A unique form of collective epithelial migration is crucial for tissue fusion in the secondary palate and can overcome loss of epithelial apoptosis.

Tissue fusion frequently requires the removal of an epithelium that intervenes distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion support their importance in MES removal. However, genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES results in complete loss of cell death and cell extrusion, but successful removal of the MES. Novel static- and live-imaging approaches reveal that the MES is removed through streaming migration of epithelial trails and islands to reach the oral and nasal epithelial surfaces. Epithelial trail cells that express the basal epithelial marker ΔNp63 begin to express periderm markers, suggesting that migration is concomitant with differentiation. Live imaging reveals anisotropic actomyosin contractility within epithelial trails, and genetic ablation of actomyosin contractility results in dispersion of epithelial collectives and failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis, and reveal a crucial and unique form of collective epithelial migration during tissue fusion.

Control of cranial ectomesenchyme fate by Nr2f nuclear receptors.

Certain cranial neural crest cells are uniquely endowed with the ability to make skeletal cell types otherwise only derived from mesoderm. As these cells migrate into the pharyngeal arches, they downregulate neural crest specifier genes and upregulate so-called ectomesenchyme genes that are characteristic of skeletal progenitors. Although both external and intrinsic factors have been proposed as triggers of this transition, the details remain obscure. Here, we report the Nr2f nuclear receptors as intrinsic activators of the ectomesenchyme program: zebrafish nr2f5 single and nr2f2;nr2f5 double mutants show marked delays in upregulation of ectomesenchyme genes, such as dlx2a, prrx1a, prrx1b, sox9a, twist1a and fli1a, and in downregulation of sox10, which is normally restricted to early neural crest and non-ectomesenchyme lineages. Mutation of sox10 fully rescued skeletal development in nr2f5 single but not nr2f2;nr2f5 double mutants, but the initial ectomesenchyme delay persisted in both. Sox10 perdurance thus antagonizes the recovery but does not explain the impaired ectomesenchyme transition. Unraveling the mechanisms of Nr2f function will help solve the enduring puzzle of how cranial neural crest cells transition to the skeletal progenitor state.

Embryonic requirements for Tcf12 in the development of the mouse coronal suture.

A major feature of Saethre-Chotzen syndrome is coronal craniosynostosis, the fusion of the frontal and parietal bones at the coronal suture. It is caused by heterozygous loss-of-function mutations in either of the bHLH transcription factors TWIST1 and TCF12. Although compound heterozygous Tcf12; Twist1 mice display severe coronal synostosis, the individual role of Tcf12 had remained unexplored. Here, we show that Tcf12 controls several key processes in calvarial development, including the rate of frontal and parietal bone growth, and the boundary between sutural and osteogenic cells. Genetic analysis supports an embryonic requirement for Tcf12 in suture formation, as combined deletion of Tcf12 in embryonic neural crest and mesoderm, but not in postnatal suture mesenchyme, disrupts the coronal suture. We also detected asymmetric distribution of mesenchymal cells on opposing sides of the wild-type frontal and parietal bones, which prefigures later bone overlap at the sutures. In Tcf12 mutants, reduced asymmetry is associated with bones meeting end-on-end, possibly contributing to synostosis. Our results support embryonic requirements of Tcf12 in proper formation of the overlapping coronal suture.

FGF signaling patterns cell fate at the interface between tendon and bone.

Tendon and bone are attached by a transitional connective tissue that is morphologically graded from tendinous to osseous and develops from bipotent progenitors that co-express scleraxis (Scx) and Sox9 (Scx+/Sox9+). Scx+/Sox9+ progenitors have the potential to differentiate into either tenocytes or chondrocytes, yet the developmental mechanism that spatially resolves their bipotency at the tendon-bone interface during embryogenesis remains unknown. Here, we demonstrate that development of Scx+/Sox9+ progenitors within the mammalian lower jaw requires FGF signaling. We find that loss of Fgfr2 in the mouse tendon-bone interface reduces Scx expression in Scx+/Sox9+ progenitors and induces their biased differentiation into Sox9+ chondrocytes. This expansion of Sox9+ chondrocytes, which is concomitant with decreased Notch2-Dll1 signaling, prevents formation of a mixed population of chondrocytes and tenocytes, and instead results in ectopic endochondral bone at tendon-bone attachment units. Our work shows that FGF signaling directs zonal patterning at the boundary between tendon and bone by regulating cell fate decisions through a mechanism that employs Notch signaling.

Frizzled10 mediates WNT1 and WNT3A signaling in the dorsal spinal cord of the developing chick embryo.

BACKGROUND: WNT1 and WNT3A drive a dorsal to ventral gradient of ß-catenin-dependent Wnt signaling in the developing spinal cord. However, the identity of the receptors mediating downstream functions remains poorly understood. RESULTS: In this report, we show that the spatiotemporal expression patterns of FZD10 and WNT1/WNT3A are highly correlated. We further show that in the presence of LRP6, FZD10 promotes WNT1 and WNT3A signaling using an 8xSuperTopFlash reporter assay. Consistent with a functional role for FZD10, we demonstrate that FZD10 is required for proliferation in the spinal cord. Finally, by using an in situ proximity ligation assay, we observe an interaction between FZD10 and WNT1 and WNT3A proteins. CONCLUSIONS: Together, our results identify FZD10 as a receptor for WNT1 and WNT3A in the developing chick spinal cord.

Eed controls craniofacial osteoblast differentiation and mesenchymal proliferation from the neural crest.

The histone methyltransferase Polycomb repressive complex 2 (PRC2) is required for specification of the neural crest, and mis-regulation of neural crest development can cause severe congenital malformations. PRC2 is necessary for neural crest induction, but the embryonic, cellular, and molecular consequences of PRC2 activity after neural crest induction are incompletely understood. Here we show that Eed, a core subunit of PRC2, is required for craniofacial osteoblast differentiation and mesenchymal proliferation after induction of the neural crest. Integrating mouse genetics with single-cell RNA sequencing, our results reveal that conditional knockout of Eed after neural crest cell induction causes severe craniofacial hypoplasia, impaired craniofacial osteogenesis, and attenuated craniofacial mesenchymal cell proliferation that is first evident in post-migratory neural crest cell populations. We show that Eed drives mesenchymal differentiation and proliferation in vivo and in primary craniofacial cell cultures by regulating diverse transcription factor programs that are required for specification of post-migratory neural crest cells. These data enhance understanding of epigenetic mechanisms that underlie craniofacial development, and shed light on the embryonic, cellular, and molecular drivers of rare congenital syndromes in humans.

Cellular transitions during cranial suture establishment in zebrafish.

Cranial sutures separate neighboring skull bones and are sites of bone growth. A key question is how osteogenic activity is controlled to promote bone growth while preventing aberrant bone fusions during skull expansion. Using single-cell transcriptomics, lineage tracing, and mutant analysis in zebrafish, we uncover key developmental transitions regulating bone formation at sutures during skull expansion. In particular, we identify a subpopulation of mesenchyme cells in the mid-suture region that upregulate a suite of genes including BMP antagonists (e.g. grem1a) and pro-angiogenic factors. Lineage tracing with grem1a:nlsEOS reveals that this mid-suture subpopulation is largely non-osteogenic. Moreover, combinatorial mutation of BMP antagonists enriched in this mid-suture subpopulation results in increased BMP signaling in the suture, misregulated bone formation, and abnormal suture morphology. These data reveal establishment of a non-osteogenic mesenchyme population in the mid-suture region that restricts bone formation through local BMP antagonism, thus ensuring proper suture morphology.

Resolving homology in the face of shifting germ layer origins: Lessons from a major skull vault boundary.

The vertebrate skull varies widely in shape, accommodating diverse strategies of feeding and predation. The braincase is composed of several flat bones that meet at flexible joints called sutures. Nearly all vertebrates have a prominent 'coronal' suture that separates the front and back of the skull. This suture can develop entirely within mesoderm-derived tissue, neural crest-derived tissue, or at the boundary of the two. Recent paleontological findings and genetic insights in non-mammalian model organisms serve to revise fundamental knowledge on the development and evolution of this suture. Growing evidence supports a decoupling of the germ layer origins of the mesenchyme that forms the calvarial bones from inductive signaling that establishes discrete bone centers. Changes in these relationships facilitate skull evolution and may create susceptibility to disease. These concepts provide a general framework for approaching issues of homology in cases where germ layer origins have shifted during evolution.

Altered bone growth dynamics prefigure craniosynostosis in a zebrafish model of Saethre-Chotzen syndrome.

Cranial sutures separate the skull bones and house stem cells for bone growth and repair. In Saethre-Chotzen syndrome, mutations in TCF12 or TWIST1 ablate a specific suture, the coronal. This suture forms at a neural-crest/mesoderm interface in mammals and a mesoderm/mesoderm interface in zebrafish. Despite this difference, we show that combinatorial loss of TCF12 and TWIST1 homologs in zebrafish also results in specific loss of the coronal suture. Sequential bone staining reveals an initial, directional acceleration of bone production in the mutant skull, with subsequent localized stalling of bone growth prefiguring coronal suture loss. Mouse genetics further reveal requirements for Twist1 and Tcf12 in both the frontal and parietal bones for suture patency, and to maintain putative progenitors in the coronal region. These findings reveal conservation of coronal suture formation despite evolutionary shifts in embryonic origins, and suggest that the coronal suture might be especially susceptible to imbalances in progenitor maintenance and osteoblast differentiation.

Requirement for Jagged1-Notch2 signaling in patterning the bones of the mouse and human middle ear.

Whereas Jagged1-Notch2 signaling is known to pattern the sensorineural components of the inner ear, its role in middle ear development has been less clear. We previously reported a role for Jagged-Notch signaling in shaping skeletal elements derived from the first two pharyngeal arches of zebrafish. Here we show a conserved requirement for Jagged1-Notch2 signaling in patterning the stapes and incus middle ear bones derived from the equivalent pharyngeal arches of mammals. Mice lacking Jagged1 or Notch2 in neural crest-derived cells (NCCs) of the pharyngeal arches display a malformed stapes. Heterozygous Jagged1 knockout mice, a model for Alagille Syndrome (AGS), also display stapes and incus defects. We find that Jagged1-Notch2 signaling functions early to pattern the stapes cartilage template, with stapes malformations correlating with hearing loss across all frequencies. We observe similar stapes defects and hearing loss in one patient with heterozygous JAGGED1 loss, and a diversity of conductive and sensorineural hearing loss in nearly half of AGS patients, many of which carry JAGGED1 mutations. Our findings reveal deep conservation of Jagged1-Notch2 signaling in patterning the pharyngeal arches from fish to mouse to man, despite the very different functions of their skeletal derivatives in jaw support and sound transduction.