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.     

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.     

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.     

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.     

Ultrastructural aspects of histolytic processes in the salivary gland cells during metamorphic stages in Rhynchosciara hollaenderi (Diptera, Sciaridae).

The cytoplasm of Rhynchosciara hollaenderi late larval, prepupal and pupal salivary gland cells was studied at the ultrastructural level. In the second half of the 4th instar, evidence of an intensive secretory activity is visible in the form of numerous secretory granules in the apical area of the cells. At the same stage, the endoplasmic reticulum cisternae adjacent to Golgi groups are active in the transfer of vesicular elements. At later stages this activity rapidly diminishes. Before the appearance of the DNA puffs, i.e. at the end of the 4th instar, mitochondria begin to show a granular deposit and normal mitochondria decrease in number. These with the granular deposit form clusters and initiate formation of single autophagic vacuoles before the appearance of the DNA puffs. Later, at the time, when the 2B puff opens, the autophagic vacuoles appear in great number. Simultaneously with the formation of the autophagic vacuoles the presence of acid phosphatase in the Golgi vesicles and in autophagic vacuoles was shown. In the last stages investigated (late pupae) acid phosphatase is present free in the cytoplasm and at the same time disappearance of free ribosomes, pycnosis of polytene chromosomes and breakage of nuclear membranes occur. It is concluded that the histolysis of the salivary gland cells begins before the large DNA puffs appear, then it becomes very intensive and continues after these puffs undergo regression.     

Developmental changes in fat body and midgut chromosomes of Drosophila auraria

Changes in puffing activity of fat body (FB) and midgut (MG) chromosomes of Drosophila auraria during late larval and white prepupal development as well as after in vitro culture with or without ecdysterone were studied and compared with those of the salivary gland (SG). The Balbiani Rings characteristic of the SG chromosomes of D. auraria, are not formed in FB and MG. Most of the inverted tandem chromosomal duplications that have been found to be common to all three tissues showed differentiation of puffing activity of the bands considered to be homologous. The major early ecdysone puffs 73A and 73B (considered to be homologues of D. melanogaster puffs 74EF and 75B, respectively), together with other early ecdysone puffs were present in all three tissues. Clear intermoult and postintermoult puffs were not evident in FB and MG chromosomes. However, a small set of late ecdysone puffs could be scored in FB, while no late ecdysone puffs were abserved in MG. Other tissue-specific puffs were identified, but a very small number of them were limited to MG.by W. Beermann     

Sequence of puff formation inRhynchosciara polytene chromosomes

The chief characteristics of the life cycle ofRhynchosciara sp. are: egg stage (12 days); three larval instars of approximately 6 days each, followed by a 4th instar of approximately 40 days duration; pupation (6 days); and adult form (5–6 days). Maps of the 4 polytene chromosomes ofRhynchosciara sp. have been prepared, and the temporal sequence of puff formation on the chromosomes described. The cocoon is synthesized during the prepupal period, and at this time major puffs are seen on all chromosomes. The largest and most numerous puffs occur on the salivary gland chromosomes during the 24 hours prior to the last or prepupal molt. Three of the puffs that occur at this time are DNA-puffs (Summary see p. 249). Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.     

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.     

Salivary gland function and chromosomal puffing patterns in Drosophila hydei

Summary The salivary glands of D. hydei larvae show differences between the cells in the distal (posterior) part and those of the proximal (anterior) part during the third instar. The first sign of these differences is an increase in cellular and nuclear volume in the distal cells of the gland, beginning at 103 hours after oviposition. After 125 hours the cytoplasm of the extreme distal cells acquires a reticulated structure, and at 130 hours these cells contain large granules or droplets of mucoprotein. From this moment up to puparium formation the number of cells containing these granules increases and the boundary of this type of cells shows a shift in the proximal direction. Just before puparium formation the granules disappear from the cells and a glue substance is secreted by the larvae. At this moment only a few cells in the extreme proximal part still lack granules. Electron-microscopical observations indicate that these cells were active in secretion, whereas all cells containing large granules are inactive in this respect during most of the third instar.During the early third instar a change in cell function occurs, i.e. from synthesis of substances presumed to be digestive enzymes which are secreted, to a synthesis of a glue substance which is stored. This change begins in the extreme distal cells of the gland.Investigation of the chromosomal puffing pattern revealed that a total number of 148 puffs were present during some period of the third instar, prepupal, and early pupal stages. The activity of 110 puffs was evaluated during a series of successive time intervals. Changes in the puffing pattern during puparium formation were compared with those observed during pupation.Proximal and distal nuclei differ in the activity level of a number of puffs, but only puff 47 B is restricted in activity to the distal cells. This puff becomes active at 119 hours and disappears 4 hours before puparium formation (156 hours). Determination of nuclear diameter and DNA in nuclei of both parts of the gland revealed a correlation between a particular DNA content and the function of the cell. Distal cells show higher nuclear diameters than proximal cells after the onset of granule production. The first differences in nuclear diameter can be seen at 103 hours. Cells in the transitional part of the gland, located between distal granulecontaining and proximal granule-negative cells, always show the same DNA content. These cells are found at different locations within the gland during the third instar. This zone of cells shows a shift in proximal direction during the third instar, identical to that of the neighbouring granule-containing cells.The possible interrelation between nuclear DNA content, the activity of puff 47 B, and the production of the glue substance were discussed.     

Cytogenetic analysis of the 2B3-4 — 2B11 region of the X chromosome of Drosophila melanogaster

Puffing patterns have been studied both in homozygotes t10/t10, a gene located in the area of the early ecdysone puff 2B5, and in a yellow (y) control stock, at the end of the third instar and during prepupal development. In mutants t10 at the end of the third instar puffing develops normally in general, however, 21 puffs (5 early and 16 late ones) underdevelop or do not develop at all, some larval intermoult puffs regressing slower. The next cycle of puffs (mid prepupal) in mutants t10 proceeds normally, but in the late prepupal cycle 21 puffs underdevelop again or are not formed at all. A model for the induction of early ecdysone puffs is proposed, assigning a key role to the 2B5 puff product in stimulating other early puffs. It is suggested that defects in the activity of early puffs in the mutant t10 may cause underdevelopment of late puffs.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

An overview of gene activity in the fat body of Drosophila gibberosa

In the larval fat body of Drosophila gibberosa, polytene chromosome structure and activity exhibit cytological differences from chromosomes of midgut and salivary glands. These differences include long-persisting puffs, transient puffs and long-persisting band modulations. Some early ecdysteroid-induced puffs are present in all three organs but few late puffs are present in the fat body. Comparative studies reveal, therefore, that late larval-early pupal puffing is enhanced in salivary glands relative to gut, fat body and Malpighian tubules. After the fat body breaks up in the prepupa, the rate of programmed cell death and the corresponding slow decline of chromosomal activity also differ from cell to cell and from other organs.by M.L. Pardue     

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.     

Ecdysteroid titers and changes in chromosomal activity in the salivary glands of Rhynchosciara americana

Titers of ecdysone and 20-OH ecdysone were measured separately in both hemolymph and salivary glands of metamorphosing Rhynchosciara larvae. Gland titers were consistently higher than hemolymph titers. Although 20-OH ecdysone was the most prominent form of the hormone, measurable quantities of ecdysone were also observed throughout development in both tissues. Changes in salivary gland replication and puffing activity could be correlated with changes in gland 20-OH ecdysone titers. This was true for both developmentally changing RNA puffs and DNA puffs, which occur during the prepupal period. The DNA puffs are tied to the final DNA replication cycle, and both this cycle and the period of amplification can be correlated with increases in gland 20-OH ecdysone content. Various aspects and possible interpretations of the above correlations are discussed.This work is dedicated to the memory of Prof. Hans D. Berendes     

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.     

Chromosomal control of foot pad development in Sarcophaga bullata

The puffing schedule of 71 puffs of chromosome B in Sarcophaga bullata polytene foot pad cells were analyzed during an 8 day period of pupal development. The number of puffs fluctuates, with maxima at about day 7 and day 10. The nucleolar volume reaches two maxima which precede the maxima in the number of puffs by approximately one day. With respect to qualitative changes in the puffing pattern, the present study shows that all puffs appear and disappear in a sequential fashion which is related to development. The pattern of potentially active loci and the sequence in which they form puffs are identical in all foot pad cells. Nevertheless, two neighboring cells may differ characteristically in the timing of the activity of a few individual loci relative to the rest of the puffing sequence. Furthermore, pulvilli cells from different pairs of legs may differ in the timing of the entire puffing sequence. It is concluded that puffing, at least in part, is under the control of intracellular processes rather than of external factors such as hormones.     

Puffing activity in the salivary gland chromosomes of Rhynchosciara under experimental conditions

By using the techniques of ligation of the larvae (brain and endocrine glands extirpation) and salivary gland implantation, the hormonal dependence of the activity of certain puffs of Rhynchosciara was investigated. Our results have shown that the puffing behaviour — activation and deactivation — varies according to the developmental stage in which the larvae were ligated. When the larvae were ligated just before the drastic changes in the puffing pattern, which occur prior to pupation, these changes fail to occur. When the larvae were ligated after the onset of these changes we have observed: a) some of the puffs active at the time of the ligature regress promptly, earlier than their normal timing observed in controls; b) others remain active indefinitely and c) there are still some which regress accordingly to the normal timing.The puff B2 which behaves as those in b was double checked by means of implantation experiments. Salivary glands which had puff B2 at its maximum expansion were implanted into younger larvae and that puff also remained active in the body cavity of these larvae. Hypotheses to explain the results obtained are discussed.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

The autosomal salivary gland chromosome puffing patterns of Drosophila simulans are described and compared with the puffing patterns of the sibling species D. melanogaster. During the late third larval instar and the prepupal period the patterns of puffing activity of these two species are similar — approximately 50% of the puffs common to both species showing identical activities. The remaining puffs differ in their timing of activity, or in their mean sizes, or in both of these parameters. A number of puffs (14) found in D. simulans have not been regularly observed in the Oregon stock of D. melanogaster but are active in other D. melanogaster strains. One puff (46 A) of D. melanogaster was absent from D. simulans and forms a heterozygous puff in hybrids, when the homologous chromosomes are synapsed. When the homologues are asynapsed a puff at 46 A is restricted to the melanogaster homologue. The puff at 63E on chromosome arm 3L is considerably smaller in D. simulans than in D. melanogaster and this size difference is autonomous in hybrids. Other puffs not common to both species behave non-autonomously in the species hybrid, even when the homologous chromosomes are asynapsed.     

Comparative study of the function of polytene chromosomes in laboratory stocks of Drosophila melanogaster and the l(3)tl mutant (lethal tumorous larvae)

A study of the puffing pattern of the salivary gland autosomes of D. melanogaster was performed through the last 24 hours of larval development and 0-hour prepupae. Since both prominent and small puffs were taken into account, the total puff number amounted to 275. Of these, 116 are almost constant in size during the 24 hours observation period, 106 increase in size or appear before pupation. 37 puffs are active in 96 hour larvae and disappear or decrease sharply in size by 115–118 hours. 12 biphasic puffs have been found with higher activity in 96 hour larvae and 0-hour prepupae and lower activity by 115–118 hours. Three extremely irregular puffs have been detected in chromosome 4. The data obtained evidence that a larger number of D. melanogaster polytene chromosome loci are active during larval development than it has been thought earlier. It has also been shown that only 38% of autosomal puffs change before the beginning of metamorphosis. The functional significance of small puffs and strain specificity of puffs are discussed.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Patterns of puffing activity during the third larval instar and the prepupal period of two different strains of D. melanogaster (Oregon and vg6) are compared. The variation in puffing activity observed is both quantitative (involving the mean size or timing of activity of individual puffs) and qualitative. The pattern of activity of 64% of the puffs is the same in the two strains, 12% show strain differences in puff size and 19% in the time of their activity. One puff (64C) is active only in one of the strains (vg6). In genetic experiments this puff segregates normally and the puff locus has been mapped genetically to a site coincident with, or at least very close to, the cytogenetic position of the puff. In heterozygotes the puff is homozygous only when the maternal and paternal homologues are synapsed. When the homologues are asynapsed only the homologue from the vg6 parent is puffed at 64C. With the exeption of some strains closely related to vg6 no other strain of D. melanogaster has been found to possess puffing activity at 64C. In vg6/In(3LR)C165 heterozygotes 64C forms a heterozygous puff even when the homologues are synapsed. In the discussion consideration is given to the various factors that control puff size.