Origins of the avian neural crest: the role of neural plate-epidermal interactions

We have investigated the lineage and tissue interactions that result in avian neural crest cell formation from the ectoderm. Presumptive neural plate was grafted adjacent to non-neural ectoderm in whole embryo culture to examine the role of tissue interactions in ontogeny of the neural crest. Our results show that juxtaposition of non-neural ectoderm and presumptive neural plate induces the formation of neural crest cells. Quail/chick recombinations demonstrate that both the prospective neural plate and the prospective epidermis can contribute to the neural crest. When similar neural plate/epidermal confrontations are performed in tissue culture to look at the formation of neural crest derivatives, juxtaposition of epidermis with either early (stages 4–5) or later (stages 6–10) neural plate results in the generation of both melanocytes and sympathoadrenal cells. Interestingly, neural plates isolated from early stages form no neural crest cells, whereas those isolated later give rise to melanocytes but not crest-derived sympathoadrenal cells. Single cell lineage analysis was performed to determine the time at which the neural crest lineage diverges from the epidermal lineage and to elucidate the timing of neural plate/epidermis interactions during normal development. Our results from stage 8 to 10+ embryos show that the neural plate/neural crest lineage segregates from the epidermis around the time of neural tube closure, suggesting that neural induction is still underway at open neural plate stages

The genesis of avian neural crest cells: A classic embryonic induction

Neural crest cells arise from the ectoderm and are first recognizable as discrete cells in the chicken embryo when they emerge from the neural tube. Despite the classical view that neural crest precursors are a distinct population lying between epidermis and neuroepithelium, our results demonstrate that they are not a segregated population. Cell lineage analyses have demonstrated that individual precursor cells within the neural folds can give rise to epidermal, neural crest, and neural tube derivatives. Interactions between the neural plate and epidermis can generate neural crest cells, since juxtaposition of these tissues at early stages results in the formation of neural crest cells at the interface. Inductive interactions between the epidermis and neural plate can also result in "dorsalization" of the neural plate, as assayed by the expression of the Wnt transcripts characteristic of the dorsal neural tube. The competence of the neural plate changes with time, however, such that interaction of early neural plate with epidermis generates only neural crest cells, whereas interaction of slightly older neural plate with epidermis generates neural crest cells and Wnt-expressing cells. At cranial levels, neuroepithelial cells can regulate to generate neural crest cells when the endogenous neural folds are removed, probably via interaction of the remaining neural tube with the epidermis. Taken together, these experiments demonstrate that: (i) progenitor cells in the neural folds are multipotent, having the ability to form multiple ectodermal derivatives, including epidermal, neural crest, and neural tube cells; (ii) the neural crest is an induced population that arises by interactions between the neural plate and the epidermis; and (iii) the competence of the neural plate to respond to inductive interactions changes as a function of embryonic age

Dorsalization of the neural tube by the non-neural ectoderm

The patterning of cell types along the dorsoventral axis of the spinal cord requires a complex set of inductive signals. While the chordamesoderm is a well-known source of ventralizing signals, relatively little is known about the cues that induce dorsal cell types, including neural crest. Here, we demonstrate that juxtaposition of the non-neural and neural ectoderm is sufficient to induce the expression of dorsal markers, Wnt-1, Wnt-3a and Slug, as well as the formation of neural crest cells. In addition, the competence of neural plate to express Wnt-1 and Wnt-3a appears to be stage dependent, occurring only when neural tissue is taken from stage 8–10 embryos but not from stage 4 embryos, regardless of the age of the non-neural ectoderm. In contrast to the induction of Wnt gene expression, neural crest cell formation and Slug expression can be induced when either stage 4 or stage 8–10 neural plates are placed in contact with the non-neural ectoderm. These data suggest that the non-neural ectoderm provides a signal (or signals) that specifies dorsal cell types within the neural tube, and that the response is dependent on the competence of the neural tissue

Analysis of cranial neural crest migratory pathways in axolotl using cell markers and transplantation

We have examined the ability of normal and heterotopically transplanted neural crest cells to migrate along cranial neural crest pathways in the axolotl using focal DiI injections and in situ hybridization with the neural crest marker, AP-2. DiI labeling demonstrates that cranial neural crest cells migrate as distinct streams along prescribed pathways to populate the maxillary and mandibular processes of the first branchial arch, the hyoid arch and gill arches 1-4, following migratory pathways similar to those observed in other vertebrates. Another neural crest marker, the transcription factor AP-2, is expressed by premigratory neural crest cells within the neural folds and migrating neural crest cells en route to and within the branchial arches. Rotations of the cranial neural folds suggest that premigratory neural crest cells are not committed to a specific branchial arch fate, but can compensate when displaced short distances from their targets by migrating to a new target arch. In contrast, when cells are displaced far from their original location, they appear unable to respond appropriately to their new milieu such that they fail to migrate or appear to migrate randomly. When trunk neural folds are grafted heterotopically into the head, trunk neural crest cells migrate in a highly disorganized fashion and fail to follow normal cranial neural crest pathways. Importantly, we find incorporation of some trunk cells into branchial arch cartilage despite the random nature of their migration. This is the first demonstration that trunk neural crest cells can form cartilage when transplanted to the head. Our results indicate that, although cranial and trunk neural crest cells have inherent differences in ability to recognize migratory pathways, trunk neural crest can differentiate into cranial cartilage when given proper instructive cues

Avian neural crest cell fate decisions: a diffusible signal mediates induction of neural crest by the ectoderm

During neurulation, a region of central ectoderm becomes thickened to form the neural plate which then folds upon itself to generate the neural tube, from which all neurons and glia cells of the central nervous system arise. Neural crest cells form at the border of the neural plate, where it abuts the prospective epidermis. The neural crest is a transient population of cells that undergo an epithelial-mesenchymal transition, become highly migratory and subsequently differentiate into most of the peripheral nervous systems as well as numerous other derivatives. The origin of neural crest cells at the epidermal–neural plate border suggests that an interaction between these two tissues may be involved in neural crest formation. By experimentally juxtaposing prospective epidermis with naive neural plate, we previously showed that an inductive interaction between these tissues can generate neural crest cells. Here, we further characterize the nature of this inductive interaction by co-culturing isolated neural plate and prospective epidermis on opposing sides of polycarbonate filters with differing pore sizes. We find that neural crest cells are generated even when epidermis and neural plate are separated by filters that do not allow cell contact. These results suggest that the epidermal inducer is a diffusible, secreted molecule. We discuss the developmental potential of neural crest precursors and lineage decisions that effect their differentiation into numerous derivatives

Origins of Neural Crest Cell Diversity

The neural crest is a population of migratory cells, arising from the ectoderm, that invades many sites within the embryo and differentiate into a variety of diverse cell types. Pigment cells, most cells of the peripheral nervous system, adrenal medullary cells, and some cranial cartilage are derived from the neural crest. Despite a wealth of knowledge concerning their pathways of migration and vast array of derivatives, little is known about the formation of neural crest cells or their acquisition of positional identity. This review focuses on the origin of neural crest cells from the ectoderm and the generation of differences in neural crest cell fates along the rostrocaudal axis. In addition, we consider the role of temporal restriction in the developmental potential of premigratory neural crest cells. While evidence for the existence of multipotent stem cells is strong, some experiments also suggest that there may be heterogeneity among neural crest cell precursors, perhaps due to differences in origin, that might explain commitment events occurring early in neural crest development

Timing and Competence of Neural Crest Formation

Neural crest cells can be induced by an interaction between neural plate and ectoderm. To clarify the timing and nature of these inductive interactions, we have examined the time of competence of the neural plate to become neural crest as well as the time of neural fold specification. The neural plate is competent to respond to inductive interactions with the nonneural ectoderm for a limited period, rapidly losing its responsive ability after stage 10. In contrast, nonneural ectoderm from numerous stages retains the ability to induce neural crest cells from competent neural plate. When neural folds are explanted to test their ability to produce neural crest without further tissue interactions, we find that folds derived from all rostrocaudal levels of the open neural plate are already specified to express the neural crest marker Slug. However, additional signals may be required for maintenance of Slug expression, since the transcript is later down-regulated in vitro in the absence of tissue interactions. Taken together, these results suggest that there are multiple stages of neural crest induction. The earliest induction must have occurred by the end of gastrulation, since the newly formed neural fold population is already specified to form neural crest. However, isolated neural folds eventually down-regulate Slug, suggesting a second phase that maintains neural crest formation. Thus, induction of the neural crest may involve multiple and sustained tissue interactions

Ex Vivo Culture of Lung Buds on the Chorioallantoic Membrane of the Avian Embryo

Rationale: Development of the vertebrate lung involves a complex series of interactions between the foregut endoderm and the adjacent mesenchyme, resulting in formation of the paired lung buds. The lung buds subsequently give rise to mature lungs through processes which include branching morphogenesis, cell commitment and differentiation. At present it remains unclear what other cell types may play a role in patterning of the developing airways and the generation of diverse cell lineages. To address this problem, we have developed an ex vivo lung culture system which allows us to experimentally manipulate the developing lung buds. The chick embryo lends itself particularly well to such manipulations and It has long served as an important model system for developmental studies. In addition, development of the respiratory system in birds has been the subject of numerous descriptive studies which can provide a useful background to more experimental approaches. Methods: We have explanted the lung buds of developing day 4 chick or quail embryos onto the chorioallantoic membranes of host embryos at a similar developmental stage. In some cases, donor king buds were taken from transgenic quail, in which all or a subset of cells were labeled constitutively through expression of fluorescent chimeric protein. The explants were cultured further before fixation and analysis by conventional and confocal laser scanning microscopy. Results: We have demonstrated that developing lung buds explanted to chorioallantoic membranes continue to develop further, in some cases surviving for a further 6 days of development. In addition, the simple airway of the explanted lung buds undergoes branching morphogenesis to form a complex airway comparable to that found in lungs of similarly-staged un-operated embryos. Moreover, the explanted lung buds develop a capillary network that appears to establish a circulation with that of the chorioallantoic membrane. Conclusions: The chorioallantoic membrane culture system can be used to sustain the growth and development of isolated chick and quail lung buds, thus permitting their experimental manipulation to determine the role of different cell populations in lung development

Ex Vivo Culture of Lung Buds on the Chorioallantoic Membrane of the Avian Embryo

Rationale: Development of the vertebrate lung involves a complex series of interactions between the foregut endoderm and the adjacent mesenchyme, resulting in formation of the paired lung buds. The lung buds subsequently give rise to mature lungs through processes which include branching morphogenesis, cell commitment and differentiation. At present it remains unclear what other cell types may play a role in patterning of the developing airways and the generation of diverse cell lineages. To address this problem, we have developed an ex vivo lung culture system which allows us to experimentally manipulate the developing lung buds. The chick embryo lends itself particularly well to such manipulations and It has long served as an important model system for developmental studies. In addition, development of the respiratory system in birds has been the subject of numerous descriptive studies which can provide a useful background to more experimental approaches. Methods: We have explanted the lung buds of developing day 4 chick or quail embryos onto the chorioallantoic membranes of host embryos at a similar developmental stage. In some cases, donor king buds were taken from transgenic quail, in which all or a subset of cells were labeled constitutively through expression of fluorescent chimeric protein. The explants were cultured further before fixation and analysis by conventional and confocal laser scanning microscopy. Results: We have demonstrated that developing lung buds explanted to chorioallantoic membranes continue to develop further, in some cases surviving for a further 6 days of development. In addition, the simple airway of the explanted lung buds undergoes branching morphogenesis to form a complex airway comparable to that found in lungs of similarly-staged un-operated embryos. Moreover, the explanted lung buds develop a capillary network that appears to establish a circulation with that of the chorioallantoic membrane. Conclusions: The chorioallantoic membrane culture system can be used to sustain the growth and development of isolated chick and quail lung buds, thus permitting their experimental manipulation to determine the role of different cell populations in lung development