The road best traveled: Neural crest migration upon the extracellular matrix

Abstract

Neural crest cells have the extraordinary task of building much of the vertebrate body plan, including the craniofacial cartilage and skeleton, melanocytes, portions of the heart, and the peripheral nervous system. To execute these developmental programs, stationary premigratory neural crest cells first acquire the capacity to migrate through an extensive process known as the epithelial-to-mesenchymal transition. Once motile, neural crest cells must traverse a complex environment consisting of other cells and the protein-rich extracellular matrix in order to get to their final destinations. Herein, we will highlight some of the main molecular machinery that allow neural crest cells to first exit the neuroepithelium and then later successfully navigate this intricate in vivo milieu. Collectively, these extracellular and intracellular factors mediate the appropriate migration of neural crest cells and allow for the proper development of the vertebrate embryo.

1. Introduction

Neural crest cells are a transient population of multipotent cells that are present during early vertebrate embryogenesis and later differentiate into a myriad of cell types throughout the body [1]. Among other tissues, neural crest cells develop into the peripheral and enteric (gut) nervous systems, skin pigment cells, portions of the heart, and bone and cartilage of the head (Figure 1A). Neural crest cells first appear at the border of the neural ectoderm (neural plate) and remain in the dorsal neural folds as pseudo-epithelial cells until they become motile in an extensive process known as the epithelial-to-mesenchymal transition (EMT) [2,3]. They first delaminate, or separate, from other neural tube cells by downregulating epithelial, cadherin-based cell adhesions, and then travel from the dorsal neural tube, sometimes quite long distances, throughout the embryo (Figure 1) [3].

Figure 1. General overview of the neural crest, including target tissues and derivatives as well as EMT.

A. A vertebrate embryo with migratory neural crest cells depicted in orange (arrows indicate direction of migration). Neural crest cells that delaminate from the cranial neural tube region (green) differentiate into bone and cartilage cells of the craniofacial skeleton, sensory neurons and glia of the cranial ganglia, and melanocytes. Neural crest cells from the vagal region of the neural tube (yellow) contribute to cardiac muscle, sympathetic and parasympathetic ganglia, and the enteric (gut) nervous system. Neural crest cells from the trunk region (gray) form neurons and glia of dorsal root ganglia, sympathetic ganglia, and chromaffin cells of the adrenal medulla. Not pictured are neural crest cells from the sacral, or most caudal, region of the neural tube, which gives rise to enteric and sympathetic ganglia. B. A representative image of cranial neural crest cells (orange), which originate in the dorsal neural tube, before (left) and after (right) the start of EMT. Before EMT, the basement membrane (red), composed of laminin, fibronectin, and collagens, is a barrier to neural crest emigration. During EMT, neural crest cells and surrounding tissues secrete several proteases (represented as scissors) of the MMP and ADAM families, which help degrade the basement membrane and process cell surface cadherins. C. A higher magnification of the boxed area in (B). Neural crest cells undergoing EMT secrete proteases into the extracellular space to promote EMT. Epithelial-like premigratory neural crest cells within the dorsal neural tube form junctions with neighboring cells through the expression of type 1 (green lines) and type II (blue lines) cadherins. Migratory neural crest cells become polarized through the planar cell polarity pathway, expressing Rac GTPases at the leading edge (yellow) and Rho GTPases at the trailing edge (red), which regulate the actin cytoskeleton to enable directional movement. Proteases in the extracellular space degrade basement membrane ECM (red), while also cleaving cadherins. Resulting extracellular fragments increase activity of proteases, providing a positive feedback loop to further enhance EMT.

Neural crest cells employ several mechanisms to migrate, which have been reviewed extensively [3–8]. While some information has come from mouse models, the vast majority of studies on neural crest migration come from chick, Xenopus, and zebrafish embryos, thanks to the relative ease of access and manipulation at early stages. Briefly, in Xenopus and zebrafish, it is well established that contact inhibition of locomotion, in which a cell stops moving forward due to contact with another cell, plays a key role during neural crest cell migration through activation of the planar cell polarity pathway, N-cadherin-mediated adhesion, and retraction of cellular protrusions upon contact [7,9,10]. Neural crest cells at the edges of the collective are polarized and possess dynamic, actin-rich protrusions called lamellipodia, but those in the center are nonpolar and lack these protrusions [9–11]. Furthermore, mutual cell attraction maintains close contact between cells during migration through Complement protein C3a in Xenopus and zebrafish [12]. Together, these cell-cell interactions mediate the directional migration observed in these species, with protrusions maintained, in part, by the presence of extracellular guidance factors [11]. Interestingly, recent live imaging studies revealed that chick neural crest cells do not use contact inhibition of locomotion for their migration, instead employing biased search with polarity refinement [13], in which dynamic, polarized protrusions are oriented in the direction of migration, even within the middle of the migratory stream [14]. However, gene expression signatures, in fact, differ between those chick neural crest cells at the front of the migrating stream versus the back. The implication is that leading neural crest cells pave the way for cells that follow, acting as “trailblazers,” though this may not occur at every axial level [15–18].

While some evidence suggests that intrinsic molecular factors control neural crest cell migration by exerting effects on gene expression (e.g. transcription factors Snail2, Sox9, Sox10, Pax3) [reviewed in 14], the extracellular milieu arguably provides much of the information driving neural crest cell EMT and migratory behavior. These include cues received from adjacent cells, soluble growth factors, extracellular matrix (ECM), and changes in tissue mechanics [7]. The ECM, comprised of many different highly conserved proteins, was once believed to merely act as structural support for cells and tissues within an organism. Now, we are beginning to recognize just how complex the role of the ECM truly is for the process of neural crest cell migration. In fact, comparative analyses of gene expression in neural crest and several other migratory cell types across diverse species indicate that the deployment of ECM and proteases (which remodel the ECM) are highly conserved mechanisms that permit long-range migration [19,20]. Indeed, neural crest cells interact with a rich, spatiotemporally dynamic ECM before and during migration. First, they must degrade the ECM-rich basement membrane at the dorsal neural tube to facilitate EMT (Figure 1B, C). Then, neural crest cells produce their own permissive ECM (in addition to ECM made by surrounding tissues) and migrate along permissive routes, largely through integrin-mediated adhesions, while also responding to inhibitory ECM cues that enhance mutual cell attraction and promote directional migration (Figure 2). Finally, neural crest cells modulate their migratory behavior in response to specific ECM signals, ECM-induced molecular gradients, and/or changes in the mechanical environment that are partially attributable to changes in ECM. Therefore, the ECM serves as a necessary substrate and mediator of environmental signals that affect neural crest cell migration.

Figure 2. Molecular interactions between migratory neural crest and ECM.

A. Diagram of collectively migrating neural crest cells (orange). Neural crest cells migrate upon permissive ECM substrates (green), including fibronectin and laminin, while avoiding inhibitory ECM substrates (purple) such as aggrecan and versican. Migratory cells are polarized, with distinct leading (yellow) and trailing (red) edges. Orange arrow indicates direction of migration. B. A higher magnification view of (A) to illustrate molecular and cellular changes occurring during migration. At the leading edge of migrating neural crest cells, integrins form focal adhesions with the ECM under lamellipodia, tethering the extracellular environment to the actin cytoskeleton (1). Cell contacts are maintained by cell adhesion molecules like Cadherin-11 during migration (2), and confinement of migrating streams by versican (3) enhances directional migration. Rac GTPase activation at the leading edge promotes actin polymerization (4), while Rho GTPase activation causes actin depolymerization at the trailing edge. Migrating neural crest cells are guided by diffusible signals (blue circles), including Sdf-1, whose local concentrations may be influenced by the ECM. Integrins also bind to growth factor receptors and other cell surface molecules to integrate signals from the extracellular environment (5).

Accordingly, this review aims to highlight the role of the ECM in neural crest migration, to describe direct and indirect interactions between neural crest cells and the ECM, and to synthesize this information in the context of other mechanisms that guide neural crest development. While several aspects of neural crest cell migration [3,6,21–23], neural crest adhesion molecules [24,25], or ECM in morphogenesis [26–28] have been recently reviewed, our goal is to consolidate the several contributions of ECM to neural crest migration, while highlighting more recent discoveries. Research examining the role of this immediate extracellular milieu or “microenvironment” on neural crest cell biology is quickly expanding due to advances in tissue imaging and the application of new biophysical technologies to the neural crest. Ultimately, studies on ECM during neural crest migration can inform translational topics including cancer metastasis, tissue repair, and nerve regeneration. Therefore, considering the ECM in the larger picture of neural crest development will be important for future studies.

2. Dismantling roadblocks: ECM remodeling by neural crest cells

Neural crest cells arise from the dorsal neural folds or neural tube, depending upon the organism, where they must delaminate from the neuroepithelium and invade the surrounding tissue to begin their journey to various target destinations (Figure 1) [3]. The neural tube and basal surface of the ectoderm are surrounded by a layer of specialized matrix proteins, broadly termed the basement membrane, which is heavily composed of laminin and other ECM proteins including fibronectin, collagen I, and collagen IV [29–31]. The basement membrane of the dorsal neural tube inhibits emigration of neural crest cells and must be degraded for neural crest cells to emerge (Figure 1B) [32]. In vivo, the basement membrane overlying the dorsal neural tube is lost at least 10 hours before EMT in the trunk and just before EMT at cranial levels and is not completely restored until all neural crest cells have left the dorsal neural tube [33–35]. This breakdown in basement membrane is facilitated, in part, by neural crest cell-expressed proteases, which degrade the basement membrane to allow entry of neural crest cells into the mesenchyme (head) or somites (trunk), where the ECM becomes more diverse and serves additional functions.

Two families of proteases, in particular, are key players in neural crest cell delamination and migration: Matrix Metalloproteinases (MMPs) and A Disintegrin and Metalloproteinases (ADAMs). Recently, functions have been described for MMPs and ADAMs in neural crest cell EMT and migration that, when taken together, implicate effective positive feedback loops for protease activation, which in turn degrades ECM, decreases cell adhesion, and facilitates migration (Figure 1B, C, Table 1). A study examining the role of MMP9 in chick hindbrain and trunk revealed that it is secreted by neural crest cells and functions during EMT and migration, correlating with a reduction in laminin [36]. MMP16, a coactivator of MMP2 and MMP9, is also essential for proper migration of neural crest cells in chick, as knockdown inhibited migration, but could be rescued with recombinant MMP16. Overexpression of MMP16 enhanced EMT through increased laminin degradation and loss of N-cadherin [37]. Proteases can also work together to enhance neural crest migration. For example, ADAM10 and ADAM19, which are critical for cranial neural crest cell EMT in the chick midbrain [38], and an MMP (likely MMP2), both cleave the extracellular domain of the premigratory neural crest cell cadherin, Cadherin-6B, at distinct sites [35]. The resulting Cadherin-6B N-terminal fragments are shed into the extracellular space, augmenting MMP2 activity and further promoting delamination through degradation of fibronectin and laminin in the neural tube basement membrane [35]. In a mouse model where ADAM10 was conditionally deleted from neural crest cells, severe craniofacial defects were observed, further indicating its role in proper neural crest development and migration [39]. In Xenopus, ADAM13 protease activity is required for neural crest migration, while nuclear translocation of the C-terminal tail activates migratory gene expression [40,41]. Furthermore, ADAM19 stabilizes ADAM13, in turn activating Wnt signaling to induce neural crest specification. [42]. Together from these studies, a common thread emerges: neural crest cells deploy proteases to both initiate migration by breaking through the neural tube basement membrane and to enhance migration once it has commenced.

Table 1. Summary of proteins expressed by neural crest cells to interact with ECM.

Molecules discussed in the text are summarized with regards to reported function, expression, and respective organism. Neural crest cells employ several proteases to enhance EMT or migration. For further review on proteases in neural crest migration, please refer to Reference 22. Moreover, neural crest cells express a variety of adhesion molecules that interact with the ECM and surrounding cells to facilitate migration. For further review of expression and functions of adhesion molecules in neural crest, please refer to References 24 and 25.

Protein	Function(s)	Expression	Species and References
Proteases
MMP2	Degrades basement membrane laminin; EMT but not migration	Neural tube & ectoderm basement membrane during EMT; secreted by neural crest	Chick35,43,44,45
MMP8	Not reported	Transcripts in cranial neural crest, mesenchyme	Mouse 51
MMP9	Degrades laminin, promotes EMT & migration	Secreted by neural crest cells	Chick 36
MMP14	Enhances migration	Premigratory and migratory neural crest	Xenopus 48
MMP15	Not reported	Cranial neural plate	Chick51,52
MMP16	Co-activates MMP2, MMP9; required for migration; degrades laminin, N-cadherin loss	Migratory neural crest	Chick37
MMP17/MMP17b	Required for migration	Premigratory & migratory neural crest	Mouse44 Zebrafish45
ADAM10	With ADAM19 and MMP, cleaves Cadherin-6B; critical for EMT; craniofacial defects when deleted in mouse neural crest	Neural crest	Chick33 Mouse37
ADAM13/33	Proteolytic fragments induce neural crest gene expression and remodel ECM	Leading neural crest	Xenopus40–42,23
ADAM19	Binds and stabilizes ADAM13	Neural crest	Chick38 Xenopus40
Adhesion Molecules
Cadherin-11	Facilitates cell adhesion in collective cell migration; facilitates adhesion to fibronectin with Syndecan-4	Migratory neural crest cells	Xenopus 23,85
Cadherin-6B	N-terminal fragments activate MMP2	Premigratory cranial neural crest cells, before and during EMT	Chick35
N-cadherin	Cell adhesion during collective migration; connects cytoskeleton at rear to facilitate actomyosin contractions for forward movement	Migratory neural crest cells	Quail109 Mouse77 Xenopus 10, 97 Chick37
Integrins	Bind laminin, fibronectin, some collagens; component of focal adhesions necessary for migration	Subunits vary	Mouse74–82 Chick82 Xenopus83–85 Several species reviewed in 24

Several other proteases are also required for neural crest migration, even if particular cleavage substrates or mechanisms have yet to be revealed. Expression of various secreted MMPs is noted not only in neural crest cells but in the tissues through which they migrate. MMP2 is expressed in the neural tube and ectoderm basement membranes during EMT [43] and is secreted by cranial [35], trunk [44], and cardiac [45] neural crest cells. Functional studies in the chick suggest a role for MMP2 specifically during neural crest cell EMT, but not in modulating neural crest cell migration [44]. Recently, the membrane-type MMPs have gained attention for their roles in neural crest cell migration. MMP17 was shown to be expressed in mouse premigratory and migratory neural crest cells [46], while the zebrafish ortholog, MMP17b, is required for proper neural crest cell migration in vivo [47]. Additionally, when MMP14, normally expressed in Xenopus and zebrafish premigratory and migratory neural crest cells, is down-regulated, neural crest cell migration is reduced. Overexpression of MMP14 led to premature migration, and MMP14 was specifically shown to be required in neural crest cells, with other MMPs needed in the surrounding mesenchyme [51,54,55]. MMP8 transcripts are present in mouse cranial neural crest cells and mesenchyme and MMP15 has been described in chick cranial and head neural plate regions; however, functions in neural crest migration have yet to be reported [51,52]. While some studies have indicated conserved utilization of certain proteases across axial levels [22,25], it is unclear whether proteases act on the same substrates across the anterior-posterior axis, begging the question of whether certain proteases are employed by specific cells or at specific times during neural crest migration. We speculate that this is the case for at least some proteases such as ADAM13, which is strongly expressed in chick leading neural crest cells and also possesses a critical function in Xenopus leading neural crest cells [15,53]. However, more studies are warranted to determine whether this is generalizable to all proteases during neural crest migration.

3. Stop and go: Permissive and non-permissive ECM signals

As neural crest cells breakout from the neural tube and become migratory, they encounter dynamic ECM environments throughout the embryo (Figure 2). Countless ECM proteins, many with several isoforms, are expressed along migratory routes in evolutionarily conserved patterns [54]. With regard to neural crest cell attachment and motility, ECM components are often categorized as permissive (supporting strong cell adhesion and locomotion), non-permissive (supporting weak cell adhesion, but not extensive locomotion), or inhibitory (directly impeding locomotion) [54,55]. For instance, permissive ECM molecules include fibronectin, laminins, and some collagen isoforms [54]. Early studies first indicated the importance for laminin and fibronectin when the use of function-blocking antibodies or antisense oligonucleotides against integrin receptors (which bind to laminin, fibronectin, and some collagens) resulted in severely perturbed neural crest cell delamination and migration in chick embryos [56–58]. While ECM proteins are produced by surrounding cells, neural crest cells indeed secrete their own permissive substrates, especially fibronectin, to facilitate their own migration [59,60]. Non-permissive molecules, including several collagens and proteoglycans, are also expressed along neural crest cell migratory routes. These molecules may support adhesion of neural crest cells in vitro, but there is little evidence to suggest they actively promote cell adhesion and directional migration in vivo [54].

Finally, inhibitory ECM components, notably proteoglycans like aggrecan and versican, are expressed in zones that exclude migrating neural crest cells (Figure 2) [reviewed in 52]. While aggrecan has long been understood to inhibit neural crest cell migration, the role of versican has only recently been settled. Previously, versican was perplexing, in that functions had been described that both directed and halted neural crest cell migration. Versican expression is evolutionarily conserved, observed in tissues surrounding neural crest cells, but not in the migrating neural crest cells themselves [54,61]. While versican does not arrest cell movement in vitro, neural crest cells cannot invade versican-rich areas in vivo, and are impeded by drastic changes in the concentration of versican in vitro [54]. Recently, however, Szabo et al. (2016) determined with elegant loss-of-function experiments in Xenopus, that versican is, indeed, an inhibitory ECM proteoglycan, which is expressed in tissues surrounding migrating neural crest cells. Loss of versican function resulted in slower, less organized migration of neural crest cells, such that there were no longer defined streams [61]. Therefore, in this model, versican contributes to impenetrable borders along migratory routes, confining motile neural crest cells to compact corridors in vivo and facilitating contact inhibition of locomotion, which drives polarization and directional movement (Figure 2) [55,61].

Intriguingly, several studies have revealed that ECM molecule permissiveness may be context-dependent. Agrin, for example, inhibits enteric neural crest cell motility toward the end of migration, but has previously been described as non-permissive in vivo, such that it neither promotes nor inhibits locomotion on its own [54,60]. While some ECM molecules may be actively inhibitory, relative amounts of permissive, non-permissive, and inhibitory molecules likely determine how well neural crest cells can locomote. In vivo, observed gradients of ECM molecules, including certain proteoglycans like versican, support this idea [62,63]. Moreover, neural crest cells may display differing affinities for specific ECM substrates at various points during the migration process [64–66]. Therefore, functions of particular ECM proteins are dynamic, and conclusions regarding neural crest cell motility should be taken in context, with consideration of other factors like concentration and the presence of additional extracellular factors.

4. Navigating the ECM: Neural crest sensing mechanisms

The ECM provides a substrate upon which neural crest cells migrate and ultimately promotes or inhibits migration by regulating the actin cytoskeleton. In order to facilitate ECM attachment and migration, neural crest cells express a variety of adhesion molecules that tether the cytoskeleton to the ECM (integrins) or adjacent cells (cadherins) (Figure 2). Specialized integrin-containing complexes called focal adhesions are highly dynamic structures that bind the cytoskeleton via intracellular adaptor proteins to create a mechanical link to the extracellular environment [67]. While focal adhesions physically tether the cytoskeleton to ECM, they also sequester other cell signaling components in close proximity, including tyrosine kinase receptors and small G-protein regulators (GTPases and guanine nucleotide exchange factors), which enzymatically control the actin cytoskeleton in response to various extracellular signals [67]. While cell-matrix adhesion is facilitated by integrins, cell-cell adhesion signals are relayed by cadherins, in conjunction with catenins, which effectively connect the cytoskeleton between adherent cells [25]. This important function allows changes in ECM adhesion to be relayed among members of a cell group. Each component of these interactions - the ECM ligands, transmembrane receptors, focal adhesion adapters, cell adhesion molecules, and actin cytoskeleton - are required for sensing the environment and for normal neural crest cell migration, and many of their roles have been reviewed previously [6,24,67,68]. The expression of matrix and cell adhesion molecules during neural crest cell migration has also been well summarized [24], but their complex functions are still being uncovered. Here, we will discuss the function of integrins and their role as signaling integrators during neural crest cell migration.

Integrins are transmembrane proteins comprised of alpha (α) and beta (β) subunits that, as heterodimers, attach extracellularly to the ECM and intracellularly to the cytoskeleton, thus acting as an anchor for the cell [69]. Integrins are appropriate molecules to interact with the changing ECM encountered by neural crest cells, as their signaling is regulated in diverse ways [26,70,71]. Binding to ligands, such as fibronectin, collagens, and laminin, can be reversibly altered by intracellular or extracellular factors causing conformational changes in integrins [69,72]. Additionally, the distribution and trafficking of specific integrin subtypes affects cell-matrix adhesion [70,73]. Moreover, integrins can activate signaling cascades within the cell to affect gene expression [69,70]. Interestingly, integrin signaling can be bidirectional and integrins can be recycled for immediate deployment during neural crest cell migration [65], making these dynamic molecules ideal mediators of cell-matrix signaling in the context of a phenomenon that occurs on a short timescale within the developing embryo.

Neural crest cells express various integrins at all axial levels and across several species, as shown in vivo and in vitro [24], yet relatively few studies have actually demonstrated functions for specific integrins in neural crest cell migration. To date, the α5 and β1 subunits have been identified as important, conserved regulators of neural crest cell migration. α5 and β1 subunits are expressed in both premigratory and migratory neural crest cells at all axial levels in chick, mouse, and Xenopus embryos, and the role of β1, in particular, has been well characterized in mouse enteric neural crest cell migration [74–77]. More recently, α5/β1 function in cardiac neural crest development has also been described in mice. Migrating cardiac neural crest cells and surrounding mesodermal cells express α5/β1 integrins, which are required to interact with fibronectin 1 (synthesized by migrating neural crest cells) to promote cardiovascular morphogenesis [78,79]. Interestingly, conditional deletion of α5 from anterior mesoderm [78], or α5 and αv from neural crest cells [80], resulted in craniofacial defects including cleft palate and improper development of neural crest cell-derived cardiac tissues, suggesting an important role for α5 integrins in facilitating neural crest cell-mesoderm interactions during migration and differentiation [78]. While α5/β1 integrin expression in cardiac neural crest cells are required for cell-autonomous signaling via fibronectin, they appear to be more important for neural crest cell differentiation or survival than migration [59,81]. Nevertheless, locally synthesized ECM by neural crest cells, combined with interactions between neural crest-generated fibronectin and mesoderm-expressed integrins, controls proper cardiac neural crest cell migration and differentiation. Future studies will need to address which subunits facilitate these functions in vivo.

Importantly, integrins have been shown to bind and facilitate functions of other cell adhesion or signaling molecules, demonstrating the role of integrins as integrators of cell-environment interactions. Gazguez et al. (2016) showed that the soluble factor endothelin-3 activates β1 integrins to increase focal adhesions and induce growth and stabilization of lamellipodia during enteric neural crest cell migration [82]. Moreover, modulating expression of Sox10 in enteric neural crest cells perturbed expression of β1 integrins, resulting in altered speed and directionality of migrating neural crest cells, indicating Sox10 may regulate cell-matrix adhesion during migration by controlling integrin expression. Furthermore, the GTPase regulator Ric8a was recently shown to be required for neural crest cell migration in Xenopus due to its role in controlling focal adhesion formation together with focal adhesion kinase [83,84]. Finally, β1 integrins work in concert with molecules that mediate intercellular adhesion, including cadherins, to support neural crest cell migration. In Xenopus, Cadherin-11 localizes to focal adhesions, and together with β1 integrins, paxillin, and the ECM proteoglycan receptor syndecan-4, promotes cell-matrix adhesion [85]. Furthermore, N-cadherin in enteric neural crest cells cooperates with β1 integrins to guide directionality and migration of neural crest cells toward the distal gut, perhaps through a similar mechanism [77]. In summary, neural crest cells encounter a rich and dynamic extracellular environment during migration, and in order to sense these changes and integrate environmental signals, they employ diverse molecular machinery (Figure 2, Table 1).

5. ECM: Policing the routes migrated by the neural crest

In the previous sections, we discussed proteins required by neural crest cells to remodel and sense the ECM to regulate migration (Table 1). Now, we will discuss ways that the ECM effects change in neural crest cells to control migratory behavior. While expression of certain integrins by neural crest cells is required for cell-matrix adhesion and migration, most integrins expressed on the cell surface are not constitutively active [71]. As previously discussed, integrins are intricately linked to other cell surface molecules, and activation of integrins by specific ECM ligands is required not only for cytoskeletal organization and motility, but also for proliferation, survival, and transcriptional regulation [70]. Moreover, many growth factor receptors require integrin activation (and therefore proper matrix adhesion) by the appropriate ligand in order to function [70,71]. For example, Fibronectin-1 activates Notch signaling to promote neural crest cell differentiation and cardiovascular morphogenesis via α5/β1 integrins [59]. Fibronectin has also been shown in cultured cells to prevent cell death via upregulation of Bcl-2 [86,87]. Additionally, ECM molecules affect integrin trafficking in a substrate-specific manner. Interestingly, when cranial neural crest cells migrate on laminin, laminin but not fibronectin receptors are internalized into recycling vesicles, while neither receptor type is internalized on fibronectin [65]. Together, these data indicate that ECM molecules activate particular intracellular signaling cascades in order to control the integrin repertoire expressed by neural crest cells, thereby affecting the ability of neural crest cells to effectively adhere to and migrate upon the matrix. Since integrins also interact with other cell adhesion molecules and growth factor receptors in a subtype-specific manner, the ECM creates not only permissive or non-permissive environments for neural crest cell adhesion and motility, but also has important functions governing the activation of several cell surface receptors that control broad aspects of neural crest development.

While the ECM can directly activate transmembrane receptors to induce intracellular responses, it can also act as a substrate to capture and present soluble chemokines and growth factors essential to neural crest cell migration and differentiation. Numerous diffusible signals have been shown to guide neural crest cell migration. For example, soluble stromal cell-derived factor 1 (Sdf-1) acts on the chemokine receptor Cxcr4 to attract trunk [88], cardiac [89], and cranial neural crest cells [11,90,91 ]. Complement C3a is important for mutual cell attraction and collective cell migration in Xenopus [12]. In addition, several growth factors, including glial cell-derived neurotrophic factor [92–95], platelet-derived growth factor [96,97], fibroblast growth factors [3,98], and vascular endothelial-derived growth factor [3,99], are essential for neural crest cell migration and survival. Notably, each of these factors has been shown to interact with ECM molecules, especially proteoglycans, resulting in a local increase in factor concentration to optimize interactions with migrating cells [100–102]. Many of these studies have been conducted in the context of inflammation or injury repair, but as cellular and molecular mechanisms are typically conserved among different migratory cell types [16,17], it seems likely that ECM interactions with soluble signals also play important roles during embryogenesis (Figure 2). Future research is needed to explore the dependence of chemokine or growth factor signaling on the ECM specifically during neural crest cell migration.

In addition to growth factors and chemokines, proteases including MMPs and ADAMs are secreted into the extracellular space, as discussed previously. Moreover, multiple adhesion molecules and membrane receptors are proteolytically processed to release peptides that act locally on the ECM and other cells to promote migration. In addition to Cadherin-6B cleavage, Cadherin-11 is processed by ADAM13 to release an extracellular fragment that binds erbB2 to facilitate neural crest cell migration in Xenopus [103]. Since cadherins have been shown to directly interact with proteoglycans and proteoglycan receptors [85], it seems plausible that the ECM may also act to locally concentrate shed cadherin fragments such as these to increase their ability to act on nearby neural crest cells. Although further investigation is necessary to understand the role of proteoglycans and other ECM substrates as ligand-presenters to migrating neural crest cells, we can speculate that several ECM components may influence local concentrations of important soluble signals, including chemokines, growth factors, and active proteolytic fragments that are shed into the extracellular space.

6. Structure matters: ECM and tissue biomechanics

While the ECM is helping to relay diffusible signals from cells at a distance, it also produces and mediates mechanical forces within the developing embryo that guide cell migration as well as other processes. During early embryogenesis, tissue stiffening occurs nonuniformly as a result of changes in cell density, cell adhesions, ECM composition, and matrix adhesion [28,104]. Several studies have investigated the role of the ECM in determining substrate stiffness and elasticity and how tissue mechanics regulate morphogenesis. During neurulation, actomyosin contractile forces lead to stiffening of the dorsal tissues that will contribute to neural crest [105,106]. A recent study in Xenopus concluded that this dorsal stiffening is sufficient to induce neural crest EMT, and that certain components of the ECM, namely fibronectin, may not actually be involved in this mechanical process [107]. Changes in the ECM over developmental time, however, do contribute to tissue stiffness and the ability of neural crest cells to migrate. Enteric neural crest cells, for example, migrate more readily upon softer substrates, and migration slows as collagen fibers increase in the embryonic gut, which is associated with tissue stiffening [108]. Our understanding of mechanisms used by neural crest cells to translate changes in the mechanical environment is only just emerging. Generally, tethering of the cytoskeleton to ECM mediates these changes, as tension produced by focal adhesions increases or decreases and affects intracellular signaling [67,109]. Intercellular adhesions are also important. For example, N-cadherin-mediated actomyosin contractions at the trailing edge of a group of cells forces movement of an entire cluster [110]. It will be interesting to determine the degree of crosstalk among cell-cell and cell-matrix adhesion molecules in vivo and how this functions to integrate global and local changes in the embryo. Furthermore, it is important to approach these questions both in vitro and in vivo using less invasive techniques that can measure tissue structure and stiffness. Recently studies have employed second harmonic generation microscopy to visualize mature ECM, but it is not clear whether this approach will be able to detect immature ECM during early embryogenesis [111]. A promising alternative method that has successfully been used in early zebrafish and mouse embryos is Brillouin microscopy, which combines confocal microscopy with light scattering to detect tissue stiffness [112–114]. Yet another recently published method involves injecting magnetic ferrofluid microdroplets into different areas of an embryo to either measure the distortion of the drop with the application of a magnetic field, or to measure how these droplets move through tissues when pulled with the magnetic field [115,116]. A final consideration is the phenomenon of convective tissue flow within the embryo. Convective tissue flow refers to global tissue changes within the developing embryo, which contribute to overall cell motility [28,117]. This tissue flow was first suggested to be ECM-induced when non-motile retinal pigment epithelial cells maintained their characteristic ‘static’ cuboidal morphology after transplantation in avian embryos [118]. This observation indicated passive propulsion of the transplanted cells, which we now understand to be a product of several developmental events, including cell proliferation, migration, and ECM production [7,28,117]. Convective tissue flow has been visualized in quail embryos by imaging of fluorescently-labeled ECM in relation to individual migratory cells, and must be taken into consideration when studying neural crest migration, as it contributes substantially to overall cell motility [117]. Recent advances in live in vivo imaging may pave the way for experiments to parse out the contributions of ECM, adhesion molecules, and tissue flow to neural crest migration, which would provide a more comprehensive picture of ECM functions within the developing embryo.

7. Conclusions and Future Directions

The ECM has diverse functions for governing neural crest cell migration, and reciprocal interactions between neural crest cells and the ECM are required for proper delamination and migration of neural crest cells throughout the embryo. Neural crest cells first must bypass the basement membrane of the dorsal neural tube by secreting proteases that degrade ECM proteins. ADAMs and MMPs generate a positive feedback loop by cleaving transmembrane adhesion molecules, including cadherins, that may further activate proteases essential to neural crest cell migration. As neural crest cells invade the mesenchyme, they secrete permissive ECM, like fibronectin and collagens, while also migrating along established ECM corridors, bordered by non-permissive or inhibitory ECM molecules, including aggrecans and versicans. Neural crest cells use various cell adhesion molecules, especially integrins, to pull along the ECM and relate mechanical feedback to the cell. The dynamic ECM environment induces changes in cell surface protein expression and intracellular signaling so that neural crest cells can alter their responses to react to their microenvironment as necessary. While it is undeniable that the ECM provides structural support to migrating neural crest cells (and to the embryo, overall), many more important functions have been observed for the ECM with regards to neural crest cell migration: 1) ECM provides a rich and dynamic substrate to which receptors on the surface of neural crest cells can tether and affect signaling cascades within the cell to adapt and differentiate as necessary; 2) ECM can influence local concentrations of soluble chemokines and growth factors that guide neural crest cell migration, proliferation, survival, and differentiation; and 3) ECM contributes to and relays mechanical changes in the embryo. Moving forward, it seems imperative to consider the complex microenvironment of a migrating neural crest cell in its entirety. While significant advances are being made with respect to molecular, cellular, and mechanical mechanisms of neural crest cell migration, it is important that future research also focus on the ways in which neural crest cells integrate these diverse signals during vertebrate development.

Abbreviations:

ADAM

A Disintegrin and Metalloproteinase

ECM

extracellular matrix

EMT

epithelial-to-mesenchymal transition

MMP

Matrix Metalloproteinase