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.     

DNA-puffing patterns in the salivary glands of Trichosia pubescens (Diptera-Sciaridae)

The puffing pattern in polytene chromosomes from salivary glands of fourth-instar larvae of Trichosia pubescens was studied. It was found that the puffing pattern is rather constant during most of larval life but changes continuously in a precise sequential order during the period preceding pupation. During this period, characterized by drastic changes in the puffing pattern, amplification of specific genes and expansion of the DNA-puffs occur. The pattern of protein synthesis in the salivary gland has also been studied by means of SDS-polyacrylamide gel electrophoresis and fluorography. It changes drastically and continuously during the phases preceding pupation. These changes in protein synthesis could be correlated with the changes in the puffing pattern, essentially with the activity of the DNA-puffs.     

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.     

Developmental studies in Drosophila

A study of the puffing patterns of the salivary gland chromosomes of D. pseudoobscura was carried out through several larval, prepupal, and pupal stages of development. A total of 176 puffs were found, 111 of which changed during the stages studied. As described in previous investigations with other Drosophila species there are two major peaks of puffing activity. These two peaks occur during puparium formation and pupation. Additionally, a minor activity-peak occurs during mid-prepupal life. Attempts have been made to establish correlations between the puffing data and those obtained from electrophoretic and ultrastructural studies.Supported in part by grants GM-16736-03 and FR-05426-09 from the U.S. Department of Health, Education, and Welfare. Ann Jacob Stocker was a holder of a University of Texas predoctoral fellowship.     

Sequential gene activation by ecdysteroids in polytene chromosomes ofDrosophila melanogaster

Summary Changes in polytene chromosome 3 L puffing patterns in the fat body ofDrosophila melanogaster larvae and prepupae are compared to those in the salivary gland. While some general features are common to the two tissues, there are differences which reflect their different developmental roles. In vitro experiments with fat body chromosomes show that they have a distinct response to ecdysteroids which is different from that of salivary gland chromosomes, and which does not,in this culture system, reproduce the changes observed in normal development. In short term culture experiments, the fat body chromosomes appear more sensitive to ecdysteroids than the salivary gland chromosomes and, although 20-OH ecdysone is more active than ecdysone in these assays, the possibility is not excluded that ecdysone has a role in normal development as it appears to alter gene activity at physiological levels in these cells.     

Developmental puffing patterns in salivary gland chromosomes of Rhynchosciara hollaenderi

An analysis of puff formation and regression has been carried out in 3 morphologically distinct regions of the Rhynchosciara hollaenderi salivary gland during mid-larval through pupal development. Puffing differences among these 3 regions have been found and analysed for both RNA and DNA puffs. The presence of such differences suggests that the gland regions may also be functionally differentiated. — Developmentally specific sequences of puffs have been distinguished and correlated with morphological and physiological events which occur during the development of Rhynchosciara. The DNA puffs as well as the RNA puffs enlarge and regress at predictably specific developmental stages. The presence of particular puffing sequences in the late larval to pupal period has been compared with the occurrence of known changes in the developmental ecdysone titre for Rhynchosciara. Certain aspects of this developmental picture appear to fit the ecdysone-stimulated puffing model for Drosophila, but other aspects indicate that the Drosophila-based model may not be completely applicable to Rhynchosciara.     

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.     

Differential induction of chromosome puffs in two cell types of Melanagromyza obtusa

The patterns of puffing activity in polytene nuclei of salivary gland (SG) and midgut (MG) tissues of Melanagromyza obtusa have been studied after heat shock (HS), 2-4-dinitrophenol (DNP) or benzamide treatment. This study has revealed that HS and DNP treatments induced the same set of puffs but in a tissue-specific pattern. Benzamide treatment was found ineffective in inducing puffing activity. Some HS genes were also found to be more or less active during normal development, indicating some function in the normal metabolism of the cells.     

Gene Activity Patterns and Cellular Differentiation

Puffing in giant chromosomes ot Diptera is considered to reflectthe pattern of active gene loci in these chromosomes. In anyone tissue only a relatively small portion of the total bands(about 10 to 20%) have been observed to form a puff at sometime or another in larval development. These patterns of "potentiallyactive" loci are tissue specific, though greatly overlapping.The actual rate of activity at these loci is controlled independentlyof each other and independently in each tissue by factors ofthe extranuclear metabolism. Puffing at some loci seems to berelated to specific cellular functions, such as secretion ofthe salivary glands. The activity of others may be related tomore basic metabolic processes. In relation to larval development,puffing patterns may change with changing cell functions orwith developmental processes in the cells themselves. In salivaryglands ofChironomus activity of DNAase and of acid phosphataseseems to change in relation to cell breakdown at the end ofthe pupal molt. Changes of acid phosphatase activity begin earlyin the last larval ins tar, but the enzyme is bound to lysosomesuntil metamorphosis. This suggests that the genes specificallyactive during metamorphosis have to interact with a longtermcontrol-system of development. The induction of metamorphosisis a sequential process, gene activations being among the firststeps in this sequence. The activation of these genes by ecdysoneis independent of protein synthesis. It is only the reactionof these genes that leads to the subsequent events in the cell,including the subsequent puff activations. This is shown bythe fact that they depend on early RNA synthesis as well ason protein synthesis. These results on puffing are discussedwith regard to the general problem of the relationships betweenpatterns of gene activity and differentiation.     

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.     

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.     

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.     

Ultrastructural changes of Drosophila larval and prepupal salivary glands cultured in vitro with ecdysone

Summary Alterations in the ultrastructure of in vitro cultured larval salivary glands of Drosophila melanogaster in response to the steroid hormone ecdysone were studied in relation to complex changes in puffing patterns. We found that the changes in the fine structure of cultured glands reflected progression of the puffing pattern, and they paralleled those seen in vivo. We observed that glue secretion by exocytosis, the main function of salivary glands, took place between puff stage 5 (PS5) and PS7. Glue could not be expectorated under culture conditions but was slowly released from the lumen through a duct into the medium. After the cultured glands reached PS13/PS14, further progress of puffing and fine structural alterations required that the ecdysteroid titer be transiently extremely low or absent. Under in vitro conditions we did not observe the putative new secretory program(s) described for glands in vivo after PS12. However, ultrastructural changes which unambiguously indicated that an autohistolytic process had begun in vitro started to appear after PS17. Many salivary gland cells developed numerous features of progressive self-degradation between PS18 and PS21. Actual degradation of salivary glands in vivo seemed to be rapid, but in vitro degradation was never completed, probably due to a lack of exogenous factors from the hemolymph. Manipulations of ecdysone titer in vitro in the culture medium, known during the larval puffing cycle to cause premature induction of developmentally specific puffing patterns, did not affect the normal development of ultrastructural features of the cytoplasm and nucleus.     

The role of the hormone ecdysterone in the control of the activity of the Balbiani rings and the other puffs of Drosophila auraria

The activity of the Balbiani rings and the other puffs of Drosophila auraria salivary gland chromosomes from various stages of development was studied in vitro in the presence or the absence of various concentrations of the hormone ecdysterone. It was found that of 81 sites affected by these conditions, 69 (including the BRs) exhibit changes during normal development as well, while the remaining 12 change only under culture conditions. The results indicate that the normal profiles of certain puffs (and the BRs) are approximated more closely by the lower concentrations of the hormone and it is suggested that such low concentrations are necessary to induce the normal course of events in vivo. Various hypotheses concerning the influence of ecdysterone on the puffing patterns are discussed in view of the data presented in this report.     

The time during which beta-ecdysone is required for the differentiation in vitro and in situ of wing imaginal discs of Drosophila melanogaster.

The time during which β-ecdysone is required for the apolysis and imaginal differentiation of wing discs of Drosophila both in vitro and in situ has been examined, and it is concluded that β-ecdysone is required as a sustained stimulus rather than as a trigger for differentiation. These results are compared with the requirement for β-ecdysone for the puffing of salivary gland polytene chromosomes during the prepupal stage (Richards, G. P., 1976, Develop. Biol.48, 191–195). It is suggested that imaginal discs and larval salivary glands require different exposures to β-ecdysone to fulfill their developmental commitments and that the drop in β-ecdysone titer during the early prepupal stage, which is necessary for the subsequent puffing of the polytene chromosomes, plays little or no part in imaginal disc differentiation.     

Deoxyribonucleases of the organs of Drosophila hydei at the onset of metamorphosis

The tissue distribution of deoxyribonucleases has been studied in the organs of Drosophila hydei at the onset of metamorphosis. The enzymes were separated by disc electrophoresis and detected directly in the gel. An extensive shift in the wide spectrum of activities that has been observed at metamorphosis indicates that deoxyribonucleases play an important role at this time in development. On the basis of the tissue distribution of these enzymes, it has been possible to assign probable functions to several of the activities. An intense activity appears in the prepupal salivary gland which is not detected in this organ in the larval stage. This observation is of particular interest in view of the changes that have been observed in the chromosomal puffing patterns of the salivary glands just prior to metamorphosis. A class of activities, which is probably of lysosomal origin, is more prevalent in the prepupal tissues. The data suggest that an increased synthesis of lysosomes is a general reaction of most larval tissues at the onset of metamorphosis irrespective of whether a tissue undergoes total histolysis. The larval intestine contains a factor which strongly inhibits Drosophila nucleases that are active at low pH. The major nuclease activities of each tissue have been tentatively characterized. A knowledge of the enzyme properties is expected to facilitate the isolation of DNA from the individual tissues.This work was performed in the Max-Planck-Institut für Biologie, Abteilung Beermann, Tübingen, Germany. The senior author was supported by the Helen Hay Whitney Foundation.     

Puffing patterns in the salivary gland chromosomes of the melonfly,Dacus cucurbitae

The puffing pattern changes in the salivary gland chromosomes of the third instar larvae of the melonfly, Dacus cucurbitae are described. Three classes of puffs were noticed over a period of development of 120 hrs. Class (1) are those which are more or less constantly found; class (2) are those which oscillate, i.e. appear and disappear at irregular time intervals; and class (3) are those that are linked to a specific developmental event. Also, 3 peaks of puffing activity have been noticed during the present study; one in the 120 hr old larva, the second in the 168 hr old larva and the third in the 240 hr old larvae. The significance of these 3 classes of puffs and the 3 peaks in puffing activity has been discussed. The puff RNA has a high rate of synthesis and incorporates ³H-cytidine within 30 secs after being offered. There is a high degree of variation in the incorporation of labelled precursors into the different nuclei of the same gland, such a variation is not noticed in the diploid and embryonic cells.