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

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

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

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
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

Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

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).

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; full text of article).

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

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

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

Enlarging the PG of larvae reared under constant light caused larvae to initiate metamorphosis earlier and at smaller sizes. Nevertheless, even though larvae starved early after the L3 molt were able to pupariate, they were unable to survive to pupation unless they had fed for at least 11.5 hr. This suggests that phm>Dp110 larvae starved prior to 11.5 hr AL3E initiated metamorphosis before surpassing the minimal 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).

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).

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).

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).

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).

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).

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).

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).

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Genes involved in tissue and organ development

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