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Cellular mechanisms in morphogenesis of the sea urchin gastrula
Experimental
361
Cell Research 21, 361-373 (1960)
CELLULAR MECHANISMS IN MORPHOGENESIS OF THE SEA URCHIN GASTRULA THE T. GUSTAFSON The Wenner-Gren
Institute
ORAL
CONTACT
and H. KINNANDER
for Experimental
Biology,
University
of StockhoEm,
Sweden
Received November 9, 1959
T IIE
morphologic development of organs and tissues is largely an expression of metabolic activities of the embryonic cells. In order to elucidate this relationship, it appears necessary to resolve the complex morphologic processes into morphologic activities at the cellular level. The morphogenesis of the sea urchin embryo has been the object of a series of investigations for this purpose. Our previous work [3, 4, 5, 61 has dealt with the different which appear to be brought about by steps in the process of gastrulation, pulsatory and pseudopodial activity of cells in the vegetal region of the larva. The present work is a study of the subsequent establishment of a contact between the archenteron tip and the ventral (oral) side of the gastrula ectoderm, a preparatory stage to formation of the mouth. In the following, this process is denoted as the establishment of an oral contact. The essential aim of the investigation was to ascertain whether the mechanisms already known from gastrulation have a general morphogenetic significance, and also the extent to which other mechanisms appear as morphogenesis proceeds. It is attractive to envisage the increasing complexity of an embryo as based on a relatively small number of different morphogenetic activities, which are released according to a simple time-space pattern, related to the polarity of the egg and creating a self-complexing system. MATERIAL
AND
METHODS
Like earlier steps in this series, the present study is based on time-lapse cinemicrographs of developing sea urchin larvae. For technical details regarding the filming equipment and film analysis, reference is made to Gustafson and Kinnander [4], and Kinnander and Gustafson [6]. In some experiments, the focus was automatically changed after each exposure, so that every second frame of the film corresponded to the same plane of focus. The films could thereby be divided into two distinct sequences, each with information from a separate plane. This “bifocal technique”, of Experimental
Cell Research 21
362
T. Gustafson and E-I. Kinnander
which an account will be given in a later paper, permits the analysis of complex processes which are enacted within a relatively large amplitude of depth. For the same purpose, some time-lapse films were made of larvae in a state of very slow, regular rotation. Exposure intervals generally ranged from 10 to 3 seconds. In order to keep the larvae in a constant position throughout the experiments, “nylon-net microaquaria” with a continuous water supply were used, as described carlicr 141. The species studied in this investigation was the same as in our previous work, I’sammechinus miliaris, the shallow and the deep form, from the west coast of Sweden. The temperature of development was about 20°C. The material analyzed included a number of abnormal cases, e.g., spontaneous exogastrulae and cases in which the process had been disturbed by mechanical obstacles. The analysis is based upon about 90 film sequences of varying length, comprising larvae in the lateral view, from the ventral (oral) and the dorsal (aboral) side and in the animal view. In order to obtain a preliminary idea of the mechanisms involved in the processes concerned, the activities observed on the running films were immcdiatcly documented with a tape recorder. On the basis of the information thus obtained, schematic preliminary diagrams of the process were drawn. These diagrams appeared to give a coherent picture of the mechanisms involved and served as a basis for a more detailed analysis. RESULTS ventral htnding During gastrulation, and especially tlurin g the suhscqucnt of the archentrron, the oral side of the ectoderm hccomcs progressivel\ flattened. IAarrae fixed in the microaquaria tenci to bc oriented \vith lhcil flattened side parallel to the slicic. Previous films of gastrulation thercfor(~ generally represent larvae seen from the ventral (oral) or dorsal (ahoral) side. In the present work, larvae seen in the lateral view \vcrc selcctctt for exl>osurc. In several cases these larvae rapidly changed their position during the experiments, due to their progressive yentral flattening. A large numhcr of cspc>rirnents \verc thereby spoiled. Many larvae could, ho\\-cxver, be kept in a relatively constant position for many hours. ‘Typical results with profile larva? are illustrated 1)~ the picture series in Figs. 1 anti 2. Although the first pseu(lopodia, formetl hi\; the presumptive
Fig. 1 .--Different stages in the rstablishmenl of oral contact in Psan7r77ecl1ir7us miliaris: picture series drawn from time-lapse films. o-d and :I, in profile; e and 1, from the animal pole. In larva {I, oral contact has been prevented by an indentation at the apical pole, and oral invagination has therefore become unusually deep. The time intervals between each stage represented in an individual series can be calculated from the frame numbers, given below each drawing, and from the time-lapse exposure intervals, which are constant in carh film: in a and P 10 seconds, in h 5 seconds, in e, tl and /, 6 seconds. Temperature of development during filming ranged from 19 to 23°C. Age at the start of each experiment (at frame 1) after cultivation at about 18°C: o and b 21 h, e 25 h, d 30 h, e 24 h, f 2.5 h. Larva 9 has developed for 24 h at 18°C and for 15 h at 21°C. Experimentnl
Cell Resenrch 21
Morphogenesis
of the sea urchin
gastrula
363
a
1
c
Experimental
Cell Research 21
364
T. Gustafson and H. Kinnander
secondary mesenchyme cells, may be emitted in any direction from the archenteron tip, there are dorsoventral differences in the rate at which the pseudopodia form bridges to the ectoderm. The young archenteron rudiment may form bridges to the ventral as \vell as to the dorsal side (Fig. 1, a and l>). As invagination proceeds, the dorsal pseudopodia are, ho\\-ever, more successful in making bridges than are the ventral ones, which mainly explore the body cavity or become retracted. Invagination, which after its initial phase is brought about by the pseudopodial activity, therefore tends to proceed more or less obliquely towards the dorsal side, and along the curvature of the dorsal ectoderm (e.g., Fig. 1, rc and h; Fig. 2, (I and 6). IVhen invagination has proceeded somewhat more than halfway, the ventrally directed pseudopodia often attain a large size and may bc repeatedly branched, but still mainly explore the body cavity (Fig. 1, 1~;Fig. 2, b). \Vhen the archentcron tip reaches the tiorsoanimal ectoderm, or somelvhat before, the pseudopodia-forming cells become more or less clearly disengaged from the archenteron tip as a cluster of secondary mesenchyme cells. ‘I’hc majority of these cells are fairly immobile and densely cro\\-(led, and appear to tic the archenteron tip to the dorsoanimal and dorsolatcral ectotlrrm (Fig. 1, c-e; Fig. 2, c-j). The densely crowded secondary mcsenchyme cells gradually begin to separate from each other, and spread on the ectoderm along its dorsal and lateral surfaces, often along the animal plate, but in the ventral direction as well. A great number of the spreading mesenchyme cells develop into pigment cells. Some of the cells in the secondary mesenchyme cluster tend to remain connected to the archenteron tip; they become elongated and form bridges to the ectoderm (Fig. 1, c-t Fig. 2, c, e, f, II, ,j). The larger ones often corrcspond to the large ventral exploring pseudopodia mentioned above. Sooner or later, this set of bridges becomes supplemented by bridges formed by pseudopodia, or thin filopodia, from cells still integrated in the archenteron tip (Fig, 2, g, i). The pattern of mesenchyme cells and of the bridges betlveen the archenteron tip and the ectoderm sho\vs great variations, both bctlveen individuals and from stage to stage. Some examples are given in Figs. 1 and 2. In some cases the bridges are formed mainly by the apical cluster of mesenchymc cells associated with the archenteron tip (cf. Fig. 1, a), but bridges arc gcnerally also formed at lower levels, i.e., by the tip itself (cf. Fig. 2, g). in a third type of variation (cf. Fig. 2, i), \vhere the mesenchyme cells spread only laterally and dorsally, the visible bridges are formed by the tip alone. The Experimental
Cell Research 21
Morphogenesis of the sea urchin gasfrula
365
most distinct bridges are more or less ventrally directed, but short dorsal bridges are also found and even some apically directed ones, partly hidden by the crowded mesenchyme cells. The individual variation appears to reflect differences in the rate at which the pseudopodia-forming cells bec,ome disengaged from the the epithelium of archenteron tip. However, for the further discussion it appears to be of minor importance whether the pesudopodia are formed by a cell in the archentcron tip itself, or by a cell in the mesenchyme closely associated with the tip. The bridges between the archenteron tip and the ectoderm contract. X number of the pseudopodia-forming cells thereby pull themselves out from the apical mesenchyme cluster and even from the actual archenteron wall (Figs. 1, c-f, and 2, c, e, f, 11-j). The liberated cells migrate in the diredion of the former bridges, either singly or in chains. The strong mechanical tension in the bridges is also reflected by the formation of large cones of attachment in the ectoderm (Figs. 1, e, and 2, ;) and by bill-like protrusions from the archenteron tip towards the ventral side (Figs. 1, c, d, and 2, e). The archenteron tip is evidently gradually liberated from its strong ectodcrma1 attachment, mediated by the secondary mesenchyme cells. It is then anchored to the ectoderm mainly by the contractile bridges. Since the ventral bridges arc most highly developed, the archenteron is moved forlvards against the ventral side. If the archenteron tip is incompletely liberated, the whole till becomes extended in the dorsoventral direction and thus still approaches the ventral side (Figs. 1, c, d, and 2, e). The role of mesenchymal pseudoFig. 2.PExamplcs of the distribution of mesenchyme cells and pseudopodia during the establishment of oral contact in Psammechinus miliuris: pictures from l&mm time-lapse reversal films of different larvae in varying stages of development. o. l.arva of about 22 h, invaginating along the dorsal curvature of the ectodcrm, and showing several pseudopodia at the archenteron tip and cones of attachment. b, The same larva as in a, but about 10’ later, with archenteron tip pseudopodia and a cone of attachment, as well as a large ventral exploring pseudopodium (arrow). c-j, Larvae of about 25 h. c-i in profile, j from the animal pole. e, Bridge formed by mesenchyme cell attached to the archenteron tip. d, Exploring pseudopod of a mesenchyme cell. e, The same larva as in (I, but 50’ later, with mesenchyme cell bridge attached to a bill-like protrusion from the archenteron tip. j, Chain of mesenchyme cells from the apical cluster, but also in contact with the archenteron tip. g, Thin pseudopods from mesenchyme cell in the state of liberation from the archenteron wall. h, Thin pseudopods from mesenchyme cells firmly attached to the archenteron tip. i, Bridge of mesenchyme cells in state of liberation from the archenteron tip, which is closely attached to the dorsoanimal ectoderm. The bridge is attached by 2 pscudopodia to the oral ectoderm, where 2 distinct cones of attachment are formed. These attachments are subsequently broken, and the cell bridge takes a more vegetal course. j, Larva seen from the animal pole; mescnchymc bridges to the oral side. If, Larva of about 30 h in profile, with oral contact established. The centripetal border of the oral invagination shows pulsatory activity, as does the whole of the archenteron tip (the archentcron in this larva is unusually large). Experimental
Cell Research 21
366
T. Gustafson and H. Kinnander
Fig. 2. Experimental
Celt Kesearch 21
Fig. 2 (continued). Experimental
Cell lkeurch
21
T. Gustafson and H. Kinnander podia in the establishment of a contact between the archenteron tip and the invaginated stomodeum rudiment has been suggested earlier in the case of Asterias [a], whereas this possibility was ruled out in the case of Astropecten [8]. As the archenteron tip approaches the ventral ectoderm, it becomes successively more strongly attached to this area by new pseudopodia, formed by cells in the archenteron tip. When direct contact between the archenteron and the ectodcrm is well established, the contact area increases and becomes oval. This process will be described in a subsequent paper on the formation of the coelomic sacs and the mouth. The aforegoing observations and our previous work [3, 4, 61 indicate that the fate of the archenteron is determined to a great extent by the secondary mesenchyme cells and by pseudopodia emitted by cells still integrated with the archenteron tip. The question then arises of the extent to which other cells of the larvae contribute to the establishment of oral contact. If the dorsal wall of the archenteron were to elongate actively and more than the ventral wall, the archenteron would bend ventrally. Certain differences in thickness no doubt appear as development proceeds, but such deformation can also occur in a tube of plastic material which is bent passively by extrinsic forces. Active bending would also occur if the cells in the dorsal and ventral archenteron wall showed differences in pulsatory activity of the kind observed in the early phase of invagination. Such pulsatory activity does not take place in the archenteron during the period of development concerned. Sometimes, there is a sharp bend in the dorsal archenteron wall, but a close examination shows that this is due to the pull by pseudopodia which are occasionally present far from the archenteron tip even at the dorsal side, and are thus analogous to the ventral bill-like protrusions mentioned above. - As a rule, the autonomous activity of the archenteron wall itself at this stage of devclopment is restricted to its tip, i.e., to the presumptive coelomic sacs and oesophagus. During the bending process, the cells in this region begin to show a moderate pulsatory activity. This is parallelled by a change in optical properties of the cells which partly reflects the disappearance of yolk granules. Although the change is striking, since the cells in other parts of the archendistributed around teron retain their yolk-loaded appearance, it is uniformly the tip, and cannot be of direct consequence for the direction of bending. .4 different way of determining the degree of autonomy of the ventral bend is to study the development of spontaneous exogastrulae. Some time-lapse films of such larvae have been made, but these films provide no definite evidence of a bending ac,henteron. A certain bend has, however, been Experimental
Cell Hesearch 21
Morphogenesis of the sea urchin gastrula
369
described [l] but this may be due largely to a stretching of the dorsal ectoderm. Even if the entotlerm shows a certain autonomous bend, this is evidently not essential for oral contact, since the archenteron is often moved forward like a straight lever during oral contact formation. The distance between the archenteron tip and the ectoderm is reduced not only by the activity of pseudopotiia, but also by a general flattening of the ventral gastrula ectoderm, a process which appears to be based on a centrifugal migration and flow of cells anti their packing in the periphery of the ventral side, the region of the large diary band and arms. This process will be described in a subsequent paper. In addition, there is an active invagination of the ectoderm, which thus meets the archenteron tip. This invagination is often preceded by passive indentation of the ectotlerm, brought about by the pseutlopodia from the archenteron tip. It is, however, largely autonomous, as it may occur in an animal egg fragment [7] and other animalized larvae devoid of mesenchymc bridges [Clj. The mechanism appears to be analogous to that of primary invagination. The ectoclcrm cells in the oral field thus become pear-shapetl, with their broad end directed towards the body cavity, and show lively pulsatory movements, cf. [6]. In larvae \vherc the archentcron bend has been prevented by means of an indentation at the apical pole, invagination becomes very deep [ll j, but in normal cases invagination is interrupted by contact Jvith the archenteron. Data on Astropectcn ([a], Fig. 17) suggest that the analogy between oral and archcnteron invagination is still more complete in this species, i.e., that oral invagination may also be complemented by the activity of pscudopotlia formed by the invaginated tip. DISCUSSION
Establishment of an oral contact is evidently brought about by a number of concurrent, but-as it seemPintlepcndent mechanisms. One is a flattening of the ventral ectoderm, a process \vhich will be discussed in a subsequent paper. Another is invagination of the oral ectoderm, mentioned earlier. In addition, a certain autonomous bending of the entotierm may occur, but is not essential. A fourth mechanism-which is the subject of the present investigation-can be regarded as a continuation of the process of invagination of the archenteron after its tip has been liberated from its firm contact with the dorsoapical ectoderm. The archrntcron tip is thus translocatetl to the oral ectoderm by the pull of pseutlopodia or filopodia formed by the tip, or by secondary mesenchyme cells associated with it. \\‘hcn the force of bending is localized, the question arises of the pattern Experimentnl
Cell Research 21
T. Gustafson and H. Kinnander of distribution of the pseudopodia-forming cells and of the course of these pseudopodia. Ho\\- great is the intrinsic complexity reflected in this pattern? The question is of general interest, since it deals with the degree of preformation of an egg, and with the mode of operation of a self-complexing system. Moreover, it is important to know to what extent the developmental behaviour of a cell reflects a specific biochemical-structural differentiation, and to what extent it merely reflects the localization of the cell in Lime and spate. The final answer to these questions requires experiments of high delicacy, and microchemical or histochemical analyses. The following discussion \vill, on the basis of time-lapse observations of normal and experimentally altered developmental patterns, try to prepare the ground for definite proof. The first question is that of the pathways of the active pscudopodia. The ventral ectoderm can evidently “guide” or at least serve as a substratum for primary mesenchyme cells, some of which move towards the apical pole pathways [lo]. The archenteron tip does not, however, along ventrolatcral follow a relatively short ventral track direct to the oral contact zone, but tends to be translocated along the dorsal ectodcrm. Is it necessary to explain the choice of the different pathways, to postulate dii’ferenws in specificity between pseudopodia of individual cell tgpcs and/or between the different parts of the ectodcrmal substratum for the pseudopodia? The diflerent pathways mentioned abovc can, to some extent, be explained in a simple way if it is assumed that the formation of successful pseudopodial contacts is determined by the relatirc distance between the archenteron tip or a mcsenchyme cell and the cctodcrm. The pseudopodia of the ventrolatcral primary mesenchyme cells arc formed in skadg contact with the vcntrolatcral wall of the blastula, and the cells will thus migrate along this \vall. The pseudopodia of the archenteron tip, on the other hand, n-ill be formed at the end of the primary phase of invagination, by cells relatircly close to the dorsal ectodcrm, and the archentcron tip therefore tends to translocatc along the dorsal ectoderm curvature. MJhen the tip aljproaches the apical pole along the curvature, it automatically also moves forward against the oral field, and becomes still closer to it when the ectodermal ventral flattening becomes pronounced. The probability of formation of pseudopodial contacts \vith the oral field thus increases. The relative distance to the ectoderm cannot account completely for the migratory pattern of the primary mesenchyme and the translocation of the archenteron tip. It must, in addition, be assumed that the ventral side has some characteristic influence on the behaviour of pseudopodia in general. Consequently, the second question is that of the nature of this influence, Experimental
Cell Research
21
Morphogenesis of the sea urchin gastrula
371
namely, whether it is necessary to postulate some kind of continuous longrang” guidance of the cells, e.g., of a chemotactic nature. If the ventral side n’cre to permit stronger pseudopodial contacts than other parts of the ectoderm, this should have great consequence for the arrangement of the primary mesenchyme, as well as for the oral contact. The ventral area would appear to attract pseudopodia within a certain radius of action. A prerequisite is, hoverer, that the pseudopodia explore in all directions. Such exploration has been observed in the case of the primary mesenchymc cells, the pseudopodia of which often branch and form an apparatus which, in less than an hour, explores the ventral side from its vegetal border up to its apical tip. This mode of exploration in all directions is also characteristic of the secondary mesenchymc cells, and of the pseudopodia emitted by the archcntcron tip itself. It must be recalled that the exploration continues even after the pseudopodia have attached to the ventral side, and cones of attachment have formed. The contacts may thus break, and new contacts form at other sites (cf. Fig. 2,i). If the stability of the ventral contacts is greatx than that of other contacts, this would contribute to a preferential ventral translocation of the archenteron tip. A third question is, however, motivated. Can the ventral dominance of the bridges and the distribution of the secondary mcsenchyme cells be csplained without referring to an intrinsic dorsoventrality of the archenteron til)? In certain cases, the difference in rate of formation of pseudopodia in tiill’crcnt directions may be only apparent, since the mesenchymc cells are apt to mask the pseudopodia in the dorsoapical region, but in other cases the xntral activity is, in fact, definitely strongest. The rate of emission of pseudopodia may be suppressed in other regions of the tip by direct contact with crowded mesenchymc cells, and n-ith the ectoderm itself. Another explanation, \\-hich is compatible with all variations observed, is that at this stage of development pseudopodia formation is normally restricted to certain cells, presumpti~c or disengaged secondary mcscnchyme cells. The dorsoapical and lateral ones soon approach the substratum for their pscudopodia, the ectodermal curvature, and leave the archenteron tip as free mrsenchyme cells. The rcntral cells, on the other hand which had no such opportunities of early, close contact \vith the ectoderm, remain in the archentcron tip and thus give the tip a preferential ventral course. The dorsoventral pattern at the young archcntcron tip may thus be largely due to its spatial relation to the ectoderm. An analysis ol this kind is incomplete as long as it is not based on strictly quantitative data. It nevertheless shows that a high degree of morphologic Experimental
Cell Research 21
372
T. Gustafson
and H. Kinnander
complexity might be explained on the basis of relatively simple assumptions. The morphogenetic movements in question are essentially brought about by pseudopodia, which are emitted and retracted, the latter process being described above as their contraction. The pseudopodia are formed in any direction of the free surfaces of the cells concerned, and the pseudopodia emitted explore in all directions. The prospect of contact betmccn pscudopodia and ectoderm is determined by the closeness of the I’seudopodia-fornling cell to the ectoderm. The specific course of the morphogenetic movements appears to be governed by some property of the ventral or ventrolateral cctoderm which increases the stahility of the pseudopodial contacts. On the other hand, it appears unnecessary to assume that the pseudopodial activity is directed chemotactically, or that the contacts of different types of pseudopodia-from primary and secondary mesenchymcare determined by different surface propcrtics. Although the l~seudopodial mechanism is not alone responsible for establishment of an oral contact, it plays a great role. One can assume that a pseudopodial mechanism has a general advantage over other mechanisms when young organ rudimenls are integrated. ‘This atlvantage may be due to the combination of an exploratory and contractile activity of the pseudopodia, which localizes the target even at a long distance and cf‘fcctuates the movement against it.
SUMMARY
On the basis of analyses of time-lapse films, an account is given of the cellular mechanisms which bring about contact between the archcnteron tip and the oral field in a sea urchin larva. This so-called oral contact is ensured by a number of concurrent but seemingly partly independent mechanisms. Contractile activity of pseudopodia at the archenteron tip plays a great role, as during the preceding phase of gastrulation. The pattern of the pseudopodia appears to hr determined by somewhat unspecific factors, such as the relative distance hct\\-een the pseudopodia-forming cells and the substrate of attachment and a shielding of certain potential pseudopodia-emitting surfaces. The pseudopodia reach their points of attachment after a lively exploration in various directions. It must, however, be assumed that the ventral cctotlerm permits stronger pseudopodial contacts than other parts of the ectodcrm. Attempts have been made to demonstrate that a high degree of morphologic complexity can be explained on the basis of simple premises. It is pointed out that a pseudopodial mechanism may have great advantages oyer other Experimental
C,‘ell Research
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Morphogenesis of the sea urchin gasfrula
373
mechanisms in bringing about the morphogenetic process of integration, as the pseudopodia both localize the target even at a long distance and effectuate the movement against it. This work was mainly carried out at Kristinebergs Zoologiska station, Fiskeb%ckskil. The authors wish to express their most cordial thanks to the Station authorities for never-failing generosity. The work has been financially supported by the Swedish Scientific Research Council. REFERENCES 1.
G~RBO~SKI,
M.
T., Bull. inlern. acad. sci. Cracouie 17, 581 (1905).
2. GEMMILL, .J. F., Phil. Trans. Roy. Sot. London B 205 (1914). 3. GUSTAFSON, T. and KISNASDER, H., Erptl. Cell Research 10, 733 (1956). 4. -ibid. 11, 36 (1956). 5. KINNANDER, H. and GUSTAFSON, T., Arkiu Zoo/. 11, 117 (1957). 6. -Exptl. CeU Reseurch 19, 278 (1960). 7. HRRSTADIUS, S., Pubbl. staz. zool. Napoli 14, 251 (1935). 8. -~~ ibid. 17, 221 (1939). 9. I.IXUAHL, P. E., Acta Zool. 17, 179 (1936). 10. Hcr~isr~ii~, J., Wilhelm Roux’ Arch. Enlwicklungsmech. Organ. 117, 123 (1929). 11. ~~~- Arkiu Zool. 17 B no. 10 (1925).
24-
6017355
Experimental
Cell Research 21
361
Cell Research 21, 361-373 (1960)
CELLULAR MECHANISMS IN MORPHOGENESIS OF THE SEA URCHIN GASTRULA THE T. GUSTAFSON The Wenner-Gren
Institute
ORAL
CONTACT
and H. KINNANDER
for Experimental
Biology,
University
of StockhoEm,
Sweden
Received November 9, 1959
T IIE
morphologic development of organs and tissues is largely an expression of metabolic activities of the embryonic cells. In order to elucidate this relationship, it appears necessary to resolve the complex morphologic processes into morphologic activities at the cellular level. The morphogenesis of the sea urchin embryo has been the object of a series of investigations for this purpose. Our previous work [3, 4, 5, 61 has dealt with the different which appear to be brought about by steps in the process of gastrulation, pulsatory and pseudopodial activity of cells in the vegetal region of the larva. The present work is a study of the subsequent establishment of a contact between the archenteron tip and the ventral (oral) side of the gastrula ectoderm, a preparatory stage to formation of the mouth. In the following, this process is denoted as the establishment of an oral contact. The essential aim of the investigation was to ascertain whether the mechanisms already known from gastrulation have a general morphogenetic significance, and also the extent to which other mechanisms appear as morphogenesis proceeds. It is attractive to envisage the increasing complexity of an embryo as based on a relatively small number of different morphogenetic activities, which are released according to a simple time-space pattern, related to the polarity of the egg and creating a self-complexing system. MATERIAL
AND
METHODS
Like earlier steps in this series, the present study is based on time-lapse cinemicrographs of developing sea urchin larvae. For technical details regarding the filming equipment and film analysis, reference is made to Gustafson and Kinnander [4], and Kinnander and Gustafson [6]. In some experiments, the focus was automatically changed after each exposure, so that every second frame of the film corresponded to the same plane of focus. The films could thereby be divided into two distinct sequences, each with information from a separate plane. This “bifocal technique”, of Experimental
Cell Research 21
362
T. Gustafson and E-I. Kinnander
which an account will be given in a later paper, permits the analysis of complex processes which are enacted within a relatively large amplitude of depth. For the same purpose, some time-lapse films were made of larvae in a state of very slow, regular rotation. Exposure intervals generally ranged from 10 to 3 seconds. In order to keep the larvae in a constant position throughout the experiments, “nylon-net microaquaria” with a continuous water supply were used, as described carlicr 141. The species studied in this investigation was the same as in our previous work, I’sammechinus miliaris, the shallow and the deep form, from the west coast of Sweden. The temperature of development was about 20°C. The material analyzed included a number of abnormal cases, e.g., spontaneous exogastrulae and cases in which the process had been disturbed by mechanical obstacles. The analysis is based upon about 90 film sequences of varying length, comprising larvae in the lateral view, from the ventral (oral) and the dorsal (aboral) side and in the animal view. In order to obtain a preliminary idea of the mechanisms involved in the processes concerned, the activities observed on the running films were immcdiatcly documented with a tape recorder. On the basis of the information thus obtained, schematic preliminary diagrams of the process were drawn. These diagrams appeared to give a coherent picture of the mechanisms involved and served as a basis for a more detailed analysis. RESULTS ventral htnding During gastrulation, and especially tlurin g the suhscqucnt of the archentrron, the oral side of the ectoderm hccomcs progressivel\ flattened. IAarrae fixed in the microaquaria tenci to bc oriented \vith lhcil flattened side parallel to the slicic. Previous films of gastrulation thercfor(~ generally represent larvae seen from the ventral (oral) or dorsal (ahoral) side. In the present work, larvae seen in the lateral view \vcrc selcctctt for exl>osurc. In several cases these larvae rapidly changed their position during the experiments, due to their progressive yentral flattening. A large numhcr of cspc>rirnents \verc thereby spoiled. Many larvae could, ho\\-cxver, be kept in a relatively constant position for many hours. ‘Typical results with profile larva? are illustrated 1)~ the picture series in Figs. 1 anti 2. Although the first pseu(lopodia, formetl hi\; the presumptive
Fig. 1 .--Different stages in the rstablishmenl of oral contact in Psan7r77ecl1ir7us miliaris: picture series drawn from time-lapse films. o-d and :I, in profile; e and 1, from the animal pole. In larva {I, oral contact has been prevented by an indentation at the apical pole, and oral invagination has therefore become unusually deep. The time intervals between each stage represented in an individual series can be calculated from the frame numbers, given below each drawing, and from the time-lapse exposure intervals, which are constant in carh film: in a and P 10 seconds, in h 5 seconds, in e, tl and /, 6 seconds. Temperature of development during filming ranged from 19 to 23°C. Age at the start of each experiment (at frame 1) after cultivation at about 18°C: o and b 21 h, e 25 h, d 30 h, e 24 h, f 2.5 h. Larva 9 has developed for 24 h at 18°C and for 15 h at 21°C. Experimentnl
Cell Resenrch 21
Morphogenesis
of the sea urchin
gastrula
363
a
1
c
Experimental
Cell Research 21
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secondary mesenchyme cells, may be emitted in any direction from the archenteron tip, there are dorsoventral differences in the rate at which the pseudopodia form bridges to the ectoderm. The young archenteron rudiment may form bridges to the ventral as \vell as to the dorsal side (Fig. 1, a and l>). As invagination proceeds, the dorsal pseudopodia are, ho\\-ever, more successful in making bridges than are the ventral ones, which mainly explore the body cavity or become retracted. Invagination, which after its initial phase is brought about by the pseudopodial activity, therefore tends to proceed more or less obliquely towards the dorsal side, and along the curvature of the dorsal ectoderm (e.g., Fig. 1, rc and h; Fig. 2, (I and 6). IVhen invagination has proceeded somewhat more than halfway, the ventrally directed pseudopodia often attain a large size and may bc repeatedly branched, but still mainly explore the body cavity (Fig. 1, 1~;Fig. 2, b). \Vhen the archentcron tip reaches the tiorsoanimal ectoderm, or somelvhat before, the pseudopodia-forming cells become more or less clearly disengaged from the archenteron tip as a cluster of secondary mesenchyme cells. ‘I’hc majority of these cells are fairly immobile and densely cro\\-(led, and appear to tic the archenteron tip to the dorsoanimal and dorsolatcral ectotlrrm (Fig. 1, c-e; Fig. 2, c-j). The densely crowded secondary mcsenchyme cells gradually begin to separate from each other, and spread on the ectoderm along its dorsal and lateral surfaces, often along the animal plate, but in the ventral direction as well. A great number of the spreading mesenchyme cells develop into pigment cells. Some of the cells in the secondary mesenchyme cluster tend to remain connected to the archenteron tip; they become elongated and form bridges to the ectoderm (Fig. 1, c-t Fig. 2, c, e, f, II, ,j). The larger ones often corrcspond to the large ventral exploring pseudopodia mentioned above. Sooner or later, this set of bridges becomes supplemented by bridges formed by pseudopodia, or thin filopodia, from cells still integrated in the archenteron tip (Fig, 2, g, i). The pattern of mesenchyme cells and of the bridges betlveen the archenteron tip and the ectoderm sho\vs great variations, both bctlveen individuals and from stage to stage. Some examples are given in Figs. 1 and 2. In some cases the bridges are formed mainly by the apical cluster of mesenchymc cells associated with the archenteron tip (cf. Fig. 1, a), but bridges arc gcnerally also formed at lower levels, i.e., by the tip itself (cf. Fig. 2, g). in a third type of variation (cf. Fig. 2, i), \vhere the mesenchyme cells spread only laterally and dorsally, the visible bridges are formed by the tip alone. The Experimental
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most distinct bridges are more or less ventrally directed, but short dorsal bridges are also found and even some apically directed ones, partly hidden by the crowded mesenchyme cells. The individual variation appears to reflect differences in the rate at which the pseudopodia-forming cells bec,ome disengaged from the the epithelium of archenteron tip. However, for the further discussion it appears to be of minor importance whether the pesudopodia are formed by a cell in the archentcron tip itself, or by a cell in the mesenchyme closely associated with the tip. The bridges between the archenteron tip and the ectoderm contract. X number of the pseudopodia-forming cells thereby pull themselves out from the apical mesenchyme cluster and even from the actual archenteron wall (Figs. 1, c-f, and 2, c, e, f, 11-j). The liberated cells migrate in the diredion of the former bridges, either singly or in chains. The strong mechanical tension in the bridges is also reflected by the formation of large cones of attachment in the ectoderm (Figs. 1, e, and 2, ;) and by bill-like protrusions from the archenteron tip towards the ventral side (Figs. 1, c, d, and 2, e). The archenteron tip is evidently gradually liberated from its strong ectodcrma1 attachment, mediated by the secondary mesenchyme cells. It is then anchored to the ectoderm mainly by the contractile bridges. Since the ventral bridges arc most highly developed, the archenteron is moved forlvards against the ventral side. If the archenteron tip is incompletely liberated, the whole till becomes extended in the dorsoventral direction and thus still approaches the ventral side (Figs. 1, c, d, and 2, e). The role of mesenchymal pseudoFig. 2.PExamplcs of the distribution of mesenchyme cells and pseudopodia during the establishment of oral contact in Psammechinus miliuris: pictures from l&mm time-lapse reversal films of different larvae in varying stages of development. o. l.arva of about 22 h, invaginating along the dorsal curvature of the ectodcrm, and showing several pseudopodia at the archenteron tip and cones of attachment. b, The same larva as in a, but about 10’ later, with archenteron tip pseudopodia and a cone of attachment, as well as a large ventral exploring pseudopodium (arrow). c-j, Larvae of about 25 h. c-i in profile, j from the animal pole. e, Bridge formed by mesenchyme cell attached to the archenteron tip. d, Exploring pseudopod of a mesenchyme cell. e, The same larva as in (I, but 50’ later, with mesenchyme cell bridge attached to a bill-like protrusion from the archenteron tip. j, Chain of mesenchyme cells from the apical cluster, but also in contact with the archenteron tip. g, Thin pseudopods from mesenchyme cell in the state of liberation from the archenteron wall. h, Thin pseudopods from mesenchyme cells firmly attached to the archenteron tip. i, Bridge of mesenchyme cells in state of liberation from the archenteron tip, which is closely attached to the dorsoanimal ectoderm. The bridge is attached by 2 pscudopodia to the oral ectoderm, where 2 distinct cones of attachment are formed. These attachments are subsequently broken, and the cell bridge takes a more vegetal course. j, Larva seen from the animal pole; mescnchymc bridges to the oral side. If, Larva of about 30 h in profile, with oral contact established. The centripetal border of the oral invagination shows pulsatory activity, as does the whole of the archenteron tip (the archentcron in this larva is unusually large). Experimental
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Fig. 2. Experimental
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Fig. 2 (continued). Experimental
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T. Gustafson and H. Kinnander podia in the establishment of a contact between the archenteron tip and the invaginated stomodeum rudiment has been suggested earlier in the case of Asterias [a], whereas this possibility was ruled out in the case of Astropecten [8]. As the archenteron tip approaches the ventral ectoderm, it becomes successively more strongly attached to this area by new pseudopodia, formed by cells in the archenteron tip. When direct contact between the archenteron and the ectodcrm is well established, the contact area increases and becomes oval. This process will be described in a subsequent paper on the formation of the coelomic sacs and the mouth. The aforegoing observations and our previous work [3, 4, 61 indicate that the fate of the archenteron is determined to a great extent by the secondary mesenchyme cells and by pseudopodia emitted by cells still integrated with the archenteron tip. The question then arises of the extent to which other cells of the larvae contribute to the establishment of oral contact. If the dorsal wall of the archenteron were to elongate actively and more than the ventral wall, the archenteron would bend ventrally. Certain differences in thickness no doubt appear as development proceeds, but such deformation can also occur in a tube of plastic material which is bent passively by extrinsic forces. Active bending would also occur if the cells in the dorsal and ventral archenteron wall showed differences in pulsatory activity of the kind observed in the early phase of invagination. Such pulsatory activity does not take place in the archenteron during the period of development concerned. Sometimes, there is a sharp bend in the dorsal archenteron wall, but a close examination shows that this is due to the pull by pseudopodia which are occasionally present far from the archenteron tip even at the dorsal side, and are thus analogous to the ventral bill-like protrusions mentioned above. - As a rule, the autonomous activity of the archenteron wall itself at this stage of devclopment is restricted to its tip, i.e., to the presumptive coelomic sacs and oesophagus. During the bending process, the cells in this region begin to show a moderate pulsatory activity. This is parallelled by a change in optical properties of the cells which partly reflects the disappearance of yolk granules. Although the change is striking, since the cells in other parts of the archendistributed around teron retain their yolk-loaded appearance, it is uniformly the tip, and cannot be of direct consequence for the direction of bending. .4 different way of determining the degree of autonomy of the ventral bend is to study the development of spontaneous exogastrulae. Some time-lapse films of such larvae have been made, but these films provide no definite evidence of a bending ac,henteron. A certain bend has, however, been Experimental
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described [l] but this may be due largely to a stretching of the dorsal ectoderm. Even if the entotlerm shows a certain autonomous bend, this is evidently not essential for oral contact, since the archenteron is often moved forward like a straight lever during oral contact formation. The distance between the archenteron tip and the ectoderm is reduced not only by the activity of pseudopotiia, but also by a general flattening of the ventral gastrula ectoderm, a process which appears to be based on a centrifugal migration and flow of cells anti their packing in the periphery of the ventral side, the region of the large diary band and arms. This process will be described in a subsequent paper. In addition, there is an active invagination of the ectoderm, which thus meets the archenteron tip. This invagination is often preceded by passive indentation of the ectotlerm, brought about by the pseutlopodia from the archenteron tip. It is, however, largely autonomous, as it may occur in an animal egg fragment [7] and other animalized larvae devoid of mesenchymc bridges [Clj. The mechanism appears to be analogous to that of primary invagination. The ectoclcrm cells in the oral field thus become pear-shapetl, with their broad end directed towards the body cavity, and show lively pulsatory movements, cf. [6]. In larvae \vherc the archentcron bend has been prevented by means of an indentation at the apical pole, invagination becomes very deep [ll j, but in normal cases invagination is interrupted by contact Jvith the archenteron. Data on Astropectcn ([a], Fig. 17) suggest that the analogy between oral and archcnteron invagination is still more complete in this species, i.e., that oral invagination may also be complemented by the activity of pscudopotlia formed by the invaginated tip. DISCUSSION
Establishment of an oral contact is evidently brought about by a number of concurrent, but-as it seemPintlepcndent mechanisms. One is a flattening of the ventral ectoderm, a process \vhich will be discussed in a subsequent paper. Another is invagination of the oral ectoderm, mentioned earlier. In addition, a certain autonomous bending of the entotierm may occur, but is not essential. A fourth mechanism-which is the subject of the present investigation-can be regarded as a continuation of the process of invagination of the archenteron after its tip has been liberated from its firm contact with the dorsoapical ectoderm. The archrntcron tip is thus translocatetl to the oral ectoderm by the pull of pseutlopodia or filopodia formed by the tip, or by secondary mesenchyme cells associated with it. \\‘hcn the force of bending is localized, the question arises of the pattern Experimentnl
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T. Gustafson and H. Kinnander of distribution of the pseudopodia-forming cells and of the course of these pseudopodia. Ho\\- great is the intrinsic complexity reflected in this pattern? The question is of general interest, since it deals with the degree of preformation of an egg, and with the mode of operation of a self-complexing system. Moreover, it is important to know to what extent the developmental behaviour of a cell reflects a specific biochemical-structural differentiation, and to what extent it merely reflects the localization of the cell in Lime and spate. The final answer to these questions requires experiments of high delicacy, and microchemical or histochemical analyses. The following discussion \vill, on the basis of time-lapse observations of normal and experimentally altered developmental patterns, try to prepare the ground for definite proof. The first question is that of the pathways of the active pscudopodia. The ventral ectoderm can evidently “guide” or at least serve as a substratum for primary mesenchyme cells, some of which move towards the apical pole pathways [lo]. The archenteron tip does not, however, along ventrolatcral follow a relatively short ventral track direct to the oral contact zone, but tends to be translocated along the dorsal ectodcrm. Is it necessary to explain the choice of the different pathways, to postulate dii’ferenws in specificity between pseudopodia of individual cell tgpcs and/or between the different parts of the ectodcrmal substratum for the pseudopodia? The diflerent pathways mentioned abovc can, to some extent, be explained in a simple way if it is assumed that the formation of successful pseudopodial contacts is determined by the relatirc distance between the archenteron tip or a mcsenchyme cell and the cctodcrm. The pseudopodia of the ventrolatcral primary mesenchyme cells arc formed in skadg contact with the vcntrolatcral wall of the blastula, and the cells will thus migrate along this \vall. The pseudopodia of the archenteron tip, on the other hand, n-ill be formed at the end of the primary phase of invagination, by cells relatircly close to the dorsal ectodcrm, and the archentcron tip therefore tends to translocatc along the dorsal ectoderm curvature. MJhen the tip aljproaches the apical pole along the curvature, it automatically also moves forward against the oral field, and becomes still closer to it when the ectodermal ventral flattening becomes pronounced. The probability of formation of pseudopodial contacts \vith the oral field thus increases. The relative distance to the ectoderm cannot account completely for the migratory pattern of the primary mesenchyme and the translocation of the archenteron tip. It must, in addition, be assumed that the ventral side has some characteristic influence on the behaviour of pseudopodia in general. Consequently, the second question is that of the nature of this influence, Experimental
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namely, whether it is necessary to postulate some kind of continuous longrang” guidance of the cells, e.g., of a chemotactic nature. If the ventral side n’cre to permit stronger pseudopodial contacts than other parts of the ectoderm, this should have great consequence for the arrangement of the primary mesenchyme, as well as for the oral contact. The ventral area would appear to attract pseudopodia within a certain radius of action. A prerequisite is, hoverer, that the pseudopodia explore in all directions. Such exploration has been observed in the case of the primary mesenchymc cells, the pseudopodia of which often branch and form an apparatus which, in less than an hour, explores the ventral side from its vegetal border up to its apical tip. This mode of exploration in all directions is also characteristic of the secondary mesenchymc cells, and of the pseudopodia emitted by the archcntcron tip itself. It must be recalled that the exploration continues even after the pseudopodia have attached to the ventral side, and cones of attachment have formed. The contacts may thus break, and new contacts form at other sites (cf. Fig. 2,i). If the stability of the ventral contacts is greatx than that of other contacts, this would contribute to a preferential ventral translocation of the archenteron tip. A third question is, however, motivated. Can the ventral dominance of the bridges and the distribution of the secondary mcsenchyme cells be csplained without referring to an intrinsic dorsoventrality of the archenteron til)? In certain cases, the difference in rate of formation of pseudopodia in tiill’crcnt directions may be only apparent, since the mesenchymc cells are apt to mask the pseudopodia in the dorsoapical region, but in other cases the xntral activity is, in fact, definitely strongest. The rate of emission of pseudopodia may be suppressed in other regions of the tip by direct contact with crowded mesenchymc cells, and n-ith the ectoderm itself. Another explanation, \\-hich is compatible with all variations observed, is that at this stage of development pseudopodia formation is normally restricted to certain cells, presumpti~c or disengaged secondary mcscnchyme cells. The dorsoapical and lateral ones soon approach the substratum for their pscudopodia, the ectodermal curvature, and leave the archenteron tip as free mrsenchyme cells. The rcntral cells, on the other hand which had no such opportunities of early, close contact \vith the ectoderm, remain in the archentcron tip and thus give the tip a preferential ventral course. The dorsoventral pattern at the young archcntcron tip may thus be largely due to its spatial relation to the ectoderm. An analysis ol this kind is incomplete as long as it is not based on strictly quantitative data. It nevertheless shows that a high degree of morphologic Experimental
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complexity might be explained on the basis of relatively simple assumptions. The morphogenetic movements in question are essentially brought about by pseudopodia, which are emitted and retracted, the latter process being described above as their contraction. The pseudopodia are formed in any direction of the free surfaces of the cells concerned, and the pseudopodia emitted explore in all directions. The prospect of contact betmccn pscudopodia and ectoderm is determined by the closeness of the I’seudopodia-fornling cell to the ectoderm. The specific course of the morphogenetic movements appears to be governed by some property of the ventral or ventrolateral cctoderm which increases the stahility of the pseudopodial contacts. On the other hand, it appears unnecessary to assume that the pseudopodial activity is directed chemotactically, or that the contacts of different types of pseudopodia-from primary and secondary mesenchymcare determined by different surface propcrtics. Although the l~seudopodial mechanism is not alone responsible for establishment of an oral contact, it plays a great role. One can assume that a pseudopodial mechanism has a general advantage over other mechanisms when young organ rudimenls are integrated. ‘This atlvantage may be due to the combination of an exploratory and contractile activity of the pseudopodia, which localizes the target even at a long distance and cf‘fcctuates the movement against it.
SUMMARY
On the basis of analyses of time-lapse films, an account is given of the cellular mechanisms which bring about contact between the archcnteron tip and the oral field in a sea urchin larva. This so-called oral contact is ensured by a number of concurrent but seemingly partly independent mechanisms. Contractile activity of pseudopodia at the archenteron tip plays a great role, as during the preceding phase of gastrulation. The pattern of the pseudopodia appears to hr determined by somewhat unspecific factors, such as the relative distance hct\\-een the pseudopodia-forming cells and the substrate of attachment and a shielding of certain potential pseudopodia-emitting surfaces. The pseudopodia reach their points of attachment after a lively exploration in various directions. It must, however, be assumed that the ventral cctotlerm permits stronger pseudopodial contacts than other parts of the ectodcrm. Attempts have been made to demonstrate that a high degree of morphologic complexity can be explained on the basis of simple premises. It is pointed out that a pseudopodial mechanism may have great advantages oyer other Experimental
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mechanisms in bringing about the morphogenetic process of integration, as the pseudopodia both localize the target even at a long distance and effectuate the movement against it. This work was mainly carried out at Kristinebergs Zoologiska station, Fiskeb%ckskil. The authors wish to express their most cordial thanks to the Station authorities for never-failing generosity. The work has been financially supported by the Swedish Scientific Research Council. REFERENCES 1.
G~RBO~SKI,
M.
T., Bull. inlern. acad. sci. Cracouie 17, 581 (1905).
2. GEMMILL, .J. F., Phil. Trans. Roy. Sot. London B 205 (1914). 3. GUSTAFSON, T. and KISNASDER, H., Erptl. Cell Research 10, 733 (1956). 4. -ibid. 11, 36 (1956). 5. KINNANDER, H. and GUSTAFSON, T., Arkiu Zoo/. 11, 117 (1957). 6. -Exptl. CeU Reseurch 19, 278 (1960). 7. HRRSTADIUS, S., Pubbl. staz. zool. Napoli 14, 251 (1935). 8. -~~ ibid. 17, 221 (1939). 9. I.IXUAHL, P. E., Acta Zool. 17, 179 (1936). 10. Hcr~isr~ii~, J., Wilhelm Roux’ Arch. Enlwicklungsmech. Organ. 117, 123 (1929). 11. ~~~- Arkiu Zool. 17 B no. 10 (1925).
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