Interaction between the neural plate and epidermis, together with signals from paraxial mesoderm, is important for the differentiation of the vertebrate neural crest (Selleck & Bronner-Fraser 1995; LaBonne & Bronner-Fraser 1999). Therefore, the neural crest is likely to have evolved in the region straddling the border between the neural plate and epidermis, not only because the neural crest occupies a homologous position, but also because this position is essential for its differentiation. In this section, the cells at the boundary between the neural plate and epidermis in protochordates are examined first, then gene expression patterns in those cells for which vertebrate homologs are involved in neural crest formation are compared.
The boundary between the neural plate and epidermis of protochordates
Protochordate surface ectoderm is believed to be relatively uniform, except for the presence of some epidermal sensory cells found in both ascidians and amphioxus, and the anterior adhesive papillae of ascidians. In the course of examining epidermis- specific gene expression, Ishida et al. (1996) found that ascidian surface ectoderm can be subdivided into six regions according to the sets of genes expressed (Fig. 2A,B). Of these, five regions are characterized as fields where epidermal peripheral neurons differentiate. Two of these regions correspond to the dorsal midline epidermis (regions 1 and 2 in Fig. 2A) and one to the ventral midline (region 5 in Fig. 2A). Two others correspond to regions where adhesive papilla and associated neurons differentiate (regions 3 and 4 in Fig. 2A). The dorsal midline populations abut the neural tube and thus are topologically homologous to the neural crest. Several cells from the dorsal midline population differentiate into primary sensory neurons, which are probably mechanosensory neurons with ciliary processes extending into the larval tunic (Torrence & Cloney 1982). Thus, ascidian larvae have a population of cells in the boundary between the neural tube and epidermis from which both neuronal cells and epidermal cells differentiate; however, little is still known about how each dorsal midline epidermal cell chooses one of the two fates. This striking similarity between the dorsal midline epidermis and the neural crest brings up the tantalizing possibility that the vertebrate neural crest may have originated from the dorsal midline epidermis of protochordates (Fig. 3).
Figure 2. Gene expression patterns of HrEpiA, HrBMPa and HrPax-37. (A) Schematic illustration of an ascidian tail-bud embryo (lateral view) indicating six epidermal territories. The dorsal midline epidermis is shown in orange. Numbering of the epidermal territories follows that used in Ishida et al. (1996). (B) Expression of HrEpiA (Ishida et al. 1996) in a tail-bud embryo (dorsal view; anterior to the left). Expression is exclusive of the dorsal midline epidermis. (C) Expression of HrBMPa in a tail-bud embryo (dorsal view; anterior to the left). Expression is observed in the dorsal midline epidermis (Miya et al. 1996) and thus is complementary to that of HrEpiA. (D–G) Expression of HrPax-37 in the gastrula (D,E; lateral view) and neurula stages (F,G; dorsal view). HrPax-37 expression at the gastrula stage is observed both in cells fated to the dorsal midline epidermis and in those fated to the dorsal neural tube, but is observed only in dorsal midline epidermis at the neurula stage (Wada et al. 1996a).
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In amphioxus, the dorsal midline epidermal cells also show similarity to the neural crest. Neurulation in amphioxus proceeds differently from that in ascidians or vertebrates and intercellular contact between the epidermis and neural plate is disrupted before the amphioxus neural plate rolls up (Fig. 4; Holland et al. 1996). The epidermal sheets from either side of the plate migrate across the dorsal surface by means of lamellipodia, so that epidermis surrounds the entire embryo prior to closure of the neural plate (Fig. 4; Holland et al. 1996). These migrating dorsal midline epidermal cells with lamellipodia are marked by expression of the Dlx gene (Holland et al. 1996), suggesting that the dorsal midline epidermal cells of the amphioxus are a distinct population that display a similar migratory property to cells in the neural crest. However, in amphioxus, sensory epidermal neurons are rather dispersed, not concentrated at the dorsal midline epidermis (Lacalli & Hou 1999). Details of this difference are discussed later.
Figure 4. Neurulation of the amphioxus embryo. (A) Schematic illustration of amphioxus neurulation. The neural plate–neural tube is shown in gray. (B) Dorsal view of a neurula embryo (anterior is to the bottom left). Epidermis is beginning to overgrow the neural plate (np). (C) Close view of cells at the edge of the epidermal sheet (top left). An arrowhead indicates lamellipodia from epidermal cells. (B,C) Reproduced from Holland et al. (1996), with permission of Nick Holland and the Company of Biologists Ltd.
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Protochordate homologs of genes involved in the differentiation of the neural crest
The BMP genes, which belong to the transforming growth factor (TGF)-β superfamily, have been proposed to be involved in the induction of the neural crest. Some BMP functions during very early development are to allow differentiation of the ventral phenotype, promote blood and kidney differentiation in the mesoderm and promote epidermal cell differentiation through suppression of neural differentiation in the ectoderm. Neural induction occurs via inhibition of BMP activity (reviewed by Hogan 1996). After neural induction, the expression of BMP4 and BMP7 is upregulated in the boundary between the neural plate and the surrounding epidermis, where neural crest cells differentiate (Liem et al. 1995). The neural crest is induced through the interaction between the epidermis and the neural plate, and arises from both epidermis and neural plate (Selleck & Bronner-Fraser 1995). BMP4 and BMP7 have been shown to be able to substitute the activity of the epidermis in inducing neural crest from the neural plate (Liem et al. 1995). BMPa, an ascidian homolog of BMP5-8 (including BMP7), displays a expression pattern similar to vertebrate BMP7 (Miya et al. 1996). Expression begins predominantly in ectodermal cells at the gastrula stage. The expression of BMPa is upregulated in the dorsal midline epidermal cells of the neurula later in the differentiation process (Fig. 2C; Miya et al. 1996) and is consistent with the idea that the neural crest originates in the dorsal midline epidermis. BMPb, the ascidian homolog of vertebrate BMP2/4, is also expressed in dorsal midline epidermal cells (Miya et al. 1996). Thus, a similar genetic mechanism may operate for differentiation in both the ascidian dorsal midline epidermis and the vertebrate neural crest. However, amphioxus BMP is not upregulated in the dorsal midline epidermis, although it is expressed in epidermal cells of the gastrula (Panopoulou et al. 1998).
Several transcription factors are involved in differentiation of the vertebrate neural crest. The Pax3 mutant mouse gene, Splotch, produces a defect in neural crest differentiation (Franz 1993) and the closely related Pax7 also causes defects in the cephalic neural crest (Mansouri et al. 1996). Pax3 and Pax7 expression is first detected throughout the entire neural plate. Subsequently, their expression is downregulated in the ventral neural tube and maintained in the dorsal neural tube and neural crest cells (Goulding et al. 1993). Pax3 and Pax7 have a single counterpart gene in protochordates. HrPax-37, the ascidian homolog of Pax3 and Pax7, exhibits a pattern of expression similar to its vertebrate counterparts (Wada et al. 1996a, 1997). It is expressed both in cells destined to form the dorsal part of the neural tube and in those destined to form dorsal midline epidermis (Fig. 2D,E). This pattern is comparable with that of vertebrate genes in the dorsal neural tube and neural crest. Just prior to closure of the neural tube, HrPax-37 expression is maintained solely in the dorsal midline epidermis (Fig. 2F,G, Wada et al. 1996a, 1997). The amphioxus homolog of Pax3/7 is expressed in the lateral part of the neural plate, which subsequently occupies the dorsal part of the neural tube and is also found in the somitic mesoderm, although no expression is detected in the epidermis (Holland et al. 1999).
Dlx genes also stand as markers for the neural crest (Robinson & Mahon 1994). AmphiDll, the amphioxus homolog of Dlx, is expressed in the dorsal neural tube and in epidermis adjacent to the neural plate (Holland et al. 1996). An ascidian homolog of Dlx is also expressed in the dorsal midline epidermis (Wada et al. 1999b).
Snail and slug are two closely related zinc finger genes that are among the most widely used markers of the neural crest (Essex et al. 1993; Mayor et al. 1995). Slug is necessary for the induction and subsequent migration of neural crest cells (LaBonne & Bronner-Fraser 2000) and its overexpression leads to expansion of the neural crest cell population (LaBonne & Bronner-Fraser 1998). Snail and slug have one counterpart in protochordates (Corbo et al. 1997; Langeland et al. 1998). The ascidian counterpart gene is expressed in the dorsal part of the anterior neural tube, from which pigment cells differentiate. The posterior neural tube of ascidian larvae consists of four rows of cells. At this level, the snail/slug homolog is expressed predominantly in the lateral row (Corbo et al. 1997). In Ciona intestinalis, a species of ascidian, expression is restricted to the lateral row (Corbo et al. 1997). In another species of ascidian, Halocynthia roretzi, this gene is expressed weakly in the dorsal row (Wada & Saiga 1999). The snail gene is not expressed in ascidian epidermis (Corbo et al. 1997; Wada & Saiga 1999). The amphioxus homolog of the snail/slug gene is expressed in the dorsal region of the neural tube, but not in the epidermis (Langeland et al. 1998).
These comparisons of gene expression patterns are not completely consistent with the idea that the neural crest originates in the dorsal midline epidermis: expression patterns differ significantly between ascidians and amphioxus, as summarized in Table 1. The amphioxus BMP2/4 and Pax3/7 homologs are not expressed in the dorsal midline epidermis (Panopoulou et al. 1998; Holland et al. 1999), possibly correlating with the scattered distribution of the sensory neuronal cells in amphioxus (Lacalli & Hou 1999), while the ascidian sensory neurons are restricted in several fields (Fig. 1A). However, the strong similarities in the patterns of gene activity in the dorsal midline epidermis of ascidians and the vertebrate neural crest are unlikely to be the result of evolutionary convergence. In addition, Dll is expressed in amphioxus dorsal midline epidermis (Holland et al. 1996). Lack of dorsal expression of amphioxus BMP2/4 and Pax3/7 is described most parsimoniously as a secondary loss that took place after the divergence of vertebrates and amphioxus. However, in order to accept this hypothesis of neural crest origin in the dorsal midline epidermis, some reasonable explanation for the amphioxus exception should be made.
Table 1. Expression of neural crest marker genes in protochordates
| ||Dorsal neural tube||Dorsal midline epidermis||Dorsal neural tube||Dorsal midline epidermis|
Another point for consideration is whether the dorsal midline epidermis is the sole origin of the neural crest, or whether the dorsal neural tube also contributes to its development. Ascidian Pax3/7 and amphioxus Dll genes are also expressed in the dorsal part of the neural tube (Holland et al. 1996; Wada et al. 1996a, 1997). Amphioxus Pax3/7 and the snail/slug genes of both ascidians and amphioxus are only expressed in the dorsal neural tube (Corbo et al. 1997; Langeland et al. 1998; Holland et al. 1999; Wada & Saiga 1999). Langeland et al. (1998) suggest that the neural crest originated in the dorsal part of the neural tube, while Holland et al. (1996) suggest that its origin may be found in both the dorsal midline epidermis and the dorsal part of the neural tube. However, it may be inappropriate to look for the origin of the vertebrate neural crest in a distinct cell population. Cell lineage analyses of cells in the vertebrate neural fold reveal that a single cell can give rise to neuronal cells, neural crest cells and epidermal cells (Bronner-Fraser & Fraser 1988, 1989; Garcia-Castro & Bronner-Fraser 1999). Moreover, because neural crest cells generate a wide diversity of cell types and because neural crest cells in the cephalic region have a quite distinct character from those in trunk region, it is not certain whether the neural crest has a single origin (Shimeld & Holland 2000). The main point stressed here is that the boundary between the neural tube and epidermis in protochordates is less well defined than has been believed in the past. In addition, there are cells that show gene activity and cell properties comparable to the vertebrate neural crest. Thus, they perhaps originate the neural crest, possibly with some contribution from the dorsal neural tube (Fig. 3). Because we know that the interaction between neural tissue and the epidermis performs a central role for neural crest induction (Selleck & Bronner-Fraser 1995), we can ask whether a similar interaction is necessary for cell differentiation or specific gene expression in the dorsal midline epidermis of protochordates. This hypothesis can also be tested by comparing the genetic mechanisms for differentiation of the neural crest and dorsal midline epidermis in greater detail.