![Effects of stretching embryonic tissues. A: a typical vector field of cells convergence-extension triggered by stationary tissue stretching in horizontal direction. Redrawn from time-lapse film 30 min duration. B (from left to right): transformation of horizontally stretched explant of SBA tissue into a dumb-bell notochord rudiment , elongated perpendicularly to its normal direction. C: a control (non-stretched) VLA explant. D-E: various shaping of VLA explants stretched on latex films. A: from Troshina et al., [18]. B: from Mansurov , [19]. C-F: courtesy of N.N. Luchinskaia.](https://www.researchgate.net/profile/Lev-Beloussov/publication/290789996/figure/fig1/AS:360885194379271@1463053143774/Effects-of-stretching-embryonic-tissues-A-a-typical-vector-field-of-cells_Q320.jpg)
![Correlations between artificially imposed shape and cell differentiation patterns in double explants of SBA tissue. A: slightly bent double explant before a complete sticking of its layers. B: a similar explant 3 h later acquiring a bent shape. Note formation of a narrow curved ingression on the concave side. C, D: in situ-hybridized 24h explants. Neural tissue (dark blue) is located on the concave side of explant, while muscle tissue (mesodermal somites) (red) form a band shifted towards convex side [21].](https://www.researchgate.net/profile/Lev-Beloussov/publication/290789996/figure/fig4/AS:360885194379274@1463053143881/Correlations-between-artificially-imposed-shape-and-cell-differentiation-patterns-in_Q320.jpg)
![Relations between curvatures and cells rearrangements. A, C, arrows: formation of columnar cell domains in the areas of maximal curvature. B: immigration of cells from the maximal curvature area. Bracket in C denotes undulations on the convex (mostly stretched) area. A: BRA sandwich; B: normally developing sponge larva. C: Xenopus embryos, artificially arrested at the early gastrula stage. From Beloussov [7].](https://www.researchgate.net/profile/Lev-Beloussov/publication/290789996/figure/fig2/AS:360885194379272@1463053143815/Relations-between-curvatures-and-cells-rearrangements-A-C-arrows-formation-of_Q320.jpg)
![A-E. Stabilization and increase of artificially imposed curvature in tissue sandwiches prepared from BRA. Times after operation are shown. Red converged arrows in (C) display active contractile forces while blue lines depict stretched surfaces which later on (E) create numerous undulations (see below for comments). IC: inflated cavities in the inner parts of tissue sandwiches [20].](https://www.researchgate.net/profile/Lev-Beloussov/publication/290789996/figure/fig3/AS:360885194379273@1463053143850/A-E-Stabilization-and-increase-of-artificially-imposed-curvature-in-tissue-sandwiches_Q320.jpg)
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Jacobs Journal of Regenerative Medicine
Simple Tools for Structuring Embryonic Rudiments
Beloussov LV*, Kremnyov SV, Luchinskaia NN
Laboratory of Developmental Biophysics, Department of Embryology, Faculty of Biology, Moscow State University 119991 Moscow
*Corresponding author: Dr. Beloussov LV, Laboratory of Developmental Biophysics, Department of Embryology, Faculty of Biology,
Moscow State University 119991 Moscow, Email: morphogenesis@yandex.ru
Received: 08-24-2015
Accepted: 11-26-2015
Published: 12-22-2015
Copyright: © 2015 Beloussov
Abstract
In this paper we review experimental approaches used in our research group for deforming embryonic tissues in amphib-
ian embryos by relaxing pre-existed tensions, stretching samples in given directions or bending them. In these experi-
ments, owing to the active tissue reactions to changes in mechanical stresses, they change their shapes in predictable ways.
In some cases the changes in geometry dictate reconstruction of cell differentiation patterns. We suggest that these re-
Keywords: Mechanical stresses; Xenopus embryos ; Cell movements; Tissue deformations ; Curvatures
Review article
Cite this article: Beloussov. Simple Tools for Structuring Embryonic Rudiments. J J Regener Med. 2015, 1(2): 008.
Introduction
It is almost trivial to remind that the samples which the re-
generative medicals are striving to construct should not be
amorphous. To give them a properly controlled shape and
spontaneously in cultivated pieces of tissue is a challenging
task. In this essay we describe some simple approaches used
in our lab for providing pieces of embryonic tissues by a de-
sired spatial structure in the hope that manipulations which
we used for apprehending the fundamental laws of morpho-
genesis may be already now of some applicatory value.
We use the term “structuring” in a broad sense as embrac-
changes in tissue structure, such as formation of columnar
cells domains (placodes) out of homogeneous epithelial
sheets, creation of smooth borders within dense cell mass-
es, collective cell migrations in desired direction(s), loss and
-
ed that all of these processes are controlled by endogenous
mechanical stresses (MS), mostly tensile ones, so that by
modulating MS at the proper stages of development we can
evidences, MS are crucial for development of all Metazoans,
tensions on the surface of yolk sac hampers the development
shape formation it is important to apprehend that it is based
upon the active mechano-chemical reactions of embryonic
tissues to MS, the latter being in normal development gener-
ated mainly by immediately preceded morphogenetic move-
-
lished within developing embryos between MS patterns and
done by a bioengineer is to modify MS patterns in the hope
to get from embryonic tissues the expected morphological
still far from being elucidated in all the details, some of its
properties are already more or less clear. We in our group
employ for this purpose a so called hyperrestoration (HR)
model which claims that embryonic tissue responds to any
-
ed towards the restoration of initial MS value but overshoot-
ing it to another side. Similarly, whenever such MS changes
are unevenly distributed or are anistropic, then the respons-
es will be directed towards reducing with an overshoot
B, C) and to multiply homologous rudiments, in the given case
Figure 1. Relaxation-induced effects in Xenopus
of a ventral tissue (pointer) is inserted in a blastula stage embryo.
-
ly. Note abnormal protuberances in B, C and a complete loss of order
-
plication (G) of neural tubes. H: 2-dimensional maps of cells height/
width (H/W) relations in the dorsal ectoderm of an intact (left) and
by most columnar cells. I: a scheme of a “remove – put it back” proce-
dure. J: a resulted chaotic arrangement of axial organs in the operated
of columnar cell areas (usually called placodes) at the expense
usually connected with cell differentiation (neuralization, de-
velopment of sensory cells) these transformations may be of a
particular value for regenerative medicine.
-
-
the crucial steps of oncogenesis.
presented below will illustrate the applications of HR model.
It is of a primary importance for bioengineering purposes to
know to what extent the cell differentiation patterns are linked
with morphogenesis in its strict sense, that is, embracing noth-
ing more than embryonic geometry and topology. In other
words, to what extent (if any) cell differentiation is mecha-
spite of a certain autonomy of the both processes, such a de-
pendence may be greater than it could be suggested before-
hand. One of below presented experimental models will be the
illustration.
following three kinds of mechanical procedures:
• Relaxation of tensions;
• Imposing tensions in abnormal directions;
Results of tensions relaxation: multiplication of anlagens, for-
mation of abnormal protuberances, enlarging of epithelial
placodes.
Tensions in amphibian embryonic tissues can be relaxed by
is to temporally reduce turgor pressure in blastocoel which
normally stretches so called blastocoel roof, later giving rise to
gastrula stage (when blastocoel is already largely reduced) is
to insert within a vegetal embryo hemisphere a sector of ho-
tensions within a small piece of tissue, a “remove – put it back”
ectoderm) should be extirpated from embryo and in about a
this brief time period the piece is contracted and wound gap
enlarged, so that the returned piece cannot establish lateral
contacts with surrounding tissues which is necessary for re-
storing the initial tensions.
In these entire cases one should take into mind that if a tissue
remains alive, a real relaxation of tensions will last no more
than for few minutes: more prolonged loss of tensions switch-
based upon cells geometry show indeed that the restoration
however that practically in no cases the initial (normal) tensile
pattern is spontaneously restored: as a rule the regular global
patterns embracing the entire embryonic body are replaced by
any of the above described procedures is a loss of a long-range
morphological order while more local processes (including
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Cite this article: Beloussov. Simple Tools for Structuring Embryonic Rudiments. J J Regener Med. 2015, 1(2): 008.
Consequences of controlled tissue stretching: triggering
convergence-extension cell movements and molding vari-
ous axi-symmetric shapes.
We use two methods for stretching embryonic tissues. By the
-
amphibian embryos of so called inner ectoderm cells, loose-
-
poses it is easier to stretch explants in a step-wise manner by
advantage of this method is that the stretching can be applied
to double explants (sandwiches) completely isolated from ex-
ternal environment, rather than to naked explants, as in the
previous case.
In the both cases the main result was stimulation of so called
convergence-extension cell movements which normally play
a leading role in formation of so called axial organs (neural
coming into more details, we have to become familiar with
gastrula stage they consist of two parts, called suprablastopo-
-
transversely) it is possible to reorient the axial organs (and in
-
cell movements (never presented normally in this area) can be
triggered as well, but in no cases this will lead to formation of
something similar to the axial organs.
are rather peculiar and various, differing from what may be
expected in the forcibly elongated samples: one can see among
stretching force is directly applied) become, contrary to expec-
model, in response to outside stretching the internal pressure
sample. This permits, by varying a total amount and periodic-
ity of stretching to obtain a large repertoire of axi-symmetric
shapes.
Figure 2.
min duration. B (from left to right): transformation of horizontal-
-
ment, elongated perpendicularly to its normal direction. C: a control
-
Artificially imposed curvature can be actively enhanced;
within a competent tissue this may affect cell differentia-
tion patterns.
-
ens of seconds it resists bending as an elastic body, attempting
to straighten back again. Within few minutes however it not
only takes an imposed shape but tries to reinforce it active-
ly by increasing the bending curvature due to contraction of
passive to active bending seems to play an important role in
making embryonic shapes and may be hence recommended
for bioengineering purposes. Moreover, while the bending of
-
neural and muscle tissue: the neural cells (dark blue) are dif-
ferentiated as a compact cluster in the concave area of a double
explant while muscle cells (red) are formed closer to the con-
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Cite this article: Beloussov. Simple Tools for Structuring Embryonic Rudiments. J J Regener Med. 2015, 1(2): 008.
Just these results are observed both in the normal devel-
In tune with
these expectations, undulations are formed along the most-
-
should be taken in consideration by the bioengineers in-
rearrangements.
Figure 5.
C, arrows: formation of columnar cell domains in the areas of maximal
curvature. B: immigration of cells from the maximal curvature area.
Bracket in C denotes undulations on the convex (mostly stretched)
-
Making tubes out of rolls
among widely spread embryonic rudiments: so called lips of
from mechanics, the transversal (meridian, or circular) sur-
-
rial ones. If applying to these bodies the above described ten-
dency towards smoothing out with an overshoot the tensions
-
placed by similarly oriented pressure stresses (as in examples
-
-
sue incompressibility) elongation of a toroid body in meridian
of intestine out of a blastoporal lip material is an example) and
-
tiating tube formation, it is enough to prepare a ring of tissue
consisting of cells able to convergent intercalation between
each other.
Figure 3A-E.-
are shown. Red converged arrows in (C) display active contractile
Relations between local curvatures and cells rearrange-
ments: bioengineering perspectives.
-
surized internal cavity and are thus transformed into vesicles.
Their shapes rarely become precisely spherical: usually the re-
walls of pressurized cavities are reversely proportional to the
curvatures: the more a given piece of the wall is curved, the
what means that the tensions in the mostly curved regions
should increase while those in less curved ones to go down.
This means that the surface of the mostly curved regions is to
be diminished while that of the less curved to be increased.
To achieve these results, the cells of the mostly curved regions
should either contract transversely (that is, become more co-
relax the surface becoming more thin; in the case of extensive
of the surface.
Figure 4.
-
ly bent double explant before a complete sticking of its layers. B: a
of explant, while muscle tissue (mesodermal somites) (red) form a
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Cite this article: Beloussov. Simple Tools for Structuring Embryonic Rudiments. J J Regener Med. 2015, 1(2): 008.
Figure 6.
depict cell convergent movements directed towards meridians for
releasing maximal tensions and exchanging them by meridionally ori-
ented pressure providing axial elongation of a body (dashed bidirec-
-
opus embryo illustrating the directions of cells convergence. The folds
arisen from the tissue ring isolated from Xenopus early gastrula em-
bryonic tissue.
Attempts to quantify the forces deforming embryonic
tissues
Several recent attempts to measure deforming forces and
which differ from each other in an order or more. By our sug-
gestion, these discrepancies are due to the lack of a common
Young modulus values in the early gastrula Xenopus embryos
-
nator the section area of a sole cell layer which could resist the
deforming force (this is so called epiectoderm)
entire section area of embryonic tissue consisting mostly of
non-supportive endoderm. In addition, we took for measure-
ments only the force values detected in few minutes after the
start of deformation (when the active reactions enhancing the
deformation have not still developed) while the mentioned
-
-
er. In general, the contribution of the active responses having
be neglected and to a large extent depreciates formally correct
imposed deformation typical for gastrula stage embryos is ex-
changed at the advanced stages by a strong oppositely directed
reaction giving the impression of enormous increase of Young
stresses during organ formation in higher Vertebrates (mostly
chicken embryos) are recommended to address the following
Conclusions
We are well aware that the above described results obtained
on amphibian embryos cannot guarantee that similar proto-
cols can effectively work as applied to mammalian (human)
embryonic tissues. By our knowledge, no such attempts has
been as yet performed, although the mechanical backgrounds
-
standartization of recommended procedures should be done.
However these latter are in their essence so simple and the
efforts seems to be not too high.
References
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chanical stresses in embryonic tissues: patterns, morphoge-
netic role and involvement in regulatory feedback. Int Rev Cy-
-
anochemical basis of morphogenesis. Nat Rev Mol Cell Biol.
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