Pum2 and TDP-43 refine area-specific cytoarchitecture post-mitotically and modulate translation of Sox5, Bcl11b, and Rorb mRNAs in developing mouse neocortex

Quantification of results from n = 3 mice of controls and Pum2 mutants in the prospective somatosensory cortex (pS) for Sox5, Bcl11b, Rorβ, and DAPI in single bins (Figure 1a) and total (Figure 1—figure supplement 5a) and Tbr1, Cux1 in single bins (Figure 1—figure supplement 6a). All markers are normalized to DAPI cells. Distribution of cells across six equal-sized bins is shown. For Bcl11b, only high-expressing neurons were counted. Data are shown as means ± standard error of the mean (SEM), n = 3 for each genotype. *p≤0.05, **p≤0.01, ***p≤0.001, two-tailed t-test. Pum2 cKO: Pum2^fl/fl; Emx1^Cre; II–IV, V, VI: layers II–IV, V, and VI.

Quantification of results from n = 3 mice of controls and TDP43^A315T in the prospective somatosensory cortex (pS) for Sox5, Bcl11b, Rorβ, and DAPI in single bins (Figure 1b) and total (or layer V for Bcl11b) (Figure 1—figure supplement 5b) and Tbr1, Cux1 in single bins (Figure 1—figure supplement 6b). All markers are normalized to DAPI cells. Distribution of cells across six equal-sized bins is shown. For Bcl11b, only high-expressing neurons were counted. Data are shown as means ± standard error of the mean (SEM), n = 3 for each genotype. *p≤0.05, **p≤0.01, ***p≤0.001, two-tailed t-test. Pum2 cKO: Pum2^fl/fl; Emx1^Cre; II–IV, V, VI: layers II–IV, V, and VI.

Quantification of results from n = 3 mice of Pum2 and TDP-43 mutants and their control littermates in the F/M for Sox5, Bcl11b, and DAPI in single bins (Figure 2b) and total (Figure 1—figure supplement 5b) and Tbr1 in single bins (Figure 1—figure supplement 6b). All markers are normalized to DAPI cells. Distribution of cells across six equal-sized bins is shown. For Bcl11b, only high-expressing neurons were counted. Data are shown as means ± standard error of the mean (SEM), n = 3 for each genotype. *p≤0.05, **p≤0.01, ***p≤0.001, two-tailed t-test. Pum2 cKO: Pum2^fl/fl; Emx1^Cre; II–IV, V, VI: layers II–IV, V, and VI.

qRT-PCR of E14.5 cortical RNA from controls (Ctrl) vs. Pum2 cKO using primers to the floxed exons. The fold change in expression levels of Pum2 mRNA normalized to GAPDH mRNA in the Pum2 cKO is shown relative to the Cre^- control (Ctrl) in Figure 1—figure supplement 1c. Data are shown as means ± standard error of the mean (SEM), n = 3 for each genotype. * p≤0.05, two-tailed t-test.

Quantification of the brain anatomy including hemisphere length, width, and area (Figure 1—figure supplement 2a), cortical thickness (Figure 1—figure supplement 2b), and nuclei size (Figure 1—figure supplement 2c) in Pum2 and TDP-43 mutants. n = 3–6 samples of each genotype. *p≤0.05, two-tailed t-test. Pum2 cKO: Pum2^fl/fl; Emx1^Cre.

Quantification of results from n = 3 mice of controls and Pum2 KO mice in the pS for Sox5 normalized to DAPI in single bins and total (Figure 1—figure supplement 7). Data are represented as means ± standard error of the mean (SEM). *p≤0.05, **p≤0.01 by two-tailed t-test. Ctrl: controls; Pum2 KO: Pum2 constitutive knockout.

Quantification of fold changes in protein levels of human TDP-43 (hTDP-43) or both mouse and human (m+h) TDP-43 normalized to total protein in nuclear or cytoplasmic fractions from three mice (n1–3) of each genotype (Ctrl, TDP43, or TDP43^A315T) (Figure 1—figure supplement 8c). Data are shown as means ± SEM, n = 3 for each genotype. *p≤0.05, **p≤0.01, ***p≤0.001 by one-tailed t-test.

Quantification of results from n = 3 animals of controls mice (Ctrl) or mice from a transgenic line expressing Prnp-TARDBP (TDP43) shown in six equal-sized bins and the total number of Sox5- or Rorβ- or Bcl11b or DAPI-positive cells (Figure 1—figure supplement 9b). Only high-expressing Bcl11b+ neurons were counted. Data are shown as means ± SEM, n = 3 for each genotype. *p≤0.05, **p≤0.01, ***p≤0.001 by two-tailed t-test. IV, V, VI: layers IV, V, and VI.

Quantification of retrogradely labeled SCPNs in equal-sized bins for the three genotypes. Analysis of bins 3 and 4 is shown separately and combined (Figure 3c). Data are shown as means ± standard error of the mean (SEM), n = 3 for each genotype. **p≤0.01, ***p≤0.001, two-tailed t-test. Pum2 cKO: Pum2^fl/fl; Emx1^Cre.

Quantification of results from n = 3 brains of controls (Ctrl), Pum2 cKO, or hTARDBP^A315T (TDP43^A315T) in the prospective somatosensory area (pS) for Sox5 and Bcl11b colocalization across six equal-sized bins (Figure 4a). Data are shown as means ± standard error of the mean (SEM), n = 3 for each genotype. *p≤0.05, **p≤0.01, two-tailed t-test. Pum2 cKO: Pum2^fl/fl; Emx1^Cre.

Quantification of results from n = 3 animals from controls (Ctrl), Pum2 cKO, and TDP43^A315T for Lmo4 and Bhlhb5 in F/M and pS areas in single bins and total. Results of F/M and pS for both markers are compared between mutants and their controls and between F/M and pS of each genotype. A summary of total cells only is shown independently comparing F/M and pS in each genotype (Figure 5a). Quantification of the number of barrels per section (Figure 5b) from n = 3 brains of controls (Ctrl), Pum2 cKO, or hTARDBP^A315T (TDP43^A315T). Data are shown as means ± standard error of the mean (SEM). *p≤0.05, **p≤0.01, ***p≤0.001, two-tailed t-test. Pum2 cKO: Pum2^fl/fl; Emx1^Cre.

Quantification of the fraction of Sox5⁺, Bcl11b⁺, or Rorβ⁺ neurons among all transfected neurons with plasmids encoding either control GFP, TDP43, or TDP43^A315T. At least 50 cells were counted for each replicate of every transfection. Data are shown as means ± standard error of the mean (SEM), n = 3 for each transfection. *p≤0.05, **p≤0.01, ***p≤0.001, two-tailed t-test.

Quantification of results from Pum2^fl/flor WT brains at P0 electroporated at E13,5 with pNeuroD-IRES-GFP as control, or with p-NeuroD-IRES-Cre-GFP to ablate Pum2 expression (Figure 7a) or p-NeuroD-TDP43-IRES-GFP or p-NeuroD-TDP43^A315T-IRES-GFP to overexpress hTDP-43 alleles (Figure 7b) only in post-mitotic neurons. The fraction of Sox5⁺, Bcl11b⁺, or Rorβ⁺ neurons among all electroporated cells was quantified. Data are shown as means ± standard error of the mean (SEM), n = 3 for each electroporation. Both p-NeuroD-IRES-Cre-GFP and hTDP-43 alleles were co-electroporated with T-dimer (red) to distinguish them from littermate control brains electroporated only with pNeuroD-IRES-GFP. For both hTDP-43 alleles, the respective control littermates for each variant were combined to a total of n = 6 for pNeuroD-IRES-GFP electroporations. **p≤0.01, ***p≤0.001, two-tailed t-test.

qRT-PCR of RNA derived from P0 somatosensory area-enriched cortical lysates for Pum2 cKO (Figure 8a) or TDP43^A315T (Figure 8b). The fold change for Sox5, Bcl11b, Rorb, and Fezf2 mRNAs normalized to GAPDH mRNA is shown for mutants relative to respective control samples (Ctrl). Data are displayed as means ± standard error of the mean (SEM) for at least n = 4 of each genotype.

Quantification of results from single-molecule fluorescent in situ hybridization (smFISH) for Sox5, Bcl11b, Rorb, and Fezf2 mRNAs on coronal sections from the prospective somatosensory area (pS) of controls (Ctrl), Pum2 cKO, and TDP43^A315T mice at P0. Distribution of cells across six equal-sized bins (Figure 8d). The number of RNA dots in the bins where they are mostly expressed is normalized to the total number of cell nuclei (DAPI) within that bin. Data are shown as means ± standard error of the mean (SEM), at least n = 3 for each genotype. *p≤0.05 by two-tailed t-test. Pum2 cKO: Pum2^fl/fl; Emx1^Cre.

Quantification of results from n = 3 experiments of polysome profiling on TDP43^A315T cortices at E14.5 (Figure 8c). Histograms depict the distribution of the Sox5, Bcl11b, Rorb, and Fezf2 mRNAs across the gradient fractions for TDP43^A315T relative to corresponding controls (Ctrl). Samples in heavier gradient fractions were virtually pooled at analysis to simplify visualization in the case of the Bcl11b B1 primer. Levels of specific mRNAs in each fraction were analyzed by qRT-PCR with normalization to an RLuc mRNA spike-in control, which was added in an equal amount to the fractions prior to RNA preparation. Data are shown as means ± standard error of the mean (SEM), n = 3 for each genotype. *p≤0.05, **p≤0.01, one-tailed t-test.

Quantification of results from n = 3 experiments of polysome profiling on Pum2 cKO prospective somatosensory area (pS)-enriched cortices at P0 (Figure 8d). Histograms depict the distribution of the Sox5, Bcl11b, Rorb, and Fezf2 mRNAs across the gradient fractions for Pum2 cKO relative to corresponding controls (Ctrl). Samples in heavier gradient fractions were virtually pooled at analysis to simplify visualization. Levels of specific mRNAs in each fraction were analyzed by qRT-PCR with normalization to an RLuc mRNA spike-in control, which was added in an equal amount to the fractions prior to RNA preparation. Data are shown as means ± standard error of the mean (SEM), n = 3 for each genotype. *p≤0.05, **p≤0.01, one-tailed t-test. Pum2 cKO: Pum2^fl/fl; Emx1^Cre.

Quantification of expression of Sox5 splicing mRNA isoforms normalized to GAPDH mRNA in P0 somatosensory area-enriched cortical lysates of Pum2 cKO (Figure 9—figure supplement 1a) and TDP43^A315T (Figure 9—figure supplement 1b) mutants and their respective control samples (Ctrl). For Sox5, 7 protein-coding isoforms were annotated. We designed primers recognizing three of them, and it was not possible to design specific qPCR primers to distinguish the other four isoforms for which we used a primer called Sox5 diff to detect the four of them simultaneously. Data are shown as means ± standard error of the mean (SEM) for at least n = 4 of each genotype. Pum2 cKO: Pum2^fl/fl; Emx1^Cre. Two-tailed t-test.

Quantification of expression of Bcl11b and Rorb splicing mRNA isoforms normalized to GAPDH mRNA is shown in P0 somatosensory area enriched cortical lysates of Pum2 cKO (Figure 9—figure supplement 1a) and TDP43 ^A315T (Figure 9—figure supplement 1b) mutants and their respective control samples (Ctrl). Data are shown as means ± standard error of the mean (SEM) for at least n = 4 of each genotype. Pum2 cKO: Pum2^fl/fl; Emx1^Cre. Two-tailed t-test.

Quantification of expression of Sox5 3′UTR mRNA isoforms normalized to GAPDH mRNA in P0 somatosensory area-enriched cortical lysates of Pum2 cKO (Figure 9—figure supplement 2a) and TDP43 ^A315T (Figure 9—figure supplement 2b) mutants and their respective control samples (Ctrl). Data are shown as means ± standard error of the mean (SEM) for at least n = 4 of each genotype. Pum2 cKO: Pum2^fl/fl; Emx1^Cre. Two-tailed t-test.

Quantification of expression of Bcl11b and Rorb 3′UTR mRNA isoforms normalized to GAPDH mRNA is shown in P0 somatosensory area-enriched cortical lysates of Pum2 cKO (Figure 9—figure supplement 2a) and TDP43^A315T (Figure 9—figure supplement 2b) mutants and their respective control samples (Ctrl). Data are shown as means ± standard error of the mean (SEM) for at least n = 4 of each genotype. Pum2 cKO: Pum2^fl/fl; Emx1^Cre. Two-tailed t-test.

Quantification of polysome/monosome (P/M) ratio from polysome profiles of E14.5 neocortices for controls (Ctrl), Pum2 cKO, and TDP43^A315T for n = 3 of each genotype (Figure 9—figure supplement 3a). Two-tailed t-test.

Quantification of polysome profiling from E13.5 neocortices of Pum2 cKO (Figure 9—figure supplement 3b). Histograms showing the distribution of the Sox5 and Bcl11b mRNAs at E13.5 across polysome gradient fractions for Pum2 cKO relative to controls. E13.5 is the peak time of birth for layer V neurons when no layer IV Rorβ+ neurons are born yet. Values were normalized to an RLuc mRNA spike-in control, which was added in an equal amount to the fractions prior to RNA preparation. Data are represented as means ± standard error of the mean (SEM). *p≤0.05 by two-tailed t-test.

Quantification of polysome profiling from E14.5 neocortices of Pum2 cKO (Figure 9—figure supplement 3b). Histograms showing the distribution of the Sox5, Bcl11b, and Rorb mRNAs at E14.5 across polysome gradient fractions for Pum2 cKO relative to controls. Values were normalized to an RLuc mRNA spike-in control, which was added in an equal amount to the fractions prior to RNA preparation. Data are represented as means ± standard error of the mean (SEM). **p≤0.01 by two-tailed t-test.

Quantification of polysome profiling from E18.5 neocortices of Pum2 cKO (Figure 9—figure supplement 3b). Histograms showing the distribution of the Sox5, Bcl11b, and Rorb mRNAs at E18.5 across polysome gradient fractions for Pum2 cKO relative to controls. Values were normalized to an RLuc mRNA spike-in control, which was added in an equal amount to the fractions prior to RNA preparation. Data are represented as means ± standard error of the mean (SEM). Two-tailed t-test.

Quantification of results from UV cross-linking immunoprecipitation (UV-CLIP) from E18.5 cortices (Figure 10c). Dissociated cells were either cross-linked with UV light or left untreated as a control. Lysates were used for immunoprecipitations with antibodies against TDP-43, Pum2, or control nonspecific IgG. RNA in the input and IP eluate were analyzed by qRT-PCR for Sox5, Bcl11b, Rorb, Fezf2, Cux1, Pum2, Tdp43, and 18S mRNAs. After verifying enrichment relative to IgG controls for UV-treated samples, histograms were generated that represent the fraction of input mRNA co-immunoprecipitated with either Pum2 or TDP-43 in the presence or absence of UV cross-linking. Statistically significant enrichment was evaluated relative to 18S rRNA, which is not known to interact significantly with either protein. Reduced signal in the absence of UV-cross-linking implies an interaction is cross-linking-dependent, that is, direct. Data are represented as means ± standard error of the mean (SEM) from n = 3–6 samples. Raw values and data normalized to 18S of each replicate are shown independently in different sheets, and a summary of consolidated results from six replicates is in the last Excel sheet. *p≤0.05, ** p≤0.01, Mann–Whitney U test.

Quantification of the fold change for Emx1 mRNA normalized to GAPDH mRNA is shown for P0 somatosensory area-enriched cortical lysates of Pum2 cKO relative to respective control samples (reviewers Figure 1a). Quantification of the fold change for Sox6 and Unc5C mRNA normalized to GAPDH mRNA is shown for P0 somatosensory area-enriched cortical lysates of Pum2 cKO and TDP43^A315T (reviewers Figure 2a and b) relative to respective control samples (Ctrl). Data are shown as means ± standard error of the mean (SEM) for n = 4-6 animals of each genotype. *p≤0.05 by two-tailed t-test.

Analysis of results of Western blot performed on nuclear fractions from three mice (N1–3) of Ctrl and Pum2 cKO for Emx1 protein. Quantification of corresponding fold changes in Emx1 protein levels normalized to total protein is shown below. Data are shown as means ± standard error of the mean (SEM), n = 3 of each genotype. two-tailed t-test.

Quantification of results of polysome profiling from P0 WT somatosensory area-enriched cortices neocortices for controls (Ctrl) and puromycin-treated samples (reviewers Figure 3). Histograms showing the distribution of the Sox5, Bcl11b, Rorb, Fezf2, GAPDH, and 18S mRNAs across polysome gradient fractions for puromycin-treated samples relative to controls. Values were normalized to an RLuc mRNA spike-in control, which was added in an equal amount to the fractions prior to RNA preparation. Data are represented as means ± standard error of the mean (SEM). *p≤0.05 by two-tailed t-test.

A zipped folder containing original and labeled bands photos for Western blots of Pum2 and tubulin as control (Figure 1—figure supplement 1e), human and mouse TDP-43 and total protein stain as control (Figure 1—figure supplement 8c), and Emx1 and total protein stain as control (reviewers Figure 1b).