The organization of honey bee embryonic cells

The

Organization

of Honey

I. Microtubules

and

Bee

Amoeboid

Embryonic

Cells

Activity

INTHODI!CTIOlY

A sophisticated technique for whole mount electron microscopy, based on the surface-spreading procedures of Kleinschmidt (1962) together with Anderson’s (1951) critical point drying method, has recently been introduced by Gall (1963). Modifications of this technique, when applied to honey bee embryonic cells, have yielded significant insights into the macromolecular organization of interphase nuclei and metaphase chromosomes ( DuPraw, 1965a,b). This report will deal with electron microscope observations of extranuclear organization, as seen in unsectioned honey bee embryonic cells, and these observations will be correlated with phase contrast and interfrrencc- microscope analysis of morphogenetic cell movements in uivo. XlATEHIALS

AND

METHODS

The events and timing of cmbryogenesis in the honey bee, as well as suitable techniques for handling the embryos, have been worked out by Nelson (1915), Schnetter (1934a,b), and DuPraw (1961, 1963a,b). Timed or untimed eggs are removed from the comb by cutting out the bases of individual hexagonal “cells,” each with a single basally attached embryo. In order to maintain the embryos to hatching, two methods are commonly used in this laboratory: the first is essentially that of Schnetter (1934a), in which the embryos (still adhering to their waxy bases) are placed in an ordinary loo-mm petri dish, moist cotton is added to maintain humidity, and the covered petri dish is incubated at 355°C for the 7%hour period of development. In the second technique, introduced by DuPraw (1963a), the embryos are immersed in paraffin oil (Fisher Scientific 53

54

E.

J.

DUPRAW

Co., viscosity 125/135, cat. no. O-119) and removed from their bases by gentle prodding with a blunt glass microtool. Immersed embryos become beautifully transparent, permitting accurate determination of developmental stages, and in the Fisher paraffin oil viability is unimpaired, making it possible to film the entire sequence of development by time-lapse techniques ( DuPraw, 1963b). Once the embryos are immersed, no special precautions are necessary to maintain humidity; in this laboratory oil-immersed embryos are usually incubated in uncovered 60-mm petri dishes, and the embryos are readily sorted into different dishes with an ordinary eyedropper pipette. Phase contrast and interference microscope observations of honey bee embryonic cells have been carried out with “squash culture” preparations of oil-immersed embryos. In this technique a single embryo is pipetted in a small drop of paraffin oil onto the surface of an ordinary, clean microscope slide; a coverslip is then added with a slight lateral “smear” motion, which ruptures and spreads the embryo. In successful preparations, such slides contain small groups of cells, often in monolayers, which are entirely surrounded by oil but separated from it by an invisibly thin layer of aqueous plasm. Provided the slide is kept warm in a microscope stage incubator, the cells remain viable for some hours, exhibiting both mitosis and conspicuous amoeboid activity. These phenomena have been photographed with an Arriflex time-lapse apparatus. The techniques for preparing electron microscope “whole mounts” with honey bee embryonic cells have been described in detail by DuPraw ( 1965). E m b r y OS maintained by Schnetter’s technique (i.e., not oil immersed) are removed from their bases and thoroughly squashed on the tip of a blunt glass microtool. The microtool is then touched to the clean air-water interface of a Langmuir trough, where surface tension forces spread and rupture the cells. Spread material is immediately picked up on a Formvar-carbon coated electron microscope grid by touching the trough surface lightly with the coated side of the grid (several such grids can be prepared from one spreading). The material may then be fixed and/or stained and/or washed by floating the grids face down on the appropriate reagents or on distilled water. After washing, the grids may also be experimentally treated by immersion in specific enzyme solutions. It should be emphasized that unfixed, unstained (and unsectioned) cells are suitable for study, presenting an appearance in the electron microscope not notably

~IICHOTUBULES

AND

AMOEBOID

ACTIVITY

55

inferior to more elaborately treated material. After fixing, washing, or further experimental treatment, grids are placed vertically in a special plastic grid holder under 25% ethanol; holder and grids are then dehydrated through an ethanol series, passed into amyl acetate, and dried bv the Anderson critical point method. The -degree and type of preservation of intranuclcar and extranuclear detail has been found to varv considerably, dcpcnding on th(s solution used for spreading in the Langmuir trough. Although some workers spread on distilled water, in this laboratory 0.25 iU sucrose is used for preparations of chromosomes and m&i, and 0.73 M sucrose (25%) is used to obtain preparations in which abundant cxtranuclcal structure is preserved. OHSEH\‘A’I‘IOhS

,4fter about 31 hours of development (at 35.5”C), the cells of intact honey bee embryos begin to exhibit marked morphogenctic movements ( DuPraw, 1963b). One of the earliest of these movements is the overgrowth of a mid-ventral mesoderm plate bv two lateral sheets of ectoderm, which meet and fuse in the ventral midline (set illustrations of sectioned embryos by Nelson, 1915). This process of overgrowth, which is typical of insect embryos, occupies about 6 hours in the honey bee, during which the advancing margins of the two rctoderm sheets are easily visible in the intact embryo (Fig. 1) (DuPraw, 1963b). In sectioned embryos at this stage (stage B), the ectoderm does not show notable mitotic activity, and the working hypothesis in this laboratory has been that ectoderm migration occurs primarily by amoeboid activitv of the individual cells. Such an interpretation is consistent with th; observation that the ectoderm boundaries, at higher magnifications , show irregularly scalloped profiles, as if the rate of progress is subject to local fluctuations. Development of a “squash culture” technique by DuPraw ( 1963a) has permitted phase contrast and interference microscope observation of living honey bee embryonic cells under conditions when mitotic activity continues for several hours. Such squash preparations, when made with stage 6 embryos, contain both isolated single cells and flattened cell monolayers, both of which exhibit conspicuous plasmatic movements. After direct studv combined with time-lapse analvsis, two

56

E. J. DU

PRAW

distinct phenomena appear to be involved in these movements: in the first place, saltatory movements of certain conspicuous, dense spheroid cytoplasmic granules of varying diameters (ranging up to 1 p) (see Figs. 2 and 3); and in the second place, the formation of hyaline pseudopodia of the classic lobate type (Fig. 3). Under some conditions, marked saltatory granule movements occur in the absence of

6. (A) Early stage 6 honey bee embryo in lateral aspect. Ectodermal FIG. 1. overgrowth first becomes visible as a bright longitudinal line in the anterior half of the embryo. (B) A somewhat later stage 6 embryo in ventral aspect. The ectoderm sheets meet first in the anterior third of the embryo, and close up toward the posterior in “zipper” fashion.

and under other conditions a cell showing pseudopod formation, extremely active pseudopod formation may simultaneously exhibit only minimal saltatory movement. Both types of movement may be reduced or absent after “chilling” at room temperature, and at these times the granules show a tendency to accumulate in the vicinity of the nucleus. Finally, after incubation both types of movement usually occur together,

.\IICROTUBULES

AND

AMOEBOID

ACTIVITY

457

The patterns of movement in stage 6 cells are distinctly different. depending on whether the cells are single or in multicellular monolayers. In a typical isolated cell, the nucleus occupies a central position and, in the absence of pseudopod formation, saltatory movements are apparently random. Each cytoplasmic particle moves independently, unpredictably, and in a straight path, but a single “jump” is

‘d 2. Diagram of three marginal cells from an isolated stage 6 monolayer. Both pseudopod formation and saltatory granule movements are oriented in the direction of the cell long axis. Approximate granule paths are indicated by the arrows. FIG.

rlsually only a few microns long. When seen in time-lapse films, these movements are also remarkably oscillatory in nature; i.e., a granule which has completed a movement in one direction shows a definite tendency to move back again in a more or less opposite direction, The overall pattern of saltation, when observed by time-lapse techniques, is sometimes reminiscent of Brownian movement, but as noted by Kebhun ( 1!364), saltatory movements in true time are much slower (and therefore seem more deliberate) than in Brownian motion. Pseudopod formation in isolated cells is also multidirectional and has a random quality; i.e., the cell surface can apparently “bleb” out independently at any point on its circumference. Sometimes a broad pseudopod progresses circumferentially around the cell periphery,

(A) Pseudopod formation at the free edge of an isolated stage 6 FIG. 3. monolayer. (B) The same cells photographed a few minutes later. Note apparent progress in the direction of inert debris (arrow). Phase contrast. Magnification: about X 1600. 58

Frc. 4. Stage G honey bee embryonic cell, spread on 0.25 M sucrose and dried by Anderson’s critical point technique. Note numerous cytoplasmic granulea. which surround the dense nucleus and are interconnected b\r an extensive svstem of extranuclear fibers. hlagnificntion: X 6900. 59

60

E. J. DU

PRAW

giving the impression in time-lapse films that the nucleus constitutus a fixed point while the mass of the cytoplasm moves around it. Narrower and longer pseudopods may double back on themselves, forming “U’s” or “S’s” in which the arms are separated from one another only by narrow extracellular channels. In such cells the saltatory movement of granules continues to be random and oscillatory, but superimposed on these saltatory movements are more massive distortions produced at points where cytoplasm suddenly flows out into a hyaline pseudopod. In cells which are not single, but form parts of a multicellular sheet or monolayer, these random patterns are notably absent. Formation of typical pseudopods, when it occurs in such monolayers, is detectable only in cells at the edge of the sheet, and these marginal cells exhibit pseudopods only at their free edges (Fig. 3). Nevertheless, it is clear that cells enclosed within the monolayer remain plastic and capable of flow; this is most apparent during mitotic division of an interior cell, when neighboring cells flow into the telophase furrow as it forms, and it is also shown by gradual changes in the shapes and relative positions of interior cells. However, active pseudopod formation at a free edge seems to confer a polarity on plasmatic flow within the entire monolayer, and this oriented amoeboid activity can be correlated, in some instances, with observable progress by the monolayer in the direction of the active free edge. Comparison of Figs. 3A and 3B, which were photographed several minutes apart, shows that in Fig. 3B the free edge has moved visibly closer to inert debris lying in the expected direction of movement; these two photographs belong to a series of five negatives, in which the distances from the cell nuclei to a fixed point on the debris become progressively shorter, and at the same time the dimensions of the nuclei themselves do not change (indicating that the apparent motion is not due to progressive flattening of the cells). During this type of oriented pseudopodal activity the marginal cells often assume forms very reminiscent of Amoeba in unidirectional movement; i.e., the cell becomes elongate, flaring slightly to a blunt tip, while the nucleus assumes a notably posterior position (Fig. 2); small hyaline pseudopods are continually formed at the blunt anterior end. Such cells, when viewed in time-lapse films, exhibit saltatory movements which are also nonrandom; i.e., there is a marked tendency for the granules to move backward or forward in the direction

\IICROTUBULES

AND

AhfOEBOID

ACTIVITY

61

of the cell’s long axis (Fig. 2). It is also very striking that the rate of saltation is distinctly more rapid than the rate of pseudopod formation; there is a definite impression that the two types of plasmatic activity reflect two different processes whose basic polarity is nevertheless the same. \Yhole

Mount Electron

Microscopy

When stage 6 honey bee embryonic cells are spread on 0.25 M sucrose, picked up on electron microscope grids, and dried by Anderson’s critical point method, interphase nuclei and metaphase chromosomes are usually preserved to the exclusion of cytoplasmic elements ( DuPraw, 1965a). However, groups of extranuclear organelles, appearing as dense spheroids, are occasionally found adhering to a nucleus by fine fibers (e.g., see Fig. 1 in DuPraw, 1965a). After spreading on 0.73 M sucrose, these extranuclear organelles are present much more frequently and in greater abundance. Figure 4 illustrates an entire cell. in which the central dense nucleus is surrounded with numerous

FIG.

5.

embryonic for saltatory

Diagram of intraand extranuclear fibers cell. Contraction of the extranuclear fibers movement of the cytoplasmic granules.

in a stage is postulated

6 honey bet to account

62

E. J. DLJ PRAW

MICHOl-UBUI,ES

AND

AMOEBOID

ACTIVITI-

63

granules, and these granules are connected to one another, as well as to the nucleus, bv an elaborate network of fine filers. The number (if granules prcscrvcd by thcsc tc&niques is highl! variable from cell to cell and may bc partly related to variations in the granule content of different cells in tiiuo (e.g., presumptive ectodermal or mesodermal cells, which cannot be distinguished after surrelationships obface spreading). In any case, the gramlle-nucleus served in many independent preparations leave little doubt that the dense, estranuclear spheroids seen in whole-mount electron microwith the saltatory particles visible by phase constop) are identical trast in living cells from embryos of the same stage (Fig. 3). Figure S illustrates these organelle relationships in a stage 6 honey bee embryonic cell, as visualized by both techniqws. The organization of the intranuclear fibers and the nuclear envelope, as shown in Fig. 5, has already been described in some detail by DuPraw (1!365a). Although the nuclear structures have not been well preserved in the particular cell shown in Fig. 4, this micrograph does emphasize the morphological distinctness between intra- and extranuclear fibers, a feature which is seen even more clearly at higher magnification in Fig. 6. As reported previously, the intranuclear chromatin fibers arc> typically “bumpy” or twisted, which gives them a filigree appearance (Fig. 6, top); tl le extranuclear fibers, on the other hand, appear very Frc. 6. Detail from Fig. 4 at higher magnification. Note the contrast hetwecn intranuclear fibers, which have a “filigree” nppearancc ( top), and the rclativch straight extranuclear fibers. Magnification: x 13,785. FIG 7. A single cytoplasmic granule from a stagy 6 hone!- bee embqwnic ccl1 (fixed in 10% formalin). Note apparent broken ends of extranuclear fibers. indicating multiple attachments between the granule and the fibr~ svstrm. Magnification: X 61,860. FIG. 8. (A) Nucleus and cytoplasmic granules from a stage 6 honey brc embryonic cell after spreading, critical point drying, and platinum shadowing. Granules are attachecl to the nucleus (top) bv a single, long fiber. hlagnification: x 2800. (B) Detail from same preparation; note that granules are npprosimatel) spherical (2: 1 platinum shadow ). hlagnification: x 5160. FIG. 9. Cytoplasmic granules and extranuclear fibers from a stage 6 hone\ bee embryonic cell. Granules are connected to nuclear cnvelopr ( top ). Note tendency to parallel orientation of fibers and “collapsed tube” configurations (bottom). 2: 1 platinum shadow. Magnification: X 26,545. FIG. 10. Cytoplasmic granule firmly attached to outside of nuclear envclopc ( stag 6). Note nonporous annuli (arrows). 2 : 1 platinum shadow. Mapnification:

K 51,750.

64

E. J. DU

PRAW

straight by contrast, and give the impression of being under tension (Fig. 6, bottom). Their diameters vary somewhat, presumably with the degree of stretching, but typical dimensions range around 185 A. These fibers are also often seen to join together in pairs, forming double strands of larger diameter. Figure 7 illustrates one of the cytoplasmic granules at high magnification. These organelles are sac-like objects, semitransparent to the electron beam, but showing no special internal structure; the granule illustrated in Fig. 7 has a maximum dimension of 0.37 p. Of particular interest are the appendages adhering to the granule, which have the aspect of broken fiber endings and indicate that each organelle may have multiple attachments to the fiber system. Also visible in Fig. 7 is a length of fiber which appears to have folded into an accordian-like configuration, suggesting that the extranuclear fibers in wioo have elastic or contractile properties. Evidence that the extranuclear fibers are not artifacts is provided by the occurrence of the broken fiber ends, as well as by the fact that the cytoplasmic granules often appear to be held on the grid primarily by their attachments to the fibers. In fact there is usually a very close correlation between the abundance of cytoplasmic granules retained in a surface-spread preparation and the preservation of the extranuclear fibers. In Fig. 8A and B, a chain of granules is connected to one another and to the nucleus by a single, long fiber. In Fig. 9, several granules are connected in a more intricate fashion to the nuclear envelope (top). In Fig. 10 a single granule is seen adhering closely to the outside of a well-preserved nuclear envelope, which shows typical (nonporous) annuli. The extranuclear fibers are completely eliminated after brief digestion in 0.001% trypsin solution (typical fibers being present on undigested control grids), even when the same treatment only partially degrades the nuclear envelope and intranuclear fibers. A very significant feature, brought out by platinum shadowing in Figs. 8B and 9, is that the heights of extranuclear fibers lying on the surface of the grid are sometimes considerably less than the heights of equivalent fibers suspended above the surface. This relationship indicates that the fibers tend to collapse after drying and suggests that they are hollow in structure; in fact, very clear “collapsed tube” configurations may be seen near the bottom of Fig. 9.

DISCI’SSIOIZ The occ’urr~~nc~~ ot: oricntcd amoeboid movement in isolated monolayers of honey bee embryonic cells strongly suggests that, in the intact embryo, the advance of lateral ectoderm over the ventral mesodcrm plate occurs by a similar process of pseudopodal activity. This interpretation is cntircly supported by the patterns of activity observed both in the isolated monolayers and in the advancing cctoderm borders of stage 6 embryos. Apparently the separation of two free cctoderm margins is sufficient stimulation to initiate creeping of the monolayers, which continues until the free margins meet ventrally and fuse into a continuous layer. In other types of embryo also, thcrc is cvidcnce that mass morphogenetic movements can be controlled or brought about by pseudopodal activity of individual cells (Dan and Okazaki, 1956; Gustafson and Wolpert, 1961; DeHaan, 1963). Although saltatory movements of cytoplasmic organclles are vcq conspicuous in honey bee embryonic cells in uioo, the rates and pattern of saltation seem to be relatively independent of the rate and pattern of psrudopod formation. During saltation the granules follow straight or angular paths, exactly as if they arc being pulled by strings, and their tendency to oscillatory movrments suggests that the same gram&! may be pulled in different directions by different strings (see Fig. 9). Electron microscope observations of the same cells, prepared as whole momlts, reveals that the cytoplasmic organellcs are indeed connected to one another and to the nuclear envelope by stringlike fibers, whose dimensions place them below the limits of resolution of the light microscope. The obvious interpretation is that, in light microscope images, resolvable cvtoplasmic granules are moved about by unresolvablc~ fibers, and this suggests that the cstranuclcar fibers detected by whole-mount electron microscopy have contractile properties. Parpart (1963) has, in fact, postulated the existence of such cytoplasmic contractile fibers to account for saltatory motion of echinochrome granules in Arbaciu. Recently the introduction of glutaraldehyde fixation for thin section electron microscopy has led to the discovery of previously unrecognized, fiberlike elements, which occur widely in the cytoplasm of both animal and plant cells. These cytoplasmic elements have the appearance of hollow tubes in cross section, and they have therefore been named “microtubules” by Lcdbetter and Porter (1963). In different

66

E. J. DU

PRAW

cell types, microtubules vary considerably both above and below 203A in diameter, and in longitudinal sections they appear to run in straight courses for indeterminate distances of at least several microns. Sandborn (1964) and Sandborn and co-workers (1964) have also found evidence that microtubules in rat neurons are connected both to mitochondria and to the nuclear envelope. It appears, therefore, that the extranuclear fibers described in the present report resemble, both in configuration and general dimensions, microtubules found in various cells after thin-sectioning. This correspondence is further supported by the occurrence of “collapsed tube” effects in the honey bee extranuclear fibers, which are similar to images observed by Pease (1963) in whole-mount preparations of flagellar fibrils; the latter fibrils have been regarded by Slautterback (1963) and Ledbetter and Porter (1964) as possibly homologous with microtubules. Preservation of the honey bee extranuclear fibers also seems to be enhanced after glutaraldehyde fixation. Since microtubules are conspicuous in regions of active cell synthesis, Ledbetter and Porter (1963) and Slautterback (1963) have postulated that they function in intracellular transport (particularly of small molecules). However these authors have also discussed a possible homology with the well-known tubular elements (of similar dimension) which comprise the 9 + 2 structure of cilia and flagella, and the fibers of the mitotic spindle. It is apparently the former relationship which suggested to Ledbetter and Porter that the microtubules in vivo may produce undulatory movements in the cytoplasm. More recently, Porter et al. (1964) have recognized a correlation between microtubule orientations and directions of plasmatic flow, and they have suggested that microtubules function as cytoskeletal elements to maintain asymmetric cell shapes. Observations reported in this paper would suggest still another type of microtubule activity, i.e., direct contractions or axial shortening to control the distribution and movements of cytoplasmic organelles. Such postulated contractile activity emphasizes and supports the homology with mitotic spindle fibers (see Rebhun, 1964), and in fact, the oscillatory movements of cytoplasmic granules in honey bee embryonic cells are often reminiscent of oscillatory movements sometimes exhibited by chromosomes during metaphase ( Mazia, 1961). In most types of amoeboid cell, oriented movements of cytoplasmic granules are closely correlated with the polarity of pseudopod forma-

hlICROTUHULES

AiN3

AhlOEBOlD

ACTI\‘ITi

67

Con. III fact, interpretations of granule movements have influenced or even suggested most current hypotheses of amoeboid movement (Mast, 1926; Allen, 1961; Jahn and Rinaldi, 1959). Even the wellknown distinction between “plasmagel” and “plasmasol” states in protoplasm is based primarily on localized differences in the freedom It is therefore particularly of movement of cytoplasmic organclles. significant that in honey bet embryonic cells, though the granule movements often show the same polarity as pseudopod formation, the rate and pattern of these movements is strikingly independent of the rate and pattern of pseudopod formation. The conclusion is very compelling in this material that the movements of cytoplasmic organelles are brought about by a process which is differcnt from the process of pseudopod formation. In these cells, a “plasmagel” condition might be thought to exist if the extranuclear fiber system were highly contracted, therebv preventing the movement of the attached cytoplasmic granules; n’“plasmasol” condition, on the other hand, would correspond to a state in which some fibers contract while others relax, leading to observable saltatory movements. In either instance, the pattern of granule movements would not necessarily be a reliable guide to the physical state or direction of flow of the hyaline plasm. These considerations may apply to other amoeboid cells as well, since the pattern of granule movements and the relative position of the nucleus during amoeboid movement are known to differ widely among different cell types (including among the various classes of human leucocyte). The classic “fountain” pattern of granule movement observed in Amoeba is not immediately evident in many amoeboid cells. Despite this perplexing variability, it is possible that the formation of hyaline pseudopods occurs by the same mechanism in all cells, while the pattern of granule movements depends on specific restrictions imposed by an extranuclcar fiber system. The same restrictions could also account for the invisible boundary which appears to exist in most amoeboid cells between the granular endoplasm and the hyaline pseudopod; this boundary may represent, not a wall in front of the particles, but a leash restraining them from behind, presumably bv fixing their positions relative to the nuclear envelope. That an extranuclear fiber system similar to that of honey bee embryonic cells occurs in other cell types is supported bv various observations in the literature. Merriam ( 1959, 1961), in his studies

68

E. J. DU

PRAW

with isolated nuclear envelopes, detected long, invisible attachments between the nucleus and cytoplasmic granules (mitochondria and yolk) in oocytes of both Rana pipiens and Chaetopterus. In Amoeba itself there is a variety of data to show that invisible structural elements exist even in the apparently fluid endoplasm (reviewed by Allen, 1961). Rebhun ( 1964) has also reviewed evidence that saltatory movements are brought about by the same mechanism wherever they occur and that this mechanism involves an invisible fiber system. Finally, the development of techniques for preserving microtubules in thin-section electron microscopy has resulted in rapid accumulation of reports showing that these fibrous elements occur widely in plant and animal cells, where they are sometimes permanent and sometimes transitory cell structures, and where their orientations are apparently related to the direction of cytoplasmic flow (Porter et al., 1964). It may be worth considering that if microtubules are, in fact, readily assembled and disassembled in many or all cell types, then amoeboid movement might be accounted for by a process of microtubule assembly at the anterior end of a cell, accompanied by microtubule disassembly at the posterior end (a mechanism analogous to that required to move a locomotive across country using only two lengths of train track). The essential event in this “disassembly-reassembly hypothesis” would be an outflow of “microtubule precursor solution” (the hyaline cap), followed by rapid assembly of new fibers and integration with the existing endoplasmic fiber-granule system (visible as a “bursting” of granules through the endoplasmic-ectoplasmic barrier). In this conception, net pseudopodal locomotion would be due to the assembly of microtubules, while the movement of cytoplasmic granules would be due to contraction of microtubules. Such a mechanism would not be fundamentally incompatible with the “fountain zone contraction hypothesis” of amoeboid movement, as developed by Allen ( 1961) . SUMMARY

In the development of the honey bee embryo, two lateral sheets of ectoderm overgrow a mid-ventral mesoderm plate, eventually meeting and fusing in the ventral midline. Cells from embryos at this stage (stage 6), isolated either singly or as multicellular monolayers, exhibit active pseudopod formation which, in the monolayers, is confined to the free edges of the marginal cells; this pseudopodal activity

hIICRO’IWBULES

AND

AMOEBOID

ACTIVITl

69

can sometimes be correlated with net locomotion in a direction normal to the free edge of the monolayer. It is therefore proposed that amoeboid movement by individual cells can account for the movement of lateral ectoderm in the intact embryo. Stage 6 embryonic cells also exhibit typical saltatory movements of relationships have been obcytoplasmic organelles. The following served: ( 1) Saltatory movements without pseudopod formation; (2) active pseudopod formation with minimal saltatory movements; (3) neither type of movement; (4) both movements together. In the latter case, when pseudopod formation has an obvious polarity (e.g., in the marginal cells of a monolayer), the saltatory movements may show a similar polarity, but they occur at a much more rapid rate. The evidence suggests that pseudopod formation and movement of cytoplasmic granules involve separate mechanisms in these cells. Whole-mount electron microscopy of stage 6 cells reveals that the cytoplasmic organelles are connected to one another and to the nuclear envelope by an extensive system of fine fibers. These fibers vary around 185 A in diameter; they run in straight courses for distances of several microns, and in general, they conform with the organization of “microtubules” (recently described in other types of cell after thin-sectioning), Saltatory movement of cytoplasmic organriles in honey bee embryonic cells would appear to be due to longitudinal contraction of the extranuclear fibers or “microtubules.” On the basis of observations in this report, a possible mechanism of amoeboid movement is proposed, which depends on assembly of microtubules at the anterior end of the cell, accompanied bv disassembly at the posterior end. In this scheme, net pseudopodal locomotion would depend on cr.ssembZr/ of microtubules, while lnovement of cytoplasmic granules would depend on contraction of microtubules. It is a pleasure to acknowledge thcb capable technical a
AIJXN, 1~. D. A. E. hlirsky, .L\NIJEHSON, T.

( 1961 ). Allleboitl movement. 111 “‘l‘lw eds. ), Vol. II, pp. 135-216. Academic ( 1951 ) Techniques for the prescrvntion

Cell” ( 1. Brachct ;ul Press, N&v York. of three-dimensional

70

E. I. DU PRAW

structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci. [III 13,, 136-134. DAN, K., and OKAZAKI, K. ( 1956). Cyto-embryological studies of sea urchins. III. Role of the secondary mesenchyme cells in the formation of the primitive gut in sea urchin larvae. BioE. Bull. 110, 2942. DEHAAN, R. L. ( 1963). Oriented cell movements in embryogenesis. In “Biological Organization at the Cellular and Supercellular Level” (R. J. C. Harris, ed. ). Academic Press, New York. DUPRAW, E. J. ( 1961). A unique hatching process in the honeybee. Trans. Am. Microscop. Sot. 80, 185-191. DUPRAW, E. J. (1963a). Techniques for the analysis of cell function and differentiation using eggs of the honeybee. Proc. 16th Intern. Congr. Zool. Washington, D.C. Vol. 2, 238. DUPRAW, E. J. ( 1963b). Analysis of embryonic development in the honeybee I. 16-mm sound film, available from The Library of Congress, Washington, D.C. DuPa~w, E. J. (1965a). The organization of nuclei and chromosomes in honeybee embryonic cells. PTOC. Natl. Acad. Sci. U.S. 53, 161-168. DUPRAW, E. J. (196513). Macromolecular organization of nuclei and chromosomes: A folded fibre model based on whole-mount electron microscopy. Nature 206, 338-343. GALL, J. ( 1963). Chromosome fibers from an interphase nucleus. Science 139, 120-121. GIJSTAFSON, T., and WOLPERT, L. ( 1961). Studies on the cellular basis of morphogenesis in the sea urchin embryo: directed movements of primary mesenchyme cells in normal and vegetalized larvae. Exptl. Cell. Res. ,24, 64-79. JAHN, T., and RINALDI, R. A. ( 1959). Protoplasmic movement in the foraminiferan, Allogromia laticokris; and a theory of its mechanism. Biol. Bull. 117, 100-118. KLEINSCHMIDT, A. K., LANG, D., JACHERTS, D., and ZAHN, R. K. ( 1962). Darstellung und Langenmessungen des gesamten Desoxyribonucleinsaure-Inhaltes von T1-Bakteriophagen. Biochim. Biophys. Acta 61, 857-864. LEDBETTER, M. C., and PORTER, K. R. (1963). A “microtubule” in plant cell fine structure. I. Cell Biol. 19, 239-250. LEDBETTER, M. C., and PORTER, K. R. ( 1964). Morphology of microtubules of plant cells. Science 144, 872-874. movement, locomotion, and stimulation in MAST, S. 0. ( 1926). Structure, Amoeba. J. Morphol. Physiol. 41, 347425. MAZIA, D. ( 1961). Mitosis and the physiology of cell division. In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. III, p. 227. Academic Press, New York. MERRIAM, R. W. (1959). Permeability and structural characteristics of isolated nuclei from Chaetopterus eggs. J. Biophys. Biochem. Cytol. 6, 353-360. MERRIAM, R. W. ( 1961). On the fine structure and composition of the nuclear envelope. J. Biophys. Biochem. Cytol. 11, 559-570. “The Embryology of the Honeybee.” Princeton Univ. NELSON, J. A. (1915). Press, Princeton, New Jersey.

A. K. [ 1964). Echinochrome granule motion in the egg of ArOacia pwdulnta. Itz “Primitive nlotilc Systems in Cc711 Biology” (R. I). Allen and IN. Kamiya, rds.), pp. 471-483. Acadcn~ic Press, New York. PEASE, D. C. (1963). The ultrastrnctlw of flagellar fibrils. 1. Cd Uid. 18, 313-326. PORTER, K. H., LEDBETTER, hi. C., and BAIIESHAUSEX, S. (1964). The microtubule in ccl1 fine structure as an accompaniment of cvtolda~mir movements. Excerptn hfcd. Intern. Congr. Siv. 77, 3C%37. REUHUN, L. I. (1964). Saltatory particle movements in ~~11s. Iti “l’rimitivc hlotile Systems in Cell Biology” (R. D. Allen ;mtl S. Kamiya, ds.), pp. 503-525. Academic Press, I\‘ew York. SANIIHOHN, I<. ( 1964). Cytoplasmic microtubulcs in the neurone. And. Record 148, 330. SANDLIOHS, E., KOEN, I'. I?., ~lcn‘anu, J. D., mtl ~IOOHE, C. ( 1964). Cytoplasmic microtubules in mammalian cells. J. Ultrrrstmct. Res. 11, 123-138. SCHNETTEH, hl. ( 1934a). Physiologische Untcrsuchungen iiber clas Differenzierungszrntrnm in cler Embryonnlentwicklung der Honigbicne. Arch. EntwickZungsnwch. Orpa. 131, 285-323. ScxrNwrEn, hi. ( 1934b). Morphologische Untersuchungen iiber clus Dill’erenzierungszentrum in cler Embryonalentwicklung der Honigbiene. Z. Alorphol. Okol. Tiere 29, 114-195. SLAUTTEHBACK, D. B. (1963). Cvtoplasmic microtubules. I. II\-elm. J. Cell Rio/. 18, 367-388. PAHPAHT,