Cooperative model of epithelial shaping and bending during avian neurulation: autonomous movements of the neural plate, autonomous movements of the epidermis, and interactions in the neural plate/epidermis transition zone.

Morphogenetic movements during neurulation cause a tissue to change shape within the plane of the epithelium (e.g., conversion of the oval neural plate into the narrow spinal plate and the wide brain plate), cause bending out of the plane of the epithelium (e.g., raise the neural folds and curl the neural plate into a tube), or contribute to both phenomena. In this study, pieces that contain neural plate alone, epidermis alone, or both tissues (with or without underlying tissues) are cut from chick embryos and allowed to develop for up to 24 hr. Examination of histological sections through such isolates allows analysis of the formation of neural folds. When the neural plate/epidermis transition zone is disrupted, neural folds do not form. Conversely, when the transition zone remains intact, neural folds form. Neural folds form even when most of the medial neural plate and lateral epidermis has been removed, leaving only the isolated transition zone. These data indicate that the transition zone is both necessary and sufficient for the formation of neural folds. The transition zone may play a number of roles in epithelial bending including organizing, focussing, and redirecting movements that are autonomous to the neural plate or epidermis. Time-lapse video recording, and sequential photographs allowed the documentation of such movements. Neural plate isolates exhibit autonomous rostrocaudal lengthening and mediolateral narrowing. Isolated strips of epidermis exhibit autonomous movements which, unlike wound-healing movements, are unidirectional (medial), and region-specific (beginning and reaching their greatest extent in the cranial region). Isolated pieces of neural plate or epidermis remain flat instead of bending, providing further evidence that the transition zone is necessary for the formation of neural folds.

Tissue boundaries and cell behavior during neurulation.

We have analyzed the dynamics of the boundaries between the neural plate and the epidermis and between the neural plate and the notoplate. Our experiments confirm that these two boundaries have important roles in neurulation. Measurements of the lengths of neural fold (the boundary between epidermis and neural plate) in embryos of axolotls and newts reveal that neural folds abutting the prospective brain decrease in length while neural folds abutting the prospective spinal cord increase in length during neurulation. We tested the proposition that boundaries of the neural plate with epidermis and with notoplate are essential for proper neurulation. Cuts made along the boundaries with epidermis or with notoplate stop, or greatly diminish, neural plate elongation and tube formation. Explanting the axolotl neural plate without any bordering epidermis stops plate elongation and prevents neural tube closure, but neural plates explanted with a rim of epidermis elongate and close into tubes. Cutting the notoplate boundary stops midline elongation in the newt embryo or diminishes it in the axolotl embryo. We conclude that the notoplate boundary and part of the boundary of the epidermis that abuts the prospective spinal cord organize cell behavior to elongate the neural plate and help close the neural tube. The boundary of the neural plate with the epidermis is essential for tube closure both because it organizes plate elongation in the spinal cord region and because cell behavior becomes organized at the boundary such that neural folds are raised and a rolling moment is produced that helps form the neural tube.

Early cranial neural crest migration in the direct-developing frog, Eleutherodactylus coqui.

Direct development is a common reproductive mode in living amphibians characterized by absence of the free-living, aquatic larval stage. In Eleutherodactylus, a species-rich genus of New World frogs, evolution of direct development from the ancestral biphasic ontogeny is correlated with a comprehensive modification in embryonic cranial patterning, including the loss of many larval-specific components and the precocious formation of many adult (postmetamorphic) structures. We use scanning electron microscopy (SEM) to examine the emergence and early migration of cranial neural crest cells in Eleutherodactylus coqui to begin to assess the possible role of the neural crest in mediating these evolutionary changes. As in metamorphosing frogs, cranial crest cells emerge prior to neural fold closure and assemble into three streams: rostral otic, and caudal otic. These streams contribute to the face and first visceral (mandibular) arch, to the second (hyoid) arch, and to posterior (branchial) arches, respectively. Rostrocaudal position, morphology, and/or migration patterns distinguish subpopulations of cells within the rostral stream and caudal otic stream. With the possible exception of the small size of the rostral otic caudal otic streams, evolution of direct development in E. coqui has not altered basic patterns of neural crest emergence or early migration as assessed by SEM. If observed evolutionary changes in embryonic cranial patterning are mediated by the neural crest, then they likely involve later aspects of crest migration or more subtle features related to pattern formation such as cell behavior and commitment, or gene expression.

The origins of neural crest cells in the axolotl.

We address the question of whether neural crest cells originate from the neural plate, from the epidermis, or from both of these tissues. Our past studies revealed that a neural fold and neural crest cells could arise at any boundary created between epidermis and neural plate. To examine further the formation of neural crest cells at newly created boundaries in embryos of a urodele (Ambystoma mexicanum), we replace a portion of the neural folds of an albino host with either epidermis or neural plate from a normally pigmented donor. We then look for cells that contain pigment granules in the neural crest and its derivatives in intact and sectioned host embryos. By tracing cells in this manner, we find that cells from neural plate transplants give rise to melanocytes and (in one case) become part of a spinal ganglion, and we find that epidermal transplants contribute cells to the spinal and cranial ganglia. Thus neural crest cells arise from both the neural plate and the epidermis. These results also indicate that neural crest induction is (at least partially) governed by local reciprocal interactions between epidermis and neural plate at their common boundary.

Neural fold formation at newly created boundaries between neural plate and epidermis in the axolotl.

According to a recent model, the cortical tractor model, neural fold and neural crest formation occurs at the boundary between neural plate and epidermis because random cell movements become organized at this site. If this is correct, then a fold should form at any boundary between epidermis and neural plate. To test that proposition, we created new boundaries in axolotl embryos by juxtaposing pieces of neural plate and epidermis that would not normally participate in fold formation. These boundaries were examined superficially and histologically for the presence of folds, permitting the following observations. Folds form at each newly created boundary, and as many folds form as there are boundaries. When two folds meet they fuse into a hollow "tube" of neural tissue covered by epidermis. Sections reveal that these ectopic folds and "tubes" are morphologically similar to their natural counterparts. Transplanting neural plate into epidermis produces nodules of neural tissue with central lumens and peripheral nerve fibers, and transplanting epidermis into neural plate causes the neural tube and the dorsal fin to bifurcate in the region of the graft. Tissue transplanted homotypically as a control integrates into the host tissue without forming folds. When tissue from a pigmented embryo is transplanted into an albino host, the presence of pigment allows the donor cells to be distinguished from those of the host. Mesenchymal cells and melanocytes originating from neural plate transplants indicate that neural crest cells form at these new boundaries. Thus, any boundary between neural plate and epidermis denotes the site of a neural fold, and the behavior of cells at this boundary appears to help fold the epithelium. Since folds can form in ectopic locations on an embryo, local interactions rather than classical neural induction appear to be responsible for the formation of neural folds and neural crest.

Structural heterogeneity in the basal regions of the teeth of the red-backed salamander, Plethodon cinereus (Amphibia, Plethodontidae).

Ultrastructural and histochemical features of marginal (monostichous) teeth associated with the jawbones are compared with those of palatal (polystichous) teeth that compose two patches in the roof of the mouth. The apices and uncalcified regions are similar in both kinds of teeth, but the basal regions display distinctive differences. While bases (pedestals) of marginal teeth are essentially hollow cylinders that attach to the jawbones by their labial faces, bases of teeth in palatal patches are fused to form two horizontal plates which lack direct attachment to underlying bone. The plates are separated from each other by a pulp-filled space containing fibroblasts, blood vessels, and vertically oriented elements resembling bony spicules. Cylindrical pedestals like those of marginal teeth project from the ventral plate. While the identity of the material composing the basal regions remains controversial, the following evidence suggests that it is similar to "bone of attachment" (Tomes, '23): most of it, unlike dentin, does not develop in direct association with an enamel organ; alcian blue stains the bases of developing teeth but stains dentin, developing dentin, enamel, or mature bone very weakly (if at all); bases of teeth in palatal patches develop in isolation from the parasphenoid bone and thus cannot be considered extensions of it; and marginal teeth attach directly to the jawbones, but the material composing their bases does not blend with the bone. Structural heterogeneity of the basal regions appears to be linked to functional differences exhibited by these two types of teeth.

Structure of the radially asymmetrical uncalcified region of the teeth of the red-backed salamander, Plethodon cinereus (Amphibia, Plethodontidae).

The teeth of the adult plethodontid salamander, Plethodon cinereus, were examined by light and electron microscopy with emphasis on the ringlike zone of uncalcified dentin that divides the calcified portion of each tooth into a proximal pedestal and a distal apex. The uncalcified region displays radial asymmetry, forming an integral part of the posterior wall of the tooth but bulging into the pulp cavity anteriorly, thus forming a hingelike structure. All portions of the dentin, including the uncalcified region, are composed predominantly of collagenous fibers but lack elastin. In scanning electron micrographs of teeth from which the oral mucosa has been removed, the location of the anterior uncalcified hinge is marked externally by a notch-like articulation of the apex and pedestal. Sites of transition between calcified and uncalcified areas of the dentin show no special modifications in transmission electron micrographs, but collagenous fibers in calcified portions are associated with more electron-dense amorphous material than are those in the uncalcified region. Odontoblasts associated with the uncalcified region possess ultrastructural features closely resembling those of odontoblasts found in calcified areas. The uncalcified region seems to afford the teeth a certain degree of flexibility, and the asymmetry of the region appears to allow the teeth to flex only in a posterior direction, thus facilitating the entry of living prey but hindering its escape. The uncalcified region also seems to permit the apex of a tooth to break away from its pedestal without damage to underlying bone.