Hwsn-in,andy-in. +++(
GENETIC CONTROL OF PATTERN DETERMINATION IN DROSOPHILA. THE ACTION OF HAIRY-WING1,2,3 FREDERICK JAY GOTI’L1,FiB Departments of Genetics and Zoology, University of California, Berkeley, and Department of Biology, University of Pittsburgh, Pittsburgh, Pennsylvania4 ‘ReceivedOctober 21, 1963
HE location and number of thoracic bristles in Drosophila melanogaster is under genetic control. These bristles fall into two major classes: macrochaetae and microchaetae. The macrochaetae are large bristles, few in number and constant in location; the microchaetae are smaller, more numerous and more variable in number and position, but have a regular gross distribution pattern. The constancy of the wild-type phenotype suggests that a homeostatic mechanism is involved. Indeed, the large number of mutant genes which affect the bristles is indicative of such a system. Adult bristle pattern is the result of regional hypodermal differentiation during development, which suggests that some control pattern is present prior to the final differentiation. The term “prepattern” (STERN1954) has been used to describe the spatial control factors which determine the differentiation of pattern in development. In the case of bristle organs, this prepattern is the potential plan of total possible bristle disposition. It thus follows that there are two major ways in which the phenotypic bristle pattern can be altered genetically. The mutant may act early in the sequence of events of differentiation and thus alter or replace the existent prepattern or it may alter the developmental competence of the differentiating tissue to respond to the existing prepattern, changing the phenotype without disturbing the prepattern. Results have been reported indicating both modes of control. HANNAH-ALAVA (1958) described a group of related cases of mutants controlling sex-comb formation on the second and third legs and suggested that the mutant pattern may be due to a genetic change in the prepattern. Genetic mosaic analyses of the control 1957), hairy mechanisms of achaete (STERN1954), scute (STERNand SWANSON (STERN1954), Theta (STERN1956) and engrailed (TOKUNAGA 1961) have been interpreted as indicative of shifts in developmental competence without neomorphic prepattern alterations. 1 A portion of a dissertation submitted to the Graduate Division of the UniTersity of California, Berkeley, in partial satisfaction of the requirements for the degree of Doctor of Philosophy. 2The major portion of the research was performed while the author was a trainee under Public Health Service Genetics Training Grant 2G-367. 3 A portion of the research was supported by an American Cancer Society Institutional Grant to the University of Pittsburgh and by Public Health Service Grant GM 1108401. 4 Present address.
Genetics 49: 739-760 M a y 1964.
740
F. J. GOTTLIEB
The present investigation is concerned with the dominant sex-linked mutant Hairy-wing ( H w ) , in particular the allele Hwlgc,which causes the appearance of numerous extra micro- and macrochaetae on various extensive areas of the body. An analysis of the control mechanism of this mutant was done by means of genetic mosaics obtained as the result of induced somatic crossing over. The mosaic technique enables us to study not only the interactions between adjacent tissues of different genotypes in the same animal, but also permits us to assess the effects of late substitution of one allele for another. MATERIALS A N D METHODS
Stocks: The Hairy-wing locus lies at O.O+ on the X chromosome. The extreme mutant allele Hw-".9c (POULSON and KING 1949) was used. The Hairy-wing homozygote is sterile and possesses large numbers of extra macrochaetae o'ver the body, notably on the head and thorax. There are large numbers of extra microchaetae on the thorax, mesopleurae, on wing vein L2 and in the wing cells. The heterozygous female is fertile, has few extra macrochaetae, and shows extra microchaetae on L2 and 3 and occasionally on the wing cells. The mutants yellow ( y ) and singed-3 (sns), at 0.0 and 21.0, respectively, on the X chromosome, served as morphological markers. The inversion chromosomes, Cy, Ins05 (OSTER1956), Zn(2LR)Pm (BRIDGESand BREHME1 9 4 ) , and Zn(SLR)Ubz'30 (LEWIS1952) and the third chromosome marker Stubble ( S b ) (BRIDGES and BREHME1944) were used in the production of co-isogenic stocks. (Since only the above mentioned alleles were used, the superscripts will be omitted in the following discussion and they will be referred to as: H w and sn.) Stocks were maintained at 26" k 1"'C, experimental animals at 25" & I". Mosaic production: The genetic mosaics in this work were obtained following BECRER'S (1957) techniques utilizing X-irradiation to increase the frequency of somatic crossing over. Larvae from the cross y / y x H w sn were irradiated with 1200, 1500, 1800, 2000, 2200 or 2400 r of hard X rays (250 kv, 15 ma, with total filtration equivalent to 3 mm aluminum f .25 mm copper) at a dose rate of 100 r per min, and at ages ranging from 15 to 70 hr after hatching. H w s n / H w sn and y / y tissue patches omn an H w sn/y background were recovered among the female offspring. All H w sn/y pro'geny were examined under a dissecting microscope and the location of mosaic patches was recorded. #Control thoraces and those mosaics to be measured were dissected and mounted on slides, employing a method which makes use of an aqueous miscible mountant ( GOTTLIEB 1963). Measurements: Because the thorax is relatively flat in the dorsocentral region and because it was felt that artificial flattening would introduce distortion, no effort was taken to alter the thoracic curvature. Thus the linear measurements, represent distances on a projected plane passing through the bristles i n question. Since the curvature does not greatly vary between genotypes, the distances measured bear a relatively constant relationship to the actual surface distances. The mounted thoraces were measured under a compound microscope. Camera lucida drawings of dorsocentral regions containing mosaic patches were made. The diagram of the thorax used in Figures 1 and 2 was derived by perpendicular projection of a mounted wild-type female thorax through a camera lucida. The relative position of the mesothoracic macrochaetae is shown in Figure 1. Twelve measurements and counts were made on each thorax (Figure 2A and B) : The width of the thorax was estimated by the distance between the two posterior supraalar bristles (PSA), and by the width of the scutoscutellar suture (SW); the length, by the distance from the tip of the scutellum to a line connecting the anterior dorsocentral bristles (L). Six measurements were made in the dorsocentral region as given in Figure 2A. All chaetae, except the four dorsocentrals, were counted in the area bounded anteriorly by a line connecting the anterior dorsocentrals, posteriorly by the scutoscutellar suture and o n the sides by lines extended from the anterior dorosocentrals, through the posterior dorsocentrals, to the suture.
DROSOPHILA P A T T E R N D E T E R M I N A T I O N
741
An estimate of the area of this region (AR, see crosshatched area i n Figure 2B) was computed from the linear measurements on the assumption that it is essentially a regular trapezoid. From this estimate and the count of the number of chaetae in this region (HI), the chaetal density (HI/AB), i n chaetae per unit area, was computed. For the count of extra macrochaetae (XTRA), the dorsocentral region was extended right and left to an imaginary line, parallel to the right
ab FIGURE 1.-Diagram of dorsal view of the pro- and mesothorax with locations of the mesothoracic macrochaetae. a-anterior, p posterior; ds-dorsocentral, np-notopleural, pa-postalar, ps-presutural, sa-supraalar, sc-scutellar.
-A-
-6
FIGURE 2.-Diagrammatic dorsal view of the thorax indicating locations of measurements and areas of counts. A. Distances measured i n the dorsocentral region. ADC-anterior dorsocentral width, PDC-posterior dorsocentral width, DICR, DCL-distance between the anterior and posterior dorsocentral bristles on the right and left sides, DCSR, DCSL-distance from posterior dorsocentral bristle to scutoscutellar suture on the right and left sides as extensions of the lines of DCR and DCL. B. Thoracic width and length measurements and the areas of counts. PSA-distance between the posterior supraalar bristles, SW-width of the scutoscutellar suture, L-distance from anterior dorsocentral line to tip of the scutellum. Crosshatched region represents AR, HI is counted in this region. (Double lines indicate the outer limits for the counts of extra macrochaetae (XTRA) .
742
F. J. GOTTLIEB
and left dorsocentral rows and just medial to the posterior postalar bristles, and extended anteriorly to include the locations of two microchaetae in the dorsocentral rows anterior to the anterior dorsocentrals (region bounded by double lines, Figure 2.B). Several additional relationships were computed from the above measurements. In the mosaic series, a number of additional measurements were made. The total area of the dorsocentral region was measured on the camera lucida drawings by means of a compensating polar planimeter. The areas of the H w s n / H w sn and y / y mosaic patches in the dorsocentral region were also measured in this manner. These area measurements, in cm‘, were converted to square scale units. The numbers of Hw s n / H w s n and y / y chaetae in mosaic patches of the same genotypes were counted. From these, three additional chaetal density ratios were computed: a corrected ratio, COR HI/AR (the total number of chaetae in the dorsocentral region less the number of H w s n / H w s n and y / y chaetae occurring in patches in this region divided by the total computed area of the region less the areas of the H w s n / H w sn and y / y patches in this region), an H w s n ratio, Hw HI/AR (the number of H w s n / H w s n chaetae occurring in patches in the dorsomcentral region divided by the area of these patches) and a y ratio, y HI/AR (the number of y / y chaetae occurring in the patches in the dorsocentral region divided by the area of these patches). The data were subjected to an analysis of variance, utilizing orthogonal (LI 1959) and nonorthogonal (LI, personal communication) contrast techniques. The results of these contrasts are listed in Tables 1 and 3 below ‘the mean values of the measures, and the contrasts made are listed under “Comparisons.” Calculations were performed on IBM 1620 and 7070 computors RESULTS
A. Control Series: In order better to understand development in the mosaics, it is desirable to characterize the effect of the H w mutant in nonmosaic animals. Therefore, females of the genotypes y / y , H w sn/y, and H w sn/Hw sn were examined. Twenty-five thoraces for each of these genotypes were measured as an unirradiated control series. The H w s n / y controls were sibs of the flies irradiated in the mosaic series. The H w s n / H w sn and y / y flies all had H w s n / y mothers and came from the crosses H w s n / y x H w sn and H w s n / y x y , respectively, where the H w sn males came from the same stock as the fathers of the H w s n / y flies and the y males were sibs of the H w s n / y females. To reduce background heterozygosity and thus phenotypic variability in the expression of H w sn/Hw sn and of y / y , stocks of these two genotypes were constructed which were coisogenic for their second and third chromosomes. The coisogenic chromosomes were derived from a single second and third chromosome from the original H w sn stock. While this procedure eliminates approximately four fifths of the background heterozygosity, a difference in response to the isogenic background is to be expected since H w s n / H w sn; Iso(2;3) differs from H w sn/Hzu sn by only one second and one third chromosome, while y / y ; Iso(2;3) differs from y / y in both of its second and both of its third chromosomes. Measurements: There are four measures of thoracic width (Table 1) . Two of them, PSA and SW give an estimate of the overall thoracic width, just anterior to and just posterior to the dorsocentral region. The other two, ADC and PDC define the width of the dorsocentral region. It can be seen (Table 1) that the substitution of the background chromosomes does not affect significantly the expression of H w in any of the four width measurements. In the case of y / y , however, the isogenic stock is significantly wider than the nonisogenic stock in
743
DROSOPHILA PATTERN DETERMINATION
TABLE 1 Comparisons between controls and mosaics
ADC Controls: Y/Y Y/Y;~SO(~;~)
Hw sn/y Hw sn/Hw sn Hw sn/Hw sn;Iso(2;3) Mosaics : both-in HW sn-in y-in neither-in
Width measurements; PDC PSA
SW
DC
Length measurements: - _ _ DCS DC+DCS
L
23.692.22 24.362.25 25.40k.40 25.772.38 25.28k.28
22.45f.23 23.54-C.23 23.962.39 23.94-C.35 24.10k.25
46.462.39 52.74f.48 52.422.68 51.03f.65 52.26k.47
26.502.24 28.20C.22 27.94k.38 27.68C.36 28.48f.24
9.532.16 11.052.17 9.682.17 9.622.20 9.762.20
6.46-C.22 7.012.24 6.842.21 6.242.26 5.34k.13
15.99f.34 18.062.37 16.52k.33 15.862.42 15.10f.27
34.592.50 38.90-C.50 36.002.61 33.862.67 33.64544
26.532.26 26.992.24 2.5.65f.39 26.652.29
25.16f.26 25.60k.26 24.21-C.38 25.26-C.27
56.35f.44 56.552.47 55.032.72 56.992.49
29.652.29 30.22C.26 28.87k.36 30.192.31
11.192.23 11.09f.22 10,192.24 10.74k.22
5.842.22 6.692.17 5.54k.22 5.872.21
17.02f.42 17.78k.33 15.73C.40 16.61-C.40
37.30f.65 38.64f.46 35.492.67 37.052.64
Comparison: Y/Y vs. y/YIs0(2;3) = ++(
