Compartments in the wing of Drosophila: A study of the engrailed gene

DEVELOPMENTAL

BIOLOGY

50, 321-337 (1976)

Compartments

in the Wing of Drosophila: of the engrailed Gene P. A. LAWRENCE

MRC

Laboratory

of Molecular

Biology,

Accepted

A Study

AND G. MORATA

Hills

Road,

December

Cambridge,

CB2 2QH,

England

15,197s

It has recently been suggested that the wildtype alleles of homeotic genes are responsible for controlling the development of compartments. Because the mutation engrailed gives the posterior wing compartment anterior characteristics, it can be regarded as such a homeotic gene. Our experiments confirm the role of the engrailed gene in development of the posterior wing compartment, results which strongly support and extend the compartment hypothesis. Clonal analysis reveals that the state of the engrailed gene is immaterial to the entire anterior compartment, and crucial to the normal development of the posterior compartment, where it controls the pattern of veins and bristles. The presence of a straight and precisely positioned compartment border is dependent on the activity of the engrailed gene until late in development. We suggest that this is due to the gene’s effects on cell affinities of the posterior compartment. The engrailed mutation increases the size and changes the shape of the posterior compartment. engrailed clones cause local wing enlargement only if they are dorsal and include the posterior margin of the wing. Wildtype cells outside the clone contribute to this change of shape. This result suggests that the postero-dorsal margin is primarily responsible for the control of shape, and that the ventral compartment is, to some extent, modeled on the dorsal. INTRODUCTION

A recent series of discoveries has strongly suggested that adult insects are subdivided into compartments (GarciaBellido, Ripoll, and Morata, 1973, 1976). Each compartment, which consists of a precisely defined region on the surface of the adult, begins existence as a small group of founder cells all of whose descendents populate the compartment. Just as the descendents of one cell constitute a clone, so the descendents of a group of cells constitute a polyclone (Crick and Lawrence, 1975). A compartment is therefore constructed by an entire polyclone. GarciaBellido et al. also showed that a polyclone (that, for example, destined to form the entire anterior wing compartment) can later become subdivided into two polyclones (one forming the dorsal, and one the ventral part of the anterior wing). This process may be reiterated several times. It has been suggested that each compartment is uniquely specified by the com321 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

bination of a small number of controlling genes, Garcia-Bellido, genes (selector 1975) active within it. One hypothesis (Garcia-Bellido, 1975; Morata and Lawrence, 1975) is that when a group of cells is split into two polyclones, each of which will form a compartment, a selector gene is permanently activated in one polyclone and inactivated in the other. The activity of that selector gene constitutes the only determining difference between the two polyclones and the compartments they each generate. This process can be repeated a number of times so that after further subdivisions several different compartments are formed, each specified by the binary coding of which selector genes are active and which are inactive within it. This hypothesis still lacks strong experimental support and there remain some important questions arising from it: we do not know how much the final form of a compartment depends on interaction with cells outside it. We do not know whether

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DEVELOPMENTAL BIOLOGY

all aspects of compartment development are under control of the selector genes. For example, there is the hypothesis that compartments are units for the control of shape and size, as well as pattern (Crick and Lawrence, 1975). Are all these properties controlled by the same selector genes? We have shown (Morata and Lawrence, 1975) that one of the roles of the engrailed gene is to instruct the posterior wing cells so that they do not mix with the cells of the neighboring anterior compartment. There is therefore the possibility that each active selector gene may express itself in a cell surface “label.” It is within this conceptual framework that we have investigated the role of the engrailed gene in wing development, that is the extent to which it is responsible for (i) the mixing behavior of wing cells, (ii) the characteristic cell-types and their arrangement into the pattern appropriate to the compartment, and (iii) the shape and size of the compartment. Some of our investigations have been helped by the temperature sensitivity of engrailed; we have found that while the phenotype is strong at 25°C; at 30°C en/en flies are almost wildtype. Our results show that the engrailed gene is critically involved in all these characteristics and to some extent illuminate the degree to which different compartments must interact to generate pattern and form. METHODS

Our basic technique has depended on the ability of X-rays to cause somatic recombination; this produces clones of homozygous cells in heterozygous individuals. In two cases we have used the Minute technique (Morata and Ripoll, 1975) in which, as a consequence of a single recombination event, a cell is simultaneously made homozygous for a cell marker mutant and for the wildtype allele of a Minute gene. Such a cell, because of the advantage it has over its neighbors, which are

VOLUME 50, 1976

heterozygous for the Minute allele, grows rapidly and its progeny frequently fill almost the whole compartment, defining the compartment boundary for hundreds, or even thousands, of cells. An additional advantage of this technique is that there is an increase in clone frequency over that found in Minute+ flies (Ferrus, 1975). The irradiated flies of interest were as follows: (i) en/en; mwh jut+. In engrailed flies clones of mwh jv cells are produced by somatic recombination (Fig. 1). C3” II e” mwh

,v

+

+

III:

FIGURE 1

(ii) enlen; M(3)iJ”lmwh jv. In engrailed Minute flies, clones of cells that are mwh jv and homozygous Minute+ are produced by somatic recombination (Fig. 2). C?” e”

II mwh

ry

+

III: +

+ tvK3h55 FIGURE 2

(iii) pwn enlM(2)c”“a; mwh jut+. These flies are Minute and because they are heterozygous for engrailed (en./+) have normal wings. Two kinds of clones are found: (a) recombination in the right arm of the second chromosome UIR) gives clones which are homozygous for engrailed and the marker, pawn, as well as being homozygous for the Minute+ allele; (b) recombination in the left arm of the third chromosome (IIIL) gives clones homozygous for mwh and jv, which remain heterozygous for the Minute (Fig. 3). Pw” en + +

II rnwh m

: +

1.v +

FIGURE 3

+ Mhc33a

LAWRENCE AND MORATA

Wing Compartments

(iv)pwn en./+-; M(3)P/mwh jv. The phenotype of these flies is similar to that of class (iii) above, but in this case the Minute is located in the third chromosome. Two kinds of clones are found in these flies: (a) recombination in the IIR produces clones homozygous for both engrailed and pawn; the Minute condition is unaffected; (b) recombination in the IIIL produces mwh jvlmwh jv clones, about 60% of which will be homozygous for Minute+ (Morata and Ripoll, 1975) (Fig. 4). pwn en

II -h lu: +

+ + 1.v : ; tv&~ FIGURE 4

All mutants used are described in Lindsley and Grell (1968) with the exceptions of pawn (2-58) (Garcia-Bellido and Dapena, 1974) and the Minute, M(3)i”’ (Garcia-Bellido, Ripoll, and Morata, 1973). pawn is a new cell marker which when homozygous, labels wing cells with a fine hair which has a distinctive basal spur and can be identified as single trichomes in the wing blade; pwnlpwn bristles are also truncated. The parents were kept in bottles for 24 hr and the larvae irradiated at different times thereafter (220 kV at 15 mA, l-mm aluminium filter, distance of 5 cm, rate 500 R/min), times being given as number of hours after egg laying (AEL). The irradiated adults were collected and the wings and thoraces, or wings alone, were mounted on slides for screening under the light microscope. For the temperature-shift experiments the flies were moved from 25 ? 0.5”C to 30.5 ? 0.5”C; newly formed pupae were collected at different times either before or after the shift. Times of the shift are therefore given as before puparium formation (BPF) or after puparium formation (APF). The cell density within a clone (say, dorsal) and in the corresponding area on the

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323

other surface of the wing (say, ventral) was measured as follows. Using a Zeiss drawing apparatus a convenient sample area was drawn within the clone, and all the trichomes in that area were counted. The microscope was then focussed onto the other wing surface and counts were made of the number of trichomes falling within the same area (which could still be seen through the drawing apparatus). The sample sizes were usually about 50 cells, the estimates being based on 20-30 sample areas. Clone sizes were measured by drawing their perimeter with the camera lucida on paper, cutting out the shape, and weighing the paper. Owing to the exponential growth of the cells, the clone sizes take up a skewed distribution. The clone sizes were therefore transformed into logs, so that normal statistical methods could be used. The cell numbers in the clones were estimated from their area, assuming an entire wing blade contains some 32,000 cells (Garcia-Bellido and Merriam, 1971a). RESULTS

The engrailed gene was chosen because of the work of Garcia-Bellido and Santamaria (1972) who showed that by a number of criteria the phenotype could best be described as a partial transformation of posterior parts of the wing into anterior ones. The discovery of the antero-posterior compartmentalization of the wing immediately suggested that the engrailed gene was a selector gene (Garcia-Bellido, Rip011 and Morata 1973), the en+ allele apparently being switched on in the posterior compartment only and controlling its development (Morata and Lawrence, 1975). Our experiments have attempted to describe the different aspects of the compartment development, and the extent to which they are all under the control of this single gene. engrailed and the Compartment Border A characteristic of the wing is the straightness of the edges of clones where

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DEVELOPMENTAL BIOLOGY

VOLUME 50, 1976

they coincide with the line separating the frequently crossed over where the line two neighboring compartments, although would normally be (Fig. 7). Although most any other clonal boundary is usually un- clones could be classified as either mainly even. One explanation for this is that the anterior, or mainly posterior, when they cells have different mixing properties, reached the boundary they did not form a cells of different compartments minimisstraight edge. enlen; M(3)Plmwh jv (age irradiated 60 ing their mutual contact (Lawrence and Green, 1975; Morata and Lawrence, 1975). ? 12, 84 ? 12 hr AEL). The size of the We have suggested that the mixing prop- posterior part of the wing is grossly enerties of the posterior polyclone may be larged (Fig. 8) and few emerge from the controlled by the engrailed gene. If the puparia. This interesting interaction between engrailed and Minute is discussed cells of the posterior polyclone in engrailed later (p. 331). wings are at least partially transformed The viability of these flies is normally into anterior ones with respect to pattern, they might also be expected to share ante- extremely low and is reduced to unworkable levels by the X-irradiation. Because of rior mixing properties. If so, they should the small number of survivors, few clones no longer form a straight compartment were found; some of these were homozyborder when they confront anterior cells. gous for Minute+ and covered a large area We have therefore examined the straightof the wing. None defined a straight line in ness of the compartment border in enthe region where the antero-posterior comgrailed wings by marking clones of cells to see how they behave in the vicinity of the partment boundary is found in en/+ flies; of seven clones three crossed the presumpborder. tive borderline. An example is shown in (i) Compartment border in engrailed wings. We have looked at clones in two Fig. 9. 30°C Culture (enlen; M(31i”“lmwh jvl. kinds of engrailed flies: enlen; mwh jvl+ At this temperature the viability is better (age irradiated 36 * 12, 60 k 12 hr AEL). and the structure of the wings approaches The normal position of the compartment having an almost boundary is shown in Fig. 5, which is a that of the wildtype, wing heterozygous for engrailed. Even perfectly normal vein pattern and very few bristles in the posterior part. though the posterior wing pattern is al- triple-row tered in en/en flies, the presumptive posi- Coordinate with this, clones now respect a border. At tion of the compartment boundary can be nearly normal compartment 30°C no clones crossed more than a few cell determined by reference to the anterior whereas at 25°C posterior diameters, region which is almost completely normal (Fig. 6). However, in en/en flies, clones clones reached vein III in several cases. A

FIG. 5. Diagram of a normal (en/+) wing. The dotted line represents the boundary between rior (A) and posterior (P) compartments. Note the presence of sensilla on vein III and the first vein (CVl). CO, costa; CV2, second crossvein; DR, double row; TR, triple row.

antecross-

LAWRENCE

AND MORATA

Wing Compartments

and Engrailed

325

TR’ FIG. 6. Diagram of an en/en wing. The posterior margin shows the presence of bristles (TR’) typical of the anterior margin (TR). Note that a vein (III’), in a similar position to vein IV in wildtype wings, bears sensilla.

FIG. 7. Scale diagram of a large mwh clone induced at 36 -t 12 hr AEL in an engrailed wing. The clone crosses the line where the antero-posterior boundary is in normal wings. / / / /, dorsal territory of clone; \\\\, ventral territory. Note the deformation of the first crossvein. subjective comparison of the straightness of the edge of clones when they reached the

border area, shows that their borders are not so straight as in clones made in enl+ flies. (ii) Local dependence of the compartment border on the engrailed gene. The previous experiments show that in engrailed wings at 25°C the compartment border is indefinable. We would like to know whether this is due to local action of the engrailed gene. To examine this we have constructed chromosomes which allow us to generate and identify engrailed clones in an otherwise wildtype (en/+) wing. We have used the Minute technique (Morata and Ripoll, 1975) so that the marked engrailed clones grow excessively. The flies used werezpwn enlM(2)Pa; mwh jv/+.

Because

of the

chromosome all pawn

gene

order

on the

clones are also en-

grailed and homozygous for M(2)c+ (apart from the unlikely event of a double cross over). Because engrailed is distal to pawn occasional engrailed clones that were pwn+ were found. Due to the very low survival of these flies at 3O”C, we were limited to studies of 25°C cultures. Following irradiation at 36 r 12 AEL, the clones were frequently very large and could fill over half the wing. The clones fell into two distinct classes: (i) those that were exclusively anterior, had no effect on the pattern of the wing, and if they reached the central area defined a straight and normally

positioned

antero-posterior

compart-

ment boundary (Fig. 10); and (ii) posterior clones which showed the engrailed phenotype and when they included the center of the wing failed to define a straight boundary and frequently crossed into territory normally occupied by anterior cells (Fig.

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DEVELOPMENTAL BIOLOGY

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FIG. 8. The phenotype of en/en; Mf3P/mwh ju at 25°C. The size of the posterior much enlarged, while the anterior is more or less normal.

compartment

is

FIG. 9. A large mwh M(3);’ clone induced at 60 2 12 hr AEL in a en/en; M(3)Plmwh jv fly. The clone crosses the line where the antero-posterior compartment boundary is in normal wings. / / / /, dorsal shading; \\\\, ventral shading. The other wing on this fly was of normal engrailed Minute phenotype (cf., Fig. 8).

FIG. 10. Drawing of a dorsalpwn en M(2)c+ clone induced in the anterior compartment of a fly of genotype pwn enIM(Z) Where it reaches the antero-posterior compartment border it defines it.

11). Later irradiations (60 k 12, 84 -+ 12 hr AEL) resulted in clones that were subsets of the above: either small anterior clones which, if they reached the border area, de-

fined a straight boundary in the normal place or posterior ones which, even if small, could cross the border and penetrate into anterior territory. The mwh control clones behaved normally; that is, they respected the compartment border. The ability of engrailed Minute+ cells to cross the boundary might be due to their growth advantage over their neighbors. We therefore examined clones in pwn en/ +; mwh jvl+ flies, and found that posterior pawn clones frequently crossed the boundary, while anterior pawn as well as anterior and posterior mwh clones never did so.

LAWRENCE AND MORATA

Wing Compartments

327

and Engrailed

FIG. 11. Drawing of a pwn en M(Z)c+ clone of posterior origin induced in a fly of genotype pwn en/ M(~)c~~. The clone eliminates the antero-posterior boundary and invades the anterior compartment. Where en+ cells remain they form normal wildtype pattern (*). Cells belonging to the clone do not make normal anterior structures, the veins are deformed, and vein II is almost eliminated by the ventral part of the clone that extends beyond it (arrow).

In flies of the genotype pwn en/+; M(3)i”,ilmwh jv (irradiated at 36 * 12 and 60 * 12 hr AEL), this point is made more forcibly, for here large mwh jv clones are frequently found and often fill the anterior or the posterior compartment. They never cross the boundary and frequently define it; it follows that the ability of posterior cells to cross the boundary must be due to the engrailed mutation. It is clear from all the results in this section that the formation of a normal compartment border at 25°C is locally dependent on the activity of an en+ allele in the posterior border cells, while its absence is immaterial to the anterior cells. engrailed

b

FIG. 12. Diagram of distal veins in normal (a) and engrailed (b) wings. Dorsal veins with the main bulge on the dorsal surface are shown in solid black, ventral veins in white. The sensilla are indicated by circles.

and Pattern

Veins and bristles. The pattern of engrailed wings can be best described as the partial substitution of the posterior part by anterior structures (compare Figs. 5 and 6) (Garcia-Bellido and Santamaria, 1972). In this view the engrailed wing has a mirror plane with two “anterior” borders, one anterior and one posterior in location. Observations by Garcia-Bellido and Santamaria supported this hypothesis: for example, the anterior vein III has characteristics which are not found in the posterior veins of en/+ wings (Fig. 12a). In enlen wings

there seems to be a substitution of the dorsal and ventral vein IV by a completely dorsal vein (III’) (Fig. 12b) which bears the characteristic sensillae of vein III (never found on vein IV in enlf flies). Likewise, the posterior margin bears bristles normally found in the anterior margin, in a similar arrangement: in the region of the alula costal-like bristles are found, in the middle part of the wing there is a welldeveloped triple row, and the typical bristles of the distal anterior type are found along the distal posterior margin.

328

DEVELOPMENTAL BIOLOGY

There are other features which do not fit with the duplication theory. Apart from vein III’ there are usually other pieces of vein in the posterior part of engrailed wings. We find that these pieces of vein, whether they are in engrailed wings, or whether they are formed by large engrailed clones in the posterior part of en/+ wings (Fig. ll), are always dorsal, and frequently bear sensilla. Thus, there seems to be no equivalent to the ventral veins I and II in the posterior part of engrailed wings (contrast Fig. 2; Garcia-Bellido and Santamaria, 1972). Garcia-Bellido and Santamaria (1972) also showed that clones of engrailed cells (labeled with straw, a bristle marker) expressed the engrailed phenotype autonomously: that is an enlen clone in an en/+ background which touched the posterior margin, instead of differentiating into double row structures, now formed straw triple-row bristles. This is true of even quite small engrailed clones, which indicates that the en+ function is required in every cell of the posterior compartment until late in development. We have confirmed and extended these observations, because the marker pawn, being a trichome and a bristle marker, can be identified anywhere on the wing surface. Even quite small clones in the posterior part of the wing may show the engrailed phenotype. Generally the pattern formed by the clone is autonomous, so that a small patch of engrailed cells may form a correspondingly small piece of the engrailed pattern in isolation. This is best shown by examination of engrailed clones produced in the region of vein IV. In normal flies the main bulge associated with this vein is ventral proximally and dorsal distally (Fig. 12a). In engrailed flies it is completely dorsal (Fig. 12b). When a ventral enlen clone extends along the region occupied by vein IV (Fig. 13a) it eliminates the vein IV proximally, but does not affect the differentiation of the distal dorsal part. By contrast a dorsal en/en clone (Fig. 13b)

VOLUME 50, 1976

FIG. 13. A ventral (a) and a dorsal (b) pzun en clone in the region of the vein IV. (a) The ventral clone almost eliminates the vein IV in the proximal region where it is normally ventral (cf., Fig. 12) and has no effect on the vein distally where it is dorsal. (b) The clone autonomously forms a dorsal vein which is characteristic of an engruiled wing in that region. Sometimes the vein runs over the underlying ventral en+ vein (arrow) and sometimes is separate from it (*).

forms a dorsal vein proximally without affecting the underlying ventral vein made by the en/+ cells there; more distally it forms a dorsal vein which may bear sensilla. Sometimes the dorsal (engrailed) vein is formed separately from the nearby ventral (wildtype) vein (Fig. 13b). Similarly small dorsal clones in the posterior proximal part of the wing may express the engrailed phenotype, forming small pieces of veins labeled withpawn, and sometimes pieces of crossveins with their characteristic sensillae. With respect to veins the autonomy is not complete, because occasionally pwn+ en+ cells become incorporated into an extra vein mostly made by a pwn en clone. The engrailed phenotype is characterised by a partial transformation of posterior into anterior patterns. Thus at the posterior margin triple-row bristles showing the transformation can be interspersed with double-row structures characteristic of wildtype cells. Garcia-Bellido and Santamaria (1972) noted that stw clones can include both triple- and double-row structures, showing that the partial expression of engrailed is not due to an early par-

LAWRENCE

AND MORATA

Wing Compartments

tition of cells into those which will give rise to transformed structures, and those which will not. We have a similar result: pawn engrailed clones, even those produced very late (132 ? 12 AEL in Minute flies) can include both triple-row and double-row structures. Clones of posterior engruiled cells can extend a long way into territory normally occupied by anterior cells. The best example is shown in Fig. 11, which illustrates the typical effects on the vein pattern. Clearly the characteristic veins of the anterior compartment are not simply displaced into a more anterior location; they have been replaced by veins made by the pawn engrailed cells. Moreover these veins are not typical of the anterior compartment but show instead characteristics of veins made by engrailed cells in the posterior part of the wing. For example, where the ventral clone extends into vein II (normally ventral) it does not form a vein II but instead almost eliminates it (Fig. 11). In the proximal part the region near the first cross vein is abnormal, associated with adventitious sensilla and extra pieces of vein. Exactly the same type of defects are associated with any extension of posterior engrailed cells into anterior territory (compare Figs. 7 and 11 with the normal wing Fig. 5). The inability of posterior engrailed cells, even when in an anterior location, to make normal anterior patterns can be contrasted with the ability of anterior engrailed cells to do so. The difference between anterior and posterior engrailed cells is clearly neither dependent on genotype, nor on position. This experiment, which amounts to a “transplantation,” shows that anterior and posterior engrailed cells are differently determined. The Duration Gene

of Function

(i) Early effect. pears partially to compartment into ask what effect it

of the engrailed

Because engrailed aptransform the posterior an anterior one, we can has on the number of

and Engrailed

329

cells in the two compartments at very early stages. The antero-posterior compartmentalization has occurred by the first larval stage ofMinute flies, and X-irradiation at that time (36 -+ 12 hr AEL) induces clones about twice as frequently in the anterior as in the posterior compartment (Garcia-Bellido, Ripoll, and Morata, 1973, 1976): their data are 35 anterior to 22 posterior clones, while ours for the similar age and genotype are 32 anterior to 20 posterior clones. For Minute+ flies we find a similar result: 26 anterior to 18 posterior clones (Table 1). Individually, these sets of numbers are not significantly different from a 1:l or a 3:l ratio, and a more accurate way to assess relative numbers of cells in the two polyclones is to measure the mean area of clones expressed as a fraction of the entire compartment (Bryant and Schneiderman, 1969). For this purpose the Minute technique cannot be used and we therefore compared mwh clone sizes in engrailed and wildtype flies following irradiation at both 36 ? 12 hr AEL and 60 c 12 hr AEL. Mean clone sizes were calculated from the log clone areas (Table 1). Since the entire wing blade is about 32,000 cells (Garcia-Bellido and Merriam, 1971a) and we have found that the areas of the anterior and posterior compartments on the wing blade of wildtype flies are almost identical and have assumed that both compartments contain 16,000 cells each. The posterior compartment of engrailed wings is some 15% larger than the anterior compartment. Using these figures we can make an approximate estimate of the mean number of cells in the polyclones at the time of irradiation. The average areas of clones (36 -C 12 hr AEL) were about onetwentieth of the total area of the anterior compartment, and about one-tenth of the area of the posterior in both engrailed and wildtype wings. This, allowing for the segregation division after mitotic recombination, suggests that in both wildtype and engrailed, approx 10 cells in the anterior polyclone and 5 in the posterior, are des-

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DEVELOPMENTAL BIOLOGY

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TABLE 1 MEAN CLONE SIZES IN NUMBER OF CELLS IN DIFFERENT COMPARTMENTS OF engrailed Anterior 36 + 12 hr AEL

en/en

en/+

60 + 12 hr AEL

en/en

enl+

AND en/+

WINGS”

Posterior

660 (2.82 k 0.08) n = 34 930 (2.97 + 0.08) n = 26)

1320 (3.12 k 0.08) n = 9) 1950 (3.29 f 0.08) n = 18

105 (2.02 + 0.05) n = 31 160 (2.20 ? 0.08) n = 31

120 (2.08 t 0.08) n = 25 135 (2.13 + 0.07) n = 22

a Log numbers + SEM within brackets. At 36 + 12 hr AEL 18/97 clones extend onto both dorsal and ventral surfaces. By 60 2 12 hr AEL nearly all clones (108/109) were confined to either dorsal or ventral surfaces.

tined to form wing blade (the notum, which is part of the prospective fate of both polyclones at that time, is not included in the estimates). These estimates do not allow for cell death induced by X-rays (which would tend to increase them) nor the possibility that the cells undergoing mitotic recombination are not randomly distributed throughout the cell cycle (which would tend to reduce them; Bryant personal communication). These results show no significant effect of engrailed on early polyclone sizes. After irradiation at 60 * 12 hr AEL (Table 1) we again found no difference between engrailed and wildtype. However, at 60 2 12 hr AEL, the clone sizes in the anterior and posterior compartments are now similar; therefore both polyclones contain about the same number of cells. We conclude that during the period between 36 and 60 hr AEL, the posterior polyclone divides faster than the anterior one. (ii) Late effect. Garcia-Bellido and Santamaria (1972) suggested that en+ allele is required throughout most of development in the posterior cells to direct development to form posterior structures. But clones produced after puparium formation failed to show the engrailed phenotype. This

phenomenon, when after a certain moment the phenotype of the cells become independent of the presence of a particular allele, is called perdurance (GarciaBellido and Merriam, 1971b). One obvious explanation of perdurance, in some cases, is persistence of a suf&lcient amount of the gene product, through a limited number of cell divisions. The amount of gene product required may well vary from gene to gene or among different cell types. In the case of engrailed, the temperature sensitivity has allowed us to determine the time when the whole wing pattern becomes independent of the temperature shift. In en/en flies reared at 30.5”C the vein pattern was nearly wildtype; the only remnant of the mutant phenotype is a few socketed bristles along the posterior wing margin. Medial triple-row bristles and other anterior structures are eliminated. At 25°C engrailed flies have an average of 39 * 2 medial triple-row bristles. Temperature shifts from 25 to 305°C were made at different times throughout development and the number of medial triple-row bristles was counted in samples of 30 wings (Fig. 14). Temperature shifts from 72 BPF to 12 APF changed the phenotype, suggesting a cumulative effect of the engrailed function

LAWRENCE AND MORATA

-

BPF

Wing Compartments

10 PF 10 APF -

FIG. 14. Effect of temperature shift experiment on engrailed flies. The temperature shift (25 -+ 30°C) at different times with respect to puparium formation. Before puparium formation (BPF) the bottles were shifted and a number of hours later the pupae were collected. After puparium formation (APFI the pupae were first collected at 25°C and then shifted. The phenotypes were measured by counting number of medial triple row bristles, which are 39 f 2 after continuous culture at 25°C (upper shaded band), and almost nil after continuous culture at 30°C (lower shaded band).

over that time. The most interesting aspect of the results is the lateness of the effects. At 12 hr APF the growth of the wing disc is nearly complete (Garcia-Bellido and Merriam, 1971a) but our experiments show that the phenotype of the posterior marginal bristles is still temperature sensitive at that stage. The results in these two sections prove that the pattern made by the posterior polyclone is dependent on the engrailed gene until very late in developmen?. They do not tell us when the engrailed gene begins to function although the temperature shift experiments suggest a role during larval growth. We could detect no effect of the engrailed mutation on polyclone sizes in first stage larval wing discs.

and Engrailed

331

which has about 50% greater area than although the anterior region normal, seems to be little affected (Fig. 8). This is an exaggeration of an effect found in engrailed Minute+ flies, for here at 25°C the posterior compartment is of 15% greater area than the posterior compartment of en/+ flies. Culture of en/en; Minute+ at 30°C reduces this to insignificance. The large Minute+ clone in a Minute fly (Fig. 9) also shows that this interaction between the two mutations is local, because the left wing containing the clone is of normal size, while right wing is of the typical engrailed Minute size and shape. Taking advantage of this interaction to investigate the control of shape and size, we have made clones of Minute and engrailed genotype in normally shaped wings. engrailed clones, marked with pawn, were produced by irradiating flies of the genotypepwn en/+; M(3)i”Jlmwh jv at 60 * 12 and 84 + 12 hr AEL. Because the frequency ofpawn clones is exasperatingly low, we have for some of the cultures resorted to irradiations of 1600 R. Our results show that small clones of engrailed cells in an en/ + wing can alter the shape of the wing (Fig. 15). They also show that in order to do so the clones have to meet three criteria: (i) They must be dorsal (with one exception). Ventral clones were not associated with changes in shape. (ii) They must include the posterior margin of the wing

The Compartment as a Unit for Control of Shape and Size In order to study the control of shape and size we have made use of the interaction between engrailed and Minute. We have found that when combined either with M(3)i”” or M(l)o”” there is a great enlargement of the posterior compartment

FIG. 15. Scale drawing to show effect of a small dorsal engrailed clone (shaded) on the shape of a pwn en/+; M(3)Pimwh ju wing. The control wing on the other side is drawn and superimposed (dotted line). Notice that the effect on shape extends beyond the border of the clone.

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DEVELOPMENTAL BIOL~CY

(Table 2). (iii) They must be larger than a certain size. Flies of this genotype, irradiated at 108 & 12 hr AEL, showed many pawn clones usually of about l-10 trichomes. Even if they were marginal dorsal clones and included one or two medial triple-row bristles they did not change the shape of the wing. The outgrowth was frequently much bigger than the clone itself (Figs. 15, 161, suggesting that the local genotype of the dorsal cells at the border was paramount in controlling the growth of cells within the wing. If so one would expect internal TABLE NUMBER OF pawn

2

CLONES IN VARIOUS CLASSES Posterior Internal

Not affecting shape Affecting shape

Marginal

D

V

D

V

29 0

17 0

3 10

18 1

(1Wings (pwn en/+; M(3)Plmwh ju) were irradiated with 1000 Rad 60 ? 12 and 84 k 12 hr AEL (some of the latter group received 1600 R); 2800 wings were screened.

FIG. 16. Photograph

VOLUME 50. 1976

engrailed clones to be under the control of a wildtype border and have no effect on the wing shape, as was found to be the case. However, the engrailed gene is not totally without effect on the growing cells away from the border. Measurements of cell density were made by counting trichomes in sample areas in the clone and comparing the number of trichomes in the same area on the other surface of the wing. Clones in the anterior compartment, where we can detect no effect ofengrailed, were used as controls. The results were expressed as the percentage increases in the pawn area over the corresponding area on the other surface. When this counting was done for normal wing regions, dorsal and ventral surfaces were found to be identical in trichome density. It appears thatpawn itself produces an increase in density, for anterior dorsal engrailed clones had more densely packed cells than the corresponding ventral surface (15 ? 2%), as did anterior ventral clones when compared with the corresponding dorsal surface (14 5 3%). Exactly the same result obtained for

of the wing shown in Fig. 15. Magnification

x 40.

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Wing Compartments

posterior ventral clones (15 ? 3%). However, posterior dorsal clones were the exception, their cell density was increased by 40 + 3%. There appears, therefore, to be an autonomous effect on cell density. However this local growth does not alter the wings’ shape, presumably because of the controlling role of the posterior dorsal margin. DISCUSSION

The Compartment

Hypothesis

In previous papers (Garcia-Bellido, Ripoll, and Morata, 1973, 1976; Garcia-Bellido, 1975; Morata and Lawrence, 1975) working hypotheses for the genetic control of compartment development have been proposed. In essence, the process depends on a partition of a polyclone (Crick and Lawrence, 1975) into two daughter polyclones, and coincidentally the permanent activation of a gene (a “selector gene”; GarciaBellido, 1975) in one daughter polyclone but not in its sister. The state of the selector gene is the only determining difference between cells of one polyclone and its sister. The reiteration of these steps generates a binary tree where the nature of the compartment is specified by which selector genes are active and which inactive. Another combinatorial system has been proposed by Kauffman (1973, 1975) on com-

FIG. 17. Hypothetical

and Engrailed

333

pletely different grounds; our scheme is similar in principle but relates to the in viva situation and includes a proposed scheme for the sequential involvement of selector genes. For example, in the case of the wing disc a hypothetical scheme could follow the tree shown in Fig. 17. Three selector genes, each performing a homologous function in every polyclone where it is active, would be sufficient to generate eight different compartments. engrailed appears to be the selector gene controlling development of the posterior polyclone. engrailed As a Selector Gene In this paper we have mostly explored the role of the engrailed gene in determining the development of the posterior compartment; we have examined its action by eliminating the en+ allele from clones at different stages of development. One prediction of the hypothesis outlined above is that the elimination of the en+ allele should affect all the cells of the posterior compartment (where it is active) and none of the cells of the anterior compartment (where it is inactive). Our experiments with large Minute+ engrailed clones prove that the elimination of the en+ allele from all the cells of the anterior polyclone is without effect (because the clone can Ii11 the whole compartment, define the normal

scheme for the successive compartmentalization

of the wing disc.

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DEVELOPMENTALBIOLOGY

boundary, and does not affect the pattern), and strongly suggest the entire’ posterior polyclone depends on the en+ .allele (even small clones show some engrailed phenotype, such as veins, socketed bristles, or cells which cross the compartment boundary) . Because enlen clones in the posterior part show only partial anterior features, and because of the temperature sensitivity of engrailed, it has been proposed that the en allele is leaky (Garcia-Bellido and Santamaria, 1972; Morata and Lawrence, 1975). Nevertheless the posterior en/en clones do have very clear anterior characteristics, which support the basic hypothesis as, apart from the marker gene (pawn), they differ from the background cells by the lack only of a single allele (en+). Clearly the en+ function must control other genes (termed “realisator genes” by Garcia-Bellido, 1975) which perform executive functions, and the action of these genes need not coincide with compartments. Obvious examples are the mwh and pawn genes which are employed in both anterior and posterior wing, as well as elsewhere. In this view, the differences between anterior and posterior wing are primarily ones that depend on modulation of the activity of overlapping sets of executive genes, and in variation in those processes controlling the arrangement of cell types into patterns, and details of growth. All these crucial differences appear to be controlled by the selector genes. The engrailed gene probably has a homologous role in the leg; there it produces a partial transformation of posterior leg into anterior leg (Tokunaga 1961) and its realm of action may well coincide with the recently discovered posterior compartment of the leg (Steiner, 1975). The genetic analysis of Garcia-Bellido and Santamaria (1972) also suggests that the engrailed gene is active in the metathoracic segment. We have suggested (Morata and Lawrence, 1975) that each active selector gene may in effect “label” the cells so that they

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will not mix with differently labeled cells of adjacent polyclones during development. This role of the engrailed gene is important and is consistent with many other observations on sorting out in insect cells. Our own observations suggest that engrailed posterior cells are intermediate in mixing properties between normal anterior (the loss of the straight compartment border shows that they will not form a straight interface with them) and normal posterior cells (engrailed clones within the posterior compartment have uneven boundaries). This is consistent with engrailed being a leaky mutation, leading to the reduction, rather than the elimination, of the activity of the label. This view is supported by previous results (GarciaBellido and Santamaria, 1972) who showed that posterior engrailed wing disc cells could mix with anterior and posterior wildtype wing disc cells. Using grafts on the pupa of Manduca and observing the behavior of cells at the interface between tissues of different origins (such as wing, leg, and eye), Nardi (1975) has described the relationship of cell affinities between these tissues. He found that this network of relationships was very similar to that produced by Kauffman (1973) from transdetermination frequencies in Drosophila. These observations are consistent with our suggestion that the activity of each selector gene may be associated with a specific “label.” The duration of activity of engrailed is unclear. We know that the en+ function is required until late, the temperature-shift experiments indicating as late as 12 hr APF. These same experiments suggest a cumulative effect of the mutation during the period 72 hr BPF to 12 hr APF. Moreover, the ability of posterior engrailed clones to cross a long way into anterior territory, suggests that the function of the en+ allele is essential to keep the growing throughout compartments separated much, if not all, of their development. This crossing cannot be a late phenomenon be-

LAWRENCE

AND MORATA

Wing Compartments

cause if it were we would expect the engrailed clone to displace rather than replace anterior structures. Moreover small engrailed clones can cross the antero-posterior compartment border, indicating that the border depends on the engrailed gene until late. Thus we believe that there is no evidence from our experiments to support the idea (Bryant, 1975) that the antero-posterior compartment border depends on the late fusion of sets of cells which have previously been developing separately. The complete lack of clones produced in larvae that extend from dorsal to ventral along the antero-posterior compartment boundary is consistent with anterior and posterior polyclones being contiguous during larval growth. Our results do not rule out the possibility that two separated anlage in the blastoderm could fuse before the first larval period. Our opinion is that the engrailed gene is probably active continuously from the time of compartmentalization, when its effects on cell affinities would be important in keeping the anterior and posterior cells from mixing. Clonal analysis also shows that in the absence of the en+ allele itself, its product soon disappears. Thus the cell miscibility and pattern properties change after the late elimination of the en+ allele. Selector Genes and Determination The concept of determination is clarified by the compartment hypothesis. As we have described, the activation of the engrailed gene in the posterior polyclone of the wing disc controls not only the development of particular cell affinities, but also the commitment to one specific pattern of differentiations to the exclusion of others. Classically determination is defined as such a stable commitment (Hadorn, 1967) and in the case of the imaginal discs has been described as consisting of discrete steps (Gehring, 1976). We are now in a position to make a more precise genetic hypothesis; at least some of these determinative steps are the processes lead-

and Engrailed

335

ing to permanent activation or inactivation of specific controlling (selector) genes. It is these genetic switches that are determinative, the maintenance of the determined state depending on the activity of the selector genes. In flies with mutated selector genes the determinative steps may be normal, but the phenotype may vary depending whether the mutation is completely lacking in wildtype function, partially lacking, or showing constitutive function. For instance, anterior engrailed cells can form normal anterior patterns; the inability of posterior engrailed cells to form normal anterior patterns (cf., Fig. 11) even when they have grown into anterior regions, shows that anterior and posterior engrailed cells are differently determined. We conclude that it is because the selector gene is leaky (a hypomorph), that the posterior compartment is a mixture of anterior and posterior characteristics, not because of any effect of the mutant on the determinative step itself. Thus it follows that the differentiation capacities of a wildtype cell are good criteria of determination, but those of a mutant cell can mislead. For example, the differentiation of aristapedia cells into either antenna or tarsus depends on the ambient temperature during the last few cell divisions prior to cuticle secretion (Schubiger and Alpert, 1975). These observations do not tell us about the determinative events in the cells, but tell us instead about such things as the temperature sensitivity and rate of turnover of the selector gene product. These ideas can be extended to other selector genes, for example, bithorax which transforms the anterior haltere compartment of the metathorax into an anterior wing compartment. Here a strong allele (bx9 causes a complete transformation, while a weaker one ( bPP) results in a mixture of wing and haltere structures in the affected disc. From both alleles we believe that the state of activation of the genes (the basis of determination in wildtype flies) is normal, but the gene product

336

DEVELOPMENTAL BIOLOGY

is either completely (bx3) or partially (bx34e) inactive. Thus clones of bithorux cells in the anterior haltere autonomously form wing cells which, because of their changed surface properties, now sort out from the surrounding haltere cells (Morata and Garcia-Bellido, 1975). For this selector gene there is evidence for an early effect of the mutation. Analysis of polyclone sizes in the first stage larva suggests that in bithorcz~ flies the anterior metathoracic polyclone is increased and approximates to that of a normal anterior mesothoracic (wing) polyclone (Morata and Garcia-Bellido, 1976). Experiments on phenocopies also suggest an early function of the bithorax genes (Capdevila and Garcia-Bellido, 1974). engrailed Size

and the Control

of Shape and

Crick and Lawrence (1975) have suggested that compartments may be the units of control of shape and size. This idea follows from the possible correlation between compartment borders and the boundaries of gradients of positional information. The relationship between gradients and size control is discussed in Bohn (1967) and Lawrence (1973). Our present study makes use of the effect of Minutes on the engrailed phenotype where the posterior wing compartment is grossly enlarged. engruiled alone produces about 15% increase in the size of the posterior compartment, so we regard the role of the Minute as augmenting an effect of engrailed rather than the reverse (Minutes have normal size wing compartments). We therefore made marked engrailed clones in enl+ Minute flies and found that such clones, over most of the wing, did not affect the wing shape. That is, they did not show autonomous expression. However, posterior dorsal clones of engrailed which included the dorso-ventral compartment border at the posterior margin of the wing did affect the shape; and this effect was nonautonomous to the extent that regions be-

VOLUME 50, 1976

yond the clone also grew excessively (Figs. 15, 16). These experiments show that the margin of the wing is crucial to the control of size and shape, the genotype of that boundary being dominant to the genotype of the cells in the wing interior. Moreover, the higher cell density of engrailed clones in the dorsal surface of Minute wings, which produce no change in size and shape, suggests that shape does not only depend on differential rates of cell division. The dorsal and ventral compartments have separate lineages throughout most of development, and yet in the adult their shapes and sizes match perfectly. This is true even when the dorsal compartment is deformed by a clone. It is difficult to avoid the conclusion that, at least to some extent, the ventral compartment is modeled on the dorsal one. We thank Sheila Green for her considerable and skilled help. We thank our colleagues in the M.R.C. lab for discussion, and Dr. P.J. Bryant for comments on the manuscript. G. Morata is supported by EMBO. REFERENCES BOHN, H. (1967). Transplantation experiments mit interkalarer Regeneration sum Nachweis eines sich segmental wiederholenden Gradienten in Beim von Leucophaea (Blattaria). 2001. Anz. (Supp.1 30, 499-508. BRYANT, P. J. (1975). Regeneration and duplication in imaginal discs. In “Cell Patterning,” Ciba Foundation Symposium 29, 71-93. BRYANT, P. J., and SCHNEIDERMAN, H. A. (1969). Cell lineage, growth and determination in the imaginal leg discs of Drosophila melanogaster. Develop. Biol. 20, 263-290. CAPDEVILA, M. P., and GARCIA-BELLIDO, A. (1974). Development and genetic analysis of bithorax phenocopies in Drosophila. Nature (London) 250, 500-502. CRICK, F. H. C., and LAWRENCE, P. A. (1975). Compartments and polyclones in insect development. Science 189, 340-347. FERRUS, A. (1975). Parameters of mitotic recombination in Minute mutants of Drosophila melanogaster. Genetics 79, 589-599. GARCIA-BELLIDO, A. (1975). Genetic control of wing disc development in Drosophila. In “Cell Patterning,” Ciba Foundation Symposium 29, 161-182. GARCIA-BELLIDO, A., and DAPENA J. (1974). Induction, detection and characterization of cell differ-

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entiation mutants in Drosophila. Mol. Gen. Genet. 128, 117-130. GARCIA-BELLIDO, A, and MERRIAM J. R. (1971a). Parameters of the wing imaginal disc development of Drosophila melanogaster. Develop. Biol. 24, 61-87. GARCIA-BELLIDO, A., and MERRIAM J. R. (1971b). Genetic analysis of cell heredity in imaginal discs of Drosophila melanogaster. Proc. Nat. Acad. Sci. USA 68, 2222-2226. GARCIA-BELLIDO, A., RIPOLL, P., and MORATA, G. (1973). Developmental compartmentalization of the wing disk of Drosophila. Nature New Biol. 245, 251-253. GARCIA-BELLIDO, A., RIPOLL, P., and MORATA, G. (1976). Developmental segregations in the dorsal mesothoracic disk of Drosophila. Develop. Biol. 48, 132-147. GARCIA-BELLIDO, A., and SANTAMARIA, P. (19721. Developmental analysis of the wing disc in the mutant engrailed of Drosophila melanogaster. Genetics 72, 87-107. GEHRING, W. (1976). Determination of primordial cells and the hypothesis of stepwise determination. In “Insect Development” (P. A. Lawrence, ed.1 Blackwells, Oxford, England. In press, HADORN, E. (1967). Dynamics of determination. Symp. Sot. Dev. Biol. 25, 85-104. KAUFFMAN, S. A. (1973). Control circuits for determination and transdetermination. Science 181, 310-317. KAUFFMAN, S. A. (1975). Control circuits for determination and transdetermination: Interpreting positional information in a binary epigenetic code. In “Cell Patterning,” Ciba Foundation Sympo-

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sium 29, 201-221. LAWRENCE, P. A. (1973). The development of spatial patterns. In “Developmental Systems: Insects” (S. J. Counce and C. H. Waddington, eds.1, Vol. 2, 157-209. Academic Press, New York. LAWRENCE, P. A., and GREEN S. M. (19751. The anatomy of a compartment border. J. Cell Biol. 65, 373-382. LINDSLEY, D. L., and GRELL, E. H. (19681. Genetic variations of Drosophila melanogaster. Publ. No. 627. Carnegie Institute, Washington. MORATA, G. AND GARCIA-BELLIDO, A. (1976) Developmental analysis of some mutants of the bithorax system of Drosophila. Wilhelm Roux’ Arch. In press. MORATA, G., and LAWRENCE, P. A. (1975). Control of compartment development by the engrailed gene of Drosophila. Nature (London) 255, 614-617. MORATA, G., and RIPOLL, P. (1975). Minutes; Mutants of Drosophila autonomously affecting cell division rate. Develop. Biol. 42, 211-221. NARDI, J. (1975). Spatial differentiation in lepidopteran wing epidermis. Harvard University, Ph. D. Thesis. SCHUBIGER, G., and ALPERT G. D. (1975). Regeneration and duplication in a temperature sensitive homeotic mutant of Drosophila melanogaster. Develop. Biol. 42, 292-304. STEINER, E. (1975). Establishment of compartments in the developing leg imaginal discs of Drosophila melanogaster. Thesis, University of Zurich. TOKUNAGA, C. (1961) The differentiation of a secondary sex comb under the influence of the gene engrailed in Drosophila melanogaster. Genetics 46, 157-176.