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. 2019 Jun 6;74(5):951–965.e13. doi: 10.1016/j.molcel.2019.03.041

Figure 1.

Figure 1

Alternative Polyadenylation of NEAT1 Induces Paraspeckle Formation upon Differentiation and Depletion of TDP-43 in Mouse and Human ESCs

(A and B) The number of paraspeckles analyzed by counting NEAT1_1, NEAT1_2 (A) or Neat1_1, Neat1_2 (B) double-labeled foci (based on single-molecule fluorescent in situ hybridization [smFISH] and criteria explained in Figure S1A).

(A) Undifferentiated hESCs, spontaneously differentiating cells, and BMP4-, CHIR99021-, WNT3A-, and retinoic acid (RA)-treated cells, promoting trophoblast, mesoderm, mesendoderm (primitive streak-like), and neuroectoderm fates, respectively.

(B) Undifferentiated mESCs and spontaneously differentiating iTDP-43-EGFP mESCs untreated or treated with doxycycline to ectopically express TDP-43; more than 250 (A) and more than 200 (B) cells analyzed per group, Mann-Whitney U test; ∗∗p < 0.001, ∗∗∗p < 0.0001. Duration of treatment was as indicated.

(C) Venn diagram depicting differentially expressed genes in hESCs exposed to the indicated differentiation stimuli for 24 h relative to untreated cells (n = 2 biological replicates of global RNA-seq per condition; adjusted p < 0.01, Fisher’s exact test, fold change ≥ 4). Bottom: representative mapping of NEAT1 RNA-seq reads aligned to the two isoforms; global and nascent RNA-seq of undifferentiated hESCs and CHIR99021-treated hESCs (n = 2 biological replicates).

(D and E) Percentage and representative maximum projection photomicrographs of h/mESCs exhibiting NEAT1_1, NEAT1_2 (D) and Neat1_1, Neat1_2 (E) isoforms analyzed as above.

(D) Undifferentiated WT and NEAT1ΔpA line and 3-day TDP-43 small interfering RNA (siRNA)-treated hESCs (knockdown [KD]) maintained under pluripotency conditions.

(E) Undifferentiated WT and Neat1ΔpA line and cTdp-43 KO (conditional knockout) mESCs 3 days following tamoxifen treatment, which leads to deletion of Tdp-43 under pluripotency conditions. Statistical analysis and counting as in (A); scale bars, 10 μm. Red, NEAT1_1, _2, Neat1_1, _2 probes; blue, DAPI (nuclear stain).

(F) Representative mapping of NEAT1 and Neat1 RNA-seq reads displaying samples from TDP-43 siRNA KD or control siRNA-treated hESCs (2 days) and untreated or tamoxifen-treated Tdp-43 KO mESCs (2 days, n = 3 biological replicates) under pluripotency conditions.