Early regulative ability of the neuroepithelium to form cardiac neural crest.

The cardiac neural crest (arising from the level of hindbrain rhombomeres 6-8) contributes to the septation of the cardiac outflow tract and the formation of aortic arches. Removal of this population after neural tube closure results in severe septation defects in the chick, reminiscent of human birth defects. Because neural crest cells from other axial levels have regenerative capacity, we asked whether the cardiac neural crest might also regenerate at early stages in a manner that declines with time. Accordingly, we find that ablation of presumptive cardiac crest at stage 7, as the neural folds elevate, results in reformation of migrating cardiac neural crest by stage 13. Fate mapping reveals that the new population derives largely from the neuroepithelium ventral and rostral to the ablation. The stage of ablation dictates the competence of residual tissue to regulate and regenerate, as this capacity is lost by stage 9, consistent with previous reports. These findings suggest that there is a temporal window during which the presumptive cardiac neural crest has the capacity to regulate and regenerate, but this regenerative ability is lost earlier than in other neural crest populations.

New methods for chicken embryo manipulations.

The capacity to image a growing embryo while simultaneously studying the developmental function of specific molecules provides invaluable information on embryogenesis. However, until recently, this approach was accomplished with difficulty both because of the advanced technology needed and because an easy method of minimizing damage to the embryo was unavailable. Here, we present a novel way of adapting the well-known EC culture of whole chick embryos to time-lapse imaging and to functional molecular studies using blocking agents. The novelty of our method stems from the ability to apply blocking agents ex ovo as well as in ovo. We were able to study the function of a set of molecules by culturing developing embryos ex ovo in tissue culture media containing these molecules or by injecting them underneath the live embryo in ovo. The in ovo preparation is particularly valuable, because it extends the period of time during which the developmental function of the molecule can be studied and it provides an easy, reproducible method for screening a batch of molecules. These new techniques will prove very helpful in visualizing and understanding the role of specific molecules during embryonic morphogenesis, including blood vessel formation.

Fate map and morphogenesis of presumptive neural crest and dorsal neural tube.

In contrast to the classical assumption that neural crest cells are induced in chick as the neural folds elevate, recent data suggest that they are already specified during gastrulation. This prompted us to map the origin of the neural crest and dorsal neural tube in the early avian embryo. Using a combination of focal dye injections and time-lapse imaging, we find that neural crest and dorsal neural tube precursors are present in a broad, crescent-shaped region of the gastrula. Surprisingly, static fate maps together with dynamic confocal imaging reveal that the neural plate border is considerably broader and extends more caudally than expected. Interestingly, we find that the position of the presumptive neural crest broadly correlates with the BMP4 expression domain from gastrula to neurula stages. Some degree of rostrocaudal patterning, albeit incomplete, is already evident in the gastrula. Time-lapse imaging studies show that the neural crest and dorsal neural tube precursors undergo choreographed movements that follow a spatiotemporal progression and include convergence and extension, reorientation, cell intermixing, and motility deep within the embryo. Through these rearrangement and reorganization movements, the neural crest and dorsal neural tube precursors become regionally segregated, coming to occupy predictable rostrocaudal positions along the embryonic axis. This regionalization occurs progressively and appears to be complete in the neurula by stage 7 at levels rostral to Hensen's node.

The presumptive floor plate (notoplate) induces behaviors associated with convergent extension in medial but not lateral neural plate cells of Xenopus.

In previous work (Elul, T., Keller, R., 2000. Monopolar protrusive activity: a new morphogenic cell behavior in the neural plate dependent on vertical interactions with the mesoderm in Xenopus. Dev. Biol. 224, 3-19; Ezin, A.M., Skoglund, P. Keller, R. 2003. The midline (notochord and notoplate) patterns the cell motility underlying convergence and extension of the Xenopus neural plate. Dev. Biol. 256, 100-114), the midline tissues of notochord and overlying notoplate were found to induce the monopolar, medially directed protrusive activity of deep neural cells. This behavior is thought to drive the mediolateral intercalation and convergent extension of the neural plate in Xenopus. Here we address the issue of whether the notochord, the notoplate, or both is essential for this induction. Our strategy was to remove the notochord, leaving the overlying notoplate intact, and determine whether it alone can induce the monopolar, medially directed cell behavior. We first establish that the notoplate (presumptive floor plate), when separated from the underlying notochord in the early neurula (stages 13-14), will independently mature into a floor plate as assayed three criteria: (1) continued expression of an early marker, sonic hedgehog, and a later, marker, F-spondin; (2) the display of the notoplate/floor plate-specific randomly oriented protrusive activity; (3) the characteristic lack of mixing of cells between the notoplate and lateral neural plate. Under these conditions, in the presence of a mature notoplate/floor plate and in the absence of the notochord, the characteristic monopolar, medially directed behavior occurred, but only locally near the midline. These results show that the notoplate/floor plate capacity to induce the medially directed motility is limited in range, and they suggest that the notochord is necessary for the normally observed longer range induction in lateral neural plate cells. This work helps to further the understanding of molecular and tissue interactions required for convergent extension.

The midline (notochord and notoplate) patterns the cell motility underlying convergence and extension of the Xenopus neural plate.

We investigated the role of the dorsal midline structures, the notochord and notoplate, in patterning the cell motilities that underlie convergent extension of the Xenopus neural plate. In explants of deep neural plate with underlying dorsal mesoderm, lateral neural plate cells show a monopolar, medially directed protrusive activity. In contrast, neural plate explants lacking the underlying dorsal mesoderm show a bipolar, mediolaterally directed protrusive activity. Here, we report that "midlineless" explants consisting of the deep neural plate and underlying somitic mesoderm, but lacking a midline, show bipolar, mediolaterally oriented protrusive activity. Adding an ectopic midline to the lateral edge of these explants restores the monopolar protrusive activity over the entire extent of the midlineless explant. Monopolarized cells near the ectopic midline orient toward it, whereas those located near the original, removed midline orient toward this midline. This behavior can be explained by two signals emanating from the midline. We postulate that one signal polarizes neural plate deep cells and is labile and short-lived and that the second signal orients any polarized cells toward the midline and is persistent.

Embryonic wound healing by apical contraction and ingression in Xenopus laevis.

We have characterized excisional wounds in the animal cap of early embryos of the frog Xenopus laevis and found that these wounds close accompanied by three distinct processes: (1) the assembly of an actin purse-string in the epithelial cells at the wound margin, (2) contraction and ingression of exposed deep cells, and (3) protrusive activity of epithelial cells at the margin. Microsurgical manipulation allowing fine control over the area and depth of the wound combined with videomicroscopy and confocal analysis enabled us to describe the kinematics and challenge the mechanics of the closing wound. Full closure typically occurs only when the deep, mesenchymal cell-layer of the ectoderm is left intact; in contrast, when deep cells are removed along with the superficial, epithelial cell-layer of the ectoderm, wounds do not close. Actin localizes to the superficial epithelial cell-layer at the wound margin immediately after wounding and forms a contiguous "purse-string" in those cells within 15 min. However, manipulation and closure kinematics of shaped wounds and microsurgical cuts made through the purse-string rule out a major force-generating role for the purse-string. Further analysis of the cell behaviors within the wound show that deep, mesenchymal cells contract their apical surfaces and ingress from the exposed surface. High resolution time-lapse sequences of cells at the leading edge of the wound show that these cells undergo protrusive activity only during the final phases of wound closure as the ectoderm reseals. We propose that assembly of the actin purse-string works to organize and maintain the epithelial sheet at the wound margin, that contraction and ingression of deep cells pulls the wound margins together, and that protrusive activity of epithelial cells at the wound margin reseals the ectoderm and re-establishes tissue integrity during wound healing in the Xenopus embryonic ectoderm.