Reporter expression in developingzebrafish. (A) Schematic of the ATF6 reporter plasmid showing five multimerized ATF6 binding sites (5XATF6RE) upstream of a c-fos minimal promoter (c-fos mp) driving expression of either destabilized GFP (d2GFP) or stable, enhanced GFP (eGFP). (B) Time-course analysis showing 5XATF6RE:d2GFP expression at 1, 3 and 5 dpf. (C) Time-course analysis showing 5XATF6RE:eGFP at 2 and 3 dpf. Scale bars: 1 mm.

Maternal/paternal inheritance of eGFP from the 5XATF6RE:eGFP transgene. Epifluorescent images showing embryos in which the 5XATF6RE:eGFP was inherited maternally (top row) or paternally (middle row). Age-matched embryos not harboring a transgene are shown as an autoflourescence control (bottom row). Scale bar: 1 mm.

Heat-shock-induced constitutive active ATF6 specifically induces reporter expression. (A) Schematic of the hsp70:GAL4 construct used to induce ubiquitous expression of UAS constructs mosaically expressing either caATF6, XBP1s or ATF4 with a T2A self-cleaving peptide and mCherry. (B,C) Representative images (B) and quantification of reporter expression (C) from embryos expressing hsp70:GAL4 and 5XATF6RE:d2GFP transgenes that were injected with mCherry-tagged UAS plasmids to overexpress caATF6 (left), XBP1s (middle) or ATF4 (right) compared to injection control (Inj Ctrl) embryos injected with all components except overexpression plasmids. 2-dpf embryos were heat shocked and confocal images were captured 12 h later. Quantification of the head region (dashed line) revealed a significant increase in 5XATF6RE:d2GFP intensity with caATF6 overexpression (P=0.0067), a significant decrease with XBP1s overexpression (P=0.0182) and no significant change with ATF4 overexpression (P=0.1055) compared to the injection control. *P≤0.05; **P≤0.01; ns, P>0.05; unpaired Student's t-test. All error bars are s.e.m. Scale bars: 1 mm.

The ATF6 target gene, Bip, is highly expressed with high ATF6 reporter expression, while XBP1 expression does not correlate with caATF6. (A-C) Embryos expressing hsp70:GAL4 and 5XATF6RE:eGFP transgenes that were injected with an mCherry-tagged UAS construct to mosaically overexpress caATF6. 2-dpf embryos were heat shocked and confocal images were captured 12 h later live (A,B) or after fixation and staining (C,D), and mean intensity was quantified. (A) 5XATF6RE:d2GFP expression positively correlates with caATF6-2a-mCherry expression (R²=0.8439, P=0.0005). (B) xbp1δ-gfp expression does not significantly correlate with caATF6-2a-mCherry expression (R²=0.0077, P=0.8099). (C) 12 h post-heat shock, embryos were fixed and stained for Bip (blue) or GFP (green). Bip expression positively correlates with 5XATF6RE:d2GFP expression (R²=0.8899, P<0.0001). (D) 60× representative images showing low versus high cellular expression patterns of Bip and 5XATF6RE:d2GFP. ***P<0.001; ****P<0.0001; Pearson's correlation coefficient. Scale bars: 1 mm (A-C); 0.02 mm (D).

Tunicamycin-induced reporter expression is blocked by inhibiting ATF6 translation and binding. (A) After injection of an atf6 morpholino (atf6 MO) at the one- to four-cell stage to inhibit ATF6 translation, 1-dpf embryos were treated with Tunicamycin (Tunic) and expression at 3 dpf in the head region (dashed line) was quantified. atf6-MO-injected embryos had significantly less 5XATF6RE:eGFP expression compared to tunicamycin-only-treated embryos (P=0.0150). Only embryos with detectable 5XATF6RE:eGFP expression were analyzed. *P<0.05; unpaired Student's t-test. (B) Embryos expressing hsp70:Gal4 and 5XATF6RE:d2GFP transgenes were injected with an mCherry-tagged UAS plasmid to mosaically overexpress dominant-negative ATF6 (dnATF6) that inhibits endogenous ATF6 binding. 2-dpf embryos were heat shocked, treated with tunicamycin, and expression of GFP and mCherry was quantified in the head region (dashed line) 12 h later. Representative images show that, compared to a DMSO-treated injection control, embryos with tunicamycin treatment show increased 5XATF6RE:d2GFP expression, which decreases with increasing dnATF6-2A-mCherry expression, resulting in a significant negative correlation (R²=0.4712, P=0.0284). *P≤0.05; Pearson's correlation coefficient. All error bars are s.e.m. Scale bars: 1 mm.

Chemical ER stressors activate reporter expression. (A) Zebrafish embryos were treated with ER stressors at 1 dpf. Quantification (dashed line) revealed that 5XATF6RE:d2GFP expression was significantly higher at 8 hpt with thapsigargin (P=0.0004) and brefeldin A (P=0.0003) treatment, at 24 hpt with tunicamycin (P=0.0001) and brefeldin A (P=0.0001) treatment, and at 48 hpt with tunicamycin (P=0.0001) and brefeldin A (P=0.0065) treatment. **P≤0.01; ***P≤0.001; ****P≤0.0001; unpaired one-way ANOVA with Dunnett's post-hoc test with respect to DMSO control group. All error bars are s.e.m. (B) Representative images showing increased 5XATF6RE:d2GFP expression in the spinal cord of embryos (at 48 hpt) treated with tunicamycin or brefeldin A compared to the DMSO-treated control. Scale bars: 1 mm (A); 0.1 mm (B).

Heat shock activates reporter expression. (A) Representative images showing increased 5XATF6RE:d2GFP expression in the spinal cord 8 h after heat shock compared to a control without heat shock. (B) Representative images and quantification of the lens (indicated by an arrow) revealed significantly higher levels of 5XATF6RE:d2GFP expression 2 days post-heat shock compared to a control without heat shock (P<0.0001). The dashed line outlines the eye. (C) Representative images and quantification of the head region (dashed line) revealed significantly higher levels of 5XATF6RE:d2GFP expression 12 h post-heat shock compared to a control without heat shock (P=0.0248). *P≤0.05; ****P≤0.0001; unpaired Student's t-test. All error bars are s.e.m. Scale bars: 0.1 mm (A,B); 1 mm (C).

Reporter expression is activated in ALS and cone dystrophy zebrafish disease models. (A) Representative images showing that embryos injected with gnat2:OPN1MW^W177R mutant opsin have elevated 5XATF6RE:d2GFP expression in photoreceptors, which is absent in injection control embryos injected with all components except the overexpression construct. (B) Representative images showing overlap of hsp70:DsRed and 5XATF6RE:d2GFP expression in sod1 G93R mutant spinal cord, which is absent in WT sod1 control spinal cord. Scale bars: 0.02 mm (A); 0.1 mm (B).

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.