Establishment of imaginal discs and histoblast nests in Drosophila

Mechanisms of Development, 34 (1991) 11 - 20 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0925-4773/91/$03.50

11

MOD 00023

Establishment of imaginal discs and histoblast nests in

Drosophila

Amanda A. Simcox 1,,, Evelyn Hersperger 1, Allen Shearn 1, j. Robert S. Whittle 2 and Stephen M. Cohen 3 * Department of Biology, The Johns Hopkins University, Baltimore, Maryland, U.S.A., : School of Biological Sciences, Sussex University, Falmer, Brighton, U.K. and 3 Howard Hughes Medical Institute and Department of Cell Biology, Baylor College of Medicine, Houston, Texas, U.S.A. (Received 12 December 1990; revision received 21 December 1990; accepted 2 January 1991)

In Drosophila the homeotic genes of the bithorax-complex (BX-C) and Antennapedia-complex (ANT-C) specify the identity of segments. Adult segment primordia are established in the embryo as the histoblast nests of the abdomen and the imaginal discs of the head, thorax and terminalia. We have used a molecular probe for the limb primordia and in vivo culture to describe the nature of the adult primordia in mutants in which the pattern of homeotic gene expression was altered. The results suggest that the histoblast or disc 'mode' of development is initiated by the extended germ band stage through activity of the BX-C and ANT-C and is relatively inflexible. Drosophila; Imaginal disc; Histoblast nest; ANT-C; BX-C; Polycomb; Distal-less

Introduction

Cell fates in the Drosophila embryo are specified by a cascade of differential zygotic gene expression initiated by maternal products (reviewed in Akam, 1987; Anderson, 1987; Niisslein-Volhard et al., 1987; Ingham, 1988; Levine, 1988; Rushlow and Arora, 1990). The earliest manifestation of cellular differences generated by this genetic activity is seen when the homogeneous sheet of blastoderm somatic cells gastrulates and a number of domains characterized by their mitotic synchrony appear (Foe, 1989). Cell commitments are realized as organogenesis, segmentation and differentiation of the larval body occurs. The pattern of the adult body is also specified during embryogenesis and we are examining how embryonic gene activity establishes the imaginal discs and histoblast nests which eventually form the adult exoskeleton (Simcox et al. 1989). By stage 14/15 of embryogenesis morphologically distinct imaginal discs are present (Bate * Present address: Dept. of Molecular Genetics, Biological Sciences, 484 West 12th Avenue, The Ohio State University, Columbus, OH 43210, U.S.A. Correspondence to: A. Simcox, Dept. of Molecular Genetics, Biological Sciences, 484 West 12th Avenue, The Ohio State University, Columbus, OH 43210, U.S.A.

and Martinez-Arias, submitted) and the leg imaginal discs express the homeobox containing gene Distal-less (Dll) (Cohen et al., 1989; Cohen et al., submitted). Dll expression serves as a molecular probe for these cells earlier in development, showing that they are already a specialized cell group at the extended germ band stage (Cohen, 1990). At the end of embryogenesis most larval cells are undergoing polyploidy but cells of the imaginal discs and histoblast nests remain diploid (Auerbach, 1936; Madhavan and Schneiderman, 1977; Whittle, 1990). Discs and histoblast nests increase in size during subsequent development but have different growth dynamics and morphologies. Disc cells divide mainly during larval growth and become organized as epithelial sacs with a central lumen, which are attached by stalks to larval structures (Madhavan and Schneiderman, 1977). In contrast, the cells of the histoblast nests remain contiguous with the larval epithelium and undergo mitosis mainly during pupal development (Madhavan and Schneiderman, 1977). During metamorphosis the disc and histoblast cells secrete the cuticle that forms the exoskeleton. Here we address how and when adult primordia in the embryo are programmed to be either the imaginal discs of the head, thorax and terminalia or the histoblast nests of the abdomen. Our results suggest that activity of the bithorax-complex ( BX-C) and Antennapedia-complex (ANT-C) genes (Lewis, 1978; Kaufman et al. 1980) programs adult

12 p r i m o r d i a as either discs or histoblast nests b y the early e x t e n d e d germ b a n d stage.

Results Adult primordia in B X - C mutants B X - C - (UBx Mxz2 abd-a M1 A b d - B M S / D f (3R) P l 1 5 ) e m b r y o s (Fig. 1) have a n o r m a l head a n d p r o t h o r a x (T1) b u t the mesothorax (T2) through the seventh abd o m i n a l segment develop with a mixed identity that is a composite of c o m p a r t m e n t s from two segments, anterior T2 a n d posterior T1 (parasegment 4) (Lewis, 1978;

Sato a n d Denell, 1985). T h e i d e n t i t y of the eighth a b d o m i n a l segment is complex; the a n t e r i o r has characteristics of b o t h the p r o t h o r a c i c (ventral) a n d mesothoracic segments (dorsal) while the i d e n t i t y of the posterior part is n o t k n o w n b u t m a y be cephalic (Lewis, 1978; Sato a n d Denell, 1985). We cultured fragments of B X - C - e m b r y o s to det e r m i n e whether they p r o d u c e d i m a g i n a l discs and, if so, i n t o what structures these differentiated. E m b r y o s were bisected through the 3rd or 4th a b d o m i n a l segm e n t s (Fig. l b ) a n d the f r a g m e n t s were cultured in vivo. W h e n posterior fragments were c u l t u r e d the a n t e r i o r

Fig. 1. Cuticle phenotypes of the embryos. (a) The ventral aspect of a newly hatched wild-type 1st instar larva. The Keilin's organs of the 3rd thoracic segment are marked ( * ). A pair of these organs is also found in the first and second thoracic segments but not in the abdominal segments. The position of the arrow head marks where cuts were made to bisect the embryos into anterior (A) and posterior fragments (P). (b) The ventral aspect of a newly hatched BX-C- (UbxMx12abd-aM1 Abd-BMS/Df (3R) Pl15) first instar larva showing the thoracic transformation of abdominal segments. The Keilin's organs of the third thoracic segment are marked (*), a pair of these organs is also found in the 1st and 2nd thoracic segments and each of the abdominal segments (A1-A8). The position of the large arrow head marks where cuts were made to bisect the embryos into anterior (A) and posterior fragments (P). The small arrow head marks where embryos were cut to produce a 'tail' fragment (T) that comprised the posterior tip (A7-A10). (c) The ventral aspect of a Pc- (Pca/Df (3L)Pc) embryo at the time of hatching showing the transformation of thoracic and abdominal segments towards the character of the last, the eighth abdominal segment. The Keilin's organs of the the thorax are variably retained, one remains in the 3rd thoracic segment (*) and both the first and second thoracic segments have a pair. The position of the large arrow head marks where cuts were made to bisect the embryos to give a posterior fragment (P). The single small arrow head marks where embryos were cut through the third thoracic segment to produce a 'head' fragment (H) that included the cephalic and thoracic segments. The pair of small arrow heads marks a midsection fragment (M) which included abdominal segments 4 and 5.

13

b

Fig. 2. lmaginal derivatives from wild-type and B X - C - embryos. (a) Wing disc from a cultured wild-type anterior embryonic fragment. The disc has been stained to report en expression (dark area). (b) Wing implant derived from cultured wild type anterior embryonic fragment. (c) Wing disc from a cultured B X - C - posterior embryonic fragment. The disc has been stained to report en expression (dark area). (d) Wing implant derived from cultured B X - C - posterior embryonic fragment. (e) Wild-type female anal plates from a cultured posterior embryonic fragment. (f) B X - C female anal plates from a cultured posterior embryonic fragment. (g) Second leg structures, from a cultured B X - C - posterior embryonic fragment, which has the characteristic bristles of the anterior second leg. A, anterior; P, posterior; N, notum; AP, apical bristle; S, spur bristles; PAP, pre-apical bristle. Bar represents 50 #m in a and c and 100 #m in b and d. f r a g m e n t was m o u n t e d so that the p o s i t i o n of the cut c o u l d be checked. A n t e r i o r f r a g m e n t s of wild t y p e a n d BX-Ce m b r y o s p r o d u c e d the s a m e range of structures

except halteres, which were n o t recovered f r o m B X - C e m b r y o s ( T a b l e 1). In c o n t r a s t , a p o s t e r i o r f r a g m e n t o f a BX-Ce m b r y o t h a t i n c l u d e d o n l y a b d o m i n a l seg-

14

a

J

b

cl

A

q

15 TABLE 1 lmaginal structures derived from B X - C - embryos Embryo fragment (n)

Wild-type anterior a (19) B X C - anterior a (9)

Wild-type posterior ~ (73) B X C - posterior a (51) BXC-

'tail' b (10)

Frequency of disc type ClypeoEyelabral antennal

Labial

Leg

Wing

Haltere

Genital

Anal

0.11 0.22

0.74 0.89

0.32 0.44

0.53 0.89

0.84 0.67

0.26 0

0 0

0 0

0 0 0

0 0 0.10

0 0 0

0 0.57 0.80

0 0.76 0.30

0 0 0

0.11 0 0

0.11 0.12 0.20

a Embryos were cut through the third or fourth abdominal segments to give an anterior fragment and a posterior fragment, b A 'tail' fragment was a small posterior fragment cut through the seventh or eighth abdominal segments.

ments produced imaginal discs that m e t a m o r p h o s e d into wing, leg and analia structures (Table 1, Fig. 2c,d,f,g) while similar fragments of wild type embryos p r o d u c e d only genital discs that m e t a m o r p h o s e d into genital and anal structures (Table 1, Figs. 2e and 3c,e). All m e t a m o r p h o s e d implants were classified according to their segmental type by identifying characteristic cuticle features. Wild-type embryos produced derivatives of all discs and the frequency of recovery of each disc type reflected relative size; derivatives of the smallest disc, the clypeolabral, were recovered from 11% of cultured embryos while derivatives of the largest discs, the wing and eye-antennal, were recovered from about 75% of cultured embryos (Table 1). Legs from each of the three thoracic segments could be distinguished in implants primarily by identifying distinctive bristles in the anterior c o m p a r t m e n t s because posterior leg structures were more difficult to identify. We found examples of anterior pro-, meso- and metathoracic leg in implants derived from wild-type embryos. Cultured anterior B X - C implants produced anterior pro- and mesothoracic leg while posterior B X - C - fragments produced anterior mesothoracic leg (Fig. 2g). Wing tissue was recognized by the wing trichomes, and the margins of the wing allowed classification according to compartment identity; the anterior margin has a triple and double row of bristles while the posterior margin lacks bristles but has a row of long hairs. We found examples of anterior and posterior wing structures derived from

wild-type and B X - C - tissue (Fig. 2b,d). Posterior fragments of wild-type e m b r y o s p r o d u c e d only derivatives of the genital disc (Table 1) (Figs. 2e and 3c,e). There were 8 embryos that p r o d u c e d these genital disc derivatives and each p r o d u c e d b o t h genital and anal structures. In contrast the 8 B X - C - embryos that p r o d u c e d derivatives of the genital disc p r o d u c e d only anal structures (Fig. 2f). Posterior fragments (' tail') cut through the 7th or 8th abdominal segments (Fig. 1, Table 1) p r o d u c e d leg tissue at a higher frequency than wing tissue and of the three wing implants none had wing blade material; two had only pleural derivatives and the other had notal tissue. One B X - C - posterior fragment in this set produced antennal tissue which m a y be an indication that the eighth abdominal segment is transformed to a cephalic segment (Lewis, 1978), but the tissue could have arisen by transdetermination and recovery of only one such implant does not exclude either possibility (Table 1). A d u l t p r i m o r d i a in Pc m u t a n t s P o l y c o m b ( P c ) is a trans-regulator of the B X - C

and and apparently acts to repress expression of the homeotic genes (Lewis, 1978); in a P c - e m b r y o homeotic genes are no longer restricted to their normal expression domains and the A b d o m i n a l - B gene is expressed throughout the b o d y (Wedeen et al., 1986; Kuziora and McGinnis, 1988; Celniker et al., 1989).

ANT-C

Fig. 3. Imaginal derivatives of wild-type and Pc- embryos. (a) Wild-type posterior embryonic fragment after culture. The tissue has been stained for en expression which is restricted to vertical stripes (dark areas). The dark central core is not fl-gal activity but the cuticle of the inverted embryo. The arrow head just anterior to the base of the hindgut marks the position where the genital disc was found (shown in c). (b) Pcposterior embryonic fragment after culture. The tissue has been stained for en expression which was restricted to weak vertical stripes (dark areas). The anal pads (A) reported strong en expression. The arrow head just anterior to the base of the hindgut marks the genital disc which was small (compared with a wild-type disc shown in panel c). Some cells in the Pc- disc express en (dark region). (c) A genital disc from a cultured wild-type embryo. The pattern of staining indicates that the animal was male. (d) Genital discs produced by a Pc- anterior fragment. Some tissue reported en expression (dark regions). (e) Some of the derivatives of the male genitalia produced by a cultured wild-type embryo; clasper bristles (CL), lateral plate (LP), genital arch (GA) and the anal plate (AP) (the penis apparatus produced by the embryo is not shown). (f) The derivatives of the male genitalia produced by a cultured Pc embryo; genital arch (GA). Bar, 100 #m in a-f.

16 TABLE 2 Imaginal structures derived from Pc- embryos Embryo fragment (n) Wild-type anterior b (19) Wild-type midsection c (14) Wild-type posterior b (73) Pc-' head' d (27) Pc- midsection c (35) Pc- posterior b (24)

Frequency of disc type Head or thoracic

Male genital

F e m a l e Basal genital cuticle a

1

0

0

0

0

0

0

0

0

0.07

0.04

0

0

0.52

0c

0.26

0

0.06

0e

0.06

0

0.08

0e

0.08

The wild-type data are the same as those in Table 1. a Basal cuticle consisted of chitin with a few bristles, b Embryos were cut through the third or fourth abdominal segments to give an anterior and a posterior fragment. CA midsection fragment was composed of abdominal segments 4 and 5. d A ' head' fragment was the anterior part of an embryo cut through the third thoracic segment, e Female Pcembryos did not produce any recognizable genital derivatives they probably produced eighth tergite tissue, which was classified as basal cuticle.

P c - embryos ( p c 3 / D f Pc) have a cuticle p h e n o t y p e (Fig. 1) in which each segment is transformed toward a segment with characteristics of the posterior seventh and anterior eighth abdominal segments (parasegment 13) (Lewis, 1978; Denell and Frederick, 1983; Sato and Denell, 1985; C a s a n o v a et al., 1986). The transformation is not complete in the head or thorax. Thoracic characteristics, such as Keilin's organs, are often retained (Fig. 1) and the mesothorax shows a variable anterior transformation (Sato and Denell, 1985). We cultured ' h e a d ' (an anterior fragment cut through the third thoracic segment), midsection (abdominal segments 4 and 5) and posterior fragments of P c - embryos (Fig. lc). Each of these fragments produced adult cuticle derivatives but at different frequencies (Table 2). We recovered no tissue characteristic of head or thoracic discs from cultured ' h e a d ' fragments. The tissue from

' h e a d ' , mid and posterior fragments was characteristic of the genital disc. The genital disc is a composite disc, thought to be derived from the last 3 abdominal segments ( A 8 - A 1 0 ) , that differentiates into the sexually dimorphic external and internal genitalia and the external analia and hind gut (Diibendorfer and NiSthiger, 1982). The cultured P c - e m b r y o s p r o d u c e d soft tissue, not analyzed in detail, which p r e s u m a b l y was part of the internal genitalia. In males the major cuticle derivative we recovered from cultured P c - embryos was genital arch tissue (Fig. 3f; Table 3) and possibly lateral plate tissue. The tissue was sometimes rather a b n o r m a l but the very dark pigmented cuticle with regions devoid of bristles was most consistent with origin from the genital arch. There were no cases where we could unambiguously identify the clasper bristles, the penis apparatus or the anal plate which were usually found in wild-type cultures (Fig. 3e; Table 3). There are fewer cuticle derivatives of the female genital disc and we found no clear examples of P c - vaginal plate, spermothecae or female anal plate tissue although these were usually found in implants derived f r o m female wild-type embryos (Table 3). A n u m b e r of P c - embryos produced some chitinous material with a few bristles which we classified as basal cuticle (Table 2). This g r o u p of embryos could represent the females and the tissue m a y be the eighth tergite (Diibendorfer and N/Sthiger, 1982), which is a female genital disc derivative comprised of a sheet of chitin with a few small bristles. The lack of overt cuticle characteristics makes this tissue difficult to classify. We observed a six-fold difference in the recovery of male genital disc derivatives between cultured ' h e a d ' and posterior fragments although each type of fragment had about the same n u m b e r of e m b r y o n i c segments with adult primordia (Table 2). As discussed below we attribute this to the different potential of anterior discs and histoblast nests to transform into genital discs. Engrailed expression in cultured discs Wild-type (en-lacZ/CyO), B X C - ( e n - l a c Z / + ; Pc 3 UBx Mx12 abd-a M1 A b d - B M S / D f ( 3 R ) P l 1 5 ) and P c ( e n - l a c Z / + ; pc3//Of Pc) e m b r y o s with an en-lacZ

TABLE 3 Genital disc derivatives in Pc- embryos Disc origin

Wild-type posterior Pc- ' head' Pc- midsection Pc- posterior a

Males (n) 5 14 2 2

Females

Frequency of structure

Penis apparatus

Clasper bristles

(n)

Anal plate

Vaginal plate

Spermotheca

1 0 0 0

1 0 0 0

3 0 0 0

1 0 0 0

1 0 0 0

0.67 0 0 0

Frequency of structure Anal plate

Genital arch

0.8 0 0 0

1 1 1 1

a

Genital arch and the lateral plate tissue were scored together.

17 reporter gene (Hama et al., 1990) were cultured in females and the implant was stained for fl-gal activity to report areas where the endogenous en gene was active. Cultured anterior fragments of wild-type embryos (n = 15) produced many discs most of which could be recognized by their morphology and pattern of en-reporter expression (Fig. 2a). 15 cultured wild-type posterior fragments were analyzed and 11 of these had a genital disc just anterior to the hind gut (Fig. 3a,c). The pattern of en-reporter expression, which was different in males and females, was like that of discs dissected from third instar larvae (Hama et al., 1990). BX-Ccultured posterior fragments (cut through abdominal segment 3 or 4) (n = 13) produced an average of 6 large discs most of which could be recognized as wings or legs by the morphology and pattern of en-reporter expression (Fig. 2c). Tissue from cultured P c - embryos had an unusual pattern of en-reporter expression. Anterior fragments (n = 16) produced a mass of disc material that expressed the en-reporter in a patchy manner (Fig. 3d). The morphology of the disc material was like that of genital discs as it had thick ridges. Busturia and Morata (1988) have reported a derepression of en in anterior P c - clones but the presence of cells not reporting en activity in the cultured discs suggest that en was not completely derepressed. Running ventrally was some amorphous tissue which also reported patchy en activity. This was apparently not neural tissue because it was still produced in embryos cultured after the brain and ventral ganglion had been removed (n = 5). The tissue was not from an imaginal disc as it did not differentiate cuticle following metamorphosis. In P c - cultured posterior fragments (n = 24) the anal pads stained strongly (Fig. 3b) and a few embryos (7/24) had small blobs of tissue which were probably defective genital discs. These were found in either the wild-type position relative to the hind gut (3/24) (Fig. 3b) or more anteriorly (4/24). Distal-less expression At the extended germ band stage cells in the leg and cephalic appendages express DII (Cohen, 1990; Fig. 4a) and we used in situ hybridization with a D// probe to monitor these cell groups in B X C - and P c - embryos. 25% of embryos produced by the triple mutant stock were B X C - embryos and these were recognized by an abnormal pattern of DII expression at the extended germ band stage. B X C - embryos had a wild-type pattern of Dll expression in the head and thorax (Cohen, 1990; Fig. 4a) but in addition each abdominal segment, A1-A9, had groups of DII expressing cells (Fig. 4b). This expression persisted in A1-A8 but faded in A9 by the end of germ band shortening (Fig. 4c). B X - C mutant embryos develop ectopic Keilin's organs (KO) (Keilin, 1915) in the abdominal segments (Lewis, 1978). Development of the KO depends on the activity of the DII

h

Fig. 4. Distal-less expression in wild-type and B X - C - embryos. (a) DII expressionin an extended germ band stage embryo. The imaginal limb primordia of the 3 thoracic segments are indicated (1, 2 and 3). (b) DII expression in an extended germ band stage B X - C - embryo (Ubx Mx12 abd-a M1 Abd-BMS). The imaginal limb primordia of the 3 thoracic segments are indicated (1, 2 and 3). Ectopicprimordia appear in each of the abdominal segments A1-A9. The A9 primordium is indicated by an arrow. (c) DII expression in an retracted germ band stage B X - C - embryo(Ubx MxI2 abd-a Ma Abd-BMS). DII expression in A9 has faded by this stage.

gene (Sunkle and Whittle, 1987; Cohen and Jiirgens, 1989) and ectopic expression of DII under control of the Ultrabithorax gene correlates well with ectopic development of KO in the mutant embryos (Cohen et al., submitted). The KO develops in intimate association with the leg imaginal disc primordia in the embryo as part of a single cluster of morphologically recognizable cells (Bate and Martinez-Arias, submitted; Cohen et al., submitted). In the B X - C mutant embryos ectopic clusters are found in A1 through A8 (not shown). In contrast, at the extended germ band stage, P c embryos had a pattern of DII expression that was indistinguishable from wild-type because all embryos from a stock in which 25% were P c - showed a wild-type expression pattern (data not shown).

18 Discussion

The head, thoracic and abdominal segments of the Drosophila adult have unique identities conferred by differential expression of the homeotic genes, the majority of which are within the bithorax and Antennapedia complexes (Lewis, 1978; Kaufman et al. 1980). Other genes required for the development of the anterior head and analia reside outside these loci. The homeotic genes are first activated in parasegmental domains in the embryo (Martinez-Arias and Lawrence, 1985) and must remain active throughout larval development to produce a normal adult (Lewis, 1964; Morata and Garcia-Bellido, 1976). The domains of expression and requirement of some homeotic genes change as development proceeds (discussed in Morata et al., 1990). This shift in deployment of some homeotic genes in imaginal tissues reflects the segmental, rather than parasegmental, morphological units of the adult. Primordia of the adult segments arise in the embryo as the imaginal discs of the head, thorax and terminalia and the histoblast nests of the abdomen. Here we describe the nature of adult primordia in embryos with homeotic phenotypes by in vivo culture (Hadorn et al., 1968) and with molecular probes. In vivo culture was used to extend the development of animals with extreme homeotic phenotypes, which would otherwise die around the time of hatching, so that we could assess how the adult primordia were affected during larval development. The results have given insights into the control the homeotic genes exert on whether, and when, a primordium becomes an imaginal disc or an abdominal histoblast nest as well as their role in dezermining the segmental character as seen in the differentiated imaginal cuticle.

Adult primordia in abdominal segments of BX-C- embryos developed as discs B X - C - embryos have a cuticle phenotype in which the head and T1 are normal but segments T2-A7 are transformed and develop as thoracic segments with an aT2 pT1 characteristic (Lewis, 1978; Sato and Denell, 1985). Ectopic expression of ANT-C genes in posterior regions causes this cuticle transformation (Hafen et al., 1984; Harding et al., 1985). We recovered imaginal discs from abdominal segments of B X - C - embryos suggesting that the adult abdominal primordia were programmed to develop as imaginal discs rather than histoblasts. This transformation of abdominal cell groups into imaginal leg primordia could be seen by the extended germ band stage because Dll, which is normally expressed in embryonic leg primordia, was expressed in two groups of cells in each abdominal segment. Our results do not show reprogramming of the same cell group directly as we do not have a molecular probe for the histoblast cells in embryos and did not

examine the cultured embryos for histoblast cells histologically. Histoblasts do not differentiate in our in vivo culture method, perhaps because of their intimate association with the larval epidermis which fails to grow well from an inverted embryo. We were also unable to recover differentiated abdominal adult cuticle from pieces of third instar wild-type larval cuticle transplanted into hosts for metamorphosis. However, Kerridge and Sang (1981) have reported that the anterior histoblast nests were reduced or absent in the BX-C mutant, bithoraxoid, and they found a leg disc ventrally. Thus, homeotic selector genes affect the programming of the adult primordia to become either a histoblast nest or an imaginal disc and this in turn activates the down stream genes responsible for elaborating the mitotic program, morphogenesis and final cuticle pattern of each type of primordium. The abdominal B X - C - discs developed into legs, wings and analia. The wings had both anterior and posterior structures confirming that Ubx function (the most anteriorly expressed BX-C gene) is not required for development of the dorsal mesothorax (Morata and Kerridge, 1981). The B X - C - legs bore characteristics of the anterior mesothorax but we could not identify posterior leg structures because these do not have readily recognizable land mark bristles that can be identified in implants. Others have shown, by examining viable BX-C alleles and using clonal analysis, that the posterior compartments of the meso- and meta-thoracic legs are transformed to a prothoracic character (Morata and Kerridge, 1981; Casanova et al., 1985). Thus the anterior limit of the Ubx domain in adults is posterior T2 ventrally and anterior T3 dorsally. The Abdominal B ( Abd-B) allele used here, Abd-B Ms, is equivalent to a deletion of the gene and hence lacks both the morphogenetic (m) and regulatory elements (r) and causes embryonic defects extending into PS14 (Lewis, 1978; Casanova et al., 1986; Tiong et al., 1987). The Abd-B gene is expressed in the genital disc (Celniker et al., 1987) and is required for the development of adult terminalia such that Abd-B mutations affect the genitalia and analia (Lewis, 1981; Sfinchez-Herrero et al., 1985; Karch et al., 1985; Casanova et al., 1986). We found that analia but not genitalia structures were produced by cultured B X - C - embryos which is consistent with a posterior limit of the BX-C in the adult A8, the genitalia deriving from segment A8, and the analia deriving from A9 a n d / o r A10 (Diibendorfer and NiSthiger, 1982; S~nchez-Herrero et al., 1985). We argue below that different parts of the genitalia derive from anterior and posterior A8. Pc- embryos produced only genital structures All body regions of cultured Pc- embryos gave rise to derivatives of the genital disc, though at different frequency. The tissue produced in males was genital

19 arch tissue and in females was the eighth tergite (probably) but we did not recover other components of the external genitalia or the anal plates from males or females. Recovering only genital tissue from Pc- embryos was consistent with the findings of others who have described the pattern made by Pc- clones in head and thoracic segments as typical of parts of the genitalia or analia (Struhl, 1981; Busturia and Morata 1988; Tiong and Russell, 1990). The wing may be an exception as Busturia and Morata (1988) described these clones as forming areas of densely packed trichomes, we found no areas of wing-like trichomes in implants. The recovery of genital disc derivatives from anterior and posterior regions is consistent with the ectopic expression of Abd-B observed throughout the body of Pcembryos (Wedeen et al., 1986; Kuziora and McGinnis, 1988; Celniker et al., 1989). Recovering only a subset of the genitalia structures suggests that, in males, the genital arch derives from anterior A8 and that other structures such as the claspers and the penis apparatus derive from posterior A8. This is consistent with a gynandromorph fate map of the genitalia (Schiipbach et al., 1978) which places the penis posterior to the genital arch and the vaginal plates posterior to the eighth tergite. Adult primordia from segments A8-A10 fuse to form the genital disc which differentiates into the adult external and internal genitalia and analia (Di~bendorfer and N~Sthiger, 1982). In Pc- embryos the posterior (A8, A9 and A10) appears normal although a rudimentary denticle belt sometimes develops in A9 (Denell and Frederick, 1983). Adult primordia from this region of Pc embryos were apparently transformed by ectopic activation of Abd-B because cultured embryos produced genital arch tissue (in males) but lacked anal derivatives. Struhl (1981) reported that clones of Pccells were 'relatively normal' in the analia but clones of genital arch tissue in the analia, would tend to look normal because of similar bristle morphology and cuticle color. Thus the differentiation of adult cells specified by gene(s) that are normally active in the telson is overridden by ectopic Abd-B expression. Discs and histoblasts Differential activity of the ANT-C and BX-C genes in the embryo programs the adult primordia in a segment to become either a histoblast nest or an imaginal disc depending upon which genes are active. In mutants lacking activity of the BX-C genes, limb primordia formed in every abdominal segment, as judged by DII expression at the extended germ band stage (Fig. 4b, c). Presumably these ectopic primordia arise because ANTC genes are expressed in posterior segments, although ectopic expression of Antp can only be detected coincident with or after we observed DO expression in abdominal segments (Hafen et al., 1984; Carroll et al.,

1986). Our culture data show that these abdominal primordia develop into mature imaginal discs which differentiate into legs and wings. In a Pc- embryo the initial domains of homeotic gene expression are normal but at the extended germ band stage Abd-B is expressed throughout the body (Wedeen et al., 1986; Kuziora and McGinnis, 1988; Celniker et al., 1989). Leg and cephalic primordia were established in normal locations in Pc- germ band stage embryos, as judged by DII expression. We have no molecular markers for the histoblast nests but assume these were established in the abdominal segments. Subsequently when Abd-B was ectopically activated throughout the body, all anterior discs were transformed into genital discs. But there was a different situation in the abdomen as we recovered very few genital derivatives. We suggest this was because the primordia in abdominal segments were committed as histoblast nests and could not respond to Abd-B expression by transforming into genital discs. These results from analyzing Pc- mutants suggest that the commitment to the disc or histoblast 'mode' of development occurs by the extended germ band stage.

Materials and Methods

Genotypes B X C - embryos were of the genotype U b x MXl2 abda MI Abd-BM8/Df (3R) Pl15 and en-lacZ/+; Pc 3 Ubx Mx12 abd-a MI Abd-BM8/Df (3R) Pl15. We were unable to use embryos homozygous for a deletion of the BX-C because these are also deficient for flanking genes which are required for cell viability, however the BX-C alleles used here are considered nulls and are described in Sfinchez-Herrero et al. (1985), Cassanova et al. (1986) and Busturia and Morata (1988). Pc- embryos were of the genotypes pc3( Df (3L) Pc and en-lacZ/ + ; Pc 3 / D f (3L)Pc. Pc 3 is the strongest Pc allele and may be slightly antimorphic (Duncan and Lewis, 1982). Wildtype embryos were heterozygous sibs derived from crosses to generate the B X C - and Pc- embryos. The en-lacZ gene is described in Hama et al. (1990) and other gene alleles and deficiencies are described in Lindsley and Grell (1968) and Lindsley and Zimm (1987, 1990). In oioo culture This was done essentially as in Simcox et al. (1989) except that the adult female hosts were o v o ° / + (Busson et al., 1983). These females have small ovaries and therefore more space in the abdominal cavity for the implanted tissues to grow. Embryos were selected by their cuticle phenotype, sliced transversely and the anterior and/or posterior fragments were inverted prior to implantation in the host. In experiments where the

20 position of the transverse cut was determined the un-injected fragment was cleared in lactic acid and methanol (9:1) and mounted in Hoyer's medium for examination. Implanted tissue was recovered from the adult hosts after about 10 days at 25°C and imaginal discs were injected into Canton S larval hosts. Implant derived cuticle structures were recovered from the host following its metamorphosis and mounted in Faure's medium. fl-galactosidase staining

Mutant and wild-type embryos that also carried an en-lacZ gene (ryxho25, Hama et al., 1990) were bisected, inverted and cultured in female hosts for 4-6 days. In this set of experiments the gut was stripped away before transplantation as this allowed easier recognition of the discs following culture. Implants of cultured embryos were stained for fl-galactosidase activity as described in O'Kane and Gehring (1987). Discs were dissected away from other tissues and mounted in Ringer's solution on slides for examination. Blue regions of fl-galactosidase activity report en expression. In situ hybridization

Embryos of genotypes described above were hybridized with a Dll probe using the method of Tautz and Pfeifle (1989) with the modifications described in Cohen (1990).

Acknowledgements We thank Drs Ernesto Sfinchez-Herrero, Gines Morata, Robert Denell and Tom Kornberg for stocks; Nick Tripoulas for reading the manuscript; and Janos Szabad for suggesting the use of ovo ° females as hosts. S.M.C. thanks Elaine McGuffin for technical assistance. This project was supported by grants from the NIH to A.S., the Science and Engineering Research Council of the U.K. to J.R.S.W. and the Howard Hughes Institute to S.M.C.

References Akam, M. (1987) Development, 101, 1-22. Anderson, K. (1987) Trends Genet., 3, 91-97. Auerbach, C. (1936) Trans. R. Soc. Edinb., 58, 787-815. Bate, C.M. and Martinez-Arias, A. Development, submitted. Busson, D., Gans, M., Komitopoulou, K. and Masson, M. (1983) Genetics, 105, 309-325. Cohen, B., Wimmer, E. and Cohen, S.M. Mech. Dev. 33, 229-240. Cohen, S.M. (1990). Nature 343, 173-177. Cohen, S.M., BriSnner, G., Kiittner, F., Jiirgens, G. and J~ickle, H. (1989) Nature, 338, 432-434. Cohen, S.M. and Jiirgens, G. (1989) EMBO J., 8, 2045-2055. Denell, R.E. and Frederick, R.D. (1983) Dev. Biol., 97, 34-47. Diibendorfer, K. and N~thiger, R. (1982). Wilhelm Roux's Arch. Dev. Biol., 191, 42-55.

Duncan, I.M. and Lewis, E.B. (1982) In Subtelny, S. (ed.), Developmental Order: Its Origin and Regulation. Alan R. Liss, New York, pp. 533-534. Foe, V. (1989) Development, 107, 1-22. Hadorn, E., Hurlimann, R., Mindek, G., Schubiger, G. and Staub, M. (1968) Rev. Suisse. Zool., 75, 557-569. Hafen, E., Levine, M. and Gehring, W.J. (1984). Nature 307, 287-289. Hama, C., All, Z. and Kornberg, T. (1990) Genes Dev., 4, 1079-1093. Harding, K., Wedeen, C., McGinnis, W. and Levine, M. (1985) Science, 229, 1236-1242. lngham, P.W. (1988) Nature, 335, 25-34. Karch, F., Weiffenbach, B., Peifer, M., Bender, W., Duncan, I., Celniker, S., Crosby, M. and Lewis, E.B. (1985) Cell, 43, 81-96. Kaufman, T.C., Lewis, R. and Wakimoto, B. (1980) Genetics, 94, 115-133. Keilin, D. (1915) Bull. Sci. France Belg., 49, 15-198. Kerridge, S. and Sang, J.H. (1981) J. Embryo. Exp. Morphol., 61, 69-86. Kuziora, M.A. and McGinnis, W. (1988) EMBO J., 7, 3233-3244. Levine, M. (1988) Cell, 52, 785-786. Lewis, E.B. (1964) In Locke, M. (ed.), The Role of Chromosomes in Development. Academic Press, New York and London, pp. 231252. Lewis, E.B. (1978). Nature, 276, 565-570. Lewis, E.B. (1981) In Brown, D.D. and Fox, C.F. (eds.), Developmental Biology Using Purified Genes. Academic Press, New York, pp. 189-208. Lindsley, L. and Grell, E.H. (1968). Genetic Variations of Drosophila melanogaster. Carnegie Inst. Washington Publ. No. 627. Lindsley, L. and Zimm, G. (1987) The genome of Drosophila melanogaster. Drosophila Information Service 65. Lindsley, L. and Zimm, G. (1990) The genome of Drosophila melanogaster. Drosophila Information Service 68. Madhavan, M.M. and Schneiderman, H.A. (1977) Roux Arch. Dev. Biol., 183, 269-305. Martlnez-Arias, A. and Lawrence, P.A. (1985) Nature, 313, 639-642. Morata, G. and Garcia-Bellido, A. (1976) Roux Arch. Dev. Biol., 179, 125-143. Morata, G. and Kerridge, S. (1981) Nature, 290, 778-781. Morata, G., Macias, A., Urquia, N. and Gonzairz-Reyes, A. (1990) In Ingham, P.W. (ed.), Seminars in Cell Biology. W.B. Saunders, Philadelphia, Vol. 1, pp. 219-227. Niisslein-Volhard, C., Frohnhofer, H.G. and Lehmann, R. (1987) Science, 238, 1675-1681. Niisslein-Volhard, C. and Wieschaus, E. (1980) Nature, 287, 795-801. O'Kane, C.J. and Gehring, W.J. (1987) Proc. Natl. Acad. Sci. USA, 84, 9123-9127. Rushlow, C. and Arora, K. (1990) In Ingham, P.W. (ed.), Seminars in Cell Biology, Vol. 1. W.B. Saunders, Philadelphia, pp. 137-150. Shnchez-Herrero, E., Vern6s, I., Marco, R. and Morata, G. (1985) Nature, 313, 108-113. Sato, T. and DeneU, R.E. (1985) Dev. Biol., 110, 53-64. Schiipbach, T., Wieschaus, E. and Nrthiger, R. (1978) Roux Arch. Dev. Biol., 185, 249-270. Simcox, A.A., Roberts, I.J.H.. Hersperger, E., Gribben, M.C., Shearn, A. and Whittle, J.R.S. (1989) Development, 107, 715-722. Struhl, G. (1981) Nature, 293, 36-41. Sunkle, C.E. and Whittle, J.R.S. (1987) Roux Arch. Dev. Biol., 196, 124-132. Tautz, D. and Pfeifle, C. (1989) Chromosoma, 98, 81-85. Tiong, S.Y.K. and Russell, M.A. (1990) Dev. Biol. 141,306-318. Tiong, S.Y.K., Whittle, J.R.S. and Gribben, C. (1987) Development, 101,135-142. Wedeen, C., Harding, K. and Levine, M. (1986) Cell, 44, 739-748. Whittle, J.R.S. (1990). In lngham, P.W. (ed.), Seminars in Cell Biology, Vol. 1. W.B. Saunders, Philadelphia. pp. 241-252.