Silencing of Rab35 in vivo results in altered cell polarity and liver tissue architecture. (a) Immunofluorescence images of liver tissue collected 4 d after in utero injection of Luciferase (siLuc) and Rab35 (siRab35) siRNAs formulated into LNP via vitelline vein in E13.5 embryos. The square on the low-magnification images (scale bar: 500 µm) shows where the high-resolution image was taken (scale bar: 20 µm). Imaged areas are located in the liver parenchyma, devoid of bile duct cell marker Sox9. The inserts (scale bar: 20 µm) in a’ and a’’ show the difference between BC and bile duct-like lumina in LNP-siRab35 injected liver. Panel a’’’ compares tubular lumina in the parenchyma to the bile duct lumina in the portal area (Sox9-positive cells near portal veins [PV]). (b) Immunofluorescence images of liver tissues from panel a show examples of hepatocyte polarity in the control tissue (a single hepatocyte forms multiple lumina per cell) and simple apico-basal polarity in LNP-siRab35 injected liver (cells have a single apical domain oriented toward a shared lumen). (c and d) 3D reconstruction of lumina labeled with an apical marker CD13 in 100-µm-thick sections of liver tissue injected with LNP-siLuc (c) and LNP-siRab35 (d). Scale bar: 30 µm. See also Video 9. (e) Quantification of the lumen radius distribution based on the 3D reconstructions such as in c and d (n = 3, error bars: SEM). (f) 3D reconstruction of a tubule in LNP-siRab35–injected livers shows a cylindrical lumen (green) surrounded by multiple cells. See also Video 10. (g) A cross-section of the reconstructed tubule in f in the original microscopy image shows organization of the cells around the lumen. Scale bar: 20 µm. (h) Quantification of number of cells surrounding the lumen in relation to lumen radius and position along the tubule.