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First published online 13 December 2006
doi: 10.1242/dev.02727

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Ascl1 defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord

¹ Center for Basic Neuroscience, UT Southwestern Medical Center, Dallas, TX 75390, USA.
² Department of Psychiatry, UT Southwestern Medical Center, Dallas, TX 75390, USA.
³ Smilow Neuroscience Program and the Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA.

View larger version (9K): [in this window] [in a new window]   Fig. 1. Diagram of modified Ascl1 BACs and fate-mapping strategy. The Ascl1 gene is located on mouse chromosome 10. The BAC RPCI 428P21 encompasses the Ascl1 coding sequence with 100 kb 5' and 200 kb 3' (orientation 5' to 3' relative to Ascl1). The genes encoding Pah4 (phenylalanine 4-hydroxylase) and Pah (phenylalanine hydroxylase) are 30 kb and 78 kb 5' of Ascl1, and are included in the BAC RPCI 428P21. Ascl1-GIC and Ascl1-CreERTM are modifications of RPCI 428P21 that replace the Ascl1 coding sequence with that encoding GFP and Cre separated by an IRES, or a tamoxifen-inducible Cre (Hayashi and McMahon, 2002) with a heterologous 3' polyA addition cassette from bovine growth hormone, respectively. The R26R-reporter mice were previously described (Soriano, 1999; Srinivas et al., 2001). Protein from these reporters is only made after the STOP sequence is recombined out by Cre recombinase activity.

View larger version (117K): [in this window] [in a new window]   Fig. 2. Ascl1-expressing cells give rise to neurons and oligodendrocytes. (A) GFP fluorescence in a whole-mount E11.5 embryo from the Ascl1-GIC transgenic line. Inset is a cross section showing expression in the sympathetic ganglia. (B-D') X-gal staining of Ascl1-GIC;R26R-lacZ E10.5, 11.5 and 12.5 embryos. Arrow in B indicates barely detectable X-gal staining in the sympathetic ganglia. (C',D') Vibratome sections through the neural tubes of embryos shown in C-D. (E-J) mRNA in situ hybridization on cross sections of E11.5 embryos with Ascl1 (E,G,I) or Cre (F,H,J) probes. (K) X-gal staining of a P30 spinal cord from an Ascl1-GIC;R26R-lacZ transgenic mouse showing labeled cells in gray and white matter. (L-O) Immunofluorescence for YFP (green) in P14 Ascl1-GIC;R26R-YFP transgenic spinal cords double-labeled in red with a marker for neurons (L, NeuN), oligodendrocytes (M, Olig2; N, APC) or astrocytes (O, GFAP). The images are from the dorsal horn gray matter regions (L and right of dotted line in M), or white matter region (left of dotted line in M,N,O). Note that YFP co-labels (yellow) with neuronal and oligodendrocyte markers (arrowheads in L-N) but not the astrocyte marker (O). di, diencephalon; dnt, dorsal neural tube; ent, enteric neurons; hb, hindbrain; mes, mesencephalon; sym, sympathetic neurons; vt, ventral telencephalon.

View larger version (87K): [in this window] [in a new window]   Fig. 3. Stage-specific fate mapping of the Ascl1-lineage. (A-D',H) X-gal staining of Ascl1-CreERTM;R26R-lacZ embryos harvested at the stages indicated and treated with tamoxifen 24 hours before harvest. Whole-mount stained and cleared embryos (A-D) and vibratome sections through the neural tube are shown (A'-D',H). Arrowheads in A,A',B indicate X-gal staining in the sympathetic ganglia. (E) Cross section of an E10.5 Ascl1-CreERTM;R26R-YFP embryo treated with tamoxifen at E9.0. Triple-label immunofluorescence with antibodies to YFP (green), Lmx1b (red) and Lhx1/5 (blue) identify the YFP+ cells as interneurons dI3, dI5 and V2. (F,I,J) Immunofluorescence showing Ascl1 in the dorsal neural tube at E11.5 (F) and broadly in the ventricular zone as well as gray and white matter in E16.5 spinal cord (I). The specificity of the Ascl1 antibody is illustrated by the absence of signal in E16.5 spinal cords of an Ascl1 null embryo (J). (G) In situ hybridization showing Cre mRNA in the neural tube at E11.5. cc, central canal; di, diencephalon; dnt, dorsal neural tube; gm, gray matter; hb, hindbrain; mes, mesencephalon; vt, ventral telencephalon; wm, white matter.

View larger version (82K): [in this window] [in a new window]   Fig. 4. Ascl1 defines lineage-restricted precursors of neurons and oligodendrocytes. (A) Time line depicting major periods of neurogenesis and gliogenesis during mouse embryonic spinal cord development. Arrowheads indicate individual time points for tamoxifen induction. (B-N) P30 spinal cords from Ascl1-CreERTM;R26R-lacZ (B,C,E-H,K-N) or Nestin-CreERT2;R26R-lacZ (D,I,J) stained for ß-gal activity (B-D,K-N) or double-label immunofluorescence for ß-gal (green) and NeuN (red) (E,G,I) or APC (red) (F,H,J). In Ascl1-CreERTM;R26R-lacZ mice, activation of Cre by tamoxifen at E10.5 results in X-gal labeled cells largely restricted to the dorsal horn gray matter (B) in cells that co-label with the neuronal marker NeuN (E, arrows). Activation of Cre at E15.5 results in X-gal labeled cells largely restricted to white matter regions (C) in cells that co-label with the oligodendrocyte marker APC (H, arrows). By contrast, in Nestin-CreERT2;R26R-lacZ, activation of Cre at E10.5 results in X-gal labeled cells in both gray and white matter throughout the dorsoventral axis (D) and cells co-label with both NeuN and APC (I,J, arrows).

View larger version (88K): [in this window] [in a new window]   Fig. 5. Ascl1-expressing cells rapidly exit the cell cycle and move laterally out of the ventricular zone. Immunofluorescence with antibodies to YFP (green) and the cell cycle marker Ki67 (red) on neural tube sections from Ascl1-CreERTM;R26R-YFP transgenic embryos with either Ascl1 wild-type (A,A') or Ascl1 null (B,B'), or Nestin-CreERT2;R26R-YFP transgenic embryos (C,C'). Tamoxifen induction was initiated at E10.5 and expression was examined 24 hours later at E11.5. A'-C' are higher magnification images of comparable regions boxed in A-C. (A,A') YFP labeled cells in Ascl1-CreERTM;R26R-YFP do not co-label with Ki67. (B,B') In the absence of Ascl1, YFP and Ki67 co-labeled cells are found in the ventricular zone (B', arrowheads). (C,C') By contrast, some YFP labeled cells in Nestin-CreERT2;R26R-YFP co-label with Ki67 and are found in the ventricular zone (C', arrowheads).

View larger version (66K): [in this window] [in a new window]   Fig. 6. Ascl1 facilitates restriction of progenitor cells to the neuronal lineage during neurogenesis. Ascl1-CreERTM;R26R-YFP transgenic spinal cords, either Ascl1 wild-type (A-F) or Ascl1 null (A'-F'), were examined at E17.5 after tamoxifen induction of Cre at E10.5. Double-label immunofluorescence for YFP (green) and for markers of neurons (NeuN; A,A'), oligodendrocytes (Sox10; B,B'), astrocytes (GFAP; C,C'), neural progenitors or astrocytes (Glast; D,D'), oligodendrocyte progenitors (Olig2; E,E') or proliferation (BrdU incorporation; F,F'). The fate of E10.5 Ascl1-expressing cells shifts from almost exclusively neurons (A, arrowheads) to cells with astrocytic or immature markers such as GFAP, Glast, Olig2 and BrdU in the Ascl1 null (C'-F', arrowheads). Quantification of these experiments is shown in the graph as the percentage of the total YFP-expressing cells that coexpress each marker. At least nine sections from three different embryos of each genotype were used. This resulted in over 350 total YFP+ cells counted in experiments for each marker.

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