Article Figures & Data
Figures
- Figure 1.
Expression of Pax6, Tbr2, and Tbr1 protein in E14.5 cortex (coronal sections). A, C, Pax6 (green) and Tbr2 (red). Boxed area in A is shown at higher magnification in C. B, D, Pax6 (green) and Tbr1 (red). Boxed area in B is shown at higher magnification in D. E, Tbr2 (green) and Tbr1 (red). All three TFs were expressed in the neocortex (nctx) and eminentia thalami (emt). Pax6 was also expressed in the ventricular zone of the dorsal thalamus (dt), hypothalamus (hy), and lateral part of the lateral ganglionic eminence (lge). Within the neocortex, Pax6 was expressed in the ventricular zone (vz) only; Tbr2 was expressed in the ventricular zone, subventricular zone (svz), and intermediate zone (iz); and Tbr1 was expressed in the intermediate zone (iz), subplate (sp), cortical plate (cp), and marginal zone (mz). Rarely, Tbr2+ cells and Pax6+ cells occupied superficial zones (arrowheads in C). Scale bar (in A): A, B, 500 μm; C-E,50 μm.
- Figure 2.
Tbr2 is expressed by NS-div IPCs. A-S, Confocal images of coronal sections through E14.5 cortex. A-D, Tbr2 (red) and BrdU (green). The bracketed area in A is shown in separate color channels in B-D. Tbr2 was expressed by most S-phase cells in the SVZ and some in the VZ (arrowheads in B-D). E-G, Tbr2 (red) and DNA (blue). Tbr2 was expressed by NS-div cells (red arrowhead in E-G) but not by S-div cells (green arrowheads). H-J, Tbr2 (red) was expressed by phosphovimentin+ (p-vim) M-phase cells (green) in the SVZ (arrowheads). K-M, Tbr2 (red) and PCNA (green). Most Tbr2+ cells contained PCNA, including mitotic figures (arrowheads). N, Tbr2 (red) and nestin (green). Most Tbr2+ cells (arrowheads) appeared to lack nestin. O, Tbr2 (red) and βIII-tubulin (β-tub; green). Most Tbr2+ cells (arrowheads) lacked βIII-tubulin. P-S, Tbr2 (red), NeuN (green), and DNA (blue). The bracketed area in P is enlarged and shown in separate color channels in Q-S. Most Tbr2+ cells did not express NeuN, but those that did included some mitotic figures (arrowheads in Q-S). T-V, E14.5 cortical cells cultured for 1 d in vitro, labeled to detect Tbr2 (red), DNA (blue), and, in green, nestin (T), βIII-tubulin (U), or NeuN (V). Few Tbr2+ cells expressed nestin (5.1%) or βIII-tubulin (12.7%), but more expressed NeuN (20.8%; arrowheads). Scale bars: (in A) A-D, P, 50 μm; (in E) E-G, 20 μm; (in H) H-J, Q-S, 10 μm; (in K) K-M, O, 10 μm; (in N) N, 20 μm; (in T) T-V, 20 μm.
- Figure 3.
Pax6 is substantially downregulated in most Tbr2+ IPCs. A-I, Confocal images of coronal sections through E14.5 cortex. A-D, Pax6 (green) and BrdU (red). The bracketed area in A is shown in separate color channels in B-D. Pax6 was not expressed by most S-phase cells in the SVZ (arrowheads in B-D) but was expressed by most S-phase cells in the VZ. E, Pax6 (green) and phosphohistone-H3 (red). Pax6 was strongly expressed in S-div mitotic figures (arrowheads) but only weakly (yellow arrow) or not at all (white arrow) in NS-div cells. F-I, Pax6 (green) and Tbr2 (red) double immunofluorescence. Most VZ and SVZ cells expressed Pax6 or Tbr2 but not both. Double-labeled cells appeared yellow (arrowheads in G). H-I, Higher magnification of mitotic figures in the SVZ (H) and VZ (I). NS-div mitoses were predominantly Tbr2+ (arrowhead in H), and S-div mitoses were Pax6+ (arrowheads in I). Scale bar (in A): A-E,30 μm; F,40 μm; G-I,12 μm.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Supplementary Figure 1. Tbr2 protein expression at different stages of neocortex development. A-H are confocal images, and I-K are standard epifluorescence images. White lines show the pial and ventricular surfaces. A, E10.5, Tbr2 (red) and reelin (green). Tbr2 was expressed in preplate (ppl) neurons, including all reelin+ Cajal-Retzius neurons. B, E11.5, Tbr2 (red) and beta-III-tubulin (green). Tbr2 was expressed in virtually all beta-III-tubulin+ ppl neurons. C-G, E12.5, Tbr2 (red), Hu-C/D (green) and DNA (blue). The bracketed area in C is enlarged and shown in different color channels in D-G. Tbr2 was expressed in most Hu-C/D+ preplate (ppl) neurons, and in NS-div mitotic figures, many of which were Hu-C/D+ (D-G). H, E14.5, Tbr2 (red), NeuN (green) and DNA (blue). Tbr2 was mainly expressed in the vz, svz, and (to a lesser extent) in the iz; NeuN was weakly expressed in the iz, and more strongly in the cortical plate (cp). Few Tbr2+ cells expressed NeuN, and those only weakly. I-K, Tbr2 (red) and DNA (blue) on E16.5 (I), E18.5 (J), and P0.5 (K). Tbr2 expression decreased rapidly after E17.5. Scale bars: (in A) A-C, 10 um; D-G, 5 um; H, 20 um; (in I) I-K, 50 um.
- supplemental material - Supplementary Figure 2. Pax6 is down-regulated in NS-div mitotic figures and Tbr2+ cells. A, Comparison of Pax6 immunoreactivity (fluorescence intensity) in S-div and NS-div mitotic figures in E12.5, E14.5, and E16.5 neocortex. Pax6 was highly expressed in most S-div cells, but low or undetectable in most NS-div cells. (Due to chromatin condensation, the maximum estimated Pax6 immunoreactivity in M-phase cells was ++, rather than +++). B, Semiquantitative analysis of Pax6 (green) and Tbr2 (red) fluorescence intensity in vz, svz, and iz cells in E14.5 cortex. Most cells expressed either Pax6 or Tbr2, but not both (pie chart). Cells that did co-express Pax6 and Tbr2 generally expressed high levels of one, and low levels of the other (3-dimensional bar graph).
