Segmental organization of neural crest migration

Mechanisms of Development 105 (2001) 37±45

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Segmental organization of neural crest migration C.E. Krull* Biological Sciences, University of Missouri-Columbia, 108 Lefevre, Columbia, MO 65211, USA Received 13 October 2000; accepted 22 December 2000

Abstract Avian neural crest cells migrate on precise pathways to their target areas where they form a wide variety of cellular derivatives, including neurons, glia, pigment cells and skeletal components. In one portion of their pathway, trunk neural crest cells navigate in the somitic mesoderm in a segmental fashion, invading the rostral, while avoiding the caudal, half-sclerotome. This pattern of cell migration, imposed by the somitic mesoderm, contributes to the metameric organization of the peripheral nervous system, including the sensory and sympathetic ganglia. At hindbrain levels, neural crest cells also travel from the neural tube in a segmental manner via three migratory streams of cells that lie adjacent to even-numbered rhombomeres. In this case, the adjacent mesoderm does not possess an obvious segmental organization, compared to the somitic mesoderm at trunk levels. Thus, the mechanisms by which the embryo controls segmentally-organized cell migrations have been a fascinating topic over the past several years. Here, I discuss ®ndings from classical and recent studies that have delineated several of the tissue, cellular and molecular elements that contribute to the segmental organization of neural crest migration, primarily in the avian embryo. One common theme is that neural crest cells are prohibited from entering particular territories in the embryo due to the expression of inhibitory factors. However, permissive, migration-promoting factors may also play a key role in coordinating neural crest migration. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Cell migration; Avian; Peanut agglutinin; Eph receptor tyrosine kinases; Ephrins; Time-lapse imaging; Somite

1. Introduction The vertebrate neural crest is a transient population of precursor cells that generates a fascinating range of derivatives including neurons, melanocytes, skeletal elements, and glia (Le Douarin and Kalcheim, 1999). Early in embryonic development, neural crest form in the dorsal portion of the neuroepithelium due to inductive events that are not yet well understood. Shortly thereafter, neural crest cells undergo an epithelial to mesenchymal transformation, changing adhesive connections with their neighbors, and emigrate from the neural tube. Neural crest cells then travel stereotypical pathways to their ®nal destinations where they give rise to speci®c cellular derivatives. The avian neural crest in particular has been well-studied by numerous investigators over many years due to its easy accessibility for embryological manipulations. Thus, a wealth of information is available concerning the timing and pathways of neural crest migration. Furthermore, the linkage of classical embryology with molecular biological approaches has provided key insights into some of the molecular factors that are required for neural crest induction, * Tel.: 11-573-882-4067; fax: 11-573-884-5020. E-mail address: [email protected] (C.E. Krull).

emigration, migration, localization, and cell fate determination. In addition, recent technological advances have permitted investigators to view neural crest migration over time in its native environment, using time-lapse videomicroscopy. These types of analyses have provided interesting clues as to the types of molecular interactions that govern neural crest migration. 2. The organization of neural crest migration In many vertebrate embryos, neural crest cells are generated in the dorsal neural tube, most likely due to signals from the adjacent ectoderm and mesoderm (Selleck and Bronner-Fraser, 1995; Ikeya et al., 1997; Bonstein et al., 1998; for review, see LaBonne and Bronner-Fraser, 1999). Members of the TGF-b family and Wnt molecules appear to play a role in neural crest formation and maintenance, although their precise functions in these events are not yet clear (Liem et al., 1997; Selleck et al., 1998; Nguyen et al., 2000). In ®sh, neural crest cells segregate as a mesenchymal cell population from the dorsal portion of the neural keel, a thickening of the ectoderm. In other vertebrates, neural crest become distinguishable from neural tube cells when they undergo a dramatic transformation from epithelial to

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C.E. Krull / Mechanisms of Development 105 (2001) 37±45

mesenchymal cells and delaminate from the neural tube. This transformation is accompanied by both morphological and molecular changes, including the loss of a neuroephithelial morphology, the acquisition of migratory capabilities characteristic of mesenchymal cells, the increased expression of cadherin-7, and downregulation of the cell adhesion molecules NCAM, cadherin-6, and N-cadherin (Akitaya and Bronner-Fraser, 1992; Newgreen and Minichiello, 1995; Inoue et al., 1997; Erickson and Reedy, 1998). Recent results indicate that changes in cadherin expression may be required for neural crest delamination: overexpression of cadherin-7 or N-cadherin by avian-speci®c retroviruses prevents neural crest emigration from the neural tube (Nakagawa and Takeichi, 1998). These results, in combination, suggest that a critical level of cadherin-7 may normally mediate adhesive contacts between neural crest that are required for emigration from the neural tube. Treatment with blockers of calcium also disrupts cell adhesion between premigratory neural crest, leading to premature emigration of neural crest from the neural tube (Newgreen and Gooday, 1985). These results also suggest that neural crest cells must alter their adhesive interactions with neighboring neuroepithelial cells to emigrate from the neural tube. In further support, electron microscopy studies have shown that premigratory neural crest are tethered together via specialized junctions. These junctions disappear coincident with the onset of crest emigration (Newgreen and Gibbins, 1982). Other recent studies have demonstrated that rhoB, a member of the Rho family of GTP-binding proteins, is expressed in neural crest cells prior to their emigration from the neural tube, and at early stages in their migration (Liu and Jessell, 1998). Furthermore, inhibition of rhoB prohibits neural crest emigration from the neural tube, suggesting that rhoB is also required for the delamination process. After neural crest cells detach from the neural tube, they migrate along stereotypical pathways to their ®nal destinations. In the trunk region of avians and mammals, neural crest travel two pathways, a ventromedial route through the somitic mesoderm, and a dorsolateral pathway, between the somites and overlying ectoderm (Rickmann et al., 1985; Le Douarin and Kalcheim, 1999; Fig. 1). Neural crest that travel ventromedially contribute to the sensory and sympathetic ganglia, and generate Schwann cells and chromaf®n cells (Le Douarin and Teillet, 1974). In contrast, neural crest that migrate dorsolaterally generate melanocytes. In addition to these differences in the cell fates of neural crest migrating on these two pathways, the organization of their migration is also distinct. Neural crest that migrate ventromedially travel in a segmental manner through the somitic mesoderm, entering the rostral half of the somitic sclerotome while avoiding the caudal half-sclerotome (Keynes et al., 1990). In contrast, dorsolaterally-migrating neural crest travel in a uniform, unsegmented fashion. Ventromedial neural crest migration is intimately linked to the maturation of the somites. Somites bud from the segmental plate, form-

ing ball-like epithelial structures. Shortly thereafter, each somite becomes compartmentalized, composed of an epithelial dermomyotome and mesenchymal sclerotome. It is at this stage of somite development that trunk neural crest invade the somite proper (Guillory and Bronner-Fraser, 1986). In zebra®sh, trunk neural crest also migrate on two pathways. However, one pathway is medial, between the neural tube and somite, whereas the other pathway is dorsolateral, between the somites and underlying ectoderm. Interestingly, the sclerotome does not appear to play a key role in the organization of neural crest migration on the medial pathway in zebra®sh (Morin-Kensicki and Eisen, 1997). Instead, the myotome appears to be prominent in the guidance of trunk neural crest migration. At the level of the avian hindbrain, neural crest migrate into the branchial arches where they form cranial ganglia and components of the craniofacial skeleton (Kontges and Lumsden, 1996). These neural crest cells migrate from the neural tube in three distinct streams: neural crest migrate from rhombomere 2 to enter the ®rst branchial arch, from rhombomere 4 to enter the second arch, and from rhombomere 6 to the third arch (Lumsden et al., 1991). Results of early transplant studies indicated that hindbrain neural crest seemed to acquire their positional identity prior to their emigration (Noden, 1983). However, recent results suggest that a combination of intrinsic and environmental factors control the migration and speci®cation of hindbrain neural crest (Saldivar et al., 1996; Trainor and Krumlauf, 2000). In Xenopus, in contrast to chick, there is not an early segregation of branchial neural crest into three migratory streams. Xenopus branchial neural crest become organized into three

Fig. 1. Avian trunk neural crest cells travel on two distinct pathways after emigration from the neural tube. Schematic diagram of a longitudinal view of the trunk region of an avian embryo. Some trunk neural crest cells (red) emerge from the dorsal neural tube and travel ventromedially, through the rostral, but not caudal, somitic sclerotome. Other neural crest cells (black) migrate dorsolaterally in a uniform manner between the somites and overlying ectoderm. DM, dermomyotome; Scl, sclerotome; No, notochord; Ao, aorta; Ec, ectoderm; NT, neural tube; R, rostral; C, caudal.

C.E. Krull / Mechanisms of Development 105 (2001) 37±45

streams only upon entering the branchial arches. Therefore, the mechanisms employed in the segmental migration of neural crest into the branchial arches in avians and Xenopus may be distinct. In avians, tissues in the migratory environment (i.e. otic vesicle) may in¯uence the segmental pattern of branchial neural crest migration. In Xenopus, mechanisms that restrict cell mixing may be key to guiding branchial neural crest to their targets (Robinson et al., 1997). Subsequent to their migration through the somitic mesoderm and other embryonic territory, neural crest cells localize to their ®nal target regions and differentiate to form a wide variety of cellular derivatives. Previous lineage studies have indicated that neural crest cells contribute to their derivatives in a ventral to dorsal order (Serbedzija et al., 1989). However, relatively nothing is known about the signals that are responsible for target localization and stopping neural crest migration. In contrast, a wide array of growth factors and transcription factors have been implicated in neural crest cell fate decisions in autonomic, sensory, melanocyte and enteric neural crest derivatives (Varley et al., 1998; Reissman et al., 1996; Schuchardt et al., 1994; Perez et al., 1999; Opdecamp et al., 1997; Pattyn et al., 1999; Enomoto et al., 1998; Britsch et al., 1998; Moore et al., 1996; Pichel et al., 1996). However, it is less clear whether cues in the environment where neural crest localize instruct these cells to adopt speci®c fates or whether precursors with distinct intrinsic developmental programs localize to particular regions in the embryo. Previous lineage analyses indicated that some dorsal neural tube cells contribute to multiple neural crest derivatives (BronnerFraser and Fraser, 1988). Recent studies support the idea that precursors for distinct neural crest derivatives are faterestricted prior to neural crest migration (Henion and Weston, 1997). Most likely, a combination of intrinsic programs and environmental factors in¯uence the diversity of neural crest fates. 3. Tissue and cellular mechanisms that guide trunk neural crest cell migration Avian neural crest cells at trunk levels emerge from the dorsal neural tube and enter a cell-free zone, rich in extracellular matrix (Newgreen and Thiery, 1980). Here, two possible pathways for the next stage of migration exist: a ventromedial route through the somite proper and a dorsolateral route, between the somites and ectoderm (Loring and Erickson, 1987). Ventromedial migration of trunk neural crest through the somitic mesoderm precedes dorsolateral migration by approximately 24 h. Inhibition of neural crest entry into the dorsolateral path correlates with the expression of two markers for inhibitory factors (Oakley et al., 1994). Peanut agglutinin (PNA)-binding glycoproteins and chondroitin-6-sulfate immunoreactivities are both present on the dorsolateral path and their expression decreases coincident with neural crest entry. Furthermore, surgical dele-

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tions of tissues expressing these two markers allow the premature entry of neural crest on the dorsolateral path. These results suggest that neural crest are transiently inhibited from migrating on the dorsolateral path due to the expression of PNA-binding glycoproteins and chondrotin6-sulfate. Interestingly, these factors also appear to play a key role in the segmental organization of trunk neural crest that migrate through the somite proper (see below). Trunk neural crest cells migrate ventromedially through the somites, entering the rostral, but not caudal, half-sclerotome, generating a segmental array of neural crest cells in the somitic mesoderm (Rickmann et al., 1985). This distinct migration pattern of neural crest in the somites contributes to the overall segmental organization of the peripheral nervous system (Goldstein and Kalcheim, 1991; Kalcheim and Teillet, 1989). How is this segmental pattern of neural crest migration established? At least two possible scenarios can be conceived: (1) At the level of the neural tube, neural crest possess intrinsic information that guides their segmental migration through the somites; or (2) cues intrinsic to the somitic mesoderm govern the segmentation of trunk neural crest migration. To determine the contribution of neural tube or somite cues to this process, microsurgical manipulations were performed whereby either neural tube tissue or segmental plate (i.e. presomitic mesoderm) were rotated (BronnerFraser and Stern, 1991; Stern et al., 1991). The subsequent effects on the segmental organization of trunk neural crest migration were later observed using an avian neural crest marker, the HNK-1 antibody. Following neural tube rotations, HNK-1-positive neural crest migrated through the rostral half-sclerotome, their normal migratory domain in the somites. However, following rotations of the segmental plate, which inverted the rostrocaudal polarity of the somites, neural crest were found in the caudal half-sclerotome (originally rostral half-sclerotome). These results suggest that cues in the somitic mesoderm guide the segmental organization of trunk neural crest migration. Interestingly, motor axons also navigate the somitic mesoderm in a similar segmental manner (Keynes and Stern, 1988). Another region of the somites, the developing myotome, possesses a basal lamina that is initially preferred by migrating trunk neural crest cells (Tosney et al., 1994). Early neural crest align in close association with the myotome's basal lamina, traveling in the lateral portion of the sclerotome, whereas later-migrating neural crest invade medial sclerotome, when a high density of neural crest laterally prevents access to the myotome. These data suggest that positive factors localized to the myotome's basal lamina may also expedite trunk neural crest migration in the rostral half-somite. An additional embryonic tissue, perinotochordal mesenchyme, has been shown to in¯uence neural crest cells during their migration to target regions that lie in ventral positions (Tosney and Oakley, 1990; Stern et al., 1991). During normal development and in embryos where the neural tube was surgically manipulated, neural crest

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C.E. Krull / Mechanisms of Development 105 (2001) 37±45

cells avoid and do not enter perinotochordal mesenchyme. Removal of the notochord abolishes the avoidance by neural crest cells, suggesting that inhibitory factors synthesized by the notochord normally prohibit neural crest migration in the perinotochordal area. 4. Attractive and inhibitory molecules: roles in patterning trunk neural crest migration Analyses of the tissue and cellular mechanisms that guide trunk neural crest migration indicate that rostral regions of somitic mesoderm may promote cell migration by means of permissive or growth-promoting molecules. In contrast, caudal regions of the somite and the perinotochordal mesenchyme are thought to prohibit cell entry by the activity of repulsive or inhibitory factors. Over the past few years, several molecules have been identi®ed as candidate guidance factors for trunk neural crest based on their spatiotemporal patterns of expression (Table 1) and functions in vitro and in vivo. In this section, I will discuss the contributions of these some of these factors to the segmentation and guidance of trunk neural crest migration. 4.1. Extracellular matrix molecules An array of extracellular matrix molecules are distributed on neural crest migratory pathways (Erickson and Perris, 1993). Some extracellular matrix molecules, including

®bronectin and laminin, are distributed diffusely in the somitic sclerotome, in both rostral and caudal halves (Newgreen and Thiery, 1980). In vitro analyses suggest that these two molecules serve as general promoters of neural crest migration through the somitic mesoderm. However, it is not likely, due to their distribution and the results of functional analyses, that they contribute to the segmental organization of trunk neural crest (Saga et al., 1992). Tenascin (tenascin-C) exhibits a dynamic distribution in the somitic mesoderm over the course of trunk neural crest migration. Initially, tenascin is associated with epithelial somites. At intermediate stages, tenascin protein is expressed in the caudal half-sclerotome, and in older somites, in rostral half-sclerotome (Stern et al., 1989). Both neural crest and somite cells synthesize tenascin protein (Tucker and McKay, 1991), suggesting that tenascin also may act as a general promoter of neural crest migration. F-spondin mRNA and protein localize prominently to the caudal half-sclerotome and perinotochordal mesenchyme, indicating they are excellent candidates to mediate the inhibitory effects of these two tissues on trunk neural crest migration (Debby-Brafman et al., 1999). Perturbations that disrupt F-spondin function in vitro, in trunk explants, and in ovo result in defects in the patterning of trunk neural crest migration. Speci®cally, trunk neural crest entered normally inhibitory territories when F-spondin protein function was blocked. Collagen IX, a chondroitin sulfate proteoglycan, is also differentially expressed in the somitic mesoderm, loca-

Table 1 Molecules implicated in the segmental organization of avian trunk neural crest migration a Molecule

Localization prior to trunk NC migration

Localization during trunk NC migration

References

Butyrylcholinesterase Collagen IX Ephrin-B1

1. Highest in rostral half-somites 1. Highest in caudal-half sclerotomes 1. Caudal half-somites

Caudal half-sclerotomes Caudal half-sclerotomes Caudal half-sclerotomes dermomyotomes

Layer et al., 1988 Ring et al., 1996 Krull et al., 1997

F-spondin

2. 1. 2. 1.

M7412 antigen PNA Glycoproteins T-cadherin

2. Highest in caudal half-sclerotome paranotochordal mesenchyme 1. Rostral half-somites 1. Diffuse in epithelial somites 2. Caudal half-somites, perinotochordal mesenchyme 1. Caudal half-somites

Tenascin

1. Diffuse in epithelial somites

Thrombospondin

1. Basal laminae: nt, noto, core of epithelial somites

Versican

1. Basal laminae: nt, som, noto, punctate in epithelial somites 2. Rostral 1 caudal sclerotomes

Fibronectin 1 laminin

a

Caudal half-sclerotomes Basal laminae: nt, som, cell-free space Diffuse in sclerotomes Diffuse in epithelial somites

nt, neural tube; som, somites, noto, notochord.

Same

Wang and Anderson, 1997 Newgreen and Thiery, 1980

Caudal half-sclerotomes paranotochordal mesenchyme dermomyotomes

Debby-Brafman et al., 1999

Rostral half-sclerotomes Caudal half-sclerotomes

Tanaka et al., 1989 Oakley and Tosney, 1991 Davies et al., 1990 Ranscht and Bronner-Fraser, 1991 Stern et al., 1989

Caudal half-sclerotomes Caudal half-sclerotomes, then rostral half-sclerotomes Dorsal sclerotomes ventral myotomes Caudal half-sclerotomes perinotochordal mesenchyme

Tucker et al., 1999 Landolt et al., 1995 Perissinotto et al., 2000

C.E. Krull / Mechanisms of Development 105 (2001) 37±45

lized to the caudal half-sclerotome, during the period of trunk neural crest migration (Ring et al., 1996). Neural crest in vitro speci®cally avoid substrate-bound collagen IX, suggesting that this factor could also contribute to the segmental organization of trunk neural crest migration in vivo. Other chondroitin sulfate proteoglycans, including versican, have also been implicated in the guidance of trunk neural crest (Oakley and Tosney, 1991; Landolt et al., 1995; Perissinotto et al., 2000). Recent results suggest that another extracellular matrix molecule, thrombospondin, exhibits the correct localization to direct neural crest migration in the rostral half-sclerotome (Tucker et al., 1999). Trunk neural crest navigate through a thrombospondin-positive matrix in vivo, and thrombospondin promotes neural crest migration and adhesion in vitro. Thrombospondin may cooperate with other factors expressed in the rostral sclerotome including tenascin, ®bronectin, or laminin, to positively promote neural crest migration rostrally. This possible scenario awaits future analyses. 4.2. T-cadherin and PNA glycoproteins T-cadherin, a calcium-dependent cell adhesion molecule, is expressed speci®cally in the caudal half-sclerotome (Ranscht and Bronner-Fraser, 1991). The timing of Tcadherin expression correlates with the migration of neural crest into the somites, suggesting that T-cadherin may be an inhibitory factor that controls the pattern of trunk neural crest migration. Although the function of T-cadherin in trunk neural crest migration has not been reported, Tcadherin as a substrate or in soluble form does inhibit neurite outgrowth by motor neurons in vitro (Fredette et al., 1996). One set of molecules that has long been implicated in the segmental organization of the nervous system includes two glycoproteins that bind the plant lectin, peanut agglutinin (PNA). PNA, speci®c for Gal-b(1±3)-GalNAc carbohydrates, binds exclusively to caudal, but not rostral, halfsclerotomal cells during the time that neural crest and motor axons navigate segmentally through the somitic mesoderm (Davies et al., 1990; Oakley and Tosney, 1991). Using PNA-af®nity chromatography, two PNA-binding glycoproteins of 48 and 55 kDa were identi®ed (Davies et al., 1990). Extracts containing these PNA-binding glycoproteins applied to neurons in culture induced growth cone collapse, suggesting these proteins may function in vivo to inhibit axons from entering the caudal portion of the somites. PNA-binding molecules are required for the segmentation of trunk neural crest migration in vivo (Krull et al., 1995). Application of PNA speci®cally disrupted the segmental route of migration taken by neural crest through the somites in avian trunk explants: neural crest entered both rostral and caudal half-sclerotomes. These data suggested that the rostral and caudal halves of the somitic sclerotomes had become uniform, with respect to migrating neural crest cells. However, time-lapse analysis

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of neural crest migrating in the PNA-treated explant environment indicated that migratory trajectories taken by neural crest in the rostral versus caudal sclerotome were distinct (see Section 6 below). Furthermore, this analysis suggested that other inhibitory factors expressed in the caudal halfsclerotome prevented neural crest entry, in combination with the PNA glycoproteins. 4.3. Eph receptor tyrosine kinases and their ligands, the ephrins Members of the Eph family of receptor tyrosine kinases (RTKs) and their ligands, the ephrins, display intriguing patterns of expression in the developing nervous system, suggesting potential roles in early neural patterning, including neural crest migration (Flanagan and Vanderhaeghen, 1998; Orioli and Klein, 1997; Robinson et al., 1997). Based on their ligand preferences and in vitro binding speci®cities, the Eph RTKs have been divided into two subclasses: EphA, which interact primarily with GPI-anchored ephrins (ephrin-As), and EphB which interact with transmembrane ephrins (ephrin-Bs) (Davis et al., 1994). Because of their membrane association, Eph RTK-ephrin interactions are thought to be involved in cell contact-mediated events. Interactions between Eph RTKs and ephrins are thought to prevent neural precursors or growing axons from entry into speci®c embryonic territories. These data, in combination, suggested that members of this family might participate in the segmental organization of trunk neural crest migration. Expression analysis of members of the EphB subclass indicated that EphB3 RTK was expressed by neural crest cells and that its ligand, ephrin-B1, localized strictly to the caudal half-sclerotome in avians (Krull et al., 1997). The non-overlapping distribution of these molecules suggested the hypothesis that interactions between EphB3 and ephrinB1 normally served to restrict neural crest entry into the caudal half-sclerotome. To test this hypothesis, exogeneous monomeric or dimeric forms of ephrin-B1 were added to neural crest migrating in trunk explants, to block signaling between endogeneous EphB3 and ephrin-B1. In ephrin-B1treated explants, neural crest migrated in both rostral and caudal portions of the somitic sclerotome. These results were similar to results from the functional analysis of PNA binding molecules in segmenting trunk neural crest migration, and suggested that EphB-ephrin interactions were also involved in this process. Together, these results suggest redundancy in the function of these guidance cues. Another interesting possibility is that a collection of inhibitory factors in the caudal half-sclerotome may be required to make this embryonic domain truly prohibitive to neural crest entry. In rodents, results of in vitro analyses suggested similar roles for EphB-ephrin-B2 interactions in trunk neural crest migration (Wang and Anderson, 1997). However, mice that lack ephrin-B2 display defects in angiogenesis, and have apparently normal patterns of neural crest migration (Wang et al., 1998; Adams et al., 1999), again

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C.E. Krull / Mechanisms of Development 105 (2001) 37±45

supporting the notion of functional redundancy between guidance cues in the somitic mesoderm. Are members of the EphA subclass also expressed in avian neural crest or in the somitic mesoderm, suggesting roles in controlling certain aspects of neural crest migration? The EphA7 RTK is expressed in the caudal half-sclerotome (Araujo and Nieto, 1997) whereas ephrinA ligands are expressed in the dorsal root ganglia (Eberhart et al., 2000). Analyses are in progress to determine whether neural crest express ephrinA ligands during early stages of their migration and the functional impact of these factors on neural crest patterning. Neural crest cells migrate in segmental streams into the branchial arches at the level of the developing hindbrain in avians, mouse, and Xenopus (Kontges and Lumsden, 1996; Sadaghiani and Thiebaud, 1987). Eph RTKs and ephrins have been directly implicated in restricting the migration of Xenopus neural crest to their particular stream (Smith et al., 1997). Speci®cally, intermingling between neural crest that migrate to the second and third branchial arch is normally prohibited by Eph-ephrin interactions. Eph family members, therefore, appear to be involved in the organization of neural crest migration at different axial levels and in different species. 5. How do these factors control neural crest migration? Insights into how these factors could direct neural crest migration come from in vitro analyses, coupled with timelapse imaging. Neural crest extend processes on the substrate-bound repulsive factor, ephrin-B1. However, soon thereafter, the leading process collapses, followed by its complete retraction, and the subsequent movement of the cell into territory in which ephrin-B1 is absent (Krull et al., 1997). Although these studies did not address potential rearrangements in the actin cytoskeleton in response to ephrinB1, they suggest that actin-bases processes are indeed involved in the patterning of neural crest migration. Previous studies have shown that the extension of actinrich processes are essential for path®nding, but not advance, by neuronal growth cones (Marsh and Letourneau, 1984; Bentley and Toroion-Raymond, 1986). Similar to neuronal growth cones, migrating neural crest likely navigate by coupling plasma membrane-associated guidance cues to rearrangements in the actin cytoskeleton. Rho GTPases are excellent candidates to mediate the effects of the guidance cues on neural crest migration (Nobes and Hall, 1999; Wahl et al., 2000). Upon activation, Rho GTPases promote the formation of ®lapodia, actin stress ®bers and actin polymerization, suggesting that these factors could play key roles in actin-driven processes such as cell migration, adhesion, and guidance. Future analyses of interactions between guidance factors, Rho GTPases, and the actin cyotoskeleton in neural crest migration should be very productive.

Neural crest cells make extensive contacts with neighboring neural crest cells throughout their migration, at the level of the trunk and hindbrain (see below). These contacts between adjacent cells could be critical for ef®cient, coordinated migration. The aforementioned guidance factors could act by promoting or disrupting adhesive contacts between neural crest, and in this way, in¯uence the organization of migratory behavior. The importance of a correct balance of adhesion in neural crest migration is suggested by results of cadherin-7 overexpression studies (Nakagawa and Takeichi, 1995). Neural crest normally upregulate cadherin-7 during their migration but overexpression of cadherin-7 disrupts neural crest emigration. These results suggest that too much cadherin-7, and thus, too much cell adhesion, negatively in¯uences neural crest movement. Future experiments designed to disrupt adhesive contacts between migratory neural crest and determine the subsequent effects on cell motility and patterning in vivo will be needed. 6. Time-lapse analyses of neural crest migratory behavior Numerous technical developments, including embryo explant systems, high resolution confocal microscopy, cell labeling methods, and in ovo imaging methods, have permitted investigators to follow the behavior of migrating neural crest cells in their native environment over time (Krull et al., 1995; Krull and Kulesa, 1998; Kulesa and Fraser, 2000). These types of analyses have provided insights into the cellular interactions that govern neural crest migration normally during development and the functions of various guidance factors. What have we learned? Trunk neural crest cells migrate normally through the rostral half-sclerotome in streams that contain several interconnecting cells (Krull et al., 1995). As neural crest migrate in these streams, they extend and retract their processes in a dynamic manner to contact their neighbors, migrating in a chain-like manner. Analyses of the trajectories taken by individual trunk neural crest revealed an overall linear organization to their migration that often contains both forward, backward, and lateral movements (Fig. 2). Rates of migration were measured and averaged approximately 12 mm/h, in the range of observations obtained previously using in vitro approaches (Rovasio et al., 1983). These data strongly suggest that cell-cell interactions between migrating neural crest may mediate cell communication that is essential for the organization of migration, in addition to interactions by neural crest with the surrounding somitic mesoderm. Time-lapse analyses revealed striking variations in the behavior of neural crest migrating in the rostral versus caudal half-somite in explants where the normal pattern of migration was disturbed (Krull et al., 1995, 1997; Fig. 2). Rostrally-migrating cells traveled in a linear manner, typical

C.E. Krull / Mechanisms of Development 105 (2001) 37±45

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of these factors is currently unknown but good candidates for maintaining adhesive contacts between neural crest that are required for migration include cadherin-7 and thrombospondin (Nakagawa and Takeichi, 1995; Tucker et al., 1999). At the level of the avian hindbrain, neural crest cells also migrate in a stream-like manner, as observed in the trunk, suggesting many opportunities for cell-cell communication (Kulesa and Fraser, 2000). Interestingly, some cells within these generally-segregated streams intermix with cells from other streams. These data add additional support to the hypothesis that dynamic interactions between neural crest may be critical for organizing their migration. 7. Future directions

Fig. 2. Trajectories taken by neural crest migrating in the rostral and caudal half-sclerotomes in ephrin-B1-treated explants. Schematic illustration showing trajectories of neural crest migrating in control (Fc-treated) and ephrin-B1-treated explants in a single somite. Images were captured every 4 min of DiI-labeled neural crest migrating in trunk explants, for 18 h. A black dot represents the start and an arrowhead marks the end of an individual cell's migration trajectory. Neural crest that migrate in the rostral (R) half somite exhibit generally linear trajectories. In the presence of ephrinB1, neural crest migrate normally in the rostral half-somite but aberrantly in the caudal (C) half-somite. In one example, a neural crest cell migrates backward from the somite into the neural tube. NT, neural tube.

of the normal migratory behavior of neural crest. In contrast, caudally-migrating neural crest cells moved in a highly disordered and tangled manner, suggesting that these cells experienced dif®culty negotiating this novel somitic territory. These results suggested that perhaps caudally-migrating cells were missing an important positive guidance cue normally present in rostral half-somites or that another disorienting cue was present in the caudal half-somite that confused caudally-migrating cells. These data and those described above suggest that several inhibitory factors present in the caudal half-somite could act collectively to segment trunk neural crest migration. Furthermore, I propose that permissive factors strictly localized to the rostral half-somite and cell-cell interactions between neural crest are responsible together for the coordination of cell migration in the rostral portion of the somite. The identity

Although studies in recent years have provided insights into some of the inhibitory factors that control neural crest migration, there are many remaining questions. What are the permissive, migration-promoting factors that in¯uence trunk neural crest cells as they navigate in the rostral halfsomite? Does each guidance factor exert distinct actions or do they act combinatorially to in¯uence neural crest migration? What are the components of the intracellular machinery that are activated when neural crest cells encounter repulsive or permissive substrates? Are there subpopulations of neural crest progenitors that respond to the migratory environment in distinct ways? What are the stop signals that halt neural crest migration? Clearly, there is much work to accomplish to fully understand the molecules and mechanisms that control the patterning of neural crest migration. The application of new technologies, including time-lapse imaging, in ovo electroporation, and targeted genetic manipulations, to this problem should help us understand how cells ®nd their way in the developing embryo. Acknowledgements Research in C.E.K.'s laboratory is supported by USPHS MH59894, a Basil O'Conner Starter Scholar Award from the March of Dimes, and the Muscular Dystrophy Association. References Adams, R.H., Wilkinson, G.A., Weiss, C., Deilla, F., Gale, N.W., Deutsch, U., Risau, W., Klein, R., 1999. Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/ venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev. 13, 295±306. Akitaya, T., Bronner-Fraser, M., 1992. Expression of cell adhesion molecules during initiation and cessation of neural crest cell migration. Dev. Dyn. 194, 12±20. Araujo, M., Nieto, M.A., 1997. The expression of chick EphA7 during segmentation of the central and peripheral nervous system. Mech. Dev. 68, 173±177. Bentley, D., Toroion-Raymond, A., 1986. Disoriented path®nding by

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