mcu inactivation renders neuroprotection against MPTP. (A) Representative images of MPTP-untreated wt (a), pink1−/− (c), (pink1; mcu)−/− (e), mcu−/− (g) and MPTP-treated wt (b), pink1−/− (d), (pink1; mcu)−/− (f) and mcu−/− (h) 3 dpf larvae after WISH using TH-specific riboprobe. MPTP-treated wt (b) and pink1−/− (d) zebrafish larvae were most susceptible to MPTP toxicity while MPTP-untreated (pink1; mcu)−/− (e) and mcu−/− (g) were most resistant to MPTP toxicity. (B) Graphical representation of chromogenic WISH. There was a significant decrease (***P<0.001) in number of dopaminergic neurons in pink1−/− zebrafish treated with MPTP when compared to untreated pink1−/− zebrafish. MPTP-treated (pink1; mcu)−/− zebrafish showed a significant increase (***P<0.001) in number of dopaminergic neurons when compared to MPTP-treated pink1−/− zebrafish. Statistical analysis with one-way anova and post hoc analysis using Tukey's multiple comparison test of two different experiments with n=20. (C) Kaplan–Meier survival curves depicting survival rate for 5 days of wt, pink1−/−, mcu−/− and (pink1; mcu)−/− zebrafish treated with 25 µg ml-1 MPTP (n=100). Scale bars:100 µm.

Mitochondrial dynamics is alteredin pink1−/− zebrafish. (A–L) Representative images of immunohistochemistry performed on wt, pink1−/−, mcu−/− and (pink1; mcu)−/− dissected 4 dpf zebrafish larval brain. DA neurons (red) are marked with anti-TH antibody, mitochondrial structures (green) are marked with anti-Tom20 antibody and DAPI (blue) is shown as nuclear stain. Arrowheads show mitochondrial morphology in muscle fibers. (M–O) Representative images of mitochondrial volumetric analysis in muscle fibers of transgenic wt, pink1−/− and (pink1; mcu)−/− 3 dpf zebrafish expressing mitochondria-localized GFP. Arrowheads point out mitochondrial structures in somites. (P) Graphical representation and statistical analysis of mitochondrial area with one-way ANOVA and post-hoc analysis using Tukey’s multiple comparison test of two different experiments with n=15. There was a significant (*P>0.05) decrease in DA neuronal mitochondrial area in pink1−/− zebrafish when compared to wt; mitochondria area was restored (**P>0.01) in (pink1; mcu)−/− zebrafish. (Q,R) Graphical representation and statistical analyses of muscle mitochondrial volume and sphericity with one-way ANOVA and post-hoc analysis using Tukey’s multiple comparison test of three different experiments with n=30. There was a slight decrease in mitochondrial volume in pink1−/− zebrafish when compared to wt. The cumulative sphericity index is significantly (***P>0.001) reduced in pink1−/− zebrafish, unlike wt and (pink1; mcu)−/− zebrafish.

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.

Nuclear distribution and shape indicate that hydrostatic pressure of the notochord is decreased in spzl mutants. ( A–B) Confocal images of cross sections of 14 dpf WT and spzl-/- larvae section. Vacuolated cells are labeled with SAG:gal4;UAS:GFP and nuclei are stained with DAPI. Scale bar = 50 µm ( C) Plot of vacuolated cell nuclear distribution for WT (red, n = 29) and spzl-/- (black, n = 32). ( D) Nuclear volume in WT and spzl-/- at 14 dpf. ( E–F) Reconstructions of nuclei in WT ( E) and spzl-/- ( F) and two different viewing angles. Scale bar = 20 µm ( G) Sphericity of WT and spzl-/- nuclei at 14 dpf. p-values were determined by an un-paired t-test using Welch’s correction.

Dopaminergic neurons are rescuedafter deleting mcu in pink1−/− zebrafish. (A–D) Representative images of wt (A), pink1−/− (B), mcu−/− (C) and (pink1; mcu)−/− (D) 3 dpf larvae after WISH using TH-specific riboprobe. (E–F) Representative images of wt (E), pink1−/− (F), mcu−/− (G) and (pink1; mcu)−/− (H) 3 dpf larvae after whole-mount FISH using TH-specific riboprobe and TSA/Cy3-based signal amplification. There was a significant decrease (P<0.001) in number of dopaminergic neurons in pink1−/− zebrafish when compared to wt. In (pink1; mcu)−/− zebrafish there was a significant increase (P<0.05) in number of dopaminergic neurons when compared to pink1−/− zebrafish. (I–L) Representative images of wt (I), pink1−/− (J), mcu−/− (K) and (pink1; mcu)−/− (L) 3 dpf larvae after immunohistochemistry using TH-specific antibody. Arrowheads show absence of dopaminergic neurons. (M) Graphical representation of chromogenic WISH. There was a significant decrease (**P<0.01) in number of dopaminergic neurons in pink1−/− zebrafish when compared to wt. In (pink1; mcu)−/− zebrafish, there was a significant increase (***P<0.001) in number of dopaminergic neurons when compared to pink1−/− zebrafish. (N) Graphical representation of immunohistochemistry. There was a significant decrease (***P<0.001) in number of dopaminergic neurons in pink1−/− zebrafish when compared to wt. In (pink1; mcu)−/− zebrafish there was a significant increase (***P<0.001) in number dopaminergic neurons when compared to pink1−/− zebrafish. The mean number of diencephalic dopaminergic neurons for wt, pink1−/−, mcu−/− and (pink1; mcu)−/− was calculated over three independent experiments (n=10 embryos per genotype and experiment). Scale bars: 100 μm.

Chronic ethanol treatment induced steatosis in the liver of adult zebrafish. (A) The relative mRNA expression of genes associated with lipid metabolism include lipogenic transcription factors, lipid synthesis, lipid uptake, and fatty acid transporting into the mitochondria. (B) Oil red O staining the section of liver in untreated control (left) and 0.2% ethanol treated wild type zebrafish. Scale bar = 50 μm. (C) The quantification of triglycerides in the liver of control and ethanol treated zebrafish. Error bars indicate standard deviation of the mean. * p < 0.05, ** p < 0.005.

Kdr Interacts with zVegfd In Vivo and Regulates Craniofacial Lymphangiogenesis but Not Development of the Thoracic Duct (A) Confocal images of 30-hpf Tg(fli1a:nEGFP) embryos injected with vegfdmRNA showing turned aISVs (arrowheads) in wild-type but not in kdrhomozygous mutant siblings. (B) Quantification of turned aISVs in wild-type (n = 39), kdruq38bh heterozygous (n = 42), and homozygous mutant (n = 15) embryos at 30 hpf. (C and C′) Analysis of thoracic duct (TD) development. Confocal images of 5-dpf Tg(fli1a:nEGFP);Tg(−5.2lyve1b:dsRed) transgenic embryos produced by in-crossing kdruq38bh carriers. TD development in kdruq38bh mutant embryos (C′) is equivalent to wild-type (C) siblings. Red denotes Lyve1-positive lymphatics and green denotes Fli1-positive nuclei. (D–F) Analysis of facial lymphatic development. Images showing the facial region of 5-dpf Tg(fli1a:nEGFP);Tg(−5.2lyve1b:dsRed) transgenic embryos. Genotypes in (D) and (D′) are kdr+/+, (E) and (E′) kdr+/−, and (F) and (F′) kdr−/−. Red denotes Lyve1-positive lymphatics and green denotes Fli1-positive nuclei. White arrow indicates mural LEC loop and white asterisk denotes its absence. (D′–F′) lyve1b:dsRed only; white anatomical structures are Lyve1-positive lymphatics. Yellow arrows indicate normal lateral facial lymphatics (LFLs), medial facial lymphatics (MFLs), and otolithic facial lymphatics (OLVs). Yellow asterisks indicate where there are reductions in LEC numbers in these vessels. (G) There was no significant difference in the number of nuclei in the TD when homozygous kdruq38bh mutants were compared to heterozygous and wild-type siblings (kdr+/+ embryos n = 11; kdr+/− n = 21; kdr−/− n = 16). (H) There was no significant difference in the number of nuclei in ISAs or ISVs when homozygous kdruq38bh mutants were compared to wild-type siblings (n = 16, 2 ISAs or ISVs from 8 embryos). (I–K) Significant differences in LFL (I), MFL (J), and OLV (K) LEC numbers were observed when homozygous kdruq38bh mutant embryos (n = 16) were compared to kdruq38bh heterozygous (n = 22) or wild-type (n = 12) embryos. ∗∗∗∗p < 0.0001. (L and M) In situ hybridization using probes complementary to kdr (L) and flt4(M) in 36-hpf embryos reveals robust expression in the region where lymphatic sprouts emerged from the common cardinal vein and the primary head sinus (arrows). (N and O) Confocal images of the TgBAC(vegfd:eYFP)uq42bh; Tg(kdrl:mcherry)transgenic line at 36 hpf (N) and TgBAC(vegfd:eYFP)uq42bh at 72 hpf (O) reveal expression of vegfd in the craniofacial region close to where lymphatic sprouts emerge from the primary head sinus (arrows). (P–U) Confocal images of 5-dpf Tg(fli1a:nEGFP);Tg(−5.2lyve1b:dsRed) transgenic embryos produced by in-crossing kdruq38bh/flt4hu4602 carriers. Genotypes in (P) are flt4+/+ kdr+/+, (Q) flt4+/+ kdr−/−, (R) flt4+/− kdr+/+, (S) flt4+/− kdr+/−, (T) flt4+/− kdr−/−, and (U) flt4−/− kdr+/+. In flt4hu4602 heterozygous embryos, loss of kdruq38bhresulted in a complete failure of the TD to form (T) and were phenotypically indistinguishable from flt4hu4602 mutant embryos. Arrows indicate TD and asterisks its absence. (V) Quantification of TD nuclei for embryos referred to in (P)–(U) (flt4+/+/kdr+/+embryos n = 11; flt4+/+/kdr+/− n = 12; flt4+/+/kdr−/− n = 4; flt4+/−/kdr+/+ n = 6; flt4+/−/kdr+/− n = 31; flt4+/−/kdr−/− n = 16; flt4−/−/kdr+/+ n = 8; flt4−/−/kdr+/− n = 14; flt4−/−/kdr−/− n = 8). All scale bars in this figure represent 100 μm except for (O), in which the bar denotes 50 μm. Data are represented as the mean ± SEM.

The expression of cathepsin L and CTSB-like during zebrafish embryogenesis. Wild-type embryos were analyzed with whole-mount  in situhybridization (WISH) at the indicated stages with antisense riboprobes against cathepsin L ( A) and CSTB-like ( B) for mRNA distribution. ( A) No appreciable signal for cathepsin L was observed in embryos before 8 hpf. Signals were significant in 12 hpf embryos at anterior prechordal plate (app) and subsequently in hatching gland (hg) at later stages. Signals were also abundant in the head region and swim bladder (sb) of 4-dpf larvae, which can be observed more clearly in a post-WISH cross-section at the trunk region (dash line and black arrow in inset). ( B) Intensive expression and homogenous distribution of CSTB-like transcripts were observed in embryos before 4 hpf. Signals became focused anteriorly in 1 dpf embryos and gradually diminished, as development proceeded, but nonetheless persistent in the trunk region along the vessels. Significant distribution appeared in the heart and the uninflated swim bladder area of 4 dpf larvae and became enriched in the region surrounding swim bladder at 5 dpf. app, anterior prechordal plate; h, heart; hg, hatching gland; sb, swim bladder.

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RyR-mediated muscle contractions are required for slow fiber maturation. (A-I) Slow fibers were visualized with F59 antibody at 20 and 24 hpf and with the slow fiber-specific S58 antibody at 48 hpf. Slow fibers, which are initially wavy, mature and align with respect to each other as wild-type embryos develop. In ryr1b mutant embryos, maturation is delayed; in paralyzed triple mutants, fiber maturation is arrested. (J) Sample image indicating how slow muscle fiber (yellow) and A-P somite (magenta) lengths were determined using ImageJ. (K) Sample image of sarcomere banding indicating how sarcomere A-P lengths (white bracket) were determined. (L) Quantification of slow fiber:somite length ratios. (M) Quantification of sarcomere lengths of slow muscle fibers. One-way ANOVA was used to determine statistical relationships at each developmental time point with Tukey's multiple comparisons test used to adjust P-values. To determine fiber length or sarcomere length, five fibers were examined in each of five different embryos for each condition. ns, not significant.