EMBO/soda-nel huggingface dataset:
The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis. (2015)
Figure 2

Characterization of the haematopoietic phenotype of <italic>Dnmt2</italic><sup>−/−</sup> mice

qPCR analysis of Dnmt2 mRNA expression in haematopoietic tissues, normalized to GAPDH.

Western blot analysis of Dnmt2 protein expression in haematopoietic tissues.

Proliferation analysis of primary bone marrow suspension cells. Primary bone marrow suspension cells isolated from the femurs of 8-day-old mice were grown together with the attached stromal cells. Dnmt2−/− population doubling levels of three biological replicates, each plated three times, were calculated relative to wild-type culture.

Reduced replating abilities of primary Dnmt2−/− bone marrow cells in a myeloid colony formation assay. For primary (1°) colony formation, 1.25 × 104 total bone marrow cells were plated in semisolid medium allowing myeloid differentiation. For secondary (2°) colony formation, cells were harvested and 0.1% of primary colonies were replated into semisolid medium. Results are from two independent experiments analyzing 9 wild-type and 13 Dnmt2−/− mice in total.

Reduced secondary CFU-preB colony formation of Dnmt2−/− cells. Numbers of CFU-preB-cell colonies are indicated per 5 × 104 wild-type or Dnmt2−/− total bone marrow cells. 10% of primary colonies were replated into secondary CFU-preB assays.

Stable engraftment (upper panel) and multi-lineage reconstitution (lower panel) of wild-type and Dnmt2−/− bone marrow transplants. CD11b: macrophages; Ly6G: granulocytes; CD45R: B cells; CD3: T cells.

Data information: Data are presented as mean ± SD. Asterisks indicate statistically significant (P < 0.05, t-test) differences.

Source data are available online for this figure.
The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis. (2015)
Figure 3

Characterization of <italic>Dnmt2</italic><sup>−/−</sup> MSCs

Flow cytometry analysis of bone marrow primary mesenchymal cells.

Quantification of fibroblast colony-forming units (CFU-F).

Quantification of alkaline phosphatase colony-forming units (CFU-ALP).

Dnmt2 expression after 3 days of mesenchymal cell differentiation.

Alizarin Red staining, indicating enhanced osteoblast differentiation of primary stromal cells from Dnmt2−/− mice. Scale bar: 50 μm. Quantification was performed with three biological replicates.

qPCR analysis of the osteoblast markers Osterix and Osteocalcin.

Oil Red O/hematoxylin staining, showing reduced adipocyte differentiation of primary stromal cells from Dnmt2−/− mice. Scale bar: 50 μm. Quantification was performed with three biological replicates.

qPCR analysis of the adipocyte markers CEBp alpha and Glut4.

Data information: qPCR expression values were normalized to GAPDH. Data are presented as mean ± SD. Asterisks indicate statistically significant (P < 0.05, t-test) differences.
The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis. (2015)
Figure 4

Dnmt2 methylates C38 of tRNA in the bone marrow and modulates the stability and fragmentation of substrate tRNAs

Bisulfite sequencing maps of 454 reads showing the average methylation status of individual cytosines (boxes) within tRNAs from bone marrow (BM), mesenchymal stromal cells (MSC), and in vitro differentiated adipocytes and osteoblasts. Methylation levels are indicated on a color scale from green (unmethylated) to red (completely methylated).

Representative Northern blot showing tRNA(Gly) and tRNA(Asp) fragmentation in bone marrow from 8-day-old and adult mice. tRNA(Ser) was included as a Dnmt2 non-substrate tRNA, and 5S rRNA was included as a loading control.

tRNA fragment coverage for the Dnmt2 substrates tRNA(Gly) and tRNA(Asp), and the non-substrate tRNA(Ser) in bone marrow, as determined by deep sequencing. Wild-type coverage is represented in red, and Dnmt2−/− coverage is indicated in blue. The plots illustrate the specific increase of 5′-halves from tRNA-Gly and 3′-halves from tRNA-Asp fragments in Dnmt2 mutants.

Source data are available online for this figure.
The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis. (2015)
Figure 5

Dnmt2 is required for specific protein synthesis in the bone marrow

Analysis of protein synthesis by dynamic SILAC. The top 10% of proteins that are translated slower in Dnmt2−/− versus wild-type cells are indicated in orange. The top 10% of proteins that are translated faster in Dnmt2−/− versus wild-type cells are shown in blue. A change of >  twofold is indicated by the black lines.

Dimethyl-labeling analysis of 8-day-old mouse bone marrow tissue. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. Nestin and periostin are marked with arrows.

Correlation between protein and mRNA expression for differentially expressed proteins. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. The Pearson correlation coefficient is −0.03.

Gene ontology analysis (top 5 categories) of mis-regulated proteins. The top 5 categories are presented.

Representative staining of nestin and periostin in femur sections from 8-day-old wild-type and Dnmt2−/− mice. The anti-periostin staining is comparable in the periosteum of wild-type and Dnmt2−/− mice, but is reduced in the bone marrow cells (compare the highlighted areas). Scale bar: 100 μm. Nuclei are counterstained with DAPI (blue).

Quantitative differences in the length of the nestin-positive trabecular zone (upper panel) between wild-type (30 measurements, three mice) and Dnmt2−/− (21 measurements, two mice) and in bone periostin-positive marrow areas (lower panel) between wild-type (26 measurements, three mice) and Dnmt2−/− (45 measurements, three mice) in femur sections from 8-day-old mice. Data are presented as mean ± SD.

Data information: Asterisks indicate statistically significant (P < 0.05, t-test) differences.
Small molecule inhibition of aging-associated chromosomal instability delays cellular senescence (2020)
Figure 1

Fig 1. Impaired k-MT error correction with advancing age.

(A, B) Representative images (A) and quantification (B) of calcium-stable k-fiber intensity levels by immunofluorescence analysis of n≥41 tubulin-stained mitotic cells of neonatal (N) and elderly (87y) human dermal fibroblasts (HDF) at prometaphase and metaphase stages. Intensity levels were normalized to neonatal samples. Scale bar, 5µm.

(C) Intra-kinetochore distance (between Hec1 and ACA immunostainings of a kinetochore) in n=50 kinetochore pairs scored from 10 elderly vs. neonatal cells in prometaphase and metaphase.

(D) Inter-kinetochore distance (between Hec1 staining of sister kinetochores) in n=50 kinetochore pairs scored from 10 elderly vs. neonatal cells in prometaphase and metaphase.

(E, F) Live cell imaging of neonatal (N) and elderly (87y) fibroblasts expressing H2B-GFP/α-Tubulin-mCherry treated with kinesin-5 inhibitor (STLC) to induce monopolar spindles, followed by washout (WO) into medium with DMSO (-) or ZM447439 (+; 500nM). (E) Quantification of anaphases with lagging chromosomes in n=cells scored and (F) representative movie frame series of a young and an elderly dividing cell that underwent correct (Normal) and incorrect (Lagging) chromosome segregation, respectively. Time, min:sec. Scale bars: 5µm (images) or 2µm (insets).

(G) Representative images of anaphases without (top) and with (bottom) mis-segregation (MS), FISH-stained for three chromosome pairs (7, 12 and 18). Arrowheads indicate micronuclei containing centromeric signal for chromosome 18. Scale bar, 10µm.

(H) Percentage of anaphases with MS in neonatal (N) vs. elderly (87y) n=cells scored by FISH analysis.

(I) Western blot analysis of total MCAK protein levels in neonatal (HDF N) and elderly (HDF 87y) fibroblasts. GAPDH is shown as loading control.

(J) Immunofluorescence analysis of MCAK levels in neonatal (HDF N) and elderly (HDF 87y) mitotic cells. Scale bar, 5µm.

Data information: All values shown are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by two tailed (B-D) Mann-Whitney test and (H) chi-square test.
Small molecule inhibition of aging-associated chromosomal instability delays cellular senescence (2020)
Figure 2

Fig 2. Overexpression of MT depolymerases, MCAK and Kif2b, restores chromosome segregation fidelity in elderly cells.

(A) GFP protein levels in FACS-sorted neonatal (N) and elderly (87y) fibroblasts transduced with empty (control, -), GFP-MCAK (+) and GFP-Kif2b (+) lentiviral plasmids. Tubulin is shown as loading control.

(B) Mitotic duration scored by time-lapse phase-contrast microscopy of n≥50 cells per condition measured from nuclear envelope breakdown (NEB) to anaphase onset.

(C, D) Representative images (C) and quantification (D) of calcium-stable k-fiber intensity levels by immunofluorescence analysis of n≥26 tubulin-stained metaphase cells transduced as indicated. Levels were normalized to the control neonatal sample. Scale bar, 5μm.

(E) Representative images of cytochalasin D-induced binucleated (BN) cells without (top) and with (bottom) mis-segregation (MS), FISH-stained for three chromosome pairs (7, 12 and 18). Scale bar, 10µm.

(F) Percentage of BN cells with MS in n=cells scored.

(G) Percentage of micronuclei in n=cells analyzed.

Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by two tailed (B, D) Mann-Whitney and (F,G) chi-square test.
Small molecule inhibition of aging-associated chromosomal instability delays cellular senescence (2020)
Figure 3

Fig 3. Overexpression of MCAK and Kif2b delays senescence in fibroblast cultures from elderly donors.

(A) Representative images of elderly cells scored as negative (-) or positive (+) for Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥1 foci; DNA damage) senescence biomarkers. Scale bar 20µm.

(B) Percentage of n=cells staining double-positive for Cdkn1a/p21 and 53BP1 in neonatal (N/N) and elderly (75/87y) human dermal fibroblasts (HDF) transduced with empty, GFP-MCAK or GFP-Kif2b lentiviral plasmids.

(C) Representative images of elderly cells scored as negative (-) or positive (+) for SA-β-galactosidase (SA-β-gal) activity. Scale bar 20µm.

(D) Percentage of n=cells positive for SA-β-gal in neonatal and elderly HDFs transduced with empty, GFP-MCAK or GFP-Kif2b lentiviral plasmids.

(E) Principle component analysis (PCA) of neonatal and elderly cells transduced with empty-vector (control) or kinesin-13 proteins (MCAK or Kif2b) based on bulk RNA expression data.

(F) Total number of genes altered (up or downregulated, as indicated) for the comparisons shown between control and kinesin-13-overexpressing young (HDF N) and/or aged (HDF 87y) cells.

(G, H) Heatmaps of differentially expressed (G) SASP and senescence genes, and (H) cGAS/STING/NF-κB-related genes of neonatal and 87y HDFs. Down and Up refer to the expected changes as reported for senescent cells (Datasets EV2 and EV4). Gene symbols highlighted in grey indicate genes that were not modulated by overexpression of Kinesin-13 proteins. Z-score row color intensities indicate higher (red) to lower (blue) expression.

Data Information: Values are mean ± s.d. of at least two independent experiments. ns p>0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square test (B,D).
Small molecule inhibition of aging-associated chromosomal instability delays cellular senescence (2020)
Figure 4

Fig 4. Small molecule agonist of MCAK activity rescues age-associated CIN and delays senescence.

(A) Mitotic duration of neonatal (N) and elderly (87y) human dermal fibroblasts (HDF) treated for 24 hrs with different concentrations of UMK57 (MCAK agonist). n≥58 cells were analyzed per condition. For all subsequent experiments UMK57 was used at 1µM for 24 hrs.

(B, C) Representative images (B) and quantification (C) of calcium-stable k-fiber intensity levels in metaphase, scored by immunofluorescence analysis of n≥34 tubulin-stained mitotic cells of neonatal and elderly samples treated with DMSO (-) and UMK57 (+). Levels were normalized to neonatal DMSO-treated condition. Scale bar, 5µm.

(D) Aneusomy index of chromosomes 7, 12 and 18 measured by interphase FISH analysis of n=cells.

(E) Percentage of cytochalasin D-induced binucleated (BN) n=cells with chromosomes 7, 12, and 18 mis-segregation (MS).

(F) Percentage of micronuclei in n=cells scored when treated with DMSO or UMK57.

(G) Percentage of n=cells staining positive for double immunostaining of Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥1 foci; DNA damage) senescence biomarkers.

(H) Percentage of n=cells staining positive for SA-β-galactosidase (SA-β-gal) activity.

Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed (A,C) Mann-Whitney and (D-H) chi-square tests.
Small molecule inhibition of aging-associated chromosomal instability delays cellular senescence (2020)
Figure 5

Fig 5. Long-term treatment with UMK57 does not lead to adaptive resistance in elderly cells.

(A) Mitotic duration of n=100 neonatal (N) and elderly (87y) human dermal fibroblasts (HDF) treated with DMSO (-) or UMK57 (+) for 96 hrs.

(B) Relative calcium-stable k-fiber intensity levels scored by immunofluorescence analysis of n≥29 tubulin-stained metaphase cells of neonatal and elderly samples treated with DMSO and UMK57 for 96 hrs. Levels were normalized to neonatal DMSO-treated condition.

(C) Aneusomy index of chromosomes 7, 12 and 18 measured by interphase FISH analysis of neonatal and elderly n=cells treated for 96 hrs.

(D) Cytochalasin D-induced binucleated (BN) n=cells with mis-segregation (MS) of chromosomes 7, 12 and 18 in neonatal and elderly samples treated for 96 hrs.

(E) Percentage of n=cells staining positive for double immunostaining of Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥1 foci; DNA damage) senescence biomarkers after 96hrs of treatment.

(F) Percentage of n=cells staining positive for SA-β-gal activity when treated for 96 hrs with DMSO or UMK57.

(G, H) Representative images (G) and quantification (H) of phospho-Aurora B Thr232 (pAuroraB T232) levels at kinetochores/centromeres in n≥75 neonatal and elderly metaphase cells treated with UMK57 for 24 hrs or 96 hrs. Intensity levels were normalized to ACA. Scale bar, 5µm.

Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed (A,B,H) Mann-Whitney and (C-F) chi-square tests.

EXPANDED VIEW FIGURE LEGENDS
Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane (2014)
Figure 1

Noc associates with the membrane in a ΔΨ‐sensitive manner

A–H Effect of CCCP on the localisation of Noc and NocNΔ10. Cellular localisation of Noc‐mYFP (DWA206) and NocNΔ10‐mYFP (DWA382) either with no additions (NA) or after CCCP treatment (5 min; 100 μM), as indicated. Scale bar, 2.5 μm.
I–L Effect of Noc overproduction on cell division. Exponentially growing cultures of DWA119 (Δnoc, Pspac(hy)‐noc) and DWA282 (Δnoc, Pspac(hy)‐nocNΔ10) were examined after growth for 1 h with 1 mM IPTG. Cell membranes and DNA were stained with FM5‐95 and DAPI, respectively. Insets show corresponding phase contrast images. Scale bar, 5 μm.
M Complementation of noc in a Δnoc ΔminCD background. Strains DWA564 (Δnoc, ΔminCD, Pxyl‐noc‐myfp) and 566 (Δnoc, ΔminCD, Pxyl‐nocNΔ10‐myfp) were streaked on nutrient agar (NA) plates in the presence of 0.5% w/v xylose and incubated at 30 and 39°C, as indicated.
N Effect of Noc and NocNΔ10 overproduction on sporulation. Strains DWA119 (Δnoc, Pspac(hy)‐noc) and DWA282 (Δnoc, Pspac(hy)‐nocNΔ10) were streaked on NA plates in the absence and presence of 1 mM IPTG, as indicated, and photographed after 48 h at 37°C.
O Western blot analysis of cellular fractions (T, total; C, cytosolic; M, membrane) of strains DWA119 and 282 expressing Noc or NocNΔ10, respectively. Proteins were detected using polyclonal antibodies against Noc, DnaA and PBP2B. Antibodies were used at a dilution of 1:10,000.
Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane (2014)
Figure 2

The N‐terminus of Noc contains an amphipathic helix

A AMPHIPASEEK prediction result for Bacillus subtilis Noc. The red “A”s indicate a putative amphipathic helical region.
B Helical wheel projection of the N‐terminus (aa 1–14) showing the presence of hydrophobic (arrow) and polar faces. Residues are coloured according to their properties, greens, hydrophobic; blues, charged; orange, polar, uncharged; and yellow, glycine. The figure was prepared using the tool available at http://rzlab.ucr.edu/scripts/wheel/wheel.cgi.
C–L Effects of N‐terminal substitutions on Noc localisation, in strains: DWA211 (F5E), 318 (F9E), 316 (K2E), 212 (R7E), 322 (F5A), 323 (F8A), 325 (F9A), 206 (WT), 328 (S4A) and 329 (S4L), as indicated. Insets show the corresponding phase contrast images. Scale bar, 5 μm.
Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane (2014)
Figure 3

A heterologous amphipathic helix can functionally substitute for the N‐terminus

A Helical wheel projection showing the amphipathic helix from hepatitis C virus protein NS4B. The hydrophobic face is indicated by an arrow. Colour scheme as in Fig B.
B Growth of strains DWA350 (Δnoc ΔminCD) and DWA307 (Δnoc ΔminCD, Pxyl‐HCVAH‐NocNΔ10) on nutrient agar plates at 30 and 48°C in the absence and presence of 0.5% w/v xylose, as indicated.
C–F Cell morphology of strains DWA350 and DWA307 following growth in LB at 30°C (C and D) and at 42°C in LB + 0.5% w/v xylose (E and F). Arrowheads indicate minicells. Cell membranes were stained with FM5–95. Insets show the corresponding phase contrast images. Scale bar, 5 μm.
G, H Cellular localisation of HCVAH‐NocNΔ10‐YFP (G) in strain DWA193 (Δnoc, Pxyl‐HCVAH‐nocNΔ10‐yfp) and overlay showing DAPI‐stained DNA (H). The strain was grown at 30°C in CH medium. Inset shows the corresponding phase contrast image. Scale bar, 5 μm.
Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane (2014)
Figure 4

Noc–membrane association requires spreading on DNA

A–F Localisation of ParB‐box mutants. Cellular localisation of (A) Noc‐mYFP (DWA206), (B) NocQ68R‐mYFP (DWA285), (C) NocG86S‐mYFP (DWA286), (D) NocR88A‐mYFP (DWA545), (E) NocR89A‐mYFP (DWA546) and (F) NocR91A‐mYFP (DWA547). Insets show the corresponding phase contrast images. Scale bar, 5 μm.
G Ability of ParB‐box mutants to rescue the growth defect of Δnoc ΔminCD. Strains DWA564 (Pxyl‐noc‐myfp), 590 (Pxyl‐nocQ68R‐myfp), 568 (Pxyl‐nocG86S‐myfp), 598 (Pxyl‐nocR88A‐myfp), 600 (Pxyl‐nocR89A‐myfp) and 602 (Pxyl‐nocR91A‐myfp) were streaked on plates containing 0.5% w/v xylose and incubated for 18 h at either 30 or 39°C, as indicated, before being photographed.
H–O ParB‐box mutants are dominant‐negative. Cells of strains DWA362 (ΔminCD, Pspac(hy)‐noc), 363 (ΔminCD, Pspac(hy)‐nocNΔ10), 364 (ΔminCD, Pspac(hy)‐nocQ68R) and 365 (ΔminCD, Pspac(hy)‐nocG86S) were examined after growth for 2 h at 42°C with either no additions (NA) (H–K) or in the presence of 1 mM IPTG (L–O), as indicated. Cell membranes were stained with FM5‐95. Insets show the corresponding phase contrast images. Scale bar, 5 μm.
Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane (2014)
Figure 5

Noc recruits NBS DNA to the cell membrane

A–C Representative images of TetR‐mCherry in cells lacking (A) or containing (B and C) the NBS plasmid pDWA117. Strains DWA427 (A) and 429 (B and C) (both strains contain Pspac‐noc) were examined after growth for 2 h in the absence (A and B) and presence (C) of 1 mM IPTG. DNA was stained with DAPI.
D–G Representative images showing the localisation of (D) Noc‐mYFP (DWA519), (E) NocNΔ10‐mYFP (DWA522), (F) NocQ68R‐mYFP (DWA520) and (G) NocG86S‐mYFP (DWA521), in the presence of the NBS plasmid pSG4929.

Data information: Insets show the corresponding phase contrast images. Scale bars, 5 ?m.
Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane (2014)
Figure 6

Noc overproduction recruits chromosomal DNA to the membrane in Escherichia coli

A, B Effects of Noc overproduction on cell division and nucleoid morphology in E. coli. Cells of strain DWA261 carrying pDWA37 (PA1/04/03‐noc) were examined after growth in LB with either no additions (A) or after induction for 1 h with 1 mM IPTG (B).
C–E Effects of overproduction of Noc variants on nucleoid morphology. Cells of strains DWA266 (PA1/04/03‐nocNΔ10) (C), 270 (PA1/04/03‐HCVAH‐nocNΔ10) (D) and 267 (PA1/04/03‐nocCΔ50) (E) carrying plasmids for the overproduction of the indicated mutants (see cartoons underneath panels) were grown in LB and examined after growth for 1 h in the presence of 1 mM IPTG. Cell membranes and DNA were stained with FM5‐95 and DAPI, respectively. Scale bar, 5 μm.
Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane (2014)
Figure 7

A Noc‐Spo0J hybrid can recruit DNA to the membrane in E. coli

A, B Effects of Spo0J and Noc30‐Spo0J overproduction on cell division and nucleoid morphology in E. coli. Cells of strains DWA271 (PA1/04/03‐spo0J) (A) and 272 (PA1/04/03‐noc‐spo0J) (B) were grown in LB in the presence of 1 mM IPTG, to induce the expression of either Spo0J (A) or the Noc‐Spo0J hybrid (B), and were examined 1 h post‐induction. Cell membranes and DNA were stained with FM5–95 and DAPI, respectively. Scale bar, 5 μm.
MYH3-associated distal arthrogryposis zebrafish model is normalized with para-aminoblebbistatin (2020)
Figure 1

Figure 1. Generation of smyhc1 mutant lines.

(A) Schematic to scale of human MYH3 gene on chromosome 17. Noncoding regions are displayed in pink. Coiled coil domain (840-1933 bp) displayed in purple. Motor domain (86-779 bp) displayed in yellow. Actin binding site (656-678 / 758-772 bp) shown in cyan. Location of R672H mutation is enlarged and labelled.

(B) Schematic to scale of zebrafish smyhc1 gene on chromosome 24. Noncoding regions are displayed in pink. Coiled coil domain (842-1929 bp) displayed in purple. Motor domain (85-778 bp) displayed in yellow. Actin binding site (655-677 bp) displayed in cyan. Location of R672H mutation is enlarged and labeled.

(C) Aligned DNA and amino acid sequences of MYH3 and smyhc1 alleles surrounding the smyhc1R673H and MYH3 R672H substitutions. The smyhc1- allele has a 7 base pair deletion that results in a frameshift in which one errant amino acid precedes a premature stop codon. The smyhc1R673H allele results from a G>A transition single point mutation.

(D) Western blot of whole zebrafish larvae, Smyhc1 stained with the F59 antibody. smyhc1+/+ larvae express Smyhc1 at 24hpf, but not at 48hpf. smyhc1-/- larvae do not express Smyhc1.

(E) Smyhc1 immunohistochemistry stain of 24 hpf (hour post fertilization) zebrafish larvae. Filamentous actin is stained with phalloidin (red). Nuclei are stained with DAPI (blue). Smyhc1 is stained with the F59 antibody (green) (Elworthy et al, 2008). smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae display Smyhc1 in the muscle fibers at 24hpf. smyhc1-/- larvae at 24 do not stain positively for Smyhc1. Scale bar represents 50 µm.
MYH3-associated distal arthrogryposis zebrafish model is normalized with para-aminoblebbistatin (2020)
Figure 2

Figure 2. Embryonic phenotypes of smyhc1 mutants.

(A) Morphologies of smyhc1+/+, smyhc1-/+ and smyhc1-/- embryos are grossly normal at 2 days post fertilization (dpf), despite complete paralysis of smyhc1-/- embryos. In contrast, the smyhc1R673H/+ embryos have notochord kinks in larval stages (arrow head), while smyhc1R673H/R673H embryos have much more severe notochord kinks that compress and distort the body axis (arrow heads). The smyhc1R673H/- embryos completely phenocopy smyhc1R673H/R673H embryos.

(B) Quantification of smyhc1R673H embryonic phenotypes at 2 dpf. Representative examples of fish in each of the four phenotypic groups (normal, mild, moderate, and severe) are shown in Figure EV3.