The Journal of Neuroscience, August 16, 2006, ():
Early Postnatal Astroglial Cells Produce Multilineage Precursors and Neural Stem Cells In Vivo
J. Neurosci. Ganat et al.
26: 8609
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. GCE-induced Cre recombination targets neurogenic radial glia during embryogenesis. R26R reporter expression at E17.5 after OHT injection at E13.5 and E14.5. A-C, Low magnification composites showing ßgal immunocytochemistry (red) and DAPI (blue) in sagittal (A,B) and coronal (C) sections. D-G, Regions of the cortical wall immunostained for ßgal and nestin (D,E) and ßgal and BIII tubulin (F,G). H, Hippocampal primordium stained for ßgal and nestin. Lv=lateral ventricle; IIIv=third ventricle. Scale bar, 200 µm in A-B, 320 µm in C, 100 µm in D,H, and 20 µm in E,G.
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Supplemental Figure 2. Characterization of the GCE-targeted lineage using Z/eG reporter mice. OHT was injected at P5 in GCE;Z/eG double transgenic animals which were analyzed at P35. A-C, GFP/ ßgal double immunostaining in cerebral cortex, showing GFP induction in three astrocytes and basal ßgal expression in GFP-negative cells. D-E, GFP/ ßgal double immunostaining and DAPI counterstaining in the cerebral cortex (D) and dentate gyrus (E), showing that most GFP-negative cells express ßgal (arrows), whereas few cells (arrowheads) are ßgal-negative. Scale bar, 20 µm.
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Supplemental Figure 3. Phenotypic characterization of GCE-targeted cell progeny 3 and 30 days post-recombination. Reporter+ cells were characterized by double immunostaining with ßgal or GFP (as indicated) and S100ß (A-C and G-I), RC1 and GFAP (D-F), Rip (J-L), and Myelin (M-O). Myel= Myelin/oligodendrocyte-specific protein (MOSP) (Dyer et al., 1991). Brain regions are indicated on the right side, and reporters and ages on the left. A-L, confocal single optical sections; M-O, epifluorescent microscopy; merged images shown in the right column. Arrows point to double immunostained cells. Scale bar, 32 µm in A-C, G-I, and P-R; 12.5 µm in D-F and 20 µm in J-L.
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Supplemental Figure 4. Cre recombinase-targeted cells are not neurons or oligodendrocyte progenitors, but generate NG2+ cells after 30 days. A-I, Immunocytochemical characterization of reporter-expressing cells three days after recombination. A-C, NG2 immunostaining; D-I, NeuN immunostaining. Reporter lines are indicated on the left side. A-C and E-H, cerebellum; D,G, hippocampal DG; F,I, cerebral cortex. Reporter+ cells negative for neuronal markers are indicated by arrowheads. J-K, characterization of reporter-expressing cells at P35, 30 days after recombination. J, X-gal/NeuN double staining of the OB. K, X-gal/NG2 double staining of the SVZ, showing a double stained cell (arrow). Dg=dentate gyrus; igl=internal granular layer; p=Purkinje cell layer; ml=molecular layer. Scale bar in D: 40 µm in A-F and H-I, and 80 µm in G. Scale bar in K:100 µm in J and 10 µm in K.
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Supplemental Figure 5. Schematic drawing of the hypothesized lineage relationships between the major cellular components of the postnatal CNS. There appears to be a major divergence among astrocytes, oligodendrocytes and neurons in the perinatal CNS, with a proportion of astrocytes retaining pluripotent NSC-like properties in the neurogenic niches. A, astrocytes; NSC, neural stem cell; NP, neuronal progenitor; OP, oligodendrocyte progenitor; O, oligodendrocyte.
