sag21k  (Tocris)

Journal: bioRxiv

Article Title: Two parallel lineage-committed progenitors contribute to the developing brain

doi: 10.1101/2025.07.02.662771

Figure Lengend Snippet: A) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours. Forebrain progenitors were then treated with Vismodegib + PD173074 for 72 hours, in the presence or absence of WNT agonist (CHIR99021, 1-6 μM) or WNT inhibitor (XAV939, 1 μM). This revealed that 2 μM of CHIR99021 (indicated by white circle) was optimal for inducing dorsal forebrain marker EMX2 . Gene expression is shown relative to reference gene YWHAZ ; 100% indicates that a gene is expressed at the same level as YWHAZ . HH inh.: HEDGEHOG inhibitor. FGFR inh.: FGF receptor inhibitor. B) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours. Forebrain progenitors were then treated with CHIR99021 (2 μM) + PD173074 (100 nM) for 72 hours, in the presence or absence of FGF2 (20 ng/mL) or FGFR inhibitor (PD173074, 100 nM). This revealed that FGF inhibition enhanced dorsal forebrain markers EMX2 and PAX6 , while preventing erroneous WNT-induced expression of midbrain marker EN1 . Gene expression is shown relative to reference gene YWHAZ ; 100% indicates that a gene is expressed at the same level as YWHAZ . FGFRi: FGF receptor inhibitor. C) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours. Forebrain progenitors were then treated with CHIR99021 (2 μM) + PD173074 (100 nM) for 72 hours, in the presence or absence of BMP4 (10 ng/mL) or BMPR inhibitors (DMH1 [250 nM] or LDN193189 [100 nM]). Gene expression is shown relative to reference gene YWHAZ ; 100% indicates that a gene is expressed at the same level as YWHAZ . BMPRi: BMP receptor inhibitor. D) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours. Forebrain progenitors were then treated with XAV939 (1 μM) in the presence or absence of increasing doses of HEDGEHOG pathway agonist 21K (0.1-5 nM) for 72 hours to generate ventral forebrain. This showed that high HEDGEHOG pathway activation, together with WNT inhibition, led to maximum expression of ventral forebrain markers. Gene expression is shown relative to the sample with the highest expression in this experiment. HH: Hedgehog pathway agonist. WNTi: WNT inhibitor. E) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours. Hindbrain progenitors were then treated with HEDGEHOG inhibitor (Vismodegib, 150 nM) and WNT agonist (CHIR99021, 2 μM) for 72 hours to generate dorsal forebrain, in the presence or absence of BMP agonist (BMP4, 10 ng/mL) or BMPR inhibitor (LDN193189, 100 nM). This showed that BMP inhibition promoted expression of dorsal forebrain markers GSX2 and PAX3 . Gene expression is shown relative to reference gene YWHAZ ; 100% indicates that a gene is expressed at the same level as YWHAZ . BMPRi: BMP receptor inhibitor. F) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours. Hindbrain progenitors were then treated with HEDGEHOG pathway inhibitor Vismodegib (150 nM) in the presence or absence of increasing doses of WNT pathway agonist CHIR99021 (1-4 μM) for 72 hours to generate dorsal forebrain. This showed that moderate WNT pathway activation, together with HH inhibition, led to maximum expression of dorsal hindbrain markers GSX2 and OLIG3 . Gene expression is shown relative to reference gene YWHAZ ; 100% indicates that a gene is expressed at the same level as YWHAZ . BMPRi: BMP receptor inhibitor. G) scRNAseq of H7 hPSC-derived day-4 hindbrain progenitors, and day-7 dorsal hindbrain or ventral hindbrain progenitors. Colors denote differentiation conditions. An asterisk (*) indicates that the legend is identical to that shown in , and is reproduced here to aid the interpretation of other images shown in this subpanel. H) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours. Hindbrain progenitors were then treated with either Vismodegib (150 nM) + CHIR99021 (2 μM) + DMH1 (250 nM) for 72 hours to generate dorsal forebrain, or alternatively, 21K (5 nM) + XAV939 (1 μM) for 72 hours to generate ventral forebrain. Gene expression is shown relative to the sample with the highest expression in this experiment. I) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours. Hindbrain progenitors were then treated with WNT pathway inhibitor XAV939 (1 μM) in the presence or absence of increasing doses of HEDGEHOG pathway agonist 21K (50 pM-5 nM) for 72 hours to generate ventral hindbrain. This showed that high doses of HEDGEHOG agonist, together with WNT inhibitor, were required to maximally induce ventral hindbrain markers. Gene expression is shown relative to the sample with the highest expression in this experiment. WNT inh.: WNT inhibitor. qPCR data depicts the mean of two biological replicates, with s.e.m. shown. In vitro scRNAseq data is from a single experiment.

Article Snippet: D5, D6, and D7 dorsal forebrain media consisted of CDM2 supplemented with XAV939 (1 μM, Tocris) and SAG 21K (5 nM, Tocris).

Techniques: Marker, Gene Expression, Inhibition, Expressing, Activation Assay, Derivative Assay, In Vitro