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.

Mesenchymal cells regulate enteric neural crest cell migration via RET-GFRA1b trans-signaling.

During early development, the enteric nervous system forms from the migration of enteric neural crest cells (ENCCs) from the foregut to the hindgut, where they undergo proliferation and differentiation facilitated by interactions with enteric mesenchymal cells (EMCs). This study investigates the impact on ENCC migration of EMC-ENCC communication mediated by GFRA1b expressed in EMCs. GFRA1-expressing cells in day 11-12 (E11-12) mouse embryos differentiated into smooth muscle cells from E12 onwards. Observations at E12-13.5 revealed high levels of GFRA1 expression on the anti-mesenteric side of the hindgut, correlating with enhanced ENCC migration. This indicates that GFRA1 in EMCs plays a role in ENCC migration during development. Examining GFRA1 isoforms, we found high levels of GFRA1b, which lacks amino acids 140-144, in EMCs. To assess the impact of GFRA1 isoforms on EMC-ENCC communication, we conducted neurosphere drop assays. This revealed that GFRA1b-expressing cells promoted GDNF-dependent extension and increased neurite density in ENCC neurospheres. Co-culture of ENCC mimetic cells expressing RET and GFRA1a with EMC mimetic cells expressing GFRA1a, GFRA1b, or vector alone showed that only GFRA1b-expressing co-cultured cells sustained RET phosphorylation in ENCC-mimetic cells for over 120 min upon GDNF stimulation. Our study provides evidence that GFRA1b-mediated cell-to-cell communication plays a critical role in ENCC motility in enteric nervous system development. These findings contribute to understanding the cellular interactions and signaling mechanisms that underlie enteric nervous system formation and highlight potential therapeutic targets for gastrointestinal motility disorders.

Inhibition of the serine protease HtrA1 by SerpinE2 suggests an extracellular proteolytic pathway in the control of neural crest migration.

We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism.SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.

Tissues and signals with true organizer properties in craniofacial development.

Transplantation experiments have shown that a true organizer provides instructive signals that induce and pattern ectopic structures in the responding tissue. Here, we review craniofacial experiments to identify tissues with organizer properties and signals with organizer properties. In particular, we evaluate whether transformation of identity took place in the mesenchyme. Using these stringent criteria, we find the strongest evidence for the avian foregut ectoderm. Transplanting a piece of quail foregut endoderm to a host chicken embryo caused ectopic beaks to form derived from chicken mesenchyme. The beak identity, whether upper or lower as well as orientation, was controlled by the original anterior-posterior position of the donor endoderm. There is also good evidence that the nasal pit is necessary and sufficient for lateral nasal patterning. Finally, we review signals that have organizer properties on their own without the need for tissue transplants. Mouse germline knockouts of the endothelin pathway result in transformation of identity of the mandible into a maxilla. Application of noggin-soaked beads to post-migratory neural crest cells transforms maxillary identity. This suggests that endothelin or noggin rich ectoderm could be organizers (not tested). In conclusion, craniofacial, neural crest-derived mesenchyme is competent to respond to tissues with organizer properties, also originating in the head. In future, we can exploit such well defined systems to dissect the molecular changes that ultimately lead to patterning of the upper and lower jaw.

Primary Cilium in Neural Crest Cells Crucial for Anterior Segment Development and Corneal Avascularity.

Purpose: Intraflagellar transport 46 (IFT46) is an integral subunit of the IFT-B complex, playing a key role in the assembly and maintenance of primary cilia responsible for transducing signaling pathways. Despite its predominant expression in the basal body of cilia, the precise role of Ift46 in ocular development remains undetermined. This study aimed to elucidate the impact of neural crest (NC)-specific deletion of Ift46 on ocular development. Methods: NC-specific conditional knockout mice for Ift46 (NC-Ift46F/F) were generated by crossing Ift46F mice with Wnt1-Cre2 mice, enabling the specific deletion of Ift46 in NC-derived cells (NCCs). Sonic Hedgehog (Shh) and Notch signaling activities in NC-Ift46F/F mice were evaluated using Gli1lacZ and CBF:H2B-Venus reporter mice, respectively. Cell fate mapping was conducted using ROSAmTmG reporter mice. Results: The deletion of Ift46 in NCCs resulted in a spectrum of ocular abnormalities, including thickened corneal stroma, hypoplasia of the anterior chamber, irregular iris morphology, and corneal neovascularization. Notably, this deletion led to reduced Shh signal activity in the periocular mesenchyme, sustained expression of key transcription factors Foxc1, Foxc2 and Pitx2, along with persistent cell proliferation. Additionally, it induced increased Notch signaling activity and the development of ectopic neovascularization within the corneal stroma. Conclusions: The absence of primary cilia due to Ift46 deficiency in NCCs is associated with anterior segment dysgenesis (ASD) and corneal neovascularization, suggesting a potential link to Axenfeld-Rieger syndrome, a disorder characterized by ASD. This underscores the pivotal role of primary cilia in ensuring proper anterior segment development and maintaining an avascular cornea.

A BMP-controlled metabolic/epigenetic signaling cascade directs midfacial morphogenesis.

Craniofacial anomalies, especially midline facial defects, are among the most common birth defects in patients and are associated with increased mortality or require lifelong treatment. During mammalian embryogenesis, specific instructions arising at genetic, signaling, and metabolic levels are important for stem cell behaviors and fate determination, but how these functionally relevant mechanisms are coordinated to regulate craniofacial morphogenesis remain unknown. Here, we report that bone morphogenetic protein (BMP) signaling in cranial neural crest cells (CNCCs) is critical for glycolytic lactate production and subsequent epigenetic histone lactylation, thereby dictating craniofacial morphogenesis. Elevated BMP signaling in CNCCs through constitutively activated ACVR1 (ca-ACVR1) suppressed glycolytic activity and blocked lactate production via a p53-dependent process that resulted in severe midline facial defects. By modulating epigenetic remodeling, BMP signaling-dependent lactate generation drove histone lactylation levels to alter essential genes of Pdgfra, thus regulating CNCC behavior in vitro as well as in vivo. These findings define an axis wherein BMP signaling controls a metabolic/epigenetic cascade to direct craniofacial morphogenesis, thus providing a conceptual framework for understanding the interaction between genetic and metabolic cues operative during embryonic development. These findings indicate potential preventive strategies of congenital craniofacial birth defects via modulating metabolic-driven histone lactylation.

Boundary cap neural crest stem cells promote angiogenesis after transplantation to avulsed dorsal roots in mice and induce migration of endothelial cells in 3D printed scaffolds.

Dorsal root avulsion injuries lead to loss of sensation and to reorganization of blood vessels (BVs) in the injured area. The inability of injured sensory axons to re-enter the spinal cord results in permanent loss of sensation, and often also leads to the development of neuropathic pain. Approaches that restore connection between peripheral sensory axons and their CNS targets are thus urgently need. Previous research has shown that sensory axons from peripherally grafted human sensory neurons are able to enter the spinal cord by growing along BVs which penetrate the CNS from the spinal cord surface. In this study we analysed the distribution of BVs after avulsion injury and how their pattern is affected by implantation at the injury site of boundary cap neural crest stem cells (bNCSCs), a transient cluster of cells, which are located at the boundary between the spinal cord and peripheral nervous system and assist the growth of sensory axons from periphery into the spinal cord during development. The superficial dorsal spinal cord vasculature was examined using intravital microscopy and intravascular BV labelling. bNCSC transplantation increased vascular volume in a non-dose responsive manner, whereas dorsal root avulsion alone did not decrease the vascular volume. To determine whether bNCSC are endowed with angiogenic properties we prepared 3D printed scaffolds, containing bNCSCs together with rings prepared from mouse aorta. We show that bNCSC do induce migration and assembly of endothelial cells in this system. These findings suggest that bNCSC transplant can promote vascularization in vivo and contribute to BV formation in 3D printed scaffolds.

Competence for neural crest induction is controlled by hydrostatic pressure through Yap.

Embryonic induction is a key mechanism in development that corresponds to an interaction between a signalling and a responding tissue, causing a change in the direction of differentiation by the responding tissue. Considerable progress has been achieved in identifying inductive signals, yet how tissues control their responsiveness to these signals, known as competence, remains poorly understood. While the role of molecular signals in competence has been studied, how tissue mechanics influence competence remains unexplored. Here we investigate the role of hydrostatic pressure in controlling competence in neural crest cells, an embryonic cell population. We show that neural crest competence decreases concomitantly with an increase in the hydrostatic pressure of the blastocoel, an embryonic cavity in contact with the prospective neural crest. By manipulating hydrostatic pressure in vivo, we show that this increase leads to the inhibition of Yap signalling and impairs Wnt activation in the responding tissue, which would be required for neural crest induction. We further show that hydrostatic pressure controls neural crest induction in amphibian and mouse embryos and in human cells, suggesting a conserved mechanism across vertebrates. Our work sets out how tissue mechanics can interplay with signalling pathways to regulate embryonic competence.

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.

A biased random walk approach for modeling the collective chemotaxis of neural crest cells.

Collective cell migration is a multicellular phenomenon that arises in various biological contexts, including cancer and embryo development. 'Collectiveness' can be promoted by cell-cell interactions such as co-attraction and contact inhibition of locomotion. These mechanisms act on cell polarity, pivotal for directed cell motility, through influencing the intracellular dynamics of small GTPases such as Rac1. To model these dynamics we introduce a biased random walk model, where the bias depends on the internal state of Rac1, and the Rac1 state is influenced by cell-cell interactions and chemoattractive cues. In an extensive simulation study we demonstrate and explain the scope and applicability of the introduced model in various scenarios. The use of a biased random walk model allows for the derivation of a corresponding partial differential equation for the cell density while still maintaining a certain level of intracellular detail from the individual based setting.

In vitro induction of patterned branchial arch-like aggregate from human pluripotent stem cells.

Early patterning of neural crest cells (NCCs) in the craniofacial primordium is important for subsequent development of proper craniofacial structures. However, because of the complexity of the environment of developing tissues, surveying the early specification and patterning of NCCs is difficult. In this study, we develop a simplified in vitro 3D model using human pluripotent stem cells to analyze the early stages of facial development. In this model, cranial NCC-like cells spontaneously differentiate from neural plate border-like cells into maxillary arch-like mesenchyme after a long-term culture. Upon the addition of EDN1 and BMP4, these aggregates are converted into a mandibular arch-like state. Furthermore, temporary treatment with EDN1 and BMP4 induces the formation of spatially separated domains expressing mandibular and maxillary arch markers within a single aggregate. These results suggest that this in vitro model is useful for determining the mechanisms underlying cell fate specification and patterning during early facial development.

A patterned human neural tube model using microfluidic gradients.

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.

Effect of valproic acid on the formation and migration of cranial neural crest cells at the early developmental stages in rat embryos.

Cranial neural crest cells (NCCs) are critical for craniofacial development. The administration of valproic acid (VPA) to pregnant females causes craniofacial malformations in offspring. However, the in vivo influence of VPA on mammalian cranial NCCs remains unclear. In this study, we aimed to elucidate the developmental stage-specific effect of VPA on cranial NCCs through the administration of a single dose of VPA to pregnant rat females immediately prior to the formation of the cranial neural crest (NC). We performed whole-mount immunohistochemistry or in situ hybridization to examine localization changes of gene transcripts associated with the epithelial-mesenchymal transition of the cranial NC (i.e., cranial NCC formation) and cranial NCC migration. The results showed that Hoxa2 mRNA was abnormally detected and Sox9 mRNA expression was decreased in the midbrain-rhombomere (R) 1/2 NC, which forms cranial NCCs that migrate to the frontonasal mass (FNM) and branchial arch (BA) 1, through VPA administration, thus reducing the formation of SNAI2-positive NCCs. Hoxa2-positive NCCs were detected normally in BA2 and abnormally in FNM and BA1, which are normally Hox-free, implying VPA-induced abnormal cranial NCC migration. In vitro verification experiments using the whole embryo culture system revealed that midbrain-R4 NCC migration was abnormal. These results indicate that VPA reduces the formation/delamination of the midbrain-R1/2 NCCs in a developmental stage-specific manner and subsequently causes the abnormal migration of R4 NCCs, which suggests that the abnormal formation and migration of cranial NCCs contribute to the inhibition of axonal elongation in the trigeminal nerve and a reduction in head size.

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.

Notch3 directs differentiation of brain mural cells from human pluripotent stem cell-derived neural crest.

Brain mural cells regulate development and function of the blood-brain barrier and control blood flow. Existing in vitro models of human brain mural cells have low expression of key mural cell genes, including NOTCH3. Thus, we asked whether activation of Notch3 signaling in hPSC-derived neural crest could direct the differentiation of brain mural cells with an improved transcriptional profile. Overexpression of the Notch3 intracellular domain (N3ICD) induced expression of mural cell markers PDGFRß, TBX2, FOXS1, KCNJ8, SLC6A12, and endogenous Notch3. The resulting N3ICD-derived brain mural cells produced extracellular matrix, self-assembled with endothelial cells, and had functional KATP channels. ChIP-seq revealed that Notch3 serves as a direct input to relatively few genes in the context of this differentiation process. Our work demonstrates that activation of Notch3 signaling is sufficient to direct the differentiation of neural crest to mural cells and establishes a developmentally relevant differentiation protocol.

Pth1r in Neural Crest Cells Regulates Nasal Cartilage Differentiation.

Neural crest cells (NCC) arise from the dorsal margin of the neural plate border and comprise a unique cell population that migrates to and creates the craniofacial region. Although factors including Shh, Fgf8, and bone morphogenetic proteins have been shown to regulate these biological events, the role of parathyroid hormone 1 receptor (Pth1r) has been less studied. We generated an NCC-specific mouse model for Pth1r and researched gene expression, function, and interaction focusing on nasal cartilage framework and midfacial development. Wnt1-Cre;Pth1rfl/fl;Tomatofl/+ mice had perinatal lethality, but we observed short snout and jaws, tongue protrusion, reduced NCC-derived cranial length, increased mineralization in nasal septum and hyoid bones, and less bone mineralization at interfrontal suture in mutants at E18.5. Importantly, the mutant nasal septum and turbinate cartilage histologically revealed gradual, premature accelerated hypertrophic differentiation. We then studied the underlying molecular mechanisms by performing RNA seq analysis and unexpectedly found that expression of Ihh and related signaling molecules was enhanced in mutant nasomaxillary tissues. To see if Pth1r and Ihh signaling are associated, we generated a Wnt1-Cre; Ihhfl/fl;Pth1rfl/fl;Tomatofl/+ (DKO) mouse and compared the phenotypes to those of each single knockout mouse: Wnt1-Cre; Ihhfl/fl;Pth1rfl/+;Tomatofl/+ (Ihh-CKO) and Wnt1-Cre;Ihhfl/+;Pth1rfl/fl;Tomatofl/+ (Pth1r-CKO). Ihh-CKO mice displayed a milder effect. Of note, the excessive hypertrophic conversion of the nasal cartilage framework observed in Pth1r-CKO was somewhat rescued DKO embryos. Further, a half cAMP responsive element and the 4 similar sequences containing 2 mismatches were identified from the promoter to the first intron in Ihh gene. Gli1-CreERT2;Pth1rfl/fl;Tomatofl/+, a Pth1r-deficient model targeted in hedgehog responsive cells, demonstrated the enlarged hypertrophic layer and significantly more Tomato-positive chondrocytes accumulated in the nasal septum and ethmoidal endochondral ossification. Collectively, the data suggest a relevant Pth1r/Ihh interaction. Our findings obtained from novel mouse models for Pth1r signaling illuminate previously unknown aspects in craniofacial biology and development.

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.

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.

SMPD3 expression is spatially regulated in the developing embryo by SOXE factors.

During epithelial-to-mesenchymal transition (EMT), significant rearrangements occur in plasma membrane protein and lipid content that are important for membrane function and acquisition of cell motility. To gain insight into how neural crest cells regulate their lipid content at the transcriptional level during EMT, here we identify critical enhancer sequences that regulate the expression of SMPD3, a gene responsible for sphingomyelin hydrolysis to produce ceramide and necessary for neural crest EMT. We uncovered three enhancer regions within the first intron of the SMPD3 locus that drive reporter expression in distinct spatial and temporal domains, together collectively recapitulating the expression domains of endogenous SMPD3 within the ectodermal lineages. We further dissected one enhancer that is specifically active in the migrating neural crest. By mutating putative transcriptional input sites or knocking down upstream regulators, we find that the SOXE-family transcription factors SOX9 and SOX10 regulate the expression of SMPD3 in migrating neural crest cells. Further, ChIP-seq and nascent transcription analysis reveal that SOX10 directly regulates expression of an SMPD3 enhancer specific to migratory neural crest cells. Together these results shed light on how core components of developmental gene regulatory networks interact with metabolic effector genes to control changes in membrane lipid content.