Lineage Diversification of the Neural Crest: In Vitro Investigations
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Abstract In recent years, research on neural crest induction has allowed the identification of several molecules as candidates for neural crest inducers. Although many of these molecules have the ability to induce neural crest in different assays, a general mechanism of neural crest induction that includes a description of the tissues that produce the inductive signals and the time and steps in which this process takes place remains elusive. To better understand the mechanism of neural crest induction, we developed an assay that has been used previously by Nieuwkoop to study anterior–posterior pattern of the neural plate. Folds of competent ectoderm were implanted in different positions of a young neurula embryo, and the induction of neural crest was analyzed using the expression of the neural crest marker Xslug . We identified a very localized region of the early neurula where it is possible to get neural crest induction, whereas all of the regions tested showed a clear induction of the neural plate marker Xsox2 . These results indicate that there is a region in the embryo with the appropriate combination of signals needed to induce neural crest cells; we called this region the neural crest competence territory. In addition, our results show that neural crest induction is always accompanied by neural plate induction, but there are many cases where neural plate was induced without neural crest. These results support the model in which the neural crest is induced by an interaction between neural plate and epidermis, but they also suggest that additional signals are required. By making grafts of different sizes and implanting them in the epidermis or the neural plate, we concluded that one of the inductive signals is produced in the dorsal region of the embryo and travels into the ectoderm. Finally, by performing gain‐ and loss‐of‐function of Wnt signaling experiments, we show that this pathway plays an important role not only in neural crest induction but also in the specification of the neural crest competence territory. Developmental Dynamics 229:109–117, 2004. © 2003 Wiley‐Liss, Inc.
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The neural crest is a transient population of embryonic cells that originates at the border between the neural plate and the non-neural ectoderm. Near the time of neural tube closure, the neural crest go through an epithelial to mesenchymal transition and start an extensive migration throughout the embryo. During migration or shortly after they reached their final position, neural crest cells differentiate to form a wealth of derivatives. The mechanisms of migration and differentiation of neural crest have been vastly studied. Comparatively, much less is known about the embryological origins of the neural crest, and the nature of the interactions that generate them. To clarify the timing and nature of these inductive interactions, I examined the time of competence of the neural plate to become neural crest as well as the time of neural fold specification in chick embryos. The neural plate is competent to respond to inductive interactions with the non-neural ectoderm for a limited period, losing its responsive ability after stage 10. In contrast, non-neural ectoderm from numerous stages retains the potential to induce neural crest cells from competent neural plate. When I tested the ability of neural folds to produce neural crest, I found that folds derived from all rostrocaudal levels of the open neural plate of stage 10 embryos can generate neural crest when cultured in isolation. To further characterize the time of neural crest specification, I isolated regions of the epiblast from stages 3 and 4 embryos and identified a region that is already specified to adopt neural crest fates at the beginning of gastrulation. I describe the early expression pattern of the paired box transcription factor Pax-7, which correlates from stage 4+ onwards with the prospective neural crest forming region. Therefore, I propose that Pax-7 is the earliest neural crest marker described in chick. Furthermore, using a morpholino-based loss of function approach, I show that Pax-7 expression is required during neural crest development in chicks. Taken together, my results suggest that specification of the neural crest begins very early in development and it requires multiple and sustained signals and tissue intractions.
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Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.
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Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.
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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.
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ABSTRACT 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 nonneural 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.
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