Parameters of the wing imaginal disc development ofDrosophila melanogaster

DEVELOPMENTAL

BIOLOGY

Parameters

24, 61-87

(1971)

of the Wing of Drosophila

lmaginal

Disc Development

rnelanogaster’

A. GARCIA-BELLIDO AND J. R. MERRIAM Centro de Investigaciones Biologicas, Institute de Genetica y Antropologia, Velazques, 144, Madrid-6, Spain; and Department of Zoology, University of California Los Angeles, California 90024 Accepted September 26, 1970

We dedicatethis paper to the memory of Alfred Henry Sturtevant, who gave to us so generously of his time and knowledge. His interest and enthusiasm for science did much to encourage us. INTRODUCTION

Any experimental approach to a developing system must be preceded by a detailed analysis of the parameters of its normal development. Such parameters include cell numbers, mitotic rates, and orientation of cell divisions. The imaginal discs of insects represent isolated and simple systems in which such analyses have been successfully attempted. The proliferation dynamics of the eye-antenna1 discs of Drosophila has been studied by measuring increase in volume of the whole disc (Medvedev, 1935), by direct cell counts in stained sections (Chevais, 1943) and in squashes (Becker, 1957). Moreover, Becker studied mitotic rates in the same system and Liibbecke (1968) working with wing discs of Ephestia, analyzed the cell cycle with autoradiographic techniques. The growth parameters can be studied further in an organism with cell marker mutants by means of somatic crossing-over. Crossing-over can be induced by X-irradiation at different developmental times and the derivative clones of cells homozygous for the cell marker mutant analyzed in terms of frequency, size, shape, and determinative characterisitics (Stern, 1936). Becker (1957) has used this method to study the clonal development of the Drosophila eye thoroughly. ’ The experimental portion of this work was performed at the California Institute of Technology in the laboratory of Dr. E. B. Lewis and was supported by a Gosney fellowship to AGB and a United States Public Health Service postdoctoral fellowship to JRM. 61

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The dorsal mesothoracic discs of Drosophila differentiate during metamorphosis into flat cuticular surfaces provided with chaetes and trichomes. They are organized into a rigid pattern which form the mesonotum and the wings. Trichomes are single cell processes (Dobzhansky, 1929), and chaetes derive from a mother cell by two differential mitoses which give rise to (two) nervous elements and to two cuticular structures, the trichogen and the tormogen (Stern 1938; Lees and Waddington, 1942). There are mutants in Drosophila which change the final differentiation of both trichomes and chaetes, and can be used as cell markers. Here we will present data on a clonal analysis of wing disc development using cell marker mutants. A summary of the present results has been published elsewhere (Garcia-Bellido, 1968). MATERIALS

AND

METHODS

The cell marker mutants were: yellow Cy, I-0.0) covered by Dp(l:3) scJ4 0, +), localized about 4 units to the left of ru and distal to mwh, javelin (ju, 3-19.2, 64C-65E) used respectively as color and shape markers, detectable only in the trichogen element of the chaetes (bristle); multiple wing hair mwh, 3- to the left of ue (GarciaBellido and Merriam, 1969c), 61E-62A (Lewis, 1969), was used as marker of the trichomes (hairs) (Peyer and Hadorn, 1966). The treated individuals were females of the genotype Of(l) SC’, y- w”/y; Dp(l:3) scJ4, y+ jvlmwh. After somatic crossing-over in the proximal part of chromosome 3L, spots which include yellow bristles and multiple-wing-hairs and twin spots marked by javelin bristle can be found (see Fig. 8, for example). Somatic crossing-over (SCO) was induced by means of X-ray irradiation at 50 kV, 20 mA, with a Be window and a 2 mm Al filter at the rate of 330 r min and a final dose of 1000 r. In order to have (1) individuals of different ages irradiated under the same conditions and, (2) an accurate timing of the irradiated stages around puparium formation (PF), the following procedure was used. Parents of the larvae to be irradiated were kept during a 4day egg-laying period on a well yeasted medium. One day after withdrawal of the parents, larvae and pupae were washed and irradiated in a plastic dish. Pupae were classified by the following stages: those of O-4 hours have an unpigmented puparium and do not float in distilled water, those of 4-8 hours have a pigmented puparium but do not float, those of 8-12 hours float but their mouthparts have not yet

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everted, and those of 12-24 hours have everted mouthparts. The larvae were transferred to new medium from which the new pupae were collected every 8 hours. The age of the larvae at the moment of irradiation is given by their delay up to PF in negative hours. In this way, the dispersion of the natural age with respect to the physiological age at PF is substantially diminished, and we may have in a single experiment comparable values of different developmental ages. Data presented on Tables 3, 4, and 5 correspond to the pooling of three experimental replicas. In order to show the internal variations of the present method, results of a single experiment are presented in Figs. 3, 5, and 6. The adults were classified according to sex and phenotype, but no selection of presumptive mosaics was done. After dissection and cooking with 10% KOH, the dorsal mesothoracic structures of both sides were mounted in euparal for microscopic examination. RESULTS

Controls Description of the adult mesothorax. The nomenclature of the bristle types, patterns, and regions of the adult mesothorax, shown schematically in Fig. 1, is the same as used elsewhere (Ferris, 1950;

DR

FIG. 1. Normal half mesothorax showing the labeling employed in this paper. Circles: macrobristles; N, notum; SC, scutellum; Hp, humeral plate; Cp, Cm, Cd, costa, proximal, medial, distal; TR, triple row; DR, double row; A-E: regions of the wing surface.

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TABLE 1 CUTICLE CELL NUMBERS’ Regions

Hairs

Notum

9,300

Scutellum Wing (dorsal) A’

1,300

Total

Bristles MB mB MB

2,700

A

1,300

B C’ C D’ D E’ E

1,800 1,000 2,200 1,000 2,100 500

2,800 26,000

MTR D + VTR DR

9 102 2 51 80 74 45 28 85 13 110 588

‘Number of cell structures in different regions of the adult mesothorax. The labeling of the regions corresponds with Fig. 1. MB, mb: macro and microbristles; TR: Triple row, D, M, V: dorsal, medial and ventral. The figures of hairs on the wing surface correspond only to the dorsal surface.

Garcia-Bellido, 1966). Table 1 shows the average number of bristles and hairs characteristic of the different regions. These figures represent average values of 10 half mesothorax of well fed Canton-S females. The same regions in the ventral and dorsal surfaces of the wing yield similar values. Each adult hair of the wing corresponds to a single process of an epidermal cell. Histological observations (Dobzhansky, 1929, Waddington, 1940) show further that all the cells are represented by a hair in the wing surface. In the notum, single mwh spots indicate that here also each hair corresponds with a cell. Notum and wing differ in density of hairs in both surfaces, the hairs of the notum being about 4-5 times more densely packed. Table 1 does not present data on the dorsal and ventral hinge hairs because of the difficulty in their evaluation. The hairs here are small and densely packed. The ventral part of the mesonotum must be small since the neighboring sternopleural bristles derive from the leg disc. Thus, we estimate the number of cells on the half adult surface to be about 52,000. The

IMAGINAL

65

IN DROSOPHILA

DISC GROWTH

number of adult cells should correspond to the number of cells in the wing disc when the last mitoses are taking place. This moment corresponds to PF + 24 hours (Stumpf, 1956). Rough estimates of these numbers by means of squash preparations yield values of 50,00054,000 final cells. To reach this number of adult cells, 15.66 dichotomic divisions are necessary (log 2 of 52,000). Timing of irradiated larvae. Ages calculated with respect to puparium formation (PF) and with respect to egg laying (EL) should yield complementary values. However, irradiation is known to affect the rate of development as well as viability. 278 Canton-S larvae were irradiated with 1000 r at given ages after egg laying and compared with nonirradiated controls in order to ascertain both their delay until PF and their mortality during the pupal stages (Table 2). Larvae irradiated at 96 and 72 hours after egg laying reach PF at about the same time as nonirradiated larvae. There is no pupal mortality. However, larvae which are younger at the time of irradiation show a delay of PF with respect to their controls. The pupal mortality also increases. This delay is very significant in early first instar larvae irradiated at 24 hours. Spontaneous frequency of SCO. The experimental interference due to spontaneous cross-over clones was studied in nonirradiated adults of the same genotype. We found 15 clones in 302 half mesonoturns (O.O5/wing disc). Among them, 3 had single y bristles and 12 had mwh clones (7 with 1 cell, 2 with 2, 1 with 32 and 1 of about 400 cells). X-ray induced malformations. Since the scoring of cross-over cells is based upon their final phenotype it is necessary to study the freTABLE IRRADIATION-INDUCED

Age after EL at irradiation 96 72 48 24

f f f f

2 2 2 2

Nonirradiated Pupae 71 70 132 101

2 DELAY’

controls Age at PF 130 130 130 130

* 10.1 f 8.3 zk 12.4 zk 8.1

Irradiated Pupae

Adults

111 210 253 220

111 210 250 212

a Delay at puparium formation (PF) of Canton-S different ages after egg laying (EL).

larvae irradidted

Delay at PF 0.0 4.9 10.5 25.4

It 10.2 f 16.1 f 16.8 f 24.0

with 1000 r at

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quency and nature of genetic-like modifications following 1000 r irradiation. This is especially important since malformations induced by X-ray are characteristic for different developmental stages (Wadding-ton, 1942; Naville, 1955). Canton-S larvae and pupae were treated and scored under the same conditions as experimental individuals. Under the present conditions we found both morphogenetic malformations and cell modifications. Among the former the most frequent (O.O7/wing disc) is a characteristic scalloping of the wing margin, which is not important in our context. Among the cell differentiation modifications the following phenocopies appear frequently: ss-like or bb-like (42%) spllike (23%) &w-like (18%), ss-&w-like (14%), and su-like (3) (for identification of these mutants, see Lindsley and Grell, 1968). y phenocopies are very rare and easily distinguishable from stw under the microscope, due to their different color. ju phenocopies were never detected. The frequencies and regional specificities for all these modifications are presented in Fig. 2. It is interesting to note that the frequency of the bristle modifications varies depending on the structure modified. Considering the number of targets affected, the more sensitive parts are the macrobristles of the notum (13%) and the bristles of the MTR (17%). The microbristles of the notum are much less sensitive (1.8%). The sensitive period is also variable, but shows two maxima at PF + 8 and PF + 16 hours for the microbristles. Macrobristles are no longer modifiable after PF + 8 hours. Hair cells are also modifiable. Their sensitivity period is very late in the pupal development (PF + 24 hours), and the frequency of modification was very low for the dose used. mwh is never found as phenocopy under the present conditions.

Hair Clones In the present experiments the induced recombinant cells are of two kinds: y; mwh and jv. The scoring of y and jv is possible only in bristles whereas mwh manifests itself only in hairs. In the notum, where both bristles and hairs appear scattered over the whole surface, all the bristles lying within the mwh clones are y. Only a small fraction (l/81) of the clones with several y bristles do not show mwh hairs between the marked bristles. Thus, mwh hairs and y bristles are different manifestations of the same genetic event. However, the analysis of the induced clones will be considered separately for hairs

IMAGINAL

l

MB

o mB A

B

DISC GROWTH

67

IN DROSOPHILA

NOTUM MTR

E 5-

t

m

2

4 \ %

1 -3b

-24

-12

DEV. TIME

Pt-

+12

I +24

(hr)

FIG. 2. Malformations induced in bristles by 1000 rX-rays. The different kinds of malformations are pooled. Each value is based on an average of 58 half mesothorax. MB, macrobristles; mB, microbristles; MTR, medial row of bristles in the triple row.

and for bristles because of the different densities, numbers, and pattern organization of these elements in different regions. Frequency of mwh clones at different developmental ages. ln the notum the density of the adult hairs and their small size hinders an accurate analysis of the frequency and size of the mwh clones, especially in clones induced later in development. mwh clones are easily storable in the wing surface. The number and size of induced mwh spots of the same wing region yields identical values for both the ventral and dorsal surfaces. Thus, we will pool the data of both surfaces. mwh hair clones appear after irradiation of heterozygous flies at all developmental stages up to PF + 21 hours old (Table 3, Fig. 3a). In the class of PF + (12-24) hours, 75% of the analysed mesonota show many mwh clones, 23% show no mwh clones, and 2% show a

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TABLE 3 FREQUENCYAND SIZE OF ~~~CLONES IN THE WING SURFACES~ Age at irradiation (hours)

Number of WD

12-21 8-12 4-8 o-4 8-O 16-8 24-16 32-24 40-32 48-40 56-48 64-56 72-64 80-72 88-80 120-88

14 13 16 4 12 12 39 84 38 157 86 90 106 48 86 36

Number of clones 2370 1311 1126 256 737 360 417 370 130 258 82 29 11 4 5 1

Clones per WD

Cells per clone

233.5 100.8 70.4 64.0 61.4 30.0 10.7 5.9 3.4 1.6 0.9 0.3 0.1 0.08 0.06 0.02

1.0 1.1 1.2 1.3 1.5 2.1 4.5 9.8 17.1 38 54 126 300 400 700 loo0

Size dispersion in n of cell divisions

Sensitivity per 100 cells

0.3 0.3 0.5 0.7 1.2 1.2 1.5 2.1 2.0 1.7 0.8 0.9 0.8 0.4 0.3 -

‘Frequency and size of mwh clones in the wing surface. Ages of induction respect to PF (0 hours). WD: wing disc.

1.6 0.8 0.5 0.5 0.6 0.4 0.3 0.4 0.4 0.4 0.3 0.3 0.2 0.2 0.2 0.1 with

very small number of them. Additional studies indicate that after 21 hours the wing cells in pupae are no longer sensitive to induced somatic crossing-over. At this time the last divisions before cell differentiation are probably occurring. Waddington (1940) reported the last visible mitosis in pupae 24 hours old, although he assumed the existence of a second mitotic period lasting until 46 hours. Stumpf (1956) detected the last mitosis at 24 hours after PF. The difference between the reported cessation of mitosis at PF + 24 hours and the last SC0 sensitive period at PF + 21 hours may be due to differences in the experimental conditions or to insensitivity of premitotic cells to induced SCO. The frequency of mwh clones increases exponentially from the youngest treated larvae (-120 hours) up to 21 hours after PF. This is to be expected on the hypothesis that the length of the mitotic cycle, from one division to the next, is constant and can be measured by the time necessary to double the number of mwh clones per wing imaginal disc. We calculate that 8.5 hours, on the average, is the time of doubling the frequency of clones per wing disc. This increase

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follows a different rate for the different regions of the wing. In prepupae the percent of clones corresponding to every wing region corresponds to the percentage of adult hairs in this region. These are region A = 10.6%, B = 14.7%, C’C = 26.2%, D’D = 25.4%, and E = 23.1%. In larval stages, region A embraces 14.6% of all the wing clones, region B 20.0%, 24.2% in C’C, 24.0% in D’D, and 17.3% in region E. Thus, region A and B have more, and region E fewer, clones than expected for equal growth rates or equal cell sensitivity to induced SCO. At a given age the observed distribution of numbers of spots on the wing fits well with the expected values according to a Poisson’s distribution. That would mean that the different wing discs WING mwh HAIRS

0

-96

-72

-48

-24

DEV. TIME (hr) s

IO 5

t

24

’ F (b)

E s 0.5 ‘\ g 0.1 z 0.05 0 d 0.01 cj .005 Z

FIG. 3. Frequency and size induced througiwut development tle (b) clones. Each value is based on 18 half mesothorax.

of mwh (a) and y bris-

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GARCIA-BELLIJIOAND MERRIAM

have a random population of sensitive cells. However, comparing the frequency of events in different regions of the same wing significant differences can be detected. Larvae treated at PF - (48-24) have an average of 0.5 spot per wing in both region AB and DF. Among 202 analyzed wings there were 43 cases with two events in region AB and 35 cases in region DE. However, we found only 40 cases with one event in AB and another in DE, although the expected value would be 78 (x’ = 18, p = 0.001). This would suggest that the different regions pass over sensitive periods at different moments. This finding is reminiscent of the detected mitotic waves in the developing pupal wing (Stumpf, 1956). Size of the mwh clones at different developmental stages. The number of mwh cells per clone decreases with the age of the treated larvae (Table 3, Fig. 3a). This is to be expected on the hypothesis that the size of a clone is inversely proportional to the number of presumptive wing cells in the imaginal disc at the time of irradiation. When irradiation is performed at the time of PF or later the clones contain only one cell. During this period Stumpf (1956) detected a very high mitotic index with a maximum at PF + 17 hours. These mitoses probably represent the last ones of the disc, since clones only contain a single mwh hair. The size of the clones decreases exponentially with increasing age of the treated larvae. However, at irradiation single clones do not necessarily contain 2” cells. This deviation from a pure exponential growth can be detected in small clones. Thus, we find clones of 1, 2, 4, and 8 cells but also clones of 3, 5, 6, and 7 cells. In this range, 2” clones are 6 to 3 times more frequent than other sizes. Deviations from the expected 2” ratio are even greater in big clones. Table 3, column 6 shows the degree of variation in the number of cells per clone at different stages. Variation in the clone size roughly follows a binomial distribution. In general 1 sigma (68% of the cases)includes sizes within the margin of a single cell division. The maximal dispersion is 4 mitotic classes. In another system, Becker (1956) found in first-instar larvae (17-23 hours old) deviations of 7 mitotic classes in the number of facets belonging to a clone. Becker also notes that an important part of this deviation is due to regional differences in the growth rate. Table 4 presents data on the number of cells per clone in the different regions of the notum and wing throughout development. In general, the notum and region A and B of the wing, which are anterior, show clones containing about half the number of cells

IMAGINAL

TABLE

AVERAGEmwh

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CLONE

SIZES’

Wing surface Age at irradiation (hours)

Number clones

,If

A

C’C

D’D

T L

E

dumber f cloner

N

SC

1.31 52 1.10 1.10 1.07 1.Y2 1.21 49 1.10 1.07 69 1.18 2.0 1.25 1.13 2.2 18 1.20 1.25 1.51 46 1.6 3.5 1.8 1.5 4.0 2.2 29 2.4 3.3 19 2.8 3.2 4.1 4.6 12 8.2 13.1 5.1 9.2 14.0 7 12.5 18.8 30.0 14 28 16 40 62 6 43 20 62 98 20 75 230 I.oo 7 3 1LOO m 5IO0 !OO I I a Size of the clones in different regions of the mesonotum and wing. Clones which embrace two or more areas are classified in the region with more marked cells. 12-21 8-12 4-8 o-4 8-O 16-8 24-16 32-24 40-32 48-40 56-48 64-56 88-64

2072 1281 1078 248 732 350 402 346 130 251 80 29 22

1.04 1.03 1.04 1.12 1.1 1.6 3.4 7.2 12.5 38 41 88 !590

1.10 1.20 1.27 1.42 1.7 2.6 4.2 10.3 15.8 38 75 180 i10

1: L

of those in the scutellum and region E, which are posterior. Central regions (C and D in the wing) yield intermediate values. Thus, the real dispersion in clone size for a given region and age is small. The largest clones found, at PF - (120-80) hours, contain about 1000 cells. Larvae irradiated soon after EL were exposed to only 500 r in order to avoid developmental delay. In 350 analyzed half notums 2 spots (with 1000 and 1200 hairs) were found. Thus, at the time of the hatching of the larvae, the clones contain about l/50 of the final number of cells. That would indicate that the imaginal disc contains at this age about 50 cells. Becker (1957) counted 150 cells in squashes of the eye-antenna1 disc at the end of the first larval instar. The spontaneous frequency of mwh spots here is 0.04 per wing disc, which is the same order as the spots induced at PF - (120-80) hours. However, the small spot size of the spontaneous spots (page 63) indicate that the majority of them arose in late stages of development. Thus, the actual spontaneous frequency at early stages, which are recognized as large clones, is significantly less than the frequency of induced spots. Cell sensitivity to SCO. Table 3 and Fig. 3a show that with increasing age of the larvae at irradiation the size of the clones de-

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creases, and, reciprocally, the number of clones per wing imaginal disc increases. The product of both values is approximately constant throughout development. Stated otherwise, we observe that somatic crossing-over induced at any age uncovers about the same total number of mwh cells per wing, although the number of crossing-over events increases in older larvae. This indicates that the same proportion of cells in the imaginal disc at each stage are “hit” (undergo crossing-over) so that the cell sensitivity to induced crossing-over is constant throughout development. Calculation of a numerical estimate of the fraction of sensitive cells (those undergoing crossing-over) can be done for larvae in any stage by dividing the average number of clones induced per wing imaginal disc by the number of presumptive hair cells present in the disc at that time. The estimate of the number of presumptive hair cells per disc is calculated from the reciprocal proportion of twice the average number of mwh cells per clone relative to the number of cells per wing in the adult, so that for a given time % sensitive cells =

2~ cell/clone x clones/WID number of adult cells

The number of adult cells of the wing surfaces is 30,800. IJsing this formula the estimates listed in Table 3 show that 0.2% to 0.4% of the presumptive hair cells are sensitive to crossing-over in the left arm of the third chromosome. This estimate is markedly lower than Becker’s (1957) estimate of the percentage of presumptive eye facet cells sensitive to crossing-over in the X chromosome (see also Becker, 1969). However, the percentage of presumptive wing hair cells sensitive to crossing-over in the 3L chromosome rises to 1.6% in the final division (after puparium formation). The reason for this change is not at all understood. The fraction of cells undergoing mitotic division in any &hour period can be followed indirectly either by computing the ratio of the average number of clones per wing in an 8-hour interval to that of the preceding interval, or by computing the ratio of the average size in number of cells per clone in an &hour interval to that of the subsequent interval. In Fig. 4 these estimates are plotted against developmental age. At the ratio of two, 100% of the cells on the average, undergo a single mitotic division. Both curves appear to oscillate

IMAGINAL

-96

-72

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-48 DEV.

IN

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-24 TIME

PF

73

+24

(hr)

FIG. 4. Rate of growth and SC0 response of wing cells at &hour intervals. derived from data in Table 3 as discussed in text.

Values

simultaneously with notable decreases at PF-72 hours, at PF-48 hours, and at PF-8 hours. These ages correspond roughly to the first, second, and prepupal molts. Since these estimates depend on different pieces of data and are thus independent, it seems likely that their agreement is significant and that molting somehow inhibits the mitotic cycle. Bristle Clones Frequency and size of clones containing bristles. The genetic setup shown in methods allows us to detect cross-over cell clones differentiating into hairs (m&z) or into bristles (y), as well as twin spot clones when they contain bristles (ju). y;mwh clones as well as ju clones may contain, besides hairs only detectable in ymwh cells, one (single clones) or several (multiple) bristles. We will deal first with y;mwh clones. Table 5 and Fig. 3b show the frequency of y spots (single or multiple) in the acrostichal bristles of the notum and medial-triple-row bristles of the wing, at different development stages. We know that two y bristles belong to the same clone when they are connected by mwh hairs. This is very clear in the acrostichal bristles of the notum but in the medial-triple-row the bristles lie next to each other and

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TABLE 5 FREQUENCY AND SIZE OF y BRISTLE CLONES’ MTB Bristles

T Microbristles

Age at ir radiation 1_

12-21 8-12 4-8 o-4 8-O 16-8 24-16 32-24 40-32 48-40 56-48 64-56 72-64 80-72 88-80

Macrobristles

Number of WD

Number of clones

Che5/ WD

Bristles/ ClOTE

163 106 76 86 126 50 70 124 75 113 90 62 76 26 14

476 220 155 196 351 69 93 90 23 45 19 10 7 3 2

2.9 2.1 2.0 2.3 2.8 1.4 1.3 0.7 0.3 0.4 0.2 0.16 0.09 0.11 0.14

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.1 1.1 1.7 2.4 5.4 6.0 11.5

Phmber of Iwistles

0 0 6 12 28 6 3 5 2 2 -

I

Bristles/ WD

Number If clones

0 0 0.18 0.14 0.21 0.12 0.04 0.04 0.03 0.02

299 199 132 118 193 38 37 40 6 11 9 4 1 1 1

-

1.8 1.9 1.7 1.4 1.5 0.8 0.5 0.3 0.08 0.09 0.10 0.06 0.01 0.04 0.07

1.0 1.0 1.0 1.0 1.0 1.0 1.3 2.0 2.5 3.5 7.2 14 36 9 19

’ Frequency and size of clones with y bristles in the notum and in the triple row. Data on macrobristles (MB) marked before PF-48 hours are included in the mB class.

are not surrounded by mwh hairs in small spots. In this case the criterion of adjacent y bristles belonging to the same clone depends on the frequency in which such events appear at a given age. Whe irradiation is performed during the pupal period, the majority of the y bristles of the medial-triple-row are single. In this case the frequency of events per wing imaginal disc is high enough so that the few cases of two adjacent bristles marked with y can be considered as due to two independent events. The frequency of induction of marked bristles increases exponentially with the developmental age. However, in both notum and medial-triple-row the exponential increase stops at about PF - 12 hours and the frequency drops at PF + 8 hours. Two alternative explanations may clarify this situation. Either there is a genuine drop in sensitivity of the prospective bristle cells or there is a decrease in the number of target cells. The second explanation would mean that the differential divisions of the bristle organ cells are actually

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restricting to one-half and then to one-fourth the storability of y trichogene structures. This interpretation is reinforced by the following facts: (1) histological size differences between epidermal and bristleforming cells are first detectable about 15 hours after PF (Lees and Waddington, 1942). (2) presumptive bristle cells are already determined at PF-8 hours (Garcia-Bellido and Merriam, 1968). (3) X-ray induced phenocopies interfering with bristle differentiation appear at the same time as the drop found in crossing-over (compare Fig. 1 and Fig. 3b). (4) neither y bristles nor mwh clones can be detected following irradiation after PF + 21 hours. The presumptive macrobristles of the notum follow a slightly different pattern with developmental age than do the acrostical bristles (Table 5). They are not sensitive to either induced somatic crossing-over of phenocopies after PF + 8 hours. It would not be surprising if it were found that their differential divisions take place earlier. The lower frequency of y bristle spots induced in the medial-triple-row compared to the acrostichal bristles might be due to the difference in the number of presumptive target cells. After irradiation at PF + 8 hours, when both kinds of cells are determined to be bristle-forming-cells, clones in the notum are 1.2 more frequent than in the MTR, but they have 1.4 times more targets to differentiate into bristles. During the larval periods when no difference between presumptive bristle-forming and presumptive hair-forming cells exists, induction of marking of cells in the notum gives about two times more bristle spots than do the prospective medial-triple-row cells. That again might be due to the fact that growing cells in the notum have a higher probability of eventually being able to differentiate into one or several bristles than do the cells in the wing, where only those cells lying in the prospective medial-triple-row will have the opportunity of differentiating into bristles. The number of bristles per clone decreases exponentially with the age of induction to reach one when irradiated before PF. Spots in the medial-triple-row induced during the larval period embrace more bristles than do spots induced at the same time in the notum. This is again easily understood by the fact that MTR bristles lie more tightly packed than acrostichal bristles. When a spot embraces two bristles in the notum, it covers the distance between with mwh hair cells.

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Twin clones. The analysis of twin clones depends first on the relative storability of the corresponding cell markers. Induced clones with several ju bristles (multiples) are about 0.63 times as frequent as multiple y clones (51/81) in the notum and wing. This may be due in part to the occurrence of somatic crossing over distal to ju on the 3L chromosome arm. When we compare the number of bristles per clone in twin y and ju clones the relation is about 1: 1, (193y : 17Oju), indicating that ju cells differentiate into bristles as frequently as y cells and that both have the same expressivity. The lower frequency of ju clones is then probably due to the existence of frequent crossovers distal to ju. Due to the lower frequency of ju clones, we will analyze the frequency of twins relative to the frequency of ju clones. Figures 5a and 5b show the appearance of y-ju twins, single or multiple, and multiple bristle ju clones induced at different stages of development. If bristle-forming lines are determined before metamorphosis (GarciaBellido and Merriam, 1968), we should expect that clones containing two or more ju-marked bristles cannot be induced as late in development as can the y-and ju-marked twin spots. In both notum and MTR this is in fact the case. However, notum and MRT differ with respect to when in development the irradiation induces twin spots or multiple bristle spots. MTR twins appear following irradiation as late as PF + 4 hours. This means that a cell marked after this time does not give rise to more than one bristle. In the notum twins are not detectable following irradiation after PF - 24 hours. (The low percentage of twins in the notum induced later are probably spurious ones, due to the probability of finding adjacent y and jv bristles not connected by mwh hairs but classifiable as possible twins). For both notum and MTR the difference between the latest times of induction of twin spots and multiple ju bristle spots is about 12 hours. This value may be significantly different from the division time (8 hours, 30 minutes) calculated for the growing presumptive hair cells. The simultaneous uncovering of a hair marker and a bristle marker in a recombinant cell allows us to analyze twin clones embracing ju bristles and mwh hairs and multiple cell mixed clones of y bristles and mwh hairs. Figure 6 shows the results of such an analysis. In order to have a bristle and a hair of the same clone, at least two cell divisions are necessary following SCO. Contrary to expectation mixed clones of bristles and hairs do not follow the theoretical

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(a) NOTUM . TWINS y-jv 0 MULTIPLE jv

-96

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-72

I

/

i!

-48

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DEV. TIME (hr)

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ptF

,_

II ,t,

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FIG. 5. Frequency of induction throughout development of ju clones with multiple bristles and ju and y twin bristle clones in the notum (a) and in the MTR (b). Data from the experiment of Fig. 3.

delay of one cell division difference in the latest times of induction. Clones of y bristles embrace mwh cells only when induced before PF - 40 hours. It is also at this age at irradiation when ju bristles have a twin mwh spot. However, irradiation at PF - 40 hours may yield clones of mwh containing about 10 hairs and the MTR clones of about 4 bristles. The preceding analysis indicates that at about PF - 40 hours cells will differentiate into two separate developmental pathways leading to several hairs and several bristles, or at least, to one bristle and four hairs. In fact, the actual minimal number of hairs which appear associated with a y bristle or in a twin with a jv bristle is 4. In the

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last case on one bristle would be equivalent to four hairs. Thus, two differential divisons would give rise to one bristle organ and two equal divisions give rise to four hairs. Cell sensitivity of bristle-forming cells. We will study the cell sensitivity of the presumptive bristle cells in the similar way to the analysis carried out with the prospective hair cells of the wing surface. Table 5 gives the frequency of y clones obtained after irradiation at different stages along development. The number of final structures for every analyzed region is given in Table 1. Using the formula on page 70, it is possible to calculate the cell sensitivity of the presumptive bristle cells. The sensitivity to induction of SC0 for bristle precursor cells at PF - 40 hours is 0.6% in the notum and 0.5% in the MTR. These values are very similar to those found in presumptive hair cells of the wing surface at the same age. This is an indication that cells are still undifferentiated. Afterward, the sensitivity of the bristle precursor cells increases and levels off during the pupal period when the bristle-forming cells undergo the differential divisions. During this period, the mean sensitivity is about 4.7% for the acrostichal bristles, 3.0% for the macrobristles of the notum, and 4.1% for the MTR bristles. Oscillations around these values may be due to

MIXED CLONES OF BRISTLES +HAlRS

-48

-24

DEV. TIME(hdPF

?

2

FIG. 6. Number of cells in pure hair clones (II) and in bristle- hair (BH) clones induced throughout development. Data from the same experiment of Fig. 3.

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CLONES

[email protected] i

ws

. . . . .

. . . . .

. . . . . . . . . . . . . . . . . . . . . . ..x......... . . . ..xxx......... .x. x..xxxx.x. ..x. .xXxXxX.. xx. -xxx. xx~~xx~~x~~~xxx~xx~ . . . . . . . . . . ..x.xxx.. . . . . . . . . . . . . ..xx... . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . ..xxxxx . . .xXxX . . . . . . . . . . . . . . ..xxxxx DR4j i

. . .._._.._.....___._. ..................... ..................... .....................

I

FIG. 7. Characteristic clone topology in different wing regions. Symbols as in Fig. 2. Dots and black symbols: normal cells; crosses: mwh cell; white signs: y bristles; dashed cricles: ju bristles.

the cells being in different periods of the mitotic cycle, but it is very difficult here to assess a sensitivity of a given period. Clone Morphology Topological characteristics of the clones. The shape of the y;mwh clones is an indication of the way the wing disc grows. The final shape of the clones, however, may come about by two distinct mechanisms. On the one hand, morphogenetic movements of the entire growing blastema could determine the passive molding and stretching of the clones. On the other hand, the final shape of the wing may result from the combined growth of the single wing cells. The following observation points toward the suggestion that a main factor in wing morphogenesis is the result of the orientations of the

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single cell divisions. Stumpf (1956) found that during the pupal period two consecutive mitotic waves occur. During the first one (1519 hours) cell divisions are oriented preferentially along a transversal axis, whereas, during the second wave (20-23 hours), the cells divide along the longitudinal axis of the wing. These observations support an internal determination of the clone shape. Large mwh clones induced in young larvae show an elongated shape with the proportions of about 10 times longer than broad. The general orientation follows the proximodistal axis, but occasionally clones deviate and cut across the wings. The limits of the clones are undetermined, as can be seen by superposing different clones of the same regions. The border line of the clones is never smooth, but

FIG. (a) a y notum. gion B.

8. Clones of y mwh and ju twin cells induced in growing wing imaginal discs. mwh clone with its ju (0) twin in the region of the dorsocentral bristles of the (b) a y (0) mwh and jv (0) clone in the MTR region. (c) a y mwh clone in reObserve the splitting of the clone.

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with many indentations of several hair rows. This presumably shows changes in the orientation of the mitotic spindle. In the notum also, the mwh clones are more irregular and their shape is preferentially square. Small clones, however, show a L- or Z-shape, again reflecting changing orientations of the cell divisions. The topology of the clones in the wing surface shows some peculiarities. y;mwh or jv clones induced during the larval period never cross over the margin separating the dorsal and ventral wing surfaces. The margin separates medial from ventral triple row in the anterior wing edge and the dorsal from ventral double row (DR) in the remaining edge (Fig. 7). Clones running along the margin on either surface are of similar in size to clones running parallel and separate from the margin. However, in gynandromorphs, following early loss of a ring chromosome, clones may overlap and cross over the wing margin (Garcia-Bellido and Merriam, 1969b). Clones marking the wing blade appear in general as compact surfaces of mwh hairs. However, sometimes these surfaces are split into two or more spots of different sizes along the direction of growth (Figs. 7 and 8). These mwh spots are separated by one or several rows of normal hairs. The origin of the so-called “split spots” is most likely not due to independent events occurring in adjacent cells, since the “split spots” may differ greatly in size. In young treated larvae the occurrence of two events is against the Poisson expectation. Normal hairs never appear in the middle of mwh spots, so this separation probably does not represent incomplete penetrance of mwh. We interpret these clones as due to partial separation of the cells of a clone during development owing to internal tissue pressures or independent cell movements. DISCUSSION

In experiments of X-ray-induced SCO, the cell phenotype is not a reliable index of genetic homozygosis unless several previous tests are undertaken. Irradiation of 1000 r induces some phenocopies in growing wing disc cells. Phenocopies of the jv and mwh markers were never found, but y phenocopies apparently form a low percentage of those modifications which change the color of the bristles. Furthermore, these modifications are restricted to a limited period of time toward the end of development. Recognition of genetic homozygosis preceded by a cross-over depends on the distance on the chromosome between the marker and

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the centromere and on the autonomy and expressivity of the markers used. Bristles lying within mwh clones of the notum were always markedly yellow. In clones with multiple bristles, the mean number of y and ju bristles is the same, suggesting a similar expressivity for ju. Among 81 multiple clones with y bristles, 1 (1.2%) did not embrace mwh hairs and 30 (37%) lacked the twin ju clones. These differences may be due to the genetic distances between the markers. These distances are about 4 units (33 salivary bands) between y’ and mwh and 19.2 units (219 bands) between mwh and jv. Analysis of the tergites of the same experimental individuals yield similar values: y alone 2%, y mwh 29%, y mwhljv twin spots 69%; 2% of the y nonmwh clones may include such events as mutation in the locus yf and induced delections of the tip of the Dp(1 :3)scJ4. The cell sensitivity of the wing disc cells to irradiation-induced SC0 proximal to mwh in the 3L chromosome in the first instar is about 0.2%. This value is about 10 times lower than the one found by Becker (1957) for presumptive facet cells in 24 hours old larvae (end of the first instar) after irradiation with 1200 r. However, the spots scored in his experiments were w facets following SC0 of first chromosomes heterozygous for w/w”. Using a comparable genetic setup in the 3L chromosomes (&se), he later found 11 times lower values (Becker, 1969). Thus, first instar eye and wing disc cells both have about the same sensitivity to SC0 in the 3L chromosome. The cell sensitivity to SC0 varies during the larval and pupal development. In general there is a slight increase during the larval development in cells not yet determined to differentiate into bristles or hairs. At the beginning of the pupal period, presumptive hair cells became 4~ and presumptive bristle-forming cells became 710X more sensitive than larval cells. During this period the clone size does not decrease, indicating that the cells do not divide except for the differential division of the bristle-forming cells. The difference in sensitivity between presumptive hair and bristle cells remains unexplained with the data at hand. It is interesting to note though, that presumptive tergite cells marked in the same way show a cell sensitivity as high as presumptive hairs in mature larvae (in preparation). The fact that presumptive tergite cells do not divide during the larval period, yet remain sensitive to induced crossing-over, indicates that they contain 4 strands per chromosome set throughout the larval period. Perhaps a long G2 phase is a prerequisite for higher sensitivity. The wing imaginal disc arises as an invagination of the embryonic

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blastoderm. Apparently it derives from about 12 preblastoderm nuclei (Stern, 1940; Garcia-Bellido and Merriam, 1969a). The wing disc is first detected in newly hatched larvae as “a small oval group of cells” (Auerbach, 1936). The biggest clones found in irradiated firstinstar larvae embrace about l/50 of the total mesothoracic cell structures. Thus, the wing bud at this age should contain about 50 cells. It follows that between the blastoderm formation and the first-instar sensitive period about 2 cell divisions take place. During the larval development up to the formation of the puparium the imaginal disc cells divide at an exponential rate. During the pupal period many cells divide but the clones contain only one cell. Divisions stop simultaneously for presumptive hair and bristle forming cells at about PF + 21 hours. Histological data of Waddington (1940) and Stumpf (1956) show that the last mitosis of the wing disc takes place at PF + 24 hours. The total number of adult cells (52,000) could be generated in 15.6 divisions. The 3.5 nuclear divisions which give rise to 12 primordial cells of the wing disc anlage occur within the first hour of development, and the anlage seems to remain stable during the embryonic development. The remaining 12 cell divisions would take 120 hours to be completed at a rate of 8.5 hours per division, This is the actual time lasting from the beginning of the larval development up to 24 hours after PF. However, significant oscillations in the division rate during the larval period apparently exist. It decreases at PF - 72, PF - 48, and PF - 8 hours (Fig. 4). This finding is consistent with the observed decrease in mitosis in the eye anlage at the end of the first and second instar observed by Becker (1957). This decrease in the division rate of the target cells corresponds with a relative decrease in the number of spots induced per wing disc. This also corroborates Becker’s finding in the eye disc. At a given age the number of cells induced per clone of the different regions of the wing disc also shows significant differences (Table 4). Regions A and B of the wing and the anterior part of the notum have clones of half the size of the clones of region E and the scutellum. The central part of the wing, regions C and D, show intermediate sizes. That would indicate that the anterior regions grow less than the posterior regions. Only during the pupal period is the relative number of cells induced per clone similar in the different regions of the wing. At this period the number of spots induced per region corresponds with the number of adult cells. Data on gynandromorphs indicate that a given clone may (in very

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few cases) embrace dorsal and ventral surfaces of the wing along the margins (Williams, 1968; Garcia-Bellido and Merriam, 1969b). Clones derived from irradiated larvae are restricted to either the ventral or the dorsal surface of the wing, even though they may contact along great portions of the entire wing margin (Fig. 7) which is over several hundred hairs. The anlage plan of mature wing discs shows that the presumptive dorsal and ventral elements of the triple and double row of bristles map together and run parallel along the wing disc margin (Hadorn and Buck, 1962). Histological data (Auerbach, 1936), do not show cell discontinuities or gaps in the cell layers. Since marginal and nonmarginal clones have the same clone size, any hypothetical intercalar mortality is improbable. This is reinforced by the histological observations of Fristrom (1969). Thus, something prevents cells of the dorsal and ventral surfaces mixing along the margin. This is especially surprising since clone splitting and mixing with normal cells appears to occur frequently in growing wing cells (Figs. 7 and 8). The situation is reminiscent of the known properties of cell recognition of disc cells following dissociation and mixing (Nothinger, 1964; GarciaBellido, 1966). We assume that a determinative event separates dorsal and ventral cells with respect both to their final differentiation and to their characteristic cell affinities. This determinative event must take place before the disc begins its larval development, i.e., when it contains less than 50 cells. Another determinative event taking place later in development separates presumptive bristle-forming cells from presumptive hairforming cells. Clones do not contain both kinds of structures when induced later than PF - 40 hours (Fig. 6). At this time of induction, clones may have as many as 4 bristles in the MTR. SCO-induced into hairy homozygous hairy cells are no longer able to differentiate bristles later than PF - 8 hours (Garcia-Bellido and Merriam, 1968). In this context it is interesting to note that presumptive bristle cells will only give rise to a bristle whereas presumptive hair cells give rise to 4 hairs (page 75). Clones of marked hairs decrease from 2 to 1 cell when induced around the time of puparium formation. Bristle-forming cells are sensitive to phenocopies and show the characteristic leveling off of their sensitivity to SC0 around this period. Lees and Waddington (1942) found the first histological signs of presumptive trichogene and tormogene cells at PF + 16 hours. It is also at about this time when presumptive trichogene cells are no longer

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able to show the y phenotype. Apparently the two differential divisions giving rise to a bristle organ take place at the same time as the two equal divisions give rise to 4 hairs. This equivalence of one bristle organ to 4 hairs is reminiscent of the “compensation law” of Henke and Pohley (1952), which seems to govern the degree of polyploidy and number of cells in scale groups of the wing of Ephestia. SUMMARY

Using X-ray-induced somatic crossing-over, a clonal analysis of the wing disc development of Drosophila melunogaster was carried out. Individuals carrying y in the X-chromosomes, and scJ4 (y+) jv and mwh in either 3L chromosome were irradiated at different developmental stages and the induced y;mwh and jv twin clones studied with respect to their frequencies, sizes, and shapes. The results indicate that the wing imaginal disc cells grow exponentially from the beginning of the larval period up to puparium formation. A total of 15.6 divisions is necessary to complete the adult number of cells. The division rate is constant in the intermolt periods, but apparently decreases during the molt periods. The average cell cycle time for the larval period is about 8 hours 30 minutes. The cell sensitivity to SC0 is more or less constant during the larval periods (0.2-0.4%). It increases during the pupal period about 4 times for the presumptive hair cells and about 5-10 times for the presumptive trichogen cells. The orientation of the mitotic spindle appears to be a major morphogenetic process. Cell divisions are preponderantly oriented along the proximodistal axis in the wing and alternating transverse and longitudinal or at random in the notum region. Clones grow in indeterminate patterns. At least two determinative events separate cell clones into different prospective developmental pathways. From the beginning of the larval period, dorsal and ventral cells do not mix over the wing margin. Forty hours before puparium formation, induced clones are able to give rise to several bristles or several hair cells but not to both in the same clone. The two differential mitoses giving rise to a bristle organ appear to be equivalent to, and to take place at the same time as, the two divisions give rise to 4 hair cells. We thank Dr. E. B. Lewis for the generous support provided oratory.

by him and by his lab-

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REFERENCES C. (1936). The development of the legs, wings and halteres in wild type and some mutant strains of Drosophila melunogaster. Tmns. Roy. Sot. Edinburgh 58,787 815. BECKER,H. J. (1957). Ueber Rbntgenmosaikflecken und Defekmutationen am Auge von Drosophila und die Entwicklungsphysiologie des Auges. Z. Verebungslehre 88, 333AUERBACH,

373.

BECKER,H. J. (1969). The influence of heterochromatin, inversion-heterozygosity and somatic pairing on X-ray induced mitotic recombination in Drosophila melanogaster. Mol. Gen. Genet. 105, 203-218.

CHEVAIS,S. (1943). Determinisme de la taille de l’oeil chez le mutant Bar de la Drosophile. Intervention d’une substance diffusible specifique. Bull. Biol. Fr. Belg. 77, 293-364.

DOBZHANSKY, T. (1929). The influence of the quantity and quality of the chromosomal material on the size of the cells of Drosophila melanogaster. Wilhelm Roux Arch. Entwicklungsmech. Organismen 115, 363-379. FERRIS,G. F. (1950). External morphology of the adult. In “Biology of Drosophila” (M. Demerec, ed.), pp. 368-418. Wiley, New York. FRISTROM,D. (1969). Cellular degeneration in the production of some mutant phenotypes in Drosophila melanogaster. Mol. Gen. Genet. 103,363-379. GARCIA-BELLIDO, A. (1966). Pattern reconstruction by dissociated imaginal disc cells of Drosophila

melanogaster,

Deuelop. Biol. 14, 278-306.

GARCIA-BELLIDO,A. (1968). Cell lineage in the wing disc of Drosophilu melunogaster. Genetics 60, 181 (Abstr.). GARCIA-BELLIDO, A., and MERRIAM,J. R. (1968). Bristles or hairs: the cell heredity of a genetic decision in Drosophila melunogaster. Proc. Nat. Acad. Sci. U. S. 61,1147. GAncIA-BELLmo, A., and MERRIAM,J. R. (1969a). Cell lineage of the imaginal discs in Drosophila Gynandromorphs. J. Exp. Zool. 170, 61-76. GARCIA-BELLmO,A., and MERRIAM,J. R. (1969b). A preliminary morphogenetic map of the wing disc. Drosophila Inf. Serv. 44, 65-66. GARCIA-BELLmO, A., and MERRIAM,J. R. (1969c). Multiple-wing-hairs: genetic order with ru and ue. Drosophila Znf. Serv. 44, 52. HADORN,E., and BUCK, D. (1962). Ueber Entwicklungsleistungen transplantierter Teilstticke von Fhigel-Imaginalscheiben von Drosophila melunogaster. Reu. Suisse Zool. 69, 302-310. HENKE, K., and POHLEY,H. J. (1952). Differentielle Zellteilungen und Polyploidie bei der Schuppenbildung der Mehlmotte Ephestia kt&niellu. Z. Nuturforsch. B 7, 65-79.

LEES, A. D., and WADDINGTON, C. H. (1942). The development of the bristles in normal and some mutant types of Drosophila melanogaster. Proc. Roy. Sot. Ser. B 131, 87110. LEWIS, E. B. (1969). Cytological location of the multiple-wing-hair (mwh) gene in Drosophila melanogaster. Drosophila Znf. Serv. 44, 188. LINDSLEY, D. L., and GRELL,E. H. (1968). Genetic variations of Drosophila melanogaster. Carnegie Inst. Washington Publ. 627.

E. A. (1968). Autoradiographische Bestimmung der DNS-Synthese-Dauer von Zellen der Fliigelimaginalanlage von Ephestia-ktihniella Wilhelm Roux Arch.

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B. (1955). Die Beeinflussung des Borstenmusters der Drosophila melcmogaster durch Rijntgenstrahlen (180 Kev und 31 MeV). Oncologia (Busel) 8,55-68. MEDVEDEV, N. N. (1935). Genes and development of characters: 1. The study of the growth of imaginal discs of the eyes of the wild type larvae and three mutants- Lobe’, glass’ and eyeless’ in Drosophila melanogaster. Z. Vererbugslehre 70, 55-72. N~THIGER, R. (1964). Differenzierungsleistungen in Kombinaten, hergestellt aus Imaginalscheiben verschiedener Arten, Geschlechter und Korpersegmente von Drosophila. Wilhelm Roux Arch. Entwicklungsmech. Organismen 155,269-301. PEYER, B., and HADORN, E. (1966). Zum Manifeststionsmuster der Mutante “multiple wing hairs” (mwh) von Drosophila melnnogaster. Arch. J&is Klaus-Stift. Vererbungsforsch. Sozidanthropol. Rassenhyg. 40, 20-26. STERN, C. (1936). Somatic crossing over and segregation in Drosophila mekmogaster. Genetics 21, 625-730. STERN, C. (1938). The innervation of the setae in Drosophila. Genetics 23, 172-173 (Abstr.). STERN, C. (1940). The prospective significance of imaginal discs in Drosophila. J. Morphol. 67, 107-122. STUMPF, H. (1956). Die Richtungen der Teilungsspindeln auf dem Puppenflugel von Drosophila in Verlaufe der Mitosenperiode. Biol. Zentrolb. 75, 17-27. WADDINGTON, C. H. (1940). The genetic control of wing development in Drosophila. J. Genet. 41, 75-139. WADDINGTON, C. H. (1942). Some developmental effects of X-rays in Drosophila. J. Exp. Biol. 19, 101-117. WILLIAMS, G. 0. (1968). Wing mosaics in Drosophila melanogaster. M. A. Thesis, Univ. of California at Berkeley.

NAVILLE,