Developmental hypoxia does not affect general CNS development.

<p>Ventral views, rostral to the top, scale bar 50 µm (except E′, 25 µm). Whole-mount embryos, shown as brightfield (A), or confocal maximum intensity z-projections (B–E). (A, A′) <i>in situ</i> hybridization for <i>dlx2</i> shows no difference in pattern. (B, B′) α-tyrosine hydroxylase (TH) antibody staining pattern is unaffected. (C, C′) Acridine orange shows similar numbers of apoptotic cells. (D, D′) Phospho-histone H3 antibody staining shows similar numbers of mitotic cells. (E) Confocal image of acridine orange stain demonstrating region for determining apoptotic cell counts. Inset shows high-magnification (E′) for counting cells in the 100 µm×100 µm area.</p

Hypoxia and survival.

<p>Table of survival percentages following hypoxia exposure in embryonic zebrafish at different ages. 30 or more embryos were examined for each time-point.</p

Summary of results for experiments involving C/L ratios.

<p>C/L axonal tract fluorescence intensity ratio analysis comparing commissural axons to longitudinal axons of embryos for the different experiments reported in this paper. Genotype is listed in the left-hand column; conditions for the experiment are listed in middle columns, and results (C/L ratios including average, standard deviation, standard error of the mean, and 95% confidence interval) are shown in the right-hand columns, as well as the two-tailed <i>t</i> test <i>p</i> value in the far right column.</p

Hypoxia disrupts axon pathfinding in the developing CNS.

<p>Confocal images are maximum intensity projections of whole-mount embryos, ventral views, rostral top, scale bars 50 µm. (A, B) 48 hpf embryos, acetylated tubulin immunohistochemistry. (B) Following 1% hypoxia (1% pO₂) from 24–36 hpf, anterior commissure and post-optic commissural tracts are disrupted (arrows). (C–I) Tg(<i>foxP2-enhancerA.2:egfp-caax</i>) embryos (abbreviated <i>foxP2-A.2:caax</i>), GFP immunohistochemistry. (C) Normoxic embryo at 72 hpf; TCPT commissure (TCPTc) (arrow). (D) Embryo following hypoxia from 24–36 hpf; TCPTc is absent (arrow). (E) Graph showing percent Tg(<i>foxP2-A.2:caax</i>) embryos with wild-type TCPTc following hypoxia exposure at different ages. Error bars standard error of the proportion; ** <i>p</i><0.0001. (F) C/L ratio determination. Confocal image of 72 hpf embryos illustrating determination of ratio of commissure intensity to intensity of longitudinal axons. (G) Quantification of TCPTc errors in Tg(<i>foxP2-A.2:caax</i>) embryos following hypoxia exposure at different ages. C/L (commissure:longitudinal) intensity ratio quantification along <i>y</i>-axis. *<i>p</i><0.05; ** <i>p</i><0.0001. ∧mark indicates significant mortality at this stage. Errors bars standard error of the mean. (H) Normoxic embryo at 96 hpf; TCPT is intact (arrow). (I) Embryo at 96 hpf following 1% hypoxia from 24–36 hpf; TCPTc is absent (arrow).</p

ephrinB2a mediates the hypoxia-induced TCPT pathfinding errors.

<p>(A–B) ephrinB2a is expressed in TCPT neurons. Confocal maximum intensity projections of whole-mount embryos, double immunohistochemistry for GFP and EfnB2a, ventral views, rostral top, scale bar 50 µm. (A–A″) TCPT neurons express ephrinB2a as they begin to extend axons. (B–B″) TCPT commissural axons express ephrinB2a as they cross the midline. (C–D) Hypoxia leads to increased expression of EfnB2a. Maximum intensity projections of 36 hpf embryos, ventral views, rostral to the top, EfnB2a immunohistochemistry. (E) Quantification of EfnB2a levels, normalized to normoxia. *<i>p</i><0.05. Error bars SEM. (F) Knockdown of EfnB2a expression with morpholino rescues TCPTc pathfinding. Tg(<i>foxP2-A.2:caax</i>) embryo at 72 hpf; confocal maximum intensity projection, ventral view, rostral top, scale bar 50 µm. (G) C/L intensity ratio quantifications show ephrin morpholino rescue of hypoxia pathfinding errors. *<i>p</i><0.01; ** <i>p</i><0.001. Error bars SEM. (H) C/L intensity ratios show <i>UAS:ephrinΔc</i> rescues hypoxia pathfinding errors. ** <i>p</i><0.001. Error bars SEM.</p

Magnesium sulfate administration protects against hypoxia-related axon pathfinding defects.

<p>(A) C/L intensity ratios of increasing magnesium concentrations (mM). *p<0.01; ** <i>p</i><0.001. Error bars SEM. (B, C) Magnesium reduces activation of the <i>hif1α</i> pathway, as shown by decreased <i>igfbp-1</i> expression following hypoxia and concurrent magnesium exposure from 24–36 hpf. Bright-field images; ventral views, rostral top, scale bar 50 µm. (D) C/L intensity ratios shows rescue by magnesium of DMOG exposure from 24–36 hpf. *p<0.05. Error bars SEM.</p