About ZFIN
The Zebrafish Information Network (ZFIN) is the database of genetic and genomic data for the zebrafish (Danio rerio) as a model organism. ZFIN provides a wide array of expertly curated, organized and cross-referenced zebrafish research data.
Learn MoreNew Data in ZFIN
Figure 3 of Lechermeier et al., 2019
Whole mount RNA in situ hybridization of slc2a3a at early embryonic stages [18–36 hours post fertilization (hpf)]. Pictures in the left column depict alternating lateral (A,C,E,G) and dorsal (B,D,F,H) views. Anterior is to the left. Higher magnifications (a1–h3) corresponding to boxes in (A–H). Arrowheads indicate examples of labeled cells or cell populations. Note staining in spinal cord, hindbrain, ventral midbrain, ventral thalamus, ventral telencephalon and epiphysis. Two bilateral rows, one medially (black arrowheads) and one laterally (white arrowheads) of positive cells are situated in the ventral hindbrain along the floor plate (f2 and h2). For abbreviations of anatomical terms see Table 1. Scale bars in (A), 100 μm and pertains to (A–H); scale bar in a1, 50 μm and pertains to a1–h3.
figure 1 of Gálvez-Santisteban et al., 2019
Endocardial Notch signaling controls myocardial reprogramming and cardiac regeneration. (A–D, L–O) Confocal microscopy imaging of heat-shocked/HS (A, B, L, M) vmhc:mCherry-NTR, (C, D) vmhc:mCherry-NTR; hsp70l:dnM and (N, O) vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM hearts reveals that (D) global or (O) endocardial-specific dnMAML (dnM) Notch inhibition inhibits CM proliferation in ventricle-ablated hearts at 48 hpt (7 dpf) when compared to (B, M) CM proliferation in control ventricle-ablated (no dnMAML) hearts. White – anti-phospho-histone H3 immunostaining; red – anti-MF-20 immunostaining. Arrows point to proliferating CMs. (E, P) Quantitation of anti-phospho-histone H3+ CMs in these hearts confirms that (E) global or (P) endocardial-specific dnM Notch inhibition prevents CMs from proliferating in injured hearts (n = 23, control dnM-; 31, MTZ dnM-; 10, control dnM+; 18, MTZ dnM+; 16, control RSdnM-; 16, MTZ RSdnM-; 15, Control RSdnM+; 15, MTZ RSdnM-). Red bars – ventricle; green bars – atrium; dark bars – control sham-ablated hearts; light bars – ventricle-ablated hearts. (F, Q) Quantitation of the percentage of heat-shocked vmhc:mCherry-NTR, vmhc:mCherry-NTR; hsp70l:dnM and vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM ventricle-ablated hearts that display recovered ventricular tissue and contractility (black bars) at 96 hpt (nine dpf) shows that (F) global or (Q) endocardial-specific Notch inhibition between 0 and 1 dpt leads to the greatest inhibitory effect on overall recovery from ventricle injury. The number of fish analyzed for each condition is indicated above each column. (G–K) To examine the effects of Notch signaling on cardiac reprogramming, (G, H) vmhc:mCherry-NTR; amhc:CreERT2; myl7:CSY and (I, J) vmhc:mCherry-NTR; amhc:CreERT2; myl7:CSY; hsp70l: dnMAML (dnM) hearts were exposed to tamoxifen at 5 dpf to genetically label atrial CMs with YFP (c-aYFP), then (G, I) sham-ablated (control) or (H, J) ventricle-ablated, and finally heat-shocked to (I, J) induce dnM expression. Confocal microscopy imaging at 72 hpt (8 dpf) reveals that (J) heat-shock induction of dnM inhibited the ability of genetically labeled c-aYFP+ atrial CMs to contribute to the regeneration of ventricle-ablated hearts when compared to (H) heat-shock control ventricle-ablated hearts. Yellow channel – (G’–J’) genetically labeled c-aYFP+ atrial CMs. (K) Quantitation of the percentage of ventricular area covered with c-aYFP+ CMs supports that dnMAML Notch inhibition prevents atrial CMs from regenerating the injured ventricle (n = 8 hsp70:dnM-, seven hsp70l:dnM+). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post MTZ/DMSO treatment. Dashed lines outline the heart. Bar: 50 μm. (E, P) Mean + s.e.m. ANOVA; (K) Mean + s.d. Student’s t-test; (F, Q) Total numbers, Binomial test (versus 0 dpt); ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 1.
Figure 4 of Lechermeier et al., 2019
Whole mount RNA in situ hybridization of slc2a3a at late embryonic and early post-hatching stages (48–120 hpf). Pictures in the left column depict alternating lateral (A,C,E) and dorsal (B,D,F) views. Anterior is to the left. Higher magnifications (a1–f3) corresponding to boxes in (A–F). Note staining in hindbrain, ventral midbrain, optic tectum, ventral thalamus, ventral telencephalon and retina. Detailed descriptions can be found in the text, for abbreviations see Table 1. Scale bar in (A), 100 μm and pertains to (A–F); scale bar in a1, 50 μm and pertains to a1–f2, d3, f3 and scale bar in a3, 50 μm and pertains to a3, b3.
Fig. 4 of Fischer et al., 2019
FHD-GFP interferes with severity of the MZsur mutant phenotype. a–dWild-type (a, b) and MZsur mutant (c, d) embryos at 24hpf. col2a1a in-situ staining in wild-type (un-injected control (a) or injected with FHD-GFP (b)) shows wild-type notochord of expected width (white brackets in enlarged sections a′ and b′). In uninjected MZsur embryos the width is reduced (c′). Injection of FHD-GFP in MZsur mutants enhances the phenotype (d/d′; note reduced size and additionally discontinuity of staining). e–l foxa2 in-situ hybridizations show reduction of axial mesoderm formation in MZsurmutants. Injection of FHD-GFP causes a broadened axial signal in 50% of wild type embryos, but no reduction of axial cells (f), and strengthens the effect in 60% of the MZsur mutants (h). dre-miR-430 morpholinos (MOs) massively reduce axial foxa2 signals in both genotypes (l, k). Co-injection of FHD-GFP and MOs also results in a decreased staining (j, l) when compared to FHD-GFP injection (f, h). Percentage of embryos showing the same phenotype as in the image is given (upper right). m–t sox17 in-situ hybridizations show only slight reduction of endoderm after injection of MOs (q–t). Number of forerunner cells (black arrow) is reduced in MZsurcontrol and after MOs injections (o, q, s). FHD-GFP lead to complete loss of forerunner cells (black arrow) in the majority of MZsur embryos (p, t; see also Additional file : Statistical analysis of forerunner cells). Numbers of sox17-positive cells seen in dorsal view and standard deviation are given (upper right) as well as number of analyzed embryos (n) (lower right). Size bars: 200 μm. γ value was changed to 0.8 in each picture
Fig. 4 of Delacruz et al., 2019
Human CNOT3 rescues intestinal defects in zebrafish apcmcr embryos. (A) Graph shows cnot3a mRNA expression relative to 18s in 72 hpf embryos. (B) apcmcr and wildtype embryos, WISH staining for gut marker fabp2 for vehicle and hCNOT3 injected apcmcr embryos. (C) WISH staining for hCNOT3 E20K and E70K-injected apcmcr embryos and vehicle-injected apcWT. Note that the red arrowhead = fabp2 staining. (D) Percentage of apcmcr fish with gut marker staining from (A and B) at 72 hpf (n > 10 per group). Statistical significance of CNOT3 mRNA injection rescue was determined using Fisher’s exact test compared to uninjected apcmcr embryos alone. ns = not significant; *** = p-value = 0.0003; **= p-value = 0.0043.
Fig. 5 of Nittoli et al., 2019
Expression of uncx during embryogenesis. Whole-mount in situ hybridization of uncx at (A) 12.5 hours post fertilization (hpf), (B–E) 18 hpf, (F) 22 hpf, (G, H) 24 hpf, (I) 34 hpf, and (J, K) 48 hpf. (A, C–E, K) Dorsal view, and (B, F–J) lateral view. Anterior to left. (A) Expression in prospective forebrain (FB) and hindbrain (HB) at 12.5 hpf. FB = forebrain, PSM = presomitic mesoderm, So = somites. (B–E) Expression of uncx at 18 hpf. HB = hindbrain, Hy = hypothalamus, No = notochord, OP = olfactory placode, SC = spinal cord, So = somites. (F) Expression at 22 hpf. Th = thalamus. (G, H) Expression at 24 hpf. Ce = cerebellum, Te = telencephalon. (I) Expression at 34 hpf. (J, K) Expression at 48 hpf.
Fig. 5 of Bagwell et al., 2020
Dstyk regulates fusion of pre-vacuolar carriers with the notochord vacuole. ( A–B) Maximum projection of a light sheet microscopy live image of WT and spzl-/- embryos at 48hpf and 65hpf, respectively. ( C–D) Still images from a 17 hr light sheet movie of a WT and spzl-/-embryos zebrafish embryo during vacuole biogenesis depicting vacuole fusion events. Vacuole membranes are labeled by NFATC:gal4;UAS:GFPrab32 expression. Arrows point to GFP-Rab32 positive vesicles. Arrowheads point to large vesicle (vacuolinos) docking events. n = 2 animals per genotype. Asterisks mark fusion events. Scale bar = 100 µm.
Fig. 4 of Fogerty et al., 2019
Survey of retinal anatomy in 5dpf mutant larvae. Transverse sections were collected near the optic nerve, which is visible in some images (asterisks), and stained with markers for different photoreceptor cell types. In all images, the ventral retina is to the left. (a) Peanut agglutinin lectin (PNA) labels the extracellular matrix surrounding cone outer segments. (b) zpr1 labels red/green cone inner segments. Inserts show higher magnification images, with double arrows indicating how inner segments were measured. (c) zpr3 labels rod outer segments, which are immature at this age and typically longer and more numerous in the ventral retina.
Fig. 1. of Soman et al., 2019
Generation, screening and validation of mcu−/− zebrafish. (A) CRISPR/Cas9-based gene editing was used to generate mcu−/− zebrafish. Schematics showing the targeted genomic sequence for the introduction of the indel mutation in the exon 3 containing the mcu coding gene. The identified 18 base pair deletion (shown in faded font) and 70 bp insertion (shown in highlighted font) is denoted in the cDNA sequence. (B) The indel mutation leads to a frameshift mutation with a premature stop codon. The predicted protein product for mcu mutant allele is shown in the lower panel. (C) RT-PCR data showing wt mcu allele in the first column; the second column shows the wt and mutant allele in heterozygous mutants and the third column shows the mcu mutant allele in mcu homozygous zebrafish. (D) q-PCR data show significant downregulation (***P<0.0001) of mcu gene expression in mcu−/− zebrafish when compared to wt, indicating mutation-induced RNA decay. Statistical analysis with t-test of three different experiments with n=3. (E) Representative images showing in situ hybridization using mcu riboprobe. mcu expression is abolished in mcu−/− 3 dpf zebrafish. (F) Impaired mitochondrial calcium ions influx/efflux in isolated mitochondria from 24 hpf larvae in mcu−/− zebrafish. Control mitochondria (green line) had an uptake in calcium added to solution, which created a drop in the fluorescence. After ∼5 min, we observed calcium leakage (rise in fluorescence). Blank control (black line) was mitochondria without added calcium. Ruthenium Red (pink line) prevented calcium uptake. CCCP (yellow line) caused leakage of calcium, observed as rising fluorescence. Uptake in mcu−/− (blue line) was completely blocked in given circumstances, being like the dynamics of Ruthenium Red treatment.
Figure 5 of Gálvez-Santisteban et al., 2019
The mechanosensitive channel Trpv4 regulates endocardial Notch activation and myocardial regeneration through Klf2a. ( A–D) Confocal imaging of vmhc:mCherry-NTR ; klf2a:H2B-GFP hearts shows that klf2a:H2B-GFP expression is activated in ( B) wild-type ( wt) ventricle-ablated hearts at 24 hpt (6 dpf) compared to ( A) control hearts; however, this activation is reduced in ( D) trpv4-/- ventricle-ablated hearts. ( F–H) Confocal imaging of vmhc:mCherry-NTR ; Tp1:d2GFP hearts further reveals that Tp1:d2GFP is decreased in ( G) klf2a-/- and ( H) trpv4-/- ventricle-ablated hearts at 24 hpt (6 dpf) when compared to ( F) wild-type ( wt) hearts. ( E, I) Quantitation of the relative average fluorescence intensity confirms reduced injury-induced ( E) klf2a:H2B-GFP activation in trpv4-/- ventricle-ablated hearts, and ( I) Tp1:d2GFP activation in trpv4-/- and klf2a-/- ventricle-ablated hearts when compared to wild-type ventricle-ablated hearts ( klf2a:H2B-GFP n = 9 control wt; 7 control trpv4-/-; 9 MTZ wt; 10 MTZ trpv4-/-. Tp1:d2GFP n = 15 control wt; 18 control trpv4-/-; 20 control klf2a-/-; 22 MTZ wt; 21 MTZ trpv4-/-; 18 MTZ klf2a-/). ( J–L) Whole-mount in situ hybridizations show that notch1b is decreased in ventricle-ablated ( K) klf2a-/- (n = 2/11) and ( L) trpv4-/- hearts (n = 6/15) at 24 hpt (6 dpf) when compared to ( J) wild-type hearts (n = 16/18). ( M–O) Confocal microscopy performed on vmhc:mCherry-NTR ventricle-ablated hearts reveals that ( N) klf2a-/- and ( O) trpv4-/-ventricle-ablated hearts display reduced CM proliferative response when compared to ( M) wild-type ( wt) ventricle-ablated hearts at 48 hpt (7 dpf). White – anti-phospho-histone H3 immunostaining; red – anti-MF-20 immunostaining. Arrows point to proliferating CMs. ( P) Quantitation of anti-phospho-histone H3+ CMs in these hearts confirms that klf2a-/- and trpv4-/- hearts fail to increase CM proliferation after ventricle-injury (n = 15 each condition). Red bars – ventricle; green bars – atrium; dark bars – control sham-ablated hearts; light bars – ventricle-ablated hearts. ( Q) Quantitation of the percentage of vmhc:mCherry-NTR ventricle-ablated hearts that display recovered ventricular tissue and contractility (black bars) at 96 hpt (9 dpf) supports that klf2a-/- and trpv4-/- mutants exhibit impaired heart regeneration. The number of fish analyzed for each condition is indicated above each column. ( R–T) Confocal microscopy imaging of vmhc:mCherry-NTR; amhc:CreERT2; β-actin2:RSGhearts at 72 hpt (8 dpf) shows that ( S) klf2a-/-and ( T) trpv4-/- ventricle-ablated hearts exhibit reduced contribution of genetically labeled atrial CMs (c-aGFP+) to the regenerating injured ventricle when compared to ( R) wild-type ventricle-ablated hearts. Green channel – ( R’–T’) genetically labeled c-aGFP+ atrial CMs. ( U) Quantitation of the percentage of ventricular area covered with c-aGFP+ CMs confirms that klf2a-/-or trpv4-/- hearts display reduced capacity to undergo injury-induced atrial-to-ventricular trans-differentiation during injury and regeneration (n = 13 wt; 8 klf2a-/-; 10 trpv4-/-). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-MTZ/DMSO treatment. Dashed lines outline the heart. Bars: 50 μm. ( E, I, P) Mean + s.e.m. ANOVA; ( Q) Total numbers, Binomial test (versus wild-type); ( U) Mean + s.d. ANOVA; ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 5.
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About ZFIN
The Zebrafish Information Network (ZFIN) is the database of genetic and genomic data for the zebrafish (Danio rerio) as a model organism. ZFIN provides a wide array of expertly curated, organized and cross-referenced zebrafish research data.
Learn MoreAdditional Resources
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Figure 3 of Lechermeier et al., 2019
Whole mount RNA in situ hybridization of slc2a3a at early embryonic stages [18–36 hours post fertilization (hpf)]. Pictures in the left column depict alternating lateral (A,C,E,G) and dorsal (B,D,F,H) views. Anterior is to the left. Higher magnifications (a1–h3) corresponding to boxes in (A–H). Arrowheads indicate examples of labeled cells or cell populations. Note staining in spinal cord, hindbrain, ventral midbrain, ventral thalamus, ventral telencephalon and epiphysis. Two bilateral rows, one medially (black arrowheads) and one laterally (white arrowheads) of positive cells are situated in the ventral hindbrain along the floor plate (f2 and h2). For abbreviations of anatomical terms see Table 1. Scale bars in (A), 100 μm and pertains to (A–H); scale bar in a1, 50 μm and pertains to a1–h3.
figure 1 of Gálvez-Santisteban et al., 2019
Endocardial Notch signaling controls myocardial reprogramming and cardiac regeneration. (A–D, L–O) Confocal microscopy imaging of heat-shocked/HS (A, B, L, M) vmhc:mCherry-NTR, (C, D) vmhc:mCherry-NTR; hsp70l:dnM and (N, O) vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM hearts reveals that (D) global or (O) endocardial-specific dnMAML (dnM) Notch inhibition inhibits CM proliferation in ventricle-ablated hearts at 48 hpt (7 dpf) when compared to (B, M) CM proliferation in control ventricle-ablated (no dnMAML) hearts. White – anti-phospho-histone H3 immunostaining; red – anti-MF-20 immunostaining. Arrows point to proliferating CMs. (E, P) Quantitation of anti-phospho-histone H3+ CMs in these hearts confirms that (E) global or (P) endocardial-specific dnM Notch inhibition prevents CMs from proliferating in injured hearts (n = 23, control dnM-; 31, MTZ dnM-; 10, control dnM+; 18, MTZ dnM+; 16, control RSdnM-; 16, MTZ RSdnM-; 15, Control RSdnM+; 15, MTZ RSdnM-). Red bars – ventricle; green bars – atrium; dark bars – control sham-ablated hearts; light bars – ventricle-ablated hearts. (F, Q) Quantitation of the percentage of heat-shocked vmhc:mCherry-NTR, vmhc:mCherry-NTR; hsp70l:dnM and vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM ventricle-ablated hearts that display recovered ventricular tissue and contractility (black bars) at 96 hpt (nine dpf) shows that (F) global or (Q) endocardial-specific Notch inhibition between 0 and 1 dpt leads to the greatest inhibitory effect on overall recovery from ventricle injury. The number of fish analyzed for each condition is indicated above each column. (G–K) To examine the effects of Notch signaling on cardiac reprogramming, (G, H) vmhc:mCherry-NTR; amhc:CreERT2; myl7:CSY and (I, J) vmhc:mCherry-NTR; amhc:CreERT2; myl7:CSY; hsp70l: dnMAML (dnM) hearts were exposed to tamoxifen at 5 dpf to genetically label atrial CMs with YFP (c-aYFP), then (G, I) sham-ablated (control) or (H, J) ventricle-ablated, and finally heat-shocked to (I, J) induce dnM expression. Confocal microscopy imaging at 72 hpt (8 dpf) reveals that (J) heat-shock induction of dnM inhibited the ability of genetically labeled c-aYFP+ atrial CMs to contribute to the regeneration of ventricle-ablated hearts when compared to (H) heat-shock control ventricle-ablated hearts. Yellow channel – (G’–J’) genetically labeled c-aYFP+ atrial CMs. (K) Quantitation of the percentage of ventricular area covered with c-aYFP+ CMs supports that dnMAML Notch inhibition prevents atrial CMs from regenerating the injured ventricle (n = 8 hsp70:dnM-, seven hsp70l:dnM+). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post MTZ/DMSO treatment. Dashed lines outline the heart. Bar: 50 μm. (E, P) Mean + s.e.m. ANOVA; (K) Mean + s.d. Student’s t-test; (F, Q) Total numbers, Binomial test (versus 0 dpt); ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 1.
Figure 4 of Lechermeier et al., 2019
Whole mount RNA in situ hybridization of slc2a3a at late embryonic and early post-hatching stages (48–120 hpf). Pictures in the left column depict alternating lateral (A,C,E) and dorsal (B,D,F) views. Anterior is to the left. Higher magnifications (a1–f3) corresponding to boxes in (A–F). Note staining in hindbrain, ventral midbrain, optic tectum, ventral thalamus, ventral telencephalon and retina. Detailed descriptions can be found in the text, for abbreviations see Table 1. Scale bar in (A), 100 μm and pertains to (A–F); scale bar in a1, 50 μm and pertains to a1–f2, d3, f3 and scale bar in a3, 50 μm and pertains to a3, b3.
Fig. 4 of Fischer et al., 2019
FHD-GFP interferes with severity of the MZsur mutant phenotype. a–dWild-type (a, b) and MZsur mutant (c, d) embryos at 24hpf. col2a1a in-situ staining in wild-type (un-injected control (a) or injected with FHD-GFP (b)) shows wild-type notochord of expected width (white brackets in enlarged sections a′ and b′). In uninjected MZsur embryos the width is reduced (c′). Injection of FHD-GFP in MZsur mutants enhances the phenotype (d/d′; note reduced size and additionally discontinuity of staining). e–l foxa2 in-situ hybridizations show reduction of axial mesoderm formation in MZsurmutants. Injection of FHD-GFP causes a broadened axial signal in 50% of wild type embryos, but no reduction of axial cells (f), and strengthens the effect in 60% of the MZsur mutants (h). dre-miR-430 morpholinos (MOs) massively reduce axial foxa2 signals in both genotypes (l, k). Co-injection of FHD-GFP and MOs also results in a decreased staining (j, l) when compared to FHD-GFP injection (f, h). Percentage of embryos showing the same phenotype as in the image is given (upper right). m–t sox17 in-situ hybridizations show only slight reduction of endoderm after injection of MOs (q–t). Number of forerunner cells (black arrow) is reduced in MZsurcontrol and after MOs injections (o, q, s). FHD-GFP lead to complete loss of forerunner cells (black arrow) in the majority of MZsur embryos (p, t; see also Additional file : Statistical analysis of forerunner cells). Numbers of sox17-positive cells seen in dorsal view and standard deviation are given (upper right) as well as number of analyzed embryos (n) (lower right). Size bars: 200 μm. γ value was changed to 0.8 in each picture
Fig. 4 of Delacruz et al., 2019
Human CNOT3 rescues intestinal defects in zebrafish apcmcr embryos. (A) Graph shows cnot3a mRNA expression relative to 18s in 72 hpf embryos. (B) apcmcr and wildtype embryos, WISH staining for gut marker fabp2 for vehicle and hCNOT3 injected apcmcr embryos. (C) WISH staining for hCNOT3 E20K and E70K-injected apcmcr embryos and vehicle-injected apcWT. Note that the red arrowhead = fabp2 staining. (D) Percentage of apcmcr fish with gut marker staining from (A and B) at 72 hpf (n > 10 per group). Statistical significance of CNOT3 mRNA injection rescue was determined using Fisher’s exact test compared to uninjected apcmcr embryos alone. ns = not significant; *** = p-value = 0.0003; **= p-value = 0.0043.
Fig. 5 of Nittoli et al., 2019
Expression of uncx during embryogenesis. Whole-mount in situ hybridization of uncx at (A) 12.5 hours post fertilization (hpf), (B–E) 18 hpf, (F) 22 hpf, (G, H) 24 hpf, (I) 34 hpf, and (J, K) 48 hpf. (A, C–E, K) Dorsal view, and (B, F–J) lateral view. Anterior to left. (A) Expression in prospective forebrain (FB) and hindbrain (HB) at 12.5 hpf. FB = forebrain, PSM = presomitic mesoderm, So = somites. (B–E) Expression of uncx at 18 hpf. HB = hindbrain, Hy = hypothalamus, No = notochord, OP = olfactory placode, SC = spinal cord, So = somites. (F) Expression at 22 hpf. Th = thalamus. (G, H) Expression at 24 hpf. Ce = cerebellum, Te = telencephalon. (I) Expression at 34 hpf. (J, K) Expression at 48 hpf.
Fig. 5 of Bagwell et al., 2020
Dstyk regulates fusion of pre-vacuolar carriers with the notochord vacuole. ( A–B) Maximum projection of a light sheet microscopy live image of WT and spzl-/- embryos at 48hpf and 65hpf, respectively. ( C–D) Still images from a 17 hr light sheet movie of a WT and spzl-/-embryos zebrafish embryo during vacuole biogenesis depicting vacuole fusion events. Vacuole membranes are labeled by NFATC:gal4;UAS:GFPrab32 expression. Arrows point to GFP-Rab32 positive vesicles. Arrowheads point to large vesicle (vacuolinos) docking events. n = 2 animals per genotype. Asterisks mark fusion events. Scale bar = 100 µm.
Fig. 4 of Fogerty et al., 2019
Survey of retinal anatomy in 5dpf mutant larvae. Transverse sections were collected near the optic nerve, which is visible in some images (asterisks), and stained with markers for different photoreceptor cell types. In all images, the ventral retina is to the left. (a) Peanut agglutinin lectin (PNA) labels the extracellular matrix surrounding cone outer segments. (b) zpr1 labels red/green cone inner segments. Inserts show higher magnification images, with double arrows indicating how inner segments were measured. (c) zpr3 labels rod outer segments, which are immature at this age and typically longer and more numerous in the ventral retina.
Fig. 1. of Soman et al., 2019
Generation, screening and validation of mcu−/− zebrafish. (A) CRISPR/Cas9-based gene editing was used to generate mcu−/− zebrafish. Schematics showing the targeted genomic sequence for the introduction of the indel mutation in the exon 3 containing the mcu coding gene. The identified 18 base pair deletion (shown in faded font) and 70 bp insertion (shown in highlighted font) is denoted in the cDNA sequence. (B) The indel mutation leads to a frameshift mutation with a premature stop codon. The predicted protein product for mcu mutant allele is shown in the lower panel. (C) RT-PCR data showing wt mcu allele in the first column; the second column shows the wt and mutant allele in heterozygous mutants and the third column shows the mcu mutant allele in mcu homozygous zebrafish. (D) q-PCR data show significant downregulation (***P<0.0001) of mcu gene expression in mcu−/− zebrafish when compared to wt, indicating mutation-induced RNA decay. Statistical analysis with t-test of three different experiments with n=3. (E) Representative images showing in situ hybridization using mcu riboprobe. mcu expression is abolished in mcu−/− 3 dpf zebrafish. (F) Impaired mitochondrial calcium ions influx/efflux in isolated mitochondria from 24 hpf larvae in mcu−/− zebrafish. Control mitochondria (green line) had an uptake in calcium added to solution, which created a drop in the fluorescence. After ∼5 min, we observed calcium leakage (rise in fluorescence). Blank control (black line) was mitochondria without added calcium. Ruthenium Red (pink line) prevented calcium uptake. CCCP (yellow line) caused leakage of calcium, observed as rising fluorescence. Uptake in mcu−/− (blue line) was completely blocked in given circumstances, being like the dynamics of Ruthenium Red treatment.
Figure 5 of Gálvez-Santisteban et al., 2019
The mechanosensitive channel Trpv4 regulates endocardial Notch activation and myocardial regeneration through Klf2a. ( A–D) Confocal imaging of vmhc:mCherry-NTR ; klf2a:H2B-GFP hearts shows that klf2a:H2B-GFP expression is activated in ( B) wild-type ( wt) ventricle-ablated hearts at 24 hpt (6 dpf) compared to ( A) control hearts; however, this activation is reduced in ( D) trpv4-/- ventricle-ablated hearts. ( F–H) Confocal imaging of vmhc:mCherry-NTR ; Tp1:d2GFP hearts further reveals that Tp1:d2GFP is decreased in ( G) klf2a-/- and ( H) trpv4-/- ventricle-ablated hearts at 24 hpt (6 dpf) when compared to ( F) wild-type ( wt) hearts. ( E, I) Quantitation of the relative average fluorescence intensity confirms reduced injury-induced ( E) klf2a:H2B-GFP activation in trpv4-/- ventricle-ablated hearts, and ( I) Tp1:d2GFP activation in trpv4-/- and klf2a-/- ventricle-ablated hearts when compared to wild-type ventricle-ablated hearts ( klf2a:H2B-GFP n = 9 control wt; 7 control trpv4-/-; 9 MTZ wt; 10 MTZ trpv4-/-. Tp1:d2GFP n = 15 control wt; 18 control trpv4-/-; 20 control klf2a-/-; 22 MTZ wt; 21 MTZ trpv4-/-; 18 MTZ klf2a-/). ( J–L) Whole-mount in situ hybridizations show that notch1b is decreased in ventricle-ablated ( K) klf2a-/- (n = 2/11) and ( L) trpv4-/- hearts (n = 6/15) at 24 hpt (6 dpf) when compared to ( J) wild-type hearts (n = 16/18). ( M–O) Confocal microscopy performed on vmhc:mCherry-NTR ventricle-ablated hearts reveals that ( N) klf2a-/- and ( O) trpv4-/-ventricle-ablated hearts display reduced CM proliferative response when compared to ( M) wild-type ( wt) ventricle-ablated hearts at 48 hpt (7 dpf). White – anti-phospho-histone H3 immunostaining; red – anti-MF-20 immunostaining. Arrows point to proliferating CMs. ( P) Quantitation of anti-phospho-histone H3+ CMs in these hearts confirms that klf2a-/- and trpv4-/- hearts fail to increase CM proliferation after ventricle-injury (n = 15 each condition). Red bars – ventricle; green bars – atrium; dark bars – control sham-ablated hearts; light bars – ventricle-ablated hearts. ( Q) Quantitation of the percentage of vmhc:mCherry-NTR ventricle-ablated hearts that display recovered ventricular tissue and contractility (black bars) at 96 hpt (9 dpf) supports that klf2a-/- and trpv4-/- mutants exhibit impaired heart regeneration. The number of fish analyzed for each condition is indicated above each column. ( R–T) Confocal microscopy imaging of vmhc:mCherry-NTR; amhc:CreERT2; β-actin2:RSGhearts at 72 hpt (8 dpf) shows that ( S) klf2a-/-and ( T) trpv4-/- ventricle-ablated hearts exhibit reduced contribution of genetically labeled atrial CMs (c-aGFP+) to the regenerating injured ventricle when compared to ( R) wild-type ventricle-ablated hearts. Green channel – ( R’–T’) genetically labeled c-aGFP+ atrial CMs. ( U) Quantitation of the percentage of ventricular area covered with c-aGFP+ CMs confirms that klf2a-/-or trpv4-/- hearts display reduced capacity to undergo injury-induced atrial-to-ventricular trans-differentiation during injury and regeneration (n = 13 wt; 8 klf2a-/-; 10 trpv4-/-). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-MTZ/DMSO treatment. Dashed lines outline the heart. Bars: 50 μm. ( E, I, P) Mean + s.e.m. ANOVA; ( Q) Total numbers, Binomial test (versus wild-type); ( U) Mean + s.d. ANOVA; ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 5.