Figure 4.

YAP Converts Neurons in yNSCs

(A) Schematic of the experiments performed with hippocampal or cortical neurons.

(B) Schematic of the genetic lineage tracing strategy used to trace neurons ex vivo.

(C) We confirmed, in primary neuronal cultures prepared from the brain of Syn1-Cre; R26-LSL-rtTA-IRES-EGFP or Syn1-Cre; R26-CAG-LSL-tdTomato transgenic mice, that expression of the lineage tracer is indeed restricted to post-mitotic neurons expressing TUJ1 (100%, n = 635 cells), TAU (100%, n = 140 cells), and NEUN (95%, n = 252 cells). None of these traced cells were ever positive for the NSCs and/or progenitor markers Nestin, Vimentin, or MASH1 or for proliferation markers (5-Ethynyl-2’-deoxyridine [EdU], Ki67) (0%, n > 4,000 cells). See Figure S4F for representative images. See also Figure S4G for similar characterizations on Thy1-Cre-traced neuronal preparations.

(D) Immunofluorescence for the synaptic markers Synaptophysin and SV2A in cortical neurons derived from Syn1-Cre; R26-CAG-LSL-tdTomato mice. Scale bars, 11 μm (top) and 5.7 μm in magnifications (bottom).

(E) Table summarizing the results obtained in different lineage tracing experiments.

(F) Lineage tracing experiment showing that yNSCs originate from neurons. The images are bright-field and tdTomato-fluorescence pictures of primary cortical neurons from Syn1-Cre; R26-CAG-LSL-tdTomato mice and yNSCs derived from them (at the indicated passages). Neurospheres from Syn1-Cre; R26-CAG-LSL-tdTomato NSCs are presented as a negative control.

(G) Panels are X-gal stainings of yNSCs derived from hippocampal neurons from Thy1-Cre; R26-LSL-LacZ mice (scale bars, 210 μm) at the indicated passages. Neurospheres from Thy1-Cre; R26-LSL-LacZ NSCs (scale bar, 210 μm) are presented as a negative control.

(H) Lineage tracing experiment with the Syn1-Cre driver showing that yNSCs originate from Syn1-traced neurons. Left: immunostaining for GFP and TUJ1 in cortical neurons obtained from Syn1-Cre; R26-LSL-rtTA-IRES-EGFP mice. Right: bright-field and GFP fluorescence pictures of neurospheres derived from yNSCs obtained from the same neurons after transduction with doxycycline-inducible YAP. See also Figures S4G–S4I for similar results with the alternative lineage tracing strategy using Thy1-Cre; R26-LSL-rtTA-IRES-EGFP mice.

(I–K) Representative images of yNSCs neurospheres (second passage, P2) derived from hippocampal (I) or cortical (J) neurons. Images from negative control transduced neurons are shown as a reference (I and J). Neurospheres from native NSCs are presented for comparison (K). Scale bars, 210 μm.

(L) P1 yNSCs were dissociated to single cells and replated at clonal density for neurosphere formation in the absence of doxycycline for further passages (P2, P3, and P4). Native NSCs are presented for comparison. Graphs are quantifications of neurospheres formed by the indicated cells. Results are representative of at least eight (P2), six (P3), and three (P4) independent experiments performed in six replicates. Data are presented as mean + SD.

See also Figure S4.