Effects Of Fam83h Mutation On Enamel Development Of Mice

Autosomal dominant hypocalcified amelogenesis imperfecta(ADHCAI)is the most severe enamel malformation,in which the enamel is cheesy-soft with normal thickness and often abrades soon after tooth eruption.Mutations located on exon 5 of family with sequence similarity 83 member H(FAM83H)are the main pathogenic factor of ADHCAI.It is generally believed that FAM83H haploinsufficiency does not cause ADHCAI,and truncated FAM83H might act in a dominant-negative manner.However,the specific pathogenic mechanism is still unclear.In this study,Fam83h c.1186C>T(p.Q396*)knock-in C57BL/6J mouse model was used to explore the effects of truncated FAM83H on teeth development of mice from the following parts.Part Ⅰ Generation of Fam83h c.1186C>T(p.Q396*)knock-in miceObjective:Truncated FAM83H encoded by mutated FAM83H on exon 5 might cause ADHCAI in a dominant-negative manner.This study aimed to construct Fam83h c.1186C>T(p.Q396*)knock-in mice.Materials and Methods:Genomic DNA from tail biopsies of wild-type,heterozygous(Fam83h+/Q396*),and homozygous(Fam83hQ396*/Q396*)mice was subjected to PCR,agarose gel electrophoresis and sequencing for genotyping and validation.Western blot analysis was performed to detect the expression of FAM83H 1-1209 protein and truncated FLAG-FAM83H 1-395 protein in protein lysates from postnatal day 1(p1)tails of mice of three genotypes.During the growth of Fam83hQ396*/Q396*mice,the body appearance,activity,growth rate,and body size were evaluated.The Alizarin red S and Alician blue compound staining was performed to detect the bone development of wild-type and Fam83hQ396*/Q396*mice.Immunohistochemistry assay was performed to detect the expression pattern of FAM83H in Fam83h+/Q396*pregnant mice.Results:The agarose gel electrophoresis showed that wild-type mice exhibited Fam83h allele band of 152 bp,Fam83hQ396*/Q396*mice exhibited Fam83hQ396*allele band of 223 bp,and Fam83h+/Q396*mice exhibited bands of both sizes.The sequencing analysis revealed that,in Fam83hQ396*/Q396*mice,a flag-tagged stop codon(GACTACAAAGACGATGACGACAAG-TGA)was introduced to Fam83h gene to replace the glutamine codon(CAG)at position 1186-1188.In Fam83hQ396*/Q396*mice,FAM83H 1-1209 protein expression was disrupted while FLAG-FAM83H 1-395 was successfully expressed.Offspring of Fam83h +/Q396*mice mating with each other(29.9%,53.6%,and 16.5%)were not born with the expected Mendelian ratio(1:2:1).The number of homozygous offspring was lower than expected;further,they were all male.Compared to control mice,Fam83hQ396*/Q396*mice exhibited a smaller body size,a sparse and scruffy coat,delayed eye-opening,and decreased general activity.And most of them died within 2 weeks after birth.Only a few survived longer than 7 weeks and were sterile.Moreover,almost all Fam83hQ396*/Q396*mice showed swollen forelimbs and dry scaly skin with exfoliated flakes approximately nine days after birth.The Alizarin red S and Alician blue compound staining showed that bone development of Fam83hQ396*/Q396*mice was inhibited.FAM83H showed moderate to strong immunohistochemistry staining in the ameloblast,urinary bladder,tongue,ovary,oviduct,and implanted embryos in uterus of Fam83h+/Q396*pregnant mice,while it showed weak immunohistochemistry staining in the submandibular salivary glands and thymus.Conclusion:The Fam83h c.1186C>T(p.Q396*)knock-in mice was successfully constructed,and it could express truncated FLAG-FAM83H 1-395 protein.FAM83H was widely expressed in many tissues.Fam83h c.1186C>T(p.Q396*)inhibited the gross development of C57/BL6 mice.Part Ⅱ Effects of truncated FAM83HQ396*on enamel development and tooth eruption of miceObjective:Truncated FAM83H might play a dominant negative role in teeth development.Truncated FAM83H of different amino acid lengths exhibits different degrees of pathological effects on ADHCAI.This study aimed to explore the effect of Fam83h c.1186C>T(p.Q396*)on teeth development of mice.Materials and Methods:Dental phenotypes of Fam83hQ396*/Q396*mice were observed by stereomicroscope.Eenergy dispersive X-ray spectroscopy(EDS)analysis and diaminobenzidine(DAB)-enhanced Prussian blue staining were used to detect the iron content of Fam83hQ396*/Q396*lower incisors.In Fam83h mutated ameloblasts,the expression of transferrin receptor(TFRC)and solute carrier family 40 member 1(SLC40A1),two iron transportation genes,was detected by quantitative real-time PCR(qPCR),western blot and immunohi stochemi stry analyses.Micro-computed tomography(micro-CT)analysis,vickers microhardness(HV)test,and scanning electron microscopy(SEM)assay were used to detect microstructural changes of Fam83hQ396*/Q396*lower incisors.The eruption time of incisors of each genotype mice was observed,and the eruption of mandibular first molar was evaluated by micro-CT analysis.Results:Fam83h Q396*/Q396*lower incisors were shorter and less sharp than those of control mice,and they lost yellow-brown pigment on the labial surface of enamel.EDS analysis revealed that the elemental composition of the lower incisors of Fam83h Q396*/Q396*mice had significantly less iron and increased calcium(Ca)and phosphorus(P),while the Ca/P ratio remained the same as that of the wild-type incisors.DABenhanced Prussian blue staining further verified the decreased iron element in Fam83h Q396*/Q396*ameloblasts of lower incisors.In addition,the mRNA and protein expression levels of Tfrc and Slc40al were decreased in Fam83h mutated ameloblasts.Fam83h Q396*/Q396*lower incisors showed enamel defects,decreased micro-hardness in enamel near dentin-enamel junction(E-DEJ),and interrupted enamel rod structure.The eruption time of Fam83hQ396*/Q396*incisors was earlier than that of wild-type and heterozygous littermates,while Fam83hQ396*/Q396*mandibular first molar showed later eruption time.Conclusion:FAM83H Q396*led to enamel defects,reduced enamel hardness,and disrupted enamel rod structure.The decreased iron on the labial surface of Fam83h Q396*/Q396*lower incisors might be caused by decreased expression of iron transportion proteins of TFRC and SLC40A1 in ameloblasts.In addition,the Fam83hQ396*/Q396*mutation promoted incisors eruption and inhibited molars eruption in C57/BL6 mice,respectively.Part Ⅲ Fam83h mutation disrupted the FAM83HCK1α-K14-AMELX interaction and cell-cell adhesion in ameloblastsObjective:Truncated FAM83H affects enamel development.However,the specific molecular biological mechanism is still unclear.This study aimed to explore the mechanism about effects of Fam83h c.1186C>T(p.Q396*)on enamel development.Materials and Methods:Western blot was used to detect the expression pattern of FAM83H,keratin 14(K14),casein kinase 1α(CK1α),and amelogenin(AMELX)in tooth germ of the wild-type mandibular first molar.Co-immunoprecipitation(co-IP)and immunofluorescence assay were used to detect the interaction of FAM83H-CK1αK14-AMELX in Fam83h mutated ameloblasts in vivo and in vitro.The expression of AMELX in the secretion-and maturation-stage Fam83hQ396*/Q396*ameloblasts was determined by immunohistochemistry assay.Histological analysis of wild-type,Fam83h+/Q396*,and Fam83hQ396*/Q396*mandibular first molars of mice on p 5,11,and 14 was performed.Lower incisors of wild-type and Fam83hQ396*/Q396*mice were subjected to standardized sequential cross-section,and the histology of the ameloblasts in different stage was analyzed.The RNA-sequencing and bioinformatics analysis in LS8-NC and LS8-flag-Fam83h-mut cells were performed.The expression of cell-cell junction indicators in Fam83h mutated ameloblasts was detected by immunofluorescence assay.Results:In embryonic day 16.5(E16.5),E17.5,postnatal day 0(p0),p1,p2,p3 and p8 tooth germ of the wild-type mandibular first molar,K14 and CK1α showed relatively stable expression,while FAM83H and AMELX showed strong expression during the secretion stage of ameloblast.In ameloblasts,FAM83H interacted with CK1α,K14,and AMELX.However,K14 and AMELX were disintegrated from the tetramer in Fam83h-mutated ameloblasts in vitro and in vivo.Immunohistochemical analysis revealed more AMELX retention in the cytoplasm of secretion stage ameloblasts in Fam83hQ396*/Q396*mice than that in wild-type mice.The secretion stage ameloblasts of mandibular first molars of Fam83hQ396*/Q396*mice on p5 exhibited a relatively increased nucleus-to-cell height ratio and were distorted and irregular.There were distinct spaces between the secretion-,transition-,and maturation-stage ameloblasts in Fam83hQ396*/Q396*incisors.124 genes were differentially expressed between LS8-NC cells and LS8-flag-Fam83h-mut cells.For the GO enrichment of differentially expressed genes,the cell adhesion pathway was the most significantly enriched.In addition,compared to the control group,the expression of DESMOGLEIN 3 was decreased in Fam83h mutated ameloblasts in vivo and in vitro.Conclusion:Fam83h Q396*/Q396*ameloblasts showed interrupted interaction of FAM83H-CK1α-K 14-AMELX,AMELX secretion retention,decreased DESMOGLEIN 3 protein expression,and abnormal cell-cell adhesion,affecting the directed secretion of enamel matrix and enamel mineralization.