Functional Study Of LRP4 In The Limb Development Process

Limb development of the vertebrate is one of the most complicated and precision process.Finding and studying genes related to limb development has been a hot topic in developmental biology..In human,three rare genetic diseases were caused by Low-density lipoprotein receptor-related protein 4 gene(LRP4)mutations,Cenani-Lenz Syndactyly Syndrome(CLS),Myasthenic syndrome,congenital 17,and Sclerosteosis 2.CLS is a kind of severe limb anomalies disease.Lrp4 deficient mice also exhibit limb syndactyly.However,the underlying molecular mechanism of how LRP4 participates in limb regulation remains unclear.To elucidate the roles of LRP4,we carried out a series of in vivo and in vitro experiments,and found that LRP4 might cross-talk with Wnt and Bmp signalling to regulate limb development.Through LRP4 siRNA knockdown in h FOB cell,when 70% knockdown efficiency was reached,expressions of Wnt/β-catenin signaling pathway and BMP pathway were upregulated significantly.Dual Luciferase Reporter Assaywas carried to further confirm that overexpression of Lrp4 could antagonize the classical Wnt signaling and Bmp signaling.Lrp4-overexpressed-MC3T3-E1 cells was constructed by lentivirus mediated way which was verified by q PCR and Western Blotting analysis.Compared with LRP4 siRNA knock down,the classical Wnt signaling pathway and Bmp signaling pathway were noticeably downregulated in Lrp4-stably-overexpressing MC3T3-E1 cells.The LRP4 antagonistic activity of WNT and BMP signaling was further verified by Dual Luciferase Reporter Assay in Lrp4-stably-overexpressing MC3T3-E1 cells,which exhibited a downregulated level of WNT and BMP signaling activity.These data demonstrated that,LRP4 is a negative regulator of Wnt/β-catenin signaling and BMP signaling in osteoblast cell lines.Concurrent with the antagonistic effects of LRP4 on BMP and Wnt signalings,markers of osteogenic differentiation were significantly augmented upon knockdown of LRP4 in h FOB cells.In contrast,the expressions of osteogenic differentiation genes were dramatically reduced in Lrp4-stably-overexpressing MC3T3-E1 cells.Taken together,these results indicate that LRP4,which bears repressive activities on BMP and Wnt/β-catenin signalings,regulates chondrogenesis in osteoblast cell lines.In mouse embryonic stem cells ES17,Lrp4 was knoch out by CRISPR/Cas9 tecnology.A 88 bp deletion was produced in Lrp4-/-ES17 cells which resulted in a frameshift mutation with a 366 AA truncated protein.Consistent with siRNA knock down anyalysis,Wnt/β-catenin signaling and BMP signaling were significantly upregulated in Lrp4-/-ES17 cells.Dual Luciferase Reporter Assay further confirm that the classical Wnt signaling and Bmp signaling were upregulated in Lrp4-/-ES17 cells.RNA-seq was carried out in Lrp4-/-ES17 cells.By GO and KEGG data analysis,we found that there were 1989 differentially expressed genes in Lrp4-/-ES17 cells compared with wildtype ES17 cells.Among that,930 genes were upregulated and 1059 genes were down regulated.To model the disorders caused by a loss of LRP4 function,we performed lrp4 MO injections in developing zebrafish embryos.Zebrafish morphants parital phenocopy human disorders with fin and kidney defects.The analysis of the expression of Wnt/β-catenin pathway and BMP pathway components by q RT-PCR indicated that Wnt/β-catenin pathway and BMP pathway activities were negatively regulated by Lrp4 in the zebrafish.Dual luciferase assay showed that the Wnt/β-cateninsignaling and the Bmp signaling were significantly downregulated in lrp4 morphants.LRP4 mutations in humans can cause CLS syndrome.To investigate whether a loss of LRP4 function causing limb defects in CLS also alters the Wnt/β-catenin and BMP signaling activity,we analyzed the effect of CLS missense mutations on the transduction and activation of BMP signaling by using a Dual Luciferase Reporter Assay in HEK293 T cells.Coexpression of each of missense mutations with Lef1 or ID1 reporter abolished the observed antagonistic Lrp4 effect on Wnt/β-catenin and BMP signaling.In a summary,these results indicate that LRP4 regulates cartilage and bone development through a crosstalk between BMP and Wnt/β-catenin signaling pathways.Abolishing the antagonistic effects of LRP4 on BMP and Wnt/β-catenin signaling leads to human limb malformations.