Effects of different doses and combinations of spotting genes in the leopard frog, Rana pipiens

DEVELOPMENTAL

Effects

BIOLOGY,

of Different Genes

in the

5,

264-295

Doses

( 1962)

and

Leopard

Combinations Frog,

Rana

of Spotting pipiens’

E. PETER VOLPE AND SANJOY DASGUPTA Department

of Zoology,

Newcomb College, New Orleans, Louisiana Accepted

Tulane

Uniuersit!y,

June 5, 1962

INTRODUCTION

One way of appraising the activity of a mutant gene is to compare the effects of the mutant locus in different doses.A given deviant gene may be curtailing the production of an essential metabolite, i.e., it produces the same substance as the wild-type allele, but not in sufficient amounts. If three doses of the mutant locus yield more of the required product than is formed by two doses, then the expectation is a quantitative shift of the mutant phenotype toward the wild type. In contrast, no improvement of the variant phenotype by extra mutant loci is to be anticipated when genie action is qualitative rather than quantitative. A qualitative mode of action is envisioned as one in which the mutant gene inhibits completely the synthesis of some metabolite. Indeed, if the presence of merely a single mutant gene is sufficient to interfere with a synthetic step, then even additional wildtype genes, as in a triploid individual with two wild-type genes and one mutant allele, could not manifest themselves. The foregoing models of genie activity, although elementary and by no means exhaustive in scope, are at least of heuristic value in imparting the view that dosage effects are to be anticipated only if genie action is of the kind that permits quantitative shifts. The objective of the present investigation was to observe the phenotypic effects of different doses of a mutant locus in the leopard 1 This investigation was supported by grants from the National Science Foundation (NSF-G16317) and the American Cancer Society (Institutional Research Award IN-24C). The authors are grateful for laboratory assistance from Mrs. Myrna A. Wilkens and Miss Sylvia Rouchell. The illustrations were drawn by Mrs. Carolyn Thorne Volpe. 264

.\CTION

OF

SPOTTING

GENES

IN

LEOPARD

FROC

265

frog (Ram pipiens), and to infer the mode of genie action. The presence or absence of dosage effects may disclose whether the mutant gene is affecting the quantity of reaction products or is inhibiting a reaction in some all-or-none fashion, whatever the reaction may be. The variant gene under consideration is the nonspotted or burnsi gene, which suppresses the formation of the black, dorsal spots that confer upon the wild-type frog its characteristic leopardlike pattern. Moore (1942) demonstrated that the burnsi gene, designated B, exerts a dominance over the wild-type or common-spotted allele ( b ) . The relation between normal and mutant action was judged by combining the wild-type and burnsi alleles in different associations in triploid individuals. Triploidy is readily induced in amphibian eggs by a cold- or heat-shock applied shortly after insemination of the eggs, a treatment which results in the retention of the chromosome set that is normally eliminated in the second polar body (Kawamura, 1941; Fankhauser, 1945; Briggs, 1947; Fischberg, 1948). The triploid embryo thus possesses two sets of maternal chromosomes-the complement of the female pronucleus and the complement that would normally enter the second polar body-and one set of paternal chromosomes contributed by the sperm nucleus. A variety of triploid embryos, differing genotypically in spotting alleles, can be obtained, depending upon the genetic constitutions of the parents. An obvious limitation in the experimental design is that an additional locus is obtained by adding a whole chromosomal set. Consequently, a given locus is not necessarily reacting on the same genetic background. The argument might be advanced that the genetic milieu is not altered appreciably in the triploid state, since a given numerical change of a specific locus is accompanied by a parallel dosage change for every other gene in the complex. Such a supposition is untenable. That the entire developmental system is affected is attested by the variable consequences of triploidy observed in plants and animals. Phenotypic alterations in triploid individuals range from general changes in body size and cell size to specific modifications in a host of morphological and physiological traits ( Stebbins, 1950). Warrantable conclusions concerning the activity, if any, of an increased dosage of a specific locus must be based on knowledge of the interplay of the locus in question and the residual genotype. This gains greater significance when one recognizes that a

266

VOLPE

AND

DASGUPTA

character such as spotting in the leopard frog has a complicated genesis which involves the interaction of numerous genes. One of the analyzable components of the residual genotype that influences the degree of spotting in the leopard frog is a system of modifiers, or minor genes with individually small effects. It is now known that the spotting phenotype is a manifestation of genie interaction between a main pigmentary locus and a complex of minor genes. The burnsi gene uncovered by Moore (1942) is a mutation at the main pigmentary locus. The burnsi mutation tends to reduce spotting markedly, but the activity of this mutant gene is altered by the minor or modifying genes (Volpe, 1960, 1961). That the spotting pattern of a triploid burnsi frog would be governed by dosage relationships at more than one locus was confidently anticipated. The prospect was to perceive differences in the mode by which the main gene and the complex of modifiers act in attaining phenotypic expression in the triploid individual. While our study was in progress, a brief report on the same subject appeared. Davison (1961) dismisses the notion that modifiers are operative and contends that different doses of the burnsi locus exercise a proportional quantitative effect upon the mutant phenotype. His findings are at considerable variance with ours. It remains to be seen which study will stand the test of time. MATERIALS

AND

GENERAL

METHODS

The burnsi mutant occurs in populations of leopard frogs (23. pipiens) in the North Central states, particularly Minnesota (Breckenridge, 1944). During the four-year period of this study ( 1958-1961), shipments of the wild type and its mutant form, collected from eastern Minnesota, were received from Mr. Steinhilber (Oshkosh Frog Farm, Oshkosh, Wisconsin). Experiments were designed to yield triploid embryos containing all possible combinations of the mutant gene (B) and its wild-type allele (b). Triploid individuals were obtained that possessed three wild-type genes ( bbb) , three burnsi genes (BBB ) , and one or two burnsi genes with two or one, respectively, wild-type alleles (Bbb and BBb). The specific crosses that were undertaken to furnish particular genotypes will be treated in detail in subsequent sections. We shall consider here only the procedures employed in inducing triploidy.

ACTION

OF SPOTTING

CENES

IN

LEOPARD

FHOG

267

In each experiment, eggs were obtained from a female by the standard technique of induced ovulation (Rugh, 1934). A sperm suspension was prepared by macerating the testes of a male in 20 ml of 10% Ringer’s solution. The Ringer’s solution and all instruments were maintained at lS.l”C, as advised by Briggs (1947). A mass of eggs from the uterus of the female was stripped into a 4-inch finger bowl containing the sperm suspension. The eggs in the bowl remained undisturbed in the sperm suspension for 20 minutes, at which time half the mass of eggs was transferred abruptly to a finger bowl, containing 200 ml of 10% Ringer’s solution, which had been kept in a 37°C “Precision Scientific” constant-temperature cabinet. After a 4minute heating period at 37”C, the eggs were returned, once again abruptly, to a finger bowl (labeled “experimental”) containing 10% Ringer’s solution at 18.1”C. The undisturbed egg mass in the original bowl (labeled “control”) was washed free of fragments of testes, and then covered with 200 ml of 10% Ringer’s solution at 18.1”C. After 2 hours, the untreated egg mass in the “control” bowl and the heat-treated egg mass in the “experimental” bowl were loosened from the bottom of the bowls with a section lifter, and each mass was divided into small clusters of 6-8 eggs. The clusters were distributed to several finger bowls, each bowl containing about 8 clusters in 200 ml of 10% Ringer’s solution. Development of the embryos at 18.1”C was observed at regular intervals. Only those experiments in which more than 10% of the heat-treated embryos proved to be triploid are reported herein. We were successful in inducing triploidy above the chosen level in 17 out of 21 trials. As Briggs (1947, p. 249) remarked, “. . . occasionally one finds a clutch of eggs to be quite refractory to heat treatment.” TRIPLOID

WILD-TYPE

FROGS

It may be reasonably surmised that the spotting pattern of the wild-type frog results from the synchronized activities of all the pigmentary genes in the diploid genome. The wild type is presumed to contain normal alleles of the main pigmentary locus (bb) and a balanced complex of modifiers. The initial step in our investigation was to ascertain whether or not the normal spotting genes, main and modifying, would act harmoniously when present in triplicate. Four sets of replicate experiments were performed. In each, eggs of a wild-type female were inseminated by sperm of a wild-type male.

268

VOLPE

AND

DASGUPTA

The egg mass was separated into two equal groups; one was untreated and the other was heat shocked, as described above. Triploid embryos resulting from the heat treatment would contain three doses of the pigmentary alleles involved in the wild-type spotting pattern. A summary of the incidence of triploidy in the four experiments is given in Table 1. Approximately 54% of the heat-treated eggs developed normally, and an average of 36% of these normal embryos

INCIDENCE

Untreated A B C D

Treated A B C D

‘I’ABLE

1

IN

WILD-TYPE

OF TRIPLOIDY

115 108 105 122

113 106 102 118

98.3 98.1 97.1 96.7

450

439

97.6 (avg.1

118 08 125 116 457

i-1 53 7% 49 248

W

0 0 0 0

i

54.1

57.6 42.2 54 :1 (avg.)

LEOPARD

0 0 0 0

FROGS

20 18 16 “1

0 0 0 0

75

10 28 27 17

31.6 F2.8 37.5 34.7

17 22 25 18

15 24 25 lfi -

88

35.5 (av!?. 1

8”

80

were triploid. The inviable heat-treated embryos were arrested principally in the gastrula and neurula stages. Some were defective at stage 11 (Shumway, 1940); the blastopore lips failed to encircle a yolk area that was atypically elongate and narrow. Other imperfect gastrulae had complete blastopores, but extruded yolk plugs. Many progressing beyond gastrulation cytolyzed as deformed neurulae with is varying degrees of open blastopores. Lethality during gastrulation probably a reflection of heat-induced chromosomal aberrations (Fankhauser, 1934; Briggs, 1947). However, no attempt was made to establish the causative factors of lethality. Our attention was focused

ACTION

OF

SPOTTING

GENES

IN

LEOPARD

FROG

269

on those heat-treated embryos that developed normally, particularly the triploid forms. The detailed description of one experiment, presented below, will serve to represent the group of triploid crosses involving the wild type* Embryonic

Period

In this representative experiment (set C, Table 1)) 72 of the 125 heat-treated embryos developed normally. Thirty-three of these normal embryos were judged to be triploid at the tailbud period (stage 17, Shumway, 1940) on the basis of size and spacing of lightly pigmented cells of the head ectoderm. This initial diagnosis proved to be correct for 82% of the embryos. Examination of aceticorcein squash preparations of clipped tail tips of 33 suspect embryos at Shumway stage 22 revealed the triploid number of chromosomes in 27 of the embryos. From 37 to 39 chromosomes (3 N = 39) were counted in most metaphase plates of tail-tip preparations of the triploid embryos. A few of the analyzable metaphase figures contained only 33 to 36 chromosomes. Judgment is reserved as to whether or not these low counts reflect true hypotriploidy, as occasional diploid (control) embryos were encountered with metaphase figures containing the subdiploid number of 21 to 24 chromosomes (2 N = 26). Smears were prepared following the procedure, slightly modified, of Hungerford and DiBerardino (1958). The tail tips were pretreated in a hypotonic medium (distilled water, in our case) for 2 minutes and then squashed in the conventional 2% solution of orcein in 60% acetic acid. Figure 1 illustrates typical diploid and triploid metaphase plates; 25 and 39 chromosomes, respectively, were discernible when these plates were examined microscopically. The triploid individuals developed normally during the late embryonic period and successfully made the transition into the larval stages. Larval

Period

The diploid (control) and triploid larvae were reared under uniform, controlled conditions so that differences, if any, in growth rates and size could be ascribed to differences in chromosomal complements. The technique described in detail by Limbaugh and Volpe

FIG. 1. Comparisons of diploid (top horizontal horizontal row) wild-type leopard frogs with respect 270

row) and triploid to spotting pattern,

(botturu chromo-

.\CTION

OF

SPOTTING

GENES

IS

LEOPARD

271

FROC

(1957) was employed. At the onset of larval development (stage I, Taylor and Kollros, 1946), 25 triploid larvae and a corresponding number of diploid larvae were placed individually into 9 by 5 by e-inch enamel pans containing 400 ml of pond water. The pans were .------

Diploid Triploid 68 Days

20 16 12

: I'

6 4

,'

,,---

---' -\

'\ 'L

,'

------Y

'\

*\

L+!/L

Length

(mm.) q

Metamorphosis FIG. wild-type

2.

Comparative leopard frogs.

rates of development See text for details.

in

diploid

and

triploid

Divloid

(da& larvae

of

maintained in an air-conditioned room, in which the temperature fluctuated from 21” to 24°C. The pond water in the individual pans was changed at 2- to 3-day intervals, and each larva was fed maximally on boiled spinach. Size determinations were made at certain intervals by anesthetizing each larva with “M.S. 222” (Sandoz Chemsome number, and Arrows are directed values of diameters

number and size of dermal melanophores of the dorsal skin. to those dorsal spots which were measured (see numerical in Fig. 3).

272

VOLPE

AND

DASGUPTA

ical Company, New York City) and measuring the distance between the snout and tip of the tail to the nearest 0.01 mm with a Filar micrometer eyepiece inserted into a stereoscopic microscope. The lengths of the 25 diploid and 25 triploid larvae were measured and recorded at the onset of larval development (“0” days) and subsequently at 27, 41, and 63 days. The results are presented in Fig. 2 in the form of frequency polygons. The diploid and triploid larvae were not distinguishable on the basis of size. During each period analyzed, the dimensions of the diploid and triploid larvae were approximately the same. The greatest difference in lengths between the two groups occurred at 63 days of larval development. The average length of the diploid larvae was 71.86 mm ‘r 0.56 (mean -+ standard error of mean) and that of the triploid larvae, 72.57 mm _t 0.48. The difference is not statistically significant (Student’s t = 1.13; P > 0.20). Repeated observations were made as the larvae approached metamorphosis, to determine accurately the times at which the larvae completed transformation into juvenile frogs. As shown in Fig. 2 (lower right-hand graph), the triploid larvae transformed no less rapidly nor no less slowly than did the diploid larvae. Moreover, the juvenile diploid and triploid frogs were of similar body lengths. At transformation, the average body length (snout to vent) of the diploid frogs was 22.48 mm -t- 0.20; that of the triploid frogs, 22.87 mm 2 0.18. Postmetamorphic

Period

The above results confirm the findings by Briggs (1947) that diploid and triploid frogs are identical in general body form and size. The individual cells of the triploid are larger, but fewer in number. Theoretically, the triploid individual should contain exactly twothirds as many cells as the diploid form. Since cells (melanophores) constitute the basic units of the spotting pattern, a comparison was made of the number of dorsal spots, the sizes of the spots, and the number of dermal melanophores in the diploid and triploid juveniles. The results are summarized in histogram form in Fig. 3. The number of dorsal spots located between the dorsolateral folds in recently transformed diploid and triploid frogs did not differ. The average number of dorsal spots in the 14.05 (Fig. 3, top diploid juveniles was 13.32; in the triploids,

ACTION

OF

SPOTTING

GENES

IN

LEOPARD

273

FROG

histogram). A cursory examination of the photographs of a diploid and a triploid frog (Fig. 1) will reveal that the sizes of the dorsal spots are essentially similar. To furnish an objective basis for this view, measurements were taken of the diameter of the dorsal spot m Diploid

fjl Triploid ii @J = 13.3 s fJ = 14.1 fj=J&jggj$-=J

fql10

11

12

13

14

15

16

17

18

19

Number of Dorsal Spots

>:. A: +. :.:. EL 2.1

: @=2.88 z&I=2.93 2.3

2.5

2.7

2.9

3.1

3.3

3.5

3.7

Diameter of Dorsal Spots %

= 25.8

z @ = 32.7

-a

II 20

22

24

26

20

Number of Interspot

30

32

34

36

38

Melanophores

FIG. 3. Comparative data on number of dorsal spots, diameters of dorsal spots, and number of dermal melanophores of the dorsal skins of diploid and triploid juveniles (recently transformed) of wild-type leopard frogs. Each cube represents a determination made on a single individual.

immediately beneath the orbit of the left eye (indicated by arrows in Fig. 1). The average diameters were virtually identical (Fig. 3, middle histogram). If the dorsal spots of a triploid are similar in size to those of a diploid frog, then one would expect to find fewer melanophores per

274

VOLPE

AND

DASGUI’TA

spot in the former than in the latter. Ordinarily the pigment (melanin) in the dermal (intracutaneous ) melanophores is dispersed, and the boundaries of the individual melanophores are not distinguishable. To cause aggregation of the melanin granules, 1 ml of a 1: 1000 solution of Adrenalin (Parke-Davis) was injected intramuscularly into the juvenile frogs. Twenty minutes later, each frog was pithed and the dorsal skin was removed, fixed in Bouin’s solution, cleared in alcohol and xylol, and mounted whole in Permount. The dermal melanophores could now be counted easily in the interspot regions of the skin, but not in the spot areas. The melanin granules in the spot regions of the skin, in sharp contrast to those in the interspot areas, were not concentrated sufficiently to permit an unbiased count of the melanophores. Our comparative study was thus confined to counts of the clearly definitive melanophores in interspot areas. The melanophores were counted in ten microscopic fields of the skin, each 0.06% mm’, chosen at random. The numbers of melanophores in the ten fields were averaged for each frog, and the average value for each frog was plotted in Fig. 3 (bottom histogram). The mean number of melanophores in triploid frogs (25.76 IL 0.61) is significantly different from that of diploid frogs (32.72 + 0.54). The calculated value of t is 8.45 and the accompanying probability is much smaller than 0.001. This difference is portrayed in Fig. 1 ( extreme right ) . It is evident that the number of melanophores in triploid frogs is reduced to approximately four-fifths of the number in diploid frogs, a value that does not approach the theoretical expectation of twothirds. The discrepancy is explicable on several grounds. First, although frogs at equivalent stages of development were analyzed, there is no assurance that the rate of formation and disintegration of melanophores is comparable in the diploid and triploid frogs. Secondly, although fields were selected at random, it is highly likely that the melanophores are not randomly arranged throughout the dorsal skin of the frog (see Baker, 1951). Thirdly, although the skins were prepared for histological examination in a uniform manner, there were undoubtedly unavoidable differences in the degree to which each skin was stretched when mounted whole. Whatever may be the factors responsible for a melanophore count in excess of expectation in the triploid frog, it is apparent that one of the conse-

.\CTION

OF

SPOTTING

GENES

IN

LEOPARD

FROG

275

quences of triploidy is a demonstrable reduction in the number of dermal melanophores. Nevertheless, the capacity of the dermal melanophores to aggregate into spots is unaffected by the additional chromosomal complement. DIPLOID

MUTANT

FROGS

The requisite foundation for an interpretation of the dorsal spotting patterns of triploid mutant frogs is an appreciation of the patterns normally encountered among diploid mutant frogs. As shown in Fig. 4, burnsi phenotypes form a graded series, ranging from those devoid of dorsal spots (type “A”) to those containing few to several spots on the appendages and back (type “En”). The variability in expression of the burnsi character has been ascribed to a complex of minor or modifying genes (Volpe, 1961). The evidence for the existence of modifiers is largely inferential. It has not proved possible to dissociate the complex of modifiers into single gene components, and thereby study their individual effects. Moreover, the extent to which the variation in spotting is nongenetic is difficult to ascertain. It is highly likely that some portion of the phenotypic variation is attributable to environmental influences. Notwithstanding these difficulties, the concept of modifiers affecting spotting is supported by the available facts and offers, at present, the most satisfactory explanation for the conspicuous variation in spotting patterns. Action of Modifiers The types and percentages of offspring recovered from four different control (diploid) crosses are presented in Fig. 5 in the form of frequency histograms. The cross between two unspotted burnsi (type “A” x type “A”) yielded strikingly different kinds and frequencies of burnsi young than the cross between two bumsi individuals possessingheavily spotted appendages (type “E” x type “E”). The bumsi parents in each cross were heterozygous at the main mutant locus (Bb), producing offspring in a ratio of 1 BB : 2 Bb : 1 bb. The variable spotting patterns of the burnsi progeny reflect the segregation of modifiers. These modifying genes possessthe property of shifting the expression of the main burnsi gene toward or away from the wild-type pattern. This twofold action is explicable as the outcome of activities of two contrasting kinds of modifiers: spotting or “f”

276

ACTION

OF

SPOTTING

GENES

IN

LEOPARD

277

FROG

modifiers, which mitigate the expression of the bumsi gene, and nonspotting or “-” modifiers, which enhance the expression of the burnsi gene. The degree of spotting of a burnsi frog is governed largely by the relative amounts of “f” and “-I’ modifiers. The results of the first of the diploid mutant crosses under consideration (Fig. 5, top row) are intelligible on the basis that non,,,---

Burnsi

Offspring

A

B

C

D

22.9

29.5

14.8

8.6

59

rnH 17.7

15.7

D--_ 7.8

-

-

-

-

-----I, E

WildEn

type

typeA typ: A

typeA Wild-“t ype Y 2 E e

52.9

type E typ:

E

28.9

39.5

23.7

25.9

48.1

typeE Wildftype FIG. 5. diploid

Types and crosses. See text

percentages for details.

of

progeny

obtained

from

four

kinds

of

spotting or “-” modifiers were predominantly present in each of the type “A” burnsi parents. The burnsi offspring comprised only types “A” to “D,” with the distribution skewed in the type “A” direction. One-quarter of the total progeny should theoretically be homozygous at the main mutant locus (BB), but these are not distinguishable phenotypically from the heterozygous (Bb) offspring. The view that the type “A” group of progeny (22.9%) represents the homozygous class (BB) is unfounded. Some of the type “A” bumsi offspring are

27s

VOLI'E

AND

DASGUPTA

probably homozygous; others must be heterozygous (Bb), like the type “A” burnsi parents utilized in the cross. Indeed, all type “‘4” adult burnsi frogs (collected from nature) thus far tested in breeding experiments have proved to be heterozygous (see also Moore, 1942). Moreover, it may be noted that type “A” burnsi offspring, known to be heterozygous, were recovered from a cross of a heterozygous type “A” burnsi and the recessive wild type (Fig. 5, second row from top). The inability to differentiate between a heterozygous and a homozygous burnsi individual suggests that the modifiers may be affecting the BB and Bb genotypes with equal intensity. In contradistinction to the type “A” burnsi parents, the type “E” burnsi parents apparently contained numerous spotting or “+” modifiers (Fig. 5, third row from top). The burnsi offspring derived from two type “E” burnsi parents were markedly altered by the presence of “+” modifiers; approximately one-half of the burnsi progeny possessed few to several spots on the back (type “E,“). Through recombinations of spotting modifiers, many of the burnsi progeny tended phenotypically in the direction of the wild-type frog. Here also, it is virtually impossible to separate the homozygous (BB) and the heterozygous (Bb) classes of burnsi progeny. The types of burnsi progeny obtained from a mating of a type “A” burnsi with the wild type (Fig. 5, second row from top) were similar to those of a mating of two type “A” burnsi frogs. However, the frequencies of the types differed; less type “A” burnsi offspring, for example, were recovered from the former than from the latter cross. Spotting or “f” modifiers contributed by the wild-type parent account for the shift in distribution of burnsi offspring away from the “A” portion of the variability spectrum. This should not be misconstrued as indicating that the wild-type strain contains predominantly “f” modifiers. It should be noted that the cross, type “E” burnsi 2 x wild-type d (Fig. 5, bottom row) yielded less type “E,” offspring than the cross of two type “E” burnsi parents. Hence, nonspotting or “-” modifiers transmitted by the wild-type parent offset to some extent the strong contribution of “+” modifiers by the type “E” burnsi parent. TRIPLOID

MUTANT

FROGS

A variety of crosseswas undertaken to obtain all possible combinations of bumsi and wild-type spotting alleles in the triplicate state.

ACTION

OF

SPOTTING

GENES

IN

LEOPAWll

ml

FHOG

A summary of the types of crosses and the nature of the resulting progeny appears in Table 2. Each kind of cross was performed twice; one particularly instructive cross was repeated a third time. All burnsi parents were heterozygous at the main locus (Bb) ; conse-

Iknt-shocked

eggs

I. \VilcMypr X t,ypr “E” II. Type “E” X wild-type III. Type “E” X type “E” IV. Wild-type X type “A”

0

x

11

burnsi C? bumsi 0 3 burnsi 0 burnsi c;” 0 burnsi ~3

u c D E F c; H I .I Ii I, 11

$1 15 1:~ 18 I!) !I 11 15 16 I3 19 16

V. Type “A” hrnsi X wild-type Cs VI. Type “A” burnsi X type “A” hrnsi

0 0 c”

I-,,t mated Diploid

Triplaid

0

12 4 3 9 ‘2 13 8 11 2 4 :4 1

eggs

D$oid

1C 16 10 II 11 1’2 II 16 lfi 12 15

15 1:i T IT 4 5 14 13 14 11 li

15

4

18

G

21 18 18 13 18 I!) 10 24 20 12 14 28 27

17 25 I!) IO 5 7 12 ‘22 27

16 18 8 10

quently, triploid wild-type progeny (bbb) were recovered in each cross. The wild-type spotting patterns will be omitted from discussion, as they were comparable to the triploid wild-type patterns previously considered.

General Efects One effect of an extra chromosomal complement was common to all triploid burnsi progeny. The mean number of dermal melanophores in a defined area of 0.0625 mm’ of the dorsal skin of a triploid burnsi frog at transformation was four-fifths of the average number in an equal area of spotless skin of diploid burnsi frog at a comparable age. In fact, the average number of melanophores in the aforementioned area of the dorsal skin of a triploid burnsi frog did not differ significantly from that in an equal area of a triploid wild-type frog (Table 3). Hence, a general consequence of triploidy per se, evident in both mutant and wild-type frogs, is a reduction in the number of individual melanophores. As discussed subsequently, the potential of the

280

VOLl’E

ANL)

DASCXJk’TA

TABLE NUMBER Lhsal

OF DERXAL

MELANOPHORES Total number individusM

Skill

Diploid W ild-t)-pe Burnsi Triploid Wild-type Burnsi

(interspot

(interspot

areas)

areas)

3 IN

WILP’YYPE of

.\NU Alrm number k standard

UURXSI

SKIS~

of mel:mophore~ error of mci,,,

22 34

33.41

+ 0.07

32.09

f 0.51

15 30

2ci.40 25.31

rfr 0.75 * 0.50

” Melanophores were count,ed in ten microscopic fields, each O.OGJ5 mm3 of the dorsal skin of each frog. The t.en values were averaged, and the average figure for each frog was used in computing the mean number (recorded in column 3) for the total number of frogs examined (recorded in column 2). b The total number is based on sample individuals from each cross listed in Table 2. Grouped data are presented, as no significant differences in melanophore cou&s were found among the corresponding types of t,he various crosses.

melanophores to congregate in presumptive spot regions is a specific effect of the number and kind of pigmentary alleles present in the triploid burnsi frog. Equational

Separation

at the Main

Pigmentary

Locus

The relative proportions of burnsi and wild-type patterns differ among the triploid progeny of reciprocal crosses. For example, in the cross involving a wild-type female and a type “E” burnsi male (set I, Table 2)) there is no significant deviation from the expected 1: 1 ratio of burnsi and wild type among the triploid progeny. However, in the reciprocal cross (set II, type “E” burnsi 0 x wild-type d ), a significant excess of triploid burnsi offspring is recovered. This disparity is traceable to the circumstance that three types of offspring may be obtained from the experimental or heat-treated eggs of a heterozygous burnsi female. If homologous loci separate from each other during the first meiotic division, then the heterozygous burnsi female (Bb) would yield heat-treated diploid eggs of constitutions BB and bb. If, however, sister loci separate (arising most likely by chromatid exchange in the region between the locus and centromere), then the resulting diploid egg would be Bb. Fertilization of the three kinds of diploid eggs (BB, bb, and Bb) with sperm (b) of a wild-type frog would result in triploid zygotes of constitutions BBb, bbb, and Bbb, respectively.

ACTION

OF

SPOTTING

GENES

IN

LEOPARD

FROG

281

The frequency of equational separation at the burnsi locus can be calculated from the numerical data obtained, but we shall defer mathematical treatment until the results of all crosses have been discussed. For the present, it should be clear from the recovery of an excess of triploid burnsi progeny in each cross involving a heterozygous burnsi female (sets II, III, V, VI, Table 2) that separation of sister loci at the first meiotic division does in fact occur. Triploidy

Involving

Type “E”

Burnsi

Frogs

In the first experimental series (set I, Table 2)) the possibility was tested that the wild-type main gene (b) might manifest some expression when present in double dose with a single major bumsi allele (B ) . Viable heat-treated eggs from a cross of a wild-type female (bb) and a heterozygous type “E” burnsi male (Bb) should produce triploid wild-type individuals, bbb, and triploids of the genetic constitution, Bbb. The kinds and percentages of triploid burnsi progeny recovered from two replicate experiments (A and B of set I) are shown in histogram form in Fig. 6. In each experiment, the triploid bumsi progeny exhibited a diverse array of spotting patterns, but the spectrum of variation was comparable to that found in the diploid progeny of the control (diploid) cross. Dorsally spotted burnsi offspring (type “E,“) were recovered with equal frequency in the diploid and triploid progeny. There was no detectable shift of the triploid bumsi phenotype toward the wild type. Apparently, the wild-type main pigmentary gene, despite two doses, is unable to mitigate the nonspotting activity of the mutant burnsi locus. Moreover, the minor genes or modifiers presumably segregated in the triploid progeny in much the same manner as in the diploid progeny. It is evident that exactly duplicable results in the two replicate experiments were not obtained. The proportions of the diploid and triploid progeny differed in the two crosses (A and B of set I). This was not unexpected. Because of a crude, man-devised classification of burnsi phenotypes and the existence of environmental variation, it is highly unlikely that the type “E” burnsi adult selected as the parent in cross A would be genetically equivalent to the type “E” burnsi parent employed in cross B. Furthermore, the wild-type parents, although both normally spotted outwardly, probably harbored different constellations of modifying genes. However, within each cross, pheno-

I. Wild-type (bb)? x Type “E” Burnsi (Bb) d*

n 60

Diploid

(Bb)

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(Bbb)

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A

B C D E Types of Burnsi Progeny

II. n

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Type “E” Burnsi (Bb) o x Wild-type (bb)g Diploid

(Bb)

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(BBb

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c

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En

Type “E” Burnsi (Bb) o x Type “E” Burnsi (Bb)d m Diploid

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C

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E

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n

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ACTION

OF

SPOTTING

GENES

IN

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283

typic differences observed between the diploid and triploid progeny of the same parents could be ascribed primarily to different genetical constitutions. Variability caused by environmental factors was minimized for a given cross by rearing the genotypically different diploid and triploid offspring of the same parents under conditions as identical as could be provided in the laboratory at any one time. The reciprocal cross, type “E” burnsi Q X wild-type CJ’, disclosed the interaction of a double dose of the mutant bumsi gene and a single wild-type allele (BBb). As a result of separation of sister loci, the mechanism having been previously discussed, a second type of triploid burnsi progeny was obtained, namely Bbb (set II, Fig. 6). The principal outcome was that burnsi triploids of the genotype BBb were not phenotypically distinguishable from burnsi triploids of constitution Bbb. In other words, two doses of the burnsi gene and one of the wild-type act in the same fashion as one dose of the burnsi gene and two of the wild-type. There was, however, a greater proportion of dorsally spotted bumsi (type “E,,“) among the triploid progeny than among the diploid offspring. Moreover, the type “E,” burnsi triploid progeny possessed more spots on the back than the type “E,” burnsi diploids. In cross D, the number of spots on the backs of type “E,” burnsi diploid progeny ranged from one to five, 29% having three or more spots on the dorsum. A greater proportion (85%) of the type “E,,” burnsi triploids possessed three or more spots on the back. This tendency toward greater dorsal spotting is attributable to the activity of a larger complement of minor spotting genes (“f” modifiers) in the triploid than in the diploid progeny. It is certainly not an expression of an additional main burnsi locus. Indeed, if the additional burnsi locus were to manifest itself in the BBb frog, then the triploid burnsi phenotype should approach the nonspotted state rather than, as occurred, the more spotted condition. There was a discernible shift in the phenotypes of triploid burnsi (set II), whereas no progeny of the crosses under consideration phenotypic change was detectable in the triploid burnsi progeny of FIG. 6. Types and percentages of diploid and triploid burnsi progeny recovered from three categories (sets I, II, and III) of crosses involving type “E” burnsi parents. The expected genotypes of diploid and triploid burnsi offspring are listed for each experimental set. Individual crosses within a set are designated bv letters (A, R, etc.). See Table 2 for pertinent numerical data.

284

VOLPE

AND

D.XSCUPT.1

the aforementioned reciprocal crosses, wild-type o X type “E” burnsi o’ (set I). This is comprehensible on the basis that the type “E” burnsi females furnishing the diploid eggs in the former crosses contained more spotting modifiers than did the wild-type females utilized in the latter crosses. Hence, more “+” modifiers recombined, and expressed themselves, in the triploid burnsi progeny of crosses involving the type “E” burnsi female (set II).

FIG. 7. Spotting patterns of three diploid burnsi progen)(top row) ancl a mating ( experiment three triploid burnsi progeny (bottom row ) derived fronl See Fig. 6 and text for details. F of set III) of two type “E” bnrnsi parents.

That the type “E” burnsi female possesses numerous spotting or “f” modifiers is substantiated by the results of the third experimental set (Fig. 6). The heat-treated eggs from a cross of two type “E” burnsi adults developed into three types of burnsi individuals, each carrying different doses of the mutant locus (BBB, BBb, and Bbb). In spite of the variety of genotypes, the triploid burnsi offspring were

ACTIOS

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GEKES

IN

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2%

not phenotypically distinguishable from each other. Different doses of burnsi locus-one, two, or three-are ineffectual in altering the burnsi pattern. That is to say, three doses of the main burnsi gene (BBB) do not shift the burnsi phenotype toward the extreme nonspotted condition. On the contrary, triploid burnsi progeny of constitution BBB contain spots on the back (type “E,,“) and, as far as can be ascertained, possess as much dorsal spotting as BBb and Bbb off spring. The results of experiment F of set III (Fig. 6) are of particular interest in that only two kinds of diploid progeny were recovered in the control cross, namely type “E” and type “E,.” This signifies that the parents transmitted a large number of spotting or “+” modifiers. Similarly, an exceptionally large amount of spotting modifiers should have recombined in the triploid burnsi progeny. Photographs of the triploid burnsi offspring (Fig. 7) reveal vividly the extreme heavily spotted condition. The wild-type phenotype is approached, but the modifiers apparently are unable to promote enough spotting to shift the burnsi phenotype completely into the normal range. Furthermore, the dorsal spots of type “E,,” burnsi frogs are haphazardly arranged (compare the irregular spotting patterns of burnsi frogs shown in Fig. 7 with the orderly patterns of spots of wild-type frogs in Fig. 1). If, as deduced, spotting of burnsi frogs is augmented by additional amounts of spotting or “+” modifiers, then it would be of interest to ascertain the consequences of increasing the dosage of nonspotting or “- x modifiers. We shall now turn to such experimentation. Triploidy

Involving

Type “A” Bumsi

Frogs

In crosses involving the type “A” burnsi and the wild type, the distribution of burnsi offspring is skewed toward the nonspotted portion of the variability spectrum (Fig. 8). Precise knowledge of the complex of modifiers harbored in the parental wild-type frogs is lacking, a factor which accounts in part for the variable frequencies of the different classes of burnsi progeny in the replicate crosses. Also, the type “A” parents probably had varying amounts of, although predominantly, “-” modifiers. It is highly likely that the wild-type parents employed in crosses G and H of set IV contained more spotting (“+“) modifiers than the wild-type parent of cross I in set IV (Fig. 8). This conjecture is founded on the phenotypic shift evidenced in triploid burnsi progeny

236

VOLPE

ITI

Wild-type

AND

DASGUPTA

(bb) 0 x Type “A”

n /i‘,‘/rJd ‘/(hl

1:

Type “A” Burnri

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n I,,] ,i,,;.i i,:i,i

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Type “A” Burnsi

iBb)P

I,i,‘i,,,d

in,: .,lS,, iii,,

d

x Type “A” Burnsi l‘ri,,lml

‘Bb) 6

I ,:,i,+. ,:,:11, I, ,,li I:hi,J

FIG. 8. Types and percentages of diploid and triploid burnsi progeny recovered from three categories (sets IV, V, and VI) of crosses involving type “A” burnsi parents. The expected genotypes of diploid and triploid burnsi offspring are listed for each experimental set. Individual crosses within a set are designated by letters (G, H, etc.). See Table 2 for pertinent munerical data.

of crosses G and H. Type “E” triploid burnsi progeny were recovered, a type which did not emerge in the corresponding diploid crosses. The greater degree of spotting in the triploid progeny is the result of activity of a strong double dose of “f” modifiers contributed by the diploid eggs of the wild-type parents. There is no support for the view that the enhanced spotting is an expression of two doses of the main wild-type gene. If the main wild-type gene were operative, then type “E” triploid bumsi offspring should have been recovered

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as well in cross I of set IV. The wild-type parent in the latter cross probably did not contain a sufficient number of “+” modifiers to counteract the “-” modifiers transmitted by the sperm of the type “A” burnsi parent. If, as the data thus far indicate, the degree of spotting is governed primarily by the number and kinds of modifiers, then no intense spotting should appear in triploid burnsi progeny when a large number of “-” modifiers are furnished by the diploid eggs. This contention is supported by the results of the reciprocal cross, type “A” burnsi 0 x wild-type d (set V, Fig. 8). In neither replicate cross were type “E” triploid burnsi offspring recovered, a finding in accordance with the thesis that the type “A” parents transmitted a heavy dose of “-” modifiers in the double chromosomal complement of the heat-treated eggs. Indeed, as might have been anticipated, the phenotypes of triploid progeny of crosses of set V were modified toward the nonspotted state. The striking contrast in the results of sets IV and V is explicable only on the basis that the burnsi mutant gene suppressesthe activity of the main wild-type allele, no matter how many of the latter are present, and that the bumsi phenotype is differentially altered by different amounts and kinds of modifying genes. The highest frequency of type “A” burnsi triploid progeny was obtained in triploid crosses involving two type “A” burnsi parents (set VI, Fig. 8). T o a double set of modifiers, predominantly nonspotting ones, contributed by the type “A” bumsi female were added a set of modifying genes, also mainly nonspotting, transmitted by the type “A” burnsi male. In cross M of set VI, more than 50% of the triploid burnsi progeny were devoid of spots. Thus, although it is impossible to disentangle all the numerous “+” and “-” modifiers involved in the gene complex, it may be asserted that modifiers possessthe property of shifting the expression of the main burnsi gene toward a more or less extreme phenotype. Question of Recovery of Different

Triploid Genotypes

Theoretically, an array of triploid burnsi genotypes can be obtained, two kinds (BBb and Bbb) from the cross, burnsi Q x wild-type d, and three kinds (BBB, BBb, and Bbb) from the cross, burnsi 0 X burnsi d (see Fig. 6 or Fig. 8). Since, as we had observed, the different triploid burnsi genotypes are not phenotypically distinguishable from each other, the question arises whether all the theoretically

288

VOLPE

AND

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possible triploid genotypes were actually recovered. It is possible, for example, that the triploid genotype BBB is poorly viable or lethal. An indirect measure of our success in recovering all triploid burnsi genotypes is obtained by comparing the two crosses, burnsi o X wildtype $ and burnsi o X burnsi d, with respect to the occurrence of separation of sister genes at the main pigmentary locus. The frequency of equational separation at the burnsi locus during the first meiotic division is equal to one minus two times the proportion of triploid recessive (wild-type) progeny obtained from the cross, bumsi 0 x wild-type d (for mathematical rationale, see Lindsley et al., 1956). Among the triploid progeny recovered in the crosses of sets II and V, the proportion of wild-type forms is 0.186 (Table 4). The frequency of equational separation is thus 0.628. EQUATIONAL

Burnsi Burnsi

0 X wild-type 3” 0 X bumsi cPh

a Comprises * Comprises

SEPARATION

57 7%

AT

THE

13 8

B~RNSI

LOWs

0.186 0.100

O.ci88

0.600

sets II and V of Table 2. sets III and VI of Table 2.

For the cross, burnsi 0 X burnsi d, the formula for calculating the frequency of equational separation at the burnsi locus becomes one minus four times the ratio of triploid wild-type progeny to the total triploid progeny. The proportion of triploid wild-type progeny in crosses of sets III and VI is 0.100; this figure corresponds to 0.600 equational separation.’ This value is comparable to the previous :‘If it is assumed that the centromere remains undivided at the first meiotic division, then separation of sister loci as a consequence of chromatid exchlnge becomes a function of centromere distance. The observed frequencies (0.600 and 0.628) for equational separation at the burnsi locals closely approach a theoretical limiting value of 0.666 (. \ee Mather, 1935), which renders it highly probable that the burnsi locus is far removed from the centromere. Three uncolor, and fluid imbalance) linked loci in the axolotl (pertaining to sex, body have been deduced, predicated on prereduction of centromeres, to be situated at great distances from the centromeres (Lindsley et t/l., 1956). It is of peculiar interest that all gene loci presently analyzed in amphibians apparently are located toward the ends of chromosome arms.

ACTION

OF

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GEXES

1X LEOPARD

FRO::

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figure of 0.628, which may be taken to indicate that all triploid burnsi genotypes (BBB, BBZ?, and Bbb) were recovered from the cross, burnsi o x burnsi d . There is thus no evidence of differential mortality among the triploid burnsi progeny of any cross. DISCUSSION

One of the rewarding methods of analysis of gene action has been the study of the effects of quantitative changes of a known mutant locus. Stern (1929) found that an increase in the number of mutant genes at the bobbed locus in Drosophila melanogaster resulted in bristles of increased length, which approximated the wild phenotype. Muller (1932) reported that additional doses of the deviant gene, eosin (or apricot), produced an eye color approaching that of the normal eye. Similarly, a tendency toward more normal development was found to be associated with additional amounts of the shaven gene in D. melanogaster (Schultz, 1935). In these and several other cases,increased dosage of the mutant gene produced an effect closer to the wild type. The interpretation is that the mutant gene and its normal allele act in the same direction but with different degrees of efficiency. The mutant gene is elaborating the same substance as the wild-type allele, but not in sufficient quantities to promote normal development. Dosage studies have also revealed that a mutant gene may act in a direction opposite to its normal allele. For example, Green (1946) studied the effects of different amounts of the recessive vestigial gene in D. melanogaster. The addition of an extra vestigial gene to a diploid heterozygote resulted in a triploid individual, whose phenotype deviated away from normal rather than toward normal. In our present study, increased dosages of the mutant burnsi gene neither enhanced nor mitigated the characteristic appearance of the mutant trait. In the tests performed, the burnsi effect, precluding the action of modifiers, was the same in BBB, BBb and Bbb individuals. It appears that the burnsi gene is so antagonistic to its normal allele that the presence of merely a single burnsi gene is sufficient to inhibit the activity of the normal allele. Our interpretation of mutant action is based upon the phenotypes of triploid individuals in which the dosage of all genes has been increased concomitantly. Since one consequence of triploidy is an increase in cell size associated with a reduction in the number of cells,

Wild - type ( Diploid) Y *

9 *

%- -x + + * Jc * 96

I,**JB*I

I**+*****]

bb

bb and +/+, +/+

Burnsi (Diploid) * I * Q + * + * * -x + * * * * 8 * * + * * x *

* * Q * +k x * * 8 * + + +

BB or Bb

* ++**9j[.*+ 96 **+*+*t *** * +* *-lk+)c r-7

I**

* *

[******~*I BB or Bb and +/+, +/+

*+ **k+ 8 * ‘y *+ t**-% * bb and

-/-7

-/-

BB or Bb and ---, -/--

Burnsi (Triploid)

BBB or BBb or Bbb

BBB or BBb or Bbb and +/+/+, +/+/+

BBB or BBb or Bbb and -/-/--,

--/-/-

FIG. 9. Pictorial model of the activity of genes governing spotting patterns in the leopard frog: diploid wild type (top horizontal row), diploid burnsi (middle horizontal row), and triploid burnsi (lower horizontal row). Each portraiture represents a piece of dorsal skin containing individual dermal melano290

ACTION

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GENES

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FHOG

291

the question arises whether some, if not all, of the alterations in the spotting patterns of the triploid mutant frogs might be directly caused by a change in cell size or in rate of cell divisions. We suspect not, since we had ascertained that the melanophores in a triploid wild type, although reduced in number, form as many dorsal spots as exist in a diploid wild-type frog. The ability of the melanophores to aggregate into spots is apparently not affected by changes in cell configuration resulting from an additional chromosomal complement. Moreover, the numerical diminution of melanophores in the triploid hurnsi frog is equivalent to that in a triploid wild-type frog. It may be safely stated that the potential of the melanophores in a triploid mutant frog to congregate into spots is a specific effect of the number and kinds of pigmentary genes present in the gene complex. The data presented in this study are integrated in Fig. 9. It may be surmised that the formation of spots in the wild type requires, at some critical stage of development, the presence of a substance which, for simplicity, and indicative of our ignorance of its true nature, can be termed a “spotting substance.” The normal alleles, bb, govern the production of a large amount of this “spotting substance.” Modifying genes, or minor genes, are capable of causing a slight increase or decrease of the “spotting substance.” Some modifiers (“+“) act in the direction of increasing the production of the “spotting substance”; other modifiers (“-“) curtail production. The slight variability in spotting that can result from the activity of modifying genes is of negligible consequence in the wild type, since the normal or main alleles, bb, produce a large quantity of the “spotting substance.” This scheme of genie action is depicted in the top row of Fig. 9. The model piece of wild-type dorsal contains four spots, and wild phenotypes containing different kinds of modifiers are indistinguishable from each other.

phores, some of which are aggregated into spots. The normal wild-type pattern is depicted as possessing four spots. The scheme represented in the first vertical column is a hypothetical situation which precludes the activity of modifiers. Gene action is envisioned solely in terms of the main wild-type gene, b, and its mutant allele, B. Actual events are closely approximated in the portrayals of the second and third vertical columns, in which the roles of spotting or “+” modifiers and nonspotting or I‘ - ,, modifiers, respectively, are brought into play. Refer to Discussion section for details.

292

VOLPE

AND

DASGIJPTA

Although the modifiers in their action are undetectable in the wild type, they do manifest themselves phenotypically in a striking manner when the main locus, b, undergoes mutational change. The amount of “spotting substance” elaborated under the influence of the dominant mutant gene, B, is lowered to a level below the minimum for the formation of spots. However, the presence of “+” modifiers ensures a sufficient amount of “spotting substance” to form some spots, whereas the “-” modifiers tend to keep the “spotting substance” below the minimum or threshold level. As revealed in the middle row of Fig. 9, the “+” modifiers are not as effective as the main spotting gene, for they can elaborate only a small amount of substance to promote a few spots (one as contrasted to four in our pictorial model j , The data on the effects of different dosages of the mutant burnsi gene not only fit into the above hypothesis of genie action, but suggest as well that the mutant burnsi gene may be producing a strong “antispotting substance.” As seen above, two wild-type alleles, bb, are necessary in the diploid individual for full expression of the spotting pattern. Normal spotting should occur in a triploid of genotype Bbb, unless the activity of one mutant locus (B) is sufficiently strong to offset the large amount of “spotting substance” normally elaborated by two doses of the wild-type alleles. It is apparent from our results that the presence of a single mutant locus is sufficient to inhibit completely, possibly through the agency of an “antispotting substance,” the activity of two wild-type alleles. Moreover, the mutant effect is all or none, for the phenotypes of triploid burnsi individuals, genetically BBB or BBb, are no more or less extreme than that of the Bbb burnsi triploid (bottom row, Fig. 9). Spotting or “f” modifiers are capable of inducing spotting on their own, but only in a limited manner. They act in a quantitative way; three doses of “f” modifiers in a triploid burnsi individual (bottom row, Fig. 9) result in more spotting than two doses in a diploid burnsi frog (middle row). However, the “f” modifiers, even when present in greater numbers in a triploid burnsi individual, cannot produce the “spotting substance” in a threshold quantity to guarantee normal development of the spotting pattern. Although no conclusive formulation of the allelic states at the modifying loci can be furnished, it appears likely that the “+” modifiers are opposed in action by nonspotting or I‘--” modifiers (Fig. 9).

ACTION

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393

The decisive point established is that the mutant burnsi gene is inhibiting some synthetic step, whatever the step may be, in a metabolic pathway leading to the phenotypic end result. There are several pathways, and innumerable steps, involved in the formation of melanin pigment and the localization of melanophores in the skin of vertebrates. Biochemical analyses by Baker (1951) have revealed that the production of at least one of the enzymes involved in melanin formation is affected by the burnsi gene. The tyrosinase activity of any region of the burnsi skin is lower than that of any region of the wild-type skin. It is tempting to suggest that the burnsi gene tends to inhibit completely the production of tyrosinase, and that the low level of tyrosinase activity in the burnsi skin is a consequence of the action of modifying genes. However, it remains to be seen whether the mutant effect is mediated solely through melanin synthesis. Other factors, presently unanalyzed, may be involved, e.g., conditions in the epidermis affecting the migration of melanophores (Dalton, 1949) or factors intrinsic to the chromatophores affecting migratory capacity (Twitty, 1945; Schumann, 1960). SUMMARY

A conspicuous variant of the common-spotted leopard frog, Rana pipiens, is the burnsi or nonspotted frog. The expression of the deviant phenotype depends not only on a main mutant gene, B, but also on a complex of minor genes or modifiers, each with a small effect. The mode of mutant action was inferred by studying the phenotypic effects of different doses and combinations of the dominant burnsi gene, B, and its wild-type allele, h. This was accomplished by adding an additional set of chromosomes to the diploid complement, i.e., by producing triploid individuals. Consequently, the numerical change at the main burnsi locus was accompanied by corresponding dosage changes at the modifying loci. Precluding the activity of modifiers, the mutant phenotype is not altered by different doses of the main burnsi gene. The action of the main burnsi gene ( B ) is so contrary to that of the wild-type allele (b) that its inhibitory effects on spotting prevail even in the presence of an increased dosage of the wild-type allele. Spotting modifiers provide a margin of stability. They are spotting genes in their own right, which serve to mitigate the otherwise strong adverse action of the main burnsi gene. The spotting or “f” modifiers

294

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ANL)

UASGUI'TA

tend to shift the expression of the burnsi phenotype toward a wildtype spotting pattern. However, even increased dosages of the “f” modifiers in a triploid burnsi frog cannot promote the formation of sufficient spots to produce the normal spotting pattern characteristic of the wild type. Other modifiers, nonspotting or “-,” act in the opposite direction; these increase the expressivity of the main burnsi gene. It is postulated that the main burnsi gene (B) is inhibiting completely the production of some substance normally elaborated by its wild-type allele. However, the “+” modifiers ensure the synthesis of a limited amount of the substance to form a few spots in the mutant phenotype, while the “-” modifiers tend to keep the substance below the minimum or threshold level. REFERENCES BAKER, A. S. ( 1951). A study of the expression of the burnsi gene in adult Rana pipiens. J. Exptl. Zool. 116, 191-229. BRECKENRIDGE, W. J. (1944). “Reptiles and Amphibians of Minnesota.” Univ. of Minnesota Press, Minneapolis. BRIGGS, R. ( 1947). The experimental production and development of triploid frog embryos. J. Ezptl. Zool. 106, 237-266. DALTON, C. H. ( 1949). Developmental analysis of genetic differences in pigmentation in the axolotl. Genetics 35, 277-283. DAVISON, J. ( 1961). A study of spotting patterns in the leopard frog. I. Effect of gene dosage. .I. Heredity 52, 301-304. FANKHAUSER, G. ( 1934). Cytological studies on egg fragments of the salamander Triton. V. Chromosome number and chromosome individuality in the cleavage mitoses of merogonic fragments. J. Exptl. Zool. 68, l-57. FANKHAUSER, G. ( 1945). The effects of changes in chromosome number on amphibian development. Quart. Rev. Biol. 20, 20-78. FISCHBEHG, M. ( 1948). Experimentelle Auslosung von Heteroploidie durch Kaltebehandlung der Eier von Triton olpestris aus verschiedenen Populationen. Genetica 24, 213-329. GREEN, M. M. (1946). A study in gene action using different dosages and alleles of vestigial in Drosophila melunogaster. Genetics 31, l-20. HUNGERFORD, D. A., and DIBERARDINO, M. (1958). Cytological effects of prefixation treatment. J. Biophys. Biochem. Cytol. 4, 391-400. KAwAhnmA, T. (1941). Triploid frogs developed from fertilized eggs. Proc. Im’J. Acad. (Tokyo) 17, 523-526. LIMBAUGH, B. A., and VOLPE, E. P. (1957). Early development of the Gulf Am. Museum Novitates No. 1842, l-32. Coast toad, Bufo valliceps Wiegmann. LINDSLEY, D. L., FANKHAUSER, G., and HUMPHREY, R. R. (1956). Mapping centromeres in the axolotl. Genetics 41, 58-64. MATHER, K. ( 1935). Reductional and equational separation of the chromosomes in bivalents and multivalents. J. Genetics 30, 53-78.

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MOORE, J, A. ( 1942). An embryological and genetical study of Rana burnsi Weed. Genetics 27, 406-416. MULLER, H. J. (1932). Further studies on the nature and causes of gene mutations. Proc. Intern. Congr. Genet. 6th Congr. Ithaco, N. Y. 1932, Vol. 1, 213-255. RUGH, R. ( 1934). Induced ovulation and artificial fertilization in the frog. Biol. Bull. 66, 22-29. SCHULTZ, J. (1935). Aspects of the relation between genes and development in Drosophila. Am. Naturalist 69, 30-54. SCHUMANN, H. (1960). Die Entstehung der Scheckung bei Miiusen mit weisser Blesse. Develop. Biol. 2, 501-515. SHUMWAY, W. ( 1940). Stages in the normal development of Rana pipiens. I. External form. Amt. Record 78, 139-144. STEBBINS, G. L. (1950). “Variation and Evolution in Plants.” Columbia Univ. Press, New York. STERN, C. (1929). Uber die additive Wirkung multipler Allele. Biol. Zentr. 49, 261-290. TAYLOR, A. C., and KOLLROS, J. J. (1946). Stages in the normal development of Rana pipiens larvae. Anat. Record 94, 7-23. TWITTY, V. C. ( 1945). The developmental analysis of specific pigment patterns, J. Exptl. Zool. 100, 141-178. VOLPE, E. P. ( 1960). Interaction of mutant genes in the leopard frog. .I. Here&u 51, 156-155. VOLPE, E. P. (1961). Variable expressivity of a mutant gene in the leopard frog. Science 134, 102-104.