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. 2017 Jun 23;6:e22477. doi: 10.7554/eLife.22477

Figure 2. Mpi loss causes cell death in zebrafish embryos via activation of p53.

(A) Whole transcriptome analysis of Mpi-depleted zebrafish embryos using RNA-seq showed increased p53 and apoptosis-related genes. Standardized fold change (FC) in mpi morphants in WT and p53 MT was calculated relative to standard control morpholino (std MO)-injected embryos. The color scheme represents gene expression changes in a log2 scale, in the range of −1.5 (blue, decreased) to 1.5 (red, increased). (B) Residual Mpi activity is significantly decreased at 4 dpf in mpimss7 MT larvae, compared to mpi+/+ siblings. (C) p53 mRNA levels are upregulated as analyzed through qPCR analysis in 24 hpf mpimss7/mss7 embryos, compared to mpi+/+ siblings. (D) p53 protein levels significantly increased at 24 hpf in mpimss7/mss7 embryos assessed by western blot, as compared to mpi+/+ siblings. Western blots quantified using densitometry analysis (ImageJ). p-Value based on two-tailed paired one sample t-test.

DOI: http://dx.doi.org/10.7554/eLife.22477.003

Figure 2—source data 1. Changes in expression of the genes involved in the N-glycosylation pathway.
Partial dataset of RNAseq analysis performed in 24 hpf zebrafish embryos injected with std or mpi MO. Log2 fold change (FC) values relative to WT + std MO. The list of showing the changes in expression of the genes involved in the N-glycosylation pathway.
DOI: http://dx.doi.org/10.7554/eLife.22477.004
elife-22477-fig2-data1.xlsx (55.6KB, xlsx)
DOI: 10.7554/eLife.22477.004
Figure 2—source data 2. Changes in expression of the genes involved in apoptosis and cell cycle arrest.
Partial dataset of RNAseq analysis performed in 24 hpf zebrafish embryos (WT and p53 null) injected with std or mpi MO. Log2 fold change (FC) values relative to WT + std MO. The list of showing the changes in expression of the genes involved in apoptosis and cell cycle arrest.
DOI: http://dx.doi.org/10.7554/eLife.22477.005
elife-22477-fig2-data2.xlsx (40.8KB, xlsx)
DOI: 10.7554/eLife.22477.005

Figure 2.

Figure 2—figure supplement 1. Mpi-depleted zebrafish embryos show few changes in N-glycosylation.

Figure 2—figure supplement 1.

(A) Residual Mpi activity in fish injected with 0.1 ng, 0.4 ng, 1 ng, and 4 ng mpi MO at t = 0. Mpi enzyme activity was measured at 4 dpf and was found to decrease in a dose-dependent manner. Mpi activity expressed as the percent of control activity is shown. p-Value based on two-tailed paired Student's t-test. (B) Heat map shows expression of genes involved in the N-glycosylation pathway in 24 hpf zebrafish embryos injected with std or mpi MO. Log2 fold change (FC) values relative to WT + std MO are listed.
DOI: http://dx.doi.org/10.7554/eLife.22477.006
Figure 2—figure supplement 2. p53 and its targets are increased in Mpi-depleted zebrafish embryos.

Figure 2—figure supplement 2.

(A) Validation of RNA-seq data by qPCR of 24 hpf zebrafish mpi morphant embryos showing transcriptional activation of candidate genes involves in apoptosis. The Ct values were normalized relative to the expression of rpp0 gene. Relative fold change (FC) was calculated as delta Ct in mpi MO compared to the std MO control. (B) and (C) Western blots showing p53 protein expression in 24 hpf and 4 dpf larvae after std MO and mpi MO injection. Western blots are quantified using densitometry analysis (ImageJ) and normalized to actin. Lines connecting std MO and mpi MO points indicate samples within the same clutch. All p-values were calculated using paired two-tailed Student’s t-test.
DOI: http://dx.doi.org/10.7554/eLife.22477.007
Figure 2—figure supplement 3. mpi mss7 MT are embryonic lethal.

Figure 2—figure supplement 3.

(A) Schematic of mpi gene with annotated exons. Diagram of partial mpi genomic DNA sequence, showing the TALEN-binding sites (in red), targeting exon 2. WT genomic and predicted protein sequences annotated (second and third rows). mss7 mutant genomic and predicted protein sequences annotated (rows four-seven), suggesting indel substitution mutation change in blue. (B) Representative genotyping of the mpi MT embryos after BsgI digestion. Genotyping described in Materials and methods. (C) Clustal alignment of MPI orthologs showing partial conservation in the region mutated in the mpimss7 mutant zebrafish. (D) Kaplan-Meier survival curves showing 50% death by day 13 in embryos produced from an mss7/+ adult incross mating, as compared to 22.7% in WT embryos. *p<0.0001 by two-tailed Fisher’s exact test. (E) Distribution of genotypes in the offspring from incross of heterozygous mpimss7/+ adults at day 13. Genotypes within the same clutch are indicated by the same symbol. (F) Mpi activity in 4 dpf embryos from wt and mss7/+ adult incross matings. p-Value was calculated using two-tailed Student’s t-test.
DOI: http://dx.doi.org/10.7554/eLife.22477.008