Fig. 4

a Dissection of KEGG data of DEGs from day 3 and day 6 control and Adnp−/− ESC-derived neurospheres, showing enrichment of the Wnt signaling pathway. Results are shown as −log10 (p value). b Heat map illustrating the expression of selected Wnt-related genes that were shown as log2 FPKM in day 6 control and Adnp−/− ESC-derived neurospheres. Each lane corresponds to an independent biological RNA-seq sample. c qRT-PCR analysis for the indicated Wnt target genes for day 3 and day 9 control and Adnp−/− ESC-derived neurospheres. qRT-PCR was based on three biologically independent experiments (n = 3 per group). d TopFlash luciferase activity assay for lysates from day 3 control and Adnp−/− ESC-derived neurospheres, in the absence or presence of Wnt3a. Data are based on two biologically independent experiments, and similar results were obtained. e Rescue of the expression of the indicated neural developmental genes and putative Wnt target genes by addition of CHIR and Wnt3a, or by restoring 3×FLAG-ADNP. Data are based on three biologically independent experiments, and similar results were obtained. Genes in black dashed box are representative neurodevelopmental genes, and genes in green dashed box are representative Wnt target genes. f Rescue of NESTIN expression by addition of CHIR. Shown is the representative IF staining of NESTIN for day 6 control and Adnp−/− ESC-derived neurospheres (n = 3 per group). g Quantification of mean fluorescence intensity of NESTIN staining using ImageJ for panel (f) based on three biologically independent experiments (n = 3–5 different regions of interest per group). Data are presented as mean values ± SEM, and p values by two-tailed unpaired t-test are shown in (c, d, e, g). Source data are provided as a Source Data file for (c, d, e).