Cranial Mesoderm Research Articles

Retinoic acid signalling regulates branchiomeric neck muscle development at the head/trunk interface.

Skeletal muscles of the head and trunk originate in distinct lineages with divergent regulatory programmes converging on activation of myogenic determination factors. Branchiomeric head and neck muscles share a common origin with cardiac progenitor cells in cardiopharyngeal mesoderm (CPM). The retinoic acid (RA) signalling pathway is required during a defined early time window for normal deployment of cells from posterior CPM to the heart. Here, we show that blocking RA signalling in the early mouse embryo also results in selective loss of the trapezius neck muscle, without affecting other skeletal muscles. RA signalling is required for robust expression of myogenic determination factors in posterior CPM and subsequent expansion of the trapezius primordium. Lineage-specific activation of a dominant-negative RA receptor reveals that trapezius development is not regulated by direct RA signalling to myogenic progenitor cells in CPM, or through neural crest cells, but indirectly through the somitic lineage, closely apposed with posterior CPM in the early embryo. These findings suggest that trapezius development is dependent on precise spatiotemporal interactions between cranial and somitic mesoderm at the head/trunk interface.

Lineage-specific requirements of Alx4 function in craniofacial and hair development.

Disruption of ALX4 causes autosomal dominant parietal foramina and autosomal recessive frontonasal dysplasia with alopecia, but the mechanisms involving ALX4 in craniofacial and other developmental processes are not well understood. Although mice carrying distinct mutations in Alx4 have been previously reported, the perinatal lethality of homozygous mutants together with dynamic patterns of Alx4 expression in multiple tissues have hindered systematic elucidation of the cellular and molecular mechanisms involving Alx4 in organogenesis and disease pathogenesis. We report generation of Alx4f/f conditional mice and show that tissue-specific Cre-mediated inactivation of Alx4 in cranial neural crest and limb bud mesenchyme, respectively, recapitulated craniofacial and limb developmental defects as found in Alx4-null mice but without affecting postnatal survival. While Alx4-null mice that survive postnatally exhibited dorsal alopecia, mice lacking Alx4 function in the neural crest lineage exhibited a highly restricted region of hair loss over the anterior skull whereas mice lacking Alx4 in the cranial mesoderm lineage exhibited normal hair development, suggesting that Alx4 plays partly redundant roles in multiple cell lineages during hair follicle development. The Alx4f/f mice provide a valuable resource for systematic investigation of cell type- and stage-specific function of ALX family transcription factors in development and disease.

Loss of Neogenin alters branchial arch development and leads to craniofacial skeletal defects.

The formation of complex structures, such as the craniofacial skeleton, requires precise and intricate two-way signalling between populations of cells of different embryonic origins. For example, the lower jaw, or mandible, arises from cranial neural crest cells (CNCCs) in the mandibular portion of the first branchial arch (mdBA1) of the embryo, and its development is regulated by signals from the ectoderm and cranial mesoderm (CM) within this structure. The molecular mechanisms underlying CM cell influence on CNCC development in the mdBA1 remain poorly defined. Herein we identified the receptor Neogenin as a key regulator of craniofacial development. We found that ablation of Neogenin expression via gene-targeting resulted in several craniofacial skeletal defects, including reduced size of the CNCC-derived mandible. Loss of Neogenin did not affect the formation of the mdBA1 CM core but resulted in altered Bmp4 and Fgf8 expression, increased apoptosis, and reduced osteoblast differentiation in the mdBA1 mesenchyme. Reduced BMP signalling in the mdBA1 of Neogenin mutant embryos was associated with alterations in the gene regulatory network, including decreased expression of transcription factors of the Hand, Msx, and Alx families, which play key roles in the patterning and outgrowth of the mdBA1. Tissue-specific Neogenin loss-of-function studies revealed that Neogenin expression in mesodermal cells contributes to mandible formation. Thus, our results identify Neogenin as a novel regulator of craniofacial skeletal formation and demonstrates it impinges on CNCC development via a non-cell autonomous mechanism.

Cxcl12a plays an essential role in pharyngeal cartilage development.

Background: Neural crest cells constitute a distinct set of multipotent cells that undergo migration along predefined pathways, culmination in the differentiation into a plethora of cell types, including components of the pharyngeal cartilage. The neurocranium is composite structure derived from both cranial neural crest and mesoderm cells, whereas the pharyngeal skeletal elements-including the mandibular and branchial arches-are exclusively formed by craniofacial neural crest cells. Previous studies have elucidated the critical involvement of the chemokine signaling axis Cxcl12b/Cxcr4a in craniofacial development in zebrafish (Danio rerio). Nonetheless, the function contribution of Cxcl12a and Cxcr4b-the homologous counterparts of Cxcl12b and Cxcr4a-remain largely unexplored. Methods: In the present study, mutant lines for cxcl12a and cxcr4b were generated employing CRISPR/Cas9 system. Temporal and spatial expression patterns of specific genes were assessed using in situ hybridization and dual-color fluorescence in situ hybridization techniques. High-resolution confocal microscopy was utilized for in vivo imaging to detect the pharyngeal arch or pouch patterning. Additionally, cartilage formation within the craniofacial region was analyzed via Alcian blue staining, and the proliferation and apoptosis rates of craniofacial neural crest cells were quantified through BrdU incorporation and TUNEL staining. Results: Our data reveals that the deletion of the chemokine gene cxcl12a results in a marked diminution of pharyngeal cartilage elements, attributable to compromised proliferation of post-migratory craniofacial neural crest cells. Subsequent experiments confirmed that Cxcl12a and Cxcl12b exhibit a synergistic influence on pharyngeal arch and pouch formation. Conclusion: Collectively, the present investigation furnishes compelling empirical evidence supporting the indispensable role of Cxcl2a in craniofacial cartilage morphogenesis, albeit cxcr4b mutants exert a minimal impact on this biological process. We delineate that Cxcl12a is essential for chondrogenesis in zebrafish, primarily by promoting the proliferation of craniofacial neural crest cells. Furthermore, we proposed a conceptual framework wherein Cxcl12a and Cxcl12b function synergistically in orchestrating both the pharyngeal arch and pouch morphogenesis.

Foxl2a and Foxl2b are involved in midbrain-hindbrain boundary development in zebrafish

Foxl2 plays conserved central function in ovarian differentiation and maintenance in several fish species. However, its expression pattern and function in fish embryogenesis are still largely unknown. In this study, we first presented a sequential expression pattern of zebrafish foxl2a and foxl2b during embryo development. They were predominantly expressed in the cranial paraxial mesoderm (CPM) and cranial venous vasculature (CVV) during somitogenesis and subsequently expressed in the pharyngeal arches after 48 h post-fertilization (hpf). Then, we compared the brain structures among zebrafish wildtype (WT) and three homozygous foxl2 mutants (foxl2a−/−, foxl2b−/− and foxl2a−/−;foxl2b−/−) and found the reduction of the fourth ventricle in the three foxl2 mutants, especially in foxl2a−/−;foxl2b−/− mutant. Finally, we detected several key transcription factors involved in the gene regulatory network of midbrain-hindbrain boundary (MHB) patterning, such as wnt1, en1b and pax2a. Their expression levels were obviously downregulated in MHB of foxl2a−/− and foxl2a−/−;foxl2b−/− mutants. Thus, we suggest that Foxl2a and Foxl2b are involved in MHB and the fourth ventricle development in zebrafish. The current study provides insights into the molecular mechanism underlying development of brain ventricular system.

TWIST1, a gene associated with Saethre-Chotzen syndrome, regulates extraocular muscle organization in mouse

Heterozygous loss of function mutations in TWIST1 cause Saethre-Chotzen syndrome, which is characterized by craniosynostosis, facial asymmetry, ptosis, strabismus, and distinctive ear appearance. Individuals with syndromic craniosynostosis have high rates of strabismus and ptosis, but the underlying pathology is unknown. Some individuals with syndromic craniosynostosis have been noted to have absence of individual extraocular muscles or abnormal insertions of the extraocular muscles on the globe. Using conditional knock-out alleles for Twist1 in cranial mesenchyme, we test the hypothesis that Twist1 is required for extraocular muscle organization and position, attachment to the globe, and/or innervation by the cranial nerves. We examined the extraocular muscles in conditional Twist1 knock-out animals using Twist2-cre and Pdgfrb-cre drivers. Both are expressed in cranial mesoderm and neural crest. Conditional inactivation of Twist1 using these drivers leads to disorganized extraocular muscles that cannot be reliably identified as specific muscles. Tendons do not form normally at the insertion and origin of these dysplastic muscles. Knock-out of Twist1 expression in tendon precursors, using scleraxis-cre, however, does not alter EOM organization. Furthermore, developing motor neurons, which do not express Twist1, display abnormal axonal trajectories in the orbit in the presence of dysplastic extraocular muscles. Strabismus in individuals with TWIST1 mutations may therefore be caused by abnormalities in extraocular muscle development and secondary abnormalities in innervation and tendon formation.

Identification of bipotent progenitors that give rise to myogenic and connective tissues in mouse.

How distinct cell fates are manifested by direct lineage ancestry from bipotent progenitors, or by specification of individual cell types is a key question for understanding the emergence of tissues. The interplay between skeletal muscle progenitors and associated connective tissue cells provides a model for examining how muscle functional units are established. Most craniofacial structures originate from the vertebrate-specific neural crest cells except in the dorsal portion of the head, where they arise from cranial mesoderm. Here, using multiple lineage-tracing strategies combined with single cell RNAseq and in situ analyses, we identify bipotent progenitors expressing Myf5 (an upstream regulator of myogenic fate) that give rise to both muscle and juxtaposed connective tissue. Following this bifurcation, muscle and connective tissue cells retain complementary signalling features and maintain spatial proximity. Disrupting myogenic identity shifts muscle progenitors to a connective tissue fate. The emergence of Myf5-derived connective tissue is associated with the activity of several transcription factors, including Foxp2. Interestingly, this unexpected bifurcation in cell fate was not observed in craniofacial regions that are colonised by neural crest cells. Therefore, we propose that an ancestral bi-fated program gives rise to muscle and connective tissue cells in skeletal muscles that are deprived of neural crest cells.

The CXCR4/SDF-1 Axis in the Development of Facial Expression and Non-somitic Neck Muscles.

Trunk and head muscles originate from distinct embryonic regions: while the trunk muscles derive from the paraxial mesoderm that becomes segmented into somites, the majority of head muscles develops from the unsegmented cranial paraxial mesoderm. Differences in the molecular control of trunk versus head and neck muscles have been discovered about 25 years ago; interestingly, differences in satellite cell subpopulations were also described more recently. Specifically, the satellite cells of the facial expression muscles share properties with heart muscle. In adult vertebrates, neck muscles span the transition zone between head and trunk. Mastication and facial expression muscles derive from the mesodermal progenitor cells that are located in the first and second branchial arches, respectively. The cucullaris muscle (non-somitic neck muscle) originates from the posterior-most branchial arches. Like other subclasses within the chemokines and chemokine receptors, CXCR4 and SDF-1 play essential roles in the migration of cells within a number of various tissues during development. CXCR4 as receptor together with its ligand SDF-1 have mainly been described to regulate the migration of the trunk muscle progenitor cells. This review first underlines our recent understanding of the development of the facial expression (second arch-derived) muscles, focusing on new insights into the migration event and how this embryonic process is different from the development of mastication (first arch-derived) muscles. Other muscles associated with the head, such as non-somitic neck muscles derived from muscle progenitor cells located in the posterior branchial arches, are also in the focus of this review. Implications on human muscle dystrophies affecting the muscles of face and neck are also discussed.

Generation of craniofacial myogenic progenitor cells from human induced pluripotent stem cells for skeletal muscle tissue regeneration

Craniofacial skeletal muscle is composed of approximately 60 muscles, which have critical functions including food uptake, eye movements and facial expressions. Although craniofacial muscles have significantly different embryonic origin, most current skeletal muscle differentiation protocols using human induced pluripotent stem cells (iPSCs) are based on somite-derived limb and trunk muscle developmental pathways. Since the lack of a protocol for craniofacial muscles is a significant gap in the iPSC-derived muscle field, we have developed an optimized protocol to generate craniofacial myogenic precursor cells (cMPCs) from human iPSCs by mimicking key signaling pathways during craniofacial embryonic myogenesis. At each different stage, human iPSC-derived cMPCs mirror the transcription factor expression profiles seen in their counterparts during embryo development. After the bi-potential cranial pharyngeal mesoderm is established, cells are committed to cranial skeletal muscle lineages with inhibition of cardiac lineages and are purified by flow cytometry. Furthermore, identities of iPSC-derived cMPCs are verified with human primary myoblasts from craniofacial muscles using RNA sequencing. These data suggest that our new method could provide not only in vitro research tools to study muscle specificity of muscular dystrophy but also abundant and reliable cellular resources for tissue engineering to support craniofacial reconstruction surgery.

Cxcr4 and Sdf-1 are critically involved in the formation of facial and non-somitic neck muscles

The present study shows that the CXCR4/SDF-1 axis regulates the migration of second branchial arch-derived muscles as well as non-somitic neck muscles. Cxcr4 is expressed by skeletal muscle progenitor cells in the second branchial arch (BA2). Muscles derived from the second branchial arch, but not from the first, fail to form in Cxcr4 mutants at embryonic days E13.5 and E14.5. Cxcr4 is also required for the development of non-somitic neck muscles. In Cxcr4 mutants, non-somitic neck muscle development is severely perturbed. In vivo experiments in chicken by means of loss-of-function approach based on the application of beads loaded with the CXCR4 inhibitor AMD3100 into the cranial paraxial mesoderm resulted in decreased expression of Tbx1 in the BA2. Furthermore, disrupting this chemokine signal at a later stage by implanting these beads into the BA2 caused a reduction in MyoR, Myf5 and MyoD expression. In contrast, gain-of-function experiments based on the implantation of SDF-1 beads into BA2 resulted in an attraction of myogenic progenitor cells, which was reflected in an expansion of the expression domain of these myogenic markers towards the SDF-1 source. Thus, Cxcr4 is required for the formation of the BA2 derived muscles and non-somitic neck muscles.

In ovo technique for cell injection in the CPM followed by bead implantation in the BA2 of chicken embryos

The major advantage of chicken embryos model is their accessibility for microsurgical manipulations and the dissection of tissues for ex vivo explant culture. Branchial arches are embryonic structure located next to the top of developing heart. Each arch is made of surface ectoderm, endoderm, myogenic mesoderm cells and cranial neural crest cells. The myogenic mesoderm originates from cranial paraxial mesoderm (CPM), which is transiently migrated to branchial arches (BAs). The first branchial arch (BA1) mesoderm contributes to formation of mastication muscles. The second branchial arch (BA2) mesoderm gives rise to facial expression muscles. This article focuses on cell injection in the CPM and bead implantation (gain of function approaches) in the BA2. In order to follow the migration of mesoderm progenitor cells from CPM to BA2, we injected quail cells in the CPM of stage HH10-11 embryos, followed by implantation of SDF-1 bead at stage HH15-16. Later the attraction of quail cells (CXCR4+) towards the SDF-1 source has been observed, using whole-mount immunostaining of a specific quail antibody (QCPN) at stage HH19-22.•Our method, which involves bead implantation followed by quail cell injection, provides useful tools for tracing migratory mesodermal cells in vivo.•The proposed method does not require any commercial kits and can be used for various developmental process.•It does not employ any complicated methods such as genetically engineered permanent cell labeling, multiplicity of fluorescent markers or clonal analysis.

Vertebrate cranial mesoderm: developmental trajectory and evolutionary origin.

Vertebrate cranial mesoderm is a discrete developmental unit compared to the mesoderm below the developing neck. An extraordinary feature of the cranial mesoderm is that it includes a common progenitor pool contributing to the chambered heart and the craniofacial skeletal muscles. This striking developmental potential and the excitement it generated led to advances in our understanding of cranial mesoderm developmental mechanism. Remarkably, recent findings have begun to unravel the origin of its distinct developmental characteristics. Here, we take a detailed view of the ontogenetic trajectory of cranial mesoderm and its regulatory network. Based on the emerging evidence, we propose that cranial and posterior mesoderm diverge at the earliest step of the process that patterns the mesoderm germ layer along the anterior-posterior body axis. Further, we discuss the latest evidence and their impact on our current understanding of the evolutionary origin of cranial mesoderm. Overall, the review highlights the findings from contemporary research, which lays the foundation to probe the molecular basis of unique developmental potential and evolutionary origin of cranial mesoderm.

The role of chick Ebf genes in the mediolateral patterning of the somites.

This study was conducted to check whether the three chick Early B-cell Factor (Ebf) genes, particularly cEbf1, would be targets for Shh and Bmp signals during somites mediolateral (ML) patterning. Tissue manipulations and gain and loss of function experiments for Shh and Bmp4 were performed and the results revealed that cEbf1 expression was initiated in the cranial presomitic mesoderm by low dose of Bmp4 from the lateral mesoderm and maintained in the ventromedial part of the epithelial somite and the medial sclerotome by Shh from the notochord; while cEbf2/3 expression was induced and maintained by Bmp4 and inhibited by high dose of Shh. To determine whether Ebf1 plays a role in somite patterning, transfection of a dominant-negative construct was carried out; this showed suppression of cPax1 expression in the medial sclerotome and upregulation and medial expansion of cEbf3 and cPax3 expression in sclerotome and dermomyotome, respectively, suggesting that Ebf1 is important for ML patterning. Thus, it is possible that low doses of Bmp4 set up Ebf1 expression which, together with Shh from the notochord, leads to establishment of the medial sclerotome and suppression of lateral identities. These data also conclude that Bmp4 is required in both the medial and lateral domain of the somitic mesoderm to keep the ML identity of the sclerotome through maintenance of cEbf gene expression. These striking findings are novel and give a new insight on the role of Bmp4 on mediolateral patterning of somites.

Gene profiling of head mesoderm in early zebrafish development: insights into the evolution of cranial mesoderm

BackgroundThe evolution of the head was one of the key events that marked the transition from invertebrates to vertebrates. With the emergence of structures such as eyes and jaws, vertebrates evolved an active and predatory life style and radiated into diversity of large-bodied animals. These organs are moved by cranial muscles that derive embryologically from head mesoderm. Compared with other embryonic components of the head, such as placodes and cranial neural crest cells, our understanding of cranial mesoderm is limited and is restricted to few species.ResultsHere, we report the expression patterns of key genes in zebrafish head mesoderm at very early developmental stages. Apart from a basic anterior–posterior axis marked by a combination of pitx2 and tbx1 expression, we find that most gene expression patterns are poorly conserved between zebrafish and chick, suggesting fewer developmental constraints imposed than in trunk mesoderm. Interestingly, the gene expression patterns clearly show the early establishment of medial–lateral compartmentalisation in zebrafish head mesoderm, comprising a wide medial zone flanked by two narrower strips.ConclusionsIn zebrafish head mesoderm, there is no clear molecular regionalisation along the anteroposterior axis as previously reported in chick embryos. In contrast, the medial–lateral regionalisation is formed at early developmental stages. These patterns correspond to the distinction between paraxial mesoderm and lateral plate mesoderm in the trunk, suggesting a common groundplan for patterning head and trunk mesoderm. By comparison of these expression patterns to that of amphioxus homologues, we argue for an evolutionary link between zebrafish head mesoderm and amphioxus anteriormost somites.

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development.

SummaryThe neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1–900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

Embryologic and Fetal Development of the Human Orbit.

To review the recent data about orbital development and sort out the controversies from the very early stages during embryonic life till final maturation of the orbit late in fetal life, and to appreciate the morphogenesis of all the definitive structures in the orbit in a methodical and timely fashion. The authors extensively review major studies detailing every aspect of human embryologic and fetal orbital morphogenesis including the development of extraocular muscles, orbital fat, vessels, nerves, and the supportive connective tissue framework as well as bone. These interdisciplinary studies span almost a century and a half, and include some significant controversial opposing points of view which the authors hopefully sort out. The authors also highlight a few of the most noteworthy molecular biologic studies regarding the multiple and interacting signaling pathways involved in regulating normal orbital morphogenesis. Orbital morphogenesis involves a successive series of subtle yet tightly regulated morphogenetic events that could only be explained through the chronological narrative used by the authors. The processes that trigger and contribute to the formation of the orbits are complex and seem to be intricately regulated by multifaceted interactions and bidirectional cross-talk between a multitude of cellular building raw materials including the developing optic vesicles, neuroectoderm, cranial neural crest cells and mesoderm. Development of the orbit is a collective enterprise necessitating interactions between, as well as contributions from different cell populations both within and beyond the realm of the orbit. A basic understanding of the processes underlying orbital ontogenesis is a crucial first step toward establishing a genetic basis or an embryologic link with orbital disease.

Expression of Nkx2.5 and Isl1 in the cardiopharyngeal mesoderm of developing Xenopus embryos

The cardiopharyngeal field (CPF) is a mesodermal field that gives rise to the first (FHF) and second heart field (SHF)‐derived myocardium and the branchiomeric musculature. Isl1 and Nkx2.5 are transcription factors expressed in the CPF. Both genes are crucial for the specification of the myocardial cells derived from the SHF and for the further development of the heart in vertebrates. The importance of these genes is supported by the fact that perturbations in their expression lead to severe heart malformations and often result in embryonic death. In order to better understand the role of these two genes and their impact on the head and heart development, we analysed their expression in Xenopus laevis, which is potentially a good model to study the CPF and craniofacial defects in terms of evolutionary developmental medicine. In contrast to basal vertebrates, such as the urochordate Ciona, Xenopus' heart has an advanced morphology with several heart chambers and in‐ and outflow tracts. Concurrently, Xenopus is easy to rear in laboratory conditions. There are various genetic tools available to work with this species (e.g., TALENs, CRISPR/Cas9, ZFNs) and there are certain advantages for the study of embryology (e.g., external fertilization and large size of eggs) that favour this species over the mouse and chicken models.Whole‐mount in situ hybridization of embryos revealed that during early stages Isl1 and Nkx2.5 are co‐expressed in cardiac progenitor cells localized near the cement gland. Isl1 was also present in the stomodeum region and other cell populations; e.g., cephalic placodes and their associated ganglia or cranial paraxial mesoderm. At stage 25 (Nieuwkoop and Faber), Nkx2.5 was expressed in the FHF, SHF, and anterior endoderm. Later, it nearly separated from the Isl1 expression and became restricted to the heart tube, derived from the FHF. Isl1 was mainly expressed in the pharyngeal arch mesoderm adjacent to the heart and in the dorsolateral heart domain. At late stages, Nkx2.5 could be detected throughout the myocardium, while Isl1 was present in the foregut and cephalic ganglia. In spite of minor differences, our results conform to early studies in X. laevis. The comparison with expression patterns in mouse and chicken shows that Xenopus is indeed a good organism to study the CPF derivatives, like the myocardium and head musculature. Therefore, Xenopus can be further extensively used for developmental, evolutionary, and medical studies of the normal and abnormal head and heart myogenesis.Support or Funding InformationJMZ is supported by a Howard University College of Medicine pilot study award (BFPSAP: U100217).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DAN (NBL1) promotes collective neural crest migration by restraining uncontrolled invasion.

Neural crest cells are both highly migratory and significant to vertebrate organogenesis. However, the signals that regulate neural crest cell migration remain unclear. In this study, we test the function of differential screening-selected gene aberrant in neuroblastoma (DAN), a bone morphogenetic protein (BMP) antagonist we detected by analysis of the chick cranial mesoderm. Our analysis shows that, before neural crest cell exit from the hindbrain, DAN is expressed in the mesoderm, and then it becomes absent along cell migratory pathways. Cranial neural crest and metastatic melanoma cells avoid DAN protein stripes in vitro. Addition of DAN reduces the speed of migrating cells in vivo and in vitro, respectively. In vivo loss of function of DAN results in enhanced neural crest cell migration by increasing speed and directionality. Computer model simulations support the hypothesis that DAN restrains cell migration by regulating cell speed. Collectively, our results identify DAN as a novel factor that inhibits uncontrolled neural crest and metastatic melanoma invasion and promotes collective migration in a manner consistent with the inhibition of BMP signaling.

Transcriptional targets of TWIST1 in the cranial mesoderm regulate cell-matrix interactions and mesenchyme maintenance

TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. We have previously shown that, in the absence of TWIST1, cells within the cranial mesoderm adopt an abnormal epithelial configuration via a process reminiscent of a mesenchymal to epithelial transition (MET). Here, we show by gene expression analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. ChIP-seq was used to identify TWIST1-binding sites in an in vitro model of a TWIST1-dependent mesenchymal cell state, and the data were combined with the transcriptome data to identify potential target genes. Three direct transcriptional targets of TWIST1 (Ddr2, Pcolce and Tgfbi) were validated by ChIP-PCR using mouse embryonic tissues and by luciferase assays. Our findings reveal that the mesenchymal properties of the cranial mesoderm are likely to be regulated by a network of TWIST1 targets that influences the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures.

Tissue interactions, cell signaling and transcriptional control in the cranial mesoderm during craniofacial development

AbstractThe cranial neural crest and the cranial mesoderm are the source of tissues from which the bone and cartilage of the skull, face and jaws are constructed. The development of the cranial mesoderm is not well studied, which is inconsistent with its importance in craniofacial morphogenesis as a source of precursor tissue of the chondrocranium, muscles, vasculature and connective tissues, mechanical support for tissue morphogenesis, and the signaling activity that mediate interactions with the cranial neural crest. Phenotypic analysis of conditional knockout mouse mutants, complemented by the transcriptome analysis of differentially enriched genes in the cranial mesoderm and cranial neural crest, have identified signaling pathways that may mediate cross-talk between the two tissues. In the cranial mesenchyme, Bmp4 is expressed in the mesoderm cells while its signaling activity could impact on both the mesoderm and the neural crest cells. In contrast, Fgf8 is predominantly expressed in the cranial neural crest cells and it influences skeletal development and myogenesis in the cranial mesoderm. WNT signaling, which emanates from the cranial neural crest cells, interacts with BMP and FGF signaling in monitoring the switch between tissue progenitor expansion and differentiation. The transcription factor Twist1, a critical molecular regulator of many aspects of craniofacial development, coordinates the activity of the above pathways in cranial mesoderm and cranial neural crest tissue compartments.