Experimental studies on two mutant genes, r and x, in the Mexican axolotl (Ambystoma mexicanum)

DEVELOPMENTAL

Experimental

BIOLOGY

62, 34-43 (1978)

Studies on Two Mutant Genes, r and x, in the Mexican Axolotl (Ambystoma mexicanum)i, 2 R. R. Department

HUMPHREY

of Biology,

Indiana

AND

HAE-MOON

University,

Received July 25,1977;

CHUNG~

Bloomington,

accepted August

Indiana

47401

9,1977

Mutant genes r and Z, discovered in two unrelated axolotl stocks, are simple recessives determining autonomous cell lethal traits. These traits become recognizable by their characteristic gill and limb patterns which appear in each at about the same period of development. The life spans of the two mutants are approximately the same. Larvae homozygous for both mutants are easily recognized by their smaller size, reduced gill development, and unusually small eyes. None of the three mutant phenotypes (r/r, x/x, rr/xz) is benefited by parabiosis with a normal larva. Transplants of the forelimb area from all three usually were soon invaded by tissues of host origin, resulting in limbs ranging from those almost normal to those reduced to functionless stumps. Those from r/r donors produced the highest percentage of useful limbs. Transplants of the gill-forming area produced gills of the mutant type which, in all cases, regressed. Distention and rupture of gill vessels led to death of some animals. In others the gills became reduced to mere stubs or even disappeared. The failure of replacement of pharyngeal structures of mutant graft origin resulted in the death of all grafted animals from vascular accidents or by prevention of normal feeding or respiration.

ents Cdld, +/r, +/xl of the spawning used in this study. It included nonviable mutant homozygotes of three different phenotypes: those of genotypes r/r and x/x and a third phenotype, that homozygous for both lethals (&XX). The rlr and XIX mutants become distinguishable from the normal and from each other by their gill types. The numerous parallel filaments of the normal gill give it a plume-like appearance (Fig. 1A). The gills of the r/r take a more horizontal position and tend to be somewhat bent or twisted, especially gill 3, with filaments more variable in length (Fig. 1B). The filaments tend to be at right angles to the rachis. The gills of the x/x mutants appear more fragile or delicate than the normal and have a tendency to be curved forward at their tips (Fig. 10. The filaments tend to come off the rachis at an acute angle, especially toward the gill tips. It should be emphasized that the two mutants, when in the same spawning, must be classified soon after they become recognizable.

INTRODUCTION

These two genes were discovered in two unrelated axolotl stocks. Gene r was found in the white (d/d) stock obtained from the Wistar Institute in 1935 (Humphrey, 1964). Gene x was discovered later in animals imported from Mexico by Dr. Louis DeLanney. A mating of a white animal heterozygous for r with a dark (DID) heterozygous for x produced darks (DIdI which carried both r and x. A mating of two such animals produced the white par’ This investigation was supported by grants from the Research Grants Division, USPHS R01 GM 05650, and from the National Science Foundation, BMS 75-17664, and constitutes Contribution No. 1056 from the Department of Biology, Indiana University. 2 The authors wish to express their appreciation to Mr. Larry Lawrence for the preparation of the photographs illustrating this paper, to Mrs. Dorothy Barone for her assistance with the manuscript, and to Dr. Robert Briggs for his valuable criticism and suggestions. 3 Present address: Pusan University, Pusan, South Korea. 34 0012-1606/78/0621-0034$02.00/O Copyright 8 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Genes r and r in the Mexican

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35

FIG. 1. (A) Normal white (d/d) axolotl larva from the same spawning as the mutants of Figs. lB-D. Note the plume-like appearance of the gills resulting from the closely placed rather uniform filaments. (B) Axolotl larva of same age as that of Fig. lA, but homozygous for gene r. Note the more irregular form of the gills and the wider spacing and more variable length of the filaments, which often are at right angles to the rachis. (0 A sibling of larvae of Figs. 1A and B, but homozygous for gene X. The gills are more upright and tend to curve forward at their tips, especially gill No. 3. Their terminal filaments make an acute angle with the rachis. (D) A larva from the same spawning as the preceding three, but homozygous for both r and n (“double homozygote,” rrlnx). Note the reduced body size and the small eyes. The gills are small with few filaments. A-D: x 7.5.

As the mutants near death their gill filaments shorten or even disappear, and the two types cannot be distinguished with certainty. In previous spawnings from matings of parents both heterozygous for any two lethal genes, the double homozygotes have seldom been recognizable. One lethal trait (that appearing earliest, as g/g, f/f, or st/ st) would usually eliminate all larvae

homozygous for it at a stage before the features characteristic of the second trait had become evident. In the case of genes r and x, the gill characteristics identifying the homozygotes appear at about the same stage in each, and the two homozygotes have about the same life span. Perhaps as a consequence of this similarity, the rr/m (“double homozygotes”) show features indicative of combined and cumulative ef-

36

DEVELOPMENTAL BIOLOGY

fects of the two genes: (1) They are smaller, the body being shorter and more slender; (2) their gills are smaller in size, each having at most only three to six filaments, and differ from those of either the r/r or the x/x, although being slightly more like the former; (3) the eyes are very much reduced in size; and (4) the limb bud never becomes more than a low dome (Fig. 1D). EXPERIMENTAL

STUDIES

Parabiosis Embryos of the spawning including XIX and r/r mutants were united in parabiosis, for the most part with embryos from a spawning without lethal traits. The operations were done at tail-bud stages. In all, 54 pairs lived to stages at which the phenotype of the mutant member was distinguishable. In 10 pairs an r/r mutant was joined with a normal, in 12 pairs an x/x mutant was similarly united, and in 3 pairs a double homozygote (n-lxx). In 29 pairs both larvae were normal. These numbers conform closely to the 9:3:3:1 distribution expected for a spawning with two mutants segregating independently (30:10:10:3). Since it was necessary to refrigerate the embryos used in these operations in order to extend the time during which tail-bud stages were available, the life span of any parabiotic combination was computed as from date of union to date of death, all pairs being kept at room temperature postoperatively. The r/r and XIX mutants in parabiosis with normal larvae, as a rule, derived little or no benefit from the combination. Their gills showed the usual degenerative and their forelimbs ceased changes, growth without forming digits. Probably as a result of their weaker heart action and lower blood pressure as compared with their normal co-twins, blood tended to accumulate in their vessels. Stasis was noted in their superficial vessels or in the liver long before death, and rupture of vessels

VOLUME 62, 1978

occurred with hemorrhage into the tissues. At death the heart and large vessels were distended with blood, while the heart and vessels of the normal twin appeared de% cient in blood cells. Death of the normal twin resulted. In eight pairs in which the mutant was r/r, the life span was either 24 or 25 days (average, 24.75). When the mutant was xl x, the life span was more variable, ranging from 22 to 29 days, with an average of 25.5 days. One r/r mutant united with a sibling (not included in the eight mentioned above) lived for 37 days. At its death the normal partner had the front foot fully developed and two digits on the hind feet. The mutant had only two digits on its front foot. The unusually long life span in this pair apparently resulted from the fact that much blood was being returned to the normal twin by way of its liver vessels. The gill filaments of the mutant finally disappeared, and at death its gills had become reduced to short stubs. Of the three twin pairs in which the mutant was a double homozygote trrfxx), one died at 24 days from a mold growth on the head. A second pair lived to 28 days, showing much stasis in the mutant at death. The third died at 42 days, likewise with much stasis in the mutant. It was short and much bent because of the more rapid growth of its normal co-twin. It is possible that, in some combinations such as this, a vigorous normal member might escape death, and its lethal twin may undergo a gradual absorption as occurs with some other mutants (g/g, milmi) in parabiosis. A lethal mutant twin remaining small in size may be absorbed by its larger normal partner whereas one of more nearly the same size cannot be absorbed. The early death of all parabiotic combinations of normal with r/r mutants is in contrast with the long survival time of such pairs reported by Humphrey (1964). The difference may result from a some-

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Genes r and x in the Mexican

what more dorsal union of the two embryos in the earlier experiment, thereby reducing the tendency for retention of blood in the viscera of the mutant.

Forelimb Area Transplants Reciprocal transplants of the forelimb area were made using white embryos of the spawning including r/r and x/x mutants and a spawning from dark parents without known mutant genes. In this combination, the transplants from normal d/d embryos produced normal white limbs on dark recipients. The transplants from r/r mutants into normal embryos gave rise to limbs the development of which in earlier stages corresponded to that of the limb in the mutant. They became slightly elongated structures showing no indication of differentiation of digits or having at their free ends only a shallow notch. Thus, the mutant graft limbs in normal recipients, by their growth rate and form, indicated that they were at first entirely of mutant origin. These graft limbs, however, did not later regress and undergo absorption but instead continued to grow and differentiate, though often in rather abnormal fashion. In time they became darkly pigmented by invasion of melanocytes. Since limbs from normal white donors remained white, it can be concluded that the melanophores appearing in these graft limbs derived from white lethals were entering territory which now consisted largely or entirely of tissues of host origin. The original limb bud of graft origin had served to determine the direction or pattern of development; the invading normal cells produced the tissue components of the limb. It is possible that the noncellular components of these graft limbs (fibers or chondromucoid) may persist for a time and influence to some extent the development that follows invasion of the limb by host cells. This may account for the fact that the humerus of the graft limb was almost invariably shorter than that of the oppo-

Axolotl

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site left forelimb. The growing tip of the original limb, relatively free of noncellular materials, may be better able to produce bones of normal length after invasion by host cells. Occasionally, at any rate, a very short humerus in a graft limb was associated with an ulna and radius of normal length. Of the 13 limbs derived from r/r grafts, none was fully equivalent to the opposite (left) limb of the host. Three approached the normal limb in length, were normally oriented (plantar surface of the foot down), and were useful or functional limbs, but had variable shortening or fusions of the toes. Seven limbs were much shorter than the normal, largely from the failure of growth in the upper portion (humerus very short; Fig. 2A). The feet were variable as a result of fusions of digits or failure in their development. One limb, for example, ended as a T-shaped structure formed by divergence of toes 2 and 3 and absence of toes 1 and 4. These limbs varied in usefulness, some being nonfunctional beThree cause of abnormal orientation. limbs were short nonfunctional stubs. Two of them had shown development of toes, but subsequent redness and swelling suggestive of hemorrhage was followed by absorption of everything below the knee. In the third animal the graft limb developed three toes, two of which disappeared. The entire limb below the knee finally consisted of a toe-like structure shorter than any toe of the opposite normal front foot. The eight limbs derived from x/x grafts showed even greater abnormality than those from r/r donors. From their dark color, all appeared to be of host origin. None was of normal length. The longest, about two-thirds normal length, turned back with the plantar surface upmost and had digits 2 and 3 fused. Other limbs, sometimes only a third the normal length, showed fusion of digits or failure to attain normal lengths. Most limbs were of little use. In three cases, after toes had differ-

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DEVELOPMENTALBIOLOGY

VOLUME 62, 1978

FIG. 2. (A) Wild-type (DID) axolotl 2 years of age with right forelimb derived from an embryonic transplant from a white r/r donor. The dark color of the limb indicates its tissues to be of host origin; its reduced length is the result of a much shortened humerus. The foot is held with the plantar surface toward the body and has only three digits. x 0.6. (B) Wild-type axolotl 2 years of age with right forelimb (at left) derived from an embryonic transplant from a white x/x donor. The limb which developed had only two toes; it became swollen and hemorrhagic and underwent partial absorption, leaving only a short stump. x 0.5. (0 Wild-type (DID) axolotl 2 years of age with right forelimb derived from a white double mutant (r-r/m). The dark color of the limb indicates its tissues to be of host origin. The foot has two instead of the normal four digits; in other transplants from such double mutants only a single digit was formed. x 0.4.

entiated, the limb underwent regressive changes in its terminal parts and finally became a short stump (Fig. 2B). Of the seven grafts of forelimb areas from double homozygotes, rrhx, four failed to produce limbs. A limb bud appeared, but never developed beyond what might be termed a low dome; this eventually flattened and disappeared. In two limbs which developed, the terminal portion formed a single long toe. In another limb graft two toes appeared. The upper part of this limb attained an almost normal length, but the knee joint was without movement, and the remainder of the limb turned caudally with the plantar surface upmost (Fig. 2C). Since the limb buds in rr/xx larvae usually remain as low mounds (these double homozygotes dying without ever eating, as a rule), it is not surprising that some of them, in grafts, failed to produce limbs. Even when kept alive in parabiosis for 42 days, one “double lethal” showed no development of the limb beyond the mound stage. In a graft of the limb area from such a mutant, prompt ingrowth of host tissue is probably essential to any further differentiation. Such invasion may fail to

occur before the limb bud has completely lost its capacity to induce limb development, in which event no limb will form. With a somewhat earlier invasion of host tissue, the mutant limb bud may still be able to induce differentiation of a limb, but may lack the capacity to induce formation of more than one or two digits. The limb buds of r/r and x/x mutants, which in grafts developed further before ceasing growth, apparently had acquired and retained the capacity to induce formation of all four digits on invasion and replacement by tissues of a normal host. Abnormalities in such limbs probably resulted largely from delays or failures in their vascularization and in the removal of the nonviable cells and other components of the limb which had developed from graft tissues. Gill Area Transplants Reciprocal transplants of the gill-forming area (branchial swelling) were made using embryos of the same white and dark spawnings used for grafts of the forelimb areas. The grafts were carefully inspected as the gills developed to determine whether a normal circulation was estab-

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Genes r and x in the Mexican

lished in each gill at the usual time. When it was, grafts from normal white individuals into darks produced normal gills which retained the character and color pattern of the gills of the white donor. When no circulation was established, the gills made little further growth and became short stubs with few or no filaments. In the nine grafts supplied by r/r donors, circulation in the gills was lacking in two. In both, the gills soon became reduced to short stubs. Both animals died, one at 3.5 months, the other at 4s13 months, apparently as a result of the pharyngeal defects incident to the development of that region from nonviable graft tissues. Some replacement of graft components by tissues of host origin doubtless occurred very early, judging by the fact that the gills and the adjacent pharyngeal territory became heavily pigmented instead of retaining the white color pattern of the donor embryos. Branchial cartilages and pharyngeal muscle of graft origin could not be replaced, however, and cessation of growth in these graft derivatives led to

Axolotl

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structural defects. Similar pharyngeal inadequacy developed in those grafted animals in which the gills did acquire a circulation at the usual time and grew to a considerable size, though never equaling the gills of the opposite side. In these functional gills, eventually, perhaps as a result of degenerative changes in the endothelium of their vessels, striking changes occurred. Vessels in the filaments became distended with blood, rupture of vessels occurred with blood then distending the gill or parts of it into bulbous expansion (see Fig. 3A). Breakdown of expanded gill filaments in this animal resulted in serious loss of blood leading to its death. Note the smaller size of the gills of graft origin. Two of the grafted animals which died of unknown causes at ages of approximately 1 and 2 months had not yet reached the age at which vascular changes and gill destruction occurred. Their gills of graft origin were still of the functional type at their deaths. Of the five gill area grafts from x/x

FIG. 3. (A) Larva of wild type (D/D) 3 months of age, with gill transplant (on right) from a white r/r donor. The circulation in the graft had become partially obstructed, and the gill filaments were distended with blood; rupture of vessels occurred with hemorrhage leading to death. Photographed shortly after death. (B) Larva of wild type (D/D) with gill transplant on right side from a white x/x donor. Photographed at 3 months. The gills of graft origin were smaller than those of Fig. 3A and had regressed earlier. Gill filaments had been absorbed, and the gills were reduced to short stubs. Note emaciated appearance of body; the animal lived only 2 weeks after the picture was taken. (0 Larva of wild type (DID) with a gill transplant on right side from a white double mutant @r/xx). The gills which developed were small, with few filaments, and were quickly reduced to short stubs which soon underwent absorption. Photographed shortly after death at 88 days after operation. A-C: x 1.6.

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DEVELOPMENTAL BIOLOGY

donors, three gave rise to gills with a good circulation. In these, however, the gill filaments very soon shortened and either disappeared or remained only as small knobs. The gill stubs curled downward (see Fig. 3B), slowly becoming shorter or even disappearing. None showed the engorgement with blood and rupture of blood vessels seen in grafts from r/r donors, in which the gills had become of functional type. One of the three grafted animals died at 2 months, but the other two reached ages of 3.5 and 4 months. Two animals with gill grafts from x/x donors failed to develop a circulation in the gills of the graft. In one, the gills became short stubs lacking filaments; the animal ceased feeding and died at about 5 months of age. In the other animal, the gill stubs remained long, and the head showed no asymmetry, but the animal died when several months of age. The two gill area grafts from rrlxx donors differed in that in one no circulation developed in the gills, which quickly became short stubs with few filaments, if any, and were absorbed before death of the animal at about 3 months (Fig. 30. The other with an rr/xx graft lived for about 4.5 months. In it, a circulation was established in all three gills of the graft, but only a few filaments developed in the first two. This is in accord with the gill development in the donors, which have only a few filaments per gill at death. In this graft, gills 1 and 2 soon became reduced to stubs without filaments and were very short at death. Gill 3 probably developed in large part from host tissue, since it had several filaments. These were still present at death, though short, and the gill was quite small. On the whole, the character of the gill grafts from the double mutants corresponds to that of the grafts from XIX donors. To summarize, gill area transplants from all three mutant phenotypes (genotypes r/r, x/x, and rrlxx) produced gills which at first corresponded closely to the

VOLUME 62, 1978

gill type of the white mutant donors before their degeneration preceding death. In the grafts, however, cells of host origin must soon have begun invasion of the graft area as its own cells ceased to multiply; this invasion was indicated by pigmentation of the graft area and gills according to the color pattern of the dark host. The development of functional gills in grafts from r/ r donors was in contrast with the more speedy reduction of the gills in grafts from x/x or rrlxx donors, but these functional gills eventually suffered degenerative changes and reduction to functionless rudiments if the grafted animal survived for a sufficient time. The animals invariably died, apparently from their unilateral pharyngeal deficiency interfering with eating or possibly also affecting respiration. DISCUSSION

The two mutant genes considered in this study, r and z, found in two unrelated axolotl stocks, have considerable similarity as to effects: (1) The abnormalities they induce become apparent at the same stage (late gill development); (2) forelimb development in the two mutants stops at about the same point; and (3) the two mutants die at about the same age and stage of development. Larvae homozygous for both genes are distinctively different from either r/r or x/x homozygotes in their reduced size and unusually small eye size. In view of the similarities between the r/r and x/x homozygotes it is not surprising to find a considerable resemblence in the results of some of the experiments undertaken with them. Neither r/r nor x/x homozygotes benefited by parabiosis, and both brought about death of the normal parabiont. Forelimb area transplants from each, after reaching a notch or early twodigit stage, were invaded and replaced by tissues of the normal hosts, producing limbs varying from those essentially normal to ones reduced to nonfunctional stumps. Those from the x/x donors were somewhat more seriously malformed than

HUMPHREY

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Genes r and x in the Mexican

those from the r/r and were for the most part nonfunctional. Of seven grafts from double homozygotes (n-lxx), four failed to produce limbs, two produced feet with only one toe, and the third produced a malformed limb with two toes (Fig. 2C). The limb buds of these rrlxx are possibly more limited in their inductive potencies than those of either r/r or x/x homozygotes. The abnormalities in graft limbs derived from lethal mutants may in part be the result of a reduced inductive capacity in the graft limb buds, especially in the double mutants. Since a few graft limbs were essentially normal, however, and the abnormalities were so variable, it seems more probable that the variations may have resulted from differences in the mixtures of graft and host tissues after the latter entered the developing limb. If the capillaries first developing in the limb are of graft origin, their eventual breakdown as their endothelial cells die may deprive more distal parts of the growing limb of adequate blood supply and may lead to reduced growth or to their partial degeneration and absorption, causing serious malformations. The reduced length of the humerus in many graft limbs may have resulted from the fact that its early formation was from cells of graft origin. Growth in the early cartilaginous humerus so produced would stop with the death of its perichondrium and chondrocytes, and its further growth would be delayed until host tissues replaced them and began to contribute to its development. Delays in the formation of blood vessels of host origin, as well as delays in death and removal of graft components in the limb, also probably contributed to some of the abnormalities observed. The gill area transplants from all three lethal mutant phenotypes proved fatal. The structures they produced (gills, cartilages, pharyngeal muscle) were not replaced by normal structures when they ceased growth and degenerated, and the grafted animals eventually died from vas-

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cular accident, respiratory failure, or starvation. None developed permanently functional gills as did animals with g/g grafts (Tompkins, 1970). The results obtained by different investigators from transplants from lethal donors into normal recipients are frequently in disagreement. For example, Humphrey (1964), reported that, in five transplants he did of the gill-forming area from white r/r mutants into dark normal recipients, the gills which developed were at first comparable in pattern to those of the mutant donor. They lagged in growth, with their filaments shortening and sometimes disappearing. Eventually, however, short filaments lengthened, and additional new ones appeared. Gills of normal type developed, though smaller than those of the normal recipient. These gills, however, became deeply pigmented, indicating their origin from host tissues. Following similar operations done by Chung (see earlier section on Gill Area Transplants), the gills from r/r donors into normal recipients developed to good size, but then regressed completely, and all recipients eventually died. All gill grafts of another lethal (X/X) done at the same time by Chung with embryos from the same spawning gave a similar result: no recipients survived. Why did host cells invade and replace the r/r gill graft in the experiments done by Humphrey (1964) and fail to do so after the operations done by Chung in the study here reported? The answer is probably to be found in a slightly greater size of the transplant in the later work. The gills in this later study apparently developed with a border zone of graft cells delaying for a time any ingrowth of host cells. Large gills developed the capillary endothelium of which was derived from cells of the mutant graft. When this endothelium died and the gill filaments regressed and disappeared, it was too late for any ingrowth of normal tissues to be stimulated to form gill structures or pharyngeal muscle. The gill transplants from Z/Z mutants

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DEVELOPMENTALBIOL~CY

reported by Chung and Briggs (1975) gave rise to functional gills of donor type which eventually degenerated and were reduced to short stumps. The gills of the recipients underwent a compensatory hypertrophy. No deaths of the grafted animals were mentioned; it is possible that a somewhat earlier death of graft components in these transplants had permitted replacement of the pharyngeal musculature of graft origin and so permitted feeding to continue. The transplants of forelimb areas of r/r mutants done by Humphrey (1964) and, for this paper by Chung agree in that the mutant graft limb, at first typical for the r/r donor, continued development and produced a limb indicated by its color to be of host origin. Here no barrier of lethal cells had prevented early ingrowth of normal tissues, and the inductive action of the lethal limb bud had stimulated these to produce limbs, often with structural or functional abnormalities yet sometimes approaching the normal. It can be readily appreciated that, in a small limb area transplant, the- limb bud which develops may lie very close to an edge of the transplant, so close, perhaps, that normal cells of the host invade it at once. If the host and donor are both normal embryos, but one white and the other dark, the limb becomes a mosaic, part dark in color and part white. If the host were a normal dark and the donor a lethal mutant white, the invasion of the limb bud would not be apparent for a time (limbs of D/D are at first nonpigmented), but when color developed the limb would be all dark, indicating replacement of the white lethal mutant tissues by the dark tissues, more favorable to melanocyte proliferation. Assume now a larger transplant in which the limb bud develops at a more central location. The bud is surrounded by a zone of mutant lethal cells which multiply for the period fixed by the life span of that lethal. The limb of the graft reaches the maximum size determined by

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its genetic type and undergoes regression and absorption. As the surrounding zone of lethal tissue also dies, normal cells of the host may finally invade the regressing mutant limb area, but by this time it will have lost all capacity for limb induction, and no limb will ever develop. The larger size of the graft area may actually have been the cause of failure of limb formation in four of the seven grafts from the double homozygotes (see section on Forelimb Area Transplants). The limb transplants from h/h mutants recently described by Humphrey and Chung (1977) are unique in that the limbs reached a four-digit stage without any admixture of host tissues, regressed, and underwent complete resorption, with no limbs of host origin ever replacing them. This mutant lives longer than any other autonomous cell lethal; grafts of limb areas from it, therefore, for a considerable time, grow about as vigorously as do surrounding host tissues and, as a result, are not readily invaded by these tissues. The eventual death and absorption of such graft limbs leaves an area without the capacity for limb induction or for replacement of the dying musculature of graft origin, a condition leading to herniation and rupture at the graft site and death of the grafted animal. It is possible that, with large transplants from cell lethal mutants such as x/x and r/r, in which all graft components die at much earlier stages, sufficient replacement by host tissues would usually occur to prevent such a fatal outcome. These mutants, however, should be further tested, with care being taken to transplant larger areas and thus prevent an early invasion of the graft limb bud. The h/h mutants may possibly stand alone in their tendency to cause death of recipients of both gill area and limb area transplants. SUMMARY

AND

CONCLUSIONS

Genes r and x, discovered in unrelated axolotl stocks, have several features in

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Genes r and x in the Mexican

common. Both determine simple recessive traits. Both are autonomous cell lethals. The features serving to distinguish the mutants from their normal siblings (peculiar gill type, cessation of growth in the forelimbs) appear at about the same stages in each, and the affected larvae have about the same life spans. Those homozygous for both mutant genes (double homozygotes, V/XX) are smaller than either the r/ r or x/x, with much smaller gills and unusually small eyes. When spawned from parents each homozygous for both mutant genes, the phenotypes of the offspring conform to the 9:3:3:1 ratio expected for two traits segregating independently. Neither rlr nor XIX homozygotes are benefited by parabiosis with normal larvae, and their deaths bring about the deaths of their normal co-twins. The blood in any pair tends to accumulate in the mutant member, probably because of its weaker heart action and lowered blood pressure as it nears death. Transplants of the forelimb area of white mutants into normal dark recipients, made at tail-bud stages, gave rise to limbs of dark color, indicating the invasion of the limb bud by host tissues. These limbs showed a great variety of abnormalities. A few approached the normal in size, form, and function, but the great majority were malformed, and some, through resorption, became reduced to functionless stumps. Those from x/x or rr/ xx were more abnormal than those from grafts from r/r donors. Transplants of the branchial swelling (gill-forming area), when a circulation was established in the gill primordia, produced gills of the type characteristic of the mutant donor. These gills eventually became dark in color, indicating ingrowth of host tissue. The vessels of the gill filaments were probably lined by endothelium of graft origin, since eventually the fdaments regressed to small knobs and finally

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disappeared, and the entire gill was reduced to a stub which also sometimes underwent absorption. All gill area grafts from mutant donors proved fatal to the recipients, the pharyngeal defects resulting from degeneration of graft components, bringing about death through vascular accident or prevention of normal feeding or respiration. Comparison of the findings of different investigators with transplants from lethal donors leads to the conclusion that the size of the area transplanted probably determines whether the resulting structure remains purely of graft origin, regresses and disappears, or whether it is invaded and replaced in varying degree by normal tissues of host origin. By using donors of one color pattern and recipients of another, the findings may be more accurately evaluated. Deeply pigmented structures do not arise from grafts from white donors unless there is invasion of the graft by tissues of the dark recipient. A graft from a lethal dark donor upon a white recipient remains dark, however, after absorption of all graft elements, since its melanocytes are of host origin, having migrated into the graft and multiplied there because tissues of dark (DID or Dl d) origin favor the multiplication of melanoblasts more than do those of the white (d/d) type. REFERENCES H-M., and BRIGGS, R. (1975). Experimental studies on a lethal gene (I) in the Mexican axolotl, Ambystoma mexicanum. J. Exp. Zool. 191, 33-47. HUMPHREY, R. R. (1964). Genetic and experimental studies on a lethal factor (r) in the axolotl which induces abnormalities in the renal system and other organs. J. Exp. Zool. 155, 139-150. HUMPHREY, R. R., and CHUNG, H-M. (1977). Genetic and experimental studies on three associated mutant genes in the Mexican axolotl: st (for stasis), mi (for microphthalmic), and h (for Hand lethal). J. Exp. Zool., in press. TOMPKINS, R. (1970). Biochemical effects of the gene g on the development of the axolotl (Ambystoma mexicanurn). Develop. Bill. 22, 59-83. CHUNG,