Tumorous Head in Drosophila*

TUMOROUS HEAD IN Drosophi/a*

.

Eldon J Gardner Department of Zoology. Utah State University. Logan. Utah

I. Introduction . . . . . . . I1. History of Tumorous-Head Strain . 111. Tumorous-Head Phenotype . .

. . . . . . . . . . . . A. Description of Head Abnormalities . . . B. Expression in Females and Males . . . . . I V . Genetics of Maternal Effects . . . . . .

.

. . . . . .

. . . . . .

. . . . . .

. . . .

. . . . . . . . .

A Maternal Effect on Tumorous-Head Phenotype . . . . . B. Maternal Effect on Male Genital Disc Defect . . . . . C. Third Chromosome Polymorphism . . . . . . . . D . Maternal Effect on Fertility . . . . . . . . . . E . Maternal Effect on Viability of Homokaryotypes . . . . V. Location of Major Genes . . . . . . . . . . . . A . Location of tu-9 . . . . . . . . . . . . . . B. Location of tu-I . . . . . . . . . . . . . . V I . Behavior of Inversion in Populations . . . . . . . . . A. Adaptive Advantage of Heterokaryotype . . . . . . B . Behavior of Inversion in Population Cage Study . . . . C. Possible Origin of tu-h Strain . . . . . . . . . . VII . Alleles of tu-I in Laboratory Stocks and Natural Populations . . A. Laboratory Stocks . . . . . . . . . . . . . B . Natural Populations . . . . . . . . . . . . . C. Gene Frequency Related to Temperature Change . . . . VIII . Modifiers of tu-3 J . . . . . . . . . . . . . . IX . Genetic Divergence in Laboratory Stocks . . . . . . . A. Divergence among Stocks Maintained in Different Laboratories B. Divergence in Population Cage Studies . . . . . . . X . Melanotic Tumors in tu-h Stocks . . . . . . . . . . Recessive Lethal Gene Associated with Failure of Pupation . XI . Developmental Studies . . . . . . . . . . . . . Ovary Transplantation . . . . . . . . . . . . XI1. Biochemical Studies on Tumorous-Head Flies . . . . . . A . Chemical Additives to the Food Medium . . . . . . B. Biochemical Composition of Tumorous-Head Flies . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . .

.

. . . . . . . .

. .

116 116 117 117 121 121 121 124 125 126 127 127 127 127 128 128 128 129 129 129 131 131 131 132 132 134 135 136 136 137 137 138 138

*The investigations conducted at Utah State University were supported by The United States National Institutes of Health. The American Cancer Society. and the Damon Runyon Memorial Fund for Cancer Research. 116

116 XIII. h l a t e d Phenotypes . A.Scarp. . . B. Eyes-Reduced C. Wi t t y. . . XIV. Summary. . . References . .

. . . . .

ELDON J

. GARDNER

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

138 139 141 142 142 143

1. Introduction

The tumorous-head genetic system in Drosophila melanogaster has provided a basis for investigating several different maternal effects and other genetic mechanisms. The study began a t the University of Utah in 1946, and much of the basic research was done a t that institution. Members of the original team of investigators later moved to Utah State University and to Arizona State University where they and their graduate students continued the research. Several other laboratories have since become involved in particular aspects of the study. Active research designed to answer unresolved questions is now in progress in several laboratories. 11. History of Tumorous-Head Strain

The tumorous head strain of Drosophila melanogaster originated from a collection of wild flies taken in 1941 near the village of Acahuizotla,

Mexico, about 50 miles north of Acapulco, by a collecting party from the University of Texas. No phenotypic deviation from wild-type flies was detected in the field nor when the sample was observed microscopically a t the University of Texas. The flies were maintained in the laboratory by the usual means of culture as a wild-type stock. Several years later, abnormal structures were observed in the head region of some of the flies that had descended from the original collection. These flies were selected and inbred. A culture was obtained by W. W. Newby a t the University of Utah in 1946 through the courtesy of Wilson Stone of the University of Texas and established in the Drosophila laboratory (Gardner, 1948a). About 64% of the adult flies showed abnormalities in the head region. The irregularities appeared amorphous growths and were located exclusively on the heads of the flies. Other parts of the body did not differ from those of wild-type flies. The descriptive name “tumorous head” was coined to characterize the phenotype, and the symbol “tu-h” was established to identify the University of Utah stock that was developed by continuous selection and inbreeding of abnormal flies. The proportion l m

TUMOROUS HEAD IN

Drosophila

117

of flies expressing the external visible trait increased within a few months to 76% and after several years of intense selection reached a level of SO-SO% of the flies expressing the external manifestations of the tumorous-head trait. 111. Tumorous-Head Phenotype

A. DMCRIPTION OF HEADABNORMALITIES Newby (1949) described the morphology and distribution on the head (Fig. 1) of abnormal growths. He found the individual irregularities to vary from small inconspicuous projections (Fig. 1, 13 ) or depressions, to massive growths (Fig. 1, 19). The growths were usually unilateral, seldom bilateral, and never symmetrical. I n many flia, both sides of the head were affected, but the individual growths did not cross the midline. Eyes, antennae, and other dorsal and lateral parts were frequently involved (Fig. 2) , but the mouth parts were described by Newby as entirely free from abnormal growths. Some flies with the trait lacked the characteristic growths, but showed an eyeless phenotype similar to that of the fourth chromosome gene ey. The arista of one or both antennae was often replaced by a massive amorphous structure, an elongated process, or a leglike growth. Replacement of an antenna with a leglike growth indicated homoeosis. Growths that appeared to be homoeotic in nature varied considerably in size and were usually crude structures by comparison with the normal appendage with which they were related (Fig. 1 ) . For example, no perfect leg was observed as a replacement for an antenna. One, two, and sometimes three leglike segments were observed (Fig. 1, 3 and 4 ) , but they were usually fleshy growths different in many respects from normal legs. Bristles and sometimes claws (Fig. 1, 11) occurred on the leglike growths replacing antennae. Most growths, however, did not follow any pattern of replacement of one part by another but were amorphous and irregular (Fig. 1, 12-20). They gave the impression of a confused developmental process preceding the origin of the visible abnormality that appears in the adult. The compound eyes are the largest structures on the head, and these were most frequently involved with abnormal growths. Eye abnormalities ranged from small depressions to massive extensions that completely obliterated one or both eyes. Growths that replaced parts of the eye frequently also involved areas surrounding the eye, particularly the tissue of the carina, the ridge beneath the eye.

118

ELDON J . GARDNER

F I ~ 1. . Different manifestations of tumorous-head expression. Normal head structures are in outline, and abnormal growths are shown in stipple. 1-19: Antennal growths showing range of expression ; 13-16: large-scale drawings of small, simple growths extending from the surface of the eye; 16-19: eye growths showing range of expression; and SO: face (carina growth also involving the eye). (From Newby, 1949.)

TUMOROUS HEAD IN &OSOphdU

119

FIQ. 2. Photographs showing different expressions of tumorous-head. (From Gardner and Gardner, 1953.)

120

ELDON J . GARDNER

Newby also described internal head structures associated with the tumorous-head trait (Fig. 3 ) . These were saclike, vesicular areas of the head wall. Some were everted and appeared as extensions of the surface (Fig. 3, B). Others were inverted and extended into the interior of the head (Fig. 3, C). Combinations were observed with some given vesicles

Fro. 3. Drawings showing internal expressions of tumorous-head. (A) Longitudinal section of a growth of the antenna. (B) Section through internal growth that was visible on the surface as a small mass on the carina. (C) Section through an internal growth not visible on the surface located at the ventral border of the right eye. (D) Section of an eye growth that appeared on the surface but also had an extensive internal portion. (From Newby, 1949.)

TUMOROUS HEAD I N D T O S O p h d U

121

partly everted and partly inverted (Fig. 3, D). Serial sections of tu-h adults that had no external abnormalities often revealed the presence of subsurface growths. These observations indicated that most, if not all members of the inbred tumorous-head stock probably expressed the trait either externally or internally.

B. EXPRESSION IN FEMALES AND MALES By comparing the proportion of eggs, larvae, and adults in the tumorous-head strain and wild-type Lausanne-S (Gardner and Ratty, 1952), it was shown that, a t 2loC, 70% of the tu-h eggs hatched compared with 86% of the wild type, and 50% compared with 71% became larvae. At 3OoC, 35% of the tu-h eggs hatched compared with 66% of the wild type, and 6% of the tu-h larvae became adults compared with 55% of the wild type (Ratty, 1949). A higher proportion of females than males expressed the tumorous phenotype, and females had more complex abnormalities than males. IV. Genetics of Maternal Effects

Initial studies on the genetics of the tumorous-head system showed that different results were obtained from reciprocal crosses. When tu-h females were crossed with wild-type males, about one-third of the F, progeny showed the trait. Some fluctuation in the proportions occurred when the cultures were maintained a t different temperatures. From the reciprocal cross between tu-h males and wild-type females, less than 1% of the F, progeny expressed the tumorous-head phenotype (Table 1). This difference in the results of reciprocal crosses suggested a genetically controlled cytoplasmic influence (Gardner, 1948b). When it was demonstrated that a factor (tu-1) in the first chromosome of the mother was largely responsible for the conspicuous difference in the results of reciprocal crosses, the mechanism was explained as a maternal effect (Gardner and Woolf, 1949; Gardner, 1949a,b, 1951).

A. MATERNAL EFFECTON TUMOROUS-HEAD PHENOTYPE The experiments that confirmed a maternal effect also showed that a basic gene (tu-3) in the third chromosome was necessary for any expression of the tumorous-head trait. It was shown repeatedly that no expression of the trait occurred in the absence of tu-9. I n the heterozygous condition, tu-3 alone (without tu-f in the mother) controlled

122

ELDON J. GARDNER

TABLE 1 Average Percentages of Abnormal Flies in the FI from Reciprocal Outcrosses between tu-h and Laboratory Stocks* ~__________________

tu-h X Laboratory stock Laboratory stock Canton Florida Wild (Turtox) W

a1 d p d b c p x s p 88'

e Bd" In(3R)C Z(3)e Muller 11* CgfPm;H / S b DIG1 M(3) 124/In(3R)C eZ(3)e eyD/ar

* Data from Gardner and Woolf

t n, Total flies.

Reciprocal cross

nt

%**

n

%

750 107 807 197 96 167 255 157 216 361 90 170 101 64

25 52 19 19 33 49 20 20 19 37 39 14 23 37

1542 220 947 583 78 598 96 53 42 289 88 132 35 27

0 1 1 1 0 0 0 0 0 0 1 0 0 0

(1949).

** Percentage of abnormality with accuracy to nearest whole number. Less than 0.5% is recorded as 0. a small expression of the tumorous-head trait. Gene tu-3 was, therefore, described as a semidominant. Less than 1% of the flies with one tu-3 gene expressed the trait. When tu-3 was homozygous, but tu-1 was not present in the mother in proper arrangement for expression, 1-3% of the flies showed the abnormality (Table 2). 1. Temperature-Sensitive Period

When tumorous-head flies were maintained a t different temperatures, the proportion of flies expressing the trait increased, parallel with increased temperatures. Gardner and Woolf (1950) showed a fairly consistent increase of expression, parallel with temperature change, in the University of Utah inbred tu-h stock, from 56% a t 15OC to 93% a t 3OOC. Maintaining the cultures at particular temperatures for different 24-hour intervals proved the first 24 hours of development in the egg

123

TUMOROUS HEAD IN D T O S O p h d U

TABLE 2 Crosses Designed to Determine the Location and Action of Genes Involved in the Expression of Abnormal Growths and the Results of these Crosses* Cross

0

d

-

1 tu-1 tu-3 *

tu-1 tu-3 tu-3

tu-1 tu-3

-

tu-3 2 tu-1 --x--

tu-1 tu-3

tu-1 tu-3 tu-1 tu-3 3 --x-' tu-1 H *

tu-1

-

H

tu-1 tu-3 tu-3

tu-1 tu-3

ClB Sb 6 --Xx--

+ tu-3

tu-3 8 tu-1 --x-*

-+ -+

tu-1 H tu-3

. tu-1 tu-3 ClB tu-3 7 --x-a

Sb

tu-1 S b

H

ClB tu-3 5 --x-'

Sb

tu-1 tu-3

tu-1 S b 4. --x--

tu-1 tu-d

9. Lausanne

tu-1 tu-3 tu-3 tu-3 tu-3 tu-3

-

-

516 291 64.0

74.0

62 57.0

70 112 38.0

46.0

275 378 42.0

161 350 23.0

32.0

0

81

-

0

Bar Non-bar

29

Bar Non-bar Bar Non-bar

149

l9 656

641

407 10 338 0

131

97 85

9

'"1

4.3 1'5] 2.9 0

21 86.0

Abn

d

+d

0

10 666

1.5

2.9

6 321

1.8

2.0

0

0.4

9

95

84

37 72.0

1 859

0.12

1 835

tu-1 Sb

-

0 814

0

0

-

0

145

0

7 + H

H

* Data from Gardner and Woolf = abnormal.

3

0

0 147

-

+ H + Sb x --Sb

t Abn

Avg. % Abn

645 126 84.0

-

-

% Abn

x -tu-3

10. Lausanne X 11. --

% Abn

Bar or Abnt Non-bar 0 +?

80.0

0.12

0.12

881

0

0

0 123

0

0

(1949).

and early larval stage to be a temperature-sensitive period (Woolf, 1949). Cultures kept at 15OC during their first 24 hours of development were lower in their ultimate proportion of flies expressing the trait than were control cultures kept a t 21OC. Cultures maintained at 31OC for the same 24-hour period had significantly more flies expressing the trait than the control cultures. F, progeny heterozygous for tu-3 from mothers homozygous for tu-1, when subjected to the high temperature for the first 24 hours of development showed the same high proportion of flies expressing the trait as the inbred tu-h stock subjected to the same tem-

124

ELDON J . GARDNER

perature treatment. This indicated that the material effect was influenced by temperature during the early developmental period. Gardner et al. (1960) subjected tu-h flies to 3OoC for intermittent 4-hour intervals to determine whether shorter intervals a t high temperature would influence the maternal effect. Four different 4-hour intervals between 8 and 24 hours were sufficient to produce approximately the same increased maternal effect as the full 24-hour period detected in the earlier studies. Abnormal sex ratios favoring males were observed a t all experimental temperatures. This may reflect the more extreme expression among females and suggest that more became lethals. 2. Influence of Modifier Genes on the Tumorous-Head Maternal Effect

Modifier genes were also found to have pronounced effect on the maternal effect and the expression of the tumorous-head phenotype. When genes tu-1 and tu-3 were homozygous and the temperature was maintained at 25OC, the frequency of flies showing external manifestations of the trait could be altered from 20% to 80% of abnormal flies. Modifiers acting as a polygenic system were mainly involved (Dearden, 1949; Gardner and Stott, 1951, 1952; Gardner et al., 1952; Mayeda, 1957).

B. MATERNAL EFFECT ON MALEGENITALDISCDEFECT Another maternal effect (Woolf, 1966, 1968) involving the development of the genital disc has been detected in the tu-h stock. During the larval and early pupal stages of Drosophilu development, the testes are ellipsoidal. Following attachment of the testes to the developing seminal vesicles, the testes elongate and become coiled (Fig. 4A). If attachment to the seminal vesicles does not occur, the testes fail to Since develop properly and appear as bean-shaped structures (Fig. a). the seminal vesicles, accessory glands, vas deferens, ejaculatory duct, sperm pump, and posterior end of the intestine develop from, or are associated with, the genital disc, many abnormalities result from abnormal development of this disc. A maternal effect associated with abnormal development of the genital disc was detected from matings between different kinds of attached-X females and tu-h males. When one kind of attached-X females (FMAS, yz/Y) was backcrossed for many generations with tu-h males about 60% of the male offspring had undeveloped testes (Table 3 ) . Attached-X females of another kind (br e c / Y ) when backcrossed with tu-h males produced male progeny with testes properly developed. Females of the first type were shown to be homozygous for tu-l+ whereas females of the second type (br ec)

TUMOROUS HEAD IN

&OSOphih

125

F I ~4.. Photographs of normal (A) and underdeveloped (B)reproductive systems of Drosophila melanogaster. (A) Normal coiled testes ( t ) attached to seminal vesicles (s), paired accessory glands ( a ) , and vas deferens (v). (B)Underdeveloped uncoiled testes ( t ) . Seminal vesicles and accessory glands were missing in this male. ~ 1 2 (From . Woolf, 1966.)

were homosygous for tu-I. Seven of nine attached-X stocks tested were homozygous for tu-1’ and produced the genital disc maternal effect. Two were homozygous for tu-1 and produced the tumorous-head maternal effect. The same basic gene tu-3 was shown to interact with both maternal effects.

C. THIRD CHROMOSOME POLYMORPHISM One member of the pair of third chromosomes designated (3B) was found by W o l f and Phelps (1960) to carry the Payne Inversion (symbolized, In(3L)P), which is a large paracentric inversion in the left arm of the third chromosome. This had been anticipated because previous linkage studies had indicated a “suppression” of crossing-over in the third chromosome. Cytological demonstrations with salivary gland chromosomes showed that the proximal break was a t 63C and the distal break was a t 72E1 in the Bridges salivary chromosome map. These breakage points coincided with those of the Payne inversion (Phelps, 1960). The Payne inversion is widely dispersed among laboratory stocks and natural populations. Woolf and Phelps (1960) found that about 85% of the flies in the University of Utah tumorous-head stock carried the inversion in heterozygous condition. Their study made use of a recessive

126

ELDON J

. GARDNER

TABLE 3 Frequency of Males with Abnormal Testes in F1 and Backcross Generations't Abnormal testes ~~

Fl Female (attached-X) Series 1 (1) zl (2) f B (3) Y 9J- f (4) ac* W' ct f (5) zlf (0) RM,ac*Bwaac*l (7) z l w f (8) br ec (9) y*au-wowa bb Series 2 (10) FMA3, y* (11) FMA3, y* (12) FMA3, ya

BCI

BCa

Male

n

%

n

%

n

%

1;2;3;4 1;2;3;4 1;2;3;4 1;2;3;4 1;2;3;4 1;2;3;4 1;2;3;4 1;2;3;4 1;2;3;4

100 01 100 112 78 100 100 100 100

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

83 85 100 87 87 102 102 100 100

13.3 23.5 42.0 14.9 20.7 7.8 17.0 0.0 0.0

90 90 109 100 92 100 103 100 100

25.0

1;+;3 +;+;3 +;2;+

82 0 . 0 88 0.0 100 0.0

100 9.0 100 7.0 100 0.0

BCs n

%

-

43.1 34.0 27.2 10.0 12.0 0.0 0.0

98 57.0 100 53.0 100 48.0 08 42.0 95 20.0 100 10.0 100 13.0 100 0.0 100 0.0

93 15.0 98 20.4 87 0.0

50 41.1 103 24.3 81 0.0

40.0

* Females resulting from each mating were backcrossed to males with the specified tumoroua-head or wild-type chromosomes. t From Woolf (1900). eye color mutant gene st (for scarlet, located a t 44 map units from the left end of the third chromosome) which was carried in the inversion chromosome. Chromosomes carrying the inversion as indicated by the marker st were the 3B chromosomes, and those not containing s t were the 3A chromosomes. Chromosome 3B was found to be homozygous lethal. The original Payne inversion described by F. Payne was a homozygous lethal like that in the tu-h stock. Examples were also found of an inversion that appears to be similar in other respects but is homozygous viable. The lethal action is not inherent with the inversion, but different lethals have been incorporated in different fly populations.

D. MATERNAL, EFFECT ON FERTILITY From the results of reciprocal crosses, Knowles (1967) found that

a maternal effect was associated with low fertility in homokaryotype females. Genes tu-1 and tu-3 were present in the tumorous-head mothers

observed and were presumed to be primarily responsible for the maternal effect on fertilify. Modifiers in the second chromosome were also involved.

TUMOROUS HEAD IN

Drosophila

127

This was demonstrated by replacing tu-h second chromosomes with those of laboratory wild and mutant stocks while keeping other factors in the tu-h genetic system constant. The tu-h second chromosomes were associated with a significant increase in sterility, and replacement with second chromosomes from other stocks increased fertility. Exchanges of other chromosomes did not alter the maternal effect. Replacement of the left arm of the third chromosome, for example, did not influence the maternal effect nor significantly change the fertility of the flies (Woolf and Knowles, 1964; Woolf et al., 1964).

E. MATERNAL EFFECT ON VIABILITYOF HOMOKARYOTYPES Results of reciprocal crosses (Woolf, 1967) involving the tu-h strain indicated a maternal effect for viability as well as for fertility. The comparison was based on the proportion of adult flies from a given number of pupae that eclosed. A smaller proportion of progeny from homokaryotype mothers (3A/3A) eclosed as compared with those from heterokaryotype (3A/3B) mothers. The proportion among maternally affected homokaryotypes was increased when the tu-h second chromosome was replaced by a second chromosome from a wild or mutant laboratory stock. Wild-type second chromosomes, however, were shown to have no influence on viability in the absence of the maternal effect (Woolf and Lott, 1965). V. location of Major Genes

A. LOCATION OF tu-3 The location of tu-3 in the third chromosome was verified by marking the different chromosomes with dominant genes (Table 2) and comparing the phenotypic effects (Gardner
B. LOCATION OF tu-1 Gene tu-1 was shown to be a special kind of a modifier for tu-3 which controlled the maternal effect. It is sex-linked and completely

128

ELDON J . GARDNER

recessive (Gardner, 1959) and has no influence on the tumorous-head trait in the absence of tu-3. Because this gene has no phenotypic expression of its own when either homoaygous or heterozygous but can be demonstrated only by special tests involving another gene, it has been considered to be a wild-type isoallele. The special test for tu-I is the expression of the trait in the presence of this gene in homoaygous condition in the mother and at least one tu-3 in the progeny. The only test for presence of tu-1 is the increased expression of the tumorous-head trait. The location of tu-1 within the X chromosome has been determined within a few map units from linkage studies. A series of four crosses was re.quired to permit sufficient crossing-over to allow detection of recombinations on the basis of phenotypic expressions. Stott and Gardner (1952) obtained data indicating that tu-1 is located within a few units of the position of the marker forked ( f ) , which is a t 56.7 map units from the left end of the X chromosome. Later studies placed tu-I a t about 64.5, a location in or near the heterochromatin region of the X chromosome. Attempts to verify the location of this gene by conventional linkage studies and by deficiency mapping are in progress. VI. Behavior of Inversion in Populations

A. ADAPTIVE ADVANTAGE OF HETEROKARYOTYPE The heterokaryotype 3A/3B has been shown (Woolf and Church,

1963; Woolf et al., 1964) to have an adaptive advantage over the 3A/3A

homokaryotype. Two fitness traits have been associated with the heterokaryotype: (1) female heterokaryotypes produce significantly more off spring than female homokaryotypes ; (2) both male and female heterokaryotypes are more viable than corresponding homokaryotypes. The difference in viability is associated with an interchromosomal effect. When a chromosome segment was substituted into the left arm of chromosome 3A, the adaptive disadvantage of the homokaryotype was decreased. B. BEHAVIOR OF INVERSION IN POPULATION CAGESTUDY Equal proportions of tu-h flies carrying the Payne inversion (3B) and those not carrying the inversion were introduced into a population cage (Berseth and Gardner, 1960). At the end of nine months, 135 males taken a t random from the cage were successfully pair-mated with scarlet

TUMOROUS HEAD IN

Drosophila

129

( s t ) females. Occurrence of the scarlet eye phenotype in the progeny was interpreted to indicate that the tu-h male from the cage contained the 3B chromosome with the inversion and the recessive gene s t . Of the 135 pair matings, 105 (78%) expressed the scarlet-eye phenotype. The increase from 50% to 78% in nine months was presumed to reflect the heterotic effect of the inversion heterozygote (Berseth and Gardner, 1961).

C. POSSIBLE ORIGINSOF tu-h STRAIN Since the tu-1 gene has been found in homozygous condition in several different natural and laboratory populations (Gardner and Stott, 1951 ; Gardner et al., 1952; Johnson and Gardner, 1965), it seems likely that the original flies collected in Mexico possessed tu-1 and were undergoing segregation for the Payne inversion, which also is widely dispersed in natural populations. Mutations producing tu-3 and s t could have occurred in the laboratory stock that was maintained a t the University of Texas. The gene s t on the other hand, could have been present on the same chromosome as the inversion in the original flies from the natural population in Mexico. Gene tu-3 has never been found in any natural or laboratory population and probably occurred only once through a single mutation when the tu-h combination originated. (Mr. Sanat Kr. Sarkar of the College of Science and Technology, University of Calcutt,a, in a personal communication indicated that a case of tumorous-head in Drosophila had occurred spontaneously in his laboratory.) Increases in expression and variations in different laboratory stocks undoubtedly occurred as a result of the pattern of selection and inbreeding that was followed in the particular laboratories since the stock was obtained at the University of Utah. VII. Alleles of tu-1

in laboratory Stocks and Natural Populations

A. LABORATORY STOCKS When reciprocal crosses were conducted with tu-h and other stocks maintained in the laboratory, maternal effects on the tumorous-head trait, different from that established for the tu-h stock, were observed. Oregon-R stock (Gardner and Woolf, 1949) was found to carry, in homozygous condition, a gene comparable with tu-1, but females from this stock were slightly less effective in producing the tumorous-head maternal effect (Table 4). The first hypothesis was that an allele of

130

ELDON J. GARDNER

TABLE 4 &ciprocel Croaeee Deeigned to Demonstrate the Maternsl Effect from Oregon-R Stock (Ore), and the Reaults of Theae Crosses’ Cross 9

d

Abn 9

-

tu-1 tu-3 1. Ore X -tu-3 tu-1 H 2. Ore x--z

3.

tu-1 lu-3 -- x Ore tu-1 tu-3

4.

tu-1 H -- X Ore tu-1 Sb

* From Gerdner and Woolf

+9

Abn 8

+cF

% Abn

17

21

19

22

40

0

177

0

187

0

03

70

47

72

44

0

187

0

191

0

(1949).

tu-I (tentatively symbolized tu-lo) was present in the Oregon-R stock. Other Oregon “wild” stocks maintained in the laboratory carried tu-I+ in homozygous condition. Thus it was demonstrated that only Oregon-R, and none others of the Oregon stocks tested, carried the gene producing the maternal effect. A survey of laboratory stocks (Gardner and Stott, 1951) resulted in the identification of several other mutant and nonmutant stocks that produced maternal effects on the tumorous-head trait. Crimea females, for example, when crossed with tu-h males were only slightly less effective in proportion of abnormal flies produced than Oregon-R females. These were postulated to carry an allele (symbolized tu-IC)in homozygous condition. Differences in proportions of flies from Oregon-R and Crimea females when crossed with tu-h males could be explained by modifiers as well as by separate alleles (Gardner, 1960). When Stephenville females were mated with tu-h males, about 90% of the F; flies expressed the tumorous-head trait. This laboratory stock was postulated to carry a powerful allele (tu-l*)in homozygous condition. When the allele was made homozygous in the mothers and tu-3 was homozygous in the progeny, nearly all the progeny expressed the tumorous-head phenotype. A weak allele of tu-l was indicated in females of a laboratory stock carrying y2 su-UP WO b b / Y . Several other laboratory stocks were found to carry alleles or other modifiers affecting the expression of tu-3 as reflected in the tumorous-head phenotype. Most of the laboratory stocks tested, however, including six melanotic tumor stocks, carried tu-I+ in homozygous condition (Gardner and Stott, 1951; Gardner et al.,1952).

TUMOROUS HEAD IN

.&OSOphih

131

B. NATURAL POPULATIONS Collections from eight natural populations in Texas, Colorado, and Utah were tested for possible alleles of tu-1. One of the eight (I11 from Provo, Utah) was interpreted to carry a tu-1 allele or other modifiers of the maternal effect were segregating in the populations. No samples were found to be homozygous for tu-1, as was common among inbred laboratory stocks, but several showed evidence for the presence of alleles or modifiers (Gardner and Stott, 1952). C. GENEFBJZQUENCY RELATEDTO TEMPERATURE CHANGE Johnson and Gardner (1965) found further evidence for the presence of tu-1 and/or alleles of tu-1 in natural populations, and they designed experiments to identify factors that might be associated with the maintenance of this gene in populations. They found evidence for a seasonal change in frequency that was consistent in the results of three consecutive annual studies (1961, 1962, and 1963) conducted in the same orchard near Brigham City, Utah. For example, the percentage of females homozygous for tu-1 increased from 4% in August, 1961, to 10% in November, 1961. Parallel changes were detected for males with tu-1 in hemizygous condition in two seasons (1962 and 1963). Twenty percent of the males collected in September were hemizygous for tu-1 compared with 35% collected in the same location in November. Samples were then taken in a linear transect pattern from Phoenix, Arizona, to Yakima, Washington. A significant linear relation was found to exist between the location in degrees north latitude a t which samples were taken and the proportion of tu-1 genes present. About 13% of the males taken at Phoenix were hemizygous, compared with more than 30% in Yakima, a t the same season of the year. Population cage experimenta designed to test the effect of temperature on the frequency of tu-1 indicated that the frequency of tu-1 was favored at a lower temperature (18OC). Investigations in the laboratory indicated that heterozygotes tu-1 and tu-1' were more prevalent than expected on the basis of the Hardy-Weinberg equilibrium. This was interpreted to indicate that a balanced polymorphism existed between the alleles tu-1 and tu-l+. VIII. Modifiers of tu-5

From the results of surveys of laboratory stocks and samples from natural populations, evidence was obtained for the presence of tu-1 and alleles of tu-1 that influenced the maternal effect. I n addition a different

132

ELDON J

. GARDNER

kind of modifier was indicated in several stocks. These modifiers affected the expression of tumorous-head and abnormal genital disc traits, but they behaved the same way in reciprocal crosses and therefore did not involve the maternal effect. Marking and replacing different chromosomes showed that the second chromosome contains modifiers that apparently influence a basic mechanism contsolled by tu-S but do not alter the maternal effects. Some modifiers alter the proportion of flies with the tumorous-head expression by at least 10%. One difference observed in the laboratory stocks as compared with natural populations was the homozygosity of laboratory stocks. Constant modifying action was produced by laboratory stocks compared with more erratic results from samples of natural populations because of segregating modifiers. IX. Genetic Divergence in laboratory Stocks

A. DIVERGENCE AMONG STOCKS MAINTAINED IN DIFFERENT LABORAT~RIES Woolf (1965) compared the genetic structure of tu-h strains that had been maintained in different laboratories over a period of several years. Soon after the tu-h stock was established a t the University of Utah, a culture was sent to the California Institute of Technology. This stock was maintained in the collection of Drosophila stocks a t that institution. I n 1949 samples were sent from the University of Utah to Louisiana State University and to the Institute for Cancer Research a t Philadelphia. Beginning in 1949, tu-h stocks were maintained independently a t Utah State University as well as a t the University of Utah. I n 1951 a sample was sent from Utah State University to Johns Hopkins University, and in 1961 the stock previously maintained a t the University of Utah was taken to Arizona State University. The following six stocks that had descended from the original tu-h strain were included in the comparative study by Woolf: tu-h (ASU) , from Arizona State University; tu-h (CT), from California Institute of Technology; tu-h (JH), from John Hopkins University; tu-h (Phil), from the Institute of Cancer Research; tu-h (60), a stock a t Utah State University that had a stock been yielding about 60% of tumorous-head flies; and tu-h (W), a t Utah State University with about 90% of tumorous-head flies. Marked differences were detected in the proportion of females and males expressing the tumorous-head trait (Table 6). Among the samples of females, tu-h (go), with 92.9% of abnormal females, showed the highest expression. The males of this same stock were highest among the six

TUMOROUS HEAD IN

133

Drosophila

TABLE 5 Percentage of Flies in the tu-h (ASU), tu-h (60), tu-h (go), tu-h (CT), tu-h (JH), and tu-h (Phil) Strains Showing the Tumorous-Head Abnormality* Females Strain tu-h tu-h tu-h tu-h tu-h tu-h

(ASU) (60) (90)

(CT) (JH) (Phil)

* From Woolf

Males

Total

n

%Abnormal

n

%Abnormal

n

%Abnormal

484 483 464 275 591 383

91.3 91.3 92.9 22.5 80.4 70.0

695 551 517 301 621 381

75.8 78.0 79.5 19.6 68.8 51.7

1179 1034 981 576 1212 764

82.2 84.7 85.8 21.0 74.4 60.9

(1965).

samples of males, with 79.5% abnormal. Lowest is expression was tu-h (CT), with 22.5% of abnormal females and 19.6% of abnormal males. When samples of males from the six stocks were tested for aspermia (Table 6 ) , tu-h (60) was high with 34.7% and tu-h (JH) was low with no aspermia. In productivity of female homokaryotypes and heterokaryotypes, a wide variation was observed (Table 7). The tu-h (Phil) stock had lost completely the 3B chromosome containing the Payne inversion and the recessive gene for scarlet eyes. This had apparently occurred by random chromosome segregation. When the 3B chromosome was added to this stock, female heterokaryotypes (3A/3B) occurred and produced more offspring than female homokaryotypes. Heterokaryotypes of both sexes were more viable than homokaryotypes. Reduced productivity of female homokaryotypes had been shown by Woolf et TABLE 6 Percentage.of Males Showing Aspermia in the tu-h (ASU), tu-h (60), tu-h (go), tu-h (CT), tu-h (JH), and tu-h (Phil) Strains* Strain tu-h tu-h tu-h tu-h tu-h tu-h

* From Woolf

(1965).

(ASU) (60) (90)

(CT) (JH) (Phil)

n

%

102 501 476 211 102 297

20.6 34.7 17.0 2.4

0.0

1.7

134

ELDON J . GARDNER

TABLE 7 Productivity of Female Homokaryotypes and Heterokaryotypes from the Various tu-h Strains* Median number of offspring produced by

Mating

Frequency of 0 homo- Homo- Heterokaryotypes karyo- karyo(%I types types

Total No. of matings

Sterile matings (%)

362

5.0

21.2

26

84.5

CO.01

263

5.3

31.6

64

81

<0.01

357

27.5

3.8

5

71

<0.01

290

32.0

3.6

11

78

<0.01

115

0.9

33.0

55

64

<0.01

523

23.3

100.0

48

1 tu-h(ASU) O X 3 bw;std d 1 tu-h(60) O X 3 bw;stdd 1 tu-h(9O) O X 3 bw;st 8 d 1 tu-h(CT) 0 X 3 bw;stdc? 1 tu-h(JH) O X 3 bw;st d d 1 tu-h(Phil) 9 X 3 bw;st d d

-

Pt

-

* From Woolf (1965). ~~~

t Probability associated with the null hypothesis of no difference in productivity

of homokaryotypes and heterokaryotypes as determined by Friedman two-way analysis of variance.

al., (1964) to be under polygenic control. Genetic divergence for this fitness component had occurred among the various stocks compared. Female homokaryotypes from tu-h (CT) and tu-h (90) were low in productivity; those from tu-h (ASU) were moderate, and those from tu-h (60) and tu-h (JH) were relatively high.

B. DIVERGENCE IN POPULATION CAGESTUDIES When Berseth and Gardner (1961) introduced into separate population cages (1) tu-h flies, (2) mixed populations of tu-h flies carrying the tu-P allele and those carrying tu-I, and (3) tu-h flies carrying only tu-18,different levels of expression were observed (Fig. 5 ) . The population from the tu-h strain that had (at that time) been selected and inbred for some 12 years and was showing about 90% expression of the trait changed within a few months of uncontrolled matings in a population cage. By the fifth month in the cage the proportion had dropped to about 20%. At the end of the tenth month only about 10%

TUMOROUS HEAD IN

100-

90-

&OSOphih

-POPULATION

I tu-h X t U - h

---POPULATION

3 tu-I'

......*POPULATION 2

135

STEPHENVILLE x t U -h x tu-I'

4 5 6 7 8 9 10 II 12 13 14 15 MONTH AFTER POPULATION WAS BEGUN

3'

FIQ. 5. Proportions of flies representing different chromosome populations and raised in population cages, expressing tumorous-head phenotype. Samples were taken at monthly intervals. (From Berseth and Gardner, 1961.)

of the flies expressed the trait. Approximately this level was maintained through the fifteenth month, when the experiment was concluded. Tumorous head flies carrying the tu-l*allele, and those with equal numbers of tu-1 and tu-lsalleles maintained their levels at about 70% and 30% of flies expressing the trait for 9 and 15 months, respectively, when the experiments were concluded. X. Melanotic Tumors in tu-h Stocks

I n 1959 Gardner and his students observed melanotic lesions in the larvae of one line of the tu-h strain. Larvae with these tumors did not pupate normally, and they died as overaged larvae. Rodman(1964) observed an unnatural stretching of the salivary gland chromosomes near the chromocenter and changes in the puffing pattern of chromosomes in these larvae. Bands 74-75 on the left arm of the third chromosome, for example, did not puff as much as those of normal larvae and appeared to be relatively inert (Rodman and Kopac, 1964; Rodman, 1967). The pathogenesis of salivary gland lesions was investigated by Rottino and Kopac (1965). These investigators observed microscopically, all internal organs of larvae containing gross melanotic lesions. The salivary glands were primarily involved. Lymph nodes were enlarged and hyperplastic. Although other glands, such as the testes, were frequently disintegrated, the abnormality was essentially a disease of the salivary glands. Tumors, which were frequently amelanotic as well as melanotic, did not arise from viscera, imaginal disc, muscle, fat, or hypodermis.

136

ELDON J. GARDNER

RECESSIVB LETHALGENE ASSOCIATED WITH FAILURE OF PUPATION Petersen and Gardner (1965) found that some tu-h third-instar larvae carrying melanotic tumors did not pupate normally, these carried a recessive gene in the left arm of the third chromosome. This gene was expressed in both sexes and was associated with the occurrence of melanotic tumors in mature larvae but was not directly responsible for the tumors. A balanced lethal condition had been established with the lethal associated with the Payne inversion carried in the third chromosome. Failure to pupate was associated with a deficiency of the hormone ecdysone. Since ecdysone was present in a normal quantity a t earlier stages, the deficiency in the overaged, nonpupating larvae was attributed to a destruction of the hormone or a malfunctioning of the ring glands. Kobe1 and van Breugel (1967) investigated further the tu-h stock bearing melanotic tumors. They identified the gene that balanced the Payne inversion as a recessive lethal It2 (lethal tumorous larvae). Preliminary crossover tests placed this gene to the left of roughened on the third chromosome. Homozygous 2tl larvae failed to pupate but survived as larvae for several days after the normal pupation age. A characteristic syndrome of abnormalities developed in the overaged larvae. Most conspicuous were melanotic pseudotumors that floated freely in the hemolymph or became associated with organs. Tumor incidence was strongly influenced by genetic modifiers. Salivary gland chromosomes were shortened and thickened, and, in advanced stages, they lost their characteristic banding pattern. XI. Developmental Studies

The developmental sequence of tu-h has become an interesting and important segment of the tumorous-head investigation. Early investigations indicated a temperature-sensitive period during the first 24 hours of development, which encompasses the egg and larval periods. Presumably, chemical and physiological changes occurring then affect the development of the tu-h phenotype. Observations have been made to discover the earliest manifestation of the visible trait in the larvae and to trace the development of the abnormality (Sorensen and Gardner, 1960). Certain structural changes apparently reflect earlier physiological irregularities. Newby and Thelander (1950) and Thelander (1951)reported irregularities of the eye-antenna1 imaginal discs of tu-h larvae as early as the 32-hour stage of development. Mulkay and Gardner (1965)found that the first morphological expression of the trait that could be com-

TUMOROUS HEAD IN

Drosophila

137

pared with the tu-h phenotype appearing in the adult was coincident with eversion of the head; that is, it occurred 107-108 hours after hatching from the egg. Remondini and Gardner (1967) also studied the first manifestations of tumorous-head abnormalities in the developing flies. To determine whether the eversion of the prepupal head is associated with the initiation of abnormal growths, they transplanted tu-h eye-antenna1 discs from late third-instar larvae to different hosts: (1) tu-h larvae of similar age and (2) wild-type, Canton-S. Transplanted discs did not undergo head eversion, and criteria were established for microscopic recognition of particular types of abnormal growths in serial sections. They interpreted these results as indicating that eversion of the prepupal head was not necessary for the initiation or development of the abnormality. They also concluded that the tu-h trait is autonomous and that the abnormalities arise from tissue already determined a t the late third-instar stage.

OVARYTRANSPLANTATION Ovary transplantation experiments were undertaken by Hansen and Gardner (1963) to determine whether the maternal effect is autonomous in the ovaries of female flies homozygous for gene tu-I. The other alternative considered in the investigation was whether the ovaries are subject to conditioning by some factor or substance in the maternal surroundings of the ovary and are thus influenced indirectly to produce the maternal effect. Reciprocal ovary transplants were made between tumorous-head and wild-type Drosophila melanogaster in the third-instar larval and in the adult stages to determine whether a diffusible substance is associated with the maternal effect. Results showed that ovaries of thirdinstar tu-h larvae are autonomous for the maternal effect. No evidence was found for a diffusible substance (Gardner and Hansen, 1962; Hansen and Gardner, 1962). XII. Biochemical Studies on Tumorous-Head Flies

The tumorous-head strain has been used for two different kinds of biochemical investigations: (1) introduction of chemicals by food additives and by injections into the body to study the influence of such agents on the expression of the tumorous-head trait and viability of the flies; and (2) assay investigations of the tumorous-head flies designed to define the basic abnormality in biochemical terms.

138

ELDON J. GARDNER

A. CHEMICALADDITIVESTO

THE

FOOD MEDIUM

In the course of several experiments, numerous chemicals, such as amino acids, hormones, drugs, were added or removed from the food medium, and the effects on the expression of the tumorous-head expression were observed. The tu-h strain has been especially favorable for such studies because fewer than 100% of the flies express the trait, and a measure of increase or decrease in expression can be obtained by noting the proportions of flies expressing the trait and observing the degree of expression in individual flies. Chemicals of various kinds have influenced the expression and presumably the origin of other kinds of abnormal growths including melanotic tumors in Drosophila. It was, therefore, deemed pertinent to test these chemicals on the tumorous-head strain (Turner and Gardner, 1960). The experiments of Gardner et al., (1957) and of Simmons and Gardner (1958) demonstrated the effect of L-tryptophan and related compounds on the tumorous-head phenotype and on the maternal effect. When flies were reared on a brewer’s yeast-agar medium to which 0.5% tryptophan was added, the proportion of tumorous-head flies was significantly reduced. The same treatment of Stephenville flies resulted in about half their progeny displaying melanotic tumors. Indole a t 0.01% concentration and m-serine a t 0.5% did not significantly affect the proportion of tumorous-head flies. The proportion of tumorous-head flies was significantly reduced when 0.1% L-cysteine was added to the medium.

B. BIOCHEMICAL COMPOSITION OF TUMOROUS-HEAD FLIES Extracts of free amino acids from the bodies of Canton, Stephenville and tumorous-head flies were separated by two-dimensional paper chromatography, phenol and lutidine being used as solvents (Gardner et al., 1957). Canton and Stephenville produced the same chromatographic pattern consisting of 20 ninhydrin-positive substances. Eighteen of these substances proved to be amino acids. Chromatograms of tumorous-head flies differed qualitatively from the two wild-type stocks but the significance of the chemical differences has not been evaluated. XIII. Related Phenotypes \

In the course of the investigation of tumorous-head, several phenotypes and genetic systems not previously described were discovered. Some of these resembled tumorous-heed in one way or another, and it seemed

"WMOROUS HEAD IN

DrOSOphikZ

139

important to determine whether the same genetic mechanism was involved. The mutant erupt described by Glass (1944), for example, was found to have a phenotype similar to one manifestation of tumoroushead, but no genetic similarity was detected.

A. SCARFFlies expressing a new eye abnormality called scarp (Fig. 6) were observed in the summer of 1960 in a wild Cockaponsett stock. A part of the eye was depressed, and the ommatidia in the depressed part were

FIQ.6. Photograph showing typical expression of scarp eye. A depression extends across the eye with the ventral part at a lower level as compared with the normal dorsal part. Tufts of vibrissae are usually observed around the eye, and abnormal growths sometimes occur in the margin around the eye. (From Hansen and Gardner, 1962.)

shorter than those in the normal part of the eye (Fig. 7). Tufts of vibrissae occurred frequently, and abnormal growths sometimes occurred in the margin around the eye. A recessive gene scrp located a t about 74 map units from the left end of the second chromosome was found to control the trait. The expression occurred only a t high temperatures (3OOC). A temperature-

FIG.7. Photomicrographs showing radial sections through normal and scarp eyes. (A) Normal eye. (B) Enlarged portion of A. (C,E, G ) Scarp eyes. (D, F, H) Enlargements of C , E, G through depressed area. The ommatidia in the depressed area are shorter than those in the nondepressed area. A, C, E, G : x 110; B, D, F,H: x 470. (From Hansen and Gardner, 1962.)

TUMOROUS HEAD IN D T O S O p h d U

141

sensitive period extending before and after hour 68 of development was detected. Several "wild" laboratory stocks and samples from natural populations were tested for the presence of scrp. Three laboratory stocks were found to carry the gene. Results of crosses with flies carrying the lobe-recessive gene showed that s q is not an allele of this gene (Hansen and Gardner, 1960).

B. EYES-REDUCED Flies with inherited structural eye abnormalities (Fig. 8 ) were obtained from experiments of Turner and Gardner (1960). These were inbred, and a stock was developed in which the trait was expressed

FIG.8. Photographs showing expression of eyes-reduced. Most of the eye is missing, and irregular growths extend from the surface normally covered by the eye. (From Edwards and Gardner, 1966.)

142

ELDON J . GARDNER

in nearly 100% of the flies. A recessive gene eyr, located about 103 map units from the left end of the third chromosome, was associated with the trait. Many modifiers were found to influence the expression; in particular, the chromosome bearing the dichaete inversion greatly influenced the expression toward wild type (Edwards and Gardner, 1966).

c. WITTY A temperature-sensitive mutant wi with some similarities to tumorous head was received through the courtesy of M. J. Whitten a t the University of Tasmania. Genetically this mutant was not related to tumorous-head. Temperatures above 25OC were necessary for expression of the trait. A recessive second-chromosome gene was indicated, and a temperature-sensitive period between 40 and 72 hours after egg deposition was identified in the experiments of Sandra H. Ely (unpublished). XIV. Summary

Four different maternal effects have been shown to be associated with the tumorous-head genetic system in Drosophila rnelanogaster. A third chromosome semidominant gene (tu-3) is basic to the entire system. In the presence of the tu-3, the maternal effect associated with the manifestation of abnormal growths in the head region is controlled by a sex-linked, recessive gene tu-1. An allele of tu-1 (tu-I+) , in the presence of tu-3 controls the maternal effect associated with a male genital disc abnormality. Fertility is influenced by a maternal effect, and the viability of one chromosome type (third chromosome homokaryotype) also responds to a maternal effect. In addition to tu-3 and tu-1 alleles, modifiers, particularly those in the second chromosome influence the maternal effects. Alleles of tu-1 and the modifiers occur in different frequencies in laboratory stocks and in natural populations. The genetic polymorphism associated with the third chromosome is dependent on the Payne inversion carrying a recessive lethal effect. Reduced productivity of homokaryotype females is a function of reduced fecundity, fertility, and longevity. Developmental studies, in which larvae a t different stages were examined , indicated that the head abnormality was present in structures arising from the eye-antenna1 disc during late third instar. Morphological changes observable microscopically at this time were presumed to reflect physiological changes that had occurred during the ternperature-sensitive period in the early stages of larval development. Autonomy of the genetic

TUMOROUS HEAD IN

DrOSOphdU

143

determination through the developmental period was demonstrated by ovary transplantation. Effects of melariotic tumors on salivary and ring glands and other internal structures were observed. Melanotic tumors were also associated with failure of larvae to pupate. Several phenotypes related in some way to tumorous head, such as scarp, eyes-reduced, and witty, were investigated. None of these were genetically related to the tumorous-head genetic system.

REFERENCES Berseth, W D., and Gardner, E. J. 1960. The behavior and influence of the alleles associated with tumorous-head in Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 37, 155. Berseth, W. D., and Gardner, E. J. 1961. The behavior in laboratory populatioas of genes associated with tumoroushead expremion in Drosophila melanogaster. Genetics 46, 1611-1617. Dearden, D. M. 1949. Genetic modifiera of tumoroushead in Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 28, 138. Edwards, J. W., and Gardnm, E. J. 1966. Genetics of the eyes-reduced mutant in Drosophila melanogaster with special reference to homoeosis and eyelessnew. Genetics 53, 785-798. Gardner, E. J. 1948a. Head tumors in Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 25, 164. Gardher, E. J. 1948b. A case of genetically controlled cytoplasmic influence in Drosophila melanogaster. Drosophila Inform. Serv. 2 5 70. Gardner, E. J. 1949a. Inheritance of tumorous-head in Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 26, 131-132. Gardner, E. J. 1949b. The significance of the genetic analysis of tumorous-head in Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 26, 137-138. Gardner, E. J. 1951. Genes producing a maternal effect in Drosophila melanogaster. Genetics 36, 551. Gardner, E. J. 1954a. Tumoroushead in Drosophila melanogaster. Proc. 9th Intern. Congr. Genetics, Bellagio, Italy, 1963. Part 11, p. 1251. (G. Montalenti, and A. Chiarugi, eds.), Caryobgia VI, Suppl. Firenze, Italy. Gardner, E. J. 1954b. Genetics of cancer and other abnormal growths. 18th Ann. Fac. Res. Lecture. Utah State Univ. Mono. Ser. 2 [No. 31, 36 pp. Gardner, E. J. 1959. Genetic mechanism of maternal effect for tumorous-head in Drosophila melanogaster. Genetics 44, 471-481. Gardner, E. J. 1960. Tumorous-head genes in fly populations. Farm & Home Sci. (Utah Agr. Ezpt. Sta.) 21, 4041. Gardner, E. J., and Gardner, M. D. 1953. Further evidence for maternal effect and modifiers of tumorous-head genes in natural populations of Drosophila melanogaster. Cancer Res. 13, 689-693. Gardner, E. J., and Hansen, A. M. 1962. Reconsideration of maternal effect transfer by injection in Drosophila melanogaster. Genetics 47, 847-853. Gardner, E. J., and Ratty, F. J. 1952. Penetrance and expremivity of tumoroushead in Drosophila melanogaster and relative viability of fiies carrying tumorous-head genes. Genetics 37, 49-61.

144

ELDON J . GARDNER

Gardner, E. J., and Stott, G. H. 1950. Survey of tumorous-head factors in “wild,” tumor bearing and other laboratory mutant stocks of Drosophila melawgaster. Utah Acad. Sci. Arts Letters Proc. 28, 67. Gardner, E. J., and Stott, G. H. 1951. Genes producing a maternal effect and modifiers of tumorous-head in “wild” and tumor bearing stocks of Drosophila melanogaster. Genetics 36, 7283. Gardner, E. J., and Stott, G. H. 1952. Tumorous-head genes in combination with modifiers from laboratory stocks and natural populations. Gemtics 37, 583-584. Gardner, E J., and Woolf, C. M. 1949. Maternal effect involved in the inheritance of abnormal growths in the head region of Drosophila melanogaster. Genetics 34, 573-585.

Gardner, E. J., and Woolf, C. M. 1950. The influence of high and low temperatures on the expression of tumorous-head in Drosophila melanogaster. Genetics 35, 4455.

Gardner, E. J., Stott, G. H., and Dearden, D. M. 1952. Modifiers of tumoroushead genes in natural populations and laboratory stocks of Drosophila melanogaster. Genetics 37, 451-456. Gardner, E. J., Simmons, J R., and Blair, P. V. 1957. Amino acids in tumoroushead stock of Drosophila melanogaster. Genetics 42, 372. Gardner, E. J., Turner, J. H., Berseth, W. D. 1960. Maternal effect transferred by injection and further analysis of temperature effective period in tumoroushead in Drosophila melanogaster. Genetics 45, 905-913. Glass, B. 1944. The effect of X-rays upon the action of a specific gene in Drosophila melanogaster. Genetics 29, 436446. Hansen, A. M., and Gardner, E. J. 1960. A phenocopy presumably induced by high temperature in laboratory stocks of Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 38, 121. Hansen, A. M., and Gardner, E. J. 1962. A new eye phenotype in Drosophila rnelanogaster expressed only a t temperatures above 25°C. Genetics 47, 587-598. Hansen, A. M., and Gardner, E. J. 1963. Ovary transplantation in the study of a maternal effect in Drosophila melanogaster. Genetics 48, 283-291. Johnson, G. R., and Gardner, E. J. 1965. Alleles tu-1 and t w l + in natural and experimental populations of Drosophila melanogaster. Genetics 51, 149-156. Knowles, B. B. 1967. Fecundity and fertility in female homo- and heterokaryotypes of the tumorous-head strain of Drosophila melanogaster. Genetics 57, 437-447. Kobel, H. R., and van Breugel, F. M. A. 1967. Observations on It1 (lethal tumorous larvae) of Drosophila melanogaster. Genetica 38, 305-327. Mayeda, K. 1957. The effect of the Y chromosome on the penetrance of the tumorous-head trait in Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 35, 173-174. Mulkay, L. M., and Gardner, E. J. 1966. Developmental expression of tumoroushead in Drosophila melanogaster. Genetics 52, 461. Newby, W. W. 1949. Abnormal growths on the head of Drosophila melanogaster. J. Movhol. 5, 177-185. Newby, W. W., and Thelander, R. P. 1950. Early development of the head in normal and tumorous-head Drosophila melanogaster. Drosophila Inform. Serv. 24, 89. Petersen, G. V., and Gardner, E. J. 1965. Development of melanotic tumors in association with failure of pupation in Drosophila melanogaster. Genetics 52, 465 *

TUMOROUS HEAD IN

DrOSOphdU

145

Phelps, L. J. 1960. An analysis of chromosome I11 in the tumorous-head stock of Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 35, 174. Ratty, F. J. 1949. The effect of viability of tumorous-head in Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 26, 139. Remondini, D. J., and Gardner, E. J. 1967. Developmental autonomy of eye-antenna1 discs in tumorous-head Drosophila melanogaster. Genetics 56, 583. Rodman, T. C. 1964. The larval characteristics of salivary gland chromosomes of a tumorigenic strain of Drosophila melanogaster. J . Morphol. 115, 419-445. Rodman, T. C. 1967. DNA replication in salivary gland nuclei of Drosophila melay nogaster a t successive larval and pupal stages. Genetics 55, 375-386. Rodman, T. C., and Kopac, M. J. 1964. Alterations in morphology of polytene chromosomes. Nature 202, 876-877. Rottino, A., and Kopac, M. J. 1965. Pathogenesis of spontaneously-occurring and induced melanotic granuloma in Drosophila melanogaster. Progr. Ezptl. Tumor Res. 8, 213-239. Simmons, J. R., and Gardner, E. J. 1958. The effect of tryptophan on the penetrance of tumorous-head in crosses among selected stocks of Drosophila melanogaster. Genetics 43, 184-171. Sorensen, W. K., and Gardner, E. J. 1960. Comparative study of larval imaginal eye antenna1 discs in wild and tumorous-head stocks of Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 3, 130. Stott, G. H. 1951. The location of the genes producing a maternal effect on tumorous-head in Drosophila melanogaster. Unpublished M.S. Thesis, Utah State University library, Logan, Utah. (Methods and results of experiments designed to locate genes tu-3 and tu-1 in the third and first chromosome, respectively, are given in this unpublished thesis.) Stott, G . H., and Gardner, E. J. 1952. The location of the two major genes responsible for the inheritance of tumorous-head in Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 29, 50. Thelander, R. P. 1951. A tumoroushead mutation in Drosophila melanogaster. Minn. Acad. Sci. Proc. 19,21-22. Turner, J. H. and Gardner, E. J. 1960. The effect of copper and iron salts and tryptophan on head abnormalities and melanotic tumors in different stocks of Drosophila melanogaster. Genetics. 45, 915-924. Woolf, C. M. 1948. Temperature effects on head tumors in Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 25, 165. Woolf, C. M. 1949. The effect of temperature treatments on an early developmental period on tumorous-head in Drosophila melanogaster. Utah Acad. Sci. Arts Letters Proc. 26, 139. Woolf, C. M. 1965. Genetic divergence among tumorous-head strains of Drosophila melanogaster. Genetics 52, 809-817. Woolf, C. M. 1966. Maternal effect influencing male genital disc development in Drosophila melanogaster. Genetics 53, 295-302. Woolf, C. M. 1967. Viability of homokaryotypes in the tumorous-head strain of Drosophila melanogaster. Genetics 57, 427-436. Woolf, C. M. 1968. Male genital disc defect in Drosophila melanogaster. Genetics, 60, 111-121. Woolf, C. M., and ChuTch, K. 1963. Studies on the advantage of heterokaryotypes in the tumorous-head strain of Drosophila melanogaster. Evolution 17, 486492. Woolf, C. M., and Knowles, B. B. 1964. Interchromosomal control of karyotype

146

ELDON J. GARDNER

fitneae traitti in tumorous-head #train of Drosophila melanagaster. Genetics 49, 166-173. Woolf, C. M., and Lott, M. 0. 1965. Relationship between penetrance and karyotype in the tumorous-head strain of Drosophila melanogaster. Am. Naturalist 99, 511-513. Woolf, C. M., and Phelps, L. J. 1960. Chromosomal polymorphism in the tumoroushead strain of Drosophila melanogaster. Science 132, 1256-1267. Woolf, C. M., Knowles, B. B. and Jarvis, M. A. 1964. Genetic analysis of fitness traits in tumorous-head strains of Drosophila melanogaster. Genetics 50, 597410.