Figure 5.
Insufficient xbp1 splicing drives the SDS phenotype in srp54-defective zebrafish embryos. (A) Graphical representation of the unconventional splicing of XBP1. Left: cell without or with moderate ER stress; right: cell under ER stress conditions. (B) Representative images of WT embryos compared with xbp1 morphants. Top row: photos showing that xbp1 morphants are incapable of hatching and breaking the chorion at 72 hpf; middle row: WISH using lyz-specific probes; bottom row: WISH using trypsin-specific probes. (C) Quantification of neutrophils using lyz-specific probes. (D) Measurement of the exocrine pancreas using trypsin-specific probes. The size of the exocrine pancreas was semi-automatically measured using ImageJ software. (E) Schematic overview of the experimental setup to assess xbp1 levels in zebrafish embryos. (F) Differences of the cycle threshold (CT) between xbp1s and the housekeeping gene gapdh (ΔCT values) of dissolved cells from dimethyl sulfoxide (DMSO)–treated WT, srp54+/−, srp54+/− injected with human G226E mRNA, and srp54−/− embryos measured by qRT-PCR. (G) ΔCT values for xbp1s of dissolved cells from Tm-treated WT, srp54+/−, srp54+/− injected with human G226E mRNA and srp54−/− embryos measured by qRT-PCR. (H) Fold change of xbp1s expression upon Tm treatment compared with DMSO treatment: WT, srp54+/−, srp54+/− injected with human G226E mRNA and srp54−/− embryos (we used 3 biological replicates with at least 2 larvae per replicate). (I) Percentage of Mpo+ cells from Tm-treated compared with DMSO-treated embryos measured by flow cytometry. Tg(srp54+/−;mpo:eGFP) zebrafish were incrossed, and their progeny were genotyped and analyzed by flow cytometry. A minimum of 5 embryos were pooled and dissociated per biological replicate. Each dot represents a biological replicate. WT, srp54+/−, srp54+/− injected with human G226E mRNA and srp54−/− embryos. (J) Representative images of WISH using lyz-specific probes performed on srp54+/− embryos either uninjected (top), injected with xbp1s mRNA (middle), or injected with xbp1u mRNA (bottom). (K) Quantification of the number of lyz+ cells (we used 4 biological replicates with at least 3 larvae per replicate). Student t test was used for statistical analysis: SR, signal receptor. *P < .05; **P < .01; ***P < .005; ****P < .0001. Bar plots and horizontal lines in the graphs represent the mean value of the replicates. Error bars indicate the standard deviation of the mean. NC, nascent chain.

Insufficient xbp1 splicing drives the SDS phenotype in srp54-defective zebrafish embryos. (A) Graphical representation of the unconventional splicing of XBP1. Left: cell without or with moderate ER stress; right: cell under ER stress conditions. (B) Representative images of WT embryos compared with xbp1 morphants. Top row: photos showing that xbp1 morphants are incapable of hatching and breaking the chorion at 72 hpf; middle row: WISH using lyz-specific probes; bottom row: WISH using trypsin-specific probes. (C) Quantification of neutrophils using lyz-specific probes. (D) Measurement of the exocrine pancreas using trypsin-specific probes. The size of the exocrine pancreas was semi-automatically measured using ImageJ software. (E) Schematic overview of the experimental setup to assess xbp1 levels in zebrafish embryos. (F) Differences of the cycle threshold (CT) between xbp1s and the housekeeping gene gapdh (ΔCT values) of dissolved cells from dimethyl sulfoxide (DMSO)–treated WT, srp54+/−, srp54+/− injected with human G226E mRNA, and srp54−/− embryos measured by qRT-PCR. (G) ΔCT values for xbp1s of dissolved cells from Tm-treated WT, srp54+/−, srp54+/− injected with human G226E mRNA and srp54−/− embryos measured by qRT-PCR. (H) Fold change of xbp1s expression upon Tm treatment compared with DMSO treatment: WT, srp54+/−, srp54+/− injected with human G226E mRNA and srp54−/− embryos (we used 3 biological replicates with at least 2 larvae per replicate). (I) Percentage of Mpo+ cells from Tm-treated compared with DMSO-treated embryos measured by flow cytometry. Tg(srp54+/−;mpo:eGFP) zebrafish were incrossed, and their progeny were genotyped and analyzed by flow cytometry. A minimum of 5 embryos were pooled and dissociated per biological replicate. Each dot represents a biological replicate. WT, srp54+/−, srp54+/− injected with human G226E mRNA and srp54−/− embryos. (J) Representative images of WISH using lyz-specific probes performed on srp54+/− embryos either uninjected (top), injected with xbp1s mRNA (middle), or injected with xbp1u mRNA (bottom). (K) Quantification of the number of lyz+ cells (we used 4 biological replicates with at least 3 larvae per replicate). Student t test was used for statistical analysis: SR, signal receptor. *P < .05; **P < .01; ***P < .005; ****P < .0001. Bar plots and horizontal lines in the graphs represent the mean value of the replicates. Error bars indicate the standard deviation of the mean. NC, nascent chain.

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