XB-ART-39567Int J Dev Biol January 1, 2006; 50 (2-3): 113-22.
Gastrulation in amphibian embryos, regarded as a succession of biomechanical feedback events.
Gastrulation in amphibian embryos is a composition of several differently located morphogenetic movements which are perfectly coordinated with each other both in space and time. We hypothesize that this coordination is mediated by biomechanical interactions between different parts of a gastrulating embryo based upon the tendency of each part to hyper-restore the value of its mechanical stress. The entire process of gastrulation in amphibian embryos is considered as a chain of these mutually coupled reactions, which are largely dependent upon the geometry of a given embryo part. We divide gastrulation into several partly overlapped steps, give a theoretical interpretation for each of them, formulate the experiments for testing our interpretation and describe the experimental results which confirm our point of view. Among the predicted experimental results are: inhibition of radial cell intercalation by relaxation of tensile stresses at the blastula stage; inversion of convergent intercalation movements by relaxation of circumferential stresses at the early gastrula stage; stress-promoted reorientation of axial rudiments, and others. We also show that gastrulation is going on under a more or less constant average value of tensile stresses which may play a role as rate-limiting factors. A macro-morphological biomechanical approach developed in this paper is regarded as complementary to exploring the molecular machinery of gastrulation.
PubMed ID: 16479480
Article link: Int J Dev Biol
Genes referenced: igf2bp3 mrc1 prph2 sult1a1
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|Fig. 1. Biomechanical interpretation of gastrulation events in amphibian embryos. (AD, E1, E2) Biomechanical �maps� of successive stages and most important regions of embryos. Active forces are displayed by red arrows, while passive stresses are indicated by blue arrows. (A) Stretching of the blastocoel (br) roof by turgor pressure in the blastocoel. (B) RCI in the blastocoel roof producing pressure forces onto vegetal regions. (C) A detailed scheme of the region framed in (B). (D) Two alternating phases of biomechanical interactions between the marginal zone (MZ) and the suprablastoporal zone (SBZ). (E1, E2) Successive stages of the involution in saggital section. Frames (ab,c,d,e) display suggested hyper-restoration responses that correspond to the upper row frames indicated by the same capital letters (ab corresponds to A and B together). Dotted arrows connect the active branches of preceding hyper-restoration responses with the shifts of stress values which they produce in the neighboring parts of embryos triggering the latter�s active responses. Pre, preinvoluted; pst, postinvoluted layer.|
|Fig. 2. Formation of a very much diminished copy of a normal amphiblastula from a ventral ectoderm explant of the early gastrula X. laevis embryo. (A) Scheme of operation. (B-D) An explant immediately after extirpation, 8 and 24 h later. From Beloussov and Petrov, 1983.|
|Fig. 3. Results of relaxation of tensile stresses at the surface of the late blastula stage of X. laevis embryos. (A) An embryo with a wedge-shaped implant causing relaxation, 1 h after operation. (B) A similar embryo, 5 h after operation. (C) Scanning electron microscopy view of a relaxed embryo, 1 day after operation. Note two extensive protrusions which stretch the surrounding tissue areas. (D) A saggital semithin section of a relaxed embryo maintained for 5 h in 50% MMR solution. (E) A similarly treated embryo maintained for the same time period in 25-fold diluted MMR, tensions being considerably restored. (F) Fragment of the blastocoel roof of an intact embryo of the same age as (B). (G,H) Similar fragments from (D,E) embryos, respectively. Note the reduction of the number of cell layers in (F) as compared with (B) and in (H) as compared with (G), indicating the renewal of RCI after incubation in hypotonic solution. (I) A diagram summarizing the results of �stress therapy� of the relaxed embryos. (A) Control group; (B) relaxation by a frontally oriented radial cut through a vegetal embryo region; (C) a similar relaxation by a saggitally oriented radial cut; (D) embryos incubated in a hypotonical (25-fold diluted) MMR after frontally oriented cut; (E) similarly treated embryos after saggitally oriented cut; (F) frontally oriented cut followed by stretching of the embryos. Figures in brackets give the number of samples in the corresponding series. Vertical axis, percent of normalized and abnormal embryos 24 h after operations. Graph 1, normal embryos; 2, exogastrulation; 3, incomplete gastrulation; 4, other anomalies. Note a substantial recovery after tension renewal. From Beloussov and Ermakov (2001).|
|Fig. 4 (Left). Relaxation-promoted formation of additional groups of bottle-shaped cells and the corresponding invaginations (shown in A, D by dense pointers) (A) 1 h after the relaxation of the suprablastoporal zone due to its separation from the underlain tissues (large bent arrow) at the early gastrula stage. blp is the normal blastopore. (B) A saggital section of the dorsal blastoporal lip of the normal mid-gastrula stage embryo. Arrows show separation directions which relax pre-existed tensions. (C) A relaxed lip immediately after separation (D) 1 h later, a new invagination pit has been formed at the site of a former dorsal blastoporal lip (pointer).|
|Fig. 5 (Right). Schemes of the operations for modulating σc and/or σm values (shown by dotted lines) in the marginal zone of early gastrula embryos and expected results. (A) A transversal view of a dorso-medial cut (vertical arrow in A) providing wound opening (diverged arrows) and hence σc relaxation, most of all in the ventral region (wavy part of a contour). Wound gap is covered by a piece of ventral ectoderm. (B) Expected cell convergence along a ventral mid-line due to increase of inequality between a preserved σm (vertical bidirectional arrow) and relaxed σc (convex arrow). View from the ventral side. (C) Enhancement of σc in the dorso-medial zone as a result of transversal stretching by two needles (arrows coming from black spots). (D) Expected result; an extensive transversal cell convergence in the area of the suprablastoporal zone and latero-ventral cell movements due to some σc relaxation in the ventral region.|
|Fig. 6. Inversion of cell convergence movements as a result of a dorso-medial cut of early gastrula stage X. laevis embryos. In the vertical panels (A,B,D), left rows are optical light views and right rows luminescent views of the same embryos at the different time periods after cuts (shown to the left). In panels (C), only luminescent views are given. In (A-C), the descendants of two ventral blastomeres at 32 cell stage are labeled, while in panel (D), the descendants of the same stage 2 dorsal blastomeres are labeled. (A) Normal dorsalwards convergent movements of a labeled ventral tissue. 24 h embryos from this panel are shown from the left side while all the others are displayed in the vegetal projection. (B) Inversion of the labeled cell movements towards ventral midline as a result of a dorso-medial cut. Note that at 24 h, an abnormal tail is formed from these cells in the ventral location. (C) If making a similar dorso-medial cut of an isolated marginal zone, no labeled cell convergence towards a ventral midline takes place. (D) After an operation similar to that shown in panel (B), the descendents of the dorsal labeled cells move to the ventral side, that is, opposite to the normal convergence direction. As a result, they spread along the lateral lips of the blastopore.|
|Fig. 7 (Left). Induction of the active lateralwards movements of the labeled descendants of dorsal cells as a result of transversal suprablastoporal zone stretching by four needles. To the left: optical light and luminescent views just after inserting needles and before stretching them (A), immediately after stretch (B) at 2.5 h (C), 3.5 h (D) and 7 h (E). In optical light views, needles are denoted by black spots. To the right are shown the positions of the needles 1-4 before stretching, immediately after stretch and 2.5 h later. Note a considerable post-stretching increase of the distance 1-2 in spite of the needles being anchored to the agarose substrate. As a result, the dorsal cell material is very much shifted to the ventral, considerably overlapping the needles� positions. This result is quite similar to that obtained by a dorso-medial cut (see Fig. 6).|
|Fig. 8 (Right). Morphological results of suprablastoporal zone (SBZ) transversal stretching, as compared with a similar stretch of neurula stage embryos. (A) Scheme of SBZ stretching by two needles. (B-F) 24 h results. (B,C) Total views (on frame B, the direction of stretching is indicated), whereas (D-F) are histological sections made in transversal embryo planes. Note a complete transversal reorientation of the embryo body including not only the axial rudiments (notochord and somite series), but also the yolk-containing compartments. In (D) and to some extent in (F) part of the notochord is shifted to the lateral blastoporal lip. (G) A scheme of a similar operation performed at the early neurula stage. (H) Its result, total view. Contrary to (A-F), the larva fully preserves its initial axis, remaining bound to one of the needles by a thin unstretched tissue thread (later on disrupted). In (H), pointers indicate the positions of the needles.|
|Fig. 9 (Left). Evidence of cell convergence on artificial folds and ectodermal �scrolls�. (A) A scheme of fold preparation; an ectodermal tissue piece situated lateral to the dorsal mid-line of a gastrula stage embryo is cut off, bent as shown by a dashed line and fixed in this position by a needle. (B,C) Protrusions formed in 10-15 h out of the bent folds (opposite to the needles). (D) If a framed region of lateral ectoderm is extirpated from the neurula stage embryo, it rolls spontaneously into a scroll, its longitudinal axis coinciding with that of embryo (E). Soon the scroll is transformed into a meridionally shrunk body with many transversal grooves and ribs (F,G). Times after scroll formation are shown.|
|Fig. 10 (Right). Residual deformations (RDs) in stretched or shrunk explants of embryonic tissues after their unloading. Vertical axes: percents of the positive (fixed elongation) or negative (fixed shrinkage ) RDs. Initial length is taken as zero. Horizontal axes: time, minutes. (A) Protocol graphs of a stretched explant (1) and two shrunk explants (2,3). Explant 1, stretched to 70% at 5 min time and unloaded 6 min later, gradually returned to its initial length. Meanwhile, explant 2 which was shrunk to 50% and kept in this state for only 2 min gave - 35% RD; explant 3 shrunk for 5 min and gave -100% RD. (B) RD after unloading 10-20% stretch in 5, 30 and 60 min after its application. (B1) Explants of the early gastrula ventral ectoderm, (B2) SBZ tissue, (B3) explants of the lateral ectoderm from neurula stage embryos. While in 5 min after force application the samples B1 and B2 undergo 20-10% contraction, (that is, negative RD), after 60 min stretch, B1 samples gave +20% RD, B2 samples just a slight RD and B3 samples no RD at all.|
|Fig. 11. Exchange of the passive stretching of the postinvoluted layer (pst) at the early-mid gastrula stage (A) by its active extension (B). Arrows show the directions of the separation of the pre-involuted (pre) and postinvoluted layers. Samples were fixed in Bowen�s fluid a few seconds after separation and sectioned saggitally. (A) An extensive contraction and curling of pst. (B) Its active extension, overlapping the layer pre. In the both frames the anteroposterior direction goes from up to down. roof, stretched for an hour up to 20% of their initial length retained, after stretch relief, just 10% of their initial length. Consequently, the rates of normal gastrulation movements roughly satisfy the condition of constant tension. One may conclude that the rate of tension relaxation is the limiting parameter of gastrulation movements. On the other hand, the lateral ectoderm explants taken from early neurula embryos (stages 13-15) did not show RD at all after any amount and duration of stretch (Fig. 10 B3). Hence, at these stages the imposed tensions are not relaxed at all. One concludes that the ability to relax tensions by cell intercalation is a stagespecific property of gastrula stage embryos only. Step 4: Extension of the postinvoluted cell material Theoretical interpretation EE feedback (see Beloussov and Grabovsky, this volume) is expected to be established between preinvoluted (pre) and postinvoluted (pst ) cell material. Namely, at the beginning of involution pst is assumed to be passively extended by the actively extending pre, while later on pst becomes an active component of the mutually promoting EE feedback (see Fig. 1 E1, E2, e). Predictions and experiments While carefully detaching the adjacent pre and pst areas from each other, one should expect that at the start of involution pst will be immediately and extensively contracted (relieving the passive tension), while at the later stages it can even overlap the overlain pre. Just this was observed: if we made such a detachment at A B|