The Interactive Fly

Genes involved in tissue and organ development

Ring Gland - The combined prothoracic glands, corpus allatum, and corpus cardiacum

Structure and function of the ring gland
Embryonic development of the corpus cardiacum, a component of the ring gland
Common origin of insect trachea and endocrine organs from a segmentally repeated precursor
Precise long-range migration results from short-range stepwise migration during ring gland organogenesis
Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation
A cell surface protein controls endocrine ring gland morphogenesis and steroid production
The histone demethylase KDM5 is essential for larval growth in Drosophila
The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues

Neuroendocrine System and Metamorphorsis
DPP-mediated TGFβ signaling regulates juvenile hormone biosynthesis by activating the expression of juvenile hormone acid methyltransferase
Regulation of onset of female mating and sex pheromone production by juvenile hormone in Drosophila melanogaster
Juvenile hormone regulation of Drosophila aging
Juvenile hormone is required in adult males for Drosophila courtship
TGF-beta signaling in insects regulates metamorphosis via juvenile hormone biosynthesis
Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila
Transcriptome analysis of Drosophila melanogaster third instar larval ring glands points to novel functions and uncovers a cytochrome p450 required for development
Deep sequencing of the prothoracic gland transcriptome reveals new players in insect ecdysteroidogenesis
Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster
Intrinsic and damage-induced JAK/STAT signaling regulate developmental timing by the Drosophila prothoracic gland
Reduction of nucleolar NOC1 accumulates pre-rRNAs and induces Xrp1 affecting growth and resulting in cell competition
Dual roles of glutathione in ecdysone biosynthesis and antioxidant function during the larval development in Drosophila
Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster
The Drosophila CCR4-NOT complex is required for cholesterol homeostasis and steroid hormone synthesis
Egfr signaling is a major regulator of ecdysone biosynthesis in the Drosophila prothoracic gland
MicroRNA miR-8 promotes cell growth of corpus allatum and juvenile hormone biosynthesis independent of insulin/IGF signaling in Drosophila melanogaster
Histone H3K27 methylation-mediated repression of Hairy regulates insect developmental transition by modulating ecdysone biosynthesis
The cytochrome P450 Cyp6t3 is not required for ecdysone biosynthesis in Drosophila melanogaster
Ecdysone coordinates plastic growth with robust pattern in the developing wing
Su(var)2-10- and Su(var)205-dependent upregulation of the heterochromatic gene neverland is required for developmental transition in Drosophila

Neuroendocrine System and Insulin Signaling
Local requirement of the Drosophila insulin binding protein Imp-L2 in coordinating developmental progression with nutritional conditions
Disruption of insulin signalling affects the neuroendocrine stress reaction in Drosophila females
Mitochondrial iron supply is required for the developmental pulse of ecdysone biosynthesis that initiates metamorphosis in Drosophila melanogaster
The Drosophila zinc finger transcription factor Ouija board controls ecdysteroid biosynthesis through specific regulation of spookier
Cooperative control of ecdysone biosynthesis in Drosophila by transcription factors Seance, Ouija board, and Molting Defective
Protein Is Required for Nuclear Localization of the Ecdysteroidogenic Transcription Factor Molting Defective in the Prothoracic Gland of Drosophila melanogaster
A local insulin reservoir in Drosophila alpha cell homologs ensures developmental progression under nutrient shortage
WAKE-mediated modulation of cVA perception via a hierarchical neuro-endocrine axis in Drosophila male-male courtship behaviour

Neuroendocrine System, Growth, and Nutrition
Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells
The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster
Snail synchronizes endocycling in a TOR-dependent manner to coordinate entry and escape from endoreplication pausing during the Drosophila critical weight checkpoint
The TOR pathway couples nutrition and developmental timing in Drosophila
Nitric oxide synthase regulates growth coordination during Drosophila melanogaster imaginal disc regeneration
Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis
A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila
Body-fat sensor triggers ribosome maturation in the steroidogenic gland to initiate sexual maturation in Drosophila
PKG acts in the adult corpora cardiaca to regulate nutrient stress-responsivity through adipokinetic hormone
Insulin signaling couples growth and early maturation to cholesterol intake in Drosophila
Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae
Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development

Neuroendocrine System and Photoperiod System
Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

Neuroendocrine System and Memory
Regulation of Drosophila Long-Term Courtship Memory by Ecdysis Triggering Hormone

Genes expressed in the ring gland

Structure and function of the ring gland

In insects, as in all animals, many aspects of development are under hormonal control. The most important insect hormones are the ecdysteroid molting hormone (EC), which is secreted from the prothoracic glands, and the sesquiterpenoid juvenile hormone (JH), which is secreted from the corpus allatum (CA). In higher dipterans, the larval prothoracic glands, CA, and corpus cardiacum are fused into a single compound structure, the ring gland. Based mainly on morphological homologies between the ring gland and the endocrine glands of larger insects, it is thought that EC is produced by the large ring gland lateral cells (LC), homologous to the prothoracic glands of other insects. JH is produced by the smaller medial cells (MC), homologous to the CA. It is generally accepted that ecdysteroid levels determine the time of molting from one instar to the next, whereas JH levels determine whether the animal molts to a larval, pupal, or adult form (Harvie, 1998 and references).

The details of the neuroendocrine control of insect development have been best characterized in lepidopterans. Many, but not all, of the biosynthetic steps and intermediates leading from dietary cholesterol to the biologically active EC 20-hydroxyecdysone (20-HE) have been identified. EC receptors (see Drosophila Ecdysone receptor) and numerous EC-responsive genes have been identified, and progress is being made in understanding the molecular nature of the EC response. One EC peak early in the last larval instar (the "commitment peak") apparently causes the epidermis to become committed to producing either larval or pupal cuticle at the next molt (see Formation of the Adult Fly), whereas a later, sharper EC peak (the "prepupal" or "molting peak") is responsible for initiating the molt itself. Five JHs have been identified in lepidopterans, and shown to be sesquiterpenoids that are synthesized from acetate and/or propionate in the CA. The main form in Drosophila is methyl 6,7;10,11-bisepoxy-3,7,11-trimethyl-(2E)-dodecenoate (Harvie, 1998 and references).

The levels of EC and JH are regulated by adenotropic neuropeptides that are produced in the developing brain and delivered to the endocrine glands via the axons of neurosecretory cells. Some of the neuropeptides (large and small prothoracicotropic hormone, or PTTH) stimulate EC production by the prothoracic glands, whereas others either stimulate [allatotropic hormone or allatotropin (ATH)] or inhibit [allatostatic hormone or allatostatin (ASH)] JH production by the CA. Immunohistochemical studies in both lepidopterans and Drosophila show that each of these peptides is produced by a small number of neurosecretory cells located in defined positions of the developing brain (Harvie, 1998 and references).

The signaling pathways leading from the adenotropic neuropeptides to the synthesis and release of EC and JH have been investigated most extensively in the tobacco hornworm Manduca sexta. For the commitment peak of EC occurring during mid-fifth larval instar, PTTH appears to act via a Ca2+/calmodulin-dependent cAMP pathway (see Drosophila Calmodulin) leading to the phosphorylation of a specific set of proteins including ribosomal protein S6 and ß-tubulin. The mechanisms involved in the stimulation of molting and metamorphosis by the later, larger peak of EC are not yet clear. In studies of the control of JH production by the CA, it has been shown that ATH induces phosphoinositide hydrolysis and that inhibition of Ca2+-ATPase, protein kinases A and C, and ATP-dependent Ca2+ sequestration inhibit production of the hormone. These results suggest that the inositol 1,4,5-triphosphate pathway may be involved in the response to ATH and possibly other neuropeptides (Harvie, 1998 and references).

Seventy-six genes have been identified that are strongly expressed in the Drosophila ring gland during development. For nine of these, further studies of expression pattern, mutant phenotype and molecular nature identify the genes as strong candidates to carry out an important role in endocrine functions controlling development. Two of the genes identified encode products that have already been implicated in the functioning of prothoracic glands in other insects. The Calmodulin gene is expressed exclusively and at high levels in the ring gland of third-instar larvae, suggesting an important, presumably endocrine function for calmodulin in that tissue, as has already been suggested for lepidopterans. Calmodulin and other Ca2+-binding proteins are integral to the transduction of a wide range of Ca2+-dependent signals; there is clear evidence for the Ca2+ dependence of EC production in the Manduca larval prothoracic gland (PTG), at least for the commitment peak early in the last larval instar. Studies of these glands in vitro show that changes in intracellular Ca2+ concentrations are both necessary and sufficient for the generation of the commitment peak of EC and that PTTH-mediated stimulation of EC production requires extracellular Ca2+. Stimulation of EC production by brain extracts on isolated Drosophila ring glands is also Ca2+-dependent. A simple interpretation is that binding of PTTH to its receptor initiates an influx of Ca2+ into the cell; this influx is thought to activate downstream elements of the Ca2+-cAMP-dependent signaling pathway. It is known that Ca2+ activates PTG adenylate cyclase both directly and as a complex when bound to calmodulin. Since cAMP phosphodiesterase activity is low at this stage, cAMP is expected to accumulate. Both large and small PTTHs stimulate increased cAMP levels in PTG; a rise in cAMP levels occurs with PTTH-stimulated EC production in early last-instar PTG (Harvie, 1998 and references).

The catalytic subunit of protein kinase A (PKA or cAMP-PK) is also expressed in the Drosophila ring gland. This protein probably functions downstream of cAMP in the Ca2+-cAMP-dependent signaling pathway. PKA is activated in M. sexta PTGs by PTTH immediately prior to EC production. This is consistent with the idea that activation of the Ca2+-cAMP-dependent signaling pathway by PTTH leads to PKA-dependent phosphorylation of key proteins, including ribosomal protein S6, and that this causes changes in selective translation leading to increased EC production (Harvie, 1998 and references).

Another enhancer trap expressed in the ring gland is inserted 30 bp 5' to the transcription start site of the gene encoding the translation elongation factor EF-1. A role for this factor in hormone production and/or secretion has not been previously suggested, but it is plausible that it plays a role downstream of ribosomal protein S6 in the Ca2+-cAMP-dependent signaling pathway. The EF-1alpha F2 gene is expressed at high levels during metamorphosis, a time of higher and prolonged levels of EC. Studies in M. sexta have shown that EC production is under translational control and that certain proteins are selectively translated and phosphorylated in response to PTTH. This selective translation could result from the production and/or activation by phosphorylation of EF-1alpha, which has been shown to be a key regulator of translational control in other systems. Rapamycin, an inhibitor of S6 phosphorylation, dramatically inhibits selective translation of both EF-1alpha and EF-2 in mammalian cells, suggesting that synthesis of these elongation factors is selectively enhanced by S6 phosphorylation (Harvie, 1998 and references).

There is another possible function for EF-1alpha in the regulation of hormone titers. This factor is structurally conserved among diverse species, including Drosophila, and probably has similar functions in all organisms. In Tetrahymena, EF-1alpha has two entirely separate functions. In addition to its role in directing the binding of aminoacyl-tRNAs to the ribosome during translation, EF-1alpha can function as a Ca2+/calmodulin-dependent F-actin bundling factor. Changes in the actin cytoskeleton have been proposed to mediate neuropeptide and hormonal secretion. Ultrastructural studies have shown an increase in smooth endoplasmic reticulum and secretory vesicles throughout the final instar in EC-producing cells of Drosophila ring glands. In flies, there is a 50-fold increase between 50 and 94 hr of development, followed by an additional 10-fold increase over the last 4 hr of the third instar. It is possible, therefore, that the hemolymph titer of EC is regulated both by biosynthetic rates and by control of secretion, and that EF-1alpha may be involved in regulating one or both of these processes (Harvie, 1998 and references).

Enhancer traps have been identified that are inserted into or very near two previously characterized genes on the third chromosome: tramtrack (ttk) and couch potato (cpo). Although these two genes are known from their roles in peripheral nervous system development, it is likely that they have other functions as well. Amorphic cpo alleles are embryonic lethal, but the homozygous embryos show no obvious developmental abnormalities. Ttk is required for embryonic glial cell development and it also functions in the assignment of cell fates during sensory organ development. If expressed early enough, both of these genes could play roles in cell fate determination during ring gland development (Harvie, 1998 and references).

The screen identified one enhancer trap with strong expression in the MC of the ring gland, which is thought to be the source of JH. The only significant reporter gene expression in these enhancer trap larvae is in the MC during the second and third instars. Low levels of expression are detected in the midgut and brain as well. Expression increases in the pupal brain but in the ring gland it remains restricted to the MC. In adults, strong expression occurs in the rectal papillae. This enhancer trap is inserted 5' to the coding sequence of the gene encoding the C subunit of V-ATPase. The main function of V-ATPase that are known in insects is to act as a proton pump to energize active transport at the apical plasma membrane of ion-transporting epithelia, for example the rectal papillae, midgut and Malpighian tubules. That this reporter gene expression represents the action of a legitimate C-subunit enhancer is supported by the strong gene expression seen in the rectal papillae. However, the specific reporter expression in the larval CA may represent a different C subunit/V-ATPase function in those cells during development. V-ATPases are known to play an important role in neurotransmission by providing the energy for the uptake of neurotransmitters into synaptic vesicles, and they may also be important in synaptic vesicle formation and in neurosecretion. It is therefore possible that the ring gland V-ATPase functions in the uptake of neuropeptides in the MC of the ring gland (Harvie, 1998 and references).

Embryonic development of the corpus cardiacum, a component of the ring gland

The development of the Drosophila neuroendocrine gland, the corpus cardiacum (CC) was investigated, along with the role of regulatory genes and signaling pathways in CC morphogenesis. CC progenitors segregate from the blastoderm as part of the anterior lip of the ventral furrow. Among the early genetic determinants expressed and required in this domain are the genes giant (gt) and sine oculis (so). During the extended germ band stage, CC progenitor cells form a paired cluster of 6–8 cells sandwiched in between the inner surface of the protocerebrum and the foregut. While flanking the protocerebrum, CC progenitors are in direct contact with the neural precursors that give rise to the pars intercerebralis, the part of the brain whose neurons later innervate the CC. At this stage, the CC progenitors turn on the homeobox gene glass (gl), which is essential for the differentiation of the CC. During germ band retraction, CC progenitors increase in number and migrate posteriorly, passing underneath the brain commissure and attaching themselves to the primordia of the corpora allata (CA). During dorsal closure, the CC and CA move around the anterior aorta to become the ring gland. Signaling pathways that shape the determination and morphogenesis of the CC are decapentaplegic (dpp) and its antagonist short gastrulation (sog), as well as hedgehog (hh) and heartless (htl; a Drosophila FGFR homolog). Sog is expressed in the midventral domain from where CC progenitors originate, and these cells are completely absent in sog mutants. Dpp and hh are expressed in the anterior visceral head mesoderm and the foregut, respectively; both of these tissues flank the CC. Loss of hh and dpp results in defects in CC proliferation and migration. Htl appears in the somatic mesoderm of the head and trunk. Although mutations of htl do not cause direct effects on the early development of the CC, the later formation of the ring gland is highly abnormal due to the absence of the aorta in these mutants. Defects in the CC are also caused by mutations that severely reduce the protocerebrum, including tailless (tll), suggesting that additional signaling events exist between brain and CC progenitors. The parallels between neuroendocrine development in Drosophila and vertebrates are discussed (De Velasco, 2004).

In the larva, the ring gland forms a large and conspicuous structure located anterior to the brain and connected to the brain by a pair of tracheal branches and the paired nerve of the corpus cardiacum (NCC). Three different glands, the corpus allatum (CA; dorsally), prothoracic gland (laterally), and corpus cardiacum (CC; ventrally) form part of the ring gland. By far, most of its volume is taken up by the prothoracic gland whose cells, the source of ecdysone, grow in size and number as larval development progresses, whereas the cells of the CC remain small and do not appear to proliferate. Both the CC and CA, as well as axons innervating the ring gland, are FasII positive from the late embryonic stage onward. Labeling of the CC is stronger and starts earlier (stage 11) than that of the CA (stage 15), which makes it easy to distinguish between the two structures in the embryo. Another convenient marker of the CC is adipokinetic hormone (AKH), which is expressed exclusively in the CC from late embryonic stages onward (De Velasco, 2004).

The ring gland of the mature embryo is situated posterior to the brain hemispheres. The CC and CA occupy their positions ventral and dorsal to the aorta, respectively. The prothoracic gland cannot yet be recognized as a separate entity, possibly due to the fact that its precursors are small and few in number. Cells of the CC number around eight on each side and are arranged in a U-shape around the floor of the aorta. All cells are spindle shaped and send short processes ventromedially where they meet and form a bundle attached to the ventral wall of the aorta (subaortic processes) (De Velasco, 2004).

The homeobox gene glass (gl) is expressed in the CC from stage 10 onward. Glass-positive CC precursors first appear as two pairs of cells located between the roof of the stomodeum and the inner surface of the brain primordium. Several populations of head mesoderm cells internalized during gastrulation as part of the anterior ventral furrow form a sheet of cells covering the inner surface of the brain primordium; the CC precursors form part of this cell group. Between stages 11 and 15, CC progenitors migrate posteriorly, gradually increasing in number (3–4 cells by stage 11; 6-8 cells by stage 13). The movement of the CC precursors parallels the invagination and elongation of the esophagus. During stages 11 and 12, the primordium of the stomatogastric nervous system (SNS) appears as three invaginating pouches in the roof of the esophagus. The CC precursors maintain a position laterally adjacent to the first SNS invagination on their posterior course. By stage 15, they have passed underneath the brain commissure and join up with CA precursor cells derived from the gnathal mesoderm to form the ring gland. On their migration, posteriorly the CC precursors are always in contact with the medial surface of the developing brain. One group of neurons transiently in contact with the CC precursors is the FasII-positive P3m cluster, which becomes part of the pars intercerebralis (PI) and is likely the source of some of the NCC axons innervating the CC. This close contact between PI and CC provides the opportunity for inductive interactions between the two structures (De Velasco, 2004).

A previously undescribed population of head mesoderm cells expressing the tinman (tin) gene represents another group of cells that surround the CC precursors during their migration. The Tin-positive cells, for which the term 'cephalic vascular rudiment' (CVR; an evolutionary vestige of the cephalic aorta which forms a prominent component of the vascular system in other insects) is suggested, form a loose cluster that extends backward dorsal of the esophagus and eventually establishes contact with the Tin-positive trunk aorta. The CC precursors are initially close to the anterior (trailing) end of the CVR, but they appear to 'catch up' and lead the CVR during later stages (De Velasco, 2004).

Based on reports from other insects, it had been anticipated that the CC is derived from the foregut as part of the invaginating stomatogastric primordium. However, this is not likely to be the case in Drosophila because the CC is present in embryos mutant for forkhead (fkh) in which both esophagus and SNS are eliminated. The expression and phenotype of numerous head gap genes were subsequently investigated to determine the origin of the CC. The results of this analysis indicate strongly that the CC originates from the anterior lip of the ventral furrow (AVF). The CC is deleted in mutations in the genes sine oculis (so), giant (gt), and twist/snail (twi/sna). Each of these genes is expressed in several domains at the blastoderm stage and during gastrulation, but the AVF is the only place of overlap between the three. Furthermore, giant expression, which is particularly strong in the AVF and persists slightly longer than expression of so or twi, visualizes the AVF cells as they spread out and form the anterior part of the head mesodermal layer that lines the inner surface of the brain primordium and includes the glass-positive CC precursors (De Velasco, 2004).

Besides sine oculis, giant, and twist/snail, one more head gap gene, tailless (tll) affects CC development. Tll is expressed in the anlage of the protocerebrum and only appears faintly, if at all, in the AVF. In tll mutant embryos, the CC is absent, whereas the SNS appears normal in size. It is speculated that the effect of tll on the CC is indirect, caused by the elimination of the protocerebrum (including the PI) in tll mutants. Another head gap gene, orthodenticle (otd), is expressed similarly to tll but leaves the CC intact. Otd mutant embryos also show a reduction in size of the protocerebrum but still possess the PI contacted by the CC precursors. Taken together, these findings (which need further follow-up analysis) hint at the possibility of inductive interactions between protocerebrum and CC (De Velasco, 2004).

In the embryo, glass is expressed in the primordium of the larval eye (Bolwig's organ), a small group of protocerebral neurons, and the CC precursors. Loss of gl in the allele gl2 results in the absence of both larval eye and the CC, as shown in labelings with anti-FasII and AKH probe. Interestingly, this phenotype is a dominant effect since 75% of embryos derived from crossing balanced gl parents have no CC or larval eye. In stages 11 and 12 gl mutant embryos, the gl probe still gives a signal in CC and larval eye precursors. The signal becomes patchy (first in CC precursors, slightly later in larval eye) during stage 12 and has disappeared by stage 13. This finding suggests that gl is required for CC migration and/or differentiation, and that the absence of the CC in gl mutants as assayed for by the differentiation marker AKH is caused by transformation and/or apoptosis of initially correctly specified CC precursors (De Velasco, 2004).

Two other regulatory genes that were found to play an important role in vertebrate pituitary development, are Lhx3 and goosecoid (gsc). The Drosophila homologs of both of these genes are expressed in the SNS and possibly the ring gland. To investigate the role of Lim3 and gsc during CC development, their expression and phenotype were analyzed. Lim3 appears in the precursors of the SNS at a relatively late stage (stage 11), following the complete separation of these cells from the esophagus. In addition, lim3 is expressed in several small clusters in the brain primordium. Comparison with the expression of gl makes it clear that the Lim3 expressing cells are distinct from the CC progenitors. No CC defects were found in lim3 mutant embryos. Goosecoid is expressed in the SNS and, in late stage embryos, the CC. However, no CC defects were detected in the gsc0534lacZ allele, which does cause structural abnormalities in the SNS. It is possible that gsc plays a role in later CC differentiation (De Velasco, 2004).

Several signaling pathways, notably Shh, BMP, and BMP antagonists, Wnt and FGF, specify the fate map of the head in vertebrates and also control later morphogenetic events shaping head structures. The same signaling pathways are active at multiple stages in Drosophila head development, and the pattern of activity and requirement of these pathways in regard to CC development was therefore investigated (De Velasco, 2004). .

The first signal acting zygotically in the Drosophila head is the BMP homolog Dpp, which forms a dorsoventral gradient across the blastoderm. The homolog of the BMP antagonist Chordin, short gastrulation (Sog), is expressed in the ventral blastoderm, overlapping with the ventral furrow. Loss of sog results in the absence of the CC, while the SNS is still present, which reflects ventral origin of the CC. Sog seems to be the only signal, of those tested, required for CC determination, since mutation of all other pathways does not eliminate the CC but merely effects its size, shape, or location (De Velasco, 2004).

Following its early widespread dorsal expression, Dpp becomes more confined during gastrulation to a narrow mid-dorsal stripe and an anterior cap that corresponds to parts of the anlagen of the esophagus and epipharynx. From this domain segregates the most anterior population of head mesoderm cells that give rise to the visceral muscle of the esophagus and which maintain Dpp expression. The visceral mesoderm of the esophagus flanks both CC and SNS. Loss of Dpp causes absence of the SNS; the CC is still present and expresses AKH but does not migrate posteriorly (De Velasco, 2004).

Both Hh and Wg are expressed from gastrulation onward in a similar pattern in the developing foregut. The pattern resolves into two domains, a posterior one covering the posterior esophagus, and an anterior one overlapping with the epipharynx. The esophageal domain, which shows a higher level of expression than the anterior domain, is located posterior to the precursors of CC and SNS. No significant abnormality in CC and SNS was obvious in Hh mutants. Wingless mutants show defects in the SNS but the CC is present, if misshapen and mislocalized, in the strongly distorted head of late wg mutant embryos (De Velasco, 2004).

Activity of the MAPK signaling pathway is widespread in the Drosophila head from gastrulation onward. Beside a wide anterior and posterior domain traversing the lateral and dorsal domain of the head ectoderm, the primordia of the foregut, including the SNS, and head mesoderm show a dynamic MAPK activity. At least two RTKs, EGFR and FGFR/heartless, drive the MAPK pathway in the embryonic head. EGFR is responsible for activation in the ectoderm and foregut. Loss of EGFR causes widespread cell death in the head and the absence of the SNS. The CC is still present, although reduced in size. Activation of MAPK by Heartless (Htl) occurs in a narrow anterior domain of head mesoderm that gives rise to the dorsal pharyngeal muscles. The foregut, SNS, and CC develop rather normally in htl mutants. However, the CC shows variable defects in shape and location, which are most likely due to the absence of the aorta and CA, both of which are derivatives of the dorsal mesoderm, which is defective in htl loss of function and to which the CC is normally attached (De Velasco, 2004).

This study has identified several early acting genes functioning in the development of the corpus cardiacum; among them sine oculis, giant, and glass are essential for its development. The apparent origin of the CC from the anterior ventral furrow, rather than the SNS placode as surmised in other studies, came as a surprise. In Manduca, CC precursors seem to delaminate from the posterior part of the neurogenic foregut ectoderm that gives rise to the SNS. In Drosophila, CC precursors are also close to the SNS placode as soon as they express the marker glass. Since this marker is not expressed during the segregation of CC precursors, it could not be directly observed from which ectodermal domain of the head they derive. It is therefore still possible that they originate from the SNS placode located in the roof of the foregut primordium. However, genetic data argue strongly against this possibility. Thus, the CC is present in a mutation of fkh, which is expressed and required in the foregut primordium and which is essential for the SNS. Similarly, the CC forms normally in mutations of EGFR, which entirely eliminate the SNS. By contrast, the CC is deleted in twist;snail and giant mutations, both of which are not expressed in the SNS placode and do not affect SNS development. The apparent discrepancy between Drosophila and Manduca indicates that the CC may originate from slightly different domains in different insect groups (the distance between presumptive SNS placode and anterior ventral furrow in the blastoderm is minimal); alternatively, the Manduca CC might also delaminate from the ventral furrow and only secondarily come to lie next to the SNS precursors (De Velasco, 2004).

The proposed origin of the CC from the anterior ventral furrow, which also gives rise to most of the anterior endoderm, underlines the close relationship between endodermal and neuroendocrine lineages. Such relationship also seems to exist in vertebrates. Numerous peptide signaling factors in vertebrates are expressed in cells of the digestive tract, in particular the pancreas, and the pituitary and/or hypothalamus. Among these are cholecystokinin (CKK), as well as the glucagon-like peptide (GLP) 1. GLPs and glucagon itself are the closest vertebrate counterparts to the CC-derived insect hormone AKH. Both AKH and glucagon, besides numerous other hormones released from the neuroendocrine system, coordinately control energy metabolism and behaviors associated with food uptake and processing. It is reasonable to assume that in the simple Bilaterian ancestor, cells that carried out the food uptake and digestive activities, that is, principal cells of the digestive tract, were identical with or spatially close to those cells that regulated these activities, among them endocrine and nerve cells (De Velasco, 2004 and references therein).

The pars intercerebralis/corpora cardiaca complex of insects has been repeatedly compared to the hypothalamus-pituitary axis in vertebrates. This comparison is usually based on clear similarities between the two on a gross anatomical and functional level. Thus, in both insects and vertebrates, neurosecretory neurons located in the anteromedial brain produce peptide hormones that are transported along axons to a peripheral gland. The axons either terminate on gland cells and modulate the release of glandular hormones, or they terminate in a separate secretory part of the gland where they release their products directly into the blood. Functional similarities include a role of both insect and vertebrate neuroendocrine factors in energy metabolism, growth, water retention, and reproduction. However, to what extent do these functional similarities represent true homologies, which would imply the presence of the homologous genes in the homologous cells in the Bilaterian ancestor (De Velasco, 2004)?

The main hormone produced by the CC is adipokinetic hormone (AKH), a peptide that acts on the fat body and mobilizes lipids and carbohydrates. AKH also stimulates the nervous system and activates locomotor activity. A peripheral feedback loop controls AKH release, in that sugars (e.g., trehalose in the hemolymph) inhibit AKH release; centrally, several PI-derived neuropeptides controlling AKH secretion have been identified, among them FMRFamide, tachykinin, and crustacean cardioactive peptide. FMRFamide inhibits AKH release, whereas cardioactive peptide and tachykinins (both of which also influence contractility of the heart and visceral muscles) stimulate AKH release (De Velasco, 2004).

AKH shares common functions with the vertebrate glucagon and has some sequence similarity with the N-terminus of glucagons. However, comparison of the genes encoding AKH and glucagons, respectively, provides no clear evidence for homology of these peptides on the molecular level. Glucagon, along with two other growth factors, GLP1 and GLP2, is encoded by the proglucagon gene for which true homologs have so far only been identified among vertebrates. The arthropod AKH gene may have been traced further back to the protostome root with the recent finding of significant sequence similarity with the mollusk gene encoding the APGWamide peptides. However, no significant sequence similarity exists between proglucagon and the AKH/APWHamide genes. The expression pattern of the proglucagon and AKH/APGWamide genes is too widespread to add meaningfully to the question of common ancestry. Glucagon is produced in the endocrine pancreas, as well as the intestinal epithelium, but the GLP growth factors (and therefore the proglucagon gene) are expressed in many cells, including neurons, of the developing and mature vertebrate. AKH is expressed mainly in the corpora cardiaca but is also found in the brain of various insect species. Thus, the molecular sequence of the specific secreted products of the CC and pituitary can currently provide no support for or against the notion that both structures are homologous (De Velasco, 2004 and references therein).

The vertebrate pituitary and Drosophila CC show significant similarities during development. Precursors of both are derived from an anterior anlage; following segregation from this anlage, CC precursors contact the part of the anteromedial forebrain primordium from which they will receive innervation. Shared regulatory genes and signaling pathways add to the overall similarity. In this regard, the role of sine oculis is particularly striking. The expression pattern of so in Drosophila is fairly restricted, including the eye field, stomatogastric anlage, and anterior lip of the ventral furrow that give rise to the CC. Another gene of the sine oculis/six family, optix, is expressed in an anterior unpaired domain close to the SNS, but not the CC. In the early vertebrate embryo, six3/6 (the ortholog of optix) is specifically expressed in the eye field and the anlage of the pituitary; six1/2, orthologs of Drosophila sine oculis, are expressed in sensory placodes of the vertebrate head, but no pituitary expression has been reported yet. In both systems, a sine oculis/six gene plays an early and essential role in the specification of the CC and pituitary, respectively. In Drosophila, both CC and SNS are absent in so mutants; in vertebrate, loss of six3/6 causes severe reduction and posteriorization of the forebrain region though not mention of the pituitary effect has been described (De Velasco, 2004).

Two other regulatory genes, goosecoid and Lhx3/lim3, are relevant in the comparison of the vertebrate and Drosophila neuroendocrine systems. Gsc appears in the ventral neural tube and foregut of postgastrula mouse embryos and is required for ventral neural tube patterning.Drosophila gsc is also expressed at an early stage, but it appears exclusively in the anlage of the SNS and comes on in the CC primordium only late. Loss of gsc results in mild structural defects in the SNS and no morphologically apparent phenotype in the CC. Lhx3 is a transcription factor of the Lim family that is triggered by Shh and FGF8 in the vertebrate pituitary primordium and required for its invagination. Drosophila lim3 is expressed at a late stage in part of the SNS primordium, but not the CC primordium. As stated for gsc, no structural phenotype associated with the SNS or CC has been noted in lim3 mutants, but more careful analysis, using additional late differentiation markers for these structures, will be required to establish the role of these two genes in Drosophila neuroendocrine development (De Velasco, 2004).

glass represents a homeobox gene expressed in the eye, nervous system, and as shown in this study, the corpus cardiacum. glass, which is absolutely required for the Drosophila CC since loss of one copy of the gene causes complete absence of the CC at late embryonic stages, has vertebrate cognates but so far no eye function of these genes has been reported (De Velasco, 2004).

Several signaling pathways are expressed in similar patterns in and/or around the developing neuroendocrine system of vertebrates and Drosophila. In both, Hh/Shh is expressed posteriorly adjacent to the CC/Rathke's pouch in the primordium of the foregut/oral epithelium. Vertebrate BMP2/4 comes on in the mesenchyme surrounding the base of Rathke's pouch. Similarly, Drosophila Dpp appears in the mesoderm flanking foregut primordium, CC, and SNS. FGF8 is derived from the hypothalamus floor; the FGF receptor homolog Htl is expressed in the myogenic head mesoderm that is anteriorly adjacent to the CC/SNS complex. In vertebrates, the ventral-to-dorsal BMP gradient and dorsal-to-ventral FGF8 gradients control the differentiation of pituitary cell types; Shh is also required in the proliferation and differentiation of the pituitary primordium. The role of these signaling pathways in Drosophila is less apparent. The CC is still present in dpp, hh, or htl mutant embryos, although it exhibits abnormalities in shape and location. This may constitute an indirect effect of these genes, given their widespread role in head morphogenesis (in case of Dpp and Hh) or mesodermal migration (for Htl). It is anticipated that with the advent of additional markers for subsets of CC cell types, the role of the Hh and Dpp signaling pathways will become clearer (De Velasco, 2004).

A mutation in the Dpp antagonist Sog was the only signaling mutant analyzed in this work that was able to completely remove the CC. This is surprising, given the relatively mild phenotype of sog mutants in the primordium of the ventral nerve cord. Here, only removal of both sog and brinker (brk) together are able to suppress the appearance of most neuroblasts. However, certain domains in the ventral head (that include the precursors of the CC) may be more sensitive to a shift in balance of the Sog-Dpp antagonism. It is speculated that the loss of the CC precursors in sog mutants results from an expanded Dpp gradient, although more experiments would be required to rule out the possibility that sog (the Drosophila homolog of chordin) directly affects CC precursor fate (De Velasco, 2004).

In conclusion, this study presents evidence for a number of conserved properties in the way the progenitors of the neuroendocrine system in vertebrate and Drosophila embryos are spatially laid out and employ cassettes of signaling pathways and fate determinants. This suggests that fundamental elements of a primordial “neuroendocrine system” were already present in the Bilaterian ancestor. Current ideas on pituitary evolution are compatible with this notion. Sensory structures proposed to represent the homologs of the vertebrate pituitary are present in cephalochordates, urochordates, and hemichordates. In amphioxus, for example, these cells form the so-called Hatschek's pit, located in the roof of the pharynx in close contact with the anterior neural tube. Molecules characteristic of the vertebrate pituitary, such as GnRH and Pit-1, are found in Hatschek's pit and in the proposed homolog in urochordates. It is thought that the pituitary originated as a chemosensory structure that senses environmental cues and produced hormones controlling gametogenesis and reproductive behavior, as well as fundamental metabolic functions. Subsequently, the pituitary lost its sensory function and was taken under the control of the CNS, which was able to assimilate sensory information more efficiently. It is likely that stage one, that is, a sensory-endocrine pituitary forerunner, was present in the Bilaterian ancestor. This forerunner probably formed part of the pharynx, which would explain the conserved developmental origin in Drosophila and vertebrates. The sensory-neuroendocrine state of the pituitary homolog is still preserved in present-day protochordates. Loss of sensory function and the taking-over of pituitary control by the CNS occurred during vertebrate evolution. In arthropods or other protostomes, evidence for a sensory forerunner of the neuroendocrine gland has not yet been described; guided by situation in protochordates, one would expect to find such a structure among the sensory organs of the head (De Velasco, 2004).

Common origin of insect trachea and endocrine organs from a segmentally repeated precursor

Segmented organisms have serially repeated structures that become specialized in some segments. The Drosophila corpora allata, prothoracic glands, and trachea are shown to have a homologous origin and can convert into each other. The tracheal epithelial tubes develop from ten trunk placodes, and homologous ectodermal cells in the maxilla and labium form the corpora allata and the prothoracic glands. The early endocrine and trachea gene networks are similar, with STAT and Hox genes inducing their activation. The initial invagination of the trachea and the endocrine primordia is identical, but activation of Snail in the glands induces an epithelial-mesenchymal transition (EMT), after which the corpora allata and prothoracic gland primordia coalesce and migrate dorsally, joining the corpora cardiaca to form the ring gland. It is proposed that the arthropod ectodermal endocrine glands and respiratory organs arose through an extreme process of divergent evolution from a metameric repeated structure (Sanchez-Higueras, 2013).

The endocrine control of molting and metamorphosis in insects is regulated by the corpora allata (ca) and the prothoracic glands (pg), which secrete juvenile hormone and ecdysone, respectively. In Diptera, these glands and the corpora cardiaca (cc) fuse during development to form a tripartite endocrine organ called the ring gland. While the corpora cardiaca is known to originate from the migration of anterior mesodermal cells, the origin of the other two ring gland components is unclear (Sanchez-Higueras, 2013).

The tracheae have a completely different structure consisting of a tubular network of polarized cells. The tracheae are specified in the second thoracic to the eighth abdominal segments (T2-A8) by the activation of trachealess (trh) and ventral veinless (vvl) (Sanchez-Higueras, 2013).

The enhancers controlling trh and vvl in the tracheal primordia have been isolated and shown to be activated by JAK/ STAT signaling. While the trh enhancers are restricted to the tracheal primordia in the T2-A8 segments, the vvl1+2 enhancer is also expressed in cells at homologous positions in the maxilla (Mx), labium (Lb), T1, and A9 segments in a pattern reproducing the early transcription of vvl. The fate of these nontracheal vvl-expressing cells was unknown, but it was shown that ectopic trh expression transforms these cells into tracheae. To identify their fate, vvl1+2-EGFP and mCherry constructs were made (Sanchez-Higueras, 2013).

Although the vvl1+2 enhancer drives expression transiently, the stability of the EGFP and mCherry proteins labels these cells during development. It was observed that while the T1 and A9 patches remained in the surface and integrated with the embryonic epidermis, the patches in the Mx and Lb invaginated just as the tracheal primordia did. Next, the Mx and Lb patches fused, and a group of them underwent an epithelial-mesenchymal transition (EMT) initiating a dorsal migration toward the anterior of the aorta, where they integrate into the ring gland. To find out what controls the EMT, the expression of the snail (sna) gene, a key EMT regulator, was studied. Besides its expression in the mesoderm primordium, it was found that sna is also transcribed in two patches of cells that become the migrating primordium. Using sna bacterial artificial chromosomes (BACs) with different cis-regulatory regions, the enhancer activating sna in the ring gland primordium (sna-rg). A sna-rg-GFP construct labels the subset of Mx and Lb vvl1+2-expressing cells that experience EMT and migrate to form the ring gland. Staining with seven-up (svp) and spalt (sal) (also known as salm) markers, which label the ca and the pg, respectively, showed that the sna-rg-GFP cells form these two endocrine glands. The sna-rg-GFP-expressing cells in the Mx activated svp, and those in the Lb activated sal before they coalesced, indicating that the ca and pg are specified in different segments before they migrate (Sanchez-Higueras, 2013).

To test whether Hox genes, the major regulators of anteroposterior segment differentiation, participate in gland morphogenesis, vvl1+2-GFP embryos were stained, and it was found that the Mx vvl1+2 primordium expressed Deformed (Dfd) and the Lb primordium Sex combs reduced (Scr), while the T1 primordium expressed very low levels of Scr. Dfd mutant embryos lacked the ca, while Scr mutant embryos lacked the pg. Dfd and Scr expression in the gland primordia was transient, suggesting that they control their specification. Consistently, in Dfd, Scr double-mutant embryos, vvl1+2 was not activated in the Mx and Lb patches, and the same was true for vvl transcription. In these mutants, the sna-rg-GFP expression was almost absent, and the ca and pg did not form. In each case, Dfd controlled the expression of the Mx patch and Scr of the Lb patch (Sanchez-Higueras, 2013).

The capacity of different Hox genes to rescue the ring gland defects of Scr, Dfd double mutants was tested. Induction of Dfd with the sal-Gal4 line in these mutants restored the expression of vvl1+2 and sna-rg-GFP in the Mx and the Lb. However, in contrast to the wild-type, both segments formed a ca as all cells express Svp. Similarly, induction of Scr also restored the vvl1+2 and sna-rg-GFP expression, but both primordia formed a pg as they activate Sal and Phantom, an enzyme required for ecdysone synthesis. The capacity of both Dfd and Scr to restore vvl expression, regardless of the segment, led to a test of whether other Hox proteins could have the same function. Induction of Antennapaedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), or Abdominal-B (Abd-B) restored vvl1+2 expression in the Mx and Lb, but these cells formed tubes instead of migratory gland primordia. These cephalic tubes are trachea, as they do not activate sna-rg, they express Trh, and their nuclei accumulate Tango (Tgo), a maternal protein that is only translocated to the nucleus in salivary glands and tracheal cells, indicating that the trunk Hox proteins can restore vvl expression in the Mx and Lb but induce their transformation to trachea (Sanchez-Higueras, 2013).

To investigate whether vvl and trh expression is normally under Hox control in the trunk, focus was placed on Antp, which is expressed at high levels in the tracheal pits. In double-mutant Dfd, Antp embryos, vvl1+2 was maintained in the Lb where Scr was present, while the Mx, T1, and T2 patches were missing. In T3-A8, vvl1+2 expression, although reduced, was present, probably due to the expression of Ubx, Abd-A, and Abd-B in the posterior thorax and abdomen. Thus, Antp regulates vvl expression in the tracheal T2 primordium. Surprisingly, in Dfd, Antp double mutants, Trh and Tgo were maintained in the T2 tracheal pit, indicating that although Hox genes can activate ectopic trh expression, in the tracheal primordia they may be acting redundantly with some other unidentified factor, explaining why the capacity of Hox proteins to specify trachea had not been reported previously (Sanchez-Higueras, 2013).

sna null mutants were studied to determine sna's requirement for ring gland development, but their aberrant gastrulation precluded analyzing specific ring gland defects. To investigate sna function in the gland primordia, the sna mutants were rescued with the sna-squish BAC, which drives normal Sna expression except in the ring gland. These embryos have a normal gastrulation and activate the sna-rg- GFP; however, the gland primordia degenerate and disappear. To block apoptosis, these embryos were made homozygous for the H99 deficiency, which removes three apoptotic inducers. In this situation, the ca and pg primordia invaginated and survived, but they did not undergo EMT. As a result, the gland primordia maintain epithelial polarity, do not migrate, and form small pouches that remain attached to the epidermis. Vvl is required for tracheal migration. In vvl mutant embryos, sna-rg-GFP expression was activated, but the cells degenerated. In vvl mutant embryos also mutant for H99, the primordia underwent EMT and migrated up to the primordia coalescence; however, the later dorsal migration did not progress (Sanchez-Higueras, 2013).

This study has shown that the ca and pg develop from vvl-expressing cephalic cells at positions where other segments form trachea, suggesting that they could be part of a segmentally repeated structure that is modified in each segment by the activity of different Hox proteins. As the cephalic primordia are transformed into trachea by ectopic expression of trunk Hox, tests were performed to see whether the trachea primordia could form gland cells. Ectopic expression of Dfd with arm- Gal4 resulted in the activation of sna-rg-GFP on the ventral side of the tracheal pits. These sna-rg-GFP0-expressing cells also expressed vvl1+2 and Trh and had nuclear Tgo, showing that they conserve tracheal characteristics. These sna-rg-GFP-positive cells did not show EMT and remained associated to the ventral anterior tracheal branch. The strength of ectopic sna-rg-GFP expression increased when ectopic Dfd was induced in trh mutant embryos. However, migratory behaviors in the sna-rg-GFP cells were only observed if Dfd was coexpressed with Sal. Thus, sal is expressed several times in the gland primordia, first at st9-10 repressing trunk Hox expression in the cephalic segments and second from st11 in the prothoracic gland. It is uncertain whether the sal requirement for migration is linked to the first function or whether it represents an additional role (Sanchez-Higueras, 2013).

These results show that the endocrine ectodermal glands and the respiratory trachea develop as serially homologous organs in Drosophila. The identical regulation of vvl in the primordia of trachea and gland by the combined action of the JAK/STAT pathway and Hox proteins could represent the vestiges of an ancestral regulatory network retained to specify these serially repeated structures, while the activation of Sna for gland development and Trh and Tgo for trachea formation could represent network modifications recruited later by specific Hox proteins during the functional specialization of each primordium. This hypothesis or alternative possibilities should be confirmed by analyzing the expression of these gene networks in various arthropod species. The diversification of glands and respiratory organs must have occurred before the split of insects and crustaceans, as there is a correspondence between the endocrine glands in both classes, with the corpora cardiaca corresponding to the pericardial organ, the corpora allata to the mandibular organ, and the prothoracic gland to the Y gland. Despite their divergent morphology, a correspondence between the insect trachea and the crustacean gills can also be made, as both respiratory organs coexpress vvl and trh during their organogenesis. Divergence between endocrine glands and respiratory organs may have occurred when the evolution of the arthropod exoskeleton required solving two simultaneous problems: the need to molt to allow growth, and the need for specialized organs for gas exchange (Sanchez-Higueras, 2013).

Precise long-range migration results from short-range stepwise migration during ring gland organogenesis

Many organs are specified far from the location they occupy when functional, having to migrate long distances through the heterogeneous and dynamic environment of the early embryo. The formation of the main Drosophila endocrine organ, the ring gland, was studied as a new model to investigate in vivo the genetic regulation of collective cell migration. The ring gland results from the fusion of three independent gland primordia that migrate from the head towards the anterior aorta as the embryo is experiencing major morphogenetic movements. To complete their long-range migration, the glands extend filopodia moving sequentially towards a nearby intermediate target and from there to more distal ones. Thus, the apparent long-range migration is composed of several short-range migratory steps that facilitate reaching the final destination. The target tissues react to the gland's proximity by sending filopodia towards it. This finding of a succession of independent migration stages is consistent with the stepwise evolution of ring gland assembly and fits with the observed gland locations found in extant crustaceans, basal insects and flies (Sanchez-Higueras, 2016).

Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation

Environmental stressors induce changes in endocrine state, leading to energy re-allocation from reproduction to survival. Female Drosophila melanogaster respond to thermal and nutrient stressors by arresting egg production through elevation of the steroid hormone ecdysone. However, the mechanisms through which this reproductive arrest occurs are not well understood. This study reports that stress-induced elevation of ecdysone is accompanied by decreased levels of ecdysis triggering hormone (ETH). Depressed levels of circulating ETH lead to attenuated activity of its targets, including juvenile hormone-producing corpus allatum and, as described in this study for the first time, octopaminergic neurons of the oviduct. Elevation of steroid thereby results in arrested oogenesis, reduced octopaminergic input to the reproductive tract, and consequent suppression of ovulation. ETH mitigates heat or nutritional stress-induced attenuation of fecundity, which suggests that its deficiency is critical to reproductive adaptability. These findings indicate that, as a dual regulator of octopamine and juvenile hormone release, ETH provides a link between stress-induced elevation of ecdysone levels and consequent reduction in fecundity (Meiselman, 2018).

Evidence presented in this study establishes a new paradigm for Drosophila reproduction, wherein stressful conditions arrest egg production via a hormonal cascade involving reciprocal ecdysone and ETH signaling. As steroid levels fluctuate in response to stress, so too does ETH, a consequence of steroid-regulated changes in Inka cell secretory competence. ETH activates two downstream targets: the JH-producing corpus allatum and modulatory OA neurons innervating the ovary and oviducts. This study characterized the nature of ETH dependence, and assigned function and context to a newly recognized hormonal axis governing reproductive responses to stress (Meiselman, 2018).

Previous report showed that ETH is an obligatory allatotropin, promoting oogenesis and fecundity through JH production; consequently, ETH deficiency results in low JH levels and arrested oogenesis (Meiselman, 2017). The present work demonstrates that ovulation of stage 14 oocytes depends upon ETH activation of OA neurons innervating the ovary and oviduct. A comprehensive explanation is offered for the change in distribution of vitellogenic oocytes reported in EcR mutants or under conditions of high or low ecdysone, depending on stress levels. ETH deficiency or ETHR knockdown results in accumulation of stage 14 oocytes in the ovary due to ovulation block, and a mechanistic link between altered endocrine state and ovulation is provided (Meiselman, 2018).

ETH promotes ovulation through activation OA neurons to induce contractions in the ovary and relaxation of the oviducts. It is interesting that ETH triggers calcium dynamics in vitro on distal axonal projections, suggesting ETH-stimulated OA release results from direct action of ETH on axons and/or nerve terminals. While ovary contractions in response to ETH exposure occur in both virgin and mated females, this study chose virgin females for analysis due to higher spontaneous contractile activity in mated females. This is likely due to actions of ovulin after insemination, which stimulate outgrowth of octopaminergic neurons innervating the oviduct. In virgin females, concentration-dependent ETH actions on the ovary are in the range predicted for activation of ETHR-A receptors (Meiselman, 2018).

Acting through OA neurons, ETH mobilizes calcium in the epithelium enveloping the ovary, initiating bursts of contractions in the peritoneal sheath at the base of the ovary associated with ovulation. Although bath-applied ETH and OA are both sufficient to induce calcium mobilization in the oviduct epithelium, they induce distinctive response patterns. OA causes a rapid, sustained calcium wave with a slowly waning plateau following the peak response. ETH actions occur with longer latency and induce oscillatory calcium dynamics, which could be a consequence of periodic synaptic reuptake of OA by nerve terminals. No changes in intensity were observed between treatments or at different doses, suggesting a possible threshold effect. It is also interesting to note that calcium waves spread through the epithelial layer, suggesting that the epithelium is a functional syncytium, which undoubtedly aids in coordination of relaxation (Meiselman, 2018).

Injection of mated females with either ETH or OA induces ovulation in vivo, whereas injected virgin females respond much more weakly. In order for ovulation to occur, OA causes follicle rupture inside the ovaries, a process requiring one to several hours ex vivo. It is hypothesized that mated females are in the proper endocrine state for ovulation, and thus follicle rupture may already be in progress before application of ETH or OA. As follicle rupture is the critical first step for egg-laying, this limiting factor would explain the length of time (up to 60 min) elapsed after physiological levels of ETH/OA are reached for in vivo ovulation to occur, given that ovary contraction and oviduct relaxation occur within seconds (Meiselman, 2018).

Agents previously implicated in oviduct contractions were also examined, including tyramine, glutamate, and proctolin. While the ineffectiveness of tyramine and glutamate is not surprising, the negative result with proctolin is at variance with prior literature. Examination of proctolin-induced contractions revealed that they are localized to the distal tip (germaria) of the ovaries. Moreover, proctolin does not stimulate ovulation in vitro. It appears that the role of proctolin in Drosophila ovaries is more limited than in the well-studied locust oviduct (Meiselman, 2018).

This study has shown that elevated ecdysone levels in response to heat and nutritional stress are associated with a drop in circulating ETH levels. It was previously hypothesized that the Inka cell secretory competence model governing ecdysis signaling during developmental stages may persist into adulthood (Meiselman, 2017). The results presented in this study support this hypothesis (Meiselman, 2018).

Both stress and ETH deficiency have similar consequences for reproduction, namely arrested oogenesis and reduced ovulation, resulting in increased stage 14 egg retention and lower egg production. Progression of mid-oogenetic oocytes is directly correlated with JH levels, while OA release from reproductive tract neurons is necessary for ovulation. This study shows that arrested oogenesis and ovulation contributing to the ovariole profile observed in heat-stressed flies can be explained by ETH deficiency, which has a dual role in regulating JH levels and activity of OA neurons innervating ovaries and oviducts. Indeed, arrest of both oogenesis and ovulation deficiencies can be rescued by ETH, either through TRPA1 activation of Inka cells or direct injection of ETH1 (Meiselman, 2018).

The mechanism through which elevated ecdysone leads to ETH deficiency was examined by performing rescue experiments designed to (1) suppress steroid signaling in Inka cells and (2) express the transcription factor βFTZ-F1, which confers secretory competence of Inka cells and is suppressed by high ecdysone levels. Although somewhat variable in their effectiveness, these manipulations resulted in clear rescue of oogenesis and ovulation in heat-stressed females, confirming that the thermal stress response operates through the influence of ecdysone on Inka cell secretion (Meiselman, 2018).

Methoprene treatment increases progression of oogenesis but does not increase oviposition in stressed animals. In fact, this study observed a significant increase in eggs retained after methoprene treatment, suggesting that synthesis of mature eggs resumes with JH treatment, but ovulation remains impaired under conditions of elevated ecdysone and ETH deficiency. This suggests that ovulation provides a gating mechanism under stressful conditions, limiting egg production while conditions are suboptimal. A recent report suggested that normal ecdysone levels stimulate follicle rupture and ovulation, but that elevated levels inhibit follicle rupture (Knapp, 2017). The present work provides an additional mechanism for suppression of ovulation associated with elevated ecdysone levels: repression of ETH release leading to reduced OA neuron activity (Meiselman, 2018).

It is interesting to note that wet starvation reduces ecdysone levels and increases ETH levels, whereas sugar starvation increases ecdysone levels and, as is shown in this study, increases ETH levels. Wet-starved females were precisely synchronized in mating on day 4, and began starvation (no nutrient source, wet KimWipe) 24 h later for an additional 24 h. mino acid-deprived females were group-raised until day 3, and groups were placed on agar + 10% sucrose for 24 h. Mating was not controlled in sugar-starved females, though it is known to influence ecdysone levels dramatically in the short term. Arguably the most interesting result is that ecdysone decrease led to elevated circulating ETH. This adds credence to the hypothesis that ETH and ecdysone levels are generally inversely correlated (Meiselman, 2018).

Unique stresses may garner different endocrine responses because different types of cues require differential behavioral adaptation. The ability of a hormone to coordinate a wide variety of target tissues to change in state makes it a perfect tool for stress adaptation. As an organism encounters a new type of stress, they may adapt a new endocrine state to coordinate a tissue-wide response. Many hormones in closely related insects play markedly different roles, which evolve as rapidly as behavioral niches, but an endocrine core in E-ETH-JH is highly conserved, similar to the hypothalamic-pituitary-gonadal (HPG) axis among vertebrates. A hormonal network with competence to adjust reproductive output in response to environmental changes is undoubtedly a common phenomenon among multicellular organisms. The discovery of a stress response hormonal axis and, more aptly, a peptide hormone with the potential to alleviate stress-induced deficits in reproduction could be of particular relevance to the honey bee Apis mellifera. In recent years, Apis reproductives have been producing fewer progeny due to a variety of stressors, including temperature extrema. While proctolin has already been found to be a short-term reproductive stimulant in Apis queens, ETH is attractive as it can alter JH levels, which in turn may rescue poor pheromone production, the proximal cause of supersedure (Meiselman, 2018).

A cell surface protein controls endocrine ring gland morphogenesis and steroid production

Identification of signals for systemic adaption of hormonal regulation would help to understand the crosstalk between cells and environmental cues contributing to growth, metabolic homeostasis and development. Physiological states are controlled by precise pulsatile hormonal release, including endocrine steroids in human and ecdysteroids in insects. This study shows in Drosophila that regulation of genes that control biosynthesis and signaling of the steroid hormone ecdysone, a central regulator of developmental progress, depends on the extracellular matrix protein Obstructor-A (Obst-A). Ecdysone is produced by the prothoracic gland (PG), where sensory neurons projecting axons from the brain integrate stimuli for endocrine control. By defining the extracellular surface, Obst-A promotes morphogenesis and axonal growth in the PG. This process requires Obst-A-matrix reorganization by Clathrin/Wurst-mediated endocytosis. Wurst (Wus) is a transmembrane protein essential for clathrin-mediated endocytosis. These data identifies the extracellular matrix as essential for endocrine ring gland function, which coordinates physiology, axon morphogenesis, and developmental programs. As Obst-A and Wurst homologs are found among all arthropods, it is proposed that this mechanism is evolutionary conserved (Pesch, 2018).

Steroid hormones are small, lipophilic compounds that can pass through cell membranes and constitute important regulators of growth, metabolism, and reproduction. To coordinate steroid production neuronal and endocrine systems need to integrate external and internal cues in the vertebrate hypothalamic-pituitary system. Furthermore, reciprocal interactions between nerves and glands maintain homeostasis and allow for responses to environmental stimuli. As a response to stimulatory and inhibitory signals coming from the hypothalamus, pituitary gland cells synthesize and secrete a variety of specific pituitary trophic hormones, such as ACTH (adrenocorticotropic hormone), Thyroid stimulating hormone (TSH), and Follicle stimulating hormone (FSH), in a pulsatile and episodic manner. Any malfunctions in the interactions between nerves and the pituitary gland can lead to multiple endocrine disorders, neurological manifestations and has substantial impact on metabolism, sexual maturation, reproduction, blood pressure and other vital physical functions (Pesch, 2018).

In Drosophila distinct neurosecretory cells from the brain stimulate hormone responses in the endocrine ring gland. Most prominent Prothoracicotropic hormone (PTTH) expressing neurosecretory cells project axons from the brain to directly stimulate ecdysone steroid production. In addition, other neuroendocrine centers in the brain send axons through the nervus corporis cardiaci (Ncc) NccI and NccII towards the ring gland. The ring gland itself is formed by the two prothoracic gland (PG, ventrally) lobes, corpora allata (CA, dorsally), and corpora cardiaca (CC, laterally). The CC cells affect metabolism by secreting Adipokinetic Hormone (AKH) for manipulating sugar levels in the hemolymph, as well as glycemic factors and heart rate accelerating peptides/hormones. Drosophila that lack the CA, which is the source of JH, pupariated at smaller size due to reduced larval growth rates and were lethal at pupal stages. Most of the ring gland volume is taken up by the PGs, whose cells grow in size, while CC cells remain small. PG cells produce and secrete the steroid hormone ecdysone. Thus, insect development and metamorphosis is coordinately controlled by the sesquiterpenoid juvenile hormones (JH) and the molting hormone 20-hydroxyecdysone (20E) (Pesch, 2018).

Ecdysone biosynthesis relies on dietary cholesterol which is processed by a number of enzymes. Npc1a protein, named after the Niemann-Pick type C disease, is required for providing cholesterol as substrate for ecdysone biosynthesis. In the PGs sterols are then converted into Ecdysone by subsequent modifications regulated by a number of ecdysteroidogenic enzymes encoded by neverland and the Halloween genes such as spook, shroud and phantom. Secreted from PGs, Ecdysone is released into the circulatory system and converted into the more active 20 hydroxyecdysone (20E) by the P450 (Halloween) enzyme Shade. 20E finally binds and controls the activity of the nuclear ecdysone receptor complex to initiate transcription of many target genes that precisely regulate larval molting and metamorphosis. The activated EcR/Usp receptor complex binds genomic response elements to initiate transcription of early response target genes, of which E74A is a prominent representative. The Cyp18a1 enzyme is involved in the termination of ecdysone pulses by feedback mechanism. Finally, ecdysis-triggering hormones (ETH) trigger centrally patterned ecdysis and accompanied behaviors by induction of the eclosion hormone (EH) and its release from neurosecretory cells (Pesch, 2018).

Obstructor (Obst)-A belongs to the Drosophila obstructor multigene family which is well-conserved among arthropods. The Obst-A protein is characterized by three Chitin-binding domains type 2 (CBDs), a specific domain composition that has been identified even in nematodes Obst-A is expressed in ectodermal epithelia and secreted towards the extracellular space where it is located at the chitinous matrix. Acting as a scaffold-like protein Obst-A binds chitin and recruits other proteins and enzymes for chitin-matrix growth. Obst-A modulates localization of proteins and enzymes at the extracellular matrix that in turn control physical and chemical properties. Thereby Obst-A affects cuticle stiffness during wound repair and integrity against numerous stresses. This study reports surprising evidence that Obst-A is needed for proper ring gland morphogenesis. Immunofluorescent studies show that Obst-A defines the extracellular matrix (ECM) of PG cells. Mutant studies further confirm that the extracellular Obst-A-matrix is required for upregulation of RNA levels of ecdysteroidogenic enzyme genes for the PG in the onset of ecdysis. Genetic studies indicate further that normal axon growth at the PG surface depends on the Obst-A defined PG matrix. This study shows that axons in obst-A null mutants and PG specific knockdown embryos are prevented from normal growth at the PG. This would be consistent with the fact that not only chemical signals but also substrate properties, such as stiffness can determine axon growth. Finally, extracellular Obst-A localization and its functional consequences for PG cells depends on its internalization via Wurst/Clathrin-mediated endocytosis. Since both Obst-A and Wurst (orthologous to vertebrate DNAJC22) proteins are largely conserved, their roles in steroid control is potentially relevant to all arthropods (Pesch, 2018).

Loss of obst-A and the PG-specific obst-A knockdown caused a range of phenotypes characteristic of ecdysone deficiency mutants. A PG specific requirement of Obst-A was further confirmed by the fact that CA (Aug21-Gal4) and CC (akh-Gal4 driven) specific obst-A knockdown did not result in ring gland defects, retained cuticles or larval lethality. Mutant studies show the requirement of Obst-A at the PG cell surface for axon formation and the pulsatile up-regulation of genes involved in ecdysone machinery. Thus, this work demonstrates the importance of the PG specific extracellular matrix for ring gland morphology and physiology during late embryonic and early larval development (Pesch, 2018).

A recent study provided evidence that the circadian clock is a key driver of steroid hormone production. Steroidogenic genes, appeared selectively expressed at night and day in the third instar ring gland. It was shown that Halloween gene expression was dependent on Timeless, which couples the circadian machinery directly to steroid synthesis. Interestingly and comparable with Halloween genes, obst-A expression is under control of circadian rhythm depending on timeless and period in the PG cells (Pesch, 2018).

obst-A mutant analysis shows that most genes representative of ecdysteroidogenesis are prevented from regular pulse-like up-regulation prior to larval ecdysis. This includes even the primary ecdysone-inducible transcription factor E74A. The findings are consistent with the observation of elevated npc1a but reduced cyp18a expression, suggesting that animals could try to raise ecdysone levels in obst-A mutants at the end of first instar development trying to restore the ability to molt. In the same context, application of the active 20E to first instar larvae had beneficial effect on survival when obst-A knocked down or completely absent, indicating that ecdysteroid production was prevented in the mutants. Although ecdysteroid application was beneficial, it did not rescue lethality to adulthood as was found for genes exclusively involved in ecdysone production. Thus, on one hand partial rescue proves ecdysteroid deficiency caused by knockdown or loss of obst-A in the PG, on the other hand it shows that larvae suffer from severe defects in the tracheal and epidermal cuticles. Obst-A is not maternally contributed (Petkau, 2012) and ring gland Obst-A expression starts at late embryonic stage 16, indicating that Ecdysone signaling in earlier embryos cannot be affected by Obst-A. By contrast, at molt from first to second instar obst-A mutants most likely become arrested when 20E levels rise. This would be consistent with data showing reduced gene expression of ecdysteroidogenic factors in obst-A mutants (Pesch, 2018).

At the cell surface Obst-A provides the capacity for binding chitin and associated proteins that modulate the chitin-matrix. Obst-A controls the proper localization of chitin-deacetylases Serpentine (Serp) and Vermiform (Verm) and the chitin protector Knickkopf (Knk). The failure in forming a normal chitin-matrix in obst-A mutants disturbs tracheal and epidermal cuticle integrity and stiffness. Larvae lacking obst-A display wrinkled trachea and epidermis, as well as deformed body shape (Pesch, 2015; Petkau, 2012; Tiklova, 2013). However, lethality at larval transition, growth arrest, and retained cuticles found in obst-A mutant is typical for defects in the ecdysone pathway. Consistent with this, a restricted Obst-A requirement in the cuticle would not explain why gene expression of serp, verm and knk failed to be up-regulated in obst-A mutant larvae in the onset of ecdysis (Pesch, 2015). In addition, none of these gene products were detected in the ring gland. This altogether provides evidence for a PG specific but chitin-independent function of the Obst-A at the ring gland. Despite its tracheal expression Serp is secreted by the fat body, transported via hemolymph and taken up by tracheal cells. Thus, levels of free Serp molecules in the hemolymph could reflect the current status of chitin-matrix maturation and accompanied cuticle formation. Whether Obst-A binds Serp also at the PG surface was not addressed, but it would be a potential mechanisms to precisely sense the current status of developmental progress (Pesch, 2018).

The ECM provides a scaffold for cellular support and mediates many processes including signaling during morphogenesis and tissue homeostasis. Tissue stiffness enhances matrix-directed differentiation for example through nuclear Lamin-A to enhance tissue specific differentiation. Local tissue stiffness is critically involved in instructing neuronal growth, and softening of tissue leads to aberrant axon growth. Axons in softened brains dispersed from their normal trajectory showing reduced directionality, while axons grown on stiff substrates were longer than those on soft substrates (Koser, 2016). These studies did not address the influences of Obst-A on PG matrix stiffness. But in analogy to its role in the cuticle (Petkau, 2012), where it modulates the chitin-matrix-properties, Obst-A may also influence extracellular matrix properties at PG cell surfaces in late embryos at a time when axons need to grow at the surfaces of the forming ring gland. It is speculated that the lack of Obst-A may alter ECM composition, leading to local changes in matrix properties at the PG surfaces contributing to normal axon growth at the ring gland. This is consistent to the findings that inhibition of endocytosis led to phenotypes that were similar to PG specific obst-A knockdown, including larval lethality and lack of Fas2 axon-like structures at the PG. Thus, depending on axonal growth progress at the PG surface, local ECM properties underlie dynamic changes via endocytosis of factors, such as Obst-A, that may contribute to matrix stiffness. Since a direct affect was excluded by a general Obst-A overexpression in the PG cells, this model would explain the observed changes in the regular pattern of Fas2 axon-like structures in obst-A mutants, PG specific obst-A knockdown, and PG specific endocytic mutants (Pesch, 2018).

This study provides evidence that genetic regulation of ecdysone production in PG cells is under control of a specific ECM. A chitin-binding protein, Obst-A, defines the surface of PG cells, thereby controlling larval survival, molting and growth. In addition to its role in the cuticle of epithelial organs, Obst-A supports function of PGs. By modulating the cell matrix Obst-A essentially contributes to axonal growth at the PG. Importantly, local matrix properties depend on Obst-A internalization by Wurst/Clathrin dependent endocytosis. Collectively, Obst-A provides a new link between the endocrine system, nervous system, and developmental growth control in insects and, due to evolutionary conservation of the obstructor gene family, potentially also in other arthropod species (Pesch, 2018).

The histone demethylase KDM5 is essential for larval growth in Drosophila

In Drosophila, the larval prothoracic gland integrates nutritional status with developmental signals to regulate growth and maturation through the secretion of the steroid hormone ecdysone. While the nutritional signals and cellular pathways that regulate prothoracic gland function are relatively well studied, the transcriptional regulators that orchestrate the activity of this tissue remain less characterized. This study shows that lysine demethylase 5 (KDM5) is essential for prothoracic gland function. Indeed, restoring kdm5 expression only in the prothoracic gland in an otherwise kdm5 null mutant animal is sufficient to rescue both the larval developmental delay and the pupal lethality caused by loss of KDM5. These studies show that KDM5 functions by promoting the endoreplication of prothoracic gland cells, a process that increases ploidy and is rate limiting for the expression of ecdysone biosynthetic genes. Molecularly, it was shown that KDM5 activates the expression of the receptor tyrosine kinase torso, which then promotes polyploidization and growth through activation of the MAPK signaling pathway. Taken together, these studies provide key insights into the biological processes regulated by KDM5 and expand understanding of the transcriptional regulators that coordinate animal development (Drelon, 2019).

This study demonstrates that KDM5 is essential for the function of the ecdysone-producing prothoracic gland during Drosophila larval development. Crucial to this conclusion was the finding that expressing kdm5 in the prothoracic gland was sufficient to rescue the lethality and developmental delay phenotypes of kdm5140> null allele homozygous mutant animals. Consistent with this observation, prothoracic gland function was defective in kdm5 mutants, with mutant larvae having low levels of ecdysone and reduced expression of downstream hormone-responsive target genes. Demonstrating the importance of KDM5-mediated regulation of ecdysone production, dietary supplementation of 20E restored normal developmental timing to kdm5 mutant larvae. At the cellular level, loss of KDM5 slowed the prothoracic gland endoreplicative cycles that increase the ploidy of these cells and are important for ecdysone biosynthesis. Restoring these endocycles by expressing cyclin E re-established normal developmental timing to kdm5140 mutants but was not able to rescue their lethality. In contrast, ectopic activation of the Torso/MAPK pathway that functions upstream of cyclin E was able to restore both developmental timing and rescue the lethality caused by loss of KDM5. It is therefore proposed that KDM5-mediated activation of the Torso/MAPK pathway in the prothoracic gland is important for larval growth regulation through its role in promoting polyploidization, and for adult eclosion by mechanisms that remain to be determined (Drelon, 2019).

Consistent with an important developmental role for KDM5-mediated regulation of Torso/MAPK, mutations in torso or ablation of the neurons that produce its ligand PTTH cause a 5-day delay to larval development similar to that observed for kdm5140. Because loss of KDM5 reduces, but does not eliminate, torso expression, the kdm5140 phenotype cannot be accounted for solely by the twofold change to Torso/MAPK signaling observed. The dysregulation of additional KDM5-regulated genes is therefore likely to contribute to the prothoracic gland phenotypes of kdm5 mutant larvae. Little is known about the transcriptional regulation of torso and other genes that make up the upstream pathways that regulate ecdysone production. One possibility is that KDM5 functions as a direct transcriptional activator of the torso gene: thus, decreased expression of this receptor would be expected in kdm5 mutant animals. Because of the small size of the prothoracic gland, it is not currently feasible to carry out ChIP experiments to examine KDM5 promoter binding in this tissue. It is, however, notable that the torso promoter was not bound by KDM5 in existing ChIP-seq datasets from larval wing imaginal discs or from whole adult flies. This could be because torso expression is largely restricted to the prothoracic gland during larval development and so may not be expected to have promoter-bound KDM5 in the tissues examined to date. Alternatively, KDM5 might regulate torso indirectly. In the silkworm Bombyx mori, expression of the torso gene is repressed in response to starvation conditions. Although the mechanism by which this occurs is unknown, it does indicate that other cellular defects caused by loss of KDM5 could lead to changes to torso transcription and subsequent decrease in MAPK activity and ecdysone production. Whether the regulation of torso by KDM5 is direct or indirect, it occurs in a demethylase-independent manner, as larvae lacking enzymatic activity show a normal developmental profile (Drelon, 2018). Consistent with this observation, components of KDM5 complexes that regulate gene expression through demethylase-independent mechanisms also affect developmental timing. For example, a development delay similar to that of kdm5140 is caused by RNAi-mediated knockdown of the histone deacetylase HDAC1 or the NuRD complex components asf1 and Mi-2. KDM5 could therefore interact with these proteins to regulate the expression of genes that are crucial to the regulation of larval development (Drelon, 2019).

Increased ploidy of prothoracic gland cells is important for optimal expression of steroidogenic genes and can be induced by activation of the Torso/MAPK pathway. Because the genes required for ecdysone biosynthesis are among the most abundantly expressed in the prothoracic gland, their mRNA levels may be entirely limited by gene copy number. A similar requirement for copy number amplification to produce peak gene expression levels has been observed in other cell types in Drosophila, including chorion gene expression in ovarian follicle cells. However, it is not known how Torso/MAPK activation promotes prothoracic gland cell cycle progression. One mechanism might be by affecting levels of cell cycle regulators such as the transcription factor E2f1, which is essential for both mitotic and endoreplicative cell cycles. This model is based on studies of the polyploid enterocytes of the adult midgut, in which activation of the MAPK pathway via the EGF receptor stabilizes E2f1 protein, leading to transcription activation of cyclin E. Although restoring correct endocycling in kdm5 mutant prothoracic glands was able to rescue their developmental timing, it did not impact their eclosion defect. This could be because loss of KDM5 leads to additional defects within the prothoracic gland that are ultimately detrimental to the function of this endocrine tissue, such as increased oxidative stress, which this study has shown to be affected in kdm5 hypomorphic mutant wing discs. Alternatively, KDM5 could have a cell cycle independent role in maintaining ecdysone levels during pupal development. kdm5140 mutant animals die as pharate adults that have no obvious morphological abnormalities but fail to eclose (Drelon, 2018). Nevertheless, these animals could have significant defects in, for example, nervous system development, which requires ecdysone and is important for eclosion (Drelon, 2019).

The observed role of KDM5 in the growth and polyploidization of larval prothoracic gland raises the possibility that it might play key roles in other cell types that use endoreplicative cycles. This could have broad consequences for understanding of KDM5 biology, as polyploidization is observed in many plant and animal cell types and is widely used during Drosophila larval development. In addition, while the role of polyploid cells in the etiology or maintenance of cancers remains a topic of ongoing research, KDM5-regulated endocycling could contribute to its tumorigenic activities in humans. Regulation of polyploidization in cells of the nervous system could also contribute to the link between KDM5 protein dysregulation and intellectual disability. This could, for example, be mediated by KDM5 function in glial cells, as polyploidization of superineurial glial cells in Drosophila is required for normal brain development. Although it is not clear the extent to which a similar phenomenon occurs during human brain development, it is interesting to note that glial cell types contribute to the severity of intellectual disability disorders such as Rett syndrome. Thus, although there is still much to be learned regarding the contribution of polyploid cells to normal development and to clinically relevant disorders, KDM5-regulated transcriptional programs could be key to the function of cells that use this variant cell cycle (Drelon, 2019).

The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues

Exocytosis is a fundamental process in physiology, communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. This study reports a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, Zda was found to be required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen, the small GTPase RalA was identified as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane (de la Riva Carrasco, 2020).

Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells

Antagonistic activities of glucagon and insulin control metabolism in mammals, and disruption of this balance underlies diabetes pathogenesis. Insulin-producing cells (IPCs) in the brain of insects such as Drosophila also regulate serum glucose, but it remains unclear whether insulin is the sole hormonal regulator of glucose homeostasis and whether mechanisms of glucose-sensing and response in IPCs resemble those in pancreatic islets. This study shows, by targeted cell ablation, that Drosophila corpora cardiaca (CC) cells of the ring gland are also essential for larval glucose homeostasis. Unlike IPCs, CC cells express Drosophila cognates of sulphonylurea receptor (Sur) and potassium channel (Ir), proteins that comprise ATP-sensitive potassium channels regulating hormone secretion by islets and other mammalian glucose-sensing cells. They also produce adipokinetic hormone, a polypeptide with glucagon-like functions. Glucose regulation by CC cells is impaired by exposure to sulphonylureas, drugs that target the Sur subunit. Furthermore, ubiquitous expression of an akh transgene reverses the effect of CC ablation on serum glucose. Thus, Drosophila CC cells are crucial regulators of glucose homeostasis and they use glucose-sensing and response mechanisms similar to islet cells (Kim, 2004).

Insect corpora cardiaca (CC) are clusters of endocrine cells in the ring gland adjacent to the prothoracic gland and corpus allatum. A principal CC product is adipokinetic hormone (AKH), a polypeptide that mobilizes stored macromolecular energy reserves to sustain energy-consuming activities, such as crawling and flight. AKH is similar to mammalian glucagon; like glucagon in pancreatic islet α-cells, AKH is synthesized as a pre-prohormone, processed, and stored in dense core vesicles. Like mammalian glucagon activity in liver, AKH has been shown to bind a G-protein-coupled transmembrane receptor and to increase lipolysis, glycogenolysis and production of trehalose in the insect fat body, a storage organ for lipid and glycogen (Kim, 2004).

Previous studies of AKH microinjection and ring gland transplantation in locusts and other insects suggest that AKH is sufficient to increase haemolymph glucose concentrations, but have not yet shown a requirement for AKH in glucose homeostasis. To examine phenotypes resulting from CC cell ablation and AKH deficiency, 1,000-base-pair DNA segment derived from sequences immediately 5' of the Drosophila akh gene was used to drive the expression of the transcriptional trans-activator GAL4 in CC cells. The akh-GAL4 construct, when crossed with a UAS-mCD8GFP (membrane-tethered green fluorescent protein, mGFP) reporter line, directed a GFP expression pattern that reflected endogenous akh expression in the ring gland corpora cardiaca of third-instar larvae. Using in situ hybridizations, it was establised that embryonic akh messenger RNA expression initiates in cells of the presumptive CC anlage and that in later larval stages it is maintained only in CC cells. To assess the role of the CC as an endocrine regulator of haemolymph glucose concentrations, akh-GAL4 lines were used to express the cell death factor Reaper in akh-expressing CC cells. This resulted in the ablation of only CC cells at high efficiency: in more than 96% of newly hatched first-instar larvae harbouring akh-GAL4, UAS-Reaper and UAS-mCD8GFP, no mGFP-labelled CC cells were detected. In contrast, mGFP was detected in CC cells within all control larvae at the same stage, and at later stages. In Drosophila , haemolymph glucose is composed of trehalose (a disaccharide of glucose) and monomeric free glucose, and the combined circulating concentration of these (hereafter referred to as total haemolymph glucose) is maintained in a narrow range for a given feeding condition. Ablation of akh-expressing CC cells in larvae raised on dextrose-supplemented medium decreased the mean total haemolymph glucose and trehalose by 50%, an effect similar to that recently reported by others. CC cell deficiency did not result in discernible growth reduction, developmental delay or lethality, phenotypes that arise after the ablation of IPCs in the brain. Thus, like glucagon, AKH is an essential regulator of energy metabolism but might be dispensable for developmental growth control (Kim, 2004).

To test whether AKH activity alone could account for the glucose-regulating action of CC cells, the ability was tested of an akh transgene with ubiquitous expression from a heat shock promoter to reverse the effect of CC ablation. Lower haemolymph glucose concentrations resulting from CC ablation were partly restored by the ubiquitous expression of an akh transgene. Thus, bioactive AKH from the akh transgene might be produced in target tissues, as has been shown for transgene-encoded neuropeptides such as Drosophila insulin. These data indicate that AKH is an essential regulator of haemolymph carbohydrate concentrations in Drosophila . It is suggested that the hyperglycaemic effects of AKH counter-regulate the activity of other systemic hormones such as insulin and that these antagonistic activities might refine the levels of circulating energy to match systemic energy requirements. If so, it is postulated that the negative energy balance accompanying starvation might worsen the hypoglycaemic effects of AKH deficiency. In comparison with starved control larvae, total haemolymph glucose was decreased by 75% in starving larvae after CC cell ablation. Thus, starvation increased the severity of hypoglycaemia in animals lacking CC cells, indicating that AKH might be required for the compensatory mechanisms that maintain circulating glucose during periods of food deprivation in Drosophila larvae (Kim, 2004).

Labelling of CC cell processes with mGFP and an antibody against AKH revealed that AKH-producing cells extend processes that terminate on the heart and on the prothoracic gland compartment of the ring gland. On the surface of the heart, CC cell processes have extensive contact with axons that project from insulin-producing cells from the brain. Labelling of CC cell processes with mGFP and an antibody against AKH revealed localization of AKH within the processes that contact the IPCs, and AKH peptide on the processes contacting the heart. These results indicate that the heart surface is the principal site of AKH release into the circulating haemolymph. Thus, like glucagon-producing cells in mammalian islets and brain, AKH-producing CC cells in the Drosophila ring gland have direct systemic vascular access, consistent with their role as endocrine regulators of metabolism (Kim, 2004).

ATP-sensitive potassium (KATP) channels regulate neuroendocrine cell function in organs such as the mammalian pancreas and brain, and this study examined whether KATP functions regulate CC cell activity. KATP channels are heteromeric protein complexes composed of sulphonylurea receptor (Sur) and inward-rectifying potassium channel (Ir; also called Kir) subunits. An ATP-binding domain in the Ir subunit regulates KATP channel activity, allowing these channels to serve as cellular energy sensors, opening or closing in response to the intracellular ADP/ATP ratio, thus influencing membrane potential and subsequent calcium currents that regulate hormone secretion. Using mRNA in situ hybridization, it was showm that larval CC cells expressed Sur (Nasonkin, 1999) and Ir (Döring, 2002), which have sequence similarity to mammalian Sur1 and Kir6 proteins, respectively. Expression of Sur or Ir was not detected in the larval brain IPCs, another group of cells known to regulate haemolymph glucose. Drosophila Sur has been shown to be sufficient to allow K+ currents that polarize membrane potentials (Nasonkin, 1999). Drosophila Ir was demonstrated to evoke an inwardly rectifying K+ current (Kim, 2004).

Tests were performed to see whether increased haemolymph glucose concentrations might result from excess AKH secretion brought about by sulphonylurea inhibition of the Sur and K+-dependent depolarization of CC cells. Glyburide and tolbutamide are representative members of the two major classes of sulphonylureas. These drugs promote the closure of KATP channels and cellular depolarization, thereby regulating secretion in mammalian neuroendocrine cells. For example, sulphonylureas stimulate glucagon secretion in diabetic patients. Glyburide has previously been shown to inhibit Drosophila Sur-mediated outward K+ currents, resulting in the depolarization of cell potential. Exposure of feeding third-instar larvae to glyburide mixed in yeast paste (standard dextrose medium did not permit drug delivery) produced a 10% increase in mean total haemolymph glucose concentration compared with controls. Exposure of larvae to tolbutamide had a greater effect, producing a 40% increase in mean total haemolymph glucose, and tolbutamide was used in subsequent studies. Average haemolymph glucose concentrations were generally decreased in animals fed with yeast paste compared with animals fed with standard dextrose medium, and this might have accentuated the hyperglycaemic effect of sulphonylureas administered in yeast paste. Moreover, the hypoglycaemic effect induced by CC cell ablation (or hyperpolarization) seemed attenuated in yeast-fed animals, further supporting the hypothesis that requirements for AKH might be altered by manipulating feeding conditions (Kim, 2004).

To test the hypothesis that Sur and Ir function in the CC to regulate haemolymph glucose concentrations in Drosophila , CC cells were ablated in larvae fed with tolbutamide. Ablation of the CC cells using Akh-GAL4 and UAS-Reaper blocked the hyperglycaemic effect of tolbutamide, indicating that CC cells must be present to support the hyperglycaemic action of tolbutamide. To determine whether the hyperglycaemic effect of tolbutamide resulted from Sur and Ir-mediated depolarization of CC cells, membrane potential was hyperpolarized in CC cells, in the presence and absence of tolbutamide. Kir2.1 is a human K+ channel that evokes an outward K+ current, independently of ATP regulation, and has previously been used to impair cellular depolarization in vivo in Drosophila by inducing persistent outward K+ current and a hyperpolarized resting potential. One indication that AKH release by CC cells requires membrane depolarization and might be regulated by K+-channel-dependent membrane potential comes from the observation that, on standard dextrose medium, third-instar larvae expressing Kir2.1 in CC cells had a 23% decrease in mean haemolymph glucose concentration, compared with controls. Expression of the Kir2.1 channel in CC cells prevented the hyperglycaemic effect of tolbutamide, indicating that K+-channel-dependent CC cell depolarization resulted from exposure to sulphonylurea. Together, these pharmacological and genetic data support the view that KATP channel activity in CC cells governs AKH release, thereby controlling concentrations of circulating glucose in Drosophila (Kim, 2004).

In pancreatic α-cells, hypoglycaemia stimulates increased intracellular calcium concentrations promoting glucagon secretion, whereas hyperglycaemia inhibits these responses. To test whether Drosophila CC cells sense glucose changes and, like pancreatic α-cells, modulate intracellular calcium concentrations, CC cells were mared with fluorescent transgene-encoded calcium sensors ('camgaroos'). The fluorescence intensity of camgaroos increases in response to elevated intracellular calcium concentration, an effect used previously to measure cytoplasmic calcium transients in depolarized Drosophila neurons. Elevation of cytoplasmic calcium concentration after CC cell depolarization stimulates AKH secretion; thus, in these experiments elevated intracellular calcium concentration in CC cells was used as an indicator of AKH secretion. Fluorescence of camgaroo-2 (cg-2) in cultured CC cells increased as extracellular trehalose or glucose concentration decreased. Direct CC cell depolarization with increased extracellular potassium concentration similarly led to increased cg-2 fluorescence. In contrast, fluorescence in cg-2-labelled CC cells decreased as extracellular trehalose concentration increased. These results corroborate previous studies of locust CC cells showing that decreases in extracellular trehalose or glucose concentration stimulated AKH secretion. Drosophila CC cells express the enzyme trehalase, raising the possibility that the sensing of trehalose by CC cells involves the hydrolysis of trehalose to glucose, a view also supported by similar effects of trehalose and glucose in in vitro studies. Thus, hypoglycaemic sensing in CC cells leads to increased concentrations of the intracellular second messenger calcium, a signal for subsequent regulated exocytosis of AKH -- a mechanism similar to those regulating glucagon secretion by mammalian pancreatic α-cells (Kim, 2004).

Thus, there are remarkable parallels in endocrine cell functions that ensure the supply of circulating glucose in Diptera and in mammals. On the basis of these parallels, it is speculated that insect CC cells and mammalian neuroendocrine cells that regulate metabolism might have arisen from an ancestral energy-sensing cell. If so, it is further speculated that pancreatic islet cells, including β-cells, might have evolved from an ancient α-cell. Similarly to pancreatic islets, insect CC cells might delaminate from embryonic epithelial cells that give rise to both gut and neuroendocrine structures. Thus, common mechanisms might regulate the development of CC and pancreatic islet cells. Understanding CC cell development could therefore accelerate the discovery of cell-replacement therapies for type 1 diabetes mellitus. This Drosophila model might also serve to elucidate the mechanisms that control stimulus-secretion coupling in CC cells, and hence the biology of hypoglycaemia. Moreover, the sensitivity of CC cells to drugs commonly prescribed for disorders such as type 2 diabetes indicates that Drosophila might provide a model system for the discovery of pharmacological agents to treat human endocrine diseases (Kim, 2004).

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

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

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 viableweight. 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).

Snail synchronizes endocycling in a TOR-dependent manner to coordinate entry and escape from endoreplication pausing during the Drosophila critical weight checkpoint

In holometabolous insects, the growth period is terminated through a cascade of peptide and steroid hormones that end larval feeding behavior and trigger metamorphosis, a nonfeeding stage during which the larval body plan is remodeled to produce an adult. This irreversible decision, termed the critical weight (CW) checkpoint, ensures that larvae have acquired sufficient nutrients to complete and survive development to adulthood. How insects assess body size via the CW checkpoint is still poorly understood on the molecular level. This study shows that the Drosophila transcription factor Snail plays a key role in this process. Before and during the CW checkpoint, snail is highly expressed in the larval prothoracic gland (PG), an endocrine tissue undergoing endoreplication and primarily dedicated to the production of the steroid hormone ecdysone. Two Snail peaks were observed in the PG, one before and one after the molt from the second to the third instar. Remarkably, these Snail peaks coincide with two peaks of PG cells entering S phase and a slowing of DNA synthesis between the peaks. Interestingly, the second Snail peak occurs at the exit of the CW checkpoint. Snail levels then decline continuously, and endoreplication becomes nonsynchronized in the PG after the CW checkpoint. This suggests that the synchronization of PG cells into S phase via Snail represents the mechanistic link used to terminate the CW checkpoint. Indeed, PG-specific loss of snail function prior to the CW checkpoint causes larval arrest due to a cessation of endoreplication in PG cells, whereas impairing snail after the CW checkpoint no longer affected endoreplication and further development. During the CW window, starvation or loss of TOR signaling disrupted the formation of Snail peaks and endocycle synchronization, whereas later starvation had no effect on snail expression. Taken together, these data demonstrate that insects use the TOR pathway to assess nutrient status during larval development to regulate Snail in ecdysone-producing cells as an effector protein to coordinate endoreplication and CW attainment (Zeng, 2020).

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

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

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

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

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

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

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

Because a similar defect in the larval-prepupal transition occurs in ecdysone-deficient larvae, it was hypothesized that the Octβ3R knockdown phenotype was due to lack of ecdysone production. Consistent with this idea, the 20E titer was much lower in phm>Octβ3RRNAi-1 larvae than in control larvae just before the larval-prepupal transition (90 hAH). Moreover, administration of 20E by feeding rescued the defect in the larval- prepupal transition caused by Octβ3R knockdown. When phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae were cultured on media containing 20E (1 mg/mL) from 48 hAH onward, approximately half of them developed to the prepupal stage, compared with only 2-3% of larvae not fed 20E. Thus, PG-specific loss of Octβ3R activity causes an arrest in the larval-prepupal transition due to lack of ecdysone (Ohhara, 2015).

Ecdysone is synthesized in the PG from dietary cholesterol through the action of seven ecdysone biosynthetic genes (neverland, spookier, shroud, Cyp6t3, phantom, disembodied, and shadow). Quantitative RT- PCR (qPCR) was performed to investigate whether loss of Octβ3R function affects expression of these genes in the PG. In control larvae, expression of these genes increased dramatically between 72 and 96 hAH, when the larval-prepupal transition occurs. By contrast, in phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae, the expression of all of these genes was significantly reduced relative to control larvae at 96 hAH. The reduced expression of ecdysone biosynthetic genes in the PG was confirmed by in situ hybridization. Furthermore, immunostaining revealed that Neverland, Shroud, Phantom, Disembodied, and Shadow protein levels were reduced in the PG of phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae. Taken together, these data show that Octβ3R function is required in the PG for proper expression of ecdysone biosynthetic genes (Ohhara, 2015).

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

To test this, PG-specific knockdowns of Tdc1, Tdc2, and Tbh were generated. To knock down Tdc2, two constructs targeting distinct regions of the Tdc2 transcript (Tdc2RNAi-1 and Tdc2RNAi-2) were expressed along with dicer2 in the PG under the control of the phm-22-Gal4 driver (phm > Tdc2RNAi-1+dicer2 and phm > Tdc2RNAi-2+dicer2). All phm > Tdc2RNAi-1+dicer2 larvae arrested at the larval stage, and phm > Tdc2RNAi-2+dicer2 larvae were significantly delayed at the larval-prepupal transition, relative to control animals. Tdc2 mRNA level was reduced in the ring gland (RG) containing the PG in both sets of knockdown animals, as demonstrated by qPCR. Moreover, octopamine and tyramine production in the PG was impaired by Tdc2 knockdown. By contrast, Tdc1 knockdown (phm > Tdc1RNAi+dicer2) caused only a subtle delay in the larval-prepupal transition and had no detectable effect on octopamine or tyramine production. These results suggest that Tdc2 is the predominant Tdc regulating octopamine and tyramine biosynthesis in the PG and the larval-prepupal transition. Contrary to these findings, a null mutation in Tdc2 does not affect metamorphosis, and these mutant flies are viable. Thus, PG-specific knockdown causes a stronger phenotype than complete loss of Tdc2 activity in whole animals. A similar situation has been reported in regulation of metamorphosis by Activin signaling. These phenomena can be explained by a model in which some compensatory changes in other mutant tissues rescue the PG-specific knockdown phenotype in null-mutant animals (Ohhara, 2015).

PG-specific Tdc2 knockdown caused a reduction in larval 20E concentration. Therefore, whether feeding 20E to Tdc2 knockdown larvae would rescue the larval- prepupal transition defect was examined. To this end, phm > Tdc2RNAi-1+ dicer2 and phm > Tdc2RNAi-2+dicer2 larvae were cultured in media with or without 20E (1 mg/mL) from 48 hAH onward. Approximately 40% of the 20E-fed phm > Tdc2RNAi-1+dicer2 larvae developed to the prepupal stage, whereas none of those larvae grown on control media progressed beyond the larval stage. Furthermore, the delay in the larval-prepupal transition in phm > Tdc2RNAi-2+dicer2 larvae was rescued by 20E feeding. These results indicate that the defect in the larval-prepupal transition in Tdc2 knockdown animals results from a lack of 20E production. Thus, octopamine/ tyramine synthesized in the PG appears to activate Octβ3R in an autocrine manner to execute the larval-prepupal transition by regulating ecdysone production (Ohhara, 2015).

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

Because ecdysone biosynthesis in the PG is under the control of Ilps and PTTH signaling, it was next examined whether Octβ3R function is required to activate these signaling pathways. To detect Ilps signaling activity, a pleckstrin-homology domain fused to GFP (PH-GFP), which is recruited to the plasma membrane when insulin signaling is activated, was used. In the PG cells of control larvae, PH-GFP was only weakly localized to the plasma membrane at 48 hAH, whereas its membrane localization became increasingly evident at 60, 84, and 90 hAH. By contrast, in PG cells of phm>Octβ3RRNAi-1 larvae, the tight localization of PH-GFP to the plasma membrane was no longer detectable, indicating that activation of Ilps signaling had been disrupted. Moreover, overexpression of a constitutively active form of the Ilps receptor InR (InRCA) was able to rescue the larval arrest in phm>Octβ3RRNAi-1 animals. Next, immunostaining was performed of the diphosphorylated form of ERK (dpERK), a downstream signaling component of the PTTH pathway. dpERK expression was found to be very weak at 48 hAH, but was activated in the PG of control larvae at 60, 84, and 90 hAH; by contrast, this activation was reduced in the PG of phm>Octβ3RRNAi-1 larvae. Expression of a constitutively active form of another downstream PTTH signaling component, Ras (RasV12), rescued the larval-prepupal transition defect in phm>Octβ3RRNAi-1 animals. These results show that Octβ3R function is required to activate Ilps and PTTH signaling in the PG and that these signaling pathways execute the larval-prepupal transition. Although activation of both the Ilps and PTTH signaling pathways requires Activin/TGFβ signaling in the PG, expression of a constitutively active form of the Activin/ TGFβ receptor Baboon (BaboCA) failed to rescue the larval-prepupal transition defect in phm>Octβ3RRNAi-1 animals. This observation suggests that Octβ3R acts downstream or independent of Activin/TGFβ signaling to regulate Ilps and PTTH signaling in the PG (Ohhara, 2015).

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

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

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

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

The TOR pathway couples nutrition and developmental timing in Drosophila

In many metazoans, final adult size depends on the growth rate and the duration of the growth period, two parameters influenced by nutritional cues. In Drosophila, nutrition modifies the timing of development by acting on the prothoracic gland (PG), which secretes the molting hormone ecdysone. When activity of the Target of Rapamycin (TOR), a core component of the nutrient-responsive pathway, is reduced in the PG, the ecdysone peak that marks the end of larval development is abrogated. This extends the duration of growth and increases animal size. Conversely, the developmental delay caused by nutritional restriction is reversed by activating TOR solely in PG cells. Finally, nutrition acts on the PG during a restricted time window near the end of larval development that coincides with the commitment to pupariation. In conclusion, this study shows that the PG uses TOR signaling to couple nutritional input with ecdysone production and developmental timing. Previously studies have shown that the same molecular pathway operates in the fat body (a functional equivalent of vertebrate liver and white fat) to control growth rate, another key parameter in the determination of adult size. Therefore, the TOR pathway takes a central position in transducing the nutritional input into physiological regulations that determine final animal size (Layalle, 2008).

Previous experiments showed that insulin/IGF signaling controls basal levels of ecdysone synthesis in the PG. This, in turn, controls the larval growth rate without modifying the duration of larval growth. These data contrast with the present observations on the role of TOR signaling in the PG and indicate that PG cells discriminate between hormone-mediated activation of InR/PI3K signaling and the nutrient-mediated activation of TOR signaling for the control of ecdysone biosynthesis. Can TOR and InR/PI3K signaling pathways function separately in Drosophila tissues? It has been established both in cultured cells and in vivo that a gain of function for InR/PI3K allows for TORC1 activation through inhibition of TSC2 via direct phosphorylation by AKT/PKB. Such crosstalk between the InR and TOR signaling pathways has important functional implications in cancer cells in which inactivation of the PTEN tumor suppressor leads to an important increase in AKT activity. Nevertheless, the physiological significance of the crosstalk between AKT and TSC2 has been challenged by genetic experiments in Drosophila, leading to the notion that, in the context of specific tissues, TOR and insulin/IGF signaling can be part of distinct physiological regulations for the control of animal growth in vivo. Although not observe in standard conditions, strong InR/PI3K activation in the ring gland shortens larval developmental timing under conditions of food limitation. In light of the present data, this suggests that, in low-food conditions, providing high PI3K activity in PG cells allows for full activation of TOR through the AKT/PKB-mediated inhibitory phosphorylation of TSC2, thus modulating developmental timing. Inversely, a severe downregulation of InR/PI3K signaling in the PG extends larval timing by preventing early larval molts. However, it was observed that strong inhibition of the InR pathway compromises the growth of PG cells, therefore interfering with their capacity to produce normal levels of ecdysone for molting. Overall, previous works as well as the present work highlight the importance of studying signaling networks in the specific contexts (tissue, development) in which these pathways normally operate. This also illustrates that only mild manipulations of these intricate pathways are suitable to unravel the regulatory mechanisms that normally occur within the physiological range of their activities. In conclusion, it is proposed that the insulin/IGF system and TOR provide two separate inputs on PG-dependent ecdysone production: the insulin/IGF system controls baseline ecdysone levels during larval life, and TOR acts upon ecdysone peaks in response to PTTH at the end of larval development (Layalle, 2008).

Important literature describes intrinsic mechanisms controlling a growth threshold for pupariation in insects. After a critical size is attained, the hormonal cascade leading to ecdysone production initiates, and larvae are committed to pupal development, even when subjected to complete starvation. Recent findings in Drosophila by using temperature-sensitive mutants for dInR have revealed that reducing the larval growth rate before the critical size is attained postpones the attainment of this threshold, but has no effect on the final size. Conversely, reducing animals' growth rate after the critical size has been attained leads to strong reduction of the final size. This highlights an important period in the determination of final size, called the terminal growth period (TGP, also called interval to cessation of growth), which spans from the attainment of critical size to the cessation of growth. Due to its exponential rate, growth during that period makes an important contribution to the determination of final size. Interestingly, the duration of the TGP is not affected by the general insulin/IGF system, which explains why reduction of the insulin/IGF system during that period leads to short adults. The present data suggest that the duration of the TGP is an important parameter in the determination of final size that is controlled by TOR. By reducing the level of TOR activity specifically in the PG, neither the growth rate or the critical size for commitment to pupariation is affected. Therefore, the time to attainment of the critical size is not changed. The observation of the developmental transitions in P0206 > TSC1/2 larvae (ectopically expressing TSC1/2) indicate that, indeed, the timing of L1/L2 and L2/L3 molts are not modified. By contrast, the L3/pupa transition is severely delayed, indicating that the interval between attainment of critical size and the termination of growth, i.e., the TGP, is increased. Interestingly, activation of TOR in the PG of fasting larvae leads to a sensible (50%) reduction of the developmental delay induced by low nutrients, whereas it has no effect in normally fed animals. This indicates that the regulation of the TGP by TOR plays an important role in the adaptation mechanisms controlling the duration of larval development under conditions of reduced dietary intake. Other mechanisms, such as the delay to attainment of the critical size due to a reduced growth rate, also contribute to timing of larval development, giving a plausible explanation for the fact that PG-specific TOR activation only partially rescues the increase in larval development timing observed under low-nutrient conditions. Despite characterization in different insect systems, the mechanisms determining the critical size remain to be elucidated. The present study shows that inhibition of TOR signaling in the PG does not modify the minimum size for pupariation. This result is in line with previous findings indicating that nutritional conditions do not modify the critical size in Drosophila. Interestingly, animals depleted of PTTH present an important shift in critical size, indicating that PTTH might participate in setting this parameter. Therefore, mechanisms determining the critical size might reside in the generation or the reception of the PTTH signal, upstream of TOR function in the cascade of events leading to ecdysone production (Layalle, 2008).

What is the limiting step that is controlled by the TOR sensor during the process of ecdysone production? Results obtained by genetic analysis in vivo are reminiscent of in vitro work on dissected PG in the M. sexta model. In these previous studies, PTTH-induced ecdysone production in the PG was shown to induce the phosphorylation of ribosomal protein S6 and was inhibited by the drug rapamycin, later identified as the specific inhibitor of TOR kinase. Interestingly, rapamycin treatment blocked PTTH-induced, but not db-cAMP-induced, ecdysone production, indicating that the drug does not act by simply inhibiting general protein translation in PG cells, but, rather, by inhibiting a specific step controlling PTTH-dependent ecdysone production. More recently, many studies mostly carried out on large insects have started unraveling the response to PTTH in the PG, leading to ecdysone synthesis. No bona fide PTTH receptor is identified yet, and the previously identified response to PTTH is a rise in cAMP, leading to a cascade of activation of kinases, including PKA, MAPKs, PKC, and S6-kinase. S6-kinase-dependent S6 phosphorylation is currently being considered as a possible bottle-neck in the activation of ecdysone biosynthesis by PTTH. The present genetic analysis of ecdysone production in the Drosophila PG now introduces the TOR pathway, the main activator of S6-kinase, as a key controller of ecdysone production and therefore provides a plausible explanation for the rise of S6-kinase in PG cells following PTTH induction. The phenotypes obtained after TOR inhibition in the PG are remarkably similar to the phenotype obtained after ablation of the PTTH neurons. Moreover, ths study shows here that PTTH expression is not altered upon starvation, and that TOR inhibition in PTTH cells has no effect on the duration of larval development, suggesting that PTTH production is not modified by a nutritional stress. Taken together, these data suggest a model whereby limited nutrients induce a downregulation of TOR signaling in the PG, abolish the capacity of PG cells to respond to PTTH and produce ecdysone, and lead to an extension of the terminal growth period (Layalle, 2008).

In conclusion, this study illustrates how the TOR pathway can be used in a specific endocrine organ to control a limiting step in the biosynthesis of a hormone in order to couple important physiological regulations with environmental factors such as nutrition (Layalle, 2008).

Nitric oxide synthase regulates growth coordination during Drosophila melanogaster imaginal disc regeneration

Mechanisms that coordinate growth during development are essential for producing animals with proper organ proportion. This study describes a pathway through which tissues communicate to coordinate growth. During Drosophila melanogaster larval development, damage to imaginal discs activates a regeneration checkpoint through expression of Dilp8. This produces both a delay in developmental timing and slows the growth of undamaged tissues, coordinating regeneration of the damaged tissue with developmental progression and overall growth. It was demonstrated that Dilp8-dependent growth coordination between regenerating and undamaged tissues, but not developmental delay, requires the activity of nitric oxide synthase (NOS) in the prothoracic gland. NOS limits the growth of undamaged tissues by reducing ecdysone biosynthesis, a requirement for imaginal disc growth during both the regenerative checkpoint and normal development. Therefore, NOS activity in the prothoracic gland coordinates tissue growth through regulation of endocrine signals (Jaszczak, 2015).

During Drosophila development, damage to larval imaginal discs elicits a regeneration checkpoint that has two effects: 1) it delays the exit from the larval phase in development to extend the regenerative period , and 2) it coordinates regenerative growth with the growth of undamaged tissues by slowing the growth rate of distal, undamaged tissues. How regenerarating tissues communicate with undamaged tissues to coordinate growth has been an open question. Damaged tissues may produce signals that directly influence the growth of undamaged tissues or may indirectly influence the growth of undamaged tissues by producing signals that alter the levels of limiting growth factors. Consistent with the latter model, this paper describes an indirect communication pathway for growth coordination during the regeneration checkpoint (Jaszczak, 2015).

An essential component of this growth coordination is the secreted peptide Dilp8, which is released by damaged tissues and is both necessary and sufficient to regulate the growth of distal tissues during the regeneration checkpoint. Dilp8 shares structural similarity to insulin - like peptides, which function to stimulate growth by activating the insulin receptor. However, in contrast to insulin - like peptides , Dilp8 acts to limit growth. A simple model explaining Dilp8 function would be that Dilp8 acts directly as an antagonist to insulin receptor activity, thus reducing growth in undamaged tissues. However, the growth response to checkpoint activation of polyploid larval tissues was shown to differ from imaginal discs. The growth of polyploid larval tissues are very sensitive to changes in insulin signaling, therefore these results are inconsistent with Dilp8 regulating imaginal disc growth by antagonizing systemic insulin signaling (Jaszczak, 2015).

NOS functions in the PG to regulate the growth of imaginal discs during the developmental checkpoint. Growth coordination during the regeneration checkpoint increases NO production in the PG, and is dependent on NOS gene function in the PG. Although constitutive expression of NOS in the PG might produce effects earlier in development that might alter the current interpretations, this study also demonstrated that transient pulses of NOS during the third instar and targeted NOS activation in the PG both produce the same effects: inhibition of imaginal disc growth by limiting ecdysone signaling. NOS activity in the PG reduces ecdysone production through the transcriptional inhibition of the P450 enzymes disembodied and spookier, which are necessary for ecdysone biosynthesis. Although it has been known that NOS activity is capable of regulating growth of imaginal discs (Kuzin, 1996), the experiments described in this study elucidate the mechanism of this growth regulation (Jaszczak, 2015).

The activity of NOS described in this study contrasts with published experiments demonstrating that NO signaling inhibits E75 activity in the PG, thus promoting larval exit (Caceres, 2011) . However, experiments from Caceres demonstrate that earlier NOS expression in the PG during larval development produces small larvae that arrest at second larval instar stage of development. This arrest can be partially rescued by either ecdysone feeding, or by reducing the level of GAL4 - UAS driven NOS expression by raising larvae at a lower temperature. Additionally, previous studies indicated that pharmacological increase of NO levels in larvae can produce larval developmental delays. Together, these observations suggest that NOS activity earlier in larval development might inhibit rather than promote ecdysone signaling during the larval growth period. Finally, this study observed that E75B is not expressed in larvae that have activated the regenerative checkpoint, suggesting that th e NOS dependent pathway that has been described by Caceres is not active during the regeneration checkpoint (Jaszczak, 2015).

This study has focused on the role of NOS during the growth phase of the third larval instar (76-104h AED) and have found that heat-shock mediated pulses of NOS activity during this period of development inhibit growth and ecdysone signaling, while pulses of NOS activity at the end of larval development do not inhibit growth or ecdysone signaling. Based on these results, it is concluded that there are distinct roles for NOS in the PG during different phases in development; NOS activity post-larval feeding promotes ecdysone synthesis through inhibition of E75, whereas NOS activity during the larval growth phase limits ecdysone synthesis and signaling by reducing the expression of ecdysone biosynthesis genes through a yet-to-be defined mechanism. Some intriguing possible mechanisms are through regulation of the growth of the PG, or via activation of cGMP-dependent pathways (Jaszczak, 2015).

Furthermore, this study demonstrated that ecdysone is essential for imaginal disc growth. Most studies have supported a model in which ecdysone acts as negative regulator of growth based on two observations: 1) the final pulse of ecdysone at the end of the third larval instar shortens developmental time and therefore reduces final organ size, and 2) increased ecdysone signaling can antagonize Dilp synthesis in the fat body. However, when measuring the effects of ecdysone on growth, many previous studies have focused on measuring either the growth of the larvae (which as this study observed does not always reflect the growth of the imaginal tissues) or measuring the final size of adults (which is a function of both growth rate and time). When one either examines clones expressing mutant alleles of ecdysone receptor or measures the growth of entire imaginal discs directly following ecdysone feeding as this study has done, ecdysone signaling can be shown to promote imaginal disc growth (Jaszczak, 2015).

During the regeneration checkpoint, both growth coordination and the delay in developmental timing are dependent on reduced ecdysone levels. Therefore, both delay and growth inhibition might be expected to be dependent on the same pathways. However, this study clearly demonstrated that the genetic requirements for these two systemic responses to damage are distinct. NOS is necessary for growth regulation following tissue damage, but is not necessary for the developmental delay. While it was observed that overexpression of NOS in the PG produces developmental delay, the results suggest that this is through a different mechanism than delays produced during the regeneration checkpoint. Therefore, Dilp8 secretion from damaged imaginal discs produce s developmental delay and growth restriction through distinct mechanisms (Jaszczak, 2015).

Finally, these observations suggest that regenerative growth, which is able to proceed despite reduced ecdysone signaling, may have different growth requirements than undamaged tissues. Understanding these differences in growth regulation could provide valuable insight s into the mechanistic distinctions between regenerative and developmental growth (Jaszczak, 2015).

DPP-mediated TGFβ signaling regulates juvenile hormone biosynthesis by activating the expression of juvenile hormone acid methyltransferase

Juvenile hormone (JH) biosynthesis in the corpus allatum (CA) is regulated by neuropeptides and neurotransmitters produced in the brain. However, little is known about how these neural signals induce changes in JH biosynthesis. This study reports a novel function of TGFβ signaling in transferring brain signals into transcriptional changes of JH acid methyltransferase (jhamt), a key regulatory enzyme of JH biosynthesis. A Drosophila genetic screen identified that Tkv and Mad are required for JH-mediated suppression of broad (br) expression in young larvae. Further investigation demonstrated that TGFβ signaling stimulates JH biosynthesis by upregulating jhamt expression. Moreover, dpp hypomorphic mutants also induces precocious br expression. The pupal lethality of these dpp mutants is partially rescued by an exogenous JH agonist. Finally, dpp is specifically expressed in the CA cells of ring glands, and its expression profile in the CA correlates with that of jhamt and matched JH levels in the hemolymph. Reduced dpp expression was detected in larvae mutant for Nmdar1, a CA-expressed glutamate receptor. Taken together, it is concluded that the neurotransmitter glutamate promotes dpp expression in the CA, which stimulates JH biosynthesis through Tkv and Mad by upregulating jhamt transcription at the early larval stages to prevent premature metamorphosis (Huang, 2011).

The functions of the TGFβ superfamily and other morphogens in regulating insect metamorphosis are rarely reported. In two independent genetic screens, it was discovered that Drosophila TGFβ signaling controls two different aspects of insect metamorphosis. In a previous study, it was found that Baboon (Babo) and dSmad2-mediated TGFβ signaling regulates larval neuron remodeling, which is part of the insect central nervous system metamorphosis induced by 20E during the pupal stage. Further investigation revealed that Babo/dSmad2-mediated TGFβ signaling controls larval neuron remodeling through regulating the expression of EcR-B1, a specific isoform of the 20E receptor (Huang, 2011).

This paper reports several findings. First, br is precociously expressed in 2nd instar tkv and Mad mutant larvae. Second, the precocious br expression phenotype in tkv and Mad mutant larvae can be suppressed by exogenous JH agonist (JHA). Third, Tkv and Mad repressed br expression in a non-cell-autonomous manner. Fourth, the presence of Mad in the CA is sufficient to repress br expression in the fat body (FB). Fifth, jhamt mRNA levels and JHAMT activity were significantly reduced in the Mad-deficient larvae. These results demonstrate that Tkv- and Mad-mediated signaling is required in the CA to activate jhamt expression and thus JH biosynthesis, which in turn controls insect metamorphosis (Huang, 2011).

The Drosophila genome encodes two TGFβ type II receptors, Punt (Put) and Wishful thinking (Wit). The genetic screen failed to identify a role for either of these receptors in the regulation of JH biosynthesis. Put and Wit are most probably functionally redundant in this biological event, as in the case of TGFβ-mediated mushroom body neuron remodeling (Huang, 2011).

Dpp is a key morphogen that controls dorsal/ventral polarity, segmental compartment determination and imaginal disc patterning. Dpp function usually depends on its gradient distribution. In an attempt to identify the ligand for Tkv/Mad-mediated TGFβ signaling in the CA, a novel, gradient-independent role for Dpp was discovered that controls JH biosynthesis. Dpp is the ligand of Tkv, which regulates jhamt transcription. Loss of Dpp, even RNAi reduction of Dpp in the CA specifically, causes precocious br expression at the early larval stages, which phenocopies tkv and Mad mutants. Phenotypes of dpp, including precocious br expression and lethality, are at least partially rescued by JHA treatment or ectopic jhamt expression in the CA. Notably, dpp-lacZ is strictly expressed in the CA cells, but not in the other two types of endocrine cells in the ring gland: the prothoracic gland and corpus cardiacum cells. The developmental expression profile of dpp in the CA is always consistent with that of jhamt. Finally, dpp expression in the CA may be directly controlled by neurotransmitter signals in the brain, which is supported by reduced dpp and jhamt transcription levels in the Nmdar1 mutant wandering larvae (Huang, 2011).

Several lines of evidence suggest that Met is a crucial regulator at or near the top of a JH signaling hierarchy, possibly acting as a JH receptor. However, null Met mutants of Drosophila are completely viable, which is unexpected if Met is a JH receptor. A recent investigation indicated that another Drosophila bHLH-PAS protein, Germ cell-expressed (Gce), which has more than 50% homology to Met, may function redundantly to Met in transducing JH signaling (Baumann, 2010). Because Met is on the X chromosome in the fly genome, it was not covered by the genetic screen. The br protein in the FBs of a Met null allele, Met27, was tested at the 2nd instar larval stage, and precocious br expression was observed. Importantly, this precocious br expression phenotype could not be suppressed by exogenous JHA. This result not only supports the previous reports regarding the function of Met in transducing JH signaling but also suggests that the precocious br expression is a more sensitive indicator for the reduced JH activity in Drosophila compared with precocious metamorphosis, lethality and other phenotypes (Huang, 2011).

Kr-h1 was reported to act downstream of Met in mediating JH action. Studies in both Drosophila and Tribolium reveal that, at the pupal stages, exogenous JHA induces Kr-h1 expression, which in turn upregulates br expression. The genetic screen successfully identified that Kr-h1 is cell-autonomously required for the suppression of br expression at young larval stages. Precocious br expression occurred in the FBs of Kr-h1 mutants and was not suppressed by JHA treatment. Therefore, these studies further suggest that Kr-h1 functions as a JH signaling component in mediating insect metamorphosis. However, the finding shows that, at the larval stages of Drosophila, the JH-induced Kr-h1 suppresses, rather than stimulates, br expression. This result is consistent with the facts that Kr-h1 functions to prevent Tribolium metamorphosis and Br is a crucial factor in promoting pupa formation (Huang, 2011).

In summary, this study has found a novel function of Dpp, Tkv and Mad-mediated TGFβ signaling in controlling insect metamorphosis. As summarized in a model, the brain sends neurotransmitters, such as glutamate, to the CA through neuronal axons. Glutamate interacts with its receptor (NMDAR) on the surface of CA cells to induce dpp expression. Dpp protein produced and secreted by CA cells forms a complex with TGFβ type I receptor (Tkv) and type II receptor on the membrane of CA cells, followed by phosphorylation and activation of Tkv. Activated Tkv in turn phosphorylates Mad, which is imported into the nucleus together with co-Smad and stimulates jhamt expression. JHAMT in CA cells transforms JH acid into JH, which is released into hemolymph. The presence of JH in young larvae prevents premature metamorphosis through Met/Gce and Kr-h1 by suppressing the expression of br, a crucial gene in initiating insect metamorphosis (Huang, 2011).

Local requirement of the Drosophila insulin binding protein Imp-L2 in coordinating developmental progression with nutritional conditions

In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. This study shows that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2 - FlyBase name: Ecdysone-inducible gene L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together these results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions (Sarraf-Zadeh, 2013).

In higher organisms, the duration of the juvenile stage needs to be variable to ensure the development of a healthy and fertile adult. Environmental stresses, such as adverse nutritional conditions, can delay development until a critical weight is reached. Additional checkpoints ensure that increased growth rates, induced by ideal nutritional conditions, do not lead to a premature passage to the adult stage. In Drosophila, the juvenile growth stage is terminated by pupae formation at the end of the third larval instar. Larval/pupal transition is induced by a pulse of the steroid hormone ecdysone produced by the PG (Sarraf-Zadeh, 2013).

Genetic manipulations of the Drosophila PG revealed the requirements of the IIS, Target of Rapamycin (TOR) and PTTH pathways to control ecdysone production . Recently, IIS dependent growth of the PG has been identified as an additional factor controlling ecdysone production. Overexpression of PI3K, a positive regulator of IIS, leads to premature, increased ecdysone production resulting in a shortened L3 stage and early pupariation. By contrast, overexpression of negative regulators of IIS in the PG delays pupariation caused by lowered and delayed ecdysone production. Reduction of whole organism IIS activity does not change critical weight but delays its attainment. In contrast, ablation of PTTH neurons induces a severe shift in critical weight, suggesting that these neurons play an important role in setting this parameter. When larvae reach the critical weight, PTTH is released on the PG and induces transcription of genes involved in ecdysone production. However, PTTH expression is not modified upon nutritional restriction, indicating that PTTH signaling does not mediate starvation induced developmental delay. Signaling via TOR, the downstream kinase of IIS, links nutritional information to ecdysone production, since starvation induced developmental delay can partially be rescued by upregulating TOR activity in the PG. This suggests that downregulating TOR signaling upon starvation desensitizes the PG for PTTH signals, resulting in delayed ecdysone production. The present study shows that increased IIS activity in the PG due to Imp L2 LOF rescues the delay caused by malnutrition to a large extent, indicating that low IIS also renders the PG irresponsive to the PTTH signal. Whether the effects of low IIS in the PG are mediated by TOR or whether the two pathways act independently remains to be elucidated (Sarraf-Zadeh, 2013).

Evidence is presented for a number of Imp L2 expressing neurons to act as possible regulators of IIS activity in the PG. High Imp L2 levels in the hemolymph can be excluded as possible inhibitors of IIS signaling in the PG, since increasing hemolymph levels of Imp L2 failed to reduce size and IIS activity of PG cells, but resulted in a strong size decrease of the whole organism. On the other hand, increasing Imp L2 levels in Imp L2 positive neurons targeting the PG causes a massive decrease in PG size and lowers IIS activity within PG cells. These results support the idea that the PG does not receive information about the nutritional state of the organism through the hemolymph but rather from Imp L2 expressing neurons. Thus, this work reveals a novel local function of the negative growth regulator Imp L2 in controlling IIS activity and ecdysone production in the PG. This finding reveals a novel mechanism for the spatial regulation of IIS: through locally restricted effects of Imp L2, diverse tissues can be effectively subjected to different levels of IIS (Sarraf-Zadeh, 2013).

Interestingly, the ability of IIS to coordinate growth with development seems to be conserved throughout evolution. In humans, the onset of puberty is linked to the nutritional state, leading to early puberty in well fed western societies. In contrast, juvenile females suffering from type I diabetes mellitus display a notable delay in menarche, indicating that decreased IIS also delays maturation in humans. Moreover, in Caenorhabditis elegans, malnutrition during the first larval stage leads to developmental arrest by inducing dauer formation, which is a larval stage best adapted for survival under adverse environmental conditions. Mutations reducing IIS pathway activity lead to dauer formation independent of the nutritional state. Hence, different phyla developed similar strategies to cope with adverse nutritional conditions during the juvenile state. When IIS activity is below a certain threshold, development is attenuated until sufficient nutrients are available, to ensure the formation of healthy and fertile adults. In Drosophila larval malnutrition leads to delayed pupariation, due to decreased IIS activity in the PG which in turn delays the production of the steroid hormone ecdysone (Sarraf-Zadeh, 2013).

Steroid hormones also play an important role in human development. In cases of human hypogonadism, puberty is prolonged, which can lead to abnormally tall adults if not treated with steroid substitutes. Referring the current data to the human system, the putative Imp L2 homolog IGFBP 7 (also known as IGFBP rP1) also displays a very diverse protein expression pattern, indicating a specialized function in different organs. Amongst other tissues, IGFBP 7 is expressed in different regions of the human brain, leading to the speculation that it might act as a local regulator of steroid production as well (Sarraf-Zadeh, 2013).

In summary, the data provides novel insights into the coupling of developmental cues to nutritional state. Since IIS and steroid hormones play evolutionarily conserved roles in regulating growth and development, the findings on the local function of the insulin binding protein Imp L2 in controlling ecdysone production might be of general interest (Sarraf-Zadeh, 2013).

Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis

Despite their fundamental importance for body size regulation, the mechanisms that stop growth are poorly understood. In Drosophila melanogaster, growth ceases in response to a peak of the molting hormone ecdysone that coincides with a nutrition-dependent checkpoint, critical weight. Previous studies indicate that insulin/insulin-like growth factor signaling (IIS)/Target of Rapamycin (TOR) signaling in the prothoracic glands (PGs) regulates ecdysone biosynthesis and critical weight. This study elucidates a mechanism through which this occurs. This study shows that Forkhead Box class O (FoxO), a negative regulator of IIS/TOR, directly interacts with Ultraspiracle (Usp), part of the ecdysone receptor. While overexpressing FoxO in the PGs delays ecdysone biosynthesis and critical weight, disrupting FoxO-Usp binding reduces these delays. Further, feeding ecdysone to larvae eliminates the effects of critical weight. Thus, nutrition controls ecdysone biosynthesis partially via FoxO-Usp prior to critical weight, ensuring that growth only stops once larvae have achieved a target nutritional status (Koyama, 2014).

A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila

Although glucose-sensing neurons were identified more than 50 years ago, the physiological role of glucose sensing in metazoans remains unclear. This study has identified a pair of glucose-sensing neurons with bifurcated axons in the brain of Drosophila. One axon branch projects to insulin-producing cells to trigger the release of Drosophila insulin-like peptide 2 (Dilp2) and the other extends to Adipokinetic hormone (AKH)-producing cells to inhibit secretion of AKH, the fly analogue of glucagon. These axonal branches undergo synaptic remodelling in response to changes in their internal energy status. Silencing of these glucose-sensing neurons largely disabled the response of insulin-producing cells to glucose and Dilp2 secretion, disinhibited AKH secretion in corpora cardiaca and caused hyperglycaemia, a hallmark feature of diabetes mellitus. It is proposed that these glucose-sensing neurons maintain glucose homeostasis by promoting the secretion of Dilp2 and suppressing the release of AKH when haemolymph glucose levels are high (Oh, 2019).

Glucose-sensing neurons respond to glucose or its metabolites, which act as signalling cues to regulate their neuronal activity. According to the glucostatic hypothesis proposed in 1953, feeding and related behaviours are regulated by neurons in the brain that sense changes in glucose levels in the blood. Despite the discovery of glucose-sensing neurons in the hypothalamus through electrophysiological methods more than ten years later, the physiological role of these neurons remained unclear until recently, when a population of glucose-excited neurons in the Drosophila brain were determined to function as an internal nutrient sensor to mediate the animal's consumption of sugar (Dus, 2015). A large number of glucose-sensing neurons appear to be present in animals; it is speculated that these neurons mediate physiological functions that are critical for the wellbeing of the animal, including glucose homeostasis. This study reports the identification of a pair of glucose-excited neurons in the Drosophila brain that maintain glucose homeostasis by coordinating the activity of the two key hormones involved in the process: insulin and glucagon (Oh, 2019).

To identify neurons that respond to sugar on the basis of its nutritional value, a two-choice assay was used to screen Vienna tiles (VT)-Gal4 Drosophila lines that had been crossed to UAS-Kir2.1, tub-Gal80ts flies (inward-rectifier potassium ion channel allele Kir2.1 with tubulin-temperature-sensitive Gal80) for defects in their ability to select nutritive D-glucose over non-nutritive L-glucose. Two independent Gal4 lines, VT58471 and VT43147-Gal4, were isolated that failed to select D-glucose after periods of starvation and appeared to contain dorsolateral cells that resemble those that are labelled by the corazonin (Crz)-Gal4 line. Flies in which Crz-Gal4-expressing neurons had been inactivated failed to select D-glucose even when starved. These results suggest that the dorsolateral neurons labelled by Crz-Gal4 and two candidate Gal4 lines mediate the behavioural response to sugar (Oh, 2019).

A Crz antibody was used to confirm the identity of the dorsolateral neurons. A previous study demonstrated that a subset of Crz-expressing neurons also express short neuropeptide F (sNPF). Immunolabelling revealed that the dorsolateral neurons expressing Crz indeed express sNPF. On the basis of these findings, these Crz+sNPF+ neurons were named CN neurons. To restrict Gal4 expression to a few cells that include the dorsolateral neurons, T58471-Gal4 was crossed to choline acetyltransferase (ChAT)-Gal80, generating CN-Gal4, which unambiguously labelled a pair of CN neurons when crossed to UAS-mCD8::GFP. Flies in which these dorsolateral neurons were inactivated using CN-Gal4 failed to select D-glucose when starved. Each CN cell body projects an axon that bifurcates to form two major branches. One branch (axon 1) projects to the pars intercerebralis (PI) region of the brain and the other branch (axon 2) projects ventrolaterally towards the corpora cardiaca (CC). An intersectional approach was used to define these projections further, thereby validating that axon 1 innervates the PI and axon 2 projects to the CC. This approach was also used to induce the expression of tetanus toxin (TNT) to silence a pair of CN neurons. These flies failed to choose D-glucose even after starvation when CN neurons were inactivated. This provided further evidence of the contribution of the pair of the dorsolateral CN neurons to glucose-evoked behaviour (Oh, 2019).

Attempts were made to determine whether CN neurons respond to glucose and other sugars. Calcium-imaging studies using ex vivo brain preparations of flies carrying the calcium indicator UAS-GCaMP6s14 and CN-Gal4 revealed that CN neurons were robustly activated by D-glucose with substantial calcium oscillations. CN neurons also responded to D-trehalose and D-fructose, which are found in the haemolymph, but failed to respond to (1) the non-nutritive sugar L-glucose; (2) the non-haemolymph sugar sucrose; and (3) the non-sugar nutrients amino acids. D-Glucose and D-trehalose are key sugars in the haemolymph, although D-trehalose stimulates the activity of CN neurons only after a substantial delay (about 12 min), possibly because it requires additional metabolic steps to be converted to glucose. D-Fructose applied at 20 mM activated CN neurons, although the concentration of D-fructose in the haemolymph is much lower (<2 mM). These findings suggest that the pair of CN neurons responds only to D-glucose under normal physiological conditions (Oh, 2019).

It was next determined whether activation of CN neurons by D-glucose requires glucose metabolism inside the cell. Exposing the brain to D-glucose mixed with 2DG, phlorizin or nimodipine, which inhibits glycolysis, glucose transport or voltage-gated calcium channels, respectively, blunted the glucose-induced stimulation of CN neurons. In the presence of pyruvate (an end product of glycolysis), the CN neurons demonstrated activity similar to that seen in the presence of other haemolymph sugars. Application of the ATP-sensitive potassium channel (KATP)16 blocker glibenclamide17 resulted in activation of CN neurons (Fig. 2b, c). Furthermore, glucose-induced calcium transients of these neurons were not abrogated by the application of the sodium-channel blocker tetrodotoxin (TTX). Using RNA-mediated interference (RNAi) lines, it was also determined that glucose transporter 1 (Glut1), hexokinase C (Hex-C), a subunit of the KATP channel (SUR1) and the voltage-gated calcium channel are required in CN neurons for the two-choice behaviour. Consistent with the behavioural results, the glucose-induced calcium response of CN neurons requires Glut1, SUR1 and a voltage-gated calcium channel, further supporting the role of the intracellular glucose metabolic pathway in stimulating CN neuronal activity (Oh, 2019).

The calcium-dependent nuclear import of LexA (CaLexA) system was used to measure cellular activity in CN neurons in intact flies; GFP signal driven by the CaLexA system in starved flies was significantly reduced compared to the signal in fed flies, and the signal was restored when starved flies were refed D-glucose. These results suggest that the activity of CN neurons is stimulated by the increase in glucose levels observed under fed conditions. In addition to the altered CaLexA signals, the effect of glucose on the number and intensity of synaptotagmin (Syt)-GPF+ puncta in fed, starved and refed animals. The Syt-GPF+ signals decreased significantly in axon 1 in starved animals and returned to normal levels after the flies were fed with D-glucose. However, this nutrient-dependent plasticity was not observed in Crz-Gal4-labelled axonal processes that did not originate from the dorsolateral CN neurons (Oh, 2019).

Next attempts were made to determine whether CN neurons are coupled with IPCs20 at the synaptic level. A modified GFP reconstitution across synaptic partners (GRASP) method was used, and the GRASP signals were found to be visible around the synapse between CN neurons and insulin-producing cells (IPCs), indicating physical coupling between CN neurons and IPCs (Oh, 2019).

To determine whether the coupling between CN neurons and IPCs is functional, ATP-gated P2X2 purine receptors were used in CN neurons and the calcium indicator GCaMP6s14 in IPCs, and then CN neurons were stimulated using ATP while recording from the IPCs. ATP-induced CN-neuron activity was accompanied by a significant increase in the amplitude of GCaMP signals in the IPCs in fed flies; this effect was reduced in starved flies. This finding supports the hypothesis that the nutrient-dependent synaptic changes observed between CN neurons and IPCs have functional consequences. The CN neurons did not appear to be functionally coupled to glucose-excited diuretic hormone 44 (Dh44) neurons. Furthermore, whether CN neuronal activity is required for Dilp2 secretion from IPCs was tested. A significant reduction in the intensity of Dilp2 immunoreactivity was observed in the IPCs of fed control flies, but not in fed flies in which CN neurons had been inactivated. These results suggest that an excitatory signal from the CN neurons contributes to the secretion of Dilp2 from IPCs in response to increased glucose levels. Using mass spectrometry and dot blot assay, it was further validated that the flies carrying CN-Gal4 and UAS-Kir2.1 had lower Dilp2 levels circulating in the haemolymph than wild-type flies, in contrast to the higher dilp2 levels found in IPCs (Oh, 2019).

To further clarify the role of CN neurons in mediating glucose-evoked activity in IPCs, CN neurons were inactivated by expressing TNT13, and then the responsiveness of IPCs to glucose was evaluated. The amplitude of calcium signals in IPCs that had been exposed to D-glucose was significantly reduced when the CN neurons were inactivated. Furthermore, it was found that IPCs harbour at least three subpopulations of neurons with distinct responses to glucose or KATP channel blocker. These findings suggest that CN neuronal activity is required for the majority of IPCs to respond to glucose (Oh, 2019).

To determine whether nutrient-dependent plasticity also occurs in axon 2 of the CN neurons, the number and intensity of Syt-GFP+ puncta was monitored before and after feeding flies with D-glucose. A significant reduction was observed in these parameters in starved flies, and a restoration to normal levels was found after refeeding starved flies with D-glucose. This raised the possibility of coupling between CN neurons and AKH-producing cells. Using a modified GRASP method, GRASP fluorescent signals were observed around AKH-producing cells. To determine whether there is any functional connectivity between these cells, the CN neurons were activated while monitoring the activity of AKH-producing cells, and calcium transients found in the AKH-producing cells appeared to decrease during activation of CN neurons (Oh, 2019).

To probe this observation further, the Arclight receptor, which increases fluorescent signals when cells become hyperpolarized, was expressed in AKH-producing cells, and P2X2 receptors were expressed in CN neurons. When the CN neurons were activated using ATP, the Arclight fluorescence intensity in fed flies increased significantly compared with that in starved flies, validating the occurrence of nutrient-dependent changes in the synapses between CN neurons and AKH-producing cells. Notably, when CN neurons were inactivated, the intracellular AKH levels decreased significantly compared with controls. Using mass spectrometry and dot blot assay, significantly higher levels of AKH were expressed in haemolymph of flies carrying CN-Gal4 and UAS-Kir2.1 compared with those in control flies. These findings suggest that CN neuronal activity inhibits the release of AKH from the CC and the increase of AKH levels in haemolymph (Oh, 2019).

Next the identities were investigated of the key neurotransmitters in axon 1 and axon 2 for regulating the functionally opposing synaptic activities. The role of Crz and sNPF was tested in the two-choice behaviour using RNAi lines, and sNPF in CN neurons and sNPF receptor in the postsynaptic IPCs, but not Crz or its receptor, were found to be important. sNPF but not Crz levels in CN neurons were significantly reduced when CN neurons were exposed to D-glucose. Approximately a half of the IPCs that had responded to glucose failed to respond glucose when the dominant-negative allele of sNPF receptor was expressed in IPCs. Furthermore, it was observed that intracellular AKH levels remained high in AKH-producing cells in fed control flies, but declined significantly in fed flies in which the function of sNPF receptor was inhibited in AKH-producing cells (Oh, 2019).

Finally, whether sNPF alters activity of IPCs and/or the CC was determined. The activity of IPCs was significantly stimulated by the application of sNPF26, whereas CC activity was significantly inhibited by sNPF. These functionally opposing effects of sNPF are probably mediated by Gq in IPCs and by Gi/o in AKH-producing cells via the sNPF receptor, which is a G-protein-coupled receptor. Exposing the brain to U73122, a PLC inhibitor that inhibits the Gq pathway, eliminated the glucose-evoked activation of IPCs, but had no effect on sNPF-induced inhibition of AKH-expressing cells. Conversely, exposing the brain to pertussis toxin, a Gi inhibitor, blunted the sNPF-induced inhibition of AKH-producing cells, but had no effect on the glucose-evoked activation of IPCs. These results indicate that axon 1 and axon 2 can have opposing synaptic activities through a mechanism involving the same neurotransmitter and receptor but with distinct downstream factors coupled with opposing outputs (Oh, 2019).

To determine whether CN neuronal activity can alter circulating sugar levels in flies, circulating concentrations of glucose and trehalose in haemolymph were monitored; they were significantly increased in flies in which CN neurons were inactivated compared with controls. This finding illustrates that dysfunctional CN neuronal input to IPCs and AKH-producing cells results in a defect in glucose homeostasis (Oh, 2019).

This study identified and characterized a pair of glucose-sensing neurons in the Drosophila brain that have an essential role in maintaining glucose homeostasis. This was achieved by counterbalancing the activities of Drosophila equivalents of insulin- and glucagon-producing cells. When food consumption leads to a rise in haemolymph sugar levels, CN neurons excite the IPCs through sNPF and its receptor, which appear to be coupled to the Gq signalling cascade to induce the secretion of Dilp2, while suppressing the release of AKH by using the same sNPF receptor, which in this case is coupled with Gi signalling pathway. It is speculated that precise control of these opposing functions is facilitated because the nutrient-dependent plastic changes arise from a single cell (Oh, 2019).

This study demonstrates how the activity of the two key endocrine systems is coordinated in metazoans and that their coordination is under the direct control of glucose-sensing neurons. Such coordination has been proposed to occur in mammals via the sympathetic and parasympathetic nerves that connect the pancreatic islets with glucose-sensing neurons in the hypothalamus and hindbrain. The finding that a large proportion of IPCs respond to glucose through CN neurons in insects raises an intriguing possibility that both direct and indirect mechanisms control endocrine function in mammals. Finally, this work may shed light on the function of glucose-sensing neurons. Further research is needed to understand how these regulatory processes are affected by excessive nutrition and other metabolic disturbances, including obesity (Oh, 2019).

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

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

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

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

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

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

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

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

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

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

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

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

At present, it is not known what the endogenous late Jak/Stat signal contributes in terms of PG function since knockdown with available reagents did not produce a significant phenotype. In Drosophila, the canonical Jak/Stat signaling pathway is commonly induced by a group of cytokines including unpaired 1-3 (Upd1-3) via their cognate receptor Domeless (Dome). However, it has also been reported that Torso and Pvr are capable of inducing Jak/Stat activation in some circumstances. Although induction of Jak/Stat signal by overexpressing wild-type Torso was not observed in the PG, this might be due to a weaker activation using wild-type Torso overexpression versus gain-of-function torY9 and torRL3 mutants as used in the previous study. Since this study observed Dome expression and endogenous activation of the 10xStat92E-GFP reporter in late L3 PGs, it is assumed to be likely to play some role at this stage. Whether the Jak/Stat activation is through Alk/Pvr or via reception of canonical Upd/Dome signals is not clear. Interestingly, note that Upd2 is secreted from the fat body into hemolymph and therefore may provide a nutrient storage signal to the PG that could be an important regulator of developmental timing, perhaps under certain types of non-standard laboratory growth conditions. It has also been recently demonstrated that inflammation-triggered release of Upd3 acts on the PG to produce developmental delay, indicating that the Jak/Stat pathway may be an important sensor for imbalance of various types of physiological processes (Pan, 2021).

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

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

Body-fat sensor triggers ribosome maturation in the steroidogenic gland to initiate sexual maturation in Drosophila

Fat stores are critical for reproductive success and may govern maturation initiation. This study reports signaling and sensing fat sufficiency for sexual maturation commitment requires the lipid carrier apolipophorin in fat cells and Sema1a in the neuroendocrine prothoracic gland (PG). Larvae lacking apolpp or Sema1a fail to initiate maturation despite accruing sufficient fat stores, and they continue gaining weight until death. Mechanistically, sensing peripheral body-fat levels via the apolipophorin/Sema1a axis regulates endocytosis, endoplasmic reticulum remodeling, and ribosomal maturation for the acquisition of the PG cells' high biosynthetic and secretory capacity. Downstream of apolipophorin/Sema1a, leptin-like upd2 triggers the cessation of feeding and initiates sexual maturation. Human Leptin in the insect PG substitutes for upd2, preventing obesity and triggering maturation downstream of Sema1a. Data shows how peripheral fat levels regulate the control of the maturation decision-making process via remodeling of endomembranes and ribosomal biogenesis in gland cells (Juarez, 2021).

PKG acts in the adult corpora cardiaca to regulate nutrient stress-responsivity through adipokinetic hormone

In Drosophila melanogaster, the adipokinetic hormone (AKH) is a glucagon-like peptide that acts antagonistically with insulin-like peptides to maintain metabolic homeostasis. AKH is biosynthesized in and secreted from the corpora cardiaca (CC). This report describes a CC-specific role for dg2 - which encodes a cGMP-dependent protein kinase (PKG) - as a regulator of AKH during adulthood. Transcriptional silencing of dg2 during adulthood decreased starvation resistance, increased sucrose responsiveness, and decreased whole body lipid content. PKG protein was localized to CC cell membranes, and starvation caused a significant decrease in CC intracellular AKH content. Strikingly, reduced CC-dg2 expression caused a significant decrease in intracellular AKH content in adults fed ad libitum. This work demonstrated that dysregulation of CC-specific dg2 expression during adult life impaired metabolic homeostasis, and that dg2 acted in the CC to regulate systemic AKH activity (Hughson, 2022).

Insulin signaling couples growth and early maturation to cholesterol intake in Drosophila

Childhood obesity is associated with precocious puberty, but the assessment mechanism that links body fat to early maturation is unknown. During development, the intake of nutrients promotes signaling through insulin-like systems that govern the growth of cells and tissues and also regulates the timely production of the steroid hormones that initiate the juvenile-adult transition. This study shows that the dietary lipid cholesterol, which is required as a component of cell membranes and as a substrate for steroid biosynthesis, also governs body growth and maturation in Drosophila via promoting the expression and release of insulin-like peptides. This nutritional input acts via the nutrient sensor TOR, which is regulated by the Niemann-Pick-type-C 1 (Npc1) cholesterol transporter, in the glia of the blood-brain barrier and cells of the adipose tissue to remotely drive systemic insulin signaling and body growth. Furthermore, increasing intracellular cholesterol levels in the steroid-producing prothoracic gland strongly promotes endoreduplication, leading to an accelerated attainment of a nutritional checkpoint that normally ensures that animals do not initiate maturation prematurely. These findings, therefore, show that a Npc1-TOR signaling system couples the sensing of the lipid cholesterol with cellular and systemic growth control and maturational timing, which may help explain both the link between cholesterol and cancer as well as the connection between body fat (obesity) and early puberty (Texada, 2022).

Nutrition is one of the most important influences on developmental growth and maturation. Malnutrition or disease can impair growth and delay puberty, whereas obese children enter puberty early. Similarly, Drosophila larvae exposed to poor nutrition, tissue damage, or inflammation delay their development, whereas rich conditions promote rapid growth and maturation. These environmental factors are coupled to the appropriate gating of steroid production via internal checkpoints, one of which is a nutrition-dependent “critical weight” (CW) required to initiate the maturation process (Texada, 2022).

This suggests that signals reflecting nutritional state and body-fat storage play a key role in activating the neuroendocrine pathways that trigger puberty. Although studies suggest that the adipokine leptin may be involved, the mechanisms linking body fat to puberty are poorly defined, and the potential involvement of lifestyles associated with excessive accumulation of cholesterol, one of the most important lipids, has not been considered. In humans, white adipose tissue is the main site of cholesterol storage and can contain over half the body's total cholesterol in obesity (Texada, 2022).

The results show that dietary cholesterol intake promotes systemic body growth through insulin-dependent pathways and that animals raised on high dietary cholesterol initiate maturation earlier. It was show nthat cholesterol is sensed through an Npc1-regulated TOR-mediated mechanism in the fat body and the glial cells of the BBB, which relay information to the IPCs within the brain to promote insulin expression and release, thus coupling growth and maturation with cholesterol status (Texada, 2022).

Insect CW likely evolved as a mechanism ensuring that maturation will not occur unless the animal has accumulated adequate nutrient stores to survive the nonfeeding metamorphosis period and has completed sufficient growth to produce an adult of proper size and thus of maximal fitness. Likewise, the link between body fat and maturation in humans probably ensures adequate stores of fat before maturation onset to support pregnancy and reproductive success (Texada, 2022).

In Drosophila, insulin signaling plays a critical role in coordinating steroidogenesis with nutritional conditions. Insulin acts upon the prothoracic gland (PG) and induces a small ecdysone peak early in L3 that is correlated with CW attainment. In combination with nutrition-related signaling mediated via insulin, nutrient availability is also assessed directly in the PG and is coupled to irreversible endoreduplication that permits ecdysone production at CW (Texada, 2022).

These findings show that accumulation of cholesterol in the PG, induced by the loss of Npc1a, drives a remarkable TOR-dependent increase in endoreduplication and leads to inappropriate attainment of CW. Taken together, these findings indicate that cholesterol sensed by the BBB glia and the fat body promotes growth through insulin signaling and that cholesterol sensed by the PG accelerates maturation through ecdysone signaling. Loss of Npc1 function in humans leads to the Niemann-Pick lysosomal storage disorder, marked by intracellular cholesterol accumulation. Although neurodegeneration is the hallmark of NPC disease, including in a Drosophila model,70 alterations in glial, adipose, hepatic, and endocrine systems are also components of NPC syndrome. In humans, Npc1 itself is strongly expressed in glia and in adipose tissues, especially in obese individuals, and variants in Npc1 are associated with obesity, type-2 diabetes, and hepatic lipid dysfunction (Texada, 2022).

These findings link glial and adipose-tissue cholesterol sensing through Npc1 to systemic growth and metabolic control through effects on insulin signaling. It was also found that intracellular cholesterol accumulation driven by Npc1 loss leads to hyperactivation of TOR that drives increases in DNA replication and cell growth. TOR activity is frequently upregulated in cancer, and the results therefore provide mechanistic insight for understanding the emerging link between cholesterol and a range of cancers. As the coupling of nutrition with growth and maturation is ancient and highly conserved, this work provides a foundation for understanding how cholesterol is coupled to developmental growth and maturation initiation in humans. These findings link a high concentration of this particular lipid in adipose tissues to the neuroendocrine initiation of maturation, which may explain the critical link between obesity (body fat) and early puberty (Texada, 2022).

Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae

The feeding of pests is one of the important reasons for losses of agricultural crop yield. This study aimed to reveal how juvenile hormone participates in larval feeding regulation of the Asian corn borer Ostrinia furnacalis. Larvae of O. furnacalis exhibit a daily circadian rhythm on feeding, with a peak at ZT18 and a trough at ZT6 under both photoperiod (LD) and constant dark (DD) conditions, which may be eliminated by application of fenoxycarb, a juvenile hormone (JH) active analogue. JH negatively regulates larval feeding as a downstream factor of neuropeptide F (NPF), in which knocking down JH increases larval feeding amount along with body weight and length. The production of JH in the brain-corpora cardiaca-corpora allata (brain-CC-CA) is regulated by the brain NPF rather than gut NPF, which was demonstrated in Drosophila larvae through GAL4/UAS genetic analysis. In addition, feeding regulation of JH is closely related to energy homeostasis in the fat body by inhibiting energy storage and promoting degradation. The JH analogue fenoxycarb is an effective pesticide to O. furnacalis that controls feeding and metabolism. The brain NPF system regulates JH, with functions in food consumption, feeding rhythms, energy homeostasis and body size. This study provides an important basis for understanding the feeding mechanism and potential pest control (Yu, 2022).

Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development

The regulation of ribosome function is a conserved mechanism of growth control. While studies in single cell systems have defined how ribosomes contribute to cell growth, the mechanisms that link ribosome function to organismal growth are less clear. This study explored this issue using Drosophila Minutes, a class of heterozygous mutants for ribosomal proteins. These animals exhibit a delay in larval development caused by decreased production of the steroid hormone ecdysone, the main regulator of larval maturation. This developmental delay is not caused by decreases in either global ribosome numbers or translation rates. Instead, this study showed that they are due in part to loss of Rp function specifically in a subset of serotonin (5-HT) neurons that innervate the prothoracic gland to control ecdysone production. These effects do not occur due to altered protein synthesis or proteostasis, but that Minute animals have reduced expression of synaptotagmin, a synaptic vesicle protein, and that the Minute developmental delay can be partially reversed by overexpression of synaptic vesicle proteins in 5-HTergic cells. These results identify a 5-HT cell-specific role for ribosomal function in the neuroendocrine control of animal growth and development (Deliu, 2022).

Disruption of insulin signalling affects the neuroendocrine stress reaction in Drosophila females

Juvenile hormone (JH) and dopamine are involved in the stress response in insects. The insulin/insulin-like growth factor signalling pathway has also recently been found to be involved in the regulation of various processes, including stress tolerance. However, the relationships among the JH, dopamine and insulin signalling pathways remain unclear. The role of insulin signalling in the regulation of JH and dopamine metabolism under normal and heat stress conditions was investigated in Drosophila melanogaster females. Suppression of the insulin-like receptor (InR) in the corpus allatum, a specialised endocrine gland that synthesises JH, causes an increase in dopamine level and JH-hydrolysing activity and alters the activities of enzymes that produce as well as those that degrade dopamine [alkaline phosphatase (ALP), tyrosine hydroxylase (TH) and dopamine-dependent arylalkylamine N-acetyltransferase (DAT)]. It was also found that InR suppression in the corpus allatum modulates dopamine, ALP, TH and JH-hydrolysing activity in response to heat stress and that it decreases the fecundity of the flies. JH application restores dopamine metabolism and fecundity in females with decreased InR expression in the corpus allatum. These data provide evidence that the insulin/insulin-like growth factor signalling pathway regulates dopamine metabolism in females of D. melanogaster via the system of JH metabolism and that it affects the development of the neuroendocrine stress reaction and interacts with JH in the control of reproduction in this species (Rauschenbach, 2014).

Regulation of onset of female mating and sex pheromone production by juvenile hormone in Drosophila melanogaster

Juvenile hormone (JH) coordinates timing of female reproductive maturation in most insects. In Drosophila, JH plays roles in both mating and egg maturation. However, very little is known about the molecular pathways associated with mating. Behavioral analysis of females genetically lacking the corpora allata (CAX), the glands that produce JH, showed that they were courted less by males and mated later than control females. Application of the JH mimic, methoprene, to the allatectomized females just after eclosion rescued both the male courtship and the mating delay. Studies of the null mutants of the JH receptors, Methoprene tolerant (Met) and germ cell-expressed (gce), showed that lack of Met in Met27 females delayed the onset of mating, whereas lack of Gce had little effect. The Met27 females were shown to be more attractive but less behaviorally receptive to copulation attempts. The behavioral but not the attractiveness phenotype was rescued by the Met genomic transgene. Analysis of the female cuticular hydrocarbon profiles showed that corpora allata ablation caused a delay in production of the major female-specific sex pheromones (the 7,11-C27 and -C29 dienes) and a change in the cuticular hydrocarbon blend. In the Met27 null mutant, by 48 h, the major C27 diene was greatly increased relative to wild type. In contrast, the gce2.5k null mutant females were courted similarly to control females despite changes in certain cuticular hydrocarbons. These findings indicate that JH acts primarily via Met to modulate the timing of onset of female sex pheromone production and mating (Bilen, 2013).

This study has shown that JH plays a critical role in the normal timing of onset of female mating and sex pheromone production. Removal of JH through genetic ablation of the CA in the developing adult female delayed the onset of mating behaviors. This change was coupled to a decrease in male courtship, suggesting a decrease in female attractiveness. Drastic changes were found in the CHC profiles in the CAX females. Some of these changes are likely due to the temperature shift regime used for the genetic allatectomy. Treating CAX females with the JH mimic (JHM) methoprene both advanced the onset of mating and increased the attractiveness of the females, apparently by increasing the production of C27 monoenes and dienes. Therefore, JH dynamically regulates the synthesis of specific cuticular hydrocarbons (CHCs), particularly the major female sex pheromones, the 7,11-C27 and 7,11-C29 dienes. These findings are consistent with previous studies showing induction of precocious mating by CA implants and reduced mating in apterous mutants and reduced female-specific pheromones in flies overexpressing the JH esterase-binding protein DmP29, both having reduced JH levels (Bilen, 2013).

Interestingly, the CAX females slowly became attractive so that by 96 h, time to onset of copulation was similar to controls. At this time the C27 monoene and the 7,11-C29 diene were still significantly reduced, whereas the C27 dienes were significantly increased, suggesting that there was a change in the CHC blend. The C27 dienes at 96 h may act alone or together with other CHCs in the pheromone blend to increase female attractiveness and decrease time to copulation in the CAX females (Bilen, 2013).

How does JH regulate CHC synthesis? CHCs are synthesized from fatty acids via elongation, desaturation, reduction to aldehyde, and oxidative decarbonylation reactions in oenocytes in D. melanogaster. The developmental appearance of the CHCs in CAX females in this study clearly shows decreases in the long-chain n-alkanes (C23- C27), the C25 and C27 monoenes, and the C27 and C29 dienes with corresponding increases in the shorter-chain dienes. Similar changes in diene profiles coupled with an increased time to copulation was seen after reduction of Elongase-F in female oenocytes (Chertemps, 2007). Reduction of Desaturase 1 in the oenocytes caused the loss of both monoenes and dienes with a large increase in the n-alkanes, whereas reduction of Desaturase-F caused a loss of the female-specific dienes with a doubling of monoenes and some increase in n-alkanes (Chertemps, 2006; Wicker-Thomas, 2009). Both manipulations significantly increased time to mating. Thus, JH apparently influences biosynthetic enzymes important in the synthesis of the long-chain alkanes as well as Elongase-F and the desaturases. These regulatory effects of JH on pheromone synthesis are similar to its effects on aggregation pheromone synthesis in bark beetles. In these beetles, JH III regulates the transcription of many of the genes encoding the pheromone biosynthetic enzymes, especially the genes encoding geranyldiphosphate synthase/myrcene synthase (GPPS/MS), CYP9T2, and an oxidoreductase (Bilen, 2013).

Application of 0.64 pmoles of the JHM methoprene just after eclosion rescued mating of the CAX females at 24 h after eclosion to the level of about 31% as seen in the parental controls, and a 10- fold higher dose caused about 58% to mate. After treatment with the higher dose of methoprene, the 7- and 9-C27 monoenes significantly increased at 24 h, but the two female-specific 7,11 dienes were not higher until 48 h. The increased C27 monoenes which have been implicated as aphrodisiacs (Marcillac, 2004) possibly made the CAX females more attractive at 24 h. Whether there is also an effect of the exogenous JH on the maturation of the female nervous system so that she becomes receptive to male courtship earlier is not known and warrants further study. At least three hormones (JH, ecdysone, and pheromone biosynthesis- activating neuropeptide) have been shown to regulate sex pheromone biosynthesis in different insects. This study has demonstrated that JH regulates sex pheromone 7,11-diene production in D. melanogaster females. Interestingly, in another dipteran, the house fly Musca domestica, the primary female sex pheromone is Z-9-tricosene. Females ovariectomized immediately after eclosion do not synthesize this compound, but synthesis is restored by either ovarian implants or multiple injections of 20-hydroxyecdysone. These two families of flies are evolutionarily distant, but the basis for this difference in hormonal regulation is unknown (Bilen, 2013).

The duplication of the JH receptor Gce occurred in the higher Diptera, and the two have partially redundant functions in the larval fat body of D. melanogaster and at metamorphosis (Abdou, 2011). Met plays a distinct role in adult optic lobe development during metamorphosis, whereas Gce has no effect. Met is also the predominant receptor required for the effects of JH on ovarian maturation, both the normal timing and normal fecundity. This behavioral analysis of female mating and attractiveness of Met and gce mutants indicates that JH is also acting primarily via Met in its regulation of mating and pheromone production. Surprisingly, in courtship assays using CS males, Met27 females lacking Met were more attractive than the wildtype CS females, whereas the CAX females were less attractive, likely due to increased C27 dienes in Met27 females and decreased 7,11-C27 and -C29 dienes in the CAX females. These findings suggest that JH acts mainly via Met in mating and pheromone synthesis similarly to the roles of Met and Gce in ovarian maturation where lack of Met delays maturation and reduces fecundity, whereas lack of Gce has relatively little effect (Bilen, 2013).

This study has demonstrated that JH acting through its receptor Met plays important roles in the initiation of sex pheromone production and the maturation of female mating behavior in Drosophila. Further investigation into these two aspects of JH action -- in the peripheral tissues involved in sex pheromone production and in the neuronal circuitry underlying the mating behavior -- is necessary to elucidate the details of its critical roles in modulation of the onset of mating. An understanding at the molecular level of this coordination in this Drosophila model should shed insights into how hormones regulate pheromone production and reproductive behavior in the vertebrates (Bilen, 2013).

Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila

Steroid hormones trigger the onset of sexual maturation in animals by initiating genetic response programs that are determined by steroid pulse frequency, amplitude and duration. Although steroid pulses coordinate growth and timing of maturation during development, the mechanisms generating these pulses are not known. This study shows that the ecdysone steroid pulse that drives the juvenile-adult transition in Drosophila is determined by feedback circuits in the prothoracic gland (PG), the major steroid-producing tissue of insect larvae. These circuits coordinate the activation and repression of hormone synthesis, the two key parameters determining pulse shape (amplitude and duration). Ecdysone has a positive-feedback effect on the PG, rapidly amplifying its own synthesis to trigger pupariation as the onset of maturation. During the prepupal stage, a negative-feedback signal ensures the decline in ecdysone levels required to produce a temporal steroid pulse that drives developmental progression to adulthood. The feedback circuits rely on a developmental switch in the expression of Broad isoforms that transcriptionally activate or silence components in the ecdysone biosynthetic pathway. Remarkably, this study shows that the same well-defined genetic program that stimulates a systemic downstream response to ecdysone is also utilized upstream to set the duration and amplitude of the ecdysone pulse. Activation of this switch-like mechanism ensures a rapid, self-limiting PG response that functions in producing steroid oscillations that can guide the decision to terminate growth and promote maturation (Moeller, 2013).

Although extensive studies have made it clear that transition to the adult stage in insects requires a high-level pulse of ecdysone, the mechanism that shapes the pulse, by determining its duration and amplitude, has remained unclear. These experiments show that the maturation-inducing pulse that coordinates the juvenile-adult transition in Drosophila is generated by ecdysone feedback control of PG steroidogenic activity. At the end of the third larval instar, ecdysone acts through EcR in a feed-forward circuit to produce the high-level pulse that triggers pupariation in response to PTTH. This illustrates an EcR-dependent positive feedback operating downstream of PTTH to generate a sustained output in terms of biosynthesis in response to neuropeptide signaling (Moeller, 2013).

The feed-forward loop described in this study provides an explanation for a number of previous observations. These studies have indicated that ecdysone can modulate PG steroidogenic activity and that PG cells undergo autonomous activation under long-term culture conditions. Interestingly, autonomous activation is prevented by juvenile hormone (JH), which inhibits br expression. During the last larval instar of holometabolous insects, a drop in JH levels eventually leads to the production of a high-level ecdysone pulse that triggers metamorphosis, although the mechanism underlying this is poorly understood. Since the decline of JH is permissive for br expression, the fact that Br promotes PG steroidogenic activity is likely to explain how the drop in JH results in the production of a high-level ecdysone pulse initiating metamorphosis. Thus, the data provide a link between JH and ecdysone that might explain how the presence of JH prevents metamorphosis (Moeller, 2013).

Observations clearly show that positive feedback is required for the transcriptional upregulation of phantom (phm), disembodied (dib) and shadow (sad), all of which encode enzymes that act at late steps in the ecdysone biosynthetic pathway. By contrast, EcR and Br activity are not necessary for the normal activity of spookier (spok), which is involved in an earlier step in the pathway and whose transcription is regulated by Molting defective, a factor that is not involved in the regulation of the other identified biosynthetic enzymes. In addition, in contrast to the other ecdysone biosynthetic enzymes, Spok is also likely to be regulated at the level of translation and phosphorylation in response to PTTH signaling. Furthermore, expression of PTTH receptor-encoding torso is not EcR and Br dependent, consistent with levels of torso not being synchronized with the ecdysone peaks. Together with the results demonstrating that the feedback is required downstream of Ras in the PG, this shows that the feed-forward loop functions downstream of PTTH to amplify the signal and not for endowing the PG with competence to respond to PTTH (Moeller, 2013).

The findings raise an important issue that challenges the classical view that ecdysone released from the PG is converted to its more active metabolite 20-hydroxyecdysone (20E) in peripheral target tissues, where it interacts with EcR. Although 20E may travel back and inform the PG, a more direct route would be that ecdysone produced by the PG acts on the gland itself or that the PG produces small amounts of 20E that control the activity of the gland. Consistent with these possibilities, reduced expression of shade, which encodes the enzyme that converts ecdysone to 20E, in the PG leads to a developmental arrest in the larval stages and all three Drosophila EcR isoforms can induce transcription in response to ecdysone. Interestingly, recent reports have demonstrated the essential function of E75, DHR3 (Hr46 – FlyBase), βFTZ-F1 and DHR4 in regulating the production of ecdysone in the PG. Although nitric oxide and PTTH regulate the activity of some of these factors, these signals alone are unlikely to explain the regulation of their function in the PG. Based on the results, an obvious possibility is that EcR controls the expression of these classical ecdysone-inducible genes in the PG. Extensive studies on these ecdysone target genes have led to the elucidation of an early response network for steroid hormone action and the molecular characterization of the genetic architecture underlying the cellular responses to steroids. Surprisingly, this study shows that this genetic program that guides the downstream cellular decisions in response to regulatory ecdysone pulses is utilized upstream to shape the pulse by setting its duration and amplitude. Thus, the same genetic components are used for coordinating the production and reception of the steroid signals that drive directional developmental progression (Moeller, 2013).

Previous experiments demonstrated that ecdysone, produced by the PG, induces an inactivation enzyme responsible for clearance of circulating ecdysone (Rewitz, 2010). This study shows that termination of the pulse requires negative feedback that represses PG steroid production activity in coordination with peripheral clearance. How does ecdysone stimulate and repress biosynthesis in the PG through EcR? The results show that EcR induces different Br isoforms, forming circuits that either increase or inhibit the activity of the biosynthetic pathway by regulating the levels of the enzymatic components. Br is required specifically for the juvenile-adulti transition and is expressed during the last instar. This study shows that the appearance of Br in the PG requires EcR and correlates with the ecdysone peak. The positive effect of EcR on ecdysone biosynthesis is mediated largely through Br-Z4, which has previously been shown to induce transcription of Niemann-Pick type C-1a (Npc1a), which encodes a key cellular component required in the PG for the delivery of cholesterol as a substrate for steroid synthesis. Together, this suggests that ecdysone-mediated positive feedback coordinates increased substrate delivery with upregulation of the biosynthetic machinery in order to produce the maturation-inducing ecdysone pulse. Conversely, the Br-Z1 isoform inhibits ecdysone synthesis, forming a negative feedback that is important for the decline of the ecdysone titer during the prepupal stage. Thus, the temporal control of these circuits relies on a dynamic switch in the PG from Br-Z4 to Br-Z1. A similar switch has been found in the imaginal discs, where Br-Z4 rapidly accumulates in response to ecdysone and then disappears several hours later when Br-Z1 is upregulated. It has been suggested that the switch from Br-Z4 to Br-Z1 is regulated at the level of alternative splicing of br transcripts. The data suggest that the switch is a hard-wired genetic timing mechanism rather than being dependent on ecdysone concentrations. This switching might also occur at the enhancer level through competition of binding to overlapping Br-Z1/Z4 regulatory sites, as was found in the phm promoter. Importantly, coupling a negative with a positive feedback through a common regulatory site ensures a self-limiting response by preventing 'run away' synthesis that would otherwise result from positive-feedback amplification alone (Moeller, 2013).

In conclusion, this study shows that the maturation-inducing ecdysone pulse is shaped by an autonomous feed-forward and feedback circuitry within the endocrine tissue that modulates the rate of hormone synthesis. The coupling of these feedback circuits ensures rapid, self-limiting hormone production that translates neuropeptide signaling into a regulatory steroid pulse which functions as a switch to drive developmental progression (Moeller, 2013).

Transcriptome analysis of Drosophila melanogaster third instar larval ring glands points to novel functions and uncovers a cytochrome p450 required for development

In Drosophila melanogaster larvae the ring gland is a control center that orchestrates major developmental transitions. It is a composite organ, consisting of the prothoracic gland, the corpus allatum and the corpora cardiaca, each of which synthesizes and secretes a different hormone. Until now, the ring gland's broader developmental roles beyond endocrine secretion have not been explored. RNA sequencing and analysis of a new transcriptome resource from D. melanogaster wandering third instar larval ring glands has provided a fascinating insight into the diversity of developmental signalling in this organ. Strong enrichment of expression was found of two gene pathways not previously associated with the ring gland: immune response and fatty acid metabolism. Strong expression was uncovered for many uncharacterized genes. Additionally, RNA interference against ring gland-enriched cytochrome p450s Cyp6u1 and Cyp6g2 produced a lethal ecdysone deficiency and a juvenile hormone deficiency respectively, flagging a critical role for these genes in hormone synthesis. This transcriptome provides a valuable new resource for investigation of roles played by the ring gland in governing insect development (Christesen, 2016).

Deep sequencing of the prothoracic gland transcriptome reveals new players in insect ecdysteroidogenesis

Ecdysteroids are steroid hormones that induce molting and determine developmental timing in arthropods. In insect larva, the prothoracic gland (PG) is a major organ for ecdysone synthesis and release. Released ecdysone is converted into the active form, 20-hydroxyecdysone (20E) in the peripheral tissues. All processes from ecdysone synthesis and release from the PG to its conversion to 20E are called ecdysteroidogenesis and are under the regulation of numerous factors expressed in the PG and peripheral tissues. Classical genetic approaches and recent transcriptomic screening in the PG identified several genes responsible for ecdysone synthesis and release, whereas the regulatory mechanism remains largely unknown. This study analyzed RNA-seq data of the silkworm Bombyx mori PG and employed the fruit fly Drosophila melanogaster GAL4/UAS binary RNAi system to comprehensively screen for genes involved in ecdysone synthesis and/or release. It was found that the genes encoding delta-aminolevulinic acid synthase (CG3017/Aminolevulinate synthase/Alas) and putative NAD kinase (CG33156) were highly expressed in the PG of both B. mori and D. melanogaster. Neither alas nor CG33156 RNAi-induced larvae could enter into the pupal stage, and they had a lower abundance of the active form ecdysteroids in their prolonged larval stage. These results demonstrated that alas and CG33156 are indispensable for ecdysteroidogenesis (Nakaoka, 2017).

Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster

Ecdysone, an essential insect steroid hormone, promotes larval metamorphosis by coordinating growth and maturation. In Drosophila melanogaster, prothoracicotropic hormone (PTTH)-releasing neurons are considered to be the primary promoting factor in ecdysone biosynthesis. Recently, studies have reported that the regulatory mechanisms of PTTH release in Drosophila larvae are controlled by different neuropeptides, including allatostatin A and corazonin. However, it remains unclear whether neurotransmitters provide input to PTTH neurons and control the metamorphosis in Drosophila larvae. This study reports that the neurotransmitters acetylcholine (ACh) affect larval development by modulating the activity of PTTH neurons. By downregulating the expression of different subunits of nicotinic ACh receptors in PTTH neurons, pupal volume was significantly increased, whereas pupariation timing was relatively unchanged. It was also identified that PTTH neurons were excited by ACh application ex vivo in a dose-dependent manner via ionotropic nicotinic ACh receptors. Moreover, in Ca(2+) imaging experiments, relatively low doses of OA caused increased Ca(2+) levels in PTTH neurons, whereas higher doses led to decreased Ca(2+) levels. It was also demonstrated that a low dose of OA was conveyed through OA *betal-type receptors. Additionally, electrophysiological experiments revealed that PTTH neurons produced spontaneous activity in vivo, which provides the possibility of the bidirectional regulation, coming from neurons upstream of PTTH cells in Drosophila larvae. In summary, these findings indicate that several different neurotransmitters are involved in the regulation of larval metamorphosis by altering the activity of PTTH neurons in Drosophila (Hao, 2021).

Intrinsic and damage-induced JAK/STAT signaling regulate developmental timing by the Drosophila prothoracic gland
Development involves tightly paced, reproducible sequences of events, yet it must adjust to conditions external to it, such as resource availability and organismal damage. A major mediator of damage-induced immune responses in vertebrates and insects is JAK/STAT signaling. At the same time, JAK/STAT activation by the Drosophila Upd cytokines is pleiotropically involved in normal development of multiple organs. Whether inflammatory and developmental JAK/STAT roles intersect is unknown. This study shows that JAK/STAT is active during development of the prothoracic gland (PG), which controls metamorphosis onset through ecdysone production. Reducing JAK/STAT signaling decreased PG size and advanced metamorphosis. Conversely, JAK/STAT hyperactivation by overexpression of pathway components or SUMOylation loss caused PG hypertrophy and metamorphosis delay. Tissue damage and tumors, known to secrete Upd cytokines, also activated JAK/STAT in the PG and delayed metamorphosis, at least in part by inducing expression of the JAK/STAT target Apontic. JAK/STAT damage signaling, therefore, regulates metamorphosis onset by co-opting its developmental role in the PG. These findings in Drosophila provide insights on how systemic effects of damage and cancer can interfere with hormonally controlled development and developmental transitions (Cao, 2022).

Reduction of nucleolar NOC1 accumulates pre-rRNAs and induces Xrp1 affecting growth and resulting in cell competition

NOC1 is a nucleolar protein necessary in yeast for both transport and maturation of ribosomal subunits. This study shows that Drosophila NOC1 is necessary for rRNAs maturation and for a correct animal development. Its ubiquitous downregulation results in a dramatic decrease in polysome level and of protein synthesis. NOC1 expression in multiple organs, such as the prothoracic gland and the fat body, is necessary for their proper functioning. Reduction of NOC1 in epithelial cells from the imaginal discs results in clones that die by apoptosis, an event that is partially rescued in a M/+ background, suggesting that reduction of NOC1 induces the cells to become less fit and to acquire a loser state. NOC1 downregulation activates the pro-apoptotic eiger-JNK pathway and leads to an increase of Xrp1 that results in Dilp8 upregulation. These data underline NOC1 as an essential gene in ribosome biogenesis and highlight its novel functions in the control of growth and cell competition (Destefanis, 2022).

This study has shown that the Drosophila homologs of yeast NOC1, NOC2 and NOC3 are required for animal development and their ubiquitous reduction results in growth impairment and larval lethality. Ubiquitous overexpression of NOC1 is also detrimental but at the pupal stage, a phenotype that is rescued by co-expression of NOC1-RNAi, which allows the animals to develop to small adults. These data suggest that NOC1 expression must be tightly regulated, as either its reduction or overexpression may be detrimental for the cells. As demonstrated in yeast, the function of Drosophila NOC1 is not redundant with the other NOC proteins, and its overexpression does not compensate for the loss of NOC2 and NOC3. The reason for this behavior might be because NOC proteins function as heterodimers (NOC1-NOC2 and NOC2-NOC3) that are necessary for proper control of rRNA processing and the assembling of the 60S ribosomal subunits. Indeed, it has been demonstrated in yeast that the NOC1-NOC2 complex regulates the activity of ribosomal RNA protein-5 (Rpr5), which controls rRNA cleavage at the internal transcribed spacers ITS1 and ITS2 sequences to ensure the stoichiometric maturation of the 40S and 60S ribosomal subunits. This function is likely to be conserved also in flies. In fact, the current results show that reduction of NOC1 induces the accumulation of the intermediate ITS1 and ITS2 immature forms of rRNAs. Moreover, a reduction was observed in the relative abundance of 18S and 28S rRNAs, suggesting that NOC1 is also required in flies for proper rRNA processing and ribosome maturation. In line with this hypothesis, this study demonstrated that NOC1 reduction results in a strong decrease in ribosome abundance and assembling, which is also accompanied by a strong reduction of the 80S and the polysomes. In addition, a mild accumulation was observed of the 40S and 60S subunits, suggesting that the mature 80S ribosome might be unstable in NOC1-RNAi animals and that a small percentage of the ribosome disassembles into the two subunits, leading to the observed increase. In addition, given that NOC1 was identified as a predicted transcription factor, and because reduction of NOC1 results in a robust decrease in global protein synthesis, it cannot be excluded that specific factors involved in the 80S assembling are reduced or missing in NOC1-RNAi animals (Destefanis, 2022).

Analysis of protein-protein interaction using STRING indicates that CG7838/NOC1 might act in a complex with other nucleolar proteins. Indeed, NOC1 was shown to colocalize in the nucleolus with fibrillarin. Moreover, NOC1 overexpression also results in the formation of large round nuclear structures, which are significantly reduced when its expression is decreased by NOC1-RNAi . Interestingly, similar structures have been shown for CEBPz, the human homolog of NOC1, as visible in images from 'The Human Protein Atlas'. CEBPz (also called CBF2 and CTF2; OMIM-612828) is a transcription factor member of the CAAT-binding protein family, which are involved in Hsp70 complex activation and are upregulated in tumors, particularly in cells from patients with acute myeloid leukemia (AML). As NOC1 also has the conserved CBP domain, this suggests that it might also act as a transcription factor, a hypothesis corroborated by data in Drosophila (CHIP-Seq and genetic screens) that demonstrates how its expression is associated to promoter regions of genes with a function in the regulation of nucleolar activity and of ribosomal proteins. This observation is important as it opens up the possibility that NOC1 can control ribosome biogenesis through alternative mechanisms in addition to its control over rRNA transport and maturation. Moreover, this function might be conserved for CEBPz, because in a bioinformatic analysis nucleolar components and ribosomal proteins were identified as being upregulated in liver and breast tumors with an overexpression of CEBPz. Interestingly, misexpression of some of these targets, like Rpl5 and Rpl35a, have been associated with ribosomopathies, suggesting that mutations in CEBPz could contribute to tumorigenesis in these genetic diseases (Destefanis, 2022).

To better characterize NOC1 functions in vivo, its expression was modified in organs that are relevant for Drosophila physiology, such as the prothoracic gland (PG), the FB and the wing imaginal discs (Destefanis, 2022).

Although the overexpression of NOC1 in the PG does not affect development, its reduction significantly decreased ecdysone production, as shown by E74b mRNA levels. This reduction is significant both at 5 and at 12 days AEL, and occurs concomitantly with the reduction of the PG size. Consequently, NOC1-RNAi animals are developmentally delayed and do not undergo pupariation but rather continue to wander until they die at ~20 days AEL. These animals feed constantly and increase their size, accumulating fats and sugars in the FB cells, which augment their size. Previous work described the presence of hemocytes (macrophage-like cells) infiltrating the FB of these animals, a condition accompanied with an increase in JNK signaling and reactive oxygen species (ROS), likely released by the fat cells under stress conditions. Interestingly, this intercellular event recapitulates the chronic low-grade inflammation, or adipocyte tissue macrophage (ATM), a pathology associated with adipose tissue in obese people that represents the consequence of impaired lipid metabolism (Destefanis, 2022).

Reduction of NOC1, NOC2 or NOC3 in the FB results in smaller and fewer cells, whereas reduction of NOC1 in the whole organ inhibits animal development. The FB regulates animal growth by sensing amino acids concentrations in the hemolymph and remotely controlling the release of DILP2, DILP3 and DILP5 from the IPCs. The FB also stores the nutrients (fats and sugars) that are necessary during the catabolic process of autophagy to allow animals to survive metamorphosis. When nutrients are limited, larvae delay their development to accumulate fats and sugars until reaching their critical size, which ensures they can progress through metamorphosis. NOC1 downregulation in the FB alters its ability to store nutrients, and larvae proceed poorly through development. In addition, these animals show DILP2 accumulation in the IPCs even in normal feeding conditions, indicating that the remote signals responsible for DILP release are greatly reduced, phenocopying animals in starvation or with reduced levels of MYC in fat cells. Interestingly, it was also observed that Cg-NOC1-RNAi animals accumulate an abnormal amount of fats in non-metabolic organs, such as gut, brain and imaginal discs. This finding suggests that these animals are subjected to inter-organ dyslipidemia, a mechanism of lipid transport activated when the FB function is impaired, which triggers non-autonomous signals to induce other organs to store fats. Interestingly, this condition recapitulates dyslipidemia in humans, where the compromised adipose tissue releases lipoproteins of the APO family, inducing fat accumulation in organs. Notably, a similar condition has also been described in flies for mutations in members of the APOE family, outlining how the mechanisms controlling the inter-organ fat metabolism are conserved among species (Destefanis, 2022).

NOC1 depletion in clones analyzed in the wing imaginal discs triggers their elimination by apoptosis. This event is partially rescued when clones are induced in the hypomorphic background of the Minute(3)66D/+ mutation. These cells also upregulate the pro-apoptotic gene Xrp1, recently shown to be responsible for controlling translation and indirectly cell competition upon proteotoxic stress. Reduction of NOC1 in the wing imaginal disc prolongs larval development with upregulation of DILP8 normally induced by cellular damage and apoptosis. The fact that NOC1-RNAi cells upregulate, in addition to Xrp1, eiger, another pro-apoptotic gene and member of the TNFα family, and activate the JNK pathway, suggests that different mechanisms are converging in these cells to induce apoptosis and DILP8 upregulation. Genetic epistasis experiments were performed to define the relationship between Eiger signaling in NOC1-RNAi cells and how this is linked to Xrp1 transcriptional upregulation in response to nucleolar stress and DILP8 upregulation. This analysis showed that reduction of Eiger did not significantly affect DILP8 expression induced upon NOC1 downregulation. Owing to the embryonic lethality induced by the simultaneous reduction of NOC1 and Xrp1 in cells of the wing imaginal discs, using both rotund and nubbin promoters, the contribute of Eiger to Xrp1 and DILP8 transcriptional regulation upon NOC1-RNAi was analyzed. These data indicate that DILP8 upregulation was not significantly affected by the reduction of Eiger seen upon NOC1 reduction, confirming the data in vivo with DILP8-GFP. In addition, it is predicted that Xrp1 acts independently of Eiger, since Xrp1 mRNA upregulation is not rescued in imaginal discs from NOC1-RNAi; eiger-RNAi animals, pointing out to a more upstream role for Xrp1 in controlling the stress response following reduction of NOC1; the function of Eiger remains to be determined (Destefanis, 2022).

In conclusion, the data corroborate the role of NOC1 in mechanisms that induce proteotoxic stress adding NOC1 as a novel component that links defects in protein synthesis with cell competition. This study also showed the relevance of NOC1 in promoting nucleolar stress and apoptosis, both leading cause of tumor formation. The data support a potential role for the human homolog CEBPz in the context of tumorigenesis. Indeed, mutations in CEBPz are described in >1.5% of tumors of epithelial origins, suggesting that it might have a role in contributing to the signals that trigger proteotoxic stress associated to tumorigenesis. CEBPz was also found, together with the METTL3-METTL14 methyltransferase complex, to control cellular growth and to have a role in the regulation of H3K9m3 histone methylation in response to sonication-resistant heterochromatin (srHC), highlighting it as a moonlighting protein for RNA and heterochromatin modifications (Destefanis, 2022).

Dual roles of glutathione in ecdysone biosynthesis and antioxidant function during the larval development in Drosophila

Ecdysteroid biosynthesis is achieved by a series of specialized enzymes encoded in the Halloween genes. Recently, noppera-bo (nobo), encoding a glutathione S-transferase (GST), has been identified. GSTs conjugate substrates with the reduced form of glutathione (GSH), a bioactive tripeptide composed of glutamate, cysteine, and glycine. It was hypothesized that GSH itself is required for ecdysteroid biosynthesis. This study reports phenotypic analysis of a mutant in the gamma-glutamylcysteine synthetase catalytic subunit (Gclc) gene in Drosophila. Gclc encodes the conserved catalytic component of the enzyme conjugating glutamate and cysteine in the GSH biosynthesis pathway. Loss of Gclc function leads to drastic GSH deficiency in the larval body fluid. Gclc-mutant animals show larval-arrest phenotype. Ecdysteroid titer in Gclc mutant larvae decreases, and the larval-arrest phenotype is rescued by oral administration of 20E or cholesterol. Moreover, Gclc mutant animals exhibit abnormal lipid deposition in the prothoracic gland, a steroidogenic organ during larval development. All of these phenotypes are reminiscent of nobo loss-of-function animals. On the other hand, Gclc mutant larvae also exhibit a significant reduction in antioxidant capacity. Ecdysteroid biosynthesis defect in Gclc mutant animals is not associated with loss of antioxidant function. It is hypothesized that a primary role of GSH in early D. melanogaster larval development is ecdysteroid biosynthesis, independent from antioxidant role of GSH (Enya, 2017).

Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster

Steroid hormones regulate life stage transitions, allowing animals to appropriately follow a developmental timeline. During insect development, the steroid hormone ecdysone is synthesized and released in a regulated manner by the prothoracic gland (PG) and then hydroxylated to the active molting hormone, 20-hydroxyecdysone (20E), in peripheral tissues. This study manipulated ecdysteroid titers, through temporally controlled over-expression of the ecdysteroid-inactivating enzyme, CYP18A1, in the PG using the GeneSwitch-GAL4 system in the fruit fly Drosophila melanogaster. Expression was monitored of a 20E-inducible glue protein gene, Salivary gland secretion 3 (Sgs3), using a Sgs3:GFP fusion transgene. In wild type larvae, Sgs3-GFP expression is activated at the midpoint of the third larval instar stage in response to the rising endogenous level of 20E. By first knocking down endogenous 20E levels during larval development and then feeding 20E to these larvae at various stages, it was found that Sgs3-GFP expression could be triggered at an inappropriate developmental stage after a certain time lag. This stage-precocious activation of Sgs3 required expression of the Broad-complex, similar to normal Sgs3 developmental regulation, and a small level of nutritional input. It is suggested that these studies provide evidence for a tissue-autonomic regulatory system for a metamorphic event independent from the primary 20E driven developmental progression (Kaieda, 2017).

The Drosophila CCR4-NOT complex is required for cholesterol homeostasis and steroid hormone synthesis

CCR4-NOT is a highly conserved protein complex that regulates gene expression at multiple levels. In yeast, CCR4-NOT functions in transcriptional initiation, heterochromatin formation, mRNA deadenylation and other processes. The range of functions for Drosophila CCR4-NOT is less clear, except for a well-established role as a deadenylase for maternal mRNAs during early embryogenesis. This study report here that CCR4-NOT has an essential function in the Drosophila prothoracic gland (PG), a tissue that predominantly produces the steroid hormone ecdysone. Interfering with the expression of the CCR4-NOT components twin, Pop2, Not1, and Not3 in a PG-specific manner resulted in larval arrest and a failure to initiate metamorphosis. Transcriptome analysis of PG-specific Pop2-RNAi samples revealed that Pop2 is required for the normal expression of ecdysone biosynthetic gene spookier (spok) as well as cholesterol homeostasis genes of the NPC2 family. Interestingly, dietary supplementation with ecdysone and its various sterol precursors showed that 7-dehydrocholesterol and cholesterol completely rescued the larval arrest phenotype, allowing Pop2-RNAi animals to reach pupal stage, and, to a low degree, even survival to adulthood, while the biologically active hormone, 20-Hydroxyecdysone (20E), was significantly less effective. Also, genetic evidence is presented that CCR4-NOT has a nuclear function where CCR4-NOT-depleted cells exhibit aberrant chromatin and nucleoli structures. In summary, these findings indicate that the Drosophila CCR4-NOT complex has essential roles in the PG, where it is required for Drosophila steroid hormone production and cholesterol homeostasis, and likely has functions beyond a mere mRNA deadenylase in Drosophila (Zeng, 2018).

The CCR4-NOT complex is an evolutionarily conserved protein complex found in eukaryotes. The functions of CCR4-NOT have been linked to the regulation of gene expression at nearly all levels, including histone modifications, transcriptional initiation, transcriptional elongation, heterochromatin formation, translational repression as well as the regulation of mRNA degradation. Given the versatility of the CCR4-NOT complex, many aspects of its functions remain unclear. Most studies on CCR4-NOT have been conducted in Saccharomyces cerevisiae or human cell culture systems, with only a few studies that have examined this complex in multi-cellular organisms. In Drosophila, the main components of CCR4-NOT are conserved, which include the homologs of yeast carbon catabolite repression 4 (CCR4, encoded by twin), Pop2 (also known as CAF1), NOT1, NOT2 (encoded by Regena), NOT3 and CAF40 (encoded by Rcd1). Two of the components, CCR4/Twin and Pop2 hydrolyze mRNA poly(A) tails through their deadenylase activities, which leads to translational repression followed by mRNA decay. Two other Drosophila poly(A)-specific ribonucleases, the Pan2-Pan3 complex and the poly(A)-specific ribonuclease (PARN), have both specialized and redundant functions when compared to the CCR4-NOT complex, and together control overall mRNA stability. Most studies on Drosophila CCR4-NOT have only focused on its role in regulating mRNA stability, especially during oogenesis and early embryonic development before the midblastula transition. Before the midblastula transition, embryonic development relies exclusively on maternal mRNAs; hence there is almost no contribution from transcriptional regulation. Therefore, studying CCR4-NOT in the context of oogenesis and early embryogenesis provides only limited evidence as to whether the Drosophila CCR4-NOT complex has additional roles, such as transcriptional initiation or transcriptional elongation. While the CCR4-NOT complex can target a wide range of transcripts, a plethora of adapter proteins ensures context-specific degradation of select mRNA targets, allowing for tissue- and pathway-specific control. Genome-wide RNA interference (RNAi) screens conducted in two different biological contexts showed that CCR4-NOT complex function is essential for Drosophila neuroblast development where loss of Pop2 or NOT1 in neuroblasts resulted in animal lethality prior to the adult stage, while regulation of intestinal stem cell homeostasis is not dependent on CCR4-NOT function. Thus, CCR4-NOT function is highly regulated and differs drastically depending on biological context, and as such does not act as a global mRNA deadenylase. This study was interested in the specific roles of CCR4-NOT in the PG, which is a central endocrine tissue that coordinates multiple development events via the secretion of the steroid hormone ecdysone (Zeng, 2018).

Like all holometabolous insects, the Drosophila life cycle has discrete developmental stages, and pulses of ecdysone trigger all developmental transitions such as molting and metamorphosis. In Drosophila larvae, ecdysone is synthesized in the principal steroidogenic tissue, the PG, which is part of the ring gland, an endocrine organ composed of three glands. In the PG, tissue-specific expression of ecdysone biosynthetic enzyme genes including neverland (nvd), spookier (spok), shroud (sro), Cyp6t3, phantom (phm), disembodied (dib) and shadow (sad) allow the conversion of suitable sterols (e.g., cholesterol) to ecdysone. spok, sro, Cyp6t3, phm, dib and sad are commonly referred to as the Halloween genes. To ensure that these developmental transitions occur at the appropriate time, the PG acts as a decision-making center to integrate systemic and environmental cues into an ecdysone pulse that advances development when the proper conditions are met. This study reports a requirement for the CCR4-NOT complex for the production of ecdysone and maintaining cholesterol homeostasis. PG-specific RNAi against five out of seven CCR4-NOT components resulted in larval arrest mainly in the third instar (L3), a phenotype consistent with a block in ecdysone production. Transcriptional profiling via RNA-Seq of PG-specific Pop2-RNAi ring gland samples revealed that ecdysone and cholesterol pathways were particularly affected, and that cholesterol feeding could rescue the larval arrest phenotype of Pop2-RNAi animals, strongly supporting the idea that CCR4-NOT has specialized tissue-specific functions (Zeng, 2018).

To determine the importance of the CCR4-NOT complex in the PG, a PG-specific Gal4 driver (phm22-Gal4, referred to as PG>) to express UAS-RNAi transgenes targeting all seven CCR4-NOT subunits individually. For each gene, all available TRiP-RNAi lines from Bloomington Stock Center were tested, and the results from a previously published genome-wide PG-specific RNAi screen using VDRC RNAi lines was also included. For Pop2, two non-overlapping RNAi lines (PG>Pop2-RNAi1 and PG>Pop2-RNAi2) caused comparable developmental arrest phenotypes, which validated the specificity of the RNAi constructs. 100% of the PG>Pop2-RNAi1 and ~93% of the PG>Pop2-RNAi2 animals failed to pupariate, and remained 3rd instar (L3) larvae for several weeks, which resulted in substantially increased body sizes. This L3 arrest phenotype is commonly observed when the major ecdysone pulse that triggers the onset of metamorphosis fails to occur. Of the ~7% PG>Pop2-RNAi2 larvae that attempted puparium formation, none completed metamorphosis and thus failed to eclose as adults. As for the other CCR4-NOT components, at least one RNAi line targeting Twin, Not1, Not3, or Not4 was also found to cause L3 arrest. Taken together, these results indicated that the CCR4-NOT complex is essential for PG function, possibly by contributing to regulating ecdysone production (Zeng, 2018).

Given the potential requirement of the CCR4-NOT complex at nearly all levels of gene expression, transcriptional profiling of hand-dissected ring glands was performed via RNA-Sequencing (RNA-Seq). Ring glands from PG>Pop2-RNAi1 and PG>w1118 control animals were collected at 24 h after the 2nd to 3rd instar molt. This time point was chosed because ring glands from these larvae are large enough for dissection while larvae undergo minimal physiological changes compared to later time points closer to puparium formation. Pop2-RNAi1 was used to disrupt CCR4-NOT function because the RNAi phenotype was 100% penetrant and because the phenotypes were validated by a second independent RNAi line. For each sample, 50 ring glands, which should average out any individual timing differences between animals, were collected. Total RNA samples were enriched for mRNAs by selectively depleting rRNAs, followed by Illumina sequencing (Zeng, 2018).

The L3 arrest phenotype in Pop2-RNAi animals suggested a defect in ecdysone biosynthesis, and it was of interest to see whether the RNA-Seq results would reveal any reduction in the expression of the ecdysone biosynthetic enzyme genes. Interestingly, two genes critical for ecdysone biosynthesis were among the top 50 downregulated genes, namely spookier (spok) and neverland (nvd), ranked #31 (8.7-fold down) and #41 (7.7-fold down), respectively. However, only spok was confirmed to be significant (p < 0.05) for both the original RNA-Seq result and the qPCR validation data. For validation via qPCR, the weaker Pop2-RNAi2 line was used at an earlier developmental time point (0 h L3). Next, whether these changes in mRNA levels translated into corresponding reductions of protein levels of Nvd and Spok was investigated. It was found via immunostaining that Spok is strongly reduced in Pop2-RNAi1 PG cells, consistent with the mRNA expression levels. However, no significant reduction of Nvd was observed upon Pop2 knockdown. A possible explanation is that the net effect of a (albeit non-significant) reduction in nvd transcript levels vs. increased nvd transcript stability (due to loss-of-Pop2) results in overall similar protein levels. Alternatively, while the immunodetection and RNA-Seq experiments were conducted at the same developmental time point (96 h AEL = 24 h post L2/L3 molt), there may be a lag between reduction in transcript and protein levels due to protein perdurance. Significant transcriptional upregulation was also found for sro and dib via qPCR, while phm was not significantly changed. To test whether this corresponded to elevated protein levels in the PG, Sro and Phm were examined via immunofluorescence. Of the two, only Sro appeared to have moderately increased protein levels, again mirroring the mRNA expression data. It is concluded that the ecdysone deficiency phenotype caused by PG-specific RNAi of Pop2 was likely caused by a reduction in spok functiom (Zeng, 2018).

If Pop2 only functions as a mRNA deadenylase, one would expect a stabilization of direct target mRNAs in cells with impaired Pop2 function. Therefore, genes that register as downregulated in the RNA-Seq data, such as nvd and spok, are likely the result of indirect effects, or a combination of direct and indirect effects. Likewise, upregulated genes could be the result of direct stabilization via loss-of-Pop2 or again caused by direct and/or indirect effects. To distinguish these possibilities, poly(A) tail-length assays were carried out to test whether Pop2 depletion affects poly (A) tail lengths of six Halloween genes. For this, ring gland mRNAs were tagged with oligo-G/I bases followed by reverse-transcription into cDNA using the tag as the priming site. PCR was then performed by using a universal reverse primer complementary to the tag and a gene-specific primer, resulting in ~150-300 bp fragments. As controls, the same gene-specific primer was used coupled to a second downstream gene-specific primer that binds upstream of the poly (A) tail. This will give rise to an amplicon without the poly (A) tail, and allows estimation of the poly (A) tail length. Lengthening of the poly (A) tail was observed for spok, phm and dib transcripts in a Pop2-RNAi1 background relative to controls, indicating that these three Halloween transcripts are direct targets of Pop2. Since spok is showing a poly(A) upshift, but strongly downregulated both at the transcriptional and protein levels, it is concluded that while spok is a direct target of Pop2, there might a superimposed transcriptional effect in a Pop2 loss-of-function background. The results for nvd (repeated in two independents assays) were inconclusive. While nvd showed a clear upshift, a second band with little or no increased length was also present. In contrast, both sro and sad showed no lengthening of the poly(A) tail, indicating that the CCR4-NOT complex regulates only select Halloween transcripts (Zeng, 2018).

Since spok transcript levels are downregulated, two possibilities are seen that may explain this. First, it is conceivable that the CCR4-NOT complex acts directly as a transcriptional modulator of spok expression, in line with reported nuclear functions of CCR4-NOT in yeast. However, it is equally possible that mRNAs encoding transcriptional regulators of the Halloween genes are regulated by CCR4-NOT, and the resulting stabilization of such transcriptional regulators upon Pop2 knockdown would then indirectly alter Halloween gene expression. In fact, many such transcription factors have been identified, the most recent example being Kr-h1, which negatively regulates the transcription of the Halloween genes. Interestingly both nvd and spok are strongly downregulated by Kr-h1 overexpression, suggesting that both genes are coordinately regulated by this and possibly other repressors. To conclude, regardless of the complex regulation and possible crosstalk between the CCR4-NOT and transcriptional regulators, the net outcome of PG>Pop2-RNAi is the specific reduction of Spok (Zeng, 2018).

Nvd catalyzes the first step of ecdysone biosynthesis where it converts cholesterol to 7-dehydrocholesterol (7DC). Spok plays a crucial role in the 'Black Box', which consists of several not yet characterized conversion steps. However, the net result of the Black Box is that 5β-ketodiol (5βkd) is derived from 7DC. To test the idea that the ecdysone deficiency phenotype observed in PG>Pop2-RNAi animals was caused directly by reduced levels of spok and possibly nvd, fly media supplemented with either 7DC, 5βkd or the biologically active form of ecdysone (20E = 20-Hydroxyecdysone) was used. Remarkably, 7DC alone rescued the L3 arrest phenotype and enabled nearly 80% of larvae to reach the pupal stage. The rescue of the L3 arrest phenotype by 7DC was observed in both PG>Pop2-RNAi lines, demonstrating the specificity of the results. Intriguingly, compared to 7DC, the rescue by 20E (the end product of ecdysone biosynthesis) was much less pronounced and not significant in PG>Pop2-RNAi1 lines. Similarly, 20E did not rescue PG>Pop2-RNAi2 animals. Moreover, combining 7DC and 20E significantly lowered pupal survival compared to 7DC alone, suggesting that 20E has some adverse effect or was neutralizing 7DC efficiency in a PG>Pop2-RNAi background. It should be noted that 20E-supplementation rescues other Halloween gene loss-of-function animals (including PG>spok-, sro-, Cyp6t3-, phm-, dib- and sad-RNAi lines) very effectively and is not considered toxic per se. For instance, 20E can rescue the L2 arrest phenotype of PG>sro-RNAi larvae all the way to adulthood (~31% adult survival). Interestingly, it was recently reported that séance mutants selectively affect nvd expression, and can be effectively rescued with 7DC, but not 20E, consistent with the observation for Pop2-RNAi lines. When 5βkd-supplementation was used, no further improvement was seen, as most of the PG>Pop2-RNAi1 animals still could not develop beyond the larval stages. Thus, similar to 20E, rescue of PG>Pop2-RNAi animals via 5βkd was inferior to 7DC. A likely explanation for this is that 7DC feeds into pathways other than ecdysone production, and that Pop2 function is required for these undefined roles (Zeng, 2018).

Given that 7DC is a cholesterol metabolite, it is of interest to note that three Niemann-Pick disease type C (NPC) genes, npc2f, npc2d, and npc2c were upregulated 6.8-fold (ranked #41), 5.1-fold and 3.9-fold respectively in Pop2-RNAi samples compared to control ring gland samples. NPC proteins play critical roles in the intracellular transport of cholesterol and cholesterol metabolites, raising the idea that 7DC-supplementation compensates for this aberrant upregulation of NPC genes. Alternatively, NPC2 upregulation may compensate for an issue with cholesterol uptake or transport in Pop2-RNAi cells, an issue that could also be rescued by 7DC feeding since 7DC is the immediate metabolites of cholesterol. Consistent with the coordinated upregulation of three NPC2 genes, 'intracellular cholesterol transport' was identified as one of the functional association subgroups in the functional interaction analysis, where a subset of genes was used from RNA-Seq data with > 3-fold upregulation in Pop2-RNAi ring glands compared to controls. To test whether cholesterol supplementation was as effective as 7DC, feeding experiments were carried out using fly media supplemented with cholesterol. Remarkably, cholesterol rescued ~33% of the Pop2-RNAi1 animals to the pupal stage, and ~3% of the RNAi larvae were even rescued to adulthood, a result that could be confirmed with the second Pop2-RNAi line. Overall, cholesterol was as effective as 7DC in rescuing the larval arrest phenotypes, indicating that nvd is functioning normally in PG>Pop2-RNAi animals since Nvd is required for converting cholesterol to 7DC. In conclusion, three NPC2 genes linked to intracellular sterol transport appear to be misregulated upon Pop2-RNAi, indicating deregulation of cholesterol homeostasis, and consistent with this, both cholesterol and its immediate downstream metabolite, 7DC, are significantly more effective than 20E in rescuing PG-specific Pop2-depleted animals (Zeng, 2018).

When significantly altered genes were analyzed in the RNA-Seq datasets, it was noticed that the number of upregulated genes in PG>Pop2-RNAi ring glands were consistently higher than the downregulated genes, regardless of which cut-offs were used. For instance, the number of > 2.5-fold upregulated genes more than doubled the number of > 2.5-fold downregulated genes. This trend is consistent with the idea that Pop2 might primarily act to negatively regulate mRNA levels through deadenylation, and a decrease in RNA degradation would thus register as a higher percentage of upregulated genes (Zeng, 2018).

Next, functional association networks were generated of differentially expressed genes using STRING database to identify processes that are affected by the loss of Pop2 function. Genes were grouped within the interaction networks according to their gene ontology (GO) terms from DAVID GO and Flybase. For the 197 > 3.5-fold upregulated genes in the Pop2-RNAi ring gland compared to control samples, this analysis revealed genes associated with several functional groups: including 'transcription' (as well as 'splicing' and 'ribosome biogenesis'), 'mitochondrial translation', 'intracellular cholesterol transport', 'proteolysis', 'lipid catabolic process' and 'glycine/serine/threonine metabolism'. To complement this functional interaction analysis, gene ontology (GO) term and KEGG pathway enrichment analysis were performed via both DAVID GO and STRING database using a less stringent cut-off of > 2.5-fold upregulated genes (total 536 genes). Consistent with the results of the functional association study, genes that are involved in transcription as well as genes encoding mitochondrialribosomal proteins and mitochondrial respiratory chain complex III component were significantly overrepresented (Zeng, 2018).

For the total of 196 > 3-fold downregulated genes, several functional groups were also identified, including: 'translation', 'mitochondria', 'splice site/transcriptional start site selection', 'actin filament-related' and 'proteolysis'. The term enrichment analysis using the> 2-fold (total 296 genes) downregulated genes confirmed that genes involved in 'cytoplasmic translation' (5 out of 8 genes are ribosomal genes) and 'mitochondrial protein complex' were significantly enriched among differentially downregulated genes. Interestingly, two genes linked to 'cytoplasmic translation' encode proteins acting in the target of rapamycin (TOR) signaling pathway, namely Rheb and the eukaryotic translationinitiation factor (eIF) subunit 4E-binding protein (4E-BP) gene (Thor in Drosophila), which are both ~3.9-fold downregulated in Pop2-RNAi ring glands. This is intriguing because recently it was shown that TOR not only acts in amino acid sensing but is also activated by cellular cholesterol, consistent with the idea that CCR4-NOT has hitherto unknown links to cellular cholesterol homeostasis. In line with the misregulation in 4E-BP and Rheb, Pop2-RNAi ring glands displayed growth defects in a nutrient-dependent manner. Pop2-RNAi ring glands were normal when larvae were reared on yeast but had a reduced size when the Nutri-Fly Bloomington formula or a standard cornmeal-based recipe were used. It is pointed out that larvae with smaller ring glands had equal (standard cornmeal) or better survival rates (Nutri-Fly) compared to those with normal-sized ring glands (yeast) (Zeng, 2018).

When PG nuclei were analyzed via DAPI staining to assess the ring gland morphology, it was found that Pop2-RNAi animals exhibited uneven and fragmented DAPI signals, resulting in a region with reduced DAPI staining. In some nuclei, the chromatin was arranged in a way that resulted in a petal-like appearance. In wild-type cells, nuclear zones that stain weakly for DAPI correspond to the nucleolus, where rRNA synthesis and ribosomebiogenesis occurs. Since this observation was consistent with the functional association analysis ('ribosome biogenesis'), propidium iodide (PI) staining was carried out to visualize the nucleolus (because PI also binds RNA in addition to DNA). Interestingly, PI staining of Pop2-RNAi PG cells revealed some nucleoli that had a distinct 'hollow sphere' appearance, a phenotype that was never observed in controls. Chromatin fragmentation and compaction were also apparent in PI staining of Pop2-RNAi nuclei, consistent with the DAPI results. These nuclear phenotypes of Pop2-RNAi PG cells are consistent with finding in fission yeast that showed involvement of CCR4-NOT in heterochromatin formation. It, therefore, appears likely that Pop2 has a direct nuclear function that is critical for accessibility of the transcriptional machinery to DNA. It is concludeed that it is very likely that the Drosophila CCR4-NOT complex also has additional functions beyond its well-documented deadenylase activity (Zeng, 2018).

This study revealed that the function of the CCR4-NOT deadenylase complex is essential for ecdysone biosynthesis and a normal nuclear structure in the PG. Disrupting CCR4-NOT function specifically in the PG via Pop2-RNAi strongly reduced mRNA and protein levels of the ecdysone enzyme gene spok. Transcriptome analysis of a Drosophila tissue with impaired CCR4-NOT function is presented in this study. Term enrichment analysis of these genomic data suggested a defect in cholesterol homeostasis, which is consistent with the finding that the L3 arrest phenotype in PG>Pop2-RNAi animals was rescued by cholesterol and 7DC feeding. Moreover, Pop2-depletion in the PG appeared to affect the structure of both chromatin and the nucleolus, suggesting CCR4-NOT also has nuclear functions, consistent with previous reports in yeast. It is concluded that CCR4-NOT has tissue-specific roles and is likely a multifunctional protein complex that has functions beyond the mRNA deadenylase in Drosophila (Zeng, 2018).

Egfr signaling is a major regulator of ecdysone biosynthesis in the Drosophila prothoracic gland

Understanding the mechanisms that determine final body size of animals is a central question in biology. In animals with determinate growth, such as mammals or insects, the size at which the immature organism transforms into the adult defines the final body size, as adult individuals do not grow. In Drosophila, the growth period ends when the immature larva undergoes the metamorphic transition to develop the mature adult. This metamorphic transition is triggered by a sharp increase of the steroid ecdysone, synthetized in the prothoracic gland (PG), that occurs at the end of the third instar larvae (L3). It is widely accepted that ecdysone biosynthesis in Drosophila is mainly induced by the activation of tyrosine kinase (RTK) Torso by the prothoracicotropic hormone (Ptth) produced into two pairs of neurosecretory cells that project their axons onto the PG. However, the fact that neither Ptth nor torso-null mutant animals arrest larval development but only present a delay in the larva-pupa transition mandates for a reconsideration of the conventional model. This study shows that Egfr signaling, rather than Ptth/torso, is the major contributor of ecdysone biosynthesis in Drosophila. Egfr signaling was found to be activated in the PG in an autocrine mode by the EGF ligands spitz and vein, which in turn are regulated by the levels of ecdysone. This regulatory positive feedback loop ensures the production of ecdysone to trigger metamorphosis by a progressive Egfr-dependent activation of MAPK/ERK pathway, thus determining the animal final body size (Cruz, 2020).

In contrast to the developmental delay phenotype observed in larvae with reduced Ptth or torso, this study foudn that specific depletion of Drosophila homolog transducers ras(ras85D), Raf oncogene (Raf), and ERK, the core components of the MAPK/ERK pathway, in the prothoracic gland (PG) using the phmGal4 driver (phm>) induced developmental arrest at L3. This result suggests that additional RTKs might play important roles in ecdysone production. To study this possibility, all known Drosophila RTKs in the PG were knocked down and found that only depletion of Egfr phenocopied L3 arrested development observed in phm > ras85DRNAi larvae. Likewise, overexpression in the PG of a dominant-negative form of Egfr (EgfrDN) or depletion of the transcription factor pointed (pnt), the principal nuclear mediator of the Egfr signaling pathway, also resulted in arrested L3 larvae. The same results were obtained upon inactivation of Egfr or different components of the MAPK/ERK pathway using an alternative PG specific driver, amnc651Gal4. Consistent with the observed phenotypes, overexpression of a constitutively activated form of either Egfr (Egfract) or Pnt (PntP2VP16) in the PG induced premature pupariation and reduced pupal size. These results are in agreement with a previous report showing that overexpression of a constitutively activated form of Ras (RasV12) in the PG produced the same phenotype. Furthermore, overexpression of RasV12 in Egfr-depleted larvae rescued the developmental arrest phenotype and forced premature pupation. These results strongly suggest that Egfr signaling in the PG is required for the synthesis of the ecdysone pulse that triggers metamorphosis. Confirming this hypothesis, ecdysone titers in larvae depleted of either Egfr or pnt in the PG were dramatically reduced. Accordingly, Hr3 and Hr4 expression, two direct target genes of the hormone that have been used as readouts for ecdysone levels, was completely abolished in phm > EgfrRNAi and phm > pntRNAi L3 larvae compared to control animals. Moreover, addition of the active form of ecdysone, 20-hydroxyecdysone (20E), to the food rescued the developmental arrest phenotype induced by inactivation of Egfr signaling in the PG. Altogether, these results indicate that Egfr signaling in the PG endocrine cells is required for the production of the ecdysone pulse that triggers pupariation and fixes adult body size (Cruz, 2020).

Since Egfr signaling is involved in cell proliferation and survival, this study analyzed whether the above-described phenotype was due to compromised viability of PG cells. Although reduced activation of Egfr signaling diminished cell size, PG cell number and viability were not affected. Interestingly, ecdysone synthesis has been recently shown to correlate with endocycle progression and therefore cell size of PG cells. PG cells undergo three rounds of endoreplication during larval development resulting in chromatin values (C values) of 32-64 C by late L3. Remarkably, a clear reduction was observed in the C value of PG cells of phm > EgfrRNAi larvae at 120 h AEL, with most cells at 8-16 C, indicating that Egfr activation is also required to promote polyploidy in the PG cells (Cruz, 2020).

This result raised the possibility that Egfr signaling regulates ecdysone production by determining the size of the PG. To analyze this hypothesis, the effect of Egfr signaling in ecdysone production was examined. Steroidogenesis in the PG cells depends on the timely expression of ecdysone biosynthesis enzyme-encoding genes that mediate the conversion of cholesterol to ecdysone. To analyze whether Egfr signaling controls ecdysone synthesis by regulating the expression of these genes, qRT-PCR was performed in early (72 h after egg laying [AEL]), mid (96 h AEL), and late (120 h AEL) phm > EgfrRNAi and phm > pntRNAi L3 larvae. Whereas expression of the six ecdysone biosynthetic genes increased gradually from mid to late L3 in control larvae, correlating with the production of the high-level ecdysone pulse that triggers metamorphosis, inactivation of the Egfr pathway in the PG resulted in a dramatic reduction in the expression levels of neverland (nvd), spook (spo), shroud (sro), and phantom (phm) in late L3 larvae. In contrast, the expression of disembodied (dib) and shadow (sad) was not significantly reduced in Egfr-depleted larvae, which suggests that compromising Egfr signaling in the PG does not result in a general reduction in the transcriptional activity by its minor C value, as previously shown, but rather by a specific transcriptional effect. Further confirming this point, the overexpression of CycE in Egfr-depleted PGs was unable to restore normal expression of ecdysteroid biosynthetic genes nor induced proper pupariation of these animals, indicating that Egfr signaling is required for proper expression of ecdysone enzyme-encoding genes independently of promoting polyploidy of PG cells (Cruz, 2020).

As the levels of circulating ecdysone are influenced by the rates of hormone production and release, whether Egfr signaling also regulates ecdysone secretion was studied. Recently, it has been shown that ecdysone secretion from the PG cells is mediated by a vesicular regulated transport mechanism. After its synthesis, ecdysone is loaded through an ATP-binding cassette (ABC) transporter, Atet, into Syt1-positive secretory vesicles that fuse to the cytoplasmic membrane for release of the hormone in a calcium-dependent signaling. To analyze the role of Egfr signaling in this process, secretory vesicles were visualized in PG cells of phm > EgfrRNAi and phm > pntRNAi L3 larvae by expressing eGFP-tagged Syt1 (Syt-GFP) in these glands. Whereas Syt-GFP vesicles accumulate at the plasma membrane with a small number of vesicles in the cytoplasm in wild-type L3 larval PGs, a dramatic accumulation of Syt-GFP vesicles in the cytoplasm was observed in PGs with reduced Egfr signaling. Similar results were obtained when the subcellular localization of the ecdysone transporter Atet-GFP was analyzed. Consistently, overexpression of rasV12 in PGs of phm > EgfrRNAi larvae restored the subcellular localization of both Syt and Atet-GFP. Furthermore, mRNA levels of several genes involved in vesicle-mediated release of ecdysone, including Syt and Atet, were dramatically downregulated in the PG of phm > EgfrRNAi and phm > pntRNAi larvae. Therefore, the results show that Egfr signaling is also required for the vesicle-mediated release of ecdysone from PG cells. Interestingly, direct effects of Egfr signaling on the endocytic machinery have been already described in Drosophila tracheal cells as well as in human cells (Cruz, 2020).

The next question was to determine which of the EGF ligands were responsible for the Egfr pathway activation in the PG. In Drosophila, Gurken (Gur), Spitz (Spi), Keren (Krn), and Vein (Vn) serve as ligands for Egfr. Expression analysis of the four ligands revealed that only vn and spi were expressed in the PGs. Consistently, the intramembrane protease rhomboid (rho), which is necessary for the proteolytic activation of Spi, was also expressed in the PG cells. A temporal expression pattern of staged L3 PGs revealed that rho expression progressively increased during the last larval stage, while the expression of spi and vn increased sequentially, with vn upregulated at mid L3 and spi at late L3. Consistent with the expression of the ligands, mRNA levels of Egfr also showed a clear upregulation by late L3. Likewise, a specific expression of PntP2 isoform was also observed in the PG of late L3 larvae. Altogether, these results suggest that Vn and Spi might activate Egfr signaling in an autocrine manner to induce ecdysone production (Cruz, 2020).

To determine the functional relevance of each ligand, vn, spi, or both simultaneously were knocked down in the PG. As in the case of phm > EgfrRNAi, depletion of spi, vn, or both ligands at the same time caused developmental arrest in L3, although Spi appeared to have a minor effect as around 40% of phm > spiRNAi larvae underwent delayed pupariation. The attenuated effect of spi-depleted animals was probably due to a weaker effect of the spiRNAi lines as depletion of the Spi-processing protease rho in the PG resulted in all phm > rhoRNAi animals arresting development at L3. Importantly, ecdysteroid levels in mid and late L3 were significantly reduced in animals depleted of either vn or spi. Consistent with their role in controlling ecdysone production, overexpression of either Vn or an active-cleaved form of Spi in the PG induced precocious pupariation and smaller pupae. Altogether, these findings show that spi and vn act in an autocrine manner as Egfr ligands in the PG to induce ecdysone biosynthesis during the last larval stage. In fact, the correlation between vn and spi expression with the occurrence of increasing levels of ecdysteroids points to a possible positive-feedback loop regulation with 20E inducing vn and spi expression. Consistent with this possibility, vn and spi mRNA levels were reduced in PGs of ecdysteroid deficient larvae that were generated by depleting spo (phm > spoRNAi) or by overexpressing a dominant-negative form of the ecdysone receptor (phm > EcRDN). Moreover, staged PGs were cultured for 6 h ex vivo in presence or absence of 20E, and vn and spi mRNA levels were found to be significantly upregulated in the presence of the hormone. Altogether, these observations demonstrate that ecdysone exerts a positive-feedback effect on PG cells amplifying its own synthesis by inducing the expression of vn and spi. This result is consistent with a previous proposed model of ecdysone regulation in an autonomous mechanism by a positive feedback and biogenic amines. Thus, a model is proposed in which increasing levels of ecdysone promote the expression of vn and spi in the PG cells, which, in turn, increases Egfr signaling in this gland in an autocrine manner to further promote the production of ecdysone. Interestingly, it has been already shown that expression of Spi and Vn in midgut cells of Drosophila depends on ecdysone activity during metamorphosis. In addition, in vertebrates, other hormones have been postulated to control Egfr activity, such as Thyrotropin-releasing hormone, which induced the phosphorylation and activation of the Egf receptor, leading to specific transcriptional events in GH3 pituitary cells. Likewise, the Growth Hormone modulates Egfr trafficking and signaling by activating ERKs (Cruz, 2020).

Thus far, the results above show that MAPK/ERK pathway is a central regulatory element in the control of ecdysone biosynthesis in the PG, with Egfr signaling chiefly contributing to its activity. However, since Ptth/torso signaling operates through the same MAPK/ERK pathway the relative contribution of this signaling pathway in the overall activity of the PG was investigated. The fact that inactivation of Egfr signaling in the PG did not affect the mRNA expression levels of either Ptth or torso points to a minor contribution of Ptth/torso signaling in the overall MAPK/ERK activity. To analyze this possibility, the levels were compared of dpERK, a readout of MAPK/ERK activity, in PGs of phm > EgfrRNAi and phm > torsoRNAi larvae. A dramatic reduction of dpERK levels was observed in PGs of phm > EgfrRNAi larvae. Importantly, dpERK levels were also reduced in phm > torsoRNAi PGs, although to a significant lesser extent when compared to phm > EgfrRNAi larvae. Similar results were observed when nuclear accumulation of dpERK was analyzed in both larvae. Consistently, the level of activity of the MAPK/ERK pathway in phm > pntRNAi and phm > torsoRNAi larvae correlated very well with expression of the biosynthetic enzyme phm and the ecdysone-responsive genes Hr3, Hr4, and Broad-Complex (BrC), although the levels of ecdysone were significantly reduced in both cases. The different level of activation of dpERK by Egfr and Ptth/torso signaling was also consistent with the respective accumulation of Syt-GFP and Atet-GFP vesicles at the cytoplasm and the reduction of the C value of PG cells. Finally, it is important to note that the level of activity of the MAPK/ERK pathway correlated with the respective phenotypes upon inactivation of each pathway, with phm > EgfrRNAi larvae arresting development at L3 and phm > torsoRNAi larvae presenting only a delay in the pupariation time. In line with this, whereas over-activation of Egfr pathway in the PG of phm > torsoRNAi larvae induced a significant advancement in pupariation, the expression of a constitutively activated form of Torso (torsoD4021 mutants) in PGs with depleted Egfr (EgfrRNAi; torso D4021) was not able to induce precocious pupariation (Cruz, 2020).

Overall, these results show that the Egfr signaling pathway plays the main role in the biosynthesis of ecdysone by activating the MAPK/ERK pathway in the PG during mid-late L3, whereas Ptth/torso signaling acts synergistically only to increase the MAPK/ERK pathway activity thus accelerating developmental timing. In this regard, it is possible that the different strength of MAPK/ERK activation by the two signaling pathways might underline this distinct requirement of each pathway. Furthermore, temporal expression of the Egfr and Torso ligands may also contribute to the difference strengths of MAPK/ERK activation, as EGF ligands vn and spi are highly expressed during L3, whereas Ptth is only upregulated at a specific developmental time, the wandering stage. Taken together, these data suggest a model in which the increasing circulating levels of ecdysone during the last larval stage are induced by a progressive Egfr dependent activation of MAPK/ERK in the PG, whereas Ptth/torso signaling further regulates ecdysone production by integrating different environmental signals such as nutritional status, crowding conditions, and light. It is important to note that, in addition to the Egfr and Ptth/torso pathways, ecdysone biosynthesis is also regulated by the insulin/insulin-like growth factor signaling (IIS)/target of Rapamycin (TOR) signaling pathway. However, in contrast to the major role of Egfr controlling ecdysteroid levels during mid-late L3, including the strong ecdysteroid pulse that triggers pupariation, the main effect of IIS/TOR pathway is to control the production of the small ecdysteroid peak that is associated to the nutrition-dependent critical weight checkpoint that occurs at the very early L3. Thus, decreasing the IIS/TOR activity in the PG delays the critical weight checkpoint, slowing development and delaying pupariation, while increasing IIS/TOR activity in the gland induces precocious critical weight and accelerates the onset of metamorphosis. Nevertheless, it is conceivable that the increasing levels of ecdysone at the critical weight checkpoint might initiate the expression of the Egf ligands, that in turn activates the ecdysone production during mid-late L3 (Cruz, 2020).

Finally, since no role of Ptth/torso signaling has been characterized in hemimetabolous insects, it is postulated that Egfr signaling might be the ancestral ecdysone biosynthesis regulator, whereas Ptth/torso signaling has probably been co-opted in holometabolous insects during evolution to fine-tune the timing of pupariation in response to changing environmental cues. Consistent with this view, depletion of Gb-Egfr in the hemimetabolous insect Gryllus bimaculatus, where no Ptth/torso has been described, results in arrested development by the last nymphal instar. Therefore, this double regulation in holometabolous insects might provide developmental timing plasticity contributing to an appropriated adaptation to a time-limited food supply (Cruz, 2020).

Juvenile hormone regulation of Drosophila aging

Juvenile hormone (JH) has been demonstrated to control adult lifespan in a number of non-model insects where surgical removal of the corpora allata eliminates the hormone's source. In contrast, little is known about how juvenile hormone affects adult Drosophila melanogaster. Previous work suggests that insulin signaling may modulate Drosophila aging in part through its impact on juvenile hormone titer, but no data yet address whether reduction of juvenile hormone is sufficient to control Drosophila life span. This study adapted a genetic approach to knock out the corpora allata in adult Drosophila melanogaster and characterized adult life history phenotypes produced by reduction of juvenile hormone. With this system potential explanations were tested for how juvenile hormone modulates aging.A tissue specific driver inducing an inhibitor of a protein phosphatase was used to ablate the corpora allata while permitting normal development of adult flies. Corpora allata knockout adults had greatly reduced fecundity, inhibited oogenesis, impaired adult fat body development and extended lifespan. Treating these adults with the juvenile hormone analog methoprene restored all traits toward wildtype. Knockout females remained relatively long-lived even when crossed into a genotype that blocked all egg production. Dietary restriction further extended the lifespan of knockout females. In an analysis of expression profiles of knockout females in fertile and sterile backgrounds, about 100 genes changed in response to loss of juvenile hormone independent of reproductive state. It is concluded that reduced juvenile hormone alone is sufficient to extend the lifespan of Drosophila melanogaster. Reduced juvenile hormone limits reproduction by inhibiting the production of yolked eggs, and this may arise because juvenile hormone is required for the post-eclosion development of the vitellogenin-producing adult fat body. These data do not support a mechanism for juvenile hormone control of longevity simply based on reducing the physiological costs of egg production. Nor does the longevity benefit appear to function through mechanisms by which dietary restriction extends longevity. Transcripts were identified that change in response to juvenile hormone independent of reproductive state and suggest these represent somatically expressed genes that could modulate how juvenile hormone controls persistence and longevity (Yamamoto, 2013).

MicroRNA miR-8 promotes cell growth of corpus allatum and juvenile hormone biosynthesis independent of insulin/IGF signaling in Drosophila melanogaster

The Drosophila melanogaster corpus allatum (CA) produces and releases three types of sesquiterpenoid hormones, including juvenile hormone III bisepoxide (JHB3), juvenile hormone III (JH III), and methyl farnesoate (MF). JH biosynthesis involves multiple discrete enzymatic reactions and is subjected to a comprehensive regulatory network including microRNAs (miRNAs). Using a high throughput sequencing approach, abundant miRNAs were identified in the D. melanogaster ring gland, which consists of the CA, prothoracic gland, and corpus cardiaca. miR-8 was identified as a potential candidate for regulation of metamorphosis and its role in the CA was further studied. Overexpression of miR-8 in the CA increased cell size of the gland and expression of Jhamt (a gene coding for a key regulatory enzyme in JH biosynthesis), resulting in pupal lethality. By contrast, sponge-mediated reduction of miR-8 in the CA decreased cell size and Jhamt expression, but did not cause lethality. Further investigation revealed that miR-8 promotes cell growth independent of insulin/IGF signaling. Taken together, these experiments show that miR-8 is highly expressed in the CA and exerts its positive effects on cell growth and JH biosynthesis (Zhang, 2021).

Mitochondrial iron supply is required for the developmental pulse of ecdysone biosynthesis that initiates metamorphosis in Drosophila melanogaster

Synthesis of ecdysone, the key hormone that signals the termination of larval growth and the initiation of metamorphosis in insects, is carried out in the prothoracic gland by an array of iron-containing cytochrome P450s, encoded by the halloween genes. This study shows that mutants in Drosophila mitoferrin (dmfrn), the gene encoding a mitochondrial carrier protein implicated in mitochondrial iron import, fail to grow and initiate metamorphosis under dietary iron depletion or when ferritin function is partially compromised. In mutant dmfrn larvae reared under iron replete conditions, the expression of halloween genes is increased and 20-hydroxyecdysone (20E), the active form of ecdysone, is synthesized. In contrast, addition of an iron chelator to the diet of mutant dmfrn larvae disrupts 20E synthesis. Dietary addition of 20E has little effect on the growth defects, but enables approximately one-third of the iron-deprived dmfrn larvae to successfully turn into pupae and, in a smaller percentage, into adults. This partial rescue is not observed with dietary supply of ecdysone's precursor 7-dehydrocholesterol, a precursor in the ecdysone biosynthetic pathway. The findings reported in this study support the notion that a physiological supply of mitochondrial iron for the synthesis of iron-sulfur clusters and heme is required in the prothoracic glands of insect larvae for steroidogenesis. Furthermore, mitochondrial iron is also essential for normal larval growth (Llorens, 2015).

Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. This study shows that two specific isoforms of TRPA1 in Drosophila are H2O2 sensitive and that they can detect strong UV light via sensing light-induced production of H2O2. Ectopic expression of these H2O2-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H2O2-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H2O2-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. Corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H2O2-sensitive dTRPA1 isoforms; they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H2O2 production but not conventional phototransduction molecules. Thsese results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H2O2. It is speculated that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response (a function of CC cells) when they encounter strong UV, an aversive stimulus for young larvae (Guntur, 2015).

This report describes the finding that UV can activate cells via signaling an H2O2-dTRPA1 photochemical pathway. Two specific isoforms of dTRPA1 in Drosophila are H2O2 sensitive, and ectopically expressing them is sufficient to confer both H2O2 and UV light sensitivity to several types of light-insensitive cells. In particular, expressing the H2O2-sensitive TRPA1 isoform in a group of proboscis extension-controlling motor neurons was as potent as ChR2 in permitting light to activate the proboscis extension response. The efficacy of the H2O2-dTRPA1 pathway in conferring UV sensitivity by was further confirmed by demonstrating that CC cells, a group of cells that express these isoforms endogenously, were sensitive to UV. CC cells' UV response were shown to critically depended on dTRPA1 and H2O2 but did not require the conventional phototransduction molecules or Gr28b. This finding is the first to show that specific isoforms of TRPA1 channels can sense UV (and also blue light) via using their H2O2 sensitivity, and it raises the intriguing possibility of using these dTRPA1 isoforms as new optogenetics tool (Guntur, 2015).

What is the mechanism that allows specific dTRPA1 isoforms to sense H2O2 and consequently UV? A comparison of the protein sequences of different dTRPA1 isoforms showed that the two H2O2-sensitive isoforms contain a stretch of 97 aa at the N terminus that is absent in the insensitive one, whereas at least one of the H2O2-sensitive isoforms shares the same C terminus as the insensitive one. Thus, it seems the critical residue(s) that confers H2O2/UV sensitivity might reside at the N terminus of the H2O2-sensitive isoforms. The cysteine residue at the N terminus is of particular interest because H2O2 is known to oxidize cysteine, and covalent modification of cysteine residues has been shown to be able to activate TRPA1. Interestingly, although structure-function analysis of mammalian TRPA1 has implicated that H2O2 and AITC may modify the same cysteine residues, the current results suggest that this rule may not apply to Drosophila TRPA1, because at least one of the dTRPA1 isoforms, dTRPA1(B)10a, showed robust AITC sensitivity but little, if any, H2O2 sensitivity (Guntur, 2015).

One natural question raised by these findings is that why the C4da neurons require Gr28b to sense UV despite the fact that they might also express the H2O2-sensitive isoforms: the 'A' promoter for dTRPA1 has been shown to be active in these neurons. One possibility is that the level of dTRPA1 expression in C4da neurons is too low, because the same dTRPA1 antibody detected a clear signal in CC cells but none in C4da neurons. Another possibility is that C4da neurons may express molecules that inhibit dTRPA1 sensitivity to H2O2, or molecules that rapidly degrade H2O2. Furthermore, it is also conceivable that C4da neurons may not express the H2O2-sensitive isoforms of dTRPA1 despite the fact that the promoter for these isoforms appears active in them. Regardless of the exact reasons for C4da neurons' lack of dependency on H2O2 for light sensing, it is speculated their reduced H2O2 sensitivity causes them to critically depend on Gr28b to activate dTRPA1 in response to light. It is noted that although recent reports have suggested that C4da can sense H2O2 at certain developmental stages, this study found that H2O2 sensitivity of C4da was significantly lower than that of CC cells (e.g., CC cells responded well to 5 μM H2O2, whereas C4da neurons showed no significant responses to 50 μM H2O2). Identifying the exact isoforms of dTRPA1 expressed in C4da neurons and determining how they interact with Gr28b are important next steps to address the question of why light-induced H2O2 production is not sufficient to confer light responses in C4da neurons (Guntur, 2015).

What is the physiological relevance of CC cells' UV response? Because CC cells are known to express and release the adipokenetic hormone (AKH) that can accelerate heart rate and mobilize sugar into the hemolymph, and that UV is a known aversive stimulus for young Drosophila larvae, it is proposed that CC cells' UV sensitivity may act to promote stress response when young larvae encounter strong UV. It is worth noting that although the sensitivity of CC cells for UV is not high -- the lowest that has been seen with GCaMP6 reporter is ∼80 μW/mm2, it may nonetheless be sensitive enough to detect sunlight on earth because one report has suggested that UV of sunlight may reach ~75 μW/mm2 in some regions on earth. In addition, although this work primarily focused on CC cells' UV sensitivity, CC cells responded to strong blue light also. Thus, CC cells likely respond to multiple spectrums of sunlight (blue, violet, UV), and their sensitivity to UV reflects only a fraction of their true light sensitivity. Indeed, electrophysiological recording showed that CC cells were capable of responding to 1 mW/mm2 white light emitted from a xenon lamp, a light source whose spectrum resembles that of sunlight received on the earth surface. While CC cells are residing in the ring gland as opposed to the body surface, given the transparency of the larval cuticle and the anatomical location of the ring gland (they are located directly above the brain lobes), it is conceivable that light can readily reach and activate these cells. It is also noted that CC cells from at least one other insect species have also been implicated to sense light as EM analysis revealed that they contained rhabdomeres, the bona fide light-sensing organelles. Thus, light sensitivity may be a common feature of these cells, especially among insects that have transparent cuticles (Guntur, 2015).

Regulation of Drosophila Long-Term Courtship Memory by Ecdysis Triggering Hormone

Endocrine state is an important determinant of learning and memory in animals. In Drosophila, rejection of male courtship overtures by mated females leads to an aversive response manifested as courtship memory. This study reports that ecdysis triggering hormone (ETH) is an obligatory enabler of long-term courtship memory (LTM). ETH deficiency suppresses LTM, whereas augmented ETH release reduces the minimum training period required for LTM induction. ETH receptor knockdown either in the mushroom body (MB) γ lobe or in octopaminergic dorsal-anterior-lateral (DAL) neurons impairs memory performance, indicating its direct action in these brain areas. Consistent with these findings, brain exposure to ETH mobilizes calcium in MB γ lobe neuropils and DAL neurons. ETH receptor (ETHR) knockdown in the corpus allatum (CA) to create juvenile hormone (JH) deficiency also suppresses LTM, as does knockdown of the JH receptor Met in the MB γ lobe, indicating a convergence of ETH and JH signaling in this region of the brain. These findings identify endocrine-enabled neural circuit components in the brain that are critical for persistent behavioral changes resulting from aversive social experience (Lee, 2021).

The Drosophila zinc finger transcription factor Ouija board controls ecdysteroid biosynthesis through specific regulation of spookier

During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. This study reports identification and characterization of the C2H2-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in Drosophila. Expression of ouib is predominantly limited to the PG, and genetic null mutants of ouib result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, ouib mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, Spookier. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of spok by binding to a specific ~15 bp response element in the spok PG enhancer element. Most remarkable, the developmental arrest phenotype of ouib mutants is rescued by over-expression of a functionally-equivalent paralog of spookier. These observations imply that the main biological function of Ouib is to specifically regulate spookier transcription during Drosophila development (Komura-Kawa, 2015).

Cooperative control of ecdysone biosynthesis in Drosophila by transcription factors Seance, Ouija board, and Molting Defective

Insect ecdysteroids are steroid hormones that control many aspects of development and physiology. During larval development, ecdysone is synthesized in an endocrine organ called the prothoracic gland (PG) through a series of ecdysteroidogenic enzymes encoded by the Halloween genes. The expression of the Halloween genes is highly restricted and dynamic, indicating that their spatiotemporal regulation is mediated by their tight transcriptional control. This study reports that three ZAD-C2H2 zinc finger transcription factors-Seance (Sean), Ouija board (Ouib), and Molting defective (Mld)-cooperatively control ecdysone biosynthesis in the fruit fly Drosophila melanogaster. Sean and Ouib act in cooperation with Mld to positively regulate the transcription of neverland and spookier, respectively, two Halloween genes. Remarkably, loss-of-function mutations in sean, ouib, or mld can be rescued by the expression of neverland,spookier, or both, respectively. These results suggest that the three transcription factors have distinct roles in coordinating the expression of just two genes in Drosophila. Given that neverland and spookier are located in constitutive heterochromatin, Sean, Ouib, and Mld represent the first example of a transcription factor subset that regulates genes located in constitutive heterochromatin (Uryu, 2017).

Protein Is Required for Nuclear Localization of the Ecdysteroidogenic Transcription Factor Molting Defective in the Prothoracic Gland of Drosophila melanogaster

In insects, ecdysteroids, like ecdysone and the more biologically-active derivative 20-hydroxyecdysone (20E). promote molting and metamorphosis. Ecdysone is biosynthesized in the prothoracic gland (PG). via several steps catalyzed by ecdysteroidogenic enzymes that are encoded by Halloween genes. The transcriptional regulatory mechanism of Halloween genes is still elusive. A previous study has found that the polyadenylated tail [poly(A)] deadenylation complex, called Carbon catabolite repressor 4-Negative on TATA (CCR4-NOT) regulates the expression of spookier (spok), which encodes one of the ecdysteroidogenic enzymes in the fruit fly Drosophila melanogaster. This study reports that poly(A) binding protein (Pabp) is involved in spok expression by regulating nuclear localization of the transcription factor molting defective (Mld). When pabp was knocked down specifically in the PG by transgenic RNAi, both spok mRNA and Spok protein levels were significantly reduced. In addition, the spok promoter-driven green fluorescence protein (GFP) signal was also reduced in the pabp-RNAi PG, suggesting that Pabp is involved in the transcriptional regulation of spok. Which transcription factors are responsible for Pabp-dependent transcriptional regulation was investigated. Among the transcription factors acting in the PG, focus was placed on the zinc-finger transcription factor Mld, as Mld is essential for spok transcription. Mld was localized in the nucleus of the control PG cells, while Mld abnormally accumulated in the cytoplasm of pabp-RNAi PG cells. From these results, it is proposed that Pabp regulates subcellular localization in the PG, specifically of the transcription factor Mld, in the context of ecdysone biosynthesis (Kamiyama, 2020).

A local insulin reservoir in Drosophila alpha cell homologs ensures developmental progression under nutrient shortage

Insulin/insulin-like growth factor (IGF) signaling (IIS) controls many aspects of development and physiology. In Drosophila, a conserved family of insulin-like peptides called Dilps is produced by brain neurosecretory cells, and it regulates organismal growth and developmental timing. To accomplish these systemic functions, the Dilps are secreted into the general circulation, and they signal to peripheral tissues in an endocrine fashion. This study describes the local uptake and storage of Dilps in the corpora cardiaca (CC), an endocrine organ composed of alpha cell homologs known to produce the glucagon-like adipokinetic hormone (AKH). Dilp uptake by the CC relies on the expression of an IGF-binding protein called ImpL2. Following their uptake, immunogold staining demonstrates that Dilps are co-packaged with AKH in dense-core vesicles for secretion. In response to nutrient shortage, this specific Dilp reservoir is released and activates IIS in a paracrine manner in the prothoracic gland. This stimulates the production of the steroid hormone ecdysone and initiates entry into pupal development. This study has therefore uncovered a sparing mechanism whereby insulin stores in CC serve to locally activate IIS and the production of ecdysone in the PG, accelerating developmental progression in adverse food conditions (Ghosh, 2022).

WAKE-mediated modulation of cVA perception via a hierarchical neuro-endocrine axis in Drosophila male-male courtship behaviour

The nervous and endocrine systems coordinate with each other to closely influence physiological and behavioural responses in animals. This study shows that Wake (encoded by wide awake) modulates membrane levels of GABA(A) receptor Resistance to Dieldrin (Rdl), in insulin-producing cells of adult male Drosophila melanogaster. This results in changes to secretion of insulin-like peptides which is associated with changes in juvenile hormone biosynthesis in the corpus allatum, which in turn leads to a decrease in 20-hydroxyecdysone levels. A reduction in ecdysone signalling changes neural architecture and lowers the perception of the male-specific sex pheromone 11-cis-vaccenyl acetate by odorant receptor 67d olfactory neurons. These finding explain why WAKE-deficient in Drosophila elicits significant male-male courtship behaviour (Chen, 2022).

Histone H3K27 methylation-mediated repression of Hairy regulates insect developmental transition by modulating ecdysone biosynthesis

Insect development is cooperatively orchestrated by the steroid hormone ecdysone and juvenile hormone (JH). The polycomb repressive complex 2 (PRC2)-mediated histone H3K27 trimethylation (H3K27me3) epigenetically silences gene transcription and is essential for a range of biological processes, but the functions of H3K27 methylation in insect hormone action are poorly understood. This study demonstrates that H3K27 methylation-mediated repression of Hairy transcription in the larval prothoracic gland (PG) is required for ecdysone biosynthesis in Bombyx and Drosophila H3K27me3 levels in the PG are dynamically increased during the last larval instar. H3K27me3 reduction induced by the down-regulation of PRC2 activity via inhibitor treatment in Bombyx or PG-specific knockdown of the PRC2 component Su(z)12 in Drosophila diminishes ecdysone biosynthesis and disturbs the larval-pupal transition. Mechanistically, H3K27 methylation targets the JH signal transducer Hairy to repress its transcription in the PG; PG-specific knockdown or overexpression of the Hairy gene disrupts ecdysone biosynthesis and developmental transition; and developmental defects caused by PG-specific Su(z)12 knockdown can be partially rescued by Hairy down-regulation. The application of JH mimic to the PG decreases both H3K27me3 levels and Su(z)12 expression. Altogether, this study reveals that PRC2-mediated H3K27 methylation at Hairy in the PG during the larval period is required for ecdysone biosynthesis and the larval-pupal transition and provides insights into epigenetic regulation of the crosstalk between JH and ecdysone during insect development (Yang, 2021).

The cytochrome P450 Cyp6t3 is not required for ecdysone biosynthesis in Drosophila melanogaster

The steroid hormone 20-hydroxyecdysone (20E) is essential for proper development and the timing of intermediary stage transitions in insects. As a result, there is intense interest in identifying and defining the roles of the enzymes and signaling pathways that regulate 20E production in the prothoracic gland (PG), the major endocrine organ of juvenile insect phases. Transcriptomics is one powerful tool that has been used to identify novel genes that are up- or down-regulated in the PG which may contribute to 20E regulation. Additional functional characterization of putative regulatory candidate genes typically involves qRT-PCR and/or RNAi mediated knockdown of the candidate mRNA in the PG to assess whether the gene's expression shows temporal regulation in the PG and whether its expression is essential for proper 20E production and the correct timing of developmental transitions. While these methods have proved fruitful for identifying novel regulators of 20E production, characterizing the null phenotype of putative regulatory genes is the gold standard for assigning gene function since RNAi is known to generate various types of "off target" effects. This study describes the genetic null mutant phenotype of the Drosophila melanogaster Cyp6t3 gene. Cyp6t3 was originally identified as a differentially regulated gene in a PG microarray screen and assigned a place in the "Black Box" step of the E biosynthetic pathway based on RNAi mediated knockdown phenotypes and rescue experiments involving feeding of various intermediate compounds of the E biosynthetic pathway. In contrast, it was found that Crispr generated null mutations in Cyp6t3 are viable and have normal developmental timing. Therefore, it is concluded that Cyp6t3 is not required for E production under typical lab growth conditions and therefore is not an obligate enzymatic component of the Black Box (Shimell, 2022).

Ecdysone coordinates plastic growth with robust pattern in the developing wing

Animals develop in unpredictable, variable environments. In response to environmental change, some aspects of development adjust to generate plastic phenotypes. Other aspects of development, however, are buffered against environmental change to produce robust phenotypes. How organ development is coordinated to accommodate both plastic and robust developmental responses is poorly understood. This study demonstrates that the steroid hormone ecdysone coordinates both plasticity of organ size and robustness of organ pattern in the developing wings of the fruit fly Drosophila melanogaster. Using fed and starved larvae that lack prothoracic glands, which synthesize ecdysone, this study showed that nutrition regulates growth both via ecdysone and via an ecdysone-independent mechanism, while nutrition regulates patterning only via ecdysone. It was then demonstrated that growth shows a graded response to ecdysone concentration, while patterning shows a threshold response. Collectively, these data support a model where nutritionally regulated ecdysone fluctuations confer plasticity by regulating disc growth in response to basal ecdysone levels and confer robustness by initiating patterning only once ecdysone peaks exceed a threshold concentration. This could represent a generalizable mechanism through which hormones coordinate plastic growth with robust patterning in the face of environmental change (Nogueira Alves, 2022).

Su(var)2-10- and Su(var)205-dependent upregulation of the heterochromatic gene neverland is required for developmental transition in Drosophila

Animals develop from juveniles to sexually mature adults through the action of steroid hormones. In insect metamorphosis, a surge of the steroid hormone ecdysone prompts the transition from the larval to the adult stage. Ecdysone is synthesized by a series of biosynthetic enzymes that are specifically expressed in an endocrine organ, the prothoracic gland. At the late larval stage, the expression levels of ecdysone biosynthetic enzymes are upregulated through the action of numerous transcription factors, thus initiating metamorphosis. In contrast, the mechanism by which chromatin regulators support the expression of ecdysone biosynthetic genes is largely unknown. This study demonstrates that Su(var)2-10 and Su(var)205, suppressor of variegation [Su(var)] genes encoding a chromatin regulator Su(var)2-10 and non-histone heterochromatic protein 1a (HP1a), respectively, regulate the transcription of one of the heterochromatic ecdysone biosynthetic genes, neverland, in Drosophila melanogaster. Knockdown of Su(var)2-10 and Su(var)205 in the prothoracic gland caused a decrease in neverland expression, resulting in a defect in larval-to-prepupal transition. Furthermore, overexpression of neverland and administration of 7-dehydrocholesterol, a biosynthetic precursor of ecdysone produced by Neverland, rescued developmental defects in Su(var)2-10 and Su(var)205 knockdown animals. These results indicate that Su(var)2-10- and Su(var)205-mediated proper expression of neverland is required for the initiation of metamorphosis. Given that Su(var)2-10-positive puncta are juxtaposed with the pericentromeric heterochromatic region, it is proposed that Su(var)2-10- and Su(var)205-dependent regulation of inherent heterochromatin structure at the neverland gene locus is essential for its transcriptional activation (Ohhara, 2022).

Juvenile hormone is required in adult males for Drosophila courtship

Juvenile Hormone (JH) has a prominent role in the regulation of insect development. Much less is known about its roles in adults, although functions in reproductive maturation have been described. In adult females, JH has been shown to regulate egg maturation and mating. To examine a role for JH in male reproductive behavior, this study generated males with reduced levels of Juvenile Hormone Acid O-Methyl Transferase (JHAMT) and tested them for courtship. JHAMT regulates the last step of JH biosynthesis in the Corpora Allata (CA), the organ of JH synthesis. Males with reduced levels of JHAMT show a reduction in courtship that can be rescued by application of Methoprene, a JH analog, shortly before performing the courtship assays. In agreement with this, reducing JHAMT conditionally in mature flies leads to courtship defects that are rescuable by Methoprene. The same result is also observed when the CA are conditionally ablated by the expression of a cellular toxin. These findings demonstrate that JH plays an important physiological role in the regulation of male mating behavior (Wijesekera, 2016).

TGF-beta signaling in insects regulates metamorphosis via juvenile hormone biosynthesis

Although butterflies undergo a dramatic morphological transformation from larva to adult via a pupal stage (holometamorphosis), crickets undergo a metamorphosis from nymph to adult without formation of a pupa (hemimetamorphosis). Despite these differences, both processes are regulated by common mechanisms that involve 20-hydroxyecdysone (20E) and juvenile hormone (JH). JH regulates many aspects of insect physiology, such as development, reproduction, diapause, and metamorphosis. Consequently, strict regulation of JH levels is crucial throughout an insect's life cycle. However, it remains unclear how JH synthesis is regulated. This study reports that in the corpora allata of the cricket, Gryllus bimaculatus, Myoglianin (Gb'Myo), a homolog of Drosophila Myoglianin/vertebrate GDF8/11, is involved in the down-regulation of JH production by suppressing the expression of a gene encoding JH acid O-methyltransferase, Gb'jhamt. In contrast, JH production is up-regulated by Decapentaplegic (Gb'Dpp) and Glass-bottom boat/60A (Gb'Gbb) signaling that occurs as part of the transcriptional activation of Gb'jhamt. Gb'Myo defines the nature of each developmental transition by regulating JH titer and the interactions between JH and 20E. When Gb'myo expression is suppressed, the activation of Gb'jhamt expression and secretion of 20E induce molting, thereby leading to the next instar before the last nymphal instar. Conversely, high Gb'myo expression induces metamorphosis during the last nymphal instar through the cessation of JH synthesis. Gb'myo also regulates final insect size. Because Myo/GDF8/11 and Dpp/bone morphogenetic protein (BMP)2/4-Gbb/BMP5–8 are conserved in both invertebrates and vertebrates, the present findings provide common regulatory mechanisms for endocrine control of animal development (Ishimaru, 2016).


Abdou, M. A., He, Q., Wen, D., Zyaan, O., Wang, J., Xu, J., Baumann, A. A., Joseph, J., Wilson, T. G., Li, S. and Wang, J. (2011). Drosophila Met and Gce are partially redundant in transducing juvenile hormone action. Insect Biochem Mol Biol 41: 938-945. PubMed ID: 21968404

Alves, A. N., Oliveira, M. M., Koyama, T., Shingleton, A. and Mirth, C. K. (2022). Ecdysone coordinates plastic growth with robust pattern in the developing wing. Elife 11. PubMed ID: 35261337

Baumann, A., et al. (2010). Paralogous genes involved in juvenile hormone action in Drosophila melanogaster. Genetics 185: 1327-1336. PubMed ID: 20498297

Bilen, J., Atallah, J., Azanchi, R., Levine, J. D. and Riddiford, L. M. (2013). Regulation of onset of female mating and sex pheromone production by juvenile hormone in Drosophila melanogaster. Proc Natl Acad Sci U S A 110: 18321-18326. PubMed ID: 24145432

Caceres, L., Necakov, A. S., Schwartz, C., Kimber, S., Roberts, I. J. and Krause, H. M. (2011). Nitric oxide coordinates metabolism, growth, and development via the nuclear receptor E75. Genes Dev 25: 1476-1485. PubMed ID: 21715559

Cao, X., Rojas, M. and Pastor-Pareja, J. C. (2022). Intrinsic and damage-induced JAK/STAT signaling regulate developmental timing by the Drosophila prothoracic gland. Dis Model Mech 15(1). PubMed ID: 34842272

Chen, S. L., Liu, B. T., Lee, W. P., Liao, S. B., Deng, Y. B., Wu, C. L., Ho, S. M., Shen, B. X., Khoo, G. H., Shiu, W. C., Chang, C. H., Shih, H. W., Wen, J. K., Lan, T. H., Lin, C. C., Tsai, Y. C., Tzeng, H. F. and Fu, T. F. (2022). WAKE-mediated modulation of cVA perception via a hierarchical neuro-endocrine axis in Drosophila male-male courtship behaviour. Nat Commun 13(1): 2518. PubMed ID: 35523813

Chertemps, T., Duportets, L., Labeur, C., Ueyama, M. and Wicker-Thomas, C. (2006). A female-specific desaturase gene responsible for diene hydrocarbon biosynthesis and courtship behaviour in Drosophila melanogaster. Insect Mol Biol 15: 465-473. PubMed ID: 16907833

Chertemps, T., Duportets, L., Labeur, C., Ueda, R., Takahashi, K., Saigo, K. and Wicker-Thomas, C. (2007). A female-biased expressed elongase involved in long-chain hydrocarbon biosynthesis and courtship behavior in Drosophila melanogaster. Proc Natl Acad Sci U S A 104: 4273-4278. PubMed ID: 17360514

Christesen, D., Yang, Y. T., Somers, J., Robin, C., Sztal, T., Batterham, P. and Perry, T. (2016). Transcriptome analysis of Drosophila melanogaster third instar larval ring glands points to novel functions and uncovers a cytochrome p450 required for development. G3 (Bethesda) [Epub ahead of print] PubMed ID: 27974438

Cruz, J., Martin, D. and Franch-Marro, X. (2020). Egfr signaling is a major regulator of ecdysone biosynthesis in the Drosophila prothoracic gland. Curr Biol 30(8): 1547-1554. PubMed ID: 32220314

de la Riva Carrasco, R., Perez Pandolfo, S., Freire, S. S., Romero, N. M., Bhujabal, Z., Johansen, T., Wappner, P. and Melani, M. (2020). The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues. Traffic. PubMed ID: 33336828

Deliu, L. P., Turingan, M., Jadir, D., Lee, B., Ghosh, A. and Grewal, S. S. (2022). Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development. PLoS Genet 18(9): e1010371. PubMed ID: 36048889

Destefanis, F., Manara, V., Santarelli, S., Zola, S., Brambilla, M., Viola, G., Maragno, P., Signoria, I., Viero, G., Pasini, M. E., Penzo, M. and Bellosta, P. (2022). Reduction of nucleolar NOC1 accumulates pre-rRNAs and induces Xrp1 affecting growth and resulting in cell competition. J Cell Sci. PubMed ID: 36314272

De Velasco, B., Shen, J., Go, S. and Hartenstein, V. (2004). Embryonic development of the Drosophila corpus cardiacum, a neuroendocrine gland with similarity to the vertebrate pituitary, is controlled by sine oculis and glass. Dev. Biol. 274: 280-294. PubMed ID: 15385159

Döring, F., Wischmeyer, E., Kuhnlein, R. P., Jäckle, H. and Karschin, A. (2002). Inwardly rectifying K+ (Kir) channels in Drosophila. A crucial role of cellular milieu factors Kir channel function. J. Biol. Chem. 277: 25554-25561. PubMed ID: 11964404

Drelon C., Belalcazar H. M. and Secombe J. (2018). The histone demethylase KDM5 is essential for larval growth in Drosophila. Genetics 209: 773-787. PubMed ID: 31862793

Drelon, C., Rogers, M. F., Belalcazar, H. M. and Secombe, J. (2019). The histone demethylase KDM5 controls developmental timing in Drosophila by promoting prothoracic gland endocycles. Development 146(24). PubMed ID: 31862793

Dus, M., Lai, J. S., Gunapala, K. M., Min, S., Tayler, T. D., Hergarden, A. C., Geraud, E., Joseph, C. M. and Suh, G. S. (2015). Nutrient sensor in the brain directs the action of the brain-gut axis in Drosophila. Neuron 87(1): 139-151. PubMed ID: 26074004

Enya, S., et al. (2017). Dual roles of glutathione in ecdysone biosynthesis and antioxidant function during the larval development in Drosophila. Genetics 207(4):1519-1532. PubMed ID: 29021278

Ghosh, S., Leng, W., Wilsch-Brauninger, M., Barrera-Velazquez, M., Leopold, P. and Eaton, S. (2022). A local insulin reservoir in Drosophila alpha cell homologs ensures developmental progression under nutrient shortage. Curr Biol. PubMed ID: 35316653

Guntur, A. R., Gu, P., Takle, K., Chen, J., Xiang, Y. and Yang, C. H. (2015). Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2. Proc Natl Acad Sci U S A 112: E5753-5761. PubMed ID: 26443856

Hao, S., Gestrich, J. Y., Zhang, X., Xu, M., Wang, X., Liu, L. and Wei, H. (2021). Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster. Front Neurosci 15: 653858. PubMed ID: 34975366

Harvie, P. D., Filippova, M. and Bryant, P. J. (1998). Genes expressed in the ring gland, the major endocrine organ of Drosophila melanogaster. Genetics 149(1): 217-231. PubMed ID: 9584098

Huang, J., et al. (2011). DPP-mediated TGFβ signaling regulates juvenile hormone biosynthesis by activating the expression of juvenile hormone acid methyltransferase. Development 138(11): 2283-91. PubMed ID: 21558376

Hughson, B. N. (2022). PKG acts in the adult corpora cardiaca to regulate nutrient stress-responsivity through adipokinetic hormone. J Insect Physiol 136: 104339. PubMed ID: 34856210

Ishimaru, Y., Tomonari, S., Matsuoka, Y., Watanabe, T., Miyawaki, K., Bando, T., Tomioka, K., Ohuchi, H., Noji, S. and Mito, T. (2016). TGF-beta signaling in insects regulates metamorphosis via juvenile hormone biosynthesis. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27140602

Jaszczak, J.S., Wolpe, J.B., Dao, A.Q. and Halme, A. (2015). Nitric oxide synthase regulates growth coordination during Drosophila melanogaster imaginal disc regeneration. Genetics [Epub ahead of print]. PubMed ID: 26081194

Juarez-Carreno, S., Vallejo, D. M., Carranza-Valencia, J., Palomino-Schatzlein, M., Ramon-Canellas, P., Santoro, R., de Hartog, E., Ferres-Marco, D., Romero, A., Peterson, H. P., Ballesta-Illan, E., Pineda-Lucena, A., Dominguez, M. and Morante, J. (2021). Body-fat sensor triggers ribosome maturation in the steroidogenic gland to initiate sexual maturation in Drosophila. Cell Rep 37(2): 109830. PubMed ID: 34644570

Kaieda, Y., Masuda, R., Nishida, R., Shimell, M., O'Connor, M. B. and Ono, H. (2017). Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster. Dev Biol [Epub ahead of print]. PubMed ID: 28782527

Kamiyama, T., Sun, W., Tani, N., Nakamura, A. and Niwa, R. (2020). Poly(A) Binding Protein Is Required for Nuclear Localization of the Ecdysteroidogenic Transcription Factor Molting Defective in the Prothoracic Gland of Drosophila melanogaster. Front Genet 11: 636. PubMed ID: 32676099

Kim, S. K. and Rulifson, E. J. (2004). Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells. Nature 431: 316-320. PubMed ID: 15372035

Knapp E, Sun J. (2017). Steroid signaling in mature follicles is important for Drosophila ovulation. Proc Natl Acad Sci 114:699-704. PubMed ID: 28069934

Komura-Kawa, T., Hirota, K., Shimada-Niwa, Y., Yamauchi, R., Shimell, M., Shinoda, T., Fukamizu, A., O'Connor, M. B. and Niwa, R. (2015). The Drosophila zinc finger transcription factor Ouija board controls ecdysteroid biosynthesis through specific regulation of spookier. PLoS Genet 11: e1005712. PubMed ID: 26658797

Koyama, T., Rodrigues, M. A., Athanasiadis, A., Shingleton, A. W. and Mirth, C. K. (2014). Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis. Elife 3 [Epub ahead of print]. PubMed ID: 25421296

Kuzin, B., Roberts, I., Peunova, N. and Enikolopov, G. (1996). Nitric oxide regulates cell proliferation during Drosophila development. Cell 87: 639-649. PubMed ID: 8929533

Layalle, S., Arquier, N., Léopold, P. (2008). The TOR pathway couples nutrition and developmental timing in Drosophila. Dev. Cell 15(4): 568-77. PubMed ID: 18854141

Lee, S. S. and Adams, M. E. (2021). Regulation of Drosophila Long-Term Courtship Memory by Ecdysis Triggering Hormone. Front Neurosci 15: 670322. PubMed ID: 33967686

Llorens, J. V., Metzendorf, C., Missirlis, F. and Lind, M. I. (2015). Mitochondrial iron supply is required for the developmental pulse of ecdysone biosynthesis that initiates metamorphosis in Drosophila melanogaster. J Biol Inorg Chem [Epub ahead of print]. PubMed ID: 26468126

Marcillac, F. and Ferveur, J. F. (2004). A set of female pheromones affects reproduction before, during and after mating in Drosophila. J Exp Biol 207: 3927-3933. PubMed ID: 15472023

Meiselman, M. R., Kingan, T. G. and Adams, M. E. (2018). Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation. BMC Biol 16(1): 18. PubMed ID: 29382341

Meiselman, M., Lee, S. S., Tran, R. T., Dai, H., Ding, Y., Rivera-Perez, C., Wijesekera, T. P., Dauwalder, B., Noriega, F. G. and Adams, M. E. (2017). Endocrine network essential for reproductive success in Drosophila melanogaster. Proc Natl Acad Sci U S A 114(19): E3849-E3858. PubMed ID: 28439025

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

Moeller, M. E., Danielsen, E. T., Herder, R., O'Connor, M. B. and Rewitz, K. F. (2013). Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila. Development 140: 4730-4739. PubMed ID: 24173800

Nakaoka, T., Iga, M., Yamada, T., Koujima, I., Takeshima, M., Zhou, X., Suzuki, Y., Ogihara, M. H. and Kataoka, H. (2017). Deep sequencing of the prothoracic gland transcriptome reveals new players in insect ecdysteroidogenesis. PLoS One 12(3): e0172951. PubMed ID: 28257485

Nasonkin, I., et al. (1999). A novel sulfonylurea receptor family member expressed in the embryonic Drosophila dorsal vessel and tracheal system. J. Biol. Chem. 274: 29420-29425. PubMed ID: 10506204

Oh, Y., Lai, J. S., Mills, H. J., Erdjument-Bromage, H., Giammarinaro, B., Saadipour, K., Wang, J. G., Abu, F., Neubert, T. A. and Suh, G. S. B. (2019). A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila. Nature 574(7779): 559-564. PubMed ID: 31645735

Ohhara, Y., Shimada-Niwa, Y., Niwa, R., Kayashima, Y., Hayashi, Y., Akagi, K., Ueda, H., Yamakawa-Kobayashi, K. and Kobayashi, S. (2015). Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis. Proc Natl Acad Sci USA [Epub ahead of print]. PubMed ID: 25605909

Ohhara, Y., Kato, Y., Kamiyama, T. and Yamakawa-Kobayashi, K. (2022). Su(var)2-10- and Su(var)205-dependent upregulation of the heterochromatic gene neverland is required for developmental transition in Drosophila. Genetics. PubMed ID: 36149288

Pan, X., Neufeld, T. P. and O'Connor, M. B. (2019). A tissue- and temporal-specific autophagic switch controls Drosophila pre-metamorphic nutritional checkpoints. Curr Biol 29(17): 2840-2851 e2844. PubMed ID: 31422886

Pan, X. and O'Connor, M. B. (2021). Coordination among multiple receptor tyrosine kinase signals controls Drosophila developmental timing and body size. Cell Rep 36(9): 109644. PubMed ID: 34469735

Pesch, Y. Y., Hesse, R., Ali, T. and Behr, M. (2018). A cell surface protein controls endocrine ring gland morphogenesis and steroid production. Dev Biol 445(1):16-28. PubMed ID: 30367846

Rauschenbach, I. Y., Karpova, E. K., Adonyeva, N. V., Andreenkova, O. V., Faddeeva, N. V., Burdina, E. K., Alekseev, A. A., Menshanov, P. N. and Gruntenko, N. E. (2014). Disruption of insulin signalling affects the neuroendocrine stress reaction in Drosophila females. J Exp Biol 217(Pt 20):3733-41. PubMed ID: 25214494

Rewitz, K. F., Yamanaka, N. and O'Connor, M. B. (2010). Steroid hormone inactivation is required during the juvenile-adult transition in Drosophila. Dev Cell 19: 895-902. PubMed ID: 21145504

Sanchez-Higueras, C., Sotillos, S. and Castelli-Gair Hombria, J. (2013). Common origin of insect trachea and endocrine organs from a segmentally repeated precursor. Curr Biol. 24(1):76-81. PubMed ID: 24332544

Sanchez-Higueras, C. and Hombria, J. C. (2016). Precise long-range migration results from short-range stepwise migration during ring gland organogenesis. Dev Biol. [Epub ahead of print] PubMed ID: 27063193

Sarraf-Zadeh, L., Christen, S., Sauer, U., Cognigni, P., Miguel-Aliaga, I., Stocker, H., Kohler, K. and Hafen, E. (2013). Local requirement of the Drosophila insulin binding protein Imp-L2 in coordinating developmental progression with nutritional conditions. Dev Biol 381: 97-106. PubMed ID: 23773803

Shimell, M. and O'Connor, M. B. (2022). The cytochrome P450 Cyp6t3 is not required for ecdysone biosynthesis in Drosophila melanogaster. MicroPubl Biol 2022. PubMed ID: 35991292

Texada, M. J., Lassen, M., Pedersen, L. H., Koyama, T., Malita, A. and Rewitz, K. (2022). Insulin signaling couples growth and early maturation to cholesterol intake in Drosophila. Curr Biol 32(7): 1548-1562. PubMed ID: 35245460

Uryu, O., Ou, Q., Komura-Kawa, T., Kamiyama, T., Iga, M., Syrzycka, M., Hirota, K., Kataoka, H., Honda, B. M., King-Jones, K. and Niwa, R. (2017). Cooperative control of ecdysone biosynthesis in Drosophila by transcription factors Seance, Ouija board, and Molting Defective. Genetics [Epub ahead of print]. PubMed ID: 29187506

Wicker-Thomas, C., Guenachi, I. and Keita, Y. F. (2009). Contribution of oenocytes and pheromones to courtship behaviour in Drosophila. BMC Biochem 10: 21. PubMed ID: 19671131

Wijesekera, T.P., Saurabh, S and Dauwalder, B. (2016). Juvenile hormone is required in adult males for Drosophila courtship. PLoS One 11: e0151912. PubMed ID: 27003411

Yamamoto, R., Bai, H., Dolezal, A. G., Amdam, G. and Tatar, M. (2013). Juvenile hormone regulation of Drosophila aging. BMC Biol 11: 85. PubMed ID: 23866071

Yang, Y., Zhao, T., Li, Z., Qian, W., Peng, J., Wei, L., Yuan, D., Li, Y., Xia, Q. and Cheng, D. (2021). Histone H3K27 methylation-mediated repression of Hairy regulates insect developmental transition by modulating ecdysone biosynthesis. Proc Natl Acad Sci U S A 118(35). PubMed ID: 34429358

Yu, Z., Shi, J., Jiang, X., Song, Y., Du, J. and Zhao, Z. (2022). Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae. Pest Manag Sci. PubMed ID: 36396604

Zeng, J., Huynh, N., Phelps, B. and King-Jones, K. (2020). Snail synchronizes endocycling in a TOR-dependent manner to coordinate entry and escape from endoreplication pausing during the Drosophila critical weight checkpoint. PLoS Biol 18(2): e3000609. PubMed ID: 32097403

Zeng, J., Kamiyama, T., Niwa, R. and King-Jones, K. (2018). The Drosophila CCR4-NOT complex is required for cholesterol homeostasis and steroid hormone synthesis. Dev Biol 443(1):10-18. PubMed ID: 30149007

Zhang, J., Wen, D., Li, E. Y., Palli, S. R., Li, S., Wang, J. and Liu, S. (2021). MicroRNA miR-8 promotes cell growth of corpus allatum and juvenile hormone biosynthesis independent of insulin/IGF signaling in Drosophila melanogaster. Insect Biochem Mol Biol 136: 103611. PubMed ID: 34182107

back to a list of Genes expressed in the ring gland

Genes involved in tissue and organ development

Home page: The Interactive Fly © 1998 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.