Journal: Developmental cell

Article Title: Hedgehog signaling activates a mammalian heterochronic gene regulatory network controlling differentiation timing across lineages

doi: 10.1016/j.devcel.2022.08.009

Figure Lengend Snippet: (A) Diagram of the SHF and HT regions of an E10.5 mouse embryo. Dotted lines indicate the boundaries of microdissection for a SHF-specific RNA-seq from Shh+/+ and Shh−/− embryos (N = 4–5). (B) Volcano plot displaying activated and repressed genes in the Shh−/− relative to Shh+/+ pSHF. Red and blue dots signify significantly upregulated and downregulated genes, respectively. (C) Gene ontology (GO) analysis of Shh−/− repressed genes. (D) Gene ontology (GO) analysis of Shh−/− activated genes. (E) Diagram of the SHF and HT regions of an E10.0 mouse embryo with dotted lines that indicate the boundaries of microdissection for RNA-seq comparing the pSHF and HT (N = 6). (F) MA plot and box plots illustrating the distribution of Shh−/− dysregulated genes superimposed on the distribution of differentially expressed genes between the wild type pSHF and HT. (G) Heatmap showing the wild type expression levels of Shh−/− repressed genes along the pseudotime differentiation trajectory from cardiac-associated Drop-seq clusters. (H) Heatmap showing the wild type expression levels of Shh−/− activated genes along the pseudotime differentiation trajectory from cardiac-associated Drop-seq clusters. (I) Immunofluorescent staining for sarcomeric myosin (MF20) in the E10.5 pSHF of Shh+/+ and Shh−/− embryos. MF20 staining is shown in red and DAPI counterstain is shown in grey. White bracket demarcates the forming DMP (N = 3–4). Scale bars = 200μm. DMP, dorsal mesenchymal protrusion. *** P ≦ 0.005.

Article Snippet: The number of beating foci was calculated as the average number of independently beating regions within 5 fields of view across two biological samples, counted from 5 second videos taken on a Zeiss Axiovert 200m inverted widefield microscope at the University of Chicago’s Integrated Light Microscopy core facility.

Techniques: Laser Capture Microdissection, RNA Sequencing, Expressing, Staining

Journal: Developmental cell

Article Title: Hedgehog signaling activates a mammalian heterochronic gene regulatory network controlling differentiation timing across lineages

doi: 10.1016/j.devcel.2022.08.009

Figure Lengend Snippet: (A) Diagram of the SHF and HT regions of an E10.0 mouse embryo and an adult mouse heart with dotted lines that indicate the boundaries of microdissection for ATAC-seq or protein isolation (N = 3). (B) Heatmap showing ATAC-seq signal Z-scores in the pSHF, HT and adult heart at GLI1 GRN enhancers. (C) Violin plot comparing mean ATAC-seq fold enrichment in the pSHF, HT and adult heart at GLI1 GRN enhancers. (D) Western blot demonstrating the expression level of GLIA and GLIR proteins in the E10.0 wild type pSHF and HT. (E) Bar plot showing the relative proportion of GLIA and GLIR proteins in the E10.0 wild type pSHF and HT, from (D). (F) Schematic of a model describing how Hh GLIA and GLIR TFs could regulate GLI1 GRN genes in a spatiotemporal manner in the mouse embryo to control SHF CM differentiation timing (G) Schematic representation of experimental design for ChAP-seq in GLI3R-FTA overexpression (GLI3 OE) mESC-derived cardiac differentiations (N = 2). (H) Heatmap showing GLI3R-FTA ChAP-seq signal Z-scores in mESC-CPs at D6 at all in vivo-accessible GLI1 GRN enhancers. (I) Bar plot depicting the proportion of GLI1 GRN genes with GLI3R-FTA binding within 200kb of the TSS. (J) Genome browser view of the Foxf1 locus showing the Foxf1 TSS and a GLI1 GRN enhancer upstream of the TSS (grey box, Fox enhancer). (K) Bar plots depicting luciferase reporter activity resulting from co-transfection of TBX5 with either GLI1 or GLI3R (N = 3). (L) Transient transgenic analysis of lacZ reporter expression in the E10.0 pSHF (red box) and HT (gold box) driven by wild type and GLI binding site mutant versions of the Fox enhancer (N = 5–8). (M) Immunofluorescent staining for MYL7 in the E10.5 pSHF from control and GLI3R OE embryos (blue = DAPI, red = MYL7). White bracket demarcates the forming DMP (N = 3). Scale bars = 200μm. (N) Histological sections of E14.5 hearts from control and GLI3R OE embryos. Black arrowheads highlight incidence of AVSD (N = 5). Scale bars = 200μm. GBS, GLI binding site. * P ≦ 0.05, **** P ≦ 0.001.

Article Snippet: The number of beating foci was calculated as the average number of independently beating regions within 5 fields of view across two biological samples, counted from 5 second videos taken on a Zeiss Axiovert 200m inverted widefield microscope at the University of Chicago’s Integrated Light Microscopy core facility.

Techniques: Laser Capture Microdissection, Isolation, Western Blot, Expressing, Control, Over Expression, Derivative Assay, In Vivo, Binding Assay, Luciferase, Activity Assay, Cotransfection, Transgenic Assay, Mutagenesis, Staining