Extended Data Fig. 3: Assessment of Cux2 Enhancer Activities Through Mouse Transgenic Reporter Assays.

a) Transgenic mouse Cux2-E1-Gfp shows no GFP expression at PCD 12.5, but strong GFP expression at PCD 14.5 GFP in the embryonic mouse forebrain. Scale bar: 1 mm (left), 2 mm (right). b) At PCD 17.5, GFP is expressed in the BCL11B negative cells in upper CP and CC in Cux2-E1-Gfp mice. Scale bar: 1 mm (whole brain), 50 µm (right). c) At PD 0, GFP is mostly expressed in BCL11B negative upper layer neurons and absent from CST as seen at pons in Cux2-E1-Gfp mice. Scale bar: 1 mm (whole brain), 50 µm (right). (d-f). At PD 15, the majority of Cux2-E1-Gfp positive cells reside in upper layers and co-label with CUX1 (closed arrows). Double open arrowheads (CUX1) and triple open arrowheads (BCL11B) again indicate cells immunolabeled for specific molecular markers but not by Cux2-E1-Gfp. CUX1 and BCL11B positive cell populations were compared using an unpaired, two-tailed t-test. The graph represents mean ± s.e.m. n = 1,116 cells from 3 biological replicates * P = 0.000352. Scale bars: 100 µm (e); inset, 50 µm (f) ;100 µm. (g) In the PCD 17.5, Cux2-E3-Gfp transgenic mice brain, GFP is mostly expressed in L1 cell and co-localize with RELN, but not with BCL11B, GFP labeled axons can be seen passing through CC. Scale bar: 100 µm (left, right), 20 µm (middle). (h) In the PCD 17.5 Cux2-E2-Gfp transgenic mice brain, GFP-positive cells are scattered throughout CP and largely co-localize with LHX6; GFP is not expressed in CC. Scale bar: 100 µm (left, right), 20 µm (middle). i) Cux2 overexpression increases neurite length of the SATB2 and ZBTB18 immunopositive developing chicken neurons. The image shows chicken neurons from E7 chicken embryonic forebrain overexpressing Cux2 using pCag-Cux2 and pCaggs-mCherry to label electroporated neurons. Scale bar: 100 µm. n = 43 neurons for control & n = 24 for Cux2 OE. j) The graph represents the length of neurites of the pCag-Cux2 + pCaggs-mCherry neurons that co-express SATB2 and ZBTB18 over controls pCagen + pCaggs-mCherry. Ordinary two-way ANOVA with Bonferroni’s multiple comparisons test, with single pooled variance, was applied. The graph represents mean ± s.e.m. n = 210 from control and 139 from Cux2 overexpressing cells from 3 experimental replicates. **** P = 0.0001. k) Luciferase reporter activity driven by the Satb2 E1 enhancer is significantly increased by ZBTB18 and reduces when the ZBTB18 binding site is mutated (Satb2 ΔE1). The graph represents mean ± s.e.m. Ordinary two-way ANOVA with Bonferroni’s multiple comparisons test, with single pooled variance, was applied. **** P = 0.0001 (Satb2 E1 (Control vs Zbtb18)), (Satb2 ΔE1 (Control vs Zbtb18)), and P = 0.003 for Satb2 E1 vs Satb2 ΔE1 (Zbtb18), n = 3.