Apical bulkhead frequency anticorrelates with BC connectivity. (a) Reconstruction of the BC network in embryonic liver tissue at E15.5 (upper) and E18.5 (lower) based on CD13 staining. Individual BC fragments are classified based on whether they are disconnected from the rest of the network (yellow) or truncated by the boundaries of the imaging volume (blue). Scale bar: 50 µm. (b) Examples of BC stained for the tight junction protein ZO-1 (cyan) containing apical bulkheads at E15.5 (left four panels) and E18.5 (middle four panels), as well as the typical BC morphology without apical bulkheads at E18.5 (right four panels). White arrows indicate apical bulkheads. Scale bar: 2 µm. (c) Example of a disconnected BC from E15.5 with apical bulkheads (white arrows). Liver tissue was stained for ZO-1 (cyan), actin (green), the apical marker CD13 (red), and DAPI (white). Scale bar: 5 µm. (d) Example of a BC from E18.5 without apical bulkheads. Liver tissue was stained for ZO-1 (cyan), actin (green), the apical marker CD13 (red), and DAPI (white). Asterisks indicate branching points in the BC network. Scale bar: 5 µm. (e) Quantification of the number of apical bulkheads in embryonic liver tissue at E15.5 and E18.5, normalized to BC length. Data is color-coded by biological replicate, dots represent the normalized apical bulkhead frequency in a single imaging volume, triangles represent the mean of each biological replicate imaged at 2–3 locations per liver, and the horizontal bars represent the average of the means ± SEM. **, P < 0.01 using an unpaired Student’s two-tailed two-sample t test of the means of each biological replicate.