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.
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Figure 7 of Klingseisen et al., 2019
Caspr Regulates Myelin Sheath Elongation and Stability, but Not Targeting(A and B) Confocal images of single oligodendrocytes labeled mosaically using mbp:mCherry-CAAX in wildtype (A) and casprsa12772 mutants at 5 dpf. Scale bar, 10 μm.(C–E) Number of myelinated cell bodies (C), number of myelin sheaths (D), and the length of myelin sheaths (D), made by individual oligodendrocytes in wildtype, het, and casprsa12772 mutants.(C) Myelination of cell bodies is increased slightly in casprsa12772 mutant oligodendrocytes (wildtype median 0.0, 25th percentile 0.0, 75th percentile 0.0, n = 15 animals; casprsa12772/+ median 0.0, 25th percentile 0.0, 75th percentile 0.0, n = 35, casprsa12772 median 0.25, 25th percentile 0.0, 75th percentile 1.5 , n = 25 animals; wildtype versus casprsa12772/+ p = 0.8056, wildtype versus casprsa12772 p = 0.0305, casprsa12772/+ versus casprsa12772 p = 0.0156, all Mann–Whitney test.(D) Number of myelin sheaths per oligodendrocytes is similar in wildtype, casprsa12772/+ and casprsa12772 (wildtype mean 11.78 ± 5.14 SD, n = 15 animals, casprsa12772/+ mean 11.19 ± 3.26 SD, n = 33 animals, casprsa12772 mean 12.00 ± 3.54 SD, n = 24; ANOVA = 0.712).(E) Myelin sheath length is reduced in casprsa12772 mutants (wildtype mean 29.14 ± 1.71 SEM, n = 15, casprsa12772/+ mean 27.58 ± 1.31 SEM, n = 33 animals, casprsa12772 mean 23.55 ± 1.30 SEM, n = 24 animals; wildtype versus casprsa12772/+ p = 0.4954, wildtype versus casprsa12772 p = 0.0124, casprsa12772/+ versus casprsa12772 p = 0.0374, all t test).(F and G) Confocal images of the dorsal horn of the spinal cord in wildtype (F) and Caspr mutant (G) mice, indicating the region indicated by dashed lines wherein myelinated cell bodies were searched for. Scale bar, 50 μm.(F′ and G′) Higher magnification views of regions within the dorsal horn of wild type (F′) and Caspr mutant (G′) animals stained with antibodies that recognize myelin basic protein (green), NeuN (red), and DAPI (blue). Scale bar, 5 μm.(H) Graph showing that essentially no myelinated cell bodies were observed in wild type or Caspr mutant animals. Wild type mean 0.2800 ± 0.049 SEM, n = 5 (10 sections per mouse), Caspr−⁄− 0.257 ± 0.083 SEM, n = 5 (10 sections per mouse), p = 0.8147, t test.(I and J) Confocal images of teased fiber preparations taken from wild type (I) and Caspr mutant (J) mice, stained with antibodies that detect Kv1.1 (red), Neurofascin 186 (green), and neurofilament 200 (red). Scale bars, 25 μm.(K) Quantitation of myelin sheath length in Caspr mutant mice. Caspr internode lengths are reduced compared to wild type; wild type mean 310.7 ± 5.32 SEM, n = 5, Caspr 226.5 ± 8.96 SEM, n = 5 animals, 50 internodes per mouse, p < 0.0001, t test.(L and L″) Confocal images of a myelin sheath elongating in a wildtype Tg(mbp:EGFP-CAAX) animal. Scale bar, 10 μm.(M and M′) Confocal images of a myelin sheath elongating in a casprsa12772 mutant. Scale bar, 10 μm.(N) Speed of myelin sheath elongation in wildtype and casprsa12772 mutants: wildtype mean 0.36 ± 0.35 SD, n = 122 sheaths from 6 animals, casprsa12772 mean 0.16 ± 0.31 SD, n = 151 sheaths from 7 animals, p < 0.0001, Mann–Whitney test. 14.8% in controls and 28.5% in nfascbue56 represent shrinking myelin sheaths.
Figure 3 of Duchemin et al., 2019
The OFT endothelium is surrounded by smooth muscle cell progenitors expressing fibronectin and elastin. (A) Staining of Fibronectin1 (magenta) on Tg(myl7:GFP; fli1a:nls-mCherry), highlighting the myocardium (white) and the endothelium (green) at 72 hpf. Scale bar: 20 μm. V: ventricle. Fibronectin1 (anti-Fn1, magenta) counterstaining on Tg(kdrl:GFP) and Elastinb (anti-Elnb, magenta) counterstaining on Tg(fli:lifeact-EGFP) showing their expressions in the OFT at 72 hpf (B), (B’) respectively) at 96 hpf (C), (C’) respectively) and at 120 hpf (D, D’ respectively). Scale bar: 20 µm. Arrows show the Fn1 localisation within the valve leaflets. Results obtained from three independent experiments. (E) Scheme of the three layers shown in A’, B, C and D (magenta, smooth muscles; green, endothelium; grey, myocardium; Fibronectin1, magenta lines) at 72hpf, 96hpf and 120hpf.
Fig. S1 of Liu et al., 2019
(A) cxcr4a expression during gastrulation. In situ hybridization of cxcr4a in embryos at the 75% epiboly stage (dorsal view with animal pole to the top) and bud stage (lateral views with animal pole to the top). Black arrowhead indicates the DFCs. (B) cxcr4a expression at the 6-somite stage. Lateral view was shown with animal pole to the top in the left panel, and dorsal view was shown in the right panel. Black arrowhead indicates the KV. (C) Confocal images depicting the formation of the lateral dorsal aorta in live Tg(flk: GFP)embryos. Scale bar, 50 μm. DFC, dorsal forerunner cell; ep, epiboly; flk, fms-like tyrosine kinase; GFP, green fluorescent protein; KV, Kupffer’s vesicle; LDA, lateral dorsal aorta; PHBC, primordial hindbrain channel; Tg, transgene.
Fig. S2 of Vogrin et al., 2019
In situ hybridisation for kdr and flt4, and analysis of vegfd expression, Related to Figure 4. (A-F) In situ hybridisation using probes designed to kdr (A-C) and flt4 (D-F) showing expression in the craniofacial region as well as in the posterior cardinal vein. (G-M) Expression of vegfd visualised using the TgBAC(vegfd:eYFP)uq42bh transgenic line. (G-I) At 36 hpf, expression is observed in the craniofacial region close to where lymphatic sprouts emerge from the primary head sinus (arrows). (J,K) At 60 hpf, vegfd expression is observed in the region close to where the otolithic lymphatic sprout emerges and in the region where mural LECs sprout from the choroidal vascular plexus (arrows). (L,M) At 72 hpf continued expression of vegfd can be observed in the craniofacial region where the first lymphatic sprouts form and in branchial arches where lymphatics sprouts emerge (arrows). Scale bars in A, D and L represent 200 m, in B, C and E-K 100 m and in M 50 m.
Fig. 7 of Bagwell et al., 2020
Loss of notochord vacuole integrity is associated with spine kinking in spzl mutants. ( A) Live confocal imaging time course of vacuole dynamics and morphology during spine development in WT zebrafish larvae. n = 3, 6, 11, 13. ( B) Live confocal imaging time course of vacuole dynamics and morphology during spine development in spzl-/- larvae. n = 3, 4, 8, 7. ( A–B) Larvae express GFP in the cytoplasm of vacuolated cells and mCherry in osteoblasts. Note the progressive loss of vacuoles in spzl-/- and the straight notochord and spine prior to the onset of concentric vertebral growth. Dotted lines mark the edge of the notochord. Arrow points to a spine kink in spzl-/-. ( C) Maximum intensity projection of a laterally oriented 29 dpf spzl-/- larva. The spine is visualized by mCherry expression in the osteoblasts. ( D–E) Maximum intensity projections showing dorsal ( D) and lateral ( E) views of a 29 dpf spzl-/- larva expressing mCherry in the osteoblasts and GFP in the notochord. The anterior spine is straight while the posterior region has developed several spine kinks ( F) Optical confocal section of the straight anterior portion of the spine. Asterisks mark intact vacuoles. ( G–I) Optical confocal sections of the posterior notochord where vacuoles are highly fragmented or absent and the spine is highly kinked. Gt(SAGFF214A:gal4); Tg(UAS:GFP); Tg(osx:NTR-mCherry). Scale bars are 100 µm.
Fig. 4 of Liu et al., 2019
Cxcr4 promotes Cyclin D1 expression through ERK signaling during DFC proliferation. (A) ERK1/2 phosphorylation levels were dramatically decreased in cxcr4aum20 mutants. WT and cxcr4a-deficient Tg(sox17:GFP) embryos were harvested at the 75% epiboly stage and subjected to immunostaining for p-ERK1/2 (red) and GFP (green). All embryos are shown in dorsal views with anterior to the top. Scale bar, 20 μm. (B–E) caMEK mRNA overexpression in DFCs rescued L–R patterning defects in cxcr4aum20 mutants. Different types of heart looping and liver laterality at 48 hpf in cxcr4aum20 mutants following midblastula injection of different caMEK mRNA doses were visualized by cmlc2 and hhex expression (B and C). The ratios are shown in (D) and (E). Underlying data can be found in S1 Data. (F) Cxcr4a-deficient Tg(sox17:GFP) embryos were injected with 60 pg caMEK mRNA at the 256-cell stage and then harvested at the 75% epiboly stage for fluorescence in situ hybridization experiments with cyclin D1 (red) and GFP (green) probes. Dorsal views with anterior to the left. Scale bar, 20 μm. (G–H) WT embryos were treated with 0.5 μM PD0332991 or 0.2 μM CY202 from the shield stage to bud stage and then analyzed for L–R patterning defects at 48 hpf by in situ hybridizations with cmlc2 and hhex probes. The proportion of treated embryos exhibiting each type of heart looping and liver laterality is shown in (G) and (H). Underlying data can be found in S1 Data. (I–J) Reintroduction of cyclin D1 into DFCs relieves DFC proliferation defects in cxcr4aum20 mutants. Cxcr4a-deficient Tg(sox17:GFP) embryos were injected with or without 300 pg cyclin D1 mRNA at the 256-cell stage, followed by coimmunostaining with anti-BrdU (red) and anti-GFP (green) antibodies at the 75% epiboly stage. Representative images are shown in (I), and the percentage of BrdU-positive DFCs is indicated in (J). Scale bar, 20 μm. Student t test, ***P < 0.001. Underlying data can be found in S1 Data. (K–M) The reduced ratios of embryos affected in KV size (K) and cmlc2 (L) or hhex (M) expression show that DFC-specific overexpression of cyclin D1 rescued the defects of KV formation (K) and L–R patterning (L and M) in cxcr4a mutants. Underlying data can be found in S1 Data. BrdU, bromodeoxyuridine; caMEK, constitutively activated version of MEK; cmlc2, cardiac myosin light chain 2; DFC, dorsal forerunner cell; ERK, extracellular regulated MAP kinase; GFP, green fluorescent protein; hhex, hematopoietically expressed homeobox; hpf, hours postfertilization; KV, Kupffer’s vesicle; L–R, left–right; ns, no significant difference; p-ERK1/2, phosphorylated ERK1/2; sox, SRY-box transcription factor; Tg, transgene; WT, wild-type
Fig. 1 of Costa et al., 2019
Mitochondrial Fitness Fine-Tunes Wnt Signaling in Human Embryonic Kidney HEK293 Cells and in Wnt-Dependent Reporter Zebrafish (A) Canonical Wnt signaling activity based on TCF-LEF-dependent transcription was assayed in HEK293 cells. These cells were either left untreated or Wnt signaling was enhanced using 3 μM CHIR99021 (CHIR). Cells were then treated for 8 h with the following compounds: 0.1% DMSO, as negative control; 10 μM XAV939 (XAV) as positive control; MgTx, ShK, ChTx (1 μM); PAP-1 (20 μM) + cyclosporin H (CSH) (4 μM); 1 μM PAPTP, 5 μM PCARBTP; 5 μM salinomycin (Salino), 0.5 μM nigericin (Nige), 1 μM valinomycin (Valino); and 2 μM FCCP; 5 μM rotenone (Rot), 0.5 mM TTFA, 1.8 μM antimycin A (Anti), and 1.2 μM oligomycin (Oligo). The luciferase signal was normalized with respect to the signal given by β-gal, which was co-transfected with TOPflash plasmid. The values are reported as the percentage of luciferase signal related to the “ref.” Values are means ± SEMs (n = 8). (B and C) Reduction of protein levels of β-catenin and its target genes LEF1 and MET are shown in representative western blots (B). Densitometric analysis (n = 6; means ± SEMs) is reported in (C). (D and E) GFP fluorescent zebrafish Tg(7xTCFX.lasiam:EGFP)ia4 Wnt-dependent reporter fishes were treated for 15 h with the following compounds: DMSO 0.2%, 20 μM XAV, 1 μM Salino, 1.5 μM PAPTP, 5 μM PCARBTP, 50 μM TTFA, and 0.12 μM Oligo. Representative bright-field and epifluorescence microscopy images are reported in (D), while fluorescence quantification is shown in (E) (means ± SEMs). The numbers reported on the graph represent the number of zebrafish embryos treated for each condition. Bars, 1.5 mm. (F) Zebrafish β-catenin (Zβ-catenin) protein level from embryos treated as (D) and (E) was determined by western blot. The quantification is reported below. The values are indicated as the percentage of the “ref” (n = 3; means ± SEMs). Statistical significance (ANOVA) was determined as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Fig. 1. of Kalasekar et al., 2019
CreLox system enables switching on activated β-catenin. (A) Schematics of DNA constructs used in this study. Grey boxes indicate promoters; red arrowheads indicate loxP sites. ABC, Activated β-catenin. (B,C) Switching in Tg(fabp10a:Cre) (HepCre),Tg(fabp10a:flox-pt-β-cat) (FloxABC) and Tg(fabp10a:Cre); Tg(fabp10a:flox-pt-β-cat) (HepCre+FloxABC) larvae imaged at 5 dpf for BFP expression, detected by immunofluorescence using an anti-GFP antibody. Successful switching is indicated by loss of BFP expression. (B) Representative images. Livers are outlined. Scale bars: 50 µm. (C) Scatter plot with bar graph quantifying switching in terms of mean intensity of GFP fluorescence, ±standard deviation (s.d.). P-values derived using Sidak's multiple comparisons test following ordinary one-way ANOVA. N values are shown above x axis. Experiment was performed three times, with similar results each time, and representative results are shown. (D,E) Wnt reporter activity in Tg(fabp10a:Cre) (HepCre),Tg(fabp10a:flox-pt-β-cat) (FloxABC) and Tg(fabp10a:Cre); Tg(fabp10a:flox-pt-β-cat) (HepCre+FloxABC) siblings imaged at 5 dpf alongside Tg(fabp10a:pt-β-cat) (HepABC) and non-transgenic siblings (NonTg). All zebrafish contain 7xTCF-Xla.Siam:mCherry transgene for visualization of Wnt reporter activity by immunofluorescence. (D) Representative images of Wnt reporter activity (7xTCF-Xla.Siam:mCherry). Livers are outlined. Scale bars: 50 µm. (E) Stacked bar graphs showing percent of zebrafish with absent (A), low (L, involving <10% of hepatocytes) or high (H, involving >10% of hepatocytes) Wnt reporter activity. P-values derived using Fisher's exact test. N values are shown above x axis. Three experiments were pooled.
Figure 7 of Duchemin et al., 2019
Klf2a and notch are necessary for valve formation. Quantification of the valve phenotypes at 72 hpf, 96 hpf and 120 hpf (normal, thick, delayed) in klf2a+/+(n = 11), and klf2a-/- (n = 12) using the Tg(fli:gal4FF/UAS:Kaede). (A) and notch1b+/+ (n = 7) and notch1b-/- (n = 20) using Tg(fli:lifeact-EGFP; kdrl:nls-mCherry) embryos. (B) Scale bar: 10 µm. N = 3 independent experiments. (C) Confocal z-sections of the Tg(tp1:dGFP) in klf2a+/+ and klf2a-/- embryos at 72 hpf, 96 hpf and 120 hpf. V: ventricle. Scale bar: 10 µm. (C’) Quantification of the fluorescent intensity of the Notch reporter (GFP over background) in the anterior versus posterior parts of the valves in in klf2a+/+ (n = 5) and klf2a-/- (n = 4) embryos. Statistical test were performed to compare the posterior intensities in klf2a+/+versus klf2a-/- at 72 hpf (p=0,05), 96 hpf (p=0,03) and 120 hpf (p=0,04). Student’s t-test. Boxplot: Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Results obtained from three independent experiments.
Fig. 7 of Gordon et al., 2019
Plvapb limits the transfer rate of blood-borne proteins into the hypophysis parenchyma. (A) Confocal maximal intensity images showing the hypophyseal capillary loop in plvapb+/+ and plvapb−/− double transgenic Tg(l-fabp:DBP-EGFP;kdrl:mCherry-caax) zebrafish larvae (5 dpf). Scale bars: 10 µm. (B,C) Morphological analysis of hypophyseal capillary loop parameters, including roundness index [width (x)/length (y)] (B) and area (C). No significant differences were found between plvapb+/+ (n=18) and plvapb−/− (n=23) fish (ns, not significant; Student's t-test). (D) Quantification of accumulated fluorescence intensity of the extravasated DBP-EGFP inside the hypophyseal capillary loop (dashed line in A, left panel) of fixed plvapb+/+ (n=16) and plvapb−/− (n=23) larvae. No significant difference was found between the fish (ns, not significant; Student's t-test). (E) Schematic representation of the fluorescence recovery after photobleaching (FRAP) of extravasated DBP-EGFP in the hypophysis of live zebrafish larval. ROI, region of interest. (F) Real-time FRAP measurements of the intensity of extravasated DBP-EGFP signal in live plvapb+/+ (n=8) and plvapb−/− (n=10) transgenic Tg(l-fabp:DBP-EGFP;kdrl:mCherry-caax) larvae (4 dpf). (G) T1/2 of the fluorescence recovery after bleaching of DBP-EGFP. T1/2 value (calculated using ImageJ FRAP Profiler tool) is significantly lower in the plvapb−/− mutant, indicating higher extravasation rate of blood-borne protein in mutant versus wild-type fish (*P<0.05; Student's t-test). a.u., arbitrary units. Data are mean±s.e.m.
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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.
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Figure 7 of Klingseisen et al., 2019
Caspr Regulates Myelin Sheath Elongation and Stability, but Not Targeting(A and B) Confocal images of single oligodendrocytes labeled mosaically using mbp:mCherry-CAAX in wildtype (A) and casprsa12772 mutants at 5 dpf. Scale bar, 10 μm.(C–E) Number of myelinated cell bodies (C), number of myelin sheaths (D), and the length of myelin sheaths (D), made by individual oligodendrocytes in wildtype, het, and casprsa12772 mutants.(C) Myelination of cell bodies is increased slightly in casprsa12772 mutant oligodendrocytes (wildtype median 0.0, 25th percentile 0.0, 75th percentile 0.0, n = 15 animals; casprsa12772/+ median 0.0, 25th percentile 0.0, 75th percentile 0.0, n = 35, casprsa12772 median 0.25, 25th percentile 0.0, 75th percentile 1.5 , n = 25 animals; wildtype versus casprsa12772/+ p = 0.8056, wildtype versus casprsa12772 p = 0.0305, casprsa12772/+ versus casprsa12772 p = 0.0156, all Mann–Whitney test.(D) Number of myelin sheaths per oligodendrocytes is similar in wildtype, casprsa12772/+ and casprsa12772 (wildtype mean 11.78 ± 5.14 SD, n = 15 animals, casprsa12772/+ mean 11.19 ± 3.26 SD, n = 33 animals, casprsa12772 mean 12.00 ± 3.54 SD, n = 24; ANOVA = 0.712).(E) Myelin sheath length is reduced in casprsa12772 mutants (wildtype mean 29.14 ± 1.71 SEM, n = 15, casprsa12772/+ mean 27.58 ± 1.31 SEM, n = 33 animals, casprsa12772 mean 23.55 ± 1.30 SEM, n = 24 animals; wildtype versus casprsa12772/+ p = 0.4954, wildtype versus casprsa12772 p = 0.0124, casprsa12772/+ versus casprsa12772 p = 0.0374, all t test).(F and G) Confocal images of the dorsal horn of the spinal cord in wildtype (F) and Caspr mutant (G) mice, indicating the region indicated by dashed lines wherein myelinated cell bodies were searched for. Scale bar, 50 μm.(F′ and G′) Higher magnification views of regions within the dorsal horn of wild type (F′) and Caspr mutant (G′) animals stained with antibodies that recognize myelin basic protein (green), NeuN (red), and DAPI (blue). Scale bar, 5 μm.(H) Graph showing that essentially no myelinated cell bodies were observed in wild type or Caspr mutant animals. Wild type mean 0.2800 ± 0.049 SEM, n = 5 (10 sections per mouse), Caspr−⁄− 0.257 ± 0.083 SEM, n = 5 (10 sections per mouse), p = 0.8147, t test.(I and J) Confocal images of teased fiber preparations taken from wild type (I) and Caspr mutant (J) mice, stained with antibodies that detect Kv1.1 (red), Neurofascin 186 (green), and neurofilament 200 (red). Scale bars, 25 μm.(K) Quantitation of myelin sheath length in Caspr mutant mice. Caspr internode lengths are reduced compared to wild type; wild type mean 310.7 ± 5.32 SEM, n = 5, Caspr 226.5 ± 8.96 SEM, n = 5 animals, 50 internodes per mouse, p < 0.0001, t test.(L and L″) Confocal images of a myelin sheath elongating in a wildtype Tg(mbp:EGFP-CAAX) animal. Scale bar, 10 μm.(M and M′) Confocal images of a myelin sheath elongating in a casprsa12772 mutant. Scale bar, 10 μm.(N) Speed of myelin sheath elongation in wildtype and casprsa12772 mutants: wildtype mean 0.36 ± 0.35 SD, n = 122 sheaths from 6 animals, casprsa12772 mean 0.16 ± 0.31 SD, n = 151 sheaths from 7 animals, p < 0.0001, Mann–Whitney test. 14.8% in controls and 28.5% in nfascbue56 represent shrinking myelin sheaths.
Figure 3 of Duchemin et al., 2019
The OFT endothelium is surrounded by smooth muscle cell progenitors expressing fibronectin and elastin. (A) Staining of Fibronectin1 (magenta) on Tg(myl7:GFP; fli1a:nls-mCherry), highlighting the myocardium (white) and the endothelium (green) at 72 hpf. Scale bar: 20 μm. V: ventricle. Fibronectin1 (anti-Fn1, magenta) counterstaining on Tg(kdrl:GFP) and Elastinb (anti-Elnb, magenta) counterstaining on Tg(fli:lifeact-EGFP) showing their expressions in the OFT at 72 hpf (B), (B’) respectively) at 96 hpf (C), (C’) respectively) and at 120 hpf (D, D’ respectively). Scale bar: 20 µm. Arrows show the Fn1 localisation within the valve leaflets. Results obtained from three independent experiments. (E) Scheme of the three layers shown in A’, B, C and D (magenta, smooth muscles; green, endothelium; grey, myocardium; Fibronectin1, magenta lines) at 72hpf, 96hpf and 120hpf.
Fig. S1 of Liu et al., 2019
(A) cxcr4a expression during gastrulation. In situ hybridization of cxcr4a in embryos at the 75% epiboly stage (dorsal view with animal pole to the top) and bud stage (lateral views with animal pole to the top). Black arrowhead indicates the DFCs. (B) cxcr4a expression at the 6-somite stage. Lateral view was shown with animal pole to the top in the left panel, and dorsal view was shown in the right panel. Black arrowhead indicates the KV. (C) Confocal images depicting the formation of the lateral dorsal aorta in live Tg(flk: GFP)embryos. Scale bar, 50 μm. DFC, dorsal forerunner cell; ep, epiboly; flk, fms-like tyrosine kinase; GFP, green fluorescent protein; KV, Kupffer’s vesicle; LDA, lateral dorsal aorta; PHBC, primordial hindbrain channel; Tg, transgene.
Fig. S2 of Vogrin et al., 2019
In situ hybridisation for kdr and flt4, and analysis of vegfd expression, Related to Figure 4. (A-F) In situ hybridisation using probes designed to kdr (A-C) and flt4 (D-F) showing expression in the craniofacial region as well as in the posterior cardinal vein. (G-M) Expression of vegfd visualised using the TgBAC(vegfd:eYFP)uq42bh transgenic line. (G-I) At 36 hpf, expression is observed in the craniofacial region close to where lymphatic sprouts emerge from the primary head sinus (arrows). (J,K) At 60 hpf, vegfd expression is observed in the region close to where the otolithic lymphatic sprout emerges and in the region where mural LECs sprout from the choroidal vascular plexus (arrows). (L,M) At 72 hpf continued expression of vegfd can be observed in the craniofacial region where the first lymphatic sprouts form and in branchial arches where lymphatics sprouts emerge (arrows). Scale bars in A, D and L represent 200 m, in B, C and E-K 100 m and in M 50 m.
Fig. 7 of Bagwell et al., 2020
Loss of notochord vacuole integrity is associated with spine kinking in spzl mutants. ( A) Live confocal imaging time course of vacuole dynamics and morphology during spine development in WT zebrafish larvae. n = 3, 6, 11, 13. ( B) Live confocal imaging time course of vacuole dynamics and morphology during spine development in spzl-/- larvae. n = 3, 4, 8, 7. ( A–B) Larvae express GFP in the cytoplasm of vacuolated cells and mCherry in osteoblasts. Note the progressive loss of vacuoles in spzl-/- and the straight notochord and spine prior to the onset of concentric vertebral growth. Dotted lines mark the edge of the notochord. Arrow points to a spine kink in spzl-/-. ( C) Maximum intensity projection of a laterally oriented 29 dpf spzl-/- larva. The spine is visualized by mCherry expression in the osteoblasts. ( D–E) Maximum intensity projections showing dorsal ( D) and lateral ( E) views of a 29 dpf spzl-/- larva expressing mCherry in the osteoblasts and GFP in the notochord. The anterior spine is straight while the posterior region has developed several spine kinks ( F) Optical confocal section of the straight anterior portion of the spine. Asterisks mark intact vacuoles. ( G–I) Optical confocal sections of the posterior notochord where vacuoles are highly fragmented or absent and the spine is highly kinked. Gt(SAGFF214A:gal4); Tg(UAS:GFP); Tg(osx:NTR-mCherry). Scale bars are 100 µm.
Fig. 4 of Liu et al., 2019
Cxcr4 promotes Cyclin D1 expression through ERK signaling during DFC proliferation. (A) ERK1/2 phosphorylation levels were dramatically decreased in cxcr4aum20 mutants. WT and cxcr4a-deficient Tg(sox17:GFP) embryos were harvested at the 75% epiboly stage and subjected to immunostaining for p-ERK1/2 (red) and GFP (green). All embryos are shown in dorsal views with anterior to the top. Scale bar, 20 μm. (B–E) caMEK mRNA overexpression in DFCs rescued L–R patterning defects in cxcr4aum20 mutants. Different types of heart looping and liver laterality at 48 hpf in cxcr4aum20 mutants following midblastula injection of different caMEK mRNA doses were visualized by cmlc2 and hhex expression (B and C). The ratios are shown in (D) and (E). Underlying data can be found in S1 Data. (F) Cxcr4a-deficient Tg(sox17:GFP) embryos were injected with 60 pg caMEK mRNA at the 256-cell stage and then harvested at the 75% epiboly stage for fluorescence in situ hybridization experiments with cyclin D1 (red) and GFP (green) probes. Dorsal views with anterior to the left. Scale bar, 20 μm. (G–H) WT embryos were treated with 0.5 μM PD0332991 or 0.2 μM CY202 from the shield stage to bud stage and then analyzed for L–R patterning defects at 48 hpf by in situ hybridizations with cmlc2 and hhex probes. The proportion of treated embryos exhibiting each type of heart looping and liver laterality is shown in (G) and (H). Underlying data can be found in S1 Data. (I–J) Reintroduction of cyclin D1 into DFCs relieves DFC proliferation defects in cxcr4aum20 mutants. Cxcr4a-deficient Tg(sox17:GFP) embryos were injected with or without 300 pg cyclin D1 mRNA at the 256-cell stage, followed by coimmunostaining with anti-BrdU (red) and anti-GFP (green) antibodies at the 75% epiboly stage. Representative images are shown in (I), and the percentage of BrdU-positive DFCs is indicated in (J). Scale bar, 20 μm. Student t test, ***P < 0.001. Underlying data can be found in S1 Data. (K–M) The reduced ratios of embryos affected in KV size (K) and cmlc2 (L) or hhex (M) expression show that DFC-specific overexpression of cyclin D1 rescued the defects of KV formation (K) and L–R patterning (L and M) in cxcr4a mutants. Underlying data can be found in S1 Data. BrdU, bromodeoxyuridine; caMEK, constitutively activated version of MEK; cmlc2, cardiac myosin light chain 2; DFC, dorsal forerunner cell; ERK, extracellular regulated MAP kinase; GFP, green fluorescent protein; hhex, hematopoietically expressed homeobox; hpf, hours postfertilization; KV, Kupffer’s vesicle; L–R, left–right; ns, no significant difference; p-ERK1/2, phosphorylated ERK1/2; sox, SRY-box transcription factor; Tg, transgene; WT, wild-type
Fig. 1 of Costa et al., 2019
Mitochondrial Fitness Fine-Tunes Wnt Signaling in Human Embryonic Kidney HEK293 Cells and in Wnt-Dependent Reporter Zebrafish (A) Canonical Wnt signaling activity based on TCF-LEF-dependent transcription was assayed in HEK293 cells. These cells were either left untreated or Wnt signaling was enhanced using 3 μM CHIR99021 (CHIR). Cells were then treated for 8 h with the following compounds: 0.1% DMSO, as negative control; 10 μM XAV939 (XAV) as positive control; MgTx, ShK, ChTx (1 μM); PAP-1 (20 μM) + cyclosporin H (CSH) (4 μM); 1 μM PAPTP, 5 μM PCARBTP; 5 μM salinomycin (Salino), 0.5 μM nigericin (Nige), 1 μM valinomycin (Valino); and 2 μM FCCP; 5 μM rotenone (Rot), 0.5 mM TTFA, 1.8 μM antimycin A (Anti), and 1.2 μM oligomycin (Oligo). The luciferase signal was normalized with respect to the signal given by β-gal, which was co-transfected with TOPflash plasmid. The values are reported as the percentage of luciferase signal related to the “ref.” Values are means ± SEMs (n = 8). (B and C) Reduction of protein levels of β-catenin and its target genes LEF1 and MET are shown in representative western blots (B). Densitometric analysis (n = 6; means ± SEMs) is reported in (C). (D and E) GFP fluorescent zebrafish Tg(7xTCFX.lasiam:EGFP)ia4 Wnt-dependent reporter fishes were treated for 15 h with the following compounds: DMSO 0.2%, 20 μM XAV, 1 μM Salino, 1.5 μM PAPTP, 5 μM PCARBTP, 50 μM TTFA, and 0.12 μM Oligo. Representative bright-field and epifluorescence microscopy images are reported in (D), while fluorescence quantification is shown in (E) (means ± SEMs). The numbers reported on the graph represent the number of zebrafish embryos treated for each condition. Bars, 1.5 mm. (F) Zebrafish β-catenin (Zβ-catenin) protein level from embryos treated as (D) and (E) was determined by western blot. The quantification is reported below. The values are indicated as the percentage of the “ref” (n = 3; means ± SEMs). Statistical significance (ANOVA) was determined as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Fig. 1. of Kalasekar et al., 2019
CreLox system enables switching on activated β-catenin. (A) Schematics of DNA constructs used in this study. Grey boxes indicate promoters; red arrowheads indicate loxP sites. ABC, Activated β-catenin. (B,C) Switching in Tg(fabp10a:Cre) (HepCre),Tg(fabp10a:flox-pt-β-cat) (FloxABC) and Tg(fabp10a:Cre); Tg(fabp10a:flox-pt-β-cat) (HepCre+FloxABC) larvae imaged at 5 dpf for BFP expression, detected by immunofluorescence using an anti-GFP antibody. Successful switching is indicated by loss of BFP expression. (B) Representative images. Livers are outlined. Scale bars: 50 µm. (C) Scatter plot with bar graph quantifying switching in terms of mean intensity of GFP fluorescence, ±standard deviation (s.d.). P-values derived using Sidak's multiple comparisons test following ordinary one-way ANOVA. N values are shown above x axis. Experiment was performed three times, with similar results each time, and representative results are shown. (D,E) Wnt reporter activity in Tg(fabp10a:Cre) (HepCre),Tg(fabp10a:flox-pt-β-cat) (FloxABC) and Tg(fabp10a:Cre); Tg(fabp10a:flox-pt-β-cat) (HepCre+FloxABC) siblings imaged at 5 dpf alongside Tg(fabp10a:pt-β-cat) (HepABC) and non-transgenic siblings (NonTg). All zebrafish contain 7xTCF-Xla.Siam:mCherry transgene for visualization of Wnt reporter activity by immunofluorescence. (D) Representative images of Wnt reporter activity (7xTCF-Xla.Siam:mCherry). Livers are outlined. Scale bars: 50 µm. (E) Stacked bar graphs showing percent of zebrafish with absent (A), low (L, involving <10% of hepatocytes) or high (H, involving >10% of hepatocytes) Wnt reporter activity. P-values derived using Fisher's exact test. N values are shown above x axis. Three experiments were pooled.
Figure 7 of Duchemin et al., 2019
Klf2a and notch are necessary for valve formation. Quantification of the valve phenotypes at 72 hpf, 96 hpf and 120 hpf (normal, thick, delayed) in klf2a+/+(n = 11), and klf2a-/- (n = 12) using the Tg(fli:gal4FF/UAS:Kaede). (A) and notch1b+/+ (n = 7) and notch1b-/- (n = 20) using Tg(fli:lifeact-EGFP; kdrl:nls-mCherry) embryos. (B) Scale bar: 10 µm. N = 3 independent experiments. (C) Confocal z-sections of the Tg(tp1:dGFP) in klf2a+/+ and klf2a-/- embryos at 72 hpf, 96 hpf and 120 hpf. V: ventricle. Scale bar: 10 µm. (C’) Quantification of the fluorescent intensity of the Notch reporter (GFP over background) in the anterior versus posterior parts of the valves in in klf2a+/+ (n = 5) and klf2a-/- (n = 4) embryos. Statistical test were performed to compare the posterior intensities in klf2a+/+versus klf2a-/- at 72 hpf (p=0,05), 96 hpf (p=0,03) and 120 hpf (p=0,04). Student’s t-test. Boxplot: Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Results obtained from three independent experiments.
Fig. 7 of Gordon et al., 2019
Plvapb limits the transfer rate of blood-borne proteins into the hypophysis parenchyma. (A) Confocal maximal intensity images showing the hypophyseal capillary loop in plvapb+/+ and plvapb−/− double transgenic Tg(l-fabp:DBP-EGFP;kdrl:mCherry-caax) zebrafish larvae (5 dpf). Scale bars: 10 µm. (B,C) Morphological analysis of hypophyseal capillary loop parameters, including roundness index [width (x)/length (y)] (B) and area (C). No significant differences were found between plvapb+/+ (n=18) and plvapb−/− (n=23) fish (ns, not significant; Student's t-test). (D) Quantification of accumulated fluorescence intensity of the extravasated DBP-EGFP inside the hypophyseal capillary loop (dashed line in A, left panel) of fixed plvapb+/+ (n=16) and plvapb−/− (n=23) larvae. No significant difference was found between the fish (ns, not significant; Student's t-test). (E) Schematic representation of the fluorescence recovery after photobleaching (FRAP) of extravasated DBP-EGFP in the hypophysis of live zebrafish larval. ROI, region of interest. (F) Real-time FRAP measurements of the intensity of extravasated DBP-EGFP signal in live plvapb+/+ (n=8) and plvapb−/− (n=10) transgenic Tg(l-fabp:DBP-EGFP;kdrl:mCherry-caax) larvae (4 dpf). (G) T1/2 of the fluorescence recovery after bleaching of DBP-EGFP. T1/2 value (calculated using ImageJ FRAP Profiler tool) is significantly lower in the plvapb−/− mutant, indicating higher extravasation rate of blood-borne protein in mutant versus wild-type fish (*P<0.05; Student's t-test). a.u., arbitrary units. Data are mean±s.e.m.