Fig. 2

HrRCs in the brain of hypoxia-exposed huORFZ embryos did not undergo apoptosis during oxygen recovery. TUNEL staining as a quantitative assay was performed to determine the hypoxia-induced zebrafish embryos apoptosis. (A) Schematic representation of the embryonic brain (72 hpf), showing the forebrain (in yellow), midbrain (m), and hindbrain (h). Forebrain is subdivided in the telencephalon (t) and the diencephalon (d). The red dotted line box indicated the observation area. Apoptotic cells were marked by red after TUNEL staining with (B) early neuron marker, HuC/HuD, and (C) NSPC marker, Sox2, were observed under a fluorescent confocal microscopy in the normoxic huORFZ embryos at 98, 108, and 120 hpf (equivalent to R24, R36, and R48, respectively) and hypoxic huORFZ embryos at R24, R36, and R48 (equivalent to 98, 108, and 120 hpf, respectively). Each figure in the lower right three panels was amplified from the area indicated by the dotted line box. Arrow indicated that the cells expressed red-colored TUNEL signals. Quantitative analyses of (D) apoptotic early neurons (HuC/HuD marker) and (E) apoptotic NSPCs-non-HrRCs (Sox2 marker) by TUNEL assay. Black and gray bars indicated the normoxic and hypoxic huORFZ embryos, respectively. Data were averaged from five examined embryos at the same position of brain. The unpaired t test showed the significant difference of apoptotic cells between two studied groups (Statistical analysis was based on t test at **P < 0.05, ***P < 0.001 significance). Error bar indicates SEM. The scale bar is 40 μm. HrRCs: hypoxia-responsive recovering cells; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling; hpf: hours postfertilization; NSPCs: neural stem/progenitor cells; VZ: ventricular zone; SEM: standard error of the mean; HuC and HuD: Neural Hu proteins.