Continuous Imp1 overexpression induced during early neurogenesis redistributes neuronal laminar organization and promotes cell accumulation at the subplate.

(A) Experimental paradigm: in utero electroporation of TEMPO. TEMPO reporters (CFP, RFP, YFP) label sequential temporal windows of neuronal birth (T1, T2, T3). (B,C) Representative cortical sections showing TEMPO distribution in control vs continuous Imp1 induction. Insets beside control setups show normal glia clusters (outlined arrowheads) (D,E) Quantification reveals disrupted laminar organization with redistribution toward deeper layers (left) while TEMPO proportions remain unchanged (right) suggesting arrested temporal progression. (F,G) High magnification images show cellular accumulations beneath layer VI. (H,I) Subplate accumulations are enriched for T2-labeled neurons (E12.5) or T1+T2 neurons (E13.5), indicating temporal window-specific sensitivity. Dashed lines: upper (II-IV), lower cortical layers (V-VI) and subplate zone (SPZ). Scale bars represent (B,C) 100 µm and (F,G) 50 µm. Data represent mean±SEM. Statistical significance was calculated by comparing each experimental condition within each temporal population with their corresponding control counterpart using two-tailed unpaired Welch’s t-test (*P < 0.05, **P < 0.01, *** P < 0.001).

Temporal window-specific Imp1 induction reveals stage-restricted effects of Imp1 on cortical laminar fate and subplate organization.

(A) Representative images of TEMPO distribution following T1- or T2-restricted Imp1 overexpression via IUE at E12.5. Insets beside each setup show normal glia clusters (outlined arrowheads). (B) T1Imp1 redistributes neurons towards deeper layers with cascading effects on T2, and reduces glia across cohorts. T2Imp1 shows restricted effects on laminar positioning without affecting glia or T3 cohorts. When SPZ is excluded, TEMPO proportions remain unchanged in T2Imp1 brains vs controls. Including SPZ foci increased %RFP+ and %YFP+ at the expense of %CFP+. (C) High magnification images of accumulations in SPZ following T2Imp1. (D) Quantification confirms increased SPZ foci only in T2Imp1. (E) Positive correlation between RFP+ SPZ foci number and %RFP+ cells (r² = 0.7536, slope = 1.544). Dashed lines: upper (II-IV), lower cortical layers (V-VI) and subplate zone (SPZ). Scale bars represent (A) 100 µm and (C) 50 µm. Data show mean±SEM. Statistics: two-tailed unpaired Welch’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

Imp1-mediated laminar redistribution reflects genuine changes in neuronal fate identity rather than migratory defects.

(A,D,G) Representative images of control, continuous Imp1 overexpression, and T1Imp1 conditions showing TEMPO reporters, Cux1/Ctip2 immunostaining and overlays. Boxed regions: Ctip2+ TEMPO neurons (dashed) or double-positive Cux1+/Ctip2+ TEMPO cells (solid). (B,E,H) High magnification images of boxed regions highlight CFP-/RFP-labeled neurons in layers V-VI colocalizing with Ctip2 (outlined arrowheads) or double-positive for both markers (solid arrowheads). (C,F,I) Quantification of marker expression in CFP+ and RFP+ neurons residing in layers V-VI. Following continuous or T1Imp1 overexpression, neurons in deep layer maintain appropriate deep-layer molecular identities (predominantly Ctip2+), demonstrating that laminar distribution reflects bona fide fate specification changes rather than mislocalization. (C) In control conditions, CFP+: Ctip2 (78.31% ± 13.82%), Cux1 (2.93% ± 2.11%), double-negative (18.76% ± 11.73%). RFP+: Ctip2 (50.35% ± 17.01%), Cux1 (1.85% ± 1.85%), double-negative (46.76% ± 17.20%), double-positive (1.04% ± 1.04%). (F) Following continuous Imp1 overexpression, CFP+: Ctip2+ (71.18% ± 3.06%), Cux1+ (7.38% ± 4.93%), double-negative (16.06% ± 1.93%), double-positive (5.38% ± 1.80%). RFP+: Ctip2+ (58.92% ± 7.18%), Cux1+ (5.84% ± 3.01%), double-negative (5.10% ± 3.12%), double-positive (30.14% ± 11.98%). (I) In T1Imp1 overexpression, CFP+: Ctip2+ (47.26% ± 4.88%), Cux1+ (14.63% ± 3.76%), double-negative (36.41% ± 5.64%), double-positive (1.68% ± 0.57%). RFP+: Ctip2+ (47.38% ± 13.91%), Cux1+ (9.17% ± 4.68%), double-negative (26.86% ± 5.03%), double-positive (16.59% ± 10.02%). Dashed lines: upper (II-IV), lower cortical layers (V-VI) and subplate zone (SPZ). Scale bars: (A,D,G) 100 µm and (B,E,H) 20 µm. Data show mean±SEM. Statistics: two-tailed unpaired Welch’s t-test (*P < 0.05, **P < 0.01, *** P < 0.001).

Continuous neuronal recruitment into the IZ-SVZ-VZ compartment underlies T2Imp1-induced RFP+ neuronal accumulation.

(A,B) Representative time-lapse sequences (48 hours) from a single control and T2Imp1 brains. (C) In control, modest and variable changes in cell populations were observed (CFP+: slope = -0.172 cells/hr, r2 = 0.619; RFP+: slope = -0.062 cells/hr, r2 = 0.306). (D) T2Imp1 exhibits highly linear increases (CFP+: slope = 0.747 cells/hr, r2 = 0.892; RFP+: slope = 1.011 cells/hr, r2 = 0.910), suggesting continuous cellular recruitment. (E) Across brains (n = 3), RFP+ accumulation rate significantly increases in T2Imp1 (*P < 0.05). RFP+ accumulation exceeds CFP+, predicting eventual RFP+ dominance.

T2Imp1 induction triggers morphologically diverse glial foci with abnormal cellular organization through HMGA2-independent pathways.

(A) Representative cortical sections showing RFP+ glial clusters at P10. (B) T2Imp1 produces significant RFP+ (10.20 ± 2.518 foci per brain) and to a lesser extent in continuous Imp1 overexpression (1.600 ± 0.6782 foci per brain), identifying T2 as a temporally sensitive window for Imp1-mediated glial clustering. T2Imp1 also shows YFP+ glial clusters (2.000 ± 0.8944 foci per brain). T2Hmga2 fails to induce significant glial clustering, demonstrating Imp1-specific mechanisms. (C-H) Morphological heterogeneity within glial clusters: Type I cells (minimal processes); Type II cells (characteristic astrocytic morphology); Type III cells (highly elaborate arbors). (I) RFP+ quantification shows dramatic expansion in T2Imp1. Glia in control brains organize into small Type II clusters (n = 105, ∼4 cells/cluster), sparse Type I (n = 6, ∼8 cells/cluster) and Type III (n = 5, ∼4 cells /cluster). Continuous Imp1 overexpression produces Type I (n = 2) and Type II foci (n = 6, ∼41 cells/focus, P < 0.05). T2Imp1 dramatically increases Type I (n = 13, ∼51 cells/focus, P < 0.001) and Type II foci (n = 38, ∼50 cells/focus, P < 0.0001) foci showing significant expansion. T2Hmga2 produces minimal foci across all types (Type I: n = 1; Type II: n = 3, ∼17 cells/focus, P < 0.05; Type III: n = 2), distinguishing Imp1 from Hmga2-mediated pathways. (J) YFP+ populations show similar RFP+ patterns with reduced frequency. Controls display Type I (n = 3, ∼8 cells/cluster) and Type II (n = 74, ∼4 cells/cluster). Continuous Imp1 and T2Hmga2 overexpression produce single YFP+ foci (∼23 and ∼14 cells per focus, respectively). T2Imp1 yields predominantly Type II foci (n = 9, ∼32 cells/focus, P < 0.05), consistent with the dramatic foci phenotype observed in RFP+ populations. (K-L) Spatial patterns for YFP+ foci: (K) nested YFP+ within RFP+ foci, and (L) layered arrangement with YFP+ near pial surface and RFP+ below. Dashed lines: upper (II-IV), lower (V-VI), SPZ. Scale bars: (A) 100 µm, (C-H) 50 µm. Data show mean ± SEM. Statistics: two-tailed unpaired Welch’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).