Role Of BMP/Wnt Pathway In Cadmium-Induced Dysplasia Of Cranial Facial Neural Crest In Chicken Embryos

Cadmium（Cd）is a toxic heavy metal,and it has no physiological activity.Amount of Cd is released into the environment by mining,smelting,decomposition of electronic waste and other industrial production activities,which poses a potential threat to hu man health.Bone is the main target organs for Cd exposure,and bone development can be affected by Cd.Craniofacial bone is one of the derivatives of cranial neural crest（CNC）cells.During embryonic development,interfere d of external stimulation on CNC cells may lead to neural tube defects（NTD）,which seriously affect the physical development and life quality of the offsprings.However,there are few reports about Cd exposure on craniofacial skeleton formation during embryonic development,and the mechanism of Cd exposure on craniofacial neural crest development in embryos has not been clarified.Breeding eggs are mature fertilized eggs which can be cultured in ovo.Moreover,the development of chicken embryos is not interfered by external factors and maternal factors,which can better reflect the toxic effects of Cd.Meanwhile,it was confirmed that most of the craniofacial bones were derived from CNC cells in chicken embryos.Based on the above research background,the experiment was conducted to investigate the cellular and molecular biological mechanisms of Cd exposure on the development of cranial and facial neural crest in chick embryos from two aspects:in ovo and in cell.In the ovo experiment,300 hy-line variety white eggs were randomly divided into three groups（100 eggs/group）:the control group（Vcon）,the vehicle control group（Con）,and the Cd treatment group（1μg/egg,Cd-treatment）.Eggs were respectively injected with sodium acetate（Na Ac）and cadmium acetate（CdAc₂）solution by injection of yolk at 4 Hamburger-Hamilton（HH）stage in the Con and Cd-treatment group,then cultured until 12HH stage.The mortality and deformity rate of chicken embryos were calculated.The morphological,structural and functional changes of the chicken embryo head.The expression of calcium adhesive proteins and related transcription factors in the process of neural crest epithelial cells-mesenchymal cells（EMT）and transcriptomics were detected.The related factors of Wnt and BMP pathways,mitochondrial damage related factors,endoplasmic reticulum stress related factors,mitochondrial mitophagy related factors and bone calcification factors were detected.In cell experiments,primary CNC cells of chicken embryo were used as the research object.The Cd concentration was 0μM,2μM,4μM or 10μM respectively in the culture medium.Cell activity,mitochondrial membrane potential and alkaline phosphatase（ALP）activity were detected after culture for 24 hours to further verify the effects of Cd exposure on the development of CNC cells.Subsequently,in order to verify the role of BMP/Wnt signaling pathway in Cd-induced abnormal of cranial facial neural crest development in chicken embryos,added BMP4 activator to interven e Cd exposure.Cell activity,mitochondrial membrane potential,expression level of cadherin and related transcription factors during EMT,ALP activity,Wnt and BMP pathways related factors,and expression level of bone calcification factors were detected.The results were shown as follow:1)Cd exposure resulted in the slower growth rate,reduced the weight of chicken embryos,led to development defects in cranial parietal（PA）and frontal（FR）bone of chicken embryos.2)Cd exposure inhibited the expression of early neural crest cell migration markers,such as Pax7 and HNK1,up-regulated the expression levels of E-cadherin,N-cadherin and Cadherin 6B,down-regulated the expression levels of Vimentin,inhibited the expression of Sema3a and transcription factors（Slug,Pax3,MMP7 and MMP9）,activated the expression of MSX1 and FoxD3,ultimately inhibited the migration of cranial neural crest cells.3)Cd exposure significantly up-regulated the expression of Caspase3 and KI67 in early neural crest cells,suggested that Cd exposure promoted the apoptosis of neural crest cells,but cell proliferation was not inhibited.The results indicated that the inhibition of Cd exposure on cell migration was not caused by cell proliferation.4)The analysis results of transcriptome about chicken embryo head tissue showed that Cd exposure could affect biological processes such as organ morphological development,phylogeny,developmental process and skeletal phylogeny,and the factors that changed these processes mainly focused on the Wnt signaling pathway.5)Cd exposure inhibited the activity of the BMP and Wnt pathway in early chicken embryo cranial neural crest,down-regulated the expression of Wnts,Frizzleds,β-catenin,DVL2,C-Myc,BMP2,BMP4,Smad1,Smad4,Smad5,Smad9,p-SMad1/4/5/9 and other related factors,and up-regulated the expression of p-β-catenin,GSK-3βand APC,thus prevented the development of craniofacial neural crest osteoblasts.6)Cd exposure inhibited the expression of bone mineralization related genes（ALP,Runx2,OC3,Col1α-1 and Col2α-1）and the activity of ALP in the primary CNC cells of chicken embryos.The results indicated that Cd exposure prevented the differentiation of CNC cells into osteoblasts,and inhibited the cranial formation.7)Cd exposure resulted in a decrease in the number of mitochondria and mitochondrial vacuolar degeneration in CNC cells;reduced mitochondrial membrane potential and cell activity and disturbed mitochondrial biogenesis and homeostasis;caused endoplasmic reticulum swelling and rupture,up-regulated the expression of HSPs,GRP78 and XBP-1s/XBP-1u,and down-regulated the expression of ATF6,which resulted in endoplasmic reticulum dysfunction.Meanwhile,the fusion of endoplasmic reticulum and mitochondrial membrane was observed,and the expression of Beclin1,LC3Ⅱ/LC3Ⅰand Parkin were up-regulated,which induced mitochondrial autophagy.These results indicated that Cd exposure induced CNC cells injury through endoplasmic reticulum stress（ERS）-unfolded protein response（UPR）-autophagy dependent pathway.8)The addition of BMP4 activator down-regulated the expression of cadherin（E-cadherin,N-cadherin,Cadherin 6B）and transcription factors（MSX1,FoxD3）,up-regulated the expression of Vimentin,transcription factors（Slug,Pax3,MMP7,MMP9）,bone mineralization related genes（ALP,Runx2,OC3,Col1α-1 and Col2α-1）,and ALP activity.These results showed that the overexpression of BMP4 alleviated cranial neural crest cytotoxicity induced by Cd.9)The addition of BMP4 activator prevented the inhibition effect of Cd exposure on BMP and Wnt pathway activity,which indicated that the overexpression of BMP4 alleviate d the inhibition of Cd exposure on osteoblasts.Meanwhile,these results showed that BMP and Wnt signaling pathway had synergistic effects on craniofacial neural crest in chick embryos.In conclusion,Cd exposure disturbed the EMT process of CNC cells in chicken embryos,inhibited the activity of Wnt and BMP pathways,cause d mitochondrial damage and endoplasmic reticulum dysfunction,led to CNC cells apoptosis,and ultimately inhibited osteoblast formation.Added BMP4 expression alleviated Cd-induced the inhibition effects on the activities of Wnt and BMP pathways and the Cd exposure-induced cranial nerve cytotoxicity,and also demonstrated the synergistic relationship between Wnt and BMP signaling pathways in the development of cranial neural crest was also demonstrated.The study provided a scientific basis for revealing the mechanism of Cd-induced neural tube malformations,and provided a theoretical basis for preventing and treating of fetal craniofacial malformations caused by Cd exposure.