Unfolded protein response transducer IRE1-mediated signaling independent of XBP1 mRNA splicing is not required for growth and development of medaka fish
- Kyoto University, Japan
- Ryukoku University, Japan
- Osaka University, Japan
- National Institute for Basic Biology, Japan
Figures
Figure 1
Construction and initial characterization of IRE1α-KO, IRE1β-KO and IRE1α/β-DKO medaka.
(A) Schematic representation of the IRE1-mediated signaling pathways. (B) Schematic representation of the WT and C156X-mutant IRE1α genes and proteins. SS, TMD, Kinase and RNase denote the signal …
Figure 2
Construction of XBP1-KO medaka and fluorescence microscopic analysis of IRE1α/β-DKO and XBP1-KO medaka.
(A) Schematic representation of the WT and Δ8-mutant XBP1 genes as well as their translational products pXBP1(U) and pXBP1(Δ8). bZIP denotes a basic leucine zipper, whereas the degron denotes a …
Figure 3
Effect of IRE1α/β-DKO and XBP1-KO on medaka development.
(A) Tail lengths of the indicated genotypes shown in Figure 2C and D were measured (n > 3) and are expressed as means with SD (error bars). IRE1α + implies IRE1α+/+ or IRE1α+/-. *p<0.05, **p<0.01, …
Figure 4
Effect on deleting IRE1α/β on phenotypes of XBP1-KO medaka.
(A) Total RNA prepared from embryos of the indicated genotypes at 3 dpf were subjected to quantitative RT-PCR to determine absolute expression levels of spliced XBP1 mRNA and β-actin mRNA (n = 3). …
Figure 5
Maternal effect on expression levels of IRE1α mRNA and spliced XBP1 mRNA.
(A) – (C) Embryos were obtained by incrossing male and female XBP1+/− medaka or by incrossing male and female IRE1α+/− IRE1β−/− medaka. (A) Level of IRE1α mRNA in embryos at indicated dpf of the …
Figure 6
Construction of mutant alleles to express various forms of XBP1.
(A) Schematic representation of the XBP1 gene as well as positions of the IRE1-mediated splice sites and three deletion mutants (Δ15, Δ26, Δ4). (B) Schematic representation of IRE1-mediated, …
Figure 7
Characterization of various forms of XBP1.
(A) HCT116 cells were transfected with pCMV-myc(-), pCMV-myc-medakaXBP1(WT), pCMV-myc-medakaXBP1(Δ8), pCMV-myc-medakaXBP1(Δ15), pCMV-myc-medakaXBP1(Δ26) or pCMV-myc-medakaXBP1 (Δ4) together with (+) …
Figure 8
Effect of expression of various forms of XBP1 on medaka development.
(A) Fluorescence microscopic analysis of XBP1+/+ and XBP1Δ4/Δ4 embryos at 5 dpf which were obtained by incrossing male and female XBP1+/Δ4 medaka, both carrying PBiP-EGFP. Scale bar: 250 μm. (B) …
Figure 9
Complete rescue of IRE1α/β-DKO medaka by constitutive expression of pXBP1(S).
(A) Male IRE1α+/− IRE1β−/− XBP1+/+ medaka were crossed with female IRE1α+/− IRE1β+/− XBP1SC/+and the 48 resulting fishes were genotyped 2 months after hatching. The genotype of IRE1β was not …
Figure 10
Survival rate and hatching rate of IRE1α/β-DKO medaka with or without the XBP1Sc allele.
(A) Male IRE1α-/- IRE1β-/- XBP1SC/+ medaka were crossed with female IRE1α+/− IRE1β-/- XBP1+/+ medaka, and the resulting 47 embryos were grown. Dead fish and fish surviving until 15 dpf were …
Figure 11
Transcriptomic comparison of IRE1β-KO, IRE1α/β-DKO XBP1+/+ and IRE1α/β-DKO XBP1SC/+ embryos.
(A) RNA prepared from embryos at 5 dpf of the indicated genotypes were subjected to RNA-seq analysis and the data obtained are expressed as MA plot. Genes whose expression was significantly altered …
Figure 12
Effect of constitutive expression of pXBP1(S) from both alleles on medaka growth and development.
(A) Fluorescence microscopic analysis of XBP1+/+, XBP1SC/+ and XBP1SC/SC embryos at 5 dpf obtained by incrossing male and female XBP1SC/+ medaka, both carrying PBiP-EGFP. Scale bar: 250 μm. (B) …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.26845.014
