The Interactive Fly
Genes involved in tissue and organ development
Embryonic development of the corpus cardiacum, a component of the ring gland
Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells
Genes expressed in 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 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).
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).
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 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).
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).
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).
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