Traffic light Hydra allows for simultaneous in vivo imaging of all three cell lineages.

We present a new transgenic Hydra vulgaris line expressing a distinct fluorescent protein in each of the three cell lineages of the adult polyp. Plasmid microinjection was used to generate a novel transgenic Hydra line expressing the yellow fluorescent protein YPet in the ectodermal epithelial cell lineage. Tissue grafting was then used to combine a YPet animal with a line that expresses DsRed2 in the endodermal epithelial lineage and eGFP in the interstitial cell (i-cell) lineage. The resulting triple-labeled ("tricolored") transgenic line provides, for the first time, a Hydra in which all three cell lineages can be imaged simultaneously in vivo. We show example confocal images of whole animals and individual cells to illustrate the imaging capabilities that this new line makes possible. We also used this line to carry out new studies of cell fate in the tentacles. Specifically, we evaluated the well-accepted notion that all tentacle cells are terminally differentiated and are displaced or migrate exclusively towards the distal end of the tentacle. We found that ectodermal and endodermal epithelial cells are displaced distally, as expected. In contrast, members of the i-cell lineage, which resembled neuronal precursors, could migrate out of a tentacle into the body column. This example illustrates how this tricolored transgenic line enables new in vivo studies of cell behaviors in Hydra.

Transmembrane H+ fluxes and the regulation of neural induction in Xenopus laevis.

It has previously been reported that in ex vivo planar explants prepared from Xenopus laevis embryos, the intracellular pH (pHi) increases in cells of the dorsal ectoderm from stage 10.5 to 11.5 (i.e. 11-12.5 hpf). It was proposed that such increases (potentially due to H+ being extruded, sequestered, or buffered in some manner), play a role in regulating neural induction. Here, we used an extracellular ion-selective electrode to non-invasively measure H+ fluxes at eight locations around the equatorial circumference of intact X. laevis embryos between stages 9-12 (˜7-13.25 hpf). We showed that at stages 9-11, there was a small H+ efflux recorded from all the measuring positions. At stage 12 there was a small, but significant, increase in the efflux of H+ from most locations, but the efflux from the dorsal side of the embryo was significantly greater than from the other positions. Embryos were also treated from stages 9-12 with bafilomycin A1, to block the activity of the ATP-driven H+ pump. By stage 22 (24 hpf), these embryos displayed retarded development, arresting before the end of gastrulation and therefore did not display the usual anterior and neural structures, which were observed in the solvent-control embryos. In addition, expression of the early neural gene, Zic3, was absent in treated embryos compared with the solvent controls. Together, our new in vivo data corroborated and extended the earlier explant-derived report describing changes in pHi that were suggested to play a role during neural induction in X. laevis embryos.

Temporal transcriptomic profiling reveals dynamic changes in gene expression of Xenopus animal cap upon activin treatment.

Activin, a member of the transforming growth factor-ß (TGF-ß) superfamily of proteins, induces various tissues from the amphibian presumptive ectoderm, called animal cap explants (ACs) in vitro. However, it remains unclear how and to what extent the resulting cells recapitulate in vivo development. To comprehensively understand whether the molecular dynamics during activin-induced ACs differentiation reflect the normal development, we performed time-course transcriptome profiling of Xenopus ACs treated with 50 ng/mL of activin A, which predominantly induced dorsal mesoderm. The number of differentially expressed genes (DEGs) in response to activin A increased over time, and totally 9857 upregulated and 6663 downregulated DEGs were detected. 1861 common upregulated DEGs among all Post_activin samples included several Spemann's organizer genes. In addition, the temporal transcriptomes were clearly classified into four distinct groups in correspondence with specific features, reflecting stepwise differentiation into mesoderm derivatives, and a decline in the regulation of nuclear envelop and golgi. From the set of early responsive genes, we also identified the suppressor of cytokine signaling 3 (socs3) as a novel activin A-inducible gene. Our transcriptome data provide a framework to elucidate the transcriptional dynamics of activin-driven AC differentiation, reflecting the molecular characteristics of early normal embryogenesis.

Oviductal fluid counterbalances the negative effect of high temperature on sperm in an ectotherm model.

Global warming is affecting biodiversity; however, the extent to which animal reproductive processes respond to predicted temperature increments remains largely unexplored. The thermal environment has a pronounced impact on metabolic rates of ectotherms; therefore, an interesting question to assess is whether temperature increase might affect specific reproductive mechanisms like sperm performance in ectotherms. Moreover, in many species, oviductal fluid (OF) is known to regulate and maintain sperm quality; however, the role of OF in relation to the effects of high temperature on sperm remains unclear. Our aim was to experimentally test the effect of increased temperature on sperm velocity, swimming path and percentage of motility in neutral conditions at ejaculation (without OF) and in female's reproductive tract fluid (with OF), in a social ectotherm lizard model, Tropidurus spinulosus, which has specific thermal requirements for reproduction. Our results suggest that a rising temperature associated with global warming (+4°C) affects negatively sperm dynamics and survival. However, OF ameliorated the harmful effects of high temperature. This is an important point, as this study is the first to have tested the role of OF in preserving sperm from a warmer pre-fertilization environment. These results contribute to our understanding of how thermal environment changes might affect post-copulatory reproductive mechanisms. This article has an associated First Person interview with the first author of the paper.

Ectoderm to mesoderm transition by down-regulation of actomyosin contractility.

Collective migration of cohesive tissues is a fundamental process in morphogenesis and is particularly well illustrated during gastrulation by the rapid and massive internalization of the mesoderm, which contrasts with the much more modest movements of the ectoderm. In the Xenopus embryo, the differences in morphogenetic capabilities of ectoderm and mesoderm can be connected to the intrinsic motility of individual cells, very low for ectoderm, high for mesoderm. Surprisingly, we find that these seemingly deep differences can be accounted for simply by differences in Rho-kinases (Rock)-dependent actomyosin contractility. We show that Rock inhibition is sufficient to rapidly unleash motility in the ectoderm and confer it with mesoderm-like properties. In the mesoderm, this motility is dependent on two negative regulators of RhoA, the small GTPase Rnd1 and the RhoGAP Shirin/Dlc2/ArhGAP37. Both are absolutely essential for gastrulation. At the cellular and tissue level, the two regulators show overlapping yet distinct functions. They both contribute to decrease cortical tension and confer motility, but Shirin tends to increase tissue fluidity and stimulate dispersion, while Rnd1 tends to favor more compact collective migration. Thus, each is able to contribute to a specific property of the migratory behavior of the mesoderm. We propose that the "ectoderm to mesoderm transition" is a prototypic case of collective migration driven by a down-regulation of cellular tension, without the need for the complex changes traditionally associated with the epithelial-to-mesenchymal transition.

Fate-Patterning of 2D Gastruloids and Ectodermal Colonies Using Micropatterned Human Pluripotent Stem Cells.

In the developing mammalian embryo, intercellular signaling allows cells to self-organize to create spatial patterns of different cell fates. This process is challenging to study because of the difficulty of observing or manipulating embryos on the spatial and temporal scales required. In vitro models can provide a complement to in vivo systems for addressing these issues. These models are also the only windows we have into early human development. Here we provide protocols for two systems based on differentiating human pluripotent stem cells in micropatterned colonies on defined size and shape. The first model replicates the patterning of the germ layers at gastrulation, while the second replicates the medial-lateral patterning of the ectoderm. These systems allow study of how signaling underlies self-organized patterning at stages of development which are otherwise inaccessible.

Loss of Grhl3 is correlated with altered cellular protrusions in the non-neural ectoderm during neural tube closure.

BACKGROUND: The transcription factor Grainyhead-like 3 (GRHL3) has multiple roles in a variety of tissues during development including epithelial patterning and actin cytoskeletal regulation. During neural tube closure (NTC) in the mouse embryo, GRHL3 is expressed and functions in the non-neural ectoderm (NNE). Two important functions of GRHL3 are regulating the actin cytoskeleton during NTC and regulating the boundary between the NNE and neural ectoderm. However, an open question that remains is whether these functions explain the caudally restricted neural tube defect (NTD) of spina bifida observed in Grhl3 mutants. RESULTS: Using scanning electron microscopy and immunofluorescence based imaging on Grhl3 mutants and wildtype controls, we show that GRHL3 is dispensable for NNE identity or epithelial maintenance in the caudal NNE but is needed for regulation of cellular protrusions during NTC. Grhl3 mutants have decreased lamellipodia relative to wildtype embryos during caudal NTC, first observed at the onset of delays when lamellipodia become prominent in wildtype embryos. At the axial level of NTD, half of the mutants show increased and disorganized filopodia and half lack cellular protrusions. CONCLUSION: These data suggest that altered cellular protrusions during NTC contribute to the etiology of NTD in Grhl3 mutants.

Pharyngeal epithelial deletion of Tbx1 causes caudal pharyngeal arch defect but not cardiac conotruncal anomaly.

TBX1 is a major disease gene of 22q11.2 deletion syndrome (22q11.2DS). It is expressed in all three germ layers of pharyngeal apparatus to control the complicated morphogenesis. The haploinsufficiency of pharyngeal endodermal or ectodermal, but not mesodermal Tbx1 causes aortic arch patterning defect. However, the mesodermal deletion of Tbx1 causes much severer pharyngeal and cardiovascular defect than either pharyngeal endodermal or ectodermal Tbx1 deletion does. It is inconsistent with the conventional thought that the invagination of pharyngeal epithelia drives pharyngeal segmentation. Therefore, we asked whether pharyngeal ectodermal and ectodermal Tbx1 can compensate the loss of each other. Here we carefully characterized pharyngeal epithelia-specific Fgf15Cre and Fgf15HspCre lines and used them to perform pharyngeal epithelia-specific deletion. Our data showed that the percentage of E18.5 Fgf15Cre;Tbx1flox/+ embryos with aortic arch patterning defects was similar to that of E10.5 Fgf15Cre;Tbx1flox/+ embryos with the 4th pharyngeal arch artery (PAA) defect, indicating that there is no significant recovery from the initial PAA defect, in contrast to germ line haploinsufficiency. Fgf15Cre;Tbx1flox/flox embryos had hypoplastic caudal pharyngeal arch and defective derivatives, but cardiac OFT development was not affected. The phenotypic spectrum of simultaneous Tbx1 deletion in both pharyngeal ectoderm and endoderm is strikingly similar to what presents with single pharyngeal endoderm or ectoderm-specific deletion of Tbx1. The absence of synergistic effect indicates intimate topographic interactions among pharyngeal endoderm and ectoderm, through which deletion of a gene in one tissue may disrupt the development of adjacent tissues and thereby lead to similar morphological phenotypes in either tissue-specific deletion.

Netrin expressed by the ventral ectoderm lineage guides mesoderm migration in epibolic gastrulation of the leech.

Netrin is a remarkably conserved midline landmark, serving as a chemotactic factor that organizes the bilateral neural architecture in the post-gastrula bilaterian embryos. Netrin signal also guides cell migration in many other neural and non-neural organogenesis events in later developmental stages but has never been found to participate in gastrulation - the earliest cell migration in metazoan embryogenesis. Here, we found that the netrin signaling molecules and their receptors are expressed during gastrulation of the leech Helobdella. Intriguingly, Hau-netrin-1 was expressed in the N lineage, which gives rise in part to the ventral midline of ectoderm, at the onset of gastrulation. We demonstrated that the N lineage is required for the entrance of mesoderm into the germinal band and that misexpression of Hau-netrin-1 in early gastrulation prevented mesoderm from entering the germinal band. Together, these results suggested that Hau-netrin-1 secreted by the N lineage guides mesoderm migration during germinal band assembly. Furthermore, ectopic expression of Hau-netrin-1 after the completion of germinal band assembly disrupted the epibolic migration of the germinal bands in a later stage of gastrulation. Thus, Hau-netrin-1 is likely involved in two distinct events in sequential stages of leech gastrulation: the assembly of germinal bands in early gastrulation and their epibolic migration in mid-gastrulation. Given that the leech netrin is expressed in the precursor cells of the ventral midline during gastrulation, we propose that a heterochronic change from the midline netrin expression had taken place in the evolution of a novel mode of gastrulation in the directly developing leech embryos.

Spawning Induction and Embryo Micromanipulation Protocols in the Amphioxus Branchiostoma lanceolatum.

In the last decades, the cephalochordate amphioxus has reached a peculiar place in research laboratories as an excellent animal model to answer Evo/Devo questions. Nevertheless, mainly due to its restricted spawning season and to the small size of its embryos, only a few basic techniques in developmental biology could be used until recently. Fortunately, these last years, and thanks to the development of high-throughput techniques, new technical approaches have been possible, such as comparative transcriptomics and/or genomics. However, classic micromanipulation techniques are still difficult to apply. Here we present simple protocols for the manipulation of amphioxus embryos. First, we present the spawning induction method used with the European amphioxus species Branchiostoma lanceolatum. Second, we explain simple methods to manipulate the developing amphioxus embryo during the first steps of its development (before the hatching stage). These methods open many technical possibilities for future functional studies. Thus, we present here a simple technique to efficiently dechorionate a large number of embryos, we detail a protocol for the dissociation of cells during the first steps of the embryonic development and, finally, we describe micromanipulation approaches for tissue isolation during the gastrula stage.

Novel mouse model of encephalocele: post-neurulation origin and relationship to open neural tube defects.

Encephalocele is a clinically important birth defect that can lead to severe disability in childhood and beyond. The embryonic and early fetal pathogenesis of encephalocele is poorly understood and, although usually classified as a 'neural tube defect', there is conflicting evidence on whether encephalocele results from defective neural tube closure or is a post-neurulation defect. It is also unclear whether encephalocele can result from the same causative factors as anencephaly and open spina bifida, or whether it is aetiologically distinct. This lack of information results largely from the scarce availability of animal models of encephalocele, particularly ones that resemble the commonest, nonsyndromic human defects. Here, we report a novel mouse model of occipito-parietal encephalocele, in which the small GTPase Rac1 is conditionally ablated in the (non-neural) surface ectoderm. Most mutant fetuses have open spina bifida, and some also exhibit exencephaly/anencephaly. However, a proportion of mutant fetuses exhibit brain herniation, affecting the occipito-parietal region and closely resembling encephalocele. The encephalocele phenotype does not result from defective neural tube closure, but rather from a later disruption of the surface ectoderm covering the already closed neural tube, allowing the brain to herniate. The neuroepithelium itself shows no downregulation of Rac1 and appears morphologically normal until late gestation. A large skull defect overlies the region of brain herniation. Our work provides a new genetic model of occipito-parietal encephalocele, particularly resembling nonsyndromic human cases. Although encephalocele has a different, later-arising pathogenesis than open neural tube defects, both can share the same genetic causation.

Self-organizing neuruloids model developmental aspects of Huntington's disease in the ectodermal compartment.

Harnessing the potential of human embryonic stem cells to mimic normal and aberrant development with standardized models is a pressing challenge. Here we use micropattern technology to recapitulate early human neurulation in large numbers of nearly identical structures called neuruloids. Dual-SMAD inhibition followed by bone morphogenic protein 4 stimulation induced self-organization of neuruloids harboring neural progenitors, neural crest, sensory placode and epidermis. Single-cell transcriptomics unveiled the precise identities and timing of fate specification. Investigation of the molecular mechanism of neuruloid self-organization revealed a pulse of pSMAD1 at the edge that induced epidermis, whose juxtaposition to central neural fates specifies neural crest and placodes, modulated by fibroblast growth factor and Wnt. Neuruloids provide a unique opportunity to study the developmental aspects of human diseases. Using isogenic Huntington's disease human embryonic stem cells and deep neural network analysis, we show how specific phenotypic signatures arise in our model of early human development as a consequence of mutant huntingtin protein, outlining an approach for phenotypic drug screening.

Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications.

OBJECTIVE: To study whether embryonic cell-free DNA (cfDNA) in spent blastocyst media is representative of the chromosomal constitution of a blastocyst. DESIGN: Pilot prospective blinded study. SETTING: In vitro fertilization center and genetics laboratory. PATIENT(S): A total of 115 trophectoderm (TE) biopsies and spent blastocyst media (SBM) from 46 patients with ages ranging from 32 to 46 years, whose indications for preimplantation genetic testing of aneuploidy (PGT-A) were advanced maternal age, recurrent miscarriage, or recurrent implantation failure. INTERVENTIONS(S): Spent blastocyst media collection and TE biopsy. MAIN OUTCOME MEASURE(S): Concordance rates, sensitivity, and specificity between TE biopsies and SBM. Clinical outcomes in cases with euploid TE biopsies and euploid SBM compared with cases with euploid TE and aneuploid SBM. RESULT(S): In general, the total concordance rate for ploidy and sex was 78.7%, and sensitivity and specificity were 94.5% and 71.7%, respectively. A significant increase for all parameters was observed for day 6/7 samples compared with day 5 samples, with day 6/7 samples showing total concordance for ploidy and sex of 84%, and sensitivity and specificity of 95.2% and 82.1%, respectively. Ongoing implantation rates in euploid TE/euploid SBM showed a threefold increase compared with euploid TE/aneuploid SBM (52.9% vs. 16.7%, respectively), without reaching significant differences. Interestingly, no miscarriages were observed when TE and SBM were euploidy concordant. CONCLUSION(S): These results offer a better understanding of the dynamics of cfDNA during embryo development and despite more basic research being needed, they are reassuring to consider in the future this noninvasive approach as an alternative to TE biopsy for PGT-A.

Mapping the Whole-Body Muscle Activity of Hydra vulgaris.

Hydra is a cnidarian polyp with an anatomically simple neuromuscular system that can offer evolutionary insights on the functional design of animal body plans. Using calcium imaging to map the activity of the entire epitheliomuscular system of behaving Hydra, we find seven basic spatiotemporal patterns of muscle activity. Patterns include global and local activation events with widely varying kinetics of initiation and wave-like propagation. The orthogonally oriented endodermal and ectodermal muscle fibers are jointly activated during longitudinal contractions. Individual epitheliomuscular cells can participate in multiple patterns, even with very different kinetics. This cellular multifunctionality could enable the structurally simple epitheliomuscular tissue of basal metazoans to implement a diverse behavioral output.

Antero-posterior ectoderm patterning by canonical Wnt signaling during ascidian development.

Wnt/ß-catenin signaling is an ancient pathway in metazoans and controls various developmental processes, in particular the establishment and patterning of the embryonic primary axis. In vertebrates, a graded Wnt activity from posterior to anterior endows cells with positional information in the central nervous system. Recent studies in hemichordates support a conserved role for Wnt/ß-catenin in ectoderm antero-posterior patterning at the base of the deuterostomes. Ascidians are marine invertebrates and the closest relatives of vertebrates. By combining gain- and loss-of-function approaches, we have determined the role of Wnt/ß-catenin in patterning the three ectoderm derivatives of the ascidian Ciona intestinalis, central nervous system, peripheral nervous system and epidermis. Activating Wnt/ß-catenin signaling from gastrulation led to a dramatic transformation of the ectoderm with a loss of anterior identities and a reciprocal anterior extension of posterior identities, consistent with studies in other metazoans. Surprisingly, inhibiting Wnt signaling did not produce a reciprocal anteriorization of the embryo with a loss of more posterior identities like in vertebrates and hemichordate. Epidermis patterning was overall unchanged. Only the identity of two discrete regions of the central nervous system, the anteriormost and the posteriormost regions, were under the control of Wnt. Finally, the caudal peripheral nervous system, while being initially Wnt dependent, formed normally. Our results show that the Ciona embryonic ectoderm responds to Wnt activation in a manner that is compatible with the proposed function for this pathway at the base of the deuterostomes. However, possibly because of its fast and divergent mode of development that includes extensive use of maternal determinants, the overall antero-posterior patterning of the Ciona ectoderm is Wnt independent, and Wnt/ß-catenin signaling controls the formation of some sub-domains. Our results thus indicate that there has likely been a drift in the developmental systems controlling ectoderm patterning in the lineage leading to ascidians.

Latrophilin2 is involved in neural crest cell migration and placode patterning in Xenopus laevis.

Latrophilin2 (Lphn2) is an adhesion-class of G protein-coupled receptor with an unknown function in development. Here, we show that Xenopus laevis lphn2 (Xlphn2) is involved in the migration and differentiation of neural crest (NC) cells and placode patterning in Xenopus laevis embryos. Although Xlphn2 mRNA was detected throughout embryogenesis, it was expressed more abundantly in the placode region. Morpholino antisense oligonucleotide-mediated knockdown of Xlphn2 caused abnormal migration of NC cells, irregular epibranchial placode segmentation, and defective cartilage formation. Transplantation of fluorescently-labeled NC regions of wild-type embryos into Xlphn2 morpholino-injected embryos reproduced the defective NC cell migration, indicating that Xlphn2 regulates the migration of NC cells in a non-cell autonomous manner. Our results suggest that Xlphn2 is essential for placode patterning and as a guidance molecule for NC cells.

Functional ectodermal organ regeneration as the next generation of organ replacement therapy.

In this decade, substantial progress in the fields of developmental biology and stem cell biology has ushered in a new era for three-dimensional organ regenerative therapy. The emergence of novel three-dimensional cell manipulation technologies enables the effective mimicking of embryonic organ germ formation using the fate-determined organ-inductive potential of epithelial and mesenchymal stem cells. This advance shows great potential for the regeneration of functional organs with substitution of complete original function in situ. Organoids generated from multipotent stem cells or tissue stem cells via establishment of an organ-forming field can only partially recover original organ function owing to the size limitation; they are considered 'mini-organs'. Nevertheless, they hold great promise to realize regenerative medicine. In particular, regeneration of a functional salivary gland and an integumentary organ system by orthotopic and heterotopic implantation of organoids clearly points to the future direction of organ regeneration research. In this review, we describe multiple strategies and recent progress in regenerating functional three-dimensional organs, focusing on ectodermal organs, and discuss their potential and future directions to achieve organ replacement therapy as a next-generation regenerative medicine.

Type I interferon response impairs differentiation potential of pluripotent stem cells.

Upon virus infection, pluripotent stem cells neither induce nor respond to canonical type I interferons (IFN-I). To better understand this biology, we characterized induced pluripotent stem cells (iPSCs) as well as their differentiated parental or rederived counterparts. We confirmed that only iPSCs failed to respond to viral RNA, IFN-I, or viral infection. This lack of response could be phenocopied in fibroblasts with the expression of a reprogramming factor which repressed the capacity to induce canonical antiviral pathways. To ascertain the consequences of restoring the antiviral response in the context of pluripotency, we engineered a system to engage these defenses in iPSCs. Inducible expression of a recombinant virus-activated transcription factor resulted in the successful reconstitution of antiviral defenses through the direct up-regulation of IFN-I-stimulated genes. Induction of the antiviral signature in iPSCs, even for a short duration, resulted in the dysregulation of genes associated with all three germ layers despite maintaining pluripotency markers. Trilineage differentiation of these same cells showed that engagement of the antiviral defenses compromised ectoderm and endoderm formation and dysregulated the development of mesodermal sublineages. In all, these data suggest that the temporal induction of the antiviral response primes iPSCs away from pluripotency and induces numerous aberrant gene products upon differentiation. Together these results suggest that the IFN-I system and pluripotency may be incompatible with each other and thus explain why stem cells do not utilize the canonical antiviral system.

ROS-induced autophagy regulates porcine trophectoderm cell apoptosis, proliferation, and differentiation.

Significant embryo loss remains a serious problem in pig production. Reactive oxygen species (ROS) play a critical role in embryonic implantation and placentation. However, the potential mechanism of ROS on porcine trophectoderm (pTr) cell fate during the peri-implantation period has not been investigated. This study aimed to elucidate the effects of ROS on pTr cell phenotypes and the regulatory role in cell attachment and differentiation. Herein, results showed that exogenous H2O2 inhibited pTr cell viability, arrested the cell cycle at S and G2/M phases, and increased cell apoptosis and autophagy protein light chain 3B and Beclin-1, whereas these effects were reversed by different concentrations of N-acetyl-l-cysteine (NAC) posttreatment. In addition, NAC abolished H2O2-induced autophagic flux, inhibited intracellular and mitochondrial ROS, and restored expression of genes important for mitochondrial DNA and biogenesis, cell attachment, and differentiation. NAC reversed H2O2-activated MAPK and Akt/mammalian target of rapamycin pathways in dose-dependent manners. Furthermore, analyses with pharmacological and RNA interference approaches suggested that autophagy regulated cell apoptosis and gene expression of caudal-related homeobox 2 and IL-1ß. Collectively, these results provide new insights into the role of the ROS-induced autophagy in pTr cell apoptosis, attachment, and differentiation, indicating a promising target for decreasing porcine conceptus loss during the peri-implantation period.

Morphomechanical reactions and mechanically stressed structures in amphibian embryos, as related to gastrulation and axial organs formation.

Reactions of embryonic tissues to a distributed and concentrated stretching are described and compared with the mechanics of the normal gastrulation movements. A role of mechanically stressed dynamic cell structures in the gastrulation, demarcation of notochord borders and in providing proportionality of the axial rudiments is demonstrated. A morphomechanical scheme of amphibian gastrulation is presented.