An Analysis of the Postgastrula Differentiation of the Hypomere I. The Influence of tissue Mass and of Endoderm in Ambystoma punctatum by CYRIL V. FINNEGAN1 From the Department of Zoology, University of British Columbia WITH TWO PLATES INTRODUCTION SINCE the publication of earlier papers (Finnegan, 1953, 1955) the investigation of the capacity of the salamander hypomeric mesoderm for histogenesis under a variety of experimental conditions has continued. It is perhaps prudent at this time to initiate a series of reports with results obtained from in vitro experiments which were designed to gain some insight into the roles of competence, tissue mass, and endodermal influence relative to hypomeric differentiation in Amby- stoma. This portion of the mesoderm is destined to undergo its differentiation far removed from the dorsal axial influences of the chorda-mesoderm but with its inner (splanchnic) material in rather intimate association with the endoderm, a tissue known to be determined at an early age and metabolically active, two conditions which lead one to suspect it of inductor potentialities (Nieuwkoop, 1947; Copenhaver, 1955). The 'synergistic' or 'additive' role of the archenteron floor in heart development as demonstrated by Ekman (1925), Bacon (1945), and Jacobson (1960), and the possible role of the posterior endoderm in germ- cell development indicated by Nieuwkoop (1947, 1950) as well as the results of this investigation, are indicative of the validity of such a supposition. Recent work (see reviews of Grobstein, 1955, 1959) has emphasized the role of supra- cellular or tissue (mass) influences on the cytodifferentiation of experimentally isolated units. The attempt at a control of mass-size plus its systematic variation in this experimentation further evidences this fact. This report then is concerned with the differentiational ability of a limited mass of Ambystoma neural-stage hypomere mesoderm when isolated in an ectodermal ball, and what occurs to this histogenesis when a small number of endoderm cells are added; when the mass of mesoderm is doubled; and, finally, when endodermal cells are added to the doubled mesodermal mass. 1 Author's address: Department of Zoology, University of British Columbia, Vancouver 8, Canada. [J. Embryol. exp. Morph. Vol. 9, Part 2, pp. 294-309, June 1961] C. V. F1NNEGAN—POSTGASTRULA DIFFERENTIATION OF HYPOMERE 295 EXPERIMENTAL PROCEDURES The A. punctatwn embryos used in this investigation were maintained, after collection and prior to operation, in pond water at 7° C. The explants prepared from these donors were cultured in modified Holtfreter or Niu-Twitty solution at 18° C. Sterile techniques were utilized throughout. In preparing the explants for the various experimental series the following procedures were used. I. Hypomeric mesoderm series (M). The donor animal was placed in the operating medium on its side, and an incision was made in the trunk flank ectoderm in the vicinity of the ventral somite region (either actual or potential) with a finely sharpened steel needle. The cut was continued for a short distance both anteriorly and posteriorly and extreme care was taken not to disturb the underlying mesoderm. The ectoderm was then peeled ventrad from this point. Prior to the complete removal of the ectoderm from the donor's flank, an incision was made in the hypomere a short distance ventral to the nephrogenic mesoderm. This cut was continued both anteriad and posteriad so that a piece approximately 0-5 mm. in length was separated and could be rolled ventrad on itself or in contact with the overlying ectoderm which had been lifted away from the flank. The sheet of hypomere separates neatly from the underlying endoderm. The distance the hypomere extended ventrally at the time of operation depended on the donor age and thus set the limit to this dimension of the explanted mass of hypomere. In practice this dimension appeared to approximate the antero- posterior dimension, exceeding it only in the older stages, particularly along the posterior edge of the explant. Considerable care was taken that no endoderm cells were clinging to the mesoderm. The superficial ectoderm was continued ventrad into the non-mesodermal area of the venter in order to provide a second 'flap' to approach and fuse with the dorsal one when the excised material balled-up in healing. In preparing the explants in all the experimental series care was taken to exclude the potential heart area as localized by Wilens (1955) for this species. Otherwise the tissue removed could be seen from later observation of the donors to reside in that portion of the hypomere which contributes material to the area lying ventral to the pronephros and posteriorly along the length of the liver; thus its somatic portion would probably include some of the limb field and peritoneum, while its splanchnic portion would have contributed to the posterior fore-gut and anterior mid-gut wall and associated ventral structures. II. Hypomeric mesoderm plus endoderm series (M+E). The operational pro- cedure described in the above paragraph was repeated. After the removal of the explant from the donor, a small group of superficial endoderm cells taken from the lateral or ventral wall of the posterior fore-gut (pharynx) was placed on the hypomere prior to the closure of the ectodermal ball. While a definite count was not made on all such endodermal contributions, the cell counts 296 C. V. FINNEGAN—POSTGASTRULA DIFFERENTIATION OF HYPOMERE made revealed that twelve to twenty endoderm cells were usually added to the explant. III. Hypomeric mesoderm doubled series (2M). To prepare each of these explants, two animals of identical age and egg-clutch were subjected to the operation described in I above, both contributions being removed from the same flank of the donors. A sandwich was constructed by juxtaposing the hypomeres centrally, the ectodermal layers remaining superficial. In such a construction, using elements from corresponding flanks, one must reverse either the dorso- ventral or the antero-posterior axes of the two pieces relative to one another. While in the event the healing frequently shifted the relationship of both these axes, the majority of explants were prepared initially with the antero-posterior axes aligned and the dorso-ventral axes reversed. IV. Hypomeric mesoderm doubled plus endoderm series (2M-\-E). The experi- mental procedure outlined in III above was repeated and an endodermal con- tribution taken from each of the donors. The two pieces of endoderm were placed separately between the two layers of hypomere when the explants were brought together to construct the sandwich. The explants remained in the operating dishes until they rounded up and any extraneous cells and debris were removed prior to transfer to the culture dishes. It should be noted that in Series I (M) and II {M-\-E) there was a tendency to lose a small quantity of mesoderm and endoderm as the balls closed. Frequently, however, this material, in particular the endoderm, remained as an epithelial layer at the surface of the explant throughout the entire culture period, thereby contributing its influence to that of the endoderm which had remained internally. The experimental results are based on observations on nearly ninety explants prepared from donors ranging from stage 13 to stage 21 inclusive, and approxi- mately one-half of these were subsequently fixed in Michaelis' solution, sectioned at 8-10 /x and stained with haematoxylin and eosin for histological examination. Macroscopic observations were made daily of the explants and illustrations prepared of their development. Individual cases were removed from culture at various intervals, typically at the end of the first week, in the middle and at the end of the second week, and in the third week, for fixation and microscopic observation. In order to facilitate comparison of the results the explants have been arranged into three major groups according to the age of the donor animals at the time of operation; stages 13-15 are considered as the stage 14 group, stages 16-18 as the stage 17 group, and stages 19-21 as the stage 20 group. EXPERIMENTAL RESULTS The experimental results concerned with histogenesis will be discussed within the framework of the operational series described in the preceding section and will be followed by an analysis of observations on the role of growth in these explants. C. V. FINNEGAN—POSTGASTRULA DIFFERENTIATION OF HYPOMERE 297 A. Histogenesis I. Hypomeric mesoderm series (19 cases). The stage 14 and the stage 17 groups developed into the second week (donor stage 35) after explantation, the former group remaining more solid while nearly one-half of the latter group became vesicular. In both groups, histological examination revealed that the more yolky cells of the splanchnic mesoderm were aggregated in the centre of the explant while the cells of the somatic mesoderm, lying more superficially, demonstrated a reduced yolk content. The latter did not, however, form a peritoneum beneath the overlying ectoderm, which remained as cuboidal cells at the surface. In both groups there appeared to be an attempt to form a haematopoietic structure in which haematoblasts could be distinguished and in the sinuses of which apparent erythroblasts were observed (Plate 1, fig. 1). The stage 17 group showed a tendency to produce a slightly thicker capsule around the splanchnic mesoderm with some separation between the somatic mesoderm and this capsule. The stage 20 group explants remained vigorous through the second week (donor stage 41) and into the third in some cases, the majority becoming vesi- cular. Examination of these made evident the existence of involutions producing a cavity or, more often, several small cavities as the superficial somatopleure material (somatic mesoderm peritoneum plus squamous ectoderm) pushed into the interior. This would appear to be an expression of the antero-posterior extension of this material which must occur in the embryos as the length of the ventral axis increases during post tail-bud stages, and the embryo appears to straighten up from its earlier C-like silhouette.