Fezf2 expression identifies a multipotent progenitor for neocortical projection neurons, astrocytes, and oligodendrocytes

Abstract

Progenitor cells in the cerebral cortex sequentially generate distinct classes of projection neurons. Recent work suggests the cortex may contain intrinsically fate-restricted progenitors marked by expression of Cux2. However, the heterogeneity of the neocortical ventricular zone as well as the contribution of lineage-restricted progenitors to the overall cortical neurogenic program remains unclear. Here, we utilize in vivo genetic fate mapping to demonstrate that Fezf2-expressing radial glial cells (RGCs) exist throughout cortical development and sequentially generate all major projection neuron subtypes and glia. Moreover, we show that the vast majority of CUX2⁺ cells in the VZ and SVZ are migrating interneurons derived from the subcortical telencephalon. Examination of the embryonic cortical progenitor population demonstrates that Cux2⁺ RGCs generate both deep- and upper-layer projection neurons. These results identify Fezf2⁺ radial glial cells as a multipotent neocortical progenitor and suggest that the existence, and molecular identity, of laminar-fate-restricted RGCs awaits further investigation.

Figure 1

Fezf2-expressing progenitors are RGCs. (A) Strategy for generation of Fezf2-CreER^T2 mice. (B-C) In situ hybridization for Fezf2 (B) and Cre (C) at E13.5. (D-F) Low magnification images of GFP⁺ cells in the cortex of Fezf2-CreER^T2; RCE-GFP mice following CRE-mediated recombination. (G-H) 24 hours after tamoxifen induction, most GFP⁺ cells expressed SOX2 (78 ± 3%) (G), and few cells expressed TBR2 (10 ± 2%) (H). (I) GFP⁺ cells dividing at the ventricular surface. (J-L) In TM @ E13.5; E16.5 brains, Fezf2⁺ RGCs gave rise to basal progenitors. (J) GFP was expressed in both VZ and SVZ progenitors (arrowhead), migrating neurons in the intermediate zone (arrow), and cortical neurons (asterisk). (K) GFP⁺ cells expressed SOX2 (54% ± 3) and showed typical RGC morphology. (L) Many GFP⁺ cells expressed TBR2 (39% ± 2). (M) Quantification of the percentages of GFP⁺SOX2⁺ RGCs and GFP⁺TBR2⁺ basal progenitors among all the GFP⁺ cells ± SEM. * P < 0.05, ** P < 0.005, *** P < 0.0001. CP, cortical plate; Ctx, cerebral cortex; IZ, intermediate zone; LV, lateral ventricle; SVZ, sub-ventricular zone. Thal, thalamus; TM; tamoxifen; VZ, ventricular zone. Scale bars: (C-F) 250 µm, (E insert) 10 µm, (H, I, J, L) 25 µm. See also Figure S1.

Figure 2

Fezf2⁺ RGCs sequentially generate projection neurons and glia. (A-F) Immunohistochemical analysis of TM @ E12.5; P21 brains. (A, B) GFP⁺ cells were present throughout the cortex. GFP⁺ cells expressed CTIP2 (C), SATB2 (D), GFAP (E) or OLIG2 (F). (G-K) Immunohistochemistry on brain sections from TM @ E14.5; P21 mice. (G, H) GFP⁺ neurons were mostly in layers 2-4. GFP⁺ cells expressed SATB2 (I), GFAP (J), or OLIG2 (K). (L) Percentage of GFP⁺ neurons in deep and upper layers ± SEM. (M-P) Immunohistochemical analysis of brains from TM @ E18.5; P21 mice. GFP⁺ cells included astrocytes (O) and oligodendrocytes (P). (Q) Percentages of GFP⁺ cells that were neurons, astrocytes or oligodendrocytes ± SEM. * P < 0.05, ** P < 0.005, *** P < 0.0001. Ctx, cerebral cortex; Hip, hippocampus; Thal, thalamus; TM, tamoxifen; WM, white matter. Scale bars: (A, G, M) 500 µm, (B, H, N) 250 µm, (F, K, P) 25 µm. See also Figure S2.

Figure 3

Clonal analysis of Fezf2⁺ RGCs. (A) GFP⁺ clone from a P21 Fezf2-CreER^T2; RCE-GFP brain that received TM at E12.5, indicating that early Fezf2⁺ RGCs generate deep- and upper-layer neurons, astrocytes and oligodendrocytes. (B-I) Fezf2-CreER^T2; Confetti mice enabled clonal anaylys of RGCs based upon flourescent protein expression. (B-D) Eamples of TM @ E12.5; P10 brains demonstrating that clones included both deep- and upper-layer neurons and glia. (E) We occasionally observed clones that contained only glia. (F-I) TM @ E14.5; P10 brains contained clones with only upper-layer neruons and glia. TM, tamoxifen. Scale Bars: (A, E, I) 100 µm, (A4) 25 µm, (E4, I4) 10 µm.

Figure 4

CUX2⁺ cells in VZ/SVZ are migrating interneurons and Cux2-Cre/CreER ^T2 labeled RGCs generate both deep- and upper-layer projection neurons. (A-B) Fezf2⁺ RGCs generated CUX2⁺ upper-layer neurons. (C-E) Immunohistochemical analysis of TM @ E10.5; E15.5 Fezf2-CreER^T2; RCE-GFP brains. Few CUX2⁺ cells in the SVZ expressed SOX2 and these cells re located at the VZ/SVZ boundary (C-D). (E) Rare CUX2⁺GFP⁺ cell in the VZ/SVZ. These CUX2⁺GFP⁺ cells did not express SOX2 (D-E). The arrowheads in C-E point to the CUX2⁺SOX2⁺ cells in VZ/SVZ, the arrows point to a rare GFP⁺CUX2⁺ cell. (F-J) Dlx1/2-Cre; Ai14 and Nkx2.1-Cre; Ai14 mice revealed that the majority of CUX2⁺ cells in the E15.5 neocotical VZ/SVZ were interneurons generated from Dlx1/2 (F-G) or Nkx2.1 (H-I) lineages. (J) Quantification of the percentage of CUX2⁺TdTomato⁺ cells among all CUX2⁺ cells in the VZ/SVZ ± SEM. (K-L) TM @ E10.5; P0 Cux2-CreER^T2; Ai9 brains contained TdTomato⁺ cells in all cortical layers. (M-O) TdTomato⁺ cells in deep-layers expressed CTIP2. (P-R) Upper-layer TdTomato⁺ cells expressed CUX1. (S) Percentage of TdTomato⁺ cells in layers 2-6 that expressed CTIP2, TBR1 or CUX1 ± SEM. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; TM, tamoxifen; VZ, ventricular zone. Scale Bars: (B, E, G, I, O, R) 25 µm, (K) 500 µm, (L) 200 µm, (close-ups from O, R) 10 µm. See also Figures S3 and S4.