Phylogenetic tree showing relationship between Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), and Danio rerio (zebrafish) PPARs (A). Percent similarity of mouse, rat, and zebrafish PPARγ relative to human PPARγ; FL, full length; DBD, DNA binding domain; and LBD, ligand binding domain (B).

Abundance of pparγ mRNA within whole zebrafish embryos from 0.75 to 96 hpf (A). Confirmation of knockdown by 24 hpf following injection of splice-blocking pparγ-MOs. Lanes 1–3: 6-hpf embryos injected with nc-MO; lanes 4–6: 6-hpf embryos injected with pparγ-MO; lanes 7–9: 24-hpf embryos injected with nc-MO; and lanes 10–12: 24-hpf embryos injected with pparγ-MO (B). Representative images of water-, nc-MO-, or pparγ-MO-injected embryos from 6 to 96 hpf before and after staining with Oil Red O (ORO) (C–FF). Arrows point to mild dorsoventral patterning defects (ventralization), whereas arrowheads point to cardiac edema and cardiac looping defects.

Initiation of ciglitazone (Cig) exposure at 4 hpf does not result in delayed epiboly by 6 hpf (N = 60 embryos per treatment) (A). Mean (± standard deviation) percent of normal, ventralized, dorsalized, or dead embryos following exposure to increasing concentrations of Cig from 4 to 24 hpf (N = 60 embryos per treatment) (B). Asterisk (*) denotes a significant difference (p < 0.05) in the percent of normal embryos relative to vehicle controls (0.2% DMSO). Representative images of (1) a normal embryo exposed to vehicle (0.2% DMSO) (C); (2) ventralized embryos exposed to 9.375 and 12.5 μM Cig (D and E); and (3) a delayed embryo exposed to 15 µM Cig (F). Arrows point to swollen yolk sac extensions, whereas arrowheads point to underdeveloped heads.

Mean (± standard deviation) percent of normal, ventralized, delayed, or dead embryos following injection of nc-MOs or pparγ-MOs at 0.75 hpf and exposure from 4 to 24 hpf to vehicle (0.2% DMSO) or 12.5 µM ciglitazone (Cig) (N = 60 embryos per treatment). Asterisk (*) denotes a significant difference (p < 0.05) in the percent of normal embryos relative to within-treatment water-injected controls (p < 0.05). Cross (†) denotes a significant difference (p < 0.05) in the percent of normal embryos relative to within-MO vehicle (0.2% DMSO) controls. chd-MO was used as a positive control for ventralization (A). Representative images of nc-MO- and pparγ-MO-injected embryos exposed to either vehicle (0.2% DMSO) or 12.5 µM Cig at 24 hpf (B–G).

Mean (+standard deviation) percent of normal, ventralized, dorsalized, delayed, or dead embryos following exposure to vehicle (0.2% DMSO), 0.078 µM DMP, 12.5 µM ciglitazone (Cig), or 0.078 µM DMP + 12.5 µM Cig (N = 60 embryos per treatment) (A). Asterisk (*) denotes a significant difference (p < 0.05) in the percent of normal embryos relative to vehicle (0.2% DMSO) controls. Representative images of embryos following exposure to vehicle (0.2% DMSO), 0.078 µM DMP, 12.5 µM ciglitazone, or 0.078 µM DMP + 12.5 µM Cig (B–E). Immunostaining with anti-phosphoSMAD-1/5/9 within 8-hpf embryos following exposure to vehicle (0.2% DMSO), 0.078 µM DMP, 12.5 µM Cig, or 0.078 µM DMP + 12.5 µM Cig; embryos exposed to 0.625 µM DMP were included as a positive control for disruption of BMP signaling gradients. Arrowheads point to elevated pSMAD 1/5/9 staining on the ventral side of the embryo (F–J).

Volcano plots showing the number of significantly different transcripts following exposure to 12.5 µM ciglitazone (Cig) (A), 0.078 µM DMP (B), or 0.078 µM DMP + 12.5 µM Cig (C). All data are relative to vehicle (0.2% DMSO) controls. Log₂ transformed fold change is plotted on the x-axis and log₁₀ transformed p-adjusted value is plotted on the y-axis. Venn diagram showing the number and percent of significantly different overlapping transcripts among treatment groups; percentage values are relative to the total number of significantly different transcripts across all treatment groups (D).

Top 5 DAVID-identified biological processes (based on significantly different transcripts) following exposure to 12.5 µM ciglitazone, 0.078 µM DMP, or 0.078 µM DMP + 12.5 µM ciglitazone (A). Venn diagram showing the number and percent of significantly altered biological processes among treatment groups (B). Significant (Benjamini < 0.05) biological processes unique to each treatment group (C).