J. Embryol. exp. Morph. 78, 23-32 (1983)
23
Printed in Great Britain © The Company of Biologists Limited 1983
Light microscope observations on actin distribution
during morphogenetic movements in the chick
embryo*
By DAVID OSTROVSKY 1 , JOSEPH W. SANGER 2 AND
JAMES W. LASH 2 ' 3
From the Department of Anatomy, University of Pennsylvania and Department
of Biology, Millersville University of Pennsylvania, Pennsylvania
SUMMARY
The cellular distribution of actin during two morphogenetic processes in the chick embryo
has been observed, using a high-resolution fluorescent technique, with heavy meromyosin as
a probe. These cytoskeletal elements have been implicated in all cell and tissue movements
in the embryo. It is now commonly accepted that microfilaments are necessary to provide the
motive force for morphogenesis. Two morphogenetic movements in the early embryo have
been studied at the light microscope level. During somitogenesis, the mesenchymal segmental
plate becomes transformed into a meristic series of transient epithelial vesicles. Again, actin
distribution is diffuse and random before the morphogenetic event. During epithelialization,
actin becomes prominent in the apical regions of the epithelial cells. Cells in the somitic
epithelial vesicles, the core cells, appear to be passive participants in this process, and
consequently show no distinct cellular localization of actin.
INTRODUCTION
There have been many reports implicating microfilaments in morphogenetic
processes. Baker & Schroeder (1967) described a role for microfilaments in
amphibian morphogenesis. Burnside (1971) also noted their role in amphibian
neurulation. Cloney (1966) and Lash, Cloney & Minor (1973), in their studies
on ascidian metamorphosis, showed that microfilaments provided the motive
force for epithelial contraction during tail resorption, and that this process could
be reversibly inhibited with cytochalasin B. There have since been many reports
analysing the role of these cytoskeletal elements in cell and tissue movements.
It is now widely accepted that microfilaments are indeed actin and myosin, and
provide the mechanism for cell and tissue movements (cf. Jacobson & Ebendal,
* Supported by NIH grants GM 25653 and HL 15835 (JWS) and HD 00380 and HD 15985
(JWL).
1
Author's address: Department of Biology, Millersville University of Pennsylvania,
Millersville, Pennsylvania 17551, U.S.A.
2
Authors' address: Department of Anatomy, School of Medicine/G3, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A.
3
Reprint requests.
24
D. OSTROVSKY, J. W. SANGER AND J. W. LASH
1978; Burgess & Schroeder, 1979; Sanger & Sanger, 1979; Pepe, Sanger &
Nachmias, 1979).
With some notable exceptions (e.g. Sadler, Greenberg, Coughlin & Lessard,
1982), actin distribution in cells and tissues during morphogenesis has been
analysed using transmission electron microscopy. Fluorescent heavy meromyosin can be used in a one-step procedure to identify actin in cells and tissues
on a light-microscope level (Sanger, 1975a,b,c). Using this technique, we have
observed with the light microscope the cellular distribution of actin during two
morphogenetic processes in the chick embryo; neurulation and somitogenesis.
MATERIALS AND METHODS
Embryos
Stage-8 (Hamburger & Hamilton, 1951) White Leghorn chick embryos were
used for studies on neurulation, and HH Stage 12 for studies on somitogenesis.
Histological preparation
These procedures were modified from those of Sainte-Marie (1962) and
Franklin & Martin (1980). The embryos were removed from the yolk, pinned to
the bottom of a paraffin-coated dish, and covered with phosphate-buffered saline
(PBS) at 4°C. The region to be analysed was marked with carbon particles (Fig.
1). After marking, the PBS was removed and replaced with 3-5 % formaldehyde
(made up fresh from paraformaldehyde) in PBS. Embryos to be treated with
cytochalasin B (Sigma, St. Louis, Mo.) were also removed from the yolk and
Fig. 1. A whole mount of a HH stage-12 chick embryo, with two carbon marks
(arrows) delimiting the area to be analysed for somitogenesis. Asterisks mark the
area analysed for neurulation. Segmental plate (a), forming somites (b), and nascent
somites (c) are indicated. Micrometer markings for measurements can be seen across
the bottom of the photograph. X1800.
Actin distribution during chick morphogenetic movements
25
placed with the drug (lOjUg/ml) for l h at room temperature (20-22 °C) before
fixation. The embryo was fixed at room temperature for l h . After the initial
fixation, the embryos were removed to a small plastic dish containing PBS, and
the carbon-marked area isolated with dissecting knives. From this point, the PBS
was deaerated to prevent small bubbles from attaching to the embryo fragments.
Immediately after isolation, the embryo fragments were transferred to 100 %
methanol (4°C) and placed in a freezer at — 20 °C. The methanol was changed
every 20min, and the total fixation time was l h . The tissues were then
rehydrated through a series of ethanol solutions at 4°C into PBS, also at 4°C.
After three 90min rinses in PBS (room temperature), the tissues were exposed
to the heavy meromyosin probe for 18 h at 4°C. The heavy meromyosin was
labelled (Sanger, 1975a,b) with lissamine rhodamine B sulphonyl chloride
(Molecular Probes, Inc., Junction City, Oregon). After this treatment, the tissue
was thoroughly rinsed with PBS (three washes, 60min each) and dehydrated
through an ethanol series before embedding in Araldite. Tissues were sectioned
at 1 /im using an LKB microtome III. The plastic sections were transferred to a
3" x 1" microscope slide with a wire loop and the slide set upon a 100 °C hotplate
for 5 min. The sections were covered with Histoclad (Clay-Adams, New York)
and a No. 1 thickness coverslip. Observations were made using a Zeiss IV Fl
epifluorescence condenser equipped with rhodamine filters. Photographs were
taken using Kodak Tri-X (ASA 400)filmwhich was then developed using Kodak
HC-110 developer.
RESULTS
Somitogenesis
Sections, were examined from three different regions of the stage-12 embryo;
the segmental plate, somites in the process of forming from the segmental plate,
and nascent somites showing the organization of an epithelial vesicle. A cross
section of the segmental plate is shown in Fig. 2. There is no detectable localization of actin at this stage of development. All cells exhibit a diffuse pattern of
actin staining. In some cells there is an asymmetric distribution which is due to
the absence of actin in the region of the nucleus. As the somite is forming, an
indication of the future epithelial vesicle can be seen (Fig. 3). Epithelialization
is more advanced in the ventrolateral portion of the somite (Fig. 3), but will soon
encompass the entire somite (Fig. 4). During somite formation there is both an
increase in cell number as the result of proliferation, and a rearrangement of the
cells from a loose mesenchyme (cf. Fig. 2) into an epithelial vesicle (cf. Figs 3 and
4). There is no developmental correlation to account for the first occurrence of
epithelialization in the ventrolateral portion of the forming somite.
When the somite is an epithelial vesicle (Fig. 3), there is a prominent localization of actin in the basal portion of the epithelial cells of the ventral and medial
somite wall (Fig. 4). This is the region of the somite that will soon migrate in a
26
D . OSTROVSKY, J. W. SANGER AND J. W. LASH
Fig. 2. A cross section through the segmental plate. There is no obvious tissue
localization of actin. In Figs 2-9, actin localization is shown as white, against a grey
or black background. Neural tube (n), epithelium (ep), and endoderm (en) designate
tissue orientation. X1800.
Fig. 3. A cross section of a somite in the process of segmentation and epithelialization. The most prominent actin localization is marked by the arrows. Neural tube (n).
X1800.
Actin distribution during chick morphogenetic movements
27
«
Fig. 4. A cross section of a nascent somite. The somite is now an epithelial vesicle,
containing core cells (c). Actin localization is most prominent in the apical portion
of the epithelioid somite cells (arrows). Neural tube (n). X1800.
ventromedial direction as the mesenchymal sclerotome. The epithelial wall is
prominently stratified at this stage. The cells in the centre of the epithelial somite
(Figs 3 and 4) are the 'core cells', and show no actin localization. These cells are
thought to contribute to the future sclerotome of the older somite.
Neurulation
Neurulation was studied at the level of the sinus rhomboidalis through the
region of the nascent somites (asterisks in Fig. 1). This enabled us to obtain
different stages in the sequence of neurulation (Figs 5-9). In the region of the
sinus rhomboidalis the neural epithelium is just beginning to fold, and in the
region of the nascent somite, neurulation is completed. In all stages of neurulation there was an increasing concentration of actin in the apical regions of the
neuroepithehum (Figs 5-9). During neurulation the apical surface (Fig. 5)
becomes the luminal surface (Fig. 9), and there is a marked concentration of
actin in the apical portion of these cells. Actin localization in the basal portion
of the neuroepithehum is sparse in the early stages (Fig. 5), and barely detectable
during the final stages of neural tube formation (Figs 7 and 9).
This is a severe disruption of the actin localization pattern after treatment with
28
D . OSTROVSKY, J. W. SANGER AND J. W. LASH
6
Fig. 5. A cross section of the neuroepithelium at the beginning of neurulation.
Whereas actin is distributed diffusely throughout the epithelium, there is a noticeable concentration at the apical surface (arrows). X1800.
Fig. 6. A later, V-shaped, stage in neurulation. The basal portion of the neuroepithelium is almost devoid of actin, whereas the apical portion of the cells show
pronounced actin localization (arrows). X1800.
Actin distribution during chick morphogenetic movements
7
8
Fig. 7. A cross section of a closure stage of neurulation where the neural tube is
almost formed. Pronounced actin localization is evident in the apical regions of the
neuroepithelial cells (arrows). Somites (s), Notochord (n). X1800.
Fig. 8. A comparable stage of neurulation as shown in Fig. 7, except this embryo had
been treated with cytochalasin B. Actin localization is completely disrupted, and the
process of tissue folding evident in Fig. 7 shows signs of reversal as the epithelium
tends to flatten. Tissue disruption is also evident in the region of the somites and
notochord. X1800.
2
EMB 78
29
30
D. OSTROVSKY, J. W. SANGER AND J. W. LASH
n
9
Fig. 9. A cross section of a completed neural tube (n). Some actin localization is still
observed in the apical regions of the neuroepithelial cells. There is, however, a much
greater concentration of actin in the apical portion (arrow) of the epithelial somite
cells in the nascent somite. A higher magnification of this nascent somite is illustrated
in Fig. 4. xllOO.
cytochalasin B (Fig. 8). Some residual localization remains in the apical region
of the cells (Fig. 8), and the folding neural tube begins to unfold (cf. Figs 7 and
8).
DISCUSSION
The patterns of actin localization during neurulation and somitogenesis are
analogous to that observed in other epithelial systems where morphogenetic
processes are accompanied by actin localization and tissue contraction at the
tissue interface which is becoming concave (Lash et al. 1973; Sadler et al. 1982;
Vakaet & Vanroelen, 1982). The process of epithelialization during
somitogenesis is poorly understood. Actin localization alone cannot be readily
understood in terms of the mechanism of somite formation; it is, however, a
significant factor in the process. It is interesting that the dorsolateral aspect of the
forming somite appears to start epithelialization after the medial and ventral
aspect. The dorsomedial surface of the somite will maintain cell-to-cell contact
for a longer period of time than the ventromedial surface. It is the most dorsal
portion (dermatome) that will migrate to the epithelium to form the dermis of
Actin distribution during chick morphogenetic movements
31
the skin whereas the cells just beneath it (the myotome) will remain in close
association as they differentiate into muscle. The relation between actin
distribution and these subsequent differentiative events is unknown. It may be
significant that the core cells, which appear to be passive participants in the
process of somitogenesis, never show indications of actin localization.
The pattern of actin localization in the neuroepithelium is also in agreement
with the observations that localization and cellular contraction occur in the
region of the epithelium that is changing its shape to become a concave structure
(cf. Karfunkel, 1972; Bancroft & Bellairs, 1975; Nagle & Lee, 1980). This
process has been described by Sadler et al. (1982) in the mouse embryo, and we
have extended their observations to obtain more direct evidence of actin involvement. Many reports have indicated that actin localization is disrupted and
morphogenetic processes cease in the presence of cytochalasin B (Karfunkel,
1972; Lash etal. 1973; Chernoff & Lash, 1981). As Chernoff & Lash (1981) have
shown this to be true for the forming somite, we see a similar phenomenon during
neurulation. Similar observations in chick embryo neurulation were made by
Karfunkel (1972). Cytochalasin B disrupts the actin pattern of localization in the
neuroepithelium, and the actin becomes randomly dispersed. Subsequently the
process of neurulation not only ceases, but the neuroepithelium was seen to
flatten again.
Thus, the pattern of actin localization during morphogenetic movements involving the bending of epithelial sheets or the meristic segmentation of segmental plate mesenchyme can be readily visualized in the light microscope using a
high-resolution technique for actin distribution. With a carefully selected series
of different developmental stages and a three-dimensional computer-assisted
reconstruction, this technique has the potential for giving useful information
concerning the mechanisms of these processes. This type of information is not
readily available using the procedure of transmission electron microscopy, the
most-commonly used method at present.
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FRANKLIN,
{Accepted 17 June 1983)