Proliferation but not apoptosis change as a function of tissue microenvironment. (a) Comparison of single-cell morphologies were made as a function of seeding site within the zebrafish larvae, each representing a distinct tissue microenvironment. The HBV is a mostly fluid-filled compartment (a), the PVS is a small, compressed region sandwiched between two membranes surrounding the yolk, and the CHT is a narrow region rich in vasculature. (b and c) TC71 cells expressing nuclear marker mCherry-H2B (red) within the HBV (inset, enlarged in b’), CHT (inset, enlarged in c’), or PVS (inset, enlarged in c’’) of fish larvae (4 dpf). Fish are immunostained for proliferation marker PH3 (blue) and apoptotic staining TUNEL (green). Measurements are made on 3D volumes, as rendered in c’’’. (d and e) Apoptotic rate is similarly low across all three regions (Kruskal-Wallis test, post-hoc Dunn’s test, P > 0.1; d), while proliferation rate appears to be highest in PVS compared to other regions (Kruskal-Wallis test, post-hoc Dunn’s test, **P < 0.05; e). Scale bars, 100 µm (b and c) and 20 µm (b’, c’, and c’’). N = 18, 27, 12 fish for PVS/CHT/HBV, respectively.