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.

gabra1 expression in the developing zebrafish. WISH was performed at 1, 2 and 3 DPF with an anti-sense gabra1 probe. Arrows indicated the expression of gabra1 at each developmental stage. Minimum of ten larvae per group/biological replicate. Abbreviations: FB, forebrain; OT, optic tectum; C, cerebellum; HB, hindbrain; MO, medulla oblongata. Scale bars: 0.2 mm.

Maternal-zygotic mutant embryos have defective chorion elevation but normal patterning. (A) Three successive stages of a single embryo from a larp6a−/−;larp6b−/− female crossed to a larp6a+/−;larp6b+/− male show that embryonic and chorionic defects do not prevent normal hatching and development. The chorion is tight. All zygotic genotypes appeared similar. Scale bar: 200 μm. (B) Yolk inclusions (arrowheads) in 24 hpf larvae of the indicated genotype, quantified in the graph. The yolk (asterisks) is yellow in double mutants. Scale bars: 500 µm. (C) Timelapse imaging of chorion elevation reveals failure in eggs derived from double mutant females. There is reduced chorion elevation (brackets) and uneven cytoplasm in unactivated egg (arrowheads), and uneven egg surface after activation (arrows). Scale bar: 200 μm. (D) Mild reduction in survival of fertilised eggs between 1 and 28 hpf in lays from mutant females. Subsequent survival did not differ between genotypes and no effect of paternal genotype was observed. Data are mean±s.e.m., number of lays is indicated on each column. (E) In situ hybridisation for the dorsal marker gene chordin and germring marker gene squint show that Mlarp6a−/− embryos are not ventralised compared with wild-type controls at 30% epiboly. Upper panels are lateral views; lower panels are animal views; arrowhead indicates dorsal. Scale bar: 200 µm.

Schwann cells develop normally in Sarm1-deficient zebrafish.a Confocal images of a double-transgenic 5dpf larva showing Schwann cells marked by expression of GFP (green) under the control of the Tg[gSAGFF202A] Gal4 driver, and lateralis afferent neurons marked by expression of mCherry under the control of the SILL enhancer (red). Wild type (top), Sarm1 mutants (bottom). Scale bar 20 μm. b Images show the indicated time points after axon transection (hours post-injury = hpi) from a videomicroscopic recording of Schwann cells (green) and their interaction with axons (red) in wild type and Sarm1−/−. White arrowheads indicate Schwann cells engulfing axonal debris in the wild type. A white arrow indicates degradation-resistant axon segment in Sarm1−/−. Please note that the proximal axon stump in Sarm1−/− is not visible in these images because it is outside the focal plane.

AICAR and Metformin Treatment Improve the Muscle Phenotype in a Zebrafish Model of Dysferlin Deficiency (A) Morpholino oligonucleotides (MOs) targeting zebrafish dysferlin. (B) A single RT-PCR product of 301 bp was seen in 4-dpf embryos injected with 1.5 ng of control MO; two differently sized RT-PCR products of 301 and 195 bp were seen in 4-dpf embryos injected with 1.5 ng of dysferlin. (C) Panels represent the results of the birefringence assay (BR). The abnormal structure of the muscles is more clearly seen in dysferlin MO-injected fish (MO, lower panel) than in controls (CMO, upper panel) under BR. Scale bar, 200 μm. (D) Histogram of the percentage of normal, affected (reduced BR), and dead fish upon MO injection. After injecting 1.5 ng of MO, approximately 30.0% of the embryos exhibited reduced birefringence. The white, black, and gray bars show the percentage of normal, reduced BR, and dead fish, respectively. WT, uninjected fish; CMO, CMO-injected fish; MO, MO-injected fish. (E) Representative western blot image of phospho-AMPK, AMPKα, and β-actin in MO-injected fish treated with AICAR for 4 days at 0, 0.1, 1.0, or 10 mM. Quantification of phospho-AMPK expression was normalized to AMPKα in the lower panels. (F) AICAR treatment reduced the percentage of BR-reduced fish. Results for metformin concentrations of 0, 0.1, 1.0, and 10 mM are the average of three independent experiments. Of the untreated MO morphants, 31.1% were BR reduced. AICAR treatment at concentrations of 0.1, 1.0, and 10 mM reduced the percentage of BR-reduced fish to 22.2%, 17.8%, and 17.8%, respectively. Statistically significant differences were determined using ANOVA followed by Tukey’s multiple comparisons test. Statistical significance was set at *p < 0.05. (G) Representative western blot image of phospho-AMPK, AMPKα, and β-actin in MO-injected fish treated with metformin for 4 days at 0, 1, and 10 mM. Quantification of phospho-AMPK expression was normalized to AMPKα in the lower panels. Results for metformin concentrations of 0, 1, and 10 mM are the average of three independent experiments and were normalized to those without metformin treatment. (H) Metformin treatment reduced the percentage of BR-reduced fish. Results for metformin concentrations of 0, 1, and 10 mM are the average of three independent experiments. Of the untreated MO morphants, 33.3% were BR-reduced fish. Metformin treatment at concentrations of 1 and 10 mM reduced the percentage of BR-reduced fish to 25.0% and 20.8%, respectively. Statistically significant differences were determined using ANOVA followed by Tukey’s multiple comparisons test. Statistical significance was set at *p < 0.05.

Genome editing generates likely null alleles of zebrafish larp6a and larp6b. (A) In situ RNA hybridisation for larp6a and larp6b at the indicated stages. (B) Schematic of larp6a and larp6b genes and proteins showing the position of kg139 and kg153 mutant alleles. The larp6akg139 frameshift mutation produces a truncated protein with the first 29 amino acids of Larp6a followed by a 72 amino acid tail lacking both La motif and RRM domains. The larp6bkg153 frameshift allele truncates Larp6b at amino acid 17 with an eight amino acid tail terminating in coding exon 1. There is a lack of in-frame ATG codons near the termination site. (C) In situ RNA hybridization for larp6a mRNA on genotyped larp6akg139 mutant and wild-type siblings from a larp6akg139/+ incross reveals nonsense-mediated mRNA decay (NMD) of mutant larp6akg139 mRNA at 24 hpf. Eleven out of 47 low expressors were shown to be mutant and 10/47 normal expressors were wild type upon sequence genotyping. As larp6b mRNA is primarily maternally expressed, NMD was analysed at the 256-cell stage by larp6b mRNA in situ RNA hybridization on lays from incrosses of larp6bkg153 F3 mutants or their wild-type siblings (see Table S1). The results shown were observed in 33/33 embryos from a wild-type female and 30/30 from a mutant female. Genotypes of mutant embryos shown were confirmed by sequencing. (D) QRT-PCR on RNA from 1k stage embryos from wild-type or larp6akg139;larp6bkg153 double mutant incrosses confirmed reduction of each mutant mRNA. Symbols indicate results from three individual RNA preparations from three separate pairs of lays. (E) Lack of larp6b mRNA upregulation in larp6akg139 mutants. All images are lateral views with anterior to the left and dorsal upwards, except 128 cell and 50% epiboly, which are animal upward. Scale bars: 200 µm.

Notochord vacuole morphology and arrangement is altered with increasing or decreasing mineralization. ( A, C) Live confocal images of fish treated with 0.0002% DMSO for 1 week at 6.0 mm and 6.5–7 mm respectively. ( B, D) Live confocal images of fish treated with 100 nM RA for 1 week at 6.0 mm and 6.5–7 mm respectively. Fish expressed nfatc:gal4;UAS:mcherry in the notochord and were stained with calcein to label centra. Scale bar = 100 µm. n = 3 for DMSO control and RA treatment. Notochord is outlined with a dotted white line. White arrows point to areas of increased mineralization, blue arrows point to the floor plate. Arrowheads indicate areas of increased vacuole fragmentation. Asterisks mark vacuolated cell stacking. ( E) Live confocal image of a 7.5 mm WT zebrafish stained with Cell Trace to visualize internal membranes and live alizarin to label mineralized bone, n = 3. ( F) Live confocal image of a 7.5 mm nobhu3718 stained with Cell Trace and alizarin red to label bone. n = 3. Black arrows point to vacuolated cells undergoing shape change. Asterisks mark stacked vacuolated cells. White arrows point to centra stained with alizarin red. v indicates rounded vacuoles that have not undergone shape change, fragmentation, or stacking. The notochord is outlined with a dotted fuchsia line. Scale bar = 100 µm. ( G–H) Cryosections of a 9.5 mm WT ( G) and nobhu3718 ( H) larvae stained with WGA (green) and alizarin red (magenta). Brackets indicate IVD domains. Arrows point to mineralized centra. The notochord is outlined with a dotted white line. Arrowheads point to mesenchymal condensations. Scale bar = 100 µm.

Cells expressing L-plastin are more abundant across the heterozygous mutant (psen1K97fs/+) zebrafish brain than in wild type siblings at 24 months.Immunostaining for the pan-leukocyte marker L-plastin supports increased numbers of microglia in the forebrain (A-B), midbrain (C-D) and hindbrain (E-F). Increased microglial abundance is evident in psen1K97fs/+ heterozygotes in both ventricular (D.i, F.i) and parenchymal (D.ii) regions compared to wild types (C, E). (G) Significant differences in MFI were observed between the forebrain, midbrain and hindbrain of psen1K97fs/+ and psen1+/+ fish; **p = 0.0048, ***p = 0.0005, ****p < 0.0001; two-way ANOVA with Sidak’s multiple comparisons test. Data presented as means with SEM. Scale bar 50 μm in all images.

Maternal effect of larp6a−/− and larp6a−/−;larp6b−/− on oogenesis. (A) Bright-field images of 5 dpf larvae from the indicated crosses. Fish are shown with anterior towards the left and dorsal upwards. Inflated swim bladders show that larvae have swimming and swallowing capacity. (B) Slow myosin immunodetection in 48 hpf larvae from a larp6a−/−;larp6b−/− female crossed to a larp6a+/−;larp6b+/− male reveal no differences in slow muscle fibre formation. Images are centred on somite 17/18 and the graph shows the number of slow fibres per myotome±s.d., tested using Welch's ANOVA on SPSS. (C) Bright-field images of lays at 3 hpf from wild-type, larp6a−/−, larp6b−/− and larp6a−/−;larp6b−/− females (upper panels) or males (lower panels) crossed to wild-type AB. Embryos from males of any genotype or wild-type females show a fully elevated chorion (ch), translucent and smooth yolk (y), tall and symmetrical blastodisc (bd), large chorion diameter (brackets), and large subchorionic space (asterisk). All embryos from maternal larp6a−/− mutants have reduced chorion diameter, reduced subchorionic space (black lines) surrounding a yolk cell of unaltered size (red lines) but more opaque and uneven. Lays from larp6b−/− females are indistinguishable from wild type. Lays from larp6a−/−;larp6b−/− females are like larp6a−/− embryos, but have even smaller chorion diameter (brackets). (D) Chorion diameter cumulative frequency distribution curves. Light colours indicate individual clutches strong colour indicates the mean for each genotype. (E) Average of the mean chorion diameters of n clutches from separate females of indicated maternal genotype±s.d. One-way ANOVA with Tukey's post-hoc test on SPSS. Scale bars: 1 mm in A,C; 100 µm in B.