bithoraxoid, postbithorax, and the determination system of the haltere disk

DEVELOPMENTAL

BIOLOGY

86,

157-169

(1981)

bifhoraxoid,

postbifhorax, and the Determination System of the Haltere Disk PAULN.ADLER

Gilmer

Hall,

Biology

Received

Department, November

University

of Virginia,

18, 1980; accepted

in revised

Charlottesville, form

March

Virginia

22901

20, 1981

Mutations at the bithoraxoid (bxd) and postbithwax (pbx) loci cause a transformation of posterior haltere to posterior wing. It has previously been shown that pbx and pbx/U!S” cause this transformation by affecting the maintenance (or cell heredity function) of determination so that the transformed cells are indistinguishable from normal wing cells, and have no “memory” of having been part of a haltere disk (Adler, 1978a). I report here that Tp(3) bxdloo/Ub~ol and bxd’ pbx e’” both cause the transformation of posterior haltere to posterior wing in the same way as @z. On the other hand , bxd’ , bxd’/lI!&“, bxd’lj, bxd5’j/Ubbo’, and bx&‘j/red pbx cause this same transformation of posterior haltere to posterior wing by interfering with the expression of the determined state so that the developmental information of posterior haltere is “misread” as posterior wing. The transformed cells in these disks retain the memory of having been part of a haltere disk; that is, these posterior cells that would secrete wing cuticle during metamorphosis regenerate anterior haltere structures. Thus it appears clear that it is possible to uncouple the expression and cell heredity functions of determination in the haltere disk of Drosophila.

INTRODUCTION

is substantial evidence, in fact, that there is a positional information system that is present in all disks (Bryant et al., 1978, Wilcox and Smith, 1977; Postlethwait and Schneiderman, 1971; Adler, 1979), and that cells in a disk use this to assess their relative position within the disk. This information is then interpreted by the cells in a disk-specific manner by the expression function of determination. The genetic basis of the imaginal disk determination system has been studied in some detail. These studies have revealed a group of mutations called homeotic mutations (see review by Ouweneel, 1976) that cause a replacement of one part of the body by another. For many of these mutations the evidence is reasonably clear that the transformation is a direct consequence of the mutation. This is particularly true for mutations in the bithorax complex, which comprises the most extensively studied group of homeotic mutations (Lewis, 1963,1964,1967,1978; Morata, 1975; Morata and GarciaBellido, 1976; Adler, 1978a, b, 1981). Mutations in this complex cause segmental transformations in the thorax and abdomen (Lewis, 1978) of both larva and adult. Indeed it seems likely that the initial embryonic segmentation is altered. Within the left part of the bithorax complex are genes bithorax+ (bx+), postbithorax+ (pbx+), and bithoraxoid+ (bxd+) that when mutated result in a transformation of parts of the haltere to wing (Lewis, 1963,1964). For these genes there is clear evidence that they function not only during the establishment of segmentation during embryogenesis but also throughout larval life (Lewis, 1963, 1964; Morata and Garcia-Bel-

The progressive and heritable restriction in the potential of cells that occurs during development is often referred to as determination. The physiological systems responsible for this phenomenon must fulfill a variety of functions. The systems must be turned on in the correct cells at the proper time. Once turned on a determination system must maintain itself during subsequent growth. This component of a determination system can be considered a cell heredity or maintenance function. The determination system must also control gene expression so that the proper type of differentiated cell (e.g., a nerve cell and not a keratinized epithelial cell) is formed. This component of a determination systern can be considered an expression function. The imaginal disks of Drosophila are one of the most attractive systems for the study of determination. The cells that make up the disks are set aside early during embryogenesis (Wieschaus and Gehring, 1976; Steiner, 1976), and grow mitotically throughout almost the entire larval period (Madhavan and Schneiderman, 1977). During metamorphosis the disk cells undergo terminal differentiation. The cells that make up any disk, such as a wing disk, do not all produce the same cuticular structure. Instead they cooperate to produce a quite complex structure in which different cells elaborate different cuticular structures. Clearly a determination system that merely specifies wing (or anterior wing) to individual cells does not provide sufficient information to produce a complex structure such as a wing. There 157

0012-1606/81/110157-13$02.00/O Copyright All rights

Q 1981 by Academic Press. Inc. of reproduction in any form reserved.

158

DEVELOPMENTAL

BIOLOGY

lido, 1976; Adler, 1981). It is of further interest that the maximum transformations caused by mutations in these genes are defined by the anterior and posterior compartments of the wing and haltere disks (GarciaBellido, 1975; Garcia-Bellido et al., 1976). Specifically, mutations at the bz locus result in the transformation of anterior haltere to anterior wing, and mutations at the pbx and bxd loci result in the transformation of posterior haltere to posterior wing. Mutations at the bxd locus also result in the transformation of the first abdominal segment to a metathoracic segment (Lewis, 1963, 1964); the transformation of posterior haltere to posterior wing due to mutations at this locus have usually been interpreted as being due to bxd exerting a polar affect on pbx (Lewis, 1963, 1964, 1967). In principle homeotic mutations could cause their phenotypes by affecting any of the previously mentioned functions of the imaginal disk determination systems. Since the functioning of bx’, pbx’, and bxd+ are all required throughout larval life, mutations in these genes cannot cause their phenotypic effects by interfering only in the establishment of the determined state. Instead these mutants must be altering either the heredity or expression functions of determination. A test to distinguish between these possibilities has been described (Adler, 1978a). Previous results have shown that pbx and pbxAlbx’“’ (Adler, 1978a) alter the cell heredity component of determination while bti, b2/ ubxlO1, b;C34e,and b$4”/Ubx101 (Adler, 1978a, 1981) alter the expression function of determination. The observation that both possible types of mutants are found argues in support of the idea that imaginal disk determination systems function along the lines described above. Nevertheless, it seemed possible, albeit unlikely, that the difference between the results obtained with pbx and the two bx alleles was due to the fact that the different mutations transform different compartments. For this reason an examination of tissue transformed by bxd alleles seemed most interesting. I report here that bxd’ bxdl/Ubx’o’, bxd51j, and bxd51j/Ubxlo1 all cause the transformation of posterior haltere to posterior wing by affecting the expression function of determination. Thus the same transformation can result from a mutation interfering with either the heredity or expression components of the determination system of the haltere disc posterior compartment. A third mutation, Tp(3) bxdl’-“‘, which results from a transposition of lJbx+ to the left arm of the third chromosome has been found to affect the cell heredity component of determination. Finally, I report that bxd51j/pbx tissue resembles bxd tissue and bxd’ pbx tissue resembles pbx tissue, in terms of which component of the determination system is altered.

VOLUME

86. 1981 METHODS

Surgical techniques. The basic procedures have been described previously (Bryant, 1975; Haynie and Bryant, 1976; Adler and Bryant, 1977; Adler, 1981). Briefly, disks are dissected out of third instar larvae in a drop of Ringers and fragmented with tungsten needles. Individual fragments are then injected into a larva for immediate metamorphosis, or cultured for 1 week in the abdomen of an adult female prior to reinjection into a larva for metamorphosis. Cuticular implants are removed from the abdomen of the host after metamorphosis and mounted between coverslips for examination under the compound microscope. To obtain regeneration across the anterior-posterior compartment line by fragments of the wing or haltere disks that are entirely composed of cells from one of these compartments it is necessary to do disk mixing experiments (Haynie and Bryant, 1978). These are done using an irradiated (Adler and Bryant, 1977) disk fragment to induce such regeneration. In this paper anterior wing disk fragments and first leg disks (Adler, 1981) from irradiated y; mwh larvae were used to induce regeneration by posterior haltere disk tissue. Regeneration test of determination. The regeneration test to distinguish between a homeotic mutation altering the expression function as opposed to the cell heredity function can probably best be explained by the use of an example. Mutations at the bxd locus result in a transformation in the adult of posterior haltere to posterior wing. The mutation could be doing this by altering the cell heredity function of determination in these cells, changing it from haltere to wing, thus making these cells indistinguishable from normal posterior wing cells. If this were the case, then when these cells regenerated to anterior positions they would produce anterior wing structures, as normal wing disk cells would. Alternatively, it is possible that the bxd mutation causes its transformation by interfering with the expression component of the determination system resulting in the developmental information of metathorax being “misread” at certain posterior positions as mesothorax. When such cells regenerate to an anterior position I would expect them to produce anterior haltere structures as the mutation does not cause a “misreading” of metathorax as mesothorax in anterior positions in situ, and the cell heredity function remains haltere in quality in the mutant. Thus the nature of the regenerated structures reveals which component of the determination system has been altered by the mutation. Cuticle markers. The haltere disks transformed by homeotic mutations were wild type with respect to cuticle markers. One exception was the bxd pbx disks that were also mutant for ebony (e). The irradiated disks

PAULN. ADLER

Determination

(or disk fragments) were 9; mwh (Lindsley and Grell, 1968) in genotype so that any cuticle produced by such cells could easily be identified. Irradiation. Mature third instar larvae (y; mwh) were irradiated with 20,000 rad of -y-rays from a “Ws (Isomedix) source at a dose rate of 500 rad/min. Disks for mixing experiments were dissected out within 2 hr of larval irradiation. Nomenclature. The nomenclature for cuticular structures is essentially that of Bryant (1975) for the wing and that of Oiiweneel and van der Meer (1973) for the haltere, modified as described previously (Adler, 19’78a; 197813). Mutant strains. Stocks carrying chromosomes of the following constitutions bxd’, cu kar bxd5’j, red pbx, sb# Sb ss bxd’ pbx, Tp(3) bxd’OO,and U&O’ (Lindsey and Grell, 1968) were all kindly provided by Dr. E. B. Lewis. Chromosomes that are bxd51j or bxd’ pbx were obtained by recombination. Several of the bxd’ pbx recombinants were also found to carry a weak allele of e (designated ew). Presumably this mutation was cryptically present on at least some of the sb@ Sb ss bxd’ pbx chromosomes. bxd pbx ew disks were used in the experiments reported in this paper. U~X’~’ is due to an inversion with a breakpoint in the bithorax complex which inactivates in cis bx+, bxdf and pbx+. TM9 is a balancer chromosome synthesized by Marsh (1979), which contains the dominant temperature sensitive mutation DTS-4. No TM9 heterozygous larvae survive extended growth at 30°C. bxd’/Ubx’“’ and bxd51j/Ubx1’l heterozygous larvae were obtained by crossing bxd males to Ub$O’/TM9 females, and raising the resulting progeny at 30°C. Tp(3) bxd”‘O/ Ubx”’ larvae were obtained by mating Ubx’O’/TM9 females and Tp(3) bxdl”“/TMg males and raising the resulting progeny at 30°C. Because bxd’ pbx e” adults are very weak, bxd’ pbx e’” larvae were obtained by mating bxd’ pbx e”/TMS males and females and raising the resulting progeny at 30°C. bxd51j/red pbx larvae were obtained by mating red pbx/TMS females (red pbx adults are very weak) and bxd51j males and raising the progeny at 30°C. bxdl and bxd5’j larvae were obtained from homozygous mutant stocks and raised at 25°C. RESULTS Fate Map of Transfomed

Disks

I have assumed that the fate map previously established for the pbx haltere disk (Fig. 3b) (Adler, 1978b) represents the fate maps of the various mutant haltere disks of interest in this paper. I have tested this by examining the cuticular structures produced by the P and AC fragments (Fig. la) of each of these disks after being injected directly into larvae for metamorphosis. In each case the cuticular structures produced were

in Drosophila

Halere

159

Disk

those predicted by the pbx fate map. I have also examined the regulative behavior of P and AC fragments that were cultured in vivo for 1 week prior to metamorphosis. For all genotypes the AC fragments regenerated and the P fragments duplicated. The P fragments are composed entirely of cells that produce wing cuticle after immediate metamorphosis. In addition, the duplicates produced by cultured P fragments are composed entirely of wing cuticle. Thus the transformed condition is stable, as long as cells remain in posterior positions. bxd’ and bxdlAJbxlol Mutant expression. The bxd’ mutation is a moderately weak allele, showing 100% penetrance with a moderate but relatively constant expressivity. The posterior compartment of the haltere is transformed so that a moderate area of wing blade is produced. Posterior marginal row bristles are evident, but the row is incomplete with occasional gaps. An alar lobe is produced, but it is small, with fewer bristles than normal. The posterior hinge region is formed, but is somewhat distorted. The axillary cord is easily identifiable. The extent of transformation is greater for bxdl/Ubx’o’ halteres. The area of wing blade is greater, and the posterior row, alar lobe, and posterior hinge are more complete and normal. The degree of transformation for bxd’AJbsc’O’ halteres is still not as extreme as that for pbxAlb3c’01 halteres. The morphology of the bxd’ and bxd’/Ub~ol haltere disks is similar to that of the pbx haltere disk (Fig. 1, Adler, 1978b), but they are perhaps slightly smaller and more haltere like (see Fig. 2 for the morphology of the wildtype wing and haltere disks). bxd’ and bxdl/Ubxlo’ qffect Determined State

the Expression of the

P fragments (Fig. la) of bxd’ haltere disks were mixed with either anterior wing disk fragments or foreleg disks from an irradiated y;mwh larvae, and then cultured in vivo for 7 days prior to reinjection into a larva for metamorphosis. Sixty-four cuticular implants were obtained (Table 1). Forty-four of these implants produced only posterior wing cuticle, and thus are uninformative. Nine implants produced posterior wing cuticle as well as capitellum, a structure which could be from either the posterior or anterior compartment of the haltere. Eleven additional implants produced cuticle that could unambiguously be identified as containing anterior haltere structures, as well as posterior wing. An example of an anterior haltere structure is the pedicellar sensilla shown in Fig. 4~. Similar experiments using the P fragments from bxd’/Vb~ol haltere disks yielded 51 cuticlular implants (Table 1). Forty of these

160

DEVELOPMENTAL

FIG. 1. A pbz haltere is shown. (b) The lines

BIOLOGY

disk is shown at a magnification of 157X. (a) The line along along which the disc is cut to generate the PM2- fragment

contained only posterior wing. Eight contained capitellum and posterior wing structures, and the remaining three contained anterior haltere structures in addition to the posterior wing cuticle and capitellum. From these data I conclude that bxd’ and bxdl/Ubx’o’ alter the expression function of determination. An additional experiment was done to confirm this. It has previously been reported (Adler, 1978b) that certain cultured, but unmixed fragments of pbx and pbx/ U~X’~’ haltere disks (such as the PM2- fragment, Fig. lb) occasionally produce anterior wing structures. This is presumably due to posterior cells (or descendants of

FIG.

2. For

VOLUME

the sake of comparison

a wild-type

wing

disk

86, 1981

which the disk is cut to generate is shown.

the P and AC fragments

posterior cells) acquiring an anterior positional value during regeneration or duplication. Since pbx affects the cell heredity of determination, such cells would be expected to produce anterior wing structures (Adler, 1978a). Three observations support this interpretation. First, it has been shown that mixing is not necessary to induce anterior compartment cells from male foreleg disk fragments to regenerate posterior structures (Schubiger, 1971; Steiner, 1976). Second, cultured fragments of bti haltere disks do not produce posterior wing structures (Adler, 1978a). This is expected since b$ alters the expression function of determination, and an-

(a) and a wild-type

haltere

disk

(b) are shown

at a magnification

of 157X.

PAUL N. ADLER

REGENERATION

Determination

TABLE 1 BY TRANSFORMED POSTERIOR

Genotype: Fragment

in Drosophila

HALTERE

Halere

FRAGMENTSO

bxd’

mixed with: No. of implants:

Ant

wing 35

161

Disk

bxd’/

1st leg 29

Total 64

29 4 11 13 7 5

62 7 24 36 18 10

Ant

wing 25

Ubd”’ 1st leg 26

Total 51

Total 115

26 11 21 14 4 2

51 24 45 29 7 2

113 31 69 65 25 12

Structure

W/Hb

A/P’

Wing blade Posterior row (PR) Alar lobe (AL) Axillary cord (AC) Posterior hinge (PH) Pleural sclerite (PS) Notum Tegula (Teg) Costa (C) Triple row (TR) Double row (DR) Dorsal proximal radius (DPR) Ventral proximal radius (VPR) Anterior hinge (AH) Pleural wing process u-w Yellow club (YC) Capitellum (Cap) Capitellum sensilla (W Pedicellar brush (PB) Ventral/dorsal pedicellar sensilla (V/DPS) Ventral scabellar sensilla (VSS) Dorsal scabellar sensilla (DSS) Metathoracic bristles (MB) Adventitious bristles (-1

W W W W W W W W W W W

AP AP P P P P A A A A A

W

A

W W

A A

W W H

A A AP

11

19

6

11

30

H H

AP AP

4 5

5 12

2 3

3 5

8 17

H

A

4

9

1

2

11

H

A

3

5

H

A

4

6

H

A

2

4

H

P

18 6 5 0

44 9 11 0

No. implants containing Post wing Post wing, Hal (A,P) Post wing, ant ha1 Post wing, ant wing

33 3 13 23 11 5

26 3 6 0

a The numbers in the table indicate the number of times a structure was seen. ‘W designates a wing disk-derived structure and H a haltere disk-derived structure. ’ A is used to designate anterior compartment structures and P posterior compartment

terior b2 haltere disk fragments have been found to regenerate only posterior haltere structures after mixing (Adler, 1978a). Third, the fragments of pbx and pbx/ Ubxio’ haltere disks that regenerated anterior wing structures at the highest frequency were those fragments where, after wound healing, posterior compartment cells from positions near the compartment boundary were juxtaposed with anterior compartment cells (French et al., 1976; Reinhardt et al., 1977). Such pos-

25 13 24 15 3

5 1

7 4

20 3 2 0

20 5 1 0

40 8 3 0

84 17 14 0

structures.

terior cells would then have to undergo only a small amount of intercalary regulation in order to form anterior (wing) structures. In previous experiments (Adler, 1978b), 37% of the PM2- fragments (Fig. lb) of red pbx haltere disks that were cultured for 1 week prior to metamorphosis produced anterior wing structures. I have therefore tested the PM2- fragment from bxdl haltere disks. None of the 30 implants of PM2- fragments obtained after 1 week of in vivo culture prior to

162

DEVELOPMENTALBIOLOGY

metamorphosis produced anterior wing structures. However, many showed evidence of regeneration of anterior haltere structures. bxd51j and bxd51j/Ubx101 Mutant expression. The bxd51jallele produces a somewhat greater transformation than bxd’, although still less extreme than pbx. The transformation of the posterior compartment of the haltere to wing is complete enough so that all of the posterior wing pattern elements are relatively normal looking, although still a little small. As would be expected, the bxd51j/Ub2” transformation is greater than the bxd51j transformation but less than the pbx/Ubx”’ transformation. bxd5’j and bxd51j/Ubx101Alter the Expression of the Determined State Seventy-four implants were obtained from P fragments (Fig. la) of bxd 51j haltere discs that had been mixed with irradiated y;mwh anterior wing or foreleg discs prior to culture and metamorphosis (Table 2). Fifty-four of these contained only posterior wing cuticle and were thus uninformative. Fourteen contained haltere cuticle (capitellum or pedicellar brush) that could have been anterior or posterior (but most likely anterior, as only anterior tissue of this disk produces haltere cuticle in situ), as well as posterior wing. Six additional implants contained haltere cuticle that could unambiguously be identified as anterior. No anterior wing cuticle was found in any implant. Similar experiments were done for P fragments obtained from bxd5’j/Ubtio1 haltere disks (Table 2). Thirty of the 39 implants contained only posterior wing structures. Six contained capitellum (presumably anterior) as well as posterior wing, and three additional implants contained cuticular structures that were clearly anterior haltere. Once again no anterior wing structures were found. The conclusion from this series of experiments is that bxd51j and bxd51j/Ubxlo1 alter the expression function of determination. Tp(3) bxd’00/Ubx”’ Mutant expression. Tp(3) bxd”“’ is a mutation that results from the transposition of bx+ and mx+ to the left arm of the third chromosome. Although there is no obvious reason for Tp(3) bxdl@’ to be lethal, I have been unable to obtain homozygous larvae even after outcrossing. I have therefore studied the mutation only as a heterozygote with ubx”‘. The Tp(3) bxd1”“/Ub201 larvae appear healthy; however, adults rarely eclose and instead die as pharate adults. An examination of

VOLUME%,~~~~

the pharate adults reveals the transformation of posterior haltere to posterior wing to be extreme and constant in expressivity, resembling pbx/Ub$‘l halteres. All of the posterior wing structures are normal in appearance and fairly large. Tp(3)bxd1w/Ubx101 Affects the Cell Heredity of Determination Forty-five implants have been obtained from P fragments (Fig. la) of Tp(3) bxd’@‘/Ubd” haltere disks that were mixed with irradiated y;mwh disk tissue prior to culture and metamorphosis (Table 3). Thirty-three implants contained only posterior wing structures, while 12 other implants contained anterior wing structures as well as posterior wing structures. An example of such an anterior structure is the notum cuticle shown in Fig. 4b. No anterior (or posterior) haltere structures were found. Thus Tp(3) bxd’@‘/Ubdol appears to alter the cell heredity of determination and resemble pbx and not bxd’ or bxd51j I have confirmed the result that Tp(3) bxd”‘O/Ubti” alters the cell heredity of determination by examining the behavior of the PM2- fragment (see Fig. lb) of this disk. Seventeen implants derived from cultured PM2fragments were obtained. Of these, 18% contained one or more anterior wing structures, thus confirming that Tp(3) bxd”“‘/UbtiO’ alters the cell heredity of determination. bxd’ pbx ew Mutant exwession. I have also analyzed haltere discs transformed due to being bxd’ pbx e”. This genotype results in an extreme and constant expressivity resembling pbx. bxd pbx ew Alters the Cell Heredity

of Determination

Seventy-two cuticular implants (Table 4) were obtained from P fragments of bxd pbx ew haltere discs that had been mixed with irradiated y;mwh tissue prior to culture and metamorphosis. Fifty-five implants consisted only of posterior wing cuticle. Seventeen implants contained anterior wing cuticle as well as the posterior wing cuticle. A good example of such anterior wing structures is the notum shown in Fig. 4a. No haltere cuticle was scored. These results show that bxd pbx e” produces its transformation by altering the cell heredity of determination. This has been confirmed by examining the regulative behavior of PM2- fragments of this disc (Table 4). Twenty-two cuticular implants have been obtained from cultured PM2- fragments of

Determination

PAUL N. ADLER

REGENERATION

Wing blade Posterior row (PR) Alar lobe (AL) Axillary cord (AC) Posterior hinge (PH) Pleural sclerite (PS) Notum Tegula (Teg) Costa (C) Triple row (TR) Double row (DR) Dorsal proximal radius (DPR) Ventral proximal radius (VPR) Anterior hinge (AH) Pleural wing process ww Yellow club (YC) Capitellum (Cap) Capitellum sensilla (CS) Pedicellar brush (PB) Ventral/dorsal pedicellar sensilla (V/DPS) Ventral scabellar sensilla (VSS) Dorsal scabellar sensilla (DSS) Metathoracic bristles (MB) Adventitious bristles CAB) No. implants Post wing Post wing, Post wing, Post wing, a As in Table

HALTERE

Halere Disk

Ant

W/H”

A/P”

W W W W W W W W W W W

AP AP P P P P A A A A A

W

A

W W

A A

W W H

wing 34

34 23 34 29 28 3

163

FRAGMENTS' bxd=/ Ubd”

bxd5”

mixed with: No. of implants:

Structure

Drosophila

TABLE 2 BY TRANSFORMED POSTERIOR

Genotype: Fragment

in

1st leg 40

Total 74

Ant

wing 18

1st leg 21

Total 39

Total 113

40 17 36 37 27 6

74 50 70 66 55 9

18 12 17 17 15 4

21 13 21 20 17 5

39 25 38 37 32 9

113 75 108 103 87 18

A A AP

10

18

4

5

9

27

H H

AP AP

6 7

12 10

4 2

2 3

6 5

18 15

H

A

3

4

1

3

7

H

A

1

1

1

2

H

A

3

3

1

3

6

H

A

1

1

2

3

H

P

16 4 1 0

30 6 3 0

84 20 9 0

1

containing Hal (A,P) ant Hal ant wing

25 7 2 0

29 7 4 0

54 14 6 0

14 2 2 0

1.

bxd pbx e’” haltere disks and 27% of these contained anterior wing structures.

degree of transformation is perhaps somewhat less than for pbx, and somewhat more than is standardly seen for bxd5lj.

bxd51i/red pbx Mutant expression. I have examined haltere disks transformed due to being bxd5’j/red pbx in genotype. The posterior compartment of the haltere in such flies shows a fairly extreme transformation to wing. The

bxd5ji/red pbx Alters the Expression of the Detemined State Forty-six cuticular implants were obtained from P fragments of bxd51j/red pbx haltere disks that were

164

DEVELOPMENTAL

BIOLOGY

FIG. 3. (a) The fate map of the wing disk (Bryant, 1975). (b) The fate map of the pbx halter-e disk (Adler, 1978b) (c) The fate map of the haltere disk (Ouweneel and van der Meer, 1973).

REGENERATION Fragment

HALTERE

86, 1981

mixed with irradiated y;mwh disk tissue prior to culture and metamorphosis. Twenty-seven of these (Table 5) consisted of only posterior wing cuticle and are uninformative. Thirteen implants contained, in addition to posterior wing, haltere structures (capitellum and pedicellar brush) that are probably anterior, but could be posterior. Six additional implants contained structures that could unambiguously be identified as being anterior haltere. A dorsal scabellar sensillum from one of these is shown in Fig. 4~. No anterior wing structures were found. Thus, I conclude that bxd51j/red pbx affects the expression of the determined state. This has been confirmed by examining the implants produced by PM2fragments that had been cultured for 1 week prior to

TABLE 3 DISK FRAGMENTS

mixed with: No. of implants:

TRANSFORMED Ant

Structure Wing blade Posterior row (PR) Alar lobe (AL) Axillary cord (AC) Posterior hinge (PH) Pleural sclerite (PS) Notum Tegula (Teg) Costa (C) Triple row (TR) Double row (DR) Dorsal proximal radius (DPW Ventral proximal radius (VW Anterior hinge (AH) Pleural wing process (PWP) Yellow club (YC) Capitellum (Cap) Capitellum sensilla (CS) Pedicellar brush (PB) Ventral/dorsal pedicellar sensilla (V/DPS) Ventral seabellar sensilla WSS) Dorsal seabellar sensilla W-W Metathoracic bristles (MB) Adventitious bristles (AB) No. implants Post wing Post wing, Post wing, Post wing,

BY POSTERIOR

VOLUME

W/H”

A/P”

W W W W W W W W W W W

AP AP P P P P A A A A A

W

A

W W

A A

W W H H H

A A AP AP AP

H

A

H

A

H H H

A A P

wing 26

BY Tp(3)

bxdlw/Ubxlo’ 1st leg 19

Total 45

26 24 24 22 20 6 6b

19 17 18 17 16 3 3

45 41 42 39 36 9 9

1

2 1 2

3

1

1

3

1

1 5

2

containing 19 7 0 0

ant wing ant Hal Hal (A,P)

a As in Table 1. b Underlined structures

would

not be produced

in situ.

14 5 0 0

33 12 0 0

PAUL N. ADLER

Determination

in Dromphila

Halere

Disk

165

a

FIG. 4. (a) Notum produced by a P fragment of the bxd pbz e”’ haltere disk that had been mixed with an irradiated foreleg disc prior to culture and metamorphosis (136X). (b) Notum produced by a P fragment of the Tp(3) bxd’w/Ub~“’ haltere disk that had been mixed with an irradiated anterior wing fragment prior to culture and metamorphosis (110X). (c) A pedicellar sensillum produced by a P fragment of a bxd’ haltere disk that had been mixed with an irradiated anterior wing fragment prior to metamorphosis and culture (320X-phase). (d) A dorsal scabella sensillum produced by a P fragment of a bxd5’j/red pox haltere disk that had been mixed with an irradiated anterior wing disc fragment prior to culture and metamorphosis (400X-phase).

metamorphosis. None of the 11 implants obtained contained any anterior wing structures, consistent with the conclusion that bxd5’j/red pbx alters the expression function of determination. DISCUSSION

The data in this paper show that the transformation of posterior haltere to posterior wing that results from flies being bxd’, bxd’/Ub~ol, bxd51j, bxd51j/Ub301 or

bxd51j/red pbx in genotype is due to an alteration in the expression function of determination; that is, the cell heredity information of posterior metathorax is “misread” as posterior mesothorax at certain positional values. The transformed cell in these discs “remember” that they are from the metathoracic segment and regenerate anterior haltere structures. The data also show that Tp(3) bxd”“‘/UbX1°1 and bxd’ pbx e’” resemble in that they cause the transforpbx and pbx/lJbxlol mation of posterior haltere to posterior wing by altering

166

DEVELOPMENTAL

BIOLOGY TABLE

VOLUME

86, 1981

4

REGENERATIONBYTRANSFORMEDPOSTERIORHALTERE

FRAGMENTS’ bxd pbx e’”

Genotype: Fragment

Structure Wing blade Posterior row (PR) Alar lobe (AL) Axillary cord (AC) Posterior hinge (PH) Pleural Sclerite (PS) Notum Tegula (Teg) Costa (C) Triple row (TR) Double row (DR) Dorsal proximal radius (DPR) Ventral proximal radius (VPR) Anterior hinge (AH) Pleural wing process (PWP) Yellow club (YC) Capitellum (Cap) Capitellum sensilla (CS) Pedicellar brush (PB) Ventral/dorsal pedicellar sensilla (V/DPS) Ventral scabellar sensilla (VW Dorsal scabellar sensilla (DSS) Metathoracic bristles (MB) Adventitious bristles (AB) No. implants Post wing Post wing, Post wing,

Ant

mixed with: No. of implants: W/H

A/P

W W W W W W W W W W W W W W W W H H H

AP AP P P P P A A A A A A A A A A AP AP AP

H

A

H H H H

A A A P

wing 38

1st leg 34

Total 72

38 30 35 30 29 1 8

34 27 31 28 30 3

72 57 66 58 59 1 11

2 5 1

1 5 1

3 10 2

2

1

3

28 10 0

27 7 0

55 17 0

containing ant wing Hal (A,P)

a As in Table

3.

the cell heredity of determination. The transformed cells in these disks have no memory of being part of the metathoracic segment and regenerate anterior wing structures. Thus, it appears clear that is is possible to uncouple the expression and cell heredity functions of determination in the haltere disk of Drosophila. The extent to which this is a general property of determination systems remains to be established. There are a number of possible models that can explain the role of the bithorax complex in the determination system of the haltere disk. Two of these are listed below, and discussed in relation to this study. (i) The first hypothesis proposes that both lJbx+ and pbx+ produce products that are involved in both the cell heredity and expression functions of determination. Mutations at the Ubx’ locus would inactivate bxd+ and pbx+ in cis via a polar effect (Lewis, 1963, 1964, 1978).

The lack of pbx+ function in pbx and pbx/lL!nP would result in the cell heredity function of determination being altered. The transformation of posterior haltere to posterior wing in bxd flies would be due to bxd mutations exerting a polar effect on pbx+. bxd+ would have no direct function in the posterior haltere. To explain the observation that bxd mutations affect the expression function of determination it is necessary to suggest that the reduction in the level of pbx+ activity due to the bxd polar affect is less severe than the reduction due to the Ubx polar effect. The intermediate level of pbx+ activity, due to the bxd polar effect, would affect the expression function, but not the cell heredity function of determination. (ii) The second hypothesis suggests that bxd+ gene product is directly involved in the expression function of determination, and that pbx gene product is involved

PAUL N. ADLER

REGENERATION Fragment

HALTERE

Wing blade Posterior row (PR) Alar lobe (AL) Axillary cord (AC) Posterior hinge (PH) Pleural sclerite (PS) Notum Tegula (Teg) Costa (C) Triple row (TR) Double row (DR) Dorsal proximal radius (DPR) Ventral proximal radius (VPR) Anterior hinge (AH) Pleural wing process (PWP) Yellow club (YC) Capitellum (Cap) Capitellum sensilla (CS) Pedicellar brush (PB) Ventral/dorsal pedicellar sensilla (V/DPS) Ventral scabellar sensilla (VW Dorsal scabellar sensilla (DSS) Metathoracic bristles (MB) Adventitious bristles (AB)

Halere Disk

TRANSFORMED Ant

BY

wing 26

167

bxd51j/?-ed pb9 1st leg 20

Total 46

W/H”

A/P”

W W W W W w W W W W W W W W W W H H H

AP AP P P P P A A A A A A A A A A AP AP AP

26 9 25 26 21

20 14 15 16 12

46 33 40 42 33

12 10 5

7 5 4

19 15 9

H

A

2

3

5

H H H H

A A A P

2

2

4

14 9 3 0

13 4 3 0

27 13 6 0

26

20

46

containing Hal (A/P) ant. Hal ant wing

Total n As in Table

in Drosophila

TABLE 5 FRAGMENTS

mixed with: No. of implants:

Structure

No. implants Post wing Post wing, Post wing, Post wing,

OF POSTERIOR

Determination

1.

in only the cell heredity function of determination. ubx’ would be directly required in cis for bxd+ and pbx+ function. A prediction of this model, that is observed, is that tissue transformed due to being bxd pbx in genotype resembles pbx. The presumed role of the expression function is to express the state of the cell heredity component of the disc determination system. Thus when pbx resulted in the cell heredity function of cells in the posterior compartment of the haltere changing to the wing heredity state, no role remained for any gene products (such as the bxd+ product) involved in expressing the haltere state, since it was no longer “on.” The observation that tissue transformed due to being bxd51j/pbx in genotype resembles bxd51j tissue has interesting consequences for this model. Since the cell heredity function of posterior haltere disk cells appears unimpaired in bxd51j/red pbx disks, it seems likely

that the pbx+ gene on the bxd5lj chromosome is active and functional. Since bxd and pbx fail to complement (Lewis, 1963,1964) one is led to the conclusion that the bxd’ gene on the pbx chromosome is nonfunctional. This would further imply that pbxf is necessary in cis for bxd+ to be functional. Since bxUpbx heterozygotes do not show a transformation in the first abdominal segment (Lewis, 1963, 1964) bxd+ is presumably active in this segment in this genotype. Thus the control or function of bxd+ would be different in different body segments. There are, of course, many other possible models. A crucial difference in the two types of models suggested above is that in the first, one gene and its product are involved in both the expression and cell heredity functions of determination. In the second, individual genes and gene products are directly involved in only one func-

168

DEVELOPMENTAL

BIOLOGY

tion, As discussed elsewhere (Adler, 1981) both types of models are plausible and attractive. Studies on multiple alleles have been undertaken, at least in part, to see if evidence could be obtained that would imply that one molecular species is involved in both the maintenance and expression functions of determination (e.g., an autogenously regulated protein). Two bxd presumed point mutations were examined in this study, and both were found to alter the expression of the determined state. Previously (Adler 1978a, 1981), similar results were obtained for two bx presumed point mutations. Thus with the possible exception of Tp(3)bxdloo (discussed below) no evidence of allele heterogeneity has been obtained. The number of alleles examined is quite small, but the laborious nature of the experiments prohibits a truly extensive survey. In the first model suggested above, a single gene (pbx) and its product are involved in both the expression and heredity functions of determination, with different activity levels required for each function. If this is correct then it seems reasonable to expect that a critical level of gene activity exists where in some posterior haltere cells the expression function would be altered, while in others the cell heredity function would be altered. When a genotype with such a level of gene activity is examined, I would expect to find evidence of both anterior wing and anterior haltere structures regenerated. I have examined nine different genotypes where posterior halter is transformed to posterior wing (Adler, 1978a; this paper). These nine genotypes exhibit a wide range in the extent of transformed tissue. In these studies, I have obtained no evidence for tissue transformed by any one genotype giving a heterogenous response in the disk type of regenerated structures. On this basis, I consider the second model presented above more likely to be correct. The transformation of posterior haltere to posterior wing due to the Tp(3) b~d’~/Ubs~~’ genotype resulted from an alteration in the cell heredity of determination. This could be taken as evidence of allele heterogeneity at the bxd locus; however, I believe that this is not a reasonable conclusion. Tp(3)bxdlm results from a transposition of bx+ and Ubxf to the left arm of chromosome three. Thus bxd and pbx are in cis with a Ubx deletion. Since the cell heredity of determination is altered in but not in pbx/bxd5’j, or bxdAJbx’ol, Ubx pbx/Ubx”‘, mutations must be inactivating pbx. Additionally, since the first abdominal segment is transformed toward metathorax in bxd/Ubx, but not in bxd/pbx (Lewis, 1963, 1964); Ubx mutations must be inactivating bxd. Thus, Tp(3)bxdlM should be inactivating both bxd and pbx to the same extent as Ubx mutations and hence cannot be considered to be a simple bxd mutation.

VOLUME

86, 1981

It is a pleasure to acknowledge the fine technical assistance of M. MacQueen. I also thank Robert Greenberg and Dr. Ann Beyer their critical reading of the manuscript. This work was supported Grant HD11763 from the National Institutes of Health, and in dition the author was supported by a Research Career Development Award (KHD o-0361) from the National Institutes of Health.

Ms. for by ad-

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genetics of homoeosis. Ad-

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WILCOX,