Expression of delta‐like 1 homologue and insulin‐like growth factor 2 through epigenetic regulation of the genes during development of mouse molar

Delta‐like 1 homolog ( D lk1 ) and insulin‐like growth factor 2 ( I gf2 ) are two of six well‐studied mouse imprinted gene clusters that are paternally expressed. Their expression is also linked to their maternally expressed non‐coding RNA s, encoded by Gene trap locus 2 ( G tl2 ) and Imprinted maternally expressed transcript ( H 19 ), co‐located as imprinted gene clusters. Using deoxyoligonucleotide microarrays and real‐time RT ‐ PCR analysis we showed D lk1 and G tl2 to exhibit a time‐course of expression during tooth development that was similar to that of I gf2 and H 19 . Western blot analysis of proteins encoded by D lk1 and I gf2 suggested that the levels of these proteins reflected those of the corresponding m RNA s. Immunohistochemical studies of DLK 1 in murine molars detected the protein in both epithelial and mesenchymal regions, in developing cusp mesenchyme, and in newly synthesized enamel and dentin tubules. IGF 2 protein was detected primarily at prenatal stages, suggesting that it may be active before birth. Analysis of methylation of cytosine–phosphate–guanine ( C p G ) islands in both D lk1 and I gf2 suggested the presence of an increasing fraction of hypermethylated bases with increasing time of development. The increased levels of hypermethylation coincided both with the diminished levels of expression of D lk1 and I gf2 and with decreased levels of DLK 1 and IGF 2 proteins in the tooth germ, suggesting that their expression is regulated via methylation of C p G islands present in these genes.