Active reinforcement of externally imposed folding in amphibians embryonic tissue

Abstract

Although the folding of epithelial layers is one of the most common morphogenetic events, the underlying mechanisms of this process are still poorly understood. We aimed to determine whether an artificial bending of an embryonic cell sheet, which normally remains flat, is reinforced and stabilized by intrinsic cell transformations. We observed both reinforcement and stabilization in double explants of blastocoel roof tissue from Xenopus early gastrula embryos. The reinforcement of artificial bending occurred over the course of a few hours and was driven by the gradual apical constriction and radial elongation of previously compressed cells situated at the bending arch of the concave layer of explant. Apical constriction was associated with actomyosin contraction and endocytosis-mediated engulfing of the apical cell membranes. Cooperative apical constrictions of the concave layer of cells produced a tensile force that extended over the entire surface of the explant and correlated with apical contraction of the concave side cells. In the explants taken from the anterior regions of the embryo, this reinforcement was more stable and the bending better expressed than in those taken from suprablastoporal areas. The morphogenetic role of cell responses to the bending force is discussed.

Full text

Active reinforcement of externally imposed folding
in amphibians embryonic tissues
Stanislav V. Kremnyov
*
, Tatyana G. Troshina, Lev V. Beloussov
Department of Embryology, Faculty of Biology, Lomonosov Moscow State University, Moscow 119991, Russia
ARTICLE INFO
Article history:
Received 24 September 2011
Received in revised form
31 January 2012
Accepted 2 February 2012
Available online 11 February 2012
Keywords:
Epithelial folding
Mechanically induced cell shape
changes
Endocytosis
Xenopus laevis
ABSTRACT
Although the folding of epithelial layers is one of the most common morphogenetic events,
the underlying mechanisms of this process are still poorly understood. We aimed to deter-
mine whether an artificial bending of an embryonic cell sheet, which normally remains
flat, is reinforced and stabilized by intrinsic cell transformations. We observed both rein-
forcement and stabilization in double explants of blastocoel roof tissue from Xenopus early
gastrula embryos. The reinforcement of artificial bending occurred over the course of a few
hours and was driven by the gradual apical constriction and radial elongation of previously
compressed cells situated at the bending arch of the concave layer of explant. Apical con-
striction was associated with actomyosin contraction and endocytosis-mediated engulfing
of the apical cell membranes. Cooperative apical constrictions of the concave layer of cells
produced a tensile force that extended over the entire surface of the explant and correlated
with apical contraction of the concave side cells. In the explants taken from the anterior
regions of the embryo, this reinforcement was more stable and the bending better
expressed than in those taken from suprablastoporal areas. The morphogenetic role of cell
responses to the bending force is discussed.
Ó2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
The folding of epithelial layers is one of the most common
and precisely regulated modes of morphogenesis throughout
the animal kingdom. This folding is a dominant means of
molding the specific shapes of organisms and has been de-
scribed by classical embryologists in great detail; however,
the motive forces and, in particular, the spatiotemporal con-
trol of the folding patterns are still poorly understood. Studies
of epithelial folding are not only of fundamental interest but
also are important in bioengineering and regenerative medi-
cine research (Davidson et al., 2010; Zartman and Shvarts-
man, 2010). Concerning motive forces, in a number of cases,
the formation of a fold was found to be mediated by the apical
constriction and radial elongation of cells located at the con-
cave side of the fold. This response is attributed to the coordi-
nated work of the cytoskeleton (actin and microtubules) and
the motor protein Myosin II (Lee and Harland, 2007; Martin
et al., 2010). Several different proteins regulate actin contrac-
tility, including Shroom3 (Haigo et al., 2003) and Lulu (Nakaj-
ima and Tanoue, 2010). However, it is unclear whether there
are any universal mechanisms of epithelial folding. For exam-
ple, apical constriction in the neural plate of chick embryos
(Schoenwolf et al., 1988) and other mammals (Ybot-Gonzalez
and Copp, 1999) can occur when microfilaments are dis-
rupted. Another unsolved problem relates to the spatiotem-
poral regulation of folding patterns. Several models have
been suggested to explain the location and arrangement of
folds under the influence of either diffusing substances or
mechanical stresses. The latter class of models, which was
0925-4773/$ - see front matter Ó2012 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mod.2012.02.001
* Corresponding author. Tel.: +7 495 939 3915; fax: +7 495 939 4309.
E-mail addresses: s.kremnyov@googlemail.com (S.V. Kremnyov), tanyatrosh@list.ru (T.G. Troshina), morphogenesis@yandex.ru
(L.V. Beloussov).
MECHANISMS OF DEVELOPMENT 129 (2012) 51–60
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/modo

first presented by Odell et al. (1981), and other morphome-
chanical approaches have recently attracted increased inter-
est (e.g., Varner et al., 2010; Martin et al., 2010; Luu et al.,
2011). Several sets of data, obtained mostly from Drosophila
embryos, suggest that mechanical forces not only drive inde-
pendently established folding processes but may also act as
triggering signals (Pouille et al., 2009; Fernandez-Gonzalez
et al., 2009a,b; Taguchi et al., 2011). These data suggest that
a mechanically based feedback circuit exists that provides
self-organization of morphological patterns (Fernandez-
Gonzalez et al., 2009a,b). In this paper, we demonstrate for
the first time the formation of a fold from embryonic material
that normally remains flat (the ventral ectoderm of Xenopus
early gastrula embryos) by an artificially imposed compres-
sive force. We also show that the primary response of a cell
to this force is the constriction and engulfment of apical cell
surfaces, which require the concerted action of endocytosis
and actomyosin contractility.
2. Materials and methods
Experiments were performed on Xenopus laevis (Daudin)
embryos obtained from hormonally stimulated adult frogs
and cultured at room temperature.
2.1. Microsurgery
Before operations, embryos were de-jellied with 2.5% cys-
teine solution and liberated by forceps from vitelline mem-
branes. During and after operation, embryos were cultured
in 2% agarose-coated plastic Petri dishes filled with Marc’s
modified Ringer’s (MMR) solution (100 mM NaCl, 2 mM KCl,
2 mM CaCl
2
, 1 mM MgCl
2
, 5 mM HEPES, pH 7.4). Experiments
were performed on X. laevis embryos at developmental stages
10–10 1/2 (early gastrula) as described by Nieuwkoop and
Faber (1956). Pieces of blastocoel roofs that were excised from
homologous regions of a pair of the same stage embryos were
fused by their inner surfaces, extensively bent along their
mid-lines and inserted vertically into a groove dig with a knife
into an agarose substrate to fix the imposed bending (Fig. 1A).
The bending plane was oriented either parallel or perpendic-
ular to the anteroposterior axis of the embryo. Because the
bending orientation did not affect the obtained results, we de-
scribe these results together. We examined the bending re-
sponse of sandwiches prepared from three different zones
of the blastocoel roof arranged in anteroposterior succession
and defined as ventral, dorsal and suprablastoporal. The bent
samples were cultured for different time periods up to 24 h
and then fixed for histology.
2.2. Evaluation of local tension by tissue incision
Assuming that the angles formed by recoiling tissue edges
immediately after local incisions are roughly proportional to
the tensions on the tissue surface (Hutson et al., 2003), we
performed incisions at the bending apex and the lateral sur-
face of the samples at different times (from 5 min to 24 h)
after artificial bending. For each time point, a minimum of
five incisions were made, one incision per sample. To study
the effect of bending on incision angles, we prepared unper-
turbed (unbent) double explants and incised them at the
same time points. All samples were fixed in Bouin’s fluid
30 s after incision and underwent routine histology for paraf-
fin sectioning. The incision angles were measured on photo-
graphs of the medial sections through the incised areas.
2.3. Cytoskeletal inhibitors
All inhibitors were diluted in MMR at the following final
concentrations: 1 lM latrunculin B, 10 lM cytochalasin D,
300 lM ML-7, 100 lM blebbistatin, 50 lM Y-26732, 15 lg/ml
nocodazole, and 20 lg/ml taxol (Lee and Harland, 2007). One
percent DMSO in MMR was used as a control solution. Each
experiment was performed on six samples.
2.4. Optical histology
Samples were fixed in 2.5% glutaraldehyde, postfixed in 1%
osmium tetroxide, embedded in epoxy resin (Epon-812) and
examined with a Carl Zeiss microscope Axiovert 25 CFL with
a Zeiss AxioCam HRc camera, in the sagittal or transverse
semi-thin sections stained by 1% toluidine blue.
2.5. Transmission electron microscopy (TEM)
For TEM, Epon-embedded ultrathin sections stained with
1% uranylacetate and lead citrate were prepared and exam-
ined using a JEM-1011 microscope with a Gatan ES500W Mod-
el 782 camera.
2.6. Scanning electron microscopy (SEM)
For SEM, total and fractured samples were fixed in 2.5%
glutaraldehyde, postfixed in 1% osmium tetroxide and dehy-
drated in ethanol and acetone. Total samples were prepared
for SEM. Samples were examined with a CamScan S-2
microscope.
2.7. Confocal microscopy
Confocal microscopy was performed on a Zeiss Axio-
vert200m LSM 510Meta with a Zeiss AxioCam HRm camera.
2.8. Morphometric measurements
Morphometric measurements were made using ImageJ.
We measured the apical indices (AI) of epithelial cells situated
at the concave or convex sides of the bending arch from pho-
tographs of microscopic sections of Epon embedded samples.
AI is the ratio L/Wbetween the maximal cell length (L) in the
direction perpendicular to its apical surface and cell width
(W), as observed on transverse sections (Fig. 1B). Each mea-
surement was performed on at least five normal (non-treated)
samples and six samples treated with any of the cytoskeletal
drugs. In each case, the measurements were made on 50 cells
per sample.
52 MECHANISMS OF DEV ELOPMENT 129 (2012) 51–60

2.9. Time-lapse filming
Time-lapse filming was performed using a digital camera
(DCM130) on an Olympus SZX9 stereomicroscope. The sam-
ples inserted into the grooves were filmed from above.
2.10. Statistics
Statistical significance was determined using the Statistica
6.0 program (Section Basic Statistics). We compared the AI
values of concave and convex surface cells belonging to sam-
ples fixed at different times after bending, prepared from dif-
ferent anteroposterior regions and treated with different
cytoskeletal inhibitors. In addition, we analyzed the differ-
ence between Land W. Using the same program, we deter-
mined a correlation coefficient between AI and incision
angles.
3. Results
3.1. Cell movements, apical indices and morphology of
bent ventral ectoderm sandwiches
Immediately after bending, the edges of the inner layer
started to roll outward, tending to fuse with the outer layer
edges. This rolling is also a part of the standard response of
an excised tissue piece, directed towards wound closure. In
a few minutes, these movements were reversed, which lasted
for a few hours and were directed toward closure of the bend-
ing slit (Fig. 2A–C and Supplement). Both the bending and slit-
closing movements correlated with an increase in the apical
indices of concave, side, outer ectodermal cells situated at
the fold arch (Fig. 3). An initial AI increase was detected as
soon as 2 min after the end of the bending procedure. Within
this brief time period, the apical surfaces of the concave side
cells were rounded and did not show any signs of reduction
(Fig. 3A). Therefore, we regarded these deformations as a
purely passive response to compression. Further AI increase
Fig. 2 – Two frames from a time-lapse film (A and B) and a
diagram of cell trajectories (C) covering the first two hours
after bending a sandwich, prepared from the ventral
ectoderm. Pink points: starting positions of a set of cells,
belonging to the inner sandwich layer; blue points: final
positions of the same cells. As observed in C, the inner layer
cells initially move outwards to close the wound gap and
subsequently become involved in an opposing movement
that closes the slit of the bent sample.
Fig. 1 – Scheme of the operation (A) and of the apical index measurements (B).
MECHANISMS OF DEVELOPMENT 129 (2012) 51–60 53

occurred gradually and was associated with the flattening and
diminishment of apical cell surfaces (Figs. 3D, 4A). Prolonged
arrays of elongated and bottle-shaped cells formed in the out-
er concave layer (Fig. 8, middle row). These events suggest an
active cell response to mechanical compression. Such a re-
sponse was in sharp contrast with the behavior of convex side
cells in bent sandwiches and blastocoel roof cells of intact
embryos. The convex side cells did not show any significant
AI changes (Fig. 3B), while the intact blastocoel roof cells dem-
onstrated statistically significant flattening (AI decrease) only
at the end of the observed time period, which we relate to
epiboly (Fig. 3G).
Immediately after artificial bending, both parts of the
sandwich, particularly the outer layer, appeared disinte-
grated, with the outer ectodermal cells taking on rounded
shapes (Fig. 5A). Within a few hours, the integrity of the outer
ectodermal layer was restored, while the inner cells created a
common mass that was gradually rearranged because of
increasing thickness (Fig. 5B and C). From several hours on-
ward, small cavities (homologous to the blastocoel) emerged
in the internal cell mass (Fig. 5D). By 8 h, the sandwich sur-
faces appeared to be extensively folded (Fig. 5D and E).
3.2. Dynamics of tension on explant surfaces, as revealed
by local incision
As observed in Figs. 6 and 7, the incision located at the
bending apex produced a substantial gap within 5 min of
bending, whereas incisions in the lateral areas reached the
same value 30 min later. Therefore, the immediate result of
artificial bending was a strictly localized stretching of the con-
vex surface and the creation of a tension inequality between
the convex and concave surfaces at the bending apex. The
subsequent spreading of a roughly uniform tension through-
out the entire external surface correlates with the fusion of
the opposite layers of outer ectoderm at the edges of a pro-
gressively narrowed slit. To test whethera pulling force is pro-
duced by apical contraction of the concave side cells that
could contribute to the tension on the outer surface, we deter-
mined whether there is a positive correlation between the
average AI of cells located within the bending groove and
the angles of incision gaps at the outer surface of the same
samples (Fig. 8). We selected a group of six samples at the
4 h time point for these measurements. Despite a restricted
number of samples, the correlation turned out to be signifi-
Fig. 4 – Scanning electron microscopy view of the concave (A) and convex (B) areas of the bent blastocoel roof sandwiches 2 h
after operation.
Fig. 3 – Dynamics of the apical indexes (AI) of the outer ectodermal cells situated at the concave and convex sides of the bent
sandwiches compared to the AI of intact blastocoel roof cells. Times after bending procedure are shown. Insets illustrate the
measurement areas of 2 min (left) and 2 h samples. Data represent the mean ± SE.
54 MECHANISMS OF DEV ELOPMENT 129 (2012) 51–60

cantly positive (r= +0.87). Consequently, by this time, the en-
tire bent explant was transformed into a mechanically inte-
grated unit with tensile forces produced by apically
contracting cells spread across the entire surface. The role
of bending-produced pulling forces in the increase of overall
tension is also supported by our finding of significantly smal-
ler values of incision angles, and hence tension values, in flat
(i.e., unbent) explants up to the 2 h time point. A sharp in-
crease in tension in 4 h flat explants was associated with
the formation of a number of inflated cavities (IC, Fig. 5),
which was typical of all ventral ectoderm samples at this time
point. The near complete loss of tension in both the bent and
flat 24 h samples (corresponding to the advanced neurula
stage) appears to be a non-specific age phenomenon.
3.3. Role of endocytosis and cytoskeleton dynamics in the
AI increase
Several studies have indicated that apical cell constriction
is accompanied by endocytosis of the apical membrane (Chua
et al., 2009; Lee and Harland, 2010). To detect whether endocy-
tosis occurred in our samples, we added the vital lipophilic
dye FM 4-64FX to 1.5 h bent explants and cultured them for
30 min. At this point, the AI difference between concave
and convex side epithelial cells have reached its maximal
value. We observed that dye absorption in concave side cells
exceeded the absorption in the convex side cells, suggesting
that extensive endocytosis occurs in concave side cells
(Fig. 9A). We additionally observed a large number of vesicles
in subapical parts of the concave side cells and numerous
outgrowths on their apical membranes, which was not ob-
served in the convex side cells (Fig. 9, cf B–E).
All the cytoskeletal drugs used significantly inhibited the
AI increase of concave side cells without noticeably affecting
the convex side cells. Inhibition was mostly due to the sup-
pression of apical constriction rather than the suppression
of radial elongation (Fig. 10, cf A–C). The most pronounced ef-
fects were generated by ML-7 or blebbistatin treatment, both
of which are known to decouple acto-myosin interactions.
Less pronounced defects were observed after treatment with
cytochalasin D or with inhibitors of microtubules assembly
(nocodazole and taxol).
3.4. Regional differences in active responses to bending
Although samples prepared from all regions of the blasto-
coel roof demonstrated an AI increase in the concave side
cells situated at the bending arch, the amount of this increase
gradually diminished in the anteroposterior direction
(Fig. 11B). This diminishment was due to a reduction in radial
cell lengths, as the amount of apical constriction remained
constant (Fig. 11C and D). The sandwiches prepared from pos-
terior regions showed a greater tendency to unbend by crawl-
ing out of the slit. After 2 h of incubation, approximately one
half of the bent sandwiches prepared from the SBA (suprabl-
astoporal area) moved out of the slit and straightened; in the
remaining explants, the imposed curvature was considerably
reduced. Most of these samples initiated elongation in the
anteroposterior direction after 2 h, and the imposed fold
stayed flattened. In the ventral samples, the imposed bending
was retained in 80% of the cases after 4 h of cultivation and in
30% after 24 h. By our observations, unbending in the resting
cases was due to the turgor pressure in newly arisen frag-
ments of the blastocoel cavity.
4. Discussion
The main result reported in this paper is that the blasto-
coel roof cells from early gastrula Xenopus embryos actively
react to mechanical compressions produced by an applied
force by gradually undergoing apical constriction and radial
elongation movements. This response occurs in a cooperative
way, as more or less prolonged arrays of elongated cells are
formed. This process increases tension on the surface of the
entire explant and produces a macromorphological result,
namely, the reinforcement and stabilization of an imposed
fold that is ectopic to this embryonic area during normal
development. Reinforcement can be interpreted in terms of
the hyper-restoration (HR) model of morphogenesis (Belous-
sov et al., 2006), which claims that an embryonic cell or tissue
affected by an external stretching or compressive mechanical
force develops an active response directed toward diminish-
ing the imposed stress. Several examples of this response
have been described, such as in cells responding to artificial
stretching by generating internal pressure forces in the direc-
Fig. 5 – Dynamics of shape changes in the bent blastocoel roof sandwiches, as observed in the medial sections. (A) 0 h, (B) 2 h,
(C) 6 h, (D) 8 h, (E) 24 h after operation. (C) displays the arrangement of active contractile forces generated by cells lining the
groove (red converged arrows) and the resulting tensile stresses (blue curved arrow). IC: inflated cavities in the inner parts of
tissue sandwiches.
MECHANISMS OF DEVELOPMENT 129 (2012) 51–60 55

tion of stretch (Beloussov et al., 2006; Troshina and Beloussov,
2009). In this study, we provide the first clear example of an
HR response to cell compression, namely the production of
tension. The cellular response to compression is confirmed
first by its gradualness, as the response lasts several hours,
and second by its sensitivity to different cytoskeletal drugs.
Among the latter, the most pronounced effects were observed
with ML-7 and blebbistatin, indicating a crucial role for acto-
myosin contractility in the response to compression. Two
other main components of the response were endocytosis
and the subsequent engulfment of the apical cell membrane,
as revealed by the use of a specific marker and transmission
electron microscopy (Fig. 7). Both components seem to be
tightly coupled with each other, as the decreasing tension in
the apical cell membrane caused by its shrinkage due to acto-
myosin contractility can directly stimulate endocytosis
(Apodaca, 2002). Previous studies have demonstrated the
involvement of endocytosis in normal and relaxation-associ-
ated morphogenetic processes (Betchaku and Trinkaus, 1986;
Ivanenkov et al., 1990).
We have also measured the relation between the incision
gap length and the total length of the explant outer surfaces,
which can be taken as a measure of the bent explant strain.
As a result, we obtained a value of approximately 15%, which
is on the same order as the strain in the blastoporal circum-
ference (24%) that is associated with normal gastrulation
and was measured using the same technique (Kornikova
et al., 2009). Therefore, the forces involved in active bending
are within the range of those involved in normal
morphogenesis.
Fig. 6 – Incisions gaps as observed in the medial sections of the samples fixed 30 s after operations. Upper row: incisions at
the bending apexes of the bent explants; middle row: incisions at the lateral surfaces of bent explants; lower row: incisions
on the surface of flat (unbent) double explants. Times of incubation are shown. Dashed lines display the gap angles. IC:
inflated cavities.
Fig. 7 – Temporal dynamics of incision gap angles at the bending apex (red), in the outer lateral ectoderm (blue) of bent
explants and in the flat (unbent) explants (green). Data represent the mean ± S.
56 MECHANISMS OF DEV ELOPMENT 129 (2012) 51–60

Fig. 9 – Endocytosis and surface dynamics in the tip regions of 2 h bent sandwiches. (A) Absorption of a lipophilic dye FM 4-
64FX in the concave side cells is increased compared to convex side cells. (B–E) Transmission electron microscopic view of the
apical surfaces of the concave (B and C) and convex (D and E) side cells. Note the extensive ruffling of the apical cell membrane
in B and C.
Fig. 8 – Correlations between the average apical indices and the angles of incision gaps in 4 h bent samples. (A) Correlation
plot (r= + 0.85). (B) Files of cells with increased AI from the groove regions of three different samples. (C) Incision gaps as seen
from the lateral outer surfaces of the same samples.
MECHANISMS OF DEVELOPMENT 129 (2012) 51–60 57

A peculiar and unexpected result of our experiments was
that the effect of bending on tissues located in the blastopore
vicinity was less pronounced and highly reversible compared
to ventrally located tissues (Fig. 11). In subsequent develop-
mental stages, the suprablastoporal area (SBA) generates the
neural fold; therefore, the bending response could be ex-
pected to be mechanically induced much easier within the
SBA than in tissues that do not typically participate in these
deformations. The observed result indicates the opposite
and suggests that SBA tissues, unlike ventral tissue, are
strictly programmed to perform morphogenetic movements
according to a precise spatiotemporal schedule and are capa-
ble of resisting interventions that would violate this pattern.
As shown by Troshina and coauthors (2011), it is much more
difficult to change the direction of cell intercalation in SBA
tissues by abnormally oriented stretching than it is to change
the direction of ventral tissue cell behavior.
Therefore, we conclude that the artificial fold can best be
reproduced on embryonic material, which, in normal devel-
opment, is not affected by any of the forces that can trigger
folding; this is the case for the ventral ectoderm of the early
gastrula Xenopus embryo. It is important to know whether
the fold-triggering situation reproduced in our experiments
is used in normal development. To that end, a recent morpho-
mechanical study analyzed the formation of the head fold in
the early chicken embryo (Varner et al., 2010). The first step in
head fold formation is a planar longitudinal compression of a
flat cell sheet, where the compression is generated by
Fig. 10 – Effects of the cytoskeletal drugs (shown at right) on the (A), (B) and (C) (lm) of the concave and convex side cells from
the tip regions of 2 h bent sandwiches prepared from the ventral parts of the blastocoel roof. For each set of measurements,
six different samples were analyzed with 50 cells per sample. Bars indicate mean value ± SE.
58 MECHANISMS OF DEV ELOPMENT 129 (2012) 51–60

convergent extension of the anterior neural plate. This longi-
tudinal compression is homologous to the artificial forces
used in our study. The second step in head fold formation is
the emergence of wedge-shaped cells on the concave surface
of the fold tip, which is comparable to our findings on the
concave surface of the Xenopus explant artificial folds. By
studying both the subsequent folding of Amniota embryo
blastoderms and the different types of extensively folded epi-
thelia of meso- and endodermal origin, similar mechanisms
of mechanical reinforcement can be revealed.
Acknowledgements
We thank Dr. Thierry Galli for his advices as concerning the
technique of endocytosis examination. This investigation
was supported by the Carl Zeiss Firm Grant ‘‘Grant for young
researchers of the leading Universities of Russia’’, the Russian
Science Support Foundation, grant for young researchers
‘‘The best postgraduate students of Russian Academy of Sci-
ences’’ and Russian Fund of Fundamental Investigations
(RFFI), Grant Number 11-04-01718.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.mod.2012.02.001.
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