Origin, Induction, and Specification

According to the most recent data, the earliest stages of neural crest induction may occur as early as gastrulation, but according to the classical model, the neural crest arises as the result of inductive actions by the adjacent non-neural ectoderm and possibly nearby mesoderm on the neural plate (Fig. 12.1). The ectodermal inductive signals are bone morphogenetic proteins (BMPs) and Wnts. Fibroblast growth factor-8 (FGF-8) from mesoderm plays a role in neural crest induction in amphibians, and it seems to be involved in mammals as well. The role of BMPs is complex and relates to a concentration gradient along the ectodermal layer as neurulation proceeds. The highest concentrations of BMP are seen in the lateral ectoderm, and cells exposed to these concentrations remain ectodermal. Cells within the neural plate are exposed to the lowest concentrations of BMP because of the local inhibitory actions of noggin and chordin (see Fig. 5.8C), and, by default, they remain neural. Cells at the border of the neural plate are exposed to intermediate levels of BMP, and, in this environment, they are induced to form neural crest precursor cells.

In response to these inductive signals, cells at the border of the neural plate activate genes coding for several transcription factors, including Msx-1 and Msx-2, Dlx-5, Pax-3/Pax-7, and Gbx-2. These and other gene products turn on a network of genes that transform the epithelial neural crest precursor cells into mobile mesenchymal cells that break free from the neuroepithelium of the neural tube.

Epitheliomesenchymal Transformation and Emigration from the Neural Tube

Within the neural tube, neural crest precursor cells are epithelial and are tightly adherent to other neuroepithelial cells through a variety of intercellular connections. Prominent among them are the cadherins. Among the new transcription factors upregulated in induced neural crest precursor cells are snail-1 and snail-2 (formerly called slug) and Foxd-3, which are instrumental in allowing the neural crest cells to break free from the neural epithelium and then migrate away as mesenchymal cells.* Under the influence of snail-1 and snail-2, the profile of cadherins produced by the neural crest precursors changes from type I cadherins (e.g., N-cadherin and E-cadherin), which are strongly adhesive, to type II cadherins, which are less adhesive.

Neural crest cells break free from the neural tube in the trunk at the level of the last-formed somite or the neural plate in the head by changing their shape and properties from those of typical neuroepithelial cells to those of mesenchymal cells. Important to this process is the loss of cell-to-cell adhesiveness. This loss is effected by the loss of cell adhesion molecules (CAMs) characteristic of the neural tube (e.g., N-CAM, E-cadherin, and N-cadherin). These molecules remain downregulated during migration, but after neural crest cells have completed their migrations and have differentiated into certain structures (e.g., spinal ganglia), CAMs are often expressed again.

In the head, where closure of the neural plate has not yet occurred, neural crest cells must penetrate the basal lamina underlying the neural plate. This is accomplished by the production of enzymes that degrade components of the basal lamina and by sending out processes that penetrate the basal lamina. In the trunk, neural crest cells do not leave the neuroepithelium until after the neural tube has formed. They do not, however, have to contend with penetrating a basal lamina because the dorsal part of the neural tube does not form a basal lamina until after emigration of the crest cells.

Neural Crest Cell Migration

After leaving the neuroepithelium, the neural crest cells first encounter a relatively cell-free environment rich in extracellular matrix molecules (Fig. 12.2). In this environment, the cells undergo extensive migrations along several well-defined pathways. These migrations are determined by intrinsic properties of the neural crest cells and features of the external environment encountered by the migrating cells.

Neural crest migration is influenced by a variety of molecules residing in the extracellular matrix. Although the presence of a basal lamina can inhibit their emigration from the neural tube, neural crest cells often prefer to migrate along basal laminae, such as those of the surface ectoderm or neural tube, after they have left the neural tube. Components of the extracellular matrix permissive for migration include molecules found in basal laminae, such as fibronectin, laminin, and type IV collagen (Fig. 12.3). Attachment to and migration over these substrate molecules are mediated by the family of attachment proteins called integrins. Other molecules, such as chondroitin sulfate proteoglycans, are not good substrates for neural crest cells and inhibit their migration.

Neural crest cells emigrate from the neural tube or neural folds in streams, with each cell in contact with neighbors through filopodial contacts. During their migratory phase, neural crest cells are exquisitely sensitive to guidance molecules, most of which are inhibitory. Among the most important of these guidance molecules are the ligand/receptor pairs Robo/Slit, Neuropilin/Semaphorin and Ephrin/Eph (see Table 11.1). Much less is known about attractive influences on neural crest cell migration. During migration, neural crest cells extend protrusions that both test the environment and are part of the propulsive mechanism. If an inhibitory influence is encountered, the protrusions collapse through signals derived from a planar cell polarity pathway (see p. 87). This mechanism acts as a brake when the cells encounter an inhibitory environment, but it is also involved in their forward propulsion. In a migrating stream of neural crest cells, contact with the cells behind also results in the pulling of protrusions at the trailing edge of the cells, thus resulting in a net forward motion of the leading cells. Specific examples of the environmental control of neural crest cell migrations are given later in this chapter. Much remains to be learned about what causes neural crest cells to stop migrating, but often they stop migrating in areas where repulsive signals are low.

Differentiation of Neural Crest Cells

Neural crest cells ultimately differentiate into an astonishing array of adult structures (Table 12.1). What controls their differentiation is one of the principal questions of neural crest biology. Two opposing hypotheses have been proposed. According to one, all neural crest cells are equal in developmental potential, and their ultimate differentiation is entirely determined by the environment through which they migrate and into which they finally settle. The other hypothesis suggests that premigratory crest cells are already programmed for different developmental fates, and that certain stem cells are favored, whereas others are inhibited from further development during migration. More recent research indicates that the real answer can be found somewhere between these two positions. Increasing evidence suggests that among migrating neural crest cells is a mix of cells whose fate has been predetermined within the neural tube and cells whose ultimate phenotype depends on environmental influences.

A correlation exists between the time of migration of neural crest cells from the neural tube and their developmental potential. Many cells that first begin to migrate have the potential to differentiate into several different types of cells. Crest cells that begin to migrate later are capable of forming only derivatives characteristic of more dorsal locations (e.g., spinal ganglia), but not sympathetic neurons or adrenal medullary cells. Crest cells that leave the neural tube last can form only pigment cells.

Several experiments have shown that the fates of some neural crest cells are not irreversibly fixed along a single pathway. One type of experiment involves the transplantation of neural crest cells from one part of the body to another. For example, many neural crest cells from the trunk differentiate into sympathetic neurons that produce norepinephrine as the transmitter. In the cranial region, however, neural crest cells give rise to parasympathetic neurons, which produce acetylcholine. If thoracic neural crest cells are transplanted into the head, some cells differentiate into cholinergic parasympathetic neurons instead of the adrenergic sympathetic neurons normally produced. Conversely, cranial neural crest cells grafted into the thoracic region respond to their new environment by forming adrenergic sympathetic neurons.

A more striking example is the conversion of cells of the periocular neural crest mesenchyme, which in birds would normally form cartilage, into neurons if they are associated with embryonic hindgut tissue in vitro. Many of the regional influences on the differentiation of local populations of neural crest cells are now recognized to be interactions between the migrating neural crest cells and specific tissues that they encounter during migration. Examples of tissue interactions that promote the differentiation of specific neural crest derivatives are given in Table 12.2.

The plasticity of differentiation of neural crest cells can be shown by cloning single neural crest cells in culture. In the same medium, and under apparently the same environmental conditions, the progeny of the single cloned cells frequently differentiate into neuronal and non-neuronal (e.g., pigment cell) phenotypes. Similarly, if individual neural crest cells are injected in vivo with a dye, greater than 50% of the injected cells will give rise to progeny with two to four different phenotypes containing the dye. By exposing cloned neural crest precursor cells to specific environmental conditions in vitro, one can begin to understand the mechanisms that determine phenotype in vivo. In one experiment, rat neural crest cells grown under standard in vitro conditions differentiated into neurons, but when they were exposed to glial growth factor, they differentiated into Schwann cells because the glial growth factor suppressed their tendency to differentiate into neurons. Similarly, the growth factors BMP-2 and BMP-4 cause cultured neural crest cells to differentiate into autonomic neurons, whereas exposure of these cells to transforming growth factor-β causes them to differentiate into smooth muscle.

Not all types of transformations among possible neural crest derivatives can occur. Crest cells from the trunk transplanted into the head cannot form cartilage or skeletal elements, although this is normal for cells of the cranial neural crest. Most experiments suggest that early neural crest cells segregate into intermediate lineages that preserve the option of differentiating into several, but not all, types of individual phenotypes. In the chick embryo, some neural crest cells are antigenically different from others even before they have left the neural tube.

Many neural crest cells are bipotential, depending on signals from their local environment for cues to their final differentiation. Cultured heart cells secrete a protein that converts postmitotic sympathetic neurons from an adrenergic (norepinephrine transmitter) phenotype to a cholinergic (acetylcholine-secreting) phenotype (see Fig. 11.22). During normal development, the sympathetic neurons that innervate sweat glands are catecholaminergic until their axons actually contact the sweat glands. At that point, they become cholinergic.

Trunk Neural Crest

The neural crest of the trunk extends from the level of the sixth somite to the most caudal somites. Three pathways of migration are commonly described (Fig. 12.4). These pathways occur in different sequences and are subject to different controls. The first neural crest cells to leave the neural tube migrate around and between the somites, which are still in an epithelial configuration. Their migratory path follows the intersomitic blood vessels, and the cells rapidly reach the region of the dorsal aorta (see Fig. 12.4, pathway 1). It may be that at this early stage no other pathway is available to these migrating cells. These cells constitute the sympathoadrenal lineage.

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