Genetica (1972) 4 3 : 2 5 - 4 2

T H E E F F E C T OF E Y E REDUCTION ON W H I T E POSITION-EFFECT VARIEGATION IN DROSOPHILA MELANOGASTER

F. M. A. VAN BREUGEL Genetisch Laboratorium der Rijksuniversiteit Kaiserstraat 63, Leiden The Netherlands

(Accepted August 30, I97I )

Position-effect variegation for the white locus was studied in normally shaped eyes and in reduced eyes of Bar (B) and Drop (Dr) flies. The average number of spots per eye is successively lower in + , B, and Dr eyes; moreover, B eyes show a relatively strong pigmentation. No simple relation seems to be present between the degree of pigmentation and the number of facets, either between + , B, and Dr eyes or within classes of Dr eyes t h a t have been analysed. The chance t h a t ommatidia will become pigmented follows a gradient across mottled eyes of wild-type shape t h a t seems fixed early in development. The gradient is less clear or absent in B eyes. The results are best interpreted on the basis of the cell-lineage theory and an early one-sided action of B on the developing eye disc after fixation of the gradient.

Introduction

The normal wild-type eye of Drosophila is rather evenly pigmented, but in cases of position-effect variegation, e.g. for the white locus (white-mottled), large spots occur in the eyes on an otherwise pale ("white") background. SURRARRER (1935), GERSH (1952), a n d BECKER (1961) n o t e d t h a t

such spots are located preferentially near the posterior rim. The occurrence of spots and, logically, the chance of pigmentation for individual ommatidia, seems to increase gradually from anterior to posterior, thus demonstrating the existence of a gradient across the eye. The relative amount of pigmentation and the gradient of mottled eyes of different size form the subject of this paper. For the analyses, the term gradient is generally applied to denote the difference between

26

F. M. A. VAN BREUGEL

the relative pigmentation of anterior and posterior halves of the respective +, Bar (B), and Drop (Dr) eyes studied. In addition to the gradient, the variegation of eyes seems based on a cell-lineage type of growth, as claimed (BECKER, 1963, 1968) for D. melanogaster, but also for D. virilis (BAKER, 1967) and D. hydei (VAN BREOGEL, 1970). BECKER (1957, 1960) concluded from the size and number of spots in mosaics that the adult eye consists of about 20 sectors. Each sector has grown from a stem cell already present in the early imaginal disc of the first larval instar. In cases of position-effect variegation these sectors are believed to react as units with respect to the pigmentation process in the pupa. So far, two main arguments have supported this cell-lineage theory. The first stems from the typical distribution and size of spots in variegated eyes and X-ray mosaics that seem to follow a more or less recognizable pattern (BECKER, 1956), the second from the coincidence of some X-ray-induced twin spots with those of position-effect variegation (BECKER, 1966; BAKER, 1967, 1968). In fact, however, many individual spots criss-crossed the borders of the supposed cell-lineage sectors and the observations on the coincidence of the two types of spots, though important, were rather scanty. In combining white position-effect variegation with mutations that considerably change the size of the eye, I wished (1) to check the validity of the cell-lineage theory; (2) to collect information about the time at which the gradient is laid down in the imaginal disc; and (3) to investigate a possible relationship between eye size and the degree of pigmentation of variegated eyes. According to DE MARINIS (1952), the mutation Bar (B) mainly suppresses the development of the prospective anterior parts of eye primordia. This conclusion was based on a study of X-ray-induced somatic mosaics. STEINBERG (1941) found smaller eye discs in homozygous B individuals 36 hours after the hatching of the larvae, as compared with the wild-type. Even earlier, in the very young second larval instar, such a difference is apparent, and furthermore not all presumptive ommatidia of the B eye primordia seem to develop (BECKER, 1957). Up to the mid-third larval instar, however, it proved possible to enlarge the number of facets in B eyes in certain experiments (CHEVAIS,1942). FRISTROM (1969), as opposed to STEINBERG (1943), found signs of degeneration in the eye primordia of late third

W H I T E V A R I E G A T I O N IN D R O S O P H I L A

27

larval instars, these signs being confined to the prospective anterior parts of the eye. Among the factors that determine the size of the B eye we thus may find local limitation of growth and differentiation, in addition to local cell death. The final result of these factors is the elimination of more than threequarters of the wild-type eye, mainly potential anterior sectors. Consequently, we should expect a lower number of sectors in the B eye and also a lower maximum number of spots. If the gradient is present before B exerts its influence, these sectors would very often be pigmented and thus the relative pigmentation of B eyes should be high; moreover, the difference in pigmentation between anterior and posterior halves of B eyes would be low. Reasoning in another way: if B eyes are not different from wild-type eyes as regards pigmentation, i.*~.show the same number of spots, have the same relative amount of pigmentation, and also show a strong gradient, we might conclude that severe objections can be raised to the cell-lineage theory. The presence or absence of a strong gradient in B might also provide information about the time at which the gradient becomes fixed, either early in development or after B has exerted its influence. To verify a simple influence of eye reduction on the pigmentation process, a point that might complicate the picture, eyes of individuals carrying the Drop mutation (Dr) instead of B were also studied. Nothing is known about the developmental effects of Dr on the growing eye disc. The number of facets of Dr eyes is, however, much smaller than that of B eyes and can be determined quite easily. In addition to a possible effect of eye reduction on pigmentation of white-variegated eyes in the series +, B, Dr, we could therefore investigate more precisely a possible relationship between facet number and pigmentation in groups of Dr individuals. Material and Methods

Stocks used. To study position-effect variegation we used a new, not previously described X-ray-induced reciprocal translocation: T(X: 4) wm Krist. A large number of the flies in the true breeding translocation stock had mottled eyes with large spots, due to variegation of the white (w+) locus. Usually, only a minor fraction of the flies had nearwild-type eyes or pale eyes without large spots. On inspection of the

28

F. M. A. V A N B R E U G E L

salivary gland chromosomes, female and male larvae were always homozygous and hemizygous, respectively, for the reciprocal translocation. Heterozygotes in crosses with w were mottled, heterozygotes with w + were wild-type. The mottled character appeared to be bound to the translocation, especially to the distal tip of the X-chromosome, which was transferred to tile centromeric region of the 4th chromosome. This part of the translocation could be isolated as an aneuploid fragment in attached-w females or males with a normal w-marked X-chromosome while retaining the mottled effect. The break in the X-chromosome was thus proximal and close to the white locus, but its exact position has not yet been determined. In addition to the original homozygous translocation stock, we made two other stocks, one homozygous for Bar (B), the other heterozygous for Drop m~o, a homozygous lethal allele of Drop (Dr) (SoBELS, 1958; LINDSLEY & GRELL, 1967) balanced over TM3. In both stocks the original translocation was present in homozygous condition; and with respect to the white locus, flies showed a mottled phenotype.

The gradient and the pigmentation indices. The gradient of wildshaped eyes was first analysed on the basis of a hundred randomly collected drawings of left eyes, the eye being divided arbitrarily into about 75 blocks and counts being made in each block to determine the number of times that more than half one surface of the block was pigmented (Fig. I). For each of the vertical rows of blocks, the average pigmentation frequency per block was plotted (Fig. 1). To compare + , B, and Dr eyes, another reticulum, with only five blocks anterior and five blocks posterior to a hypothetical longitudinal axis was used (Fig. 2). Paired anterior and posterior blocks had roughly the same surface. Instead of the all-or-nothing estimates, the degree of pigmentation of blocks 1-10 was now judged separately, according to the following scale: k ---- 0: meaning no pigment (only background colour, nearly white); k = 1: a spot smaller than ¼ of the total surface of the block in question ; k = 2: spots covering between ¼ and ~; k = 3" covering between ½ and ~" k = 4: covering between ~ and all of the surface of a block with wild-type pigment; k ---- 5 : full pigmentation.

W H I T E VARIEGATION IN DROSOPHILA

35

29

79

30,

~5 u ~ h b 20 / /

~

h

~

'L h

i h b b

15

10

__ __ ~ ~'e///j

~

35,

h

..41_

_

_

_e..

~

~

-4)-

-- -- -e"

dd

30,

cO

.m

~25, 4-J

/.1: : :,:'

~J 20

,.-I,

Q.

~

Ors,

c

0~ lO

o-

i

i

3

1 ~ ~ I1//1/1111 4

'

g '

g " i

, 11 11

..... i,./7 ]f~

'-V~

Area Fig. 1. The distribution of p i g m e n t spots in 100 white-mottled eyes. The black area within each block shows how often over 50% of the block was p i g m e n t e d (16~C). Besides an antero-posterior gradient, differences in dorso-ventral direction are evident. The graphs show p i g m e n t a t i o n frequencies at 16°C (dashed lines) and 25eC (solid lines), t h e latter corrected (see text).

30

F. M. A. VAN B R E U G E L

From the protocols, a pigmentation index (I) was counted for each eye separately, for both the anterior and posterior halves (Ia and Ip, respectively) in which I = Nk/5 and 0 ~< I ~ 5. Further, the averages (ia and Ip) based on a hundred eyes were calculated. In this way various blocks contribute numerically to the I-value (cf Discussion).

/

a

/

~

Fig. 2. A b o v e : a, b, c: p h o t o g r a p h s of + , B, a n d Dr eyes w i t h position-effect v a r i e g a t i o n of t h e w+-locus. Below: s c h e m a t i c a l d r a w i n g s of c o m p a r a b l e eyes s u b d i v i d e d into anterior a n d posterior fields (odd a n d e v e n n u m b e r i n g , respectively) as used for d e t e r m i n i n g p i g m e n t a t i o n values. T h e n o n - f a c e t t e d b u t p i g m e n t e d zone of t h e Dr eye is clearly visible in c.

WHITE VARIEGATION IN DROSOPHILA

31

The arithmetic mean (IT) of Ia and ip gives an impression of the pigmentation of the eye as a whole. The i values could be compared in pairs by a t-test. For a and p, the i values of 50 flies were used and compared with the i values of 50 other randomly taken flies.

Counts o//acets and spots. Counts on facets of Drop eyes were made with a dissecting microscope in separate groups of individuals ( ~ and dT~, reared at two temperatures). The counts were based on the same individuals as used for counting the number of spots and for estimating the degree (index) of pigmentation. In Drop eyes a very low number of facets (about 10) is present. In a few cases the individual corneal facets were less clear or very small, which led to small errors, one or more facets having been overlooked. Only spots reaching a certain minimum size, i.e. about one-twentieth of the total eye surface, were counted; therefore, "pepper-and-salt" spots were excluded. The numbers of spots and facets, when used, were compared in the same way as the pigmentation indices. Results

The pigmentation o/+, B, and Dr eyes. Figure 1 shows the results of the analysis of a large number of eyes of wild shape submitted to position-effect variegation, based on a large number of blocks in which the eye was subdivided. The pigmentation is much stronger at 25°C that at 16°C and the gradient steeper at the former temperature. The 25°C values (summed k values per block) were in fact much higher than would appear from the graph: all female 25°C values were reduced by 45 and all male 25°C values b y 35 to facilitate comparison. I t is clear that the anterior-posterior gradient is not straight. The semischematic drawing of the eye, moreover, also shows differences in the dorsoventral direction, a point not further elaborated here. From twelve classes of a hundred eyes each (+, B, Dr; males as well as females; two temperatures) the values were plotted graphically in Figure 3, (cf: Methods) based on Table l and the schemes in Figure 2. The line connecting ia and Ip gives a rough estimate of the course of the gradient. In fact, as we have seen, it is not straight. Half way the line we find iT, as the average pigmentation of the whole eye. The differences between the various r values were tested for significance. Table 1

32

F. M. A. V A N B R E U G E L

TABLE

1

AVERAGE DEGREE OF PIGMENTATION "OF ANTERIOR (Ta) AND POSTERIOR (ip) HALVES OF EYES IN 100 LEFT EYES PER SAMPLE; AND t-TEST BASED ON THE COMPARISON BETWEEN 50 ANTERIOR AND 50 POSTERIOR HALVES OF DIFFERENT EYES CHOSEN AT RANDOM FROM EACH SAMPLE ( S i g n i f i c a n c e : * = P < 0.05,**

= P < 0.01)

T y p e of flies T o t a l p o p u l a t i o n 1) (100)

t-test ½ pop.

½ pop.

1(50)

2(50)

ia

ip

ia

Ip

+ +

~ 25 ° ~ 16 °

2.53

3.33

2.14

3.66

--5.59**

0.31

0.51

0.20

0.56

--2.09*

+ +

~' 25 ° ~' 16 °

2.09 0.16

2.89 0.52

1.92 0.20

3.17 0.58

--4.01'' --3.75**

B B

~ 25 ° ~ 16 °

4.19 3.43

4.16 3.51

4.07 3.20

4.08 3.74

--0.04 --0.14

B B

~' 25 ° ~' 16 °

4.13 1.88

4.24 2.09

4.07 1.81

4.13 2.26

--0.54 -- 1.20

Dr Dr Dr Dr

? ? ~ (~

25 °

3.75

4.23

3.16

4.50

--6.03**

16 ° 25 °

0.62 3.60

1.04 4.30

0.68 3.50

0.96 4.25

--1.19 --3.29**

16 °

0.47

0.96

0.63

0.77

--0.75

1) U s e d a l s o i n T a b l e s 2, 4 a n d 5.

gives the results as well as the basic information for the other Tables. Tables 2 a n d 3 comparegenotypes ( + , B, and Dr). Table 4 compares 25°C and 16°C cultures, and Table 5 deals with the difference between males and females. --The gradients. In wild-type-shaped eyes the posterior i values were higher than the anterior, but in B eyes the difference between l~ and ip was always slight. Table 1 shows that in wild-type eyes for both temperatures and both sexes and in Dr eyes for 25°C, the anteriorposterior differences were significant whereas this was not the case for B eyes and Dr eyes from 16°C cultures. In the case of B a rather small difference between Ia and lp could be expected if the B eye represents a small dorso-ventral strip of the wild-type eye. So far, the results seem

WHITE

VARIEGATION

DROSOPHILA

IN

TABLE

33

2

COMPARISON OF D E G R E E OF P I G M E N T A T I O N B E T W E E N F L I E S W I T H D I F F E R E N T T Y P E S OF E Y E S

(For I values, Type

cf. Table

1) ( t - t e s t .

Significance:

of flies

* =

P <

0.05, ** =

P < 0.01)

Anterior

Posterior

t

+

+

versus B 25 °

--

16 °

--14.86"*

--14.77"*

25 °

--

9.71"*

--

7.50**

16 °

--

9.05**

--

9.23**

8.17"*

--

4.30**

250

--

4.95**

--

5.29**

16 °

--

2.14"

--

3.53**

25 °

--

versus Dr

16 °

B

t

-

7.19"*

--

3.20**

-

-

-

7.83** 3.67**

versus Dr 25 °

--

3.65**

16 °

12.78"*

25 °

2.94**

C~ 16°

6.71"*

COMPARISON

OF

THE

AVERAGE

---

3

NUMBER

OF

PIGMENTED EYES (PIGMENTATION INDEX 2 <

Type

of flies

* =

Average

N °

P <

0.41 5.28**

TABLE

(t-test. Significance:

0.41 11.23"*

SPOTS IT <

0.05,

BETWEEN

MODERATELY

3) OF VARIOUS G E N O T Y P E S

** =

P <

0.01)

n

~(S--S)2

of spots

+

2.16

58

117.6

B

1.51

39

23.7

Dr

1.23

39

8.9

Comparison

t

+

versus B

2.54**

+

versus Dr

3.84**

B

versus Dr

1.84*

F. M. A. VAN B R E U G E L

34

to fit quite well with the conception concerning the mode of action of B on the imaginal disc. The Dr data seem difficult to interpret. Dr eyes of 25°C resemble the wild-type because they show a gradient, TABLE COMPARISON

OF

THE

DEGREE

OF

4

PIGMENTATION

BETWEEN

25°C

AND

16°C

CULTURES. (For i values, cf. Table 1) (t-test. All differences are significant at t h e 0.005 level) Type of flies

Anterior t

Posterior t

+ +

9 ~

11.81 12.21

B

?

3.50

B

~

9.87

10.05

17.25 20.59

20.95 23.03

Dr ~_ Dr (~

Ta

IT

dd

~P

17.09 15.00

2.88

~

~T i

~P Dr

l i

B

4.

:

__2,'

....

;-

--

~2 Dr

4-

~i

Anterior

;:::

Posterior

~--

Aoterior

;_:::

Posterior

---

Fig. 3. R e p r e s e n t a t i o n of t h e Ia and ip values, based on Table 1, with t h e average p i g m e n t a t i o n index iT on t h e connecting line, giving an impression of t h e gradient in + , B, and D r eyes. I n B, the gradient is absent or weak. Slightly different values were obtained after correction, as indicated b y separate marks.

35

W H I T E V A R I E G A T I O N IN DROSOPHILA

but for 16°C there is no demonstrable gradient. This divergence might be due to inadequacies of our analysis or might be the result of a real temperature dependence.

Relative pigmentation o/ the eyes. B eyes have a relatively large pigmented area (Fig. 3). Table 2 shows that the anterior half of B eyes not only has relatively more wild-type pigment than that part of wild-type-shaped eyes, but also more than Dr eyes. This seems to hold for the posterior half as well, except that at 25°C the difference between B and Dr is not significant. It is conceivable that here full

2 15 10

~ 20I

+,,-

iil lllr

IIII

I I

}

,_Number ef faJ~ts (F) Fig. 4. H i s t o g r a m s h o w i n g t h e f r e q u e n c y d i s t r i b u t i o n of t h e n u m b e r of facets in g r o u p s of 100 Dr flies each. B l a c k c o l u m n s : 25°C cultures. W h i t e c o l u m n s : 16°C cultures.

36

F. M. A. VAN B R E U G E L

pigmentation of the eye eliminates the difference. The high degrees of relative pigmentation of B eyes can be explained on the basis of the cell-lineage theory: the B eye can be seen as a small dorso-ventral strip consisting of the most posteriorly situated (most often pigmented) sectors of the wild-type eye. If this were true one could predict a smaller average number of spots (sectors) in B eyes as compared with wild-type eyes of comparable degree of pigmentation (cf. below). In Dr eyes of 25°C a gradient still exists but the I values are somewhat higher than those of eyes of wild-type shape (Table 2). It is possible that Dr eyes have a small number of sectors with great differences in pigmentation (gradient) but an iT differing little from that of the wild-type. Alternatively, it is possible that in Dr almost the same number of sectors is present as in the wild-type but that the sectors stop growing at an early stage. To evaluate these two possibilities, the number of spots of Dr eyes were compared with eyes of wild-type shape having comparable degrees of pigmentation.

The number o[ spots and the size o[ the eye. If B eyes represent only some sectors of the wild-type eye, one would expect a lower number of spots, but perhaps (not necessarily) the size of the spots may reach that of wild-type eyes. The number of spots will, however, depend on the degree of pigmentation, for instance with high degrees of pigmentation the number of spots will decrease, because neighbouring sectors will fuse as regards pigmentation. At midvalues of pigmentation the number of spots will reach a maximum. Therefore, I determined the number of spots of B eyes with midclass pigmentation (2 < I~ < 3) and compared them with midclass eyes of + and Dr phenotype. B eyes had fewer spots than the wild-type but more than the Dr eyes. Thus indeed it seems that the numbers of sectors of B and Dr eyes successively are lower. In B eyes these sectors most probably stem from the posterior parts, in Dr eyes there possibly is a more equal contribution from anterior and posterior fields. The influence o] temperature. Table 4 gives the result of an analysis of the pigmentation of 25°C and 16°C cultures separately. For all types, flies reared at 25°C were significantly more heavily pigmented than those reared at 16°C. This was even true for anterior and posterior halves of the eye individually. The effect agrees with what is generally

WHITE VARIEGATION

37

IN DROSOPHILA

known about the influence of temperature on position-effect variegation, but as such differs from the observations of SURRARRER (1935) on white-variegation in D. melanogaster and of VAN BREUGEL (1970) on the m u t a n t wm2 of D. hydei.

Di//erences between the sexes. Whereas temperature and genotype had clearcut effects on the degree of pigmentation, the i values of males and females only incidentally differed significantly as shown by Table 5. No explanation can be offered for the erratic differences, which occurred only between flies having midvalues of pigmentation. TABLE

5

COMPARISON OF THE DEGREE OF PIGMENTATION BETWEEN FEMALES AND MALES ( F o r I v a l u e s , cf. T a b l e 1) ( t - t e s t . S i g n i f i c a n c e : * = P < 0.05, ** = P < 0.01)

Type of flies

Anterior t

Posterior t

+

25 °

1.96"

2.17"*

+

16 °

1.54

0.92

B 25° B 16°

0.34 5.96**

0.48 5.46**

Dr 25 ° D r 16 °

0.17

0.50

0.91

0.43

The number o/[acets o] + , B, and Dr eyes. The facets of wild-type eyes number about 800 in females and 750 in males; for B eyes, these numbers are about 70 and 90, respectively (STURTEVANT, 1925). There is considerable variation, depending also on genetic background and culture conditions. For Dr eyes, the frequency distribution for the number of facets is given in Figure 4. At a low temperature (16°C) on the average there were more facets than at a higher temperature (25°C). The average number of facets was, at 25°C and 16°C respectively, 9.18 and 10.82 in females, and 7.39 and 8.22 in males. These temperature differences are significant at the 5~o level. The difference, at both temperatures, between male and female facet numbers is significant at the 1% level.

38

F. M. A. VAN BREUGEL

An attempt was made to determine whether there was a preference for an even number of facets in Dr eyes. The ratio even/odd number of facets within the group of Dr individuals could be considered homogeneous (X ~ = 2.45, DF = 3, P > 0.05), but in total (217/183) did not differ significantly from a 1 : 1 ratio (X ~ = 2.89, DF = 1, P > 0.05). In a typical Dr eye the pigmented area is much larger than the facetted area. In all likelihood the non-facetted region is composed of incomplete ommatidia in which pigment cells are present but not corneal structures. B eyes, too, have a small pigmented but nonfacetted region showing a degenerate structure (WoLsKY ~: HUXLEY, 1936). The edge of the non facetted but potentially pigmented region of variegated Dr eyes is, however, rather sharply delimited by a suture in the head capsule, which makes it possible to estimate the ratio of the pigmented surface in relation to the potential maximum of "eye" surface (cf. Fig. 2).

The number o] ]acets and the degree o/pigmentation. The observations on + , B and Dr eyes suggest that no simple relation is present between facet number and the degree of pigmentation, since B flies with an intermediate number of facets (70-90) have darker eyes than either flies with wild-type-shaped eyes (about 700 facets) or Dr eyes (about 10 facets), whereas both B and Dr flies have eyes darker than wildtype. Within classes of Dr eyes (males only were used) the number of facets that could be counted precisely also did not show a simple relationship with the degree of pigmentation, as can be seen from Figure 5. At a low temperature (16°C), however, the average number of facets increases (Fig. 4), whereas a decrease in the degree of pigmentation was noted (Fig. 5). The development of the corneal facet structure therefore seems to be independent of the pigmentation (read: positioneffect variegation) of primary and secondary pigment cells of the ommatidia. The effect of temperature on the number of facets in Dr is in the same direction as that found for wild-type (HERsH, 1924) and B (SEYSTER, 1919 ; KRAFKA, 1920 ; MARGOLIS, 1935).

Conclusions and Discussion

In morphologically normal eyes with position-effect variegation for the white locus a clear gradient could be demonstrated as regards

W H I T E VARIEGATION IN DROSOPHILA

:......

•

•

•

.,"

~..

•

eo

•

•

•

:

39

-\

,

~

oO

3,

•

o° e

•

2.

5,

16 °

4.

~3. C 0

~2 E n-

•

:"

:

~..

:o :K

1 •

•

2

*

i

~'

/

/

•

eo

°°

: "-

,

"

•

-~

~

~-~---#-~...~.I

*.

"

~ Number

of

facets(F)

Fig. 5. The degree of pigmentation (IT) plotted against the number of facets in 100 left eyes of Dr males reared at two temperatures. No relation seems to be present.

pigmentation. For wild-shaped eyes this gradient has been worked out in detail, but a simplified abstraction of it was used for comparison with the gradient of Bar and Drop eyes. For this purpose, separate pigmentation indices were calculated for the anterior and posterior halves of the respective kinds of eye, thus providing a measure for the relative pigmentation of both areas. Since the blocks into which the eye was subdivided were not equal in size (Fig. 2), the mean pigmentation values (i) might be biased. For instance, where the gradient

40

F. M. A. VAN B R E U G E L

appeared to be somewhat steeper, this could be spurious if larger blocks, especially 4 and 6 in wild-type, by their size had a greater chance of being pigmented. However, when a correction for differences in size was applied by calculating for each of the anterior or posterior blocks its percentage Of the total surface of the anterior or posterior halves of the eye, the corrected values were almost the same as the old ones (Fig. 3) and the total picture seemed unchanged. Therefore, only uncorrected i values were used. The gradient of the eyes could conceivably be based on the decline of a morphogenetic substance that has an influence on the switching mechanism of the w+ locus submitted to position-effect variegation. As BECKER (1966) has claimed, it is not the time of switching but only the chance of a +-switch that seems to be influenced under these circumstances (cf. also VAN BREUGEL, 1970). The analysis of Bar (B) eyes showed that in B the gradient is lacking or at least is no longer demonstrable with the method used. In all probability the gradient has been laid down in the eye disc before B exerts its influence. Given the early time of action of B, it seems likely that the gradient of the eyes is even already present in the embryo or the young first larval instar. Because in B discs only the most posteriorly situated sectors develop, rather dark eyes and a low absolute difference between la and Ip must be expected, as can be extrapolated from the graphs (Fig. 1 and 3). Since B eyes indeed have a relatively high degree of pigmentation and probably only a fraction of the number of sectors in wild-type eyes, as judged from the maximum number of spots, the results are consistent with the cell-lineage theory. An important objection that could be raised to this interpretation is that smaller eyes might in general have a higher degree of pigmentation due for instance to an abundance of pigment precursors. The data on variegation of Drop (Dr) eyes prove, however, that at least in this case much smaller eyes have less, not more, pigmentation than the B eyes. But a small effect of eye size on pigmentation may nevertheless exist, since Dr eyes are on the average slightly darker than wild-shaped eyes. Because we know nothing about the develomental effects of Dr on the eye disc, this question remains open for the present. From the observations on the number of facets and the degree of pigmentation of Dr eyes it may, however, be concluded that there is no direct or simple relation between eye size and pigmentation.

W H I T E VARIEGATION IN DROSOPHILA

41

D r e y e s s h o w a s m a l l e r n u m b e r of sectors t h a n do e i t h e r B o r w i l d

t y p e eyes, b u t - in c o n t r a s t to B e y e s - a s t r o n g g r a d i e n t for 25°C. I t m a y t e n t a t i v e l y be c o n c l u d e d t h a t D r eyes h a v e h a d a m o r e b a l a n c e d c o n t r i b u t i o n of a n t e r i o r a n d p o s t e r i o r fields of t h e w i l d - t y p e eye. I n t h e a b s e n c e of c o n t r a d i c t o r y e v i d e n c e , t h e d a t a a r e m o s t s u s c e p t i b l e to i n t e r p r e t a t i o n a c c o r d i n g t o t h e cell-lineage h y p o t h e s i s a n d on t h e basis of t h e e x i s t e n c e of an e a r l y g r a d i e n t in t h e d e v e l o p i n g e y e in a d d i t i o n to a o n e - s i d e d a c t i o n of B on t h e i m a g i n a l disc. T h e a n a l y s e s s h o w t h a t , d e p e n d i n g on t h e m o d e of a c t i o n of e y e - r e d u c i n g f a c t o r s , t h e v a r i a b l e e x p r e s s i o n of p o s i t i o n - e f f e c t v a r i e g a t i o n c a n be modified. I wish to thank Prof. H. GLOOR, Dr. W. VOLKERS,Dr. H. J. FREI and Drs. B. J. M. ZONN~VELI) for their valuable criticism, as well as Mrs. I. SEEGgRWOLF who read the English test and Miss Q. van der Aart who offered technical assistance.

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