Homologies of positional information in thoracic imaginal discs ofDrosophila melanogaster

Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.     

Regenerative interactions between Drosophila imaginal discs of different types.

We have tested the ability of fragments of one type of imaginal disc to stimulate regeneration of another type. It has been shown by others that, when extreme proximal and distal fragments of the wing disc are combined, intercalary regeneration of the missing tissue ensues. Each fragment, if cultured alone, will merely duplicate its structures. We now find that distal fragments of other thoracic discs, haltere and leg, while retaining their autonomy for differentiation, also interact with proximal wing tissue to promote regeneration of more distal wing structures. The proximal wing tissue used in these experiments was the wingless abnormal wing disc which, in the absence of interaction, yields only proximal wing structures. These results suggest that spatial organization is controlled by similar systems in the various thoracic discs. In contrast, head and genital disc material provided no regenerative stimulus to the mutant wing disc tissue.     

The Transcription Factor Optomotor-Blind Antagonizes Drosophila Haltere Growth by Repressing Decapentaplegic and Hedgehog Targets

In Drosophila, decapentaplegic, which codes for a secreted signaling molecule, is activated by the Hedgehog signaling pathway at the anteroposterior compartment border of the two dorsal primordia; the wing and the haltere imaginal discs. In the wing disc, Decapentaplegic and Hedgehog signaling targets are implicated in cell proliferation and cell survival. However, most of their known targets in the wing disc are not expressed in the haltere disc due to their repression by the Hox gene Ultrabithorax. The T-box gene optomotor-blind escapes this repression in the haltere disc, and therefore is expressed in both the haltere and wing discs. Optomotor-blind is a major player during wing development and its function has been intensely investigated in this tissue, however, its role in haltere development has not been reported so far. Here we show that Optomotor-blind function in the haltere disc differs from that in the wing disc. Unlike its role in the wing, Optomotor-blind does not prevent apoptosis in the haltere but rather limits growth by repressing several Decapentaplegic and Hedgehog targets involved both in wing proliferation and in modulating the spread of morphogens similar to Ultrabithorax function but without disturbing Ultrabithorax expression.     

Participation of lethally irradiated imaginal disc tissue in pattern regulation in Drosophila

When fragments of tissue derived from opposite ends of an imaginal disc are mixed prior to culture, intercalary regeneration occurs so that structures are produced which neither of the fragments would have made when cultured alone. We report here that, if a fragment is irradiated so heavily with X rays that its cells do not survive culture and metamorphosis, it nevertheless can stimulate regeneration by a fragment with which it is mixed.     

Enzyme patterns in D. melanogaster imaginal discs: distribution of glucose-6-phosphate and 6-phosphogluconate dehydrogenase

Distribution of glucose-6-phosphate dehydrogenase (G6PD) and 6-phospho-gluconate dehydrogenase (6PGD) in imaginal discs of Drosophila melanogaster was determined. Differential patterns of staining were found in all discs examined, i.e., eye-antennal, wing, leg, labial and genital. By using null mutants for either G6PD or 6PGD, the enzymes were shown to have the same distribution patterns. Staining with glucose-6-phosphate as a substrate resulted in the detection of both G6PD and 6PGD. Results of staining discs from homoeotic mutants indicate that the enzyme distribution patterns are under genetic control. In the presence of the homoeotic engrailed (en) mutation which transforms posterior wing compartment into anterior, the G6PD pattern of the posterior compartment of the wing disc was specifically transformed toward that of the anterior compartment. The bithorax series of homoeotic mutants was similarly investigated. The bithorax (bx3) mutation transforms the anterior part of the haltere to anterior wing blade. Similarly the G6PD pattern in the anterior haltere disc transforms to that of anterior wing disc. The complimentary transformation, postbithorax (pbx) results in a change of the posterior part of the haltere to posterior wing, which is likewise reflected in an altered staining pattern for G6PD in the posterior portion of the haltere disc. The combination of the bx3 and pbx resulted in a staining pattern of the haltere disc virtually indistinguishable from the normal wing disc.     

Protein synthesis and accumulation in Drosophila melanogaster imaginal discs: Identification of a protein with a nonrandom spatial distribution

Proteins from Drosophila imaginal discs and disc fragments were analyzed on two-dimensional electrophoretic gels following labeling in vitro with [³⁵S]methionine. The protein synthetic pattern in autoradiograms is very complex and parallels the pattern of protein accumulation visualized in silver-stained gels. We find no reproducible qualitative differences in the proteins synthesized or accumulated by different disc types. Additionally, analysis of the proteins synthesized by different fragments of wing and haltere discs has resulted in the identification of a polypeptide which is synthesized preferentially in homologous regions of these two imaginal discs. Scanning densitometry of our autoradiograms corroborates these findings. This protein, therefore, has some of the properties one would predict for a molecule involved in the imaginal disc positional information system.     

Protein synthesis during imaginal disc pattern regulation

We have examined the pattern of protein synthesis during wing disc pattern regulation. Although in vivo culture dramatically alters the pattern of abundant protein synthesis in wing discs, only one protein--RG38--changes specifically in response to pattern regulation. This polypeptide, previously identified as being nonuniformly distributed in wing and haltere discs, is synthesized in a graded distribution across the wing disc. During wing disc pattern regulation, it acts as a molecular marker for regeneration of particular wing disc regions. Thus, the rate of RG38 synthesis increases during regeneration (by fragments with initial low levels) with kinetics that parallel those for regeneration as scored by the presence of adult cuticular structures.     

Early development of the thoracic discs ofDrosophila

Summary We estimate the number of blastoderm cells which generate the thoracic imaginal discs ofDrosophila. At hatching the wing disc is twice the size of the haltere disc, but the results suggest that both discs develop from a similar number of blastoderm cells. Two homeotic mutations, which transform the haltere into wing, affect embryonic growth but not the primordial number. All the segmental primordia may be of similar size and each may be similarly subdivided into a larger anterior, and a smaller posterior polyclone.     

Regulative Properties of Wing Discs from the Vestigial Mutant of Drosophila melanogaster

The vestigial (vg) mutant of Drosophila melanogaster shows reduced wing size and lacks margin structures from the wing blade. The expressivity is temperature-sensitive, more structures being formed at 29°C than at 25°C. There is cell death in the third instar wing disc which to some extent parallels the fate map locations of the structures absent in the adult.
Vestigial wing discs are unable to regenerate margin structures even when given extra time for growth by culturing them in an adult abdomen before metamorphosis. If the region of cell death is excised from the disc before culture, there is still no regeneration of margin structures, indicating that the dead cells do not physically prevent regulation. Furthermore, by metamorphosing young vg wing discs, it was discovered that cells never acquire competence to make margin during wing disc development. Experiments mixing fragments of vg wing disc with non- vg wing disc fragments of ebony multiple wing hairs (e mwh) genotype showed that the vg cells interacted with the e mwh cells and wing blade was intercalated of both genotypes. However, structures such as wing margin, and alar lobe, usually affected in vg wings, were always made from e mwh cells and not from vg cells. Analysis of mutants which are unable to differentiate particular cell types may help us to understand the mechanism of pattern establishment in developing imaginal discs.     

Dissociation and sorting out of Drosophila imaginal disc cells

Previous attempts to study sorting out of Drosophila imaginal disc cells have been hampered by an inability to thoroughly dissociate these cells and the need to use cuticular markers which require several days of in vivo culture. This study overcomes these limitations by using a new dissociation procedure and a genetic marker for undifferentiated cells, the succinate dehydrogenase8 (sdh8) mutation. Dissociated and reaggregated cells from wing and leg imaginal discs segregated or "sorted out" from one another after only 24 hr of in vivo culture. It was also found that leg cells from different body segments may sort out, but to a lesser degree than wing and leg cells. Mixtures of wing and haltere cells did not sort out, in contrast to previous reports. These results constitute the first unambiguous study of sorting out with Drosophila imaginal disc cells and indicate that dorsally situated imaginal cells share a recognition specificity which is different from that of ventral imaginal cells.     

Regulative interaction of mature imaginal disc Fragments with embryonic and immature disc tissues inDrosophila

Summary These experiments examined whether inDrosophila immature imaginal disc tissue and tissues from embryonic stages can influence pattern regulation in a disc fragment in the same way as can mature imaginal discs. Immature imaginal discs, or the cells of whole embryos, were mixed with a test fragment (presumptive notum) from a mature wing disc. The immature tissues in each mixture were genetically marked and had been heavily irradiated (25 Kr gamma) prior to mixing to prevent growth and maturation during subsequent culture in vivo. Alteration of the regulative behavior of the test fragment (that is, regeneration of wing) thus provided an assay for the communication of positional information by the immature tissues. The results suggest that this capacity arises well before competence to metamorphose, as early as the 16th hour of embryonic development, whereas prior to 16 h, essentially no stimulation of regeneration occurred. It is suggested that the imaginal disc (or presumptive disc) cells of the embryo may have been responsible for this early stimulatory capacity.     

Sensory Projections from Ectopic Appendages in an insect: Inherent Specificity and Influence of Location

Peripheral and central pathfinding by sensory axons from appendages was investigated in the fly Sarcophaga bullata . (a) Supernumerary appendages (haltere, wing, antenna and leg) were produced by imaginal disc transplantation at various ectopic sites, (b) Leg neuropil was deafferented by leg disc extirpation and in its place another leg disc was implanted. (c) The basal stalk of a leg disc connecting it with the thoracic ganglion was transected. Using cobalt chloride and HRP backfilling methods the pathways taken by the afferents from these experimentally altered appendages was examined. The results indicate that the larval nerves and the imaginal disc stalks act as guides for growing axons to locate their correct entry sites within the ventral ganglion. In the absence of these guides the axons follow any peripheral nerve, such as abdominal nerve, and enter the ganglion at inappropriate sites. However, within the ganglion they take particular routes, almost identical to those taken by axons from in situ appendages suggesting the existance of some kind of a labelled pathway. Deafferentation does not make the leg neuropil more attractive to ingrowing ectopic sensory axons.     

Mutants of the bithorax complex and determinative states in the thorax of Drosophila melanogaster.

Homoeotic mutations of the bithorax complex cause segmental transformations. The genes in which these mutations occur are good candidates for genes that are involved in determination. The determination system in imaginal discs must have at least two functions. One is a cell heredity function that is responsible for maintaining the determined state during growth and development. A second is the expression of the determined state (e.g., different imaginal discs have different morphologies). The homoeotic mutations of the bithorax complex could be affecting either of these two functions. I have found that when posterior haltere disc cells, that are transformed by the mutation postbithorax so that they form wing cuticle in situ, regenerate anterior structures, these structures are anterior wing. This is the same result as that seen when wild-type posterior-wing disc cells regenerate anterior structures. On the other hand, when anterior haltere disc cells transformed by the mutation bithorax³, so that they produce wing cuticle in situ, regenerate, they produce posterior haltere structures. This is unlike wild-type anterior-wing disc cells, which regenerate posterior-wing structures. From these results, I conclude that bithorax³ affects the expression of the determined state and postbithorax affects the cell heredity of determination.     

Aldehyde oxidase distribution in haltere discs of homoeotic bithorax mutants in Drosophila melanogaster.

Summary Distribution of the enzyme aldehyde oxidase in transformed haltere discs from the homoeotic bithorax series of mutants was investigated by histochemical means. The bithorax (bx) mutant, which transforms the anterior part of the haltere into an alterior with blade, possesses in the haltere disc an aldehyde oxidase staining pattern similar to that of the anterior side of the wing disc. The postbithorax (pbx) mutant, which transforms the posterior haltere into a structure resembling the posterior wing blade, reveals an aldehyde oxidase staining pattern in the haltere disc characteristic of the posterior side of the wing disc pouch. When both (bx ³ (pbx) mutants are present the haltere develops into a metathoracic wing. It is shown here that the transformed haltere disc closely resembles the previously established pattern in the wing disc with respect to aldehyde oxidase distribution. Change in the pattern of aldehyde oxidase in bithorax mutants signals alteration in gene expression which at least for this particular enzyme correlates well with the morphological transformation from haltere to wing. A possible correlation between pattern of enzyme activity and developmental compartmentalization has been discussed.     

Positional information in imaginal discs transformed by homoeotic mutations

Summary Mutations of the bithorax complex result in segmental transformations in the thorax and abdomen ofDrosophila. The haltere discs from larvae homozygous forbx ³ orpbx are transformed so that the discs contain cells that will produce wing cuticle as well as cells that produce haltere cuticle. The pattern regulation behavior of these discs has been examined. The fate maps of the two discs were established, and then the regulative behavior of a number of fragments from both types of mutant discs was established by culturing the fragments in vivo prior to metamorphosis. The most important conclusion from this work is that the cells producing, haltere cuticle and wing cuticle within the same disc share the same positional information and that they communicate during pattern regulation.     

Regulative interactions between cells of wing discs from different dipteran species

The mechanism by which patterns are produced appears to be repeated in each segment of an animal, and it has been proposed that it may even have been conserved in evolution so that different species would have the same system of positional information. This idea has been tested by mixing cells of a defined fragment of the wing disc of Drosophila melanogaster with wing disc fragments of five other dipteran species to assay the ability of these disc fragments to stimulate intercalary regeneration of the D. melanogaster cells. The genetically marked (y; mwh) D. melanogaster fragment was mechanically mixed with wing discs or wing disc fragments of four drosophilids (D. melanogaster as a control, D. virilis, D. hydei, Zaprionus vittiger), of Musca domestica, and of Piophila casei. The mixed aggregates were cultured in vivo for 7 days, then metamorphosed in D. melanogaster larval hosts. The D. melanogaster fragments were only stimulated to regenerate when combined with complementary fragments from D. melanogaster or D. virilis wing discs. In the combination between D. melanogaster and D. hydei, the tissue formed integrated mosaic patterns, but no regeneration ensued. The one positive result (D. melanogaster mixed with D. virilis) shows that positional cues can be exchanged and correctly interpreted between cells of different species. The negative results do not prove that the mechanism for establishing patterns is different in the tested species, but may be due to incompatibilities that are not related to pattern formation.     

In vitro culture of Drosophila imaginal disc cells: aggregation, sorting out, and differentiative abilities

This paper describes the aggregation in vitro of cells dissociated from imaginal discs and demonstrates the sorting out of undifferentiated cells from different imaginal discs and from differently determined regions of the same imaginal disc, as well as the abilities of such cells to undergo pattern reconstruction when injected into larvae. Dissociated cells begin to aggregate by 1.5 hr of rotation. By 5 hr of rotation, large aggregates of loosely associated cells appear. By 18 hr the aggregates have condensed and taken on a characteristic epithelial structure. To study sorting out in undifferentiated cells, we combined a histochemical stain for acid phosphatase with the use of the acid phosphatase null mutant acphn-11. We performed cell mixing experiments with 0-2 (prospective notum) and 2-8 (prospective wing) fragments, with the A and P (prospective anterior and posterior) fragments of the dorsal mesothoracic disc and with mixtures of cells from ventral prothoracic and dorsal mesothoracic discs. We found that prospective anterior and posterior dorsal mesothoracic cells do not sort out, but that prospective notum and wing and leg and wing cells do. The results from differentiated implants are consistent with those from undifferentiated mixes.     

Dynamics of decapentaplegic expression during regeneration of the Drosophila melanogaster wing imaginal disc

Regeneration of an imaginal disc involves highly ordered proliferation and pattern regulation of the newly formed tissue. Although the general principles of imaginal disc regeneration have been extensively studied, knowledge of the underlying molecular mechanisms is far from complete. Results from other model organisms suggest that regeneration is the result of local recapitulation of the normal patterning genes. To analyze the dynamics of one major Drosophila patterning gene, decapentaplegic (dpp), in wing imaginal disc regeneration, a vital GFP reporter together with iontophoretic cell labeling were used. Our observations reveal that the restoration of compartment-border-specific dpp expression is a common event in imaginal disc regeneration. However, we did not find evidence of an upregulation of dpp expression during the regeneration process.