Estadios normales

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DEVELOPMENTAL BIOLOGY 42, 391-400 (1975)

Normal Stages of Development of the Axolotl,Ambystoma mexicanum

G. M. SCHRECKENBERGDepartment of Biology, Fairleigh D ickenson Uniuersity, Rutherford, New Jersey 07070

AND

A. G. JACOBSONDepartment of Zoology, University of Texas, Austin, Texas 78712

Accepted October 29, 1974Illustrations and descriptions of normal stages of the axolotl Ambystomamexicanumre given.

The time to reach each morphological stage is compared with similar stages in two other specie s,Ambystoma maculatum and Taricha torosa.

INTRODUCTION 14-19 follow one another rapidly while daysThe Mexican axolotl, Ambystoma may separate successive larval stages.mexicanum (also called Siredon The time between the stages is illus-

mericanum), has long been used in labora- trated in Fig. 1. The data for Fig. 1 weretories around the world. This convenient compiled by rearing embryos in a constant-laboratory animal probably will be used temperature box (18 f 0,5C), inspectingincreasingly for experimental studies of frequently, and recording the time sincedevelopment. We give here a series of stage 1 that each stage first appears. Evennormal stages for this species and data on at constant temperature and using eggsthe timing of the stages. from the same mating, there is variation inWe have tried to make these normal timing of stages. Differences in egg sizestages of the axolotl as comparable as may contribute to this timing variation.possible to Harrisons (1969) staged series We have timing data for the Californiaof Ambystoma maculatum (Amblystoma newt Taricha torosa (staged by Twitty andpunctatum). The Twitty and Bodenstein Bodenstein, 1962), which we present here(1962) staged series of Taricha torosa (Tri- for comparison (Fig. 2). The T. torosa dataturus torosus), which was also modeled is taken from time-lapse movies. The mov-after Harrisons series, served as a useful ies were made at an exposure interval of 1supplementary guide. Gastrulation and min. The movies were analyzed using aneurulation of the axolotl looks more like stop-motion projector equipped with athat of T. torosa than of A. maculatum, frame counter. A movie was run forwardbut the timing of stages is most similar in and backward until a decision could bethe two Ambystoma species. reached as to what frame was the midpointDevelopment is, of course, continuous of each stage. The time since stage 1 couldand the designated stages gradually grade then be read directly in minutes from theinto one another. The stages are based on frame counter. The timing in differentchanging external morphological features. movies was similar except through neurulaThe amount of time between different stages.stages varies. For example, neurula stages After cleavage stages, T. torosa lags

391Copyright 0 1975 by Acade mic Press, Inc.All rights of reproduction in any form reserved.


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392 DEVELOPMENTAL BIOLOGY VOLUME 42. 19751 Ambystomo mexiconum

at 18'C

FIG. 1. Most points on this graph represent the average of several determinations. The horizontal linesthrough a point indica te the entire observed range of time that embryos reached that stage. The number ofobservations for each point varied from 1 to 22, the average is 7. Points without lines were single observations.

1 Ambvstoma I

DAYS5 IO ,5 20 250 30 60 90 120 240 360 480 600HOURS

FIG. 2. Taricha torosa stage times were comp iled from time-lapse movies of development of this spec ies. T hepoints represent the midpoint of each stage. Va riation in time of stage is greatest during neurula stages and datafrom two films (open and closed circles) is given through this period. For comparison, we show our A.mexicanurn time-stage curve, and Harrisons (1969) time-stage curve for A. maculatum (Anblystomapunctatum).

considerably in rate of development com-pared to A. mexicanum. Stage 28, for ex-ample, occurs about 5 days later in T.torosa. By stage 40, T. torosa has caughtup.Comparing the development of A.mexicanum to Harrisons (1969) time-stagegraph for A. maculatum (Fig. Z), there isan almost exact parallel between the twospecies from stage 1 through stages 37-38.A. maculatum stages, measured at ZOC,

are consistently a few hours earlier than A.mexicanum stages, measured at 18C. A.maculatum reaches stage 37 after about195 hr; A. mexicanum reaches stage 37after 197 hr. After stage 37-38, A.mexicanum stages are very slow comparedto A. macalatum. For example,mexicanum takes 488 hr to reach stage 40while maculatum reaches stage 40 in just265 hr.We have illustrated the staged series


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BRIEF NOTES 393only through stage 40 when the larva 17 days development (at 2OC). Hindlimbshatches from the membranes and turns first appear in the axolotl on the 56th dayonto its belly. Older stages vary greatly of development (at 18C). The limb devel-depending on crowding, food, and other opment of A. maculatum seen at stage 46environmental factors. (22 days at 20C) is attained in the axolotlLimb development of the axolotl is very at 18C after 64 days of development (Fig.much retarded compared to A. 3). The hind limbs of the axolotl larva aremaculatum. Forelimb development of the well developed with toes after 84 days ofstage 40 axolotl compares to that of the development (Fig. 3).stage 36 A. maculatum. Hindlimbs first A discussion of the care and breeding ofappear in A. macdatum at stage 43 after axolotls is presented by Fankhauser (1967).

FIG. 3. A Dorsal view of A. mexicanurn larva after64 days of development at 18C. B. Ventral view oflarva shown in A. Note co nical hindlimb buds andwell-formed operculum. Limb characte ristics aresimilar to a stage 46 A. maculatum, but the axolotllarva takes more than 40 days longer to attain thesame stage. C. An axolotl larva after 84 days develop-ment at 18C. H indlimb digits are not well developed.A-C are at the same magnification.


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394 DEVELOPMENTAL BIOLOGY VOLUME 42, 1975The cleaving axolot l egg has been studied this stage. Polar bodies may often be seenin detail (Skoblina, 1965; Meinertz, 1970; near the animal pole.Signoret and Lesfresne, 1971; Hara, 1971; Stage 2. Two cells. The cleavage furrowRott, 1973). Ignateva (1968) described ax- extends from pole to pole.olot l gastrulation and compares it to the Stage 3. Four cells. The second cleavagesturgeon. C.-O. Jacobson (1959, 1962) de- furrow also extends from pole to pole atscribed and mapped axolot l neurulation. right angles to the first.

The illustrations of stages that follow Stage 4. Eight cells. The third cleavage(Fig. 4A-C) are photographs of liv ing em- furrow forms along a latitude above thebryos, al l at the same magnif ication. Ex- equator. The animal hemisphere quartet ofcept as noted below, al l embryos are cells is thus smaller than the quartet be-viewed from above as they naturally orient low.themselves when they are removed from Stage 5. Sixteen cells. Stage 5 animalthe jel ly capsules and placed unrestrained hemisphere cells appear about half the sizein a dish of water. The only exceptions are of stage 4 cells.stages 9, 10, and 11 whose diagnostic fea- Stage 6. About 32 cells around a centralture, the forming blastopore, is visible only cavity.from beneath. These three embryos were Stage 7. The upper cells appear aboutphotographed through an inverted micro- half the size of stage 6. Cleavage is becom-scope. The view of them is of their vegetal ing asynchronous even among the animalhemispheres. hemisphere cells. Stage 7 may be consid-

Through cleavage stages (l-7), blastula ered the end of cleavage stages and thestages (7-g), and early gastrula stages beginning of blastula stages.(g-11), the embryos rest with their animal Stage 8. Blastula stage. The upper cellspoles uppermost. Between the end of stage are about half the size of those at stage 7 at11 and the beginning of late gastrula stage the beginning of stage 8. Stage 8 lasts 9-1612, the embryo rotates, turning the future hr and the cells continue to divide, becom-dorsal side uppermost. This change in ing too small to distinguish indiv iduallyorientation coincides with the formation of without magnification (Stage 8+).the archenteron and the disappearance of Stage 9. Vegetal pole view. The firstthe blastocoel. The embryo retains this signs of gastrulation are pigment concen-orientation from gastrula stage 12 through tration (indicative of cell elongation per-neurula stages 13-19. As neural tube for- pendicular to the surface) and invaginationmation approaches complet ion at stage 19, at the mid line of the future dorsal l ip of thethe embryo begins to lie on its side, is blastopore. An animal pole view does notpartially on its side at stage 20, and corn- appear much dif ferent from stage 8+.pletely on its side from stage 21 through 39. Stage 10. Vegetal pole view. The dorsalStage 39 lasts for many days and gradually lip of the blastopore is fully formed as agrades into stage 40. At stage 40, larvae crescent and involution has begun.have turned onto their bellies. They hatch Stage 11. Vegetal pole view. The lateralfrom the membranes about this time. lips have formed making the blastopore

In the list that follows, each stage is into a semicircle.briefly characterized to call attention to Stage 12. Dorsal view. As the archen-diagnostic features in staging. Note the teron forms and the blastocoel recedes, theprogressive increase in length of the em- embryo rotates until it rests dorsal side up.bryo from stages 25-40. This brings the blastopore into view fromStage 1. Uncleaved, single-celled egg. above. The ventral lip has formed to makeCytoplasmic rearrangements occur during the blastopore a complete circle surround-


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FIG. 4 AFIG. 4. Cleavage, blastula, gastrula, and neurula stages (see text for descriptions). B. Late neurula and

tail-bud stages (see text for descriptions). C. Late tail-bud and early larval stages (see text for descriptions).395


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FIG. 4 B396


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397BRIEF NoTEs

4

FIG. 4 c


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398 DEVELOPMENTAL BIOLOGY VOLUME 42, 1975ing a large yolk plug.Stage 13. The yolk plug is much reduced.Neurulation has commenced and is evidentin the flattening of the dorsal hemisphereand the thickening of the prospective neu-ral plate.Stage 14. The yolk plug is withdrawn;the blastopore is a slit. The neural plate isoutlined by pigmentation concentration inelongated cells. By stage 14-t, the neuralfolds are evident. Between stages 14 and19, the length of the nervous system in-creases rapidly pushing both head and tai lregions around the curve of the embryo.

Stage 15. The neural plate has distortedinto a keyhole shape.Stage 16. The spinal cord region of theneural plate has narrowed and the neuralfolds appear more prominent.Stuge 17. The brain plate is still flatopen, but the neural folds are touching, orabout to touch, where the spinal cord joinsthe brain.Stage 18. The brain plate is still open,but rolling into a tube. The neural folds arenearly apposed along the length of thespinal cord.Stage 19. The neural folds are touchingone another in the spinal cord region. Thebrain piate is nearly closed into a tube. Theembryo begins to turn on its side.Stage 20. The neural folds are closedalong their length. A few embryos may beslightly open in the brain area. The embryolies almost on its side. The epidermis hasdrawn in along and below the nervoussystem so the first three or four somites canbe seen through the epidermis.Stage 21. The embryo lies on its sidewith neural folds closed. Continued elonga-tion of the nervous system has brought theanterior tip of the head in line with thebelly or slightly extending beyond. A simi-lar protrusion at the tai l end is the begin-ning of the tai l bud. The optic vesicles arebecoming distinct. The mandibular arch isapparent as a band below the brain.Stage 22. The head is larger and pro-

trudes more past the line of the belly.Further nervous system e longation is ap-parent.Stage 23. The head protrudes well pastthe line of the belly. The tail end is slightlymore developed. The pronephric swelling isbecoming visible.

Stage 24. The head is more prominentand ,more clearly separated from the rest ofthe embryo by grooves anterior and poste-rior to the mandibular arch. The gil l bulgeis beginning. About 9 somites are visiblethrough the epidermis.Stage 25. The head and the mandibulararch now extend past the belly line . Gi lland pronephric bulges are prominent. Thisstage is made quite distinctive by the largehead protruding almost at right angles pastthe belly line.Stage 26. The nervous system is furtherelongated and the head region is lifting tobecome more in line with the long axis ofthe rest of the embryo. The smooth curve ofthe top of the head of stage 25 is nowinterrupted by the abrupt bend of thecephalic flexure.Stage 27. The tail end has become quiteprominent and flexes ventrally. The headhas further straightened and the branchialgrooves appear more distinct.Stage 28. The nervous system has furtherelongated and the head appears relativelylarger. The pronephric duct can be seenrunning posteriorly beneath the somitemargin.Stage 29. The g ill bulge is especiallyprominent. The tail end is also more no-ticeably distinct.Stage 30. The head has straightenednoticeably. The tail end is more promi-nent. Head features become more deline-ated. Stages 30-34 show a continued in-crease in nervous system length andstraightening of the head and tai l regions.These stages also show marked changes inthe length and shape of the tail end.Stage 31. Tail end longer and headstraighter.


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BRIEF NOTES 399Stage 32. Tail end is longer, head is

straighter. The gill bulge is more circum-scribed.

Stage 33. Tail and head both straighter.Groove posterior to gill bulge more promi-nent. At this stage the body muscles be-come responsive. If pricked in the side, theanimal bends toward the tormentor.

Stage 34. The nervous system is astraight line from tip of the tail to cephalicflexure. The heart begins to beat. Threedivisions of the gills are first apparent.

Stage 35. The three gills are more promi-nent. Fins appear on tail and back. A fewpigment cells make their appearance onthe flank.

Stage 36. The three gill buds and thenasal pit lie in an almost straight line. Finsand tail have enlarged.

Stage 37. Pigmentation is more exten-sive. The gills protrude farther. Tail andfins are larger.

Stage 38. Gills are longer, finger-like,and project ventrally.

Stage 39. Gills are longer, point morehorizontally and posteriorly, and begin tobranch. Flanks and eye are much morepigmented. Fins are very extensive. Thecloaca is more distinct. This stage lasts along time and during it all these featureschange, especially the size and elaborationof the gills.

Stage 40. The distinguishing feature isthe turning of the larva onto its belly.Hatching from the membranes occurs atthis time. The gills are long and branched.Pigmentation is very extensive. Forelimbs

are just beginning to become visible asbulges behind the gills.REFERENCES

FRANKHAUSER, G. (1967). Urodele s. In Methods inDevelopmental Biology (F. H. Wilt and N. K.We ssells , eds.), pp. 95-97. Crowell, New York.

HARA, K. (1971). Cinematographic observation ofsurface contraction waves (SCW) during theearly cleavage of axolotl eggs. Wilhelm Roui Arch.Entwichlung smech . Organismen 167, 183-186.

HARRISON , R. G. (1969). Organ ization and Develop-ment of the Embryo (S. Wilens , ed.), pp. 44-66.Yale Univ. Press, New Haven, CT.

IGNATEVA, G. M. (1968) (In Rus sian) Dyna mics ofmorphogenetic movements at the period of gastru-lation in axolotl embryos. Dokl. Akad. Nauk SSSR179, 1005-1008.

JACOBSON, C.-O. (1959) Th e localization of the pre-sumptive cerebral regions in the neural plate of theaxolotl larva. J. Enbtyo l. Exp. Morphol. 7, l-21.

JACOBSON, C.-O. (1962) Cell migration in the neuralplate and the process of neurulation in the axolotllarva. zool. &drag Uppsala 35, 433-499.

MEINERTZ, T. (1970) Eine Untersuchung tiber dieDauer der Zellteilung und die Teilungfrequenz inaxolotlen-E iern. 2. Mikrosk. Anat. Forsch . 81,405-412.

Ron, N. N. (1973) (In Russian) Correlation betweenkaryokinesis and cytokinesis during the first cleav-age divisions in the axolotl (Ambystoma mer-icanum, Cope). Ontogenez 4, 190-192.

SIGNORET, J., and LESFRESNE, J. (1971). Contributiona letude de la segmentation de loeuf daxolotl: I.Definition de la transition blastuleenne. Ann. En-bryol. Morphogen. 4, 113-123.

SKOBLINO, M. N. (1965) (In Russian) Dime nsionles scharacterization of the duration of the mitoticphases of the first cleavage divisions in the axolotl.Dokl. Akad. Nuuk SSSR 160, 700-703.

Twrrrv, V. C., and BODENSTEIN, D. (1962). In Experi-mental Embryology (R. Rugh ed.), p. 90, Burgess,Minneapolis, MN.

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