Development of subcellular mRNA compartmentation in hippocampal neurons in culture

Abstract

Neurons possess an RNA transport system that is present in dendrites (but not axons) and sort mRNAs so that some mRNAs are restricted to cell bodies while a few others (like the mRNA for MAP2) are present in dendrites. The present study evaluates when dendrite-specific RNA transport and mRNA sorting into cell body and somatodendritic compartments first appear in developing hippocampal neurons maintained in culture. A 3H-uridine pulse-chase paradigm was used to evaluate transport of newly synthesized RNA from the site of synthesis in the nucleus into the developing neurites. The intracellular distribution of mRNAs encoding actin, tubulin, GAP-43, and MAP2 as well as polyA RNA and rRNA was evaluated by in situ hybridization at different stages of development. Newly synthesized RNA was translocated into both developing axons and dendrites early in development, but only into dendrites as the neurons matured. Tubulin, GAP-43, and actin mRNAs, which are restricted to cell bodies in mature neurons, were found exclusively in neuronal cell bodies at all developmental stages. MAP2 mRNA, which is present in the dendrites of mature neurons, was present at very low levels in neurons at 2 or 3 d in culture and was not detectable within dendrites. The overall levels of MAP2 mRNA increased over time, and by 5-7 d in culture, MAP2 mRNA was detectable in some dendrites. PolyA RNA and rRNA were detectable in developing neurites including axons. Levels of polyA and rRNA increased in dendrites as neurons matured while labeling of axons diminished. By 10 d in culture, axonal labeling for polyA and rRNA had virtually disappeared. The increase in the levels of polyA, rRNA, and MAP2 mRNA in dendrites between 5 and 7 d in culture corresponds roughly with the appearance of other dendritic characteristics and the beginning of dendritic outgrowth.