Prothoracicotropic hormone: Biological Overview | References
Gene name - Prothoracicotropic hormone
Cytological map position - 21E2-21E2
Function - hormone
Keywords - developmental timing cue
Symbol - Ptth
FlyBase ID: FBgn0013323
Genetic map position - 2L:575,777..576,586 [-]
Classification - cysteine knot-type structure
Cellular location - extracellular
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).
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 torsoRL3 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, torsoRL3 is activated, and heterozygous torsoRL3/+ 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 (fig. S6), 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).
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).
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).
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).
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
Adachi-Yamada, T., et al. (1994). Structure and expression of the gene for the prothoracicotropic hormone of the silkmoth Bombyx mori. Eur. J. Biochem. 220: 633-43. PubMed ID: 8125124
Caldwell, P. E. Walkiewicz, M. and Stern, M. (2005). Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release. Curr. Biol. 15: 1785-1795. PubMed ID: 16182526
Colombani, J. et al. (2005). Antagonistic actions of ecdysone and insulins determine final size in Drosophila, Science 310: 667-670. PubMed ID: 16179433
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
Edgar, B. A. (2006). How flies get their size: genetics meets physiology. Nat. Rev. Genet. 7: 907-916. PubMed ID: 17139322
Gilbert, L. I., et al. (2000). Dynamic regulation of prothoracic gland ecdysteroidogenesis: Manduca sexta recombinant prothoracicotropic hormone and brain extracts have identical effects. Insect Biochem. Mol. Biol. 30: 1079-1089. PubMed ID: 10989295
Girgenrath, S. and Smith, W. A. (1996). Investigation of presumptive mobilization pathways for calcium in the steroidogenic action of big prothoracicotropic hormone. Insect Biochem. Mol. Biol. 26(5): 455-463. PubMed ID: 8763164
Ghosh, A., McBrayer, Z. and O'Connor, M. B. (2010). The Drosophila gap gene giant regulates ecdysone production through specification of the PTTH-producing neurons. Dev. Biol. 347(2): 271-8. PubMed ID: 20816678
Gu, S. H., et al. (1996). A deficiency in prothoracicotropic hormone transduction pathway during the early last larval instar of Bombyx mori. Mol. Cell. Endocrinol. 120(2): 99-105. PubMed ID: 8832568
Gu, S. H., Chow, Y. S. and Yin, C. M. (1998). Involvement of juvenile hormone in regulation of prothoracicotropic hormone transduction during the early last larval instar of Bombyx mori. Mol. Cell. Endocrinol. 127(1): 109-116. PubMed ID: 9099906
Ishizaki, H. and Suzuki, A. (1994). The brain secretory peptides that control moulting and metamorphosis of the silkmoth, Bombyx mori. Int. J. Dev. Biol. 38: 301-310. PubMed ID: 7981038
Kataoka, H., et al. (1991). Prothoracicotropic hormone of the silkworm, Bombyx mori: amino acid sequence and dimeric structure. Agric. Biol. Chem. 55: 73-86. PubMed ID: 1368675
Kim, A. J., et al. (1997). Purification and characterization of the prothoracicotropic hormone of Drosophila melanogaster. Proc. Natl. Acad. Sci. 94: 1130-1135. PubMed ID: 9037018
Kulesza, P., Lee, C. Y. Watson, R. D. (1994). Protein synthesis and ecdysteroidogenesis in prothoracic glands of the tobacco hornworm (Manduca sexta): stimulation by big prothoracicotropic hormone. Gen. Comp. Endocrinol. 93: 448-58. PubMed ID: 8194744
McBrayer, Z., et al. (2007). Prothoracicotropic hormone regulates developmental timing and body size in Drosophila. Dev. Cell 13(6): 857-71. PubMed ID: 18061567
Mirth, C., Truman, J. W. and Riddiford, L. M. (2005). The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster. Curr. Biol. 15: 1796-1807. PubMed ID: 16182527
Mirth, C. K. and Riddiford, L. M. (2007). Size assessment and growth control: how adult size is determined in insects. Bioessays 29(4): 344-55. PubMed ID: 17373657
Nijhout, H. F. (1981). Physiological control of moulting in insects. Am. Zool. 21: 631-640
Nijhout, H. F. (2003). The control of body size in insects. Dev. Biol. 261: 1-9. PubMed ID: 12941617
Noguti, T., et al., (1995). Insect prothoracicotropic hormone: a new member of the vertebrate growth factor superfamily. FEBS Lett. 376: 251-256. PubMed ID: 7498553
Rewitz, K. F., et al. (2009). The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis. Science 326: 1403-1405. PubMed ID: 19965758
Riehle, M. A. (2002). Neuropeptides and peptide hormones in Anopheles gambiae. Science 298: 172-175. PubMed ID: 12364794
Rybczynski, R. and Gilbert, L. I. (1995). Prothoracicotropic hormone elicits a rapid, developmentally specific synthesis of beta tubulin in an insect endocrine gland. Dev. Biol. 169: 15-28. PubMed ID: 7750634
Rybczynski, R. and Gilbert, L. I. (2003). Prothoracicotropic hormone stimulated extracellular signal-regulated kinase (ERK) activity: the changing roles of Ca(2+)- and cAMP-dependent mechanisms in the insect prothoracic glands during metamorphosis. Mol. Cell. Endocrinol. 205: 159-168. PubMed ID: 12890578
Rybczynski, R. Prothoracicotropic hormone. In: L.I. Gilbert, K. Latrou and S. Gill, Editors, Comprehesive Molecular Insect Science Volume 3, Elsevier, Oxford (2005), pp. 61-123
Rybczynski, R., Bell, S. C. and Gilbert, L. I. (2001). Activation of an extracellular signal-regulated kinase (ERK) by the insect prothoracicotropic hormone. Mol. Cell. Endocrinol. 184: 1-11. PubMed ID: 11694336
Siegmund, T. and Korge, G. (2001). Innervation of the ring gland of Drosophila melanogaster. J. Comp. Neurol. 431: 481-491. PubMed ID: 11223816
Smith, W. A., Varghese, A. H. and Lou, K. J. (1993). Developmental changes in cyclic AMP-dependent protein kinase associated with increased secretory capacity of Manduca sexta prothoracic glands. Mol. Cell. Endocrinol. 90(2): 187-195. PubMed ID: 8495800
Smith, W. A., et al. (1996). Cyclic AMP is a requisite messenger in the action of big PTTH in the prothoracic glands of pupal Manduca sexta. Insect Biochem. Mol. Biol. 26(2): 161-170. PubMed ID: 8882659
Song, Q. and Gilbert, L. I. (1994). S6 phosphorylation results from prothoracicotropic hormone stimulation of insect prothoracic glands: a role for S6 kinase. Dev. Genet. 15(4): 332-8. PubMed ID: 7923936
Song, Q. and Gilbert, L. I. (1997). Molecular cloning, developmental expression, and phosphorylation of ribosomal protein S6 in the endocrine gland responsible for insect molting. J. Biol. Chem. 272(7): 4429-4435. PubMed ID:
Truman, J. W. (1972). Physiology of insect rhythms. I. Circadian organization of the endocrine events underlying the molting cycle of larval tobacco hornworms. J. Exp. Biol. 57: 805-820.
Truman, J. W. and Riddiford, L. M. (1974). Physiology of insect rhythms. 3. The temporal organization of the endocrine events underlying pupation of the tobacco hornworm. J. Exp. Biol. 60: 371-382. PubMed ID: 4832987
Warren, J. T., et al. (2006). Discrete pulses of molting hormone, 20-hydroxyecdysone, during late larval development of Drosophila melanogaster: correlations with changes in gene activity. Dev. Dyn. 235: 315-326. PubMed ID: 16273522
Watson, R. D., et al. (1996). Involvement of microtubules in prothoracicotropic hormone-stimulated ecdysteroidogenesis by insect (Manduca sexta) prothoracic glands. J. Exp. Zool. 276(1): 63-69. PubMed ID: 8828185
Yamanaka, N., et al. (2005). Identification of a novel prothoracicostatic hormone and its receptor in the silkworm Bombyx mori. J. Biol. Chem. 280: 14684-14690. PubMed ID: 15701625
Yamanaka, N., et al. (2006). Regulation of insect steroid hormone biosynthesis by innervating peptidergic neurons. Proc. Natl. Acad. Sci. 103: 8622-8627. PubMed ID: 16707581
Yoshida I., et al. (1998). A novel member of the bombyxin gene family: structure and expression of bombyxin G1 gene, an insulin-related peptide gene of the silkmoth bombyx mori. Dev. Genes Evol. 208(7): 407-10. PubMed ID:
date revised: 15 December 2011
Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.