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Further studies on the cellular basis of gastrulation in the sea urchin larva
Experimental
278
Cell Research
FURTHER STUDIES ON THE CELLULAR GASTRULATION IN THE SEA URCHIN H. KINNANDER The Wenner-Gren
Institute
for
and
Experimental Received
(1960)
BASIS OF LARVA
T. GUSTAFSON Biology,
April
19, 27X-290
University
of Stockholm,
Sweden
21, 1959
THE metabolic background of morphogenesis in embryos has been the object of investigation for a fairly long time. The morphologic events have, however, been used mainly as indicators of the kind of metabolism which takes place, e.g., during the “phases of determination” in early developmental stages. Little is known, on the other hand, of the way in which the metabolic processes operate in moulding the shape of the embryo. The gap between the biochemical level and the morphologic level is thus wide. As a contribution to bridging this gap, it would be appropriate to resolve the morphologic events in organs and tissues into morphologic processes at the cellular level, so simple that their biochemical background could be discussed and analyzed in the future. Time-lapse cinemicrography of the simple, transparent sea urchin larva during different phases of gastrulation and subsequent stages opens up the possibility of making such a resolution. In the sea urchin larva, gastrulation involves formation of the primary mesenchyme and a subsequent invagination of the presumptive archenteron material. In a preceding paper [5] these processes were discussed on the basis of data obtained by means of time-lapse cinemicrography. An analysis of the films suggested that the primary mesenchyme cells might be more or less passively squeezed out from the blastula wall into the blastocoel. This process will be further elucidated in the present paper. During a second phase of translocation of the mesenchyme cells, they migrate actively along the blastula wall by means of contractile pseudopodia or filopodia, until they reach their final point of attachment, cf. [lo]. The analysis also revealed two main phases in the subsequent invagination of the archenteron material, each characterized by a separate mechanism of translocation. The onset of the second phase of invagination could be correlated with the appearance of pseudopodia or filopodia from cells in the archenteron tip, the presumptive secondary mesenchyme cells. It is evident from the films and the diagrams of analysis that a causal relation exists betjveen these pseuExperimental
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dopodia and the process of invagination. The pseudopodia, in fact, pull the tip of the archenteron rudiment through the blastocoel, just as the pseudopodia of the primary mesenchyme cells pull themselves along the blastula wall. Most of the cells of the archenteron rudiment are thus passively translocated during the second phase of invagination [5]. The mechanism during the first phase of invagination has hitherto been obscure. The running films thus far only revealed a diffuse “boiling” activity at the vegetal pole of the advanced blastula and at the tip of the archenteron rudiment [5]. The primary aim of the work described in the present paper was to resolve this activity into its components, on the assumption that this may throw light on the mechanism of the first phase of invagination. In addition, the relation between mesenchyme formation and invagination will be discussed, as well as the possible relation between the different mechanisms involved.
TIME-LAPSE
TECHNIQUE
AND
MATERIAL
In order to obtain detailed information from the time-lapse recordings, the exposure intervals between the single frames had to be reduced from the 18 or 10 secondspreviously used to e.g., 3 or 1 second. For 16 mm time-lapse cinemicrography we used a double installation, relatively easily transportable, consisting mainly of the equipment described earlier [5], and of Cine-Kodak Special II (CKS) camera mounted on the vertical supporting column of a boring-machine stand, and adapted over the microscope (Leitz Ortholux). For making single-frame exposures, the CKS camera has a “Dreh-Magnet” (Wilhelm Nass, Hannover, West Germany) for 6 volt DC connected to the eight-frame shaft. The exposure-interval timer is driven by a synchronous motor. The source of light was a low-voltage filament bulb, 6 volt, 5 amp. Wild KG 1 (3 mm) heat filters for microscopy were used, and the larvae were constantly illuminated for more than 20 hours at 3.5 amp. The development was normal, as with the intermittent illumination previously used. The microcine-adaptor for the CKS camera was a Reichert Kinekonnex. The microscope objectives used in the two equipments were Leitz apochromates 2410.65and 4010.95.“Nylon-net microaquaria”, as earlier described, were used to keep the larvae in a constant position, and the “cuvette slides” for the continuous supply of water were mainly of the type previously used [5]. The film material was Kodak Super X (now discontinued), Kodak Plus X and Agfa Isopan F, all reversal films. The films were analyzed as earlier in a G. B. Bell and Howell projector, especially adapted for time and motion studies. The use of a tape recorder during the analysis made it easy to document even minute, rapid, transient changesin the films. The observations were noted on key sort cards, which greatly facilitated the analysis of the abundant film material. The speciesusedin the investigation wasPsammechinus miliaris, the shallow form from the west coast of Sweden, and the temperature of development was about 20°C. 19
603702
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CONSTRUCTION
and T. Gustafson OF THE
DIAGRAMS
The film material analyzed consisted of films with various exposure intervals. The 3- to l-second interval films, about 50 per cent of the material analyzed, gave a distinct picture of details which were poorly resolved by films with longer intervals. Invagination was described by curves, invagination curves, obtained by plotting the height of the archenteron lumen (microns) against time (hours), cf. Fig. 1. The essential new information given by the short-exposure interval films, i.e., information on the “boiling” activity at the vegetal pole of the blastula and at the archenteron tip, could not, however, be analyzed in an exact quantitative way in a reasonable time. It was therefore necessary to make provisional use of a more subjective description of the process. The “boiling” proved to be resolvable into the pulsations of a great number of plasma lobes protruding from the surface of the cells in the most vegetal region of the young gastrula, finally concentrated to the apex of the archenteron rudiment and directed centripetally, i.e., against the blastocoel. The lobes were of different shape and size, and varied with respect to their amplitude and frequency of pulsation. Taken together, these characteristics, which were closely interrelated, can be said to express a cellular pulsatory activity, denoted in the following as CPA. The CPA varied from cell to cell and with the course of gastrulation. Symbols for the CPA were drawn close to the invagination curves in diagrams in the following way, cf. Fig. 1. The CPA in 7 points, each corresponding to one or a few cells, and evenly distributed along the archenteron rudiment, was used to represent the whole of the archenteron surface against the blastocoel. One point was stituated at the apex of the rudiment, two points at the base, on the left and the right, and four points were evenly distributed in the intermediate segment (see Fig. 1). In earlier stages, the test points were distributed along the vegetal blastula wall, mainly in an area corresponding to the presumptive archenteron. The CPA in the points selected was recorded while running the film at a constant speed during time intervals of constant length in each experiment. The length of the relevant time interval, e.g., 30 minutes, is given in the legends to the figures. It corresponded to the micron intervals, usually indicated by the horizontal lines to the right of the invagination curve in the diagrams. The profile of the archenteron rudiment at the beginning of each time interval was indicated by dotted contours. Although the intervals were long enough to disclose alterations in the CPA in the individual points, no difficulty \vas Experimental
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7Hourr
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8 Hours
7
90 I 80 I 70
Liberation
I
60-so-40-30-20-IO-
d
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8
I I
Fig. l.-Diagrams of the course of invagination in 4 larvae of Psammechinus miliaris. The height of the archenteron lumen is plotted against the relative age in hours, the first stage being arbitrarily denoted as 0 hours. For detailed explanation of the construction of the diagrams and the symbols used, see text p. 280. Dashed segment of curves: interpolated values. In d, this segment is only theoretical, since the pseudopodia-forming cells pulled themselves out from the archenteron tip before it had reached the animal pole. Length of the time intervals marked by the horizontal lines to the right of the curves (and by letters A-E to the left of the rising curve segments in b and d): a, b and c, 25 minutes; d, 41 minutes. Experimental
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encountered in estimating the mean activity, and repeated estimations showed good agreement. The CPA was not expressed after studying all its components separately, but by observing the shape of the lobes at their maximal size, and by subjective estimation of their motility, corresponding to components of amplitude and frequency. The shape and motility components of the CPA were indicated by symbols denoting the changes observed at the surface against the blastocoel. The symbols were inserted between the outer and inner contours of the archenteron rudiments, sketched in the diagrams with dotted lines. The shape component of the CPA was indicated by schematic profiles of the lobes at their maximal size. The motility component of the CPA (strong, moderate or low) was indicated by differences in the thickness of these contours, the greatest thickness denoting the highest motility. In a case with no tendency to lobe formation and consequently no local motility, the point selected was marked by a cross. The pseudopodia which appeared during the second phase of invagination were indicated by symbols resembling thick pseudopodia, and directed from their site of formation towards their point of attachment at the ectoderm. In addition, the first visible pseudopodium was marked by an arrow above the invagination curve. In order to obtain an integrated picture of the CPA and the course of invagination, the motility component of the CPA was also expressed by short lines crossing the invagination curve. These lines were of three different lengths, corresponding to strong (longest line), moderate, and low motility, respectively. A point with no motility was indicated by a gap in the sequence of cross-lines. An over-all picture of the motility during a time interval was obtained by repeating the sequences of cross-lines corresponding to the seven points. RESULTS
The material analyzed represents 9 films (about 700 m of film with various exposure intervals) of the whole of gastrulation, and a large number of shorter sequences in early gastrulation. Four of the complete cases are presented in Fig. 1, in the form of diagrams constructed on the principles described above. Among the features shown by the diagrams are the course of invagination and the variation in cellular pulsatory activity (CPA) in time and space. During liberation of the primary mesenchyme cells from the most vegetal part of the blastula wall, lively CPA occurred in this region (Fig. 1, b and d). Most of the pulsatory lobes corresponded to the whole free surface of cells bordering on the blastocoel (cf. Fig. 3), but cells with several lobules could Experimental
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Fig. 2.-Pulsatory activity of plasma lobes (CPA) in the archenteron rudiment miliaris, as revealed by a series of frames from a 16 mm reversal film. Interval vidual frames is 22 sec. The larva is of an aberrant type, since it lacks primary
of Psummechinus between the indimesenchyme cells.
also be discerned. Certain centrifugal pulsations were sometimes visible. The rate of pulsation varied, but 10 to 40 seconds for a complete cycle can be given as examples. The CPA in the primary mesenchyme cells decreased when they had left the blastula wall and had piled up at the vegetal pole (Fig. 1 b, d). The piled-up cells soon began to form pseudopodia or filopodia, Experimental
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H. Kinnander
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by which they moved radially, as described earlier [5]. Translocation of the primary mesenchyme cells could thus be divided into a first phase with high CPA, and a second phase with high pseudopodial activity. An intermediate phase of low activity was sometimes lacking in the pile of cells as a whole, in which case the two movement patterns overlapped to some extent. In suc.h cases, there was sometimes high CPA in the centre of the pile of mesenchyme cells at a time when the cells at the surface had already produced their pseudopodia. In other cases, where the pseudopodial activity started still earlier, pseudopodia were produced as soon as the cells had left their site in the blastula wall. The remaining cells in the blastula wall stayed fairly inactive until the time of invagination (cf. Fig. 1 d). Occasionally, pulsatory cells of an aberrant type occurred in the mesenchyme-blastula before invagination started. Such a case is symbolized in Fig. 1 b, detail c-e, in the left part of the vegetal blastula wall. In this event, both the rate and amplitude of pulsations were greater than ordinarily, and several giant lobes could be present concurrently. These aberrant cases were generally restricted to the periphery of the presumptive archenteron. Another type of aberrant case is shown in Fig. 1 d, detail D (cf. Fig. 3). In these cases, the CPA involved a large area of the vegetal hemisphere-even far beyond the area of the presumptive archenteron-and consisted of a large number of small, densely crowded, rapidly pulsating lobules. This activity, lvhich had a duration of e.g., 25 minutes, occurred shortly before the first, faint signs of beginning invagination. The curves of invagination (Fig. 1) showed a two-phase rise, separated by a more or less plateau-like segment, as described in an earlier paper [5]. This was fully evident, despite the fact that the time scale in the diagrams in Fig. 1 is more compressed than in invagination curves published earlier [5]. The plateau between the two rising curve segments was sometimes lacking, but the border between the two segments was then indicated by a break in the curve or a point of inflexion. The plateau or break regularly corresponded to the arrow indicating the onset of pseudopodial activity. From the very beginning of the first phase of invagination, a new wave of strong CPA took place in the blastula wall at the vegetal pole, this time in an area corresponding to the archenteron rudiment (Fig. 2). This ne\\- \vave of CPA lasted throughout the first phase of invagination, remained undiminished and even increased when the speed of invagination decreased (during the period corresponding to the plateau in the inragination curve). A considerable variation could be noted in the properties of the CPA. Some lobes corresponded to the whole centripetal surface of the cells; in other cases, Experimental
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Fig. 3.-Mesenchyme blastula stages of Psommechirrus miliaris from a 16 mm reversal film, showing the first faint signs of inragination, and an area with densely crowded pulsating lobules appearing for 25 minutes (in h, area indicated by arrows). The pear-shaped, centripetally pulsating cells in the presumptive archcnteron are visible in c. The picture series corresponds to the experiment in Fig. ld.Relative ages, the start of the experiment dcnoled as zero: o, 1 h 48’; b and c. 19 and 33 min. later, respectively.
Fig. 4. - ~hlitl-gastrula stages of Psammechimts miliuris from a 16 mm reversal film, showing rapid formation of a pseudopodium at the site indicated by an arrow in o. Two cones of attachment ran be seen in 5. The pirture series corresponds to the experiment in Fig. Id and the same larva as in Fig. 3. Relative ages in hours, minutes, and seconds of the three stages, at start of the cxperimcnt denoted as zero: o, 6 h 35’15”; h and c, 59” and 1’ 15” later, respectively.
several small lobules Fig. 2). The activity applied both to the plasmic movements. During the second began to rise steeply
or a single small lobe protruded from a single ccl1 (cf. \vas highest at the tip of the archenteron rudiment; this number of active cells and to the intensity of their cytocurve phase of invagination, i.e., \\-hen the invagination after the plateau, the CPA, still focused to the archenteron Experimentul
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tip, became essentially reduced. This did not, however, occur abruptly, but the CPA was in a sense inversely proportional to the number of long pseudopodia which appeared at the archenteron tip during this phase of invagination (cf. [5]). No systematic analysis was made of the mode of formation of the pseudopodia. It can, however, be mentioned that some of the most vigorously pulsating lobes finally threw out string-like “cascades” of cytoplasm into the blastocoel (cf. Fig. 4). These cascades were still attached to the cells from which they were given off. The “unsuccessful” cascades, i.e., those failing to reach the blastula mall, were retracted into the cell body, but sometimes such a cascade became more or less completely fragmented into a number of droplets, which remained in the blastocoel. The number of droplets of this kind often increased during the second phase of invagination, and formed a measure of the number of “unsuccessful” cascades. The “unsuccessful” cascades were perhaps guided by the very scanty network of a jelly-like substance, which was indirectly detectable by the restricted movements of rows of granules in the blastocoel, but which was sufficiently wide-meshed to allow occasional broken fibrils to bend freely. The cytoplasmic bridge between the archenteron tip and the blastula wall was classified as a pseudopodium or filopodium. The pseudopodia pulled the tip of the archenteron rudiment through the blastocoel. Fairly often; the tension in the pulling pseudopodia was clearly reflected by the formation of conical protrusions from the ectoderm, at the point of attachment of the pseudopodia, “cones of attachment”, cf. [2, 7] and Fig. 4. Sometimes the pseudopodia were extremely thin, even so thin that they were detectable only by the rows of granules attached to them or by the presence .of cones of attachement, which emphasizes the high mechanical resistance of these delicate fibrest Sometimes at least, these thin fibres were remnants of thicker pseudopodia. The pseudopodia were often branched at their ends, and thus had the possibility of shifting their site of attachment by a successive change in the points of attachment of the individual end Maments. The life time of the whole pseudopodium was, however, often very short, e.g., 15 minutes or less, cf. [5], and after being retracted into the cell body, it was sometimes replaced by a new pseudopodium with a more favourable direction. In certain cases there was a delay in formation of the primary mesenchyme, or even an invagination in blastulae lacking a mesenchyme. These cases, as well as some observations on exogastrulation, will be discussed in the following. Experimental
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Cellular
basis of gasfrulafion DISCUSSION
It is evident that both translocation of the primary mesenchyme cells and invagination are two-phase processes. In both cases, the first phase is characterized by high cellular pulsatory activity (CPA) in the translocating cells, and the second phase by high pseudopodial activity. The second phase of invagination is dependent on the pull exerted by pseudopodia formed by cells at the tip of the archenteron, cf. [5]. The same mechanism is active in migration of the primary mesenchyme cells along the blastula wall, from their site of entry into the blastocoel towards their site of final attachment, cf. [5, lo]. Other mechanisms must, however, be responsible for the translocations during the first phase of mesenchyme formation and invagination. A definite correlation exists between a wave of high CPA and translocation of the primary mesenchyme cells into the blastocoel, as well as between such a wave and early invagination. This suggests that the CPA or pulsatory activity of the centripetal plasma lobes plays an important role in these processesof translocation. It is therefore pertinent to discuss the cytologic nature of the CPA. Time-lapse films of cell cultures frequently show the formation of a great number of pulsatory plasma lobes at the two poles of cells in mitosis, this often being denoted as a “bubbling” activity. One could suggest that the CPA in the archenteron rudiment corresponds to such mitotic “bubbling”. In this event, it is, however, necessary to assume that the “bubbling” is restricted by some mechanism to the centripetal part of the cells in mitosis. The centrifugal surface of the cells is probably strongly attached to the hyaline layer, which may explain this restriction. However this may be, a connexion between mitotic activity and the occurrence of centripetal pulsatory lobes has been observed in the early sea urchin hlastula by Kuhl [8], using about the same exposure intervals and the same sea urchin species as in the present study. This activity reached its maximum at the time of separation of the blastomeres. Similar observations in early blastula stages have been made by the present authors (unpublished data). According to the aforegoing interpretation, the intense CPA in the’vegetal region indicates a high mitotic activity at this site. This is in agreement with the large number of mitotic figures observed in the vegetal region of the fixed and stained mesenchyme blastula [l]. Furthermore, in the time-lapse films, cell cleavage at the site of formation of primary mesenchyme cells can sometimes be observed, and in some cases it is evident that two cell layers have Experimental
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been formed in the archenteron tip at the end of the phase of primary invagination. This hypothetical interpretation can be concluded as follows. During a late blastula stage, a mitotic activity in the vegetal region forms primary mesenchyme cells, which are translocated into the blastocoel by a process related to a mitosis-associated CPA. During a subsequent phase, one can postulate a second wave of mitotic activity, associated with formation of the secondary mesenchyme cells; the CPA during this process causes a primary invagination. The formation of pulsatory plasma lobes is an indication of a local weakness in the cellular wall, which is not restricted to cells in mitosis but is of fairly widespread occurrence, e.g., in amoeboidic cells. As an alternate and better grounded interpretation of the CPA, the process can thus be regarded as the result of a weakening of the centripetal cell surfaces, unassociated with mitotic activity. This alternative avoids certain difficulties involved in the first one. It is namely difficult and conflicting with facts to assume that every invagination and folding in an embryo is associated with mitotic activity. Moreover, the scarcity of mitoses observed in the films makes the role of mitoses more than doubtful in this case. The question arises of how the CPA brings about the translocation of cells in the vegetal region. Invagination would be attained in the vegetal blastula wall if the cells there were to undergo a swelling of their centripetal ends; cf. the mechanisms of invagination discussed in the review of Moore [9]. The CPS involves a centripetal translocation of cytoplasmic material, which causes the cells to become much thicker at their centripetal than at their centrifugal ends. On mechanical grounds, this results in an invagination if the cellular interconnexions are strong. If, on the other hand, the cellular interconnexions are weak, the CPA of the presumptive mesenchyme cells may translocate the cell actively but more or less individually from the blastula wall into the blastocoel, in a way reminiscent of an amoeboidic movement. Halfway into the blastocoel, the cells may be squeezed out completely by the remaining cells in the vegetal plate. Early translocation of the primary mesenthyme cells and early invagination are thus closely related, CPA-dependent processes. This conclusion is further underlined by the occurrence of the following two anomalous types, which appeared spontaneously. In certain larvae, the CPA in the vegetal area became intense before weakening of the interconnexions between the future primary mesenchyme cells had taken place. In such cases a slight, transient “premature” invagination occurred, but ceased when the cells finally became free. Occasionally, there was no weakening of the interconnexions between the future primary mesenExperimental
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thyme cells. In this event, no primary mesenchyme appeared, but the CPA gave rise directly to an invagination. These observations show, in addition, that the ring of primary mesenchyme cells is not a prerequisite for the process of invagination, cf. [5]. It is possible that the CPA in the region of the presumptive mesenchyme cells and the presumptive archenteron is not the only mechanism of translocation of mesenchyme cells and early invagination. The field of rapidly pulsating lobules outside the regions mentioned (cf. above and Fig. 3) may sometimes contribute to creating pressure in the centripetal surface of the vegetal wall. This phenomenon will be further analyzed in a later paper. Exogastrulation in vegetalized, animalized or untreated larvae can be expected to result from centrifugal CPA in the presumptive archenteron. Several of our films show such aberrantly directed CPA in connexion with exogastrulation. This must be considered as strong support for the role of CPA in the process of invagination. The CPA described can bring about only comparatively shallow invagination. Further CPA will merely serve to make the archenteron rudiment more spherical; theoretically, a slight elongation may occur if individual cells in the archenteron tip slide against and climb upon each other. A second, efticient, mechanism is thus needed to stretch the archenteron rudiment across the blastocoel. This is the pseudopodial activity described above and in [5]. Although quite different in appearance from the pulsatory lobes, the pseudopodia may be the expression of qualitatively the same basic phenomenon as that underlying the CPA, i.e., weakening of the centripetal cell wall. In other words, the pseudopodial activity can be regarded as the result of an exaggerated CPA. Formally, contractile activity of the pseudopodium can be interpreted as related to the pulsations, but restricted to a single dimension. The number of “successful” cascades of cytoplasm emitted by cells in the archenteron tip increases with decreasing distance between the tip and the blastula wall. An indentation in the blastula wall will therefore appear to attract the archenteron tip, even in the absence of guiding agents in the blastocoel. In such cases, invagination proceeds obliquely towards the indented region, and the archenteron may even slide along the blastula wall, cf. [5]. These indentations may explain some of the variations between the curves of invagination. Another but probably very important cause of variation consists of differences in the relation between animal and vegetal strength during the period of determination. Such differences are manifested by variations in the relative size of the archenteron rudiment. In larvae which are slightly animalized, the pull in the pseudopodia cannot elongate the rudiment Experimental
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the I’seudopodia-forming: cells pull further or rapidly enough. Consecluently, This is the probable explanation themsclvcs out of the tip of the archentcron. of the diagram in Fig. 1 d in the present paper, as well as of curve 0 rl in i;i]. All the separate processes concerned in gastrulation are dependent on the cells being in a highly dynamic state. Rigid structural elements, e.g., coarse protein fihres, in the translocating cells would prevent the process. ‘This is in agreement with the view that protein anabolism, e.g., the formation of structural proteins from yolk, is more or less concentrated to the ectodermal areas of the larvae in the stage of development concerned [3, 4, 61, whereas the vegctal cells remain at a less advanced stage of development from a biochemical point of view. SUMMARY ‘I’hc cellular basis of early gastrulation in the sea urchin Psammechinus miliaris has been studied by means of time-lapse cinemicrography with relatively short intervals between individual exposures. The two main comof the primary mesenchyme and invaponents of gastrulation, i.e., formation gination, appear to depend on similar sequences of cellular changes. Pulsatory plasma lobes are formed centripetally by cells in the vegetal region of the larvae. Both the translocation of primary mesenchyme cells into the blastocoel and the primary phase of invagination are probably dependent on this cellular pulsatory activity (CPA). Further translocation of the mesenchyme cells and elongation of the archenteron rudiment are both the result of the contractile activity of pscudopodia, the formation of which is described. The nature and mode of action of the pulsatory lobes are discussed, as well as the relation between the pulsatory plasma lobes and the contractile pseudopodia. This work was partly carried out at Kristinebergs Zoologiska station, Fiskebgckskil. We wish to express our most cordial thanks to the station authorities, Professor John Runnstrijm and Fil.Dr. Gunnar Gustafson, for never-failing generosity. Our thanks are also due to Mr. Tore Dalberg, for the design and construction of certain elements in our equipment. The work has been supported by the Swedish Natural Science’Research Council. REFERENCES AGRELL, I., Arkiv Zool. 6, 13 (1953). DAN, K. and OKAZAKI, K., Biol. Bull. 110, 29 (1956). GUSTAFSON, T. and HASSELBERO, I., Ezpll. Cell Research 2, 642 (1951). GUSTAFSON, T. and HGRSTADIUS, S., ibid. Suppl. 3, 170 (1955). GUSTAFSON, T. and KmNmom, H., ibid. 11, 36 (1956). GUS~AFSON, T. and LENICQUE, P., ibid. 3, 251 (1952). KINSANDER, H. and GLXTAFSON, T., Arkiv Zool. 11, 117 (1957). KUHL, M’. and KUHL, G., Zool. Jahrb. 70, 1 (1949). 9. MOORE, A. R., Protoplasma 9, 25 (1930). 10. v. UBISCH, L., Z. Wiss. Biol. 149, 402 (1937). 1. 2. 3. 4. 5. 6. 7. 8.
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FURTHER STUDIES ON THE CELLULAR GASTRULATION IN THE SEA URCHIN H. KINNANDER The Wenner-Gren
Institute
for
and
Experimental Received
(1960)
BASIS OF LARVA
T. GUSTAFSON Biology,
April
19, 27X-290
University
of Stockholm,
Sweden
21, 1959
THE metabolic background of morphogenesis in embryos has been the object of investigation for a fairly long time. The morphologic events have, however, been used mainly as indicators of the kind of metabolism which takes place, e.g., during the “phases of determination” in early developmental stages. Little is known, on the other hand, of the way in which the metabolic processes operate in moulding the shape of the embryo. The gap between the biochemical level and the morphologic level is thus wide. As a contribution to bridging this gap, it would be appropriate to resolve the morphologic events in organs and tissues into morphologic processes at the cellular level, so simple that their biochemical background could be discussed and analyzed in the future. Time-lapse cinemicrography of the simple, transparent sea urchin larva during different phases of gastrulation and subsequent stages opens up the possibility of making such a resolution. In the sea urchin larva, gastrulation involves formation of the primary mesenchyme and a subsequent invagination of the presumptive archenteron material. In a preceding paper [5] these processes were discussed on the basis of data obtained by means of time-lapse cinemicrography. An analysis of the films suggested that the primary mesenchyme cells might be more or less passively squeezed out from the blastula wall into the blastocoel. This process will be further elucidated in the present paper. During a second phase of translocation of the mesenchyme cells, they migrate actively along the blastula wall by means of contractile pseudopodia or filopodia, until they reach their final point of attachment, cf. [lo]. The analysis also revealed two main phases in the subsequent invagination of the archenteron material, each characterized by a separate mechanism of translocation. The onset of the second phase of invagination could be correlated with the appearance of pseudopodia or filopodia from cells in the archenteron tip, the presumptive secondary mesenchyme cells. It is evident from the films and the diagrams of analysis that a causal relation exists betjveen these pseuExperimental
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dopodia and the process of invagination. The pseudopodia, in fact, pull the tip of the archenteron rudiment through the blastocoel, just as the pseudopodia of the primary mesenchyme cells pull themselves along the blastula wall. Most of the cells of the archenteron rudiment are thus passively translocated during the second phase of invagination [5]. The mechanism during the first phase of invagination has hitherto been obscure. The running films thus far only revealed a diffuse “boiling” activity at the vegetal pole of the advanced blastula and at the tip of the archenteron rudiment [5]. The primary aim of the work described in the present paper was to resolve this activity into its components, on the assumption that this may throw light on the mechanism of the first phase of invagination. In addition, the relation between mesenchyme formation and invagination will be discussed, as well as the possible relation between the different mechanisms involved.
TIME-LAPSE
TECHNIQUE
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In order to obtain detailed information from the time-lapse recordings, the exposure intervals between the single frames had to be reduced from the 18 or 10 secondspreviously used to e.g., 3 or 1 second. For 16 mm time-lapse cinemicrography we used a double installation, relatively easily transportable, consisting mainly of the equipment described earlier [5], and of Cine-Kodak Special II (CKS) camera mounted on the vertical supporting column of a boring-machine stand, and adapted over the microscope (Leitz Ortholux). For making single-frame exposures, the CKS camera has a “Dreh-Magnet” (Wilhelm Nass, Hannover, West Germany) for 6 volt DC connected to the eight-frame shaft. The exposure-interval timer is driven by a synchronous motor. The source of light was a low-voltage filament bulb, 6 volt, 5 amp. Wild KG 1 (3 mm) heat filters for microscopy were used, and the larvae were constantly illuminated for more than 20 hours at 3.5 amp. The development was normal, as with the intermittent illumination previously used. The microcine-adaptor for the CKS camera was a Reichert Kinekonnex. The microscope objectives used in the two equipments were Leitz apochromates 2410.65and 4010.95.“Nylon-net microaquaria”, as earlier described, were used to keep the larvae in a constant position, and the “cuvette slides” for the continuous supply of water were mainly of the type previously used [5]. The film material was Kodak Super X (now discontinued), Kodak Plus X and Agfa Isopan F, all reversal films. The films were analyzed as earlier in a G. B. Bell and Howell projector, especially adapted for time and motion studies. The use of a tape recorder during the analysis made it easy to document even minute, rapid, transient changesin the films. The observations were noted on key sort cards, which greatly facilitated the analysis of the abundant film material. The speciesusedin the investigation wasPsammechinus miliaris, the shallow form from the west coast of Sweden, and the temperature of development was about 20°C. 19
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DIAGRAMS
The film material analyzed consisted of films with various exposure intervals. The 3- to l-second interval films, about 50 per cent of the material analyzed, gave a distinct picture of details which were poorly resolved by films with longer intervals. Invagination was described by curves, invagination curves, obtained by plotting the height of the archenteron lumen (microns) against time (hours), cf. Fig. 1. The essential new information given by the short-exposure interval films, i.e., information on the “boiling” activity at the vegetal pole of the blastula and at the archenteron tip, could not, however, be analyzed in an exact quantitative way in a reasonable time. It was therefore necessary to make provisional use of a more subjective description of the process. The “boiling” proved to be resolvable into the pulsations of a great number of plasma lobes protruding from the surface of the cells in the most vegetal region of the young gastrula, finally concentrated to the apex of the archenteron rudiment and directed centripetally, i.e., against the blastocoel. The lobes were of different shape and size, and varied with respect to their amplitude and frequency of pulsation. Taken together, these characteristics, which were closely interrelated, can be said to express a cellular pulsatory activity, denoted in the following as CPA. The CPA varied from cell to cell and with the course of gastrulation. Symbols for the CPA were drawn close to the invagination curves in diagrams in the following way, cf. Fig. 1. The CPA in 7 points, each corresponding to one or a few cells, and evenly distributed along the archenteron rudiment, was used to represent the whole of the archenteron surface against the blastocoel. One point was stituated at the apex of the rudiment, two points at the base, on the left and the right, and four points were evenly distributed in the intermediate segment (see Fig. 1). In earlier stages, the test points were distributed along the vegetal blastula wall, mainly in an area corresponding to the presumptive archenteron. The CPA in the points selected was recorded while running the film at a constant speed during time intervals of constant length in each experiment. The length of the relevant time interval, e.g., 30 minutes, is given in the legends to the figures. It corresponded to the micron intervals, usually indicated by the horizontal lines to the right of the invagination curve in the diagrams. The profile of the archenteron rudiment at the beginning of each time interval was indicated by dotted contours. Although the intervals were long enough to disclose alterations in the CPA in the individual points, no difficulty \vas Experimental
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Fig. l.-Diagrams of the course of invagination in 4 larvae of Psammechinus miliaris. The height of the archenteron lumen is plotted against the relative age in hours, the first stage being arbitrarily denoted as 0 hours. For detailed explanation of the construction of the diagrams and the symbols used, see text p. 280. Dashed segment of curves: interpolated values. In d, this segment is only theoretical, since the pseudopodia-forming cells pulled themselves out from the archenteron tip before it had reached the animal pole. Length of the time intervals marked by the horizontal lines to the right of the curves (and by letters A-E to the left of the rising curve segments in b and d): a, b and c, 25 minutes; d, 41 minutes. Experimental
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encountered in estimating the mean activity, and repeated estimations showed good agreement. The CPA was not expressed after studying all its components separately, but by observing the shape of the lobes at their maximal size, and by subjective estimation of their motility, corresponding to components of amplitude and frequency. The shape and motility components of the CPA were indicated by symbols denoting the changes observed at the surface against the blastocoel. The symbols were inserted between the outer and inner contours of the archenteron rudiments, sketched in the diagrams with dotted lines. The shape component of the CPA was indicated by schematic profiles of the lobes at their maximal size. The motility component of the CPA (strong, moderate or low) was indicated by differences in the thickness of these contours, the greatest thickness denoting the highest motility. In a case with no tendency to lobe formation and consequently no local motility, the point selected was marked by a cross. The pseudopodia which appeared during the second phase of invagination were indicated by symbols resembling thick pseudopodia, and directed from their site of formation towards their point of attachment at the ectoderm. In addition, the first visible pseudopodium was marked by an arrow above the invagination curve. In order to obtain an integrated picture of the CPA and the course of invagination, the motility component of the CPA was also expressed by short lines crossing the invagination curve. These lines were of three different lengths, corresponding to strong (longest line), moderate, and low motility, respectively. A point with no motility was indicated by a gap in the sequence of cross-lines. An over-all picture of the motility during a time interval was obtained by repeating the sequences of cross-lines corresponding to the seven points. RESULTS
The material analyzed represents 9 films (about 700 m of film with various exposure intervals) of the whole of gastrulation, and a large number of shorter sequences in early gastrulation. Four of the complete cases are presented in Fig. 1, in the form of diagrams constructed on the principles described above. Among the features shown by the diagrams are the course of invagination and the variation in cellular pulsatory activity (CPA) in time and space. During liberation of the primary mesenchyme cells from the most vegetal part of the blastula wall, lively CPA occurred in this region (Fig. 1, b and d). Most of the pulsatory lobes corresponded to the whole free surface of cells bordering on the blastocoel (cf. Fig. 3), but cells with several lobules could Experimental
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Fig. 2.-Pulsatory activity of plasma lobes (CPA) in the archenteron rudiment miliaris, as revealed by a series of frames from a 16 mm reversal film. Interval vidual frames is 22 sec. The larva is of an aberrant type, since it lacks primary
of Psummechinus between the indimesenchyme cells.
also be discerned. Certain centrifugal pulsations were sometimes visible. The rate of pulsation varied, but 10 to 40 seconds for a complete cycle can be given as examples. The CPA in the primary mesenchyme cells decreased when they had left the blastula wall and had piled up at the vegetal pole (Fig. 1 b, d). The piled-up cells soon began to form pseudopodia or filopodia, Experimental
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by which they moved radially, as described earlier [5]. Translocation of the primary mesenchyme cells could thus be divided into a first phase with high CPA, and a second phase with high pseudopodial activity. An intermediate phase of low activity was sometimes lacking in the pile of cells as a whole, in which case the two movement patterns overlapped to some extent. In suc.h cases, there was sometimes high CPA in the centre of the pile of mesenchyme cells at a time when the cells at the surface had already produced their pseudopodia. In other cases, where the pseudopodial activity started still earlier, pseudopodia were produced as soon as the cells had left their site in the blastula wall. The remaining cells in the blastula wall stayed fairly inactive until the time of invagination (cf. Fig. 1 d). Occasionally, pulsatory cells of an aberrant type occurred in the mesenchyme-blastula before invagination started. Such a case is symbolized in Fig. 1 b, detail c-e, in the left part of the vegetal blastula wall. In this event, both the rate and amplitude of pulsations were greater than ordinarily, and several giant lobes could be present concurrently. These aberrant cases were generally restricted to the periphery of the presumptive archenteron. Another type of aberrant case is shown in Fig. 1 d, detail D (cf. Fig. 3). In these cases, the CPA involved a large area of the vegetal hemisphere-even far beyond the area of the presumptive archenteron-and consisted of a large number of small, densely crowded, rapidly pulsating lobules. This activity, lvhich had a duration of e.g., 25 minutes, occurred shortly before the first, faint signs of beginning invagination. The curves of invagination (Fig. 1) showed a two-phase rise, separated by a more or less plateau-like segment, as described in an earlier paper [5]. This was fully evident, despite the fact that the time scale in the diagrams in Fig. 1 is more compressed than in invagination curves published earlier [5]. The plateau between the two rising curve segments was sometimes lacking, but the border between the two segments was then indicated by a break in the curve or a point of inflexion. The plateau or break regularly corresponded to the arrow indicating the onset of pseudopodial activity. From the very beginning of the first phase of invagination, a new wave of strong CPA took place in the blastula wall at the vegetal pole, this time in an area corresponding to the archenteron rudiment (Fig. 2). This ne\\- \vave of CPA lasted throughout the first phase of invagination, remained undiminished and even increased when the speed of invagination decreased (during the period corresponding to the plateau in the inragination curve). A considerable variation could be noted in the properties of the CPA. Some lobes corresponded to the whole centripetal surface of the cells; in other cases, Experimental
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Fig. 3.-Mesenchyme blastula stages of Psommechirrus miliaris from a 16 mm reversal film, showing the first faint signs of inragination, and an area with densely crowded pulsating lobules appearing for 25 minutes (in h, area indicated by arrows). The pear-shaped, centripetally pulsating cells in the presumptive archcnteron are visible in c. The picture series corresponds to the experiment in Fig. ld.Relative ages, the start of the experiment dcnoled as zero: o, 1 h 48’; b and c. 19 and 33 min. later, respectively.
Fig. 4. - ~hlitl-gastrula stages of Psammechimts miliuris from a 16 mm reversal film, showing rapid formation of a pseudopodium at the site indicated by an arrow in o. Two cones of attachment ran be seen in 5. The pirture series corresponds to the experiment in Fig. Id and the same larva as in Fig. 3. Relative ages in hours, minutes, and seconds of the three stages, at start of the cxperimcnt denoted as zero: o, 6 h 35’15”; h and c, 59” and 1’ 15” later, respectively.
several small lobules Fig. 2). The activity applied both to the plasmic movements. During the second began to rise steeply
or a single small lobe protruded from a single ccl1 (cf. \vas highest at the tip of the archenteron rudiment; this number of active cells and to the intensity of their cytocurve phase of invagination, i.e., \\-hen the invagination after the plateau, the CPA, still focused to the archenteron Experimentul
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tip, became essentially reduced. This did not, however, occur abruptly, but the CPA was in a sense inversely proportional to the number of long pseudopodia which appeared at the archenteron tip during this phase of invagination (cf. [5]). No systematic analysis was made of the mode of formation of the pseudopodia. It can, however, be mentioned that some of the most vigorously pulsating lobes finally threw out string-like “cascades” of cytoplasm into the blastocoel (cf. Fig. 4). These cascades were still attached to the cells from which they were given off. The “unsuccessful” cascades, i.e., those failing to reach the blastula mall, were retracted into the cell body, but sometimes such a cascade became more or less completely fragmented into a number of droplets, which remained in the blastocoel. The number of droplets of this kind often increased during the second phase of invagination, and formed a measure of the number of “unsuccessful” cascades. The “unsuccessful” cascades were perhaps guided by the very scanty network of a jelly-like substance, which was indirectly detectable by the restricted movements of rows of granules in the blastocoel, but which was sufficiently wide-meshed to allow occasional broken fibrils to bend freely. The cytoplasmic bridge between the archenteron tip and the blastula wall was classified as a pseudopodium or filopodium. The pseudopodia pulled the tip of the archenteron rudiment through the blastocoel. Fairly often; the tension in the pulling pseudopodia was clearly reflected by the formation of conical protrusions from the ectoderm, at the point of attachment of the pseudopodia, “cones of attachment”, cf. [2, 7] and Fig. 4. Sometimes the pseudopodia were extremely thin, even so thin that they were detectable only by the rows of granules attached to them or by the presence .of cones of attachement, which emphasizes the high mechanical resistance of these delicate fibrest Sometimes at least, these thin fibres were remnants of thicker pseudopodia. The pseudopodia were often branched at their ends, and thus had the possibility of shifting their site of attachment by a successive change in the points of attachment of the individual end Maments. The life time of the whole pseudopodium was, however, often very short, e.g., 15 minutes or less, cf. [5], and after being retracted into the cell body, it was sometimes replaced by a new pseudopodium with a more favourable direction. In certain cases there was a delay in formation of the primary mesenchyme, or even an invagination in blastulae lacking a mesenchyme. These cases, as well as some observations on exogastrulation, will be discussed in the following. Experimental
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Cellular
basis of gasfrulafion DISCUSSION
It is evident that both translocation of the primary mesenchyme cells and invagination are two-phase processes. In both cases, the first phase is characterized by high cellular pulsatory activity (CPA) in the translocating cells, and the second phase by high pseudopodial activity. The second phase of invagination is dependent on the pull exerted by pseudopodia formed by cells at the tip of the archenteron, cf. [5]. The same mechanism is active in migration of the primary mesenchyme cells along the blastula wall, from their site of entry into the blastocoel towards their site of final attachment, cf. [5, lo]. Other mechanisms must, however, be responsible for the translocations during the first phase of mesenchyme formation and invagination. A definite correlation exists between a wave of high CPA and translocation of the primary mesenchyme cells into the blastocoel, as well as between such a wave and early invagination. This suggests that the CPA or pulsatory activity of the centripetal plasma lobes plays an important role in these processesof translocation. It is therefore pertinent to discuss the cytologic nature of the CPA. Time-lapse films of cell cultures frequently show the formation of a great number of pulsatory plasma lobes at the two poles of cells in mitosis, this often being denoted as a “bubbling” activity. One could suggest that the CPA in the archenteron rudiment corresponds to such mitotic “bubbling”. In this event, it is, however, necessary to assume that the “bubbling” is restricted by some mechanism to the centripetal part of the cells in mitosis. The centrifugal surface of the cells is probably strongly attached to the hyaline layer, which may explain this restriction. However this may be, a connexion between mitotic activity and the occurrence of centripetal pulsatory lobes has been observed in the early sea urchin hlastula by Kuhl [8], using about the same exposure intervals and the same sea urchin species as in the present study. This activity reached its maximum at the time of separation of the blastomeres. Similar observations in early blastula stages have been made by the present authors (unpublished data). According to the aforegoing interpretation, the intense CPA in the’vegetal region indicates a high mitotic activity at this site. This is in agreement with the large number of mitotic figures observed in the vegetal region of the fixed and stained mesenchyme blastula [l]. Furthermore, in the time-lapse films, cell cleavage at the site of formation of primary mesenchyme cells can sometimes be observed, and in some cases it is evident that two cell layers have Experimental
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been formed in the archenteron tip at the end of the phase of primary invagination. This hypothetical interpretation can be concluded as follows. During a late blastula stage, a mitotic activity in the vegetal region forms primary mesenchyme cells, which are translocated into the blastocoel by a process related to a mitosis-associated CPA. During a subsequent phase, one can postulate a second wave of mitotic activity, associated with formation of the secondary mesenchyme cells; the CPA during this process causes a primary invagination. The formation of pulsatory plasma lobes is an indication of a local weakness in the cellular wall, which is not restricted to cells in mitosis but is of fairly widespread occurrence, e.g., in amoeboidic cells. As an alternate and better grounded interpretation of the CPA, the process can thus be regarded as the result of a weakening of the centripetal cell surfaces, unassociated with mitotic activity. This alternative avoids certain difficulties involved in the first one. It is namely difficult and conflicting with facts to assume that every invagination and folding in an embryo is associated with mitotic activity. Moreover, the scarcity of mitoses observed in the films makes the role of mitoses more than doubtful in this case. The question arises of how the CPA brings about the translocation of cells in the vegetal region. Invagination would be attained in the vegetal blastula wall if the cells there were to undergo a swelling of their centripetal ends; cf. the mechanisms of invagination discussed in the review of Moore [9]. The CPS involves a centripetal translocation of cytoplasmic material, which causes the cells to become much thicker at their centripetal than at their centrifugal ends. On mechanical grounds, this results in an invagination if the cellular interconnexions are strong. If, on the other hand, the cellular interconnexions are weak, the CPA of the presumptive mesenchyme cells may translocate the cell actively but more or less individually from the blastula wall into the blastocoel, in a way reminiscent of an amoeboidic movement. Halfway into the blastocoel, the cells may be squeezed out completely by the remaining cells in the vegetal plate. Early translocation of the primary mesenthyme cells and early invagination are thus closely related, CPA-dependent processes. This conclusion is further underlined by the occurrence of the following two anomalous types, which appeared spontaneously. In certain larvae, the CPA in the vegetal area became intense before weakening of the interconnexions between the future primary mesenchyme cells had taken place. In such cases a slight, transient “premature” invagination occurred, but ceased when the cells finally became free. Occasionally, there was no weakening of the interconnexions between the future primary mesenExperimental
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thyme cells. In this event, no primary mesenchyme appeared, but the CPA gave rise directly to an invagination. These observations show, in addition, that the ring of primary mesenchyme cells is not a prerequisite for the process of invagination, cf. [5]. It is possible that the CPA in the region of the presumptive mesenchyme cells and the presumptive archenteron is not the only mechanism of translocation of mesenchyme cells and early invagination. The field of rapidly pulsating lobules outside the regions mentioned (cf. above and Fig. 3) may sometimes contribute to creating pressure in the centripetal surface of the vegetal wall. This phenomenon will be further analyzed in a later paper. Exogastrulation in vegetalized, animalized or untreated larvae can be expected to result from centrifugal CPA in the presumptive archenteron. Several of our films show such aberrantly directed CPA in connexion with exogastrulation. This must be considered as strong support for the role of CPA in the process of invagination. The CPA described can bring about only comparatively shallow invagination. Further CPA will merely serve to make the archenteron rudiment more spherical; theoretically, a slight elongation may occur if individual cells in the archenteron tip slide against and climb upon each other. A second, efticient, mechanism is thus needed to stretch the archenteron rudiment across the blastocoel. This is the pseudopodial activity described above and in [5]. Although quite different in appearance from the pulsatory lobes, the pseudopodia may be the expression of qualitatively the same basic phenomenon as that underlying the CPA, i.e., weakening of the centripetal cell wall. In other words, the pseudopodial activity can be regarded as the result of an exaggerated CPA. Formally, contractile activity of the pseudopodium can be interpreted as related to the pulsations, but restricted to a single dimension. The number of “successful” cascades of cytoplasm emitted by cells in the archenteron tip increases with decreasing distance between the tip and the blastula wall. An indentation in the blastula wall will therefore appear to attract the archenteron tip, even in the absence of guiding agents in the blastocoel. In such cases, invagination proceeds obliquely towards the indented region, and the archenteron may even slide along the blastula wall, cf. [5]. These indentations may explain some of the variations between the curves of invagination. Another but probably very important cause of variation consists of differences in the relation between animal and vegetal strength during the period of determination. Such differences are manifested by variations in the relative size of the archenteron rudiment. In larvae which are slightly animalized, the pull in the pseudopodia cannot elongate the rudiment Experimental
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the I’seudopodia-forming: cells pull further or rapidly enough. Consecluently, This is the probable explanation themsclvcs out of the tip of the archentcron. of the diagram in Fig. 1 d in the present paper, as well as of curve 0 rl in i;i]. All the separate processes concerned in gastrulation are dependent on the cells being in a highly dynamic state. Rigid structural elements, e.g., coarse protein fihres, in the translocating cells would prevent the process. ‘This is in agreement with the view that protein anabolism, e.g., the formation of structural proteins from yolk, is more or less concentrated to the ectodermal areas of the larvae in the stage of development concerned [3, 4, 61, whereas the vegctal cells remain at a less advanced stage of development from a biochemical point of view. SUMMARY ‘I’hc cellular basis of early gastrulation in the sea urchin Psammechinus miliaris has been studied by means of time-lapse cinemicrography with relatively short intervals between individual exposures. The two main comof the primary mesenchyme and invaponents of gastrulation, i.e., formation gination, appear to depend on similar sequences of cellular changes. Pulsatory plasma lobes are formed centripetally by cells in the vegetal region of the larvae. Both the translocation of primary mesenchyme cells into the blastocoel and the primary phase of invagination are probably dependent on this cellular pulsatory activity (CPA). Further translocation of the mesenchyme cells and elongation of the archenteron rudiment are both the result of the contractile activity of pscudopodia, the formation of which is described. The nature and mode of action of the pulsatory lobes are discussed, as well as the relation between the pulsatory plasma lobes and the contractile pseudopodia. This work was partly carried out at Kristinebergs Zoologiska station, Fiskebgckskil. We wish to express our most cordial thanks to the station authorities, Professor John Runnstrijm and Fil.Dr. Gunnar Gustafson, for never-failing generosity. Our thanks are also due to Mr. Tore Dalberg, for the design and construction of certain elements in our equipment. The work has been supported by the Swedish Natural Science’Research Council. REFERENCES AGRELL, I., Arkiv Zool. 6, 13 (1953). DAN, K. and OKAZAKI, K., Biol. Bull. 110, 29 (1956). GUSTAFSON, T. and HASSELBERO, I., Ezpll. Cell Research 2, 642 (1951). GUSTAFSON, T. and HGRSTADIUS, S., ibid. Suppl. 3, 170 (1955). GUSTAFSON, T. and KmNmom, H., ibid. 11, 36 (1956). GUS~AFSON, T. and LENICQUE, P., ibid. 3, 251 (1952). KINSANDER, H. and GLXTAFSON, T., Arkiv Zool. 11, 117 (1957). KUHL, M’. and KUHL, G., Zool. Jahrb. 70, 1 (1949). 9. MOORE, A. R., Protoplasma 9, 25 (1930). 10. v. UBISCH, L., Z. Wiss. Biol. 149, 402 (1937). 1. 2. 3. 4. 5. 6. 7. 8.
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