Developmental Biology 344 (2010) 543–554

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Developmental Biology

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Review

Cranial neural crest migration: New rules for an old road

Paul M. Kulesa a,b,⁎, Caleb M. Bailey a, Jennifer C. Kasemeier-Kulesa a, Rebecca McLennan a

a Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110, USAb Department of Anatomy and Cell Biology, University of Kansas School of Medicine, USA

⁎ Corresponding author. Stowers Institute for MedicKansas City, MO 64110, USA.

E-mail address: pmk@stowers.org (P.M. Kulesa).

0012-1606/$ – see front matter © 2010 Elsevier Inc. Adoi:10.1016/j.ydbio.2010.04.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received for publication 23 February 2010Revised 6 April 2010Accepted 9 April 2010Available online 23 April 2010

Keywords:Neural crestCranialMigrationCell communication

The neural crest serve as an excellent model to better understand mechanisms of embryonic cell migration.Cell tracing studies have shown that cranial neural crest cells (CNCCs) emerge from the dorsal neural tube ina rostrocaudal manner and are spatially distributed along stereotypical, long distance migratory routes toprecise targets in the head and branchial arches. Although the CNCC migratory pattern is a beautifullychoreographed and programmed invasion, the underlying orchestration of molecular events is not wellknown. For example, it is still unclear how single CNCCs react to signals that direct their choice of directionand how groups of CNCCs coordinate their interactions to arrive at a target in an ordered manner. In thisreview, we discuss recent cellular and molecular discoveries of the CNCC migratory pattern. We focus onevents from the time when CNCCs encounter the tissue adjacent to the neural tube and their travel throughdifferent microenvironments and into the branchial arches. We describe the patterning of discrete cellmigratory streams that emerge from the hindbrain, rhombomere (r) segments r1–r7, and the signals thatcoordinate directed migration. We propose a model that attempts to unify many complex events thatestablish the CNCC migratory pattern, and based on this model we integrate information between cranialand trunk neural crest development.

al Research, 1000 E. 50th St.,

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© 2010 Elsevier Inc. All rights reserved.

Introduction

The vertebrate embryo regulates the programmed invasion of theneural crest, a cell population that makes important contributions tostructures that include the head, heart, and peripheral nervoussystem. In the head, cranial neural crest cells (CNCCs) emerge fromthe hindbrain (rhombomere (r) segments r1–r7) and are spatiallydistributed along discrete migratory pathways (Fig. 1). During theirdorsolateral migration, CNCCs may interact with and receive signalsfrom multiple sources. CNCCs may touch the ectoderm and crawlthrough microenvironments rich in cranial mesenchyme and extra-cellular matrix (ECM). Signals arising fromwithin the hindbrain, fromother CNCCs, or from the local microenvironments traversed bymigratory CNCCs together establish neural crest cell-free zones(Fig. 1). Failure of CNCC migration leads to significant morphologicalabnormalities of the face, neck and cardiovascular system (Hutsonand Kirby, 2007; Tobin et al., 2008), making this an important modelsystem to better understand birth defects.

The long history of NCC tracing and cell behavior analyses by staticimaging and time-lapse cinematography (Davis and Trinkaus, 1981;Newgreen et al., 1982), respectively, have provided invaluable data on

the CNCC migratory pattern (summarized in Le Douarin andKalcheim, 1999). From early in vitro studies, neural crest biologistsrealized the complexity of cell migratory behaviors and struggledwithdetermining whether the CNCC migratory streams were composed ofindividual cell movements or collective migration in sheets, and towhat extent cells responded to growth of the embryo (Erickson, 1985;Erickson et al., 1980; Le Douarin, 1982; Noden, 1975; Thiery et al.,1982; Tosney, 1982). Detailed investigations of the local ECM in theCNCC microenvironment transitioned studies from mapping cellpathways to providing a basis for how cell microenvironmentalinteractions influenced neural crest cell direction (Bronner-Fraser,1993; Newgreen, 1989). From these data and influence from mentorsin the cell migration field, such as J.P. Trinkaus and MichaelAbercrombie, who also elegantly described cell movements inFundulus (Trinkaus, 1973) and fibroblasts (Abercrombie and Heays-man, 1954), neural crest biologists derived several models to explaindirected cell migration. However, concern that the inability of anysingle model to explain the CNCC migratory problem suggested thatthe mechanisms in effect were more complex.

In this review, we report recent insights into the molecularsignals that direct CNCC behaviors and more detailed cell dynamicsanalyses that produce the CNCC migratory pattern. First, we willdefine features of the migratory CNC and cell-to-cell contactdynamics. We will describe participating structures of the CNCC-rich microenvironment and the heterogeneity of cell morphologyand proliferative activity that depend on cell position within amigratory stream. Next, we will characterize the selection and

Fig. 1. The cranial neural crest cell migratory pattern; cellular features and signaling pathways. (A) A schematic representation showing recently discovered key guidance cuesinvolved in CNCC migration. (B) The cranial NCCs migrate in 3 distinct streams as seen by membrane (Gap43-GFP) and nuclear (H2B-mCherry) labeling (introduced intopremigratory NCCs by electroporation delivery) reduced to grayscale for clarity. (C) CNCCs that emerge from mid-r3 and more rostral migrate in a broad wave and display multiplefilopodial protrusions. (D) CNCCs that emerge mid-r3 to mid-r5 are sculpted into a tight stream adjacent to r4 that spreads out at the front (E) Post-otic NCCs that emerge frommid-r5 and more caudal migrate as an initial wave, followed by NCCs that form chain-like arrays. The arrows point to cells that travel in a chain-like array. (F) A schematic representationof the molecules guiding the r4 NCC stream. The r4 NCCs express neuropilins, Plexin A1 and VEGFR2. The overlaying ectoderm expresses VEGF, which is a NCC chemoattractant. R3and r5 secrete semaphorin-3A, which is a NCC inhibitor. A guidance cue that prevents the r4 NCCs migrating ventromedially is as yet unknown (?). r, rhombomere; ba, branchialarch; OV, otic vesicle, NT, neural tube, N, notochord. The scale bars are 20 μm in (B) and 10 μm in (C–E).

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plasticity of the CNCC migratory routes and acquisition of orienta-tion and direction after cells leave the hindbrain. Then, we will detailthe signaling pathways that have emerged to regulate the CNCCmigratory pattern. We will contrast results obtained at multiple

spatial scales, from a single cell to populations, and propose a unifiedmodel for cranial neural crest development. Finally, we will comparecranial and trunk neural crest development in order to highlightcommon mechanisms.

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Cranial neural crest migratory route selection

Three phases of cranial neural crest migration

The segmented nature of the hindbrain, into rhombomeres (r),r1–r7, provides a structural and anatomical framework to describethe emergence and early sculpting of CNCCs. The relationshipbetween patterns of gene expression in the hindbrain and branchialarches have been discussed separately (Santagati and Rijli, 2003;Trainor and Krumlauf, 2001).We focus here on defining the phases ofCNCC migration after cells exit the neural tube. The initiation of CNCmigration begins with inductive cues from non-neural ectoderm andmesoderm that converge at the lateral plate border (Basch andBronner-Fraser, 2006). These inductive signals initiate signal cas-cades that result in a remodeling of cellular architecture and adhesiveproperties characterized by an epithelial-to-mesenchymal transition(EMT) and exit from the neural tube. Alterations to cellular adhesion,mediated principally by changes in the expression of multiplecadherin family members, facilitate delamination from the neuraltube (Taneyhill, 2008).

The CNCC invasion program consists of at least three distinct phasesof migration that form the basis for our discussion of recent cellular andmolecular developments of the CNCC migratory pattern. Acquisition ofdirected migration along the dorsolateral pathway defines the initialphase of CNCC migration (Figs. 2A,B). After CNCCs leave the hindbrain,they come into intimate contact with the surface ectoderm and cranialmesenchyme adjacent to the hindbrain. The second phase of CNCCmigration, homing to the branchial arches, consists of the maintenanceof cells in loosely connected streams along the dorsolateral pathway(Figs. 2C,D). The last phase of CNCCmigration is entry into and invasionof the branchial arches (Figs. 2E,F).

Acquisition of directed migration along the dorsolateral pathway

Filopodial dynamics and cell contact behaviors: evidence for cellcommunication

There is an impressive body of literature detailing the spatio-temporal emergence and pathway selection of an aggressive neuralcrest cell, and more recently its cellular features and cell-to-cellcontact dynamics (Birgbauer et al., 1995; Epperlein et al., 2000; Kulesaand Fraser, 1998; Lumsden et al., 1991; Schilling and Kimmel, 1994;Sechrist et al., 1993; Serbedzija et al., 1992) (Fig. 2). For example, useof fluorescent reporters targeted to the cell membrane and nucleus,combined with in vivo chick time-lapse confocal microscopy haverevealed the unexpected finding that an individual CNCC extends longfilopodia well beyond its center of mass to reach distant cells andectoderm overlying the migratory route (Teddy and Kulesa, 2004)(Fig. 2). CNCC morphologies vary depending on cell position within amigratory stream. Chick CNCCs at the migratory front of a streamdisplay protrusive activity in multiple directions, and trailing cellshave a bipolar shape with equal leading and trailing edge protrusiveactivity aligned along the migratory route (Kasemeier-Kulesa et al.,2008) (Fig. 2). Furthermore, CNCC filopodial extensions appear early,as cells exit the neural tube, as visualized in vivo in the zebrafishembryo (Berndt et al., 2008). Membrane blebbing activity, character-istic of CNCCs in the neural tube, transitions to lamellipodial andfilopodial extensions when cells exit the neural tube (Berndt et al.,2008). Extensive filopodial dynamics have also been described duringNC-derived cell migration in the embryonic mouse gut (Druckenbrodand Epstein, 2005; Young et al., 2004). In the gut, enteric NCCs travelrostrocaudally in strands and display filopodial extensions as cellscrawl over each other and reach into the unpopulated tissue to extendthe strand. Enteric NCCs have a subpopulation of cells that aredisconnected from the migratory front, called advance cells (Druck-enbrod and Epstein, 2007). Advance cells have multiple filopodial

extensions, some of which extend backward and appear to guidebipolar shaped cells within the strands (Druckenbrod and Epstein,2007).

Cell contact between migratory CNCCs has provided evidence forcell communication. Contact between two CNCCsmay be local (withina cell diameter or two) or non-local, up to 70 μm away (Teddy andKulesa, 2004) (Fig. 2). Detailed analysis of filopodial dynamics in thechick CNCC migratory streams has shown that cell-to-cell contactoccurs when a thin process extends to contact the trailing edge of alead cell. Then, either the process remains near that trailing edge andthe cell body moves forward, or the process retracts back to the cellbody and the cell body moves forward to the position of contact(Teddy and Kulesa, 2004) (Fig. 2B). CNC cell-to-cell contact may alsotake the form of more membrane surface area contact and result incell movement away from the contact, termed contact inhibition ofmovement (Abercrombie and Heaysman, 1954), recently visualized inXenopus CNCCs (Carmona-Fontaine et al., 2008). In vivo evidence tothe contrary in the zebrafish trunk has shown that NC cell-to-cellcontact does not result in filopodial retraction, but a continued contactand movement in the direction of the contact (Jesuthasan, 1996).Whether this is due to behavioral differences in the cranial versustrunk NC or species differences is unclear.

Communicating positional information within the cranial microenvironment

The CNC are a heterogeneous population of stem and progenitorcells. CNCCs contribute to a variety of cell types that are neural andnon-neural. Derivatives that include bone, cartilage and othermesenchymal structures, neurons, glia, and melanocytes, arise fromthe same regions of the CNC. Extensive experimental data suggest theCNCC-rich microenvironment contains powerful signals that mayreprogram cells enroute to head and neck targets (reviewed in Craneand Trainor, 2006; Le Douarin et al., 2008; Le Douarin et al., 2004). Theneural crest microenvironment can also reprogram small numbers ofneural crest cells transplanted from a different axial level (Noden andTrainor, 2005; Sandell and Trainor, 2006; Trainor et al., 2002a).Together with single cell tracing data of chick trunk NCCs (Bronner-Fraser and Fraser, 1988), this suggests the subpopulation of CNCCswithin a migratory stream consists of a mix of multipotent cells andmore restricted progenitors whose developmental potential may beinstructed by local microenvironmental signals. Indeed, the ability ofhighly aggressive CNCCs to express multiple cellular phenotypes andengage in functional plasticity, such as changes in trajectory andproliferative activity, defines their multipotency.

Contributions of cranial neural crest cells from rhombomeres 3 and 5

It is well known that CNCC migratory streams are composed ofcells from multiple rhombomeres. Each CNCC migratory stream issculpted by a combination of extrinsic and intrinsic cues. Thecontribution of r3- and r5-CNCCs to neighboring migratory streamshas been previously described in detail (Graham et al., 2004; Kulesaet al., 2004). Briefly, discrete CNCCmigratory streams are separated byNCC-free zones adjacent to both r3 and r5. Prevailing explanations forthe lack of CNCC migration into the regions flanking r3 and r5 includediminished CNCC production or increased apoptosis associated withthese odd-numbered rhombomeres, or the restricted movement ofCNCCs generated by r3 and r5.

Each of these processes may play an important role in the earlystages of neural crest segmentation, but may vary widely amongspecies. For example, analyses in avian embryos demonstratedincreased apoptosis in neural crest populations residing in r3 andr5, resulting in part from BMP-4-induced Msx2 expression in theserespective rhombomeres at the time of neural crest induction andmigration (Graham et al., 1994; Lumsden et al., 1991). Similarly,studies in Xenopus have demonstrated an important role for msx1 in

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regulating cell death in specific areas of the neural folds as a means ofgenerating precise neural crest territorial boundaries (Tribulo et al.,2004). However, this report contradicts previous studies in Xenopusthat demonstrated an absence of rhombomere-specific apoptosis(Hensey and Gautier, 1998). Furthermore, blocking neural crest celldeath in r3 and r5 is not sufficient to disrupt neural crest migratorysegregation (Ellies et al., 2002). Considering that studies in mouse andzebrafish have revealed an absence of rhombomere-specific apoptoticpatterning (reviewed in Kulesa et al., 2004), these findings suggestthat the potential role of apoptosis in segmenting neural crestmigratory streams is widely variable across species and remainspoorly understood. Alternatively, there may be diminished CNCCproduction in r3 or r5; this mechanism appears to be species-specificand is discussed inmore detail in Kulesa et al., 2004. Lastly, CNCCs thatemerge from r3 and r5 may have restrictive lateral movement. Staticand time-lapse analysis of chick r3- and r5-CNCCs shows that CNCCsmay enter these regions lateral to the hindbrain, but CNCCs eitherchange direction to move towards a neighboring migratory stream orcollapse filopodia and stop (Sechrist et al., 1993; Birgbauer et al.,1995; Kulesa and Fraser, 1998).

Homing to the branchial arches

Each cranial neural crest cell migratory stream has its own characteristics

What is striking and worth describing in more detail is that eachCNCCmigratory stream is distinct in terms of the shape and populationdensity of the migratory front and individual cell migratory behaviors(Fig. 1). For example, CNCCs that emerge from the midbrain to mid-r3and mid-r5 through r7 emerge in a wide front (the width of 2–3rhombomeres) of cells thatmove in a directedmanner to the periphery(Figs. 1B,C,E). After an initial wavefront of individual migratory cells,trailing cells from r7 (fewer in number) form chain-like arrays (Fig. 1E)that follow-the-leader to the trailing edge of the wavefront (Rupp andKulesa, 2007). In contrast, the CNCC migratory stream that emergeslateral to r4 (comprised of CNCCs from mid-r3 to mid-r5) consists of amigratory front (the width of one rhombomere) that narrows inrostrocaudal width, back towards the neural tube (Figs. 1B,D). Analysisof chick CNCC trajectories within the migratory front reveals that cellsthat emerge from rostral- or caudal-r4 tend tomigrate near the bordersof the stream and NCC-free zones (Kulesa et al., 2008). In contrast, cellsthat emerge first from mid-r4 spread out into all regions of themigratory front (Kulesa et al., 2008). This suggests that a permissivecorridor lateral to r4 emerges. CNCCs may travel to the branchial archeswithout contact with neural crest cell-free zones. Indeed, whenpremigratory r4 CNCCs are reduced by ablation and replaced by asmaller number of r4 NCCs, fewer numbers of cells emerge and traveldirectly down themiddle of the region lateral to r4, then spread out intothe ba2 (McLennan and Kulesa, 2007).

Cranial neural crest cell proliferative activity along the migratory route

CNCCs proliferate along their migratory route and the activity iskey to the complete invasion of the branchial arches and formation

Fig. 2. The three phases of cranial neural crest cell migration and characteristic cell behavioNCC migration starts around Hamburger and Hamilton (HH) Stage 11 in chick. During Phcranial NCCs do not exhibit directed orientation (orange NCCs), but within a short distanceNCCs communicatewith each other and themicroenvironment, by touch. First, a NCC touchbehavior, where one NCC touches another, and then with or without filopodia retractiondirectionality, cranial NCCsmigrate in a directedmanner and exhibit a bipolar phenotype ((red NCCs). As they invade the target site, the cranial NCCs extendmultiple filopodia in all doverlaying ectoderm and local microenvironment. (D) Cranial NCCs continue their migratiohighly directed manner towards their target site, in this case branchial ach 2. Phase III: EntrHH St 17, they have invaded and colonized their target sites. During Phase III, NCCs transitarget site, branchial arch 2. (F) As the cranial NCCs enter the arch, they spread out from ontube; NC, neural crest.

of head and neck structures. The proliferation of cranial NCCsappears to occur in a regulated manner that involves the FGF/TGF-beta signaling pathways (see Table 1 for a list of known signals andreferences). Specifically, a subpopulation of CNCCs within the frontportion of a typical migratory stream proliferate at a higher ratethan the trailing cells (Kulesa et al, 2008). Higher cell proliferationwithin the migratory front may be triggered by space availability inthe local microenvironment and less physical limitations, in theform of cell crowding, on the lead CNCCs. Lead CNCCs may in turnrespond to molecular signals that stimulate proliferative activity.Alternatively, lead CNCCs may possess an intrinsic mechanism thatregulates cell proliferative activity, independent of microenviron-mental signals or cell crowding. Further investigation of differencesin CNC proliferative activity depending on cell position within astream and cell orientation during division will help to shed lighton mechanisms that regulate proliferation of CNCCs along themigratory route.

Differences in NCC proliferative activity depending on cellposition within a migratory stream have been revealed duringenteric NC migration. In the mouse gut, NCCs within the front of thecell strands actively proliferate more than trailing cells (Simpsonet al., 2007). This spatial bias of cell proliferative activity was not dueto intrinsic mechanisms within NCCs at the front, but rather afunction of proximity to uninvaded tissue (Simpson et al., 2007).Therefore, cell proliferation is an important component of directedNCC target invasion.

Cranial neural crest cells are not restricted to a particularmigratory stream

CNCCs that enter a particular discrete migratory stream are notrestricted to remaining in this stream and to populating the segmentalbranchial arch target directly lateral to the rhombomere level atwhich the cell exited. Cell contact between CNCC migratory streamstends to occur near the branchial arches. CNCCs that reach thebranchial arches, only to find the arch full of other NCCs may changedirection to contact a neighboring migratory stream (Kulesa andFraser, 2000). The cell contact between streams is not a wholesalerearrangement, but rather a small number of cells form a cellularbridge between the streams by entering typically neural crest cell-freezones at the entrances to the branchial arches. This cell-to-cellinteraction between CNCC migratory streams can be exacerbatedwhen either the cell's ability to read local inhibitory signals of themicroenvironment are disrupted or the surface ectoderm adjacent tothe presumptive neural crest cell-free zones is ablated (we discussthis in a later section).

Plasticity of cranial neural crest cell trajectories

CNCC trajectories are plastic and cells may respond to changes intheir local microenvironment. CNCCs may alter their trajectorieswhen physical barriers are introduced in advance of the migratoryfront. Interestingly, chick CNCCs can overcome a physical barrier orablation of neighboring cells and re-target to branchial archdestinations. When a barrier is placed in front of a CNCC migratory

rs. Phase 1: Acquisition of directed migration along the dorsolateral pathway. (A) Cranialase I, the lead cranial NCCs emerge from the dorsal neural tube (beige). Initially thefrom the dorsal neural tube, they acquire directionality (yellow NCCs). (B) The craniales the ectoderm and receives direction information. Second, there is follow-the-leader, follows the lead NCC. Phase II: Homing to the branchial arches. (C) After acquiringgreen NCCs). Along themigratory route, cranial NCCs stop, retract filopodia and divideirections (light blue NCCs). Themigrating cranial NCCs have intimate contact with then toward their target sites throughHH St14 in chick. During Phase II, NCCsmigrate in ay into and invasion of the branchial arches. (E) Cranial NCCs continue to migrate and bytion from being loosely connected with one another to spreading out to fill the entiree another and display multiple filopodia in all directions (dark blue NCCs). NT, neural

Table 1

Phase of migration Cue Proposed role Reference

Delamination Slug Involved in the epithelial-to-mesenchymal transition (Nieto et al., 1994)Delamination RhoB Necessary for correct delamination of NCCs (Liu and Jessell, 1998)Delamination Cadherins Cell–cell adhesion molecules that control the timing of

emigration, delamination and migration(Borchers et al., 2001; Coles et al., 2007; Kashef et al.,2009; McCusker et al., 2009; Taneyhill, 2008)

Migration Hox genes Maintain segmental identity of cranial NCCs Reviewed by (Trainor and Krumlauf, 2000)Migration Integrins Mediate NCC motility on fibronectin in avian, Xenopus and mouse (Alfandari et al., 2003; Strachan and Condic, 2003;

Strachan and Condic, 2008)Migration Chemokines Regulate cell migration and patterning in zebrafish (Olesnicky Killian et al., 2009)Migration EphA4, EphB1 and ephrin-B2 Prevent intermingling of third and second arch Xenopus NCCs (Smith et al., 1997)Migration Multiple Ephs and ephrins Restricts avian and murine NCCs into streams by inhibiting

migration into NCC-free zones(Adams et al., 2001; Davy et al., 2004;Mellott and Burke, 2008)

Migration Neuropilin-1 andSemaphorin-3A, -3F

Avian and murine cranial NCCs express neuropilin-1 and arerepelled by semaphorin-3A

(Eickholt et al., 1999; Gammill et al., 2007;Osborne et al., 2005; Schwarz et al., 2008)

Migration Neuropilin-1a,-1b, -2a, -2band Semaphorin-3Fa, -3Ga

Restricts zebrafish NCCs into streams by inhibiting migrationinto NCC-free zones

(Yu and Moens, 2005)

Migration Wnt11r Promotes Xenopus cranial NCC migration (Matthews et al., 2008)Migration Myosin-X Promotes migration and segregation of Xenopus cranial NCCs (Hwang et al., 2009; Nie et al., 2009)Induction, migrationand differentiation

BMPs Multiple roles Reviewed by (Nie et al., 2006)

Migration Retinoic Acid Mediates the segmental migration of cranial NCCs (Dupe and Pellerin, 2009; Menegola et al., 2004);(Lee et al., 1995); (Pratt et al., 1987)

Migration RhoA Influences migration rate and filopodia dynamics (Rupp and Kulesa, 2007)Migration anddifferentiation

Laminin alpha5 Required for proper migration and timely differentiation of asubset of murine cranial NCCs

(Coles et al., 2006)

Migration anddifferentiation

Disc1 Represses transcription of foxd3 and sox10 (Drerup et al., 2009)

Migration ErbB4 Maintains the r3-adjacent NCC-free zone (Golding et al., 2004; Golding et al., 2000)Migration Chokh/rx3 Mutant chokh/rx3 zebrafish lack eyes and have disorganized

NCC dorsal anterior migration(Langenberg et al., 2008)

Target invasion Neuropilin-1 and VEGF VEGF attracts neuropilin-1 expressing NCCs into branchial arch 2 (McLennan and Kulesa, 2007; McLennan et al., 2010)Trigeminal ganglionformation

Neuropilin-2 andSemaphorin-3F

Mice with null mutations in either molecule display improperlyformed ganglia

(Gammill et al., 2007)

Trigeminal ganglionformation

Robo2 and Slit1 Disruption of either molecule results in disorganized ganglia (Shiau et al., 2008)

Palatogenesis PDGF and MicroRNAMirn140

PDGF is required for NCCs to contribute to cranial mesenchymeand attracts zebrafish NC-derived palatal precursors

(Eberhart et al., 2008; Tallquist and Soriano, 2003)

Target invasion FGFR1 Provides a permissive environment for NCC migration intobranchial arch 2

(Trokovic et al., 2005)

Target invasion Endothelin-1 and endothelinA receptor

Required for proper migration into or within the arches (Abe et al., 2007; Clouthier et al., 2003;Pla and Larue, 2003); (Clouthier et al., 2000)

Survival andproliferation

Msx1 and Msx2 Mouse mutants display impaired cranial NCC patterning,survival and proliferation

(Han et al., 2003; Ishii et al., 2005)

Survival and/ordifferentiation

B-catenin Conditional inactivation of B-catenin results in increasedapoptosis in mouse cranial NCCs and craniofacial malformations

(Brault et al., 2001)

Survival Sonic Hedgehog Reduction in sonic hedgehog signaling leads to increased neuraltube and NCC death

(Ahlgren and Bronner-Fraser, 1999;Jeong et al., 2004)

Survival anddifferentiation

Dlx2 Involved in survival of zebrafish cranial NCCs and differentiationof sensory ganglia

(Sperber et al., 2008)

Survival, proliferationand differentiation

Pinch1 Required for multiple steps for the development of murinecranial NCC-derived structures

(Liang et al., 2007)

Proliferation TGF-beta Mediates FGF signaling which is required for cranial NCCproliferation

(Iwata et al., 1999; Oka et al., 2008;Sasaki et al., 2006)

Proliferation anddifferentiation

FGF2 Depending on the concentration of FGF2, either proliferation isenhanced or cartilage differentiation is induced

(Sarkar et al., 2001)

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stream, lead cells encounter the barrier and some lead and trailingcells are able to re-orient around the barrier and move towards thebranchial arch target (Kulesa et al., 2005). CNCCs also respond toablation of premigratory neighboring CNCCs and alter theirtrajectories to fill into less populated targets (Saldivar et al., 1997).When r5–r6 premigratory chick NCCs are ablated, neighboring r7NCCs reroute their trajectories and fill in the second branchial arch, atarget they do not normally invade (Kulesa et al., 2000). CNCCs alsofill in for missing ablated neighbors in response to the addition offactor-soaked beads placed in the microenvironment (Creuzet et al.,2004). When mesencephalic and r1–r2 premigratory NCCs areablated in chick, r3 NCCs are stimulated by exogenous Fgf8 toproliferate and migrate in a rostrolateral direction to fill in formissing neighboring cells in the first branchial arch (Creuzet et al.,2004). Thus, CNCC cell trajectories are not hardwired.

Cranial and trunk neural crest cells share migratory behaviorcharacteristics

Features of CNCC migratory behaviors are mimicked in thetrunk. In the trunk, spatio-temporal signals in the somites along thelength of the neural tube guide NCCs within multiple migratorypathways that travel adjacent to, through, and dorsal to the somitesto organize structures such as the peripheral nervous system (seethe review by Gammill and Roffers-Agarwal in this volume). TrunkNCCs may migrate in chain-like arrays (Kasemeier-Kulesa et al.,2005; Krull et al., 1997) or reverse direction back towards theneural tube (Kasemeier-Kulesa et al., 2005). Interestingly, trunkNCC trajectories are also unpredictable. Whether trunk NCCs alsospread out along the dorsal neural tube before selecting a ventralmigratory pathway or are restricted to a particular axial level is not

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clear. Cell labeling studies of premigratory trunk NCCs and endpointanalysis at the sympathetic ganglia show discrete ganglia arecomposed of NCCs that emerged from multiple axial levels (Yip,1986). One explanation for this has been provided by data that showtrunk NCCs that travel from the dorsal neural tube within a discretemigratory stream reach the dorsal aorta and spread out in theanteroposterior direction before ending up in a particular sympa-thetic ganglia (Kasemeier-Kulesa et al., 2005). Additionally,exchange of trunk NCC cells between neighboring discrete migra-tory streams may also take place within the somites along themigratory route (Kasemeier-Kulesa et al., 2005).

Thus, advances in cell labeling and improved in vivo imagingtechniques have yielded two important facts: first, many factorscontribute to the segmentation and the shaping of the caudal hindbrainstreams; and second, significant species-specific differences exist inhow the neural crest streams are shaped. The culmination of thesefeatures builds the picture of loosely connected, individually migratingCNCCs that display local and non-local cell contacts and complex cellcontact behaviors that influence a cell's choice of direction.

Models of directed cell migration applied to the neural crest

There are several model mechanisms that have been proposed toexplain the CNCC migratory pattern (reviewed in Weston, 1982). Wefocus on the set of model mechanisms that seek to explain how CNCCsare directed from the hindbrain to the branchial arches along discretemigratory pathways. These model mechanisms include cell chemo-taxis, cell nudging, population pressure and contact inhibition ofmovement, and polarized cell movement. In this section, we brieflydescribe details of the models and fill in emerging molecular data thatsupport aspects of each model. Although it is conceivable that oneunique model mechanism could account for the complex dynamics ofthe CNCC migratory pattern, we consider a scenario in which severalmodel mechanisms could function in a coordinated manner topropagate the typical CNCC stream and produce the global migratorypattern.

First, contact inhibition of movement suggests that cells will moveaway from densely populated regions into less populated regions bydirect cell-to-cell contact. Contact inhibition of movement was firstdescribed in fibroblasts in culture in the 1950s (Abercrombie andHeaysman, 1953; Abercrombie and Heaysman, 1954) and has recentlybeen discovered as an in vitro and in vivo behavior in Xenopus CNCCs(Carmona-Fontaine et al., 2008). Time-lapse imaging has shown thatXenopus CNCCs move away from each other upon contact. Bycombining a fluorescent reporter for RhoA activity, the authors wereable to show an increase in RhoA during CNCC neighbor contact(Carmona-Fontaine et al., 2008). The authors show that CNCC contactleads to contact inhibition of movement that in turn activates theplanar cell polarity signaling pathway (Carmona-Fontaine et al.,2008). The authors suggest that propagation of a CNCC migratorystream is the result of contact inhibition of movement.

It is evident that short-term interactions between NCCs may resultin contact inhibition of movement, but whether this is the drivingmechanism is unclear. In order for contact inhibition of movement topropagate a CNCC stream along a particular migratory pathway, theremust be an additional mechanism(s), otherwise emerging CNCCswould spread out concentrically from a high-to-low density. Localinhibitory signals, that exist adjacent to r3 and r5 (Farlie et al., 1999;Trainor et al., 2002b) could restrict CNCC movements to permissivecorridors adjacent to r1–r2, r4, and r6–r7. When population pressurefrom newly emerging cells is combined with this restriction of APmovement, contact inhibition of movement would act to propagatethe CNCCs towards the branchial arches. Although this modelmechanism is conceivable, there is evidence that chick, zebrafish,and mouse NCCs use cell contact to promote movement, shown bothin vivo (Jesuthasan, 1996; Schilling and Kimmel, 1994; Teddy and

Kulesa, 2004) and in vitro (Davis and Trinkaus, 1981; Druckenbrodand Epstein, 2005; Young et al., 2004), and convey guidanceinformation by touch (Figs. 2B,C). Additionally, cell-to-cell contact isnecessary to propagate the stream forward in a contact inhibition ofmovement model. However, imaging of migratory NCCs in the mousegut (Druckenbrod and Epstein, 2005; Young et al., 2004) and in vivo inaxolotl (Keller and Spieth, 1984) have shown that directed migrationcan occur in absence of contact between NCCs. So, alternatively, CNCCcontact inhibition of movement behaviors in the Xenopus embryo ascompared to other animal model systems could be due to speciesdifferences.

CNCC contact with neighboring cells can lead to forwardmovement by a process called cell nudging, first described inFundulus (Tickle and Trinkaus, 1976). In this model mechanism,CNCCs would exert a mechanical influence on each other (tension)that would cause membrane blebbing on the opposite side of thecell contact. Membrane blebbing would lead to lamellipodiaprotrusive activity and directed migration would follow. It isplausible that cell-to-cell contact would cause either intracellularsignaling or even exchange of information through membranechannels tomove a CNCC forward in themanner suggested by Tickleand Trinkaus (1976). Interestingly, Jesuthasan (1996) found thatNCCs can propel beads in culture and hypothesized that the sameforce could be exerted on other NCCs by the observation that theycan adhere to one another and their protrusions thicken aftercontact. Further investigation with fluorescent activity reporterswill be necessary to decipher whether this model mechanism playsa role in CNCC migration.

The thirdmodel suggests that NCCs have an intrinsic cell polaritythat drives their directed migration towards the branchial arches. Apolarized cell type has been linked to directed cell migration sincethe 1960s (Trelstad et al., 1967). In vitro culture assays of the eye(Bard and Hay, 1975) showed that NCCs move in a directed mannerwith a polarized morphology. This model mechanism is dependenton the ability of CNCCs to emerge from the neural tube and adopt apolarized morphology that is sustained over long distances. Time-lapse imaging of zebrafish CNCCs has recently shown that cellsemerge and acquire direction shortly after delaminating from theneural tube (Berndt et al., 2008). However, other time-lapseimaging results have shown that CNCCs can reverse direction,adopt a diagonal trajectory from the neural tube to join aneighboring stream, or change direction after encountering a localinhibitory region (Kasemeier-Kulesa et al., 2005; Kulesa and Fraser,1998; Kulesa and Fraser, 2000), that would be contradictory tosustained polarized cell movement. Also, it is not clear whether cellpolarity results in directed migration or whether polarity is aconsequence of directed migration. For example polarized mor-phologymay be a feature adopted by cells within a particular regionof the migratory stream and influenced by local microenviron-mental or cell-to-cell communication. This has been observed as afeature in trailing cells within chick CNCC streams that display abipolar cell morphology whereas lead cells have multiple filopodiaextending in many directions (Teddy and Kulesa, 2004).

The last model mechanism proposed for CNCC migrationinvolves receptor–ligand mediated guidance cues and the chemo-tactic response of cells to microenvironmental signals. Recentexciting data have discovered evidence for NCC chemoattractantsand inhibitory signals in the head, gut, and trunk. CNCCs mayrespond to non-permissive cues present in the NCC-free zones(Farlie et al., 1999; Kulesa and Fraser, 1998). When tissue overlyingthe CNCC migratory pathway is removed, cells may pass throughthese NCC-free zones and form cellular bridges between neighbor-ing migratory streams (Golding et al., 2000). Alternatively,attractive or permissive cues could directly guide the NCCs totheir final destinations, the branchial arches (McLennan et al.,2010). These ideas were supported by studies in which regions of

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the neural tube was rotated (Sechrist et al., 1994). When a portionof the neural tube was rotated 180°, so that rhombomeres 3 and 4were transposed, cranial neural crest cells followed their normalmigratory streams (Sechrist et al., 1994). Thus, the CNCC migratorystreams may be sculpted by a combination of attractive andinhibitory cues.

The real challenge is to better understand how individual CNCCsrespond to guidance cues and each other, and to develop strategiesthat include the interrogation of cell behaviors across multiplescales. The presence of both multipotent and more restrictedprogenitors within a migratory stream and differences in cellmorphologies and filopodial dynamics make it clear that we shouldassess whether every cell responds in a similar manner to the samesignal. The first step is to identify the input and output of anindividual cell and how spatial position is communicated betweenneighbors. Toward this goal, new technologies such as photoactiva-tion cell labeling, fluorescent activity reporters, and targetedelectroporation have enabled neural crest biologists to specificallyvisualize, and perturb CNCCs within different microenvironmentsalong their migratory routes. We are now in a position to addresskey questions of the CNCC migratory pattern. First, what signals areprovided by the CNC microenvironment to drive directed migra-tion? Second, how are local signals communicated between CNCCsto produce a coordinated migratory stream?

Mechanisms of cranial neural crest cell migration

Signals that sculpt the early CNCC migratory streams

Many guidance molecules have been shown to play a role in cranialNCCmigration (see Table 1 for a list of known cues). To review themainmolecular players in cranial NCC migration, we will evaluate theseparate phases of migration. Several guidance cues found within thelocal microenvironment have been reported to sculpt and maintain theearly aspects of the CNCC streams. These include ErbB4 (Golding et al.,2004; Golding et al., 2000), Eph/ephrin interactions (Adams et al., 2001;Davy et al., 2004; Mellott and Burke, 2008; Smith et al., 1997),chemokines (Olesnicky Killian et al., 2009) and neuropilin/semaphorininteractions (Gammill et al., 2007; Schwarz et al., 2008).

ErbB4, a receptor for neuregulin that is typically expressed withinr3 and r5, is involved in maintaining the NCC-free zone adjacent torhombomere 3 in both mouse and chick (Golding et al., 2004, 2000).Mouse embryos lacking ErbB4, as well as chick embryos electro-porated with a dominant negative form of ErbB4, display misroutedneural crest cells into dorsolateral r3 mesenchyme (Golding et al.,2004, 2000).

Ephs and ephrins have been shown to be involved in themaintenance of the cranial NCC streams in the chick as well asXenopus and mouse (Adams et al., 2001; Davy et al., 2004; Mellottand Burke, 2008; Smith et al., 1997), however their specific functionin different species is poorly conserved. In Xenopus, cranial NCCsoriginating from different rhombomeres express different Ephs and/or ephrins and it is this expression that plays a role in preventingintermingling between the cranial NCC streams (Smith et al., 1997).In the chick, neural crest cells express a variety of Eph receptors andmembrane-bound ephrin ligands, which interact in a repulsivemanner with cognate Eph/ephrins expressed in the mesenchyme todemarcate stream boundaries (Mellott and Burke, 2008). In themouse, mutations in ephrin-B1 and ephrin-B2 disrupt neural crestguidance, resulting in NCCs breaching the borders that separate thestreams (Adams et al., 2001; Davy et al., 2004). Such inconsistenciesrelated to specific protein function combined with significantspecies-specific differences in function bring into question the extentto which the role of these guidance molecules is conserved in cranialNCC migration.

Signals that influence neural crest cell homing to the branchial arches

The most recently discovered key guidance receptors for directedCNCC migration are chemokines and neuropilins and their ligands.Chemokines, a family of small secreted cytokines, are found to playmany roles during embryonic development to shepherd cells overlong distances (Raz and Mahabaleshwar, 2009). Recently, Artingerand colleagues have shown an important role for CXCR4/SDF-1signaling in the condensation and patterning of CNCCs in thepharyngeal arches (Olesnicky Killian et al., 2009). Neuropilin-1 andneuropilin-2 are expressed by cranial NCCs in both mouse and chick(Chilton and Guthrie, 2003; Eickholt et al., 1999; Gammill andBronner-Fraser, 2002; Gammill et al., 2007; McLennan and Kulesa,2007; Osborne et al., 2005; Schwarz et al., 2008). Cranial NCCs alsoexpress Plexin-A1 transcripts, a co-receptor for neuropilin, whilesemaphorin-3A and semaphorin-3F transcripts are expressed in odd-numbered rhombomeres (Eickholt et al., 1999; Osborne et al., 2005).Finally, neuropilin-1, neuropilin-2, semaphorin-3A or semaphorin-3Fmutant mice display NC-cellular bridges directly adjacent to rhom-bomere-3, linking the r1/r2 stream to the r4 stream (Gammill et al.,2007; Schwarz et al., 2008). Chick CNCCs avoid substrates containingsemaphorin-3A in vitro (Eickholt et al., 1999), suggesting thatsemaphorin–neuropilin interactions play a role in the initial sculptingCNCC streams.

Signals that influence neural crest cell entry into and invasion of thebranchial arches

After the CNCCs undergo their initial migration in the segmentalstreams, they must invade their target destinations and then properlyassemble into differentiated structures. For example, the NCCs in therhombomere 4 stream must invade branchial arch 2 before formingfacial bone and cartilage as well as cranial ganglia. Recently it has beenshown that this is not a passive event but rather a highly regulatedone that involves multiple guidance cues (see Table 1 for a list ofknown cues).

Neuropilins not only play a role in the initial sculpting of the CNCCstreams, but also in the invasion of target sites. When neuropilin-1expression was knocked down in chick r4 NCCs (using Np-1 siRNABron et al., 2004), their initial migration was normal, but then theyfailed to properly invade branchial arch 2 (McLennan and Kulesa,2007). This phenotype was specific to branchial arch 2 invasion aswhen premigratory Np-1 siRNA transfected CNCCs were transplanteddirectly into the branchial arch 2 microenvironment, they failed tomigrate from the transplant site (McLennan and Kulesa, 2007).Furthermore, Np-1 siRNA CNCCs that had migrated to the entrance ofbranchial arch 2 and were transplanted back into r4, regained theirmigratory abilities (McLennan and Kulesa, in preparation). Recently, ithas been shown that the ectoderm of branchial arch 2 expresses theneuropilin-1 ligand, vascular endothelial growth factor (VEGF) andVEGF has been shown to be a strong attractive cue for CNCCs both invitro and in vivo (McLennan et al., 2010). This is an exciting exampleof chemoattraction-mediated NCC target invasion.

Recently, other CNCC chemoattraction factors have been identi-fied. One example of CNCC attraction involves platelet-derivedgrowth factor (PDGF). Experiments in zebrafish have demonstratedthat PDGF attracts CNCCs to the oral ectoderm, and that thismechanism is modulated by the microRNA Mirn140 (Eberhartet al., 2008). The role of PDGF signaling in mouse CNCCs is lessclear, but it is suggested to be involved not in migration but indifferentiation or extracellular matrix deposition (Tallquist andSoriano, 2003). Finally, the branchial arch ectoderm expressesfibroblast growth factor receptor 1 (FGFR1) and at a lower level,FGFR2 (Trokovic et al., 2005). In hypomorphic FGFR1mousemutants,CNCCs fail to invade branchial arch 2 in a non-cell-autonomousmanner (Trokovic et al., 2005).

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Future perspectives

The CNC model for cell migration includes complex cell behaviorsthat lead to programmed cell invasion in at least three phases ofmigration that include the acquisition of directed migration along thedorsolateral pathway, homing to the branchial arches, and entry intoand invasion of the branchial arches (Figs. 1,2). We have learned thatCNCCs sample their microenvironment and each other with short-and long-range filopodial dynamics. Cell contact between CNCCspromotes local follow-the-leader behavior or contact inhibition ofmovement. Filopodial dynamics and their consequence in CNCCbehaviors offer overwhelming evidence for cell communicationduring migration that imparts direction information.

We also learned that there are differences in CNCCmorphologies andcell behaviors depending on position with respect to the migratoryfront. This appears to be a common feature of NCC migration in otherregions of the vertebrate embryo, including gut and trunk. It is possiblethat lead cells experience microenvironmental signals and transmitinformation to trailing cells through filopodial contact or alteration ofthe microenvironment. In this way, cell behaviors may be distinctdepending on cell position and acquisition, transmission, or receiving ofguidance information. In contrast, phenotypic differences betweenmigratory cells that begin to adopt a particular fate may alter theirbehaviors. Indeed, immature enteric neurons migrate slower anddisplay a long-leading process that is distinct from the multipolar,multiple short filopodial extensions on neighboring, other enteric NCCs(Hao et al., 2009). Given the heterogeneous composition of the CNCCmigratory streams asmultipotent andmore restricted progenitors cells,it is possible that differences in cell migratory behaviors are manifestedalong the migratory routes to the branchial arches. Thus, it will be veryexciting to correlate differences in cell migratory behaviors with bothcell position and cell fate.

The identification of the neuropilin/semaphorin/VEGF signalingpathway and interplay of distinct co-receptors at different phases ofthe CNCC migration program suggest a strategy of chemoattractionand local inhibition (Gammill et al., 2007; McLennan and Kulesa,2007;McLennan et al., 2010). It is conceivable that the CNC respond tochemoattraction and cell contact guidance that in combination withlocal inhibitory cues provide directional information for cells tomaintain discrete streams and reach long distance targets. However, itis important to recognize that expression of VEGF in the surfaceectoderm is not a typical gradient form until near the entrance to the2nd branchial arch (McLennan et al., 2010). Thus, VEGF signal mayplay a role to stimulate non-directed CNCC movement to the 2ndbranchial arch entrance, then play a chemotactic role to direct CNCCsinto and within the branchial arch. A similar model mechanismappears in the trunk. Early emerging trunk NCCs are restricted tomigrate through the rostral somites by an interplay of neuropilin/semaphorin signaling (Gammill et al., 2006) and are thought to beguided over long distances to the dorsal aorta by chemokine signaling(Kasemeier-Kulesa, in preparation; Y. Takahashi, personal communi-cation). When neuropilin/semaphorin signaling is disrupted inmouse, cells continue to reach and are sculpted into discretesympathetic ganglia (Gammill et al., 2006); the latter morphogenesisby local molecular mechanisms in the normal embryo (Kasemeier-Kulesa et al., 2006). This evidence demonstrates that local inhibitorycues and chemoattraction may work in unison to maintain a discreteNCC migratory stream to a precise location.

ForVEGF toplay a CNCCchemoattractant role all along themigratoryroute, an interesting issue is thenhowdoes aVEGF chemotactic gradientresponse becomeestablished fromauniformly expressedVEGF signal inthe ectoderm? One explanation is that lead CNCCs may simply bindVEGF ligand to create a VEGF sink, proximal to the migratory front.CNCCs within the migratory front would sense the distal gradient andcontinue to move forward. Guidance information would have to becommunicated from cells within the migratory front to trailing cells.

Thismodel assumes the lack of resupply of VEGF ligand to themigratoryroute microenvironment proximal to the migratory front, within thetimeframe of CNCC migration. Alternatively, mesenchymal tissue ortrailing CNCCsmay sequester VEGF ligand to create a sink, such that leadcells sense a gradient and continue proper directed migrationdownstream. In support of this model, recent data in zebrafishprimordial germ cell migration has shown that ubiquitously expressedSDF-1a ligand is sequestered by somatic cells expressing CXCR7(Boldajipour et al., 2008), thus revealing a local source-sinkmechanismto direct CXCR4b-expressing cells toward their targets. Whether thistype of mechanism is mimicked during CNCC migration and whatmolecular mechanisms control the distribution of VEGF chemoattrac-tant in vivo are for future investigation.

We presented several potential model mechanisms that mightregulate the CNCC migratory pattern. What is emerging is thepotential for multiple model mechanisms to co-exist, but play acritical role during the different phases of CNCC migration. This issupported by changes in cell morphology, migratory behaviors, andproliferative activity depending on cell position within a CNCCmigratory stream. We suggest a unified model in which chemoat-tractant and repulsive mechanisms integrate with cell contactguidance and contact inhibition of movement to generate the CNCCmigratory pattern. One possibility is that contact inhibition ofmovement may be an active mechanism for CNCCs at the migratoryfront. This would allow lead CNCCs to survey larger subregions ofuninvaded tissue, without hindrance from cell-to-cell contact, andselect a direction of migration from external cues. Once lead CNCCdirection is chosen, this information may be communicated to trailingCNCCs that rely on cell-to-cell contact guidance information fordirection. Future studies to dissect out how information is propagatedbetween CNCCs will be important.

The discovery and further investigation of cell guidance and fatedetermination signals within the CNC microenvironment have thepotential to provide significant value to cancer biology (Fig. 3). Wehave discussed how multipotent NCCs demonstrate the ability tospatially distribute along programmed pathways and undergo cellfate specification. Interestingly, two of the most aggressive cancer celltypes include NC-derived melanoma and neuroblastoma, however itis unclear whether these cancer cell types recapitulate aspects of theirembryonic invasion program during metastatic events (Hendrix et al.,2007; Yang and Weinberg, 2008). During cancer progression, multi-potent melanoma cells secrete and receive molecular cues thatpromote tumor growth and metastasis (Postovit et al., 2006; Uongand Zon, 2010). Whether signals within the embryonic NC microen-vironment may reprogram the metastatic phenotype of theirancestrally-related cancer cell types is a fertile area of research withpotential for differentiation and anti-metastatic therapies (Abbottet al., 2007). In vivo and in vitro studies are underway to understandthe embryonic signals that downregulate and silence the expressionof genes associated with the metastatic phenotype(Hendrix et al.,2007; Kasemeier-Kulesa et al., 2008; Kulesa et al., 2006). Furthermore,information is emerging on signals that regulate CNC stem andprogenitor cells (Dupin et al., 2010; Le Douarin, 2004; Sieber-Blumand Hu, 2008; Sommer, 2006). Together, it will be exciting todetermine the potential for reprogramming the metastatic phenotypeby working at the interface between embryonic and tumorigenicsignaling pathways associated with the NC and NC-derived cancers.

In summary, we have gained new insights into the potential of theCNC microenvironment to sculpt discrete cell migratory streams anddirect cells into specific branchial arches throughout the three phasesof CNCC migration. What has clearly emerged is a picture wherebysingle models proposed over 30 years ago may be brought together incombinations to explain how complex mechanisms of CNCC migra-tion are coordinated in space and time to produce the CNC migratorypattern. Data from in vivo imaging and targeted molecular perturba-tion have begun to add a molecular basis to the complexity of CNCC

Fig. 3. Common features of the multipotent neural crest cell and neural crest-derived cancer cell metastatic program. The neural crest migration program shares many similarities tomelanoma metastasis. (A) A cartoon depicting the neural crest migration program and developmental potential. Neural crest stem cells give rise to a multipotent neural crest cellpopulation that emigrates to a specific, defined site of differentiation and gives rise to diverse cell types including pigment cells. Following neoplastic transformation, melanocytesdisplay many stem-cell-like traits, suggesting that melanoma cells reacquire specific neural crest attributes. The neural crest migratory program parallels many aspects of melanomametastasis, and when aggressive human melanoma cells are transplanted into the chick embryonic neural crest microenvironment, they exhibit behaviors typical of neural crestmigration. (B) GFP-labeled c8161 human melanoma cells transplanted into the chick neural tube at the rhombomere 4 (r4) axial level exit the dorsal neural tube and migrate alongthe r4 neural crest migratory pathwaywhile generally avoiding the NC-free zones. (C) The schematic shows that humanmelanoma cells respect the host embryonic neural crest cell-free zones adjacent to r3 and r5, and a subset of the invading human melanoma cells may be influenced by the host embryonic neural crest microenvironment to express genescharacteristic of a neural crest-like phenotype (data in Kulesa et al., 2006). The neural tube region of r4 and the boundaries between the host r4 NCC migratory stream and neuralcrest cell-free zones are highlighted. (D) In comparison, a schematic representation of in vivo metastatic dissemination highlights the unprogrammed invasion of NC-derived tumorcells in the human microenvironment.

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migratory behaviors. Now that some of the CNCC guidance signals areknown, future migration studies will need to determine how dynamicchanges in the guidance signals are regulated in space and time. Thediscovery of key signaling pathways that underlie CNCC migrationmay help to devise new therapeutic strategies to migration-derivedbirth defects and allow us to better understand events of NC-derivedcancer cell invasion.

Acknowledgments

We thank Laura Gammill for helpful discussions and criticalreading of the manuscript. We also thank Cameron Cooper and JessicaTeddy for images of the cranial neural crest. PMK would like to thankthe National Institutes of Health (1R01HD057922) and the StowersInstitute for Medical Research for funding.

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