Developmental Studies in Drosophila

The salivary gland chromosomes of 3rd instar Drosophila pseudoobscura larvae were observed for puffing changes after injection of larvae with ecdysterone solution. Chromosomes from the salivary glands of 3rd instar larvae and prepupae were similarly examined after incubation in ecdysterone-containing medium. The larvae, after treatment, showed advancement of the puffing process with the occurrence of a pattern similar to that observed during the pre-spiracle eversion period of normal development. At least 92 puffs showed changes in size. For the prepupae, the puffing changes resembled those occurring normally during the late prepupal period. A group of puffs were selected for detailed study. Among these were four puffs on the XR chromosome which exhibited large increases before spiracle eversion and pupation in normal development. As in normal development, two of these became the most prominent puffs observed within h after hormone treatment. In chromosomes from larval glands, the other two XR chromosome puffs were among the largest puffs to appear later in the sequence. However, in chromosomes from prepupal glands one of these later puffs failed to appear. The significance of this large number of hormone-inducible puffing changes at two different periods in development is discussed.     

The apparent requirement of two hormones, alpha- and beta-ecdysone, for molting induction in insects

Puff formations at loci I-18-C and IV-2-B of the salivary gland chromosomes are early indications of a beginning molting process in Chironomus tentans larvae. The effectiveness of the two ecdysone analogs, α- and β-ecdysone, in inducing these puffs was compared. Incubation of salivary glands in vitro with β-ecdysone causes only puff IV-2-B to appear; incubation with α-ecdysone stimulates initially puffing at only I-18-C. After an injection of α-ecdysone, puffing at I-18-C begins within less than 15 min, whereas puffing at IV-2-B is delayed for more than 30 min. Following an injection of β-ecdysone, puffing at IV-2-B begins within less than 15 min, whereas puffing at I-18-C is delayed. Injected ³H-α-ecdysone is converted to β-ecdysone and a polar compound. Injected ³H-β-ecdysone is converted to a compound less polar than α-ecdysone and a polar metabolite which stimulates puffing at I-18-C, like α-ecdysone. It is suggested that the two ecdysones have different targets in the cell, that they can be rapidly converted to compounds with the activity of the other analog, and that the induction of a complete molt requires the action of both hormones.     

Identification of the spI products of Balbiani ring genes in Chironomus thummi

The spI fraction of high molecular weight secretory proteins was analysed in Chironomus thummi. These proteins are encoded by giant Balbiani ring (BR) genes which develop specifically in salivary gland cells. Each component of the spI fraction was studied electrophoretically from early and middle 4th instar larvae and prepupae, as well from galactose-treated larvae where changes in the relative puffing pattern of BR1 and BR2 are known to occur. The spI fraction consists of at least two bands with electrophoretic mobilities slower than those of the spI components of Camptochironomus. The slow migrating component remains throughout the 4th larval instar, while the amount of the faster component changes, being abundant in early 4th instar and prepupae, but not present (or very weak) in middle 4th instar. The correlated shifts in BR puffing pattern during these developmental stages suggest that the slow and fast components are encoded by BR2 and BR1. The spI fraction is modified by galactose treatment, the fast component being induced in parallel with a decrease in the slow component. These changes are correlated with changes in the steady-state levels of RNA: an increase in BR1 RNA and a decrease in BR2 RNA, and of proteins. These proteins could correspond to the spIb and spIa fractions allocated to BR2 and BR1, respectively, in Camptochironomus. After galactose treatment a new faster band sometimes appears, that could correspond to the spIc fraction of Camptochironomus. A possible spId equivalent was also identified. In conclusion the main features of the spI family in C. thummi are similar to those of spI in Camptochironomus.Abbreviations BR Balbiani ring - spI family of M_r=10⁶ secretory polypeptides     

Heat shock phenomena in Chironomus tentans

Incubation of 4th instar larvae of Chironomus tentans at elevated temperatures leads in salivary and Malpighian chromosomes to the appearance of 4–5 new puffs. Previously present puffs, particularly Balbiani rings in salivary chromosomes, become drastically reduced. The reactions of region IV-5C and Balbiani ring 1 and 2 in salivary glands are quantitatively analyzed. Statistically significant heat shock effects are observed already after 5 min and reach a maximum between 30 and 60 min. The effective temperature range is small (between 33 to 40 ° C) with an optimum at 37 ° C. Above 40 ° C, i.e., at overheat shock temperatures, heat shock reactions are suppressed. Larvae heat or overheat shocked for 1–7 h or 15–30 min, respectively, survive when returned to normal culturing temperatures. The recovery from heat shock of the puffing pattern occurs in two phases: a fast one (10–20 min) and a slow one (up to 5 h) sometimes separated by a period of backlash. Quenching of overheat shocked larvae does not result in a delayed heat shock reaction.     

Comparative characteristics of the puffing pattern and the endocrine system in an oregon laboratory stock and in l(2)gl Drosophila melanogaster mutants differing in the time of their death

This study shows that homozygotes for different alleles of the lethal mutant, l(2)gl, differing in the time of death also vary in the state of their endocrine system and the puffing patterns of their salivary gland chromosomes. Homozygotes which die at the larval stage have underdeveloped prothoracic glands and normal corpora allata (CA); in those dying at the prepupal stage both the prothoracic glands and the CA are equally underdeveloped. — All the early third instar larval puffs (96–110 h., PS 1–2) develop in homozygotes; however, the reduction of some early larval puffs, normally occurring before pupariation or at puparium formation, is delayed. Some puffs are more developed than normal. — The differences in puffing patterns chiefly concerned puffs which normally appear 4–5 h before puparium formation and at puparium formation. In homozygotes lethal as larvae some of the puffs normally active at this time did not develop. However, along with some of the late larval puffs, there appeared many puffs characteristic of prepupae. — In homozygotes lethal as prepupae only the time and sequence of puff appearance was altered. Many late larval puffs were active in prepupae rather than in larvae, whereas some of the puffs, normally appearing in prepupae, were active in the larval stage.Accordingly, we propose to distinguish two groups of puff loci. 1) Hormone dependent puffs: These do not develop in larval lethals and are active only after puparium formation in pupariated lethals. 2) Autonomous puffs: Their appearance depends more on the time of development, than on hormonal background. It is suggested that the induction of hormone dependent puffs and of puparium formation is possible at low ecdysone levels, provided that the juvenile hormone level is also low.     

Dependence of Balbiani ring puffing on protein synthesis

The possible dependence of puffing of the Balbiani rings (BRs) on the protein synthesis has been investigated by studying the response of these structures to protein synthesis inhibition induced by cycloheximide and anisomycin. When larvae of Chironomus thummi belonging to middle IV instar (BR1 repressed, BR2 expanded) are subjected to short treatment (3–6 h) with these drugs, BR1 and BR2 puffing states remain essentially unaffected. But when the same treatments are applied to galactose-pretreated larvae (BR1 expanded, BR2 repressed), selective reactivation of the collapsed BR2 occurs. These observations suggest that maintenance of a given puffing state can be dependent, to a variable extent, on the supply of newly synthesized proteins. In particular, selective re-expansion of galactose-repressed BR2 induced by the drugs seem to indicate the existence of repressor-like factor whose activity would be triggered by the galactose treatment.     

Puffing activities and binding of ecdysteroid to polytene chromosomes of Drosophila melanogaster

Salivary glands of third instar Drosophila melanogaster larvae were incubated in vitro in the presence of 5 x 10(-6) M 20-hydroxy-ecdysone. Steroid hormone was localized on the polytene chromosomes of the salivary gland by a combination of photoaffinity-labeling and indirect immunofluorescence microscopy. Steroid hormone binding to chromosomal loci and their puffing activity was correlated for the larval/prepupal puffing cycle characterized by puff stages 1-10. In general, there was a good correlation between the sequential and temporal puffing activity induced by 20-hydroxy-ecdysone and the binding of ecdysteroid hormone to these puffs. Ecdysteroid hormone was detected at intermolt, and at early and late puffs with two notable exceptions. Ecdysteroid was not detected at the two well-studied puffs at 23E and at 25AC, the former being an early puff, which is activated in the presence of 20-hydroxy-ecdysone, and the latter being an intermolt puff, which regresses more rapidly in the presence of hormone. Ecdysteroid hormone was present at puffs as long as the respective puff was active. Also, it apparently accumulated at late puff sites after induction. Since ecdysteroid binding to chromosomal loci is temporal as well as sequential during the larval/prepupal puffing cycle, additional factors besides steroid hormone are necessary for sequentially regulating puffing and concomitant gene activity during development from larvae to prepupae.     

The control of prepupal puffing patterns in vitro; Implications for prepupal ecdysone titres in Drosophilia melanogaster

In late third instar larvae and prepupae of Drosophila melanogaster there is a complex change in puffing patterns in the salivary gland chromosomes. There are two peaks of activity in this period. The first, in larvae, is known to be under the control of the moulting hormone ecdysone. The second, in prepupae, is now shown by the in vitro culture of prepupal glands to be under the specific control of β-ecdysone in a manner similar to the first. A new class of puffs, active between these two peaks, whose induction is inhibited by ecdysone in vitro, is described. The behaviour of these puffs, exemplified by 75CD and 63E, suggests a period of very low ecdysone titre in vivo. The developmental significance of the role of ecdysone during prepupal development is discussed.     

Wirkungen von L-glutaminsäure auf grösse und puff-muster der Larvalen Speicheldrüsenchromosomen von Drosophila melanogaster in vivo

The ratio DNA: RNA in the dry substance of Drosophila melanogaster larvae changes when L-glutamic acid is added to the substratum. The number of cells in the salivary glands is not affected (Fahrig et al., 1967). The present paper deals with the effect of this altered ratio on the puffing pattern of the salivary gland chromosomes.Compared to controls of the same game age, glutamic acid fed prepupae younger than 15 min have five additional puffs. In all, 98 pairs of homologous puffs were studied. In 54 of these, size differences were tested statistically; in the glutamic acid series, 20 puffs were larger and 18 smaller than in the controls whereas 16 had the same size. Size reduction was stronger in the more prominent puffs. In prepupae reared at lower temperatures, chromosomes were significantly longer. Glutamic acid causes a significant increase in chromosome width. Combined measurements of both effects were made by determining the surface area of tested segments. Lowering the temperature adds higher size classes, whereas addition of glutamic acid causes a significant shift of the distributional peak towards the higher size classes. This excludes the possibility of an additional replication cycle. In salivary glands of glutamic acid fed prepupae the majority of nuclei have reached the highest degree of polyteny, which in controls is never found at 25° C but sometimes at 18° C. The agent causing both additional puffing and enhanced growth is effective only via the alimentary tract of the larva.     

Heat-shock phenomena in Chironomus tentans

Isolated salivary glands of fourth instar larvae of Chironomus tentans were incubated at different above-normal temperatures for various lengths of time. Size changes in Balbiani ring 2 (BR2) and in the heat-inducible chromosome region IV-5C were quantified. These chromosome regions behaved in vitro very much the same as in vivo: BR2 was repressed rapidly by heat shock (37° C), whereas under overheat-shock conditions (42° C) it stayed decondensed. Region IV-5C showed the opposite responses. After a return from heat shock to normal temperatures the puffing pattern recovered. This process depended strictly on the integrity of the gland and on a change of medium. In injured glands a recovery process occurring under heat-shock conditions was discovered.     

Developmental changes in the fine structure of the salivary gland of Trichosia pubescens (Morgante) (Diptera : Sciaridae) in relation to puffing activity

Two differentiated sections (S₁ and S₂) of the salivary gland of Trichosia pubescens (Morgante) (Diptera : Sciaridae) have been examined by electron microscopy for fine structural alterations that occur in the cell cytoplasm during larval development. Such changes have been correlated with the puffing patterns of the polytene chromsomes. During stage 1 (end of the 3rd instar to mid 4th instar), the puffing pattern and the ultrastructure of S₁ and S₂ cells are rather constant. Nevertheless, marked differences are noted when the puffs and the fine structure of the 2 sections are compared. In S₁, secretory material is concentrated and eliminated as membrane-bound granules, while in S₂, secretory granules are not detected and the elimination of secretion seems to occur continuously. At stage 2 (end of the 4th instar), the puffing pattern undergoes considerable alterations simultaneously with the appearance of many ultrastructural modifications. In S₁, the morphological aspect of the secretory granules is altered, while in S₂ a decline in the secretory activity is detected. At stage 3 (older 4th-instar larvae), most of DNA puffs are active, there being no striking differences in the puffing pattern between S₁ and S₂. This stage is marked by the onset of gland histolysis, with the appearance of an intense autophagic activity of lysosomes in S₁ and S₂. As histolysis progresses during stage 4 (prepupae and early pupae) the activity of the polytene chromosomes decreases; most of the cells present a large number of autophagic vacuoles and an increasing disorganization of the cytoplasm, leading to the final lysis of the gland.     

Developmental puffing activity in the salivary gland and Malpighian tubule chromosomes ofAcricotopus lucidus (Diptera,Chironomidae)

A detailed map of the salivary gland chromosomes ofAcricotopus lucidus is presented. Differences in puffing and developmental Puffing sequences of the three salivary gland lobes were investigated from mid fourth larval instar to pupation and compared with the puffing pattern of the Malpighian tubules. The intraglandular differentiation is quite extensive; the differences in the pattern of gene activity between the anterior lobe and the main and side lobes are as great as between the salivary gland and the Malpighian tubules. In the main and side lobes all developmental puffing changes proceed synchronously whereas in the anterior lobe both asynchronous and synchronous changes occur. In the anterior lobe the asynchronous regression of BR 3 and BR 4 is followed by a characteristic sequence of activation and inactivation of puffs.     

Veränderungen im Puffmuster und das Wachstum der Riesenchromosomen in Speicheldrüsen von Drosophila melanogaster aus spätlarvalen und embryonalen Spendern nach Kultur in vivo

Salivary glands from late third instar larvae of Drosophila melanogaster were transplanted into the abdomens of adult female and male flies and were kept in this medium from 6 to 120 h. Changes in the puffing pattern of chromosome arm III L were studied after the culture in vivo. Two noticeable puffs are induced. They are located in 68 B and 78 E. Neither of these loci show activity during normal development. — Front halves of embryos (6 to 9 h of age) were also transferred into adults. After 5 to 13 days in vivo they are able to develop and differentiate larval structures. Salivary glands, imaginal discs, fat body, Malpighian tubules and muscle fibers could be identified. Even 4 h old embryos can form polytene salivary gland chromosomes after a 13 day culture. These chromosomes can reach sizes comparable with the maximal size in normal development. In some nuclei an extensive growth leads to “supergiant” chromosomes. The puffs in 68B and 78E are formed in the polytenic chromosomes from embryonic implants as in cultured larval salivary gland chromosomes.     

Die experimentelle Beeinflussung des Puffmusters von Riesenchromosomen

By treating larvae and prepupae of Ch. thummi with 2 mg/ml oxytetracycline (OTC) about 30 puffs not present in normal development are induced in the salivary gland chromosomes. Already existing puffs become enlarged (cf. Fig. 4). A considerable number of induced puffs appeared in heterozygous condition (cf. Fig. 1a-c). The species Ch. strenzkei does not react in any way to the same treatment. Other inhibitors of protein synthesis such as cycloheximide and chloramphenicole do not influence the puffing pattern in both species. — Animals which had been treated with OTC for 2 hrs show the first signs of puffing. Fully developed OTC-induced puffs are detectable 20 hrs after treatment. At this time the Balbiani rings and the nucleolus are mostly regressed. — Both the induced puffing pattern and the number of heterozygous puffs depend on the genetic constitution of the animals. Animals derived from different locations can be shown to possess different specific spectra of induced puffs. The induced puffing pattern of animals bred from single egg masses is reduced, and heterozygous puffs are rare or absent. — OTC-induced puffs show a strong uptake of tritiated uridine (cf. Fig. 2). Heterozygous puffs are labelled only in the puffed half of the band (cf. Fig. 3).     

Puff induction in larval salivary gland chromosomes ofDrosophila hydei sturtevant

The puffing pattern in salivary chromosomes of third instar larvae ofDrosophila hydei was studied following treatment with various gases, potassium cyanide, or vacuum. It was found that a number of specific puffs appear when anaerobiosis is followed by exposure to air or oxygen. These puffs seem to be independent of larval age, and are identical with some of those puffs which can be induced by raised temperature. It is suggested that the chromosomal loci involved, are connected with respiration.     

Die puff-Musterfolgen der Speicheldrüsenchromosomen aus wanderlarven und vorpuppen von Drosophila melanogaster in vitro

Late larval salivary glands of D. melanogaster of an exactly defined developmental stage (VP 0, i.e. prepupae ot later than 15 min after formation of the puparium) are cultured under sterile conditions in three standard media for insect tissue culture and in Ringer solution. In chromosomes II and III, variations in puff number and size are the same in vivo and in vitro, and almost all changes in puffing pattern are very similar to those appearing in normal development. They are the same in the four media. No additional puff is ever induced due to the medium. By contrast, salivary gland chromosomes from larvae of the late third instar before pupation do show different alterations in vitro than in vivo. This points to a threshold in the course of the puffing pattern between puff stage 8/9 and 10/11. The appearance of a substance causing prepupal changes in puffing is strictly correlated with the formation of the pupanium and the beginning of the intermoult phase in the prepupa. Comparing the results of the experiments it can be stated that the new control system is not based solely on the absence of ecdysone, but also on the existence of another inducer. Immediately after puparium formation the control by ecdysone is still active, together with the control by the supposed inducer. Later, control by ecdysone respectively by the puffs of the ecdysone cycle is substituted by the new control system, up to the next moult. As far as the chemical nature of the puffing inducer in the intermoult phase is concerned, further investigations are necessary.