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By virtue of its smallness, hardiness, rapid rate of reproduction, ease of rearing, and presence of various mutants, the medaka (Oryzias latipes ) is unparalled material in various fields of biology, especially in developmental biology and genetics, among fishes.
Brown wild type inhabits Japan, Korea, China and Formosa. In addition, domesticated orange-red (golden) and other mutants in Japan lend themselves excellent to fish genetics. In fact, among fishes, it is the first in which the Mendelian laws have been proven to be valid (Ishikawa, 1913; Toyama, 1916). Aida (1921) in Oryzias shares with Schmidt (1920) and Winge (1922) in Lebisles the credit for the first discovery of Y-linked inheritance in organisms.
The medaka is, also, the first animal in which complete reversal of sex differentiation in both directions was deliberately induced by administration of sex hormones during larval stage (Yamamoto, 1953, 1955, 1958). In view of these facts, the writer has presented earlier an article entitled "Medaka" dealing with hints on procurement, maintenance, care, sexing, breeding technique, artificial fertilization, etc. (Yamamoto, 1967).
The breeding season of the medaka extends from mid-April to early September in Nagoya district. In outdoor culture, the period for attainment of sexual maturity depends on season in which fry are raised. Fry hatched in May and early June may reach maturity in August. However, fry hatched in August may reach maturity in the next spring. When breeding begins in spring, therefore, two generations can be reared per annum. When kept indoors at 25-28 degree C, they reproduce all the year round, even in the winter season.
Chromosomes in the medaka
Goodrich (1927) counted haploid number of 22-24 chromosomes in the spermatocyte metaphases. Iriki (1932) first established that 2n=48. Katayama (1937) confirmed this.
Aida ( 1921) demonstrated genetically that male is heterogametic (XY) and female homogametic (XX) in the medaka. It is an interesting question whether or not a heteromorphic pair of sex chromosomes can be cytotologically distinguished. Using genetically analyzed materials supplied by the writer, Ojima and Hitotsumatsu ( 1969) studied the karyotype of males known to be XrYR , YrYr and androgen-induced XrXr males. The chromosome number was decided to be n=24 in the primary spermatocytes (Fig. 12-1A) and 2n=48 in somatic metaphases.
Autosomal and sex-linked genes
Key genes and their symbols in the medaka are listed in Table 12-1. Other new mutant genes are given in the chapter 21 . The inheritance of body colors in this fish has been studied by Ishikawa (1913), Toyama (1916), Ishiwara (1916, 1917) and Aida (1921).
b-Alleles
The triple autosomal alleles B, B', b control the formation of melanin in melanophores in non-albino types. The gene B, which is dominant to both B' and b, permits full melanin formation. The B' is dominant to b and causes variegation or mottled pigmentation. In the orange-red or golden (b, R) and white (b, r) fish, melanin formation is so scanty that 'melanophores' are almost colorless in adults and usually invisible in the 'expanded' state. However, test with adrenalin to concentrate melanin granules shows the presence of 'colorless' melanophores (Goodrich, 1927).
Hishida, Tomita and Yamamoto (1961) found that incubation of isolated scales of light colored varieties (orange-red and white) carrying b genes homozygously in the mixture of the following solution for 24 hours results in the full formation of melanin in 'colorless' melanophores.
(A) Tyrosine (0.1%) + NaHCO3 (0.04%) in M/7.5 NaC1. . . . .6 parts
(B) M/l0 phosphate buffer (pH7.3). . . . . . . . . . . . . . . . . . . . . . . . 3 parts
(C) M/l0 Iodoacetamide solution. . . . . . . . . . . . . . . . . . . . . . . . . .1 part
This tyrosinase reaction revealed that the number of 'colorless' melanophores in the light colored varieties is significantly greater than that of the melanic melanophores in the dark colored types. Tyrosinase does exist in an inhibited state in the 'colorless' melanophores of the light colored varieties. Iodoacetamide seems to remove inhibitor.
Graded dominance in multiple alleles is generally interpreted on the assumption that each preceding allele advances the biochemical reaction one step further as compared to the allele that follows, so that the presence of an allele is masked when combined with another allele that promotes the sequential biochemical reaction one more step. The way of action of the B' gene, however, is peculiar because it causes regional difference of state of melanophores. Melanin content in the eye of types carrying B, B' or b looks the same at least in gross appearance.
r-Alleles
The gene R and r govern the doposition of carotenoids in xanthophores (see Color Plates 15-18) and partially sex-linked. Both the X and the Y chromosome have a locus for either R or r. The types carrying R gene possess well developed xanthophores and those having the recessive r have xanthophores with little or no pigment.
In cultivated fishes available from dealers, white fish (bbrr) are rare (about 1/200). Furthermore, rare white fish are usually females with the constitution bb XrXr. The r gene in domesticated medaka on the market is usually restricted to the X chromosome while the R is carried by either X or Y. This might be because of the fact that the gene r must have arisen from mutation from the R in the X but not in the Y chromosome. Hence, white males (bbXrYr) are even rarer. White males that appeared in both Aida's (1921) and the writer's breeds (see Yamamoto 1955 and later papers) were the result of crossing over of the r gene from the X to the Y chromosome. When we obtain even a single white male, it is easy to establish a pure white strain, bbXrXr females and bbXrYr males.
Color interferer (ci)
In 1951, Eiji Nakabori, an employee of Kyoto University collected a pair of gray fish among a population of the wild (brown, B,R) medaka at northern outskirts of Kyoto. Phenotype of the gray is shown in Color Plate 7. He offered the fish to the late Aida for genetical studies. According to Aida's oral communication to the writer in 1955, he performed two matings, viz. gray ´ white (b,r) and variegated white (B',r) ´ gray. The F1 hybrids of both cases were all brown (B,R). Segragation of the F2 were of a trihybrid type. Evidently Aida found that the new mutant gene behaved as a Mendelian autosomal recessive and non-allelic to and independent from color genes known by that time.
He designated it as ci (color interferer) because its effect in homozygotes was to suppress manifestation of R intensively and B less significantly. He intended to read a paper entitled 'On the newly discovered white in the medaka' before the 148th meeting of Kyoto genetic conversion held Dec. 4th in 1954. However, a disease prevented him to attend it. So that his result was unpublished eventually.
Before his death in 1957, he asked to the writer and Takeuchi (Okayama) to keep the strain and gave them some gray medaka. The gray (ci) strain of the Nagoya colony has been established since 1955 from the fish received from Aida.
In 1955, he personally communicated to the writer that F1 of gray ( ci/ci; B/B; R/R) ´ white (+ci/+ci, b/b, r/r) were all wild type brown (ci/+ci, B/b, R/r). In F2 he observed 100 brown +ci, B, R, 60 'pale blue' (gray ci, B, R; Blue +ci, B, r; pale blue ci, B, r), 26 orange-red +ci, b, R and 23 'white' (cream ci, b, R ; white +ci, b, r; milky ci, b, r) totalling 209, whereas expected ratio would be 27 : 21(9+9+3) : 9 : 7(3+3+1), respectively. According to him 26 orange-red to 23 'white' is approximately 9 : 7. Phenotypes included by parentheses were scored together by him without discriminating each other, so that cream, white and milky were all called 'white' by him. He told the writer that there were a certain 'white' hybrids which produced orange-red (+ci, b, R) fish in mating with the white (+ci, b, r). It seems that such 'white' might be cream (ci, b, R). In this connection it may also be stated that he called the phenotype of ci, B, R 'pale blue'. Actually, however, the phenotype looks gray. Furthermore, to avoid confusion with ci, B, r (pale blue), it is preferable to call ci, B, R gray.
Following Aida, Takeuchi (1969) studied genetic behavior of the ci-gene carefully. In the F2 of a mating of white (+ci/+ci, b/b, r/r) ´ gray (ci/ci, B/B, R/R), eight color phenotypes appeared: brown (+ci, B, R), gray (ci, B, R), blue (+ci, B, r), orange-red (+ci, b, R), pale blue (ci, B, r), cream (ci, b, R), white (+ci, b, r) and milky (ci, b, r) in the ratio of a 27 : 9 : 9 : 9 : 3 : 3 : 3 : 1. Color phenotypes of ci-medaka are illustrated in Color Plates 8-11.
Albino gene (i)
In the spring of 1961, our laboratory of Nagoya University bought about 4,000 orange-red medaka from a dealer at Yatomi near Nagoya. The stock is known to have been transferred from Koriyama district, Nara Prefecture, after extermination of the original stock at Yatomi by the terrible flood caused by the Ise-bay typhoon in 1959. Tomita, my research associate, selected some pale orange-red fish from this population. Among the offspring from the selective matings, a number of albino with pink eyes appeared. Phenotype of albino medaka is illustrated in Color Plate 12. Sections of albino and normal eyes are shown in Fig. 12-2.
When an original albino fish (i, b) is crossed with a wild type (+i, B), the segregating ratio in the F2 advanced embryos (Fig. 12-3) is 9 : 3 : 4 for +i, B, +i, b and albinos (i, B plus i, b) characteristic for two genes interacting by recessive epistasis. By test crosses of the F2 adult albinos, B-carrying and b-carrying albinos are actually detected. The albino gene shows pleiotropic effects. When homozygous, it not only inhibits melanin formation but partially suppresses xanthic pigmentation governed by the sex-linked R and Rd genes. For the expression of the characteristic colors of races, the presence of the normal allele (+i) of i is indispensable. If thei is homozygous, all colors of other color varieties do not appear and all fish turn out to be albino even though they are carrying various color genes. For the color of the wild type (brown) to appear, at least a dozen normal alleles of recessive color genes are necessary. Of these, the +i gene is the most essential.
As stated before, albino medaka are semi-lethal. However, they are all viable during the embryonic stages. Formerly, the two embryonic phenotypes +i, +ci, B and +i, ci, B were scored together as 'dark' without discriminating them. Later, it was able to discriminate them in advanced embryos. Hence, the following four types can be scored in the F2 advanced embryos from the mating of albino (ii, +ci +ci, bb) ´ gray (+i+i, cici, BB).
Dark (+i, +ci, B): Eyes are pigmented. Deeply pigmented corolla-type melanophores on the head are large and contact each other, so that they cannot be counted in the 'expanded' condition.
Pale dark (+i, ci, B): Eyes are pigmented. Deeply pigmented corolla-type melanophores on the head are smaller, so that they are countable even in the 'expanded' condition.
Light (+i, -, b): Eyes are pigmented. Faintly pigmented corolla-type melanophores are scarcely visible.
Albino (i, -, -): Eyes are non-pigmented. No melanophores in all parts of the body.
(Hyphen-represents any one of respective alleles)
If the i and ci had been independent, the expected ratio in advanced embryos would have been 27 : 9 : 12 : 16 for dark, pale dark, light, and albino, viz. a trihybrid one modified mainly by recessive epistasis of the albino gene (i). If, on the other hand, the two were linked, the expected ratio would be approximately 6 : 3 : 3 : 4, viz. a modified dihybrid one. Observed results coincide fairly with the expectation based on the linkage hypothesis. Single backcross data, also, support this.
Originally, the two were linked in the repulsion phase, viz. i, +ci in albinos and +i, ci in the color interferer (ci) stock. A number of F2 albino adult males (i, -, -, -) were singly tested by mating with milky females (+i, ci, b, r), where hyphen (-) represents any one of respective alleles. Among these males, two recombinant fish in which one chromosome is linked in the coupling phase, i, ci, were detected. Using these fish as progenitors, the i, ci homozygotes were produced. Employing appropriate mating types, recombinants, +i, +ci, were observed in matings of i, ci/i, ci ´ i, +ci/+i, ci. The recombination frequency is found to be 4.6 +/- 1.2 percent. This is the first instance of autosomal linkage in teleostean fishes.
Fused (f) and wavy (w) genes
Two autosomal genes pertinent to vertebral abnormality in the domesticated stock were found by Aida (1930). The f (fused) manifests itself fusion or ankylosis of vertebrae here and there while the w (wavy) results in hunchback or lordosis of vertebral column. Both were found to be simple recessive characters and independent from color genes and sex. Morphological features and genetic behavior of the fused were studied by Yamamoto, Tomita and Matsuda ( 1963) in connection with fused wild fish found in nature and lordosis artificially induced by phenylthiourea in embryonic stage. Over-all appearance of the fused is shown in Fig. 12-5 and Color Plate 13.
A phenogenetics of the fused was carefully studied by Ogawa (1965) in our laboratory. She found that the sign of ankylosis of vertebrae is noticeable at the time of hatching as the irregular arrangement of rudiments of centra. The expressivity varies significantly even among the same sibsib groups reared at a constant temperature. This suggests the presence of modifiers of the major gene (f). The expressivity is modified by external temperatures at which embryos are reared. The expressivity increases with increase of temperature.
The temperature sensitive period is from stage 27 to 32.5 and is subdivided into the first period (27 to 29) and the second (31 to 32.5). The former seems to be associated with ankylosis of the posterior two-thirds region of the trunk and the latter that of the caudal region. Expressivity of ankylosis displayed by an individual is determined not only heredity, but in part also by temperature prevailing during the sensitive developmental period.
Ogawa (1971) studied the relationship between temperature (20-28 oC) and the number of vertebrae before ankylosis using fry of the fused. She found that temperature prevailing during development has a slight but significant effect on the number of centra. The number decreases with increase of temperature.
Takeuchi (1966) showed that the w and f genes are independent. The F1 offspring between them are all normal. In the F2 , he was able to obtain some wavy-fused (w, f) fish.
Sex linked inheritance
Sex-linked inheritance has been masterly worked out by Aida (1921). A dihybrid cross white female (bb XrXr) ´ brown male (BB XRYR) produces F2 offspring composing of the 9 : 3 : 3 : 1 ratio for brown (B, R), Blue (B, r), orange-red b, R), and white (b, r) types. However, all the blue and white fish are only females because no Yr chromosome is present in this cross. As stated before, the Yr chromosome has appeared as the result of crossing over between the Xr and the YR chromosomes and a white male (bb XrYr) was produced. The F2 offspring of a dihybrid cross brown female (BB XR XR) ´ white male (bb XrYr) shows also the 9 : 3 : 3 : 1 ratio for the respective phenotypes. However, all the blue and white fish are only males in this case.
In the paragraph that follows orange-red (golden) and white pheno-types are represented by R and r, respectively, since they possess the common bb. The F1 of a cross r female (XrXr) ´ R male (XRYR) are R daughters (XrXR) and R sons (XrYR). In the F2 generation, r females (XrXr), R females (XRXr) and R males (XrYR and XRYR) appear in a 1 : 1 : 2 ratio. The backcross of an F1 R male (XrYR) to a r female (XrXr) produce r daughters (XrXr) and R sons (XrYR) in a l : 1 ratio (Aida, 1921, 1930; Yamamoto, 1953 and later papers). This mode of inheritance has been referred to as one-sided masculine inheritance in the guppy by Winge (1922b). It is preferable to call it simply Y-linked. In crosses of this type rare exceptional R daughters (XrXR) and exceptional r sons (XrYr) appear. Matings of r females (XrXr) with heterozygous R males (XR Yr) produces R daughters (XrXR) and r sons (XrYr) in a 1 : 1 ratio, the typical crisscross inheritance.
The cross of R female (XRXR) ´ r male (XrYr) gives rise to F1 R daughters (XRXr) and R sons (XRYr). In the F2 ,R females (XRXR, XRXr),R males (XRYr) and r males (XrYr) appear in a 2 : 1 : 1 ratio (Aida, 1921). In this case, all white fish are males. The backcross of F1 R female (XRXr) to r male (XrYr) results in the production of R females (XRXr), r females (XrXr), R males (XRYr) and r males (XrYr) in a 1 : 1 : 1 :1 ratio. If we read R as red-eye and r as white eye instead of actual orange-red and white body colors, these results are superficially the same as the famous Morgan's case in Drosophila (Morgan, 1910). The difference lies in the fact that while the Y chromosome of the medaka possesses a homologous segment including ther-locus, that of Drosophila is almost empty if any at all.
Morgan (1910) used the term 'sex-limited' inheritance in his classic paper. As commented by Winge (1923) the term is not adequate because it is generally assigned to secondary sex characters manifested by one of the two sexes and dependent on autosomal genes. For this reason 'sex-limited' manifestation would be better. Winge (1923) commented also that the term 'sex-linked' ought to be applied to conditions of inheritance in which the gene in question is carried by the X- and the Z chromosomes which are normally found in both sexes. He further stressed that the term 'one-sided' should be used only in such cases where the gene is present in the Y- and the W chromosomes which are normally found in one sex alone. In the latter cases, he recommended to add male or female so as to specify whether said gene belongs to the Y- or the W chromosome. The father-to-son type of the guppy was called by him 'one-sided masculine' inheritance.
In order to unify terminology, the present writer wants to recommend to call all the modes of allosomal inheritance sex-linked. Thus, sex-linked inheritance includes various modes or types. Morgan's 'sex-limited', Winge's 'sex-linked' and crisscross types are all X-linked. Winge's 'one-sided masculine' or father-to-son type would simply be called Y-linked inheritance.
It is noteworthy that all the modes of both X- and Y-linked inheritance are manifested by the medaka. In other words the medaka seems to represent the most generalized organism in the mode of sex-linked inheritance.
In teaching and writing sex-linked inheritance, it is advisable to refer the medaka first, the mouse secondly and the human thirdly. These forms are not only phylogenetically linked but are common in having epistatic M-gene (s) in the Y-chromosome. Then, other specialized types would be referred to.
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