| ObjectiveSubmucous cleft palate caused by decreased palatal osteogenesis is a common birth defect.Currently,there are few etiology studies about the submucous cleft palate,and the main reason is the lack of knowledge about the anatomy of the hard palate and the normal palatal osteogenesis process.Osteogenesis is a complex process,involving condensation of mesenchymal cells,osteoblasts differentiation and maturation,extracellular matrix deposition and mineralization,osteoclasts differentiation and bone remodeling,and angiogenesis.These osteogenic events are regulated by specific genes.It is known that the osteogenesis mechanisms of the palatal process of the maxilla and palatal process of the palatine bone are different,but the known is still insufficient.BMP(Bone Morphology Protein)type Ⅰ receptor includes Bmpr1a(Bone morphogenetic protein receptor type 1A),Bmpr1b(Bone morphogenetic protein receptor type 1B),and Acvr1(Activin A receptor type 1).It is known that Bmpr1 a plays an important role in the early differentiation of osteoblasts in the palatal mesenchyme,while it is unknown whether Bmpr1 b and Acvr1 function in palatal osteogenesis and whether BMP type Ⅰ receptor is involved in other osteogenic events.Taken together,this study intends to describe the osteogenesis process of the hard palate and analyze the gene expression patterns of the palatal process of the maxilla and palatal process of the palatine bone.Finally,the functions and regulated mechanisms of the BMP type Ⅰ receptor during palatal osteogenesis are elucidated.Contents and Methods1.Observed the anatomy of the hard palate of E(Embryonic day)18.5 embryos and adult mice through Micro-CT.Alizarin red staining and von Kossa staining of the hard palate of E14.5 to E18.5 were used to describe the calcification process of hard palate.The immunofluorescence stainings of Runx2(Runt-related transcription factor-2),Col1(type Ⅰ collagen),Osx(Sp7 transcription factor),Opn(Osteopontin),Ocn(Osteocalcin),and CD31(platelet endothelial cell adhesion molecule-1)together with TRAP(Tartrate-Resistant Acid Phosphatase)staining were used to outline the process of palatal osteogenesis and angiogenesis.2.RNA-seq analysis on the palatal process of the maxilla and palatal process of the palatine bone of E14.5 to E15.5 was conducted.GO(Gene Ontology)annotation on the genes with fpkm(fragments per kilobase of transcript per million mapped reads)≥ 1 at E15.5 was performed and the osteogenic and angiogenic genes were summarized.The expression of osteogenic and angiogenic genes between the palatal process of the maxilla and palatal process of the palatine bone was analyzed and the differently expressed genes were summarized.The alteration in the expression of osteogenic and angiogenic genes during E14.5 to E16.5 was analyzed.3.RNA-seq(RNA-sequencing)and RT-q PCR(Real-Time Quantitative polymerase chain reaction)were used to examine the expression patterns of Bmpr1 a,Bmpr1b,and Acvr1 in the hard palate.The inhibitor of Bmpr1 a and Acvr1,LDN-193189,was used to induce the submucous cleft palate.Immunofluorescence stainings of Runx2,Col1,Osteirx,Opn,Ocn and CD31,together with TRAP staining,between the control group and administered group was performed to elucidate the role of BMP signaling pathway during palatal osteogenesis.RNA-seq analysis on the hard palate at E15.5 between the control group and administered group was conducted.GO annotation on the downregulated genes was performed.Results1.The mouse hard palate is comprised of the palatal process of the maxilla and palatal process of the palatine bone.The palatal process of the maxilla connected with the maxilla at the first molar and then extended toward the primary palate in partial palatal plate,while the palatal process of the palatine bone was formed by the horizontal extension of palatine bone.The active period of palatal osteogenesis was E14.5 to E16.5.Bone remodeling in the hard palate mainly occurred on the nasal side of the hard palate.The osteoclasts between the palatal process of the maxilla and palatal process of the palatine bone were heterogeneous in many aspects.The osteoclast in the palatal process of the maxilla was giant and multinucleated but few,which was located on the bone surface at the lateral-nasal side of the palatal process of the maxilla.The osteoclasts in the palatal process of the palatine bone were small with less nuclear but more and on the bone surface adjacent to blood vessels.During the vascularization of the hard palate,blood vessels in the loose mesenchyme invaded the palatal mesenchymal condensation by sprouting fashion.2.RNA-seq analysis on the palatal process of the maxilla and palatal process of the palatine bone at E14.5 to E16.5 showed that the differently expressed genes at E14.5were mainly associated with the muscle development.Nerve-associated differently expressed genes started to be detected at E16.5.Osteogenesis-associated differently expressed genes were detected at E15.5.Analysis of the expression of the osteogenic and angiogenic genes in the hard palate at E15.5 indicated that there were 78 differentially expressed genes between the palatal process of the maxilla and palatal process of the palatine bone.Analysis of the expression of osteogenic and angiogenic genes in the hard palate during E14.5 to E16.5 showed that there were 46up/downregulated genes.3.In the hard palate,the expression of Bmpr1 a was the strongest,followed by Acvr1,while Bmpr1 b was rarely expressed.There was no difference in the expression of Bmpr1 a and Acvr1 between the palatal process of the maxilla and palatal process of the palatine bone.But the expression of Bmpr1 a and Acvr1 was decreased with time.The compound inhibition of Bmpr1 a and Acvr1 by LDN-193189 demonstrated that Bmpr1 a mainly functioned in osteogenesis in the palatal mesenchyme,while Acvr1 was mainly involved in palatal shelves development.During palatal osteogenesis,BMP signaling pathway played a key role in the final differentiation and maturation of osteoblasts and therefore the synthesis and mineralization of the extracellular matrix.In addition,BMP signaling pathway had crosstalk with the Wnt signaling pathway in regulating the palatal osteogenesis.ConclusionThe palatal process of the maxilla and palatal process of the palatine bone differ in histological origin,osteogenic process,and gene expression patterns.Bmpr1 a is the primary BMP type Ⅰ receptor involved in palatal osteogenesis.BMP signaling pathway has crosstalk with the Wnt signaling pathway in regulating the final differentiation and maturation of osteoblasts together with the deposition and mineralization of the extracellular matrix.Our study helps better understand the anatomy of hard palate,normal osteogenesis process,and gene expression patterns,which is significant for the subsequent studies on the submucous cleft palate.We comprehensively explore the role and corresponding mechanisms of BMP signaling pathway during palatal osteogenesis to provide a theoretical basis for the etiology and treatment of the submucous cleft palate. |
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