Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells.

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

Functional and Genetic Analyses Unveil the Implication of CDC27 in Hemifacial Microsomia.

Hemifacial microsomia (HFM) is a rare congenital genetic syndrome primarily affecting the first and second pharyngeal arches, leading to defects in the mandible, external ear, and middle ear. The pathogenic genes remain largely unidentified. Whole-exome sequencing (WES) was conducted on 12 HFM probands and their unaffected biological parents. Predictive structural analysis of the target gene was conducted using PSIPRED (v3.3) and SWISS-MODEL, while STRING facilitated protein-to-protein interaction predictions. CRISPR/Cas9 was applied for gene knockout in zebrafish. In situ hybridization (ISH) was employed to examine the spatiotemporal expression of the target gene and neural crest cell (NCC) markers. Immunofluorescence with PH3 and TUNEL assays were used to assess cell proliferation and apoptosis. RNA sequencing was performed on mutant and control embryos, with rescue experiments involving target mRNA injections and specific gene knockouts. CDC27 was identified as a novel candidate gene for HFM, with four nonsynonymous de novo variants detected in three unrelated probands. Structural predictions indicated significant alterations in the secondary and tertiary structures of CDC27. cdc27 knockout in zebrafish resulted in craniofacial malformation, spine deformity, and cardiac edema, mirroring typical HFM phenotypes. Abnormalities in somatic cell apoptosis, reduced NCC proliferation in pharyngeal arches, and chondrocyte differentiation issues were observed in cdc27-/- mutants. cdc27 mRNA injections and cdkn1a or tp53 knockout significantly rescued pharyngeal arch cartilage dysplasia, while sox9a mRNA administration partially restored the defective phenotypes. Our findings suggest a functional link between CDC27 and HFM, primarily through the inhibition of CNCC proliferation and disruption of pharyngeal chondrocyte differentiation.

[Research progress of genetic research on Char syndrome].

Char syndrome is a rare autosomal dominant genetic disorder characterized by patent ductus arteriosus, facial dysmorphism, and dysplasia of fingers/toes. It may also be associated with multiple papillae, dental dysplasia, and sleep disorders. TFAP2B has proven to be a pathogenic gene for neural crest derivation and development, and several variants of this gene have been identified. Bone morphogenetic protein signaling plays an important role in embryonic development by participating in limb growth and patterning, and regulation of neural crest cell development. TFAP2B is an upstream regulatory gene for bone morphogenetic proteins 2 and 4. Variants of the TFAP2B gene may lead to abnormal proliferation of neural crest cells by affecting the expression of bone morphogenetic proteins, resulting in multiple organ dysplasia syndrome. In addition, TFAP2B variants may only lead to patent ductus arteriosus instead of typical Char syndrome.

A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system.

The vertebrate enteric nervous system (ENS) is a crucial network of enteric neurons and glia resident within the entire gastrointestinal tract (GI). Overseeing essential GI functions such as gut motility and water balance, the ENS serves as a pivotal bidirectional link in the gut-brain axis. During early development, the ENS is primarily derived from enteric neural crest cells (ENCCs). Disruptions to ENCC development, as seen in conditions like Hirschsprung disease (HSCR), lead to the absence of ENS in the GI, particularly in the colon. In this study, using zebrafish, we devised an in vivo F0 CRISPR-based screen employing a robust, rapid pipeline integrating single-cell RNA sequencing, CRISPR reverse genetics, and high-content imaging. Our findings unveil various genes, including those encoding opioid receptors, as possible regulators of ENS establishment. In addition, we present evidence that suggests opioid receptor involvement in the neurochemical coding of the larval ENS. In summary, our work presents a novel, efficient CRISPR screen targeting ENS development, facilitating the discovery of previously unknown genes, and increasing knowledge of nervous system construction.

Making developmental sense of the senses, their origin and function.

The primary senses-touch, taste, sight, smell, and hearing-connect animals with their environments and with one another. Aside from the eyes, the primary sense organs of vertebrates and the peripheral sensory pathways that relay their inputs arise from two transient stem cell populations: the neural crest and the cranial placodes. In this chapter we consider the senses from historical and cultural perspectives, and discuss the senses as biological faculties. We begin with the embryonic origin of the neural crest and cranial placodes from within the neural plate border of the ectodermal germ layer. Then, we describe the major chemical (i.e. olfactory and gustatory) and mechanical (i.e. vestibulo-auditory and somatosensory) senses, with an emphasis on the developmental interactions between neural crest and cranial placodes that shape their structures and functions.

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.

Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.

Neural crest origin of sympathetic neurons at the dawn of vertebrates.

The neural crest is an embryonic stem cell population unique to vertebrates1 whose expansion and diversification are thought to have promoted vertebrate evolution by enabling emergence of new cell types and structures such as jaws and peripheral ganglia2. Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates3-8. Here, by contrast, we report the presence of trunk sympathetic neurons in the sea lamprey, Petromyzon marinus, an extant jawless vertebrate. These neurons arise from sympathoblasts near the dorsal aorta that undergo noradrenergic specification through a transcriptional program homologous to that described in gnathostomes. Lamprey sympathoblasts populate the extracardiac space and extend along the length of the trunk in bilateral streams, expressing the catecholamine biosynthetic pathway enzymes tyrosine hydroxylase and dopamine ß-hydroxylase. CM-DiI lineage tracing analysis further confirmed that these cells derive from the trunk neural crest. RNA sequencing of isolated ammocoete trunk sympathoblasts revealed gene profiles characteristic of sympathetic neuron function. Our findings challenge the prevailing dogma that posits that sympathetic ganglia are a gnathostome innovation, instead suggesting that a late-developing rudimentary sympathetic nervous system may have been characteristic of the earliest vertebrates.

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.

Diphthamide deficiency promotes association of eEF2 with p53 to induce p21 expression and neural crest defects.

Diphthamide is a modified histidine residue unique for eukaryotic translation elongation factor 2 (eEF2), a key ribosomal protein. Loss of this evolutionarily conserved modification causes developmental defects through unknown mechanisms. In a patient with compound heterozygous mutations in Diphthamide Biosynthesis 1 (DPH1) and impaired eEF2 diphthamide modification, we observe multiple defects in neural crest (NC)-derived tissues. Knockin mice harboring the patient's mutations and Xenopus embryos with Dph1 depleted also display NC defects, which can be attributed to reduced proliferation in the neuroepithelium. DPH1 depletion facilitates dissociation of eEF2 from ribosomes and association with p53 to promote transcription of the cell cycle inhibitor p21, resulting in inhibited proliferation. Knockout of one p21 allele rescues the NC phenotypes in the knockin mice carrying the patient's mutations. These findings uncover an unexpected role for eEF2 as a transcriptional coactivator for p53 to induce p21 expression and NC defects, which is regulated by diphthamide modification.

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.

GFP-labeled Schwann cell-like cells derived from hair follicle epidermal neural crest stem cells promote the acellular nerve allografts to repair facial nerve defects in rats.

BACKGROUND: Acellular nerve allografts (ANAs) have been successfully applied to bridge facial nerve defects, and transplantation of stem cells may enhance the regenerative results. Up to now, application of hair follicle epidermal neural crest stem cell-derived Schwann cell-like cells (EPI-NCSC-SCLCs) combined with ANAs for bridging facial nerve defects has not been reported. METHODS: The effect of ANAs laden with green fluorescent protein (GFP)-labeled EPI-NCSC-SCLCs (ANA + cells) on bridging rat facial nerve trunk defects (5-mm-long) was detected by functional and morphological examination, as compared with autografts and ANAs, respectively. RESULTS: (1) EPI-NCSC-SCLCs had good compatibility with ANAs in vitro. (2) In the ANA + cells group, the GFP signals were observed by in vivo imaging system for small animals within 8 weeks, and GFP-labeled EPI-NCSC-SCLCs were detected in the tissue slices at 16 weeks postoperatively. (3) The facial symmetry at rest after surgery in the ANA + cells group was better than that in the ANA group (p < 0.05), and similar to that in the autograft group (p > 0.05). The initial recovery time of vibrissal and eyelid movement in the ANA group was 2 weeks later than that in the other two groups. (4) The myelinated fibers, myelin sheath thickness and diameter of the axons of the buccal branches in the ANA group were significantly worse than those in the other two groups (P < 0.05), and the results in the ANA + cells group were similar to those in the autograft group (p > 0.05). CONCLUSIONS: EPI-NCSC-SCLCs could promote functional and morphological recovery of rat facial nerve defects, and GFP labeling could track the transplanted EPI-NCSC-SCLCs in vivo for a certain period of time. These may provide a novel choice for clinical treatment of peripheral nerve defects.

The domestication of the larynx: The neural crest connection.

Wolves howl and dogs bark, both are able to produce variants of either vocalization, but we see a distinct difference in usage between wild and domesticate. Other domesticates also show distinct changes to their vocal output: domestic cats retain meows, a distinctly subadult trait in wildcats. Such differences in acoustic output are well-known, but the causal mechanisms remain little-studied. Potential links between domestication and vocal output are intriguing for multiple reasons, and offer a unique opportunity to explore a prominent hypothesis in domestication research: the neural crest/domestication syndrome hypothesis. This hypothesis suggests that in the early stages of domestication, selection for tame individuals decreased neural crest cell (NCCs) proliferation and migration, which led to a downregulation of the sympathetic arousal system, and hence reduced fear and reactive aggression. NCCs are a transitory stem cell population crucial during embryonic development that tie to diverse tissue types and organ systems. One of these neural-crest derived systems is the larynx, the main vocal source in mammals. We argue that this connection between NCCs and the larynx provides a powerful test of the predictions of the neural crest/domestication syndrome hypothesis, discriminating its predictions from those of other current hypotheses concerning domestication.

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.

The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

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.

From neural tube to spinal cord: The dynamic journey of the dorsal neuroepithelium.

In a developing embryo, formation of tissues and organs is remarkably precise in both time and space. Through cell-cell interactions, neighboring progenitors coordinate their activities, sequentially generating distinct types of cells. At present, we only have limited knowledge, rather than a systematic understanding, of the underlying logic and mechanisms responsible for cell fate transitions. The formation of the dorsal aspect of the spinal cord is an outstanding model to tackle these dynamics, as it first generates the peripheral nervous system and is later responsible for transmitting sensory information from the periphery to the brain and for coordinating local reflexes. This is reflected first by the ontogeny of neural crest cells, progenitors of the peripheral nervous system, followed by formation of the definitive roof plate of the central nervous system and specification of adjacent interneurons, then a transformation of roof plate into dorsal radial glia and ependyma lining the forming central canal. How do these peripheral and central neural branches segregate from common progenitors? How are dorsal radial glia established concomitant with transformation of the neural tube lumen into a central canal? How do the dorsal radial glia influence neighboring cells? This is only a partial list of questions whose clarification requires the implementation of experimental paradigms in which precise control of timing is crucial. Here, we outline some available answers and still open issues, while highlighting the contributions of avian models and their potential to address mechanisms of neural patterning and function.

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.