Objectives: To determine whether En2 ontogenetic functions in cerebellar granule neuron precursors (GNPs) exhibit age-dependent effects on proliferation and differentiation.
Methods: En2 cDNA was cloned into an EGFP-containing over expression vector, as described (Benayed et al 2005). Cultured mouse or rat GNPs harvested at postnatal (P) day 4, P7, and P10 were transfected with GFP alone (control) or En2+GFP (En2), and fixed 24h later. Proliferation was assessed using thymidine analog BrdU immunocytochemistry. Neuronal differentiation was defined as cells bearing processes ³ 2-cell bodies. GFP+ cells were immunostained for proliferation and cytoskeletal markers, and assessed by fluorescence microscopy.
Results: En2 overexpression reduced mitotic marker BrdU 75% and 67% at P4 and P7, respectively, but had no effect at P10. Further, overexpression elicited a 2-fold increase in GNPs exhibiting neuronal processes at P4 and P7, but only a 25% increase at P10. Additionally, En2 overexpression reduced BrdU labeling and increased differentiation identically in P7 wildtype and En2 knockout mouse GNPs.
Conclusions: Overexpression of En2 facilitates GNP cell cycle exit and neuronal process outgrowth, indicating the gene promotes the transition from proliferation to differentiation, consistent with postnatal patterns of gene expression. The magnitude of these effects diminished with increasing postnatal age, suggesting En2 functions are modulated by cell-intrinsic signals. Additionally, since En2-naïve and WT GNPs respond identically to En2-overexpression, postnatal function is not dependent on earlier En2 expression. Therefore these data imply that En2 functions should be studied in each developmental epoch in which it is expressed to characterize potentially diverse roles.