Control of polytenic replication in dipteran larvae. I. Increased number of cycles in a mutant strain of Drosophila melanogaster

Previous studies have demonstrated that a consistent maximum number of polytenic replication cycles occurs in the salivary gland nuclei of a wild-type strain of D. melanogaster. Since that number is achieved within the larval period, the DNA synthesis of the prepupal period is believed to be that of propagation of the final cycle. Photometric determinations have been made, in this study, of the salivary gland nuclei of larvae and prepupae of the tu-h strain in which the larval period has been extended as a consequence of delay or failure of pupation. The DNA values indicate that a higher maximum number of polytenic replications is achieved in such nuclei. It is inferred, thereby, that initiation of polytenic replication is a function of the larval state and, since it is terminated by the intervention of metamorphosis, a hormonal dependence is suggested.     

DHR3 is required for the prepupal-pupal transition and differentiation of adult structures during Drosophila metamorphosis.

Pulses of the steroid hormone ecdysone activate genetic regulatory hierarchies that coordinate the developmental changes associated with Drosophila metamorphosis. A high-titer ecdysone pulse at the end of larval development triggers puparium formation and induces expression of the DHR3 orphan nuclear receptor. Here we use both a heat-inducible DHR3 rescue construct and clonal analysis to define DHR3 functions during metamorphosis. Clonal analysis reveals requirements for DHR3 in the development of adult bristles, wings, and cuticle, and no apparent function in eye or leg development. DHR3 mutants rescued to the third larval instar also reveal essential functions during the onset of metamorphosis, leading to lethality during prepupal and early pupal stages. The phenotypes associated with these lethal phases are consistent with the effects of DHR3 mutations on ecdysone-regulated gene expression. Although DHR3 has been shown to be sufficient for early gene repression at puparium formation, it is not necessary for this response, indicating that other negative regulators may contribute to this pathway. In contrast, DHR3 is required for maximal expression of the midprepupal regulatory genes, EcR, E74B, and betaFTZ-1. Reductions in EcR and betaFTZ-F1 expression, in turn, lead to submaximal early gene induction in response to the prepupal ecdysone pulse and corresponding defects in adult head eversion and salivary gland cell death. These studies demonstrate that DHR3 is an essential regulator of the betaFTZ-F1 midprepupal competence factor, providing a functional link between the late larval and prepupal responses to ecdysone. Induction of DHR3 in early prepupae ensures that responses to the prepupal ecdysone pulse will be distinct from responses to the late larval pulse and thus that the animal progresses in an appropriate manner through the early stages of metamorphosis.     

Photoinduced bonding of endogenous ecdysterone to salivary gland chromosomes of Chironomus tentans

Endogenous ecdysterone has been bonded to chromosomal loci by irradiation of Ch. tentans salivary glands. The hormone has been localized on the polytene chromosomes by indirect immunofluorescence microscopy. Hormone binding to chromosomes is stage-specific. Seven chromosomal loci could be identified which specifically bound hormone in larval salivary glands, and 21 chromosomal loci which specifically bound hormone in prepupal salivary glands. All puffs that have been described by Clever (1961) as being inducible by ecdysterone have been found to contain irreversibly bound ecdysterone in prepupal salivary gland chromosomes. A small number of puff sites in larval salivary gland chromosomes exhibited varying amounts of bound ecdysterone, (as judged by fluorescence intensity) most notably 117B and Balbiani rings 1 and 3 on chromosome IV. In addition to stage specific binding sites, there were many others showing equal binding of the hormone in both, larval and prepupal, stages of development. — Fluorescence intensities (reflecting the amount of bonded hormone) at puff sites along the tip section of the prepupal salivary gland chromosome arm IR have been computed indicating that differences between fluorescence intensities of different puffs can be expressed as multiples of a basic fluorescence intensity. Thus, the amount of fluorescence intensity (bonded hormone) in the various puffs may be quantized. — The data indicate that in Ch. tentans salivary glands ecdysterone acts, at the chromosomal level. The development of larvae into prepupae generates more puff sites and more hormone binding. This is discussed in the light of current models of hormone-receptor function.     

Glucosamine metabolism in Drosophila virilis salivary glands: Ontogenetic changes of enzyme activities and metabolite synthesis

In Drosophila virilis salivary glands the in vitro activities of enzymes involved in the glucosamine pathway were examined during the third larval instar and in the prepupa. While glutamine-fructose-6-phosphate aminotransferase (EC 5.3.1.19) becomes inactive at the time of puparium formation, glucosamine-6-phosphate isomerase (EC 5.3.1.10) and glucosamine-6-phosphate N-acetyltransferase (EC 2.3.1.3) show maximal activities in the prepupal gland. The activity of UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) may also decrease prior to puparium formation. Incubation of larval and prepupal glands in medium containing [³H]glucose + [¹⁴C]-uridine or [¹⁴C]glucosamine and subsequent separation of intermediates of the glucosamine pathway by chromatographic procedures reveal that the capacity of the glands to incorporate the isotopes into these intermediates decreases significantly at the time of puparium formation. The results suggest that in D. virilis salivary glands the formation of aminosugars is mainly controlled by the activities of the two enzymes glutamine-fructose-6-phosphate aminotransferase and UDP-N-acetylglucosamine pyrophosphorylase.     

Developmental plasticity of neuropeptide expression in motoneurons of the moth,Manduca sexta: Steroid hormone regulation

Developmental changes in the expression of a FMRFamide-like (Phe-Met-Arg-Phe-NH₂) peptide or peptides in motoneurons of the tobacco hornworm, Manduca sexta, were demonstrated using immunohistochemical techniques. The onset of FMRFamide-like immunoreactivity (FLI) was gradual during larval growth but by the final larval stage, immunoreactivity was present in the majority of motoneurons. FLI then declined during metamorphosis and was absent in all identified adult motoneurons. We used a novel in vivo culture system to demonstrate that the steroid hormone, 20-hydroxyecdysone, regulates the loss of FLI in motoneurons during metamorphosis. The small commitment peak of ecdysteroid appears to shut off the program of neuropeptide accumulation that is characteristic of the larval state of the motoneurons. The prepupal peak of steroid then causes the rapid loss of stored FLI. This steroid-induced change in the neuropeptide content of motoneurons may reflect major changes in neuromuscular functions between the larval and adult stages. © 1996 John Wiley & Sons, Inc.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

The patterns of puffing activity have been studied during the late larval and prepupal stages of Drosophila melanogaster. On the major salivary gland autosomes (chromosomes 2 and 3) 108 loci form puffs at some time during these developmental stages. The timing and pattern of activity of 83 of these puffs is found to be strictly dependent upon the age of the animals. Two major peaks in puffing activity occur. The first of these is at the time of puparium formation and the second in 8 hr. old prepupae. Both of these puffing peaks precede a moult by 4 hrs. 30 puffs are active before or at the time of both of these two moults. However, the sequence of appearance and regression of many of this group of puffs is different at the prepupal moult than at the pupal moult. 12 puffs occur only before or at the time of the prepupal moult and 13 puffs only before or at the time of the pupal moult. The functional significance of these periods of puffing activity is discussed and it is concluded that one function of this genetic activity in the salivary glands of metamorphosing Drosophila is the production of substances to be utilised during the histogenesis of the adult tissues.     

Hormonal regulation and developmental role of Krüppel homolog 1, a repressor of metamorphosis,in the silkworm Bombyx mori

Juvenile hormone (JH) has an ability to repress the precocious metamorphosis of insects during their larval development. Krüppel homolog 1 (Kr-h1) is an early JH-inducible gene that mediates this action of JH; however, the fine hormonal regulation of Kr-h1 and the molecular mechanism underlying its antimetamorphic effect are little understood. In this study, we attempted to elucidate the hormonal regulation and developmental role of Kr-h1. We found that the expression of Kr-h1 in the epidermis of penultimate-instar larvae of the silkworm Bombyx mori was induced by JH secreted by the corpora allata (CA), whereas the CA were not involved in the transient induction of Kr-h1 at the prepupal stage. Tissue culture experiments suggested that the transient peak of Kr-h1 at the prepupal stage is likely to be induced cooperatively by JH derived from gland(s) other than the CA and the prepupal surge of ecdysteroid, although involvement of unknown factor(s) could not be ruled out. To elucidate the developmental role of Kr-h1, we generated transgenic silkworms overexpressing Kr-h1. The transgenic silkworms grew normally until the spinning stage, but their development was arrested at the prepupal stage. The transgenic silkworms from which the CA were removed in the penultimate instar did not undergo precocious pupation or larval–larval molt but fell into prepupal arrest. This result demonstrated that Kr-h1 is indeed involved in the repression of metamorphosis but that Kr-h1 alone is incapable of implementing normal larval molt. Moreover, the expression profiles and hormonal responses of early ecdysone-inducible genes (E74, E75, and Broad) in transgenic silkworms suggested that Kr-h1 is not involved in the JH-dependent modulation of these genes, which is associated with the control of metamorphosis.     

Three‐dimensional fate mapping of larval tissues through metamorphosis in the glass sponge Oopsacas minuta

The tissue of glass sponges (Class Hexactinellida) is unique among metazoans in being largely syncytial, a state that arises during early embryogenesis when blastomeres fuse. In addition, hexactinellids are one of only two poriferan groups that already have clearly formed flagellated chambers as larvae. The fate of the larval chambers and of other tissues during metamorphosis is unknown. One species of hexactinellid, Oopsacas minuta, is found in submarine caves in the Mediterranean and is reproductive year round, which facilitates developmental studies; however, describing metamorphosis has been a challenge because the syncytial nature of the tissue makes it difficult to trace the fates using conventional cell tracking markers. We used three‐dimensional models to map the fate of larval tissues of O. minuta through metamorphosis and provide the first detailed account of larval tissue reorganization at metamorphosis of a glass sponge larva. Larvae settle on their anterior swimming pole or on one side. The multiciliated cells that formed a belt around the larva are discarded during the first stage of metamorphosis. We found that larval flagellated chambers are retained throughout metamorphosis and become the kernels of the first pumping chambers of the juvenile sponge. As larvae of O. minuta settle, larval chambers are enlarged by syncytial tissues containing yolk inclusions. Lipid inclusions at the basal attachment site gradually became smaller during the six weeks of our study. In O. minuta, the flagellated chambers that differentiate in the larva become the post‐metamorphic flagellated chambers, which corroborate the view that internalization of these chambers during embryogenesis is a process that resembles gastrulation processes in other animals.     

Developmentally regulated localization of endosymbiotic dinoflagellates in different tissue layers of coral larvae

In adult cnidarians, symbiotic dinoflagellate Symbiodinium are usually located in the gastrodermis. However, the onset of this endosymbiotic association and its regulation during larval development are unclear. This study examined the distribution of the Symbiodinium population in tissue layers of planula larvae released from the stony coral Euphyllia glabrescens. Symbiodinium were redistributed from the epidermis to the gastrodermis, at a rate that was fastest during early planulation and then decreased prior to metamorphosis. This process indicates that the endosymbiotic activity of coral tissues is developmentally regulated. During the early larval stage, both the epidermis and gastrodermis contained Symbiodinium; then, as the larvae developed toward metamorphosis, the numbers in the epidermis gradually diminished until they were only found in the gastrodermis. The mechanism of redistribution remains unknown, but may be due to a direct translocation and/or change in the proliferation of symbionts in different tissue layers.     

Some autophagic and apoptotic features of programmed cell death in the anterior silk glands of the silkworm,Bombyx mori

Programmed cell death has been subdivided into two major groups: apoptosis and autophagic cell death. The anterior silk gland of Bombyx mori degenerates during larval-pupal metamorphosis. Our findings indicate that two types of programmed cell death features are observed during this physiological process. During the prepupal period, pyknosis of the nucleus, cell detachment and membrane blebbing occur and they are the first signs of programmed cell death in the anterior silk glands. According to previous studies, all of these morphological appearences are common for both cell death types. Autophagy features are also exhibited during the prepupal period. One of the lysosomal marker enzymes, acid phosphatase, levels are high during this period then decrease gradually. Vacuole formation begins to appear first at the basal surface of the cell, then expands to the apical surface just before the larval pupal ecdysis. After larval-pupal ecdysis, DNA fragmentation, which is the obvious biochemical marker of apoptosis, is detected in agarose gel electrophoresis which also shows that caspase-like enzyme activities occur during the programmed cell death process of the anterior silk glands. Apoptosis and autophagic cell death interact with each other during the degeneration process of the anterior silk gland in Bombyx mori and this interaction occurs at a late phase of cell death. We suggest that only apoptotic cell death not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.

Addendum to: Goncu E, Parlak O. Morphological changes and patterns of ecdysone receptor B1 immunolocalization in the anterior silk gland undergoing programmed cell death in the silkworm, Bombyx mori. Acta Histochem 2008; In press.     

Ecdysterone responsive functions in the mutantl(1)su(f) ts67g ofDrosophila melanogaster

Summary Transferring the temperature sensitive mutantl(1)su(f) ^ts67g from 25° C to 30° C before or early in the third larval instar blocks the increase in the ecdysterone titer that normally occurs at the end of the larval period. Feeding exogenous ecdysterone to these hormone-deficient larvae results in the formation of pseudopupae. The mutant was used to study ecdysterone-inducible functions in late larval salivary glands by preparing three animal samples with different hormone titers: the titer was low in one sample because of an earlier temperature shift, high in a second sample because the larvae were subsequently transferred to ecdysterone-supplemented food, and also high in a third sample that was kept at 25°C, providing a control for normal development. The effect of the different hormone conditions was studied by³⁵S-methionine labeling of the salivary gland proteins during the larval to prepupal transition and the prepupal period. The results indicate that synthesis of several of the proteins normally appearing during the transition and prepupal period is induced by exogenous ecdysterone.     

Ecdysone-induced changes in glycoprotein synthesis and puff activities in Drosophila virilis salivary glands

During five hours after the injection of -ecdysone into the hemolymph of D. virilis late third instar larvae the formation of larval glycoproteins in the salivary glands is terminated and the synthesis of a different set of glycoproteins which is characteristic for the prepupal gland is initiated. The data presented suggest that products from early puffs inhibit the formation of larval glycoproteins while the induction of late puffs may be responsible for the appearance of prepupal glycoproteins.     

The timing of drosophila salivary gland apoptosis displays an l(2)gl-dose response

During Drosophila metamorphosis, larval tissues, such as the salivary glands, are histolysed whereas imaginal tissues differentiate into adult structures forming at eclosion a fly-shaped adult. Inactivation of the lethal(2)giant larvae (l(2)gl) gene encoding the cytoskeletal associated p127 protein, causes malignant transformation of brain neuroblasts and imaginal disc cells with developmental arrest at the larval-pupal transition phase. At this stage, p127 is expressed in wild-type salivary glands which become fully histolysed 12 - 13 h after pupariation. By contrast to wild-type, administration of 20-hydroxyecdsone to l(2)gl-deficient salivary glands is unable to induce histolysis, although it releases stored glue granules and gives rise to a nearly normal pupariation chromosome puffing, indicating that p127 is required for salivary gland apoptosis. To unravel the l(2)gl function in this tissue we used transgenic lines expressing reduced ( approximately 0.1) or increased levels of p127 (3.0). Here we show that the timing of salivary gland histolysis displays an l(2)gl-dose response. Reduced p127 expression delays histolysis whereas overexpression accelerates this process without affecting the duration of third larval instar, prepupal and pupal development. Similar l(2)gl-dependence is noticed in the timing of expression of the cell death genes reaper, head involution defective and grim, supporting the idea that p127 plays a critical role in the implementation of ecdysone-triggered apoptosis. These experiments show also that the timing of salivary gland apoptosis can be manipulated without affecting normal development and provide ways to investigate the nature of the components specifically involved in the apoptotic pathway of the salivary glands.     

Hormonally regulated expression of arginine kinase in Drosophila melanogaster

Summary Arginine kinase (AK) is present throughout the life cycle of Drosophila melanogaster, but there is a sharp, transient peak of AK activity during the prepupal period and a second period of elevated activity at the time of eclosion of the adult. Imaginal discs show the greatest increase in AK activity at the prepupal stage of those tissues assayed. The prepupal peak is not seen when the temperature-sensitive ecdysoneless mutant ecd-1 is shifted to 29° C at mid-third instar larval stage. The peak in activity reappears when ecd-1 is either shifted back to 20° C after 60 h at 29° C or is fed 20-hydroxyecdysone. At the restrictive temperature, imaginal discs from ecd-1 larvae progressively lose AK activity, whereas discs from 20-hydroxyecdysone-fed larvae have a marked increase in AK activity at stage P3 of the prepupal period. These data suggest that the prepupal peak is regulated by the hormone 20-hydroxyecdysone.     

A novel hemocyte-specific membrane protein of Sarcophaga (flesh fly).

Extensive tissue remodeling takes place during metamorphosis of holometabolous insects. It has been shown that hemocytes play crucial roles in the recognition and elimination of apoptotic cells and larval tissue fragments produced during metamorphosis. We report the immunoaffinity purification, cDNA cloning, and characterization of a prepupal hemocyte membrane protein of Sarcophaga (flesh fly) with a molecular mass of 120 kDa. This protein is a novel type I transmembrane protein with 18 repeats of an epidermal growth factor-like domain in the predicted extracellular region. Expression of the protein was restricted exclusively to prepupal hemocytes. This protein is suggested to be a scavenger receptor for tissue remodeling.     

Patterns of protein synthesis in salivary glands ofDrosophila melanogaster during larval and prepupal development

Summary Patterns of protein synthesis in the salivary glands ofDrosophila melanogaster have been studied throughout late larval and prepupal development by pulse labelling the tissues with³⁵S-methionine. Specific changes to the pattern of proteins synthesized during development are found and the significance of these changes is discussed in view of the known changes in gene (puffing) activity which occur at the same times. We review the problem of salivary gland function in prepupalDrosophila.     

Characteristics of the endocrine system in Drosophila melanogaster 1 (2)gl mutants, differing in time of death]

A comparative study of the state of the endocrine system and one of the larval organs, the salivary gland, has been carried out in larvae homozygous for the 1(2)gl gene. They differ in time of death (death at the third larval instar--larval allele, and at the prepupal state--prepupal allele). It is shown that homozygotes for the larval and prepupal allele have underdeveloped prothoracal glands. Corpora allata in homozygotes for the larval allele does not differ from the norm. Corpora allata in homozygotes for the prepupal allele is decreased proportionally to the decrease of prothoracal glands. A decrease of gland size is due to a decrease of the volume of cell but not to their number; this decrease is accompanied by the decrease of their relative DNA content. Salivary glands in homozygotes are reduced and comprise 80% of the normal size in homozygotes for the prepupal allele and 50% for homozygotes for the larval allele. Polyteny level in the salivary gland nuclei is much decreased as compared with the normal level. DNA level is more reduced in larvae homozygous for the larval allele.