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Cellular mechanisms in the morphogenesis of the sea urchin embryo
Experimenfal
570
CELLULAR
CELL
Cell Research
32, 570-589
(1963)
MECHANISMS IN THE MORPHOGENESIS OF THE SEA URCHIN EMBRYO
CONTACTS
WITHIN
THE
MESENCHYME
AND
T. The
ECTODERM ECTODERM
AND
BETWEEN
CELLS
GUSTAFSON
Wenner-Gren Institute for Experimental University of Stockholm, Sweden Keceived
January
Biology,
23, 1963
purpose of the investigations in this series is to define the main cellular activities that are responsible for changes in shape of an embryo and to account for the development of complicated patterns. Changes in adhesion and tension in the cell membrane bring about changes in packing of the cells and hence a deformation of the cell sheet [15, 17, 311. Pseudopodal activity, another main mechanism in morphogenesis, is responsible for the local extension of the cell sheets, e.g. of the archenteron, and for the migration of mesenchyme cells [B, 9, 10, 12, 13, 171. The direction ofthe movements can be accounted for in terms of random exploration and a variation in the stability of their attachments, reflecting a variation in the adhesiveness of the substrate [7, 13, 141. The ectoderm thus serves as a “template” for the mesenthyme pattern in the sense that the mesenchyme cells finally line up along the most adhesive zones [13, 15, 171. The present investigation is focused on the mutual adhesion between ectoderm cells and the way in which pseudopods adhere to the ectoderm (cf. [8]). The detailed questions we want to answer can be listed as follows: (1) What is the basis of the varying cellular adhesion within the different regions of the wall of the gastrula ectoderm (highest in the animal plate, somewhat lower in the ciliated band and a ring around the lower part of the ectoderm, and still lower in other regions [15])? (2) IVhy do the primary mesenchyme pseudopods preferentially attach to the zones of high mutual adhesion between the ectoderm cells, but not to the animal plate, where the mutual adhesion between the ectoderm cells must be particularly high [ 13, 15, 271:’ (3) Why do other pseudopods attach to some other regions, e.g. the archenteron tip pseudopods to a great extent to the dorsal ectoderm and, later on, to the oral zone [lo]? Are we forced to postulate more than one type of
THE
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“stickiness” in the body wall, one specific for the primary mesenchyme pseudopods, another for the archenteron tip pseudopods, and so on? In order to facilitate the reading some of the different regions considered are defined by the schematic drawing of a gastrula (Fig. 1).
Archenteron-tIppseudopods Secondary
mesenchyme
Main ciliated band ventral side Ventro-lateral primary Ventro-lateral primary
cells
around
chain of mesenchym cluster mesenchyme
Ring of primary
of,
mesenchyme
Fig. l.-Schematic drawings to define some of the regions of an advanced gastrula where the ventral side has become flattened as the result of an increase in contact between the cells at its margin (which form the main ciliated band and the animal plate). The increase in cell contact in a zone around the lower region of the ectoderm is also indicated. The primary mesenchyme cells line up along zones with high contact between the ectoderm cells with the exception of the animal plate. The terms “upper corner” and “arm buds” are non-conventional. MATERIAL
AND
METHODS
The establishment of cell contacts has been studied by means of time-lapse cinematography, which not only gives a static picture of the distribution of contacts at a certain time but a dynamic picture of the formation and breaking of contacts and thus of their stability. The basic technique is the same as in our previous morphogenetic studies [9]. Some essential modifications have, however, been necessary. Thus we have used an interference-contrast microscope (Barbier, Benard et Turenne, Paris), in order to obtain better contrast than with the ordinary phase microscope. Most of our observations were made with the 100 x objective. Another essential change in our technique was the mounting of the larvae. The microaquarium technique used previously [9] was not applied, but the larvae were injected below a drop of paraffin oil on the slide. By adjusting the size of the water drop, the oil drop, and the coverslip, a preparation was obtained in which the larvae were compressed and kept in a constant position for several hours. Although the larvae were morphologically disturbed to a certain extent, some of the major features of development could proceed fairly normally, e.g. gastrulation occurred and the mesenchyme cells distributed themselves to form a more or less normal pattern. A disadvantage is that the compressed larvae gradually adjust themselves to the narrow space and begin to Experimental
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move. On the other hand, in spite of the long diffusion way for 0, and CO,, the gaseous exchange was generally good enough to permit continued development up to the pluteus stage. In short-term experiments the beat of the cilia were used as an indicator for an appropriate gaseous exchange. The compressed state of the body increased the chance of getting the pseudopods in the plane of focus. In addition, since the larvae were compressed the ectoderm was extended and its cells partly separated from each other in many regions and only kept together by their attachment to the hyaline membrane and by bridges between the cells. This increased the possibility of investigating the ability of the pseudopods to attach to different regions of the cells. The intervals between the exposures were generally 1 and 3 seconds. The film used was Kodak Plus X, a reversal 16 mm film. Since the making of a picture involves the making of a negative from the reversal film and an enlarged positive, followed by the conventional printing procedure, all stages involving loss of contrast and resolution, the figures in this paper are all drawings. The species used in this investigation was Psammechinus miliaris, both the shallow and the deep water form, both from the west coast of Sweden. Our observations are based upon about 1000 m of film.
RESULTS
cells.--When a gastrula becomes flattened by compression, the ectoderm sheet becomes extended and its cells tend to separate, but remain interconnected at certain points of their surfaces. When seen in surface view, the cells are therefore joined up by bridges into a net-like structure (cf. [28]) (Fig. 2). The cells are also indirectly interconnected, since they all attach to a “basal membrane”, the hyaline membrane, covering the outside of the larva. A thin inner layer also seems to occur (see the following text). The affinity for the hyaline membrane is evidently high, and the cells tend to flatten to cover it. The bridges and the contacts to the hyaline membrane may break to a varying extent. Pseudopod-like processes may, however, persist and join up to form new bridges [15]. If a cell breaks all its contacts, however, it tends to round up and begins to migrate in the spaces between still-attached cells. This is frequently observed when the ectoderm in the gastrula is greatly extended. A rounding up also occurs during mitosis and the daughter cells push apart, but soon normal contacts are re-established [151. When seen in optical cross-section, the cells in the extended sheet have the shape of hour-glasses standing on the hyaline membrane and tapering off onto it, touching each other at their broad inner and outer ends (Figs. 3, c-h and j-l; 4, b and c; 5; 6, d, f; and g). For convenience we may refer to these contacts (bridges) as “inner” (toward the blastocoel) and “outer” (toward the hyaline membrane). Some additional bridges occasionally occur The
Experimental
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at intermediate levels (Fig. 4, c). Pseudopods of the primary and secondary mesenchyme may easily enter the meshwork between the inner bridges and explore the spaces between the cells (Fig. 3, j). The meshes are, however, small enough to prevent detached ectoderm cells from passing through and entering the body cavity.
Fig. 2.-Surface view of an ectoderm where the cells have been partly separated from each other by extension of the cell sheet, showing bridges between the cells. The contact between the cells gradually increases, apparently due to the contraction of the bridges, but also due to an increased attachment of the cells to the hyaline membrane.
The description above refers to the main ciliated band or the ring-like thickening around the larva, where the cell contacts are of an intermediate strength [15]. In a slightly compressed larva oriented with the ventral side upward, regions of different contact relations can be seen, ranging from broad contacts between adjacent cells to minute contacts (Fig. 3). To a great extent this reflects the contact relations in the normal larva. The broadest contacts occur within the animal plate (Figs. 3, a; 6, a-c, and e). Even in a strongly compressed larva the cells in most of the plate are in contact with each other along the whole of their length and no bridges can be observed. The strength of the contacts within the plate decreases towards its periphery, as revealed by the decrease in its thickness and sometimes a tendency to bridge formation (Figs. 3, a; 6, a and c). The plate gradually tapers down and continues as the main ciliated band around the ventral side, where both inner and outer bridges regularly occur. This picture of the transition between the animal plate and main ciliated band is somewhat simplified, however. Where the plate merges with the band (at the “upper corner”, see Fig. l), clefts between the cells are common in normal larvae, and in compressed larvae the plate (Figs. 3, a; 6, b and e) and the band (Fig. 6, d) are abruptly delineated from the “upper corner”, Experimental
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T. Gustafson where the cells have no inner bridges but round up onto the hyaline membrane. Further downwards along the main ciliated band, at the level of the ventrolateral cluster of primary mesenchyme, the inner contacts appear to be particularly stable and do not break, even if the larva is strongly compressed (Fig. 3, e). The cells also seem to join up at their inner ends to form a continuous sheet rather than to be joined into a network. In any case, pseudopods apparently cannot enter the spaces between the cells, as they may do in other regions. Since the cells do not break their inner contacts and retract, the cells in strongly compressed larvae become greatly extended (Fig. 3, e). Geometric conditions may contribute to the marked elongation of the cells here, when the larvae become compressed. In some regions of the ectoderm the contacts between the cells are rather weak, at least in a slightly compressed larva, i.e. mainly restricted to outer contact points (Figs. 3, i; 6, h). The contacts within the mesendoderm will be considered in a later paper. During the formation of the thickened animal plate and the main ciliated band, the increased packing of the cells within the zones concerned (see Fig. 1) are sometimes accompanied by transient ruptures between the cells in the adjacent zones [15]. The spaces between the cells close up after a varying period of time, as the cells tend to increase their contacts with the hyaline membrane. The spaces are more common in the deep form of Psammerhinus miliaris than in the shallow form, where the increases and decreases in cellular contacts in adjacent zones appear to compensate each other more smoothly. But even if large spaces do not appear, there is a tendency to separation of the cells, when cell packing increases in adjacent zones. Some observations were made of the ectoderm contacts in larvae treated xvith Ca-Mg-free sea water, which partly dissolves the hyaline membrane and also breaks the direct contact between the cells. A mesenchyme blastula returned into normal sea water underwent a strong shrinkage, and the dissociated cells were pressed out through a rupture in the lveakened hpaline membrane. The cells piled up, but soon spread out and formed a sheet of densely packed hexogonal cells on the slide. After varying periods of time some of the cells began to round up and showed a rotating movement shortly before death. The cells concerned are probably cells that also normally form a cell sheet. Mesenchyme cells were also seen, but made only transient contacts with the slide and moved around. Contacts between primary mesenchyme and ectoderm.-The activity of the pseudopods (filopods) of the primary mesenchyme described in a previous Experimental
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k Fig. 3.--Cell contacts in different regions of the ectoderm in a somewhat compressed gastrula. a, part of the animal plate (to the left) with secondary mesenchyme below and part of the “upper corner” (to the right); b, “upper corner”; c, upper part of the main ciliated band ending abruptly at the “upper corner”; d, intermediate part of the main ciliated band; e, part of the main ciliated band at the level of the clusters of primary mesenchyme cells (note the great extension of the cells and their close attachments at the inner ends); f and g, transition between the region in e and the dorsal ectoderm; h, dorsal ectoderm at the level of the ring of primary mesencbyme; i, about same level and region as in h, but the inner contacts have broken (note the attachment of two mesenchyme cells); j, k, and I, the ectoderm somewhat above the region in e; j, shows how a filopod of the primary mesenchyme explores the space between two cells; k, from a strongly compressed ectoderm and 1 from the same larva after the release of compression when the cells have been allowed to shorten; the cells in k and I appear to be connected all the time at their inner edge, suggesting the presence of some thin fibrous or membraneous elements to which the cells adhere. Experimental
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paper can be more easily and better studied by means of the interference contrast microscope than with an ordinary microscope. The pseudopods shoot out from the cell bodies as apparently rigid, thin rods, which often begin to bend at their basis or at their tips, an activity which increases their ability to explore the body wall. Pseudopods which do not attach to the body wall
Fig. 4.-Attachment of primary mesenchyme mesenchyme cluster in non-compressed larva; compressed larva; c, ventro-lateral mesenchyme between the two main levels of contact).
pseudopods to the ectoderm. a, ventro-lateral bi ventro-lateral mesenchyme cable in a slightly cluster (note bridges between ectoderm cells
sooner or later collapse, as described earlier [13]. In compressed larvae it is easy to see how the tips of the pseudopods explore the inner surface of the ectoderm cells and even pass between the inner bridges and explore the space between the cells (Fig. 4, a) and the lateral cell surfaces. When a pseudopod tip comes in contact with a junction between cells, the mid-point of a bridge, it attaches to this region (Figs. 3, j; 4). In a few cases a pseudopod was seen to attach to contact points, which sometimes occur between the two main levels. In the region of the dorsal segment of the primary mesenthyme ring, where the inner bridges may be lacking in compressed gastrulae, the pseudopods were occasionally seen to attach to the cell junction close to the hyaline membrane (Fig. 3, i), which indicates that the outer contact points are not completely devoid of adhesiveness for pseudopods. On the other hand, the pseudopods have never been seen to attach to regions of the cell surface that are not directly involved in the mutual contact between the ectoderm cells, i.e. the main inner surface, or to the surfaces bordering the spaces between the cells. The contacts between the mesenchymal pseudopods and the ectoderm appear to be more labile than the contacts between the ectoderm cells. The Experimental
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Morphogenesis pseudopods i.e. there which is cable, but cells form
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thus often detach, whereas new contacts are steadily formed, is a “turnover” of the pseudopod-ectoderm contacts (cf. [7, 13]), particularly intense at the terminal branches of the ventro-lateral much slower in the ventro-lateral region, where the mesenchyme clusters.
Fig. 5.-Attachments of archenteron tip pseudopods to the ectoderm. a, simple attachment and slight “attachment cone” formation; b, two pseudopods attaching to the same site -one of the pseudopods has an additional attachment at the junction between two ectoderm cells somewhat below the plane of focus; c, and e, double and triple attachment of branched pseudopods; d, attached pseudopod continuing its exploration with a branch; f, attachment. of thin pseudopod and the formation of a “cone of attachment”.
An interesting observation is that once a pseudopod attaches, its active elongation stops, so that it remains straight. This phenomenon might be related to contact inhibition described in other systems, e.g. [l]. The observations described above agree well with observations in noncompressed larvae. It is here quite easy to see that the pseudopods only attach at the border between cells (Fig. 4, a). In one case a pseudopod was seen to penetrate deeply between two cells and apparently made a transient contact lower down between the ectoderm cells, probably at a contact point between them. The terminal branches of the ventro-lateral primary mesenchyme cable sometimes explore the animal plate and may make some contacts, but evi38
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dently not firm ones. However, when they occur, it is always at the border between the cells. Contacts of the archenteron tip pseudopods and the secondary mesenchyme cells with the ectoderm.--‘l’he interference contrast microscope also gives an excellent opportunity of studying the activity at the archenteron tip. Pseudopods shoot out from the tip where the pulsatory activity-the “lobopodal” activity-is strongest (cf. [22]). They form as apparently rigid rods, sometimes from the pulsatory lobes themselves, although it is difficult at present to make any generalisations. The activity of the pseudopods bears close resemblance to those of the primary mesenchyme. However, they do not fuse with each other to form a syncytium, and in later stages of gastrulation many of them attain a considerable thickness and gradually taper off into the cell body. Their tips frequently branch, and may form a net-like structure, and the individual branches explore, attach, and detach independently. Also the archenteron tip pseudopods attach only to the contact points between the ectoderm cells (Fig. 5). “Attachment cones” at the point of contact are quite frequent, reflecting their strong pull (Fig. 5, a and c). The cones are generally made up of the bridges between two or more adjacent cells that are pulled inwards. \Vhen a pseudopod has several terminal branches which attach independently, the cone may be rather complex (Fig. 5, b). The attachments are transient, but the break of a contact need not mean the retraction of the pseudopod, since branches of the same filopodal tip may attach separately. In addition, the pseudopods are sometimes able to continue to explore and find another point of attachment before they retract. The course of the invagination of the archenteron tip has been described earlier [9, 10, 221. It may be recalled that the archenteron tip pseudopods attach mainly to the dorsal and lateral ectoderm sheet. It is quite striking that the attachments to the animal plate are scarce or at least very unstable, particularly in its central region, although quite frequent at the edges of the plate bordering the “upper corner” (cf. Fig. 1, Fig. 6, b and e). Furthermore, at this time the cells, lvhich may anchor to the plate, begin to detach from the tip as free secondary mesenchyme cells, and do not contribute to a further invagination toward the plate. Many of the pseudopods that still occur at the tip orient themselves ventro-laterally, as well as towards the oral region, and bend the archenteron forwards, so that the oral contact is established [lo]. The secondary mesenchyme cells that leave the archenteron tip also attach at the cell junction within the bridges (Fig. 6, f and g), and are sometimes seen to enter between the inner bridges and explore the spaces between the Experimental
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(Fig. 6, b). “Cones of attachment” cells, particularly in the “upper corner” do not generally form, as the tension in the pseudopods is only moderate, but may occur if pseudopods from the same cell pull in opposite directions. The contacts are more or less transient. The pseudopods of the free secondary mesenchyme cells show the same low affinity for the animal plate as other pseudopods, and the same holds for the coelom pseudopods [16]. When attachments to the plate can be seen, holvever, it is always at the junction between its cells (Fig. 6, c). Better contacts are formed with the sharp lateral edges of the plate (Fig. 6, h), and with the upper end of the ciliated band (Fig. 6, d) that delineate the “upper corner” in compressed larvae.
Fig. K-Attachments of secondary mesenchyme pseudopods to the ectoderm. a, secondary mesenchyme cells close to the animal plate, making little contact with the animal plate but with attachments at “upper corner” (outside the figure) towards which the cells move; b, edge of the animal plate and the “upper corner” region, showing no contact with the central region of the plate, but contacts with its edge and with a cell at the “upper corner”, and strong exploration of the clefts between the cells; c, giant secondary mesenchyme cell suspended between left and right “upper corner” and making only few contacts with the plate; d, attachment of a mesenchyme cell at the upper abrupt end of the ciliated band close to the “upper corner”; e, attachment of archenteron tip pseudopod (from cell that has almost detached from the tip) to the edge of the animal plate in a compressed larva; f and g, attachments of secondary mesenchyme to the ectoderm; h, secondary mesenchyme pseudopod exploring the surface of an ectoderm cell and attaching with one of its pseudopodal branches at the border between two cells. Experimental
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Many of the secondary mesenchyme cells will therefore move laterally, and may thereby slide along the animal plate without being trapped by pseudopodal connections to it (Fig. 6, a), and their first destination is the edges that delineate the “upper corner”. The large secondary mesenchyme cells commonly extend parallel to the plate between their attachments to the right and left and with only few and weak contacts with the plate (Fig. 6, c). Thus, during their early dispersion, many secondary mesenchyme cells have the same destination as the ventro-lateral primary mesenchyme cables. The pseudopods of the two types of mesenchyme evidently attach to the same regions, and cases have been observed where pseudopods of each kind attach to the same point. On the other hand, there is no adhesion between the pseudopods of the two types of mesenchyme. Secondary mesenchyme cells may thus migrate through the primary mesenchyme-pseudopod meshwork without sticking to it. During their further migration the secondary mesenchyme cells appear to maintain their tendency to attach to the same regions as the pri-
Fig. 7.-a, segment of a radial larva seen from the anal side showing a net of secondary mesenchyme (in black) in the same plane as the primary mesenchyme cells and the ciliated band around the lower part of the ectoderm. b, dorsal view of a larva (prisma stage) to show the distribution of the secondary and the primary mesenthyme. Experimental
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mary mesenchyme cells, hut many cells migrate along the dorsal ectoderm and the archenteron. The final pattern of secondary mesenchyme is not as easily characterized as that of the primary mesenchyme, and it is not easy to find similarities in the two patterns, although the pseudopods of both types of mesenchyme may attach to the same types of contact points. It is important for the following discussion that in a radial larva, where the primary mesenchyme cells are lined up to form a ring parallel with the main ciliary band, a large number of secondary mesenchyme cells formed a network in the same plane (Fig. 7). Also in normal larvae there is a tendency for the secondary mesenchyme to line up along the ciliated band.
DISCUSSION
The problem dealt with in this paper is how contact is established between different cell types in the developing sea urchin embryo and to what extent the variation in thickness and curvature of the ectoderm and the distribution of various mesenchymal cell types can be accounted for in a simple way. The specific questions involved have already been listed in the introduction. Our observations indicate that the cells of the gastrula ectoderm in general are mutually connected only at certain points. The points of high adhesion are concentrated at two main levels, one along the inner border of the cells, another close to the hyaline membrane. For convenience we have referred to the contacts at the two levels as inner contact points and outer contact points. In regions with low contact between the cells one may say that the contact is restricted to outer contact points, whereas in the animal plate the cells are connected along the whole of their length. The pseudopods of various mesenchymal elements adhere only at the points of contact between the ectoderm cells, generally to the inner but also to the outer, provided the pseudopods can reach them. In the animal plate, if pseudopods adhere at all, the attachments are made only at the inner end of the junction between the cells. Pseudopods have never been seen to adhere to the free cell surfaces not involved in mutual ectoderm cell contact, neither the inner one nor the lateral ones, although pseudopods can explore these surfaces. The nature of the cell contacts in the early sea urchin development have been investigated by means of electron microscopy by Balnisky [a], Endo (see Dan [S]), Wolpert and Mercer [32], and Larje [23]. Apart from general contact and some interdigitating processes, clusters of intercellular connecting Experimental
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T. Gusfafson bars reminiscent of the desmosomal organization in vertebrates have been observed at the outer (distal) end of the cells, close to the hyaline membrane. In a large number of cases there is a mitochondrium close to the connecting bars [23]. The desmosomes begin to appear at about 54 hr after fertilization [32]. The proximal end of the cells has no ultrastructural organisation of desmosomal or any other specialized type. Preliminary investigations in our laboratory of the gastrula !23] also indicates a lack of such an organization at the inner points of contact at this stage. The long contacts between the cells of the animal plate also appear to lack such an organization [23]. The attached pseudopodal tips also seem to lack any defined ultrastructural patterns at their tips. The electron micrographs also reveal the relation between the cells and extracellular layers. The cells are attached to the hyaline membrane by numerous microvilli (see [5]). From about 84 hr after fertilization the inner surfaces, bordering the blastocoel are covered by a layer, the “fuzzy layer”, which has a thickness of about 0.1 p and contains particles and fibres [32]. There is no evidence of any similar substance tilling the spaces between the cells [32]. On the basis of Immers’ [21] histochemical and radioautographic studies Mercer and Wolpert [32j conclude that the “fuzzy layer” contains a mucopolysaccharide-protein complex secreted by the cells. The spatial distribution of the “fuzzy layer” and its occurrence in later stages still remains to be elucidated. Okazaki, Fukushi and Dan [2i] emphasize the work of Motomura [25], who demonstrated the presence of mucopolysaccharides secreted by the cells in the regions where the ectoderm cells pack closely and the primary mesenchyme cells collect. Our observations also indicate that the inner ends of the cells in these regions tit closely together and are not only connected by bridges, i.e. as if they were spreading on some membraneous secretion. The observations in Fig. 3, k and 1 on a strongly flattened gastrula also suggest that the inner ends of the cells, at least in some regions, are connected by some material, although the direct contact between the cells are partly broken. Taken together, these observations suggest that the “fuzzy layer” occurs also after the blastula stage, at least in zones \vith relatively strong cell contact. The contact relations discussed may be summarized as in Pig. 8. On the basis of the electron-microscopical investigations we may conclude that the outer points of contacts correspond to the regions with a desmosome-like organization which, however [32], may extend all around the outer border of the cells as a more or less continuous ring-like zone. Other surfaces involved in strong contact are devoid of a desmosome-like organization. Experimental
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One may ask to what extent the cell surface as a whole has a certain adhesiveness or if there is an intercellular cement covering the cells all around. Balinsky [2] assumes that the narrow spaces between the cells in the blastula wall are filled with a cementing substance (cf. [24]). In the cleaving blastula there is, however, no strong general attachment of the cells to each other,
Fig. S.-Diagram summarizing the contact relations within the ectoderm and between primary mesenchyme and ce,ls. ectoderm in regions of moderate and low contact between the ectoderm
since they round up and push apart at each cleavage, nor in the mesenchyme blastula of Echinocardium, where pigment cells easily migrate between the cells within the wall [18]. Also in Psammechinus there is no marked “general” adhesiveness, since the spaces between the cells occur in many sites even in the unextended sheet, and the spaces do not appear to be filled with any cementing substance, since pseudopods can freely explore these spaces. Furthermore, detached ectoderm cells can move around in the clefts between the attached cells without sticking to them, and this is not compatible with a high general adhesiveness, nor with the presence of a cementing substance. The long contacts within the animal plate, however, indicate a high general adhesion or possibly a cementing substance. Since there is, in general, no evidence for any general, high adhesion between the cells, with the exception of the animal plate, one may suggest that the hyaline membrane and the “fuzzy layer” play some part in the establishment and distribution of the contact points. Wolpert and Mercer [32] have in fact discussed the possible part played by the hyaline membrane in the development of desmosomes at the sites here denoted as the outer contact points. It seems attractive to suppose that a “fuzzy layer” plays a similar part in the establishment of the contacts at the inner points. If the cells have high adhesion for these two layers (hyaline and fuzzy), formed as secretions from the cells, they would tend to spread on these layers and therefore come in close contact with each other at their inner and outer ends. Where the “fuzzy layer” is lacking, there would be only outer contact points. \&‘hen dissociated cells of a mesenchyme blastula form a thin sheet on the slide, Experimental
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T. Gustafson this may reflect their attachment and tendency to spread on the glass surface. That the cells in the young blastula also form a single layer certainly reflects their strong attachments to the hyaline layer [31]. The close mutual contact at the inner and outer ends of the cells may result in a direct sticking together of the cells at these points, even if the mutual adhesion is rather low. The sticking together may be favoured by the fact that the adhesion of the cells to the hyaline and the fuzzy layer increases the length of time when the cells are close together; the movements of the other surfaces may prevent a contact between the cells of sufficient duration to allow a sticking together. The adhesion at the inner and outer ends may also be influenced by the strong curvature of the cell surface (cf. [29]), that follows from the attachment of the cells to the layers. Local differences in tension of the cell membrane may also be important (cf. [J]). Although the reason for the adhesiveness in the points concerned remains unknown, the fact that the cells in the animal plate have broad mutual contacts, those in the ciliated band both inner and outer point contacts and those in the thin regions only outer point contacts might be discussed in terms of differences in general adhesiveness of the cell surface and the distribution and properties of the fuzzy layer. Why do the pseudopods attach only at the junction between cells and not at any point of the cell surface, and what determines the differences in adhesiveness for pseudopods between different regions of the ectoderm? At present we may only, as a basis for further investigations, hint at some factors that may be involved. The tension in the cell sheet of the body wall may tend to break up the inner point contacts and the fuzzy layer to some extent and part of the former contact surface would therefore become available for the pseudopodal tips. Such a local uncovering of adhesive surfaces would not occur within the animal plate, where the cells adhere to each other along their whole length, and this could explain why pseudopods attach so weakly to the plate. In fact, where ruptures do occur within the plate and clefts are formed, mesenchyme cells regularly attach there. Hut why do their pseudopods attach only at the sharp edges or corners of the cells and not to their lateral and inner borders? In addition, why do pseudopods never attach to the rounded cells at the corners that have lost their inner point or long contact with adjacent cells and ought to have their adhesive surfaces exposed? It seems after all that adhesiveness for pseudopods is not only a question of a static molecular configuration of the ectoderm cell surface but also a function of movement, extension, and curvature of the cell surface, concepts discussed by Curtis [3, 41 and by I’ethica [29]. Experimental
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Another problem that is difficult to answer is why mesenchyme cells only attach with the tips of their pseudopods. Does the “stickiness” of the tip reflect, e.g. the motility, extension or curvature of its surface or “biochemical” peculiarities? Still another problem is why the tips of the pseudopods do not attach to each other in spite of their ability to attach to the ectoderm. This is illustrated by the secondary mesenchyme cells, which frequently move through a web of primary mesenchyme pseudopods. The answer may be that it takes some time for a contact to become established, and that two moving tips have little chance to meet for long enough. Later on, when pseudopods attach to points close enough, the probability of fusion would increase, and this may be part of the explanation of the formation of syncytia as development proceeds. The morphogenetic consequences of the contact relationships are easier to discuss than the basis for the establishment of the different types of cell contacts. The occurrence of inner contact points or broad contacts may determine the extent of packing of the cells and hence the curvature of the body wall (cf. [lS]). In fact, the distribution of these contacts agrees well with the changes in curvature of the ectoderm, the formation of the main ciliated band, “arm buds” and the animal plate (cf. [ 151). The consequences of a decrease in cell contact in the presumptive primary mesenchyme and in the archenteron rudiment during early invagination and the release of the secondary mesenchyme are also quite clear [ 15, 1 T]. The “upper corner” is a region at the border between a region of cells with broad contacts and with inner point contacts. If their mutual adhesion is not high enough and the “fuzzy layer” not strong enough, one may visualize that the cells easily separate and round up as a result of the deformation of the flattened larva. (Geometrical conditions may also make the deformation particularly strong there.) Another consequence of the distribution of inner contact points of ectoderm cells is that the primary mesenchyme cells will distribute themselves along the zones where the mutual contact between the ectodermal cells is relatively high, i.e. where the ectoderm is thickened and its curvature is increased (cf. [15, 26]). The mesenchyme pattern corresponds to that of the main ciliated band and the ring-like thickened zone around the young gastrula. In other words, the so-called graded stickiness of the ectoderm that determines the distribution of the pseudopodal contacts [ 131 reflects the distribution of inner points of contact within the ectoderm. More correctly, it is rather number of contact points per unit volume than per unit surface that determines the mesenchyme pattern. The cells therefore tend to collect at sharp curves or corners of the ectoderm. Such regions of strong curvature, Experimental
Cell Reseurch
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T. Gustafson
586
however, coincide with the zones of strong contact, the curvature being a consequence of increased contact between the cells (cf. [151). Since pseudopods from different types of cells all appear to be able to attach to the points of contact between the ectoderm cells, one may ask why the statistical pathways and destinations of different kinds of cells and cell groups are not identical and do not correspond in detail to the thickened zones of the ectoderm. Is it after all necessary to postulate that the specificity of the point of contacts varies \vithin the sea urchin embryo, or is it possible to account for the differences in terms of a single but graded type of adhesiveness that may vary with time? The second alternative, which is closely related to the concept of Curtis [-I-], may be worth preliminary consideration with regard to our system. If the probability of the attachment of a pseudopod not only reflects the number of preformed contact points present but also their availability, it is easier to understand why the archenteron tip pseudopods attach so \vell to the dorsal side of the larva, whereas the primary mesenchyme forms the typical ring with the two ventro-lateral clusters, which extend upwards along the ciliated band. The primary mesenchyme cells, which move along the wall, faithfully redistribute themselves when the number of contact points available increases in certain zones (cf. [13,11,30]), and therefore the rentro-lateral chains extend when the main ciliated band appears. The pseudopods may not only respond to the appearance of new contact points, but also to pre-existent ones that become more available than before, due to partial separation of the ectoderm cells. The transient ruptures that tend to occur close to the ciliated band and behind the animal plate in connection with changes in cell packing and changes in shape of the larva ‘151 indicate that the inner contact points are partly uncovered there; the extension of the cell sheet may also change the adhesiveness for other reasons. These contact surfaces may be used as points of attachments for various pseudopods. Those along the ciliated band may easily be used by the primary mesenchyme cells that already attach along the band. The dorsal branch of the main ventral mesenchyme chain that occurs in some species [3O] indicates that also contact points behind the animal plate become available for the primary mesenchyme pseudopods. Rut since the tip of the archenteron rudiment is close to the same region, due to the obliqueness of the ventrally flattening larva [lo], it would mainly be the archenteron tip pseudopods that attach to the adhesive points behind the plate, and so the invagination tends to proceed along the dorsal side. Later on, when most of the mesenchyme cells involved leave the archenteron tip, which is no\v not far from the ventral side, its pseudopods will have a Experimental
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greater chance than before to attach to the ventro-lateral region and particularly to the oral ectoderm that for unknown reasons is very adhesive and bend the archeteron towards the oral region (cf. [lo, 161). An experimental way to test this concept is to extend the ectoderm to see if the pattern of pseudopodal attachments alters. In fact it does to a certain extent (cf. Fig. 6 e). The concept may be wrong, but still it illustrates how a complex pattern could arise, although the basic principles arc few. The secondary mesenchyme pattern is not so distinct, but appears irregular and does not clearly correspond to that of the primary mesenchyme, but even this may be explicable without assuming specific differences in adhesion between the different cells. The observed attachment of primary and secondary mesenchyme pseudopods to the same cell, or even to the same contact point, favours this idea. A similarity between the pattern of the two mesenchymal classes may in fact appear if only the points of attachment of the secondary mesenchyme cells are considered, and not the position of the cell bodies. The irregularity of the pattern of secondary mesenthyme cells may follow from the ability of these multipolar cells to attach with their pseudopods simultaneously to widely distant points between which the cell body will be suspended. The primary mesenchyme cells, on the other hand, closely attach to a syncytial cable formed by fused Iilopods with innumerable pseudopodal branches and therefore with a high probability of lining up in a more regular way [13]. It is too early to try to account for the distribution of other mesenchymal elements in the terms used above. The regular appearance of pigment cells at the site of future arm formation is particularly provocative, but this problem will be considered in a later paper. Our observations indicate that the general pattern of points of contact within the ectoderm also plays a part in the development of the coelom. The pseudopods that pull out the coelom thus never make stable contacts with the animal plate, the coelom being extended laterally. However, the coelom pseudopods and the secondary mesenchyme are also capable of attaching to the endoderm. The nature of these contacts will be considered in a later paper. The observation that different cell types can join up at the same contact points, which hence are rather unspecific, appears to agree with the hypothesis of Curtis [3, -I]. A more detailed consideration of his ideas on an adhesiveness that changes with time may be fruitful when dealing with the problems of pattern dealt with above. The molecular basis of the cell contacts has hardly been dealt with in this study. It is, however, worth mentioning that the pseudopods of the meExperimental
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32
T. Gusfafson senchyme cells may attach to glass, and that the pseudopods of an exocoelom may also do so, but even better to a nylon surface [16]. The contact relations in a larvae can furthermore be changed by treatment of early stages with anionic detergents, i.e. the contact pattern of the ectoderm and hence of the mesenchyme becomes altered [ll, cf. 201, the first ring of early high adhesiveness [15] tends to predominate, as it seems, at the expense of the ventral ciliated band. That the sensitivity towards the detergent abruptly drops before the contacts involved appear may be accounted for by a drop in permeability of the hyaline membrane (cf. [5]). Another observation of interest in this connection is that treatment with glucose and sucrose in fairly high concentrations favours the later packing of cells, so that the plate and the ciliated band increase at the expense of the thinner regions of the between different regions for cells reectoderm [ 191. (This “competition” minds us of that which may be supposed to occur in radialized larvae.)
SUMMARY
The contacts between various pseudopodal elements and the ectoderm in the sea urchin gastrula have been investigated by means of time-lapse cinematography and interference contrast microscopy on slightly compressed larvae, where the ectoderm is more or less extended and the cells tend to separate to a varying extent. In zones of the ectoderm where cell contact is moderate, the cells are joined up by points of attachment which are restricted to the margin of the inner and outer end of the cells. Where the contact is low, only the outer ends are involved in these mutual attachments, whereas in the animal plate, where the contact is strongest, the cells are attached to each other along the whole of their length. An increased packing of the cells can thus be correlated with the occurrence of inner contact points or broad contacts. The pseudopods of the primary mesenchyme, the archenteron tip, and the free secondary mesenchyme cells only attach at the points where the ectoderm cells attach to each other, and particularly to the inner points of attachment. The distribution of the inner points of attachment explains the correlation between the location of the primary mesenchyme cells and the thickened zones within the ectoderm. The inability of pseudopods to make strong contacts with the animal plate and the tendency of the archenteron tip pseudopods to attach to the dorsal side during gastrulation are tentatively explained in terms of availability of free contact surface within the ectoderm, but other factors may also be involved. The basis of the apparent differences between the pattern of primary and Experimental
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secondary mesenchyme is discussed. The inner and the outer contact points appear to be structurally different. The nature and origin of the cell contacts within the ectoderm is discussed, and it is suggested that the mutual adhesion between the cells is rather low, but that the cells tend to spread at the hyaline membrane and an inner layer which promotes a direct adhesion between the cells. The broad contact within the animal plate, however, indicates a high mutual adhesiveness of the cell surface as a whole. The results suggest that many of the complex morphological features of the young sea urchin embryo have a simple background. Part of this work was carried out at Kristinebergs Zoologiska Station, Fiskebgckskil. The author wishes to express his most cordial thanks to the Station authorities for unfailing generosity. The work has been financially supported by the Swedish Scientific Research Council.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
M., Exptl CeZZ Res. Suppl. 8, 188 (1961). B. I., ibid. 16, 429 (1959). S. G., Am. Naturalist 94, 37 (1960). Biol. Reu. >7, 82 (1962). DAN, K., Intern. Rev. Cytol. 9, 321 (1960). DAN, K. and OKAZAKI, K., BioZ. Bull. 110, 29 (1956). GUSTAFSON. T. in Proc. 1st IUBlIUBS Svmn., Biolonical Structure and Function p. 49j, Acad. Press, 1961. ” _ 8. -ZooZ. Bidrag Uppsala In press. 9. GUSTAFSON, T. and KINNANDER, H., ExptZ Cell Res. 11, 36 (1956). 10. _ ibid. 21, 361 (1960). 11. GUSTAFSON, T. and S~VHAGEN, R., Arkiv Zool. 42 A, no 10 (1950). 12. GUSTAFSON, T. and WOLPERT, L., ExptZ CeZZ Res. 22, 437 (1961). 13. -ibid 24, 64 (1961). 14. -ibid. 25, 311 (1961). 15. _ ibid. 27, 260 (1962). 16. -ibid. In press. 17. -Intern. Rev. Cytol. In press. 18. --In preparation. 19. HGRSTADIUS, S., J. ExptZ ZooZ. 142, 141 (1959). 20. H~RSTADIUS, S. and GUSTAFSON, T., J. Embryol. Exptl MorphoZ. 2, 216 (1954). 21. IMMERS, J., Expfl Cell Res. 24, 356 (1961). 22. KINNANDER, H. and GUSTAFSON, T., ExptZ Cell Res. 19, 278 (1960). 23. LARJE, R., In preparation. 24. MOSCO.NA,‘A. A. in 18th Growth Symp., p. 45. New York, Ronald Press, 1960. 25. MOTOMURA, I., Bull. Mar. Biol. Stat. Asamushi 10, 165 (1960). ~ I 26. OKAZAKI, K., Embryologia 5, 283 (1960). 27. OKAZAKI, K., FUKUSHI, T. and DAN, K., Acta Embryol. Morphol. ExptZ 5, 17 (1962). 28. OVERTON, J., Developmental BioZ. 4, 532 (1962). 29. PETHICA, B. A., ExptZ Cell Res. Suppl. 8, 123 (1961). 30. v. UBISCH, L., Verhandl. Koninkl. Ned. Akad. Wetenschap-Afd. Naturskde. Sect. (1950). 31. WOLPERT, L. and GUSTAFSON, T., ExpfZ Cell Res. 25, 374 (1961). 32. WOLPERT, L. and MERGER, E. H., ibid. In press. ABERCROMBIE, BALINSKY, CURTIS, A.
ExperimentaZ
vol.
Z. 47,
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CELLULAR
CELL
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(1963)
MECHANISMS IN THE MORPHOGENESIS OF THE SEA URCHIN EMBRYO
CONTACTS
WITHIN
THE
MESENCHYME
AND
T. The
ECTODERM ECTODERM
AND
BETWEEN
CELLS
GUSTAFSON
Wenner-Gren Institute for Experimental University of Stockholm, Sweden Keceived
January
Biology,
23, 1963
purpose of the investigations in this series is to define the main cellular activities that are responsible for changes in shape of an embryo and to account for the development of complicated patterns. Changes in adhesion and tension in the cell membrane bring about changes in packing of the cells and hence a deformation of the cell sheet [15, 17, 311. Pseudopodal activity, another main mechanism in morphogenesis, is responsible for the local extension of the cell sheets, e.g. of the archenteron, and for the migration of mesenchyme cells [B, 9, 10, 12, 13, 171. The direction ofthe movements can be accounted for in terms of random exploration and a variation in the stability of their attachments, reflecting a variation in the adhesiveness of the substrate [7, 13, 141. The ectoderm thus serves as a “template” for the mesenthyme pattern in the sense that the mesenchyme cells finally line up along the most adhesive zones [13, 15, 171. The present investigation is focused on the mutual adhesion between ectoderm cells and the way in which pseudopods adhere to the ectoderm (cf. [8]). The detailed questions we want to answer can be listed as follows: (1) What is the basis of the varying cellular adhesion within the different regions of the wall of the gastrula ectoderm (highest in the animal plate, somewhat lower in the ciliated band and a ring around the lower part of the ectoderm, and still lower in other regions [15])? (2) IVhy do the primary mesenchyme pseudopods preferentially attach to the zones of high mutual adhesion between the ectoderm cells, but not to the animal plate, where the mutual adhesion between the ectoderm cells must be particularly high [ 13, 15, 271:’ (3) Why do other pseudopods attach to some other regions, e.g. the archenteron tip pseudopods to a great extent to the dorsal ectoderm and, later on, to the oral zone [lo]? Are we forced to postulate more than one type of
THE
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“stickiness” in the body wall, one specific for the primary mesenchyme pseudopods, another for the archenteron tip pseudopods, and so on? In order to facilitate the reading some of the different regions considered are defined by the schematic drawing of a gastrula (Fig. 1).
Archenteron-tIppseudopods Secondary
mesenchyme
Main ciliated band ventral side Ventro-lateral primary Ventro-lateral primary
cells
around
chain of mesenchym cluster mesenchyme
Ring of primary
of,
mesenchyme
Fig. l.-Schematic drawings to define some of the regions of an advanced gastrula where the ventral side has become flattened as the result of an increase in contact between the cells at its margin (which form the main ciliated band and the animal plate). The increase in cell contact in a zone around the lower region of the ectoderm is also indicated. The primary mesenchyme cells line up along zones with high contact between the ectoderm cells with the exception of the animal plate. The terms “upper corner” and “arm buds” are non-conventional. MATERIAL
AND
METHODS
The establishment of cell contacts has been studied by means of time-lapse cinematography, which not only gives a static picture of the distribution of contacts at a certain time but a dynamic picture of the formation and breaking of contacts and thus of their stability. The basic technique is the same as in our previous morphogenetic studies [9]. Some essential modifications have, however, been necessary. Thus we have used an interference-contrast microscope (Barbier, Benard et Turenne, Paris), in order to obtain better contrast than with the ordinary phase microscope. Most of our observations were made with the 100 x objective. Another essential change in our technique was the mounting of the larvae. The microaquarium technique used previously [9] was not applied, but the larvae were injected below a drop of paraffin oil on the slide. By adjusting the size of the water drop, the oil drop, and the coverslip, a preparation was obtained in which the larvae were compressed and kept in a constant position for several hours. Although the larvae were morphologically disturbed to a certain extent, some of the major features of development could proceed fairly normally, e.g. gastrulation occurred and the mesenchyme cells distributed themselves to form a more or less normal pattern. A disadvantage is that the compressed larvae gradually adjust themselves to the narrow space and begin to Experimental
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move. On the other hand, in spite of the long diffusion way for 0, and CO,, the gaseous exchange was generally good enough to permit continued development up to the pluteus stage. In short-term experiments the beat of the cilia were used as an indicator for an appropriate gaseous exchange. The compressed state of the body increased the chance of getting the pseudopods in the plane of focus. In addition, since the larvae were compressed the ectoderm was extended and its cells partly separated from each other in many regions and only kept together by their attachment to the hyaline membrane and by bridges between the cells. This increased the possibility of investigating the ability of the pseudopods to attach to different regions of the cells. The intervals between the exposures were generally 1 and 3 seconds. The film used was Kodak Plus X, a reversal 16 mm film. Since the making of a picture involves the making of a negative from the reversal film and an enlarged positive, followed by the conventional printing procedure, all stages involving loss of contrast and resolution, the figures in this paper are all drawings. The species used in this investigation was Psammechinus miliaris, both the shallow and the deep water form, both from the west coast of Sweden. Our observations are based upon about 1000 m of film.
RESULTS
cells.--When a gastrula becomes flattened by compression, the ectoderm sheet becomes extended and its cells tend to separate, but remain interconnected at certain points of their surfaces. When seen in surface view, the cells are therefore joined up by bridges into a net-like structure (cf. [28]) (Fig. 2). The cells are also indirectly interconnected, since they all attach to a “basal membrane”, the hyaline membrane, covering the outside of the larva. A thin inner layer also seems to occur (see the following text). The affinity for the hyaline membrane is evidently high, and the cells tend to flatten to cover it. The bridges and the contacts to the hyaline membrane may break to a varying extent. Pseudopod-like processes may, however, persist and join up to form new bridges [15]. If a cell breaks all its contacts, however, it tends to round up and begins to migrate in the spaces between still-attached cells. This is frequently observed when the ectoderm in the gastrula is greatly extended. A rounding up also occurs during mitosis and the daughter cells push apart, but soon normal contacts are re-established [151. When seen in optical cross-section, the cells in the extended sheet have the shape of hour-glasses standing on the hyaline membrane and tapering off onto it, touching each other at their broad inner and outer ends (Figs. 3, c-h and j-l; 4, b and c; 5; 6, d, f; and g). For convenience we may refer to these contacts (bridges) as “inner” (toward the blastocoel) and “outer” (toward the hyaline membrane). Some additional bridges occasionally occur The
Experimental
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between
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at intermediate levels (Fig. 4, c). Pseudopods of the primary and secondary mesenchyme may easily enter the meshwork between the inner bridges and explore the spaces between the cells (Fig. 3, j). The meshes are, however, small enough to prevent detached ectoderm cells from passing through and entering the body cavity.
Fig. 2.-Surface view of an ectoderm where the cells have been partly separated from each other by extension of the cell sheet, showing bridges between the cells. The contact between the cells gradually increases, apparently due to the contraction of the bridges, but also due to an increased attachment of the cells to the hyaline membrane.
The description above refers to the main ciliated band or the ring-like thickening around the larva, where the cell contacts are of an intermediate strength [15]. In a slightly compressed larva oriented with the ventral side upward, regions of different contact relations can be seen, ranging from broad contacts between adjacent cells to minute contacts (Fig. 3). To a great extent this reflects the contact relations in the normal larva. The broadest contacts occur within the animal plate (Figs. 3, a; 6, a-c, and e). Even in a strongly compressed larva the cells in most of the plate are in contact with each other along the whole of their length and no bridges can be observed. The strength of the contacts within the plate decreases towards its periphery, as revealed by the decrease in its thickness and sometimes a tendency to bridge formation (Figs. 3, a; 6, a and c). The plate gradually tapers down and continues as the main ciliated band around the ventral side, where both inner and outer bridges regularly occur. This picture of the transition between the animal plate and main ciliated band is somewhat simplified, however. Where the plate merges with the band (at the “upper corner”, see Fig. l), clefts between the cells are common in normal larvae, and in compressed larvae the plate (Figs. 3, a; 6, b and e) and the band (Fig. 6, d) are abruptly delineated from the “upper corner”, Experimental
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T. Gustafson where the cells have no inner bridges but round up onto the hyaline membrane. Further downwards along the main ciliated band, at the level of the ventrolateral cluster of primary mesenchyme, the inner contacts appear to be particularly stable and do not break, even if the larva is strongly compressed (Fig. 3, e). The cells also seem to join up at their inner ends to form a continuous sheet rather than to be joined into a network. In any case, pseudopods apparently cannot enter the spaces between the cells, as they may do in other regions. Since the cells do not break their inner contacts and retract, the cells in strongly compressed larvae become greatly extended (Fig. 3, e). Geometric conditions may contribute to the marked elongation of the cells here, when the larvae become compressed. In some regions of the ectoderm the contacts between the cells are rather weak, at least in a slightly compressed larva, i.e. mainly restricted to outer contact points (Figs. 3, i; 6, h). The contacts within the mesendoderm will be considered in a later paper. During the formation of the thickened animal plate and the main ciliated band, the increased packing of the cells within the zones concerned (see Fig. 1) are sometimes accompanied by transient ruptures between the cells in the adjacent zones [15]. The spaces between the cells close up after a varying period of time, as the cells tend to increase their contacts with the hyaline membrane. The spaces are more common in the deep form of Psammerhinus miliaris than in the shallow form, where the increases and decreases in cellular contacts in adjacent zones appear to compensate each other more smoothly. But even if large spaces do not appear, there is a tendency to separation of the cells, when cell packing increases in adjacent zones. Some observations were made of the ectoderm contacts in larvae treated xvith Ca-Mg-free sea water, which partly dissolves the hyaline membrane and also breaks the direct contact between the cells. A mesenchyme blastula returned into normal sea water underwent a strong shrinkage, and the dissociated cells were pressed out through a rupture in the lveakened hpaline membrane. The cells piled up, but soon spread out and formed a sheet of densely packed hexogonal cells on the slide. After varying periods of time some of the cells began to round up and showed a rotating movement shortly before death. The cells concerned are probably cells that also normally form a cell sheet. Mesenchyme cells were also seen, but made only transient contacts with the slide and moved around. Contacts between primary mesenchyme and ectoderm.-The activity of the pseudopods (filopods) of the primary mesenchyme described in a previous Experimental
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k Fig. 3.--Cell contacts in different regions of the ectoderm in a somewhat compressed gastrula. a, part of the animal plate (to the left) with secondary mesenchyme below and part of the “upper corner” (to the right); b, “upper corner”; c, upper part of the main ciliated band ending abruptly at the “upper corner”; d, intermediate part of the main ciliated band; e, part of the main ciliated band at the level of the clusters of primary mesenchyme cells (note the great extension of the cells and their close attachments at the inner ends); f and g, transition between the region in e and the dorsal ectoderm; h, dorsal ectoderm at the level of the ring of primary mesencbyme; i, about same level and region as in h, but the inner contacts have broken (note the attachment of two mesenchyme cells); j, k, and I, the ectoderm somewhat above the region in e; j, shows how a filopod of the primary mesenchyme explores the space between two cells; k, from a strongly compressed ectoderm and 1 from the same larva after the release of compression when the cells have been allowed to shorten; the cells in k and I appear to be connected all the time at their inner edge, suggesting the presence of some thin fibrous or membraneous elements to which the cells adhere. Experimental
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paper can be more easily and better studied by means of the interference contrast microscope than with an ordinary microscope. The pseudopods shoot out from the cell bodies as apparently rigid, thin rods, which often begin to bend at their basis or at their tips, an activity which increases their ability to explore the body wall. Pseudopods which do not attach to the body wall
Fig. 4.-Attachment of primary mesenchyme mesenchyme cluster in non-compressed larva; compressed larva; c, ventro-lateral mesenchyme between the two main levels of contact).
pseudopods to the ectoderm. a, ventro-lateral bi ventro-lateral mesenchyme cable in a slightly cluster (note bridges between ectoderm cells
sooner or later collapse, as described earlier [13]. In compressed larvae it is easy to see how the tips of the pseudopods explore the inner surface of the ectoderm cells and even pass between the inner bridges and explore the space between the cells (Fig. 4, a) and the lateral cell surfaces. When a pseudopod tip comes in contact with a junction between cells, the mid-point of a bridge, it attaches to this region (Figs. 3, j; 4). In a few cases a pseudopod was seen to attach to contact points, which sometimes occur between the two main levels. In the region of the dorsal segment of the primary mesenthyme ring, where the inner bridges may be lacking in compressed gastrulae, the pseudopods were occasionally seen to attach to the cell junction close to the hyaline membrane (Fig. 3, i), which indicates that the outer contact points are not completely devoid of adhesiveness for pseudopods. On the other hand, the pseudopods have never been seen to attach to regions of the cell surface that are not directly involved in the mutual contact between the ectoderm cells, i.e. the main inner surface, or to the surfaces bordering the spaces between the cells. The contacts between the mesenchymal pseudopods and the ectoderm appear to be more labile than the contacts between the ectoderm cells. The Experimental
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Morphogenesis pseudopods i.e. there which is cable, but cells form
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embryo
thus often detach, whereas new contacts are steadily formed, is a “turnover” of the pseudopod-ectoderm contacts (cf. [7, 13]), particularly intense at the terminal branches of the ventro-lateral much slower in the ventro-lateral region, where the mesenchyme clusters.
Fig. 5.-Attachments of archenteron tip pseudopods to the ectoderm. a, simple attachment and slight “attachment cone” formation; b, two pseudopods attaching to the same site -one of the pseudopods has an additional attachment at the junction between two ectoderm cells somewhat below the plane of focus; c, and e, double and triple attachment of branched pseudopods; d, attached pseudopod continuing its exploration with a branch; f, attachment. of thin pseudopod and the formation of a “cone of attachment”.
An interesting observation is that once a pseudopod attaches, its active elongation stops, so that it remains straight. This phenomenon might be related to contact inhibition described in other systems, e.g. [l]. The observations described above agree well with observations in noncompressed larvae. It is here quite easy to see that the pseudopods only attach at the border between cells (Fig. 4, a). In one case a pseudopod was seen to penetrate deeply between two cells and apparently made a transient contact lower down between the ectoderm cells, probably at a contact point between them. The terminal branches of the ventro-lateral primary mesenchyme cable sometimes explore the animal plate and may make some contacts, but evi38
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dently not firm ones. However, when they occur, it is always at the border between the cells. Contacts of the archenteron tip pseudopods and the secondary mesenchyme cells with the ectoderm.--‘l’he interference contrast microscope also gives an excellent opportunity of studying the activity at the archenteron tip. Pseudopods shoot out from the tip where the pulsatory activity-the “lobopodal” activity-is strongest (cf. [22]). They form as apparently rigid rods, sometimes from the pulsatory lobes themselves, although it is difficult at present to make any generalisations. The activity of the pseudopods bears close resemblance to those of the primary mesenchyme. However, they do not fuse with each other to form a syncytium, and in later stages of gastrulation many of them attain a considerable thickness and gradually taper off into the cell body. Their tips frequently branch, and may form a net-like structure, and the individual branches explore, attach, and detach independently. Also the archenteron tip pseudopods attach only to the contact points between the ectoderm cells (Fig. 5). “Attachment cones” at the point of contact are quite frequent, reflecting their strong pull (Fig. 5, a and c). The cones are generally made up of the bridges between two or more adjacent cells that are pulled inwards. \Vhen a pseudopod has several terminal branches which attach independently, the cone may be rather complex (Fig. 5, b). The attachments are transient, but the break of a contact need not mean the retraction of the pseudopod, since branches of the same filopodal tip may attach separately. In addition, the pseudopods are sometimes able to continue to explore and find another point of attachment before they retract. The course of the invagination of the archenteron tip has been described earlier [9, 10, 221. It may be recalled that the archenteron tip pseudopods attach mainly to the dorsal and lateral ectoderm sheet. It is quite striking that the attachments to the animal plate are scarce or at least very unstable, particularly in its central region, although quite frequent at the edges of the plate bordering the “upper corner” (cf. Fig. 1, Fig. 6, b and e). Furthermore, at this time the cells, lvhich may anchor to the plate, begin to detach from the tip as free secondary mesenchyme cells, and do not contribute to a further invagination toward the plate. Many of the pseudopods that still occur at the tip orient themselves ventro-laterally, as well as towards the oral region, and bend the archenteron forwards, so that the oral contact is established [lo]. The secondary mesenchyme cells that leave the archenteron tip also attach at the cell junction within the bridges (Fig. 6, f and g), and are sometimes seen to enter between the inner bridges and explore the spaces between the Experimental
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(Fig. 6, b). “Cones of attachment” cells, particularly in the “upper corner” do not generally form, as the tension in the pseudopods is only moderate, but may occur if pseudopods from the same cell pull in opposite directions. The contacts are more or less transient. The pseudopods of the free secondary mesenchyme cells show the same low affinity for the animal plate as other pseudopods, and the same holds for the coelom pseudopods [16]. When attachments to the plate can be seen, holvever, it is always at the junction between its cells (Fig. 6, c). Better contacts are formed with the sharp lateral edges of the plate (Fig. 6, h), and with the upper end of the ciliated band (Fig. 6, d) that delineate the “upper corner” in compressed larvae.
Fig. K-Attachments of secondary mesenchyme pseudopods to the ectoderm. a, secondary mesenchyme cells close to the animal plate, making little contact with the animal plate but with attachments at “upper corner” (outside the figure) towards which the cells move; b, edge of the animal plate and the “upper corner” region, showing no contact with the central region of the plate, but contacts with its edge and with a cell at the “upper corner”, and strong exploration of the clefts between the cells; c, giant secondary mesenchyme cell suspended between left and right “upper corner” and making only few contacts with the plate; d, attachment of a mesenchyme cell at the upper abrupt end of the ciliated band close to the “upper corner”; e, attachment of archenteron tip pseudopod (from cell that has almost detached from the tip) to the edge of the animal plate in a compressed larva; f and g, attachments of secondary mesenchyme to the ectoderm; h, secondary mesenchyme pseudopod exploring the surface of an ectoderm cell and attaching with one of its pseudopodal branches at the border between two cells. Experimental
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Many of the secondary mesenchyme cells will therefore move laterally, and may thereby slide along the animal plate without being trapped by pseudopodal connections to it (Fig. 6, a), and their first destination is the edges that delineate the “upper corner”. The large secondary mesenchyme cells commonly extend parallel to the plate between their attachments to the right and left and with only few and weak contacts with the plate (Fig. 6, c). Thus, during their early dispersion, many secondary mesenchyme cells have the same destination as the ventro-lateral primary mesenchyme cables. The pseudopods of the two types of mesenchyme evidently attach to the same regions, and cases have been observed where pseudopods of each kind attach to the same point. On the other hand, there is no adhesion between the pseudopods of the two types of mesenchyme. Secondary mesenchyme cells may thus migrate through the primary mesenchyme-pseudopod meshwork without sticking to it. During their further migration the secondary mesenchyme cells appear to maintain their tendency to attach to the same regions as the pri-
Fig. 7.-a, segment of a radial larva seen from the anal side showing a net of secondary mesenchyme (in black) in the same plane as the primary mesenchyme cells and the ciliated band around the lower part of the ectoderm. b, dorsal view of a larva (prisma stage) to show the distribution of the secondary and the primary mesenthyme. Experimental
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mary mesenchyme cells, hut many cells migrate along the dorsal ectoderm and the archenteron. The final pattern of secondary mesenchyme is not as easily characterized as that of the primary mesenchyme, and it is not easy to find similarities in the two patterns, although the pseudopods of both types of mesenchyme may attach to the same types of contact points. It is important for the following discussion that in a radial larva, where the primary mesenchyme cells are lined up to form a ring parallel with the main ciliary band, a large number of secondary mesenchyme cells formed a network in the same plane (Fig. 7). Also in normal larvae there is a tendency for the secondary mesenchyme to line up along the ciliated band.
DISCUSSION
The problem dealt with in this paper is how contact is established between different cell types in the developing sea urchin embryo and to what extent the variation in thickness and curvature of the ectoderm and the distribution of various mesenchymal cell types can be accounted for in a simple way. The specific questions involved have already been listed in the introduction. Our observations indicate that the cells of the gastrula ectoderm in general are mutually connected only at certain points. The points of high adhesion are concentrated at two main levels, one along the inner border of the cells, another close to the hyaline membrane. For convenience we have referred to the contacts at the two levels as inner contact points and outer contact points. In regions with low contact between the cells one may say that the contact is restricted to outer contact points, whereas in the animal plate the cells are connected along the whole of their length. The pseudopods of various mesenchymal elements adhere only at the points of contact between the ectoderm cells, generally to the inner but also to the outer, provided the pseudopods can reach them. In the animal plate, if pseudopods adhere at all, the attachments are made only at the inner end of the junction between the cells. Pseudopods have never been seen to adhere to the free cell surfaces not involved in mutual ectoderm cell contact, neither the inner one nor the lateral ones, although pseudopods can explore these surfaces. The nature of the cell contacts in the early sea urchin development have been investigated by means of electron microscopy by Balnisky [a], Endo (see Dan [S]), Wolpert and Mercer [32], and Larje [23]. Apart from general contact and some interdigitating processes, clusters of intercellular connecting Experimental
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T. Gusfafson bars reminiscent of the desmosomal organization in vertebrates have been observed at the outer (distal) end of the cells, close to the hyaline membrane. In a large number of cases there is a mitochondrium close to the connecting bars [23]. The desmosomes begin to appear at about 54 hr after fertilization [32]. The proximal end of the cells has no ultrastructural organisation of desmosomal or any other specialized type. Preliminary investigations in our laboratory of the gastrula !23] also indicates a lack of such an organization at the inner points of contact at this stage. The long contacts between the cells of the animal plate also appear to lack such an organization [23]. The attached pseudopodal tips also seem to lack any defined ultrastructural patterns at their tips. The electron micrographs also reveal the relation between the cells and extracellular layers. The cells are attached to the hyaline membrane by numerous microvilli (see [5]). From about 84 hr after fertilization the inner surfaces, bordering the blastocoel are covered by a layer, the “fuzzy layer”, which has a thickness of about 0.1 p and contains particles and fibres [32]. There is no evidence of any similar substance tilling the spaces between the cells [32]. On the basis of Immers’ [21] histochemical and radioautographic studies Mercer and Wolpert [32j conclude that the “fuzzy layer” contains a mucopolysaccharide-protein complex secreted by the cells. The spatial distribution of the “fuzzy layer” and its occurrence in later stages still remains to be elucidated. Okazaki, Fukushi and Dan [2i] emphasize the work of Motomura [25], who demonstrated the presence of mucopolysaccharides secreted by the cells in the regions where the ectoderm cells pack closely and the primary mesenchyme cells collect. Our observations also indicate that the inner ends of the cells in these regions tit closely together and are not only connected by bridges, i.e. as if they were spreading on some membraneous secretion. The observations in Fig. 3, k and 1 on a strongly flattened gastrula also suggest that the inner ends of the cells, at least in some regions, are connected by some material, although the direct contact between the cells are partly broken. Taken together, these observations suggest that the “fuzzy layer” occurs also after the blastula stage, at least in zones \vith relatively strong cell contact. The contact relations discussed may be summarized as in Pig. 8. On the basis of the electron-microscopical investigations we may conclude that the outer points of contacts correspond to the regions with a desmosome-like organization which, however [32], may extend all around the outer border of the cells as a more or less continuous ring-like zone. Other surfaces involved in strong contact are devoid of a desmosome-like organization. Experimental
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One may ask to what extent the cell surface as a whole has a certain adhesiveness or if there is an intercellular cement covering the cells all around. Balinsky [2] assumes that the narrow spaces between the cells in the blastula wall are filled with a cementing substance (cf. [24]). In the cleaving blastula there is, however, no strong general attachment of the cells to each other,
Fig. S.-Diagram summarizing the contact relations within the ectoderm and between primary mesenchyme and ce,ls. ectoderm in regions of moderate and low contact between the ectoderm
since they round up and push apart at each cleavage, nor in the mesenchyme blastula of Echinocardium, where pigment cells easily migrate between the cells within the wall [18]. Also in Psammechinus there is no marked “general” adhesiveness, since the spaces between the cells occur in many sites even in the unextended sheet, and the spaces do not appear to be filled with any cementing substance, since pseudopods can freely explore these spaces. Furthermore, detached ectoderm cells can move around in the clefts between the attached cells without sticking to them, and this is not compatible with a high general adhesiveness, nor with the presence of a cementing substance. The long contacts within the animal plate, however, indicate a high general adhesion or possibly a cementing substance. Since there is, in general, no evidence for any general, high adhesion between the cells, with the exception of the animal plate, one may suggest that the hyaline membrane and the “fuzzy layer” play some part in the establishment and distribution of the contact points. Wolpert and Mercer [32] have in fact discussed the possible part played by the hyaline membrane in the development of desmosomes at the sites here denoted as the outer contact points. It seems attractive to suppose that a “fuzzy layer” plays a similar part in the establishment of the contacts at the inner points. If the cells have high adhesion for these two layers (hyaline and fuzzy), formed as secretions from the cells, they would tend to spread on these layers and therefore come in close contact with each other at their inner and outer ends. Where the “fuzzy layer” is lacking, there would be only outer contact points. \&‘hen dissociated cells of a mesenchyme blastula form a thin sheet on the slide, Experimental
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T. Gustafson this may reflect their attachment and tendency to spread on the glass surface. That the cells in the young blastula also form a single layer certainly reflects their strong attachments to the hyaline layer [31]. The close mutual contact at the inner and outer ends of the cells may result in a direct sticking together of the cells at these points, even if the mutual adhesion is rather low. The sticking together may be favoured by the fact that the adhesion of the cells to the hyaline and the fuzzy layer increases the length of time when the cells are close together; the movements of the other surfaces may prevent a contact between the cells of sufficient duration to allow a sticking together. The adhesion at the inner and outer ends may also be influenced by the strong curvature of the cell surface (cf. [29]), that follows from the attachment of the cells to the layers. Local differences in tension of the cell membrane may also be important (cf. [J]). Although the reason for the adhesiveness in the points concerned remains unknown, the fact that the cells in the animal plate have broad mutual contacts, those in the ciliated band both inner and outer point contacts and those in the thin regions only outer point contacts might be discussed in terms of differences in general adhesiveness of the cell surface and the distribution and properties of the fuzzy layer. Why do the pseudopods attach only at the junction between cells and not at any point of the cell surface, and what determines the differences in adhesiveness for pseudopods between different regions of the ectoderm? At present we may only, as a basis for further investigations, hint at some factors that may be involved. The tension in the cell sheet of the body wall may tend to break up the inner point contacts and the fuzzy layer to some extent and part of the former contact surface would therefore become available for the pseudopodal tips. Such a local uncovering of adhesive surfaces would not occur within the animal plate, where the cells adhere to each other along their whole length, and this could explain why pseudopods attach so weakly to the plate. In fact, where ruptures do occur within the plate and clefts are formed, mesenchyme cells regularly attach there. Hut why do their pseudopods attach only at the sharp edges or corners of the cells and not to their lateral and inner borders? In addition, why do pseudopods never attach to the rounded cells at the corners that have lost their inner point or long contact with adjacent cells and ought to have their adhesive surfaces exposed? It seems after all that adhesiveness for pseudopods is not only a question of a static molecular configuration of the ectoderm cell surface but also a function of movement, extension, and curvature of the cell surface, concepts discussed by Curtis [3, 41 and by I’ethica [29]. Experimental
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Another problem that is difficult to answer is why mesenchyme cells only attach with the tips of their pseudopods. Does the “stickiness” of the tip reflect, e.g. the motility, extension or curvature of its surface or “biochemical” peculiarities? Still another problem is why the tips of the pseudopods do not attach to each other in spite of their ability to attach to the ectoderm. This is illustrated by the secondary mesenchyme cells, which frequently move through a web of primary mesenchyme pseudopods. The answer may be that it takes some time for a contact to become established, and that two moving tips have little chance to meet for long enough. Later on, when pseudopods attach to points close enough, the probability of fusion would increase, and this may be part of the explanation of the formation of syncytia as development proceeds. The morphogenetic consequences of the contact relationships are easier to discuss than the basis for the establishment of the different types of cell contacts. The occurrence of inner contact points or broad contacts may determine the extent of packing of the cells and hence the curvature of the body wall (cf. [lS]). In fact, the distribution of these contacts agrees well with the changes in curvature of the ectoderm, the formation of the main ciliated band, “arm buds” and the animal plate (cf. [ 151). The consequences of a decrease in cell contact in the presumptive primary mesenchyme and in the archenteron rudiment during early invagination and the release of the secondary mesenchyme are also quite clear [ 15, 1 T]. The “upper corner” is a region at the border between a region of cells with broad contacts and with inner point contacts. If their mutual adhesion is not high enough and the “fuzzy layer” not strong enough, one may visualize that the cells easily separate and round up as a result of the deformation of the flattened larva. (Geometrical conditions may also make the deformation particularly strong there.) Another consequence of the distribution of inner contact points of ectoderm cells is that the primary mesenchyme cells will distribute themselves along the zones where the mutual contact between the ectodermal cells is relatively high, i.e. where the ectoderm is thickened and its curvature is increased (cf. [15, 26]). The mesenchyme pattern corresponds to that of the main ciliated band and the ring-like thickened zone around the young gastrula. In other words, the so-called graded stickiness of the ectoderm that determines the distribution of the pseudopodal contacts [ 131 reflects the distribution of inner points of contact within the ectoderm. More correctly, it is rather number of contact points per unit volume than per unit surface that determines the mesenchyme pattern. The cells therefore tend to collect at sharp curves or corners of the ectoderm. Such regions of strong curvature, Experimental
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however, coincide with the zones of strong contact, the curvature being a consequence of increased contact between the cells (cf. [151). Since pseudopods from different types of cells all appear to be able to attach to the points of contact between the ectoderm cells, one may ask why the statistical pathways and destinations of different kinds of cells and cell groups are not identical and do not correspond in detail to the thickened zones of the ectoderm. Is it after all necessary to postulate that the specificity of the point of contacts varies \vithin the sea urchin embryo, or is it possible to account for the differences in terms of a single but graded type of adhesiveness that may vary with time? The second alternative, which is closely related to the concept of Curtis [-I-], may be worth preliminary consideration with regard to our system. If the probability of the attachment of a pseudopod not only reflects the number of preformed contact points present but also their availability, it is easier to understand why the archenteron tip pseudopods attach so \vell to the dorsal side of the larva, whereas the primary mesenchyme forms the typical ring with the two ventro-lateral clusters, which extend upwards along the ciliated band. The primary mesenchyme cells, which move along the wall, faithfully redistribute themselves when the number of contact points available increases in certain zones (cf. [13,11,30]), and therefore the rentro-lateral chains extend when the main ciliated band appears. The pseudopods may not only respond to the appearance of new contact points, but also to pre-existent ones that become more available than before, due to partial separation of the ectoderm cells. The transient ruptures that tend to occur close to the ciliated band and behind the animal plate in connection with changes in cell packing and changes in shape of the larva ‘151 indicate that the inner contact points are partly uncovered there; the extension of the cell sheet may also change the adhesiveness for other reasons. These contact surfaces may be used as points of attachments for various pseudopods. Those along the ciliated band may easily be used by the primary mesenchyme cells that already attach along the band. The dorsal branch of the main ventral mesenchyme chain that occurs in some species [3O] indicates that also contact points behind the animal plate become available for the primary mesenchyme pseudopods. Rut since the tip of the archenteron rudiment is close to the same region, due to the obliqueness of the ventrally flattening larva [lo], it would mainly be the archenteron tip pseudopods that attach to the adhesive points behind the plate, and so the invagination tends to proceed along the dorsal side. Later on, when most of the mesenchyme cells involved leave the archenteron tip, which is no\v not far from the ventral side, its pseudopods will have a Experimental
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greater chance than before to attach to the ventro-lateral region and particularly to the oral ectoderm that for unknown reasons is very adhesive and bend the archeteron towards the oral region (cf. [lo, 161). An experimental way to test this concept is to extend the ectoderm to see if the pattern of pseudopodal attachments alters. In fact it does to a certain extent (cf. Fig. 6 e). The concept may be wrong, but still it illustrates how a complex pattern could arise, although the basic principles arc few. The secondary mesenchyme pattern is not so distinct, but appears irregular and does not clearly correspond to that of the primary mesenchyme, but even this may be explicable without assuming specific differences in adhesion between the different cells. The observed attachment of primary and secondary mesenchyme pseudopods to the same cell, or even to the same contact point, favours this idea. A similarity between the pattern of the two mesenchymal classes may in fact appear if only the points of attachment of the secondary mesenchyme cells are considered, and not the position of the cell bodies. The irregularity of the pattern of secondary mesenthyme cells may follow from the ability of these multipolar cells to attach with their pseudopods simultaneously to widely distant points between which the cell body will be suspended. The primary mesenchyme cells, on the other hand, closely attach to a syncytial cable formed by fused Iilopods with innumerable pseudopodal branches and therefore with a high probability of lining up in a more regular way [13]. It is too early to try to account for the distribution of other mesenchymal elements in the terms used above. The regular appearance of pigment cells at the site of future arm formation is particularly provocative, but this problem will be considered in a later paper. Our observations indicate that the general pattern of points of contact within the ectoderm also plays a part in the development of the coelom. The pseudopods that pull out the coelom thus never make stable contacts with the animal plate, the coelom being extended laterally. However, the coelom pseudopods and the secondary mesenchyme are also capable of attaching to the endoderm. The nature of these contacts will be considered in a later paper. The observation that different cell types can join up at the same contact points, which hence are rather unspecific, appears to agree with the hypothesis of Curtis [3, -I]. A more detailed consideration of his ideas on an adhesiveness that changes with time may be fruitful when dealing with the problems of pattern dealt with above. The molecular basis of the cell contacts has hardly been dealt with in this study. It is, however, worth mentioning that the pseudopods of the meExperimental
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T. Gusfafson senchyme cells may attach to glass, and that the pseudopods of an exocoelom may also do so, but even better to a nylon surface [16]. The contact relations in a larvae can furthermore be changed by treatment of early stages with anionic detergents, i.e. the contact pattern of the ectoderm and hence of the mesenchyme becomes altered [ll, cf. 201, the first ring of early high adhesiveness [15] tends to predominate, as it seems, at the expense of the ventral ciliated band. That the sensitivity towards the detergent abruptly drops before the contacts involved appear may be accounted for by a drop in permeability of the hyaline membrane (cf. [5]). Another observation of interest in this connection is that treatment with glucose and sucrose in fairly high concentrations favours the later packing of cells, so that the plate and the ciliated band increase at the expense of the thinner regions of the between different regions for cells reectoderm [ 191. (This “competition” minds us of that which may be supposed to occur in radialized larvae.)
SUMMARY
The contacts between various pseudopodal elements and the ectoderm in the sea urchin gastrula have been investigated by means of time-lapse cinematography and interference contrast microscopy on slightly compressed larvae, where the ectoderm is more or less extended and the cells tend to separate to a varying extent. In zones of the ectoderm where cell contact is moderate, the cells are joined up by points of attachment which are restricted to the margin of the inner and outer end of the cells. Where the contact is low, only the outer ends are involved in these mutual attachments, whereas in the animal plate, where the contact is strongest, the cells are attached to each other along the whole of their length. An increased packing of the cells can thus be correlated with the occurrence of inner contact points or broad contacts. The pseudopods of the primary mesenchyme, the archenteron tip, and the free secondary mesenchyme cells only attach at the points where the ectoderm cells attach to each other, and particularly to the inner points of attachment. The distribution of the inner points of attachment explains the correlation between the location of the primary mesenchyme cells and the thickened zones within the ectoderm. The inability of pseudopods to make strong contacts with the animal plate and the tendency of the archenteron tip pseudopods to attach to the dorsal side during gastrulation are tentatively explained in terms of availability of free contact surface within the ectoderm, but other factors may also be involved. The basis of the apparent differences between the pattern of primary and Experimental
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secondary mesenchyme is discussed. The inner and the outer contact points appear to be structurally different. The nature and origin of the cell contacts within the ectoderm is discussed, and it is suggested that the mutual adhesion between the cells is rather low, but that the cells tend to spread at the hyaline membrane and an inner layer which promotes a direct adhesion between the cells. The broad contact within the animal plate, however, indicates a high mutual adhesiveness of the cell surface as a whole. The results suggest that many of the complex morphological features of the young sea urchin embryo have a simple background. Part of this work was carried out at Kristinebergs Zoologiska Station, Fiskebgckskil. The author wishes to express his most cordial thanks to the Station authorities for unfailing generosity. The work has been financially supported by the Swedish Scientific Research Council.
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