Figure 3.

Effects of En2 misexpression on neurogenesis. A, No En2 expression in rat cerebral cortex. En2 expression was detected by RT-PCR of RNAs obtained from freshly dissected E14.5 rat cortex (Ctx) and hindbrain (HB). The primers amplified the same 220-bp region of the En2 3′ UTR from both genomic mouse (Ms) and rat DNAs, consistent with the high degree of nucleotide homology. No expression was found in rat cortex, whereas a high level was detected in the hindbrain, recapitulating the known expression pattern of En2. +/− RT = with/without reverse transcriptase; Con = control. B–D, Transfected cortical cells exhibiting morphologies of undifferentiated precursors and differentiated neurons. The arrowheads denote cells with precursor morphology, whereas the arrows identify cells with neuronal morphology. Magnification 40×; scale bar = 30 μm. B, Phase photomicrograph demonstrating cells with both precursor and neuronal morphologies. Note the large, flat cell bodies and the thick, tapering, and short processes of the precursors (arrowhead). In contrast, neuron-like cells exhibit round cell bodies with long, thin, and uniform-diameter processes (arrow). C, Examples of both precursors and neurons transfected with the En2 vectors and identified for gene expression by immunostaining for EGFP marker protein. D, Cultures that were double labeled to detect EGFP and neuron-specific cytoskeletal protein βIII-tubulin (TuJ1), an early marker of neuronal differentiation. TuJ1 expression was only detected in cells exhibiting neuronal morphologies, a finding consistent with our previous studies (Nicot and DiCicco-Bloom 2001). E, Increase in the proportion of GFP⁺ precursor cells, noted 24 h after En2 transfection. F, Corresponding decrease in the proportion of En2-transfected cells exhibiting neuronal morphology. G, En2 transfection elicited a >50% reduction in TuJ1-immunoreactive cells, a change that parallels the decrease in neuronal morphology. **P<.0005, N=9; ***P<4×10^-8, N=15.