In recent years, the cascades of inductive interactions leading to neuralization and/or mesodermalization of naive embryonic tissues have been studied in great detail (Niehrs, 2004; Heasman, 2006). However, the problem of precise positioning and proportionality of neural and mesodermal rudiments is far from a solution. According to a widely accepted point of view, such positioning is fully provided by the balance between opposing concentration gradients of dorsal- inducing substances and the ventral factors, such as secreted proteins of the BMP superfamily (Reversade et al., 2005; Khokha et al., 2005). From this perspective, neural and mesodermal patterns should be firmly established already before the start of gastrulation (and, moreover, of neurulation), such that the role of morphogenetic movements is limited to placing predetermined rudiments in their proper locations. There are, however, several sets of arguments suggesting that this point of view is an oversimplification of a real situation. First, invagination movements, especially those of lateromedial cell convergence, are far from being precise when observed on the individual cell level; rather, they exhibit substantial stochasticity (Keller and Danilchik, 1988). Under these conditions, if cell fates were strictly determined before the start of morphogenetic movements, the resulting patterns would be highly variable, which is not the case. Next, Zaraisky (1991) showed that after extirpation of a substantial portion of non-axial tissues from X. laevis, embryos the final volumes of notochords, somites, and neural tissues were diminished in proportion to the reduced embryo volume, even if extirpations were made well after the start of gastrulation movements. The latest extirpation stages compatible with the proportional volume reduction were early gastrula for the notochord, midgastrula for somite tissue, and early-mid neurula for neural tissue. These results indicate that even up to the neurulation period, the borders between the axial and non-axial tissues as well as between different axial organs are not strictly determined and, therefore, gastrulation and neurulation movements can somehow participate in their precise positioning. Although this positioning can, in principle, be interpreted on the basis of BMP gradients without any involvement of morphogenetic movements (Ben-zvi et al., 2008), the corresponding models are essentially non-robust and applicable only to the interventions made at earlier developmental stages. Meanwhile, more robust pattern-generating models could be constructed by focusing on the tissue geometry created by morphogenetic movements (Beloussov and Grabovsky, 2006).
In our previous experiments (Kornikova et al., 2009), we arrested gastrulation movements by relaxing mechanical tensions in the suprablastoporal area (SBA) and traced the locations of tissue-specific genes expression sites in arrested embryos. These locations turned out to be rather variable and discontinuous and were far from coinciding with the maps of presumptive anlagen for pre-gastrula-stage embryos. These results argue for a direct participation of gastrulation movements in determining the precise patterns of axial organs. In addition, we noticed that neural rudiments and somite series were arranged as parallel arches along the extended lateral lips of the opened and curved blastopores, with the neural rudiments located along the inner (concave) surface and the somite series lining the outer (convex) side of the lips (Kornikova et al., 2009; see Fig. 7d). This neuro-mesodermal arrangement is the reverse of that predicted by anlagen maps, and led us to suggest that the mutual arrangement of axial rudiments is not strictly determined prior to gastrulation and can be influenced by experimental modulations of embryo geometry. The aim of this study was to test this idea in a more direct way. We prepared double explants of SBA ectoderm (later on defined as explants), artificially bent them either parallel or perpendicular to their antero-posterior (AP) axes, and compared the mutual arrangements of neural and mesodermal rudiments (identified by tissue-specific gene expression and conventional histology) in deformed explants with that of intact (non-deformed) tissues. We found that in spite of certain variability, the neuro-mesodermal patterns of intact explants obeyed a distinct axi-symmetric AP arrangement, with the neural rudiments shifted anteriorly. On the contrary, in the deformed explants that preserved the initial bending, the axi-symmetric patterns were completely disturbed: the neural tissue showed a definite bias towards the concave surface, while the mesodermal tissue tended toward the convex side, encircling the neural rudiment in a horseshoe-shaped fashion. In addition, some of explants, both intact and bent, took on a spherical shape. In these, the arrangement of the neural and mesodermal tissue was highly irregular and variable. By tracing cell movements and early morphogenesis in the artificially bent explants, we demonstrate that the imposed deformations trigger active extension of the explants' convex surface and active contraction/folding of the concave surface. We speculate that these movements play an important role in establishing a mutual neuro-mesodermal positioning in both SBA explants and during normal development.