Osteoclast differentiation and Dynamic mRNA expression during Mice embryonic palatal bone development
This study is the first to investigate the process of osteoclast differentiation, its potential functions, and the associated mRNA and signaling pathway during embryonic palatal bone. Our findings suggest that osteoclasts may be involved in bone remodeling, bone marrow cavity formation, and blood vessel formation in embryonic palatal bone. We observed Trap-positive osteoclasts at E16.5, E17.5, and E18.5 at the palatal process of the palate (PPP), Posterior and anterior part of the palatal process of the maxilla (PPMXP, and PPMXA), respectively, with osteoclast differentiation starting 2 days prior to TRAP positivity. By comparing the key periods of osteoclast differentiation between PPMX and PPP (E14.5, E15.5, and E16.5) using RNA-seq data of the palates, we found that the PI3K-AKT and MAPK signaling pathways were sequentially enriched, which may play critical roles in osteoclast survival and differentiation. Csf1r, Tnfrsff11a, Ctsk, Fos, Tyrobp, Fcgr3, and Spi1 were significantly upregulated in both PPMX and PPP, while Pik3r3, Tgfbr1, and Mapk3k7 were significantly downregulated in both. Interestingly, Tnfrsff11b was upregulated in PPMX but downregulated in PPP, which may regulate the timing of osteoclast appearance. These results contribute to the limited knowledge regarding mRNA specific steps in OLCs differentiation in the embryonic palatal bone. Overall design: E14.5-E16.5 pregnant mice were euthanized by decapitation and soaked in 100% alcohol for one minute. Afterward, embryos were obtained and transferred to ice-cold Hanks liquid. The mandibles and skulls of the embryos were removed. The ppmx and ppp of E14.5 to E16.5 embryos (n = 3 for each group) were dissected out under a stereomicroscope and then stored in RNAlater (BI, Israel) for RNA sequencing (RNA-seq) as described in our previous study.(9) Expression patterns of OLCs differentiation genes expression from E14.5 to E16.5 in PPP and PPMX were compared. The log2 (fold-change cut-off) was set at 0 or higher for analyses of differential expression, and the Q-value was set to less than 0.05. And there were E15.5 vs E14.5 pairwise, E16.5 vs E15.5 pairwise, and E16.5 vs E14.5 pairwise with the E14.5 group, E15.5 group, E14.5 were used as the control groups, respectively. We performed KEGG enrichment analysis of the total differentially expressed genes (DEGs). GSEA enrichment analysis of pathways related to osteoclast differentiation was performed, and the heat map and volcano map of differential genes were displayed, and the corresponding KEGG pathway map was depicted.