Article Title: Lhx8 Mediated Wnt and TGFβ Pathways in Tooth Development and Regeneration
Figure Lengend Snippet: Lhx8 knockdown accelerates mesenchyme differentiation ( A ) Schematic representation of the Lhx8 intervention and dental epithelium and dental mesenchyme recombination procedures. Dental epithelium and mesenchyme of mandibular first molar at E16.5 are separated and then the dental mesenchyme is placed onto upper chamber of Transwell cell culture insert and further incubated with either RNAi or lentivirus for 1 hr at RT. Meanwhile the dental epithelium is kept in ice cold DPBS. Then the mesenchyme and epithelium are recombined and cultured for up to 7 days. ( B and C ) Representative data showing the location ( B ) and morphology ( C ) of the tooth germ of E16.5 mandibular first molar. ( D–F ) The top view of the recombined tooth germ right after recombination ( D ), 1 day culture ( E ), 3 days culture ( F ) and 7 days culture ( G, control group; K , siLhx8 group). ( H–N ) The recombined tooth germs from control ( G–J ) and siLhx8 (K–N) were harvested at day 7 and chemically stained with HE ( H , I , L and M ) and immunostained with Dspp ( J , N ) ( n=5) . ( O–Q ) Immunofluorescence of the isolated E16.5 dental mesenchyme transfected with Lipofectamine RNAiMAX coated Cy3 labeled RNAi duplex before recombination. Strong positive Cy3 dots indicate high transfection efficiency. ( R ) Knockdown efficiency of Lhx8. ( S , T ) Quantification data of dentin area ( S ) and thickness ( T ). ( U ) Tooth development related gene expression were examined by qPCR in the recombined tooth germs 4 days after in vitro culture from both control and siLhx8 groups. Data presented are a representative of 3 experiments. de, dental epithelium; dm, dental mesenchyme; d, dentin; od, odontoblast; am, ameloblast; e, enamel. Scale bars: 1mm ( B–F, Q and K, O–Q) ; 500 μm ( I and M ); 250 μm ( J and N ); Data represent mean ± SD. * P
Article Snippet: In overexpression experiments, dental mesenchyme was incubated with Lhx8 over-expressing lentivirus, whereas dental epithelium was incubated in DPBS (HyClone, Thermo Scientific) followed by reconstitution[ ], and culture for up to 7 days[ ].
Techniques: Cell Culture, Incubation, Staining, Immunofluorescence, Isolation, Transfection, Labeling, Expressing, Real-time Polymerase Chain Reaction, In Vitro