Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: Increased SOX2 expression in CP tumors in humans and mice. (A) Immunohistochemistry of SOX2 is shown in the upper rhombic lip/roof plate (red dotted lines) and hindbrain CP (arrow) in wild-type mice, NOTCH-driven CPP and CPC (arrowheads) in Lcre;NICD1 and Lcre;Ptch cko ;NICD1 animals at embryonic (E) day 14.5 (E14.5). Scale bar, 50 µm. Black dotted line marks the border of the ventricle with SOX2-expressing ependymal cells. (B) Immunofluorescence of SOX2 and OTX2 is shown in Rb1/Trp53 -deficient CPC in adult Lcre;p53 cko ;Rb cko mice. Nuclei are labeled with DAPI. Scale bar, 50 µm. (C) RT-qPCR analysis of Sox2 expression in wild-type CP, NOTCH-driven CPP, and Rb1/Trp53 -deficient CPC ( n = 5 per tissue type, mean ± SEM, 1-way ANOVA, **** P < 0.0001). (D) RNAscope of Sox2 and Myc expression in hindbrain CP (black arrow) in adult wild-type mice, and NOTCH-driven CPP (arrowhead) in adult Lcre;NICD1 animals. The dotted line marks ventricular walls with ependymal cells (red arrow). Scale bar, 50 µm. (E) Immunofluorescence of SOX2 and ARL13B is shown in NOTCH-driven CPP in adult Lcre;NICD1 animals. DAPI labels nuclei. Scale bar, 20 µm. (F) Immunofluorescence of SOX2 is shown in NOTCH-driven CP tumor cells treated with DMSO or RIN-1 (10 µM). DAPI labels nuclei. Scale bar, 50 µm. Quantification of fluorescence intensity is shown ( n = 333 [DMSO]; n = 101 [RIN-1], mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). (G) Immunohistochemistry of SOX2 in human CP tumors are shown. Scale bar, 50 µm. (CPC: n = 14; CPP and atypical CPP: n = 26). All results were obtained from 3 independent experiments. (H) RT-qPCR analysis of gene expression in human CP organoids and CP tumors ( n = 6 [CP organoid], n = 9 [CPP], n = 13 [CPC], mean ± SEM, 1-way ANOVA, * P < 0.05; ** P < 0.01; *** P < 0.001; NS, nonsignificant). (I) CosMx analysis of the expression of NOTCH1 , NOTCH2 , NOTCH3 , and SOX2 in a human CPC sample. Boxed region is shown in higher magnification on the right. (J) RNAscope studies of SOX2 and HES5 expression in a human CPP. Scale bar, 200 µm.

Article Snippet: Gt(ROSA)26Sor tm 1 .Notch1Dam /J ( Rosa26-NICD1 ) mice, B6N.129- Ptch1 tm1Hahn /J ( Ptch flox/flox ) mice, Sox2 tm 1 .1Lan /J ( Sox2 flox/flox ) mice, B6.129P2- Trp53 tm1Brn /J ( Trp53 flox/flox ) mice, Rb1 tm2Brn /J ( Rb flox/flox ) mice, and C57BL/6 mice (all from Jackson Laboratory), and Tg(Lmx1a-cre)1Kjmi ( Lmx1a-Cre ) mice were maintained by breeding with C57BL/6 mice.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Labeling, Quantitative RT-PCR, RNAscope, Fluorescence, Gene Expression

Journal: Neuro-Oncology

Article Title: SOX2 commands LIM homeobox transcription factors in choroid plexus development and tumorigenesis

doi: 10.1093/neuonc/noaf085

Figure Lengend Snippet: SOX2 regulates transcription factors LMX1A and LMX1B in NOTCH-driven CP tumors. (A) UMAP of 6428 single-nucleus profiles from a NOTCH-driven CPP colored by Lmx1a and Lmx1b expression, respectively. (B) t-distributed stochastic neighbor embedding (t-SNE) plot shows the annotated scATAC-seq profiles of different cell populations in NOTCH-driven CPP. Different subclusters of cells are marked by different colors. (C) Violin plots show the activity of different genes in each subcluster of cells. (D) Western blot analysis of the expression of SOX2, LMX1A, and LMX1B in CP in wild-type mice, and NOTCH-driven CPP in Lcre;NICD1 animals ( n = 3 per group, mean ± SEM, 2-tailed unpaired t test, ** P < 0.01; *** P < 0.001). Three independent experiments were conducted; also see . (E, F) Immunofluorescence of LMX1A (E) and LMX1B (F) is shown in Rb1/Trp53 -deficient CPC cells infected with viruses expressing SOX2. SOX2 labels infected cells. Scale bars, 50 µm. Three independent experiments were conducted. (G) Immunofluorescence of LMX1A is shown in TP53 -deficient human CPC cells infected with viruses expressing SOX2. SOX2 labels infected cells. Scale bar, 50 µm. Three independent experiments were conducted. (H, I) Immunofluorescence of LMX1A (H) LMX1B (I) is shown in CPP in Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals at postnatal (P) day 7 (P7). Arrowheads point to tumor cells, arrows point to SOX2-deficient tumor cells. DAPI labels nuclei. Scale bars, 50 µm. Fluorescence intensity is quantified ( n = 1101 [LMX1A], n = 754 [LMX1B] for NOTCH-driven CPP cells; n = 457 [LMX1A], n = 563 [LMX1B] for Sox2 -deficient tumor cells; mean ± SEM, 2-tailed unpaired t test, **** P < 0.0001). The experiments were repeated 3 times independently; also see and . (J) RT-qPCR analysis of the expression of Lmx1a and Lmx1b in CP in wild-type mice, and CPP from Lcre;NICD1 and Lcre;NICD1;Sox2 cko animals ( n = 8 [CP], n = 6 [CPP], mean ± SEM, 1-way ANOVA, *** P < 0.001; **** P < 0.0001; NS, nonsignificant). Results were obtained from 1 experiment. (K) t-SNE plots show that motifs of LMX1A and LMX1B are enriched in tumor cell subclusters in NOTCH-driven CPP. Colors represent average gene activity score of cells in each subcluster. Dark red means high gene activity score, blue means low gene activity score.

Article Snippet: Gt(ROSA)26Sor tm 1 .Notch1Dam /J ( Rosa26-NICD1 ) mice, B6N.129- Ptch1 tm1Hahn /J ( Ptch flox/flox ) mice, Sox2 tm 1 .1Lan /J ( Sox2 flox/flox ) mice, B6.129P2- Trp53 tm1Brn /J ( Trp53 flox/flox ) mice, Rb1 tm2Brn /J ( Rb flox/flox ) mice, and C57BL/6 mice (all from Jackson Laboratory), and Tg(Lmx1a-cre)1Kjmi ( Lmx1a-Cre ) mice were maintained by breeding with C57BL/6 mice.

Techniques: Expressing, Activity Assay, Western Blot, Immunofluorescence, Infection, Fluorescence, Quantitative RT-PCR