Journal: bioRxiv

Article Title: Sox9 links biliary maturation to branching morphogenesis

doi: 10.1101/2024.01.15.574730

Figure Lengend Snippet: (A) Bulk RNA-seq identifies DEGs between Sox9cKO and control BECs. (B) GSEA demonstrates significant enrichment of TGF-β signaling in Sox9cKO BECs relative to control BECs. (C) Co-localization of pSMAD2 and EpCAM confirms elevated TGF-β signaling in Sox9cKO BECs (scale bar represents 5μm; white asterisks indicate portal vein). (D) IF demonstrates elevated Activin A in EpCAM+ BECs in Sox9cKO livers (scale bar represents 50μm; white asterisks indicate portal vein). (E) GSEA demonstrates that Sox9cKO BECs are de-enriched for adult BEC gene signature (NES: −2.70; p = 3.67E-10) and enriched for adult hepatocyte gene signature (NES: 2.31; p = 3.67E-10). (F) GSEA of liver development gene sets shows that the Sox9cKO BEC transcriptome resembles embryonic BECs (NES: 1.23; p = 6.06E-6) and embryonic hepatocytes (NES: 2.32; p = 3.00E-10), but not hepatoblasts (NES: 0.99; p = 0.55). (G) Co-localization of NCAM1 and EpCAM is significantly elevated in Sox9cKO compared to controls (scale bar represents 5μm; white asterisks indicate portal vein). (H) scRNA-seq of FACS-isolated EpCAM+ BECs reveals subpopulation-specific transcriptomic changes in Sox9cKO. (I) Upregulation of Inhba and Ncam1 in Sox9cKO samples is specific to Sox9cKO ductule-like cells (cluster 6).

Article Snippet: To neutralize Activin A, control and Sox9cKO neonates were administered 2 µg/g anti-Activin A (Biotechne; MAB3381) or mouse IgG 1 (Biotechne; MAB002) at P1 and P3 by intraperitoneal injection.

Techniques: RNA Sequencing Assay, Isolation

Journal: bioRxiv

Article Title: Sox9 links biliary maturation to branching morphogenesis

doi: 10.1101/2024.01.15.574730

Figure Lengend Snippet: (A) Single Sox9cKO BECs cultured for 7d fail to form lumens typical of control mICOs. (B) Sox9cKO BECs form significantly fewer mICOs, a majority of which (C) exhibit non-cystic morphology. (D) mICO area is unchanged between control and Sox9cKO. (E) 50ng/mL Activin A is sufficient to induce Sox9cKO morphology in WT BECs, but (F) unlike Sox9cKO (Sox9 Δ/Δ) did not impact mICO organoid formation. (G) Rates of non-cystic mICO morphology were similar between Sox9cKO and Activin A treated WT BECs. (H) Organoid area remained unchanged between all groups. (I) Ncam1 was significantly upregulated in Sox9cKO mICOs by RT-qPCR, and induced by Activin A in WT mICOs. (J) Activin A upregulates Sox9 expression in WT mICOs (letters indicate grouping by significance , p < 0.05; scale bars represent 50μm).

Article Snippet: To neutralize Activin A, control and Sox9cKO neonates were administered 2 µg/g anti-Activin A (Biotechne; MAB3381) or mouse IgG 1 (Biotechne; MAB002) at P1 and P3 by intraperitoneal injection.

Techniques: Cell Culture, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Sox9 links biliary maturation to branching morphogenesis

doi: 10.1101/2024.01.15.574730

Figure Lengend Snippet: (A) Co-localization of Ki-67 and EpCAM in IgG and α-ActA treated livers demonstrates that Activin A neutralization in vivo (B) increases BEC numbers per portal field while (C) inhibiting BEC proliferation (scale bar represents 50μm; white asterisks indicate portal vein). (D) Whole tissue imaging of EpCAM+ IHBDs reveals that Activin A neutralization partially rescues Sox9cKO ductal paucity, resulting in increased branching near the hilum. (E) Sholl analysis supports increased proximal branching and decreased distal branching in α-ActA treated livers, which is corroborated by (F) AUC demonstrating increased hilar branch complexity (0.00-0.25a.u.), no change at 0.25-0.50a.u., and decreased branching distally (0.50-1.00a.u.). (G) Sholl decay coefficient is increased following Activin A neutralization reflecting increased localized branching near the hilum.

Article Snippet: To neutralize Activin A, control and Sox9cKO neonates were administered 2 µg/g anti-Activin A (Biotechne; MAB3381) or mouse IgG 1 (Biotechne; MAB002) at P1 and P3 by intraperitoneal injection.

Techniques: Neutralization, In Vivo, Imaging