Pattern formation in the wing veins of the fused mutant (Drosophila melanogaster)

DEVELOPMENTAL

63.358-369

BIOLOGY

Pattern Formation

(1978)

in the Wing Veins of the Fused Mutant (Drosophila metanogaster) ANNE

FAUSTO-STERLING

with the assistance of Lily Hsieh Brown

University,

Box G, Division

Received April

of Biology

and Medicine,

Providence,

Rhode Island

02912

6, 1977; accepted in revised form December 9, 1977

In order to analyze the effects of the mutation fused (fu) on vein pattern formation, wings mosaic for fused and non-fused tissue were obtained. Analysis of these wings (1) confirmed that fused does not involve the production of a freely diffusible substance; (2) showed that a genotypically fu fourth longitudinal vein (LV 4) develops a normal phenotype nonautonomously if the third longitudinal vein (LV 3) and most of the first posterior cell are non-fused, (3) showed that in the reciprocal situation, a non-fused fourth longitudinal vein often exhibited autonomous differentiation; (4) demonstrated that small groups of cells (either fu or non&) could be incorporated into structures characteristic of the opposite genotype; and (5) offered evidence that the dorsal wing surface may play an important role in the control of vein formation. Additionally, the fused phenotype itself was examined in some detail, and the anterior-posterior compartment border was defined. This examination suggests that in the more extreme cases LV 4 does not fuse with LV 3, but simply fails to form. INTRODUCTION

Stern (see review, 1968) pioneered the study of pattern formation in Drosophila by introducing the use of genetic mosaics as an analytical tool. He, as well as many others (cited in Tokunaga and Gerhart, 1976), used mosaics to analyze a variety of mutations affecting the arrangement of bristles on the cuticle of the adult fly. In most cases, bristle pattern formation was autonomous. That is, the decision made by an individual cell to differentiate into a bristle depends solely on its own genotype and is uninfluenced by the genetic makeup of the surrounding tissue. Based on this and other work, Wolpert (1969, 1971) has developed an inclusive theory of pattern formation. His concept of positional information is now generally used as a framework for analyzing pattern formation. The arrangement of veins on the wing of Drosophila is a simple and constant pattern formed by the cooperative action of thousands of cells (Waddington, 1940). Waddington (1940) analyzed the development

of a large number of mutations affecting vein patterns. He found that these could be separated into two categories: those in which the change in vein pattern is visible as soon as the wing disc evaginates, and those in which the alteration becomes apparent somewhat later in development. From the effects of operating on the pupal wing at critical development stages, Lees (1941) concluded that cells on the dorsal surface may induce vein formation in the underlying ventral cells. Recent studies of genetically mosaic wings done on a variety of different wing vein mutations (Santamaria and Garcia-Bellido, 1975; Garcia-Bellido, 1977) have provided further evidence for the existence of developmental interactions between the dorsal and ventral wing surfaces. Thus it is likely that in comparison with bristle pattern formation, different or additional types of developmental controls may be important in wing development. In order to better understand the mechanisms of pattern formation in the veins of the wing, we examined a mutation

358 OOlZ-1606/78/0632-0358$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

ANNE FAUSTO-STERLING Pattern Formation in Fused Mutant affecting the shape and location of two of the major longitudinal veins. The mutant fused affects the anterior crossvein and the third and fourth longitudinal veins (LV 3 and LV 4) (see Fig. 1A). While analyzing gynandromorphs composed of fused and non-fused tissues, we observed a number of mosaic wings with atypical vein patterns (a combination of fused and wild type) (Fausto-Sterling, 1970). In the work presented here, we describe in detail the fused wing phenotype

359

and analyze vein pattern formation in wings mosaic for fused and non-fused tissue. MATERIALS

AND

METHODS

Detailed descriptions of the genetic markers used may be found in Lindsley and Grell (1968). Flies were raised on standard cornmeal-sugar-agar medium at 26°C (+ 1°C). Gynandromorph production and analysis. Gynandromorphs mosaic for fused and non-fused tissue were obtained by mating SUBMARGINAL

CELL

A VEIN

8 CELL

ALULA I ST BASAL CELL Bi NTERIOR CROSSVEIN ZND POSTERIOR CELL 8 POSTERIOR CROSSVEIN THICKENED

3RD

POSTERIOR

CELL

LV III

FRAGMENT OF LV Ip

FIG. 1. (A) A tracing of a wing from a ~u~~/CZB female made using a Wild drawing tube. (B and C) Tracings of wings from fS/Y; murh/M”““’ males. The location of the anterior-posterior compartment horder is shown for each wing.

360

DEVELOPMENTAL BIOLOGY

VOLUME 63.1978

females homozygous for the ring X chromosome wvcwith yellow, singed-3, fused-59 (y sn3 fu”“) males. Loss of the ring X chromosome during the first or second cleavage division (Hall et al., 1976) produced flies in which the male (X0) component expressed the fu5s phenotype and could be recognized by the presence of yellow, singed bristles and wing hairs; the female component (XX) was phenotypically wild type. Figure 2 shows a photograph of wing tissue containing y sn3 fu5’, and non-singed tissue. It can be seen that in these crosses one can distinguish between wing hairs of these two genotypes. With the aid of a dissecting microscope, flies containing male tissue on the thorax were selected, and their wings were mounted on slides and subsequently studied in detail using a compound microscope.

irradiated at 24-36,36-48, or 48-60 hr after being laid. In all cases, a dose of 1000 R (100 kV, 10 mA) was used in combination with a l-mm aluminum and a O.&mm copper filter. The wings from fu”“/Y; mwh/M(3) hS3’ males were mounted on coverslips and examined using a compound microscope. Those wings containing large mwh clones (see Morata and Ripoll, 1975) were drawn using a Wild drawing tube and the exact location of the mwh clone fried in on the drawing.

Analysis of compartment borders in fu5” wings. Females of the genotype fu5’/ClB; mwh/mwh (mwh = multiple wing hair) were crossed with Minute (3)hS37/Moire’ (M(3)hS37/Me) males. Embryos from this

stages of development. An X chromosome bearing yellow and the wild-type allele of mwh was kindly supplied by Dr. J. R. Merriam. The forked bristle marker, which is severe enough to be distinguished on wing hairs, was added to the chromosome in this

cross were collected at 12-hr intervals

and

Production of mosaic wings duringpostembryonic development. Animals of the genetic composition yellow, multiple wing hair-plus, forked-36a/fused-59, and homozygous on the third chromosome for mulwing hair (y mwh+fasa/fu5’; tiple mwh/mwh) were X-irradiated at various

FIG. 2. A region of a wing from a Xc’/y sn3 fuss gynandromorph. The black line drawn in separates a region ofy sti’ fuS9 tissue from heterozygous (phenotypically wild-type tissue).

ANNE FAUSTO~TERLING

Pattern Formation in Fused Mutant

laboratory. A more detailed explanation of this cross is presented in the Results section. To obtain larvae during the first 48 hr after hatching from the egg, eggs were collected during B-hr periods following the mating of fu5’/C1B; mwh/mwh females with y mwh+fGa; mwh/mwh males and irradiated at known intervals thereafter. For older larval stages, larvae of mixed ages were irradiated, and the amount of time elapsed until prepupa formation was recorded (Garcia-Bellido and Merriam, 1971). All larvae were irradiated with a dose of 1000 R. RESULTS

The Fused Phenotype Before interpreting the data from wings mosaic for fused and non-fused tissue, the fused phenotype was analyzed in further detail. A tracing of a wing from a fu5’/ClB (phenotypically wild type) female is shown in Fig. 1A. Anatomical features are labeled according to the terminology used by Ferris (1965). In addition, the location of the boundary which separates the anterior and posterior compartments is shown (GarciaBellido et al., 1973). Lindsley and Grell (1968) describe both an extreme and a mild form of the fu phenotype. For the allele fu, for example, they state that LV 3 and LV 4 are fused from the base to beyond the anterior crossvein, with the elimination of the anterior crossvein and the first basal cell In addition, LV 3 and LV 4 are fused at the wing tip. In more extreme cases, this fusion may reach back to the basal cell. Figures 1B and 1C show drawings of wings demonstrating the mild (1B) and the extreme (1C) phenotype of fused. The experimentally determined anterior-posterior compartment border for these individual wings is also shown. A close study of these and other fused wings suggests that the description of the fused phenotype obtained from Lindsley and Grell is not accurate. For example, the proximal portion of LV 4 is frequently formed (Fig. 1B) cre-

361

ating a small basal cell. Just as often, this portion of LV 4 is only partially present (Fig. 1C). The frequency and extent of formation of the proximal portion of LV 4 appear to vary with the genetic background. We thus suggest (see additional evidence below) that LV 3 and LV 4 do not fuse in this region. Instead, LV 3 forms in its normal position but is much thicker than usual, while LV 4 is frequently incomplete. Similarly, what appears in the extreme phenotype (Fig. 1C) to be complete fusion of LV 3 and LV 4 along the length of the wing is better interpreted as the failure of LV 4 to form and the formation of a thickened and in many cases vesiculated LV 3. In the less extreme phenotype (Fig. lB), LV 4 develops completely, but both it and LV 3 are slightly vesiculated, most frequently at the wing tip where they fuse. The dorsoventral polarity (see Santamaria and Garcia-Bellido, 1972) of veins 3 and 4 in fused wings is consistent with this viewpoint. Our interpretation of the fused phenotype suggests that the gene products involved do not actually control the location of the veins on the wing surface, but rather influence the mechanism of wing vein differentiation. Experimental evidence in support of the above interpretation of the fused phenotype has been obtained by a more detailed analysis of the location of compartment borders in fused wings. As described in the Materials and Methods section, clones of mwh cells on a fused background were induced, and their location was recorded on drawings of individual wings. The presence or absence of mwh cells on the proximal portion of LV 3 (i.e., that part defining the basal cell), on the proximal portion (or fragment) of LV 4, the distal portion of LV 3 (i.e., that part running from the distal end of the basal cell to the wing tip), and the distal portion (or fragment) of LV 4 were recorded. Wings with clones in the anterior compartment were scored separately from those with clones in the posterior compartment. The data demonstrate that the proximal portion of LV 3 is always found in the

362

DEVELOPMENTAL BIOLOGY

anterior compartment, a strong indication that this vein is not shifted toward a central midline. A similar conclusion may be reached for the distal portion of LV 3. Conversely, the proximal portion of LV 4 is always found in the posterior compartment. With only a few (3/39) exceptions, the data also demonstrate that, the distal portion of LV 4, when formed, is found in the posterior compartment. Finally, it is striking that in flies of this particular genetic make-up the proximal portion’of LV 4 is present to some degree in three-quarters of the wings examined. Gynandromorph

Analysis

More than 100 mosaic wings were obtained from gynandromorphs in which the female cells were heterozygous fused (w”‘/y .~n.~fu5g) and the male cells hemizygous fused (y sn3 fu”“). These were examined and separated on the basis of phenotype into one of five categories: (1) those with completely wild-type venation; (2) those with completely fused venation; (3) those in which the distal portion of LV 4 was thickened, branched, or vesiculated; (4) those in which the distal portion of LV 3 was thickened, branched, or vesiculated; and (5) those in which vein formation was badly disrupted. Each group is analyzed in turn below. (1) Mosaic wings with wild-type vein pattern. The distribution of male and female tissue on phenotypically wild-type wings was recorded on standard diagrams. The frequency with which different regions or structures within the wing were either entirely male (fused), entirely female (nonfused), or mosaic (containing both male and female tissue) was tabulated (Table 1). If certain regions of the wing are critical for the formation of a wild-type vein pattern, then one would expect that in wild-type wings such critical regions would only rarely be genotypicahy male (fused). Examination of the data in Table 1 shows that the submarginal cell, the first posterior cell, the third longitudinal vein, and the first

VOLUME 63,1978

basal cell and anterior crossvein are, in fact, only rarely composed of male tissue (fewer than 5 out of 31 cases). The first posterior and basal cells, and LV 3 are structures which are considerably altered by the fused mutation. In contrast, the fourth longitudinal vein, which is also changed in fused flies, is nonautonomous; i.e., in a majority of the wings in this category (20 out of 31), this vein was genotypically fused, yet phenotypically wild type. The third longitudinal vein was never found to be male in phenotypically wildtype wings and was mosaic in only two out of 31 cases. An example is shown in Fig. 3A. In contrast, both the first posterior and basal cells and the anterior crossvein were mosaic in greater than 50% of the cases. In 21 out of the 24 examples in which mosaic first posterior and basal cells were observed, male and female tissue was separated by the anterior/posterior compartment border (e.g., Fig. 3A). Finally, the wing shown in Fig. 3A strikingly illustrates that the genotype of the first posterior cell and its bordering veins is critical for the expression of the fused phenotype. Phenotypically wild-type wings were also analyzed with regard to the frequency with which anterior and posterior compartments are male (fused), female (non-fused), or mosaic. No phenotypically wild-type wings were found for which the anterior compartment was entirely male. The anterior compartment is, however, frequently mosaic, often including rather large patches of male tissue (Fig. 3A). (2) Mosaic wings with a fused vein pattern. Twenty-one of the mosaic wings obtained from gynandromorphs were phenotypically fused. Table 1 gives the distribution of male and female tissue within them. The data show that the first posterior cell and LV 3 are never entirely female (nonfused). Interestingly, this is also true of LV 4, a contrast with the reciprocal case, in which the LV 4 from phenotypically wildtype wings is often genotypically fused (male). Not surprisingly, the anterior com-

TABLE

1

I

29 32

43 19

33 42

38 23

29 58

43 23

33 48

29 24

43 61

36 10

33 55

390

33 61

390

715

260

330

100 0

07

940

33 13

360

19 10

260

45 0

455

615

260

24 45

29 16

33 29

14 16

33 36

14 19

- - -

23 16 -

19

- - I

“When the percentages do not. add up to IOOO., the missing numbers represent cases in which the st.ructure was not formed. ‘, LV = longitudinal vein. ’ Total fu phenotype = 21; total for + phenotype = 31.

Percent.age mosaic

Percentage female ;I,

23

383

- - -

29 39

38 10

- -.

53

57 17

s

$

$I

5’

8

% ? 2

- . .- -

53

52 13

TIIE FIIEQIIISN(‘Y WITI! WHK*H DIFFENRNT WINI; REGIONS AHE MAIX (y sd fil”!‘), FF.MAI.E (y ~n~‘fil”~/u~“‘), OH MOSAIC (CONTAINING BETH MALE ANT> FF.MAI.F. TISSIIF.) IN WINGS WHICH AI~F. EITHEH PHF.NOTYI~IVAI.I.Y W1l.r) TYPF: (+) OH fused (fu)” Alula Discal Third LV3 First .Second First posLV 4 Costal Submaryfezl cell posterior basal cell posterior terior cell cell and ginal cell cell and cell and and antecostal LV Ih and LV 2 LV5 nor crossposterior vein vein crossvein --___ ~ ~ ___ -___ DVDVDVDVDVDVDVDVDVDVDV ---___Percentage male 45 65 52 74 80 83 0 3 0 0 5:! 65 10 13 39 55 +’ 48 35 19 23 3 7 52 57 33 38 38 43 62 90 67 100 43 57 14 14 48 52 fu’ 29 29 29 43 48 67

-

_^

FIG. 3. Unshaded, XPa/y sd fu5’ (wild type) tissue; stippled, dorsally locatql y sna fu” tissue; striped, ventrally located y en3 /iP’ tiaaue. (A) Distribution of y sn3 fu” tissue on a mosaic wing with wild-type vein pattern. (B) Distribution of y un3 fu” tissue on a mosaic wing with a ficsed vein pattern. (C and D) A photograph and drawing of a wing with a noti LV 3 and a fu LV 4. The distribution of y ~9 fi” tissue is shown on the drawing. (E and F) A photograph and diGi& of a wing with fi LV 3 and wild-type LV 4. The distribution of .y sn3 fitbe tksue 2 shown on the drawing.

Pattern

ANNE FAUSTO-STERLING

partment of these phenotypically fused wings is never entirely female. Figure 3B provides an illustration of a mosaic fused wing. mixed wings. (3) Phenotypically Twenty-six of the mosaic wings exhibited phenotypes which were neither entirely fused, nor entirely wild type. It was possible to divide these into two groups. The first, of which there were 19, had a relatively normal LV 3 but a fused LV 4 (an example is shown in Figs. 3C and 3D); the remaining seven had a fused LV 3 but a relatively normal LV 4 (an example is shown in Figs. 3E and 3F). The distribution of male and female tissue in these wings is shown in Table 2. Only those regions relevant to the formation of the fu phenotype are described. The data show that the dorsal genotype of fused-like structures are never entirely wild type, and vice versa. There are, however, a number of cases in which the ventral cells of a phenotypically fused structure may be genotypically wild type. The converse is also true. Careful examination of such veins under the compound microscope shows that they are made from cells on both the dorsal and ventral wing surfaces. This implies that fused cells on the dorsal surface of the wing can influence

ventrally located wild-type cells to engage in vein formation in places where a wildtype vein would normally not be found. The conclusion drawn from the data in the previous sections that LV 4 may be nonautonomous under certain circumstances, while LV 3 is always autonomous is supported by the data in Table 2. In 14% of the cases where LV 4 was wild type, it was nevertheless entirely male on its dorsal surface. However, in none of the cases for which LV 3 was wild type was it entirely male. All 26 mixed phenotype wings are mosaic for tissue in the first posterior cell and the bordering veins. Since the anterior-posterior compartment border runs through the first posterior cell, it is not surprising that the anterior and posterior compartments of all of the mixed phenotype wings are mosaic, both dorsally and ventrally (data not shown). Finally, the mosaicism observed in the mixed phenotype wings provides a clear indication of the fact that wild-type cells can take part in the formation of fused structures and vice versa. The data suggest that while there is some sort of regional autonomy with regard to the control of vein pattern formation, no such autonomy exists at the level of the individual cell. (4) Mosaic wings with incomplete vein

WING (CONTAINING

OR MOSAIC

First posterior cell -___ D

\

(y WI ’

LV4

First basal cell and anteriot crossvein” v

D

0 0

0 14

47 14

53 “9

11 14

11 0

68 0

53 14

0 14

0

11

29

“9

29

79 loo

3% loo

47 71__--

53 71

32 4:3

79 14

47 14

11 0

Percentage female LV 4 “ficsed-like” LV 3 “fused-like”

0 0

Percentage mosaic L\’ 4 “fused-like” LV 3 “fused-like”

95 100

D

FI-:MAI.E

Trssrw~

D

5 0

are absent.

LV 3

(y sn ’ fit”!‘),

v

Percentage male LV 4 “fused-like”* LV 3 “fused-like”’

____-_~ ” In some cases these structures ” Total = 19 wings. Total = seven wings.

365

in Fused Mutant

TABLE 2 STINK~T~~I{W AIE MALE BOTH MAW r\Nr) FEMALE

FIWX~IIEN(.Y WITH WHICH Mrxc~n PHENOTWE fu”“/u”),

Formation

lfj

~~~~~~.~-

v 3’2 14

DEVELOPMENTAL BIOLOGY

366

formation. Among the mosaic wings from gynandromorphs were 11 in which large segments of LV 3 and/or LV 4 were missing. Five of the 11 (5/11) formed veins on neither the dorsal nor the ventral wing surfaces, four had some regions in which there was vein formation only on the dorsal side, and two had partial vein formation on nonoverlapping portions of either the dorsal or the ventral surface. All of these wings were profoundly mosaic in the critical region bounded by LV 3 and LV 4. The existence of this group of wings suggests that it is not always possible for wild-type and fused tissue to cooperate in making a normal vein. Mosaic wings obtained from the induction of somatic crossing over. The gynandromorph studies discussed above describe expression of fused in large patches of mosaic tissue, induced very early in development. We wished also to look at small patches of fused tissue formed during the postembryonic period. Therefore, we induced homozygous fused clones in a heterozygous background at different stages of development. The specific chromosomal arrangement used is diagrammed in Fig. 4. Briefly, females were used which were homozygous for the autosomal cellular wing marker multiple wing hair (mwh); they also had one X chromosome bearing the mutation fu5’, and the other bearing yellow (y), a small duplication of the wild-type allele of mwh (mwh+), and a severe allele of the cell marker forked (p). Such females were phenotypically wild type. For the most frequently expected class of crossovers (Garcia-Bellido, 1972), homozygous

MWH MWH

)vruol*IA lMuynrl

FIG. 4. The chromosome

1

constitution for experiments with X-ray-induced somatic crossing over. The location of the most frequent crossover point is shown as well as the resultant twin spots.

VOLUME 63.1978

fused tissue should be recognized as mwh spots, while the sister clone should be y, f. In fact, y, f-mwh twin spots were observed frequently. We found 21 wings containing large (15 or more cells) clones in the region of the wing bounded by and including LV 3 and LV 4. Sixteen of these were phenotypically wild type. The patches of fused tissue were either small and in the central portion of the first posterior cell or were larger and ran along the length of LV 4. The latter group is consistent with the conclusion already drawn that a fused LV 4 is nonautonomous in a predominantly wild-type background. The five mosaic wings expressing a partially fused phenotype are drawn in Fig. 5. Four of these (Figs. 5A-D) have clones of fu mwh tissue on the dorsal surface only. Three (Figs. 5A-C) show limited areas of “fused-like” vein formation. The veins are formed from both the dorsal and ventral cells. The smallest clone (Fig. 5B) associated with an area of extra vein formation involved only 24 cells. Figure 5D shows a wing with a large stripe of mwh cells running the length of the wing. At the tip of the wing a fused vein pattern is observed, while in the central portion of the first posterior cell there is no proper vein formation. Instead, there are areas of dorsal vein-like chitin deposition. Lastly, the wing in Fig. 5E has mwh tissue on both the ventral and dorsal wing surfaces. In that portion of the wing which is fu mwh on the reciprocal dorsal/ventral surfaces, there is a clear-cut fused-like vein. Where there are only ventrally located fu cells, one sees a fused-like structure which is in actuality nothing more than a heavy deposition of chitin confined to the ventral surface. Such observations were also made for some of the mosaic wings from gynandromorphs (see previous section). DISCUSSION

A number of conclusions and observations may be made about the studies presented in this paper:

ANNE FAUSTO-STERLING

Pattern Formation in Fused Mutant

367

FIG. 5. Phenotypically and genotypically mosaic wings resulting from X-ray-induced somatic crossing over in heterozygous fused flies. The distribution of mosaic tissue is shown. Unshaded, wild type; stippled, dorsally located fu59;mwh tissue; striped, ventrally located fu59;mwh tissue; dashed lines, incomplete dorsal vein formation; dotted lines, incomplete ventral vein formation. Wings A and B come from larvae X-rayed between 61 and 77 hr of development (counting from the time of egg-laying), C from a larva X-rayed between 40 and 60 hr of development, and D and E from X-irradiation at 12-36 hr of development.

(1) The fused wing phenotype may be expressed even when a large proportion of the body of the fly is genotypically nonfused. Furthermore, large portions of an individual wing may be genotypically nonfused but nevertheless have a fu vein pattern. These observations confirm and extend earlier work (Clancy and Beadle, 1937; Fausto-Sterling, 1971) demonstrating that the fu locus does not involve the production of a diffusible substance at a distant site. (2) The fu locus affects the formation of LV 4 as well as the differentiation of LV 3. The third longitudinal vein may be thick-

ened and branched to varying degrees. While LV 4 may be partially or completely present in less extreme cases, it is not formed at all in the most extreme expression of fi. (3) A large number of phenotypically wild-type wings were obtained in which the posterior compartment, including the entire fourth longitudinal vein, was genotypicaIly fused. In these wings, fu cells in the first posterior cell and LV 4 behaved nonautonomously. In contrast, phenotypically fused wings never had fourth longitudinal veins which were entirely non-fused in gen-

368

DEVELOPMENTALBIOLOGS

otype. We conclude that when LV 4 is entirely or predominantly wild type, it behaves autonomously. The data obtained from wings of mixed phenotype also support this view. However, patches of wildtype tissue on LV 4 were on occasion incorporated into a fused LV 4. Thus, small groups of wild-type cells may behave nonautonomously in a predominantly fused genetic background. (4) The data obtained in this study provide evidence that prospective longitudinal vein-forming cells on the dorsal wing surface influence the underlying ventral cells to participate in vein formation. Mosaic wings of mixed phenotype show this most clearly. For example, when LV 3 expressed a fused phenotype, it was never found to be entirely female (+) on the dorsal side, although in 14% of the cases it was completely female on the ventral side. Similarly, fusedlike LV 4’s were never entirely wild type on the dorsal surface, but in 16% of the cases were entirely so on the ventral side. Additional supporting evidence comes from an examination of the mosaic wings obtained from somatic crossover induction. (5) Among the mosaic wings which failed to form veins, an occasional vein-like deposition of chitin occurred on either the dorsal or the ventral surface. In his study of certain mutations of vein formation, Waddington (1940) made similar observations. This phenomenon suggests that the heavy chitin deposition characteristic of vein forming cells can occur independently of vein formation. After studying mosaics from a number of mutations which cause extra veins, failure of vein formation, or extra chaetae, GarciaBellido (1977) concluded that vein determination was a two-step process. He suggests that the approximate location of the veins is first laid down autonomously on both wing surfaces; the final location of the veins is then determined by interaction between the dorsal and ventral wing surfaces, the cells of the ventral wing surface exhibiting a nonautonomous behavior with respect

VOLUME 63.1978

to vein formation. We have observed a significant number of mosaic wings which formed a wild-type LV 4 in spite of the fact that this vein was both dorsally and ventrally fused. Such events suggest that the failure in fused wings to form LV 4 is not a cell-autonomous characteristic. Rather it appears to depend on the genotype of cells which lie in between LV 3 and LV 4 (see Fig. 3A as an example). If this observation were fit into Garcia-Bellido’s scheme it would imply t,hat the initial roughing out of the vein pattern in the developing wing is not solely dependent on the genot.ype of the vein-forming cells. We have also presented evidence for the idea that the dorsal wing surface influences the development of cells on the ventral side. This confirms the observations of a number of workers (Lawrence and Morata, 1976; Lees, 1941; Santamaria and Garcia-Bellido, 1975; Garcia-Bellido, 1977). The data presented in this paper suggest that in order for proper vein formation to occur the fu’ gene must be active in some minimum number of cells in the first posterior cell. Large blocks of wild-type tissue in the first posterior cell result in the formation of a wild-type or wild-type-like LV 4 even when the cells comprising LV 4 are fused. In cases where there is a finer intermingling of fused and wild-type tissue in the region of the presumptive first posterior cell, we observed a complete disruption of vein formation. Lawrence and Morata (1976) found that non-engrailed cells can participate in the formation of a largerthan-normal posterior compartment if they are influenced by en clones which are larger than a certain minimum size. Our observations that small numbers of wild-type cells can participate in the formation of fused structures is consistent with their observation. It is also consistent with the idea that the forces involved in the wing contraction and vein formation are probably supracellular, representing the sum of individual cell forces such as the strength of individual intercellular adhesions.

ANNE FAUSTO-STERLING

Pattern

Currently we have no real idea of what the wild-type gene product coded by the fu locus might be. We do, however, know several things about the effects of mutation at this locus: On the basis of the data described in this paper we suggest that fu affects certain quantitative properties involved with wing contraction during the process of vein formation; King et al. (1957) have shown that homozygous fused females have tumorous ovaries and often produce egg chambers which fuse together; finally, Counce (1956) described the “sliding” apart of lateral halves of lethal fused embryos. Pufting together these observations, we speculate that the fu’ gene may act in some manner to affect the cell surface properties of at least those cells which show effects from the fu mutation. Hopefully, we will in the future be able to put this speculation to experimental test. Different portions of this work have been supported by the following grants GM 16538, RR-05664, R01 HD 07918, and from the National Science Foundation, BMS 74-19691. We wish to thank Ms. L. Pearlman and Mr. M. Maguire for their technical assistance. REFERENCES CLANCY, C. W., and BEADLE, G. W. (1937). Ovary transplants in Drosophila melanogaster: Studies of the characters singed, fused, and female-sterile. Biol. Bull. 72, 47. COUNCE, S. J. (1956). Studies on female-sterility genes in Drosophila melanogaster. II. The effects of the gene fused on embryonic development. 2. Indukt. Abstamm. Vererbungsl. 87,462-481. FAUSTO-STERLING, A. (1970). Developmental and genetic studies on the female-sterile mutant fused of Drosophila melanogaster. Brown University, Ph.D. thesis. FAUSTO-STERLING, A. (1971). On the timing and place of action during embryogenesis of the female-sterile mutants fused and rudimentary of Drosophila melanogaster. Develop. Biol. 26, 452-463. FERRIS, G. F. (1965). External morphology of the adult. In “Biology of Drosophila” (M. Demerec,

Formation

in Fused Mutant

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