Fig. 1: Differentiation of GBX2-/- and 4X cells using a caudal neural progenitor protocol.

a Schematic gene expression profile of genes along the A-P axis that define regions from the telencephalon (Tel) to rhombomere (r)5 during embryonic development. b Schematic diagram of the DIV4 CNP differentiation protocol. c Representative immunofluorescence images of H9 and GBX2^-/- cells at DIV4 showing OTX2-/CDX2+ cells. A few 4X cells were positive for OTX2, but no cells were positive for CDX2. Scale bars, 20 µm. QPCR analysis of OTX2 (d) and CDX2 (e) expression in H9, GBX2^-/- and 4X cells at DIV4. The data are presented as the mean ± SD; n = 3 biological replicates. One-way ANOVA showed statistical significance, and then an unpaired t test comparing two groups was performed. f Heatmap of the expression of pluripotent and neural genes representing the forebrain, midbrain, hindbrain and spinal cord regions in H9, GBX2^-/- and 4X cells at DIV4. g The top 10 downregulated (blue) and upregulated (red) genes (and additional selected gene in bold) between 4X and H9 cells, GBX2^-/- and H9 cells, and 4X and GBX2^-/- cells at DIV4. The threshold bar (white line) indicates a fold change of ±2. h Schematic diagram of the DIV11 CNP differentiation protocol. i RNA expression analysis of the midbrain genes (orange) OTX2, EN1 and PAX8; the hindbrain genes (gray) MAFB, EGR2, HOXA2, HOXB1, HOXA3, HOXB2, and HOXA4; and the spinal cord genes (purple) HOXB8 and HOXC10. Nanostring data shown as RNA count and QPCR as fold change. The data are presented as the mean ± SD; n = 3 biological replicates. One-way ANOVA followed by Tukey’s multiple comparisons test. j Representative immunofluorescence analysis of OTX2/EN1 double-positive cells among DIV11 4X cells. No OTX2/EN1 double-positive cells were detected among H9 cells. Scale bars, 10 µm. Diencephalon: Di, rostral midbrain: rM, caudal midbrain: cM, caudal neural progenitor: CNP. Source data are provided as a Source Data file.