DNA-guided transcription factor cooperativity shapes face and limb mesenchyme.

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.

An immune cell atlas reveals the dynamics of human macrophage specification during prenatal development.

Macrophages are heterogeneous and play critical roles in development and disease, but their diversity, function, and specification remain inadequately understood during human development. We generated a single-cell RNA sequencing map of the dynamics of human macrophage specification from PCW 4-26 across 19 tissues. We identified a microglia-like population and a proangiogenic population in 15 macrophage subtypes. Microglia-like cells, molecularly and morphologically similar to microglia in the CNS, are present in the fetal epidermis, testicle, and heart. They are the major immune population in the early epidermis, exhibit a polarized distribution along the dorsal-lateral-ventral axis, and interact with neural crest cells, modulating their differentiation along the melanocyte lineage. Through spatial and differentiation trajectory analysis, we also showed that proangiogenic macrophages are perivascular across fetal organs and likely yolk-sac-derived as microglia. Our study provides a comprehensive map of the heterogeneity and developmental dynamics of human macrophages and unravels their diverse functions during development.

Migratory Neural Crest Cells Phagocytose Dead Cells in the Developing Nervous System.

During neural tube closure and spinal cord development, many cells die in both the central and peripheral nervous systems (CNS and PNS, respectively). However, myeloid-derived professional phagocytes have not yet colonized the trunk region during early neurogenesis. How apoptotic cells are removed from this region during these stages remains largely unknown. Using live imaging in zebrafish, we demonstrate that neural crest cells (NCCs) respond rapidly to dying cells and phagocytose cellular debris around the neural tube. Additionally, NCCs have the ability to enter the CNS through motor exit point transition zones and clear debris in the spinal cord. Surprisingly, NCCs phagocytosis mechanistically resembles macrophage phagocytosis and their recruitment toward cellular debris is mediated by interleukin-1ß. Taken together, our results reveal a role for NCCs in phagocytosis of debris in the developing nervous system before the presence of professional phagocytes.

Single Amino Acid Change Underlies Distinct Roles of H2A.Z Subtypes in Human Syndrome.

The developmental disorder Floating-Harbor syndrome (FHS) is caused by heterozygous truncating mutations in SRCAP, a gene encoding a chromatin remodeler mediating incorporation of histone variant H2A.Z. Here, we demonstrate that FHS-associated mutations result in loss of SRCAP nuclear localization, alter neural crest gene programs in human in vitro models and Xenopus embryos, and cause craniofacial defects. These defects are mediated by one of two H2A.Z subtypes, H2A.Z.2, whose knockdown mimics and whose overexpression rescues the FHS phenotype. Selective rescue by H2A.Z.2 is conferred by one of the three amino acid differences between the H2A.Z subtypes, S38/T38. We further show that H2A.Z.1 and H2A.Z.2 genomic occupancy patterns are qualitatively similar, but quantitatively distinct, and H2A.Z.2 incorporation at AT-rich enhancers and expression of their associated genes are both sensitized to SRCAP truncations. Altogether, our results illuminate the mechanism underlying a human syndrome and uncover selective functions of H2A.Z subtypes during development.

Seq Your Destiny: Neural Crest Fate Determination in the Genomic Era.

Neural crest stem/progenitor cells arise early during vertebrate embryogenesis at the border of the forming central nervous system. They subsequently migrate throughout the body, eventually differentiating into diverse cell types ranging from neurons and glia of the peripheral nervous system to bones of the face, portions of the heart, and pigmentation of the skin. Along the body axis, the neural crest is heterogeneous, with different subpopulations arising in the head, neck, trunk, and tail regions, each characterized by distinct migratory patterns and developmental potential. Modern genomic approaches like single-cell RNA- and ATAC-sequencing (seq) have greatly enhanced our understanding of cell lineage trajectories and gene regulatory circuitry underlying the developmental progression of neural crest cells. Here, we discuss how genomic approaches have provided new insights into old questions in neural crest biology by elucidating transcriptional and posttranscriptional mechanisms that govern neural crest formation and the establishment of axial level identity.

FGF signaling regulates development by processes beyond canonical pathways.

FGFs are key developmental regulators that engage a signal transduction cascade through receptor tyrosine kinases, prominently engaging ERK1/2 but also other pathways. However, it remains unknown whether all FGF activities depend on this canonical signal transduction cascade. To address this question, we generated allelic series of knock-in Fgfr1 and Fgfr2 mouse strains, carrying point mutations that disrupt binding of signaling effectors, and a kinase dead allele of Fgfr2 that broadly phenocopies the null mutant. When interrogated in cranial neural crest cells, we identified discrete functions for signaling pathways in specific craniofacial contexts, but point mutations, even when combined, failed to recapitulate the single or double null mutant phenotypes. Furthermore, the signaling mutations abrogated established FGF-induced signal transduction pathways, yet FGF functions such as cell-matrix and cell-cell adhesion remained unaffected, though these activities did require FGFR kinase activity. Our studies establish combinatorial roles of Fgfr1 and Fgfr2 in development and uncouple novel FGFR kinase-dependent cell adhesion properties from canonical intracellular signaling.

Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway.

Hirschsprung disease (HSCR), one of several neurocristopathies in children, is characterized by nerve loss in the large intestine and is mainly treated by surgery, which causes severe complications. Enteric neural crest-derived cell (ENCC) transplantation is a potential therapeutic strategy; however, so far with poor efficacy. Here, we assessed whether and how fecal microbiota transplantation (FMT) could improve ENCC transplantation in a rat model of hypoganglionosis; a condition similar to HSCR, with less intestinal innervation. We found that the hypoganglionosis intestinal microenvironment negatively influenced the ENCC functional phenotype in vitro and in vivo. Combining 16S rDNA sequencing and targeted mass spectrometry revealed microbial dysbiosis and reduced short-chain fatty acid (SCFA) production in the hypoganglionic gut. FMT increased the abundance of Bacteroides and Clostridium, SCFA production, and improved outcomes following ENCC transplantation. SCFAs alone stimulated ENCC proliferation, migration, and supported ENCC transplantation. Transcriptome-wide mRNA sequencing identified MAPK signaling as the top differentially regulated pathway in response to SCFA exposure, and inhibition of MEK1/2 signaling abrogated the SCFA-mediated effects on ENCC. This study demonstrates that FMT improves cell therapy for hypoganglionosis via short-chain fatty acid metabolism-induced MEK1/2 signaling.

Piezo1 and Piezo2 collectively regulate jawbone development.

Piezo1 and Piezo2 are recently reported mechanosensory ion channels that transduce mechanical stimuli from the environment into intracellular biochemical signals in various tissues and organ systems. Here, we show that Piezo1 and Piezo2 display a robust expression during jawbone development. Deletion of Piezo1 in neural crest cells causes jawbone malformations in a small but significant number of mice. We further demonstrate that disruption of Piezo1 and Piezo2 in neural crest cells causes more striking defects in jawbone development than any single knockout, suggesting essential but partially redundant roles of Piezo1 and Piezo2. In addition, we observe defects in other neural crest derivatives such as malformation of the vascular smooth muscle in double knockout mice. Moreover, TUNEL examinations reveal excessive cell death in osteogenic cells of the maxillary and mandibular arches of the double knockout mice, suggesting that Piezo1 and Piezo2 together regulate cell survival during jawbone development. We further demonstrate that Yoda1, a Piezo1 agonist, promotes mineralization in the mandibular arches. Altogether, these data firmly establish that Piezo channels play important roles in regulating jawbone formation and maintenance.

Zebrafish raptor mutation inhibits the activity of mTORC1, inducing craniofacial defects due to autophagy-induced neural crest cell death.

The mechanistic target of rapamycin (mTOR) coordinates metabolism and cell growth with environmental inputs. mTOR forms two functional complexes: mTORC1 and mTORC2. Proper development requires both complexes but mTORC1 has unique roles in numerous cellular processes, including cell growth, survival and autophagy. Here, we investigate the function of mTORC1 in craniofacial development. We created a zebrafish raptor mutant via CRISPR/Cas9, to specifically disrupt mTORC1. The entire craniofacial skeleton and eyes were reduced in size in mutants; however, overall body length and developmental timing were not affected. The craniofacial phenotype associates with decreased chondrocyte size and increased neural crest cell death. We found that autophagy is elevated in raptor mutants. Chemical inhibition of autophagy reduced cell death and improved craniofacial phenotypes in raptor mutants. Genetic inhibition of autophagy, via mutation of the autophagy gene atg7, improved facial phenotypes in atg7;raptor double mutants, relative to raptor single mutants. We conclude that finely regulated levels of autophagy, via mTORC1, are crucial for craniofacial development.

Cranial suture lineage and contributions to repair of the mouse skull.

The cranial sutures are proposed to be a stem cell niche, harbouring skeletal stem cells that are directly involved in development, homeostasis and healing. Like the craniofacial bones, the sutures are formed from both mesoderm and neural crest. During cranial bone repair, neural crest cells have been proposed to be key players; however, neural crest contributions to adult sutures are not well defined, and the relative importance of suture proximity is unclear. Here, we use genetic approaches to re-examine the neural crest-mesoderm boundaries in the adult mouse skull. These are combined with calvarial wounding experiments suggesting that suture proximity improves the efficiency of cranial repair. Furthermore, we demonstrate that Gli1+ and Axin2+ skeletal stem cells are present in all calvarial sutures examined. We propose that the position of the defect determines the availability of neural crest-derived progenitors, which appear to be a key element in the repair of calvarial defects.

BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

Cornelia de Lange syndrome (CdLS) is a congenital disorder featuring facial dysmorphism, postnatal growth deficits, cognitive disability and upper limb abnormalities. CdLS is genetically heterogeneous, with cases arising from mutation of BRD4, a bromodomain protein that binds and reads acetylated histones. In this study, we have modeled CdLS facial pathology through mouse neural crest cell (NCC)-specific mutation of BRD4 to characterize cellular and molecular function in craniofacial development. Mice with BRD4 NCC loss of function died at birth with severe facial hypoplasia, cleft palate, mid-facial clefting and exencephaly. Following migration, BRD4 mutant NCCs initiated RUNX2 expression for differentiation to osteoblast lineages but failed to induce downstream RUNX2 targets required for lineage commitment. BRD4 bound to active enhancers to regulate expression of osteogenic transcription factors and extracellular matrix components integral for bone formation. RUNX2 physically interacts with a C-terminal domain in the long isoform of BRD4 and can co-occupy osteogenic enhancers. This BRD4 association is required for RUNX2 recruitment and appropriate osteoblast differentiation. We conclude that BRD4 controls facial bone development through osteoblast enhancer regulation of the RUNX2 transcriptional program.

TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway.

Cranial neural crest development is governed by positional gene regulatory networks (GRNs). Fine-tuning of the GRN components underlies facial shape variation, yet how those networks in the midface are connected and activated remain poorly understood. Here, we show that concerted inactivation of Tfap2a and Tfap2b in the murine neural crest, even during the late migratory phase, results in a midfacial cleft and skeletal abnormalities. Bulk and single-cell RNA-seq profiling reveal that loss of both TFAP2 family members dysregulates numerous midface GRN components involved in midface morphogenesis, patterning and differentiation. Notably, Alx1, Alx3 and Alx4 (ALX) transcript levels are reduced, whereas ChIP-seq analyses suggest TFAP2 family members directly and positively regulate ALX gene expression. Tfap2a, Tfap2b and ALX co-expression in midfacial neural crest cells of both mouse and zebrafish implies conservation of this regulatory axis across vertebrates. Consistent with this notion, tfap2a zebrafish mutants present with abnormal alx3 expression patterns, Tfap2a binds ALX loci and tfap2a-alx3 genetic interactions are observed. Together, these data demonstrate TFAP2 paralogs regulate vertebrate midfacial development in part by activating expression of ALX transcription factor genes.

BET activity plays an essential role in control of stem cell attributes in Xenopus.

Neural crest cells are a stem cell population unique to vertebrate embryos that retains broad multi-germ layer developmental potential through neurulation. Much remains to be learned about the genetic and epigenetic mechanisms that control the potency of neural crest cells. Here, we examine the role that epigenetic readers of the BET (bromodomain and extra terminal) family play in controlling the potential of pluripotent blastula and neural crest cells. We find that inhibiting BET activity leads to loss of pluripotency at blastula stages and a loss of neural crest at neurula stages. We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition and find that they lead to similar cellular outcomes through distinct effects on the transcriptome. Interestingly, loss of BET activity in cells undergoing lineage restriction is coupled to increased expression of genes linked to pluripotency and prolongs the competence of initially pluripotent cells to transit to a neural progenitor state. Together these findings advance our understanding of the epigenetic control of pluripotency and the formation of the vertebrate neural crest.

SoxB1 transcription factors are essential for initiating and maintaining neural plate border gene expression.

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

Zebrafish Pigment Pattern Formation: Insights into the Development and Evolution of Adult Form.

Vertebrate pigment patterns are diverse and fascinating adult traits that allow animals to recognize conspecifics, attract mates, and avoid predators. Pigment patterns in fish are among the most amenable traits for studying the cellular basis of adult form, as the cells that produce diverse patterns are readily visible in the skin during development. The genetic basis of pigment pattern development has been most studied in the zebrafish, Danio rerio. Zebrafish adults have alternating dark and light horizontal stripes, resulting from the precise arrangement of three main classes of pigment cells: black melanophores, yellow xanthophores, and iridescent iridophores. The coordination of adult pigment cell lineage specification and differentiation with specific cellular interactions and morphogenetic behaviors is necessary for stripe development. Besides providing a nice example of pattern formation responsible for an adult trait of zebrafish, stripe-forming mechanisms also provide a conceptual framework for posing testable hypotheses about pattern diversification more broadly. Here, we summarize what is known about lineages and molecular interactions required for pattern formation in zebrafish, we review some of what is known about pattern diversification in Danio, and we speculate on how patterns in more distant teleosts may have evolved to produce a stunningly diverse array of patterns in nature.

Disruption of DNA methylation-mediated cranial neural crest proliferation and differentiation causes orofacial clefts in mice.

Orofacial clefts of the lip and palate are widely recognized to result from complex gene-environment interactions, but inadequate understanding of environmental risk factors has stymied development of prevention strategies. We interrogated the role of DNA methylation, an environmentally malleable epigenetic mechanism, in orofacial development. Expression of the key DNA methyltransferase enzyme DNMT1 was detected throughout palate morphogenesis in the epithelium and underlying cranial neural crest cell (cNCC) mesenchyme, a highly proliferative multipotent stem cell population that forms orofacial connective tissue. Genetic and pharmacologic manipulations of DNMT activity were then applied to define the tissue- and timing-dependent requirement of DNA methylation in orofacial development. cNCC-specific Dnmt1 inactivation targeting initial palate outgrowth resulted in OFCs, while later targeting during palatal shelf elevation and elongation did not. Conditional Dnmt1 deletion reduced cNCC proliferation and subsequent differentiation trajectory, resulting in attenuated outgrowth of the palatal shelves and altered development of cNCC-derived skeletal elements. Finally, we found that the cellular mechanisms of cleft pathogenesis observed in vivo can be recapitulated by pharmacologically reducing DNA methylation in multipotent cNCCs cultured in vitro. These findings demonstrate that DNA methylation is a crucial epigenetic regulator of cNCC biology, define a critical period of development in which its disruption directly causes OFCs, and provide opportunities to identify environmental influences that contribute to OFC risk.

A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages.

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

Schwann cell precursors represent a neural crest-like state with biased multipotency.

Schwann cell precursors (SCPs) are nerve-associated progenitors that can generate myelinating and non-myelinating Schwann cells but also are multipotent like the neural crest cells from which they originate. SCPs are omnipresent along outgrowing peripheral nerves throughout the body of vertebrate embryos. By using single-cell transcriptomics to generate a gene expression atlas of the entire neural crest lineage, we show that early SCPs and late migratory crest cells have similar transcriptional profiles characterised by a multipotent "hub" state containing cells biased towards traditional neural crest fates. SCPs keep diverging from the neural crest after being primed towards terminal Schwann cells and other fates, with different subtypes residing in distinct anatomical locations. Functional experiments using CRISPR-Cas9 loss-of-function further show that knockout of the common "hub" gene Sox8 causes defects in neural crest-derived cells along peripheral nerves by facilitating differentiation of SCPs towards sympathoadrenal fates. Finally, specific tumour populations found in melanoma, neurofibroma and neuroblastoma map to different stages of SCP/Schwann cell development. Overall, SCPs resemble migrating neural crest cells that maintain multipotency and become transcriptionally primed towards distinct lineages.

De novo variants implicate chromatin modification, transcriptional regulation, and retinoic acid signaling in syndromic craniosynostosis.

Craniosynostosis (CS) is the most common congenital cranial anomaly. Several Mendelian forms of syndromic CS are well described, but a genetic etiology remains elusive in a substantial fraction of probands. Analysis of exome sequence data from 526 proband-parent trios with syndromic CS identified a marked excess (observed 98, expected 33, p = 4.83 × 10-20) of damaging de novo variants (DNVs) in genes highly intolerant to loss-of-function variation (probability of LoF intolerance > 0.9). 30 probands harbored damaging DNVs in 21 genes that were not previously implicated in CS but are involved in chromatin modification and remodeling (4.7-fold enrichment, p = 1.1 × 10-11). 17 genes had multiple damaging DNVs, and 13 genes (CDK13, NFIX, ADNP, KMT5B, SON, ARID1B, CASK, CHD7, MED13L, PSMD12, POLR2A, CHD3, and SETBP1) surpassed thresholds for genome-wide significance. A recurrent gain-of-function DNV in the retinoic acid receptor alpha (RARA; c.865G>A [p.Gly289Arg]) was identified in two probands with similar CS phenotypes. CS risk genes overlap with those identified for autism and other neurodevelopmental disorders, are highly expressed in cranial neural crest cells, and converge in networks that regulate chromatin modification, gene transcription, and osteoblast differentiation. Our results identify several CS loci and have major implications for genetic testing and counseling.

POLR1A variants underlie phenotypic heterogeneity in craniofacial, neural, and cardiac anomalies.

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies.