J. Embryol. exp. Morph. Vol. 23, 2, pp. 491-507, 1970
Printed in Great Britain
491
A light and electron
microscopic study of cell behavior and
microtubules in the embryonic
chicken lens using Colcemid
By T H O M A S L. P E A R C E 1 AND J O H A N Z W A A N 1
From the Department of Anatomy, School of Medicine,
University of Virginia, Charlottesville, Virginia
The first sign of differentiation of the lens rudiment is a change in cellular
shape, as in many other embryonic systems (Tilney, 1968«). This occurs some
time before the beginning of overt chemical differentiation, the appearance of
specific proteins (Zwaan, 1968). From stage 11 (Hamburger & Hamilton, 1951),
just after the optic cup contacts the surface ectoderm, to stage 13, before the
onset of invagination, the lens Anlage is rapidly transformed from a cuboidal
to a high columnar epithelium, a process described by McKeehan (1951) as
palisading. This is slightly preceded in time by nuclear rearrangement. First
irregular in outline and randomly oriented, the nuclei become smooth and oval,
and aligned at right angles to the surface. They take up positions in the basal
portions of the cells (McKeehan, 1951).
The development of a highly asymmetric cell form probably leads to the
creation of tensile forces at the cell surface. Shape-stabilizing mechanisms,
extra- or intracellular, must therefore initiate and maintain the cellular elongation. While palisading, which appears not to involve an increase in cell volume,
leads to a larger cell density in the placode area, this is not due to a localized
augmentation of the mitotic rate but to a drawing of adjacent ectodermal cells
into the region (McKeehan, 1951). Thus crowding seems to be the result, rather
than the cause, of the elongation. Pressure from spreading neighboring cells can
also be excluded, because the lens rudiment palisades even in culture (Dorris,
1938; Langman, 1956). Direct contact with the underlying optic cup is not
required; when the components of the lens-optic cup system are cultured on
opposite sides of a Millipore filter, lens-cell elongation is not prevented (Muthukkaruppan, 1965). The mechanisms behind the shift in form thus may well reside
within the placode cells. Byers & Porter (1964) have related the appearance
of large numbers of cytoplasmic microtubules, parallel to the long axis of the
1
Authors' address: Department of Anatomy, School of Medicine, University of Virginia,
Charlottesville, Virginia, 22901, U.S.A.
492
T. L. PEARCE AND J.
ZWAAN
cell, to the palisading of cells in the induced lens rudiment. Numerous
reports on a variety of systems have now demonstrated a correlation in time and
in place between the presence of microtubules and the elongation of cells and
cell extensions (see reviews by Porter, 1966; Tilney, 1968a).
We have recently shown (Zwaan, Bryan & Pearce, 1969) that asymmetry of the
lens placode cells is a dynamic condition, the cells going through a cycle of
elongation and contraction with a periodicity of 8-10 h, correlated with phases
of the replicative cycle. It occurred to us that this behavior might be reflected
in the state of the microtubules. We therefore studied the fine structure of lens
placode cells in interphase and in mitosis. Colcemid (iV-desacetyl-iV-methylcolchicine), a stathmokinetic agent, was used to disrupt the normal cell cycle.
Cell cycle parameters were reported elsewhere (Zwaan & Pearce, 1970).
MATERIALS AND METHODS
Light microscopic technique
Colcemid (CIBA, Fairlawn, N.J., U.S.A.) in saline was dropped on the vitelline membrane of stage 13 chick embryos at 50-52 h of incubation through a
hole in the egg shell. On the basis of pilot experiments a dosage of 0125 ml
of a 5-4 x 10 _ 6 M solution, which gave consistent results, was chosen for the
present work. The eggs were returned to the incubator for 1-5 h. Embryos were
fixed in Bouin's fluid and embedded in paraffin wax. Sections of 4 JLL were
stained with Delafield's hematoxylin and alcoholic eosin.
Staging of the degree of lens development was done as previously described
(Zwaan et ai, 1969).
Electron microscopic technique
Chicken embryos of 56-58 h (stage 16) were exposed to Colcemid for 2 h,
removed from the egg with the surrounding membranes intact, and placed
immediately in a 3 % glutaraldehyde solution buffered with cacodylate at pH
7-5. After 2 h fixation the embryos were washed in cacodylate-buffered 10 %
sucrose for 1 h and then post-fixed in 1 % osmium tetroxide buffered with
phosphate to pH 7-3 for 1 h. The specimens were dehydrated in an alcohol
series and embedded in a 1 : 1 mixture of Epon and Araldite. Thin sections were
cut with a diamond knife on a Porter-Blum ultramicrotome (MT2), stained
with uranyl acetate and lead citrate, and examined in a Philips EM-300 electron
microscope. Control specimens were prepared in the same fashion.
RESULTS
Light microscopic observations
The normal stage 15 lens placode in the chick embryo formed a pseudostratified columnar epithelium (Fig. 1 A). Most of the nuclei were located in the
basal portion of their cells; those found at the luminal surface of the tissue were
Effect of Colcemid on lens rudiment
493
in various stages of mitosis. In normal animals the latter constituted about
2-7 % (range 2-4-4-3) of the total number. After exposure to Colcemid striking
changes in the normal pattern were observed. The number of nuclei at the
luminal surface increased greatly with time, indicating that the treatment led to
arrest in metaphase (Fig. IB).
Several other abnormal phenomena were seen in the structure of the right
lens of the experimental embryos (Fig. 1 B). Left lenses were unaffected by the
drug because at the time of treatment the head was turned on its left side,
exposing only the right eye. Patches of amorphous material similar in appearance to cytoplasm of the placode cells were present in the area of the invagination of the lens pit. There seemed to be an increase of this extracellular material
with longer exposure to Colcemid. The lumen of the lens cup also enclosed
small, dark, round structures which stained deeply with hematoxylin. These
were thought to be either free cells with very little cytoplasm and pyknotic
nuclei or nuclei extruded from cells of the lens placode. This material is very
similar to pyknotic debris found in the neurocoel of mouse embryos following
irradiation and interpreted as 'damaged material' released from neuroepithelial
cells (McDonald & Gatz, 1969). These bodies increased until after 4 h their
accumulation completely disrupted and disorganized the luminal surface of the
lens.
In spite of all this, invagination of the placode proceeded normally. The
central portion of the placode became progressively depressed and the peripheral portions approximated each other with time, as in normal lenses. The
pseudostratified columnar arrangement of the tissue was preserved. Cells did
not appear to collapse to the luminal surface immediately following treatment
with Colcemid, but remained elongate, rounding up to the lumen only as they
entered mitosis in the normal progress of the cell cycle.
Electron microscopic observations
Normal lenses. Since our observations of the fine structure of normal presumptive lens cells in the chick embryo agree essentially with previous reports (Hunt,
1961 ; Byers & Porter, 1964), detailed descriptions of all subcellular systems are
not included in the following paragraphs. Rather, attention is focused on the
effects of Colcemid on microtubules in these cells.
Figs. 2 and 3 are examples of electron micrographs of cells in the posterior
pole of a normal stage 18 lens. Fig. 2 shows the apical portions of several cells,
joined by junctional complexes ('terminal bars') and bordering the lumen or
lens cavity. 'Terminal bars' were frequent, but only at the luminal surface of the
cells. Plasma membranes of adjacent cells in the mid-region of the posterior pole
and at the posterior surface, while often closely approximated, were never
observed to be involved in tight junctions. Numerous free ribosomes and polyribosomes were present in both normal and experimental embryos. These
structures were scattered throughout all lens cells in the high concentration
494
T. L. PEARCE AND J. ZWAAN
* ^ . *\
•"r-y
T-'Z'^V
I
b'-Ji
B WV
^
• ' r J ' V ' .i*
K
*.
'\r
ir^fck
::.'••
>\
8/i
.
I
Effect of Colcemid on lens rudiment
495
typical of embryonic tissues. Rough endoplasmic reticulum was seen very
frequently (Figs. 2, 6 A). Mitochondria, present in all cells, in general appeared
to be oriented with their longest dimension in the long axis of the cell (Fig. 2),
although in some cases, especially in mitotic cells, they were oriented more
randomly (Fig. 3). Large intercellular spaces which have been described before
(McKeehan, 1951 ; Hunt, 1961 ; Byers & Porter, 1964) were prominent throughout the posterior pole of the lens vesicle (Fig. 6 A). Lipid droplets of various
sizes were found in many cells (Fig. 2).
Cytoplasmic microtubules, about 240 Â in diameter and varying in length
from 480 Â to 2-1 /*, were seen in great numbers in normal interphase lens cells.
In general, they were oriented in the long axis of the cell, and were most frequent
in the paranuclear and apical regions (Figs. 2, 4A, C). They appeared to be
associated with no other organelle in particular, but usually were seen in groups,
quite often near the plasma membrane. Cilia, as expected, contained microtubules (Fig. 2B). There was no evidence that microtubules were 'inserted into'
or otherwise associated with a 'terminal web' area in the apical cytoplasm (Fig.
2). Microtubules, while present in dividing cells, were greatly reduced in both
number and length and were oriented more randomly than in non-dividing
cells (Fig. 3).
Colcemid-treated lenses. Fig. 5 is an electron micrograph of several posterior
lens epithelial cells after 2 h exposure to Colcemid in vivo. At the apical side
they are joined, like untreated cells, by junctional complexes which appeared
undisturbed by the drug. Other subcellular systems apparently unaffected by
Colcemid were numerous free ribosomes and polyribosomes and rough endoplasmic reticulum (Figs. 4B, 5). A normal-appearing basement membrane was
seen adjacent to the posterior end of treated lenses (Fig. 6B), and lipid droplets
were also present in experimental material (Fig. 5). Preliminary data indicate
that the number of lipid droplets may actually increase as a result of Colcemid
treatment. In general, mitochondria seemed greatly disturbed following
Colcemid treatment (Fig. 6B). Many more 'membranous whorls' of the type
described by Jurand & Yamada (1967) in degenerating mitochondria were seen
inside mitochondria in cells treated with Colcemid than in normal tissue.
FIGURE 1
(A) Normal lens placode at stage 15, a pseudostratified columnar epithelium closely
approximated to the underlying optic vesicle (ov). Most of the cells are in interphase,
with the nucleus located basally. Mitotic figures {mf) are found exclusively at the
surface of the lens placode, x 500.
(B) Stage-15 lens placode after 3 h exposure to Colcemid. Nuclei which have migrated
to the surface to undergo mitosis have been arrested in metaphase {am) and prevented from returning to their interphase position. Also present are dark, pyknoticlooking bodies which are presumably extruded nuclei {en), and cytoplasmic debris {d),
both of which increase with time of exposure to Colcemid. In spite of an accumulation
of nuclei arrested at the luminal surface, lens invagination has proceeded normally,
x 400.
496
.«»j*. i i i-
T. L. PEARCE AND J. ZWAAN
».
..».*;».«.
• -«*•*
w*-*v
>V
Vf*
*•-
* *$•*& „"^ JKJL
*Ä/.
A**.
«K^.
Effect of Colcemid on lens rudiment
497
The most striking change following Colcemid treatment was the virtually
complete disappearance of cytoplasmic microtubules (Figs. 4B, D; 5, and 6B).
The few that remained in treated cells were randomly oriented and shorter than
in untreated cells, but were of the same diameter as microtubules in normal
cells, about 240 Â (Fig. 5B). A quantitative comparison of both the frequency
and the length of cytoplasmic microtubules in normal and treated lenses is given
in Fig. 7.
It is clear (Fig. 6B) that normal tissue architecture in the posterior pole of the
lens was maintained following Colcemid treatment; a pseudostratified columnar
epithelium persisted, in spite of almost total disappearance of microtubules
from the cells.
DISCUSSION
The results presented show that nuclei arrested in metaphase by Colcemid
treatment are located only at the luminal surface of the lens rudiment; others, in
interphase, were seen at various places throughout this epithelium, but not
adjacent to the lumen. Zwaan et al. (1969) found earlier by pulse-labeling with
tritiated thymidine that the DNA-synthetic phase of the cell cycle occurs while
the nucleus is in the basal position. Tracing the labeled nuclei over a period of
time showed that the normally columnar lens cells round up towards the lumen
for division. Following mitosis the cells elongate again and the daughter nuclei
return to the basal zone close to the optic cup. The two sets of experiments
together prove the existence of interkinetic nuclear migration in the lens
placode. The possible role of this process in induction has been discussed
(Zwaan et al., 1969).
Exposure of early chicken embryos to the stathmokinetic drug vincristine
sulfate for 4 h caused eversion of the dorsal lips of the neural groove. This led
Langman, Guerrant & Freeman (1966) to conclude that undisturbed interkinetic nuclear migration, causing the bulk of the neuroepithelial cells to be
wedge-shaped, is necessary for normal invagination of the neural tube. Despite
the treatment with Colcemid the morphology of the lens rudiment progressed
FIGURE 2
(A) Electron micrograph of the apical ends of several cells of the posterior pole of a
stage-18 lens. Typical features of these cells are junctional complexes (jc) at the apical
end of contiguous cells, rough endoplasmic reticulum (rer), mitochondria (m),
plasma membrane (pm), lipid droplets (Id), terminal web (tw) and numerous long
microtubules (mt). Microtubules are about 240 Â in diameter and from 480 Â to
2-1/1 long, and are oriented parallel to the long axis of these elongated cells,
x31500.
(B) Lower-power electron micrograph of the same region as in Fig. 2 A, showing
the apical surfaces of these cells bordering the lumen (/c), and a rare cilium (ci),
x 14500.
32
E M B 23
498
T. L. PEARCE AND J. ZWAAN
.H«
mm^mm
*»
ȀI** ^ \ %
%
bA
* / .>
',f
*^/% fc '•* > £
t
!•« v
FIGURE
.-s, v s,
3
Electron micrograph of a dividing cell and an adjacent cell in the posterior pole
of a normal stage-18 lens. Condensed chromatin (chr) identifies the dividing cell,
which is separated from its non-dividing neighbor by a plasma membrane (pm).
Both cells border an intercellular space (is). Microtubules of the dividing cell (mh)
have largely been lost as the cell has rounded up for mitosis, but several remain.
They are markedly shorter and oriented more randomly than microtubules of the
non-dividing cell (mt2). x 31500.
*•>*
Effect of Colcemid on lens rudiment
FIGURE
499
4
(A, C) Electron micrographs of the apical parts of normal stage-18 lens cells,
approximately halfway through the posterior pole. Large intercellular spaces (is)
surround the apical region of these cells, and the cytoplasm apical to the nucleus
(nu) contains typical microtubules (mt), oriented in the long axis of the cell, x 31500.
(B, D) Electron micrographs of sections, similar to those in Fig. 4 A and C, from a
Colcemid-treated lens. Intercellular spaces (is), nucleus (nu), nuclear membrane
(nm), and mitochondria (m) appear the same as in normal cells, but no microtubules
are present, x 31500.
32-2
500
»
T. L. PEARCE AND J. ZWAAN
' .»'-A » " • *•':''**
'1 •
L
y^ PS
m
të^îi. '«•**
1
WM
^
Effect of Colcemid on lens rudiment
501
in the current work from placode to deeply invaginated cup over a 4 h period,
without evidence for eversion of the edges. Thus, at least in this system, invagination does not appear to be interrupted by arrest of cells in metaphase. The
objection might be raised that neural plate and lens placode are not strictly
comparable, because the latter is strongly attached to the surrounding optic
cup. In fact, Jacobson (see Källen, 1965) has shown that the Axolotl neural
plate is firmly adherent to the underlying mesoderm. The tendency of this substratum to invaginate is even stronger than that of the neural plate itself. On the
other hand, the lens placode will invaginate independently from the optic cup
(Coulombre, 1965). Thus, whatever factors are involved in lens invagination,
interkinetic nuclear migration and attachment to the optic cup do not seem to be
essential.
Our observations confirm the work of Byers & Porter (1964) on the existence in all elongated embryonic lens cells of numerous cytoplasmic microtubules, consistently oriented in the long axis of the cell. We have found that
there are few microtubules in dividing cells, and that those which remain during
mitosis are oriented irregularly. Apparently, as the cell rounds up for division
its microtubules disappear; following cytokinesis each daughter cell elongates
and microtubules reappear in the cytoplasm. This suggests that the cycle of cell
elongation and rounding-up is a direct result of the appearance and disappearance, respectively, of cytoplasmic microtubules.
Borisy & Taylor (1967 a, b) have suggested that the microtubules consist of
subunits whose state of polymerization and depolymerization is dependent on an
equilibrium process. In the lens placode cells the Gx phase takes less than 30
min (J. Zwaan & T. L. Pearce, in preparation). Within this time span the cell
shape alters from virtually spherical to maximally elongated and the nucleus
travels the length of the cell from apex to base. It is reasonable to assume that
cytoplasmic microtubules are dissociated into subunits during mitosis, and then
re-associated during the brief Gx phase, when the cell becomes elongated once
again. Apparently all cells in the S and G2 phases (elongated cells) contain
abundant microtubules. That the reappearance of microtubules is accomplished
FIGURE 5
(A) Electron micrograph of the apical ends of several cells of the posterior pole of
a stage 18-lens exposed to Colcemid for 2 h in vivo. These cells border the lens cavity
(/c), and exhibit junctional complexes (y'c), lipid droplets {Id), rough endoplasmic reticulum (rer), terminal web (tw), and a desmosome or macula adhaerens (d) identical
to those found in normal lenses. Only a few of the cytoplasmic microtubules (mt)
found in a similar section of an untreated embryo still remain (cf. Fig. 2). The
diameter of these microtubules is the same as in normal tissue, but the length is greatly
reduced, x 31500.
(B) Electron micrograph similar to Fig. 5 A, showing a microtubule (mt) remaining
after Colcemid treatment. The few such microtubules seen were often oriented
randomly, not consistently parallel to the long axis of the cell, x 31500.
502
Ä '
' bm
T. L. PEARCE AND J. ZWAAN
A<$r%
Effect of Colcemid on lens rudiment
503
so quickly favors a mechanism of re-assembly from a pre-existing pool of
subunits rather than a de novo synthesis of microtubules.
Treatment of the lens placode with Colcemid clearly leads to disassembly of
cytoplasmic microtubules, as in cultures of chick embryo muscle (Ishikawa,
BischofT & Holtzer, 1968). Its action is comparable to that of colchicine (R.obbins & Gonatas, 1964; Tilney, 19686). The effect of the latter on interphase
cells has been attributed to its property of complexing with a protein (Borisy &
Taylor, 1967 A, b) that at least in some cases has been shown to be a microtubule
precursor (Shelanski & Taylor, 1967; Stephens, 1968). Despite the virtual
absence of microtubules the pseudostratified architecture of the treated lens
epithelium did not change. Only cells ready to enter mitosis in the normal
progression of their cell cycle rounded up and approached the lumen. This
indicates that cytoplasmic microtubules, while probably involved in the initiation
50 -
•5 40
|
30
20 -
10
0-500
1000-1500
500-1000
n
2000-2500
1500-2000
2500-5000
5000-7500
10000-15000
7500-10000
15000-Longer
Length (Â)
FIGURE
7
Graph of the length and frequency of microtubules found in an equal number of
comparable cross-sections of normal (white bars) and Colcemid-treated (black bars)
cells. In all cases the diameter of microtubules was the same, about 240 Â.
FIGURE
6
(A) Low-power electron micrograph of several cells of the posterior pole of a normal
stage-18 lens. Large intercellular spaces (is) are frequently found in this tissue.
This micrograph shows a continuity between the nuclear membrane (nm) and rough
endoplasmic reticulum (rer) of the cytoplasm. A mitochondrion (m) is also indicated,
x 14500.
(B) Low-power electron micrograph of the posterior end of a stage-18 lens after 2 h
exposure to Colcemid in vivo. Even though microtubules are almost entirely absent
from these cells, the tissue has not lost its pseudostratified columnar arrangement.
Other features which parallel normal lenses are the basement membrane (bm) found
between lens cells and the future vitreous region (vr), a mitochondrion (m) which
contains 'membranous whorls', and a lipid droplet (ld), x 8300.
504
T. L. PEARCE AND J . ZWAAN
of lens cell elongation, are not required for maintenance of the asymmetric
shape. The same is true for the morphogenesis of primary mesenchyme in the
sea urchin (Tilney & Gibbins, 1969). Conversely, our data show that the disappearance of microtubules from G2 cells does not form the critical step in the
contraction of the cell to a sphere, in preparation for division.
This focuses attention on other factors that could be involved in the regulation
of cell form. Cell-to-cell adhesions, either directly through junctional complexes
and lateral cell coats or indirectly through the basement lamina, come primarily
to mind. The terminal web may influence the shape of the apical cell surface.
Colcemid does not alter the ultrastructure of any of these devices, which may
therefore well be responsible for the maintenance of cell shape even during
treatment with this drug. Normally the lens placode adheres very strongly to the
optic cup during the period studied here (McKeehan, 1951), presumably through
an intercellular matrix of which basement laminae are a part (Cohen, 1961 ;
Hunt, 1961; Weiss & Fitton-Jackson, 1961; Porte, Stoeckel & Brini, 1968).
Moreover, work in our laboratory has demonstrated that lens placode cells
actively synthesize a glycoprotein which is incorporated into the lateral cell
coats and the interface between Jens Anlage and retinal Anlage', a model will be
proposed to explain the possible role of this glycoprotein in cell-to-cell and
lens-optic cup adhesiveness (R. W. Hendrix & J. Zwaan, in preparation). So far
we have no reason to assume a cyclic change in adhesiveness and we have to postulate the generation of a force in the G2 phase of the cell cycle which is sufficient
to overcome this interaction of the cell with its environment. It is well known that
increase of tension in the pericellular membrane plays an important role in the
initiation of cell division in other systems such as the cleavage of sea-urchin
eggs (Wolpert, 1966). Cone (1969) has recently shown that the volume of restrain fibroblasts in cultures increases appreciably prior to mitosis, leading to a
decrease of surface/volume ratio, which in turn may cause an increase in membrane tension. It may be pertinent that lens placode cells rounding up for division always display a distinct PAS-positive plaque at their basal surface, which
gives the impression of having been separated forcibly from the basement
membrane (R. W. Hendrix & J. Zwaan, in preparation).
Holmes & Choppin (1968) have implicated microtubules in the nuclear
movements which take place in virus-induced syncytia. These organelles are
also in a position to influence interkinetic nuclear migration in the lens placode.
In the apical parts of the cells they are crowded together, while they spread out
and run in the cortical cytoplasm down to beyond the level of the nucleus. They
are less frequent in the most basal area of the cells (Byers & Porter, 1964;
present observations). Thus, they form an inverted basket over the nucleus. It is
interesting to note that undulations have frequently been noted in cells possessing microtubules (Byers & Porter, 1964; Branson, 1968). It is easy to see how
undulatory motions of a basket-like array of microtubules could extrude the
nucleus along the long axis of the cell.
Effect of Colcemid on lens rudiment
505
In conclusion, while microtubules are not responsible for the maintenance of
the columnar shape of the lens placode cells, they are probably involved in the
establishment of cell elongation and/or the nuclear migration that occur in this
tissue in a cyclic fashion. So far, we have been unable to differentiate between
the latter two possibilities. It may be significant, however, that considerable
nuclear orientation and positioning, concomitant with the appearance of large
numbers of microtubules (Byers & Porter, 1964), precedes palisading of the
presumptive lens cells (McKeehan, 1951).
SUMMARY
1. Treatment of embryonic chick lens placodes in vivo with Colcemid, a
mitotic inhibitor, caused a steady accumulation of metaphase nuclei at the
luminal surface of this tissue, confirming the theory of interkinetic nuclear
migration in the lens placode.
2. Electron microscopic studies on the posterior pole of lens vesicles showed
that microtubules, arranged in the long axis of the cells, were abundant in
apical and paranuclear parts of the cytoplasm of all untreated cells in interphase. They were scarce, and randomly oriented in dividing cells.
3. Microtubules were almost totally absent after 2 h of Colcemid treatment.
4. The finding that the pseudostratifled nature of the tissue was not destroyed
at once following Colcemid treatment in spite of the virtual absence of microtubules is taken as evidence that microtubules alone are not responsible for
maintenance of cell elongation in this system. Their absence during mitosis and
presence in other phases of the cell cycle indicates, however, that they may be
required for the development of the elongate cell shape and/or the migration of
nuclei to the basal zone after completion of cell division.
RÉSUMÉ
Une étude aux microscopes photonique et électronique du comportement
cellulaire et des microtubules dans le cristallin de l'embryon de Poulet,
après l'usage de colcémide
1. Le traitement in vivo par de la colcémide, un inhibiteur mitotique, des
placodes cristalliniennes d'embryons de Poulet provoque une accumulation
progressive de noyaux en métaphase à la surface de la lumière de ce tissu, ce
qui confirme la théorie de la migration nucléaire intercinétique dans la placode
du cristallin.
2. Des études au microscope électronique au pôle postérieur des vésicules
cristalliniennes ont démontré l'existence, le long de l'axe longitudinal des
cellules, de nombreux microtubules dans les parties apicales et paranucléaires
de toutes les cellules non traitées, en interphase. Ces microtubules étaient rares
et orientés de façon aléatoire dans les cellules en division.
506
T. L. PEARCE AND J. Z W A A N
3. Les microtubules étaient totalement absents après 2 h de traitement à la
colcemide.
4. Le fait que la pseudostratification du tissu n'a pas été détruite immédiatement après le traitement à la colcemide en dépit du fait de l'absence virtuelle de
microtubules, est considéré comme une preuve que les microtubules seuls ne
sont pas responsable du maintien de l'élongation cellulaire dans ce système.
L'absence des microtubules au cours de la mitose et leur présence au cours des
autres phases du cycle cellulaire indique, cependant, qu'ils peuvent être requis
pour le développement de la forme allongée de la cellule et/ou la migration vers
la zone basale après completion de la division.
We express our appreciation to Mr Harold Friedman of our department for help in taking
the electron micrographs. We are indebted to Miss Sharon Olson for valuable technical
assistance. This research was supported by grant no. GB-3236 of the National Science
Foundation, grant no. NB-08218-01 of the U.S. Public Health Service (National Institute for
Neurological Diseases and Blindness), and U.S. Public Health Service predoctoral research
fellowship no. 1-F1-GM-39, 219-01 (National Institute of General Medical Sciences).
REFERENCES
G. G. & TAYLOR, E. W. (1967a). The mechanism of action of colchicine: binding of
colchicine-3H to cellular protein. /. Cell Biol. 34, 525-35.
BORISY, G. G. & TAYLOR, E. W. (19676). The mechanism of action of colchicine: colchicine
binding to sea urchin eggs and the mitotic apparatus. /. Cell Biol. 34, 535-48.
BRANSON, R. J. (1968). Orthogonal arrays of microtubules inflatteningcells of the epidermis.
Anat. Rec. 160, 109-22.
BYERS, B. & PORTER, K. R. (1964). Oriented microtubules in elongating cells of the developing lens rudiment after induction. Proc. natn. Acad. Sei. U.S.A. 52,1091-9.
COHEN, A. I. (1961). Electron microscopic observations of the developing mouse eye. I.
Basement membranes during early development and lens formation. Devi Biol. 3, 297-316.
CONE, C. D. (1969). Electroosmotic interactions accompanying mitosis initiation in sarcoma
cells in vitro. Trans. N. Y. Acad. Sei. 31, 404-27.
COULOMBRE, A. J. (1965). The Eye. In Organogenesis (eds. R. L. DeHaan and H. Ursprung),
pp. 219-51. New York: Holt, Rinehart and Winston.
DORRIS, F. (1938). Differentiation of the chick eye //; vitro. J. exp. Zool. 78, 385-415.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
HOLMES, K. V. & CHOPPIN, P. W. (1968). On the role of microtubules in movement and
alignment of nuclei in virus-induced syncytia. /. Cell Biol. 39, 526-43.
HUNT, H. H. (1961). A study of the fine structure of the optic vesicle and lens placode of the
chick embryo during induction. Devi Biol. 3, 175-209.
ISHIKAWA, H., BISCHOFF, R. & HOLTZER, H. (1968). Mitosis and intermediate-sized filaments
in developing skeletal muscle. /. Cell Biol. 38, 538-55.
JURAND, A. & YAMADA, T. (1967). Elimination of mitochondria during Wolffian lens regeneration. Expl Cell Res. 46, 636-8.
KALLEN, B. (1965). Early morphogenesis and pattern formation in the central nervous
system. In Organogenesis (eds. R. L. DeHaan and H. Ursprung), pp. 107-28. New York:
Holt, Rinehart and Winston.
LANGMAN, J. (1956). Certain morphological aspects in the development of the crystalline
lens in chick embryos. Acta morph. neerl.-scand. 1, 81-92.
LANGMAN, J., GUERRANT, R. L. & FREEMAN, B. G. (1966). Behavior of neuroepithelial cells
during closure of the neural tube. J. comp. Neurol. 121, 399-412.
BORISY,
Effect of Colcemid on lens rudiment
507
MCDONALD, T. F. & G ATZ, A. J. (1969). Sequential changes in the neural tube of X-irradiated
rats: a study with light and electron microscope. Anat. Rec. 163, 22.
MCKEEHAN, M. S. (1951). Cytological aspects of embryonic lens induction in the chick.
/ . exp. Zool. 117, 31-64.
MUTHUKKARUPPAN, V. (1965). Inductive tissue interaction in the development of the mouse
lens in vitro. J. exp. Zool. 159, 269-88.
PORTE, A., STOECKEL, M. E. & BRINI, A. (1968). Formation de l'ébauche oculaire et différenciation du cristallin chez l'embryon de poulet. Archs Ophthal. Rev. gén. Ophthal. 28, 681 —
706.
PORTER, K. R. (1966). Cytoplasmic microtubules and their functions. In Ciba Foundation
Symposium on Principles of Biomolecular Organization (eds. G. E. W. Wolstenholme and
M. O'Connor), pp. 308-56. London: Churchill.
ROBBINS, E. & GONATAS, N. K. (1964). The ultrastructure of mammalian cells during the
mitotic cycle. J. Cell Biol. 21, 429-64.
SHELANSKI, M. L. & TAYLOR, E. W. (1967). Isolation of a protein subunit from microtubules. / . Cell Biol. 34, 549-54.
STEPHENS, R. E. (1968). Reassociation of microtubule protein. / . molec. Biol. 33, 517-19.
TILNEY, L. G. (1968a). The assembly of microtubules and their role in the development of
cell form. In The Emergence of Order in Developing Systems (ed. M. Locke), pp. 63-102.
New York: Academic Press.
TILNEY, L. G. (19686). Studies on the microtubules in Heliozoa. IV. The effect of colchicine on
the formation and maintenance of the axopodia and redevelopment of pattern in Actinosphaerium nucleofilum (Barrett). / . Cell Sei. 3, 549-62.
TILNEY, L. G. & GIBBINS, J. R. (1969). Microtubules in the formation and development of the
primary mesenchyme in Arbacia punctulata. II. An experimental analysis of their role in
development and maintenance of cell shape. / . Cell Biol. 41, 227-50.
WEISS, P. & FITTON-JACKSON, S. (1961). Fine structural changes associated with lens determination in the avian embryo. Devi Biol. 3, 532-54.
WOLPERT, L. (1966). The mechanical properties of the membrane of the sea urchin egg during
cleavage. Expl Cell Res. 41, 385-96.
ZWAAN, J. (1968). Lens-specific antigens and cytodifferentiation in the developing lens.
J. cell. Physiol. 72, (suppl. 1), 47-72.
ZWAAN, J., BRYAN, P. R., Jun. & PEARCE, T. L. (1969). Interkinetic nuclear migration during
the early stages of lens formation in the chicken embryo. / . Embryol. exp. Morph. 21, 71-83.
ZWANN, J. & PEARCE, T. L. (1970). Mitotic activity in the lens rudiment of the chicken embryo
before and after the onset of crystallin synthesis. I. Results of treatment with Colcemid.
Wilhelm Roux Arch. EntwMech. Org. (in the Press.)
(Manuscript received 26 May 1969, revised 1 October 1969)