Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: ( A ) A horizontal cladogram of eif2b1-5 gene sequences shows that zebrafish have a single ortholog of each human EIF2B gene, and a relative conservation of amino acid sequence between orthologs. Scale bar equals an evolutionary distance of 0.1 amino acid changes per position in the sequence (Phylodendron). ( B ) Comparison of zebrafish and human EIF2B protein sequences shows conserved domain architectures. ( C–L ) Whole-mount in situ expression analysis in zebrafish embryos and larvae, brightfield microscopy, rostral left, dorsal top. ( C–E ) eif2b subunit genes at 24 hpf shows predominantly brain and eye expression, with lower levels of expression throughout the embryo. ( H–L ) eif2b subunit genes at 72 hpf shows higher expression throughout the animal and in the brain compared to 24 hpf. ( M–Q ) qRT-PCR expression of eif2b subunit genes from 24 hpf through 7 dpf, normalized to 24 hpf expression demonstrates variable expression changes across development. Error bars, standard error of the mean. Figure 1—source data 1. Quantification of qRT-PCR results of eif2b subunits. Figure 1—source data 2. Statistical analysis of changes in subunit expression.

Article Snippet: Genetic reagent ( D. rerio ) , eif2b1 sa12357 , Zebrafish International Resource Center (ZIRC) , ZIRC Catalog ID: ZL8971.02; RRID: ZIRC_ZL8971 .02 , .

Techniques: Sequencing, Comparison, In Situ, Expressing, Microscopy, Quantitative RT-PCR

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: ( A-D ) Depiction of zebrafish eif2b subunits exon structure and the location and nucleotide change for each mutant. ( A ) eif2b1 harbors a T/A transversion resulting in an early stop in exon 8. ( B ) eif2b2 has a G/A transition in exon 5, mutating an essential splice site. ( C ) eif2b4 has a G/A transition in exon 12 mutating an essential splice site. ( D ) eif2b5 exon one was targeted for mutagenesis using a gRNA (red). Six distinct alleles were recovered (described in text). ( E ) Chromatograms of cDNA confirm presence of predicted mutations for eif2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 . ( F ) eif2b5 zc103/zc103 mutants survive until adulthood in Mendelian ratios, but show grow defects compared to their heterozygous and wild-type siblings. ( G ) Adult eif2b5 zc103/zc103 lengths are significantly shorter compared to their wild-type and heterozygous siblings. ( H ) Bright-field (BF) images of 6 dpf eif2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 larva. eif2b2 sa17223/sa17223 and eif2b5 zc102/zc102 have no swim bladder (arrowhead) and a small head. ( I ) Kaplan-Meyer survival curves from an eif2b2 sa17223/+ heterozygous in-cross shows 1% (n = 3) homozygote survival at 10 dpf (total n = 302); however no homozygotes live past 2 weeks of age. ( J ) Kaplan-Meyer survival curves from an eif2b5 zc102/+ heterozygous in-cross shows that all homozygotes were dead by 10 dpf (total n = 62). ( K ) Kaplan-Meyer survival curves from an eif2b5 zc103/+ heterozygous in-cross show no mortality of homozygotes. ( L ) Motor swimming analysis shows impaired swimming behavior in mutants. Distance moved, time spent moving, and velocity, for wild-type controls, and eif2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 mutants, at 5, 6, and 7 dpf. Mean shown with 95% confidence intervals. Figure 2—source data 1. Quantification of lengths. Figure 2—source data 2. Quantification of behavior results. Figure 2—source data 3. Quantification of behavior results of eif2b4 sa17367/sa17367 allele. Figure 2—source data 4. qRT-PCR for ISR transcripts for eif2b4 sa17367/sa17367 allele. Figure 2—source data 5. Survival quantification for eif2b4 sa17367/sa17367 allele. Figure 2—source data 6. Survival quantification for eif2b5 zc103/zc103 allele.

Article Snippet: Genetic reagent ( D. rerio ) , eif2b1 sa12357 , Zebrafish International Resource Center (ZIRC) , ZIRC Catalog ID: ZL8971.02; RRID: ZIRC_ZL8971 .02 , .

Techniques: Mutagenesis, Quantitative RT-PCR

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet:

Article Snippet: Genetic reagent ( D. rerio ) , eif2b1 sa12357 , Zebrafish International Resource Center (ZIRC) , ZIRC Catalog ID: ZL8971.02; RRID: ZIRC_ZL8971 .02 , .

Techniques: Generated, Recombinant, Transfection, Construct, Labeling, Plasmid Preparation, SYBR Green Assay, Software, Staining

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: ( A-D ) Depiction of zebrafish eif2b subunits exon structure and the location and nucleotide change for each mutant. ( A ) eif2b1 harbors a T/A transversion resulting in an early stop in exon 8. ( B ) eif2b2 has a G/A transition in exon 5, mutating an essential splice site. ( C ) eif2b4 has a G/A transition in exon 12 mutating an essential splice site. ( D ) eif2b5 exon one was targeted for mutagenesis using a gRNA (red). Six distinct alleles were recovered (described in text). ( E ) Chromatograms of cDNA confirm presence of predicted mutations for eif2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 . ( F ) eif2b5 zc103/zc103 mutants survive until adulthood in Mendelian ratios, but show grow defects compared to their heterozygous and wild-type siblings. ( G ) Adult eif2b5 zc103/zc103 lengths are significantly shorter compared to their wild-type and heterozygous siblings. ( H ) Bright-field (BF) images of 6 dpf eif2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 larva. eif2b2 sa17223/sa17223 and eif2b5 zc102/zc102 have no swim bladder (arrowhead) and a small head. ( I ) Kaplan-Meyer survival curves from an eif2b2 sa17223/+ heterozygous in-cross shows 1% (n = 3) homozygote survival at 10 dpf (total n = 302); however no homozygotes live past 2 weeks of age. ( J ) Kaplan-Meyer survival curves from an eif2b5 zc102/+ heterozygous in-cross shows that all homozygotes were dead by 10 dpf (total n = 62). ( K ) Kaplan-Meyer survival curves from an eif2b5 zc103/+ heterozygous in-cross show no mortality of homozygotes. ( L ) Motor swimming analysis shows impaired swimming behavior in mutants. Distance moved, time spent moving, and velocity, for wild-type controls, and eif2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 mutants, at 5, 6, and 7 dpf. Mean shown with 95% confidence intervals. Figure 2—source data 1. Quantification of lengths. Figure 2—source data 2. Quantification of behavior results. Figure 2—source data 3. Quantification of behavior results of eif2b4 sa17367/sa17367 allele. Figure 2—source data 4. qRT-PCR for ISR transcripts for eif2b4 sa17367/sa17367 allele. Figure 2—source data 5. Survival quantification for eif2b4 sa17367/sa17367 allele. Figure 2—source data 6. Survival quantification for eif2b5 zc103/zc103 allele.

Article Snippet: Genetic reagent ( D. rerio ) , eif2b2 sa17223 , Zebrafish International Resource Center (ZIRC) , ZIRC Catalog ID: ZL10609.15; RRID: ZIRC_ZL10609 .15 , .

Techniques: Mutagenesis, Quantitative RT-PCR

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: Confocal images, z-stack maximal projections. ( A–I ), dorsal views of the brain, rostral to the top, scale bar 50 μm; ( K–N ), lateral views of spinal cord, dorsal to the top, scale bar 50 um. ( P–S ), dorsal views of brain, rostral to top, scale bar 50 μm. *p<0.05. ( A–D ) TUNEL and DAPI staining shows increased apoptosis in homozygous mutant alleles compared to controls (wild-type and heterozygous siblings) in ei f2b5 zc103/zc103 eif2b5 zc102/zc102 and eif2b2 sa17223/sa17223 mutants. ( E ) Quantification of mean TUNEL+ cell counts in ei f2b5 zc103/zc103 eif2b5 zc102/zc102 and eif2b2 sa17223/sa17223 mutants compared to sibling controls. ( F–I ) Phospho-histone 3 and DAPI staining shows decreased cell proliferation in 5 dpf eif2b2 sa17223/sa17223 mutants compared to controls, while ei f2b5 zc103/zc103 eif2b5 zc102/zc102 mutants show a change in proliferation pattern, specifically in the optic tectum. ( J ) Quantification of mean number pH3+ cells counts in ei f2b5 zc103/zc103 eif2b5 zc102/zc102 and eif2b2 sa17223/sa17223 mutants compared to sibling controls. ( K–N ) Olig2dsRed and DAPI staining shows no change in OPC counts in the spinal cords of 5 dpf ei f2b5 zc103/zc103 eif2b5 zc102/zc102 and eif2b2 sa17223/sa17223 mutants compared to controls. ( O ) Quantification of mean number Olig2dsRed+ counts in ei f2b5 zc103/zc103 eif2b5 zc102/zc102 or eif2b2 sa17223/sa17223 mutants compared to sibling controls. ( P–S ) Co-labeled Olig2dsRed+/TUNEL+ cell counts staining shows increase in Olig2dsRed+ cells undergoing apoptosis in brains of 5 dpf ei f2b5 zc103/zc103 , eif2b5 zc102/zc102 , or eif2b2 sa17223/sa17223 mutants compared to sibling controls. ( O ) Quantification of mean number of co-labeled Olig2dsRed+/TUNEL+ cell counts in ei f2b5 zc103/zc103 , eif2b5 zc102/zc102 , or eif2b2 sa17223/sa17223 mutants compared to sibling controls. Figure 3—source data 1. Quantification of TUNEL, pH3, olig2, and olig2/TUNEL results. Figure 3—source data 2. Quantification of TUNEL, pH3, olig2, and olig2/TUNEL results. Figure 3—source data 3. Quantification of TUNEL, pH3, olig2, and olig2/TUNEL results. Figure 3—source data 4. Quantification of TUNEL, pH3, olig2, and olig2/TUNEL results.

Article Snippet: Genetic reagent ( D. rerio ) , eif2b2 sa17223 , Zebrafish International Resource Center (ZIRC) , ZIRC Catalog ID: ZL10609.15; RRID: ZIRC_ZL10609 .15 , .

Techniques: TUNEL Assay, Staining, Mutagenesis, Labeling

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: Confocal images of brain, z-stack, rostral to the top, double-labeling for TUNEL and olig1 (in situ probe), in WT, eif2b5 zc103/zc103 , eif2b5 zc102/zc102 or eif2b2 sa17223/sa172233 larvae. Region used for quantification shown by box. Inset in each panel shows example of double-labeled cell (TUNEL, olig1 ) for each genotype (except WT), single confocal slice image. Middle panels: no change in apoptosis of differentiated oligodendrocytes, co-labeled with myelin associated glycoprotein ( mag ) (in situ probe) and TUNEL. Confocal z-stack images of spinal cord, rostral to the left; quantified in lower right panel. Figure 4—source data 1. Quantification of olig1 , TUNEL and mag results.

Article Snippet: Genetic reagent ( D. rerio ) , eif2b2 sa17223 , Zebrafish International Resource Center (ZIRC) , ZIRC Catalog ID: ZL10609.15; RRID: ZIRC_ZL10609 .15 , .

Techniques: Labeling, TUNEL Assay, In Situ

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: ( A ) Confocal images of brain, z-stack, rostral to the top, in WT, eif2b5 zc103/zc103 , eif2b5 zc102/zc102 or eif2b2 sa17223/sa172233 larvae. Confocal images of brain, z-stack, rostral to the top. Top row, labeling for Zrf1, DAPI, and pH3. Middle row, labeling for HuC/D, DAPI, and pH3. Bottom row, labeling for apoeb , DAPI, and pH3. ( B ) Quantification of cell counts in ( A ). ( C ) Confocal images of brain, z-stack, rostral to the top, in WT, eif2b5 zc103/zc103 , eif2b5 zc102/zc102 or eif2b2 sa17223/sa172233 larvae. Confocal images of brain, z-stack, rostral to the top. Top row, labeling for Zrf1, DAPI, and TUNEL. Middle row, labeling for HuC/D, DAPI, and TUNEL. Bottom row, labeling for apoeb , DAPI, and TUNEL. ( D ) Quantification of cell counts in ( C ). Figure 5—source data 1. Quantification of Zrf1, HuC/D, and apoeb , double-labeling with pH3, results in tectum, midline, and eyes in eif2b5 zc103/zc103 , eif2b2 zc102/zc102 , and eif2b2 sa17223/sa172233 . Figure 5—source data 2. Quantification of Zrf1, HuC/D, and apoeb , double-labeling with TUNEL, results in tectum, midline, and eyes in eif2b5 zc103/zc103 , eif2b2 zc102/zc102 , and eif2b2 sa17223/sa172233 .

Article Snippet: Genetic reagent ( D. rerio ) , eif2b2 sa17223 , Zebrafish International Resource Center (ZIRC) , ZIRC Catalog ID: ZL10609.15; RRID: ZIRC_ZL10609 .15 , .

Techniques: Labeling, TUNEL Assay

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: ( A ) 11.1 kb zebrafish eif2b2 gene structure. ( B ) 9.5 kb human EIF2B2 gene structure. ( C ) Conservation of amino acid sequence between zebrafish eif2b2 and human EIF2B2. ( D ) Schematic of rescue construct containing Tol2, β-actin, human EIF2B2, and eGFP. ( E ) Genotype results (at adult age), of an eif2b2 sa17223/+ heterozygous incross, and an eif2b2 sa17223/+ heterozygous zebrafish crossed with two different transgenic alleles (#3 and #4): eif2b2 sa17223/+ ;Tg 3 ( β-actin:EIF2B2:2A:eGFP ) or eif2b2 sa17223/+ ;Tg 4 ( β-actin:EIF2B2:2A:eGFP ) heterozygous zebrafish. ( F ) Bright-field and immunofluorescent images of eif2b2 sa17223/sa17223 ;β-actin:EIF2B2:2A:eGFP mutant fish: showing a swim bladder and regular-sized head; or showing GFP expression in eif2b2 sa17223/sa17223 ;β-actin:EIF2B2:2A:eGFP mutant fish. ( G–J ) TUNEL and DAPI antibody staining of wild-type control; e if2b2 sa17223/sa17223 mutant; eif2b2 +/+ ;β-actin:EIF2B2:2A:eGFP wild-type control; and eif2b2 sa17223/sa17223 ;β-actin:EIF2B2:2A:eGFP mutant. ( K ) Quantification of TUNEL+ cells. Figure 7—source data 1. Quantification of TUNEL results. Figure 7—source data 2. Quantification of behavior results.

Article Snippet: Genetic reagent ( D. rerio ) , eif2b2 sa17223 , Zebrafish International Resource Center (ZIRC) , ZIRC Catalog ID: ZL10609.15; RRID: ZIRC_ZL10609 .15 , .

Techniques: Sequencing, Construct, Transgenic Assay, Mutagenesis, Expressing, TUNEL Assay, Staining, Control

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet: ( A ) Schematic showing integrated stress response activated intron 12 retention of eif2b5 resulting in premature stop codon and truncated form of EIF2B5. ( B ) Fold change of eif2b5 intron 12 expression with qRT-PCR in ei f2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 mutants relative to controls. ( C ) qRT-PCR for intron 12 expression in a VWM patient and their control unaffected father. ( D ) Control-injected larvae, compared to those injected with truncated eif2b5 construct, shows impaired swimming behavior; distance moved, time spent moving, and velocity, at 5 dpf. ( E ) qRT-PCR for atf4 , bip , chopII , and perk ISR transcripts shows increased expression in ei f2b5 zc103/zc103 , eif2b5 zc102/zc102 , and eif2b2 sa17223/sa17223 mutants. ( F ) qRT-PCR for ISR transcripts ( atf 4 , bip , chop II , and perk ) shows increased expression following injection of truncated eif2b5 construct. Figure 8—source data 1. qRT-PCR for intron 12 expression changes ( eif2b5 , zebrafish). Figure 8—source data 2. qRT-PCR for intron 12 expression changes ( eif2b2 , zebrafish). Figure 8—source data 3. qRT-PCR for intron 12 expression changes (human). Figure 8—source data 4. Behavior data for truncated eif2b5 effects. Figure 8—source data 5. qRT-PCR for ISR transcript expression. Figure 8—source data 6. qRT-PCR for ISR transcript expression changes following injection with truncated eif2b5 .

Article Snippet: Genetic reagent ( D. rerio ) , eif2b2 sa17223 , Zebrafish International Resource Center (ZIRC) , ZIRC Catalog ID: ZL10609.15; RRID: ZIRC_ZL10609 .15 , .

Techniques: Expressing, Quantitative RT-PCR, Control, Injection, Construct

Journal: eLife

Article Title: Vanishing white matter disease expression of truncated EIF2B5 activates induced stress response

doi: 10.7554/eLife.56319

Figure Lengend Snippet:

Article Snippet: Genetic reagent ( D. rerio ) , eif2b2 sa17223 , Zebrafish International Resource Center (ZIRC) , ZIRC Catalog ID: ZL10609.15; RRID: ZIRC_ZL10609 .15 , .

Techniques: Generated, Recombinant, Transfection, Construct, Labeling, Plasmid Preparation, SYBR Green Assay, Software, Staining

Journal: eLife

Article Title: High hedgehog signaling is transduced by a multikinase-dependent switch controlling the apico-basal distribution of the GPCR smoothened

doi: 10.7554/eLife.79843

Figure Lengend Snippet:

Article Snippet: Genetic reagent ( D. melanogaster ) , UAS SNAP-smo PKA-SD FU-SA , This paper , , Generated by BestGene Inc using the PhiC31 integration system.

Techniques: Generated, Knock-Out, Mutagenesis, CRISPR, Recombinant, Amplification, Sequencing, Blocking Assay, Software