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Intrauterine infection would harm a developing embryo/fetus, thereby increasing the risk of developmental malformation. But, whether or not the infection-induced inflammation affects neural crest development still remains obscure. In this study, we employed meta-analysis to demonstrate the potential correlation between infection-induced inflammation and craniofacial anomalies, which was usually derived from the problems in neural crest cell development. The correlation was further verified by inflammatory cytokine release and the activation of nuclear factor kappa-light-chain enhancer of activated B cells signaling in lipopolysaccharide-treated HH10 chicken embryos. In such an inflammatory condition, AP-2α- and Pax7-labeled pre-migratory and migratory neural crest cells in HH10 chicken embryos were significantly less than the ones in control. The bioinformatics analysis of RNA-seq data demonstrated that the principal differential gene expression occurred in transforming growth factor-beta (TGF-β) signaling pathway, which was confirmed by the subsequent experimental results of quantitative PCR and immunofluorescent staining. Under this inflammatory circumstance, whole-mount in situ hybridization, immunofluorescence, and quantitative PCR showed the gene expression changes of key EMT-related transcription factors including upregulated Msx1, downregulated Slug, and FoxD3, as well as adhesion molecules and extracellular matrix protein including upregulated Cadherrin6B, E-cadherin, N-cadherin, and Laminin at the dorsal portion of neural tube of HH10 chicken embryos. Meanwhile, the bioinformatics analysis of RNA-seq data also manifested the differential gene expressions relevant to cell proliferation, which was confirmed by proliferating cell nuclear antigen Western blot data and co-immunofluorescence staining of human natural killer-1 and phosphorylated histone H3. In brief, this study revealed for the first time that the double-edged sword role of TGF-β signaling pathway between intrauterine inflammation (protective role) and cranial neural crest development (harmful role)., (© 2021 Federation of American Societies for Experimental Biology.)

Zinc oxide Nanoparticles (ZnO NPs) are widely used as emerging materials in agricultural and food-related fields, which exists potential safety hazards to public health and environment while bringing an added level of convenience to our original life. It has been proved that ZnO NPs could be taken up by pregnant women and passed through human placental barrier. However, the toxic potential for embryo development remains largely unanswered. In this study, we discovered that ZnO NPs caused the cytotoxicity in vitro. Inhibition of free Zn 2+ ions in solution by EDTA or inhibition of Zn 2+ ions absorption by CaCl 2 could partially eliminate ZnO NPs-mediated cell toxicity, though not redeem completely. This indicated that both nanoparticles and the release of Zn 2+ ions were involved in ZnO NPs-mediated cytotoxicity. In addition, we also found that both nanoparticles and Zn 2+ ion release triggered reactive oxygen species (ROS) production, which further induced cell toxicity, inflammation and apoptosis, which are mediated by NF-κB signaling cascades and the mitochondria dysfunction, respectively. Eventually, these events lead to the suppressed production and migration of cranial neural crest cells (CNCCs), which subsequently prompts the craniofacial defects in chicken embryos. The application of the antioxidant N-Acetyl-L-cysteine (NAC) rescued the ZnO NPs-induced cell toxicity and malformation of the CNCCs, which further verified our hypothesis. Our results revealed the relevant mechanism of ZnO NPs exposure-inhibited the development of CNCCs, which absolutely contribute to assess the risk of nanoparticles application., Competing Interests: Declaration of competing interests The authors report no conflict of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

As a main marine phycotoxin, okadaic acid (OA) is mainly responsible for diarrheic shellfish poisoning (DSP), through specifically inhibiting phosphatase (PP1 and PP2A). It has been shown that isotope labelled-OA could cross the placental barrier in mice. However, it remains obscure how OA exposure could affect the formation of neural crest cells (NCCs), especially cranial NCCs in early embryo development. Here, we explored the effects of OA exposure on the generation of neural crest cells during embryonic development using the classic chick embryo model. We found that OA exposure at 100 nM (80.5 μg/L) could cause craniofacial bone defects in the developing chick embryo and delay the development of early chick embryos. Immunofluorescent staining of HNK-1, Pax7, and Ap-2α demonstrated that cranial NCC generation was inhibited by OA exposure. Double immunofluorescent staining with Ap-2α/PHIS3 or Pax7/c-Caspase3 manifested that both NCC proliferation and apoptosis were restrained by OA exposure. Furthermore, the expression of Msx1 and BMP4 were down-regulated in the developing chick embryonic neural tubes, which could contribute the inhibitive production of NCCs. We also discovered that expression of EMT-related adhesion molecules, such as Cadherin 6B (Cad6B) and E-cadherin, was altered following OA exposure. In sum, OA exposure negatively affected the development of embryonic neural crest cells, which in turn might result in cranial bone malformation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Zika virus (ZIKV) infection during gestation is deemed to be coupled to birth defects through direct impairment of the nervous system during neurogenesis. However, in this study, our data showed that ZIKV infection dramatically suppressed cranial osteogenesis, shown by Safranin O/Fast Green and alizarin red staining, in chick embryos, which provides another possibility that craniofacial bone malformation caused by ZIKV may be a major cause of ZIKV-mediated birth defects. By immunofluorescent staining and electron microcopy, we confirmed ZIKV infection in chick embryo neural tubes and sites of neural crest. Next, in vivo (chick embryos) and in vitro [primary culture of neural crest cells (NCC)] ZIKV and HNK-1 double immunofluorescent staining demonstrated that ZIKV infection inhibited the production of migratory NCC. The reduction of both AP-2α- and Pax7-positive NCC in HH10 chick embryos infected by ZIKV confirmed that abnormal development of cranial NCC also occurred in the migratory process. Whole mount in situ hybridization demonstrated that cadherin 6B expression was elevated and Slug, FoxD3, and BMP4/Msx1 expressions decreased in ZIKV-infected HH10 chick embryos, implying that epithelial-mesenchymal transition (EMT) of neural crest production was blocked by ZIKV infection. Moreover, in vivo and in vitro pHIS3 and Pax7 double immunofluorescent staining showed that NCC proliferation was repressed by ZIKV infection. C-caspase-3 and AP-2α double immunofluorescent staining in HH10 chick embryos and western blotting showed that NCC apoptosis increased following ZIKV infection. Finally, electron microscopy showed multiple autophagosomes in ZIKV-infected embryos, and western blot and LC3B immunofluorescent staining demonstrated that autophagy-related genes were activated by ZIKV infection. Taken together, our data first showed that ZIKV infection during embryogenesis could interfere with cranial neural crest development, which in turn causes aberrant cranial osteogenesis. Our results provided new insights into brain malformations induced by ZIKV infection., (Copyright © 2019. Published by Elsevier B.V.)

SummaryFibroblast growth factor (FGF) signalling acts as one of modulators that control neural crest cell (NCC) migration, but how this is achieved is still unclear. In this study, we investigated the effects of FGF signalling on NCC migration by blocking this process. Constructs that were capable of inducing Sprouty2 (Spry2) or dominant-negative FGFR1 (Dn-FGFR1) expression were transfected into the cells making up the neural tubes. Our results revealed that blocking FGF signalling at stage HH10 (neurulation stage) could enhance NCC migration at both the cranial and trunk levels in the developing embryos. It was established that FGF-mediated NCC migration was not due to altering the expression of N-cadherin in the neural tube. Instead, we determined that cyclin D1 was overexpressed in the cranial and trunk levels when Sprouty2 was upregulated in the dorsal neural tube. These results imply that the cell cycle was a target of FGF signalling through which it regulates NCC migration at the neurulation stage.

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7 + neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7 + and HNK-1 + cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1 + neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU + neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

Slit/Robo signaling plays an important role in the guidance of developing neurons in developing embryos. However, it remains obscure whether and how Slit/Robo signaling is involved in the production of cranial neural crest cells. In this study, we examined Robo1 deficient mice to reveal developmental defects of mouse cranial frontal and parietal bones, which are derivatives of cranial neural crest cells. Therefore, we determined the production of HNK1 + cranial neural crest cells in early chick embryo development after knock-down (KD) of Robo1 expression. Detection of markers for pre-migratory and migratory neural crest cells, PAX7 and AP-2α, showed that production of both was affected by Robo1 KD. In addition, we found that the transcription factor slug is responsible for the aberrant delamination/EMT of cranial neural crest cells induced by Robo1 KD, which also led to elevated expression of E- and N-Cadherin. N-Cadherin expression was enhanced when blocking FGF signaling with dominant-negative FGFR1 in half of the neural tube. Taken together, we show that Slit/Robo signaling influences the delamination/EMT of cranial neural crest cells, which is required for cranial bone development., (Copyright © 2017. Published by Elsevier Inc.)

Excess alcohol consumption during pregnancy could lead to fetal alcohol syndrome (FAS). However, the molecular mechanism leading to craniofacial abnormality, a feature of FAS, is still poorly understood. The cranial neural crest cells (NCCs) contribute to the formation of the craniofacial bones. Therefore, NCCs exposed to ethanol was investigated - using chick embryos and in vitro explant culture as experimental models. We demonstrated that exposure to 2% ethanol induced craniofacial defects, which includes parietal defect, in the developing chick fetus. Immunofluorescent staining revealed that ethanol treatment downregulated Ap-2ɑ, Pax7 and HNK-1 expressions by cranial NCCs. Using double-immunofluorescent stainings for Ap-2ɑ/pHIS3 and Ap-2ɑ/c-Caspase3, we showed that ethanol treatment inhibited cranial NCC proliferation and increased NCC apoptosis, respectively. Moreover, ethanol treatment of the dorsal neuroepithelium increased Laminin, N-Cadherin and Cadherin 6B expressions while Cadherin 7 expression was repressed. In situ hybridization also revealed that ethanol treatment up-regulated Cadherin 6B expression but down-regulated slug, Msx1, FoxD3 and BMP4 expressions. In summary, our experimental results demonstrated that ethanol treatment interferes with the production of cranial NCCs by affecting the proliferation and apoptosis of these cells. In addition, ethanol affected the delamination, epithelial-mesenchymal transition (EMT) and cell migration of cranial NCCs, which may have contributed to the etiology of the craniofacial defects., (Copyright © 2017. Published by Elsevier B.V.)

Dexamethasone (Dex) is commonly used in the treatment of a variety of benign and malignant conditions. Unfortunately, although it has a variety of teratogenic effects, it remains used in clinical practice for pregnant women mainly due to limited alternatives. However, there is limited knowledge of the mechanisms that lead to the observed teratogenic effects. In this study, the effects of Dex during embryogenesis on neural crest development were evaluated in the early chick embryos. First, we demonstrated that 100 µl 10-6 M Dex treatment leads to craniofacial developmental defects, and also retards embryo growth and plausibly can cause embryo demise. Second, we demonstrated that Dex represses the production of HNK-1, PAX7, and AP-2α labeled cranial neural crest cells, the progenitor cells of the craniofacial skeleton. Third, double immunofluorescent staining of pHIS3/PAX7 and AP-2α/c-Caspase3 revealed that Dex promotes cell apoptosis but does not change cell proliferation rates. Last, fibroblast growth factor signaling molecules were inhibited by Dex treatment. Dex also inhibited neural crest cells production by repressing Msx1 expression in the developing neural tube and by altering expression of epithelial-mesenchymal transition-related adhesion molecules and cell migration genes. Overall, we obtained experimental evidence that Dex treatment during embryogenesis disrupts cranial neural crest development which in turn causes defective cranial bone development., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

High glucose levels induced by maternal diabetes could lead to defects in neural crest development during embryogenesis, but the cellular mechanism is still not understood. In this study, we observed a defect in chick cranial skeleton, especially parietal bone development in the presence of high glucose levels, which is derived from cranial neural crest cells (CNCC). In early chick embryo, we found that inducing high glucose levels could inhibit the development of CNCC, however, cell proliferation was not significantly involved. Nevertheless, apoptotic CNCC increased in the presence of high levels of glucose. In addition, the expression of apoptosis and autophagy relevant genes were elevated by high glucose treatment. Next, the application of beads soaked in either an autophagy stimulator (Tunicamycin) or inhibitor (Hydroxychloroquine) functionally proved that autophagy was involved in regulating the production of CNCC in the presence of high glucose levels. Our observations suggest that the ERK pathway, rather than the mTOR pathway, most likely participates in mediating the autophagy induced by high glucose. Taken together, our observations indicated that exposure to high levels of glucose could inhibit the survival of CNCC by affecting cell apoptosis, which might result from the dysregulation of the autophagic process.

Neural crest stem cells (NCSCs), a population of multipotent cells that migrate extensively and give rise to diverse derivatives, including peripheral and enteric neurons and glia, craniofacial cartilage and bone, melanocytes and smooth muscle, have great potential for regenerative medicine. Non-human primates provide optimal models for the development of stem cell therapies. Here, we describe the first derivation of NCSCs from cynomolgus monkey embryonic stem cells (CmESCs) at the neural rosette stage. CmESC-derived neurospheres replated on polyornithine/laminin-coated dishes migrated onto the substrate and showed characteristic expression of NCSC markers, including Sox10, AP2α, Slug, Nestin, p75, and HNK1. CmNCSCs were capable of propagating in an undifferentiated state in vitro as adherent or suspension cultures, and could be subsequently induced to differentiate towards peripheral nervous system lineages (peripheral sympathetic neurons, sensory neurons, and Schwann cells) and mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). CmNCSCs transplanted into developing chick embryos or fetal brains of cynomolgus macaques survived, migrated, and differentiated into progeny consistent with a neural crest identity. Our studies demonstrate that CmNCSCs offer a new tool for investigating neural crest development and neural crest-associated human disease and suggest that this non-human primate model may facilitate tissue engineering and regenerative medicine efforts., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Gene transfection is an indispensable approach for studying gene function since it provides important information on gain- and/or loss-of-function. Chick embryos are also extensively employed for studying biological function since they are easily accessible and can be maintained alive after manipulation. The combination of both techniques presents a powerful approach to understanding how genes regulate embryo development. Furthermore, combining these approaches with tissue transplant techniques make even more attractive for elucidate gene function. Electroporation, employing parallelly fashioned electrodes, has been widely used in chick embryos. However, experimenters have been frustrated by unsuccessfully transfection in some embryonic tissue of interest because the electrodes were improperly positioned. We presently demonstrated the different patterns of organizing and positioning the electrodes, in combination with tissue transplantation, to efficiently and specifically transfect the chick embryonic head, trunk neural tube, heart tube, somites and neural crest cells with the GFP reporter gene. [ABSTRACT FROM AUTHOR]

• Nano-sulforaphane attenuates PhIP-induced neural tube defects. • Nano-sulforaphane efficiently reversed PhIP-inhibited neural cell differentiation. • Nano-sulforaphane improved PhIP-suppressed NCCs generation and migration. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyrimidine (PhIP), one of the most abundant heterocyclic aromatic amines (HAA) formed by cooking meat at high temperatures, may modify humans and rodents through the metabolic process prior to affecting nervous system development. In humans and rodents may be modified by metabolic processes and then affecting nervous system development. In this paper, PhIP was used to prepare a chicken embryo model with abnormal embryonic nervous system defects. Sulforaphane (SFN) is a derivative of a glucosinolate, which is abundant in cruciferous vegetables, and can pass through the placental barrier. Moreover, SFN has antioxidant and anti-apoptotic functions and is considered as a bioactive antioxidant with significant neuroprotective effects. Nano-sulforaphane (Nano-SFN, sulforaphane nanoparticles) was prepared by self-assembly using biocompatible, biodegradable methoxy polyethylene glycol 5000-b-polyglutamic acid 10,000 (mPEG5K-PGA10K) as the substrate, to explore the new application of Nano-SFN and its modified compounds as leading compounds in protecting against the abnormal development of the embryonic nervous system. The results show that Nano-SFN could protect against PhIP-induced central nervous system (CNS, derived from neural tube) and peripheral nervous system (PNS, derived from neural crest cells, NCCs) defects and neural tube defects (NTDs), and increase the embryo survival rate. This study indicates that Nano-SFN can effectively alleviate the developmental defects of embryonic nervous system induced by PhIP in the microenvironment and has a protective effect on embryonic development. It not only helps with expanding the application of SFN and improving its medicinal value, but also provides a possibility of SFN being developed as a novel drug for neuroprotection. [ABSTRACT FROM AUTHOR]