Prospective fates and regulative capacities of fragments of the female genital disc of Drosophila melanogaster

70, 127-148 (1979)

DEVELOPMENTAL

BIOLOGY

Prospective

Fates and Regulative Capacities of Fragments Genital Disc of Drosophila melanogaster C.LYNNELITTLEFIELD'AND

Center for Pathobiology

and Department

of the Female

PETERJ. BRYANT

of Developmental and Cell Biology, Irvine, California 92717

University

of California,

Received May 3, 1978; accepted in revised form September 27, 1978 A fate map of the female genital disc of Drosophila melanogaster was established by examining the derivatives of fragments transplanted into host larvae for metamorphosis. The fate map is presented as a two-dimensional projection, but for several reasons it is proposed that the anal plates originate from the dorsal epithelial layer whereas the genitalia are produced from the ventral layer. Fragments produced by cuts parallel to the axis of symmetry of the disc undergo regeneration during culture in adult hosts if the fragments comprise more than half of the disc, or duplication if they comprise less than half. Most of the fragments generated by bilaterally symmetrical cuts across the line of symmetry of the disc undergo neither regeneration nor duplication during culture, but with some such fragments there is a low frequency of regeneration. It is argued that the usual lack of regeneration in these fragments results from wound healing which confronts identical positions from right and left sides, giving no growth stimulation. The fragments which regenerate might do so as a result of healing between dorsal and ventral surfaces, providing the discontinuity in positional information which is thought to be involved in growth stimulation. INTRODUCTION

During the development of embryonic fields a series of complex and largely mysterious events results eventually in the production of reproducible and often intricate spatial patterns of differentiation. It is now widely believed that this process of pattern formation involves the perception by cells of their physical positions with respect to other cells occupying the same’ field, by some kind of intercellular communication (Wolpert, 1969). It has further been suggested (Wolpert, 1971) that the patterns generated in different parts of an organism may be controlled by similar pattern-forming mechanisms, the unique patterns produced by different fields resulting from differences in the way their constituent cells interpret their positions. It has been proposed by French et al. (1976), based on the regulative behavior of ’ Present address: Zoologisch-Vergl. Anatomisches Institut der Universitat Ziirich, 8006 Ztirich, Switzerland.

regenerating amphibian limbs and insect appendages, that within these fields cells determine their positions by means of a two-dimensional array of polar coordinates and that this mechanism for specifying position is identical for all such fields. Support for the idea of a universal mechanism comes from studies on the interactions of cells from different imaginal discs of Drosophila (Wilcox and Smith, 1977; Strub, 1977a; Bryant et al., 1978). When cells of one disc type are brought into contact with cells of another disc type, by tissue mixing or by cell dissociation and reaggregation, regeneration can be induced in disc fragments which otherwise only duplicate, and this change in regulative behavior can be explained by the communication of positional information between cells of the heterotypic disc types. It seems likely, therefore, that the different imaginal discs in Drosophila have a common mechanism for specifying and communicating positional information, and it should be possible to account for the regulative behavior of all of OO12-1606/79/05OOOl-12$02.00/O Copyright D 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

128

DEVELOPMENTALBIOLOGY

the imaginal discs using the same principles. The model originally proposed to account for the regulative behavior of the imaginal wing disc (French et al., 1976) has been modified to account for the regulative behavior of fragments of the first leg disc (Strub, 1977b) by altering the spacing of positional values, and for the bilaterally symmetrical male genital disc (Bryant and Hsei, 1977) by assigning bilaterally symmetrical positional values to the field. To test the validity of the proposed bilaterally symmetrical model, we have investigated the regulative behavior of the only other bilaterally symmetrical disc in Drosophila, the female genital disc. In the studies presented here we show that while the model proposed for the male genital disc is consistent with the regulative behavior of most of the fragments of the female genital disc, there are also some fragments whose behavior conflicts with that predicted by the model. We therefore propose a new model utilizing polar coordinates which is consistent with the regulative behavior of fragments of both the male and female genital discs and which is in even closer agreement with the proposal of a common pattern-forming mechanism in all of the imaginal discs of Drosophila. MATERIALS

AND

METHODS

Morphology of the male and female genital discs. For histological analysis, genital discs were removed in buffered Ringer’s solution (Schubiger, 1971), from late third instar male and female larvae of the OreRC strain of Drosophila melanogaster. They were fixed in Kahle’s fixative (formaldehyde-alcohol-acetic acid-water, 3:7.5: 1:15), dehydrated, and embedded in paraffin. Sagittal sections 4 pm thick were stained with Schiffs reagent and counter-

VOLUME 70. 1979

stained with fast green. The sections were analyzed with a compound microscope at 600 x. Genital discs were observed in situ using scanning electron microscopy. The discs and associated larval epidermis were removed from late third instar larvae without disturbing the connecting stalks, fixed in 5% glutaraldehyde in 0.1 M cacodylate buffer, processed according to the modified thiocarbohydrazide-osmium tetroxide procedure (Reinhardt et al., 1977), dehydrated to 100% acetone, transferred to 100% amylacetate, and dried at room temperature. Scanning electron micrographs were taken with an Etec Autoscan at 10 kV. Isolation and transplantation of fragments of the female genital disc. Female genital discs were removed from late third instar larvae of the Ore-RC strain aged 120 f 4 hr after oviposition at 25°C. Care was taken to select only those discs which had attained a size and morphology approximating that shown in Fig. la. The discs were removed in buffered Ringer’s solution and cut into fragments with electrolytically sharpened tungsten needles. They were cut horizontally across the axis of symmetry at levels 1, 2, or 3, or vertically, parallel to the axis of symmetry, at levels B, B’, or C (Fig. la). Fragments are designated by the bounding levels; for example, 02 is the material between the anterior edge and the level 2 cut. To obtain a fate map of the female genital disc, horizontally cut fragments, 01, 14, 02, 24, 03, 34, and vertically cut fragments, AB, AC, and BB’, were injected for metamorphosis into late third instar male larvae following the technique of Ephrussi and Beadle (1936). Intact female genital discs were similarly transplanted in order to assess the exact inventory of structures pro-

FIG. 1. (a) Photograph of the female genital disc with lines superimposed to indicate the levels cut to generate the fragments used in these studies. B, C, and B’ represent vertical cuts, parallel to the axis of symmetry of the disc, and 1,2, and 3 represent cuts in the horizontal direction, transecting the axis of symmetry. (b) Two-dimensional fate map of the female genital disc showing the approximate regions of the disc from which the adult anal and genital structures are derived. LB, long bristle; ST, sensilla trichodea.

LITTLEFIELD

(Dorsal

AND BRYANT

Female Genital Disc of Drosophila

anal plate)

(Ventral

melanogaster

(Dorsal

anal plate)

1Ventral

Semmal

129

anal plate)

anal plate )

receptacle

Oviduct Spermatheca

(Parovarlum) Spermatheca

Anterior FIG. 1.

b

130

DEVELOPMENTAL BIOLOGY

duced after transplantation. To determine their regulative capacities, female genital disc fragments were injected into l- to 4-day-old adult females (Ore-RC) where they were cultured for 7 days, and then reinjected into late third instar male larvae for metamorphosis. When culturing the small AR, 02, and 34 fragments, virgin females were used as hosts and they were mated immediately after injection. This prevented the implants from becoming incorporated into the ovary, allowing a much higher frequency of recovery of implants after culturing (J. Haynie, personal communication). The metamorphosed implants were removed in Ringer’s solution, and the cuticular tissue was separated from the soft parts and dehydrated in absolute alcohol. The soft parts were separated from any fat body, stained with 2% aceto-orcein and 0.12% fast green, cleared with absolute alcohol, and mounted with the cuticular tissue in Euparal between coverslips. After staining, the various internal genital structures are distinguishable because of differences in the size and shape of the cells and their associated nuclei (Miller, 1950). Structures were identified in the implants using a compound microscope at 600 X. Observation of wound healing and growth in cultured 02 and 24 fragments. Individual 02 and 24 fragments were photographed immediately after cutting and then implanted into adult hosts. After 1 and 2 days they were removed, photographed, and implanted into new hosts. After 7 days they were removed, photographed, and injected into late third instar larvae for metamorphosis. Structures produced by the female genital disc. The derivatives of the female genital disc have been described by Dobzhansky (1930), Gleichauf (1936), Hadorn and Gloor (1946), and Mindek (1968). More detailed descriptions of the female terminalia including the 8th tergite, and external anal and genital structures, have been published

VOLUME 70, 1979

by Ferris (1950). The internal reproductive system has been described in detail by Miller (1950). The structures are further described below and diagrammed in Figs. 2 and 3. The female genital disc in Drosophila melanogaster gives rise to the analia, including the dorsal and ventral anal plates and the hindgut, the 8th tergite, the external genitalia, and the internal genitalia excluding the ovaries. Externally the genitalia consist of paired chitinous vaginal plates which symmetrically border the vulva. Each plate bears a row of approximately 15 short, thick, thorn bristles with a single long bristle (LB) and three to five sensilla trichodea (ST) at the dorsal end, slightly medial to the thorn bristles. In the dorsal region the vulva bears chitinous papillae whereas in the ventral region it consists of transparent rilled cuticle. Attached to the vulva is the most posterior part of the internal genitalia, the vagina. The vagina constitutes a very small part of the tube which anteriorly becomes the thickened, muscular uterus. Anteriorly the uterus runs into the common oviduct, which splits distally to form the two lateral oviducts which attach to the ovaries in the adult. Attached to the dorsal side of the uterus near the uterus-oviduct junction are four tubes which end in a pair of accessory glands called parovaria and a pair of mushroom-shaped capsules called spermathecue. Ventral to these structures, attached to the uterus, is a compactly coiled tube called the seminal receptacle. The analia consist of two chitinous anal plates, a dorsal one bearing two large bristles and an average of 16 smaller bristles, and a ventral one bearing four large bristles and an average of 15 smaller bristles (Mindek, 1968). The hindgut opens between the dorsal and ventral anal plates. Encircling the anal plates is the lightly chitinized eighth tergite with four or five unpigmented bristles on each side.

LITTLEFIELD

AND BRYANT

Ventral

Dorsal anal plate

Sensilla

Female Genital Disc of Drosophila

melanogaster

131

anal plate

trichodea

Thorn

bristles

FIG. 2. The cuticular derivatives RESULTS

Morphology of the Male and Female Genital Discs The male and female genital discs are bilaterally symmetrical and are located in the 8th larval abdominal segment just anterior to the anus. Each is attached to the larval epithelium by a single stalk located medially at the posterior edge of the disc as shown in the scanning electron micrographs in Fig. 4. The stalks of these discs are quite wide and occupy about one quarter the

of the female genital disc.

width of each disc, making them much larger in proportion to the size of the disc than those of other disc types (Poodry and Schneiderman, 1970; Reinhardt et al., 1977). Histological sections of the female genital disc (Fig. 5a) show that the ventral side consists of a thickened columnar epithelium with adepithelial cells adhering to its ventral surface. The dorsal side is also composed of a columnar epithelium in the posterior half, but toward the anterior it is thinner and resembles the peripodial membrane as described in other imaginal discs

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DEVELOPMENTAL BIOLOGY

VOLUME 70, 1979

Lateral

Common

Seminal

oviduct

,A

oviduct

:\

receptacle. .mathecae

Anal

FIG. 3. The internal

structures

plates

produced from the female genital disc.

LITTLEFIELD

AND BRYANT

Female Genital Disc of Drosophila

melanogaster

FIG. 4. Scanning electron micrographs of the (a) male and (b) female genital discs in (S) which connects the disc to the larval epidermis.

133

situ showing the stalk

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DEVELOPMENTALBIOLOGY

VOLUME 70, 1979

LITTLEFIELD AND BRYANT

Female Genital Disc of Drosophila melanogaster

(Ursprung, 1972). The epithelium is again thickened at the anterior end of the disc, and this region is associated with a few adepithelial cells. Enclosed by the epithelium is the disc lumen. Histological sections of the male genital disc show a similar arrangement of cell types (Fig. 5b). This disc is less flattened than that of the female and contains large folds of tissue. The posterior layer near the stalk consists of columnar epithelium similar to that found in the dorsal side of the female disc. This is joined dorsally to a region of peripodial-membrane-like tissue, which joins a very thick layer of epithelium at the dorsal side. The epithelium in this region is about twice as thick as in the thickest part of the female genital disc, and appears to be multilayered, although this may be pseudostratification (as discussed by Ursprung, 1972). This region also contains a relatively large number of adepithelial cells. The entire anterior side also contains columnar epithelium and adepithelial cells resembling the cells in the ventral region of the female genital disc. The disc epithelium encloses a lumen. Whole Female Genital Discs Subjected to Immediate Metamorphosis Whole female genital discs were injected into late third instar male larvae for immediate metamorphosis in order to ascertain the range of structures produced by transplanted discs and the extent to which the disc may be damaged during the operational procedure. The data presented in Table 1 show that all unpaired structures were present in all implants, and that all paired structures, with the exception of the long bristles of the vaginal plates and parovaria, were present at least once in every metamorphosed implant. In most cases the loss of a structure from one side of the disc occurred in only one implant in the series. The long bristles and parovaria were the most variable of the structures scored and in rare cases were missing entirely. However, in these experiments the complete

135

inventory of structures was present in at least 80% of the implants. In metamorphosed implants from the genital disc the external genital structures and analia are contained in a cuticular vesicle with the bristles facing inward. The hindgut and internal genitalia project out from this cuticular vesicle into the haemocoel of the metamorphosed host, with the ducts of the internal reproductive system in the same relative orientation as in the adult female. One of the lateral oviducts is often found attached to a host testis. Fate Map of the Female Genital Disc The structures produced by immediately metamorphosed fragments are summarized in Tables 1 and 2. In general, complementary pairs of fragments produce complementary sets of structures, indicating that the data are not complicated by any significant regeneration or loss of structures due to cell death caused by the cutting procedure. Complementarity of derivatives was verified when pairs of fragments from single discs were analyzed individually. Structures not present in one of the two complementary fragments were, in most cases, present in the other, although with certain pairs of fragments such as the AC pairs, and in the AB fragment, the number of certain structural elements was consistently higher than expected. This will be discussed later. The fate map derived from our data is shown in Fig. lb. It is, in general, similar to the fate map produced by Hadorn and Gloor (1946). The internal reproductive organs are derived from the anterior portion of the disc whereas the cuticular structures (analia, external genitalia, and 8th tergite), and hindgut are derived from the posterior region nearer the stalk. The presumptive cuticular structures occupy the most lateral positions in the fate map with all the presumptive soft parts at or near the midline. The most anterior internal structure of the adult, the oviduct, occupies the medial region near the anterior edge followed in order by the uterus and the vagina. The

>2

1

Not cultured [n = 181

0.43 0.21

1.83 1.94

was incomplete

0.32 0.14

0.5

0.93

0.05 0.02

1.0 1.0 1.0

0.21

(0.05

0.02 0.41

(1.0)

0.43

1.0

(0.21)

.O .25

0.75 0.04 0.8: 0.58 ,: 0.1;

0.41 0.93 (0.75

0.72

1.94

0.014 0.8: i

Recovered

0.88 0.08

in SO-100% of the implants.

0.21 )

1.0 1.0 1.0

structure

3.46 0.46 3.08 0.92

1.0 1.0 1.0

1.0 1.0 1.0

D.2: I( I.67 3.3$ 1( I.62

1( i (

1

a - f :22

1

C ultured rl2 = IS]

0 0

0.3: 70 .37 O.l( $0 .68

1.0 1.0 1.0

1.0 1.0 1.0

O.l( IO ‘.79

0.2t 50 8.42

0.9:5

O.l( SO

O.l( IO

BA I

.16

was present singly (I), paired (2), as a fused

0.1 0.5

0.3 0.t

1.0 1.0 1.0

1.0 1.0 1.0

1.0 1.0

0.04 ‘0.96

0.3 0.i 0.2 0.i

1.0

0.2 0.f 0.1 0.:

0.73

0.96

1.0 1.0

Q.61L( j.38

1.0

2

LO8

1.0

Culture !d [n = l(

1.0

0.01

Not cultured [n = 261

AC

0.04 0.04

ID.2E

0.29

.02

2 I0.71

0.88

0.12

Cultured [n = 241

(0.28)

0.98

[n = 441

Not cultured

AB

.O

0.21

2

1

I

(0.28)

1

Not cultured [n = 41

B-B’

1.94

2

1

I

Whole disc

(LNumbers in parentheses indicate the structure pair (2f), or more than one pair (>2).

Half dorsal anal plate Half ventral anal plate Hindgut 8th tergite Row of thorn bristles Long bristle Group of sensilla trichodea Dorsal vulva Ventral vulva Seminal receptacle Vagina Uterus Common oviduct Parovarium Spermatheca

T

TABLE 1 FREQUENCIES OF STRUCTURES PRODUCED BY WHOLE FEMALE GENITAL DISCS AND ASYMMETRICAL (VERTICALLY CUT) FRAGMENTS OF THE FEMALE GENITAL DISC SUBJECTED TO IMMEDIATE METAMORPHOSIS (NOT CULTURED) OR CULTURED FOR SEVEN DAYS IN ADULT HOSTS PRIOR TO TRANSFER TO LARVAL HOSTS FOR METAMORPHOSX?

LITTLEFIELD AND BRYANT

Female Genital Disc of Drosophila

spermathecae and parovaria are bilaterally arranged in the anterior part of the disc with the spermathecae located nearest to the anterior edge. The spermathecae must be derived from a position close to the midline as evidenced by the high frequency of pairs of these structures found in implants from lateral halves. Since we did not find an equal number of fragments containing no spermathecae, we conclude that the high incidence of paired spermathecae is not due to cutting mistakes but results from duplication of this primordium before or during metamorphosis of the larval host. Duplication of structures was also found in AB fragments. This effect was most pronounced for the thorn bristles which duplicated 25% of the time in noncultured AB fragments. The ventral anal plates also duplicated occasionally in these fragments, suggesting that these structures originate near the wound edge or are bisected by the cut. This would place the thorn bristles near the B level, the ventral anal plate slightly lateral to this and the 8th tergite and dorsal anal plates farthest from the cut. The external genitalia in the fate map are inverted with respect to the position of the anal plates, as compared to their orientation in the adult. The presumptive dorsal ends of the vaginal plates are positioned toward the anterior end of the disc next to the internal genital anlage with the presumptive ventral ends of the vaginal plates towards the posterior. This is clearly indicated by the presence of dorsal vulva and of sensilla trichodea, marking the dorsal vaginal plate, in derivatives of the 02 fragments and the lower frequency of sensilla trichodea and of dorsal vulva, as compared to ventral vulva, in the 24 fragments. We have obtained nearly complete anal plates from 34 fragments indicating that these structures should be localized in the posteriormost region of the fate map. However, the only fragment which completely excludes the anal plate bristles is 01. The presumptive anal plate region therefore appears to overlap the presumptive genitalia

melanogaster

137

in the fate map, and for this reason it is shown in parentheses in Fig. lb. As previously reported for the male genital disc (Bryant and Hsei, 1977), when a horizontal cut through the female genital disc produced a fragment containing a partial anlage on the left side and a corresponding one on the right side, the derivatives of these anlagen were often found fused together in the metamorphosed implant. For example, the groups of sensilla trichodea were fused in 50% of the 02 fragments which contained them. With the 24 fragments a similar result was obtained for the dorsal region of the vaginal plates, as shown in Fig. 6a. In the 03 fragments the 8th tergite bristle groups were often fused and the 34 fragments showed fusion of the partial anal plates. Although this phenomenon could not, of course, be recognized for median, unpaired structures, it might have been responsible for the abnormal appearance of the seminal receptacle from 02 fragments, which appeared shortened and approximately twice as thick as the corresponding structure from intact genital discs. Regulation in Cultured Female Genital Disc

Fragments

of the

Fragments generated by cuts in the vertical direction were found to undergo either regeneration or duplication in a predictable manner. AC fragments underwent regeneration, restoring the bilaterally symmetrical organization of all the paired structures, as previously shown by Ursprung (1959). Similarly, about 90% of the BA’ fragments regenerated those structures (anal plates, 8th tergite, thorn bristles) which would have been produced by the complementary AB fragments. The AB fragment, in contrast, failed to regenerate medial structures but duplicated the anal plates and 8th tergite bristles in the majority of the implants. Duplication of the thorn bristles was apparently not significantly more frequent after culturing in an adult than when the fragments were metamorphosed immedi-

138

DEVELOPMENTAL BIOLOGY

VOLUME 70, 1979

h! 0

1.0 1.0 1.0

0.22 0.28 0.16

0.3 0.2

0.75 0.25 0.9

0.44 0.22

0.31 0.15 0.1)

0.28 0.44 0.22

0.05 0.17

1.2E

).05

0.05 0.11 was incomplete

).29

0.55 0.44

3.35i

2f

0.31 0.61

1.0 0.95 1.0

0.74 0.26 0.95

0.53 0.31

0.16 0.16 0.16)

0.63)

0.63)

1

T

0.31 0.26

0.21

0.16 (0.W

Recovered

(0.16)

(0.16)

2

Cultured [n = 191

in 90-100% of the implants.

2

-

[n = 201

Not cultured

YO.8) LO5 ‘0.7)

>2

n Numbers in parentheses indicate the structure pair (2f), or more than one pair (>2).

0.29 0.29

0.78 0.17 :o.ll)

2f

f

).05 0.1 0.05

3.23 3.41

3.23 3.35

D.88 cl.12 0.88 D.06 0.53

0.82

).41

2

10.05) 0.78

1

9.18

21

10.05) 0.83

2

Cultured [n = 181

D.18 0.82

1

-

Not cultured [n = 171

0.05

Half dorsal anal plate Half ventral anal plate Hindgut 8th tergite Row of thorn bristles Long bristle Group of sensilla trichodea Dorsal vulva Ventral vulva Seminal receptacle Vagina Uterus Common oviduct Parovarium Spermatheca

!

24

structure

0.64)

>2

0.07

0.13 0.07

0.07 0.27

0.27

T [n = 151

Cultured

was present singly (l), paired (2), as a fused

0.07 0.07

0.07

0.07 0.07

(0.36)

(0.36)

2f

Not cultured [n = 141

34

z % 2

FIG. 6. Female genital structures from (a) immediately metamorphosed and (b) cultured 24 fragments showing no growth and (c) immediately metamorphosed and (d) cultured AB fragment showing duplication of thorn bristles and 8th tergites in the cultured fragment with (e) an immediately metamorphosed whole disc for comparison. TB, thorn bristles; LB, long bristle; AP, anal plate; 8T, 8th tergite.

LITTLEFIELD

AND BRYANT

Female Genital Disc of Drosophila

ately, but this is at least partly due to the difficulty of scoring duplications in these implants which produced only a small part of the thorn bristle pattern. In implants with more than three thorn bristles, duplication could often be recognized by the presence of separated groups of bristles (Fig. 6d). A significant number of AB implants contained incomplete spermathecal capsules indicating that the primordia for these structures must extend into the lateral parts of the disc. Similar results were not obtained for the parovaria, probably because of the difficulty in recognizing a partial parovarium without the advantage of identifiable cuticular tissue as seen in spermathecae. Some bilaterally symmetrical fragments showed no detectable regulation when cultured in adult hosts. For example, there was no significant difference between the derivatives of cultured and noncultured 01 fragments except for a slight increase in paired parovaria. There was also no detectable regulation in the 02 and 03 fragments. In fact, cultured 03 fragments appeared to lose certain presumptive structures during the culture period, especially the 8th tergite, accessory glands, and spermathecae. The complementary 14, 24, and 34 fragments, however, underwent a certain amount of regeneration during culture. The 24 fragment regenerated the seminal receptacle, vagina, uterus, and oviduct in approximately 20% of the implants and the more distant internal structures, the parovaria and spermathecae, in lo-15% of the implants. In the 14 fragments, parovaria and spermathecae were regenerated in 50-60% of the implants, whereas in the 34 fragment regulation often resulted in the duplication of structures. In one cultured 34 implant there was regeneration of some soft parts, including two spermathecae. Cultured 34 fragments also displayed a high frequency of thorn bristles not found in the uncultured fragments.

141

melanogaster

Wound Healing and Growth 02 and 24 Fragments

in Cultured

Photographs of a 24 fragment immediately after cutting and of the same implant after 1, 2, and 7 days of culture are shown in Fig. 7. The fragment appears to have healed right to left, maintaining this configuration throughout the entire culture period. All of the implants followed in this manner appeared to heal in the same way, and upon metamorphosis none showed any evidence of regeneration. DISCUSSION

Fate Map of the Female Genital Disc Structures produced by the female genital disc are derived from specific regions as shown in Fig. lb. The posterior region, nearest the stalk, gives rise to the anal plates, hindgut, and 8th tergite, the midposterior region gives rise to the vaginal plates and associated ventral and dorsal portions of the vulva, and the anterior region gives rise to the internal genital structures. The internal genitalia are organized in the disc in approximately the same anterior-posterior sequence as in the adult. All unpaired structures are located at the midline and all paired structures are arranged bilaterally. Earlier fate maps of the female genital disc were constructed by Hadorn and Gloor (1946) who studied metamorphosed fragments of the disc, and by Ursprung (1957, 19591, who analyzed defects produced by ultraviolet irradiation of specific regions of the disc. In the fate map proposed by Hadorn and Gloor the anal plates, external genitalia, and internal genitalia were localized in approximately the same regions of the disc as our studies have indicated, but the organization of structures within these areas was not clearly established. Ursprung was able to map some of the internal genital structures within these regions in more detail, and concluded that the parovaria originated from bilateral anlagen but that the

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DEVELOPMENTAL BIOLOGY

VOLUME 70, 1979

b

d FIG. 7. Photographs of a 24 fragment of the female genital disc (a) immediately fragment after (b) 1, (c) 2, and (d) 7 days of culture in an adult host.

spermathecae originated in the anterior medial region of the disc. From the results of immediately metamorphosed 01 and AC fragments presented here it is clear that the spermathecal primordium lies at or very near the midline in the anteriormost region of the disc. However, normal spermathecae were occasionally produced by immediately metamorphosed lateral (AB) fragments, indicating that the primordia for these structures extend to the lateral regions of the disc and are bilaterally organized. When sagittal halves of the female genital disc were subjected to immediate metamorphosis, they produced one-half of the normal derivatives of the disc, but in many cases a second spermatheca was present. On the basis of results similar to these, Hadorn and Chen (1956) concluded the spermathecal anlage possessed a high capacity for regulation and proposed a model to account for this regulation based on the

after cutting and the same

observation by Graber (1949) that both spermathecae developed from an unpaired median anlage. In our experiments, we found that immediately metamorphosed sagittal halves (AC pieces) give rise to either one or two spermathecae, but never produced partial spermathecae as would be expected if the cut had bisected a median anlage. Cultured AB pieces did, however, produce partial spermathecae as would be expected if the spermathecal anlagen were bilateral. The high frequency of paired spermathecae in immediately metamorphosed sagittal halves therefore probably results from duplication of this primordium during the short period (less than 18 hr) between injection of the fragment into the larva and the subsequent metamorphosis of the host. A high frequency of pairs, similar to that found for spermathecae in sagittal halves was observed for groups of thorn and anal

LITTLEFIELD

AND BRYANT

Female Genital Disc of Drosophila

plate bristles from lateral (AB) fragments of the disc. Yet these structures are clearly bilateral in origin since they are produced singly from sagittal half-discs. This indicates that duplication can occur relatively quickly in those structures derived from cells at or near the wound site in these fragments and supports the proposal by Ursprung (1959), Luond (1961), and Schubiger (1971) that regions nearest the wound site are the first to regulate when there is an opportunity for growth. Although we have shown the genital disc fate map as a two-dimensional representation, it seems likely that in this imaginal disc both dorsal and ventral epithelia might contribute to adult structures. In this case, our map would be a two-dimensional projection of the layout of anlagen in the threedimensional disc. The proposed three-dimensional organization of the female genital disc is shown in Fig. 5c. Several pieces of evidence support the arrangement shown: First, histological sections show that both dorsal and ventral epithelia consist for the most part of columnar cells, the squamous cells which are usually designated “peripodial membrane” in other discs and which are thought not to produce adult structures (Poodry and Schneiderman, 1970). being restricted to a small anterior dorsal region. Second, if one surface of the disc produced the anal plates, while the other produced the genital structures, this would explain why anal plate bristles were found in implants derived from all fragments except the anterior quarter of the disc. Third, the three-dimensional organization can explain why the orientation of the vaginal plates is reversed in our map as compared to their orientation in the adult. In the first leg disc (Poodry and Schneiderman, 1970), the disc epithelium everts through the stalk which widens during metamorphosis. If the same mechanism were to occur in the genital discs, the anal plates would move out from the dorsal side of the opening whereas the external genitalia would evert from the ventral side such that

melanogaster

143

the vaginal plate region nearest the anal plates in the fate map (and nearest the stalk in the disc) would assume the most ventral final position. This would result in a reversal of the dorso-ventral polarity of the genitalia relative to the anal plates, compared to that shown in the two-dimensional fate map. Fourth, Dubendorfer (1977) has shown that the anal plates and parovaria are clonally isolated from the external and other internal genitalia at the blastoderm stage and that this isolation persists throughout the embryonic and larval life of the animal. Clonal isolation can be due to physical isolation, so that Dubendorfer’s results are consistent with the view that the anal plates, and probably the parovaria, are derived from the dorsal epithelial disc tissue whereas the 8th tergites, vaginal plates, and remaining internal genital structures are derived from the disc epithelium on the ventral side. This might further suggest that some property of the disc edge prohibits clones from overlapping the dorsal and ventral epithelia. An alternative explanation is that these primordia arise from separate regions in the blastoderm as suggested by Nothiger et al. (1977). Fifth, Emmert (1972) has shown that in the female genital disc of Calliphora the anal plates are derived from the dorsal side of the disc, whereas most of the genital structures are derived from the ventral side. Sixth, the male genital disc is also apparently organized in a manner similar to that proposed for the female. Ehrensperger and Nothiger (personal communication) constructed a three-dimensional fate map for the male genital disc in which the anal plates and hindgut are mapped to a region near the stalk on the posterior side of the disc and the external genitalia to the anterior side, opposite the anal plates. The similarities in the general organization of structures within the two discs further support the proposed three-dimensional organization of the female genital disc. A comparison of the proposed general arrangement of genital and anal structures in the male and female

144

DEVELOPMENTALBIOLOGY

genital disc is diagrammed d. Regulation ments

in Figs. 5c and

of Female Genital

Disc Frag-

In a recently proposed model for epimorphic regulation in amphibian and insect appendages and imaginal discs (French et al., 1976) it was suggested that cells acquire positional information in a two-dimensional array based on a system of polar coordinates. According to this model the position of each cell is determined by a value around the circumference of a circle represented numerically as 1-12 and by a radial value represented by a series of concentric circles at levels A through E. For the imaginal wing disc, the outermost circle (A) represents the disc edge, and the central point (E) the presumptive distal tip of the wing. Regulation within such epimorphic fields is assumed to be governed by two rules: The first rule is that when normally nonadjacent positional values along the circular sequence (l-12) are confronted, growth occurs to intercalate all the intermediate values using the shorter of the two possible routes (shortest intercalation rule). This accounts for many cases of regeneration and duplication in segments and sectors of imaginal discs, when it is recognized that soon after such a fragment is produced, the cut edge is eliminated by wound healing (Reinhardt et al., 1977) which brings into contact positional values from different parts of the disc. In general, if a segment or sector contains more than half of the positional values in the circular sequence it will regenerate, whereas fragments with less than half will duplicate. The radial sequence of positional values is also assumed to show intercalation, but of course in this case the question of alternative routes does not arise. The second rule proposed in this model is that when an entire circular sequence is either exposed by amputation or generated by intercalation at some proximal level, this results in distal transformation from that

VOLUME 70, 1979

level, generating all of the more distal positional values within the field. This feature of the model allows it to account for the duplication of a central square of the wing disc, and regeneration of the complementary annulus (French et al., 1976). The polar coordinate model effectively accounts for much of the pattern of regulation which is seen in asymmetrical fields such as in the imaginal wing (Bryant, 1975; Haynie and Bryant, 1976) and first leg (Schubiger, 1971; Strub, 1977b) discs of Drosophila. There are several ways of adapting the model to cases of symmetrical fields such as the genital discs. For example, Bryant and Hsei (1977) modified the circular sequence itself to be bilaterally symmetrical, and this led to the prediction that bilaterally symmetrical fragments produced by cuts across the axis of symmetry of the disc would not regulate. This is based on the assumption that these fragments would heal left to right, confronting identical positional values from right and left sides. This confrontation of identical values would represent a stable situation and would not lead to stimulation of growth of the fragment, so no regeneration or duplication would occur. This two-dimensional bilaterally symmetrical model was sufficient to account for the absence of detectable growth in most bilaterally symmetrical fragments of the male genital disc. It was based on the implicit assumption that the genital disc is organized as are the leg and wing imaginal discs, with a columnar epithelium on one side and a thin peripodial membrane on the other, with only the former producing adult derivatives. However, the analysis of the female and male genital discs presented here indicates that in these discs both layers of epithelium might produce adult derivatives and therefore might carry positional information. For this reason we propose a different adaptation of the polar coordinate model to include both layers of the disc and to account for the variability in modes of regulation displayed by bilaterally symmetrical fragments of the

LITTLEFIELD

AND BRYANT

of Drosophila

Female Genital Disc

145

melanogaster

0a VERTICAL

CUT

/

\ C’ I2 D’ II

2

IO 3 8

9

E’

8

0

b

J

DISTAL TRANSFORMAT

4

0

7 5

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s

‘ION=

REGENERATION

DUPLICATION c’

(El I

E’

0 C

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E’)

0e

FIG. 8. Polar coordinate model specifying position in a bilaterally symmetrical field. (a) Following a cut in the vertical direction, parallel to the axis of symmetry, regulation proceeds by the rule for distal transformation with the formation of the more distal radial values from the proximal wound surface. A cut at level C’ leads to regeneration of D’E’ in the larger fragment (b-c) and duplication of D’E’ in the smaller fragment (d-e).

female genital disc. This model is presented in Fig. 8 and 9 for fragments generated by vertical and horizontal cuts, respectively. Although the field is represented in these diagrams as a three-dimensional spheroid, the position of each cell within the field is still determined by the two-dimensional,

polar coordinate system operating within the single-cell layer of the disc epithelium. We suggest that the genital disc can be represented as two polar coordinate fields fused together at their proximal boundaries in the mid-sagittal plane, with more “distal” levels in the field corresponding to more

GROWTH

\

INTERCALATION= REGENERATION

DUPLICATION

(El

WOUND HEALS FRONT TO BACK

INTERCALATION=

WOUND HEALS FRONT TO BACK

CUT

(E)

\

I

1

E’

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NO

WOUND HEALS LEFT TO RIGHT

GROWTH

A

the

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.II~IIPE

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FIG. 9. Polar coordinate model specifying position in a bilaterally symmetrical field. (a) Following a cut in the horizontal direction, transecting the atis of symmetry, the mode of regulation is dependent upon the mode of wound healing of the fragment. Wound healing from right to left leads to the confrontation of cells having identical positional values and no stimulation of growth (b-c and f-i). Wound healing from front to back leads to regulation which proceeds by intercalation along the shortest route. Fragments having fewer than one-half the circular values (d-e) duplicate whereas fragments having greater than one-half

NO

/

WOUND HEALS LEFT TO RIGHT

HORIZONTAL

LITTLEFIELD

AND BRYANT

Female Genital Disc

of Drosophila

melanogaster

147

sible for the observed variability in regulalateral parts of the disc. Fragments genertive behavior of some fragments (especially ated by a cut parallel to the axis of symmetry (vertical cut) regulate according to the 24 fragment) of the female genital disc. the rule for distal transformation (Figs. 8b The more consistent lack of regulation shown by other fragments such as 02 may and d). In both fragments, a complete circle be due to a more consistent mode of healing is exposed by the cut, and distal transformation will result in reestablishment of all in these fragments. of the more distal (=lateral) radial values. This work was supported by Grant No. HD06082 When the field is bisected mid-sagittally (as from the National Institutes of Health. We thank in the AC fragment of the genital disc), Becky Hsei for technical assistance and Maureen Kildistal transformation ensues from level A lacky for artwork. in both fragments regenerating levels B-E REFERENCES and reestablishing the bilateral symmetry BRYANT, P. J. (1975). Pattern formation in the imagiof the field. Bisection of the field at level C nal wing disc of Drosophila melanogaster: Fate map, regeneration and duplication. J. Exp. 2001. also results in distal transformation, which 193,49-78. corresponds to regeneration of structures in BRYANT, P. J., ADLER, P. N., DURANCEAU, C., FAIN, the larger fragment and duplication in the M. J., GLENN, S., HSEI, B., JAMES, A. A., LITTLEsmaller fragment. These predictions are in FIELD, C. L., REINHARDT, C. A., STRUB, S., and agreement with the observed regulative beSCHNEIDERMAN, H. A. (1978). Regulative interactions between cells from different imaginal discs of havior of equivalent fragments of both feDrosophila melanogaster. Science 201,928-930. male and male genital discs, except that a certain amount of regeneration is seen in BRYANT, P. J., and HSEI, B. W. (1977). Pattern formation in asymmetrical and symmetrical imaginal duplicating lateral fragments of the male discs of Drosophila melanogaster. Amer. Zool. 17, genital disc (Luond, 1961). 595-611. DOBZHANSKY, TH. (1930). Studies on the intersexes The regulative behavior of fragments and supersexes in Drosophila melanogaster. Bull. generated by cuts across the axis of symBureau Genet. 8,91-158. metry of the field is less predictable in that D~~BENDORFER, K. (1977). “Die Entwicklung der this behavior is dependent on how the mannlichen und weiblichen Genital-Imaginalwound surfaces heal after cutting. The scheibe von Drosophila melanogaster: Eine klonale Analyse.” Ph.D. Thesis, University of Zurich. three-dimensional organization of the tisEMMERT, W. (1972). Experimente zur Bestimmung des sue allows two alternative modes of healAnlageplans der mannlichen und der weiblichen ing with different developmental conseGenital-Imaginalscheibe von Calliphora (Insecta, quences. One possibility is for the bilatDiptera). Wilhelm Roux Arch. Entwicklungsmech. erally symmetrical fragment to heal left to Organismen. 171, 109-120. right, confronting cells with similar posi- EPHRUSSI, B., and BEADLE, G. W. 1936). A technique of transplantation for Drosophila. Amer. Natur. 70, tional values and resulting in no regulation 218-225. (Figs. 9c and i). Alternatively, the tissue FERRIS, G. F. (1950). External morphology of the can heal “front to back” (dorsal to ventral adult. In “Biology of Drosophila” (M. Demerec, or anterior to posterior) confronting cells ed.), pp. 409-412. Hafner, New York. with different positional values in the cir- FRENCH, V., BRYANT, P. J., and BRYANT, S. V. (1976). Pattern regulation in epimorphic fields. Science 183, cular sequence. In this case the shortest 969-981. intercalation rule would apply; fragments GLEICHAUF, R. (1936). Anatomie und Variabilitat des containing more than half of the circular Geschlechtsapparates von Drosophila melanogaster (Meigen). Z. Wiss. Zool. 148, l-66. sequence of positional values would regenerate the missing parts (Fig. 9g), whereas GRABER, H. (1949). Genetische, entwicklungsphysiologische und morphogenetische Untersuchungen an fragments containing fewer than half the der Mutante “spermatheca” (Spt) von Drosophila positional values would duplicate (Fig. 9d). melanogaster. Z. Vererbungslehre 83.106-135. We suggest, therefore, that alternative HADORN, E., and CHEN, P. S. (1956). Die Feldorganimodes of wound healing might be responsation der Spermatheken Anlage bei Drosophila

148 melanogaster. HADORN, E.,

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and GLOOR, H. (1946). Transplantationen zur Bestimmung des Anlagemusters in der weiblichen Genital-Imaginalscheibe von Dro-

sophila. Rev. Suisse Zool. 53,495~501. HAYNIE, J., and BRYANT, P. J. (1976). Intercalary regeneration in imaginal wing disk of Drosophila melanogaster. Nature (London) 259,659-662.

Lijo~~, H. (1961). Untersuchungen zur Mustergliederung in fragmentierten Primordien des mannlichen Geschlechtsapparates von Drosophila seguyi. Develop. Biol. 3, 615-656. MILLER, A. (1950). The internal reproductive systems of male and female. In “Biology of Drosophila” (M.

Demerec, ed.), pp. 518-530. Hafner, New York. MINDEK, G. (1968). Proliferations-und Transdetermi-

nationsleistungen der weiblichen Genital-Imaginalscheiben von Drosophila melanogaster nach Kultur in vivo. Wilhelm Roux Arch. Entwicklungsmech. Organismen 161,249-280.

N~THIGER, R., D~BENDORFER, A., and EPPER, F. (1977). Gynandromorphs reveal two separate primordia for male and female genitalia in Drosophila melanogaster. Wilhelm Roux Arch. Entwicklungsmech. Organismen 181, 367-373. POODRY, C. A., and SCHNEIDERMAN,H. A. (1970). The ultrastructure of the developing leg of Drosophila melanogaster. Wilhelm Roux Arch. Entwicklungsmech. Organismen. 166, l-44.

REINHARDT, C., HODGKIN, N., and BRYANT, P. J. (1977). Wound healing in the imaginal discs of Drosophila. I. Scanning electron microscopy of normal and healing wing discs. Develop. Biol. 60, 238-257.

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SCHUBIGER, G. (1971). Regeneration, duplication and

transdetermination in fragments of the leg disc of Drosophila. Develop. Biol. 26, 277-295. STRUB, S. (1977a). Pattern regulation and transdetermination in Drosophila imaginal leg disc reaggregates. Nature (London) 269,688-691.

STRUB, S. (1977b). Developmental potential of the cells of the male foreleg disc of Drosophila. I. Pattern regulation in intact fragments. Wilhelm Roux Arch. Entwicklungsmech. Organismen. 181, 309320. URSPRUNG, H. (1957). Untersuchungen zum Anlagemuster der weiblichen Genitalscheibe von Drosophila melanogaster durch UV Strahlenstich. Rev. Suisse Zool. 64,303-311.

URSPRUNG,H. (1959). Fragmentierungs-und Bestrahlungsversuche zur Bestimmung von Determinationszustand und Anlageplan der Genitalscheiben von Drosophila melanogaster. Wilhelm Roux Arch. Entwicklungsmech. Organismen. 151, 504-558. URSPRUNG, H. (1972). The fine structure of imaginal discs. In “Biology of Imaginal Discs” (H. Ursprung

and R. Nothiger, eds.), pp. 93-107. Springer, New York. WILCOX, M., and SMITH, R. J. (1977). Regenerative interactions between Drosophila imaginal discs of different types. Develop. Biol. 60, 287-297. WOLPERT, L. (1969). Positional information and the spatial pattern of cellular differentiation. J. Theoret. Biol. 25, l-47. WOLPERT, L. (1971). Positional information and pattern formation. Curr. Top. Develop. Biol. 6, 183224.