Non-recurrent duplications on chromosome 4p16.1 involving cis-regulatory elements affecting neural crest development in patients with isolated bilateral microtia.

We have used a quantitative cell attachment assay to compare the interactions of cranial and trunk neural crest cells with the extracellular matrix (ECM) molecules fibronectin, laminin and collagen types I and IV. Antibodies to the beta 1 subunit of integrin inhibited attachment under all conditions tested, suggesting that integrins mediate neural crest cell interactions with these ECM molecules. The HNK-1 antibody against a surface carbohydrate epitope under certain conditions inhibited both cranial and trunk neural crest cell attachment to laminin, but not to fibronectin. An antiserum to alpha 1 intergrin inhibited attachment of trunk, but not cranial, neural crest cells to laminin and collagen type I, though interactions with fibronectin or collagen type IV were unaffected. The surface properties of trunk and cranial neural crest cells differed in several ways. First, trunk neural crest cells attached to collagen types I and IV, but cranial neural crest cells did not. Second, their divalent cation requirements for attachment to ECM molecules differed. For fibronectin substrata, trunk neural crest cells required divalent cations for attachment, whereas cranial neural crest cells bound in the absence of divalent cations. However, cranial neural crest cells lost this cation-independent attachment after a few days of culture. For laminin substrata, trunk cells used two integrins, one divalent cation-dependent and the other divalent cation-independent (Lallier, T. E. and Bronner-Fraser, M. (1991) Development 113, 1069-1081). In contrast, cranial neural crest cells attached to laminin using a single, divalent cation-dependent receptor system. Immunoprecipitations and immunoblots of surface labelled neural crest cells with HNK-1, alpha 1 integrin and beta 1 integrin antibodies suggest that cranial and trunk neural crest cells possess biochemically distinct integrins. Our results demonstrate that cranial and trunk cells differ in their mechanisms of adhesion to selected ECM components, suggesting that they are non-overlapping populations of cells with regard to their adhesive properties.

While neural crest development is known to be transcriptionally controlled via sequential activation of gene regulatory networks (GRNs), recent evidence increasingly implicates a role for post-transcriptional regulation in modulating the output of these regulatory circuits. Using available single-cell RNA-sequencing datasets from avian embryos to identify potential post-transcriptional regulators, we found that Elavl1, which encodes for an RNA-binding protein with roles in transcript stability, was enriched in the premigratory cranial neural crest. Perturbation of Elavl1 resulted in premature neural crest delamination from the neural tube as well as significant reduction in transcripts associated with the neural crest specification GRN, phenotypes that are also observed with downregulation of the canonical Wnt inhibitor Draxin. That Draxin is the primary target for stabilization by Elavl1 during cranial neural crest specification was shown by RNA-sequencing, RNA immunoprecipitation, RNA decay measurement, and proximity ligation assays, further supporting the idea that the downregulation of neural crest specifier expression upon Elavl1 knockdown was largely due to loss of Draxin. Importantly, exogenous Draxin rescued cranial neural crest specification defects observed with Elavl1 knockdown. Thus, Elavl1 plays a critical a role in the maintenance of cranial neural crest specification via Draxin mRNA stabilization. Together, these data highlight an important intersection of post-transcriptional regulation with modulation of the neural crest specification GRN.

Cranial and trunk neural crest cells produce different derivatives in vitro. Cranial neural crest cultures produce large numbers of cells expressing fibronectin (FN) and procollagen I (PCol I) immunoreactivities, two markers expressed by mesenchymal derivatives in vivo. Trunk neural crest cultures produce relatively few FN or PCol I immunoreactive cells, but they produce greater numbers of melanocytes than do cranial cultures. Treatment of trunk neural crest cultures with transforming growth factor-beta 1 (TGF-beta 1) stimulates them to express both FN and PCol I immunoreactivities at levels comparable to those normally seen in cranial cultures and simultaneously decreases their expression of melanin. These observations raised the possibility that endogenous TGF-beta is involved in specifying differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro. Consistent with this idea, we found that treatment of cranial cultures with a function-blocking TGF-beta antiserum inhibits the development of FN immunoreactive cells and stimulates the development of melanocytes. Cranial and trunk neural crest cells express approximately equal levels of TGF-beta mRNA. However, trunk neural crest cells are significantly less sensitive to the FN-inducing effect of TGF-beta 1 than are cranial neural crest cells. These results suggest that: (1) endogenous TGF-beta is required for the expression of mesenchymal phenotypes by cranial neural crest cells, and (2) differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro result in part from differences in the sensitivities of these two cell populations to TGF-beta.

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Collective cell migration plays an essential role in vertebrate development, yet the extent to which dynamically changing microenvironments influence this phenomenon remains unclear. Observations of the distribution of the extracellular matrix (ECM) component fibronectin during the migration of loosely connected neural crest cells (NCCs) lead us to hypothesize that NCC remodeling of an initially punctate ECM creates a scaffold for trailing cells, enabling them to form robust and coherent stream patterns. We evaluate this idea in a theoretical setting by developing an individual-based computational model that incorporates reciprocal interactions between NCCs and their ECM. ECM remodeling, haptotaxis, contact guidance, and cell-cell repulsion are sufficient for cells to establish streams in silico, however, additional mechanisms, such as chemotaxis, are required to consistently guide cells along the correct target corridor. Further model investigations imply that contact guidance and differential cell-cell repulsion between leader and follower cells are key contributors to robust collective cell migration by preventing stream breakage. Global sensitivity analysis and simulated gain- and loss-of-function experiments suggest that long-distance migration without jamming is most likely to occur when leading cells specialize in creating ECM fibers, and trailing cells specialize in responding to environmental cues by upregulating mechanisms such as contact guidance. Editor's evaluation This important study presents predictions from a computational model demonstrating the impact of the extracellular matrix on collective cell migration in the neural crest. The evidence supporting the claims of the authors is solid, and the study is interesting to cell biologists exploring cell migration in different contexts. https://doi.org/10.7554/eLife.83792.sa0 Decision letter eLife's review process Introduction Vertebrate neural crest cells (NCCs) are an important model for collective cell migration. Discrete streams of migratory NCCs distribute throughout the embryo to contribute to nearly every organ (Le Douarin and Kalcheim, 1999; Tang and Bronner, 2020). Consequently, mistakes in NCC migration may result in severe birth defects, termed neurocristopathies (Vega-Lopez et al., 2018). In contrast to well-studied tightly adhered cell cluster models, such as border cell and lateral line migration (Peercy and Starz-Gaiano, 2020; Olson and Nechiporuk, 2021), much less is known about how ‘loosely’ connected cells, such as NCCs, invade through immature extracellular matrix (ECM) and communicate with neighbors to migrate collectively. Moreover, several invasive mesenchymal cancers resemble collective NCC migration, with cells moving away from the tumor mass in chain-like arrays or narrow strands (Friedl et al., 2004; Friedl and Alexander, 2011). Therefore, utilizing the neural crest experimental model to gain a deeper knowledge of the cellular and molecular mechanisms underlying collective cell migration has the potential to improve the repair of human birth defects and inform strategies for controlling cancer cell invasion and metastasis. Time-lapse analyses of NCC migratory behaviors in several embryo model organisms have revealed that leader NCCs in discrete migratory streams in the head (Teddy and Kulesa, 2004; Genuth et al., 2018), gut (Druckenbrod and Epstein, 2007; Young et al., 2014), and trunk (Kasemeier-Kulesa et al., 2005; Li et al., 2019) are highly exploratory. Leading NCCs extend thin filopodial protrusions in many directions, interact with the ECM and other cell types, then select a preferred direction for invasion. Trailing cells extend protrusions to contact leaders and other cells to maintain cell neighbor relationships and move collectively (Kulesa et al., 2008; Ridenour et al., 2014; Richardson et al., 2016). These observations suggest a leader-follower model for NCC migration (McLennan et al., 2012) in which NCCs at the front of migrating collectives read out guidance signals and communicate over long distances with trailing cells. This model challenged other proposals that NCCs respond to a chemical gradient signal and sustain cohesive movement via an interplay of local cell co-attraction and contact inhibition of locomotion (Theveneau and Mayor, 2012; Szabó et al., 2016; Merchant et al., 2018; Merchant and Feng, 2020). The precise cellular mechanisms that underlie leader-follower migration behavior, for example, the involvement with ECM or the nature of communication signals created by leaders, have not been elucidated. One paradigm that has emerged suggests that loosely connected streams of NCCs move in a collective manner by leaders communicating through long-range signals that are interpreted and amplified by follower NCCs. In support of this, chick leader cranial NCCs show enhanced expression of secreted factors, such as angiopoietin-2 (Ang-2) (McLennan et al., 2015a) and fibronectin (FN) (Morrison et al., 2017). Knockdown of Ang-2 reduces the chemokinetic behavior of follower chick NCCs, resulting in disrupted collective cell migration (McKinney et al., 2016). These observations suggest that follower NCC collective movement depends on signals deposited in the microenvironment by leaders or the adjacent mesoderm. Signals in the FN-rich ECM may also provide microenvironmental cues for NCC collective migration. NCCs cultured in FN-rich ECM move with thin, anchored filopodia through a field that is punctate immediately distal to the migrating front, while ‘pioneer’ NCCs within the front appear to form radially oriented bundles of FN-containing filaments (Rovasio et al., 1983). Later work on the NCC epithelial-to-mesenchymal transition showed that inhibition of ECM-integrin receptors resulted in delamination of NCCs into the neural tube lumen (Kil et al., 1996). Moreover, FN is abundant in the mouse head and neck (Mittal et al., 2010), and there is a dramatic reduction in the number of migrating NCCs that reach the heart in FN null mice (Wang and Astrof, 2016). Thus, FN is critical for NCC to reach their targets, although its precise role in collective cell migration remains unclear. Mathematical modeling of NCC migration can help provide insight into the role of FN and identify the origins of multiscale collective behaviors (Giniūnaitė et al., 2020; Kulesa et al., 2021). Previous models for NCC migration demonstrated that contact guidance, a mechanism by which cells align themselves along ECM fibers, can establish collective behavior and create single-file cell chains (Painter, 2009). Later models incorporating ECM degradation and haptotaxis, a process by which cells migrate up adhesive ECM gradients, demonstrated how matrix heterogeneity and cell-cell interactions could determine cell migratory patterns (Painter et al., 2010; Wynn et al., 2013). Models for other collectively migrating cells, such as fibroblasts, suggest that ECM remodeling by motile cells can enable anisotropic collective movement, with migratory directions dictated by fiber orientation (Dallon et al., 1999; Groh and Louis, 2010; Chauviere et al., 2010; Azimzade et al., 2019; Wershof et al., 2019; Pramanik et al., 2021; Suveges et al., 2021; Metzcar et al., 2022). No models have yet addressed how collective migration arises from cell remodeling of an initially isotropic, immature ECM. In this paper, we develop an agent-based model (ABM; also known as an individual-based model) of chick cranial NCC migration that considers a cell-reinforced migratory cue in which ‘leader’ cells remodel an initially punctate and immature FN matrix to signal ‘follower’ cells. Other processes detailed in the model include cell-cell repulsion, haptotaxis, and contact guidance. This framework incorporates new observations of FN distribution in the chick cranial NCC microenvironment and simulates gain- and loss-of-function of FN. Detailed simulations of the model over parameter space, coupled with global sensitivity analysis of ABM parameters, identifies mechanisms that dominate the formation of migrating streams and other NCC macroscopic behavior. Through this analysis, we find that migration is most efficient when leading NCCs specialize in remodeling FN to steer the collective, as cells otherwise enter a ‘jammed’ state in which migration is greatly reduced and cells are densely packed together (Sadati et al., 2013). The addition of ‘guiding signals’ that direct NCCs along a target corridor limits excessive lateral migration in the ABM but concurrently promotes separation between leader and trailing cells in the stream. We survey ABM parameter combinations that recover robust collective migration without such stream breaks, which underscore the potential importance of contact guidance. Results FN protein expression within the mesoderm is unorganized and punctate prior to cranial NCC emigration, but filamentous after it has been traversed by leader cells We confirmed that FN throughout the head, neck and cardiovascular regions in the chick embryo is distributed in patterns that overlap with NCC migratory pathways (Figure 1; Duband and Thiery, 1982). To assess the in vivo distribution of FN protein at higher spatial resolution, we examined transverse cryosections (Figure 1A; Hamburger and Hamilation, 1951) at developmental stages (HH12–13) midway through cranial NCC migration to reveal that NCCs are in close association with a heterogeneous meshwork of FN (Figure 1B). Punctate FN, not yet fibrils, are found both distal to the invasive NCC migratory front and adjacent to the leader NCC subpopulation (Figure 1C). By contrast, elongated FN fibrils are located behind the leading edge of invasive NCC streams (Figure 1D). FN fibrils proximal to the invasive NCC migratory front did not reveal a preferred directional orientation. Similar punctate FN was also visible in regions outside the NCC migratory pathway, subjacent to the surface ectoderm (Figure 1C). Figure 1 with 1 supplement see all Download asset Open asset In vivo observations of fibronectin (FN) and results from gain/loss-of-function experiments. (A) Schematic of a typical neural crest cell (NCC) migratory stream in the vertebrate head of the chick embryo, at developmental stage HH12–13 (Hamburger and Hamilation, 1951) at the axial level of the second branchial arch (ba2). (B) Transverse section through the NCC migratory stream at the axial level of rhombomere 4 (r4) triple-labeled for FN (green), nuclei (DAPI-blue) and migrating NCCs (HNK1-red). NCCs migrate subjacent to the surface ectoderm after emerging from the dorsal neural tube (NT). The arrowhead points to the surface ectoderm. The yellow boxes highlight the tissue subregions that contain the leader NCCs (box 1) and are distal to the leader NCCs (box 2), marking distinct shapes of the FN in each box with an asterisk (box 1) and open circle (box 2). (C) FN only. (D) The fibrous (box 1-asterisk) and punctate (box 2-open circle) appearance of FN in the NCC microenvironment. (E) NCC distance migrated in FN morpholino-injected embryos and (F) percentage area of the NCC migratory stream after microinjection of soluble FN into the r4 paraxial mesoderm prior to NCC emigration. NT = neural tube. The scale bars are 50 µm (B–C) and 10 µm (D). Gain- or loss-of-function of FN leads to reduced NCC migration Functional analysis confirms FN is required for normal NCC migration. Knockdown of FN expression introduced into premigratory NCCs within the neural tube led to a significant reduction (nearly 30%) in the total distance migrated by NCCs enroute to BA2 (Figure 1E). Microinjection of soluble FN into the cranial NCC migratory domain, for example, into the paraxial mesoderm adjacent to rhombomere 4 (r4) prior to NCC delamination, also led to a dramatic reduction (by 70%) in NCC migration as indicated by a decrease in the area typically covered by the invading NCCs (Figure 1F). Thus, decreasing the expression of FN – presumably by decreasing the rate at which FN is secreted by motile NCCs – or increasing the FN density in the microenvironment led to significant changes in the NCC migration pattern. We conclude that a balance of FN within the migratory microenvironment is required to promote proper migration. An individual-based model of NCC migration and ECM remodeling produces collectively migrating streams in silico Our observations led us to hypothesize that migrating NCCs remodel punctate FN into a fibrous scaffold for trailing cells. We evaluated this idea in a theoretical setting by constructing a mathematical model in which NCCs are represented as discrete off-lattice point masses, that is, agents, that freely move in a two-dimensional (2D) plane (Figure 2—figure supplement 1). Each agent responds to, and influences, the remodeling of an initially punctate ECM (Figure 2; Figure 2—figure supplement 2). Their velocities are determined using an overdamped version of Newton’s second law. The three types of forces that alter agent trajectories arise from friction (which is proportional to the cell velocity), cell-ECM interactions (specifically, those from haptotaxis and contact guidance), and cell-cell repulsion. Neighboring NCCs, FN puncta, and FN fibers generate the latter two forces and affect the motion of an agent only when they are within a user-specified distance, Rfilo , of the agent center (this distance represents the length of cell filopodia protrusions). Figure 2 with 13 supplements see all Download asset Open asset Integration of extracellular matrix (ECM) within an agent-based model (ABM) of neural crest cell (NCC) migration. Individual snapshots (A) of an example ABM realization reveals that the model can generate a single anisotropically migrating stream over a simulated time of 12 hr. Different realizations generated using the same parameter values but different random seeds (B), however, may produce streams that exhibit the formation of multiple branches and demonstrate the range of possible model behaviors. Black circles denote leader cells, which can secrete new FN, red cells signify follower non-secretory cells, and blue squares correspond to FN puncta. Arrows denote cell velocities or fiber orientations. The extra column of FN at the right boundary is an artifact of visualization. Sobol indices (C) and scatter plots (D) of the horizontal distance traveled by NCCs indicate that this metric is most sensitive to the haptotaxis-contact guidance weight, χ, and the filopodial length, Rfilo , but is less dependent on parameters related to cell-cell repulsion (ci). Statistical significance in (C) is determined from a two-sample t-test that compares the Sobol indices produced by the parameter of interest to those obtained from a dummy parameter (red asterisks indicate significant first-order indices, black asterisks indicate significant total order indices, p<0.01). Each data point in (D) represents the average of 20 ABM realizations. The cell-repulsion (resp. cell-ECM) force accounts for the collective interactions that an agent experiences from neighboring NCCs (resp. FN puncta and fibers). The magnitude of the total cell-cell repulsion force is determined from the sum of radially oriented forces from all neighboring NCCs, whose strength, modulated by a user-defined constant, ci , decreases quadratically with respect to the distance from the agent center of mass. To represent the finding that chick cranial NCCs do not always repel each other upon contact (Kulesa and Fraser, 1998; Kulesa et al., 2004), we adjust the direction of the resultant force by stochastically sampling from a von Mises distribution (Mardia and Jupp, 1999). The parameters of the distribution depend on the number and location of NCCs sensed by the agent. They ensure it is uniform when few NCCs are present but cause it to resemble a periodic normal distribution biased in the direction of lowest NCC density when many cells are sensed (for details, see the Materials and methods section). Thus, when cells sense each other at longer ranges, they may align. When cells are close enough to be overlapping, however, they are more likely to repel each other along the direction of contact, similarly to other implementations of cell-cell repulsion (Colombi et al., 2020). We have verified that increasing the magnitude of the cell-cell repulsion forces increases the average nearest neighbor distance between cells in the ABM (Figure 2—figure supplement 5; Figure 3—figure supplement 4). The magnitude of the total cell-ECM force is similarly determined by summing radially oriented forces originating from neighboring FN puncta and fibers, with the strengths of such forces decreasing quadratically with respect to distance from the agent center. The resultant cell-ECM force direction is determined by a linear combination of signals arising from haptotaxis and contact guidance, which are weighted by a parameter, χ. The haptotaxis and contact guidance cues are both represented as unit vectors. Haptotaxis biases the total cell-ECM force toward increasing FN densities and its cue is sampled from a von Mises distribution whose parameters depend on the number and location of neighboring FN puncta and fibers sensed by the cell. The distribution is biased such that the cell is likely to travel toward the greatest FN concentration sampled. Contact guidance aligns the cell-ECM force along the direction of FN fibers. The unit vector corresponding to this cue is computed from the average orientation of FN fibers sensed by the agent. Cells migrate through an initially isotropic, equi-spaced, square lattice of FN puncta that approximates the matrix distribution prior to NCC migration. Leader cells are represented by a fixed number of ‘secretory’ cells that generate new FN puncta at their centers according to times drawn from an exponential distribution with user-specified mean. The cells start at the left-hand boundary of the lattice, which we take to represent the section of the neural tube located along r4. Non-secretory cells, which cannot create new FN puncta, enter the domain at later times, provided sufficient space is available. We note that the terms ‘secretory’ and ‘non-secretory’ refer only to the ability of cells to secrete new FN puncta, as both cell types can create and align fibers emanating from puncta they pass over. Observations of individual ABM simulations indicate that ECM remodeling, haptotaxis, contact guidance, and cell-cell repulsion can generate stream-like patterns, with secretory cells able to maintain their positions at the front of the stream (Figure 2A; Figure 2—video 1). Cells can migrate the FN lattice is isotropic, and trailing cell velocities are oriented toward leader cells (for the average that the follower cell vector with the horizontal is or for the in Figure patterns most ECM fibers to the horizontal that is, the direction of the target corridor (Figure 2—figure supplements Figure 2—video NCCs do not always maintain a single mass as they migrate collectively. In ABM streams into two or more that regions along the (Figure Figure 2 and 4). there is signal NCCs in a direction to the horizontal Thus, in cells may sense and travel toward FN puncta located to the target corridor. We conclude that additional signals are required to NCC stream In later we how such signals affect the of patterns by NCCs. Global sensitivity analysis suggests a key role for contact guidance in long-distance NCC migration To determine how collective migration depends on ABM parameters, we sensitivity et al., to for collective migration. This analysis Sobol indices (Figure which the by which the of a is to changes in a parameter of interest values indicate that the is more sensitive to the The analysis produces two types of Sobol first-order indices, which the of to a parameter of and indices, which include additional that arise when other parameters are The resulting Sobol indices indicate that the distance NCCs travel in the horizontal direction along the target is most sensitive to χ, the parameter the to which NCC directional migration in response to the FN matrix is by haptotaxis contact guidance or a linear combination of the the of this parameter decreases the distance cells travel The cell filopodial Rfilo , the Sobol indices and a increasing with the This as increasing the number of FN puncta and fibers that the cell be to generate more and migration in the The parameter for the cell-cell repulsion force strength, ci , presents a but significant first-order analysis of its scatter (Figure reveals that when contact guidance the cell-ECM force increasing the cell-cell repulsion decreases the distance that the streams migrate When haptotaxis the direction of the force this is but remains decreasing the distance traveled by NCCs is most sensitive to the mechanism by which cells respond to the with contact guidance longer while haptotaxis and cell-cell repulsion are with the distance that NCCs We results for a the extent of the migrating stream in directions to the target that the lateral of cells in the ABM from the target corridor (Figure 2—figure supplement we refer to this as the The cell-ECM weight, χ, the Sobol indices for this and a that lateral migration increases with of contact guidance. By contrast, scatter plots suggest this is with the filopodial and cell-cell repulsion of Sobol indices for the average distance between neighboring cells reveals the importance of haptotaxis and cell-cell repulsion on cell The cell-ECM and the cell-cell repulsion parameter both significant first-order Sobol indices (Figure 2—figure supplement with the latter parameter the Sobol is significant for the filopodial which that this parameter cell-cell separation via its interactions with the other two This that the cell-ECM and cell-cell repulsion forces are by changing the number of FN puncta and NCCs plots (Figure 2—figure supplement suggest a decreasing between the nearest neighbor distance and the cell-ECM weight, a increasing with the cell-cell repulsion parameter, ci and for the filopodial Leading NCCs collective migration by and remodeling ECM To identify combinations of parameters that collective migration, we simulated experimental that affect cell of new FN (Figure see Materials and while cell of fibers from puncta When leader secretory cells secrete FN puncta more we find that the stream by By contrast, cell new FN puncta, then the average distance the stream decreases by about migration cells can respond to both cell types secrete FN, such that there is between cell then the average distance migrated by cells decreases from the of at the rate or is Similar results for the lateral of NCCs (Figure 3—figure supplement 1). Figure with 4 supplements see all Download asset Open asset distance traveled by neural crest cells (NCCs) after 12 hr. plots for experiments in which (A) fibronectin (FN) is (B) and fiber are and (C) cells invade a FN lattice suggest that the extracellular matrix (ECM) plays an important role in NCC migration. experiments decrease the average over which a cell new FN from to 10 Contact guidance and cell-cell repulsion in (C) correspond to and indicate are different from that of the parameter using a agent-based model (ABM) realizations are to generate each We how cells alter the FN by both FN and fiber in leader follower cells (Figure When leading secretory cells both cell are to remodel the the average distance traveled by the NCC stream decreases by (resp. When follower ‘non-secretory’ cells are from and FN fibers, however, the is with only a reduction in distance Similar results are for the nearest neighbor distance and the lateral of NCCs (Figure 3—figure supplement 2). These results demonstrate within the FN remodeling by leading NCCs plays a key role in long-distance collective migration. By contrast, are when trailing cells cannot remodel the Our suggest that collective migration may be more when leading and trailing NCCs with leading cells remodeling the ECM. The FN matrix distribution is to long-distance collective cell migration ECM remodeling can direct collective cell migration, but the to which the ECM cell trajectories remains unclear. the lattice between FN puncta from 20 to µm collective migration in the decreasing the distance that the stream by The lateral of NCCs is similarly reduced within (Figure 3—figure supplement We conclude that NCCs are less likely to sense FN in their local then they are less likely to migrate as the nearest neighbor distance between cells decreases (Figure 3—figure supplement an cell density and state a in which cells are tightly to movement, and a (Sadati et al., 2013). These lead us to cells in the ABM as in a ‘jammed’ state when they collectively travel less µm over 12 as we have found a but significant between the nearest neighbor distance and the distance traveled in the horizontal direction (Figure 2—figure supplement Figure 3—figure supplement 4). Cells however, mechanisms to for FN and long-distance collective migration (Figure contact guidance is in the ABM the decreases from to then the NCC migration distance in FN increases by The distance that obtained in the FN lattice by the cell-cell repulsion or the rate at which secretory cells produce FN also cause NCCs to travel distances within the FN matrix (by and but changes do not migration to distances to those in These results highlight the importance of contact guidance, ECM remodeling, cell-cell repulsion in long-distance migration, when cells are in a state migration is otherwise directional guidance cells along their target but the model sensitive to stream and cell separation mechanisms that generate collective migration do not that cells travel along a single

Sox9 has essential roles in endochondral bone formation during axial and appendicular skeletogenesis. Sox9 is also expressed in neural crest cells, but its function in neural crest remains largely unknown. Because many craniofacial skeletal elements are derived from cranial neural crest (CNC) cells, we asked whether deletion of Sox9 in CNC cells by using the Cre recombinase/loxP recombination system would affect craniofacial development. Inactivation of Sox9 in neural crest resulted in a complete absence of cartilages and endochondral bones derived from the CNC. In contrast, all of the mesodermal skeletal elements and intramembranous bones were essentially conserved. The migration and the localization of Sox9-null mutant CNC cells were normal. Indeed, the size of branchial arches and the frontonasal mass of mutant embryos was comparable to that of WT embryos, and the pattern of expression of Ap2, a marker of migrating CNC cells, was normal. Moreover, in mouse embryo chimeras Sox9-null mutant cells migrated to their correct location in endochondral skeletal elements; however, Sox9-null CNC cells were unable to contribute chondrogenic mesenchymal condensations. In mutant embryos, ectopic expression of osteoblast marker genes, such as Runx2, Osterix, and Col1a1, was found in the locations where the nasal cartilages exist in WT embryos. These results indicate that inactivation of Sox9 causes CNC cells to lose their chondrogenic potential. We hypothesize that these cells change their cell fate and acquire the ability to differentiate into osteoblasts. We conclude that Sox9 is required for the determination of the chondrogenic lineage in CNC cells.

Author response: Heritability enrichment in context-specific regulatory networks improves phenotype-relevant tissue identification

The expression pattern of the tyrosine kinase gene Pag in whole-mount preparations of Gastrotheca riobambae embryos and the immunostaining of embryos against the proteins vimentin, NCAM, Pax-2, Hoxd9, and antigen 2G9 allowed detection of migrating streams of cranial neural crest (NC) cells, the isthmus, the hindbrain boundaries, rhombomeres, cranial nerves, and the developing spinal cord. Expression patterns of these genes and the basic neural morphology of Gastrotheca have been conserved in comparison with other vertebrates. However, as in Xenopus, a prominent stream of migrating cranial NC cells from rhombomere 5 was found in Gastrotheca embryos. By contrast, in chick embryos, premigratory NC cells from rhombomeres 3 and 5 undergo extensive apoptosis, which suggests that in anurans, apoptosis of the cranial NC may deviate from the chick pattern. The branchial-anterior and branchial-posterior masses of cranial NC cells, that populate the gill arches, are very large in G. riobambae. We cannot distinguish whether this feature corresponds to an anuran trait related to development of the tadpole pharyngeal skeleton, or is related to development of the peculiar external bell gills of this frog. This work provides the descriptive groundwork for lineage studies of the NC in G. riobambae embryos. Gastrotheca embryos are large and flat, have prominent streams of cranial NC cells, and develop very large external bell gills. These unique characteristics may facilitate future comparative analysis of the role of apoptosis in patterning the amphibian NC cell streams, and the contribution of the NC to development of the gills.

BackgroundCollective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood.ResultsUsing in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells.ConclusionsTogether, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0323-9) contains supplementary material, which is available to authorized users.

The different effects on cranial and trunk neural crest cell behaviour following exposure to a low concentration of alcohol in vitro

Compared to placental mammals, marsupials have short gestation period, and their neonates are relatively immature. Despite these features, marsupial neonates must travel from the birth canal to the teat, suckle and digest milk to complete development. Thus, certain organs and tissues of marsupial neonates, such as forelimbs to crawl and jaw elements to suckle, must develop early. Previous reports showed that cranial neural crest (CNC) cells, as the source of ectomesenchyme of jaw elements, are generated significantly early in gray short-tailed opossum (Monodelphis domestica) compared to other amniote models, such as mouse. In this study, we examined the expression of genes known to be important for neural crest formation, such as BMP2/BMP4 (neural crest inducer), Pax7 (neural border specifier), Snail1 and Sox9/Sox10 (neural crest specifier) in Monodelphis domestica, and compared the expression patterns with those in mouse, chicken, and gecko embryos. Among those genes, the expression of Sox9 was turned on early and broadly in the premigratory CNC cells, and persisted in the ectomesenchyme of the cranial anlagen in opossum embryos. In contrast, Sox9 expression diminished in the CNC cells of other animals at the early phase of migration. Comparison of the onset of Pax7 and Sox9 expression revealed that Sox9 expression in the prospective CNC was earlier and broader than Pax7 expression in opossum, suggesting that the sequence of border specification and neural crest specification is altered. This study provides the first clue for understanding the molecular basis for the heterochronic development of the CNC cells and jaw elements in marsupials.

A non-canonical JAGGED1 signal to JAK2 mediates osteoblast commitment in cranial neural crest cells

In vertebrate embryos, neural crest cells emigrate out of the neural tube and contribute to the formation of a variety of neural and nonneural tissues. Some neural crest cells undergo apoptotic death during migration, but its biological significance and the underlying mechanism are not well understood. We carried out an in vitro study to examine how the morphology and survival of cranial neural crest (CNC) cells of the mouse embryo are affected when their actin cytoskeleton or anchorage-dependent cell spreading is perturbed. Disruption of actin fiber organization by cytochalasin D (1 microg/ml) and inhibition of cell attachment by matrix metalloproteinase-2 (MMP-2; 2.0 units/ml) were followed by morphologic changes and apoptotic death of cultured CNC cells. When the actin cytoskeleton was disrupted by cytochalasin D, the morphologic changes of cultured CNC cells preceded DNA fragmentation. These results indicate that the maintenance of cytoskeleton and anchorage-dependent cell spreading are required for survival of CNC cells. The spatially and temporally regulated expression of proteinases may be essential for the differentiation and migration of neural crest cells.

The cranial neural crest (CNC) is a transient cell population that originates at the crest of the neural fold and gives rise to multiple cell types during craniofacial development. Traditionally, researchers have used tissue explants, such as the neural tube, to obtain primary neural crest cells for their studies. However, this approach has inevitably resulted in simultaneous isolation of neural and non-neural crest cells as both of these cells migrate away from tissue explants. Using the Wnt1-Cre/R26R mouse model, we have obtained a pure population of neural crest cells and established a primary CNC cell culture system in which the cell culture medium best supports the proliferation of E10.5 first branchial arch CNC cells and maintains these cells in their undifferentiated state. Differentiation of CNC cells can be initiated by switching to a differentiation medium. In this model, cultured CNC cells can give rise to neurons, glial cells, osteoblasts, and other cell types, faithfully mimicking the differentiation process of the post-migratory CNC cells in vivo. Taken together, our study shows that the Wnt1-Cre/R26R mouse first branchial arch provides an excellent model for obtaining post-migratory neural crest cells free of any mesodermal contaminants. The cultured neural crest cells are under sustained proliferative, undifferentiated, or lineage-enhanced conditions, hence, serving as a tool for the investigation of the regulatory mechanism of CNC cell fate determination in normal and abnormal craniofacial development.

During tooth development, cranial neural crest (CNC) cells represent a population of pluripotent stem cells that give rise to various dental tissues. This study aimed to investigate whether CNC cells could differentiate into odontoblast-like cells by in vitro induction. CNC cells were isolated from explants of cranial neural tubes and cultured in serum-free Dulbecco's modified Eagle's medium (DMEM)/F12 medium which contained fibroblast growth factor 8 (FGF8) and dentin non-collagen proteins (DNCP). The initiation of controlled differentiation was determined using histological assays, and the expression of specific gene phenotypes was detected using immunocytochemical staining and reverse transcription--polymerase chain reaction (RT--PCR). The first branchial arch phenotype of the CNC cells demonstrated negative Hoxa2 expression and positive vimentin expression in the presence of 100 ng/ml FGF8. Following DNCP induction, the CNC cells became bipolar, demonstrated high alkaline phosphatase (ALP) activity, and formed mineralized nodules. In addition, the expression of DSPP, DMP1, and collagen type I confirmed the odontoblast phenotype. The results indicate that the tissue-specific cellular differentiation (odontoblast-like cells) of early-stage embryonic-derived cells (such as CNC cells) can be induced by adult extracellular matrix proteins (such as DNCP). CNC cells may be used as a valuable cell model for research on dental tissue differentiation and regeneration.

There is a significant difference between the developmental patterns of cranial and trunk neural crest cells in the amniote. Thus, whereas cranial neural crest cells generate bone and cartilage, trunk neural crest cells do not contribute to skeletal derivatives. We examined whether mouse trunk neural crest cells can undergo chondrogenesis to analyze how the difference between the developmental patterns of cranial and trunk neural crest cells arises. Our present data demonstrate that mouse trunk neural crest cells have chondrogenic potential and that fibroblast growth factor (FGF) 2 is an inducing factor for their chondrogenesis in vitro. FGF2 altered the expression patterns of Hox9 genes and Id2, a cranial neural crest cell marker. These results suggest that environmental cues may play essential roles in generating the difference between developmental patterns of cranial and trunk neural crest cells.