Hormonal control of growth and differentiation of insect tissues cultured in vitro

Summary This paper reviews the effects of insect hormones on lepidopteran imaginal discs cultured in vitro.β-ecdysone stimulated both evagination and cuticle deposition of wing discs ofPlodia interpunctella (Hübner). However, evagination required a shorter exposure to ecdysone than did cuticle deposition. Cuticle deposition was obtained under the following conditions: (a) a 24-hr pulse ofβ-ecdysone (0.5–5.0μg/ml); (b) continuous treatment with 0.2μg/mlβ-ecdysone; or (c) continuous treatment with 0.5 to 50.0μg/mlβ-ecdysone in medium conditioned with larval fat body. Investigations of some biochemical effects of ecdysone showed that RNA and protein synthesis was required for evagination and cuticle deposition. In particular, studies with actinomycin D and cycloheximide (at nontoxic levels) showed that RNA and protein synthesis during the ecdysone-dependent period was essential for subsequent development. These findings support the hypothesis that stimulation of macromolecular synthesis is fundamental to the action of ecdysone on imaginal discs. The influence of beta-ecdysone on chitin synthesis was also examined.β-ecdysone stimulated uptake and incorporation of tritiated-glucosamine by culturedP. interpunctella wing discs. Addition of hexosamines to the culture medium had no influence on ecdysone-induced cuticle deposition, but inhibition of glucose-uptake by cytochalasin B prevented the formation of cuticle. The action of ecdysone on particular enzymes in the chitin pathway remains to be elucidated. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Electrophoretic analysis of soluble proteins specifically synthesized under phosphate deficiency in the mycelia ofPholiota nameko

Mycelial soluble proteins ofPholiota nameko labeled in vivo during the Pi-supplied (P⁺) and the Pi-depleted (P⁻) cultures were separated by SDS-polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis, and visualized by fluorography. A comparison of protein profiles from the P⁺ and P⁻ cultures showed that Pi deficiency induces the synthesis of 15 polypeptides and an increase in the relative amount of 29 polypeptides. These result suggests that many proteins may be specifically synthesized de novo under Pi deficiency as part of the adaptive mechanism for this condition.     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.     

Identification of newly synthesized wing imaginal disc proteins induced by 20-hydroxyecdysone inGalleria mellonella

Summary Wing imaginal discs from 7th instarGalleria mellonella L. larvae evaginate and exhibit tracheolar elongation when exposed to 20-hydroxyecdysone in vitro. This response was elicited within 24 h of treatment as was a greater than fourfold stimulation of the incorporation of [³H]leucine into disc proteins. Autoradiographic analyses of [³⁵S]methionine labeled polypeptides separated on two-dimensional gels, however, revealed no differences in protein profiles between control and treated discs until 48 h following exposure to molting hormone. At this time, wing imaginal discs exposed to 1 μg/ml 20-hydroxyecdysone synthesized four unique polypeptides not detected either in controls or in discs treated for 24 h. These four new proteins were also found to be synthesized by imaginal discs that had evaginated in vivo. These results suggest that these proteins are normally synthesized subsequent to evagination and do not play a role in the morphological events necessary for evagination. Mention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA. S. G. M. is employed through a cooperative agreement between the Insect Attractants, Behavior and Basic Biology Laboratory and the Department of Entomology, University of Florida.     

Determination of Optimal Protein Quantity Required to Identify Abundant and Less Abundant Soybean Seed Proteins by 2D-PAGE and MS

Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (75, 100, 125, 150, and 200 μg) by 2D-PAGE. Following 2D-PAGE and spot excision, proteins were identified by mass spectrometry analysis. The number of visible protein spots was increased with an increase in the amount of protein loaded. The intensity of highly abundant proteins [β-conglycinin β-homotrimer and glycinin G4 (A5A4B3) precursors] increased linearly between 75 and 125 μg, whereas the proglycinin G3 (A1ab1b) homotrimer showed linearity between 75 and 150 μg. The spot intensity of less abundant proteins, glycinin G2 (A2b1a) precursor and proglycinin G3 (A1ab1b) homotrimer, increased linearly with an increase in the amount of protein through 200 μg, whereas spot intensity of β-conglycinin β-homotrimer and the allergen Gly m bd 28K increased linearly until 150 μg and did not increase further at 200 μg. These results suggest that 150 μg protein was a suitable amount for the separation of abundant proteins, and 200 μg protein was suitable for the separation of less abundant proteins prepared from soybean seeds. Mention of trade name, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or imply its approval to the exclusion of other products or vendors that also may be suitable.     

Effects of fat body on α-ecdysone induced morphogenesis in cultured wing disks of the wax moth, Galleria mellonella

Fat body promoted ecdysone induced morphogenesis in Galleria wing disks cultured in vitro. Medium preincubated with fat body and α-ecdysone from any of the first 5 days of the final larval instar enhanced tracheal migration and elongation in the disks. Disks from the third, fourth, and fifth days of the final larval instar responded equally well to the fat body and α-ecdysone. We suggest a physiological rôle for Galleria fat body as an intermediary in the stimulation of wing disk development by α-ecdysone.     

Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation

Differentiating imaginal hypodermal cells of Drosophila melanogaster form adult cuticle during the second half of the pupal stage (about 40 to 93 hr postpupariation). A group of proteins with molecular weights of 23,000, 20,000, and 14,000 is identified as putative major wing cuticle proteins with the following biological properties: These proteins are abundant components of cuticle and are major synthetic products of cuticle-secreting hypodermal cells. They are leucine-rich and methionine-free and are the most prominent proteins of this type synthesized by wing hypoderm at 65 hr, during the period of procuticle formation. Electron microscopic autoradiography shows that leucine-rich, methionine-free proteins specifically localize to the apical cell surface and newly secreted cuticle of 65-hr wing cells. This strongly suggests the export of these proteins to the cuticle. Lastly, these proteins undergo a reduction in extractability just after eclosion, during the period of cuticle protein crosslinking (sclerotization). The synthesis of these major hypoderm proteins is temporally regulated in development. In wing cells, the 14-kDa proteins are synthesized first, from 53 to 78 hr, and the 20- and 23-kDa proteins are synthesized from 63 to 93 hr. The pattern of synthesis for these proteins is similar in abdominal cells but delayed by 6 to 10 hr. Two-dimensional gel electrophoresis shows that each of the 23-, 20-, and 14-kDa size classes contains at least two component polypeptides. Patterns of protein synthesis in cells of the imaginal hypodermis are regulated in a precise temporal sequence during the production of adult cuticle. Their study yields a useful system for the analysis of molecular events in gene control and cell differentiation.     

Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by β-ecdysone

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.     

The effect of fat body on the differentiation in vitro of wing imaginal discs ofDrosophila melanogaster

Summary The effect of suboptimal levels of -ecdysone on the differentiation in vitro ofDrosophila melanogaster wing discs was enhanced by the addition of larval fat body to the cultures. However, similar experiments with -ecdysome showed no enhancement. It is suggested that a partial conversion of -ecdysone to -ecdysone by the fat body may well account for these results.     

Time-dosage studies of juvenile hormone action on the development of Plodia interpunctella

The degree of inhibition of larval-pupal ecdysis of Indian meal moths, Plodia interpunctella, by juvenile hormone (JH) treatment depended upon the dosage of hormone and time of treatment. During the last larval instar, the timing aspect operated independently of dosage and had two essential components for effectiveness, (a) early initiation of exposure and (b) maintenance of exposure. The effects of JH treatments could be reversed by removing the insects from the JH diet. In vitro tests with wing disks indicated that JH reversibly inhibited disk development only during the early part of the last larval instar, a time when disks are insensitive to β-ecdysone. After disks acquire full sensitivity to β-ecdysone, they lose their ability to respond to JH.     

Integument and sensory nerve differentiation ofDrosophila leg and wing imaginal discs in vitro

Summary An ultrastructural analysis is presented of the cuticular and neural structures formed by the prothoracic leg and wing imaginal discs of maleDrosophila melanogaster larvae during culture in vitro with 0.2 g/ml of -ecdysone. A pupal cuticle, and subsequently an imaginal cuticle with a well-defined epicuticle and a laminated endocuticle is formed. The ultrastructure of the epidermis and of cuticular structures such as bristles, trichomes, apodemes, and tracheoles is very similar to that found in situ. Dendrites and nerve cell bodies are formed in vitro, and sensory axons form nerve bundles similar to those of normal appendages in situ, despite their isolation from the central nervous system. It is concluded that at the ultrastructural level, differentiation in vitro closely parallels the normal course of development.     

Synthesis of salt-soluble proteins in barley. Pulse-labeling study of grain filling in liquid-cultured detached spikes

The accumulation of salt-soluble proteins in the endosperm of developing barley (Hordeum vulgare L.) grains was examined. Detached spikes of barley were cultured at different levels of nitrogen nutrition and pulse-labeled with [¹⁴C] sucrose at specific times after anthesis. Proteins were extracted from isolated endosperms and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Fluorography revealed an early, middle and late synthesis of specific proteins during grain filling. Synthesis of proteins appearing at the later stages responded to increased nitrogen nutrition. Two major components, -amylase and protein Z in particular, had a synthesis profile almost identical to that of the endosperm storage protein, hordein.Abbreviations CIE Crossed immunoelectrophoresis - SDSPAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae

Exoglucanase (exo-1,3-β-D-glucan glycohydrolase, EC 3.2.1.56) activity secreted by Saccharomyces cerevisiae into the culture medium was separated by ion exchange chromatography into two glycoprotein isoenzymes which contributed 10% (exoglucanase I) and 90% (exoglucanase II) towards the total activity. Analysis of the “in vitro” deglycosylated products by polyacrylamide gel electrophoresis under native or denaturing conditions indicated that the protein portions of both exoglucanases exhibited identical mobility, each one consisting of two polypeptides with M _r of 47000 and 48000. The same profile was shown by the exoglucanase secreted in the presence of tunicamycin. Antibodies raised against the protein portion of exoglucanase II did react with both native exoglucanases and their deglycosylated products with a pattern indicative of immunological identity. Digestion of the “in vitro” deglycosylated products of both exoglucanases with Staphylococcus aureus V-8 protease or trypsin generated the same proteolytic fragments in each case. Only exoglucanase II was secreted by protoplasts. These and previously reported results indicate that the protein portions of both isoenzymes may be the product of the same gene (or a family of related genes), and that exoglucanase I is a product of enzyme II, modified by a process occurring beyond the permeability barrier of the cell.     

Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by beta-ecdysone.

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.     

Changes in synthesis of specific proteins following axotomy: Detection with two-dimensional gel electrophoresis

Changes in protein synthesis during development and following axotomy were analyzed by two-dimensional gel electrophoresis. The two major postganglionic nerves emerging from the superior cervical sympathetic ganglia (SCSG) of adult rats were either cut or crushed unilaterally. At intervals ranging from 1 to 112 days after surgery both SCSG were removed and incubated for 1 hr in the presence of ¹⁴C-leucine. Proteins were extracted and subjected to two-dimensional electrophoretic separation and autoradiography. With this technique, proteins are separated on the basis of isoelectric point and molecular weight. Also, intact SCSG from 1, 2, 7, and 14 day old rats were labeled and analyzed. It was found that a minority of the separated proteins exhibited some detectable change in relative rate of synthesis following axotomy. Actin exhibited a slight (< 20%) increase in relative synthesis rate while tubulin did not change significantly. There were small but significant differences in the protein patterns following nerve crush, as opposed to nerve cut. Comparison of protein synthesis patterns from developing rat SCSG with those from intact and from axotomized adult SCSG failed to demonstrate any marked similarity between the developmental and the axotomy patterns.     

Stage-specific protein synthesis during early embryogenesis in Drosophila melanogaster

The changes in protein species synthesized during early Drosophila embryogenesis were characterized by two-dimensional electrophoresis. Of the 261 proteins scored, 68 (26%) show dramatic changes in rates of synthesis during the first 8 h of embryogenesis. These stage-specific proteins can be classified into three categories: early, detected at 1, 2 and 3 h but not later; late, not detected at 1 h, but appearing later; and discontinuous, detected before and after, but not at 3 and 4 h. RNA was extracted from three representative stages, translated in vitro, and the translation products separated on two-dimensional gels. There was a strong correlation between the patterns of synthesis in vivo and in vitro, suggesting that the early proteins are translated from maternal mRNA, and the late proteins from zygotic mRNA. A thorough comparison was made between the proteins synthesized in wild-type and dorsal embryos, in which virtually only dorsal hypoderm differentiates. The first observed difference was a reduced synthesis of actin I at 8 h, indicating that the absence of mesodermal and endodermal tissues is not detectable at the level of moderately abundant protein until the onset of differentiation.     

Further observations on the effect of ethionine on the synthesis of β-galactosidase inEscherichia coli: Formation of an immunologically cross-reacting protein

It was found that ethionine partially inhibits the transport of the inducer (TMG) of β-galactosidase into the cells ofEscherichia coli ML-30. The synthesis of β-galactosidase-specific messenger RNA is not inhibited. Ethionine appears to be incorporated into proteins synthesized by the strains used. The incorporation of ethionine into the molecule of β-galactosidase results in the synthesis of an enzymically inactive, immunologically cross-reacting protein.