In Drosophila, there is a variation on this general theme (Riddiford, 1993). The ecdysone is released by the ring gland (which is a structure having regions similar to both the corpus allatum and prothoracic gland). A high titre pulse of ecdysone, at the end of the third instar period, signals the onset of metamorphosis. The larva ceases movement, everts its spiracles, and allows the larval cuticle to harden into a puparium (pupal case) that surrounds the organism for the time of its metamorphosis. At this time, the imaginal discs evert to form the basic outline of the adult body, but with the head still tucked within the body cavity. After 12 hr (at 25°C), a brief pulse of ecdysone triggers the eversion of the head from the thorax and the transition from "prepupa" to pupa. The head is pushed out by the contraction of abdominal muscles which push an air bubble up to the anterior to make room for the head to evert (Fristrom and Fristrom, 1993). A subsequent burst of ecdysone brings about the final differentiation of adult form in the Drosophila pupa immediately prior to eclosion, the "hatching" of the adult from the pupal case. Like other insects, Drosophila have an eclosion hormone, which initiates the movements and behaviors that enable the adult to wiggle from its pupal case and out into the larger world.
From the on-line B. A. Honors thesis of Anna Gniadek, Mount Holyoke College
Drosophila eggs are produced in the female's ovaries and fertilization occurs in the female oviduct (St. Johnston and Nusslein-Volhard, 1992). The adult female fly typically lays several hundred eggs. Embryonic development consists of 16 stages identified by the events taking place and the time after fertilization at which they occur. The embryonic stage lasts from 21 to 22 hours and is followed by the hatching of the first larva (a process mediated by high ecdysone titer). Larval development progresses through three instars which are separated by molting. Each moltingoa process of cuticle apolysis (separation) and consequent shedding of the old cuticle (Kaznowski et al., 1985)ois triggered by an ecdysone pulse. During the 1st and 2nd instar, each lasting one day, the larva grows in size and constantly consumes food. The 3rd instar, leaves the food source, urged by a need to find a solid surface suitable for adhesion. This "wandering stage" lasts for 48 hours.
The larva is built of 12 segments: one head segment, three thoracic segments and eight abdominal segments. The inner body is surrounded by an inner cellular epidermis and an outer, noncellular cuticle. This cuticle consists of the exocuticle, the outer layer, and endocuticle, the lamellar layer.
The body wall of a larva is very soft and provides movement flexibility (Bodenstein, 1965). The larva contains the imaginal discs, groups of imaginal cells, which are primordia of adult structures. These cells remain diploid throughout the developmental phase and undergo mitotic divisions. The larval tissues become polyploid and most of them undergo histolysis (Wilder and Perrimon, 1996).
Puparium formation—a process of attachment to a solid surface—is triggered also by an ecdysone pulse, the larval pulse. Puparium formation is the onset of metamorphosis. At this stage the larva shortens, becomes broader, and everts anterior spiracles used for gas exchange. There is a 15 minute period after puparium formation, during which the cuticle is white. At this stage, the animal is called a "white prepupa." This stage is referred to as the 0 hour prepupa stage. Animals before this event may be referred to as negative hour prepupae, and animals after this event can be referred to as positive hour prepupae. Following this period, the animal cuticle tans and hardens, forming a protective pupal case (Chiara et al., 1982).
For the next 12 hours, the animal is headless, with no external legs or wings, and is surrounded by a fine cuticle (Bodenstein, 1965). At ca. 6 hours after puparium formation, the ecdysone titre drops to a low level. At this point the cuticle starts apolysis, and both imaginal and larval cells begin to secrete another cuticle (Fechtel et al., 1989). At approximately 9 hours after puparium formation, imaginal discs are visible in the thorax.
At 12 hours after puparium formation, ecdysone titer rises again (the prepupal ecdysone pulse) to induce head eversion, which marks the prepupal/pupal transition. The abdominal muscles push the fly's head into the air bubble at the anterior end. Interestingly, this air bubble is formed at the posterior end and then moves into the anterior area during cuticle apolysis (Demerc, 1994). One hour after head eversion the imaginal discs begin to evaginate. Then, 24 hours after puparium formation, the pupal cuticle is separated, and at around 48-50 hour after puparium formation, the adult cuticle is formed (Chiara et al.,1982). The 18 hour period before formation of the adult cuticle is necessary for the free movement of cells and the complete evagination of imaginal discs (Fristrom and Fristrom, 1993). In the following 2 days the process of adult formation continues. At the end of metamorphosis all larval organs have been histolysed and adult structures have been formed. After 3.5 days of existing in the pupal stage, the fly uses its legs to push its way out of the cuticle. This process is called eclosion (Thummel, 1995).
Hormones in Drosophila Life Cycle
Steroid hormones are derivatives of cholesterol and play important roles in many biological processes. Hormones are chemicals produced in one organ and transported by the circulatory system to act at another place (cells or organs) in the body. Unlike water-soluble peptide hormones, steroid hormones are lipid-soluble hormones that pass through a plasma membrane bilayer into the cytoplasm without special receptors on the plasma membrane. These hormones move through the cytoplasm and bind to the specific protein receptors in the cytoplasm. (Some hormones bind receptors in the nucleus of the cell.) The active hormone-receptor complex formed in the cytoplasm moves to the nucleus. Binding of a hormone to its receptor induces an allosteric change. This structural change gives the hormone-receptor complex high affinity for binding to specific sites in chromatin. This binding initiates the transcription of specific genes.
20-hydroxyecdysone, the only known physiologically active steroid hormone in Drosophila melanogaster, serves as a temporal signal to stimulate developmental events. A-ecdysone is synthesized in the prothoracic gland, which resides in the tissue of the ring gland, located anterior to the larval brain. A-ecdysone is converted in the fat body, and other target organs, to 20-hydroxyecdysone (with an additional hydroxyl group at position 20), 20,26-dihydroxymakisterone, 3-dehydroecdysteroids, makisterone A, and several other metabolites (Heftmann and Mosettig, 1970; Ridifford, 1993; Sliter and Gilbert, 1992).
Ecdysone is the most active of the endogenous ecdysteroids. Like all steroid hormones, ecdysone is lipid-soluble, as it is a polyhydroxylated cholesterol derivative with a four ring cyclical structure. Pulses of ecdysone hormone direct Drosophila development. The first peak in the hormone level occurs during embryogenesis. The next two pulses of ecdysone appear in the 1st and 2nd instar. Here they trigger larval ecdysis, molting of the larval cuticle defining the end on 1st and 2nd instar, by controlling the synthesis of proteins which occur in the epidermis and the internal organs (Riddiford, 1993). A high titer of the hormone at the end of the third larval instar induces puparium formation, and marks the beginning of the prepupal stage and the onset of metamorphosis.
Ecdysone titer drops drastically just after ecdysis. It starts to rise about 5 hours prior to puparium formation. The titer level drops just before a formation of a stationary white prepupa with everted anterior spiracles (Richards, 1981; Baehrecke, et al., 1993) and reaches its lowest level at 4-6 hours after pupariation. After this drop, ecdysone titer rises and reaches its maximum in a 10-12 hour prepupae (Handler, 1982; Silter and Gilbert, 1992; Baehrecke et al., 1993). This pulse initiates the pupal development and the eclosion of the adult fly.
Prothoracicotropic hormone (PTTH) plays an essential role in Drosophila development since it initiates the synthesis and release of ecdysone from the prothoracic glands and, by doing so, contributes to ecdysone titer changes critical to development. PTTH is produced by a set of neurosecretory cells in the brain (Kopec, 1922; Agui et al., 1979). In other insects, PTTH release is dictated, in an unknown manner, by animal size (Nijhout, 1981). In Drosophila, however, the size of the animal does not seem to play a critical role in PTTH release (Beadle et al., 1938: Bakker, 1959)
Juvenile hormone (JH) is a sesquiterpenoid, synthesized in the corpora allata. It is necessary at the time of ecdysone release for larval molting and the prevention of metamorphosis (Wigglesworth 1934; Nijhout and Wheeler 1981; Richards, 1981; reviewed by Riddiford, 1993). Perhaps the most interesting fact about JH titer is that there is an increase in JH just before eclosion, reaching its peak just after eclosion. A few more pulses of JH appear just after the eclosion (Bownes and Rembold, 1987).
Eclosion hormone (EH) is important, as in other insects, for ecdysis in Drosophila (reviewed by Riddiford, 1993). EH has been shown to appear between 1-3 hours before ecdysis; it tends to appear however, only at a certain time of day. Besides causing ecdysis, EH is believed to initiate apoptosis in the abdominal intersegmental muscles, which happens just after the ecdysis (Kimura and Truman, 1990).
Ashburner's 1974 Model and the Current Model
In the 1960's, Ashburner studied the molecular mechanism by which ecdysone affects Drosophila melanogaster metamorphosis. Ashburner (1967) observed a pattern of puff formation in the larval salivary gland polytene chromosome. Two distinct sets of puffs are induced by the late-larval ecdysone pulse. Ashburner et al. (1974) performed a series of studies on ecdysone dose response, hormone removal and the re-addition, and drug inhibition with cycloheximide. A few puffs are called early puffs since they are directly induced by the ecdysone pulse. These puffs persist for several hours in the presence of the hormone and then regress. As the early puffs peak in size, many more so-called "late puffs" appear and remain transcribed throughout puparium formation. The protein synthesis inhibitor cycloheximide blocks regression of early gene (genes within early puffs) transcription and late gene induction, but does not affect early gene induction. Based on his studies, Ashburner proposed a model of ecdysone regulation. In this model, ecdysone binds to the ecdysone receptor complex (EcR). This EcR-ecdysone complex acts in three ways:
- it positively stimulates early gene transcription,
- it represses late gene transcription,
- it represses intermolt puffs.
The early gene products, early proteins, also have dual roles; they repress the transcription of their own genes and induce late gene transcription.
The late gene products play more direct roles in directing metamorphosis.
Almost 40 years of research into Drosophila development has provided significant progress in the understanding of this biological process. Even though the proposed, oversimplified model has been significantly improved, the complete understanding of the gene regulatory hierarchy has not been yet achieved.
The current model for the ecdysone regulation mechanism includes almost all intra-communications between ecdysone, EcR, genes and the transcribed proteins proposed by Ashburner. A new concept of early-late gene proteins has been recently introduced. These proteins contribute to a positive stimulation of late genes as well as they negatively regulate intermolt puffs (Huet et al., 1995). Presented below is a description of the gene regulatory hierarchy that mainly applies to salivary glands. Other tissues do present different patterns of gene expression.
Ecdysone-Ecdysone Receptor Complex
Ashburner's model has been supported by numerous studies, including those identifying the ecdysone receptor protein (EcR) in heterodimeric combination with the ultraspiricle gene product (Yao et al.,1992). Ecdysone prompts the development regulation machinery via this ecdysone-ecdysone receptor complex. This receptor is a heterodimer of EcR and usp products (Thummel, 1995). Both of these genes belong to a nuclear hormone receptor superfamily. The nuclear receptor superfamily includes over 150 known proteins that mediate extracellular signals into transcriptional responses. Nuclear receptors bind to the specific DNA sequences known as hormone response elements (HREs) (Evans, 1988; Beato, 1991; Mangelsdorf et al., 1995). The nuclear receptors are characterized by a DNA-binding domain (DBD), located in N-terminal C region, that binds to HREs. Steroid hormone nuclear receptors have a 66-68 amino acid DBD that contains two highly conserved zinc fingers, which pull the nuclear receptor apart from other DNA binding proteins (Green and Chambon, 1988). The E region of receptors contains a domain of approximately 225 amino acids that is a hormone binding domain (Kumar and Chambon, 1988). The EcR receptor belongs to Class II of the superfamily. These receptors heterodimerize with RXR (retinoid X receptors) and bind to direct repeats or symmetrical repeats (Mangelsdorf et al., 1995).
The EcR gene is induced directly by ecdysone. There are three EcRs: EcR-A, EcR-B1, and EcR-B2. They encode different protein isoforms (EcRA, EcR-B1, and EcR-B2), which have common DNA and hormone binding domains (LBD), but which differ in their N-terminal regions. Different ecdysone target tissues express different isoforms (Talbot et al., 1993). All three isoforms must heterodimerize with Ultraspiracle in order to bind DNA (Koelle, 1992, Yao et al.,1992, Thomas et al., 1993). The usp gene was first identified in a study of lethal mutant phenotypes. This gene encodes only one protein. USP is a homolog of vertebrate RXRs. USP protein can bind to several RXR partners, including: retinoic acid receptor, thyroid hormone receptor, or vitamin D receptor (Thummel, 1995). All high-affinity hormone binding to ecdysone responsive elements (EcREs) requires the presence of the heterodimer EcR/USP. The ligand binding also stabilizes the EcR/Usp heterodimer (Yao et al., 1992).
The genes responsible for intermolt puffs encode salivary gland secretion (Sgs) proteins. These proteins are one of the components of the glue secreted by the larva used to attach itself to a solid surface for puparition. The 3C and 68C glue genes are repressed by the late larval pulse of ecdysone (Hansson and Lambertsson, 1989).
The first pulse of ecdysone, at the beginning of the prepupal stage (0 hour prepupa), induces the expression of a set of early genes. Three of them have been defined at the molecular level: Broad Complex (BR-C), E74, and E75. Early genes are located within early puffs 2B5, 74EF, 75B, respectively (Burtis et al., 1990; Seagraves and Hogness, 1990; DiBello et al., 1991). In the presence of ecdysone, early genes are transcribed for the next 4 hours. Most interestingly, the second ecdysone pulse, around 10 hours after puparation, induces the same early genes BR-C, E74, E75, but also induces the initial formation of the 93F puff in the polytene chromosomes (Richards 1976a). The early gene E93 is encoded within this puff.
The molecular structures of BR-C, E74, and E75 are very similar; they are unusually long (60-100 kb in length), for Drosophila genes, and are complex genes containing multiple promoters that enable the transcription of several mRNA transcripts. Each of the transcripts encodes a family of related DNA binding proteins. As a consequence, early proteins can therefore be transcription regulators (Anders and Thummel, 1992). Mutations in early genes lead to lethality during the larval, prepupal, or pupal stage. This evidence reinforces the critical role of early genes in metamorphosis.
All three genes have been studied in detail.
All three genes have been studied in detail.BR-C encodes several zinc-finger proteins. A zinc-finger is a structure that allows DNA binding (DiBello et al., 1991). BR-C appears to play an important role in imaginal disc evagination and fusion (Kiss et al., 1988), histolysis of the salivary gland, optic lobe organization, and proper development of thoracic muscle (Restifo and White, 1991; 1992). E74 encodes two major products: E74A and E74B. E74A hemizygous mutants have delayed pupal cuticle tanning and do not eclose. Therefore, this protein is thought to play a role in puparium formation (Fletcher and Thummel, 1995). Most of E74B homozygous mutants die as prepupae and retain their larval body shape, while some fail to evert at the beginning of the pupal stage. Based on these results, E74B has been postulated to play a role in: 1) prolonging larval muscles into the pupal stage until they are required for a successful head eversion and body shortening during puparium formation; 2) ecdysone induction of imaginal discs evagination (Fletcher and Thummel, 1995).
E75 encodes three proteins that are isoforms of one another. The N-terminal sequences are different, but the C-terminal sequences carry an identical DNA binding domain and ligand binding domain (Segraves and Hogness, 1990). All three early genes BR-C, E74, and E75 have binding sites for both early genes and late genes (Hill et al., 1993). This finding reinforces a model of early proteins effecting the late and the early gene transcription.
The early-late genes, directly induced by ecdysone, are considered to be a subclass of the late genes (Ashburner et al., 1974). They differ from late genes in their immediate regression when ecdysone is withdrawn. Three studied early-late genes, E78B, DHR39 and DHR3 belong to the receptor family (Ohno and Petkovich, 1992). The low level induction of E78B and DHR39 occurs between-6 and 0 hour prepupa. There is a second pulse of induction in 6 hour prepupae. DHR3 transcripts appear starting in 5 hour prepupae and reach their maximum levels in 2 hour prepupae. DHR3 transcript levels then decrease and return to initial levels in 6 hour prepupae (Huet et al., 1995).
In the midpupal stage the 63E and 75 CD puffs appear. The mid-prepupal genes increase their activity from 4 to 5 hours after pupation, reaching maximum levels in the 8 hour prepupa (Richards, 1976b). Their transcription requires ecdysone withdrawal as well as the synthesis of the late-larval ecdysone-induced proteins (Baechrecke et al,. 1995; Richards 1976a)
A set of the late gene transcripts appears during the late larval and prepupal pulse of ecdysone (Ashburner et al., 1974). This observation logically follows a proposed model of the ecdysone control mechanism, as early genes are activated prior to late gene transcription. One of the late genes is L71. The set of five L71 genes encodes polypeptides resembling defensins and venom toxins. These are secreted from the salivary glands into the space between the imaginal discs' hypoderm and prepupal cuticle which may protect the animal from bacterial infections (Wright et al., 1996).
For a more detailed account of the genes involved in this process, look at "Formation of the adult fly" in the Interactive Fly.
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