal stretch receptors is thought to be the key event that signals for PTTH release and the molting response. In Lepidoptera and Drosophila, however, artificial inflation of larvae with air does not induce a molt, suggesting that similar stretching of the larval abdomen cannot, by itself, stimulate PTTH release in these insects. Instead, a more complex nutritional assessment is made in which larvae must pass through several checkpoints to ensure that they have achieved an appropriate size and amassed sufficient nutritional storage, primarily in the fat body, to survive the prolonged period of nonfeeding during metamorphosis (reviewed in Edgar, 2006; Nijhout, 2003; McBrayer, 2007).

Attainment of 'minimal viable weight' is the first checkpoint that must be attained to survive metamorphosis. Starvation before this time results in a prolonged larval stage and eventual death without pupation or a partial attempt at pupation. A second checkpoint, referred to as 'critical weight,' is reached when starvation no longer affects the time to pupation (Nijhout, 2003). Attainment of minimal viable weight ensures that there is enough nutrient storage for the animals to undergo metamorphosis, whereas reaching critical weight is thought to initiate the metamorphic process (McBrayer, 2007).

In Lepidoptera, achieving critical weight is thought to be a key factor in stimulating PTTH release (Nijhout, 1981). Superimposed on this control is a photoperiodic gating mechanism in which PTTH can only be released during a specific 8 hr window each day (Truman, 1972; Truman, 1974]. If critical weight is achieved outside this time frame, then the larvae continue to feed until the next photoperiodic gate is reached the next day (McBrayer, 2007).

Although PTTH is considered to play a key role in regulating ecdysone production and release, and therefore in the timing of insect development, this hypothesis has never been rigorously tested by genetic loss-of-function studies. This report describes the identification and characterization of a Drosophila PTTH-related gene. Similar to its lepidopteran homologs, Drosophila PTTH is a secreted factor that is produced by a pair of bilateral neurosecretory cells in the brain. In Drosophila, however, these neurons directly innervate the prothoracic gland, instead of the corpus allatum, to regulate ecdysone production. Using the Gal4/UAS system, the PTTH-producing neurons were specifically ablated and the developmental consequences were examined. Surprisingly, ablation does not completely halt development. Rather, loss of PTTH substantially increases the time required to pass through the larval period, especially the third-instar stage. This prolonged developmental period results in a longer duration of larval feeding and eclosion of larger adult flies with more cells. Feeding 20E to PTTH-producing, neuron-ablated larvae can reverse these phenotypes. These results indicate that PTTH directs proper temporal progression through larval stages and contributes to the determination of final body size in insects by regulating the duration of growth through the control of ecdysteroid production (McBrayer, 2007).

Previous biochemical attempts to purify a factor from Drosophila with PTTH-like ecdysteroidogenic activity led to the identification of two partial peptide amino acid sequences, neither of which showed significant similarity to lepidopteran PTTH (Kim, 1997). Searching the Drosophila database with NCBI Blast and several different lepidopteran amino acid sequences as templates revealed no gene with a p value lower than 0.01. However, the predicted gene CG13687 was similar in length to the moth sequences and showed high conservation in the spacing pattern of 7 cysteine residues. In lepidopteran PTTH, six of these cysteines form intramolecular disulfide bridges, creating a cysteine knot-type structure similar to those found in NGF, PDGF, and TGF-β-type factors, whereas the seventh cysteine links two monomers together to form a homodimer. Phylogenetic comparisons by using a neighbor-joining method indicate that the Drosophila sequence, as well as related sequences found in mosquitoes, are distant relatives of the lepidopteran sequences. The similarity of this sequence to PTTH has been noted previously (Rybczynski, 2005; Riehle, 2002), and it is called here Drosophila PTTH (McBrayer, 2007).

The predicted Drosophila PTTH contains a hydrophobic stretch of amino acids at its N-terminal end that likely serves as a signal peptide or a type II transmembrane segment, suggesting that it is secreted. It also contains a dibasic (KR) sequence just before the first of the conserved cysteines. Proteolytic cleavage at this dibasic sequence would release a C-terminal fragment from the precursor protein, as found for lepidopteran PTTH (McBrayer, 2007).

This PTTH-like sequence is conserved in other Drosophilidae species. However, the methionine identified by Flybase as the start codon for the D. melanogaster sequence was not conserved. Instead, a valine is substituted at this position, and an open reading frame continues upstream for an additional 24 amino acids to a second methionine codon that is preceded by an in-frame stop codon. In D. melanogaster, the reading frame also remains open upstream of the predicted start codon, suggesting that the upstream Met may indeed represent the true N terminus (McBrayer, 2007).

To determine if Drosophila ptth is transcribed and to determine its molecular structure, several cDNAs were isolated. This analysis revealed three isoforms that differ only in the region upstream of the probable signal peptide, suggesting that all transcripts will likely produce the same mature, secreted protein (McBrayer, 2007).

To examine the expression of Drosophila ptth, in situ hybridization to embryos and dissected larvae was carried out. ptth expression was first seen in stage-17 embryos in a pair of bilaterally symmetric central brain neurons. This expression continues through all larval stages and is prominent in wandering third-instar larvae, but not in sense-probe controls (McBrayer, 2007).

Since it is difficult to survey all developmental times and stages by in situ hybridization, a promoter/enhancer Gal4 fusion was created by using ~1 kb of intergenic DNA that spans the region upstream of CG13687 to the next identified gene, Pph13 (CG2819). When crossed to a strain containing a UAS-cd8 membrane-bound GFP reporter, expression is observed in two central brain neurons starting in late embryos, similar in position to those identified by in situ hybridization. Also transient expression is occasionally seen during the first- and second-instar stages in several additional central brain neurons of unknown identity. During the third-instar stage, the same two prominent neurons and their dendritic arbors are seen. To trace the axon projections of these neurons, a genomic PTTH construct tagged with a hemagglutinin (HA) epitope was created. In this case, PTTH-HA localization is seen prominently in the axons and in terminal varicosities on the prothoracic gland. Since the dendritic arbors of these neurons extend in the same direction as the axons, the neurons are unipolar and appear to correspond to the PG neurons identified in a Gal4 enhancer trap screen for neurons that innervate the prothoracic gland (Siegmund, 2001). Consistent with this assignment, it was also found that axons projecting from the pigment dispersion factor PDF-producing neurons terminate in close proximity to the dendritic arbors of the PTTH-producing neurons, similar to what has been described previously for the PDF axons. To confirm that these are the PG neurons, ptth transcripts were localized to the GFP-positive cells of the Feb211-Gal4 line described by Siegmund (2001) (McBrayer, 2007).

Since in Lepidoptera the prothoracic gland undergoes apoptosis during pupal stages, it was interest to know about whether Drosophila PTTH is expressed in the adult, particularly since Bombyx PTTH was originally extracted from adult heads. No expression was found in any tissue other than the brain, where several neurons per hemisphere were seen that continue to express PTTH, and some axons from these neurons appear to innervate the ellipsoid body, a circular structure with roles in regulating walking and flight behavior (McBrayer, 2007).

To more closely examine the transcriptional profile of ptth, semiquantitative RT-PCR was carried out on RNA isolated from carefully staged third-instar larval brains. ptth was found to be expressed throughout the third-instar stage. Its expression is not uniform, but instead shows an unusual cyclic pattern with an ~8 hr periodicity. In addition, expression shows a dramatic upregulation ~12 hr before pupariation. Attempts were made to examine protein levels on western blots by using the genomic ptth-HA-tagged lines, but the HA epitope was not detected in brain/ring gland extracts, probably because of the low expression level (McBrayer, 2007).

Since the PDF-producing neurons synapse with the PG neuron dendritic arbors, attempts were made to determine if PDF influenced the expression of ptth. RT-PCR on RNA extracted from pdf ⁰¹ mutants displayed an altered periodicity, and the overall levels of ptth transcripts were significantly increased, suggesting that PDF signaling may contribute to the transcriptional periodicity and acts as a general negative regulator of ptth transcription or message stability (McBrayer, 2007).

To gain insight into the potential function of Drosophila ptth, the consequence of its loss on larval/pupal development was examined. At present, no loss-of-function mutants are available for this gene. Several different RNAi constructs were expressed in PG neurons, but none showed significant knockdown of PTTH-HA protein expression. As an alternative, the PTTH-producing neurons were specifically ablated by using the Gal4/UAS system. Such a method has been used successfully to examine the function of eclosion hormone-producing neurons as well as the functions of several other neuropeptide-producing neurons (McBrayer, 2007).

To achieve cell-specific ablation, UAS-Grim was expressed in the PG neurons by using the ptth > Gal4 driver. A cd8GFP reporter or a PTTH-HA genomic transgene was included in the background, enabling monitoring of the the timing and extent of ablation. It was found that larvae containing two copies of UAS-Grim and two copies of ptth > Gal4 showed no detectable GFP or PTTH-HA at any stage, suggesting effective killing of PG neurons at an early age. Despite the complete loss of the PG neurons and PTTH production, some viable adults emerged from the ablation crosses. The females showed reduced fecundity, and the males exhibited male-on-male courtship behaviors similar to that seen in fruitless mutants. It was also observed that these adults, as well as the pupae and wandering third-instar larvae, were larger than wild-type. Female ablated pupae were, on average, 32% and 21% longer than UAS-Grim and ptth > Gal4 pupae, respectively; ablated male pupae were 26% and 15% longer, on average, than UAS-Grim and ptth > Gal4 controls, respectively. The adult females and males ranged from 50% to 70% heavier than the controls (McBrayer, 2007).

Not only were the bodies of adults larger and heavier, their wings were also larger. To determine if this increased size was due to an increase in cell number or to an increase in cell size, wing-hair numbers were counted within a defined 100 μ² area on each wing. Since each wing epidermal cell produces only one wing hair, the density of hairs is a useful indicator of cell size. This analysis revealed that there is no change in cell size, leading to the conclusion that the larger wings are produced by an increase in cell number (McBrayer, 2007).

To determine if the increased body size is the result of an alteration in the rate of mass accumulation during feeding or the duration of feeding, the time period spent in each larval instar stage was measure, as well as the rate of weight gain during the third larval instar as a function of time. It was found that the duration of each larval instar was lengthened in ablated animals compared to controls. The average time to ecdysis from the first- to the second-instar stage for larvae in which the PG neurons were ablated increased by ~8 hr. Interestingly, during the second- to third-instar stages, the growth delay in larvae with ablated PG neurons did not become any more pronounced and stayed ~8 hr behind the controls. During the third-instar stage, however, there is an additional dramatic developmental delay of 5 days. The average total time from egg deposition to pupariation increased from 5.5 days for controls to ~10.5 days for larvae in which the PTTH-producing neurons were ablated. After pupation, the time to eclosion for ablated animals is not different from the controls and averages about 5 days. It should be noted that not all ablated animals were able to complete development. The death rate of ablated larvae varied between trials. Approximately 5% of ablated larvae died between the first and second instar. By the third instar, 20% of the larvae had died, and by puparium formation 50% of the larvae had died. Lastly, not all animals that succeeded in puparium formation were able to complete metamorphosis, and up to 50% of puparia died before eclosion. These dead puparia included elongated prepupae, ones with head-eversion defects, and some with other phenotypes characteristic of reduced ecdysone titers (McBrayer, 2007).

Although phylogenetic analysis suggests that this study has identified a Drosophila homolog of lepidopteran PTTH, it was not possilbe to directly stimulate ecdysone production and/or release in isolated prothoracic glands. Purified Manduca PTTH is able to stimulate ecdysone production/release ~4- to 10-fold when added to isolated glands (Gilbert, 2000). Similar studies were tried with Drosophila glands and recombinant Drosophila PTTH produced in S2 cells with variable success. The inconsistent stimulation might result from several factors. First, active lepidopteran PTTH is derived from the C-terminal portion of the protein by proteolytic processing (Kataoka, 1991; Kawakami, 1990). It is not known if endogenous Drosophila PTTH is similarly processed since it has not been detected on a western blot from brain extracts. In S2 cells, no evidence is seen for PTTH processing, but these cells might not express the appropriate maturation enzyme (McBrayer, 2007).

While the inability to produce active PTTH in S2 cells might be caused by lack of proper processing, an alternative explanation for its lack of activity in a ring gland assay is that Drosophila PTTH may require a specific route of delivery that precludes it from working effectively when added exogenously to glands. Unlike lepidopteran PTTH, which is released into the hemolymph from specialized nerve endings in the corpus allatum, Drosophila PTTH is expressed in neurons that directly innervate the prothoracic gland itself, where it may function more like a neurotransmitter rather than a circulating hormone. If PTTH receptors are primarily clustered in specialized regions around the terminal varicosities, then exogenously added PTTH may not have effective access to them (McBrayer, 2007).

The axons of PTTH-positive neurons in Lepidoptera terminate on the corpus allatum, whereas, in Drosophila, the PG neurons send out processes that terminate on the prothoracic gland. Although this might represent differences between the two species in the wiring of an equivalent set of neurons, it seems more likely that it reflects a difference in the identity of the neurons that express PTTH. It is noted in this regard that both Lepidoptera and Drosophila have a pair of bilaterally symmetric brain neurons, referred to as the CAs, that specifically innervate the corpus allutum. Likewise, both species have a pair of bilateral neurons that innervate the prothoracic gland, and, intriguingly, in Lepidoptera these neurons express several prothoracicostatic peptides (Yamanaka, 2005; Yamanaka, 2006). Furthermore, some Drosophila Gal4 enhancer trap lines show expression in neurons that innervate both the PGs and CAs, suggesting that these neurons may be functionally or developmentally related (McBrayer, 2007).

To fully understand the developmental timing mechanism, it is essential to identify and characterize the signals that regulate PTTH production and release. The ptth transcriptional profile during the third-instar stage was examined; it shows an unusual periodicity and a dramatic upregulation ~12 hr prior to metamorphosis. Interestingly, this periodicity is similar to that seen in the 20E titers of carefully staged third-instar larvae when using a highly sensitive RP-HPLC/RIA assay (Warren, 2006). In this study, several small ecdysone peaks were observed at 8, 20, and 28 hr after ecdysis to the third instar, and these peaks roughly correspond to the temporal periodicity of ptth transcriptional fluctuations that are reported here. Similar small increases in levels of molting hormone have been described in the last larval stage of Lepidoptera and have been termed 'commitment' peaks. Commitment peaks have long been thought to initiate reprogramming of the larva in preparation for the subsequent larval-pupal transition. Consistent with this view is the observation that, in Drosophila, these small ecdysteroid peaks temporally correlate with large-scale transcriptional profile changes that take place during the third-instar stage (see Warren, 2006). It is speculated that the observed periodic fluctuations in PTTH transcriptional levels precede an increased burst of PTTH release at the terminal varicosities on the prothoracic gland that then determine the temporal progression of transcriptional responses during the third larval-instar stage by stimulating small, periodic increases in the basal ecdysteroid titer (McBrayer, 2007).

How the periodic PTTH transcriptional profile is generated remains unclear. In some species, including Drosophila, photoperiod gating of PTTH release has been inferred (see Mirth, 2005) as one type of regulatory input, but no studies on the role of circadian cycles in regulating the transcription of ptth have been documented. PDF is thought to play a role in coupling circadian outputs to downstream neurons to control rhythmic outputs. The close apposition of the PDF-expressing axon terminals within the PG neuron dendritic field prompted an examinination of whether PDF influenced the periodicity of PTTH transcription. It was found that, in pdf null mutant larvae, the cycle changed in complicated ways. The ~8 hr transcriptional periodicity was lost and was replaced with a modulated cycle that varied in length from 12 to 16 hr. In addition, the sharp upregulation in transcription prior to metamorphosis was attenuated, and the rise was spread out over a period of time beginning at ~20 hr instead of 12 hr prior to metamorphosis. These results suggest that not only is there a complex interaction between PTTH transcription and the circadian cycle, but also that other unknown processes likely influence PTTH production to account for its unique transcriptional periodicity (McBrayer, 2007).

Although photogating is one mechanism that regulates PTTH release, it is not the primary means by which larval developmental timing is regulated. One current model, based primarily on data from moths, suggests that an important factor in this regulation is attainment of critical weight (Mirth, 2007; Nijhout, 2003). Critical weight is operationally defined by the way larval size dictates the response to starvation (Nijhout, 2003). Prior to attainment of critical weight, starvation prevents metamorphosis, whereas after reaching critical weight, metamorphosis takes place in the majority of animals. The final adult size and the length of the metamorphic process can vary depending on the manipulations and species of insect involved (Mirth, 2007). Critical weight is assumed to reflect a neuroendocrine timing switch that signals the readiness of the larva to begin the metamorphic process. Although the means by which critical weight is assessed is not clear, correlative timing studies have suggested a model for how critical weight initiates the metamorphic developmental program in moths and other insects (Mirth, 2007). Juvenile hormone (JH) levels need to drop below a threshold for the process to begin in lepidopteran insects. In addition, it has been observed in Manduca that if JH levels are artificially raised by injection of hormone, PTTH secretion is delayed and a prolonged larval feeding phase ensues, producing larger adults. Whether reaching critical weight triggers the JH drop, or the JH drop is simply the operative signal indicating that critical weight has been achieved, is not clear. Once JH levels dip below this critical threshold, PTTH is then released, triggering the rise in ecdysteroid titers that initiate metamorphosis (McBrayer, 2007 and references therein).

The role of JH in regulating Drosophila PTTH release and metamorphosis is not as clearly defined as in Lepidoptera. If an equivalent scenario applies to Drosophila, then PTTH release should be downstream of critical weight and should respond to it. However, the data show that loss of Drosophila PTTH results in a dramatic increase in critical weight and a prolonged developmental delay. Therefore, rather than responding to critical weight, Drosophila PTTH appears to act upstream to set the critical weight threshold. In this scenario, critical weight is not an active developmental timing switch. Instead, it is an indication that the developmental program has progressed past a certain point. It is proposed that the actual timing switches are the minor pulses of PTTH and subsequent small ecdysteroid peaks that occur prior to the major rise in ecdysteroid titer that initiates metamorphosis. The idea that critical weight responds to small changes in ecdysteroid titers is also consistent with recent observations that slightly enhancing basal ecdysteroid levels by manipulating insulin signaling in the prothoracic gland shifts critical weight to a smaller size and leads to precocious metamorphosis (Caldwell, 2005; Colombani, 2005; Mirth, 2005). An alternative view, however, is that critical weight acts as the operative timing signal, but since in ablated animals there is no PTTH, they cannot respond properly and continue to feed, resulting in an apparent shift in critical weight (McBrayer, 2007).

In vivo data suggest that the primary function of Drosophila PTTH is to regulate the ecdysteroid level, especially during the third-instar stage, to properly time metamorphosis. A surprising finding from these studies is the observation that loss of PTTH does not result in a complete block to development. While up to 60% of the progeny in which the PG neurons are ablated die during larval and pupal stages, the remainder are able to eclose after a prolonged developmental period. Although viable, the flies that do eclose have reduced fecundity and likely cannot compete well with wild-type flies for limited resources. The nonviable animals likely die due to the asynchronous expression of 20E-regulated genes (McBrayer, 2007).

During the extended developmental delay in ablated larvae, the ecdysteroid titer remains very low. However, it eventually rises in white prepupae, suggesting that an alternative mechanism for triggering metamorphosis is in place. Consistent with this idea is the observation that extracts prepared from Drosophila ventral ganglia, which should not contain the PG neurons, possess an ecdysteroidogenic activity. One likely candidate is an insulin-like peptide. In Lepidoptera, the insulin-like peptide bombyxin was originally identified as a small molecule with PTTH-like activity (Ishizaki, 1994). While the role of bombyxin-like peptides in regulating ecdysteroid levels in Lepidoptera remains unclear, recent evidence from Drosophila points to a role for insulin in regulating ecdysteroid signaling (Caldwell, 2005; Colombani, 2005; Mirth, 2005) and developmental timing. These investigators found that increased insulin signaling in the prothoracic gland results in small flies, whereas reduced insulin signaling produces large flies. Effects on size are likely caused by changes in basal levels of ecdysteroids. Similar to the current findings, higher ecdysteroid titers decrease body size by reducing cell number, whereas lower ecdysteroid levels lead to more cells and larger flies (McBrayer, 2007).

How insulin-like factors modulate the ecdysteroid level is not entirely clear. However, of note is the observation that the levels of both disembodied (dib) and phantom (phm) transcripts were shown to moderately increase in response to activation of insulin signaling (Colombani, 2005). Curiously, no an increase is seen in the transcription of dib, phm nvd, or spok in white prepupae of ablated animals, despite the fact that ecdysteroid titer does rise. Therefore, it does not appear that transcription of these enzymes is the rate-limiting step in ecdysteroid production. It is possible, however, that the transcriptional level of shadow (sad), which does rise, or some other ecdysteroidogenic enzyme is rate-limiting. Another possibility is that rate-limiting control is exerted at the level of enzymatic activity, not transcription (McBrayer, 2007).

The suggestion that both PTTH and insulin may control ecdysone production via separate pathways is consistent with the finding that manipulating the Ras/Raf pathway in the PGs also affects developmental timing and size. Increased Ras or Raf activity increased ecdysteroid levels and resulted in small flies, whereas expression of dominant-negative Ras or Raf lowered ecdysteroid levels, prolonged larval development, and produced large flies (Caldwell, 2005). Although the mechanism of PTTH signal transduction remains elusive, in part because the receptor has not been identified, it is interesting to note that addition of exogenous PTTH to Manduca PGs leads not only to enhanced Ca⁺ and cAMP signaling, but also to the rapid phosphorylation of ERK (Rybczynski, 2001; Rybczynski, 2003). The common phenotypes produced in Drosophila by the manipulation of PTTH levels and Ras/Raf suggest that, in this organism, a major component of the PTTH signal likely involves activation of a MAP kinase cascade. It is likely that the PTTH signal is subsequently integrated with nutritional signals via the insulin pathway (Colombani, 2005; Mirth, 2005) and modulated by prothoracicostatic signals (Yamanaka, 2006) to determine developmental timing and final body size. In this respect, insect metamorphosis shows remarkable similarity to mammalian reproductive development, in which production and release of the neuropeptide kisspeptin gates the timing of puberty in conjunction with input from nutritional and metabolic sensors (McBrayer, 2007).

In addition to the role of PG neurons in controlling developmental timing, it is also noted that PTTH expression continues in a limited number of neurons in the adult brain. Similarly, Manduca PTTH expression is also expressed in the adult brain, but it appears to be the same l-NSCs that innervate the CA. Since the prothoracic gland degenerates during the pupal stage, the Drosophila PTTH-positive neurons would have to undergo developmental pruning and rewiring if they are the direct descendants of the larval PG neurons. No projections from the adult PTTH-positive neurons to the ovaries were seen, suggesting that PTTH does not directly regulate ecdysteroidogenesis in the ovary, although it may still act indirectly via the hemolymph. It is noted, however, that adult males in which PTTH-expressing neurons are ablated exhibit male-on-male courtship behavior. Since ecdysteroid signaling is needed to remodel many neuronal connections during metamorphosis, it is possible that the developmental delay and/or generally low ecdysteroid titers that result from ablation of the PG neurons might alter axon guidance and connectivity during metamorphosis. Alternatively, the PTTH-positive neurons in the adult male brain might directly affect the expression or activity of genes involved in determining courtship rituals. Other functions for PTTH in the adult brain are also possible (McBrayer, 2007).

The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis.

Holometabolous insects undergo complete metamorphosis to become sexually mature adults. Metamorphosis is initiated by brain-derived prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone via an incompletely defined signaling pathway. This study demonstrates that Torso, a receptor tyrosine kinase that regulates embryonic terminal cell fate in Drosophila, is the PTTH receptor. Trunk, the embryonic Torso ligand, is related to PTTH, and ectopic expression of PTTH in the embryo partially rescues trunk mutants. In larvae, torso is expressed specifically in the prothoracic gland (PG), and its loss phenocopies the removal of PTTH. The activation of Torso by PTTH stimulates ERK phosphorylation, and the loss of ERK in the PG phenocopies the loss of PTTH and Torso. It is concluded that PTTH initiates metamorphosis by activation of the Torso/ERK pathway (Rewitz, 2009).

Many organisms undergo distinct temporal transitions in morphology as a part of their normal life process. In humans, for example, passage through puberty is accompanied by changes in body mass and the acquisition of sexual maturity. Likewise, in all holometabolous insects, metamorphosis transforms the immature larva into a completely new body form that is capable of reproductive activity. In both cases, neuropeptide signaling in response to environmental and nutritional cues triggers the transition process. In insects, the process is initiated by the neuropeptide known as prothoracicotropic hormone (PTTH). PTTH signals to the prothoracic gland (PG), the primary insect endocrine organ, which triggers the production and release of ecdysone, the precursor of the active steroid molting hormone 20-hydroxyecdysone (20E). The increased level of 20E provides a systemic signal that ends the larval growth period and initiates metamorphosis (Rewitz, 2009).

PTTH has been proposed to be structurally similar to certain mammalian growth factors that are ligands for receptor tyrosine kinases (RTKs). Previous studies have also indicated that PTTH signaling results in the phosphorylation of cellular signaling molecules that are linked to the mitogen-activated protein kinase (MAPK) pathway in the PG. In light of the potential involvement of MAPK pathway components in PTTH signaling, the expression of all Drosophila RTKs was examined in the PG to determine whether any showed a tissue-specific expression profile that was consistent with a possible role as a PTTH receptor. It was found that after early embryogenesis, the RTK encoded by torso is expressed specifically in the PG (Rewitz, 2009).

The gene torso belongs to the so-called terminal group of genes that are required for the correct patterning of anterior and posterior structures during early embryogenesis. The presumed ligand for Torso during terminal patterning is Trunk (Trk), which contains a cysteine knot-type motif in the C-terminal region similar to the motif in PTTH. Also like PTTH, Trk is thought to be proteolytically processed from a precursor molecule to generate an active C-terminal fragment that is comparable in length to that of PTTH. Alignment of the protein sequences of Trk and PTTH reveals that they share some conserved structures in the C-terminal region that compose the mature peptide, including all of the six cysteines that are important for intramonomeric bonds of the PTTH homodimeric molecule. Previously, it was noted that Trk is related to Spatzle, but a phylogenetic analysis of different insect cysteine knot-type proteins shows that Trk and PTTH form a separate cluster and that PTTH is the closest paralog of Trk. These results raise the possibility that Trk and PTTH share a conserved three-dimensional structure enabling both to activate Torso despite the modest conservation of primary sequence. Expression of trk is not detected in the wandering third-instar larval (L3) stage using real-time polymerase chain reaction (PCR) (no product after 30 PCR cycles) or by in situ hybridization to the brain-PG complex, supporting the idea that PTTH, and not Trk, is a ligand for Torso in post-blastoderm stages (Rewitz, 2009).

To investigate possible post-embryonic roles of Torso in Drosophila development, RNA interference (RNAi) was used to knock down torso specifically in the PG. The PG-specific phantom (phm)-Gal4 line (phm>) was used to drive expression of RNAi constructs under control of upstream activator sequences (UASs) in the PG. This expression of a torso RNAi construct produced a phenotype that was almost identical to the one created by the loss of PTTH-expressing neurons. Reduction of torso expression in the PG of phm>torso-RNAi larvae delays the onset of pupariation by 5.8 days as compared with the phm> + control animals, similar to the 5.4-day delay of pupariation in animals lacking PTTH. As with the loss of the PTTH-producing neurons, torso silencing in the PG also leads to excessive growth during the prolonged L3 stage, resulting in increased pupal size. To test the specificity of the RNAi, it was confirmed that torso mRNA levels are reduced in phm>torso-RNAi larvae and that the PG cells are morphologically normal, although slightly smaller (Rewitz, 2009).

Because torso is a maternal-effect gene, homozygous mutants derived from heterozygous parents are viable. Therefore, the developmental profile and adult size were examined of animals homozygous and transheterozygous for three different torso mutations. Larvae with mutations in torso exhibited substantial developmental delays, although not as long as those seen by RNAi knockdown, in the time to pupariation as compared with heterozygous controls, and the mutants produced larger adults. The difference in time delay may result from residual maternally loaded torso mRNA. In contrast, trk mutants developed on a normal time scale, and adults were similar in size to heterozygous control adults, demonstrating that the phenotype of torso mutants is independent of early embryonic signaling (Rewitz, 2009).

In animals lacking PTTH-producing neurons, it is the low level of the active molting hormone 20E that causes the developmental delay and tissue overgrowth. To investigate whether the torso loss-of-function phenotype is also caused by low 20E levels, 20E was fed to phm>torso-RNAi larvae. Similar to what was found when the PTTH-producing neurons were removed, feeding these larvae with 20E completely rescued the developmental delay and overgrowth. Taken together, these results demonstrate that reducing Torso signaling in the PG alone phenocopies the loss of PTTH, which is consistent with the notion that Torso mediates PTTH signaling in the PG. If this is the case, it would be expected that the constitutively active torso^RL3 allele might produce precocious pupation, as would overexpression of PTTH. Consistent with this conjecture, it was found, using the daughterless (da)-Gal4 driver (da>), that ubiquitous overexpression of PTTH advances the onset of pupariation by 11.5 hours as compared with (da> +) balancer controls and produces smaller adults. At 25°C, torso^RL3 is activated, and heterozygous torso^RL3/+ animals pupariate 9.2 hours before controls and form smaller adult males (Rewitz, 2009).

To establish whether PTTH can activate Torso in vivo, it was reasoned that if PTTH is a ligand for Torso, then ectopic expression of PTTH in the embryo might elicit partial rescue of trk mutants. To examine this, the maternal nanos (nos)-Gal4 line (nos>) was used to drive ubiquitous early embryonic expression of a UAS-PTTH-hemagglutinin (HA)-tagged transgene in trk mutant embryos. In the blastoderm-stage embryo, activation of Torso by Trk induces expression of the downstream target gene tailless (tll) in the anterior and posterior regions. The inability to activate this target gene in trk or torso mutants leads to the loss of structures posterior to the seventh abdominal segment. Early embryonic expression of PTTH was observed in 13% of blastoderm-stage embryos derived from trk¹/trk¹; nos>PTTH females. Ectopic expression of PTTH in these embryos was sufficient to activate tll in the posterior part of the embryos. Although PTTH expression did not fully restore wild-type tll expression, the partial rescue elicited by PTTH was sufficient to restore posterior structures, such as the Filzkörper, in several trk mutant embryos. These results provide genetic evidence that PTTH functions as a ligand for Torso in vivo (Rewitz, 2009).

In the embryo, Torso signaling is transduced through the canonical MAPK pathway that includes the Drosophila homologs of Ras (Ras85D), Raf (Draf), MAPK kinase (MEK), and extracellular signal-regulated kinase (ERK). If Torso is indeed the PTTH receptor, it would be expected that disrupting MAPK signaling in the PG would result in a phenotype similar to that resulting from loss of the PTTH-producing neurons and Torso signaling. So far, the role of the MAPK pathway in transduction of the PTTH signal has been determined only by in vitro studies of lepidopteran PG. In Drosophila, the expression of dominant negative forms of Ras and Raf is known to delay development. To further examine the importance of the MAPK pathway in mediating PTTH/Torso signaling, RNAi was used to reduce the expression of several core components of this pathway, including Ras, Raf, and ERK, in the PG. Loss of either Ras, Raf, or ERK delayed pupariation by 4.3, 2.7, and 6.1 days, respectively. ERK silencing in the PG delays pupariation as severely as the reduction of Torso signaling or the complete loss of the PTTH-producing neurons does. The increase in size of phm>ERK-RNAi pupae and adults was also similar to the increase caused by the loss of PTTH or loss of Torso. The developmental delay, as well as the size increase caused by ERK silencing, were negated by 20E feeding. The less-severe phenotypes produced by the loss of Raf and Ras may result from less-efficient knockdown or, in the case of Ras, may reflect partial redundancy with Rap1. Consistent with Ras being downstream of torso, it was also found that expression of constitutively active Ras in the PG completely rescued the torso-RNAi-induced delay and overgrowth phenotype. Taken together, these results indicate that, as during embryonic terminal patterning, Torso regulation of ecdysone production in the PG is primarily mediated by the MAPK pathway, resulting in the activation of ERK (Rewitz, 2009).

To test directly whether stimulation of Torso by PTTH could lead to ERK phosphorylation, a cell culture-based signaling assay was developed. Because active Drosophila PTTH has not been produced in tissue culture, the silkworm Bombyx mori full-length Bombyx torso cDNA was cloned. As in Drosophila, the Bombyx torso ortholog is expressed predominantly in the PG of the final (fifth)-instar larvae. Stimulation of Drosophila S2 cells transfected with Bombyx torso and Drosophila ERK with 10^-9 M PTTH led to robust phosphorylation of ERK. PTTH stimulation of ERK phosphorylation was not detected in control S2 cells, either incubated in the absence of PTTH or those stimulated with PTTH but not expressing Bombyx torso. Bombyx PTTH did not stimulate activation of ERK through Drosophila Torso or through the insulin receptor, demonstrating that ERK stimulation by Bombyx PTTH is specific to Bombyx Torso. These results demonstrate that Torso is a functional PTTH receptor that is able to mediate PTTH signaling through the activation of the ERK pathway (Rewitz, 2009).

These observations define another role for the terminal system, which is the initiation of metamorphosis at the end of larval growth. Therefore, insects apparently use the same core system for two developmentally distinct processes: the establishment of terminal cell fate in the embryo and the termination of larval growth at the correct time to ensure an appropriate final adult body size. This identification of the PTTH receptor will facilitate further characterization of the system that determines body size in insects. It will be of interest to ascertain just how similar this system is in overall design to the hypothalamus-pituitary-gonadal axis, which controls the timing of puberty in mammals (Rewitz, 2009).

Conserved and divergent elements in Torso RTK activation in Drosophila development

The repeated use of signalling pathways is a common phenomenon but little is known about how they become co-opted in different contexts. This study examined this issue by analysing the activation of Drosophila Torso receptor in embryogenesis and in pupariation. While its putative ligand differs in each case, Torso-like, but not other proteins required for Torso activation in embryogenesis, is also required for Torso activation in pupariation. In addition, it was demonstrated that distinct enhancers control torso-like expression in both scenarios. It is concluded that repeated Torso activation is linked to a duplication and differential expression of a ligand-encoding gene, the acquisition of distinct enhancers in the torso-like promoter and the recruitment of proteins independently required for embryogenesis. A combination of these mechanisms is likely to allow the repeated activation of a single receptor in different contexts (Grillo, 2012).

This study provided evidences that Tsl participates in Tor activation both in the embryo and in the prothoracic gland. In the embryo, the Trk^C108 cleaved form activates Tor in the absence of tsl function, thereby suggesting that the latter is directly or indirectly involved in the processing of the Trk protein. Given the similarity between Trk and Ptth, the effect of Tsl in dpERK accumulation in the prothoracic gland and the effect of Trk^C-108 and Tsl in advancing and delaying pupariation respectively, it is proposed that Tsl is similarly involved in Ptth processing in the prothoracic gland. It should be noted that in the prothoracic gland Tsl and Tor proteins are produced in the same cells while during embryogenesis Tor accumulates in the embryo upon synthesis while Tsl is synthesized and secreted from cells surrounding the oocyte. However, tor and tsl expression in distinct cell types is not an absolute requirement for Tor activation in embryogenesis, it has been shown that tsl expression in the germline is also functional in Tor activation. Thus, Tsl is detected in the cytoplasm of the cells where it is synthesised both in the ovary and in the prothoracic gland, although the presence of a signal peptide in the protein suggests that it is secreted in both cases. Indeed, secreted Tsl is detected, upon specific processing, in the vitelline membrane, a particular type of extracellular matrix in the early embryo; yet, it has not been possible to detect Tsl at the extracellular matrix of the prothoracic gland cells (Grillo, 2012).

As Tsl lacks any feature indicating that it has protease activity, it has been suggested that this protein participates in the activation or nucleation of such an enzymatic complex. In this scenario, similar proteins that could equally be activated/nucleated by Tsl could carry out the processing of Trk and Ptth. In this case, Tsl would be the common module in both events of Tor activation. Alternatively, the same players could be involved in both Trk and Ptth processing, in which case, the common module for Tor activation should be enlarged to also encompass the same processing complex. Final clarification of these two possibilities awaits the identification of the Trk (and Ptth) processing mechanism, which still remains elusive (Grillo, 2012).

Conversely, fs(1)N, fs(1)ph and clos are required for Tor activation only in the early embryo indicating that Tsl does not need the function of these gene products to exert its function outside the embryo. Indeed, a relevant function of these three proteins is in vitelline membrane morphogenesis. Therefore, it is likely that these proteins are recruited to anchor Tsl at the vitelline membrane and thus they participate in Tor activation exclusively in embryonic patterning. Of note, several observations have led to the proposal that anchorage of Tsl in the vitelline membrane serves to store it in a restricted domain until Tor activation in the early embryo (Grillo, 2012).

As for Tor, Toll signalling is a transduction pathway that was initially thought to act only in early embryonic patterning but does in fact participate in other signalling events. However, in the case of Toll signalling, a single putative ligand, Spätzle (spz), acts both during embryonic patterning and in immunity, while for Tor signalling different putative ligands are responsible for its activation in embryonic patterning and in the control of pupariation. Spz triggers Toll activation in many scenarios because the spz promoter drives its expression in several groups of cells, possibly by distinct enhancers. In contrast, in the case of the Tor pathway a likely duplication might have generated two genes each with a distinct expression pattern and encoding the corresponding ligand for one of the two Tor activation events. The observation that Coleoptera but not Hymenopthera possess both trk and ptth orthologues suggest the putative duplication to have occurred at the origin of holometabolous insects. However and regarding tsl as the key element in ligand activation, multiple usage of the Tor pathway appears to have evolved by recruiting independent enhancers responsible for the distinct expression of the same gene (Grillo, 2012).

In summary, Tor activation in oogenesis and in the prothoracic gland is linked to the following: a duplication and subsequent differential expression of trk and ptth; the acquisition of independent specific oogenesis and prothoracic gland enhancers in the tsl promoter; and the recruitment of proteins independently required for organ morphogenesis, in particular for eggshell assembly. The Drosophila EGFR resembles the case of Tor as another example of the repetitive use of the same receptor by different ligands in different contexts: Gurken in oogenesis and Spitz, Vein, and Keren during other stages of development. Thus, it is proposed a combination of gene duplication, enhancer diversification and cofactor recruitment to be common mechanisms that allow the co-option of a single receptor-signalling pathway in distinct developmental and physiological functions (Grillo, 2012).

Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis

In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). This study shows that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, β3-octopamine receptor (Octβ3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octβ3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulated ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. The study proposes that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant (Ohhara, 2015).

In many animal species, the developmental transition is a well-known biological process in which the organism alters its body morphology and physiology to proceed from the juvenile growth stage to the adult reproductive stage. For example, in mammals, puberty causes a drastic change from adolescent to adulthood, whereas in insects, metamorphosis initiates alteration of body structures to produce sexually mature adults, a process accompanied by changes in habitat and behavior. These developmental transitions are primarily regulated by steroid hormones, production of which is regulated coordinately by developmental timing and nutritional conditions. How these processes are precisely regulated in response to developmental and environ mental cues is a longstanding question in biology (Ohhara, 2015).

In holometabolous insects, the steroid hormone ecdysone plays a pivotal role in metamorphosis. In Drosophila, metamorphic development from the third-instar larva into the adult, through the prepupa and pupa, initiates 90-96 h after hatching (hAH) at 25°C under a standard culture condition. At the onset of the larval-prepupal transition, ecdysone is produced in the prothoracic gland (PG) and then converted into its active form, 20-hydroxyecdysone (20E), in the peripheral organs. The activities of 20E terminate larval development and growth and initiates metamorphosis. Ecdysone biosynthesis is regulated in the PG by neuropeptides, enabling modulation of the timing of 20E pulses during development. The best-known stimulator of ecdysone biosynthesis is prothoracico-tropic hormone (PTTH), which is produced by neurons in the CNS. PTTH activates the receptor tyrosine kinase Torso in the PG to stimulate expression of ecdysone biosynthetic genes through the Ras85D/Raf/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Insulin-like peptides (Ilps), members of another class of neuron-derived factors, activate PI3K in the PG, resulting in production of ecdysone biosynthetic proteins. The Activin/transforming growth factor-β (TGF-β) signaling pathway is also required in the PG for the expression of PTTH and Ilps receptors, although to date it remains unclear which organ produces the ligand that acts on the PG (Ohhara, 2015).

In addition to these neuropeptides, the larval-prepupal transition is modulated by environmental cues such as nutritional conditions that influence larval body size. For example, at 56 hAH, early third-instar larvae attain the minimal viable weight (MVW), at which sufficient nutrition is stored in larvae to ensure their survival through metamorphosis. After attaining MVW, larvae pass another checkpoint, critical weight (CW), defined as the minimum larval size at which starvation no longer delays the larval-prepupal transition. In Drosophila, both checkpoints occur almost simultaneously, making it difficult to distinguish them. However, CW is regarded as a body size checkpoint that initiates metamorphosis and is therefore believed to ultimately modulate ecdysone production in the PG. However, its downstream effectors and signaling pathway remain elusive (Ohhara, 2015).

Based on data obtained in Manduca and Bombyx, a G protein-coupled receptor (GPCR) has long been postulated to be essential for ecdysone biosynthesis in the PG. However, this GPCR and its ligand have not yet been identified. This study shows that monoaminergic autocrine signaling through a GPCR, β3-octopamine receptor (Octβ3R), plays an essential role in ecdysone biosynthesis to execute the larval-prepupal transition. Octβ3R is also required for activation of PTTH and Ilps signaling. It is proposed that this autocrine system acts downstream of the CW checkpoint to allow the larval-prepupal transition. It is speculated that monoamines play an evolutionarily conserved role in the regulation of steroid hormone production during developmental transitions (Ohhara, 2015).

Previously studies have shown that the GPCR Octβ3R is expressed in multiple larval tissues, including the PG. To determine whether Octβ3R is involved in ecdysone biosynthesis and metamorphosis, RNAi was used to knock down Octβ3R function specifically in the PG, using the Gal4-upstream activation sequence (UAS) system. Two different UAS-Octβ3RRNAi constructs targeting distinct regions of the Octβ3R mRNA (Octβ3R^RNAi-1 and Octβ3R^RNAi-2) were used to knock down Octβ3R in the PG with the help of a phantom (phm)-22-Gal4 driver. Strikingly, larvae expressing Octβ3RRNAi in the PG never developed into adult flies, and 96% of phm>Octβ3R^RNAi-1 animals and 34% of phm>Octβ3R^RNAi-2 animals arrested at the larval stage. When UAS-dicer2 was introduced into phm>Octβ3R^RNAi-2 larvae (phm>Octβ3R^RNAi-2+dicer2) to increase RNAi activity, all of these animals arrested at the larval stage. Using in situ hybridization, a significant reduction was confirmed in the Octβ3R mRNA levels in the PG of knockdown animals relative to control larvae. These data suggest that Octβ3R expression in the PG is essential for executing the larval-prepupal transition in metamorphosis (Ohhara, 2015).

Because a similar defect in the larval-prepupal transition occurs in ecdysone-deficient larvae, it was hypothesized that the Octβ3R knockdown phenotype was due to lack of ecdysone production. Consistent with this idea, the 20E titer was much lower in phm>Octβ3R^RNAi-1 larvae than in control larvae just before the larval-prepupal transition (90 hAH). Moreover, administration of 20E by feeding rescued the defect in the larval- prepupal transition caused by Octβ3R knockdown. When phm>Octβ3R^RNAi-1 and phm>Octβ3R^RNAi-2+dicer2 larvae were cultured on media containing 20E (1 mg/mL) from 48 hAH onward, approximately half of them developed to the prepupal stage, compared with only 2-3% of larvae not fed 20E. Thus, PG-specific loss of Octβ3R activity causes an arrest in the larval-prepupal transition due to lack of ecdysone (Ohhara, 2015).

Ecdysone is synthesized in the PG from dietary cholesterol through the action of seven ecdysone biosynthetic genes (neverland, spookier, shroud, Cyp6t3, phantom, disembodied, and shadow). Quantitative RT- PCR (qPCR) was performed to investigate whether loss of Octβ3R function affects expression of these genes in the PG. In control larvae, expression of these genes increased dramatically between 72 and 96 hAH, when the larval-prepupal transition occurs. By contrast, in phm>Octβ3R^RNAi-1 and phm>Octβ3R^RNAi-2+dicer2 larvae, the expression of all of these genes was significantly reduced relative to control larvae at 96 hAH. The reduced expression of ecdysone biosynthetic genes in the PG was confirmed by in situ hybridization. Furthermore, immunostaining revealed that Neverland, Shroud, Phantom, Disembodied, and Shadow protein levels were reduced in the PG of phm>Octβ3R^RNAi-1 and phm>Octβ3R^RNAi-2+dicer2 larvae. Taken together, these data show that Octβ3R function is required in the PG for proper expression of ecdysone biosynthetic genes (Ohhara, 2015).

Octβ3R is thought to be activated by octopamine and tyramine binding. Octopamine is synthesized from tyramine by tyramine β-hydroxylase (Tbh), and tyramine is synthesized from tyrosine by tyrosine decarboxylase (Tdc). In Drosophila, two Tdc genes (Tdc1 and Tdc2) and one Tbh gene have been identified, and all of them are expressed in the larval CNS. Tdc1, Tdc2, and Tbh are also expressed in the PG. Furthermore, octopamine and tyramine were detected in the PG by immunostaining. Thus, octopamine and/or tyramine synthesized in the PG may activate Octβ3R in an autocrine manner to induce ecdysone production (Ohhara, 2015).

To test this, PG-specific knockdowns of Tdc1, Tdc2, and Tbh were generated. To knock down Tdc2, two constructs targeting distinct regions of the Tdc2 transcript (Tdc2^RNAi-1 and Tdc2^RNAi-2) were expressed along with dicer2 in the PG under the control of the phm-22-Gal4 driver (phm > Tdc2^RNAi-1+dicer2 and phm > Tdc2^RNAi-2+dicer2). All phm > Tdc2^RNAi-1+dicer2 larvae arrested at the larval stage, and phm > Tdc2^RNAi-2+dicer2 larvae were significantly delayed at the larval-prepupal transition, relative to control animals. Tdc2 mRNA level was reduced in the ring gland (RG) containing the PG in both sets of knockdown animals, as demonstrated by qPCR. Moreover, octopamine and tyramine production in the PG was impaired by Tdc2 knockdown. By contrast, Tdc1 knockdown (phm > Tdc1RNAi+dicer2) caused only a subtle delay in the larval-prepupal transition and had no detectable effect on octopamine or tyramine production. These results suggest that Tdc2 is the predominant Tdc regulating octopamine and tyramine biosynthesis in the PG and the larval-prepupal transition. Contrary to these findings, a null mutation in Tdc2 does not affect metamorphosis, and these mutant flies are viable. Thus, PG-specific knockdown causes a stronger phenotype than complete loss of Tdc2 activity in whole animals. A similar situation has been reported in regulation of metamorphosis by Activin signaling. These phenomena can be explained by a model in which some compensatory changes in other mutant tissues rescue the PG-specific knockdown phenotype in null-mutant animals (Ohhara, 2015).

PG-specific Tdc2 knockdown caused a reduction in larval 20E concentration. Therefore, whether feeding 20E to Tdc2 knockdown larvae would rescue the larval- prepupal transition defect was examined. To this end, phm > Tdc2^RNAi-1+ dicer2 and phm > Tdc2^RNAi-2+dicer2 larvae were cultured in media with or without 20E (1 mg/mL) from 48 hAH onward. Approximately 40% of the 20E-fed phm > Tdc2^RNAi-1+dicer2 larvae developed to the prepupal stage, whereas none of those larvae grown on control media progressed beyond the larval stage. Furthermore, the delay in the larval-prepupal transition in phm > Tdc2^RNAi-2+dicer2 larvae was rescued by 20E feeding. These results indicate that the defect in the larval-prepupal transition in Tdc2 knockdown animals results from a lack of 20E production. Thus, octopamine/ tyramine synthesized in the PG appears to activate Octβ3R in an autocrine manner to execute the larval-prepupal transition by regulating ecdysone production (Ohhara, 2015).

To determine which Octβ3R ligand is responsible for this autocrine signaling, Tbh was knocked down in the PG to prevent conversion of tyramine into octopamine. To knock down Tbh, two constructs targeting distinct regions of the Tbh transcript (Tbh^RNAi-1 and Tbh^RNAi-2) were expressed along with dicer2 under the control of phm-22-Gal4 (phm > Tbh^RNAi-1+ dicer2 and phm > Tbh^RNAi-2+dicer2). Although the Tbh knockdown caused a reduction in octopamine production in the PG, these larvae did not exhibit any obvious defects in the larval-prepupal transition or subsequent metamorphosi. These data suggest that tyramine, rather than octopamine, is the Octβ3R ligand that activates ecdysone production in the PG (Ohhara, 2015).

Because ecdysone biosynthesis in the PG is under the control of Ilps and PTTH signaling, it was next examined whether Octβ3R function is required to activate these signaling pathways. To detect Ilps signaling activity, a pleckstrin-homology domain fused to GFP (PH-GFP), which is recruited to the plasma membrane when insulin signaling is activated, was used. In the PG cells of control larvae, PH-GFP was only weakly localized to the plasma membrane at 48 hAH, whereas its membrane localization became increasingly evident at 60, 84, and 90 hAH. By contrast, in PG cells of phm>Octβ3R^RNAi-1 larvae, the tight localization of PH-GFP to the plasma membrane was no longer detectable, indicating that activation of Ilps signaling had been disrupted. Moreover, overexpression of a constitutively active form of the Ilps receptor InR (InRCA) was able to rescue the larval arrest in phm>Octβ3R^RNAi-1 animals. Next, immunostaining was performed of the diphosphorylated form of ERK (dpERK), a downstream signaling component of the PTTH pathway. dpERK expression was found to be very weak at 48 hAH, but was activated in the PG of control larvae at 60, 84, and 90 hAH; by contrast, this activation was reduced in the PG of phm>Octβ3R^RNAi-1 larvae. Expression of a constitutively active form of another downstream PTTH signaling component, Ras (RasV12), rescued the larval-prepupal transition defect in phm>Octβ3R^RNAi-1 animals. These results show that Octβ3R function is required to activate Ilps and PTTH signaling in the PG and that these signaling pathways execute the larval-prepupal transition. Although activation of both the Ilps and PTTH signaling pathways requires Activin/TGFβ signaling in the PG, expression of a constitutively active form of the Activin/ TGFβ receptor Baboon (BaboCA) failed to rescue the larval-prepupal transition defect in phm>Octβ3R^RNAi-1 animals. This observation suggests that Octβ3R acts downstream or independent of Activin/TGFβ signaling to regulate Ilps and PTTH signaling in the PG (Ohhara, 2015).

The observations described above demonstrate that phm>Octβ3RRNAi affects Ilps and PTTH signaling in the PG as early as 60 hAH, raising the question of when Octβ3R function is required in the PG for execution of the larval-prepupal transition. To address this issue, the Gal80ts and Gal4/UAS system, which restricts expression of Octβ3R dsRNA in the PG at 18^oC, but allows its expression at 28^oC, was used. The results of temperature upshift and downshift experiments revealed that the larval-prepupal transition was impaired only when Octβ3R dsRNA was expressed in the PG at around 60 hAH. Notably, 60 hAH is the critical period during which larvae attain CW under nutrient-rich conditions. As noted above, when larvae are starved before attainment of CW, they are unable to transit into the prepupal stage. By contrast, starved larvae can successfully transit to prepupal/pupal stage without developmental delay once they have attained CW by growing beyond the critical period (~56 hAH) under nutrient-rich conditions in standard Drosophila medium. Thus, it is hypothesized that Octβ3R signaling acts downstream of the body-size checkpoint, or attainment of CW, to allow the larval-prepupal transition (Ohhara, 2015).

Several lines of evidence support this hypothesis. First, Octβ3R function is required for activation of Ilps and PTTH signaling detected in the PG at 60 hAH. By contrast, at 48 hAH, before the attainment of CW, neither signaling pathway is active in the PG. Second, Ilps and PTTH signaling was not activated in the PG when the larvae were starved from 48 hAH onward (early starvation), whereas these signaling pathways were active when the larvae were starved after 60 hAH (late starvation). Finally, a ligand for Octβ3R, tyramine, was detectable in the PG at 60 hAH, but decreases after this stage under a nutrient-rich condition. This decrease in tyramine was abrogated by early starvation but not by late starvation. Assuming that this decrease in tyramine in the PG is due to its secretion from PG cells, it is reasonable to propose that attainment of CW causes tyramine secretion from the PG at around 60 hAH, which in turn activates Octβ3R to regulate the Ilps and PTTH pathways, leading to the larval-prepupal transition (Ohhara, 2015).

This study demonstrates that monoaminergic regulation plays a pivotal role in ecdysone biosynthesis to induce metamorphosis and that Octβ3R acts as an upstream regulator essential for the Ilps and PTTH signaling. In addition, the data indicate that Octβ3R ligands are produced in the PG to stimulate ecdysone biosynthesis in an autocrine manner. Autocrine signaling has been proposed to mediate the community effect, in which identical neighboring cells are coordinated in their stimulation and maintenance of cell type-specific gene expression and their differentiation, as observed in muscle development of amphibian embryos. Thus, it is proposed that monoaminergic autocrine signaling among PG cells acts to increase their responsiveness to Ilps and PTTH, thereby allowing coordinated ex- pression of ecdysone biosynthetic genes within a time window following exposure to neuropeptides (Ohhara, 2015).

These findings raise the larger question of whether monoamine acts as part of an evolutionarily conserved mechanism of steroid hormone production. In vertebrates, there is limited evidence of monoaminergic regulation of steroid hormone biosynthesis. For example, in cultured adrenal glands, catecholamine stimulates the biosynthesis of the steroid hormone cortisol in a paracrine manner to elicit a stress reaction. Another example is the Leydig cells of the mammalian testes, in which the steroid hormone testosterone is produced mainly in response to pituitary gonadotropin. However, catecholamine signaling through β-adrenergic receptors, the orthologs of Octβ3R, also promotes the production of testosterone from cultured fetal Leydig cells, which may be the site of catecholamine synthesis in the fetal and mature human testes. Thus, monoamines may play a conserved role in modulating and/or stimulating steroid hormone production during physiological and developmental transitions (Ohhara, 2015).

Ecdysone-dependent feedback regulation of prothoracicotropic hormone controls the timing of developmental maturation

The activation of a neuroendocrine system that induces a surge in steroid production is a conserved initiator of the juvenile-to-adult transition in many animals. The trigger for maturation is the secretion of brain-derived neuropeptides, yet the mechanisms controlling the timely onset of this event remain ill-defined. This study shows that a regulatory feedback circuit controlling the Drosophila neuropeptide Prothoracicotropic hormone (PTTH) triggers maturation onset. The Ecdysone Receptor (EcR) in the PTTH-expressing neurons (PTTHn) was identified as a regulator of developmental maturation onset. Loss of EcR in these PTTHn impairs PTTH signaling, which delays maturation. The steroid ecdysone dose-dependently affects Ptth transcription, promoting its expression at lower concentrations and inhibiting it at higher concentrations. These findings indicate the existence of a feedback circuit in which rising ecdysone levels trigger, via EcR activity in the PTTHn, the PTTH surge that generates the maturation-inducing ecdysone peak toward the end of larval development. Because steroid feedback is also known to control the vertebrate maturation-inducing hypothalamic-pituitary-gonadal axis, these findings suggest an overall conservation of the feedback-regulatory neuroendocrine circuitry that controls the timing of maturation initiation (Christensen, 2020).

The activation of a neuroendocrine signaling cascade triggers maturation onset in most animals. This activation is associated with body-size gating to ensure the fitness of the reproductive adult. In insects, attainment of 'critical weight' during the last larval instar is the main such checkpoint gating the transition to adulthood. After this checkpoint, a larva becomes committed to maturing on a fixed schedule irrespective of further nutrition. Thus, critical weight likely reflects energy stores sufficient to survive the non-feeding maturation process (metamorphosis) and obtain a final adult body size that maximizes fitness. Nutritional status is likewise a main factor permitting the entry into maturation in mammals. In humans, body weight correlates with the timing of menarche, which led to the use of the term 'critical weight' for the onset of reproductive cycles in humans. Obese children enter puberty earlier than height-matched non-obese children, and malnutrition and lack of body fat can lead to delayed puberty. These observations suggest that the maturation gate reflects not body size per se but rather the amount of body fat, and thus that the neuroendocrine system controlling the timing of this process somehow assesses nutritional and energetic stores. Interestingly, the adipokine leptin regulates pubertal maturation in mammals. Circulating leptin levels correlate with adiposity, and leptin-deficient humans and mice fail to undergo puberty. Leptin may therefore communicate body-fat levels to the neuroendocrine system controlling puberty, which could explain the link between childhood obesity and early onset of puberty. In insect larvae, the fat body is the main nutrient-storage and -sensing organ, releasing numerous nutrient-dependent insulin-regulating hormones. Insulin is a stimulator of ecdysone production, thus coupling adipose-tissue nutrient sensing to the neuroendocrine maturation axis in Drosophila. Among the insulinotropic adipokines is Unpaired 2 (Upd2), which is structurally and functionally similar to human leptin. Upd2 acts through the JAK/STAT receptor Domeless (Dome) in GABAergic neurons that regulate insulin secretion from the insulin-producing cells (IPCs) in the brain, which are the primary source of circulating insulin. Thus, related adiposity hormones that signal nutrition and energy storage influence the neuroendocrine events that lead to the onset of maturation in divergent systems (Christensen, 2020).

Mammalian GnRH-producing neurons regulate the timing of puberty onset, and these cells are activated by the neuropeptide KISS1. The PTTHn, activated by the KISS1 ortholog AstA and its receptor AstA-R1 (Deveci, 2019), serve this function in Drosophila. This suggests conservation of the overall neuroendocrine architecture of the maturation-initiation system. AstA is regulated by nutritional intake, providing another potential link between energy status and maturation onset. Furthermore, PTTHn-specific knockdown of Insulin receptor (InR) or dome, encoding the Upd2 receptor, produced size phenotypes in the screen carried out in this study, suggesting that the PTTHn integrate systemic nutrition-regulated signals and may also receive input via insulin from the IPCs themselves. Because PTTH controls developmental timing, and insulin is the main growth-regulatory factor, these results suggest that Upd2 may link growth and maturation by coordinating the activity of both the IPCs and the PTTHn. Knockdown of the amino-acid transporters Polyphemus and Minidiscs also induced strong growth effects in the screen, suggesting that the PTTHn may also sense nutrient status autonomously; in the IPCs, Minidiscs is required for inducing insulin secretion after intake of the amino acid leucine (Christensen, 2020).

This raises the key question of how these nutritional cues lead to the surge mode of GnRH/PTTH release that initiates maturation. The current findings suggest that ecdysone feedback, via EcR in the PTTHn, is the mechanism that induces the PTTH and ecdysone surge towards the end of larval development. This is further reinforced by EcR-mediated positive feedback on ecdysone production in the PG. It is proposed that the triggering event that begins the feedback cycle is a small nutrient-dependent ecdysone peak early in the L3 stage. Nutritional signaling via insulin acts directly on the PG and is required for ecdysone production pre-critical weight but not post-critical weight. Furthermore, PTTH secretion is also controlled by nutrition and is required for normal attainment of critical weight, suggesting that PTTH acts together with insulin before attainment of critical weight to generate a small nutrient-dependent rise in ecdysone production at the beginning of L3. This small ecdysone peak upregulates Ptth via EcR and, under this scenario, corresponds to critical weight, which occurs ~10 h after the L2-L3 transition. Thus, when ecdysone reaches the threshold corresponding to critical-weight attainment, it generates an irreversible, self-sustaining feedback activation of the neuroendocrine system by promoting the PTTH surge that triggers the maturation-inducing ecdysone pulse towards the end of L3. This model is supported by findings showing that a small nutrient-sensitive ecdysone peak early in L3 does indeed signal critical weight (Christensen, 2020).

The main feature of this model is ecdysone feedback onto the PTTHn via a mechanism requiring EcR in these cells. EcR/Usp may regulate Ptth expression by direct binding to the Ptth enhancer or through downstream target transcription factors regulated by this complex. Many transcription factors are known to be targets of EcR, and EcR may indirectly regulate Ptth expression by altering the expression of one or more of these. Indeed, RNAi against certain known EcR-induced transcription factors, such as Hr39, Hr3 and ftz-f1, produced phenotypes in the screen, consistent with a possible role in Ptth regulation. Hr3 and Ftz-F1 are also known to participate in ecdysone regulation in the PG, as is another nuclear receptor, Knirps, which was also identified in the screen as a potential regulator of PTTH. Clarifying the precise mechanism by which EcR controls Ptth expression will be an interesting topic for future investigation (Christensen, 2020).

Early maturation is associated with smaller adult size in both flies and humans, as this event limits the juvenile growth period. The prevalence of precocious puberty has been linked with the increasing rates of childhood obesity; however, the mechanisms that gate GnRH secretion at the time of puberty are poorly understood. The mammalian HPG axis controlling the onset of puberty is regulated by feedback control in which steroid hormones act to regulate the GnRH-expressing neurons, but whether these neurons themselves are direct steroid targets is still debated. However, the KISS1-expressing neuronal population has also emerged as a possible link between sex steroids and the GnRH neurons (Christensen, 2020).

Many studies in Drosophila and other insects have explored the neuroendocrine PTTH-PG-ecdysone axis. The existence of feedback control between ecdysone and PTTH has been hypothesized for decades and is supported by studies of PTTH in other insects, especially in lepidopterans, in which hemolymph titers of PTTH and ecdysone are clearly correlated during the last larval instar. A PTTH surge immediately precedes a rise in ecdysone levels, and gradual increases in ecdysone levels appear to reinforce the peak levels of circulating PTTH, suggesting that ecdysone might positively influence PTTH release. This is supported by findings that injection of ecdysone before an endogenous PTTH peak induces a premature rise in PTTH, whereas injection of ecdysteroid-22-oxidase, a potent enzymatic inactivator of ecdysteroids, inhibits this rise. This study has demonstrated an EcR-dependent positive-feedback mechanism, operating specifically within the PTTHn, that regulates the transcription of Ptth. The findings show a mechanism by which steroid-mediated feedback signaling triggers the PTTH surge at the onset of metamorphosis, suggesting that feedback control is an evolutionarily conserved regulator of the neuroendocrine signaling that initiates the onset of maturation (Christensen, 2020).

Coordination among multiple receptor tyrosine kinase signals controls Drosophila developmental timing and body size

In holometabolous insects, metamorphic timing and body size are controlled by a neuroendocrine axis composed of the ecdysone-producing prothoracic gland (PG) and its presynaptic neurons (PGNs) producing PTTH. Although PTTH/Torso signaling is considered the primary mediator of metamorphic timing, recent studies indicate that other unidentified PGN-derived factors also affect timing. This study demonstrates that the receptor tyrosine kinases anaplastic lymphoma kinase (Alk) and PDGF and VEGF receptor-related (Pvr), function in coordination with PTTH/Torso signaling to regulate pupariation timing and body size. Both Alk and Pvr trigger Ras/Erk signaling in the PG to upregulate expression of ecdysone biosynthetic enzymes, while Alk also suppresses autophagy by activating phosphatidylinositol 3-kinase (PI3K)/Akt. The Alk ligand Jelly belly (Jeb) is produced by the PGNs and serves as a second PGN-derived tropic factor, while Pvr activation mainly relies on autocrine signaling by PG-derived Pvf2 and Pvf3. These findings illustrate that a combination of juxtacrine and autocrine signaling regulates metamorphic timing, the defining event of holometabolous development (Pan, 2021).

Body size is one of the most important traits of a multicellular organism. In species whose growth is determinate, the body growth of an individual is largely completed when it matures into an adult. A good example of determinate growth is found among holometabolous insects, such as the fruit fly Drosophila melanogaster. During development, the size of a Drosophila larva increases 100-fold during its three molts, but it does not change after metamorphosis, the developmental stage that transitions the juvenile larval form into the sexually mature adult fly. Therefore, the control of metamorphic timing is a key factor that regulates final body size (Pan, 2021).

In the past decades, numerous studies in Drosophila and other holometabolous insect species have demonstrated that the onset of metamorphosis is regulated through a neuroendocrine signaling axis composed of two central information processing nodes: the prothoracic gland (PG), which produces the metamorphosis inducing steroid hormone ecdysone (E), and a bilateral pair of brain neurons, the PG neurons (PGNs), that innervate the PG and release the neuropeptide PTTH that stimulates E production. After release into the hemolymph, E is taken up by peripheral larval tissues through a specific importer (EcI) and then converted into its active form, 20-hydroxyecdysone (20E), by the enzyme Shade. Subsequently, 20E stimulates metamorphosis via activation of the EcR/Usp receptor complex and stimulation of tissue-specific downstream transcriptional cascades (Pan, 2021).

In this scheme, PTTH functions as a trophic hormone to stimulate PG growth and E synthesis. In PG cells, PTTH binds to Torso, a receptor tyrosine kinase (RTK) family member, and stimulates the E biosynthetic pathway via Ras/Erk signaling. As the two central nodes on the neuroendocrine axis, both the PG and the PGNs receive additional diverse internal and external signals to modulate their output appropriately. For instance, the PG cells respond to insulin signals reflecting the general nutritional state. In addition, systemic bone morphogenetic protein (BMP) signals help coordinate metamorphosis with appropriate imaginal disc growth. The PGNs in turn, receive presynaptic inputs from various upstream neurons that regulate circadian and pupation behaviors. They also respond to tissue damage signals to delay maturation onset until the damage is resolved (Pan, 2021).

Although it is widely accepted that PTTH is the key neuropeptide that triggers developmental maturation in holometabolous insects, several studies indicate that additional timing signals are also likely. The first suggestion that PTTH is not the sole prothoracicotropic signal came from PGN ablation studies in Drosophila where it was found that up to 50% of animals with no PGNs still undergo metamorphosis, but after a prolonged ~5-day developmental delay. Subsequently, it was found that genetic null mutations in the Drosophila PTTH gene only produced a 1-day developmental delay and had little effect on viability. In this case electrical stimulation of the mutant PGNs restored proper timing while inactivation produced a more substantial 2-day delay. Ptth null mutants have also been generated in Bombyx mori, and while most animals arrest development at late larval stages, a fraction still escape and produce adults. Taken together, these studies strongly indicated that the PGNs produce additional timing signals besides PTTH (Pan, 2021).

RTK family receptors have been speculated to mediate the additional PGN signal, since blocking the Ras/Erk pathway in the PG causes strong developmental defects, phenocopying the PGN ablation model rather than the ptth mutant. Epidermal growth factor receptor (Egfr) has recently been implicated in regulating PG tissue growth, E synthesis, and secretion. However, the Egfr pathway is activated by autocrine signals from the PG, which does not involve the activity of PGNs. In the present study, two additional RTK family receptors, anaplastic lymphoma kinase (Alk) and PDGF and VEGF receptor-related (Pvr), were identified that play important roles in the PG controlling metamorphic timing. Interestingly, the Alk ligand Jelly belly (Jeb) and Pvr ligand Pvf3 are both expressed in the PGNs, verifying that the prothoracicotropic function of PGNs is mediated by multiple signaling molecules, while Pvf2 and Pvf3 are also expressed in the PG itself and likely provide additional autocrine signals that also contribute to metamorphic timing control (Pan, 2021).

In previous studies, RTKs, that is, Torso, and Egfr, have been demonstrated to be crucial in the PG for the control of pupariation and body size. This work identified two additional RTKs, Alk and Pvr, that are also required for proper timing and body size control. Suppression of either Alk or Pvr compromises E synthesis in the PG, delays pupariation, and increases pupal size, while moderate activation of Alk or Pvr accelerates development. The biological functions of Alk/Pvr in the neuroendocrine pathway are similar to those of the other RTKs, indicating likely signal coordination among the receptors. Downstream signaling from Torso, Egfr, Alk, and Pvr all involve activation of Ras/Erk signaling, while InR and Alk can also stimulate the PI3K/Akt pathway. Consistent with the signaling pathway convergence, suppression of Alk and Pvr simultaneously or suppression of Alk/Pvr in ptth mutants exhibits prolonged delay of developmental timing and larger pupal size. In addition, activation of Alk/Pvr rescues the developmental defects of ptth mutants, while activated Alk rescues the delay of InRDN overexpression. In total, both the downstream signaling pathway convergence and the additive effects of receptor activation/suppression support the coordination of signaling among these RTKs (Pan, 2021).

Cellular level coordination of receptor-mediated signals is very common during development. The PG is a good example of this coordination, which integrates a large variety of signals, such as insulin, PTTH, Hedgehog, Activin, BMP, serotonin, and octopamine, to precisely control hormonal output. The coordination among receptors of the same class is of special interest. At least five RTKs (InR, Torso, Egfr, Alk, and Pvr) are expressed in the PG, all of which activate the Ras/Erk pathway. Although PTTH/Torso has been considered the key tropic signal for PG function, it appears that three of the other RTKs can partially replace Torso to maintain some level of PG E production. Loss of either the Torso, Alk, or Pvr signal causes developmental delay but does not block pupariation. Even considering that loss of Egfr in the PG causes arrest at the L3 stage, Egfr is still dispensable during the first two molts, which also require production of E pulses by the PG. These observations lead to an open question: why does the PG utilize multiple signals that appear to function redundantly (Pan, 2021)?

An obvious possibility is that multiple timing signals provide both robustness and flexibility in response to variable developmental conditions. For example, given a choice of diets, Drosophila larvae chose one that maximizes developmental speed over other life-history traits. This is not surprising given the ephemeral nature of rotting fruit, a primary food source for Drosophila. Thus, multiple signals may enable larvae to maximize developmental speed. Another possibility is that the different signals contribute to different temporal aspects of the developmental profile. For example, perhaps none of the receptors alone can achieve a strong enough Ras/Erk activation in late-stage larva that meets the demand for the large rise in E production that triggers wandering and initiation of pupation. Interestingly, the expression of Egfr, Alk, and Pvr all increase remarkably during the late L3 stage when both Halloween gene expression and E synthesis ramps up, suggesting that the three receptors may function as supplements to Torso in order to achieve robust Ras/Erk activation and stimulation of E production (Pan, 2021).

Yet another possibility is that in addition to Ras/Erk signaling, each receptor may induce other downstream pathways. For instance, it has been previously reported that regulated autophagy induction in the PG is a key mechanism that prevents precocious non-productive pupation by limiting E availability if larva have not achieved critical weight (CW) (Pan, 2019). In that report, it was also demonstrated that after CW, autophagy inducibility is greatly repressed. This makes sense from a developmental perspective because if food becomes limiting after CW is achieved, it is likely disadvantageous to slow development down by limiting E production. Therefore, a mechanism to shut down autophagy inducibility after attainment of CW may be beneficial and, in this study, it was found that Alk activation is, in part, responsible for shut down of autophagy activation in the PG after the CW nutrient checkpoint has been surpassed (Pan, 2021).

Manipulations of Alk and Pvr, but not Torso, signaling in the PG led to the discovery that Jak/Stat activation can also affect developmental timing. A distinct feature of Alk and Pvr is that they can exert opposite effects on development likely depending on the activation strength. Weak activation of Alk or Pvr in the PG facilitates pupariation, while strong activation results in the arrest of development at various larval stages due to Jak/Stat activation. Using a weak spok-Gal4 driver led to overgrowth of the PG and to atypical morphology. Tissue overgrowth is commonly observed when either PI3K/Akt or Ras/Erk is hyperactivated in the PG; however, neither pathway induces atypical morphological change in the overgrown PGs or developmental arrest, which was observed when Alk or Pvr are hyperactivated, especially with the strong phm-Gal4 driver. Since suppression of Jak/Stat rescues the developmental arrest caused by phm-Gal4-driven Alk/Pvr hyperactivation, it appears that Jak/Stat signaling is the key factor that mediates the side effect of Alk/Pvr activation on PG morphology and developmental timing. At lower levels of activation as found in the spok>AlkCA and spok>PvrCA, many larvae still manage to pupariate, suggesting that larvae can tolerate a certain level of ectopic Jak/Stat signaling caused by Alk/Pvr activation. What goes wrong at a high level of activation of Jak/Stat is still not clear (Pan, 2021).

At present, it is not known what the endogenous late Jak/Stat signal contributes in terms of PG function since knockdown with available reagents did not produce a significant phenotype. In Drosophila, the canonical Jak/Stat signaling pathway is commonly induced by a group of cytokines including unpaired 1-3 (Upd1-3) via their cognate receptor Domeless (Dome). However, it has also been reported that Torso and Pvr are capable of inducing Jak/Stat activation in some circumstances. Although induction of Jak/Stat signal by overexpressing wild-type Torso was not observed in the PG, this might be due to a weaker activation using wild-type Torso overexpression versus gain-of-function tor^Y9 and tor^RL3 mutants as used in the previous study. Since this study observed Dome expression and endogenous activation of the 10xStat92E-GFP reporter in late L3 PGs, it is assumed to be likely to play some role at this stage. Whether the Jak/Stat activation is through Alk/Pvr or via reception of canonical Upd/Dome signals is not clear. Interestingly, note that Upd2 is secreted from the fat body into hemolymph and therefore may provide a nutrient storage signal to the PG that could be an important regulator of developmental timing, perhaps under certain types of non-standard laboratory growth conditions. It has also been recently demonstrated that inflammation-triggered release of Upd3 acts on the PG to produce developmental delay, indicating that the Jak/Stat pathway may be an important sensor for imbalance of various types of physiological processes (Pan, 2021).

Since its discovery, PTTH has been recognized as the most important prothoracicotropic neuropeptide that triggers metamorphosis in holometabolous insects. In some species, such as Bombyx mori, additional prothoracicotropic neuropeptides such as orcokinin and FXPRL-amide peptides have been discovered; however, PTTH, insulin-like peptides (Ilps), and serotonin are the only known brain-derived PG tropic hormones in Drosophila. Nevertheless, analysis of the Drosophila ptth null mutant phenotype verses PGN ablation and PGN electrical manipulation provided evidence that there are other tropic signals derived from the Drosophila PGNs. The observations described in this study demonstrate that the Alk ligand Jeb and the Pvr ligand Pvf3 are produced in the PGNs. Knockdown of jeb in the PGNs causes delay of pupariation and increased pupal size, phenocopying the phm>AlkRNAi animals and showing that the PGNs are the major source of Jeb that functions in the PG. Depletion of Pvf3 in the PGNs does not significantly affect developmental timing, which is not a surprise since it was found that Pvf2 and Pvf3 are also produced in the PG itself. Overexpression of Jeb or Pvf3 in the PGNs did not influence timing either, indicating that the neural activity of PGNs and/or the temporal regulation of Alk/Pvr expression plays the dominant role in the regulation of signaling by these factors. It is also pointed out that the combined knockdown of both ptth and jeb or ptth, jeb, and Pvf3 in the PGNs still does not produce the ~4- to 5-day developmental delay exhibited by larvae in which the PGNs are ablated, likely signifying that the additional developmental delay produced by PGN ablation is due to elimination of some other non-RTK-mediated neuropeptide signals (Pan, 2021).

Besides the well-established role of the PGNs in regulating developmental timing and body size, several recent studies also indicate that autocrine signaling within the PG itself provides important developmental regulatory cues. This signaling was first documented for biogenic amine signaling but more recently was extended to include the RTK Egfr and its ligands Vein and Spitz. Interestingly, the expression levels of Vein and Spitz in the PG increase in middle to late L3 and may not contribute to CW determination, but instead they respond to it to form part of a E feedforward circuit that helps ramp up hormone production during late L3 in anticipation of the large pulse that drives pupation. Similarly, since expression of both Pvf2 and Pvf3 was observed in the late L3 PG, and since knockdown of Pvf2 and Pvf3 simultaneously in the PG causes delay of pupariation and larger pupal size, these ligands together with their receptor Pvr also appear to form an autocrine signaling pathway. Expression of Pvf2/3 has also been observed in other tissues/cell types such as fat body, salivary gland, and hemocytes. Whether these sources also provide some input to the PG is not clear. This study also found that overexpression of Pvf2 or Pvf3 did not cause accelerated development. This is in stark contrast to the case of Egfr signaling in which overexpression of Vein or Spitz advances pupariation significantly. This finding indicates that the activity of Pvr signaling may depend on the expression of Pvr receptor and/or the release of ligands, rather than ligand expression. Endogenous Pvf2 expression is limited to the late L3 stage, yet Pvf3 is constitutively expressed in the L3 stage. The biological significance of the differentially regulated Pvf ligand expression is still an open question. It is noteworthy that there are three Pvr isoforms produced by alternative splicing among the exons coding the ligand-binding domain. Thus, reception of different Pvf ligand signals could very much depend on the levels and timing of receptor isoform expression in the PG. Lastly, it is noted that neither Alk nor Pvr accumulates to substantial levels on the PG membrane until after CW. Thus, similar to Egfr signaling, their primary functions likely control post-CW events. What regulates the post-CW membrane localization of these receptors is not yet clear, but it is interesting to speculate that the process might be one of the first downstream responses to surpassing the CW checkpoint that prepares the PG gland for a major acceleration in hormone production (Pan, 2021).

Kang, P., Liu, P., Kim, J., Bolton, M., Kumar, A., Miao, T., Shimell, M., O'Connor, M. B., Powell-Coffman, J., Bai, H. (2023). Ptth regulates lifespan through innate immunity pathway in Drosophila. bioRxiv, PubMed ID: 37873203

Ptth regulates lifespan through innate immunity pathway in Drosophila

The Prothoracicotropic hormone (Ptth) is well-known for its important role in controlling insect developmental timing and body size by promoting the biosynthesis and release of ecdysone. However, the role of Ptth in adult physiology is largely unexplored. This study shows that Ptth null mutants (both males and females) show extended lifespan and healthspan, and exhibit increased resistance to oxidative stress. Transcriptomic analysis reveals that age-dependent upregulation of innate immunity pathway is attenuated by Ptth mutants. Intriguingly, this study found that Ptth regulates the innate immunity pathway, specifically in fly oenocytes, the homology of mammalian hepatocytes. It was further shown that oenocyte-specific overexpression of Relish shortens the lifespan, while oenocyte-specific downregulation of ecdysone signaling extends lifespan. Consistently, knocking down torso, the receptor of Ptth in the prothoracic gland also promotes longevity of the flies. Thus, these data reveal a novel function of the insect hormone Ptth in longevity regulation and innate immunity in adult Drosophila (Kang, 2023).

In Drosophila, Ptth (prothoracicotropic hormone) is secreted by a few of neuroendocrine cells located in each brain hemisphere. These neuronal cells are also called PG neurons, which is in accordance with their innervation pattern. The PG neuronal cells project their axons to terminate on the PG (prothoracic gland). Ptth ligand binds to Torso receptor in PG cells and initiates the biosynthesis and release of ecdysone. Ecdysone is the steroid hormone that regulates molting and metamorphosis. Loss of function mutation Ptth flies delay L3 larval molting to pupa, around 24 hours. Interestingly this study found that this delayed development from L3 to pupa also slows down aging through repressing age-induced Imd pathway, especially in oenocyte tissue (Kang, 2023).

Innate immunity pathway is evolutionally conserved between Drosophila and mammal. The transcription factor, Relish, in Drosophila innate immunity pathway is homolog to the transcription factor, NF-κB in mammal innate immunity pathway. These data suggests that loss of Ptth reduces the Relish translocation from cytoplasm to nucleus that is usually induced by aging. Thus reducing growth factor, such as Ptth, could reduce age induced immunity response, further lead to reduced inflammation during aging, also known as inflammaging (Kang, 2023).

Ptth is one of the key hormones in the insect endocrine system. The current findings suggest that endocrine growth factor like Ptth plays a role in regulating tissue aging, especially oenocyte aging, by modulating innate immunity pathway. Repressing growth factors can attenuate age-dependent induction of innate immunity response and inflammation. Like mammalian liver, insect oenocyte is the center for lipid metabolism. Recent studies show that oenocytes function similarly to mammal liver. In mammal, liver is the target tissue of growth hormone. Together with IGF1 (Insulin-like Growth Factor 1), growth hormone promotes muscle mass and lipid metabolism. Lack of growth hormone in mice results in smaller body size (Ames Dwarf Mouse) and prolonged lifespan. Consistent with these data, growth hormone also regulate innate immunity pathway. Studies in macrophages also show that growth hormone receptor (GH-R) plays a role in maintaining immune system homeostasis in aging (Kang, 2023).

As early as in 1995, it was reported that in Bombyx Ptth shares a common molecule with the vertebrate growth factors Beta-nerve growth factor (beta-NGF), transforming growth factor-beta2 (TGF-beta22) and platelet-derived growth factor- BB (PDGF-BB) and belongs to the growth factor superfamily. The current findings suggest that insect Ptth may share many common mechanisms with mammalian growth hormone in longevity regulation (Kang, 2023).

Neuroendocrine control of Drosophila larval light preference

Animal development is coupled with innate behaviors that maximize chances of survival. This study shows that the prothoracicotropic hormone (PTTH), a neuropeptide that controls the developmental transition from juvenile stage to sexual maturation, also regulates light avoidance in Drosophila melanogaster larvae. PTTH, through its receptor Torso, acts on two light sensors (the Bolwig's organ and the peripheral class IV dendritic arborization neurons) to regulate light avoidance. PTTH was found to concomitantly promote steroidogenesis and light avoidance at the end of larval stage, driving animals toward a darker environment to initiate the immobile maturation phase. Thus, PTTH controls the decisions of when and where animals undergo metamorphosis, optimizing conditions for adult development (Yamanaka, 2013)

Animal development is associated with multiple primitive, innate behaviors, allowing inexperienced juveniles to choose an environment that maximizes their survival fitness before the transition to adulthood. In insects, this transition is timed by a peak of ecdysone production induced by the prothoracicotropic hormone (PTTH). In the larval brain of Drosophila, PTTH is produced by two pairs of neurosecretory cells projecting their axons onto the prothoracic gland (PG), where ecdysone is produced. Transition to adulthood is associated with drastic changes in larval behavior: Feeding larvae remain buried in the food, whereas wandering larvae (at the end of larval development) crawl out and find a spot where they immobilize and pupariate. Mechanisms allowing proper coordination of these behavioral changes with the developmental program remain elusive (Yamanaka, 2013)

Two pairs of neurons in the central brain were recently reported to control larval light avoidance. Using specific antibodies to PTTH, this study established that these neurons labeled by the NP0394-Gal4 and NP0423-Gal4 lines correspond to the PTTH-expressing neurons. Moreover, silencing the ptth gene by using NP0423-Gal4 or a ubiquitous driver (tub-Gal4) impaired light avoidance, indicating that PTTH itself controls this behavior. PTTH activates Torso, a receptor tyrosine kinase whose knockdown in the PG prevents ecdysone production and induces a developmental delay. In contrast, knocking down torso in the PG did not cause any change in light avoidance, indicating that the role of PTTH in ecdysteroidogenesis is functionally distinct from its role in light avoidance behavior (Yamanaka, 2013)

Because in Drosophila the PTTH-producing neurons only innervate the PG, it was reasoned that PTTH is secreted into the hemolymph and reaches the cells or organs involved in light avoidance. Consistent with this, inactivation of PTTH-expressing neurons affects light avoidance with 8 to 10 hours delay, arguing against PTTH neurons projecting directly on their target cells to control light avoidance. PTTH peptide is present in the PTTH-expressing neurons throughout larval development and shows a marked increase before wandering, correlating with the rapid increase of ecdysteroidogenesis at this stage. Using an enzyme-linked immunosorbent assay (ELISA), it was found that PTTH is readily detected in the hemolymph with a fluctuation pattern similar to that of its accumulation in the PTTH-expressing neurons. Furthermore, hemolymph PTTH levels were significantly decreased upon RNA interference (RNAi)–mediated knockdown of ptth in the PTTH-expressing neurons, suggesting that in addition to the paracrine control of ecdysteroidogenesis in the PG, PTTH also carries endocrine function (Yamanaka, 2013)

Pan-neuronal knockdown of torso (elav>torso-RNAi^GD) recapitulates the loss of light avoidance observed upon torso ubiquitous knockdown (tub>torso-RNAi^GD), suggesting that PTTH acts on neuronal cells to control light avoidance. The potential role of torso was specifically tested in two neuronal populations previously identified as light sensors in Drosophila larvae: (1) the Bolwig's organ (BO) and (2) the class IV dendritic arborization (da) neurons tiling the larval body wall. An enhancer trap analysis of torso, as well as in situ hybridization using a torso antisense probe, confirmed torso expression in class IV da neurons. In parallel, torso transcripts were detected by means of quantitative reverse transcription polymerase chain reaction in larval anterior tips containing the BO, and their levels were efficiently knocked down by using the BO-specific drivers Kr5.1-Gal4 and Rh5-Gal4, demonstrating torso expression in the BO. The knockdown of torso in the BO (Kr5.1>torso-RNAi^GD and GMR>torso RNAi^GD) or in the class IV da neurons (ppk>torso-RNAi^GD) abolished larval light avoidance (motoneurons serve as a negative control: OK6>torso-RNAi^GD). Knocking down torso in both neuronal populations (ppk>, GMR>torso-RNAi^GD) mimicked the effect observed with the BO driver or class IV da neuron driver alone. A similar loss of light avoidance was observed when these neurons were separately inactivated by expressing the hyperpolarizing channel Kir2.1 (GMR>Kir2.1 and ppk>Kir2.1), suggesting that both of these light sensors are necessary for light avoidance behavior. Down-regulation of PTTH/Torso signaling did not lead to any neuronal morphology or locomotion defect, further indicating its direct effect on light sensing. The knockdown of torso in class IV da neurons or in the BO had no effect on the pupariation timing. Taken together, these results indicate that PTTH/Torso signaling is required for light avoidance behavior in two distinct populations of light-sensing neurons and that this function is separate from its role in controlling developmental progression (Yamanaka, 2013)

Drosophila light-sensing cells use photosensitive opsins that upon exposure to light, activate transient receptor potential (TRP) cation channels, thus depolarizing the membrane and triggering neural activation. Although the BO and class IV da neurons use different photosensitive molecules and TRP channels, one can assume that PTTH/Torso signaling regulates the phototransduction pathway through a similar mechanism in both types of neurons. Immunohistochemical detection of Rh5, the opsin involved in light avoidance behavior in the BO, showed no difference in protein level in torso mutant background. PTTH/Torso signaling knockdown did not change the expression level of Gr28b, a gustatory receptor family gene that plays an opsin-like role in class IV da neurons. These results strongly suggest that PTTH affects signaling components downstream of the photoreceptors (Yamanaka, 2013)

The neural activity of the light sensors was investigated using the calcium indicator GCaMP3 for live calcium imaging. torso mutant class IV da neurons showed a 25% reduction of their response to light as compared with that of control. This was accompanied by a loss of light avoidance, indicating that such partial reduction of the GCaMP3 signal corresponds to a reduction of neural activity strong enough to exert a behavioral effect. Indeed, blocking the firing of class IV da neurons by using TrpA1-RNAi caused a similar 25% reduction of the GCaMP3 signal and behavioral effect. This suggests that in da neurons, PTTH/Torso signaling exerts its action upstream of TrpA1 channel activation. Accordingly, a strong genetic interaction was observed between torso and TrpA1 mutants for light preference. A genetic interaction between torso and Rh5 mutants was also detected, further supporting that PTTH/Torso signaling affects a step in phototransduction between the photoreceptor molecule and the TRP channel. Collectively, these data are consistent with the notion that PTTH/Torso signaling acts to facilitate TRP activation downstream of photoreceptor-dependent light sensing (Yamanaka, 2013)

A previous study suggested that larval photophobic behavior diminishes at the end of larval development, perhaps facilitating larval food exit and entry into the wandering phase. The present finding and the increase of PTTH at the beginning of the wandering stage appear to contradict such a hypothesis. Indeed, a sustained larval light avoidance mediated by PTTH was detected that persisted through the wandering stage. These results imply that wandering behavior is triggered by a signal distinct from light preference. Consistent with this notion, the timing of wandering initiation in ppk>torso-RNAi^GD or Kr5.1>torso-RNAi^GD larvae was found comparable with that of control animals, despite the fact that these animals are not photophobic (Yamanaka, 2013)

As found in other insects, wandering is either directly or indirectly triggered by PTTH- induced ecdysone production. Therefore, concomitant PTTH-mediated photophobicity could ensure that wandering larvae maintain a dark preference for pupariation site, providing better protection from predators and dehydration during the immobile pupal stage. To test this hypothesis, a light/dark preference assay was developed for pupariation. When exposed to a light/dark choice, larvae indeed showed a strong preference to pupariate in the dark. This behavior was abolished either by inactivating PTTH-expressing neurons (ptth>Kir2.1), by silencing ptth in the PTTH-expressing neurons (NP0423>ptth-RNAi, dicer2), or by introducing a torso mutant background (torso[e00150]/[1]). Dark site preference for pupariation was observed in Drosophila populations collected in the wild, confirming that this innate behavior was selected in a natural environment (Yamanaka, 2013)

This work illustrates the use of a single biochemical messenger, PTTH, for the concomitant control of two major functions during larval development. PTTH establishes a neuroendocrine link between distinct neuronal components previously shown to be involved in light avoidance. In contrast to previous interpretations but consistent with another study, this study showed that wandering is independent of light preference and that PTTH maintains a strong light avoidance response through to the time of pupariation. High levels of circulating PTTH during the wandering stage could reinforce the robustness of light avoidance, which might otherwise be compromised by active roaming. This eventually promotes larvae to pupariate in the dark, a trait potentially beneficial for ecological selection. PTTH is thus at the core of a neuroendocrine network, promoting developmental progression and appropriate innate behavioral decisions to optimize fitness and survival (Yamanaka, 2013)

Regulation of Drosophila circadian rhythms by miRNA let-7 is mediated by a regulatory cycle

MicroRNA-mediated post-transcriptional regulations are increasingly recognized as important components of the circadian rhythm. This study identified microRNA let-7, part of the Drosophila let-7-Complex, as a regulator of circadian rhythms mediated by a circadian regulatory cycle. Overexpression of let-7 in clock neurons lengthens circadian period and its deletion attenuates the morning activity peak as well as molecular oscillation. Let-7 regulates the circadian rhythm via repression of Clockwork Orange (Cwo). Conversely, upregulated cwo in cwo-expressing cells can rescue the phenotype of let-7-Complex overexpression. Moreover, circadian Prothoracicotropic hormone (PTTH) and Clock-regulated 20-OH ecdysteroid signalling contribute to the circadian expression of let-7 through the 20-OH Ecdysteroid receptor. Thus, this study has found a regulatory cycle involving PTTH, a direct target of Clock, and PTTH-driven miRNA let-7 (Chen, 2014).

The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster

The timely onset of metamorphosis in holometabolous insects depends on their reaching the appropriate size known as critical weight. Once critical weight is reached, juvenile hormone (JH) titers decline, resulting in the release of prothoracicotropic hormone (PTTH) at the next photoperiod gate and thereby inducing metamorphosis. How individuals determine when they have reached critical weight is unknown. Evidence is presented that in Drosophila, a component of the ring gland, the prothoracic gland (PG), assesses growth to determine when critical weight has been achieved. The GAL4/UAS system was used to suppress or enhance growth by overexpressing PTEN or Dp110 (Pi3K92E), respectively, in various components of the ring gland. Suppression of the growth of the PG and CA, but not of the CA alone, produced larger-than-normal larvae and adults. Suppression of only PG growth resulted in nonviable larvae, but larvae with enlarged PGs produced significantly smaller larvae and adults. Rearing larvae with enlarged PGs under constant light enhanced these effects, suggesting a role for photoperiod-gated PTTH secretion. These larvae are smaller, in part as a result of their repressed growth rates, a phenotype that could be rescued through nutritional supplementation (yeast paste). Most importantly, larvae with enlarged PGs overestimated size so that they initiated metamorphosis before surpassing the minimal viable weight necessary to survive pupation. It is concluded that the PG acts as a size-assessing tissue by using insulin-dependent PG cell growth to determine when critical weight has been reached (Mirth, 2005; full text of article).

These manipulations of insulin-dependent PG growth showed that this growth is inversely related to larval growth. Suppressing the growth of the PG (P0206>PTEN - ectopically driven PTEN) produced larvae that spent more time in each instar and were larger than normal. These effects are presumably due to a combination of reduced ecdysteroid biosynthesis, which is known to delay development, and increased growth rate. Conversely, larvae with enlarged PGs (phm>Dp110; phm is a phantom GAL4 line which was used to drive expression of Dp110) showed accelerated development in the L3. Their growth rate was dependent on nutritional conditions. Whereas phm>Dp110 larvae reared on suboptimal food grew slowly, well-fed phm>Dp110 larvae grew at the same rate as controls. Together, these data indicate that the growth of the PG negatively regulates the growth rate of the whole animal and that this regulation is modulated by nutrition (Mirth, 2005).

In addition, decreasing PG size in P0206>PTEN larvae resulted in premature metamorphosis and the formation of L2 puparia. Similar L2 puparia have been described in larvae with mutations that affect the regulation of ecdysteroid biosynthesis or signaling and in larvae where the Broad isoform Z3 was overexpressed in the ring gland, resulting in its apoptosis. L2 puparia are seen in situations where ecdysone synthesis is compromised because larvae cross the threshold weight for metamorphosis prior to the production of sufficient ecdysone to initiate a larval molt, redirecting their development to the metamorphic pathway (Mirth, 2005).

Reducing PG size resulted in reduced ecdysteroid biosynthesis; P0206>PTEN larvae showed reduced ecdysteroid titers at 44 hr AEL3, and phm>PTEN larvae only molted to L2 when fed 20E. Under conditions of low ecdysteroid synthesis, fast-growing larvae could surpass the threshold for metamorphosis before the ecdysteroid titer was sufficient to induce a molt, resulting in L2 prepupae. Slower-growing larvae would be unable to reach this threshold weight before the rise in ecdysteroid titer induced the molt to L3. Indeed, undernourished, and presumably slow-growing, P0206>PTEN L2 larvae all molted to L3, whereas only 33% of the well-fed P0206>PTEN larvae molted to L3 (Mirth, 2005).

Enlarging the PG of larvae reared under constant light caused larvae to initiate metamorphosis earlier and at smaller sizes. Nevertheless, even though larvae starved early after the L3 molt were able to pupariate, they were unable to survive to pupation unless they had fed for at least 11.5 hr. This suggests that phm>Dp110 larvae starved prior to 11.5 hr AL3E initiated metamorphosis before surpassing the minimal viable weight. Furthermore, although in control larvae, critical weight and minimal viable weight are apparently attained at the same time, they are uncoupled in phm>Dp110 larvae. Therefore, the assessment of critical weight is dependent on PG growth, whereas the minimal viable weight is not (Mirth, 2005).

In Drosophila, the PGs are responsible for a size-assessment event, early in the L3, that induces the onset of metamorphosis once critical weight is surpassed. Enhancing PG growth resulted in an overestimation of body size, thereby causing the larva to initiate metamorphosis early, at a subnormal size. Under LL, the effects of enlarging the PG were enhanced, producing individuals that pupariated even earlier at even smaller sizes, suggesting that when PTTH release was unconstrained by circadian gating, the PTTH delay period was reduced. These data provide the first indication in Drosophila that the post-critical-weight PTTH release may be under photoperiod control, as it is in Manduca (Mirth, 2005).

There has been some discussion in the literature as to whether critical weight as described in Drosophila is the same as critical weight as defined in Manduca. This discussion has arisen because the definition for Manduca states that critical weight is the minimal size at which starvation can no longer delay the onset of metamorphosis, but when Drosophila larvae are starved before critical weight is reached, they die. The current data suggest that this is due to a tight relationship between minimal viable weight and critical weight in Drosophila. Effects more similar to those observed in Manduca can be obtained when pre-critical-weight Drosophila larvae are starved for an interval and then re-fed. Under these conditions, they delay metamorphosis for a period greater than the period of starvation. Much of the confusion surrounding critical weight in Drosophila has arisen because in wild-type larvae, minimal viable weight and critical weight are achieved at the same time (Mirth, 2005).

After critical weight has been surpassed, the metamorphic pathway appears to be partially suppressed by continued feeding in Drosophila. Hence, the nutrition pathway appears to promote growth and suppress metamorphosis, whereas insulin-dependent PG growth suppresses larval growth and promotes differentiation (Mirth, 2005).

The effects of increased growth in the PG are not simply due to increasing cell size, but rather are specific to the nutrition-dependent InR signaling pathway. Studies have indicated that when either dMYC or cyclinD/cdk4 are used to enlarge the PG cells, there is no reduction in overall body size. Overexpression of dMYC, of cyclinD/cdk4, and of Dp110 all enhance cell growth, but they do so in fundamentally different manners by using separate cascades. Whether the size-assessment mechanism operates via increased intracellular PIP3 levels in the PG cells or the accumulation of some other downstream component of the InR cascade in these cells is unknown (Mirth, 2005).

Although no difference in was detected ecdysteroid titers in larvae with enlarged PGs, there is evidence that increased InR signaling in the PG cells can produce mild increases in ecdysteroidogenesis and ecdysone signaling, increases that are below the level of detection of ecdysteroid-titer assays. Larvae with enlarged PGs showed both a mild increase in the transcription of phantom during feeding stages and an increase in the transcription of the early ecdysone response gene E74B. These subtle differences in ecdysteroid titers may be important for determining growth rates and for size assessment. A gradual rise in ecdysteroid titers is coincident with the time that critical weight is reached in Drosophila. Also, subtle shifts in 20E concentrations are important for growth. Basal concentrations of 20E in combination with bombyxin enhance the growth of wing imaginal tissues in vitro; slightly higher concentrations of 20E suppress growth (Mirth, 2005).

Mutations that cause imaginal disc and larval overgrowth often cause delayed pupariation and, in some cases, show low L3 ecdysteroid titers. In the case of the mutant lethal (2) giant larvae, the ring glands are smaller than normal and have the ultrastructural appearance of glands that have low rates of ecdysteroid biosynthesis. Delayed pupariation in these larvae can be rescued by implanting wild-type ring glands. Lastly, hypomorphic mutations in DHR4, a repressor of ecdysone-induced early genes, cause reductions in critical weight and early-pupariation phenotypes similar to those described in this study. Thus, the size-assessment mechanism is likely to involve surpassing a threshold ecdysteroid titer above which the activation of the ecdysone cascade occurs (Mirth, 2005).

These data allow construction of the following model for size assessment in Drosophila. As PG cells grow in response to increased InR signaling, they increase their basal level of ecdysteroid biosynthesis. Critical weight is then reached when systemic ecdysteroid concentrations surpass a threshold that sets into motion the endocrine events that will end the growth phase of larval development and allow the larva to begin metamorphosis (Mirth, 2005).

Studies in the mid-1970s defined a size-assessment event during the final instar of the moth Manduca sexta; termed critical weight, it is the minimal size required for the timely initiation of metamorphosis. How insect larvae determine when they have reached critical weight has long been a mystery. It is hypothesized that a size-assessing tissue determines when critical weight had been reached. Suppressing growth in this size-assessing tissue would cause an underestimation of body size, resulting in metamorphosis at larger than normal sizes, whereas enlarging this tissue would result in subnormal sizes. Studies in Drosophila have shown that manipulation of the growth of the PG via the InR pathway produced these types of effects. Furthermore, larvae with enlarged PGs metamorphosed at even smaller sizes when reared under LL, suggesting a role for PTTH circadian gating in this response. Smaller size arose both as a result of a reduction in growth rate, an effect that could be rescued via nutritional supplementation, and the early onset of metamorphosis. Most importantly, larvae with enlarged PGs had a remarkably reduced critical weight, suggesting that they are severely overestimating their own body size. These results offer a very new perspective on the problem of size control in insects, uniting the recent data exploring the role of nutrition and the insulin-receptor pathway on growth with the classical physiological experiments that defined critical weight (Mirth, 2005).

Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release

In Drosophila, each of the three larval instars ends with a molt, triggered by release of steroid molting hormone ecdysone from the prothoracic gland (PG). Because all growth occurs during the larval stages, final body size depends on both the larval growth rate and the duration of each larval stage, which in turn might be regulated by the timing of ecdysone release. This study shows that the expression of activated Ras, PI3 kinase (PI3K), or Raf specifically in the PG reduces body size, whereas activated Ras or PI3K, but not Raf, increases PG cell size. In contrast, expression of either dominant-negative (dn) Ras, Raf, or PI3K increases body size and prolongs the larval stages, leading to delayed pupariation, whereas expression of dn-PI3K, but not of dn-Raf or dn-Ras, reduces PG cell size. To test the possibility that altered ecdysone release is responsible for these phenotypes, larval ecdysone levels were measured indirectly, via the transcriptional activation of two ecdysone targets, E74A and E74B. It was found that the activation of Ras within the PG induces precocious ecdysone release, whereas expression of either dn-PI3K or dn-Raf in the PG greatly attenuates the [ecdysone] increase that causes growth cessation and pupariation onset. It is concluded that Ras activity in the PG regulates body size and the duration of each larval stage by regulating ecdysone release. It is also suggested that ecdysone release is regulated in two ways: a PI3K-dependent growth-promoting effect on PG cells, and a Raf-dependent step that may involve the transcriptional regulation of ecdysone biosynthetic genes (Caldwell, 2005; full text of article).

The Drosophila gap gene giant regulates ecdysone production through specification of the PTTH-producing neurons

In Drosophila, hypomorphic mutations in the gap gene giant (gt) have long been known to affect ecdysone titers resulting in developmental delay and the production of large (giant) larvae, pupae and adults. However, the mechanism by which gt regulates ecdysone production has remained elusive. This study shows that hypomorphic gt mutations lead to ecdysone deficiency and developmental delay by affecting the specification of a pair of bilaterally symmetric neurons (PG neurons) located in the cerebral labrum portion of the brain that produce prothoracicotropic hormone (PTTH). The gt¹ hypomorphic mutation leads to random loss of PTTH production in one or more of the 4 PG neurons in the larval brain. In cases where PTTH production is lost in all four PG neurons, delayed development and giant larvae are produced. Since immunostaining shows no evidence for Gt expression in the PG neurons once PTTH production is detectable, it is unlikely that Gt directly regulates PTTH expression. Instead, it was found that innervation of the prothoracic gland by the PG neurons is absent in gt hypomorphic larvae that do not express PTTH. In addition, PG neuron axon fasciculation is abnormal in many gt hypomorphic larvae. Since several other anteriorly expressed gap genes such as tailless and orthodenticle have previously been found to affect the fate of the cerebral labrum, a region of the brain that gives rise to the neuroendocrine cells that innervate the ring gland, it is concluded that gt likely controls ecdysone production indirectly by contributing the peptidergic phenotype of the PTTH-producing neurons in the embryo (Ghosh, 2010).

In many insects, the regulation of ecdysone production in larvae involves two major components: a pair of bilaterally symmetric neurons (PG neurons) and the prothoracic gland, the endocrine organ that actually produces and secretes ecdysone. In Drosophila, the PG neurons directly innervate the prothoracic gland and induce production and secretion of ecdysone by releasing an adenotropic peptide hormone called prothoracicotropic hormone (PTTH). PTTH signals through the receptor tyrosine kinase Torso to activate a RAS/ERK cascade that ultimately stimulates transcription of ecdysone biosynthetic enzymes. Intriguingly, elimination of PTTH signaling delays the rise in ecdysone titer and the onset of pupation by approximately 5 days resulting in large pupae and adults, similar to those produced by gt hypomorphs. The similarity in phenotype between gt hypomorphs and loss of PTTH signaling prompted an investigation of whether gt in some way controls PTTH signaling. This paper reports that rather than directly regulating PTTH production in the PG neurons, gt indirectly controls PTTH and subsequent ecdysone production by influencing the development of the PTTH-producing PG neurons (Ghosh, 2010).

In the absence of any evidence supporting a role for Gt in regulating ptth transcription, attempts were made to determine if loss of Gt affects the specification of PG neurons. Besides ptth, the only other described marker for PG neuron fate is the Feb211-Gal4 enhancer trap line that contains an insertion into an unknown gene on chromosome 3. Analysis of expression from this enhancer line in gt¹ mutant larvae revealed a similar stochastic loss of GFP expression in different numbers of PG neurons as seen for ptth expression itself. The all or none response observed for both ptth and Feb211-Gal4 expression in gt hypomorphs is consistent with a stochastic loss of PG neurons in these mutants (Ghosh, 2010).

The gt¹ mutation has been shown to be associated with two spontaneous insertions, one near the 5' region of the gene and the other in the 3' region. It was predicted that these insertions likely affect gt expression levels during embryogenesis, and altered gt expression may affect the specification of different neuron subtypes within the brain including precursors that give rise to the PG neurons. To examine this issue in more detail, attempts were made to determine if Gt expression is reduced or if fewer cells express Gt in gt¹ mutant animals compared to wild type embryos. Under identical staining and exposure conditions, Gt staining intensity in the control embryo is stronger compared to the gt¹ embryo. The primary staining is in an anterior medial position that is anatomically close to or overlapping with the pars intercerebralis (PI) and pars lateralis (PL) region of the brain that gives rise to a number of neurosecretory cells including several neurons that innervate the corpus cardaicum and corpus allatum, two other portions of the ring gland. The PI placode derives from neuroepithilium that expresses tailless and orthodenticle, two anteriorly expressed gap genes. The exact origin of the PG neurons has not been established, but they may be derived from two other placodes that reside more posterior to the PI region (see de Velasco, 2007). Interestingly, a prominent cluster of approximately 5 bilateral posterior midline neurons is noted that express Gt in stage 13 embryos. In equivalently staged gt¹ mutant embryos, the number of cells in this cluster that express Gt is reduced to two to four cells. Similarly, staining of a cluster of three cells positioned anteriorly on the midline axis is also dramatically reduced in the gt¹ sample (Ghosh, 2010).

These results suggest that the specification of multiple neuron subtypes in the brain is likely affected in the gt¹ mutant animals. Since no lineage tracers are available to directly determine if the PG neurons are derived from earlier precursors that express Gt, an indirect assay was used to determine if PG neurons are mis-specified in gt¹ hypomorphs. Previous axon tracing experiments have revealed that the PG neurons are the only neurons that innervate the prothoracic gland. To determine if gt affects the specification of PG neurons, cysteine string protein (Csp) distribution was examined on ring gland cells. Csp is enriched in synaptic boutons. Csp co-localizes with PTTH in axon terminals and boutons on the surface of wildtype prothoracic gland cells as well as in gt¹ mutant larvae that still show PTTH expression. In contrast, developmentally delayed gt¹ larvae in which PTTH expression is absent from all 4 PG neurons, no Csp-containing boutons are observed on prothoracic gland cells. In these same larvae however, Csp-containing axons and boutons are still seen within the corpus cardiacum and corpus allatum, two regions of the ring gland that are innervated by different sets of neurons H, yellow arrowheads. It is concluded that loss of Gt affects the development of the PG neurons since its absence leads to a loss of prothoracic gland innervation. At this point, it cannot be distinguished if Gt directly affects the specification of PG neurons, or if it affects PG neuron development in a cell non-autonomous manner, perhaps by affecting cell-cell interactions during early cortex development. Nevertheless, these experiments add gt to the list of anteriorly expressed gap genes that affect the specification of the proto-cerebrum (Ghosh, 2010).

In Manduca sexta PTTH is believed to have a tropic effect on the larval prothoracic gland as it has been shown to induce general protein synthesis. Similar to Manduca sexta, prothoracic gland cells in Drosophila are mitotically quiescent during larval stages. Nevertheless, the gland cells exhibit substantial growth during the three larval stages and this growth is characterized by the formation of polytene chromosomes and an increase in size of the gland cells. It was observed that gt¹ mutant larvae exhibiting unilateral innervation of the prothoracic glands consistently produced an asymmetrically sized gland in which the innervated portion was significantly larger than the non-innervated side. Measuring the diameter of DAPI stained nuclei revealed that cells on the non-innervated side contained nuclei that are significantly smaller compared to the innervated side. This difference was consistently observed in all samples that failed to innervate one of the prothoracic glands indicating that DNA synthesis is likely reduced in absence of prothoracic gland innervation. Curiously, when both sides lacked innervation, the ring gland did not appear substantially smaller than wild type. However these glands are from developmentally delayed larvae in which the extra growth time likely enables them to 'catch up' to the wildtype in terms of prothoracic gland size. Ultimately it will be necessary to examine PTTH null mutants to prove that PTTH, and not some other factor, is the tropic signal secreted from the PG neurons. However, the recent finding that PTTH signals through the Drosophila receptor tyrosine kinase (RTK) Torso is certainly consistent with the idea that PTTH is the tropic factor since the Torso signal is transduced through the canonical Ras-Raf-ERK pathway (Rewitz, 2009) which is known to regulate cell proliferation in many systems (Ghosh, 2010).

In addition to the absence of prothoracic gland innervation in many gt¹ hypomorphic larvae, it was noted that there is an enhanced frequency of axon misrouting in gt mutant larvae that still show ptth-HA expression in one or more of their PG neurons. In wild type larvae, the polarized PG neurons in the left lobe of the brain send out their axons from the cell body across the central axis of the CNS to the right brain lobe. There the axon forms a loop with a left hand twist and then projects anteriorly to innervate the prothoracic gland cells on the right half of the ring gland. Similarly the neurons in the right lobe extend their axons into the left lobe and innervate the left half of the ring gland. This innervation pattern is most clearly revealed in gt¹ mutant larvae retaining one pair of the bilateral PG neurons. For example, in a gt¹ mutant larva that retains the right side set of PG neurons, there is innervation only within the left prothoracic gland. In wild type larvae, the axon tracts from each pair of PG neurons are almost parallel to each other at the base of the ring gland and rarely exhibit cross (only 1 out of 23 CNSs from wt controls showed branching). However, in the gt¹ CNSs containing one or two PG neurons in only one brain lobe, the axons are often seen branching at the base of the ring gland and innervating both prothoracic glands. Interestingly similar branching events were observed in gt¹ samples that have all four PG neurons. This suggests that the cross innervations are not likely to be caused by a mechanism that tries to compensate for the lack of innervation on one side of the prothoracic gland. Consistent with this view, it was found that in certain cases such branching events caused excessive innervation of one of the prothoracic glands at the cost of the other. Approximately 24% of gt¹ CNSs that retained at least one PG neurons showed cross innervation events with clear branching at the base of the ring gland (Ghosh, 2010).

These results suggest that gt is required not only for correct specification of the PG neurons, but also influences the projection of PG neurites to their target tissue. At present, it is not possible distinguish if these axon guidance defects represent reduction in the expression of intrinsic factors within the PG neurons that respond to guidance cues or whether Gt not only affects the specification of the PG neurons themselves, but also surrounding neurons that might provide guidance cues. Ultimately, lineage tracing experiments will be required to determine which neurons are descendent from Gt-expressing cells in order to address these issues (Ghosh, 2010).

Bombyx and Manduca prothoracicotropic hormone

The prothoracicotropic hormone (PTTH) stimulates the prothoracic glands to synthesize and release ecdysone, and is therefore a key hormone for the regulation of insect moulting and metamorphosis. Bombyx PTTH is a 30 kDa homodimeric glycoprotein, whose carbohydrate moiety is not essential for the biological function. The Bombyx genome contains a single copy of the PTTH gene. PTTH is produced by four dorsolateral neurosecretory cells of brain. Another Bombyx brain peptide exerting prothoracicotropic activity to a heterologous moth Samia cynthia ricini but no activity to Bombyx has been identified and termed bombyxin. Bombyxin is a 5 kDa heterodimeric peptide that shows a high similarity to insulin in the amino acid sequence. The bombyxin gene structure also shows a high similarity with the insulin gene structure. The Bombyx genome contains more than 30 copies of the bombyxin gene. Bombyxin is synthesized by eight dorsomedial neurosecretory cells of brain (Ishizaki, 1994).

Two allelic variants were cloned of the gene for the Bombyx mori prothoracicotropic hormone, a homodimeric 30-kDa brain secretory protein. These PTTH genes contain five exons that encode a precursor protein consisting of 224 amino acid residues whose C-terminal 109 residues represent the PTTH subunit. The Bombyx haploid genome contains a single copy of the PTTH gene. The major site of PTTH expression is the brain but expression at a very low level occurs in the gut. One Bombyx brain at day 0 of the fifth larval instar contained 2.4-2.8 pg PTTH mRNA, and this amount did not change markedly during larval-pupal development (Adachi-Yamada, 1994).

Bombyxin G1 gene, a novel insulin-related peptide gene of the silkmoth Bombyx mori, has been identified. The G1 gene encodes a precursor peptide that shows 41%-56% and 28% sequence identities with preprobombyxins previously characterized and human preproinsulin, respectively. The G1 gene forms a pair with bombyxin C2 gene with opposite transcriptional orientation in a bombyxin gene cluster. The bombyxin G1 mRNA in Bombyx brain has been shown to locate in four pairs of medial neurosecretory cells (Yoshida, 1998).

The 28-kDa size variant of prothoracicotropic hormone (big PTTH) stimulates ecdysteroidogenesis by prothoracic glands of Manduca sexta. Big PTTH stimulates in vitro incorporation of [35S]methionine into proteins of prothoracic glands from Day 7 last instar larvae. In 2-hr incubations, big PTTH elicited an approximately 2-fold increase in total protein-specific activity. The effect appeared to be tissue specific, as big PTTH had no effect on incorporation of label into proteins of control tissue (fat body). Electrophoretic separation of tissue homogenates, followed by autoradiography and densitometric analysis, reveals increased incorporation of radiolabel into numerous glandular proteins. The result suggest that the effect of big PTTH was a general stimulation of protein synthesis, not specific stimulation of a subset of glandular proteins. Big PTTH-stimulated ecdysteroidogenesis was inhibited by cycloheximide, indicating that the increase in protein synthesis is a requisite for enhanced hormone production. Analysis of gland incubation media revealed numerous radiolabeled proteins. The effect of big PTTH on incorporation of [35S]methionine into media proteins was considerably more variable than the effect of big PTTH on tissue incorporation. The result is consistent with the hypothesis that prothoracic glands may release proteins in addition to ecdysteroids (Kulesza, 1994).

Prothoracicotropic hormone (PTTH) is a brain neurosecretory protein that controls insect development. PTTH of the silkmoth Bombyx mori is a homodimeric protein, the subunit of which consists of 109 amino acids. Clear-cut sequence similarity to any other proteins has not been observed. By disulfide-bond pattern analysis and modeling of the PTTH structure based on the known three-dimensional (3D) structures of growth factor family with cystine-knot motif, it is proposed that the PTTH protomer adopts the fold unique to the structural superfamily of the growth factors, beta-nerve growth factor (beta-NGF), transforming growth factor-beta 2 (TGF-beta 2), and platelet-derived growth factor-BB (PDGF-BB). The insect neurohormone PTTH appears to be a member of the growth factor superfamily, sharing a common ancestral gene with the three vertebrate growth factors, beta-NGF, TGF-beta 2 and PDGF-BB (Noguti, 1995).

PTTH also stimulates the specific synthesis of three proteins in the prothoracic glands of the tobacco hornworm Manduca sexta. One of these proteins, p50 is identified as a beta tubulin. The ability of PTTH to stimulate beta tubulin synthesis increased dramatically late on Day 3 of the 10-day fifth larval instar. At this time and later, beta tubulin synthesis in response to PTTH in vitro could be detected in some prothoracic glands 5-10 min after the onset of stimulation, and newly synthesized beta tubulin entered the microtubule pool within 12 min. Levels of beta tubulin in the glands of fifth instar larvae, measured by immunoblot, changed in a tissue-specific manner that paralleled or presaged circulating ecdysteroid levels. The accumulation of beta tubulin in PTTH-stimulated prothoracic glands resulted from increased transcription and translation and not from a decreased protein turnover rate. Pulse-chase experiments indicate that the newly synthesized beta tubulin had a very short half-life in vitro (approximately 0.5 hr). Studies with cycloheximide and actinomycin D indicated that beta tubulin synthesis and ecdysteroid synthesis are coregulated and that beta tubulin synthesis is regulated in a unique manner relative to most other prothoracic gland proteins. Beta tubulin levels may play an important role in ecdysteroidogenesis, perhaps by influencing the dynamics of microtubule-dependent secretion or interorganelle movement of ecdysteroid precursors (Rybczynski, 1995).

Secretion of ecdysteroid molting hormones by insect prothoracic glands is stimulated by neuropeptide prothoracicotropic hormones (PTTH). Studies reported here were conducted to assess the effects of microfilament and microtubule inhibitors on in vitro ecdysteroidogenesis by prothoracic glands of Manduca sexta. Microfilament inhibitors (cytochalasins B and D) have no effect on basal or big PTTH-stimulated ecdysteroidogenesis. Microtubule inhibitors (colchicine, podophyllotoxin, nocodazole) have no effect on basal ecdysteroid secretion, but suppress PTTH-stimulated secretion in a concentration-dependent manner. The effect of nocodazole is partially reversible, suggesting it is not due to nonspecific toxicity. Colchicine has no effect on glandular ecdysteroid levels, indicating that inhibition is not due solely to blockage of secretion. The combined results are consistent with the hypothesis that microtubule-mediated transport of ecdysteroid precursors plays a critical role in stimulation of ecdysteroidogenesis by PTTH (Watson, 1996).

The insect prothoracic glands are the source of steroidal molting hormone precursors and the glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH). PTTH acts via a cascade including Ca2+/calmodulin activation of adenylate cyclase, protein kinase A, and the subsequent phosphorylation of a 34 kDa protein (p34) hypothesized, but not proven, to be the S6 protein of the 40S ribosomal subunit. The immunosuppressive macrolide, rapamycin, is a potent inhibitor of cell proliferation, a signal transduction blocker, and also prevents ribosomal S6 phosphorylation in mammalian systems. Rapamycin inhibits PTTH-stimulated ecdysteroidogenesis in vitro by the prothoracic glands of the tobacco hornworm, Manduca sexta, with half-maximal inhibition at a concentration of about 5 nM. At concentrations above 5 nM, there is a 75% inhibition of ecdysteroid biosynthesis. Similar results are observed with the calcium ionophore (A23187), a known stimulator of ecdysteroidogenesis. Most importantly, the inhibition of ecdysteroid biosynthesis is accompanied by the specific inhibition of the phosphorylation of p34, indicating that p34 indeed is ribosomal protein S6. In vivo assays reveal that injection of rapamycin into day 6 fifth instar larvae results in a decreased hemolymph ecdysteroid titer and a dose-dependent delay in molting and metamorphosis. When S6 kinase (S6K: see Drosophila RPS6-p70-protein kinase) activity is examined using rapamycin-treated prothoracic glands as the enzyme source and a synthetic peptide (S6-21) or a 40S ribosomal subunit fraction from Manduca tissues as substrate, the data reveal that rapamycin inhibits S6K activity. It is concluded that S6 kinase plays a role in prothoracicotropic hormone stimulation of insect prothoracic glands by targeting ribosomal protein S6 (Song, 1994).

Phosphorylation of ribosomal protein S6 is requisite for prothoracicotropic hormone (PTTH)-stimulated specific protein synthesis and subsequent ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm, Manduca sexta. To better understand the role of S6 in regulating ecdysteroidogenesis, S6 cDNA was isolated from a Manduca prothoracic gland cDNA library and sequenced. The deduced protein is comprised of 253 amino acids, has a molecular weight of 29,038, and contains four copies of a 10-amino acid motif defining potential DNA-binding sites. This Manduca S6 possesses a consensus recognition sequence for the p70(s6k) binding domain as well as six seryl residues at the carboxyl-terminal sequence of 17 amino acids. Phosphoamino acid analysis reveals that the phosphorylation of Manduca prothoracic gland S6 is limited exclusively to serine residues: although alterations in the quantity of S6 mRNA throughout the last larval instar and early pupal-adult development are not well correlated with the hemolymph ecdysteroid titer, developmental expression and phosphorylation of S6 are temporally correlated with PTTH release and the hemolymph ecdysteroid titer. These data provide additional evidence that S6 phosphorylation is a critical element in the transduction pathway leading to PTTH-stimulated ecdysteroidogenesis (Song, 1997).

Development of the corpora allata

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce ecdysteroids, which initiate moulting and metamorphosis. During the last larval instar of holometabolous insects, a reduction in the hemolymph juvenile hormone levels is a necessary step in initiating larval-pupal transformation. Very low ecdysteroid levels in the early last larval instar of Bombyx mori initiate the complete inactivation of corpora allata. PTTH signal transduction pathways undergo specific developmental changes, with a deficiency in transduction in prothoracic gland cells occurring during the early last instar. Glands from the early last instar show no increase in either cAMP levels or steroidogenesis as a result of PTTH stimulation, indicating the absence of the PTTH receptors in gland cells. It is proposed that this absence of PTTH receptors plays a critical role in directing larval-pupal transformation (Gu, 1996).

A deficiency in prothoracicotropic hormone (PTTH) transduction during the early last larval instar of Bombyx mori has been found to play a role leading to very low ecdysteroid levels in the hemolymph, inactivation of corpora allata, as well as larval-pupal transformation. In the present study, the role of juvenile hormone (JH) in the regulation of PTTH transduction has been clarified. When JH analog (hydroprene) is applied to early last instar larvae, the development of larvae is greatly inhibited. It is not PTTH release, but rather prothoracic gland competency in both cAMP generation and ecdysteroidogenesis that is developmentally inhibited by hydroprene application, as a result of PTTH stimulation. Glands in hydroprene-treated larvae show no response in ecdysteroidogenesis to either PTTH or 1-methyl-3-isobutylxanthine (MIX) until day 7, 4 days later than those of control larvae. JH-I application shows the same effects as those of hydroprene. By contrast, allatectomy on day 0 of the last instar accelerates development, and glands show the activation response to either PTTH or MIX in both cAMP generation and ecdysteroidogenesis 1 day after allatectomy. From these results, it is concluded that the absence of JH is a prerequisite for successful PTTH transduction and for acquisition of the cAMP generating system of gland cells (Gu, 1998).

Ca2+ signaling and PTTH action

Ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm Manduca sexta is stimulated by the cerebral neuropeptide prothoracicotropic hormone (PTTH). PTTH-stimulated cAMP synthesis and ecdysone secretion are dependent on the presence of extracellular calcium, suggesting that PTTH enhances calcium entry into the cytosol. Such entry into the cytosol might involve the opening of a plasma membrane calcium channel, or a mechanism dependent on prior inositol triphosphate (IP3)-mediated release of intracellularly stored calcium. In pupal prothoracic glands, PTTH does not increase IP3 or other inositol phosphates over the course of times ranging from seconds up to 30 min, even in the presence of lithium. However, the L-type calcium channel antagonist nitrendipine completely prevents PTTH-stimulated ecdysone synthesis. A 41 kDa G-protein in prothoracic glands is ADP-ribosylated by pertussis toxin. However, PTTH-stimulated ecdysone synthesis is unaffected by prior exposure to pertussis toxin, indicating that the 41 kDa protein is not involved in the acute stimulation of steroidogenesis. By contrast, cholera toxin has a stimulatory effect on ecdysone secretion, suggesting the involvement of a Gs-like protein (see Drosophila G protein salpha 60A). Based on the absence of PTTH-stimulated inositol phosphate formation in pupal prothoracic glands, it is suggested that calcium mobilization may occur through the opening of a calcium channel, possibly regulated by Gs (Girgenrath, 1996).

Prothoracicotropic hormone (PTTH), a peptide produced by the insect brain, stimulates the prothoracic glands to secrete ecdysteroids. The big form of this peptide (25.5 kDa) has been postulated to act through cyclic AMP in larval Manduca sexta, but the role of the cyclic nucleotide in the action of PTTH in pupal glands has been less clear. PTTH-stimulated ecdysteroid secretion and protein phosphorylation by glands removed from pupal Manduca sexta are blocked by two inhibitors of cAMP-dependent protein kinase: Rp-cAMPS (see Drosophila cAMP-dependent protein kinase 1), an antagonist of cAMP binding to the regulatory subunit of the kinase, and H-89, an inhibitor of the catalytic subunit of the kinase. Further, PTTH stimulates significant accumulation of cAMP in pupal glands, although less than that previously seen in PTTH-stimulated larval glands. Cyclic AMP-dependent protein kinase is found in cytoplasmic and membrane-associated glandular subfractions, as measured by incorporation of radioactively labeled cAMP into the regulatory subunit of the kinase. PTTH enhances cytoplasmic cAMP content and appears to increase the amount of cAMP bound to a cytoplasmic type II regulatory subunit of cAMP-dependent protein kinase. The results indicate that cAMP plays a requisite role in PTTH action in pupal glands, thus arguing in favor of a uniform mechanism of action for the peptide during Manduca development (Smith, 1996).

In Manduca sexta, levels of basal and PTTH-stimulated secretion of ecdysteroids by prothoracic glands in vitro increase with time from day 1 to day 4 of the fifth larval stage. Glandular content of cAMP-dependent protein kinase was analyzed to determine if the enzyme changes in concert with increased secretory response. Photoaffinity labeling with radioactively labeled cAMP reveals a 55-kDa cAMP-binding protein characteristic of the regulatory subunit of type-II cAMP-dependent protein kinase (RII). It appears that RII is one of a limited number of cellular proteins that is phosphorylated in the presence of [gamma-35S]ATP: the thiophosphorylated protein and the photoaffinity-labeled regulatory subunit possess the same M(r) and pI, and thiophosphorylation is blocked by mammalian cAMP-dependent protein kinase inhibitor. From day 1 to day 4 of the fifth instar, glandular content of RII increases in conjunction with increased ecdysteroid secretory capacity. Application of JH analog on day 1 significantly inhibits the observed increase in RII. Catalytic subunit activity does not change from days 1 to 4 of the fifth instar, nor does cellular content of a 34-kDa protein previously shown to be phosphorylated in response to PTTH. While it is unlikely that increased content of RII is solely responsible for enhanced ecdysteroid secretion by the prothoracic glands, it may serve as a convenient marker for investigating the mechanism by which steroidogenic capacity is regulated (Smith, 1993).

Search PubMed for articles about Drosophila Ptth

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de Velasco, B., et al. (2007). Specification and development of the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain. Dev. Biol. 302(1): 309-23. PubMed ID: 17070515

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