Gene expression during early murine odontogenesis

Objective: Investigation of gene expression in the murine 1st. molar tooth germ from 12.5dpc to 17.5dpc using 30K murine microarrays, RT-PCR and in situ hybridisation. We have in particular focused on three genes involved in mineralisation occurring post partum, i.e. Ambn, Amlx, Enam.

Methods: Tooth germs were isolated at 24hr. intervals from phenotypical embryos. Total RNA isolated from separate tooth germs used for cDNA synthesis. The resulting cDNA was labelled with either Cy3 or Cy5 using the Genisphere 900 labelling kit. Analysis of gene expression was carried using triplicate 30K microarrays (NTNU Microarray Core Facility, Trondheim, Norway). Real-time RT-PCR was carried out using Ampliqon III mastermix, with primer designed using Primer 3.In situ hybridisation was carried out with DIG-labeled oligo-probes using a Ventana Discovery XT slide processor.

Results: Using microarrays we established a time-course of expression ranging from 12.5dpc to 6DPN.These revealed a dramatic increase in expression starting about 18.5dpc accelerating rapidly thereafter, the increase abating after 2DPN. The data also suggested an intermittent peak of expression at 14.5 to 15.5dpc for several genes, including Ambn, Amlx and Enam. These findings were confirmed using real-time RT-PCR. ISH results suggested these genes to be expressed in surface oral epithelium at 13.5dpc. At 14.5 and 15.5dpc the genes were expressed both epithelium and condensing mesenchyme of the tooth germ and in cells surrounding the tooth germ. At 17.5dpc expression in primarily found in the developing ameloblasts. Other genes, with an expression profile similar to these three genes, also exhibited a similar transient peak of expression.

Conclusions: Genes coding for enamel proteins, and some other genes exhibiting similar expression profiles, exhibit a transient peak of expression during early odontogenesis.