recombinant human sonic hh protein shh n  (R&D Systems)

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

Journal: iScience

Article Title: The WAVE regulatory complex interacts with Boc and is required for Shh-mediated axon guidance

doi: 10.1016/j.isci.2024.111333

Figure Lengend Snippet: Nckap1 and Cyfip1/2 are required for Shh-mediated growth cone turning (A) Dunn chamber schematic from top (left) and side (right) views from Yam et al. 2009. The inner well is filled with media containing no chemoattractant, whereas the outer well is filled with media containing chemoattractant. Diffusion of the chemoattractant from the outer well to the inner well forms a gradient of the chemoattractant over the bridge region. The neurons cultured on a coverslip and exposed to the gradient in the bridge region are imaged. (B and G) Time-lapse imaging of commissural neurons electroporated either with scrambled shRNA or shRNA targeting Nckap1 or Cyfip1/2 and exposed to a Shh gradient in a Dunn chamber. Axons of commissural neurons electroporated with scrambled shRNA turned toward high concentrations of Shh, whereas axons of Nckap1 or Cyfip1/2 knockdown neurons did not change their direction of growth. The Shh gradient increases along the y axis in the images. Scale bar: 20 μm. (C) Scatterplots of the angle turned versus the original angle between the axons and the direction of the Shh gradient for neurons under the indicated conditions. Positive angles represent turning of axons toward the Shh gradient. (D) The mean angle turned (±SEM) of axons of commissural neurons in a Shh gradient. Nckap1 knockdown inhibits the turning of axons up a Shh gradient. Welch’s t test. n = 100 and n = 134 axons for scrambled and Nckap1 shRNA electroporated commissural neurons, respectively. (E) Scatterplots of the angle turned vs. the original angle and (F) the mean angle turned (±SEM) of axons of commissural neurons in a Shh gradient under the indicated conditions. Expression of NCKAP1 sm -Flag rescues the inhibitory effect of Nckap1 knockdown on the turning of axons up a Shh gradient. One-way ANOVA, n = 84, n = 46, and n = 45 axons for scrambled shRNA + empty vector, Nckap1 shRNA + empty vector, and Nckap1 shRNA + NCKAP1 sm -Flag electroporated commissural neurons, respectively. (H) Scatterplots of the angle turned vs. the original angle and (I) the mean angle turned (±SEM) of axons of commissural neurons in a Shh gradient under the indicated conditions. Cyfip1/2 knockdown inhibits the turning of axons up a Shh gradient. Unpaired t test, n = 114 and n = 101 axons for scrambled and Cyfip1/2 shRNA electroporated commissural neurons, respectively. (J) Net extension (±SEM) of the axons during the 2 h exposure to a Shh gradient. Mann-Whitney test, n = 100 and n = 134 axons for scrambled and Nckap1 shRNA electroporated commissural neurons, respectively. Kruskal-Wallis test, n = 84, n = 46, and n = 45 axons for scrambled shRNA + empty vector, Nckap1 shRNA + empty vector, and Nckap1 shRNA + NCKAP1 sm -Flag electroporated commissural neurons, respectively. Mann-Whitney test, n = 114 and n = 101 axons for scrambled and Cyfip1/2 shRNA electroporated commissural neurons, respectively. Error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01; n.s., not significant. See also Figures S9–S11 .

Article Snippet: Gradients were generated in the Dunn chamber with 0.1–0.2 μg/mL Shh (R&D Systems, 1845-SH or 8908-SH) in the outer well.

Techniques: Diffusion-based Assay, Cell Culture, Imaging, shRNA, Knockdown, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: iScience

Article Title: The WAVE regulatory complex interacts with Boc and is required for Shh-mediated axon guidance

doi: 10.1016/j.isci.2024.111333

Figure Lengend Snippet:

Article Snippet: Gradients were generated in the Dunn chamber with 0.1–0.2 μg/mL Shh (R&D Systems, 1845-SH or 8908-SH) in the outer well.

Techniques: Virus, Recombinant, Activity Assay, Protease Inhibitor, Molecular Weight, Concentration Assay, Sequencing, Modification, Affinity Purification, Expressing, shRNA, Generated, Plasmid Preparation, Software, Blocking Assay