/. Embryol. exp. Morph. Vol. 40, pp. 23-34, 1977 23 Printed in Great Britain © Company of Biologists Limited 1977 The blastomere pattern in echinoderms: cleavages one to four By JOHN W. PROTHERO1 AND ARNOLD TAMARIN From the Departments of Biological Structure and Oral Biology, University of Washington SUMMARY The results of a longitudinal study of the blastomere pattern in six embryos during the first four cleavages are reported. At each cleavage stage optical sections through an embryo, taken at vertical intervals of 5 or 10/*m, were recorded on 35 mm film: digitization of the blastomere contours and computer analysis allow calculation of the center, radius, surface area and volume of each blastomere. The subjective impression of exquisite regularity seen in normal echinoderm blastulae acquires a quantitative dimension from the present study. For example, the individual angles formed by the various quartets of blastomeres depart from right angles by at most a few degrees. The egg volume was found to be conserved up to the fourth cleavage. At the 16-cell stage, unlike the earlier stages, the blastomere positions cannot be ascribed solely to the position and orientation of the respective cleavage planes. Finally, a few features of a formal model of these early cleavages are sketched. INTRODUCTION Blastula formation, by virtue of its apparent simplicity, may provide a useful model system for the study of morphogenesis. In principle it is possible to provide a relatively complete quantitative description of the process at the cellular level. The transparency of the early blastulae, the ease of cultivation and the exten- sive background information combine to make echinoderm embryos especially inviting material for the study of blastula formation (Horstadius, 1973). A semiquantitative study of blastula formation was the subject of an interesting paper by Wolpert & Gustafson (1961). The present paper seeks to contribute to the construction of a detailed quantitative model of the early blastomere pattern in echinoderms. MATERIALS AND METHODS The procedure employed in this study has been described in some detail in a prior paper, hereafter denoted PTP (Prothero, Tamarin & Pickering, 1974). Essentially, the procedure is to record on film, following each cleavage, a 1 Author's address: Department of Biological Structure SM-20, University of Washington, School of Medicine, Seattle, Washington 98195, U.S.A. 24 J. W. PROTHERO AND A. TAMARIN B Fig. 1. Blastomere nomenclature at 3rd and 4th cleavages. (A) an and vg refer to animal and vegetable blastomeres at the 8-cell stage. (B) me, ma and mi refer to meso- meres, macromeres and micromeres respectively at the 16-cell stage. In each case, blastomeres are drawn to represent approximately the measured average dimensions. series of optical sections through a developing embryo. Digitization and com- puter techniques are used to reconstruct the size, shape and position of all the blastomeres, up to and including the 16-cell stage. In the present study, as distinct from the prior one (PTP), the reference co- ordinates on each 35 mm frame are derived from the image of a calibrated reticule inserted at the field diaphragm of the ocular. The animals used in this study {Dendraster excentricus) are maintained in a laboratory tank. For obscure reasons our success in obtaining the classical three-tiered embryo characteristic of the 16-cell stage has been quite variable. (In the previous study (PTP) the blastomeres were shifted with respect to one another.) To circumvent this difficulty the development of several score of embryos was recorded. From the resultant data the film strips for six developing embryos, the 16-cell stages of which seemed normal by inspection, were selected for data processing. The contours for the six embryos were digitized at a total of about 115000 points. RESULTS The blastomere pattern in six embryos (derived from five pairs of parents) was reconstructed (see Table 1). The data base is incomplete in several respects. Only three embryos had been photographed at the 1-cell stage, and two of these were photographed at the (coarse) vertical interval of 20 /urn. In two cases, the 8-cell stage was not reconstructed because the animal and vegetable quartets ,were not distinguishable in the film (see Fig. 1). Furthermore, the quartet of micromeres could not be discerned in two cases. In consequence of these short- comings, there are only two embryos in this series for which the data are com- plete (i.e. from the 1-cell to the 16-cell stage). The blastomere pattern in echinoderms 25 Table 1 Cleavage ... 0 1 Micromeres Sample size 3 6 6 4 6 4 Time(min) 80-5±599 139-3±29-9 1933±37-7 229-1 ±33-5 2908±43-4 — Total points 4022 16117 16504 13551 63785 113979 A summary of some features of the data base. Sample size refers to the number of embryos analyzed at each cleavage stage. The last column indicates that in two cases the micromeres could not be distinguished at the 16-cell stage. The time after fertilization at which each embryo was photographed is given in the third row and the total number of digitized points for all embryos in the fourth row. Plus-minus figures in this and other tables represent one standard deviation. Table 2 3 Cleavage ... 0 1 2 an veg Horizontal 66±5 51±3 39±3 30-5±l-5 33 ±1 radius (/*m) Vertical 78±2 66±8 544+11 33-O±2-5 36±5 radius (/*m) Surface area 63 ±10 41 ±6 28±5 14±2 16±2 (/*m2 x 10-3) Volume 136±30 69 ±14 37±8 13±2 17±4 Om3 x 10-4) Total 4th cleavage Meso meres Macromeres Micromeres Blastocoel volume Horizontal 26±4 32±2 12-5±2 — — radius (/*m) Vertical 36±5 33±6 20±4 — — radius (/*m) Surface area 18±11 22 ±14 5±4 — — Om2 x 10-3) Volume 10±3 13-5±4 l-3±O-5 11 ±6 147 ±34-5 Om3xl0-4) Mean values of the horizontal radius (Rs), vertical radius (Rv), surface area (S) and volume (V)at each cleavage stage. The parameters which have been computed for each blastomere include: the center, horizontal (RH) and vertical (Rv) radii, surface area (S) and volume (V) (see Table 2). Note that the horizontal radius is computed at the largest cross-section of the blastomere, which need not coincide with an equatorial plane. For each blastomere one may compute a hypothetical surface area (Sc) and a hypothetical volume (Vc) from the radii (RH, Rv), assuming that the blasto- 26 J. W. PROTHERO AND A. TAMARIN 120 110 "5 100 90 80 L 2 4 8 16 Number of blastomeres Fig. 2. The conservation of total cellular volume. The volume (V) of each embryo is normalized to 100 % at the 1-cell or 2-cell stage (whichever was recorded initially). The vertical bars represent one standard deviation. meres are simply prolate spheroids. The regression lines fitted to (Sc, S) and (Vc, K)are given by: 3 2 Sc = 1-00S+1-55 x 10 Om ), (1) 4 3 Vc = 1 -05 V+1 -00 x 10 Om ). (2) For both equations the correlation coefficient is 0-98. The total blastomeric volume is simply the sum of the volumes of the blasto- meres. To facilitate the comparison of total volumes at successive cleavages we have normalized the volume (V) to that at the 1-cell stage (three cases) or the 2-cell stage (three cases). The comparison, expressed in percent, is shown in Fig. 2. Another approach to the question of the conservation of cellular volume entails a comparison of the volume of each pair of daughter cells with that of the mother cell. Taking the volume of each mother cell as 100 %, we find (for 60 cases) that the mean volume of the daughter cells is 101 ±22 %. (Throughout this paper plus-minus figures denote one standard deviation from the mean.) It proves useful to compute the regression of Rv on RH. The result (see Fig. 3) is given by: Rv = l-16RH + 3-36(jim) (3) with a correlation coefficient of 0-92. As a corollary we find that the axial ratio (RHIRV) varies systematically from 0-67 for the micromeres to 0-82 for the egg. This fact may be important in understanding the control of blastomere packing (see Discussion). Study of the pattern formed by the array of blastomere centers provides some insight into the organization of the embryo as a whole. To this end we need to The blastomere pattern in echinoderms 27 80 1 cell 60 2 cell on Mesomeres / 1 40 4 cell Micromeres 20 20 40 60 80 Horizontal radius (j.im) Fig. 3. Mean values of Kv are plotted against mean values of R# at each cleavage stage. The bars represent one standard deviation. The solid line is the regression line fitted to all the data. The point for the macromeres is omitted for reasons of clarity (see Table 2). distinguish between center-to-center spacings in the horizontal (DH) and vertical (Dv) planes. In addition, we require, for an adequate description, a specification of the angle (a) enclosed by lines joining those blastomere centers lying in a horizontal plane. In this regard note that for a (planar) polygon having n sides (generally of unequal length) the average angle (a) subtended at the vertices is given by: a = 180 (n-2)jn degrees. (4) Thus the mean angle is 90° for a quadrilateral and 135° for an octagon (in- dividual angles may, of course, deviate widely from these values for a given arbitrary polygon). At the 2-cell stage the horizontal center-to-center spacing (DH) is 84 ± 5 jum.