Tsc2 coordinates neuroprogenitor differentiation

Abstract

Neural stem cells (NSCs) of the ventricular-subventricular zone (V-SVZ) generate numerous cell types. The uncoupling of mRNA transcript availability and translation occurs during the progression from stem to differentiated states. The mTORC1 kinase pathway acutely controls proteins that regulate mRNA translation. Inhibiting mTORC1 during differentiation is hypothesized to be critical for brain development since somatic mutations of mTORC1 regulators perturb brain architecture. Inactivating mutations of TSC1 or TSC2 genes cause tuberous sclerosis complex (TSC). TSC patients have growths near the striatum and ventricles. Here, it is demonstrated that V-SVZ NSC Tsc2 inactivation causes striatal hamartomas. Tsc2 removal altered translation factors, translatomes, and translational efficiency. Single nuclei RNA sequencing following in vivo loss of Tsc2 revealed changes in NSC activation states. The inability to decouple mRNA transcript availability and translation delayed differentiation leading to the retention of immature phenotypes in hamartomas. Taken together, Tsc2 is required for translational repression and differentiation.

Keywords: Cell biology; Molecular biology; Molecular mechanism of gene regulation; Neuroscience; Stem cells research; Transcriptomics.

Graphical abstract

Figure 1

NSC Tsc2 deletion generates striatal hamartomas (A) Schematic diagram of conditional Tsc2 and inducible RFP genes. Tsc2 is mutated and RFP is expressed when CRE recombinase is present. (B) Schematic diagram of CAG-CRE and CAG-GFP (Green) plasmid electroporation which induces genomic CAG-RFP leading to co-expression (yellow). (C) Timeline of electroporation and analysis. (D) Long-range PCR of Tsc2 wild-type and mutant alleles following electroporation of CRE-recombinase with varying starting amounts and low exposure to confirm precise size (left, approximately 1090 base pairs) and high exposure demonstrating efficient recombination (right). (E) 5x Tsc2^wt/wt coronal section demonstrating labeling along the V-SVZ, cortex, and striatum at P30. (F and G) 20x Tsc2^w/w coronal section stained for p4E-BP at P30. (H) Quantification of p4E-BP1 in the V-SVZ at P30. Tsc2^wt/wt, N = 6, n = 49, mean = 1.00 ± 0.057 SEM vs. Tsc2^mut/mut, N = 8, n = 53, mean = 1.51 ± 0.081 SEM. (I) Tsc2^mut/mut coronal sections demonstrating labeling along the V-SVZ, cortex, and striatum at P30. Dotted squares denote hamartomas. (J and K) 20x Tsc2^mut/mut coronal section stained for p4E-BP at P30. (L and M) Quantification of p4E-BP1 in the striatum at P30. Tsc2^wt/wt, N = 6, n = 29, mean = 1.00 ± 0.06 SEM vs. Tsc2^mut/mut, N = 8, n = 79, mean = 1.898 ± 0.013 SEM M) Quantification of hamartoma size Type I, N = 8, n = 13, mean 1,764 μm = 216.8 ± SEM vs. Type II, N = 16, n = 92, mean = 5,273 μm ± 333.7 SEM. (N) Quantification of hamartoma circularity. Type I, N = 8, n = 13, mean = 0.368 ± 0.027 SEM vs. Type II, N = 16, n = 92, mean 0.1022 = ± 0.005 SEM. (O and P) Example of Neu-N positive heterotopic neurons within striatal hamartomas at P30. (Q and R) Sample tracing showing cellular morphology. Note that traces in P are from the section shown in M-N. (S) Sholl analysis of Tsc2^wt/wt and Tsc2^mut/mut neurons at P30. (T) Total number of dendrite crossings at P30. Tsc2^wt/wt, n = 39, mean = 32.64 ± 3.046 SEM vs. Tsc2^mut/mut, n = 55, mean = 54.98 ± 3.682 SEM ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. Data are represented as mean ± SEM. Scale bar = 75 μm. See also Figures S1–S3.

Figure 2

NSC Tsc2 deletion alters ribosome biogenesis (A) Upper-Timeline of NSC culturing and RNA sequencing. Lower-Heatmap and hierarchical clustering of mRNA transcripts from Tsc2^w/w and Tsc2^mut/mut NSCs. (B) Example of topmost increased and decreased mRNA transcripts from Tsc2^w/w and Tsc2^mut/mut NSCs. (C) Network analysis of differentially expressed transcripts from Tsc2^w/w and Tsc2^mut/mut NSCs. Green circles highlight proteins critical for translation. (D) Translation-related genes in top 100 most up-regulated genes by Tsc2 deficiency (red). (E) G.O. analysis indicating top biological processes represented in differentially expressed transcripts. See also Figures S4–S7.

Figure 3

NSC Tsc2 deletion alters mRNA translation dynamics (A) Upper-Timeline of NSC culturing and RNA sequencing. Lower-Heatmap of topmost differentially translated polyribosomal mRNAs. (B) Heatmap of topmost differentially translated mRNAs. (C) Scatterplot of mRNA transcript availability compared to translatome abundance. Each diamond represents an mRNA transcript plotted on the x axis which represents the total mRNA abundance (transcriptome) and the y axis represents the polyribosome abundance (translatome). A gray diamond indicates there is no change between Tsc2^wt/wt and Tsc2^mut/mut. A blue diamond indicates changes to the total mRNA abundance only without translational changes. A yellow diamond indicates changes only to the translatome. A green diamond denotes changes for both transcriptome and translatome that occur in the same direction (i.e., more total mRNA correlates with more polyribosome mRNA = homodirectional). A red diamond reflects changes in the opposite direction (more total abundance but less polyribosome abundance). (D) Histogram of transcript and translatome abundance and changes. (E) G.O. analysis of most highly expressed mRNAs with altered translation efficiency. (F) G.O. analysis of mRNAs having the most highly increased translational efficiency. Green highlights those terms mentioned in text.

Figure 4

Tsc2 mutant neuroprogenitors exhibit altered transitional states (A) U-MAP clustering of V-SVZ microdissected cells from four mice for each genotype (Tsc2^wt/wt and Tsc2^mut/mut) and represented as a composite. Data represent an N of 8 mice. (B) Percentage of cells represented in respective clusters. (C) Summary of top 10 transcripts (Identifiers) enriched in the indicated clusters 15, 8, 4, and 2. Each row indicates a cluster type and their representative activation states (left) with cluster 15 being the least activated and cluster 2 being the most activated. See Table S2 for additional clusters and respective differentially enriched transcripts. (D) Each row represents the indicated cluster (15, 8, 4, and 2). Each cluster (row) contains the differentially expressed transcripts in Tsc2^mut/mut mice compared to Tsc2^wt/wt mice. Each listed transcript is significantly decreased in the Tsc2^mut/mut condition. Note that the transcripts also happen to be important identifiers of clusters listed in C. For reference, cluster numbers were placed at the bottom of D to identify the clusters that the transcripts are identifiers of. The decrease in transcripts reflect changes in activation states, most notably, a reduction in transcripts found in cluster 2 indicating a change in activation dynamics. Columns represent the clusters that the transcripts are enriched in. (E–I) 20x images of CRE and GFP electroporated brains with Tsc2^mut/mut RFP (red) and DCX (E, blue), Sox2 (F, blue), Glutamine Synthetase (GS) (G, blue), Nestin (H, blue), or NeuN (I, blue) stained hamartomas and nodules. (J) Quantification of hamartoma size in Tsc2^wt/wt, Tsc2^wt/mut and Tsc2^mut/mut mice at P30 and P60. (K) Quantification of cell markers in hamartomas. p4E-BP, N = 9, n = 24, mean = 100 ± 0 SEM; DCX, N = 7, n = 19, mean = 67.04 ± 4.496 SEM; Sox2, N = 11, n = 32, mean = 93.79 ± 1.618 SEM; GS, N = 4, n = 7, mean = 48.74 ± 4.429 SEM; NeuN, N = 9, n = 23, mean = 54.18 ± 2.722 SEM. N = mice, n = hamartoma. Data are represented as mean ± SEM. Scale bar = 75 μm for E-H and 7.5 μm for I-K. See also Figures S8 and S9.