Neural Crest Markers Research Articles

CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos

The chick embryo is a classical model system commonly used in developmental biology due to its amenability to gene perturbation experiments. Pairing this powerful model organism with cutting-edge technology can significantly expand the range of experiments that can be performed. Recently, the CRISPR-Cas13d system has been successfully adapted for use in zebrafish, medaka, killifish, and mouse embryos to achieve targeted gene expression knockdown. Despite its success in other animal models, no prior study has explored the potential of CRISPR-Cas13d in the chick. Here, we present an adaptation of the CRISPR-Cas13d system to achieve targeted gene expression knockdown in the chick embryo. As proof-of-principle, we demonstrate the knockdown of PAX7, an early neural crest marker. Application of this adapted CRISPR-Cas13d technique resulted in effective knockdown of PAX7 expression and function, comparable to knockdown achieved by translation-blocking morpholino. CRISPR-Cas13d complements preexisting knockdown tools such as CRISPR-Cas9 and morpholinos, thereby expanding the experimental potential and versatility of the chick model system.

Retinoic acid, an essential component of the roof plate organizer, promotes the spatiotemporal segregation of dorsal neural fates.

Dorsal neural tube-derived retinoic acid promotes the end of neural crest production and transition into a definitive roof plate. Here, we analyze how this impacts the segregation of central and peripheral lineages, a process essential for tissue patterning and function. Localized in ovo inhibition in quail embryos of retinoic acid activity followed by single-cell transcriptomics unraveled a comprehensive list of differentially expressed genes relevant to these processes. Importantly, progenitors co-expressed neural crest, roof plate and dI1 interneuron markers, indicating a failure in proper lineage segregation. Furthermore, separation between roof plate and dI1 interneurons is mediated by Notch activity downstream of retinoic acid, highlighting their crucial role in establishing the roof plate-dI1 boundary. Within the peripheral branch, where absence of retinoic acid resulted in neural crest production and emigration extending into the roof plate stage, sensory progenitors failed to separate from melanocytes, leading to formation of a common glia-melanocyte cell with aberrant migratory patterns. In summary, the implementation of single-cell RNA sequencing facilitated the discovery and characterization of a molecular mechanism responsible for the segregation of dorsal neural fates during development.

CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos.

The chick embryo is a classical model system commonly used in developmental biology due to its amenability to gene perturbation experiments. Pairing this powerful model organism with cutting-edge technology can significantly expand the range of experiments that can be performed. Recently, the CRISPR-Cas13d system has been successfully adapted for use in zebrafish, medaka, killifish, and mouse embryos to achieve targeted gene expression knockdown. Despite its success in other animal models, no prior study has explored the potential of CRISPR-Cas13d in the chick. Here, we present an adaptation of the CRISPR-Cas13d system to achieve targeted gene expression knockdown in the chick embryo. As proof-of-principle, we demonstrate the knockdown of PAX7, an early neural crest marker. Application of this adapted CRISPR-Cas13d technique resulted in effective knockdown of PAX7 expression and function, comparable to knockdown achieved by translation-blocking morpholino. CRISPR-Cas13d complements preexisting knockdown tools such as CRISPR-Cas9 and morpholinos, thereby expanding the experimental potential and versatility of the chick model system.

Abstract 145: Unraveling SMARCB1 depletion in hiPSC-derived neural crest cells: Implications for ATRT cell of origin

Abstract Atypical teratoid rhabdoid tumors (ATRTs) are highly aggressive pediatric brain cancers that lack standardized treatment regimens and are associated with poor prognoses. Despite being driven by biallelic inactivation of a single gene, SMARCB1, ATRTs exhibit three epigenetic subgroups (SHH, TYR, and MYC), each with different clinical outcomes. Like many pediatric brain tumors, ATRTs share the fundamental underlying mechanism of stalled differentiation, suggesting that it may arise from neural progenitor cells that fail to terminally differentiate. However, unlike other brain tumors, ATRTs share molecular similarities with malignancies found outside of the CNS, especially in the kidney and soft tissues. This unique feature implies a putative cell of origin that is not restricted to the CNS: neural crest cells, a transient embryonic cell population that emerges from the neuroectoderm but then migrates throughout the embryo during development. To study SMARCB1 loss in a genetically defined neural progenitor cellular context with isogenic controls, the Furnari lab engineered a doxycycline inducible SMARCB1 knockdown system into human induced pluripotent stem cells (hiPSCs). After simultaneous SMARCB1 knockdown with directed neural differentiation, the resulting SMARCB1-depleted neural progenitor cells exhibited transcriptomic profiles that were similar to ATRTs, particularly to the SHH subgroup. Building upon these findings, the engineered hiPSCs were differentiated into neural crest cells using a modified version of a published protocol. Via RT-qPCR, these hiPSC-derived neural crest cells were confirmed to express typical neural crest marker genes and lose expression of SMARCB1 in the presence of doxycycline. Remarkably, ATP assays demonstrated that neural crest cells that were differentiated without SMARCB1 acquired enhanced proliferation in comparison to those differentiated with SMARCB1 intact. Further work aims to tease apart this proliferative phenotype in the neural crest cellular context via RNAseq, evaluate the impact of cell identity upon subgroup status, and to determine whether SMARCB1 depletion post-differentiation also enhances proliferation. Together, these findings feature the utility of hiPSC-derived models for investigating mechanisms underlying ATRTs to hopefully identify novel subgroup-specific targets. Citation Format: Clark Wang, Frank Furnari. Unraveling SMARCB1 depletion in hiPSC-derived neural crest cells: Implications for ATRT cell of origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 145.

Histological characterisation of the horn bud region in 58 day old bovine fetuses.

The presence of horns in domestic ruminants, such as cattle, sheep and goats, has financial and welfare implications. The genetic interactions that lead to horn development are not known. Hornless, or polled, cattle occur naturally. The known causative DNA variants (Celtic, Friesian, Mongolian and Guarani) are in intergenic regions on bovine chromosome 1, but their functions are not known. It is thought that horns may be derived from cranial neural crest stem cells and the POLLED variants disrupt the migration or proliferation of these cells. Relaxin family peptide receptor 2 (RXFP2) is more highly expressed in developing horns in cattle compared to nearby skin and has been shown to play a role in horn development in sheep. However, the role of RXFP2 in horn formation is not understood. Histological analyses of cranial tissues from homozygous horned and polled cattle fetuses at day 58 of development was carried out to determine the differences in the structure of the horn bud region. Condensed cells were only observed in the horn bud mesenchyme of horned fetuses and could be the progenitor horn cells. The distribution of neural crest markers (SOX10 and NGFR) and RXFP2 between horned and polled tissues by immunohistochemistry was also analysed. However, SOX10 and NGFR were not detected in the condensed cells, and therefore, these cells are either not derived from the neural crest, or have differentiated and no longer express neural crest markers. SOX10 and NGFR were detected in the peripheral nerves, while RXFP2 was detected in peripheral nerves and in the horn bud epidermis. Previous research has shown that RXFP2 variants are associated with horn phenotypes in cattle an sheep. Therefore, the RXFP2 variants may affect the development of the epidermis or peripheral nerves in the horn bud.

Analyzing Sex-Specific Dimorphism in Human Skeletal Stem Cells.

Sex-related differences are a current topic in contemporary science. In addition to hormonal regulation, cell-autonomous mechanisms are important in bone homeostasis and regeneration. In this study, human skeletal stem cells (SSCs) from female and male adults were cultured and analyzed with immunological assays and osteogenic differentiation assessments. Female SSCs exhibited a mean doubling time of 100.6 h, whereas male SSCs displayed a mean doubling time of 168.0 h. Immunophenotyping revealed the expression of the stem cell markers Nestin, CD133, and CD164, accompanied by the neural-crest marker SOX9. Furthermore, multiparameter flow cytometric analyses revealed a substantial population of multipotent SSCs, comprising up to 80% in both sexes. An analysis of the osteogenic differentiation potential demonstrated a strong mineralization in both male and female SSCs under physiological conditions. Recognizing the prevailing association of bone diseases with inflammatory processes, we also analyzed the osteogenic potential of SSCs from both sexes under pro-inflammatory conditions. Upon TNF-α and IL-1β treatment, we observed no sexual dimorphism on osteogenesis. In summary, we demonstrated the successful isolation and characterization of SSCs capable of rapid osteogenic differentiation. Taken together, in vitro cultured SSCs might be a suitable model to study sexual dimorphisms and develop drugs for degenerative bone diseases.

ScRNA-sequencing in chick suggests a probabilistic model for cell fate allocation at the neural plate border.

The vertebrate 'neural plate border' is a transient territory located at the edge of the neural plate containing precursors for all ectodermal derivatives: the neural plate, neural crest, placodes and epidermis. Elegant functional experiments in a range of vertebrate models have provided an in-depth understanding of gene regulatory interactions within the ectoderm. However, these experiments conducted at tissue level raise seemingly contradictory models for fate allocation of individual cells. Here, we carry out single cell RNA sequencing of chick ectoderm from primitive streak to neurulation stage, to explore cell state diversity and heterogeneity. We characterise the dynamics of gene modules, allowing us to model the order of molecular events which take place as ectodermal fates segregate. Furthermore, we find that genes previously classified as neural plate border 'specifiers' typically exhibit dynamic expression patterns and are enriched in either neural, neural crest or placodal fates, revealing that the neural plate border should be seen as a heterogeneous ectodermal territory and not a discrete transitional transcriptional state. Analysis of neural, neural crest and placodal markers reveals that individual NPB cells co-express competing transcriptional programmes suggesting that their ultimate identify is not yet fixed. This population of 'border located undecided progenitors' (BLUPs) gradually diminishes as cell fate decisions take place. Considering our findings, we propose a probabilistic model for cell fate choice at the neural plate border. Our data suggest that the probability of a progenitor's daughters to contribute to a given ectodermal derivative is related to the balance of competing transcriptional programmes, which in turn are regulated by the spatiotemporal position of a progenitor.

Two induced pluripotent stem cell (iPSC) lines derived from patients affected by Waardenburg syndrome type 1 retain potential to activate neural crest markers

Waardenburg syndrome type 1 (WS1), a rare genetic disease characterized by pigmentation defects and mild craniofacial anomalies often associated with congenital deafness is caused by heterozygous mutations in the PAX3 gene (2q36.1). We have generated two induced pluripotent stem cell lines (PCli029-A and PCli031-A) from two patients from the same family both carrying the same heterozygous deletion in PAX3 exon 1 (c.-70_85 + 366del). These cells are pluripotent as they can differentiate into ectoderm, mesoderm and endoderm. They also can activate the early neural crest marker SNAI2. These cells will be useful for studying the human neural crest-derived pigment cells.

An analysis of lateralized neural crest marker expression across development in the Mexican tetra, Astyanax mexicanus.

The biological basis of lateralized cranial aberrations can be rooted in early asymmetric patterning of developmental tissues. However, precisely how development impacts natural cranial asymmetries remains incompletely understood. Here, we examined embryonic patterning of the cranial neural crest at two phases of embryonic development in a natural animal system with two morphotypes: cave-dwelling and surface-dwelling fish. Surface fish are highly symmetric with respect to cranial form at adulthood, however adult cavefish harbor diverse cranial asymmetries. To examine if lateralized aberrations of the developing neural crest underpin these asymmetries, we used an automated technique to quantify the area and expression level of cranial neural crest markers on the left and right sides of the embryonic head. We examined the expression of marker genes encoding both structural proteins and transcription factors at two key stages of development: 36hpf (∼mid-migration of the neural crest) and 72hpf (∼early differentiation of neural crest derivatives). Interestingly, our results revealed asymmetric biases at both phases of development in both morphotypes, however consistent lateral biases were less common in surface fish as development progressed. Additionally, this work provides the information on neural crest development, based on whole-mount expression patterns of 19 genes, between stage-matched cave and surface morphs. Further, this study revealed 'asymmetric' noise as a likely normative component of early neural crest development in natural Astyanax fish. Mature cranial asymmetries in cave morphs may arise from persistence of asymmetric processes during development, or as a function of asymmetric processes occurring later in the life history.

The heparan sulfate modification enzyme, Hs6st1, governs Xenopus neuroectodermal patterning by regulating distributions of Fgf and Noggin

The nervous system has various types of cells derived from three neuroectodermal regions: neural plate (NP), neural crest (NC), and preplacodal ectoderm (PPE). Differentiation of these regions is regulated by various morphogens. However, regulatory mechanisms of morphogen distribution in neural patterning are still debated. In general, an extracellular component, heparan sulfate (HS), is essential to regulate morphogen gradients by modulating morphogen binding. The present study focused on an HS modification enzyme, heparan sulfate 6-O-sulfotransferase 1 (Hs6st1), which is highly expressed during the neurula stage in Xenopus. Our present in situ hybridization analysis revealed that Hs6st1 is expressed in the lateral sensorial layer of neuroectoderm. Overexpression of Hs6st1 expands Sox3 (NP marker gene) expression, and slightly dampens FoxD3 (NC marker) expression. Hs6st1 knockout using the CRISPR/Cas9 system also expands the neural plate region, followed by retinal malformation. These results imply that 6-O sulfation, mediated by Hs6st1, selectively regulates morphogen distribution required for neuroectodermal patterning. Among morphogens required for patterning, Fgf8a accumulates on Hs6st1-expressing cells, whereas a secreted BMP antagonist, Noggin, diffuses away from those cells. Thus, cell-autonomous 6-O sulfation of HS at the sensorial layer of neuroectoderm also affects neuroectodermal patterning in neighboring regions, including neural plate and neural crest, not only through accumulation, but also through dispersal of specific morphogens.

Human Olfactory Ecto-mesenchymal Stem Cells Displaying Schwann-cell-like Phenotypes and Promoting Neurite Outgrowth in Vitro.

Strategies of Schwann cell (SC) transplantation for regeneration of peripheral nerve injury involve many limitations. Stem cells can be used as alternative cell source for differentiation into Schwann cells. Given the high potential of neural crest-derived stem cells for the generation of multiple cell lineages, in this research, we considered whether olfactory ectomesenchymal stem cells (OE-MSCs) derived from neural crest can spontaneously differentiate into SC lineage. OE-MSCs were isolated from human nasal mucosa and characterized by the mesenchymal and neural crest markers. The cells were cultured in glial growth factors-free medium and further investigated in terms of the phenotypic and functional properties. Immunocytochemical staining and real-time PCR analysis indicated that the cultured OE-MSCs expressed SCs markers, SOX10, p75, S100, GFAP and MBP, differentiation indicative. It was found that the cells could secrete neurotrophic factors, including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Furthermore, after co-cultured with PC12, the mean neurite length was enhanced by OE-MSCs. The findings indicated that OE-MSCs could be differentiated spontaneously into SC-like phenotypes, suggesting their applications for transplantation in peripheral nerve injuries.

Keratinocyte Growth Factor Stimulates Growth of p75+ Neural Crest Lineage Cells During Middle Ear Cholesteatoma Formation in Mice

During development, cranial neural crest (NC) cells display a striking transition from collective to single-cell migration and undergo a mesenchymal-to-epithelial transformation to form a part of the middle ear epithelial cells (MEECs). While MEECs derived from NC are known to control homeostasis of the epithelium and repair from otitis media, paracrine action of keratinocyte growth factor (KGF) promotes the growth of MEECs and induces middle ear cholesteatoma (cholesteatoma). The animal model of cholesteatoma was previously established by transfecting a human KGF-expression vector. Herein, KGF-inducing cholesteatoma was studied in Wnt1-Cre/Floxed-enhanced green fluorescent protein (EGFP) mice that conditionally express EGFP in the NC lineages. The cytokeratin 14-positive NC lineage expanded into the middle ear and formed cholesteatoma. Moreover, the green fluorescent protein-positive NC lineages comprising the cholesteatoma tissue expressed p75, an NC marker, with high proliferative activity. Similarly, a large number of p75-positive cells were observed in human cholesteatoma tissues. Injections of the immunotoxin murine p75-saporin induced depletion of the p75-positive NC lineages, resulting in the reduction of cholesteatoma invivo. The p75 knockout in the MEECs had low proliferative activity with or without KGF protein invitro. Controlling p75 signaling may reduce the proliferation of NC lineages and may represent a new therapeutic target for cholesteatoma.

S3438 Malignant Gastrointestinal Neuroectodermal Tumor of the Small Intestine: A Rare Cause of Iron Deficiency Anemia

Introduction: Gastrointestinal neuroectodermal tumors (GNET) are a type of high-grade sarcoma of the gastrointestinal (GI) tract that share features with clear cell carcinoma of tendon sheaths and aponeuroses. Patients typically present with clinical signs and symptoms of small bowel obstruction, intussusception or abdominal mass. These tumors can arise in the stomach, colon, as well as metastasis to liver or regional lymph nodes. We present a case of ileal GNET in an otherwise asymptomatic patient with Iron-deficiency Anemia (IDA) diagnosed using video capsule endoscopy (VCE). Case Description/Methods: A 58-year-old Hispanic male with a past medical history of GERD and obesity presented for evaluation of chronic microcytic anemia. Workup revealed: hemoglobin 8.6 g/dl, MCV 62.2, iron saturation 2.6%, transferrin 307 mg/dl, ferritin < 5 ng/ml, reticulocyte index of 1.34; serum folate, vitamin B12 and TSH levels were within normal limits. He was taking oral ferrous sulfate and vitamin C for IDA. He reported heartburn but denied melena, hematochezia, hematemesis, weight loss and had an unremarkable colonoscopy four years prior. Upper endoscopy revealed mild erosive esophagitis and mild chronic active gastritis with negative biopsies. Colonoscopy was unremarkable in the setting of optimal bowel preparation. VCE revealed an ulcerated mass occupying 35% of the ileal lumen. CT of the abdomen noted asymmetric wall thickening with enhancement of the small bowel. The patient underwent laparoscopy-assisted enteroscopy with rendezvous technique allowing direct visualization of a 3 cm proximal ileum mass requiring open segmental resection with primary anastomosis. Pathology suggested malignant GNET or clear cell sarcoma-like tumor of the GI tract. Immunohistochemistry was positive for neural crest markers (S100, SOX10) and Vimentin; negative for CD117, DOG1, HMB45, MART1, MAA, chromogranin, and synaptophysin. Fluorescence in-situ hybridization was positive for EWSR1 (22q12) rearrangement. Given the clear surgical margins, absent regional lymph node spread and lack of standard guidelines for this rare pT1N0M0 tumor, surveillance imaging every three months without adjuvant radiation or systemic chemotherapy was recommended. (Figure) Discussion: This case highlights the importance of VCE for small bowel assessment after negative bidirectional endoscopy to identify the etiology of IDA per American Gastroenterology Association guidelines. In this case, an ulcerated GNET was the etiology of IDA persisting despite iron supplementation.Figure 1.: A-Luminal view of small bowel tumor on Video Capsule Endoscopy (VCE) B–Close up view of ulceration of small bowel tumor on VCE C–Histopathology slide showing epithelioid cells with eosinophilic cytoplasm, pleomorphic nuclei with prominent nucleoli and vesicular nucleoli. Scattered mitotic figures identified.

Ash2l, an obligatory component of H3K4 methylation complexes, regulates neural crest development

The vertebrate nervous system develops from embryonic neural plate and neural crest. Although genetic mechanisms governing vertebrate neural development have been investigated in depth, epigenetic regulation of this process remains less understood. Redundancy of epigenetic factors and early lethality of animals deficient in critical epigenetic components pose major challenges in characterization of epigenetic factors in vertebrate neural development. In this study, we use the amphibian model Xenopus laevis to investigate the roles of non-redundant, obligatory components of all histone H3K4 activating methylation complexes (COMPASS, also known as SET1/MLL complexes) in early neural development. The two genes that we focus on, Ash2l and Dpy30, regulate mesendodermal differentiation in mouse embryonic stem cells and cause early embryonic lethality when removed from mouse embryos. Using targeted knockdown of the genes in dorsal ectoderm of Xenopus that gives rise to future nervous system, we show here that ash2l and dpy30 are required for neural and neural crest marker expression in Xenopus late neurula embryos but are dispensable for early neural and neural plate border gene expression. Co-immunoprecipitation assays reveal that Dpy30 and Ash2L associate with the neural plate border transcription factors, such as Msx1 and Tfap2a. Chromatin immunoprecipitation (ChIP) assay further demonstrates that Ash2L and the H3K4me3 active histone mark accumulate at the promoter regions of the neural crest gene sox10 in a Tfap2a-dependent manner. Collectively, our data suggest that Ash2l and Dpy30 interact with specific transcription factors to recruit COMPASS complexes to the regulatory regions of neural crest specification genes to control their expression and influence development of the nervous system during vertebrate embryogenesis.

PEDOT: PSS promotes neurogenic commitment of neural crest-derived stem cells.

Poly (3,4-ethylendioxythiophene) polystyrene sulphonate (PEDOT:PSS) is the workhorse of organic bioelectronics and is steadily gaining interest also in tissue engineering due to the opportunity to endow traditional biomaterials for scaffolds with conductive properties. Biomaterials capable of promoting neural stem cell differentiation by application of suitable electrical stimulation protocols are highly desirable in neural tissue engineering. In this study, we evaluated the adhesion, proliferation, maintenance of neural crest stemness markers and neurogenic commitment of neural crest-derived human dental pulp stem cells (hDPSCs) cultured on PEDOT:PSS nanostructured thin films deposited either by spin coating (SC-PEDOT) or by electropolymerization (ED-PEDOT). In addition, we evaluated the immunomodulatory properties of hDPSCs on PEDOT:PSS by investigating the expression and maintenance of the Fas ligand (FasL). We found that both SC-PEDOT and ED-PEDOT thin films supported hDPSCs adhesion and proliferation; however, the number of cells on the ED-PEDOT after 1 week of culture was significantly higher than that on SC-PEDOT. To be noted, both PEDOT:PSS films did not affect the stemness phenotype of hDPSCs, as indicated by the maintenance of the neural crest markers Nestin and SOX10. Interestingly, neurogenic induction was clearly promoted on ED-PEDOT, as indicated by the strong expression of MAP-2 and —Tubulin-III as well as evident cytoskeletal reorganisation and appreciable morphology shift towards a neuronal-like shape. In addition, strong FasL expression was detected on both undifferentiated or undergoing neurogenic commitment hDPSCs, suggesting that ED-PEDOT supports the expression and maintenance of FasL under both expansion and differentiation conditions.

MITF deficiency and oncogenic GNAQ each promote proliferation programs in zebrafish melanocyte lineage cells.

Uveal melanoma (UM) is the most common primary malignancy of the adult eye but lacks any FDA‐approved therapy for the deadly metastatic disease. Thus, there is a great need to dissect the driving mechanisms for UM and develop strategies to evaluate potential therapeutics. Using an autochthonous zebrafish model, we previously identified MITF, the master melanocyte transcription factor, as a tumor suppressor in GNAQQ209L‐driven UM. Here, we show that zebrafish mitfa‐deficient GNAQQ209L‐driven tumors significantly up‐regulate neural crest markers, and that higher expression of a melanoma‐associated neural crest signature correlates with poor UM patient survival. We further determined how the mitfa‐null state, as well as expression of GNAQQ209L, YAPS127A;S381A, or BRAFV600E oncogenes, impacts melanocyte lineage cells before they acquire the transformed state. Specifically, examination 5 days post‐fertilization showed that mitfa‐deficiency is sufficient to up‐regulate pigment progenitor and neural crest markers, while GNAQQ209L expression promotes a proliferative phenotype that is further enhanced by YAPS127A;S381A co‐expression. Finally, we show that this oncogene‐induced proliferative phenotype can be used to screen chemical inhibitors for their efficacy against the UM pathway. Overall, this study establishes that a neural crest signature correlates with poor UM survival, and describes an in vivo assay for preclinical trials of potential UM therapeutics.

Acetaminophen Disrupts the Development of Pharyngeal Arch-Derived Cartilage and Muscle in Zebrafish.

Acetaminophen is a common analgesic, but its potential effects on early embryonic development are not well understood. Previous studies using zebrafish (Danio rerio) have described the effects of acetaminophen on liver development and physiology, and a few have described gross physiological and morphological defects. Using a high but non-embryonic lethal dose of acetaminophen, we probed for defects in zebrafish craniofacial cartilage development. Strikingly, acetaminophen treatment caused severe craniofacial cartilage defects, primarily affecting both the presence and morphology of pharyngeal arch-derived cartilages of the viscerocranium. Delaying acetaminophen treatment restored developing cartilages in an order correlated with their corresponding pharyngeal arches, suggesting that acetaminophen may target pharyngeal arch development. Craniofacial cartilages are derived from cranial neural crest cells; however, many neural crest cells were still seen along their expected migration paths, and most remaining cartilage precursors expressed the neural crest markers sox9a and sox10, then eventually col2a1 (type II collagen). Therefore, the defects are not primarily due to an early breakdown of neural crest or cartilage differentiation. Instead, apoptosis is increased around the developing pharyngeal arches prior to chondrogenesis, further suggesting that acetaminophen may target pharyngeal arch development. Many craniofacial muscles, which develop in close proximity to the affected cartilages, were also absent in treated larvae. Taken together, these results suggest that high amounts of acetaminophen can disrupt multiple aspects of craniofacial development in zebrafish.

RNF152 negatively regulates Wnt/β-catenin signaling in Xenopus embryos

The Wnt/β-catenin signaling plays crucial roles in early development, tissue homeostasis, stem cells, and cancers. Here, we show that RNF152, an E3 ligase localized to lysosomes, acts as a negative regulator of the Wnt/β-catenin pathway during Xenopus early embryogenesis. Overexpression of wild-type (WT) RNF152 inhibited XWnt8-induced stabilization of β-catenin, ectopic expression of target genes, and activity of a Wnt-responsive promoter. Likewise, an E3 ligase-defective RNF152 had repressive effects on the Wnt-dependent gene responses but not its truncation mutant lacking the transmembrane domain. Conversely, knockdown of RNF152 further enhanced the transcriptional responses induced by XWnt8. RNF152 morphants exhibited defects in craniofacial structures and pigmentation. In line with this, the gain-of-RNF152 function interfered with the expression of neural crest (NC) markers, whereas its depletion up-regulated NC formation in the early embryo. Mechanistically, RNF152 inhibits the polymerization of Dishevelled, which is key to Wnt signaling, in an E3 ligase-independent manner. Together, these results suggest that RNF152 controls negatively Wnt/β-catenin signaling to fine-tune its activity for NC formation in Xenopus embryo.

Investigating Zmym2 and Zmym4 as Putative Six1 Co‐Factors

Branchio‐oto‐renal syndrome (BOR) is a condition that presents with variable craniofacial, otic, and/or renal malformations. Half of the BOR patients have a mutation in SIX1or its co‐factor EYA1. As the other 50% of cases are of unknown cause, it is important to identify other putative co‐factors of SIX1 that may contribute to BOR. A screen of the fly interactome identified zinc‐finger MYM‐containing proteins, Zmym2 and Zmym4, as putative co‐factors of Six1. Recent studies have established that Zmym2 is a transcriptional repressor that interacts with Six4 during renal development. The function of Zmym4 has not yet been well characterized. In situ hybridization revealed that they are co‐expressed with six1and eya1 in the pre‐placodal ectoderm (PPE) and are later expressed in the otic vesicle (OV) and neural crest (NC) of Xenopus. Luciferase assays indicate that Zmym4 enhances Six1‐Eya1 transcriptional activity whereas Zmym2 has a mild repressive effect. Loss of either Zmym2 or Zmym4 affects neural border, NC, and PPE marker genes, suggesting that both genes are important for craniofacial development. At later stages, loss of either Zmym2 or Zmym4 results in a reduction in the size of the OV as well as the branchial arch NC; the later results in altered craniofacial cartilages. Current experiments aim to decipher whether Zmym2 and Zmym4 interact with Six1 and/or Eya1 through co‐immunoprecipitation experiments. Collectively, these data indicate that Zmym4 modulates Six1 transcriptional activity, Zmym2 and Zmym4 are required for craniofacial development, and suggests they may contribute to BOR phenotypes.

Neural Crest-Derived Stem Cells (NCSCs) Obtained from Dental-Related Stem Cells (DRSCs): A Literature Review on Current Knowledge and Directions toward Translational Applications.

Evidence from dental-related stem cells (DRSCs) suggests an enhanced potential for ectodermal lineage differentiation due to their neural crest origin. Growing evidence that DRSC cultures can produce cells with a neural crest-derived stem cell (NCSC)-like phenotype supports their potential for future therapeutic approaches for neurodegenerative diseases and nerve injuries. However, most of the evidence is limited to the characterization of DRSCs as NCSCs by detecting the expression of neural crest markers. Only a few studies have provided proof of concept of an improved neuro-glial differentiation or direct applicability in relevant models. In addition, a current problem is that several of the existing protocols do not meet manufacturing standards for transferability to a clinical scenario. This review describes the current protocols to obtain NCSCs from DRSCs and their characterization. Also, it provides important considerations from previous work where DRSCs were established and characterized as mesenchymal stromal cells but studied for their neuro-glial differentiation potential. The therapeutic advancement of DRSCs would depend on establishing protocols that can yield a neural crest-like phenotype efficiently, using appropriate manufacturing standards and testing them in relevant models of disease or injury. Achieving these conditions could then facilitate and validate the therapeutic potential of DRSC-NCSCs in regenerative therapies.