Fig. 2. Isolated E14.5 neural stem cell colonies express region-specific genes. (A) Gene expression analysis was determined using RT-PCR. RNA was isolated from neural stem cell colonies derived from cortical (CTX), medial ganglionic eminence (MGE), midbrain/rostral hindbrain (MB/rHB) or caudal hindbrain (cHB) germinal zone dissections, and generated after 7 days of culture. Primers were designed to detect Emx1 (229 bp), Dlx2 (310 bp), En1 (567 bp), Hoxb1 (307 bp), Otx1 (347 bp) and Emx2 (151 bp). To normalize for the amount of cDNA present in the sample, the cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (401 bp) was amplified and band intensity was comparable to the band intensities from sphere colonies and tissue (not shown). Data are representative of at least 3 separate experiments. (B) Single sphere RT-PCR analyses of E14.5 primary sphere colonies were performed and representative examples (lanes 1-9) are shown. Some of MGE-derived sphere colonies (lane 6 in this figure) as well as all of the MB/rHB colonies expressed En1 as revealed by 40 cycles of PCR amplification. However, the expression of the En1 gene in the MGE sphere colonies was relatively weaker than that in the MB/rHB colonies; the expression in the single MGE colonies was below the level of detection after 35 PCR cycles.