Enhancement of neural crest formation by mechanical force in Xenopus development.

In vertebrate development, ectoderm is specified into neural plate (NP), neural plate border (NPB), and epidermis. Although such patterning is thought to be achieved by molecular concentration gradients, it has been revealed, mainly by in vitro analysis, that mechanical force can regulate cell specification. During in vivo patterning, cells deform and migrate, and this applies force to surrounding tissues, shaping the embryo. However, the role of mechanical force for cell specification in vivo is largely unknown. In this study, with an aspiration assay and atomic force microscopy, we have demonstrated that tension on ectodermal cells decreases laterally from the midline in Xenopus early neurula. Ectopically applied force laterally expanded the neural crest (NC) region, a derivative of the NPB, whereas force relaxation suppressed it. Furthermore, force application activated both the FGF and Wnt pathways, which are required for NC formation during neuroectodermal patterning. Taken together, mechanical force is necessary for NC formation in order to regulate signaling pathways. Furthermore, molecular signals specify the NP and generate force on neighboring tissue, the NPB, with its closure. This force activates signals, possibly determining the appropriate width of a narrow tissue, the NC.

Neural tube-associated boundary caps are a major source of mural cells in the skin.

In addition to their roles in protecting nerves and increasing conduction velocity, peripheral glia plays key functions in blood vessel development by secreting molecules governing arteries alignment and maturation with nerves. Here, we show in mice that a specific, nerve-attached cell population, derived from boundary caps (BCs), constitutes a major source of mural cells for the developing skin vasculature. Using Cre-based reporter cell tracing and single-cell transcriptomics, we show that BC derivatives migrate into the skin along the nerves, detach from them, and differentiate into pericytes and vascular smooth muscle cells. Genetic ablation of this population affects the organization of the skin vascular network. Our results reveal the heterogeneity and extended potential of the BC population in mice, which gives rise to mural cells, in addition to previously described neurons, Schwann cells, and melanocytes. Finally, our results suggest that mural specification of BC derivatives takes place before their migration along nerves to the mouse skin.

Neuro-mesodermal assembloids recapitulate aspects of peripheral nervous system development in vitro.

Here we describe a novel neuro-mesodermal assembloid model that recapitulates aspects of peripheral nervous system (PNS) development such as neural crest cell (NCC) induction, migration, and sensory as well as sympathetic ganglion formation. The ganglia send projections to the mesodermal as well as neural compartment. Axons in the mesodermal part are associated with Schwann cells. In addition, peripheral ganglia and nerve fibers interact with a co-developing vascular plexus, forming a neurovascular niche. Finally, developing sensory ganglia show response to capsaicin indicating their functionality. The presented assembloid model could help to uncover mechanisms of human NCC induction, delamination, migration, and PNS development. Moreover, the model could be used for toxicity screenings or drug testing. The co-development of mesodermal and neuroectodermal tissues and a vascular plexus along with a PNS allows us to investigate the crosstalk between neuroectoderm and mesoderm and between peripheral neurons/neuroblasts and endothelial cells.

Shared reproductive disruption, not neural crest or tameness, explains the domestication syndrome.

Altered neural crest cell (NCC) behaviour is an increasingly cited explanation for the domestication syndrome in animals. However, recent authors have questioned this explanation, while others cast doubt on whether domestication syndrome even exists. Here, we review published literature concerning this syndrome and the NCC hypothesis, together with recent critiques of both. We synthesize these contributions and propose a novel interpretation, arguing shared trait changes under ancient domestication resulted primarily from shared disruption of wild reproductive regimes. We detail four primary selective pathways for 'reproductive disruption' under domestication and contrast these succinct and demonstrable mechanisms with cryptic genetic associations posited by the NCC hypothesis. In support of our perspective, we illustrate numerous important ways in which NCCs contribute to vertebrate reproductive phenotypes, and argue it is not surprising that features derived from these cells would be coincidentally altered under major selective regime changes, as occur in domestication. We then illustrate several pertinent examples of Darwin's 'unconscious selection' in action, and compare applied selection and phenotypic responses in each case. Lastly, we explore the ramifications of reproductive disruption for wider evolutionary discourse, including links to wild 'self-domestication' and 'island effect', and discuss outstanding questions.

Colec12 and Trail signaling confine cranial neural crest cell trajectories and promote collective cell migration.

BACKGROUND: Collective and discrete neural crest cell (NCC) migratory streams are crucial to vertebrate head patterning. However, the factors that confine NCC trajectories and promote collective cell migration remain unclear. RESULTS: Computational simulations predicted that confinement is required only along the initial one-third of the cranial NCC migratory pathway. This guided our study of Colec12 (Collectin-12, a transmembrane scavenger receptor C-type lectin) and Trail (tumor necrosis factor-related apoptosis-inducing ligand, CD253) which we show expressed in chick cranial NCC-free zones. NCC trajectories are confined by Colec12 or Trail protein stripes in vitro and show significant and distinct changes in cell morphology and dynamic migratory characteristics when cocultured with either protein. Gain- or loss-of-function of either factor or in combination enhanced NCC confinement or diverted cell trajectories as observed in vivo with three-dimensional confocal microscopy, respectively, resulting in disrupted collective migration. CONCLUSIONS: These data provide evidence for Colec12 and Trail as novel NCC microenvironmental factors playing a role to confine cranial NCC trajectories and promote collective cell migration.

RNA-binding protein Elavl1/HuR is required for maintenance of cranial neural crest specification.

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.

As an embryo develops, different genetic programs become activated to give cell populations a specific biological identity that will shape their fate. For instance, when certain sets of genes get switched on, cells from the outermost layer of the embryo start to migrate to their final destination within the body. There, these 'neural crest cells' will contribute to bones and cartilage in the face, pigmented skin spots, and muscles or nerves in the gut. When genes responsible for the neural crest identity are active, their instructions are copied into an 'RNA molecule' which will then relay this information to protein-building structures. How well the RNA can pass on the message depends on how long it persists within the cell. Certain RNA-binding proteins can control this process, but it is unclear whether and how this regulation takes place in neural crest cells. In their work, Hutchins et al. therefore focused on identifying RNA-binding proteins involved in neural crest identity. Exploratory searches of genetic data from chick embryos revealed that, even before they started to migrate, neural crest cells which have recently acquired their identity produced large amounts of the RNA-binding protein Elavl1. In addition, these cells did not behave normally when embryos were deprived of the protein: they left the outer layer too soon and then switched off genes important for their identity. Genetic studies of neural crest cells lacking Elavl1 revealed that this effect was due to having lost the RNA molecule produced from the Draxin gene. Introducing an additional source of Draxin into mutant embryos missing Elavl1 was enough to restore normal neural crest behaviour. Further biochemical experiments then showed that the RNA for Draxin decayed quickly in the absence of Elavl1. This suggests that the protein normally allows Draxin's RNA to persist long enough to pass on its message. These results reveal a new mechanism controlling the identity and behaviour of the neural crest. Since many cancers in adulthood arise from the descendants of neural crest cells, Hutchins et al. hope that this knowledge could lead to improved therapies in the future.

Species-specific sensitivity to TGFß signaling and changes to the Mmp13 promoter underlie avian jaw development and evolution.

Precise developmental control of jaw length is critical for survival, but underlying molecular mechanisms remain poorly understood. The jaw skeleton arises from neural crest mesenchyme (NCM), and we previously demonstrated that these progenitor cells express more bone-resorbing enzymes including Matrix metalloproteinase 13 (Mmp13) when they generate shorter jaws in quail embryos versus longer jaws in duck. Moreover, if we inhibit bone resorption or Mmp13, we can increase jaw length. In the current study, we uncover mechanisms establishing species-specific levels of Mmp13 and bone resorption. Quail show greater activation of and sensitivity to transforming growth factor beta (TGFß) signaling than duck; where intracellular mediators like SMADs and targets like Runt-related transcription factor 2 (Runx2), which bind Mmp13, become elevated. Inhibiting TGFß signaling decreases bone resorption, and overexpressing Mmp13 in NCM shortens the duck lower jaw. To elucidate the basis for this differential regulation, we examine the Mmp13 promoter. We discover a SMAD-binding element and single nucleotide polymorphisms (SNPs) near a RUNX2-binding element that distinguish quail from duck. Altering the SMAD site and switching the SNPs abolish TGFß sensitivity in the quail Mmp13 promoter but make the duck promoter responsive. Thus, differential regulation of TGFß signaling and Mmp13 promoter structure underlie avian jaw development and evolution.

Cellular plasticity in the neural crest and cancer.

In vertebrates, neural crest cells (NCCs) are a multipotent embryonic population generating both neural/neuronal and mesenchymal derivatives, and thus the neural crest (NC) is often referred to as the fourth germ layer. NC development is a dynamic process, where NCCs possess substantial plasticity in transcriptional and epigenomic profiles. Recent technical advances in single-cell and low-input sequencing have empowered fine-resolution characterisation of NC development. In this review, we summarise the latest models underlying NC-plasticity acquirement and cell-fate restriction, outline the connections between NC plasticity and NC-derived cancer and envision the new opportunities in studying NC plasticity and its link to cancer.

Chick cranial neural crest cells release extracellular vesicles that are critical for their migration.

The content and activity of extracellular vesicles purified from cell culture media or bodily fluids have been studied extensively; however, the physiological relevance of exosomes within normal biological systems is poorly characterized, particularly during development. Although exosomes released by invasive metastatic cells alter migration of neighboring cells in culture, it is unclear whether cancer cells misappropriate exosomes released by healthy differentiated cells or reactivate dormant developmental programs that include exosome cell-cell communication. Using chick cranial neural fold cultures, we show that migratory neural crest cells, a developmentally critical cell type and model for metastasis, release and deposit CD63-positive 30-100 nm particles into the extracellular environment. Neural crest cells contain ceramide-rich multivesicular bodies and produce larger vesicles positive for migrasome markers as well. We conclude that neural crest cells produce extracellular vesicles including exosomes and migrasomes. When Rab27a plasma membrane docking is inhibited, neural crest cells become less polarized and rounded, leading to a loss of directional migration and reduced speed. These results indicate that neural crest cell exosome release is critical for migration.

Progress on the regulation of neural crest and the genetics in craniofacial development.

The craniofacial features endow vertebrates with unparalleled evolutionary advantages. The craniofacial is composed of bone, cartilage, nerves, and connective tissues mainly developed from cranial neural crest cells (cNCCs). These tissues form complex organs which enable vertebrates to have powerful neural and sensory systems. NCCs are groups of migratory and pluripotent cells that are specific to vertebrates. The specification, premigration and migration, proliferation, and fate determination of the NCCs are precisely and sequentially controlled by gene regulatory networks, to ensure the ordered and accurate development of the craniofacial region. The craniofacial region represents a combined set of highly heritable phenotypes, which could be illustrated by the inherited facial features between relatives but perceptible differences among non-relatives. Such phenomena are termed heredity and variation, which are in accordance with the precision and plasticity of cNCCs gene regulatory network, respectively. Evidence has shown that genetic variations within the regulatory network alter the proliferation and differentiation of NCCs within a tolerable range, while deleterious mutations will lead to craniofacial malformations. In this review, we first summarize the development procedure of NCCs and their gene regulatory networks and then provide an overview on the genetic basis of the facial morphology and malformations. This review will benefit the understanding of craniofacial development and the prevention of craniofacial diseases.

SOX10: 20 years of phenotypic plurality and current understanding of its developmental function.

SOX10 belongs to a family of 20 SRY (sex-determining region Y)-related high mobility group box-containing (SOX) proteins, most of which contribute to cell type specification and differentiation of various lineages. The first clue that SOX10 is essential for development, especially in the neural crest, came with the discovery that heterozygous mutations occurring within and around SOX10 cause Waardenburg syndrome type 4. Since then, heterozygous mutations have been reported in Waardenburg syndrome type 2 (Waardenburg syndrome type without Hirschsprung disease), PCWH or PCW (peripheral demyelinating neuropathy, central dysmyelination, Waardenburg syndrome, with or without Hirschsprung disease), intestinal manifestations beyond Hirschsprung (ie, chronic intestinal pseudo-obstruction), Kallmann syndrome and cancer. All of these diseases are consistent with the regulatory role of SOX10 in various neural crest derivatives (melanocytes, the enteric nervous system, Schwann cells and olfactory ensheathing cells) and extraneural crest tissues (inner ear, oligodendrocytes). The recent evolution of medical practice in constitutional genetics has led to the identification of SOX10 variants in atypical contexts, such as isolated hearing loss or neurodevelopmental disorders, making them more difficult to classify in the absence of both a typical phenotype and specific expertise. Here, we report novel mutations and review those that have already been published and their functional consequences, along with current understanding of SOX10 function in the affected cell types identified through in vivo and in vitro models. We also discuss research options to increase our understanding of the origin of the observed phenotypic variability and improve the diagnosis and medical care of affected patients.

Cell Fate Decisions in the Neural Crest, from Pigment Cell to Neural Development.

The neural crest shows an astonishing multipotency, generating multiple neural derivatives, but also pigment cells, skeletogenic and other cell types. The question of how this process is controlled has been the subject of an ongoing debate for more than 35 years. Based upon new observations of zebrafish pigment cell development, we have recently proposed a novel, dynamic model that we believe goes some way to resolving the controversy. Here, we will firstly summarize the traditional models and the conflicts between them, before outlining our novel model. We will also examine our recent dynamic modelling studies, looking at how these reveal behaviors compatible with the biology proposed. We will then outline some of the implications of our model, looking at how it might modify our views of the processes of fate specification, differentiation, and commitment.

Single-cell RNA analysis identifies pre-migratory neural crest cells expressing markers of differentiated derivatives.

The neural crest is a migratory population of stem-like cells that contribute to multiple traits including the bones of the skull, peripheral nervous system, and pigment. How neural crest cells differentiate into diverse cell types is a fundamental question in the study of vertebrate biology. Here, we use single-cell RNA sequencing to characterize transcriptional changes associated with neural crest cell development in the zebrafish trunk during the early stages of migration. We show that neural crest cells are transcriptionally diverse and identify pre-migratory populations already expressing genes associated with differentiated derivatives, specifically in the xanthophore lineage. Further, we identify a population of Rohon-Beard neurons in the data. The data presented identify novel genetic markers for multiple trunk neural crest cell populations and Rohon-Beard neurons providing insight into previously uncharacterized genes critical for vertebrate development.

Insulin Promotes Schwann-Like Cell Differentiation of Rat Epidermal Neural Crest Stem Cells.

Schwann cells (SCs) are considered potentially attractive candidates for transplantation therapies in neurodegenerative diseases. However, problems arising from the isolation and expansion of the SCs restrict their clinical applications. Establishing an alternative Schwann-like cell type is a prerequisite. Epidermal neural crest stem cells (EPI-NCSCs) are well studied for their autologous accessibility, along with the ability to produce major neural crest derivatives and neurotrophic factors. In the current study, we explored insulin influence, a well-known growth factor, on directing EPI-NCSCs into the Schwann cell (SC) lineage. EPI-NCSCs were isolated from rat hair bulge explants. The viability of cells treated with a range of insulin concentrations (0.05-100 µg/ml) was defined by MTT assay at 24, 48, and 72 h. The gene expression profiles of neurotrophic factors (BDNF, FGF-2, and IL-6), key regulators involved in the development of SC (EGR-1, SOX-10, c-JUN, GFAP, OCT-6, EGR-2, and MBP), and oligodendrocyte (PDGFR-α and NG-2) were quantified 1 and 9 days post-treatment with 0.05 and 5 µg/ml insulin. Furthermore, the protein expression of nestin (stemness marker), SOX-10, PDGFR-α, and MBP was analyzed following the long-term insulin treatment. Insulin downregulated the early-stage SC differentiation marker (EGR-1) and increased neurotrophins (BDNF and IL-6) and pro-myelinating genes, including OCT-6, SOX-10, EGR-2, and MBP, as well as oligodendrocyte differentiation markers, upon exposure for 9 days. Insulin can promote EPI-NCSC differentiation toward SC lineage and possibly oligodendrocytes. Thus, employing insulin might enhance the EPI-NCSCs efficiency in cell transplantation strategies.

Altered sacral neural crest development in Pax3 spina bifida mutants underlies deficits of bladder innervation and function.

Mouse models of Spina bifida (SB) have been instrumental for identifying genes, developmental processes, and environmental factors that influence neurulation and neural tube closure. Beyond the prominent neural tube defects, other aspects of the nervous system can be affected in SB with significant changes in essential bodily functions such as urination. SB patients frequently experience bladder dysfunction and SB fetuses exhibit reduced density of bladder nerves and smooth muscle although the developmental origins of these deficits have not been determined. The Pax3 Splotch-delayed (Pax3Sp-d) mouse model of SB is one of a very few mouse SB models that survives to late stages of gestation. Through analysis of Pax3Sp-d mutants we sought to define how altered bladder innervation in SB might arise by tracing sacral neural crest (NC) development, pelvic ganglia neuronal differentiation, and assessing bladder nerve fiber density. In Pax3Sp-d/Sp-d fetal mice we observed delayed migration of Sox10+ NC-derived progenitors (NCPs), deficient pelvic ganglia neurogenesis, and reduced density of bladder wall innervation. We further combined NC-specific deletion of Pax3 with the constitutive Pax3Sp-d allele in an effort to generate viable Pax3 mutants to examine later stages of bladder innervation and postnatal bladder function. Neural crest specific deletion of a Pax3 flox allele, using a Sox10-cre driver, in combination with a constitutive Pax3Sp-d mutation produced postnatal viable offspring that exhibited altered bladder function as well as reduced bladder wall innervation and altered connectivity between accessory ganglia at the bladder neck. Combined, the results show that Pax3 plays critical roles within sacral NC that are essential for initiation of neurogenesis and differentiation of autonomic neurons within pelvic ganglia.

Profiling NSD3-dependent neural crest gene expression reveals known and novel candidate regulatory factors.

The lysine methyltransferase NSD3 is required for the expression of key neural crest transcription factors and the migration of neural crest cells. Nevertheless, a complete view of the genes dependent upon NSD3 for expression and the developmental processes impacted by NSD3 in the neural crest was lacking. We used RNA sequencing (RNA-seq) to profile transcripts differentially expressed after NSD3 knockdown in chick premigratory neural crest cells, identifying 674 genes. Gene Ontology and gene set enrichment analyses further support a requirement for NSD3 during neural crest development and show that NSD3 knockdown also upregulates ribosome biogenesis. To validate our results, we selected three genes not previously associated with neural crest development, Astrotactin 1 (Astn1), Dispatched 3 (Disp3), and Tropomyosin 1 (Tpm1). Using whole mount in situ hybridization, we show that premigratory neural crest cells express these genes and that NSD3 knockdown downregulates (Astn1 and Disp3) and upregulates (Tpm1) their expression, consistent with RNA-seq results. Altogether, this study identifies novel putative regulators of neural crest development and provides insight into the transcriptional consequences of NSD3 in the neural crest, with implications for cancer.

Symmetry and fluctuation of cell movements in neural crest-derived facial mesenchyme.

In the face, symmetry is established when bilateral streams of neural crest cells leave the neural tube at the same time, follow identical migration routes and then give rise to the facial prominences. However, developmental instability exists, particularly surrounding the steps of lip fusion. The causes of instability are unknown but inability to cope with developmental fluctuations are a likely cause of congenital malformations, such as non-syndromic orofacial clefts. Here, we tracked cell movements over time in the frontonasal mass, which forms the facial midline and participates in lip fusion, using live-cell imaging of chick embryos. Our mathematical examination of cell velocity vectors uncovered temporal fluctuations in several parameters, including order/disorder, symmetry/asymmetry and divergence/convergence. We found that treatment with a Rho GTPase inhibitor completely disrupted the temporal fluctuations in all measures and blocked morphogenesis. Thus, we discovered that genetic control of symmetry extends to mesenchymal cell movements and that these movements are of the type that could be perturbed in asymmetrical malformations, such as non-syndromic cleft lip. This article has an associated 'The people behind the papers' interview.

Trio cooperates with Myh9 to regulate neural crest-derived craniofacial development.

Trio is a unique member of the Rho-GEF family that has three catalytic domains and is vital for various cellular processes in both physiological and developmental settings. TRIO mutations in humans are involved in craniofacial abnormalities, in which patients present with mandibular retrusion. However, little is known about the molecular mechanisms of Trio in neural crest cell (NCC)-derived craniofacial development, and there is still a lack of direct evidence to assign a functional role to Trio in NCC-induced craniofacial abnormalities. Methods:In vivo, we used zebrafish and NCC-specific knockout mouse models to investigate the phenotype and dynamics of NCC development in Trio morphants. In vitro, iTRAQ, GST pull-down assays, and proximity ligation assay (PLA) were used to explore the role of Trio and its potential downstream mediators in NCC migration and differentiation. Results: In zebrafish and mouse models, disruption of Trio elicited a migration deficit and impaired the differentiation of NCC derivatives, leading to craniofacial growth deficiency and mandibular retrusion. Moreover, Trio positively regulated Myh9 expression and directly interacted with Myh9 to coregulate downstream cellular signaling in NCCs. We further demonstrated that disruption of Trio or Myh9 inhibited Rac1 and Cdc42 activity, specifically affecting the nuclear export of ß-catenin and NCC polarization. Remarkably, craniofacial abnormalities caused by trio deficiency in zebrafish could be partially rescued by the injection of mRNA encoding myh9, ca-Rac1, or ca-Cdc42. Conclusions: Here, we identified that Trio, interacting mostly with Myh9, acts as a key regulator of NCC migration and differentiation during craniofacial development. Our results indicate that trio morphant zebrafish and Wnt1-cre;Triofl/fl mice offer potential model systems to facilitate the study of the pathogenic mechanisms of Trio mutations causing craniofacial abnormalities.

The Special Developmental Biology of Craniofacial Tissues Enables the Understanding of Oral and Maxillofacial Physiology and Diseases.

Maxillofacial hard tissues have several differences compared to bones of other localizations of the human body. These could be due to the different embryological development of the jaw bones compared to the extracranial skeleton. In particular, the immigration of neuroectodermally differentiated cells of the cranial neural crest (CNC) plays an important role. These cells differ from the mesenchymal structures of the extracranial skeleton. In the ontogenesis of the jaw bones, the development via the intermediate stage of the pharyngeal arches is another special developmental feature. The aim of this review was to illustrate how the development of maxillofacial hard tissues occurs via the cranial neural crest and pharyngeal arches, and what significance this could have for relevant pathologies in maxillofacial surgery, dentistry and orthodontic therapy. The pathogenesis of various growth anomalies and certain syndromes will also be discussed.

Runx2-Twist1 interaction coordinates cranial neural crest guidance of soft palate myogenesis.

Cranial neural crest (CNC) cells give rise to bone, cartilage, tendons, and ligaments of the vertebrate craniofacial musculoskeletal complex, as well as regulate mesoderm-derived craniofacial muscle development through cell-cell interactions. Using the mouse soft palate as a model, we performed an unbiased single-cell RNA-seq analysis to investigate the heterogeneity and lineage commitment of CNC derivatives during craniofacial muscle development. We show that Runx2, a known osteogenic regulator, is expressed in the CNC-derived perimysial and progenitor populations. Loss of Runx2 in CNC-derivatives results in reduced expression of perimysial markers (Aldh1a2 and Hic1) as well as soft palate muscle defects in Osr2-Cre;Runx2fl/fl mice. We further reveal that Runx2 maintains perimysial marker expression through suppressing Twist1, and that myogenesis is restored in Osr2-Cre;Runx2fl/fl;Twist1fl/+ mice. Collectively, our findings highlight the roles of Runx2, Twist1, and their interaction in regulating the fate of CNC-derived cells as they guide craniofacial muscle development through cell-cell interactions.