The Mexican Axolotl, Ambystoma Mexicanum: Its Biology and Developmental Genetics, and Its Autonomous Cell-Lethal Genes

AMER. ZOOL., 18:195-206(1978).

The Mexican Axolotl, Ambystoma mexicanum: Its Biology and Developmental Genetics, and Its Autonomous Cell-lethal Genes

GEORGE M. MALACINSKI

Department of Biology, Indiana University, Bloomington, Indiana 47401

SYNOPSIS. The Mexican axolotl, a neotonous salamander, has found widespread use as an experimental animal for studies in embryology and physiology. The convenience with which the axolotl can be maintained as a laboratory animal and the high quality of the eggs it Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 produces contribute to its popularity in laboratory research. A large array of mutant genes has been recognized in the axolotl. These genes affect one or another process during development of the organism, and have provided a basis for carrying out research in developmental genetics. Approximately three dozen mutant genes in the axolotl are known. These genes have been grouped into 5 different categories which reflect, for the most part, the developmental stage at which the mutant phenotype can first be recognized. One group of mutant genes, the autonomous cell-lethals, is discussed in detail. These genes share the common feature that homozygous embryos cannot be rescued by parabiosis with normal embryos. In addition, grafts of mutant tissue (e.g., gill or limb primorida) do not survive on normal hosts. These genes probably are responsible for metabolic or regulatory defects which affect all cells and tissues in the organism. The mutant phenotypes and potential usefulness of these genes are discussed in detail.

HISTORY OF THE VARIOUS STRAINS The name "axolotl" has subsequently been The Mexican axolotl, Ambystoma translated by various scholars of Aztec mexicanum shaw, a neotonous salamander linguistics to mean "water-slave" or "water (Fig. 1) is native to a large fresh-water lake dog" (Smith, 1969). Some 300 years later, in the vicinity of Mexico City. It is most in 1864, a shipment of 33 animals was easily distinguished from other salaman- received in Paris at the Jardin des Plantes ders, especially its close relative, Ambystoma (Dumeril, 1866). These animals were tigrinum (the tiger salamander), by its ex- probably from Lake Xochimilco, for their ternal morphological characters. Most of descriptions resemble stock more recently these distinguishing features, e.g., promi- imported from that region (Smith, 1969). nent gills, broad trunk, and undulating Animals from that original shipment ap- tail, are essential components of its parently caused somewhat of a sensation in neotonous existence. the biological community. They mated, It was the Europeans who first brought and produced offspring, and several of the axolotl to the attention of the scientific that original stock eventually underwent community. When the Spaniards first ar- spontaneous metamorphosis. Descendants rived at Mexico City, approximately 450 of this original stock, and animals which years ago, they observed that the Aztecs arrived in a second shipment a few years were fond of eating these salamanders. later, were distributed to museums, The axolotl was described at about that zoological institutes, and to the courts of time by F. Hernandez in a publication on royalty (as pets) throughout Europe. Dur- the natural history of Mexico, and named ing those early years of excitement, in by him as well (reviewed by Newth, 1960). approximately 1870, the white phenotype—the product of the first mutant The author dedicates this paper to the memory of gene recognized in the axolotl—appeared R. R. Humphrey. R. R. Humphrey very generously (Newth, 1960). Several of the strains of provided G.M.M. with an introduction to many of the axolotl in widespread use today are appar- topics discussed in this review. ently actually descendants of the original The author's research described in this paper is Paris stock (Table 1). The irony of this supported by grants from the N.S.F.

195 106 GEORGE M. MALACINSKI

and morphology {e.g., extent of development of gills). Some of these characteristics of the strains maintained in the Indiana University Axolotl Colony are summarized in Table 1.

BIOLOGY OF THE AXOLOTL

The life cycle of the axolotl is diagram- Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 med in Fig. 2. Axolotl eggs are considered to be relatively large when compared to the eggs of other common laboratory am- phibia, including Xenopus and Rana. The rate of development of the eggs, being temperature dependent, can be relatively FIG. 1. Normal wild-type adult with dark pigmenta- slow, a situation that facilitates microinjection (left). Note the prominence of the gills and the tion or microsurgical manipulations. After broad tail. Adult which has completed metamorphosis (right). Note the absence of gills, and the hatching (approximately 3 weeks at room slender body and tail. temperature) young larvae are usually reared on a diet of washed, freshly point is clear—the original Paris stock hatched brine shrimp. By the age of ap- apparently provided most of the breeding proximately two months the larvae are material available today, rather than more capable of ingesting larger pieces of food, recent importations from the fresh-water and are switched to a diet of ground beef lakes around Mexico City. If this is indeed liver or heart. Since axolotls retain their true, then it follows that many of the more aquatic mode of existence throughout life, common laboratory strains of the axolotl they are maintained as adults in laboratory are substantially more selectively inbred aquaria. They are fed only once every 3-4 than might be assumed from information days. The life span of the axolotl is rela- reviewed in Table 1. Among vertebrates, tively long. It is not at all uncommon for the axolotl may indeed have achieved one animals to survive for 7-10 years, but by of the higher extents of selective, closed that age they are usually considered too colony breeding yet achieved for a old for breeding purposes. A detailed de- laboratory maintained experimental ani- scription of the anatomical and histological mal. And to push this point even one step features of the axolotl is available, which further, it should be appreciated that Am- might be useful for guiding preliminary bystoma mexicanum from the Xochimilco characterizations of new mutant pheno- area are themselves highly inbred, for that types (Brunst, 19556). population is reported to differ substan- Adult animals are mated by placing a tially from axolotls from other nearby male and female together in a small areas (Smith, 1969). aquarium which contains a layer of coarse The officially recognized name — gravel at the bottom. The sexes are distin- Ambystoma — has only in recent years come guished by examination of the cloacal re- into common usage. In fact, the previous gion (photographs are included in Brunst, names—Amblystoma, Siredon, and 1955a; Chung and Malacinski, 1977). A Axolotus—have been placed on the Official courtship routine between the male and Index of Rejected and Invalid Genetic female takes place in which the pair may names in zoology (Wake, 1976). Among make physical contact as they perform a the various stocks in common use in re- "waltz." This courtship pattern is not un- search laboratories today, several charac- like the courtships displayed by other teristics differentiate the various strains. urodeles (Salthe, 1967). The male deposits These include pigmentation pattern, size, one or more (up to 2 dozen) sper- INTRODUCTION TO THE AXOLOTL 197

TABLE 1. Characteristics of various strains of the Mexican axolotl maintained at the Indiana University Axolotl Colony. Mutant Strain Original source Morphological genes Notes on breeding Wistar" Morris Biological White (light) Inbred Wistar animals are Farm, 1935. Origi- pigmentation r.y.e, not particularly good nally imported from an,d breeders. Animals of Cracow, Poland mixed background (e.g., with English) are, however, good breeders

Holtfreter" Dr. J. Holtfreter, Dark pigmentation 0 Frequently difficult to obtain Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 1951. Originally fertile spawnings from inbred imported from Holtfreter animals Europe

DeLanneyb Imported from Dark pigmentation m,x,c Mexico in 1961, p,ax by Dr. L. DeLanney Dutch" Dr. P. Nieuwkoop, Corrugated skin nucleolar Good breeders the Netherlands, marker, in 1962, via Dr. ut, cl, DeLanney d Tompkinsb Imported from Dark pigmentation as,ph,q, Good breeders Mexico in 1966 st,t,h,p, by Dr. R. mi,nc,sp Tompkins

English8 Dr. Newth, Great White, with gray I, also Britain, in 1968, or mottled pattern. possibly via Dr. DeLanney Generally small, a variant slender animals ofrf Albino Artificial hybrid- Lack melanin Fair to good breeders in ization of A. a season mexicanum X A. tigrinum in 1963 Brandon" Imported from Dark pigmentation b,x,mi Fair breeders in season Mexico in 1968, by Generally small Dr. R. Reese body size

Xochimilco Imported from Dark pigmentation 1 Out-crossed with Wistar Mexico in 1970, by strain Dr. Brandon

" Probably direct descendants of the 1864 Paris stock (Dumeril, 1866). b Descendants of stock recently imported from Mexico. matophores (Fig. 3) which adhere to the through the cloaca, they are fertilized by gravel substrate. Eventually the female the sperm stored in the tubules. A typical picks up a batch of spermatozoa by press- spawning yields a few hundred to several ing her cloacal opening onto a sper- hundred fertile eggs. Axolotl eggs are fre- matophore. The spermatozoa are stored in quently polyspermic, and display promi- spermathecal tubules in the roof of the nent surface pits which mark the site of cloacal chamber. One or more of these entry of the sperm (Chung and Malacinski, steps in the courtship pattern apparently 1977). The quality of the eggs is usually stimulates ovulation in the female. As the quite high, although the percentage of the female expels eggs from the oviducts eggs which are successfully fertilized in any 198 GEORGE M. MALACINSKI

Morphology Age* General Features numbers of eggs is possible (Brunst,

2 _. 1955a; Humphrey, 1962). ••* ™ iTwn •g*i fraa^antlr a'i.ala., Characteristically, laboratory bred and 0 hrs sawacal worm pit* reared axolotls do not undergo metamor- oogonasis ^ phosis. Observations at the Indiana Uni- claovoga con occur in versity Axolotl colony indicate that first cloawaga \_J a hrs onwclootad aggs perhaps 1 or 2 out of every several

36 hrs hundred adult animals undergo spontane- gastrulation xj*' induction of primary em.lt ous metamorphosis (R. R. Humphrey, per-

sonal communication, 1977). Those few Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 3 dors organ primordia prtiant, idoal sraga for groftt cases of spontaneous transformation (Fig. limb* ihow ramarhobU 1) have frequently been associated with roganaration whan ramavad diet changes or major alterations in the

odwll is naolonows, rttoins laboratory fresh water supply. Such a low 12 toll gilli and lorga toil frequency of metamorphosis is apparently months r* 20 Clfi a] not the case for animals caught in their FIG. 2. Life cycle of the axolotl. Relatively large eggs natural habitat near Mexico City. One re- which lend themselves to various experimental man- port indicates that spontaneous metamor- ipulations {e.g., microinjection, nuclear transplanta- phosis occurs with an astonishingly high tion, and microsurgery) are shed in abundance. frequency (up to 75%) in freshly caught *20°C. animals. These observations have led Smith (1969) to challenge the prevalent single spawning may vary between 10 and notion that neotony is obligatory in the 90%. Adult animals reach sexual maturity axolotl. at an age of 10 to 18 months. Considering the previously mentioned The large eggs, their slow development, possibility that most common laboratory and the ease with which larvae are raised stocks are selectively inbred to a higher and adult animals are maintained in the laboratory in an aquatic environment provide distinct advantages for the axolotl as experimental material. Perhaps the only real disadvantage of the axolotl is the difficulty of obtaining frequent fertile spawnings throughout the 12 months of the year. A partial regression of the sex organs takes place during the summer months. Attempts with the use of hormone injections have been made (Newrock and Brothers, 1973), yet the results remain inconclusive (Humphrey, 1977). The re- productive physiology of a urodele such as the axolotl is more complicated than that of a typical anuran. The female picks up part of a spermatophore, and fertilization of the eggs, in contrast to the process in anurans, takes place within the cloaca. The presence of the sperm within the female might stimulate the hypophysis to induce ovulation. A simple, reliable method for the simultaneous artificial insemination of large numbers of eggs has not yet been developed, although under some cir- FIG. 3. Axolotl spermatophore. The gelatinous cone serves to adhere to the gravel substrate. The cumstances artificial insemination of small white mass (arrow) contains spermatozoa. INTRODUCTION TO THE AXOLOTL 199

degree than is generally appreciated, the quired animals with ones of known difference in metamorphosis frequencies is genotype. The progeny from such matings easily accounted for. Metamorphosed were then backcrossed with the parent animals being more difficult to breed, an being tested, or used in random sib mat- inadvertent selection for the neotonous ings. One wild type male recently acquired phenotype might have occurred. In the from Mexico City was employed in this process of evolution of the laboratory systematic manner and found to carry 4 axolotl, the capacity for transformation previously unknown lethal traits. Hum- might, therefore, be considered a vestigial phrey began his selective breeding pro-

character. Under laboratory conditions, gram at the University of Buffalo prior to Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 however, several treatments, including World War II, and expanded it when he daily injections of thyroid stimulating moved his axolotl colony to Indiana Uni- hormone or thyroxine will induce versity in 1957. At Indiana University the metamorphosis in highly inbred strains inbreeding program was expanded. New (Taurog, 1974). In one well documented breeding stock was acquired, and the case, metamorphosed axolotls have actu- scope of the operation of the axolotl colony ally been bred in the laboratory. The prog- substantially enlarged. At the present time eny did not, needless to say, differ from approximately 1,000 adult animals are the progeny of neotonous adults. The jelly maintained at the Indiana University capsules surrounding the eggs of the Axolotl Colony. A personal account of the metamorphosed pair were, however, early history of Humphrey's axolotl colony somewhat thinner (Thomas, 1976). has been recorded (Humphrey; Axolotl Despite numerous considerations, in- Newsletter, Spring, 1976). cluding the following: (1) the apparent Since axolotl spawnings usually produce straightforwardness of the biology of the several hundred fertile eggs, new mutant axolotl; (2) the fact that formal descrip- phenotypes are frequently very readily de- tions of its life cycle appeared in scientific tected. Genetic ratios are also easily estab- journals as early as the late 1800's (for lished from the large number of eggs from example, Chauvin, 1876); and (3) the ap- a single spawning. Once a new mutant pearance of literally thousands of articles phenotype has been recognized, a variety in which the axolotl is discussed (Smith and of procedures is employed to characterize Smith, 1971), several misconceptions of it. These procedures are outlined in the animal persist. Perhaps the review ar- Fig. 4, and will be briefly reviewed here. In ticles which make up this collection of pa- the simplest case — maternal effects — pers will eventually serve as source material matings of animals which are heterozygous and clarify some of those problems. for a maternal effect gene produce progeny which develop to sexual maturity in an apparently normal manner. The female MUTANT GENES IN THE AXOLOTL offspring which are homozygous for such a Recognizing the advantages of the gene spawn eggs which display characteris- axolotl as a laboratory animal, and ap- tic patterns of developmental arrest. In preciating its potential for embryological other cases some of the mutant genes do analysis, R. R. Humphrey set out over 25 not give rise to easily observed gross mor- years ago to discover mutant genes in this phological abnormalities. In the case of animal. He began with an assortment of genes which affect the size of the nucle- animals collected from various laboratories olus, routine cytological analyses provide in the U.S.A. and Europe. As mentioned valuable information about the nature of previously, most of the breeding stock the mutant phenotypes. probably traced back to the original Paris Should a spawning provide approxi- stock. Humphrey's approach was to set up mately 25% of its embryos displaying a random matings, and look for the expres- consistent developmental arrest pattern, a sion of new mutant phenotypes among the variety of embryological manipulations are progeny. In addition, he mated newly ac- performed to characterize the phenotype 200 GEORGE M. MALACINSKI

moting of possible heterozygotes of those genes. Most of them are presently maintained in stocks of the Indiana Uni- ? x c? */- . */- versity Axolotl Colony. In order to facili- tate an understanding of the potential usefulness of these genes for studies in de- careful analysis of development of progeny velopmental genetics, I have listed each gene in one of a total of 5 groups. These groupings are rather arbitrary, but in the Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 mating of $ s pigmentation organ or tissue Forelimb primordium with +/+

observation of progeny tissue or for developmental arrest organ grafts

Parabiosis Gill primordium

discovery of maternal characteristics of effect genes mutont phenotype FIG. 4. Procedure employed to characterize the phenotype of new mutant genes. (Figs. 4 and 5). At early developmental (e.g., neural fold or tailbud) stages, reciprocal tissue grafts and parabiosis are carried out. For those mutant genes that are apparently expressed in all tissues, a predictable set of results is obtained. Fig. 5 displays the results from the characterization of one of the autonomous cell-lethal genes (ut). In the case of mutant genes that exert apparent organ specific effects, reciprocal grafts or transplantations of organ primordia suspected to be defective are made between normal and mutant em- FIG. 5. Diagramatic scheme for characterizing phenotypes of possible cell-lethal mutants. Results bryos. Analysis of the results (rescue of the shown here were obtained for several mutant genes, homozygous mutant) is facilitated by including ut, I. At the tail bud stage the gill and employing embryos that bear known pig- forelimb primordia are transplanted between mutant ment markers (dark, white, or albino) as and normal embryos. As well, parabiosis is established hosts for the operation. The relative ease (a). Embryos which are homozygous for an autonomous cell-lethal gene invariably die during parabiosis. and simplicity of techniques with which In a typical case illustrated in (b) the mutant member many of these operations can be carried of the parabiont pair dies and its tissues are absorbed out form the foundation of the science of by the surviving host, (arrow). In the other cases, e.g., amphibian developmental genetics. genes x, h, and q, the normal co-twin dies as well. These results can be contrasted with the results of The extensive selective breeding pro- parabiosis of animals which are homozygous for gram mentioned above, and the characteri- organ specific mutant genes. In the case of the gene c, zation schemes illustrated in Figs. 4 and 5, for example, both mutant and normal host survive. have permitted the accumulation of a rela- The results of the forelimb and gill transplants are shown in (c): mutant limb fails to develop on normal tively large number of mutant genes in the host; (d): mutant gills fail to develop on normal host; axolotl. These genes are all simple reces- (e): normal gill displays partial development on mu- sives, and were discovered, for the most tant host; and (f): normal limb displays partial de- part, by Humphrey. Table 2 contains a list velopment on mutant host. INTRODUCTION TO THE AXOLOTL 201

TABLE 2. List of mutant genes in the Mexican axolotl."

Gene Description Gene Description Group I: Oogenesis Group IV: Cell and tissue function 0 Ova deficient; arrest at gastrulation P Premature death due to an ooplasmic deficiency st Stasis of blood circulation cl Abnormal cleavage 1 Quivering behavior f Fluid imbalance; swelling of t Twisted gills the blastocoel ut Slow growth; abnormal gills V Arrest at blastula stage; variable mi Microphthalmic lethal nc No cleavage g Gill lethal Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 Group II: Early development I Small eyes r Renal insufficiency 1 n Small nucleolus X Fragile gills 2 n Small nucleolus y Limb development arrested n3 Small nucleolus (mutation left no h Foot malformation descendents b Slow development of front limbs n* Reduced amount of rDNA n5 Small nucleolus Group V: Adult Group III: Organogenesis d White Eyeless; sterile a Albino e m Melanoid c Cardiac nonfunction ax Axanthic an Transitory anemia micro Microphthalmia (semilethal) s Short toes ph Phocomelia sp Spastic as Ascites *Genes are placed in groups according to the developmental stage at which the phenotype is unequivocally established. case of some genes, especially several of These include the Im (lethal-mitotique), u those in Groups I and III, the apparent (ulcere), m (melanoide), and ac (ascite stage specificity of the mutant phenotype is caudale) genes. The phenotypes of those well established. Representative genes mutations are briefly described in Hum- from each of the groups will be discussed phrey's review article (1975) and in more in detail in other papers in this volume, so detail in recent papers from the a review of those phenotypes will not be laboratories in which the genes were dis- presented here. In addition, several com- covered (Fernandez and Beetschen, 1975; prehensive reviews of the gene list given in Gounon and Collenot, 1974, 1975; Table 2 have recently appeared (Hum- Beetschen, 1970; Lacroix and Capuron, phrey, 1975; Briggs, 1973; Malacinski and 1970). Each of these genes appears to be a Brothers, 1974). The reader is directed to simple autosomal recessive. those papers for descriptions of the indi- vidual genes, and extensive lists of the primary references which contain accounts AUTONOMOUS CELL-LETHAL GENES of the initial discoveries of the mutant The largest number of genes by far fall genes. The histocompatability factors in into Group IV: autonomous-cell and tissue the axolotl are described by DeLanney lethals. The phenotypes displayed by ani- elsewhere in this volume and are not in- mals which are homozygous for such mu- cluded in Table 2. tant genes are extremely diverse. In some cases, for example, the genes mi, or q, MUTANT GENES IN OTHER URODELES homozygotes die at about the time of hatching. Those embryos display diver- Several mutant genes have been iden- gent phenotypes, ranging from microph- tified in another urodele, Pleurodeles waltlii. thalmia {e.g., gene mi) to altered be- 202 GEORGE M. MALACINSKI

death syndrome sets in (Fig. 6), usually first observed as a necrosis of peripheral tissues, including the gills and limbs (Fig. 7). In some cases embryos display an apparent early tissue specificity of the mutant gene. Embryos which are homozygous for the gene /, for example, are first recognized at the time of feeding by their small eyes, in which the pupil is of an abnormally small size. Eventually, however, the "gen- Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 eral death" syndrome sets in. Mutant genes in this category have been given FIG. 6. Top view of normal larva at the time of names which, in many cases, reflect the feeding (Top). Side view of its sibling of the same age primary and/or most prominent effects of which is homozygous for the geneg". Note that growth the genes. The effects of the mutant genes of the mutant has ceased, and development of exter- in this group are, however, considered to nal structures, such as the gills has been arrested be manifested by most, if not all, of the (Bottom). cells of the organism, hence, the name havior patterns (e.g., gene q). In other autonomous-cell lethals. This assump- cases, for example, the genes g, r, ut, at tion— that all cells of an organism which is approximately the time of feeding, the homozygous for one of these genes will homozygotes cease growing, and a general display altered properties—has naturally

•y*.

FIG. 7. Larvae which are homozygous for cell lethal larvae. Bottom photographs are normal siblings of genes frequently display cessation of growth, arrest of the same age. Arrows point to forelimbs, which show limb development, and necrosis of gill tissue. Top arrested development in ullut larvae. photographs are dorsal and ventral views of ullut INTRODUCTION TO THE AXOLOTL 203 not been rigorously tested. It is not ex- a possibility that genes from some of the perimentally feasible to examine all cells of other groups might eventually be assigned a mutant organism. An abbreviated to Group IV, making this group even scheme, in which some tissues are larger than it already is. examined, has been routinely employed. It A rigorous analysis of the mechanism of is outlined in Fig. 5. Further experiments action of these autonomous cell-lethal should perhaps be directed towards an genes is difficult to accomplish. Since the analysis of cells from more tissues, includ- homozygotes are the product of a mating ing muscle, and various internal organs. between two heterozygous animals, only

Such experiments would more rigorously 25% of the progeny of any such spawning Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 establish the autonomous cell-lethal will display the mutant phenotype. In sev- character of the mutant phenotype. eral cases, analysis of mutant tissues and Could other genes, for example, some of organs prior to the stage at which the those included in Group III, also be true cell lethals? The answer to this question is not immediately available, and will have to await further characterization of several of the genes in Group III. In some cases, e.g., genes e and c, it appears that the effects are indeed relatively organ specific. In other cases, however, it remains a formal possibility that a mutant gene which at one stage most profoundly affects the development of a specific organ or tissue, might, had the ">% organism survived, have had direct effects on all tissues at a later stage. The gene s (short toes) is a case in point. The mutant phenotype can be first recognized in homozygous larvae at about one month of age (Humphrey, 1967). The forelimbs develop abnormally. Development of both the limb and toes is drastically affected. At a later stage incomplete development of the Mullerian ducts can be detected. r Nevertheless, the mutant continues to develop and in some cases survives to adult- hood. Figure 8 displays an adult s/s axolotl. The digit malformation which persists is shown in Fig. 9. Eventually renal pathology and dysfunction lead to death of all s/s animals. The meaning of such diverse effects is not clear. Transplants of the forelimb primordium (Fig. 5) between normal and 5/5 embryos yields limbs of the donor type. Mullerian ducts derived from a pronephric transplant from an s/s into a normal embryo attain their full length. The example of the gene 5, and other examples, such as the gene ph (phocomelia), serve to point out the fact that the full extent and true nature of the effect of FIG. 8. Top view of adult sis animal. The trunk and some of many mutant genes in the axolotl limbs are swollen. Fluid withdrawn from the are not completely established. It remains peritoneal cavity is usually replaced within a few days (Humphrey, 1967). 204 GEORGE M. MALACINSKI

pletely unique mechanism of action. The action of literally thousands of genes might be required for normal development from fertilization through to the larval stages at which arrest occurs for most cell-lethal genes. Alternations in the effects of several of those genes, if not any one of them, could conceivably give rise to the various mutant phenotypes displayed by the genes

in Group IV. Potential experimentation Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 should, therefore, first be directed at nar- rowing down that large number of possibilities to a finite number, each of which could then be systematically tested. In the cases of those mutants that display developmental arrest at relatively early stages (e.g., hatching), it is entirely possible that developmental information, derived FIG. 9. Forelimb of animal shown in Figure 8. Ex- treme shortness of digits, first observed at one month first in embryogenesis from the heterozy- of age, persists throughout development. gous female during oogenesis, is required at all stages of growth. In homozygous em- mutant phenotype can be visually recog- bryos the supply of the essential compo- nized is extremely difficult. By the time the nent might be exhausted, and not replaced mutant phenotype can be recognized, sec- because of the alteration in the mutant ondary effects of the mutant gene may be gene. Humphrey (1975) first proposed this prominent. The possibility therefore exists idea as a possible explanation for the that those analyses are actually inadver- phenotypes of some of the axolotl mutants. tently directed at the physiology of death, It is certainly surprising that if this expla- rather than at primary gene effects. That nation is correct, development of some of is, the establishment of a "cause-effect" the mutants proceeds as far as it does (e.g., relationship between phenotypic charac- to feeding stages). But it is impossible to teristics and primary gene effects is ex- exclude the possibility that subtle effects of tremely difficult in the case of several au- the mutant genes first occur at much ear- tonomous cell-lethal genes. lier stages, and that cascading secondary In addition, the possibility exists that effects which lead to death are simply not these genes may display variable pene- manifested until a much later stage. If this trance from one tissue to another is correct, analysis of the primary effect of (Hadorn, 1961). A set of minor alterations the gene can be expected to be very com- in the metabolism of one cell type might be plicated indeed. A precedent for this pos- expressed, for example, as a set of major sibility exists. In Xenopus laevis, a nucleolar alterations in the same metabolic pathway mutation (1-nu), when in the homozygous in another cell type. condition (0-nu), gives rise to embryos In the case of one mutant gene in the which cannot synthesize ribosomal RNA. axolotl, the geneg, homozygotes appear to Yet, remarkably, homozygotes survive on a display an abnormal cell membrane store of maternally derived ribosomal metabolism. The cell membranes of gig RNA from fertilization through to the embryos have a reduced capacity for bind- hatching stage (Brown and Gordon, 1964). ing proteins (Tompkins, 1970). Perhaps In other cases, the activity of the normal other cell-lethal genes may also lead to alleles of the mutant genes might be re- such general effects on cell metabolism. quired in all cells at around the larval stage The diverse phenotypes displayed by at which development arrests. Lacking the mutants in Group IV present the possibility expression of the mutant gene, homozy- that each cell-lethal gene may have a com- gous embryos may fail to complete the dif- INTRODUCTION TO THE AXOLOTL 205 ferentiation of each of the cell types which Urodele Pleurodeles waltlii homozygotes pour le facteur recessif ac (ascite caudale) C. R. Hebd. employ the normal gene as part of their Seances Acad. Sci. Ser. D. Sci. Nat. 270: 855-858. developmental or growth program. Con- Briggs, R. 1973. Developmental genetics of the ceivably, differentiation pathways for di- axolotl. In F. H. Ruddle (ed.), Genetic mechanisms of verse organs and tissues (e.g., gills, limbs, Development, 31st. Symp. Soc. Dev. Biol., pp. 169- liver, eyes, etc.) may share an enormous 199. Academic Press, New York. Brown, D. D. and J. B. Gurdon. 1964. Absence of number of common steps. The expression ribosomal RNA synthesis in the anucleolate mutant of the cell-lethal genesjn Group IV may be of Xenopus laevis. Proc. Natl. Acad. Sci. U.S.A. 51: common to most of the differentiation 139-146. pathways employed in early em- Brunst, V. V. 1955a. The axolotl (Siredon mexicanum) Downloaded from https://academic.oup.com/icb/article/18/2/195/205366 by guest on 23 September 2021 bryogenesis. Naturally, different cell types I. As material for scientific research. Lab. Invest. 4: 45-64. may employ the common pathway at dif- Brunst, V. V. 19556. The axolotl (Siredon mexicanum) ferent times in development of the whole II. Morphology and pathology. Lab. Invest. 4: organism. Failure of the development of 429-449. several organs or tissues might then have a Chauvin, M. 1876. Ueber die Verwandlung der cascading effect on the growth of the mexicanischen Axolotl in Amblystoma. Zeitschrift fur Wissenschaftliche Zoologie 27: 522-535. whole organism. Chung, H-M. and G. M. Malacinski. 1977. The Mexi- Finally, since cell death in embryonic can axolotl (Ambystoma mexicanum) as experimental systems is a well established phenomenon material for studies in embryology. I. General introduction. Korean J. Zool. 19: 49-56. (Saunders, 1966), it is entirely possible that Dumeril, A. 1866. Observations faites a la menagerie some of the cell-lethal genes may represent des reptiles du Museum d'Histoire Naturelle sur alterations in cell-death regulatory genes. reproduction des axolotls, batraciens urodeles a Rather than being confined to specific branchies exterieures et sur les metamorphoses tissues—such as the interdigital tissue of qu'ils y ont subies. Bull. Soc. Imp. Zool. Acclim. 2: 79-89. limbs—these genes may be active in all Fernandez, M. and J. C. Beetschen. 1975. Recherches cells. Alternatively, the cell death which sur le role de la temperature dans la realisation du has been postulated to be a normal com- phenotype chez des embryons de l'Amphibien ponent of the life cycle of each cell type Pleurodeles waltlii homozygotes pour la mutation (Hayflick, 1965) might be altered by some thermosensible ac (ascite caudale.) J. Embryol. Exp. Morph. 34: 221-252. of these cell-lethal genes. The alteration Gounon, P. and A. Collenot. 1974. Analyse ex- might result in the premature occurence of perimentale de l'expression de la mutation lethale a regulatory event which turns back a recessive "lethal mitotique" chez Pleurodeles waltlii biological cell-death "clock." The cell- (Amphibien Urodele). Experientia 30: 1079-1081. death syndromes displayed by some of the Gounon, P. and A. Collenot. 1975. Analyse ex- perimentale de l'expression de la mutation lethale larvae which are homozygous for these recessive ("ulcere") chez l'embryon de Pleurodeles cell-letha'l genes could easily be reconciled waltlii (Amphibien Urodele). Experientia 31: with such a possible model. 1461-1464. Hadorn, E. 1961. Developmental genetics and lethal Designing experiments to provide in- factors. Methuen, London. formation that would permit a choice Hayflick, L. 1965. The limited in vitro lifetime of among some of these possibilities, and human diploid cell strains. Exp. Cell. Res. 37: others not mentioned above, produce chal- 614-636. lenges which are not easily overcome. Ap- Humphrey, R. R. 1962. Mexican axolotls, dark and white strains; care of experimental animals. Bull. preciating, however, the vast potential for Phila. Herpetol Soc. 10: 21-25. experimental manipulation which the Humphrey, R. R. 1967. 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