Bioinformatic and expression analysis of the zebrafish orthologues of Cacna2d4. (A) Maximum likelihood phylogenetic tree of the Cacna2d family. Eight members were identified in the zebrafish genome, two of which are clear orthologues of mammalian Cacna2d4 (dashed box). (B) Schematic representation of predicted protein structure applying to both members of the Cacna2d4 subclade (dashed box in A). Cacna2d4 proteins contain Van-Willebrand factor (VWA) and VWA N-terminal (VWA-N) domains, as well as one Cache domain, an N-terminal signal peptide and a ω site adjacent to a short C-terminal hydrophobic region, predicted to mediate proteolytic cleavage and GPI anchoring. Proteolytic cleavage (dashed line) of the Cacna2d4 pro-form gives rise to α2 and δ peptides, which remain attached to each other through disulfide bonds in mature α2δ proteins. (C) In situ hybridization of zebrafish cacna2d4a and cacna2d4b. In 3 dpf zebrafish, cacna2d4a is expressed throughout the retinal INL and in photoreceptors (Phs) of the retinal periphery (arrowheads). In the adult retina, cacna2d4a is expressed in the INL and the distal half of the Ph layer. In larval zebrafish, cacna2d4b shows strong expression in Phs, weak expression in the INL and additional signal in the cerebellum. In the adult retina, cacna2d4b is expressed across the Ph layer and in the INL. Arrows in (B) indicate proteolytic cleavage. aa, amino acids; CB, cerebellum; Dr, Danio rerio; GCL, ganglion cell layer; Hs, Homo sapiens; Mm, Mus musculus. Scale bars correspond to 100 μm in images of 3 dpf zebrafish and 50 μm in adult sections.

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.

The p53 pathway mediates cell development in HCO cells and zebrafish. a Expression of the FAS gene in cells transfected with different lentiviruses (sh2, sh3, and shNT as a scramble control) at 3 days before cell confluence. HCO cells subjected to EFTUD2 knockdown (sh2 and sh3) exhibited increased FAS levels. b The expression of CASP3 in the sh2 and sh3 groups was dramatically increased compared with that in the shNT group. cSTEAP3 mRNA levels in the sh2 and sh3 groups were markedly higher than that in the negative control. dP21 mRNA levels were also highly increased in the sh2 and sh3 groups. eSESN1 was highly expressed in the sh2 and sh3 groups. f HCO cells transfected with sh2 had a higher expression of phosphorylated P53 protein (53 kDa) than the nontransfected (control) and shNT groups. g Expression of relevant genes involved in the P53 pathway in the EFTUD2 knockout (−/−) and WT (+/+) zebrafish. h The expression of phosphorylated P53 in EFTUD2-mutated larvae (−/−) with curved bodies and WT larvae at 4 dpf. i The survival rate among the curved F3 generation hybridizing from EFTUD2 heterozygous mutants (EF3 control), EF3 controls injected with EFTUD2 normal mRNA (EN mRNA), and p53 morpholino (P53-MO) during the early developmental stage (*P < 0.05, **P < 0.01, and ***P < 0.001)

Whole mount in situ and transverse section hybridization analysis of rab11a in zebrafish embryos. A 64 cell, lateral view. B 12 hpf, dorsal view, red arrowheads indicate the ubiquitous expression. C 20 hpf, lateral view, red arrowheads indicate endoderm. C’ 20 hpf, dorsal view, red arrowheads indicate brain. D 24 hpf, dorsal view, red arrowheads indicate brain. D’ 24 hpf, dorsal view, red arrowheads indicate hindbrain. E 36 hpf, lateral view, red arrowhead indicates brain. F 48 hpf, lateral view, red arrowhead indicates brain. G 72 hpf, lateral view, red arrowhead indicates brain. H 4 dpf, lateral view, red arrowhead indicates brain; green arrowheads indicate pronephric duct. H’ 4 dpf, dorsal view, arrowhead indicates brain. H” 4 dpf, lateral view, green arrowheads indicate pronephric duct. I 48 hpf, the trunk transverse section of the embryonic forebrain, arrowheads indicate cerebral cortex. I’ 48 hpf, the trunk transverse section of the embryonic midbrain, arrowheads indicate cerebral cortex. I” 48 hpf, the trunk transverse section of the embryonic hindbrain, arrowheads indicate cerebral cortex. J 48 hpf, lateral view, embryos stained with the sense rab11a probe

csf1r genes show combinatorial effects in regulating patterning of the dentition and tooth number. (A-D,F) Alizarin Red stained pharyngeal arches from 4 month post-fertilization (mpf) csf1r mutants and wild-type siblings. Numbers on the bottom represent the range of tooth numbers on each arch (n=7). Arrows indicate buildup of bone surrounding base of teeth in csf1ra and csf1rb mutants (B,C). (E) Quantification of the number of teeth represented in csf1r mutants, double mutants and wild-type siblings (****Pcsf1ra;csf1rb double mutants showing organization of supernumerary teeth into distinct tracts (yellow line) associated with a row of normal tooth germ patterning in wild-type dentitions (Fig. S6). (G-J) Localization of ctsk-positive cells (magenta) on the dentary of zebrafish csf1r mutants. Bottom, overlay with calcein stain (cyan).

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.

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.

In situ hybridisation detects OCRL expression in the glomeruli of zebrafish and adult human kidney tissue. a Top panels show OCRL expression in a whole-mount zebrafish embryo (left panel, lateral view, anterior to the left) and in transverse section through the glomerulus (right panel, highlighted with white dotted outline) at 5 days post fertilisation (dpf). b In situ hybridisation of normal adult human kidney tissue showing OCRL expression within the glomerulus. The left panel shows a low magnification image with glomeruli and proximal tubules labelled. The right panel shows a close-up view of the glomerulus with OCRL expression labelled in podocytes (black arrow), mesangial cells (green arrow) and endothelial cells (red arrow)

(a) Whole-mount in situ hybridization showing the expression of PGC marker genes, including nanos1 and vasa, after morpholino injection at 25 hpf. The reduction of nanos1 and vasa expression intensity was obviously observed in MOaug-injected embryos. The nanos1 expression was also decreased in MOsp-injected embryos and it was recovered when mRNA was co-injected. The expression levels of nanos1 (b) and vasa (c) were graded as the normal and abnormal percentage. The mean pixel intensity of nanos1 and vasa gene expression of the normal embryos were 24.7 ± 8.7 and 22.6 ± 8.2, respectively. An abnormal level of the gene expression was taken as one with the value less than the mean minus one standard deviation of the gene expression of the wild-type, i.e., less than 16 for nanos1 and 14.4 for vasa gene. The number of animals quantified in each group are shown on top of the bars.