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 and neural crest movement in the Mexican salamander Ambystoma mexicanum

In studies of amphibian neurulation, the terms "neural ridge," "neural fold," and "neural crest" are sometimes used as synonyms. This has occasionally led to the misconception that grafting of the neural crest is equivalent to grafting of the neural fold. The neural fold, however, is composed of three parts: the neural crest, prospective neural tube tissue, and epidermis. In order to investigate how these neural fold components move during neurulation, time-lapse photography, electron microscopy, and grafting were performed. Ambystoma mexicanum embryos were photographed during neurulation at regular intervals. The photographs were analyzed to find the position of those cells at beginning of neurulation that end up on the line of fusion as the neural folds close. Posteriorly, these cells are already on the emerging neural fold. In the anterior neural folds, however, these cells are located in the lateral epidermis. Electron microscopy of the neural folds confirms the presence of epidermis. To follow the movement of the cells differentiating into melanophores (neural crest), neural fold parts were grafted into albino hosts. The crest cells differentiating into melanophores following ectopic grafting are located in the flank of the neural fold that is in contact with the neural plate. In grafts from the outside (distal) flank, no melanophores developed. Semithin sections show that the third part of the neural fold consists of apically constricted cells known to differentiate into neural tissue. Because the neural folds consist of epidermis, neural tissue, and neural crest, neural fold and neural crest cannot be used as synonyms.     

Experimental Analyses of the Shaping of the Neural Plate and Tube

Understanding the changing morphology of an embryo presentsspecial challenges. Analyses of neurulation in vertebrate embryosdescribed here required observation from sectioned materialand from time-lapse movies, modeling, computer simulation, andexperiments. All these approaches were essential, and each approachhelped guide the use of the others. Experiments have the specialrole of letting the embryo decide between our alternative hypotheses. In the newt embryo, induction and patterning events establishin the ectoderm boundaries between epidermis and neural plate,and between neural plate and the notoplate at its midline. Thedifferentdomains of cells thus established—epidermis, neural plateand notoplate—develop different adhesive properties suchthat cell motility behavior along the notoplate boundary andalong the spinal cord/epidermis boundary produces forceful intercalationof cells which lengthens the boundaries and distorts (lengthens)the neuroepithelium. Neural plate cells also attempt to crawlbeneath the epidermis along their common boundary, raising neuralfolds and producing a rolling moment directed mediad that islargely responsible for neural tube formation. Both cell motilitythat leads to columnarization of neural plate cells and contractionof organized subapical microfilament bundles reduce the apicalsurface area of the neural plate cells and produce an apicaltension that aids neural tube formation. Cell relocation reducesthe width of the neural plate and increases its length, andthe Poisson buckling forces resulting from this elongation ofthe plate also aid neural tube formation. The newt embryo accomplishes neurulation without growth, butbird and mammal embryos grow during neurulation. Understandingthe organization of the products of growth in the amniote neuralplate is critical in determining whether growth helps or hindersneurulation.     

Neural crests are actively precluded from the anterior neural fold by a novel inhibitory mechanism dependent on Dickkopf1 secreted by the prechordal mesoderm

It is known the interactions between the neural plate and epidermis generate neural crest (NC), but it is unknown why the NC develops only at the lateral border of the neural plate and not in the anterior fold. Using grafting experiments we show that there is a previously unidentified mechanism that precludes NC from the anterior region. We identify prechordal mesoderm as the tissue that inhibits NC in the anterior territory and show that the Wnt/beta-catenin antagonist Dkk1, secreted by this tissue, is sufficient to mimic this NC inhibition. We show that Dkk1 is required for preventing the formation of NC in the anterior neural folds as loss-of-function experiments using a Dkk1 blocking antibody in Xenopus as well as the analysis of Dkk1-null mouse embryos transform the anterior neural fold into NC. This can be mimicked by Wnt/beta-catenin signaling activation without affecting the anterior posterior patterning of the neural plate, or placodal specification. Finally, we show that the NC cells induced at the anterior neural fold are able to migrate and differentiate as normal NC. These results demonstrate that anterior regions of the embryo lack NC because of a mechanism, conserved from fish to mammals, that suppresses Wnt/beta-catenin signaling via Dkk1.     

Molecular mechanisms of neural crest induction

The neural crest is an embryonic cell population that originates at the border between the neural plate and the prospective epidermis. Around the time of neural tube closure, neural crest cells emigrate from the neural tube, migrate along defined paths in the embryo and differentiate into a wealth of derivatives. Most of the craniofacial skeleton, the peripheral nervous system, and the pigment cells of the body originate from neural crest cells. This cell type has important clinical relevance, since many of the most common craniofacial birth defects are a consequence of abnormal neural crest development. Whereas the migration and differentiation of the neural crest have been extensively studied, we are just beginning to understand how this tissue originates. The formation of the neural crest has been described as a classic example of embryonic induction, in which specific tissue interactions and the concerted action of signaling pathways converge to induce a multipotent population of neural crest precursor cells. In this review, we summarize the current status of knowledge on neural crest induction. We place particular emphasis on the signaling molecules and tissue interactions involved, and the relationship between neural crest induction, the formation of the neural plate and neural plate border, and the genes that are upregulated as a consequence of the inductive events.     

Formation and distribution of neural crest mesenchyme to the first pharyngeal arch region of the mouse embryo

Murine neural crest mesenchyme begins its escape from columnar epithelium near the tips of the midbrain-rostral hindbrain neural folds at 4+ to 5 somites of age. At that time the tip of each fold is located dorsolateral to the pharynx. Once crest formation is complete at this earliest site, it leaves behind both crest mesenchyme and overlying squamous epithelium. Crest formation then progresses medially, into the lateral margin of the neural plate. At the same time, this lateral margin elevates as the tip of the neural fold. By the time crest formation ceases at approximately 10 somites, the result of these simultaneous activities is to passively distribute the earliest mesenchyme, formed from the lateralmost epithelium, dorsolateral to the pharynx and the later, more medially derived mesenchyme lateral to the neural tube. Once formed, the crest mesenchyme dorsolateral to the pharynx is displaced ventromedially in a narrow, transient subectodermal space functionally similar to that observed in the chick embryo. Displacement might result from cell motility or the formation of matrix-filled spaces between cells of the mesenchyme. Displaced cells are closely associated with the overlying columnar epithelium. This association precedes their subsequent induction and may reflect preliminary patterning. The crest mesenchyme passively distributed lateral to the neural tube is subsequently displaced medially. Here the formation of enlarged (matrix-filled?) spaces is clearly involved in the initial displacement. Displaced cells proliferate to form the anlage of the trigeminal ganglion. The other major contributor to this ganglion is the trigeminal placode. The placodal epithelium is located dorsolateral to the pharynx of the 12-somite embryo. If the epithelia of the head maintain their relative positions, this placode is derived from the squamous epithelium formed together with the earliest crest mesenchyme. If not, an alternative source is the columnar epithelium located ventromedial to the tip of the 4+- to 5-somite neural fold.     

Combined intrinsic and extrinsic influences pattern cranial neural crest migration and pharyngeal arch morphogenesis in axolotl

Cranial neural crest cells migrate in a precisely segmented manner to form cranial ganglia, facial skeleton and other derivatives. Here, we investigate the mechanisms underlying this patterning in the axolotl embryo using a combination of tissue culture, molecular markers, scanning electron microscopy and vital dye analysis. In vitro experiments reveal an intrinsic component to segmental migration; neural crest cells from the hindbrain segregate into distinct streams even in the absence of neighboring tissue. In vivo, separation between neural crest streams is further reinforced by tight juxtapositions that arise during early migration between epidermis and neural tube, mesoderm and endoderm. The neural crest streams are dense and compact, with the cells migrating under the epidermis and outside the paraxial and branchial arch mesoderm with which they do not mix. After entering the branchial arches, neural crest cells conduct an "outside-in" movement, which subsequently brings them medially around the arch core such that they gradually ensheath the arch mesoderm in a manner that has been hypothesized but not proven in zebrafish. This study, which represents the most comprehensive analysis of cranial neural crest migratory pathways in any vertebrate, suggests a dual process for patterning the cranial neural crest. Together with an intrinsic tendency to form separate streams, neural crest cells are further constrained into channels by close tissue apposition and sorting out from neighboring tissues.     

Rohon-Beard sensory neurons are induced by BMP4 expressing non-neural ectoderm in Xenopus laevis

Rohon-Beard mechanosensory neurons (RBs), neural crest cells, and neurogenic placodes arise at the border of the neural- and non-neural ectoderm during anamniote vertebrate development. Neural crest cells require BMP expressing non-neural ectoderm for their induction. To determine if epidermal ectoderm-derived BMP signaling is also involved in the induction of RB sensory neurons, the medial region of the neural plate from donor Xenopus laevis embryos was transplanted into the non-neural ventral ectoderm of host embryos at the same developmental stage. The neural plate border and RBs were induced at the transplant sites, as shown by expression of Xblimp1, and XHox11L2 and XN-tubulin, respectively. Transplantation studies between pigmented donors and albino hosts showed that neurons are induced both in donor neural and host epidermal tissue. Because an intermediate level of BMP4 signaling is required to induce neural plate border fates, we directly tested BMP4′s ability to induce RBs; beads soaked in either 1 or 10 ng/ml were able to induce RBs in cultured neural plate tissue. Conversely, RBs fail to form when neural plate tissue from embryos with decreased BMP activity, either from injection of noggin or a dominant negative BMP receptor, was transplanted into the non-neural ectoderm of un-manipulated hosts. We conclude that contact between neural and non-neural ectoderm is capable of inducing RBs, that BMP4 can induce RB markers, and that BMP activity is required for induction of ectopic RB sensory neurons.     

Bimodal functions of Notch-mediated signaling are involved in neural crest formation during avian ectoderm development

Neural crest is induced at the junction of epidermal ectoderm and neural plate by the mutual interaction of these tissues. In previous studies, BMP4 has been shown to pattern the ectodermal tissues, and BMP4 can induce neural crest cells from the neural plate. In this study, we show that epidermally expressed Delta1, which encodes a Notch ligand, is required for the activation and/or maintenance of Bmp4 expression in this tissue, and is thus indirectly required for neural crest induction by BMP4 at the epidermis-neural plate boundary. Notch activation in the epidermis additionally inhibits neural crest formation in this tissue, so that neural crest generation by BMP4 is restricted to the junction.     

Epiblast origin and early migration of neural crest cells in the chick embryo

Chick embryos carrying transplants labeled with tritiated thymidine demonstrate that the neural crest originates in the anterior epiblast, at the junction of areas destined for epidermis and neural tube. As the neural tube begins to fold and the axis lengthens, cells along this junction are drawn dorsomedially; at the seven-somite stage they begin to separate from the epithelium of the head, and migrate into the angle between the epidermis and the neural tube. The paraxial mesoderm already populating this angle originates in more posterior and medial portions of the epiblast than do the neural crest cells; after invagination at the primitive streak, it migrates anterolaterally, ventral to the ectoderm layer, until it too is folded dorsomedially into the angle between the epidermis and the neural tube.     

Xerl is a secreted protein required for establishing the neural plate/neural crest boundary in Xenopus embryo

We have previously isolated a CNS-specific gene, Xerl. The prospective amino acid sequence and functional analysis had shown that Xerl might act as the secretory protein for determining the neural plate/neural crest boundary. However, we had not yet characterized the Xerl protein. In the present study we examined the distribution and function of Xerl protein using anti-Xerl polyclonal antibody. Western blot analysis revealed that Xerl exists as 150 kDa protein in soluble fraction from the neurula stage. In comparison with gene expression of Xerl, Xerl protein showed a diffusive distribution from the neural tissue to the neighboring notochord and somite. Immunostaining of endogenous Xerl protein and subcellular localization of GFP-tagged Xerl demonstrated the extracellular secretion of Xerl protein. With functional blocking by antibody injection, the injected anti-Xerl antibody caused an inhibitory effect on the neural plate formation, whereas neural crest formation was promoted in the antibody-injected embryo. These results suggest that Xerl is a secreted protein required for establishing the neural plate/neural crest boundary in Xenopus embryo.     

Anterior patterning by synergistic activity of the early gastrula organizer and the anterior germ layer tissues of the mouse embryo.

Fragments of the germ layer tissues isolated from the early-primitive-streak (early-streak) stage mouse embryos were tested for axis induction activity by transplantation to late-gastrula (late-streak to early-bud) stage host embryos. The posterior epiblast fragment that contains the early gastrula organizer was able to recruit the host tissues to form an ectopic axis. However, the most anterior neural gene that was expressed in the ectopic axis was Krox20 that marks parts of the hindbrain, but markers of the mid- and forebrain (Otx2 and En1) were not expressed. Anterior visceral endoderm or the anterior epiblast alone did not induce any ectopic neural tissue. However, when these two anterior germ layer tissues were transplanted together, they can induce the formation of ectopic host-derived neural tissues but these tissues rarely expressed anterior neural genes and did not show any organization of an ectopic axis. Therefore, although the anterior endoderm and epiblast together may display some inductive activity, they do not act like a classical organizer. Induction of the anterior neural genes in the ectopic axis was achieved only when a combination of the posterior epiblast fragment, anterior visceral endoderm and the anterior epiblast was transplanted to the host embryo. The formation of anterior neural structures therefore requires the synergistic interaction of the early gastrula organizer and anterior germ layer tissues.     

Effects of antibodies against N-cadherin and N-CAM on the cranial neural crest and neural tube.

We have examined the distribution and function of the defined cell adhesion molecules, N-cadherin and N-CAM, in the emigration of cranial neural crest cells from the neural tube in vivo. By immunocytochemical analysis, both N-cadherin and N-CAM were detected on the cranial neural folds prior to neural tube closure. After closure of the neural tube, presumptive cranial neural crest cells within the dorsal aspect of the neural tube had bright N-CAM and weak N-cadherin immunoreactivity. By the 10- to 11-somite stage, N-cadherin was prominent on all neural tube cells with the exception of the dorsal-most cells, which had little or no detectable immunoreactivity. N-CAM, but not N-cadherin, was observed on some migrating neural crest cells after their departure from the cranial neural tube. To examine the functional significance of these molecules, perturbation experiments were performed by injecting antibodies against N-CAM or N-cadherin into the cranial mesenchyme adjacent to the midbrain. Fab' fragments or whole IgGs of monoclonal and polyclonal antibodies against N-CAM caused abnormalities in the cranial neural tube and neural crest. Predominantly observed defects included neural crest cells in ectopic locations, both within and external to the neural tube, and mildly deformed neural tubes containing some dissociating cells. A monoclonal antibody against N-cadherin also disrupted cranial development, with the major defect being grossly distorted neural tubes and some ectopic neural crest cells outside of the neural tube. In contrast, nonblocking N-CAM antibodies and control IgGs had few effects. Embryos appeared to be sensitive to the N-CAM and N-cadherin antibodies for a limited developmental period from the neural fold to the 9-somite stage, with older embryos no longer displaying defects after antibody injection. These results suggest that the cell adhesion molecules N-CAM and N-cadherin are important for the normal integrity of the cranial neural tube and for the emigration of neural crest cells. Because cell-matrix interactions also are required for proper emigration of cranial neural crest cells, the results suggest that the balance between cell-cell and cell-matrix adhesion may be critical for this process.     

Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy

The Xenopus borealis quinacrine marker and scanning electron microscopy have been used to study the appearance, migration, and homing of neural crest cells in the embryo of Xenopus. The analysis shows that the primordium of the neural crest develops from the nervous layer of the ectoderm and consists of three segments at early neurula stages. This primordium is located in the lateral halves of the neural folds behind the prospective eye vesicles. The histological and experimental evidence shows that the neural crest cells also originate from the medial portion of the neural folds. The neural crest segments in the cephalic region start to migrate just before the closure of the neural tube. Isotopic and isochronic unilateral grafts of X. borealis neural crest into X. laevis embryos were performed in order to map the fate of the cranial crest segments and the vagal-truncal neural crest. The analysis of the X. laevis host embryos shows that the mandibular crest segment contributes to the lower jaw (Meckel's cartilage), quadrate, and ethmoid-trabecular cartilages, as well as to the ganglionic and Schwann cells of the trigeminus nerve, the connective tissues, the mesenchymal and choroid layers of the eye, and the cornea. The hyoid crest segment is located in the ceratohyal cartilage and in ganglia VII and VIII. The branchial crest segment migrates from the caudal part of the otic vesicle and divides into two portions which contribute to the cartilages of the gills. The vagal-truncal neural crest starts to migrate later at stage 25. It migrates by means of the vagus complex in a ventral direction and penetrates into the splanchnic layer of the digestive tract. The trunk neural crest cells disperse into three different pathways which differ from those of the avian embryo at this level.