The mechanism of pattern reconstruction by dissociated imaginal discs of Drosophila melanogaster

DEVELOPMENTAL BIOLOGY 26, 464-417 (1971)

The Mechanism of Pattern Reconstruction by Dissociated

lmaginal Discs of Drosophila melanogaster

CLIFTON A. POODRY,’ PETER J. BRYANT, AND HOWARD A. SCHNEIDERMAN

Developmental Biology Laboratory, and Center for Pathobiology, University of California at Irvine, Irvine, California 92664

A new analysis of the process of pattern reconstruction by dissociated imaginal disc cells of Drosophila is presented. Imaginal leg discs of two genotypes were dissociated and reaggregated in the presence of increasing quantities of wing discs. The leg disc cells cooperated to reconstruct vesicles of leg tissue surrounded by wing. Some vesicles contained leg cells of both genotypes (mosaics), but a substantial proportion contained only one of the two genotypes of leg (monotypic). The high frequency of monotypic vesicles indicates that pattern reconstruction does not occur by the migration of individually determined cells to preassigned positions in the pattern, as has been previously suggested. A more likely interpretation is that a considerable amount of “repatteming” occurs among the randomly reassociated cells.

INTRODUCTION

When embryonic organs of vertebrates are dissociated and their cells are recom- bined in random fashion, they are often able to reconstruct tissues (Moscona and Moscona, 1952; Townes and Holtfreter, 1955; Trinkaus and Groves, 1955; Mos- cona, 1957; Weiss and Taylor, 1960; Stein- berg, 1964; Trinkaus and Lentz, 1964). Furthermore, when the cells from two embryonic tissues are brought together, with or without prior dissociation, they segregate from one another in predictable fashion and separately reconstruct their respective tissues (Townes and Holtfreter, 1955; Trinkhaus and Groves, 1955; Steinberg, 1964). These sorting out processes have been interpreted as reflecting inherent locomotor and adhesive properties of the cells, which are important in morphogenesis and which are retained upon dissociation and reaggregation in vitro (Townes and Holtfreter, 1955; Trinkaus, 1966). Experi- ments with Drosophila indicate that cells from dissociated embryos of insects can also take up characteristic positions in a reaggregate (Lesseps, 1965).

Dissociated and reaggregated imaginal discs of Drosophila can also reconstruct

* Present address: Department of Zoology, Univer- sity of British Columbia, Vancouver 8, B.C.

spatial patterns related to those developed in situ (Hadom et al., 1959; Ursprung and Hadorn, 1962). By mixing cells from imaginal discs of different genotypes, it has been found that cells from homonomous imaginal discs (e.g., leg and leg) can cooperate to form integrated patterns, but that cells from heteronomous discs (e.g., leg and wing) do not (Niithiger, 1964; Tobler, 1966; Garcia-Bellido, 1966a,b, 1967, 1968). Interpretation of these experiments is made difficult by the fact that the reaggregate must be cultured in the abdo- men of an adult, since in uitro culture methods have not been worked out. Direct observations of the reconstruction process are therefore impractical, and it is possible to observe only the differentiated end product of the reconstruction process. Hadorn et al. (1959) and Ursprung and Hadorn (1962) believed that during reconstruction a new pattern of differentiation was imposed upon the randomly aggregated cells by a mechanism analogous to that which occurs during normal differentiation of imaginal disc cells in situ. The alternative view was that pattern reconstruction might occur through the directed migration of individual cells, which would take up their preassigned specific positions in the new pattern (see Niithiger ( 1964, Tobler (1966), Garcia-Bellido

464

POODRY ET AL. Mechanism of Pattern Reconstruction 465

(1966a,b, 1967, 1968) and Ursprung, 1966). The hypothesis of directed migration of individual cells in the aggregate or in the epithelium is one that we find difficult to accept, for several reasons.

First, the imaginal discs of Drosophila are composed of a single-layered epithelium which is contiguous with the epithelium of the larval epidermis (Poodry and Schneiderman, 1970). When imaginal discs are dissociated, the integrity of the epithelium is destroyed. A priori it may be expected that cells possessing the general quality “epidermal epithelium” would behave as do other epithelia (see, e.g. Chiakulas, 1957) and would initially re- unite to reform an epithelium with little or no regard for relations of homonomy. Such general epithelial affinity is indicated by the behavior of imaginal disc transplants of other insects (Bhaskaran and Sivasu- bramanian, 1969). Second, Poodry and Schneiderman (1970) have shown that cells of the imaginal discs are connected by gap junctions and septate desmosomes, which suggest firm attachments and a lack of cell mobility. Third, experiments on cell sorting out in other systems involve segregation of different tissues. In systems other than Drosophila imaginal discs, only one claim of segregation within a single tissue type has been made (Zwilling, 1968). Therefore, sorting out of imaginal disc cells, which differ only in their determination to form epidermis of different parts of the body, would represent an unparalleled degree of specificity of cellular recognition. Fourth, autografts of epidermis in other insects (Wigglesworth, 1940; Locke, 1959, 1960; Lawrence, 1966; Stumpf, 1966) in which a small piece of integument is rotated by 90” or 180°, lead to a distorted pattern. The center of the autograft behaves autono- mously with respect to its determination. The cells of the graft do not appear to migrate to reconstruct the original pattern even though considerable time (several molts) may be allowed for such migration (Locke, 1960; Lawrence, 1966).

Since the nature of the mechanism of pattern reconstruction by dissociated imaginal disc cells bears directly on problems of determination and pattern formation, we feel that the hypothesis of migration and sorting out should be critically tested. We present here the first step in such a test.

The approach we have taken is to ex- amine the behavior of a mixture of two types of genetically marked leg disc cells in a background of increasing quantities of cells from wing discs. We believe that, in principle, such a progressive dilution experiment could distinguish between the repatterning and migration hypotheses.

MATERIALS AND METHODS

The procedure followed for dissociating and reaggregating imaginal discs was es- sentially that used by Garcia-Bellido (1968). The genetic markers used were yellow Cy, l-0.0, yellow bristles and cuticle), singed (sn 3, l-21.0, gnarled bristles), ebony (e”, 3-70.7, ebony bristles and cuticle) and multiple-wing-hairs (mwh, 3-0.0, two to five trichomes per cell instead of one as in the wild-type). Male first leg discs and distal parts of wing discs (anlage for wing spread, Hadorn and Buck, 1962) from mature larvae (120 hr old at 25°C) were dissected free from other larval tissues under a drop of buffered Ringer’s solution of the following composition: NaCl, 55 mM; KCl, 40 mM; MgSO,.7H,O, 15 mM; CaC1,.2H,O, 5 mM; tricine (Cal- biochem, Los Angeles, California), 10 mM; glucose, 20 mM; sucrose, 50 mM; ficoll (Pharmacia, Upp=la, Sweden) 0.2:;; phenol red, 1.2 mg/l; pH 7.0 (modified after Gehring, personal communication). The discs were washed by serial transfer through several drops of buffered Ringer’s solution on siliconized slides, then trans- ferred by micropipette to a siliconized microcentrifuge tube which had an inside diameter of 2.4 mm tapering to about 0.5 mm. The discs were treated for 5-15 min at room temperature with 0.25’;’ trypsin

466 DEVELOPMENTAL BIOLOGY VOLUME 26, 1971

(Sigma 2 x crystallized) in buffered Ringer’s solution. They were then washed several times with buffered Ringer’s solution, dissociated mechanically by means of a poly- ethylene microstirrer (0.5-0.8 mm in diameter) powered by a small direct-cur- rent motor, and centrifuged in a Beckman/ Spinco Microfuge for 1.5 min at about 1000 g. The pellet was washed several times with buffered Ringer’s solution, drawn up into a micropipette and trans- ferred to a drop of buffered Ringer’s solution on a siliconized microscope slide. The washing steps are very important since trypsin affects the survival of the adult hosts. The clump was then divided into smaller fragments for implantation by standard methods into the abdominal hemocoels of mated adult female hosts (see Ursprung, 1967). After 3-5 days, the implants were transplanted into third instar larval hosts. Differentiated implants were dissected from the abdomens of these hosts after metamorphosis and mounted between coverslips in Gurr’s water mounting medium.

Table 1 shows the combinations of different discs used. The proportion of leg cells to wing cells was varied while the proportion of one genotype of leg to the other genotype of leg was kept constant at one to one. As a control we also mixed y sn3 first leg discs with mwh first leg discs without adding wing discs. We have also performed experiments involving two different genotypes of wing and one of leg. The re-

TABLE 1 COMBINATIONS OF IMAGINAL DISCS USED IN

THIS STUDY

y sn3 leg

discs

mwh Number leg snags’ Frwon of experi- discs discs ments

8 8 8 0.667 3 6 6 12 0.50 1 8 8 16 0.50 1 4 4 12 0.40 2 4 4 24 0.25 2 2 2 28 0.125 1

covery rate from adult hosts was 7370, and from larval hosts 52%.

The method of dissociation of imaginal discs used in this study yielded single cells and many clumps of cells. The usual size of the clumps was 2-10 cells but some contained up to 100 cells. Many single cells stained positively when tested for uptake of dyes such as nigrosin indicating that they were dead or severely injured. Some cells on the outer edges of clumps also stained but cells within clumps generally appeared healthy. If mechanical disruption was con- tinued so as to eliminate clumps, most of the single cells were killed. We have shown earlier (Poodry and Schneiderman, 1971) that as much as 4 hr of tryptic digestion did not remove the tight connection afforded by the septate desmosomes and gap junctions at the apical portions of the cells, but that it did reduce nonjunctional adhesivity. Therefore, the dissociation procedure used by us and by earlier workers must mechanically disrupt the septate desmcsomes and gap junctions, or the cell membranes. It should also be noted that the adepithelial cells (myoblasts) of the discs do not form an epithelium and are not held together by strong adhesive junctions (Poodry and Schneiderman, 1970). Therefore, many of the apparently healthy single cells ob- served after dissociation may be of this cell type and not derived from the chitogenous epithelium.

Several other enzymes were tested in an effort to disrupt chemically the adhesive junctions. Each was dissolved in buffered Ringer’s solution at pH 7.0, and discs were exposed to the solution for periods of up to 4 hr. The enzymes included 0.1% pronase (Calbiochem grade B); 0.2% collagenase (Sigma, tme I); 0.2% hyaluronidase (Sigma, type I); 0.2% neuraminidase (Sig- ma, cholera filtrate); 0.2% a-amylase (Sigma, type III-A, crude); 0.2% diastase (Nutritional Biochemical Co., U.S.P.); 0.2% pectinase (Sigma, purified); 0.2% chitinase (Calbiochem); 0.2% cellulase (Nutritional Biochemical Co.); 0.2% ribo-


nuclease (Nutritional Biochemical Co., crystalline). Collagenase had an effect similar to that of trypsin. Pronase rapidly caused death but still did not remove the tight adhesion. The other enzymes were without noticeable effect when used singly, nor did they have any further effect when tested on discs pretreated with trypsin.

RESULTS

We recovered 167 differentiated implants with monotypic (one genotype) or mosaic (more than one genotype) arrays of bristles and hairs similar to those found by Tobler (1966) and Garcia-Bellido (1966a,b). Sometimes the tissue was in the form of single isolated vesicles of leg specific cuticle, but in most cases the implant was a large complex vesicle in which leg specific cuticle was found as islands surrounded by wing cuticle (Figs. 1 and 2). We do not have evidence for a complete segregation of wing and leg tissue as reported by Tobler (1966)

and Garcia-Bellido, (1966a). In the vesicles the length of the border between wing cuticle and leg cuticle was much smaller than the border between different genotypes of leg. Figures 3 and 4 show parts of differentiated implants from a mixture of y sn3 leg discs with an equal number of mwh discs. No wing discs were added. In these examples it can be seen that all the elements of the tarsus of the male first leg are present, including claws, sex combs, and transverse rows, but the overall pattern does not resemble a normal tarsus. The mosaic sexcomb illustrated in Fig. 3 is associated with a large yellow-singed area and a large multiple wing hair area. We agree with Garcia-Bellido (1966b, p. 385) that “the differentiated reaggregates show better reconstructed and more complete patterns the longer the culture time”, and that “good pattern reconstruction is also usually associated with a greater extent of ‘monotypic’ territories, made up of only

FIG. 1. Diagrammatic representation of the organization of a typical implant containing both wing and leg elements. The implant has been opened on the left side, exposing the bristles and hairs which are on the inside surface of the large vesicle. Several leg areas, 1, some of which have bristles of two genotypes, are present as islands surrounded by wing tissue.

468 DEVELOPMENTAL BIOLOGY VOLUME 26, 19’71

FIG. 2. A leg vesicle mosaic for y sn3 and mwh tissue surrounded by ma; e’l wing tissue. The actual area of contact between leg and wing is limited to a small opening on one side of the leg vesicle (delineated by arrows).

one marker cell type.” While the leg vesi- quency of mosaic leg vesicles. When an cles contained many elements of femur, implant contained 67% leg discs and 33% tibia, and especially tarsus, only a few im- wing discs, about 80% of the adult leg plants were found that had sensilla groups vesicles were mosaic; that is, they con- characteristic of the trochanter or coxa. mined both y sn3 and mwh tissue. On the

Figure 5 records the frequency with other hand, when the percentage of leg to which vesicles of leg-specific cuticle wing disc was changed such that the im- (whether in isolated vesicles or as islands plant contained 50% leg discs (mixed 1: 1 surrounded by wing) were genetically with respect to the two leg genotypes) to mosaic, as a function of the amount of wing 50% wing discs, the frequency of mosaicism tissue added. It is seen that the more the in the adult tissue was about 50%. We used leg discs (always mixed 1: 1 with respect for this analysis a total of 111 implants con- to the two genotypes of leg) were diluted mining varying proportions of leg and with wing discs, the lower was the fre- wing, and found 187 monotypic and 228


mosaic leg vesicles. These numbers include only those cases that were clearly identifi- able with respect to the genetic markers used.

We obtained many examples of transdetermination from sn3; ell wing to leg and y sn3 or mwh leg to wing. The patterns which resulted from the transdetermina- tions of leg to wing were usually well formed, and resembled the patterns made by control implants (Fig. 6b). However, in several cases, the pattern of bristles specific to the wing border was somewhat distorted (Fig. 6a). The bristles were separated by greater distances than is normally found between the bristles of the double or triple row on the wing, just as in Fig. 5 of Ursprung and Hadorn (1962). The signifi-

cance of this observation will be discussed later.

DISCUSSION

Mosaic and Monotypic Vesicles

These observations demonstrate that cells from dissociated imaginal discs can cooperate to reconstruct recognizable tissue- and organ-specific structures such as sex comb, claw organs, wing border bristles, transverse bristle rows, etc., and confirm the similar findings of Hadorn et al. (1959), Ursprung and Hadorn (1962), Nijthiger (1964), Tobler (1966) and Garcia- Bellido (1966a,b, 1968). When dissociated leg and wing discs are mixed together, islands or vesicles of leg tissue are formed which are surrounded by wing tissue. If

FIG. 3. Part of a mosaic vesicle which includes two rows of sex comb teeth, one of which has both yello wildtype teeth, and other tarsal bristles.

wand


FIG. 4. Numerous tarsal elements including claws, sex comb teeth, and other tarsal bristles. Notice that the monotypic areas (one genotype), either y sn3 or nwh, are large.

two genetically distinguishable types of leg disc cells are mixed with the wing disc cells, then the leg vesicles can be classified as either mosaic or monotypic. Inclusion of wing disc cells as a “substrate” for the interactions of leg disc cells thus provides us with a useful aid in the analysis of this problem, by separating the leg tissue into vesicles which are convenient units for analysis.

Even when most of the cells in the aggregate were leg cells, the frequency of mosaic leg vesicles was much less than 100%. For example, as Fig. 5 shows, with 67CO leg discs we still obtained 20’:, monotypic

vesicles. Furthermore, dilution of the leg cells with wing cells resulted in a progressive lowering of the mosaicism frequency (Fig. 5).

These observations seem difficult to reconcile with the hypothesis of pattern reconstruction by directed migration of individual cells derived from a suspension of single cells. According to this theory each leg vesicle would be expected to be derived from the reassociation of many cells from the suspension. Since the leg cells in the suspension are 50Yb of each genotype, then large samples of such cells would be expected to contain cells of both genotypes


in a high proportion of cases. Furthermore, if the cells could migrate extensively to find homonomous cells, then dilution of leg cells with wing cells should not reduce the expected high mosaicism frequency. Only when the dilution increased the distances between leg cells to beyond that distance over which they could interact, would we expect a reduction of mosaicism frequency. Our results therefore cast considerable doubt on the hypothesis of pattern reconstruction by cell migration.

Experiments of the kind we have performed do not permit conclusions about selective adhesivity of homonomous cell types. The cells are forcibly reaggregated by centrifugation, and in the absence of cell migration, would have no opportunity to show selective adhesion.

100

9c

SC

70

6C

1c

Number of Units Which Originate a Vesicle

We have made our analysis quantitative, in the following way. If each leg vesicle were derived from a large number of cells in the suspension, we would expect a high mosaicism frequency. If each vesicle were derived from only one cell in the suspension, we should expect to find no mosaic vesicles. Thus it is possible to calculate the average number of cell clusters, in the suspension, which gives rise to a vesicle. Since the suspensions contained clumps of cells as well as single cells, we shall define each as a unit. We will let n represent the number of units which aggregate to form a single vesicle.

First we shall assume that n is constant.

1 130 I

I I I I I r I I 10 20 30 40 50 60 70 80 90 lb0

PERCENT LEG COMPOSITION OF IMPLANT

lb0 do do 7b do io do B 2b lb 6

PERCENT WING

FIG. 5. Plot of percent mosaic vesicles versus the total composition of the implant in terms of percent wing and percent leg. Vertical lines show 95% confidence interval for sample number (under point).

DEVELOPMENTAL BIOLOGY VOLUME 26, 1971

FIG. 6. A piece of murh leg tissue and mwh wing which arose by transdetermination. The pieces in 6A and 6B were continuous in the implant but were separated in preparation. The portion of triple row seen in 6A has an interbristle space greater than that found in situ. In contrast, the portion in 6B shows a good triple row pattern.

POODRY ET AL. Mechanism of Pattern Reconstruction

.- - -. q:(l-e-“y) ( ,-enb)

0-o q.l-(y”+ b”)

n

FIG. 7. Two plots of the relationship between the frequency with which a sample is mosaic (q) and sample size (n).

The probability q of a sample of n units containing units of both genotypes is given byq = 1 - (y” + bn) whereyandbarethe fractions of the total implants which are yellow and black, respectively (y + b = 1). The relation between q and n generated by this equation is given in Fig. 7. From this curve we can estimate n for any value of q. For the experiments reported in Fig. 5, this gives values of n which are less than 4. If n is not constant, the predictions are slightly different.

A more likely possibility for the distribution of n is that it follows a Poisson distribution. The probability q of mosaicism for samples whose size varies according to a Poisson distribution with mean n, is given by q = (1 ~ e-nY) (1 - e-12*) (M. Finkel- stein, personal communication). The curve generated by this equation is also shown in Fig. 7. Just as in the previous estimates, we can then deduce n from the experimental q values. This gives values of n between 1 and 6 for the experiments of Fig. 5.

From this analysis we conclude that most of the vesicles of leg tissue found in our implants are derived from small numbers of cells or cell clumps in the suspension. This

is inconsistent with the supposition that reconstruction of these patterns is achieved by the migration of the many cells of the vesicle to preassigned positions in the pattern.

Computer Simulation of Reaggregation

A second approach we have adopted to enable us to analyze our data was to per- form a computer simulation2 of the mixture of cells in the epithelium. The method was as follows. Each position on an 18 X 18 rectangular grid is assigned, at random, one of the states y, b, or w (yellow leg, black leg or wing), in various ratios. Left and right, and upper and lower edges of the grid are made confluent, so that we are dealing with a surface with no edges. Hence we are simulating an epithelium with no free edges-a condition we believe to be attained rather quickly by the aggregate in the experiment. Areas of “leg units” which are completely surrounded by “wing units” are then identified and classified as

‘The details of the computer program are on file and may be obtained from the Center for Pathobiology, University of California, Irvine, California 92664.


monotypic or mosaic. The frequency of mosaic vesicles is then calculated.

A variable we introduce into this program is the number of units which a given unit recognizes as adjacent (“interactive neighbors”). In a rectangular array, the number of truly adjacent neighbors is eight. We have simulated this situation, and modified the program to consider fewer interactive neighbors by restricting the directions of interaction. Furthermore, we have simulated situations where the number of interactive neighbors is 24, by allowing interactions over 2 “unit diameters.” This latter situation simulates sorting out involving cellular recognition beyond adjacent cells. Each simulation was performed with various dilutions of “wing units” and the results, in terms of mosaicism frequencies, are shown in Fig. 8. We have also plotted

100

80

70

60

COkif~0slT10~ I

on this diagram the actual experimental results from the progressive dilution experiment.

Simulations which most closely approxi- mate the experimental situation are those where each unit has 6 or 8 interactive neighbors. Interaction with 24 neighbors seems to be excluded in this comparison. This result is consistent with the supposition that the cells are arranged in a single layered epithelium, and do not reassociate with homonomous cells at remote locations.

Patch Size in Reaggregates

The calculations reported in the first part of this discussion indicate that each leg vesicle is initiated by a small number of units in the suspension. Since the vesicles must contain hundreds of cells, it follows either that only large clumps of cells sur-

I 1 I 1 1 I I I I 1 10 20 30 40 50 60 70 80 90 100

PERCENT LEG

OF IMPLANT 1 lb0 &I do 70 do 5b 40 $I 2il lb b

PERCENT WING

FIG. 8. Plots of relationship between mosaic frequency and total composition of implant. Experimental results (same as Fig. 5) are represented by solid circles; computer simulations of 6 interactive neighbors, solid squares; 8 interactive neighbors, open circles; 24 interactive neighbors, triangles.

POODKY ET AL. Mechanism of Pattern Reconstruction 475

vive (see Materials and Methods) or that a considerable amount of growth and cell division occurs prior to differentiation, or both. The occurrence of large continuous areas of yellow, singed, or multiple wing hair tissue within the mosaic vesicles, de- scribed in this report (Figs. 3 and 4) and in those of Ursprung and Hadorn (1962) and Garcia-Bellido (1966b), support both sug- gestions. It is already known that dissociation of imaginal disc cells stimulates their proliferation (Tobler, 1966).

“Incomplete” Patterns.

A phenomenon common to both auto- typically differentiating cells from dissociated wing imaginal discs (see Fig. 5 in Ursprung and Hadorn, 1962) and to groups of cells which have transdetermined from leg to wing (Fig. 6) is an “incomplete pattern” in which the distance between the border bristles is much greater than in the in situ pattern. There is, however, a general tendency for these border bristles to form rows. This observation in reaggregated wing discs suggested to Ursprung and Hadorn (1962) that “the border bristle prepattern was newly organized under the influence of the state of the field of the blastema only after the conclusion of cell division.” An alternative interpretation which would account for the “incomplete pattern” in recombinates after dissociation might be that the border bristle cells differentiated according to their prospective significance and that they were trapped among hair cells before they could reach their final destinations. An analogous argument is presented for the “randomly agglomerated” sex comb teeth and claw elements by Garcia-Bellido (1966a).

The second possibility is made less likely by the finding of this phenomenon in transdetermined tissue. It has been shown by Gehring (1967) that the process of transdetermination occurs in groups of cells rather than in single cells. Borderlines between y sn and wild-type tissue or between mwh and wild-type tissue crossed the border-

lines between auto- and allotypic tissue without extensive peregrinations. A similar observation was made by Postlethwait and Schneiderman (1969) for homeotic determination. Gehring’s (1967) observations rule out the possibility of extensive cell migration during or following the transdetermination event. Patterns formed by cells after transdetermination, therefore, are unlikely to be formed by migration of individually determined cells to previously assigned positions. The pattern, whether normal or “incomplete,” must be super- imposed upon the cell population.

Borderlines in Mosaics

If we exclude the possibility of directed migrations of cells in the epithelium, there remains to be explained the observation that the borderlines between leg tissue of different genotypes are more extensive than borderlines between leg and wing tissue. One possibility is that the morphogenetic processes occurring in the leg tissue are different from those in the wing tissue. Wing tissue might tend to form sheets and leg tissue to form tubes, as they do in situ. The tubular leg structure (vesicle) would contact the wing tissue only at one end of the tube, and would therefore show a rather limited region of contact. Geneti- cally marked homonomous tissues, on the other hand, might show extensive borderlines as they both participate in the same morphogenetic events. Antenna discs and leg discs, whose morphogenesis is closely related (Bryant and Schneiderman, 1969; Postlethwait and Schneiderman, 1971) show extensive heteronomous associations (Garcia-Bellido, 1968).

Conclusions

We consider that pattern reconstruction in the leg vesicles does not need to be explained in terms of the migrations of individual cells. Although such an interpretation was attractive at a time when determination in individual imaginal disc cells was thought to be somewhat rigid

476 DEVELOPMENTAL BIOLOG

(see, e.g., Hadorn, 1966; Ursprung, 1966; Gehring, 1968), recent findings of regen- erative phenomena in this tissue (Schu- biger, 1971; Bryant, 1971) favor an interpretation based on repatterning of the cell population (see also Uhich, 1971; Postle- thwait et al., 1971).

Pattern reconstruction in our experience is never very extensive. We have numerous examples of mosaic claw organs, sex combs, transverse rows, triple rows, double rows, wing spread, etc., but the pattern reconstruction is usually limited to one of these areas. Mosaics of complete leg or even complete leg segments have never been found. The reconstruction of small patterns might depend on fortuitous association of those regions. Chance association with random orientation of two homonomous regions might be followed by repatterning over small areas as originally suggested by Ursprung and Hadorn (1962). Such repat- terming is known to occur in transplants in other insects (Marcus, 1962; Stumpf, 1968; see also review by Lawrence, 1970) when different regions of epidermis within or between segments are exchanged. Simi- larly, some repatterning of epidermal cells occurs in Rhodnius (Locke, 1967) after small regions are destroyed by microcau- tery.

If pattern reconstruction involves, to some extent, the imposition of a new pattern on randomly associated cells and groups of cells, it takes on a new interest because positional information (Wolpert, 1969) must be reestablished. In this way pattern reconstruction may be considered as a model of the process of pattern formation in developing imaginal discs.

We wish to thank Drs. S. V. Bryant, R. D. Camp- bell, G. Schubiger, and E. R. Vyse for their helpful comments on the manuscript. We are indebted to Dr. A. Garcia-Bellido for kindly demonstrating to one of us (C. A. P.) the technique of dissociation and reaggregation of imaginal discs. This investigation was supported by Grant GB-16690 from the National Science Foundation and by Grant HE-13194-01 from the National Institute of Health awarded to Howard A. Schneiderman. Clifton A. Poodry was supported by

1Y VOLUME 26, 1971

a Developmental Biology Training Grant 8Tl-HD20 from the National Institute of Health.

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The mechanism of pattern reconstruction by dissociated imaginal discs of Drosophila melanogaster