In this study we investigate the induction of the cell behaviors underlying neurulation in the frog, Xenopus laevis. Although planar signals from the organizer can induce convergent extension movements of the posterior neural tissue in explants, the remaining morphogenic processes of neurulation do not appear to occur in absence of vertical interactions with the organizer (R. Keller et al. , 1992, Dev. Dyn. 193, 218-234). These processes include: (1) cell elongation perpendicular to the plane of the epithelium, forming the neural plate; (2) cell wedging, which rolls the neural plate into a trough; (3) intercalation of two layers of neural plate cells to form one layer; and (4) fusion of the neural folds. To allow planar signaling between all the inducing tissues of the involuting marginal zone and the responding prospective ectoderm, we have designed a "giant sandwich" explant. In these explants, cell elongation and wedging are induced in the superficial neural layer by planar signals without persistent vertical interactions with underlying, involuted mesoderm. A neural trough forms, and neural folds form and approach one another. However, the neural folds do not fuse with one another, and the deep cells of these explants do not undergo their normal behaviors of elongation, wedging, and intercalation between the superficial neural cells, even when planar signals are supplemented with vertical signaling until the late midgastrula (stage 11.5). Vertical interactions with mesoderm during and beyond the late gastrula stage were required for expression of these deep cell behaviors and for neural fold fusion. These explants offer a way to regulate deep and superficial cell behaviors and thus make possible the analysis of the relative roles of these behaviors in closing the neural tube.

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In this paper we examine the contributions of planar and vertical signaling to the patterning of gene expression in neural development and we examine the routes of this neural induction. We have examined how the expression of Xenopus homeobox gene, Hoxb-1, is regulated by instruction from the mesoderm and/or endoderm and ask whether this instruction is by the vertical or planar routes. We investigated normal expression patterns of Hoxb-1 during early Xenopus development and Hoxb-1 expression in sandwich explants of the dorsal marginal zone, which putatively allow only planar signals to pass from the mesodermal and endodermal tissue (Spemann's organizer) to the prospective neural tissue. In the latter case we found significant variability of expression. Observations during dissections suggested that variable degrees of invasion of the mesodermal-endodermal tissue at the leading edge of the mesodermal mantle might be the cause of this variability. Alternatively, differing lengths of time that the prospective neural region spends in planar contact with tissues of the lateral or ventral regions of the embryo could also contribute to this variability. Analysis of staged Keller sandwich explants, "skewered" sandwiches, in which the degree of contact with underlying, involuted mesoderm-endodermal tissues was marked, and "over-the-pole" and "giant" sandwich explants, in which the degree of planar contact with lateral or ventral tissues was normalized, suggests that both planar and vertical signals are involved in induction and patterning of Hoxb-1 expression. The shift in Hoxb-1 expression from a broad, diffuse pattern to a local, focused pattern, characteristic of the ultimate expression pattern in vivo, does not reflect variable degrees of contact with ventral or lateral tissues, but rather reflects early vertical contact with underlying mesodermal-endodermal tissues. We observed such contact at early gastrula stages (stages 10 to 10+), stages commonly assumed not to have the potential for vertical signaling. As the bottle cells first begin to form, at stage 10-, a massive rotation of the lower involuting marginal zone occurs around an internal lip ("levre interne," Nieuwkoop and Florschutz, 1950). This rotation initiates the formation of the Cleft of Brachet from the floor of the blastocoele and brings the prospective mesoderm and endoderm at the leading edge of the marginal zone into vertical apposition with the prospective neural region quite early in gastrulation. The consequence and importance of recognizing these early internal rearrangements are that it pushes backward the time at which potential vertical inductive interactions between mesoderm and neurectoderm can occur. This means that a purely planar inductive situation can cease to exist as early as the inception of bottle cell formation and that neural patterning through vertical induction starts at the very beginning of gastrulation.