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.

Neural stem cell colonies from E14.5 medial ganglionic eminence germinal zone show a unique migratory potential. Isolated neural stem cell colonies derived from cortical (CTX) or medial ganglionic eminence (MGE) germinal zone of GFP transgenic mice were deposited on CD1 coronal slices and observed under phase contrast (A) and fluorescent (B) microscopy. After 48 hours co-culturing, cells migrating out of sphere colonies derived from CTX (C) or MGE (D) were observed using fluorescence microscopy. (E) Higher magnification image of the boxed area in D is shown and the numbers of cells that had migrated into the three 0.25 mm × 0.25 mm areas indicated (I-III) were counted. Distance migrated: I, 0.25-0.5 mm; II, 0.5-0.75 mm; III, 0.75-1.0 mm. (F) The number of cells in zones I, II or III (derived from CTX (n=9) or MGE (n=12) sphere colonies after 48 hours co-culturing with CD1 coronal slice) were quantified. Scale bar, 0.5 mm (A-D).

The expression of a ganglionic eminence-specific late marker gene in vivo and in neural stem cell colonies. (A) A coronal slice (400 μm thick) of E14.5 zfdlx4/dlx6lacZ transgenic mice brain was subject to X-gal staining, and showed robust transgene expression in subventricular zone and mantle zone, but not in ventricular zone, of the ganglionic eminence. Bar, 1.0 mm. (B) The expression of the endogenous Dlx5 gene was determined using RT-PCR. RNA was isolated from E14.5 primary 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 in culture. Positive amplification of 438 bp bands was demonstrated in the neural stem cell colonies from CTX and MGE, as well as in the corresponding primary tissues.

Ganglionic eminence-specific late marker gene expression in both forebrain and midbrain/hindbrain neural stem cell colonies. Neural stem cell colonies derived from E14.5 zfdlx4/dlx6lacZ transgenic mice were co-cultured with E14.5 CD1 tissues. (A,B) Sphere colonies derived from cortex, ganglionic eminence (GE) or midbrain/rostral hindbrain (MB/rHB) were placed (A, dotted areas) on the GE of host in the CD1 coronal slices. After 5 days in vitro (B), although some distortion of the tissue was seen, robust transgene expression (blue arrow pointing to blue staining) could be observed only when sphere colonies were placed in the region of the ventral forebrain. (C) Higher magnification of the boxed area in B showing transgene expression. (D,E) Transgene expression was not induced in cortical, GE and MB/rHB colonies (originally located in dotted areas) when placed on the germinal layer of CD1 cortical tissue (D) or midbrain tissue (E) after 5 days in vitro. (F-H) Neurosphere colonies were placed on the isolated VZ (F, dotted areas on top slice) from E14.5 CD1 coronal slice and on the remaining GE in the slice (F, dotted areas on bottom slice). Transgene expression was not observed in the neural stem cell colonies (0/4 co-cultures) on isolated VZ tissue (G, arrowhead), but was expressed (4/4 co-cultures) in the neural stem cell colonies on the remaining GE tissue, composed of subventricular zone and mantle zone (H, blue arrow pointing to blue staining). Data are representative of at least 3 separate experiments in each case. Scale bar, 1.74 mm (A), 1.42 mm (B), 0.39 mm (C), 12.1 mm (D,E), 2.2 mm (F), or 1.2 mm (G,H).