Leg regeneration in insects

DEVRLOPMENTAL

BIOLOGY

69,

31-45 (1979)

Leg Regeneration An Experimental

Analysis

in Drosophila SIEGWARD

Center for Pathobiology.

in Insects

Unir~ersity

and A New Interpretation

STRUB'

of California,

Iwine.

California

Y2717

Receicaed May 22, 1978; accepted in rer:ised form September 28. 1978 Fragments from prospective distal regions of Drusophilu male foreleg imaginal discs failed to undergo proximal intercalary regeneration across leg segment borders when mechanically intermixed and cultured for F(days with various fragments from prospective proximal disc regions. The failure of the distal cells to regenerate proximal leg segments was not due to a general restriction in their developmental potentials: Distal fragments, when deprived of their distal-most tips. regenerated in the distal direction at a high frequency. It is concluded that there exist in Drosophila leg discs the same restrictions with respect to regeneration along the proximodistal leg axis as had been previously observed in legs of several hemimetabolous insect species: Intersegmental discontinuities between grafted tissue pieces are not eliminated by intercalation. Based on the available evidence in hemimetabolous insects and in Drosophila, a new interpretation of the different aspects of regeneration in insect legs is offered. It is proposed that the two caregories of regulative fields observed in insect Iegs, the leg segment fields and the whole leg Md, represent the units of regulation for two fundamentally different regulative pathwa.vs that a cell at a wound edge can follow, the intercalative pathway and the terminal pathway. respectively. It is suggested that the criterion used by cells at healing wounds to choose between the two pathways is the difference in circumferential positional information between juxtaposed cells. The intercalative regulative pathway is switched on when cells with disparities in their axial positional information, or cells with less than maximal disparities in their circumferential information, conlact OIW another. The terminal regulative pathway is triggered whenever cells with maximal circumferential disparities come into contact. INTRODUCTION

Many animals possess the ability to fully regenerate distal parts of legs or leg primordia that are lost due to an injury or an experimental amputation. Even the removed distal pieces, if allowed to survive by appropriate grafting procedures, undergo complete distal regeneration from their free proximal cut surfaces (for detailed lists of references, see Bohn, 1976; French et al., 1976). This type of regenerative behavior of leg fragments, which will be referred to as “terminal regeneration” (see Bohn, 1976), can be prevenled from occurring if fragments from different parts of an appendage are grafted together. Instead of regenerating all distal leg structures, the ’ Present address: Department of Biology, State (Jniversity of New York at Stony Brook, Stony Brook, New York 11794

cells at the apposed cut edges then undergo “intercalary regeneration,” replacing only the pattern elements that RW normally located between them. In hemimetabolous insects grafting experiments have demonstrated that leg fragments can undergo intercalary regeneration along both the proximodistal axis (Bohn, 1965, 1970a, b, 1971; Bullike and Sengel, 1970; Bull&e, 1971; Shaw and Bryant, 1975a; French, 1976a, b) and the circumferential axis (French and Bulk-e, 19’iSa. b; French et al., 1976) of the leg. However, in contrast to intercalary regeneration along the leg circumference, for which no restricting borderlines have been detected so far, there appeared to be strict limitations on the capacity of leg fragments to undergo intercalary regeneration along the proximodistal axis. When fragments from differ-

32

DEVELOPMENTAL BIOLOGY

ent levels within a leg segment were grafted together, the resulting positional discontinuities were fully eliminated by both proximal and distal intercalary regeneration (Bohn, 1971; Bull&e, 1971). But when fragments from different leg segments (e.g., coxa and femur) were juxtaposed, the intervening leg segments were not regenerated. Instead, intercalation by each fragment again resulted only in intrasegmental proximal or distal regeneration (Bohn, 1970a, b, 1976; Bulliere, 1971). As a result of this and of the fact that terminal regeneration replaces only distal, and not proximal, leg segments (see above), no case of proximal regeneration across leg segment borders in hemimetabolous insects has so far been reported (see Bohn, 1976). As with hemimetabolous insects, Drosophila leg imaginal disc fragments, cultured in vivo, can undergo terminal regeneration of removed distal leg structures (Schubiger, 1971, 1973; Lee and Gerhart, 1973; &rub, 1977b, d). Furthermore, as demonstrated for the wing disc by Haynie and Bryant (1976) and others (Adler and Bryant, 1977; Wilcox and Smith, 1977; Bryant et al., 1978), imaginal disc fragments can also undergo intercalary regeneration, when grafted together with fragments from different locations in the disc. Using the grafting technique introduced by Nothiger (1964) and modified by Haynie and Bryant (1976), I have examined the imaginal foreleg capacity of Drosophila disc tissue for intercalation in the proximodistal (radial) axis of the disc, by testing the ability of distal fragments to undergo proximal intercalary regeneration across leg segment borders. The results agree with those obtained in legs of hemimetabolous insects and suggest that also in Drosophila leg imaginal discs, leg segmental disparities cannot be eliminated by intercalation in the proximodistal axis. Based on the evidence available from both hemimetabolous insects and Drosophila, a new interpretation of regeneration in insect appendages is presented.

VOLUME 69, 1979 MATERIALS

AND

METHODS

General procedures. All animals were kept at 25°C on standard medium. Discs were dissected in Ringer’s solution (Schubiger, 1971) and fragmented with tungsten needles. Fragments were mechanically intermingled using tungsten needles (for details, see Nothiger, 1964; Haynie and Bryant, 1976), and the combinations were transplanted into young fertilized adult females. After 8 days of culture, the implants were injected into larval hosts for metamorphosis. The differentiated implants were mounted in Euparal and scored under a compound microscope. Irradiations were carried out with a y-ray source (CSI~~;Isomedix) at a dose rate of 1000 R/min. Developmental stages of discs and fiagments. Male foreleg imaginal discs from

animals at two different developmental stages were used: (i) unevaginated discs from mature third-instar larvae, 116-120 hr after oviposition; and (ii) evaginated discs from prepupae, 6-8 hr after pupariation. In the unevaginated discs, the central “endknob” fragment (Di), containing most of the presumptive tarsal material (Schubiger, 1968,1971), and the complementary peripheral fragment (Pr) were tested (Fig. 1). A subscript “i” is used to indicate that a fragment had been irradiated prior to mixing. In the evaginated discs, cuts were made at six different proximodistal levels (Fig. 2). The fragments are designated either by the tip present (P, proximal; D, distal) and the number of the cut level, e.g., PI, D5, or by the two cut levels in intermediate pieces, e.g., 35. Genotypes.

One of the two intermixed fragments in each implant was wild type (Ore-R), and the other y w sn3;mwh (for a description of these mutations and their phenotypes, refer to Lindsley and Grell, 1968), which allowed an unambiguous distinction between the cuticular structures produced from either fragment. In every combination tested, unless otherwise noted, reciprocal experiments with the two geno-

SIEGWARDSTRUB

Leg Regeneration

in Znsects

FIG. 1. Fragments of the third-in&u Drosophila male foreleg disc tested (cut lines are dashed), superimposed on a simplified fate map of the disc (modified from Strub, 1977d; after Schubiger, 1968). Fragments: Di, central “endknob” fragment, containing most of the presumptive tarsal material; Pr, complementary proximal fragment, Leg segments: PT prothorax; CX, coxa; TR, trochanter; FE, femur; TI, tibia; TA, tarsus. Cuticular landmarks that (together with other features; see Material and Methods) were used to determine the leg segment specificity of vesicles: Stl, St& GSt, St5, and RSt, groups of sensilla trichodea; Scl, Sc5, Sell, Sc3, and Sc8, groups of sensilla campaniformia; BH- and EB, single conspicuous bristles; Tvr, transverse row of bristles; JTr and JTh. conspicuous joints; Set, sex comb teeth; Cl, claws.

types were performed. Since the genotype in no case significantly altered the performances of fragments, the data have been pooled. In control experiments (intermixture of genetically marked identical fragments) the structures formed by each partner fragment in an implant were scored separately, giving in these experiments two sets of data per cultured implant. Scoring. Only implants containing rec-

ognizable cuticular structures from both intermixed fragments were scored. Implants consisted of a mass of leg segment-specific vesicles (Garcia-Bellido, 1966; Strub, 1977b, d), whose segment specificity was determined by differences in presence, type, shape, and arrangement of specific bristles, bracts, trichomes, and landmarks (sensilla, joints, and others; see Fig. 1) [for details, see Schubiger (1968); tarsal sensilla cam-

34

DEVELOPMENTAL BIOLOGY

paniformia, described by Russell et al. (1977), were often useful for distinguishing tarsal and tibia1 tissue]. RESULTS

Fragments

from Unevaginated

Discs

In control experiments, either two proximal (Pr) or two distal (Di) fragments were intermixed and cultured. In the Pr/Pr combinations, terminal regeneration (judged by the regeneration of claws which represent the distal tip of the leg) occurred in 57% of the fragments (Table 1, b). In the Di/Di combinations, the cells of each fragment only yielded structures typical for tarsus (Table 1, a). When distal and proximal fragments were intermixed (Di/Pr combinations; Table 1, c), the tissue derived from the Pr fragments underwent claw regeneration in all 13 implants, whereas the tissue derived from the Di fragments again formed only tarsal structures. The frequency of Di fragments containing basitarsal sex comb teeth, whose anlagen are located close to

VOLUME 69, 1979

the medial (proximal) edge of the Di fragment (Schubiger, 1968, 1971), was not significantly higher than in the Di/Di controls. Two factors in these experiments might have prevented the tarsal cells from undergoing proximal intercalary regeneration across leg segments. The positional discontinuity between distal and proximal cells could have been filled in by “unilateral” distal intercalary regeneration by the more readily proliferating Pr cells alone. Alternatively, the average positional discontinuity between juxtaposed Di and Pr cells could have been too small to stimulate the Di cells to intercalate proximally, since Pr fragments are known to contain some tarsal material (Schubiger, 1968, 1971). To test the first possibility, Pr fragments were irradiated with 17,500 R prior to mixing them with the Di fragments. This dosage presumably rendered the Pri cells mitotically inactive and eventually led to their disappearance during culture in adults. It has been shown, however, that heavily ir-

FIG. 2. Camera lucida drawing of an evaginated male foreleg primordium, 6 hr after pupariation. Cut levels, in places where they do not coincide with a natural fold, are indicated by dashed lines. Approximate fate map positions of cuts in the proximodistal leg axis, as determined by the direct injection of fragments into larvae: 1, within coxa; 2, within femur; 3, within tibia; 4, within first tarsal segment; 5, within second tarsal segment; 6, between fourth and fifth tarsal segments. P, proximal tip of the primordium; D, distal tip. Fragments are denoted either by the tip they contain and the cut level (e.g., P2) or by the two cut levels (e.g., 35).

SIEGWARD

&RUB

Leg Regeneration TABLE

PERCENTAGE OF IMPLANTS DAYS in Vito CULTURE

Combination”

iP

Origin of tissue

28’

(c) Dz/Pr

13

1

IN WHICH SPECIFIC STRUCTURES WERE FORMED AFTER INTERMIXTURE AND 8OF PROXIMAL AND DISTAL FRAGMENTS OF THIRD-INSTAR Drosophila MAI.E FORELEG DISCS ~~~__

Leg structures --__ (si)’ ‘j Set (X k SD)” TA FE TI

PT

CX

TR

93

82

68

75

89

-

-

77

100

92

--___(a) Dz/Di (b) Pr/Pr

35

in Insects

-

24’

loo Rfi

13 (0.8 + 82 (15.8 f 23 (0.5 * 92 (23.2 + -

2.1) 19.2) 1.2) “0 3)

.__Clta~ ? SD)’

Transdet ntrurt.”

83 il.7 -c 1.1)

57(1.6 t 2.1)

!A 85 (2.2 c 1.6) 100 3 100 !oJ (4.5 * 4.2) 77 77 91 13.65 2.41 100 (dl Di/Pr, 11 ~ ~.__ ” Refer to Fig. 1 for designation of fragments Pr, refers to Pr fragments of? u’ sn ‘: mw h genotype that had hren wradiaced with 17,500R prior to the intermixture. ” Number of fragments scored (NJ. ’ Abbreviations of leg segments and tarsal structures as in Fig. 1. ” Underlining indicates that this structure would not have been formed by the original fragment after immediate melamorphosis. “Average numbers (X) of sex comb teeth and claws per fragment, and standard deviation (SD). calculated from the total number of fragments, including those without sex comb teeth or claws. ‘In combinations of identical fragments (controls), each partner fragment WBSwored independently. giving ~MOsrt~ of rlat;i per cultured implant. From Di From Pr From Di

radiated imaginal disc cells retain the ability to convey information about their position in the disc to intermixed nonirradiated cells, thereby causing marked changes in the regenerative behavior of the latter (see Adler and Bryant, 1977; Strub, 1977b, d; Wilcox and Smith, 1977; Bryant et al., 1978). Despite their proliferative superiority over the Pr, cells, the Di cells nonetheless failed to form any structures other than tarsal ones in the Di/Pri combinations (Table 1, d). Fragments

from Evaginated

Discs

Increasing proximodistal discontinuities. To test the second possibility mentioned, evaginated prepupal leg primordia were used instead of third-instar discs since cuts at precise proximodistal levels could be made (Fig. 2). The distal test fragments used in the main experimental series (D5 fragments) contained the presumptive tarsal material up to the middle of the second tarsal segment. These fragments were grafted together with proximal fragments which contained the leg material from the proximal tip down to the second tarsal segment, (P5 fragments), to approximately the middle of the femur (P2 fragments), or to approximately the middle of the coxa (Pl fragments).

In control experiments, the regenerative behavior of intermixed identical proximal (P5/P5, P2/P2, and PI/PI combinations) and distal (D5/05 combinations) fragments was analyzed. The cells of each of the proximal fragments regenerated the removed distal structures with a high frequency (Table 2, b, d, and f). The cells of the 05 fragments (Table 2, a), on the other hand, differentiated only tarsal structures. Sex comb teeth, however, which are located in the distal part of the in situ basitarsus, were formed by 29% of the cultured 05 fragments. The D5 fragments were then combined with each of the three proximal fragments (D5/P5, D.5/P2, and D5/Pl combinations; Table 2, c, e, and g). In none of these combinations were recognizable structures of leg segments other than tarsus produced by the cells derived from the D5 fragments. There was one exception. In a D5/P2 implant, a mosaic coxal vesicle was found that contained two bristles of 05 genotype. Although proximal regeneration cannot be excluded in this case, another likely possibility seems to be that one of the D5 fragments, in the course of the dissection and excision, became contaminated with some coxal cells. In none of the combinations was there a significant difference in the fre-

36

VOLUME 69, 1979

DEVELOPMENTAL BIOLOGY

quency of sex comb tooth formation by the D5 fragments compared to the frequency observed in the D5/D5 controls. In a further experiment, I tested whether proximal regeneration from the tarsal fragments failed to occur as a result of the cut position 5 being too far away from the tarsal-tibial border. 04 fragments, cut in the middle of the presumptive basitarsus, were mixed with Pl fragments. Among 10 storable implants, the cells of the 04 fragments formed only structures typical of the tarsus, as with D5 fragments. The result is especially noteworthy, since in six of the implants, for unknown reasons, the Pl fragments failed to undergo distal regeneration and only formed prothoracic and coxal structures. In these implants, the large proximal-distal disparity between the intermixed fragments was therefore present throughout the entire culture time. If the stimulation of proximal intercalary regeneration in tarsal cells required a prolonged and exclusive contact with tissue from the proximal-most leg parts, the circumstances for its occurrence would seem to have been especially favorable in these six implants. Tibiotarsal test pieces. The evaginated prepupal foreleg provided the opportunity to test also the ability of tibial cells to regenerate structures of more proximal leg segments. Fragments were isolated whose

proximal cut edges ran approximately through the middle of the prospective tibia (03 fragments; Fig. 2). In the D3/D3 control combinations (Table 3, a) as well as in the combinations with the complementary P3 fragments (Table 3, c), the cells derived from the 03 fragments produced only tibia1 and tarsal structures. It is thus obvious that the tibial cells, as the tarsal cells, do not undergo intercalary regeneration across the proximal segment boundaries under these conditions. In two further experimental series, the possibility was studied whether in mature leg imaginal discs the cells in the distal segments might have lost their regenerative ability altogether. However, if distal cells were indeed rigidly determinated for a specific developmental fate, regeneration in the distal direction should not occur either. In the above experiments, many fragments formed claws in numbers that considerably exceeded the usual number formed in situ (Tables 1 and 3). It was impossible to decide, however, whether they had resulted from a proliferative enlargement of specific leg regions (“multiplication of units”; Schubiger, 1971) or had been produced by regenerative events. It was therefore necessary to study the regenerative behavior of distal fragments which were deprived of their distal-most tips.

TABLE

2

STRUCTURES FORMED AFTER INTERMIXTURE AND ~-DAY in Viva CULTURE OF TARSAL FRAGMENTS AND PROGRESSIVELY MORE PROXIMAL FRAGMENTS FROM EVAGINATED PREPUPAL FORELEG PRIMORDIA Combination”

N

(a) D5/D5 P5/P5 (c) D5/P5

28’ 14’ 14

(d) P2/P2 (e) D5/P2

14

(b)

Origin of tissue

Leg structures PT

CX

TR

FE

TI

TA

86

100 100

14 43

93 93

86 93

100 79 100 100

86

93

14

86

9_3

m

86

From05 From P5

Set (x f SD) 29 (1.9 64 (10.5 3_6 (1.4 100 (32.9

+_ 3.7) +_ 19.5) zk 2.8) f 28.2)

8_s (13.1 + 10.3) 15 (0.3 +_ 0.8)

Cl (fr f SD) 89 (2.7 + 2.0)

-

g (1.7 + 2.1) 79 (1.6 +_ 1.3)

14 -

8 (3.6 k 4.2) 79 (1.5 f 1.3)

43 7

8 1M) 100 (3.4 77 100 69 100 92 92 7_7(19.2 + 16.6) 1_ (1.9 (t-l PI/PI 18 100 83 2s !2 12 89 50 (4.2 -c 6.1) 56 (0.8 (g) D5/PZ 15 From05 100 33 (2.5 f 3.8) 80 (2.0 From PI 93 93 2_3 93 73 82 @ (17.1 f 18.7) @g (2.5 a For designation of fragments and their approximate fate map contents, see Fig. 2. * Percentage of fragments in which a specific structure was found. For further explanations, see Table ‘See Table 1, footnote f 13

From 05 From P2

Transdet. struct.h

(%)”

+ 2.7) +_ 1.6)

23

+ 0.9) + 1.6)

11 -

f

33

2.2)

1 footnotes.

SIECWARD STRUB

Leg Regeneration TABLE

37

in Insects

3

PERCENTAGE OF IMPLANTS IN WHICH SPECIFIC STRUCTURES WERE FORMEDAFTERINTERMIXTUREAND 6% DAY in Vivo CULTURE OF TIBIOTARSAL AND PROXIMAL FRAGMENTS FROM EVAGINATED PREPUPAL FORELEG PRIMORDIA” Combination

N

(a) 03/03 (b) P3/P3 (c) D3/P3

22h 24’ 21

(d) 35/35 (e) 35/P3

26* 17

Leg structures

Origin of tissue

From 03 From P3 From 35 From P3

Pt

CX

TR

FE

TI

TA

79 90 _ 82

100 100 88

38 48 47

100 100 82

41 96 71 95 62 76 88

100 ss 95 95 88 94 E!

Transdet. struct.

(o/o) Set (2 t SD) 82 (13.5 s_S(40.3 81 (13.9 95 (52.4 65 (10.0 82 (14.7 94 (59.5

k 11.4) + 26.5) + 15.1) + 60.4) +- 10.1) + 21.5) zk 72.4)

Cl (a%‘* SD, 77 (2.8 2_9(3.2 81 (1.7 76 (2.7 73 (1.2 5J (1.2 82 (3.4

+- 3.3) -t 2.8) -e 1.6) f 2.1) + 1.0) * 1.4) + 3.1 I

g &

41

” For explanations, see footnotes to Tables 1 and 2. ” See Table 1, footnote

fi

Fragments containing the leg material from midtibia to tarsal segment 2 (35 fragments) underwent frequent claw regeneration both in control combinations (35/35; Table 3, d) and in combinations with P3 fragments (Table 3, 3), but in no case produced any structures from segments proximal to the tibia. Likewise, 56 fragments, which consisted only of tarsal segments 2-4, regenerated the excised claws in four of seven mixtures with PI fragments, but showed no signs of proximal intercalary regeneration across leg segments (data not shown). Thus, the distal fragments still possess a pronounced ability to regenerate in the distal direction. Interactions between Intermixed Proximal and Distal Fragments Many of the structures formed by the cells of the distal fragments were present in leg segment-specific vesicles which also contained structures derived from cells of the originally proximal fragments (mosaic vesicles). Table 4 presents the data for those combinations in which the tarsal fragments Di and 05 were involved. In combinations with their complementary proximal fragments (Di/Pr, D5/P5), both the Di and the D5 cells formed mosaic tarsal vesicles with cells of their partner fragments at frequencies as high as in the Di/Di and D5/D5 controls (around 70%). In the combinations with an initial intersegmental gap between the mixed fragments, a lower frequency of

TABLE

4

PERCENTAGE OF TARSAL VESICLES CONTAINING STRUCTURES PRODUCED BY BOTH THE TARSAL AND THE PROXIMAL FRAGMENTS IN A COMBINATION Fragments intermixed Di with

Di Pr D5 with 05 P5 P2 PI

n”

mh

23 23 20 18 20 29

16 18 13 13 10 7

m/n (%) -.-~ 70 78 65 7””

50” 24** -__~ a Total number of tarsal vesicles containing structures formed by the tarsal fragment in the mixture. h Number of mosaic vesicles, i.e., those that contained tarsal structures formed by both the fragments in the mixture. * Frequency of mosaic vesicles not significantly different from D5/D5 control (x2 test: for D5/P5, 0.9 > P > 0.5; for D5/P2, 0.5 > P > 0.1). * * Frequency of mosaic vesicles significantly different from D5/D5 control (0.01 > P > 0.001) and from D5/P5 (0.01 > P z O.OOl), but not from D5/P2 (0.1 > P > 0.05).

mosaic tarsal vesicles (D5/P2, 50%; D5/Pl, 24%) was observed; the difference was statistically significant only for the D5/Pl combination, however. These data strongly indicate that the cells from intermixed proximal and distal fragments could interact, since they formed mosaic vesicles provided that the proximal cells had acquired an identical leg segmental specification as the distal cells. Transdetermination All proximal fragments, from both une-

38

DEVELOPMENTAL BIOLOGY VOLUME69. 1979

vaginated (Pr) and evaginated (P5, P3, P2, Pl) foreleg primordia, formed wing disc structures by transdetermination (Hadorn, 1965) at low to moderately high frequencies (Tables l-3). No transdetermined structures were formed by any of the distal test fragments (Di, 04, D5,03,35,56), however. This suggests that the region in the male foreleg disc in which the transdetermination-competent cells are clustered (located within the anterior half of the unevaginated disc; Strub, 1977c) does not extend distal to the midtibial level (see also &rub, 1977b). But the exact dimensions of the transdetermination-competent foreleg disc region still remain to be determined. DISCUSSION

Terminal Regeneration It has repeatedly been shown that presumptive proximal parts of Drosophila male foreleg primordia can regenerate removed distal parts (Schubiger, 1971, 1973; Lee and Gerhart, 1973; Strub, 197713,d). In the present study, I confirmed and extended these findings. I furthermore demonstrated that distal fragments, lacking their distal-most tips, are also able to regenerate distally. It is unlikely that in any of the fragments tested terminal regeneration was simply a consequence of mechanical intermixing. Two of the fragments used in this study, Pr (Schubiger, 1971, 1973; “R pieces”) and P2 (Lee and Gerhart, 1973, as deduced from their Fig. lB), had been shown to undergo frequent terminal regeneration when cultured as nonintermixed pieces. All proximal fragments tested underwent terminal regeneration both in proximal/proximal and in proximal/distal graft combinations. The frequencies at which specific distal leg segments or claws were regenerated were, for each of the proximal fragments, not significantly different in the two graft situations. The finding that proximal fragments underwent terminal regeneration in graft combinations with distal fragments contrasts

sharply with results obtained in legs of hemimetabolous insects. There terminal regeneration by a proximal leg stump is blocked when a distal leg part is grafted to it (Bohn, 1970a, b; Bulliere and Sengel, 1970; Bull&e, 1971). This discrepancy probably reflects the differences in the grafting techniques employed. With the random intermixing method used here, it was not possible to precisely appose proximal and distal cut edges as has been done with the experiments on hemimetabolous insect legs. It is likely therefore that the cut edges of most proximal fragments were essentially “free.” Terminal regeneration could easily proceed from them after they had undergone internal wound closure. Even in graft combinations with hemimetabolous insect legs, occasionally both apposed fragments undergo terminal regeneration from their cut surfaces at the host/graft junction. The interposed terminal regenerates develop in mirror symmetry and are usually fused with one another at an intermediate proximodistal level. These formations, called “excessive regenerates” by Bohn (1970b), “segmented intercalary regenerates” by Bull&e and Sengel (1970) and Bulliere (1971), and “partially or completely autonomous regenerates” by French (1976a, b), have generally been interpreted as cases in which the wound healing process between the apposed cut surfaces either was not occurring at all or was incomplete or delayed. The two free cut surfaces were thought to heal internally, allowing terminal regeneration to proceed from both of them. The frequent occurrence of terminal regeneration in my proximal/distal combinations thus appears to be equivalent to the cases of autonomous terminal regeneration after grafting in hemimetabolous insect legs.

Failure of the Overall Proximodistal Axis to Intercalate

Leg

Results previously obtained by Tobler (1966) and Strub (1977b) suggested that

SIEGWARD STRUB

Leg Regeneration in Insects

tarsal tissue of Drosophila leg discs might be restricted in acquiring developmental programs of proximal leg segments. Tobler combined fragments identical to the Di and Pr pieces used here (Fig. l), but the graft combinations had only a very short time for proliferation since they were injected directly into young third-instar larvae. I cultured dissociated tarsal cells from fragments similar to the 04 pieces (Fig. 2) in the presence of irradiated wing disc tissue, using a technique introduced by GarciaBellido and Niithiger (1976). In both cases, the cells derived from the distal fragments produced almost exclusively tarsal structures. In the present study, the question about the possibility of intercalary regeneration across leg segmental boundaries in Drosophila leg discs was reexamined. Several prospective distal fragments, with t,heir proximal cut edges within both the tarsus and the tibia, were grafted to fragments from the prospective proximal leg regions. The results obtained (Tables 1-3) provide strong indications that cells do not regenerate across proximal leg segment boundaries, even when they are combined with the most proximal leg parts. The question of course arises as to whether the cells of the Drosophila male foreleg disc, with the grafting technique employed here, generally do not undergo intercalary regeneration in any direction. To test this possibility, I have performed experiments in which the capacity of the male foreleg disc tissue to intercalate in the circumferential direction was tested. To this end, the four disc quadrants (see Strub, 1977b, d) were mixed with the quadrants from the opposite edges of the disc; in control experiments, identical quadrants were intermixed. All quadrants that were unable to regenerate circumferentially missing structures in the control experiments underwent intercalary regeneration in the circumferential direction when mixed with their respective opposite quadrants (Strub, in preparation). These results indicate that

39

the failure of foreleg disc fragments to undergo intercalary regeneration across proximal leg segment borders was not a consequence of the mixing procedure used, but rather reflects a restriction in the developmental potentials inherent to the Drosophila male foreleg disc cells. An identical restriction in their developmental capacities has been observed in the leg cells of all hemimetabolous insects investigated. In graft combinations involving cut surfaces from different leg segments, the distal cells never regenerated any structures beyond the proximal segment border. Equally importantly, the leg segmental discontinuities were not replaced by distal intersegmental intercalary regeneration from the proximal cut surfaces (Bohn, 1970a, b; Bullike and Sengel, 1970; Bulliiire, 1971; Bart, 1971b; French and Bullike, 1975b). The latter result is especially noteworthy, since the proximal cut surfaces would have regenerated all missing distal leg segments had their internal wound closure (see Bullike, 1972) not been prevented by the apposition of the grafts. A similar demonstration in Drosophila leg primordia has to wait until a grafting technique allowing exact appositions is available (see above). Recent findings by Haynie and Schubiger (personal communication) demonstrate that legs are not the only insect appendages in which restrictions with respect to intercalation in the proximodistal axis exist. When central fragments of Drosophila wing discs, containing presumptive distal wing blade parts, were intermixed and cultured with the peripheral (=proximal) wing disc portions, the distal cells failed to regenerate intermediate (wing hinge) or proximal (pleura, notum) structures. The authors interpret their results as indicative of a segmental organization in insect wings. similar to that found in the legs. The above data provide new insights with respect to the applicability of the “shortest intercalation rule” of the polar coordinate model by French et ~1. (1976). The model assumes that each cell’s position in an in-

40

DEVELOPMENTAL BIOLOGY VOLUME69, 1979

sect appendage is specified by’ a unique combination of two positional values, a circumferential and a radial value. Regeneration is proposed to be stimulated by the apposition, during wound healing or by grafting, of cells with disparate positional values. According to the shortest intercalation rule, the apposed cells then proliferate until all intervening circumferential and radial values are replaced. In the case of circumferential intercalation, the rule demands that the shorter of the two possible routes is chosen (French and Bull&e, 1975a, b). The rule’s validity for intercalation in the circumferential sequence of leg segments (French and Bullibre, 1975a, b; French et al., 1976; see also Schubiger, 1971; Strub, 1977a) and the proximodistal axis within a leg segment (Bohn, 1970a, 1971; Bull&e and Sengel, 1970; Bulliere, 1971; Shaw and Bryant, 1975a; French, 1976a, b) is well documented. However, the rule implies that also juxtaposed tissue pieces with disparities in the overall proximodistal axis of an appendage, that is, from different segments in legs or from proximal and distal parts in wing primordia, should inter&ate the intervening structures. As discussed above, in none of the insect systems thus far investigated has this occurred. It can therefore be inferred that no intercalation rules, including the one proposed by the polar coordinate model, can be applied to the overall proximodistal axes of legs and wing primordia in insects.

A Dual System of Regulative Pathways in the Insect Leg; A New Interpretation One of the most important theoretical contributions to the causal understanding of development has been the concept of fields (Weiss, 1924, 1939; Wolpert, 1969). Although fields are primarily conceived of as autonomous self-organizing units in normal development, the evidence for their existence and for the location of their boundaries necessarily requires an analysis

of the effects of experimental disturbances of normal development. A field is thus always characterized as a regulative field, that is, as a body region that behaves as an independent unit during regulative processes (Weiss, 1939; Wolpert, 1969; Bryant et al., 1977). It has been known for some time (see, e.g., Bohn, 1970b; Bulliere, 1971) that two categories of regulative units exist in insect legs, the leg as a whole and the individual leg segments. Furthermore, there is ample experimental evidence which suggests that the regulative fields of the different leg segments are identical: Grafting between axially or circumferentially homologous positions in different leg segments did not stimulate any intercalary growth (Bohn, 1970a, b; Bart, 1971b; Bulliere and Sengel, 1970; Bullibre, 1971; French and Bulliere, 1975a, b). It was not fully understood, however, under what conditions regulating cells referred to their leg segment as the unit of regulation and under what conditions the whole leg was the regulative unit. A new interpretation of insect leg regeneration, to be presented now, can account for these phenomena. An analysis of the experimental data suggests that the two categories of fields represent the units of regulation for the two kinds of regeneration which cells in insect legs can undergo. In all experiments where terminal regeneration occurred, the whole leg behaved as the unit of regulation. This is shown by the fact that, from any axial leg level, all missing distal structures and segments were replaced (Bohn, 1965, 1974; Bulliere, 1971; Bar-t, 1971a, b; Shaw and Bryant, 1974; this report). However, whenever intercalary regeneration occurred, the leg segments invariably represented the units of regulation. This is seen clearly in those experiments in which tissue pieces were grafted to circumferentially or axially nonhomologous positions in different leg segments. In these combinations, only intrasegmental intercalation occurred, as if

SIEGWARD STRUB

Leg Regeneration in Insects

the graft pieces had been transplanted to the corresponding nonhomologous positions within the original leg segments; the segment borders were not transgressed (Bohn, 1970a, 1971; Bullikre and Sengel, 1970; Bulli&re, 1971; Bart, 1971b; French and Bull&e, 1975b). From the above analysis, it then follows that any rules proposed to govern the intercalative behavior of leg cells, such as the shortest intercalation rule (French et al., 1976), apply only to the leg segmental regulative fields. On the other hand, rules with respect to terminal regeneration of cells, such as Rose’s (1962) “rule of distal transformation” or French, Bryant, and Bryant’s (1976) “complete circle rule for distal regeneration,” are applicable only to the whole leg regulative field, and only to terminal distal regeneration, and not to intercalary distal regeneration. The two categories of regulative fields appear to be but the reflection of two fundamentally different regulative pathways which cells in insect legs can follow: a terminal and an intercalative pathway. But what is the mechanism by which leg cells at wound edges decide on which pathway to embark? Based on a large body of data, discussed below, the following scheme is proposed: (i) The only criterion used by cells in insect legs to choose between the intercalative and the terminal regulative pathways is the disparity in the circumferential positional information of the cells with which they become juxtaposed during normal wound healing or as the result of grafting. (ii) The intercalative regulative pathway is activated when cells with disparate axial positional information, or cells with less than maximally disparate circumferential information, contact one another. Regeneration of axially or circumferentially intervening structures then occurs within the leg segmental fields of the apposed cells. (iii) However, whenever cells with maximally disparate circumferential information (regardless of their axial positional in-

41

formation) contact one another, their terminal regulative pathway is activated. Certain additional conditions provided (see below), the cells then regenerate all structures which are located more distally in the appendage. Ample evidence has been provided in hemimetabolous insect leg experiments showing that the grafting together of any axially nonhomologous cut surfaces causes intrasegmental intercalation to occur, but does not trigger the terminal pathway (Bohn, 1970a, b, 1971, 1976; Bulliitre and Sengel, 1970; Bullikre, 1971; Shaw and Bryant, 1975a; French, 1976a). Several authors have likewise demonstrated that confrontation of cut edges from nonopposite circumferential positions, although stimulating intercalary regeneration, does not lead to terminal regeneration. These experiments included ipsilateral grafting of amputated legs to 90°-rotated stumps (Bohn, 1965; Bull&e, 1970; Shaw and Bryant, 1975b) or grafting of rectangular strips of integument to less than maximally disparate circumferential locations (French and Bull&e, 1975a, b). However, when circumferentially maximally opposed cells, regardless of whether they were derived from the same or from different axial levels, were experimentally juxtaposed to one another, terminal regeneration was induced. Grafting of amputated legs to stumps such that either the anteroposterior axes or the dorsoventral axes of stumps and grafts were reversed (by contralateral transplantation with or without a 180” rotation of the graft) caused the cells at the two sites of greatest circumferential disharmony to produce lateral supernumerary regenerates (Bohn. 1965, 1972; Bulli&re, 1970; Bart, 1971a; Shaw and Bryant, 397513; French, 1976b). The same result was obtained when small rectangular pieces of integument were grafted to opposite circumferential positions in the same or a different leg segment (Bohn, 1965; Bart, 1971b; French and Bulli&re, 1975a). Ipsilateral

42

DEVELOPMENTAL BIOLOGY VOLUME69, 1979

grafting of amputated legs to MO’-rotated stumps, which causes maximal circumferential disharmony all along the host/graft junctions, led to the production of a variable number of supernumerary regenerates at various circumferential positions (Bohn, 1972; Bart, 1971a; Bulliere, 1970; Shaw and Bryant, 1975b; French, 1976b). After simple amputation, cells from circumferentially opposite locations are automatically juxtaposed during the process of wound closure by the epidermal cell layer (see Bull&e, 1972, for instance). The proposed trigger mechanism for the terminal regulative pathway implies that the presence of a complete set of circumferential positional information at a given radial level is not a prerequisite for the initiation of terminal regeneration, as has been proposed by the complete circle rule for distal regeneration of the polar coordinate model (French et al., 1976). This view finds good support in data from hemimetabolous insects. In cockroaches, terminal regeneration was started from bases where a complete set of circumferential information was neither present at the beginning nor later regenerated. Bohn (1965) obtained frequent biventrally symmetric axial outgrowths from experimentally constructed doubleventral tibiae. The same author, later confirmed by French (1976a), observed that simply cutting V-shaped wedges, covering at their widest parts about half of the leg circumference, out of the ventral sides of tibiae stimulates the production of biventrally symmetric lateral supernumeraries. However, the circumferentially grossly deficient bases which were present both in double-ventral tibiae and after excision of V wedges, appeared to limit the distal extent of the terminal regenerates. In both situations, the biventrally symmetric outgrowths mostly tapered off and failed to regenerate the most distal segments of the tarsus and the claws. The reason for this phenomenon is not understood. It appears, however, as if some correlation existed between the circumferential completeness of

a terminal regenerate and the distal completeness it can achieve. Bart (1969, 1971a, b), based on experiments with legs in the walking stick Carau&s, proposed a trigger mechanism for terminal regeneration very similar to one put forth here. He suggested that terminal regeneration is induced whenever cells from physically opposite circumferential locations meet. The present hypothesis makes the additional assumption that in certain appendages the circumferential positional information can be unevenly spaced (French et al., 1976). As a consequence, cells with maximally disparate circumferential values need not necessarily be located in physically opposite locations in the leg. Unequal spacing of circumferential positional values has been proposed for the Drosophila foreleg disc (French et al., 1976; Strub, 1977a) and seems also to be likely in certain cockroach legs (Strub, in preparation). Juxtaposition of cells with very large circumferential disparities has also been proposed to trigger transdetermination in transdetermination-competent cells of the Drosophila male foreleg disc (Strub, 1977c). Indeed, in none of the foreleg disc blastemas tested so far has transdetermination been reported without terminal regeneration occurring in the same blastema type (although not necessarily in the same implant) (Schubiger, 1971; Lee and Gerhart, 1973; Strub, 1977b, d). The striking similarities between the two trigger mechanisms proposed might indicate a relation between terminal regeneration and transdetermination. A detailed account of this hypothesis will be given elsewhere (Strub, in preparation). Is the Tarsus a Single Regulative

Field?

There is conflicting evidence in the literature as to whether the tarsus of legs of hemimetabolous insects represents one single regulative field [proposed by Bulliere (1971) for Blabera] or whether each of the

SIEGWARD STRUB

Leg Regeneration

tarsal subsegments is an independent field [proposed by Bohn (1970b) for Leucophaea]. In the present study, I tried to approach this question in the prepupal male foreleg primordium of Drosophila. D5 fragments, lacking basitarsal material completely, were tested for their ability to regenerate the sex comb teeth (located in the distal portion of the in situ basitarsus) by proximal intercalary regeneration when grafted together with various proximal fragments (P5, P2, PI). In none of these combinations did the 05 fragments form sex comb teeth significantly more frequently than in the D5/D5 control implants (Table 2). These results suggest that sex comb tooth formation by D5 cells in the distal/ proximal combinations was caused by a process other than proximal (intrasegmental) intercalary regeneration (see the following). The data thus seem to favor Bohn’s concept of the organization of the tarsus, but a final conclusion is not possible yet. I still have to account for the fact, however, that even in the D5/D5 control combinations, sex comb teeth were differentiated in 30%. of the fragments. The following alternatives can be considered. (i) Occasional cutting errors: This possibility seems improbable because the direct injection of 05 and complementary P5 fragments into metamorphosing larvae caused no sex comb teeth to result in 9 05 fragments, while 7 of 10 P5 fragments contained a sex comb. (ii) Spontaneous regeneration of basitarsal material from more distal levels: This possibility cannot be discarded, but there is also no experimental support for it. (iii) A dormant ability of cells in the second, and perhaps even the third, tarsal segment to form sex comb teeth, which can manifest itself only under certain conditions. I shall discuss three lines of evidence in support of this notion. First, in the vast majority of species of the Drosophila melanogaster species group, the males carry sex comb teeth on the second, and in some species even on the

in Inserts

43

third, tarsal segment of the forelegs (Bock and Wheeler, 1972). Second, several mutations in Drosophila melanogaster cause the formation of sex comb teeth on the second and third tarsal segments of male forelegs:

esc/esc;ssCr/ssn(esc, extra sex comb, 2-S; ss I’, aristapedia, 3-58; Stern, 1953), sn (sparse arista, 1-14; Rayle, 1968), fi (four jointed, 2-81; Tokunaga and Gerhart, 1976), d (dachs, 2-31; Tokunaga and Gerhart, 1976), and 1(3)c43”“’ (3-49; Martin et ai., 1977). Third, sex comb formation on the second tarsal segments of male forelegs could be induced by treatments which most probably caused cell death in the developing leg discs, such as heat pulses in the temperature-sensitive cell-lethal mutation I (1) ts-504 (Simpson and Schneiderman, 1975) or nitrogen mustard feeding of wildtype larvae (Tobler and Huber, 1972). From the foregoing, it can be speculated, therefore, that Drosophila melanogaster. or its ancestors, only in recent evolutionary times abandoned sex comb formation on the second tarsal segment of the in situ foreleg, but that the capacity to do so is still present in a latent form. The sex comb teeth formed by the D5 fragments in both distal/distal and distal/proximal graft combinations can then be interpreted as cases of “atavistic regeneration” (see Needham, 1965) caused by cell death due to the relatively rough intermixing technique, which was followed by compensatory growth. I am indebted to Dr. Howard Schneiderman for his enthusiastic support and encouragement, and to Drs. Susan and Peter Bryant for many stimulating discussions. In addition, I wish to thank these persons and also Drs. Richard Campbell. Margerv Fain. Lewis Held, and Thomas Wilson for valuable suggestions and comments on the manuscript. This research was supported by Grants AI 10527 and HD 06082 from the National Institutes of Health, DHEW, and by grants from the Swiss National Science Foundation and the Julius Klaus-Stiftung. Zdrich.

REFERENCES ADLER, P. N., and BRYANT, P. J. (1977). Participation of lethally irradiated imaginal disc tissue in pattern

44 regulation

DEVELOPMENTAL BIOL! 3GY in

Drosophila.

Develop.

Biol.

60,

298-304. BART, A. (1969). Conditions locales du declenchement et du developpement de la regeneration dune patte chez 1’Insecte Carausius morosus Br. C. R. Acad.

Sci. Paris 269,473-476. BART, A. (1971a). Morphogenbse surnumeraire au niveau de la patte du phasme Carausircs morosus Br. Wilhelm Roux Arch. 166,331-364. BART, A. (1971b). Modalites de la formation et de developpement dun centre morphogenetique surnumeraire chez Carausius morosus Br. Wilhelm Roux Arch. 168,97-124. BOCK, I. R., and WHEELER, M. R. (1972). The Drosophila melanogaster species group. In “Studies in Genetics” (M. R. Wheeler, ed.), Vol. VII, pp. I-102. University of Texas, Austin, Publ. 7213. BOHN, H. (1965). Analyse der Regenerationsfahigkeit der Insektenextremitat durch Amputationsund Transplantationsversuche an Larven der Afrikanischen Schabe (Leucophaea maderae Fabr.). II. Achsendetermination. Wilhelm Roux Arch. 156, 449-503. BOX-IN, H. (1970a). Interkalare Regeneration und segmentale Gradienten bei den Extremitaten von Leucophaea-Larven (Blattaria). I. Femur und Tibia. Wilhelm Roux Arch. 165,303-341. BOHN, H. (1970b). Interkalare Regeneration und segmentale Gradienten bei den Extremitaten von Leucophaea-Larven (Blattaria). II. Coxa und Tarsus.

Develop. Biol. 23, 355-379. BOHN, H. (1971). Interkalare Regeneration und segmentale Gradienten bei den Extremitaten von Leucophaea-Larven (Blattaria). III. Die Herkunft des interkalaren Regenerates. Wilhelm Roux Arch. 167, 209-221. BOHN, H. (1972). The origin of the epidermis in the supernumerary regenerates of triple legs in cockroaches (Blattaria). J. Embryol. Exp. Morphol. 28, 185-208. BOHN, H. (1974). Extent and properties of the regeneration field in the larval legs of cockroaches (Leuexperiments. J. cophaea maderae). I. Extirpation Embryol. Exp. Morphol. 31,557-572. BOHN, H. (1976). Regeneration of proximal tissues from a more distal amputation level in the insect leg (Blaberus craniifer, Blattaria). Develop. Biol. 33,

285-293. BRYANT, P. J., ADLER, P. N., DURANCEAU, C., FAIN, M. J., GLENN, S., HSEI, B., JAMES, A. A., LITTLEFIELD, C. L., REINHARDT, C. A., STRUB, S., and SCHNEIDERMAN, H. A. (1978). Regulative interactions between cells from different imaginal discs of Drosophila melanogaster. Science 201,928-930. BRYANT, P. J., BRYANT, S. V., and FRENCH, V. (1977). Biological regeneration and pattern formation. Sci.

Amer. 237,66-81. BULLIBRE, multiples

D. (1970). Interpretation des regenerats chez les Insectes. J. Embryol. Exp. Mor-

VOLUME 69, 1979

phol. 23,337-357. BULLI~RE, D. (1971). Utilisation de la regeneration intercalaire pour l’etude de la determination cellulaire au tours de la morphogenbse chez Blabera Develop. Biol. 25, craniifer (Insecte Dictyoptere).

672-709. BULLI~RE, D. (1972). Etude de la regeneration d’appendice chez un Insecte: stades de la formation des regenerats et rapports avec le cycle de mue.

Ann. Embryol. Morphol. 5,61-74. BULLI~RE, D., and SENGEL, PH. (1970). Nouvelles don&es sur la determination qualitative des cellules au tours de la regeneration chez les Insectes. C. R.

Acad. Sci. Paris 270,2556-2559. FRENCH, V. (1976a). Leg regeneration in the cockroach, Blattella germanica. I. Regeneration from a congruent tibia1 graft/host junction. Wilhelm Roux Arch. 179, 57-76. FRENCH, V. (1976b). Leg regeneration in the cockroach, Blattella germanica. II. Regeneration from a non-congruent tibia1 graft/host junction. J. Em-

bryol. Exp. Morphol. 35,267-301. FRENCH, V., BRYANT, P. J., and BRYANT, S. V. (1976). Pattern regulation in epimorphic fields. Science 193, 969-981. FRENCH, V., and BULLI~RE, D. (1975a). Nouvelles don&es sur la determination de la position des cellules Cpidermiques sur un appendice de Blatte. C.

R. Acad. Sci. Paris 280, 53-56. FRENCH, V., and BULLI~RE, D. (1975b). Etude de la determination de la position des cellules epidermiques: Ordonnancement des cellules autour dun appendice de Blatte; demonstration du concept de generatrice. C. R. Acad. Sci. Paris 280, 295-298. GARCIA-BELLIDO, A. (1966). Pattern reconstruction by dissociated imaginal disk cells of Drosophila melanogaster. Develop. Biol. 14,278-306. GARCIA-BELLIDO, A., and NBTHIGER, R. (1976). Maintenance of determination by cells of imaginal discs of Drosophila after dissociation and culture in vivo. Wilhelm Roux Arch. 180, 189-206. HADORN, E. (1965). Problems of determination and transdetermination. Brookhaven Symp. Biol. 18, 148-161. HAYNIE, J. L., and BRYANT, P. J. (1976). Intercalary regeneration in imaginal wing disk of Drosophila

melanogaster. Nature (London) 259,659-662. LEE, L.-W., and GERHART, J. C. (1973). Dependence of transdetermination frequency on the developmental stage of cultured imaginal discs of Drosoph-

ila melanogaster. Develop. Biol. 35, 62-82. LINDSLEY, D. L., and GRELL, E. H. (1968). Genetic variations of Drosophila melanogaster. Carnegie Institute of Washington Publ. No. 627. MARTIN, P., MARTIN, A., and SHEARN, A. (1977). Studies of 1(3)~43~“‘,a polyphasic, temperature-sensitive mutant of Drosophila melanogaster with a variety of imaginal disc defects. Develop. Biol. 55, 213-222.

S~EGWARD STRUB

Leg Regeneration

NEEDHAM, A. E. (1965). Regeneration in the arthropoda and its endocrine control. In “Regeneration in Animals and Related Problems” (V. Kiortsis and H. A. L. Trampusch, eds.), pp. 283-323. North-Holland, Amsterdam. N~THIGER, R. (1964). Differenzierungsleistungen in Kombinaten, hergestellt aus Imaginalscheiben verschiedener Arten, Geschlechter und Kdrpersegmente von Drosophila. Wilhelm Roux Arch. 155, 269-301. RAYLE, R. E. (1968). Report. Drosophila Inform. Serv. 43, 62. ROSE, S. M. (1962). Tissue-arc control of regeneration in the amphibian limb. Symp. Sot. Study Develop. Grouth 20, 153-176. RUSSELL, M. A., GIRTON, J. R., and MORGAN, K. (1977). Pattern formation in a ts-cell-lethal mutant of Drosophila: The range of phenotypes induced by larval heat treatments, Wilhelm Roux Arch. 183, 41-59. SCHUBIGER, G. (1968). Anlageplan, Determinationszustand und Transdeterminationsleistungen der mannlichen Vorderbeinscheibe von Drosophila melanogaster. Wilhelm Roux Arch. 160, 9-40. SCHUBIGER, G. (1971). Regeneration, duplication, and transdetermination in fragments of the leg disc of Drosophila melanogaster. Develop. Biol. 26, 277-295. SCHUBIGER, G. (1973). Regeneration of Drosophila melanogaster male leg disc fragments in sugar fed female hosts. Experientia 29, 631. SHAW, V. K., and BRYANT, P. J. (1974). Regeneration of appendages in the large milkweed bug, Oncopeltus fasciatus. J. Insect. Physiol. 20, 1849-1857. SHAW, V. K., and BRYANT, P. J. (1975a). Intercalary leg regeneration in the large milkweed bug Oncopeltus fasciatus. Deoelop. Biol. 45, 187-191. SHAW, V. K., and BRYANT, P. J. (1975b). Supernumerary regeneration in the large milkweed bug Oncope&us fasciatus. Develop. Biol. 45, 221-230. SIMPSON, P., and SCHNEIDERMAN, H. A. (1975). Isolation of temperature sensitive mutations blocking clone development in Drosophila melanogaster, and the effects of a temperature sensitive cell lethal

in Insects

45

mutation on pattern formation in imaginal discs. Wilhelm Roux Arch. 178, 247-275. STERN, C. (1953). Genes and developmental patterns. In “Proceedings, 9th Int. Congr. Genet.,” Vol. 1, pp. 355-369. STRUB, S. (1977a). Developmental potentials of the cells of the male foreleg disc of Drosophila. I. Pattern regulation in intact fragments. Wilhelm Roux Arch. 181, 309-320. STRUB, S. (1977b). Developmental potentials of the cells of the male foreleg disc of Drosophila. II. Regulative behaviour of dissociated fragments. Wilhelm Roux Arch. 182, 75-92. STRUB, S. (1977c). Localization of cells capable of transdetermination in a specific region of the male foreleg disk of Drosophila. Wilhelm Roux Arch. 182,69-74. STRUB, S. (1977d). Pattern regulation and transdetermination in Drosophila imaginal leg disk reaggregates. Nature (London) 269, 688-691. TOBLER, H. (1966). Zellspezifische Determination und Beziehung zwischen Proliferation und Transdetermination in Bein- und Fliigelprimordien von Drosophila melanogaster. J. Embrvol. Exp. Morphol 16, 609-633. TOBLER, H., and HUBER, S. (1972). Effects of nitrogen mustard (HN-2) on the development of the male foreleg of Drosophila melanogaster. Drosophila In form. Sew. 49, 99-100. TOKUNA~A, C., and GERHART, J. C. (1976). The effects of growth and joint formation on bristle pattern in D. melanogaster. J. Exp. Zool. 198, 79-96. WEISS, P. (1924). Unabhingigkeit der Extremitatenregeneration vom Skelett (bei Triton cristatus). Arch. Mikrosk. Anat. Entu~icklungsmech. 104, 359-394. WEISS, P. (1939). “Principles of Development.” Hafner, New York. WILCOX, M., and SMITH, R. J. (1977). Regenerative interaction between Drosophila imaginal discs of different types. Dec>elop. Biol. 60, 287-297. WOLPERT, L. (1969). Positional information and the spatial pattern of cellular differentiation. J. Theo, Biol. 25, I-47.