Novel Lhx8 Roles in Odontoblastic Lineage Specification via TGFβ/Wnt Signaling

Objectives: Lhx8 (LIM homeobox 8) plays pivotal roles in craniofacial development, especially palatogenesis and odontogenesis. However, little is known how Lhx8 mediates tooth development. We hypothesized that Lhx8 functions importantly in odontoblastic lineage specification and tooth specific extracellular matrix gene expression.
Methods: Lhx8 expression was determined by in situ hybridization and qPCR analysis of laser captured dental mesenchyme. Lhx8-knocked down E16.5 dental mesenchyme was recombined with dental epithelium for organ culture. Lhx8 overexpression and knockdown were induced in human dental pulp stem/progenitor cells (hDPSCs) for qPCR and Western blot, with mineralization assayed by ALP and alizarin red. Proliferation and migration of hDPSCs were analyzed by CCK8 and Transwell assays respectively. Genome wide profiling was performed and analyzed by Ingenuity Pathway Analysis. Putative direct targets of Lhx8 were further confirmed by ChIP. Lhx8-overexpressed hDPSCs seeded porous scaffolds were implanted in nude mice (n=24) subcutaneously. All quantitative data were analyzed statistically by one-way ANOVA and Bonferroni adjustment upon confirmation of normal distribution.
Results: Lhx8 expression was restricted in dental mesenchyme of E12.5 and gradually decreased afterwards, suggesting its stage-specific roles in dentinogenesis. Lhx8 overexpression in hDPSCs robustly affected ALP and alizarin red staining over the tested 21 day. Recombination assay and in vivo transplantation further confirmed the role of Lhx8 in dentinogenesis. Overexpression of Lhx8 could also promote the proliferation and migration. RNA-Seq analysis revealed that a total of 846 genes were differentially expressed by Lhx8 knockdown, and bioinformatics revealed that TGFβ and Wnt were key signals for the altered gene expression profile. Further ChIP analysis showed that Lhx8 directly bound promoters of Wnt3a, Smad6, Smurf2 and others. Besides these Wnt/TGFβ regulators, Lhx8 can also transcriptionally activate matrix genes related to tooth development.
Conclusions: Lhx8 is likely an early but pivotal regulator of odontoblastic lineage specification and plays important roles in dentinogenesis.