Analysis of haploid mosaics in Drosophila

I)EVELOPMENTAI,

RIOLO(;Y

96,

285-85

(1983)

Analysis of Haploid Mosaics in Drosophila

Adult chimeric epidermal structures were obtained following transplantation of haploid nuclei from haploid donor embryos of Drosophila into genetically marked diploid embryos. The haploid nuclei either remained haploid or became diploid. Where possible, physical measurements indicated that the haploid cells were smaller and produced smaller cuticular structures than did diploid cells. An increase in the number of pattern elements was observed in many patches which, by various criteria, were judged to be formed by haploid cells. The observation of altered pattern element spacing in haploid patches is in agreement with the conclusion, reached by L. I. Held (1979, Wilhelm Rouxk Arch. 187, 105-12’7) in triploid flies, that hristle spacing is a function of cell size.

INTRODIJCTION

MATERIAL

The first indication that haploid tissues can survive in Drosophila was provided by the experiments of Bridges (192513, 1930) and Morgan et al. (1930). Five individuals were recovered which, on the basis of genetic markers and cell size, were apparently diploid-haploid mosaics. Subsequently, Waletzky (193’7), Sturtevant (1929), Hinton and McEarchen (1963), and Hall et al. (1976) reported additional cases of haploid-diploid mosaics. It is possible that these mosaics were not haploid but the result of simultaneous mitotic recombination (for review, see Hall et al., 19’76), although this mechanism does not account for the smaller cell size in the putative haploid patches. It could be of genetic interest to have haploid differentiated tissues. The absence of a complete “trans” set of genes in an animal in which the genetics is as well known as it is in Drosophila would help us to understand several aspects of regulation. Jack and Judd (1979) have shown recently that the control of gene activity can depend upon close proximity between alleles. There are examples of asynapsis affecting a gene’s function (Lewis, 1954; Ashburner, 1967, 1969; Korge, 1977), so that haploid tissues could help to analyze cis-trans regulatory phenomena. Chimeras were obtained with patches of donor tissue by the transplantation of haploid nuclei obtained from haploid embryos (Gans et al., 1975; Zalokar et al., 1975) into diploid embryos. A previous note about these results was published by Santamaria and Gans (1980). In the present paper, I have studied the morphogenesis and differentiation of haploid cells.

Experiments

AND

METHODS

were done with the following

Hosts

Donors.

strains:

y w slrl Y Y(‘; m wh jv

xmh x u,lm mh: Dw(l:Ei I,o+"'~" z w+ mh; Dp(l$) w+“‘~~‘, I

-,,,

where y, IX, sn3, f9”‘, mwh, and jv were employed as marker mutations (Lindsley and Grell, 1968; GarciaBellido, 1972). Maternal haploid (mh) is the name given (Santamaria and Gans, 1980) to the mutation fs (1) 1182 described by Gans et al. (1975) that produces the haploid embryos. wllEq is a mutant equivalent of a white deletion (Jack and Judd, 1979) and Dp(l$) wi51b7 is a nonvariegating insertional translocation of the wild-type white locus and some adjacent genes into chromosome 2. The breakpoints are 3Cl-2, 3 D6, and 52F in the polytene chromosome map. The eggs were collected, prepared, and injected according to the technique of Zalokar (1971,1981), slightly modified as described in Santamaria (1975). The donor embryos were all in the syncytial blastoderm stage and were arranged with their long axis parallel to the needle. The hosts were freshly collected eggs (15-45 min old) arranged perpendicularly to the needle. One donor egg provided nuclei to inject a mean of 20 host eggs; each host received one to six nuclei. Both the adults which eclosed and those which failed to eclose (the latter dissected from the puparium) were scored for patches of donor tissue. Animals with donor

285 0012-1606/83 (‘orwright All rights

$3.00

0 1983 by Academic Press, Inc. of reproduction in any form reserved

286

DEVELOPMENTAL TABLE

BIOLOGY

The Nature

1

RESULTS OF THE INJECTIONS OF DIPLOID MARKED NUCLEI INTO HAPLOID EMBRYOS AND OF HAPLOID NUCLEI INTO VIRGIN EGGS (*)

DOMX

Host

g w sn’

mh

mh

1/ w my*)

NO. e&Y 239 35

Note. The adult obtained

Blastoderm

Unhatched larvae

232

10

Hatched 1aIWie

127

3

3

0

in the first series (**) was completely

Adults 1 (**) 0

y w sn’.

tissue in the eye were photographed with color film. Afterwards they were dried by the critical-point method, surface-coated with gold, and photographed in the scanning electron microscope. The rest of the individuals were mounted in Euparal. Camera lucida drawings and photographs were employed to analyze the variation in the number of elements in the pattern. RESULTS

The Maternal-Hap&d

Mutant

VOLUME

(mh)

One of the X-linked female sterile mutants isolated by Gans et al. (1975), fs(1)1182, is a fecund recessive mutant; the genotype of the father does not affect the phenotype of the progeny. Zalokar et al. (1975) described it as a thermosensitive, maternal effect mutant producing haploid embryos which die before the end of organogenesis. In a preliminary note, Santamaria and Gans (1980) renamed the mutant maternal-haploid (mh) and showed that homozygous females of a nonthermosensitive strain of this mutant (isolated by A. Debec) produce haploid gynogenetic embryos (in which the male pronucleus does not participate). Haploid embryos laid by mh mothers die. In order to test whether this phenomenon was of a zygotic or maternal origin, normal diploid nuclei from the strain y w sn’ were injected into eggs laid by mh homozygous mothers (Table 1). Some larvae hatched and one developed completely to produce a y w atis adult. On the other hand (Table l), haploid nuclei from eggs laid by mh homozygous mothers, injected into virgin eggs, developed to form the characteristic haploid embryos which show no head involution and have abnormal segments. That this anomalous development is due to the genotype of the transplanted nuclei is suggested by the observation (Illmensee 1972; our own unpublished observations) that diploid nuclei, transplanted into virgin eggs, can support normal development at least up to larval stages. These results indicate that the eggs laid by homozygous mh mothers are capable of undergoing normal diploid development, but that haploid nuclei are unable to do so.

96, 1983

of the Donor Tissue: Hap&d

ur Dip&d

Nuclei from eggs laid by homozygous mh females crossed to Kinked ebony males were injected into eggs from the marked strains y f”“; mwh jv or y w sn”. One hundred and ninety-seven chimeras were obtained from the different series of injections. The fact that none of these chimeras was marked in the donor territory by Kinked indicates that fertilized diploid eggs were not employed as donors. Since cytological examination (Santamaria and Gans, 1980) has shown that the nuclei of eggs laid by mh homozygous mothers are inevitably haploid, the injected nuclei were in all likelihood haploid when transplanted. However, the nuclei may not have remained haploid during development. In the chimeras described here there were organs formed by cells of donor origin which were, by various criteria, of normal or of small size. This suggests that the donor territory can consist of haploid and diploid cells. There are several possible criteria that could be employed to determine the final ploidy. The karyotype is a possible criterion, but since only 10% of the adults (Table 3) were chimeric and only half of these were suspected to have haploid cells, this approach was not practical. The generally smaller size of haploid cells (Fankhauser, 1955) is a satisfactory criterion on which to judge ploidy. Indeed, the Drosophila, comparison of the cell size in males, females, superfemales (3X;ZA), supermales (lX;3A), intersexes (2X;3A), and triploid females has shown (Dobzhansky, 1929) that the size of a cell is directly correlated with the number of chromosomes contained in the nucleus. The clonal origin of the injected nuclei from the female pronucleus of the donor egg (Santamaria and Gans, 1980) precludes the possibility that the different sizes of the cells could be due to genetic differences such as spontaneous mutations. Since cell size could not be directly measured in all organs, in some cases the size of bristles or the density of hairs was taken as a measure of cell size (Table 2). However, the reduced size of an organ such as a limb (Table 2) is not a good criterion for haploidy. Even though in plants, organs are, in general, smaller in haploids than in diploids, in amphibians, increased ploidy results in size regulation of organs with fewer larger cells (Fankhauser, 1955). The normal form of the organ is preserved in spite of large differences in cell size. Size regulation is accomplished by reduction in cell number (Ibidem). The strongest argument that a patch of donor tissue included haploid cells came from the experiments with zeste. The xeste phenotype is expressed only in the presence of two doses of white+ (Gans, 1953). Haploid xeste cells with a single w+ dose should, thus, have a wild-

Hap&d

PEDRO SANTAMARIA TABLE 2 THE EFFECTS OF HAPLOIDY ON CELL SURFACE, BRISTLE SIZE, AND LENGTH OF FIRST LEG BASITARSUS Diploid mh females”

Individual examples in haploid patches

Eye-facet surface area (m2) b

239 f

4.5

140

125

155

Wing-cell (kdc

231 f

4.6

125 134

143 167

120 141

287 k

7.9

200 233

213 146

193 200

Posterior dorso central bristle length (pm)

444 * 19.8

306 300

346 200

286 300

Anterior bristle

scutellar length (Km)

472 k 12.0

300 333

280 280

Posterior bristle

scutellar length (gm)

479 * 10.1

373 280

173 393

233 +

216 233 186

160 193 180

surface area

Anterior bristle

dorsocentral length (pm)

First leg basitarsus length (Km)

5.4

186 226 166

Chimeras

in

287

Drosophila

W+ to the second chromosome (Experiment III). A donor spot should be xeste in diploid or triploid cells, as well as in occasional X0;2A cells in Experiment III. It should be wild type in true haploid cells and in occasional X0;2A cells in Experiment II. In Experiment III, inbalance for the second chromosome being lethal (Poulson, 193’7), only haploid cells should give rise to a wild-type spot. In both experiments all the spots in the eyes that were of wild-type color were formed by smaller facets and the spots that were zeste were all formed by facets of normal size (Fig. 1). The ommatidium of a fly which underlies each facet is composed of a fixed number of cells regularly arranged in a neurocrystalline lattice (Benzer, 1973). Thus it is probable that the facet size is an accurate index of the size of the cells composing the ommatidium. Thus t.he injected haploid nuclei can remain haploid or become diploid. Based on these criteria, 89 chimeras were considered to be formed by diploid cells in all the donor tissue and 108 to be partial or complete haploids.

The Repression of Unpaired Alleles

Jack and Judd (1979) have suggested that the mutant of paired alleles at the white+ locus and does not do so with unpaired alleles. According to this hypothesis, one would predict that haploid xeste cells having only one w+ locus would be wild type in phenotype. The second and third experiments (Table 3) confirm this prediction. The fourth series was intended to test if, in the haploid cells, mutant for zeste but with two homologous wt regions, the normal w+ in the X and the duplication of w’ in the second chrotype eye color. This theory was tested (Table 3) using mosome, the two w+ loci were able to pair. If both pair, either a x w+ mh stock (Experiment II), or a z w11E4 then a xeste pigmentation is expected when these cells mh; Dp(l$)~‘“‘~~ stock equivalent to a translocation of populate the eye. Out of 23 eye mosaics, 5 were x, 16

a All controls are means * 1 standard deviation (N = 10). ‘Average area of the facet surface. Density of facets measured in a rectangular area of 12,725 pm2 in the region nearest the center. From Bridges’ (1925) data, the facet area of haploid females is decreased by 31.8% relative to diploid females; the average decrease observed in mosaic patches reported here was 41.4% ‘Average area of the wing cell surface. Hair density measured in a rectangular area of 12,725 pm2 between first and second veins on the dorsal side of the wing. The average decrease in wing-cell area was 40.3%.

xeste represses the activity

TABLE RESULTS OF THE INJECTION

OF HAPLOID

3 NUCLEI

INTO MARKED

STRAINS

Experiments I Donor Host ems

larvae adults chimeras zeste zeste and zeste+ zeste+ Note. markers of donor Expt II,

II

mh Y

f SBa;mLvh 1868 1128 726 c*;

jv

z mh y w my

994 622 488 22

(6) (1) (4)

III 2 u’- mh; Dp(1.2) y II‘ S??

1295 862 665 60 (16) (2) (10)

IV IA’+

z w’ mh; Dp(l:2)

w+

y 7A).S?l” 776 465 371 38 15)

(2) (16)

The genotype of the donors refers to the mother and also to the gynogenetic haploid embryos. In the first experimental series the employed did not allow recognition of donor tissue in the eye (*). In the second, third, and fourth experimental series, all the patches tissue that were Z+ were formed by small ommatidia and those that were 2 were formed by ommatidia of normal size. One fly in two in Expt III, and two in Expt IV were z in one region and Z’ in an other.

288

DEVELOPMENTAL BIOLOGY

FIG. 1. Two examples of mosaics produced In a ommatidia derived from donor nuclei are of the donor patch is zeste’, the ommatidia (The phenotype z or Z+ is not distinguishable

by injection of z ui- nh; ZL’+nuclei from haploid donor embryos into diploid y w a,’ receptors. zeste, normal in size and indistinguishable from those formed by diploid cells. In h the phenotype are reduced in size and the bristle pattern is disturbed, probably due to haploidy of the cells. in these SEM pictures, but is clear in the living flies.)

were x+, and 2 contained x and xi areas. The facets forming the Z+ areas were small and so, likely to be haploid; the x facets were of normal size and likely to be diploids. The existence of the 16 + 2 wild-type mosaics implies that the two doses of homologous wi loci in a heterologous background were not repressed and were therefore probably not paired. The Morphology

of the Haploid

VOLUME 96, 1983

Fly

Haploid donor nuclei can form any cuticular region of the fly. Here I describe how the small size of the

haploid cells affects the cuticular patterns of various organs. (a) The arista. In the anterior part of the head the third antenna1 segment bears the arista. This organ is probably formed by the fusion of a few hairs (GarciaBellido, 1968) and differentiates in homozygous mh mothers a mean of 15.8 tips with a range of 14 to 1’7. The 29 aristae made by donor tissue (wild type in a background yellow) (Fig. 2) were distributed in two groups, one with a mean value of 15.5 tips and another with a mean of 24.3, with a maximum of 30 tips. The

PEDRO SANTAMARIA

Haploid

Chimeras

in Drosophila

289

two kinds of aristae are probably made by two different kinds of cells, diploid cells making normal and haploid cells making more feathered aristae. The range of the number of tips is wider in haploid than in diploid aristae. Undetected mosaics of haploid and diploid donor tissue cannot be excluded and their existence could explain the wider range of the number of tips in the second group of aristae. Two aristae, which were clearly mosaics of donor and receptor tissues, had 21 and 26 tips. The haploid aristae do not look like the aristae of mwh or Xwh (Poodry, 1980). These mutants increase the number of hairs produced per cell and, consequently, each secondary thread of the arista is feathered, while the haploid arista bears more threads originating directly from the principal rachis (Fig. 3). This morphology indicates that the haploid arista is formed by more cells, each one producing its individual thread. (b) First leg basitarsus. I have focused my attention on the first leg basitarsus but many of the characteristics of this leg segment can be found in other legs and leg segments. Of the 197 chimeras, 38 bore donor tissue in the first leg basitarsus and at least 10 showed a reduction in bristle size and an increase in the number of bristles (Fig. 4). These 10 flies all bore donor tissue in the anterior compartment of the leg and all donor cells differentiated as female (but see below). The morphology of a first leg basitarsus in a diploid female is schematized in Fig. 4a. Some characteristic haploid first leg female basitarsus are shown in Figs. 4b-d. The increase in the number of bristles is the result of the differentiation of extra bristles in both the longitudinal and the circumferential axes, leading to an increase in the number of rows as well as in the number of bristles per row. The number and positions of the

FIG. 3. Camera lucida drawings of (a) normal arista of a mh diploid female, (b-d) some examples of aristae with increased number of branches.

0 0

b

25

N’

of

lips

FIG. 2. Distribution of aristae by the number of branch tips formed by diploid homozygous mh females (a) and the 29 aristae formed by donor tissue (b).

seven bractless bristles were affected in an erratic fashion. (c) The notum. Forty-two heminota were totally or partially formed by mh cells. Among them, 28 showed smaller bristles and a higher density of trichomes. Two were partially haploids; 24 of these 30 also showed an increase in the density of brisltes (Fig. 5). The manner in which bristle density was increased in haploid patches differed for the microchaetae and for the macrochaetae. In microchaetae, increased density

290

DEVELOPMENTALBIOLOGY

0 2

Oo -

0 omo,O 0

‘0

CcoQo

oooo~

-0

O

T

oo”.“oo

-b

0

0

0000

o”

-% O-20

a

0

0

0

o@“,

0

‘0 00 Ofi o. 0 %c& O.00 b

C

FIG. 4. Schematically diagramed bristle patterns of the first leg basitarsus. Open circles = bristles with bracts; full circles = bractless bristles. (a) mh, diploid female. (b-d) examples of first leg basitarsus formed entirely by donor cells.

was due to closer spacing in both the anteroposterior and the mediolateral axes. The spacing of macrochaetae was decreased only in the anterior-posterior axes which is similar to the way in which extra-chaetes are added either in experiments of selection (Rendel, 1967; Fraser, 1967; Latter, 1970) or in “atavic” species (Garcia-Bellido, 1981), e.g., Leptoceratidae. Up to four dorsocentrals and five scutellars were formed by one hemithorax composed of haploid cells. These bristles were always in line and maintained approximately equal separation. (d) The tergites. Any of the abdominal segments could be formed by haploid cells. Tergites were considered to be formed by haploid tissue when the trichomes and microchaetae were increased in density and reduced in size. Macrochaetae formed in these tergites were, however, reduced in number, although this apparent reduction may, in fact, have been due to the unavoidable confusion of the unusually small macrochaetae with the microchaetae. In homozygous mh diploid females the fourth hemitergite had a mean of 46.0 microchaetae and

VOLUME96, 1983

9.0 macrochaetae. Complete haploid fourth hemitergites in the chimeras had 96 and 8, 87 and 6, 84 and 4, 85 and 4, 77 and 7 micro- and macrochaetae, respectively. The higher number of microchaetae in the tergites was coincident with decreases in both anteroposterior and mediolateral spacing. The pigment normally found in the posterior margin of the tergite was also present in haploid tergites. Bristles resembling split (Lindsley and Grell, 1966) were found in the haploid tergites (Fig. 6a). They appeared also in the medial triple row of the wing and, less frequently, in the notum and legs. (e) The sexually dimorphic organs. Thirty-two chimeras bore donor tissue in the terminalia (9 on both sides). Of these, 13 were considered to be composed of haploid cells based on the bristle size, one was haploid on one side, diploid on the other. The remaining 18 were diploid by this criterion. All the haploid tissues differentiated as female (Fig. 6b) in agreement with the balance theory of Bridges (1925a). X/O;ZA individuals arise in parthenogenetic Drosophila species (Stalker, 1954) in which the diploid constitution is reached in virgin eggs by incomplete diploidization or by loss of an X after diploidization. Diploidization must also have been incomplete in some of the chimeras obtained by injection of haploid nuclei. Among the 38 chimeras with donor tissue in the first leg basitarsus, 30 had donor tissue in the anterior compartment. Twenty-eight of these thirty patches were female (10 classified as haploids and 18 as diploids) and 2 were male. One of these male forelegs had nine sex comb teeth, four mh, and five y sd, and the other had seven teeth of which all were mh. Both were classified as diploids by the size of the bristles. These sex combs bore fewer bristles than the mean of 11.6 found in normal mh males. Of the 32 chimeras with donor tissue in the terminalia, 27 showed female differentiation (13 haploids, 1 haploid and diploid, and 13 diploids), 4 differentiated male structures, and 1 individual contained male and female diploid donor genitalia. All the male structures differentiated bristles of normal size and thus were considered as diploids X/O;ZA. It is interesting to point out that the female structures (haploids or diploids) appeared as frequently in male hosts as in female hosts, but male differentiation occurred only in male hosts. The five male genitalia and the two male sex combs differentiated in male hosts. DISCUSSION

It is not known why haploid animals fail to survive to adult stages. In anurans and urodeles, in which abnormal combinations of nuclear and cytoplasmic com-

292

DEVELOPMENTAL BIOLOGY

VOLUME 96. 1983

b Frc. 6. Tergite formed by donor cells with 80 microchaetae and lacking clear macrochaetae. The arrows point to split bristles. (b) Chimeric terminalia in which the vaginal plates (VP) and partial anal plates (AP) are formed by small female donor cells. Genital arch (GA), posterior lobes (PL), lateral plates (LP), claspers (CL), and penis apparatus (PA) formed by male host cells.

ponents have been studied, favorite explanations for the causes of death have been either the unmasking of recessive lethal genes or the altered nucleocytoplasmic ratio (for review, see Moore, 1955). Moore (1955) has shown that, on theoretical grounds, it is extremely unlikely that recessive lethals can account adequately for the “haploid syndrome” (Briggs, 1952), and Subtelny (1958) has demonstrated this directly in Rana pipiens. An alteration of the nucleocytoplasmic ratio is probably also not the cause of lethality of the haploid embryos of Drosophila. Since haploid cells can develop and form all cuticular regions of the adult when present in a chimera, it is unlikely that any fundamental property is

disturbed by haploidy in Drosophila. The haploid blastoderm in eggs laid by homozygous mh mothers comprises more cells than normal (Zalokar et al., 1975), and the cells are smaller. Thus, it is possible that the failure of these eggs to develop normally results from an interference with determination due to small cell size. There may be a misfunction in the formation of segments, compartments, or even later in gastrulation or in germ band extension movements due only to increased cell number and an inadequate mechanism for compensation of this variable. Jack and Judd (1979) have proposed a model to account for the interactions observed between the zeste

PEDRO SANTAMARIA

Huploid

and white loci. This model may have more general application for chromosome organization and gene regulation in eukaryotes. They proposed that gene activity might depend upon some type of interaction between alleles and their data suggest that this communication requires close proximity of alleles. The result of a xeste+ phenotype in haploid x w+ cells presented here supports their hypothesis. Also, in the haploid cells of the genetic constitution x w+; w+ (Experiment IV, Table 2) the formation of wild-type eye color is an indication that the two white+ loci were not paired and thus escaped repression by zeste. The possibility exists that a duplication of UJ+present in another region, for instance in a region of ectopic pairing (Kaufmann and Iddles, 1964), would result in the formation of zeste pigmented cells. cells (Morata In mosaics of Minute and non-Minute and Ripoll, 1975) the different rates of cell division allow the progeny of individual Minute+ cells to fill, and thus define, whole compartments. Also, the slower growing population of Minute cells tends to be eliminated. If haploid and diploid cells had different rates of division, then one would expect one of these phenomena to be observed in the haploid/diploid mosaics described here; this was not the case as haploid/diploid boundaries in chimeras were seldom found to coincide with compartment borders over long stretches. Thus, haploid and diploid cells probably have similar rates of division or haploid cells divide faster to fill the same area as would larger diploid cells. Since the frequency with which haploid/diploid chimeras were obtained was high, it is possible that haploid cells may even be favored in competition with diploid cells. In any case the small size of cells itself does not lead to a disadvantage in cell competition. The increase in density of pattern elements in haploid tissue is one of the most interesting results reported here. In plants, with some exceptions, haploid individuals are smaller than diploids (Kimber and Riley, 1963). Haploid/diploid chimeras have been obtained in watermelon (Swaminathan and Singe, 1958), but unfortunately, although haploids are frequently produced in plants, their pattern formation have rarely been analysed. Interestingly the hypohaploid plants produced by Tran Thanh Van (1977) in N. tabacum have one, two, three, or even four more than the normal five anthers, and the pistil is formed by the coalescence of two or three haploid pistils. In amphibians, Muto (1951), Moore and Moore (1953), Subtelny (1958), and Hamilton (1963) have obtained haploid chimeras; in Drosophila such chimeras have also been found (see review by Hall et ~1. (1976)). In general, in the animal kingdom, haploid and polyploid individuals maintain the same body and organ size although the cell sizes are noticeably reduced or increased respectively (Fankhauser, 1955). For the haploid mosaics discussed here, there was a

Chimeras

ix Drosophila

293

general coincidence of smaller cell size (as judged by bristle size and hair density) and increased density of pattern elements. Dubious cases were probably due to the small size of the patch of haploid tissue. Thus, the rule seems to be that in haploid mosaic patches, the pattern elements derived from the smaller haploid cells were more closely spaced than those elements derived from the larger diploid cells. Haploid bristles in chimeras were, as a rule, surrounded by epidermal cells of the same donor origin. However, in one mosaic tergite, where two diploid bristles of host origin were found within a patch of haploid cells, these diploid bristles were more closely spaced than were normal diploid bristles in the adjacent region. This indicates that, although cell and bristle size is autonomous and correlated with ploidy, bristle spacing is more strongly influenced by the size and ploidy of the intervening cells, in the pattern. Wigglesworth (1940) has proposed a model that can explain the increased number of bristles in a field formed by small cells. According to the mechanism proposed by him for Rhodnius (a hemimetabolous insect) a diffusible substance might originally be distributed uniformly throughout the epidermis, be absorbed by extant bristles and be essential for the initiation of new bristle differentiation. The epidermal cells, which originally produce this substance, when sufficiently distant from existing morphogen consuming bristles, respond to a threshold concentration of morphogen by becoming determined to form bristles and then rapidly absorb the substance from the nearby cells, thereby inhibiting determination of additional bristles in their vicinity. This model assumes that bristle determination can occur in any cell within a competent area. It is clear that the rate of consumption (or production) of this morphogen or its rate of diffusion might be changed by the size of the epidermal cells and so distance between bristles could be reduced in a tissue formed by small cells. The fact that each patch of haploid tissue is autonomous for the density of bristles in the notum, legs, and tergites, even if it is completely surrounded by diploid tissue, is relevant to our understanding of how the pattern is controlled. A system of positional information (Wolpert, 1969) in which the positions of pattern elements are specified by thresholds of a gradient does not provide for interferences due to cell size. Bristles resembling split were found in the haploid organs. We can discard the presence of the mutant in the strain since none of the homozygous diploid mh flies of the strain showed these characteristics. In the chimeras studied here, the split phenotype was always restricted to the haploid cells, even when these cells were very close to the border with diploid cells. Lees and Waddington (1942) indicate that the split effect might be caused by an extra division of the initial bristle form-

294

DEVELOPMENTAL BIOLOGY

VOLUME 96, 1983

ing cell. I therefore think that the small size of cells FANKHAUSER, G. (1955). The role of nucleus and cytoplasm. In “Analysis of Development” (B. H. Willier, P. A. Weiss, and V. Hamburger, provokes the phenocopy of the mutant split, perhaps by eds.), pp. 126-150. Hafner, New York. allowing the initiation of an extra division of the bristle FRASER, A. (1967). Variation of scutellar bristles in Drosophila XV mother cell. System of modifiers. Genetics 57, 919-934. GANS, M. (1953). Etude genetique et physiologique du mutant z de Lindsley et al. (1972) identified eleven haplo-abnormal Drosophila melanogaster. Bull. Biol. FT. Belg (Suppl.) 38, l-90. loci in aneuploids, one on the X chromosome (N), four GANS, M., AUDIT, C., and MASSON, M. (1975). Isolation and characon the second (S, b, vg, and Px) and six on the third terization of sex-linked female-sterile mutants in Drosophila melchromosome (Ubx, Iw, Dl, H, WD, Spl) apart from the anogaster. Genetics 81, 683-704. Minutes and the region 83D-83E that are also haploGARCIA-BELLIDO, A. (1968). Cell affinities in antenna1 homeotic mutants of Drosophila melanogaster. Genetics 59, 487-499. insufficient. None of these mutant phenotypes appeared GARCIA-BELLIDO, A. (1972). Some parameters of mitotic recombinain haploid spots. Only Star (S), which shows the artion in Drosophila melanogaster. Mol. Gen. Genet. 115, 54-72. rangement of bristles on the surface of the eye irregGARCIA-BELLIDO, A. (1981). From the gene to the pattern: Chaete ular, and Delta (Dl), which increases the number of acdifferentiation. I?[ Cellular Control in Differentiation (C. W. Lloyd rostical bristles, could be suspected to appear, but Star and D. A. Rees, eds.), pp. 257-304. Academic Press, London/New York/San Francisco. or Delta have other effects that did not appear in hapHALL, J. C., GELBART, W. M., and KANKEL, D. R. (1976). Mosaic sysloid patches. We must conclude that the haplo-insuffitems. 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