Journal: Developmental cell

Article Title: Neuronal-epithelial cross-talk drives acinar specification via NRG1-ERBB3-mTORC2 signaling.

doi: 10.1016/j.devcel.2022.10.011

Figure Lengend Snippet: Figure 4. NRG1 rapidly upregulates a network of transcription factors involved in cell differentiation and genes related to mTOR signaling (A) Schematic of E13 and E14 SG epithelia dissection and in vitro culture with NRG1. (B) Hierarchical clustering of differentially expressed genes from E13 and E14 epithelial cultured for 4 h with NRG1 or without (control = C). Plot highlights up- regulated genes (green) and downregulated genes (red) with NRG1 stimulation. p adj < 0.05. (C) Venn diagram representing upregulated genes in E13 and E14 NRG1-stimulated end buds. (D) GO biological processes for all upregulated TFs in E13 and E14 NRG1 stimulated epithelia. (E) (right) Cluster map of 12 master TFs (highlighted by the red box) with binding motifs in downstream TFs in the 4 h NRG1 E13/14 cultures. (left) Subset of the master regulator TFs putatively regulating genes in the acinar secretory and polarity programs. (F) GO KEGG results of genes upregulated with NRG1 stimulation in both E13 and E14 epithelia. (G) Cluster map showing E2f1 and Irf6 with downstream targets involved in mTOR signaling. Green icons indicate log2-fold change R 0.5, padj < 0.05 in NRG1 E13/14 samples versus WT (D–F).

Article Snippet: E13 SG epithelia were freshly isolated as previously described.11,12 Briefly, the SG epitheliumwas separated from themesenchyme using dispase II (Gibco) treatment followed by mechanical dissection and cultured in 15ml of laminin-1 (R&D Systems) on a nucleopore filter floating above 200 mL of serum-free media (DMEM/F12; Thermo Fisher Scientific) containing penicillin-streptomycin (Gibco), 50mg/ml holo-transferrin (HT), 150mg/ml ascorbic acid (AsA) and 200ng/ml human recombinant NRG1 isoform SMDF (R&D Systems), or 20ng/ml HB-EGF (R&D Systems) for up to 120 h. For inhibitor experiments, epithelia were cultured at 37 C in a humidified 5% CO2/95% air atmosphere in complete media in the presence of 0.2% DMSO (Sigma-Alrdrich), 5mM of mTORC1 inhibitor rapamycin (Biotang Inc.), or 1mM mTORC1/2 inhibitor Torin-1 (Cell Signaling Technologies).

Techniques: Cell Differentiation, Dissection, In Vitro, Cell Culture, Control, Binding Assay

Journal: Developmental cell

Article Title: Neuronal-epithelial cross-talk drives acinar specification via NRG1-ERBB3-mTORC2 signaling.

doi: 10.1016/j.devcel.2022.10.011

Figure Lengend Snippet: Figure 6. ERBB3-mediated mTOR signaling is essential for acinar specification (A) Schematic of E13 SG epithelia isolation and culture mTOR inhibitor. (B) 48 h control and Torin-1 E13 epithelia immunostained for MIST1, pAKTSer473, and SOX10. Acini are outlined with dashed lines. Scale bars, 50 mm. (C) Quantification of MIST1+ and SOX10+ cells as a percentage of total epithelial cells per field of view for 48 h control and Torin-1 epithelia. n = 6 rudiments per treatment, averaged from three images. Data are shown as mean ± SD; Student’s t test. **p < 0.01, ***p < 0.001. (D) Brightfield images of control and Torin-1 E13 epithelia cultured with NRG1 for 120 h (left, scale bars, 200 mm). Cultures were immunostained for ECAD, AQP5, ZO-1, MIST1, and PSP. Scale bars, 50 mm. (E) Heatmaps displaying differential expression of acinar secretory program, polarity program, and cell lineage TF genes in control and Torin-1 cultured epithelia (72 h). Levels of gene expression shown as relative Z score (red = upregulated, blue = downregulated). Asterisks indicate significantly differentially expressed genes (log2-fold change R 0.5, padj < 0.05).

Article Snippet: E13 SG epithelia were freshly isolated as previously described.11,12 Briefly, the SG epitheliumwas separated from themesenchyme using dispase II (Gibco) treatment followed by mechanical dissection and cultured in 15ml of laminin-1 (R&D Systems) on a nucleopore filter floating above 200 mL of serum-free media (DMEM/F12; Thermo Fisher Scientific) containing penicillin-streptomycin (Gibco), 50mg/ml holo-transferrin (HT), 150mg/ml ascorbic acid (AsA) and 200ng/ml human recombinant NRG1 isoform SMDF (R&D Systems), or 20ng/ml HB-EGF (R&D Systems) for up to 120 h. For inhibitor experiments, epithelia were cultured at 37 C in a humidified 5% CO2/95% air atmosphere in complete media in the presence of 0.2% DMSO (Sigma-Alrdrich), 5mM of mTORC1 inhibitor rapamycin (Biotang Inc.), or 1mM mTORC1/2 inhibitor Torin-1 (Cell Signaling Technologies).

Techniques: Isolation, Control, Cell Culture, Quantitative Proteomics, Gene Expression