Journal: bioRxiv
Article Title: Mapping the temporal and functional landscape of Sonic Hedgehog signaling reveals new insights into early human forebrain development
doi: 10.1101/2025.05.20.654466
Figure Lengend Snippet: (A) Schematic illustration of the differentiation protocol for generating dorsal (dAN) and ventral (vAN) anterior neuroectoderm from hiPSCs over a 12-day period. Neural induction and ventralization are initiated by a combination of small molecules: LDN193189 (500 nM), SB431542 (10 µM), from day 0 to day 12, and XAV939 (5 µM) from day 0 to day 5. Additional exposure to human recombinant Sonic Hedgehog (SHH, 500 ng/ml) from day 0 to day 6 selectively drives ventral patterning in vAN cultures. (B) Representative Immunofluorescent images at day 12 show distinct marker expression in dAN and vAN cells. Markers analyzed include OTX2 (anterior neuroectoderm marker), PAX6 and EMX2 (dorsal forebrain markers), EOMES (early neural cortical marker), MEIS2 (forebrain marker), NKX2.1 (ventral forebrain marker), FOXA2 (floor plate marker), and SHH (ventral marker). Expression levels are quantified as the percentage of positively stained cells relative to DAPI (mean values from 5 independent images). Scale bar: 40 µm. (C) Principal Component Analysis (PCA) of day 12 dAN and vAN samples (LON and WTC lines) from 5 replicates revealed clear segregation by identity and lineage. PC1 (52% total variance) strongly distinguished dAN versus vAN conditions (one-way ANOVA p.value of 8.9e-12), while PC2 (30% total variance) separates samples by cell lineage origin (one-way ANOVA p.value of 3.1e-12). (D) Heatmap displaying normalized RNA-seq expression profiles of day 12 dAN and vAN samples. Samples were manually ordered by type (vAN/dAN) and cell line (LON/WTC). Marker genes sets specific for hiPSCs, forebrain, ventral/dorsal Forebrain, Midbrain and Hindbrain identity were curated from literature. (E) Heatmap of scaled normalized RNA-seq data showing differentially expressed genes (DEGs) between day 12 dAN and vAN samples (|log2FC| ≥ 1, FDR < 0.01), across 2 hiPSC lines (LON, WTC). Both genes and samples were hierarchical clustered (cluster vAN and cluster dAN), two cluster were identified and then delimited using cutree R function with k=2. (F) Brain development-related Gene Ontology terms among the top 20 most enriched (in term of gene ratio) biological process for both DEGs clusters (dAN and vAN). Color scale indicates gene sets enrichment statistical significance.
Article Snippet: To induce ventralisation and generate ventral anterior neuroectoderm 500ng/ml of human recombinant SHH (C24II, #78065, STEMCELL) was added to media mix from day 0 to day 6.
Techniques: Recombinant, Marker, Expressing, Staining, RNA Sequencing
