TABLES FOR THE NORMAL DEVELOPMENT O F RANA SYLVATICA ARTHUR W. POLLISTER AND JOHN A. MOORE Department of Zolology, Columbia University INTRODUCTION To any student of anuran embryology it is probably uiinecessary to point out the need f o r a description of a series of normal stages in the development. The usefulness of such data is shown by the frequency of references to the widely known, though unpublished, series of Amblystoma punctatum by Harrison. The need for a similar ‘common language’ for anuran development has been keenly felt in this laboratory and in others f o r some time, and the present study is an attempt to meet this demand. It has been found most useful to define all stages in the series by at least two characteristics : the external morphology and the age a t a constant temperature (hours after fertilixation at 18°C.). The changes in external form are relatively simple up to the beginning of the growth of the tail. Accordingly no additional data, beyond age and form, are needed on stages 1 to 17, which comprise the successive phenomena of cleavage, gastrulation and neurula formation. I n the later period of embryology, however, there a r e many simultaneous processes that are readily identifiable externally. The most obvious are the following: growth of tail; elongation of the body ; growth of external gills, development of operculum ; development of transparency of epidermis ; coiling of gut. Furthermore, there are physiological features of development that appear as the embryo approaches larval condition, eg., beginning of muscular movement and of swimming ability, onset of heart beat, of gill circulation, and of tail fin circula489 T H E ANATOMICAL RECORD, VOL. 68, N O . 4 490 A. W. POLLISTEE AND J. A. MOORE tion, and spontaneous hatching of the embryo. There are interspecific differences in the time of occurrence of these various features of later development. F o r example, the operculum is nearly completed in a Rana pipiens embryo which, as classified by other features, is at a stage when it is just beginning to grow backward over the gills i n Rana sylvatica. Because of such differences one cannot describe a series of stages for even these two species of R a m , which will be strictly alike in every one of the features mentioned above. Hence it has been found more widely useful t o define each of stages 18 to 23 by a group of characteristics. The total length of' the embryo is widely used as an exclusive basis for definition of stages in later development of Anura. I n wide practice this is not very reliable because of variability of egg size, both iiitraspecifically and interspecifically. A much more nseful criterion, likewise based on measurcmciits, is the ratio of tail length (measured from the anus) t o the length of the remainder of the embryo, the body. Data on total length are, nevertheless, included in the tables. They are based on measurements of eggs of approximately average size. The study has been made partly from eggs collected in nature and partly from those artificially fertilized after orulation had been induced by pituitary administration. There are no differences in the development of eggs obtained by the two methods. I n the r i c h i t y of New York, R a m splvatica tisnally lays its eggs about the middle of March in water a t a temperature of about 11". I n the laboratory eggs will develop normally a t from 2 to 22". At the lower limit development is extremely slow, yet when eggs are removed to higher temperatures they develop normally in every respect. This unusual tolerance of such a low temperature is of practical advantage in that one can place eggs collected in the field in heat-insulated containers with ice cubes and thus bring the embryos to the laboratory in very nearly the stage in which they were collected. Data on the time f o r development at different temperatures between stages 3 and 20 are given N O R M A L DEVELOPMENT O F R A N A SYLVATICA 491 in table 4. Such data are very accurately reproducible for the early stages, but from stage 18 onward differences between embryos become increasingly significant. The total elapsed time for development to stage 23 may vary as much as 10% among embryos from a single batch of eggs. DESCRIPTION OF STAGES * Stages 1 t o 17 (table I) 1. E g g at fertilization. 2. Establishment of gray crescent area a s first external evidence of development, sharply defined at 1 hour. 3 t o 6. Age given is time of appearance of cleavage furrow that establishes the number of cells drawn for the stage. 7 to 9. Later stages in cell multiplication, best determined by comparison of size of cells a t vegetal pole. 10. Appearance of dorsal lip. 11. Blastopore approximately a semicircle. 12. Complete blastopore (yolk-plug) stage. 13. Slit blastopore or neural plate stage. 14. Neural fold stage. 15. Beginning of closure of neural folds, beginning of elongation. Cilia begin to rotate the embryo a t about this stage. 16. Closurc! of neural folds comnleted. 17. Beginning of development o'f tail bud, marlicd off from body by ventral notch when embryo is viewed laterally. Stages 18 t o 23 (tables 2 and 3 ) The figures are ventral and lateral views of all but stage 22, which shows dorsal instead o€ ventral aspect. 18. Stage begins with development of capacity for mnscular movement, i.e., simple unilateral flexure in response to mechanical stimulation. This is very suddenly acquired and is closely correlated with attainment of the external form figured. The stages illustrated and defined by age are of course esseiltially a n arbitrary series of readily identifiable points in the coiitinuous process of development. Defined in terms of these points, the total developmelit is comprised i n a series of periods, each extellding from oiic stage until the nest. In practice the points and the periods are iiot sharply distinguished and it is convenient t o describe each period in terms of the stage t h a t initiates it. Thus the development from onset of the heart beat to the beginning of gill circulation would be the period of stage 19. 492 A. W. POLLISTER A N D J. A. N O O R E 19. Time given indicates onset of heart beat which appears very suddenly and is accordingly a most useful marker for this stage. (Use of strong reflected light is necessary f o r identification of this early pulse.) Tail equals one-third the length of the body. 20. Beginning of circulation of blood corpuscles through a capillary loop of anterior gill is closely correlated with gill morphology, and is the best indication of the beginning of this stage, Shaking will hatch embryos early in this stage; they hatch spontaneously late in 20. Swimming ability is acquired in the latter part of this stage. Tail equals one-half the body length. TABLE 4 H o u r s f r o m first cleavage required l o reach variozts s l a p s at different teniperatzlrrs STAGE 3 4 5 30.4"C. 0 2+ 5 ti 7 8 9 10 11 12 13 14 15 16 17 18 19 20 15.4"C. 0 1.3 2.2 3.0+ 11 24 36 45 60 72 96 112 124 141 168 180 216 27.5 4.7 14.0 19.5 24.0 32.0 37.0 52.0 .XO 63.0 72.0 83.0 90.0 108.0 130.0 18.5'C. 0 1.0 2.0 3.0 3.5 9.5 13.5 16.5 21.0 25.0 33.0 3 7.0 42.0 47.0 55.0 62.0 72.0 87.0 21. Cornea becoming transparent so lens is visible as light spot. Body and tail nearly equal in length. 22. Developmeiit of posterior beiid in gut niakes trunk appear asymmetrical from dorsal aspect. A few capillary loops are fiinctioiial in the tail fin. Epidermis rapidly becoming trans parent. 23. Triiiik and head liave rounded out and embryo assumes trne lnrval or 'tadpole ' shape. Horny larval teeth developed. Posterior limb bud identifiable. Opercixlar fold beginning to develop. Active spoiitaiieous swimming begins. X O R N A L DEVELOPMENT O F R A N A SYLVATICA TABLE 1 493 494 A. W. POLLISTElZ A N D J. A. MOORE TABLE 2 NORMAL DEVELOPMENT O F R A N A SYLVATICA TABLE 3 495 3-96 A. W. POIJ,ISTEit A N D J. A. MOORE In t e r nal a n at o ~1 y It has been found useful to have some meails of readily identifying sectioned material in terms of the series of stages described above. There arc, of c’ourse, no difficulties in doing this with cnibryos up to the time of closure of thc n e u i d folds TABLE 5 EYE EAR EYE EAR 6 6 __ 7 12 -- 8 18 (stage 15). F o r recognition of stages 16 t o 21 table 5 was constructed. The number of somites was counted from frontal or sagittal sections. Tlie development of the eye and ear, as seen in (TOSS sections, are shown by the series of drawings made at a commoii magiiification by a projection method.