Fig 2: ß-catenin regulates Class I and III genes during mESC differentiation downstream of seeding density(A) Representative confocal images (63× oil objective) of Tcf/Lef reporter mESCs cultured in either self-renewing (left, middle left) or differentiating (middle right, right) conditions at the densities indicated. Green indicates H2B-GFP. Nuclei were counterstained with NucBlue (Hoechst 33,342). Scale bar: 40 µm.(B) Quantification of fluorescent intensity of H2B-GFP in (A). Individual nuclei were traced and quantified in ImageJ. For +LIF at HD, n = 84 nuclei; for -LIF, n = 121 nuclei for HD and 77 for LD. Significance stars indicate p values from the Kruskal-Wallis test for difference among all samples as well as the t test with Bonferroni-corrected p values between samples. * is 0.05 > p value > 0.01. ** is 0.01 > p value > 0.001. *** is 0.001 = p value.(C) Representative confocal images (63× oil objective) of immunocytochemistry of mESCs grown in +LIF at HD or -LIF at HD or LD. Green represents ß-catenin, blue represents the nucleus. White box represents the zoom in area. Scale bar: 40 µm.(D) Boxplots showing weighted colocalization coefficients between nuclei and ß-catenin in mESCs for several fields of view quantified using ZEISS ZEN. n = 5 for +LIF, 11 for -LIF LD, and 12 for -LIF LD. Significance stars indicate p values from the Kruskal-Wallis test for difference among all samples as well as the t test with Bonferroni-corrected p values between samples. * is 0.05 > p value > 0.01. ** is 0.01 > p value > 0.001. *** is 0.001 = p value.(E) RT-qPCR of Classes I-IV density-sensitive genes after 72 h differentiation in -LIF at high or low density upon KD of ß-catenin (Ctnnb1) or control KD. All samples were normalized using the 2–??Ct method relative to Tcp1 expression and HD shCon (log2). Error bars indicate standard deviations (n = 2 biological replicates). Red text indicates presence of the Tcf7l1 motif ±10 kb of the TSS, determined using TFmotifView (http://bardet.u-strasbg.fr/tfmotifview/). Significance stars indicate p values from two-tailed t test of KD vs. shCon. * is 0.05 > p value > 0.01. ** is 0.01 > p value > 0.001. *** is 0.001 = p value.(F) RT-qPCR of Class I-IV density-sensitive genes after 72 h differentiation in -LIF at high or low density upon 48 h dox-induced OE at 125 or 500 ng/mL of 4A ß-catenin or no induction. All samples were normalized to no induction at HD (log2). Error bars indicate standard deviations (n = 2 biological replicates). Red text indicates presence of the Tcf7l1 motif ±10 kb of the TSS, determined using TFmotifView (http://bardet.u-strasbg.fr/tfmotifview/).

Fig 3: Molecular model of FMRP-mediated regulation in GSCs.FMRP is upregulated in GSCs and regulates the expression of WNT5B and CTNNB1 mRNAs, pivotal in stem cells maintenance. Increased levels of FMRP lead to the aberrant activation of the WNT canonical and non-canonical signalling pathways affecting the transcription of genes related to cell proliferation.

Fig 4: Low seeding density enables efficient mESC differentiation(A) Heatmap of RNA-seq performed on J1 mESCs seeded with serial dilutions starting at a concentration of 2.63 × 105 cells/cm2 and ending at a concentration of 4.11 × 103 cells/cm2 and grown in -LIF for 96 h. Transcripts per million (TPM) was calculated using kallisto and transcripts were consolidated into genes. After normalizing expression to the average (log2TPM+1 to avoid undefined results when TPM in any sample = 0, hereafter simply stated as log2), only genes showing a range of 1.5 log2 difference in TPM across samples are shown. Line graphs depict median gene expression ± interquartile range.(B) Boxplots depicting expression of genes involved in naive and primed pluripotency as well as primary gene layer formation.(C) Venn diagram showing overlap of dHDA, dLDA, and differentiation-induced or differentiation-down genes using top 1000 differentially expressed genes (based on TPM compared to control).(D) Scatter plot of 13,797 transcripts depicting log2 TPM ratio of low vs. high density in -LIF (x-axis) and -LIF and +LIF (y-axis) showing a positive correlation between low density and successful induction of the -LIF transcriptional program. Only transcripts where at least one of their q-values (associated with either LD vs. HD or -LIF vs. +LIF) was below 0.05 were included. Color indicates the -log of the higher (worse) q-value.(E) Western blot of proteins whose expression is impacted by seeding density after 72 h of differentiation in -LIF at HD or LD. For Gata3, FL = full length, SV = splice variant.(F) RT-qPCR of lineage markers corresponding to trophectoderm, ectoderm, endoderm, and mesoderm. mESCs were differentiated in LIF HD or LD for 72 h or maintained at LD in +LIF for 48 h. Data were normalized using the 2–??Ct method relative to Tcp1 expression and +LIF LD samples (log2). Error bars indicate standard deviations (n = 3 biological replicates for -LIF, 2 for +LIF).(G and H) RT-qPCR of Class I, II, III, and IV density-sensitive genes after 72 h of differentiation in -LIF, either (G) LD normalized to HD or (H) -LIF relative to +LIF. Data were normalized using the 2–??Ct method relative to Tcp1 expression and HD samples (log2). Error bars indicate standard deviations (n = 4 biological replicates for (G), and 3 for (H)). Significance stars indicate p values from two-tailed t tests of LD vs. HD (G) or -LIF LD vs. +LIF LD (H). * is 0.05 > p value > 0.01. ** is 0.01 > p value > 0.001. *** is 0.001 = p value.

Fig 5: FMRP modulates the WNT pathways regulating the WNT5B and CTNNB1 mRNA metabolism.A Enrichment analysis of known FMRP target mRNAs and mutated in GBM according to The Cancer Genome Atlas. Shown are pathways that are significantly enriched in the PANTHER database (FDR < 0.05). B Cluster heat map represents the log2 fold change (FC) for the 156 genes differentially expressed in all the three shFMR1 GSCs. Values on a log2 scale are color-coded as indicated on the right. C Left panel, representative western blot of total extract (input), FMRP immunoprecipitation (IP-FMRP) and mock immunoprecipitation (IP-IgG). FMRP and its interactor FXR2P are detected. Right panel, HPRT1, CTNNB1, and WNT5B mRNAs were quantified by RT-qPCR. Shown is the enrichment immunoprecipitation/total, relative to H3 mRNA (mean ± SEM, n = 3, *P < 0.05, **P < 0.01, Student’s t-test). HPRT1 mRNA was used as a negative control; CTNNB1 mRNA is also a well-known FMRP target and was used as a positive control. D The histogram represents WNT5B and CTNNB1 mRNA levels quantified in shNTC and shFMR1 #148, #163, and #169 GSC cell lines. HPRT1 mRNA was used as normaliser (mean ± SEM, n = 3, **P < 0.01, Student’s t-test). E WNT5B and CTNNB1 mRNA levels in CTRL siRNA (siCTRL) and FMR1 siRNA (siFMR1) T89G cells detected by RT-qPCR (mean ± SEM, n = 3, **P < 0.01, ***P < 0.001, Student’s t-test). F mRNA stability assay in CTRL siRNA (siCTRL) and FMR1 siRNA (siFMR1) T89G cells. RNA was isolated at the indicated time points after Actinomycin D treatment and the stability of WNT5B and CTNNB1 mRNAs was analysed by RT-qPCR (mean ± SEM, n = 3, ***P < 0.001, ****P < 0.0001, two-way ANOVA).