[ 130 ] STUDIES ON THE MORPHOGENESIS OF THE MOUSE STERNUM III. EXPERIMENTS ON THE CLOSURE AND SEGMENTATION OF THE STERNAL BANDS BY JUI MING CHEN Strangeways Research Laboratory, Cambridge INTRODUCTION The study of the normal embryonic development of the mouse sternum and the experiments on its differentiation in vitro reported in Parts I and II of the present series of studies (Chen, 1952a, b) indicated two points of special interest in relation to the developmental mechanics of this bone, namely the closure of the two sternal bands and the segmentation of the sternum. The bilateral, dual origin of the sternum is perhaps an unusual feature in morpho- genesis, for very few organs in the vertebrate have a similar ontogenetic course, but in all classes the true sternum originates in the same way (Gladstone & Wakeley, 1932). So far, the mechanism by which the two widely separated mesodermal bands are brought together has only been investigated experimentally in birds (Fell, 1939). Fell has shown that, of the factors possibly involved, i.e. (i) the expansion of the sternal bands themselves, (ii) the elongation of the ribs, (iii) the shrinkage of the mid-ventral body-wall and (iv) the movement of the dorso-lateral tissue of the thoracic wall, the last one is primarily responsible, while the shrinkage of the ventral body-wall may have a secondary effect. The study of the normal development of the mouse sternum has shown that all these factors except the first may operate in the mouse also. By analogy, it seemed possible that the same mechanism was involved in both species, but it was advisable to investigate the matter by experimental means. Section A accordingly is devoted to investigations made to find (a) whether the closure of the bands is due to the elongation of the ribs, (b) whether there is a movement of the undifferentiated tissue in the ventral body-wall in a median direction, (c) if so, whether it is due to the shrinkage of the ventral body-wall or to an active migration, and (d) whether the sternal bands can be displaced by this tissue movement. The problem of the segmentation of the sternum pertains only to the mammals whose sternum ossifies as a series of bony segments separated by bands of cartilage. As briefly mentioned in the discussion on the normal development of the mouse sternum (Chen, 1952a), this segmentation seemed secondary in nature and due to the influence of the ribs. Some workers (viz. Bryson, 1945) believe that it is a result of the inhibition by the rib tips of hypertrophy of the cartilage cells at the sterno- costal junctions. However, no definite proof has been presented and arguments both for and against this theory are available. The intimate contact of the ribs with the sternal tissue during development, and the fact that the sternal ends of the ribs do not ossify, support the possibility that the rib tissue might exert a mechanical or Studies on the morphogenesis of the mouse sternum 131 a chemical effect on the sternal cartilage cells. On the other hand, in birds and certain species of reptiles the attachment of ribs does not result in the segmentation of the sternum (Gladstone & Wakeley, 1932). It seemed obvious that the problem could not be solved by morphological studies alone and an experimental approach was therefore made, the results of which are reported in section B. A. THE CLOSURE OF THE STERNAL BANDS TECHNIQUE To see whether the closure of the sternal bands was due to the elongation of the ribs, the same technique was used as that for the study of the effect of ribs on segmenta- tion which is fully described in section B (p. 136). Briefly, explants were made of embryonic sterna of mice at stage 3 (Chen, 1952a) when the bands had not yet fused. The sternal bands were dissected free of all ribs, cultivated by watch-glass method in a medium composed of equal parts of plasma and embryo extract and compared with control explants with ribs attached. To study the movement of tissues in the ventral body-wall, embryos of slightly younger age (stage 2) were used. The explants were cultivated similarly by watch- glass method. Since the purpose of the experiment was to measure and compare the movement of the structures and of vital marks, subcultivation which usually causes disturbances and contraction of the explants was not practicable. All observations were therefore made on the undisturbed culture during the first 2 or 3 days of growth. Drawings of the explants were made with the aid of a camera lucida at various intervals. Photographs were also taken of some of the cultures. EXPERIMENTS AND RESULTS (1) THE ELONGATION OF THE RIBS As a full account on the effect of the removal of the ribs on the development of the sternum in vitro is given in section B (p. 136), it suffices to say here that in the com- plete absence of the ribs, the two sternal bands still moved medially toward each other; after 24 hr. they were very close together and by the end of the third day had completely fused. As compared with sternal bands with ribs attached, the rate of movement seemed to be slightly faster in the explants without ribs. Conclusion The elongation of the ribs is not essential for the closure of the sternal bands. (2) TISSUE MOVEMENT IN THE VENTRAL BODY-WALL Experiments were designed to find whether the undifferentiated tissue in the ventral body-wall can move towards the mid-line in the absence of all or various amounts of the lateral differentiated tissues. Material and methods The ventral body-wall of 13-day embryos were excised with very little of the differentiated tissues, i.e. pectoral muscles, sternal bands and clavicles, attached to the edges of the explants. In a few explants, rather more of this differentiated tissue was deliberately left. Since it is difficult to remove the skin at this stage without 132 Jui Ming Chen damaging the tissue underneath, the explants were sometimes cultivated with the ectoderm intact. To detect tissue movement, the method of using gas black as reference marks was employed. When the carbon particles were first deposited, a few floated in the medium, but most of them were quickly incorporated in the tissue. Gross tissue movement could thus be readily followed by observing the behaviour of these black marks. To make sure that nutrients from the medium to the tissue were not seriously blocked by the keratinization of the ectoderm, some explants were planted with the skin surface uppermost and others with the skin surface downwards. As will be described below, no essential difference could be detected between the two with regard to the movement of the carbon marks. For controls, the lateral body-wall of the costal region was cultivated, as normally there is no converging median movement in this region. The skin surface was placed uppermost in all seven cultures. In both the experimental and control groups, four patches of sterile gas black were laid on each explant. They were arranged in two pairs one behind the other just within the margin (PI. 1, figs. 1 a, Id). All the explants were fixed in acetic Zenker's solution after 36-48 hr. of cultivation and were sectioned. The sections were stained with haematoxylin and chromotrope. Photographs or camera lucida drawings were made of each culture after the explants had lain on the clot for 2 hr. Owing to the initial contraction of the explants in vitro, these were found to serve more accurately than the fresh tissue as the initial standard for comparison with subsequent drawings. Other drawings were made on the first and second day of cultivation. Results Successive drawings showed that although there was active outgrowth of cells in the explants of the ventral body-wall, the carbon marks moved towards the mid-line (PI. 1, figs. 1 a, 1 b), provided enough dorso-lateral tissue had been included. In the controls, on the other hand, the carbon marks remained stationary or even moved apart (PI. 1, figs. ld, le). (a) Observations on the living cultures. The typical changes observed in thirty-three cultures during the 2 days' growth may be summarized as follows: During the first few hours, the explants in both groups contracted a little as usual. The outgrowth of marginal cells migrating radially into the surrounding medium, began after about 10 hr. and continued throughout the period of cultivation. The outline of the original explant, however, could still be readily detected, since the outgrowing marginal tissue was much thinner and less opaque. Apart from this initial contraction and the migration of the marginal tissues, the two groups of explants behaved quite differently. In the ventral body-wall the carbon marks moved medially so that by the end of the second day of cultivation, the anterior pair of black patches had either fused into one or lay very close together. The degree of their approximation varied a little according to a slight difference in the ages of the embryos from which the explants were taken. Movement was usually faster in the older rudiments and the patches of gas black fused more often. In all Studies on the morphogenesis of the mouse sternum 133 the cultures of this series, however, both pairs of carbon marks moved a considerable distance from the margin of the explant toward the mid-line. This active movement of the tissue in a median direction was further evidenced by the change in size and shape of the explants. In spite of the active migration of marginal cells into the medium which should have caused an increase in area, at the end of the second day in vitro, the explants of ventral body-wall remained almost the same size as they were when first explanted.