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. Author manuscript; available in PMC: 2021 Nov 1.
Published in final edited form as: FASEB J. 2020 Sep 12;34(11):14850–14862. doi: 10.1096/fj.201902308RR

Figure 2. Expression of alox12 mRNA in pancreatic development.

Figure 2.

(A) Quantitative PCR showing expression levels of alox12 mRNA and pdx1 mRNA throughout embryonic stages 1 dpf through 4 dpf. Levels are normalize to elfa mRNA and depicted as the relative expression level at 1 dpf. Two-way ANOVA was used to compare 2, 3, and 4 dpf levels with 1dpf. (B-B’’) Merged (B), single channel (B’), and single channel inset (B’’) confocal image of 3 dpf Tg(ptf1a:GFP)jh1 transgenic embryo with expression of alox12 marked by fluorescent whole mount in situ hybridization (red). Green ptf1a-GFP signal is detected in the retina (r), pancreas (p), liver (L), heart (H), endoderm (E) and central nervous system. Red signal for alox12 is detected throughout the green fluorescent pancreatic region. DNA is marked by TOPRO-3 (blue). (C) Tg(ptf1a:GFP)jh1 zebrafish embryos injected with 0 ng or 4 ng of alox12 antisense morpholino (alox12 MO1) as zygotes shown at 3 dpf. (C, right panel) Brightfield-only image shows no overt aberrant phenotypes in the knockdown embryos. (C, left panel) Merged image showing GFP expression (green) in the exocrine pancreas (xp), hindbrain (hb), and retina (rt) and the H2B-RFP marker of injection; zebrafish injected with 4 ng MO1 show shortened pancreas phenotype. (D) Quantification of GFP+ exocrine pancreas length shows a significant shortening by 33.6% (p < 0.0001). (E) 3 dpf Tg(ptf1a:GFP)jh1 zebrafish embryos treated with vehicle control or 5 μM of the ALOX12 inhibitor ML127 for 48 hr (from 12 h post fertilization to 3 dpf). ML127 treated embryos show slight developmental delay, but substantially truncated pancreas length (arrows), phenocopying the alox12-morpholino knockdown embryos. (F) Quantification of the GFP+ exocrine pancreas length shows a dramatic and highly significant shortening by 52.8% (p<0.0001). (G-I) Lipidomic analysis of products generated by 12-LOX (G), 15-LOX (H), and 5-LOX (I), in zebrafish injected with 2 ng or 4 ng of alox12 morpholino and 0 ng controls.(t-test, p value <0.05 *). (G) All tested 12-LOX products, 12-HETE, 12-HEPE, 14-HDHA, and 8-HETE were essentially eliminated in animals injected with either dose of morpholino (showing one order of magnitude difference between control and injected). (H) The 15-LOX product, 15-HEPE, was expressed at lower levels but not statistically significant (t-test, p = 0.0583 for control vs 4 ng). 13-HODE remained unchanged (t-test, p = 0.116 for control vs 4ng). 15-HETE was undetectable in all 4ng samples and in. some control and 2 ng samples, between the detected samples no statistical difference was found (t-test, p = 0.1393). 17-HDHA was undetectable. (I) The 5-LOX product remained unchanged (t-test, p = 0.3379). (J-L) Lipidomic profile of LOX products from 3 dpf embryos treated with 5 uM ML127 for 12 hrs. T-test was used to compared treatment with control group. (J) Statistically significant reduction was observed for all 12-LOX products. (K) 15-LOX products, 15-HEP and 13-HODE, are significant reduced. 15-HETE does not changes and 17-HDHA was undetectable. (L) 5-LOX product, 5-HETE, is significantly reduced. 5-HEPE, 9-HODE and 4-HDHA were not detected.