Loss of Myof affects the skeletal muscle of zebrafish embryos. (A)myof knockdown was achieved by injecting a splice-target morpholino (myof-MO). (B) Loss of Myof results in wavy and abnormally patterned myofibers (Myosin, red), and disruption of the myosepta, seen by a decreased intensity in the staining and increased width (Dystrophin and Dystroglycan, green). (C)Analysis of the myofiber ultrastructure by electron microscopy shows disruption of myofiber structure (C4) and their sarcomeres (arrows, C5, C6), abnormally shaped and distributed mitochondria (arrowheads, C5), when compared to control mitochondria (arrowheads, C1), and Z-disc thickening (arrowheads, C6).  

WISH analysis of ackr3b and cxcr4a expression during zebrafish embryo development. WISH analysis of ackr3b(A–H) and cxcr4a(I–J) mRNA expression was performed at the indicated stages of development. (A–C,E,G,I,J) Lateral view of whole-mount embryos. (D) Coronal cross section through the trunk highlighted as a dashed vertical bar in (C), anterior to the top; arrows indicate somitic ackr3b expression. (F,H) Transverse cross sections through the trunk highlighted as dashed bars in (E,G), respectively. Arrows in (F,H) indicate ackr3b-positive developing gut and the basal part of the somites, respectively. (A–C,E,G,I,J) Scale bar: 100 μm. (D,F,H) Scale bar: 25 μm.

RA signaling regulates irx2a expression during nephrogenesis. (A) WISH analysis at the 28 ss demonstrates alterations in the irx2a pronephros domain after either treatment of RA or DEAB. The black arrowheads show the distance from distal irx2a expression to the cloaca, and the black lines highlight irx2a expression. (B) Quantification of irx2a length (μm) at the 28 ss. Each dot represents one pronephros. (C) Quantification of the length from irx2a expression to the cloaca (μm), where each dot represents one pronephros. For each graph, data is represented +/− SEM and significance was determined with an ANOVA. WISH – whole mount in situ hybridization, ss- somite stage, RA – retinoic acid, DEAB - N,N-diethylaminobenzaldehyde.

Blocking formation or function of primary cilia impairs hematopoietic stem and progenitor cell (HSPC) development. a Confocal imaging of cilia in the aorta-gonad-mesonephros (AGM) region in kdrl:mCherry/βact:Arl13b–GFP double-transgenic line with fli1a:ift88-cKO-functional injection, or ciliobrevin D (CBD) treatment at 28 hpf. White arrow denotes the blood flow direction. Scale bar, 5 µm. b, c The quantification of primary cilia number and length in the blood vessels of AGM in a. Data in b, c represent the analysis results of one-way ANOVA–Dunnett test. Error bars, mean ± s.d., n = 10 embryos. ns non-significant, *P < 0.05, ****P < 0.0001. d WISH result of runx1 (red arrowheads) in control and CBD-treated embryos at 31 hpf. The red arrowheads indicate the expression of HSPC marker runx1. Scale bar, 100 µm. e The kdrl:mCherry+/cmyb:EGFP+ HE cells (white arrowheads) in the AGM region in fli1a:ift88-cKO-injected embryos (left panel) with quantification (right panel) at 36 hpf. Cmlc2:EGFP-negative embryos were used as a negative control (control). Scale bar, 50 µm. Error bars, mean ± s.d., n = 14 embryos. **P < 0.01, Student’s t-test

Combined loss of cep290 and cc2d2a does not exacerbate cone degeneration or rhodopsin trafficking defects. Panels show immunohistochemical analysis of dorsal retinas from wild-type (top), cep290fh297/fh297 (middle), and cep290fh297/fh297;cc2d2a+/- mutants (bottom) at 6 months of age stained with (A) PNA (red) and Zpr-1 (green) to label cone photoreceptor; (B) Zpr-3 to label rhodopsin; or (C) GRK1 to label rhodopsin kinase. ROS, rod outer segments; COS, cone outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cells. Scale bar: 50 μm. (D) Quantification of cone outer segment density or (E) rhodopsin mislocalization from the indicated genotypes at 6 months of age. See methods section for details on quantification. Removing one allele of cc2d2a from a cep290fh297/fh297mutant background had no effect on cone degeneration or rhodopsin mislocalization. At least 10 unique fish over at least 2 independent experiments were evaluated. *** P < 0.0005; **** P < 0.0001 as determined by a 1-way ANOVA with a Multiple Comparisons test and Tukey corrections. Data represented as means ± s.e.m.

Otic expression of fgf genes following mis-expression of fgf3 or inhibition of Hh signalling.(A–F’) In situ hybridisation for otic expression of fgf genes in Tg(hsp70:fgf3) embryos following a 30-minute heat shock (HS) at the 10-somite stage (14 hpf). Controls (left-hand panels of each pair of images) were sibling non-transgenic (Non-Tg) embryos subjected to the same heat shock. Numbers of embryos shown in the dorsal view panels indicate the number showing the phenotype from a mixed batch of transgenic and non-transgenic embryos in each pair of panels; 75% of the batch is expected to be transgenic. A–B’ show staining with a probe specific to the 3’ UTR of fgf3: note the ectopic patch of endogenous fgf3 expression at the posterior otic pole (B,B’; arrowheads) and disruption to fgf3 expression ventral to the otic vesicle (B’; asterisks) after heat shock in transgenic embryos. Expression of fgf10a is strengthened in the otic vesicle of transgenic embryos after heat shock (E–F’). (G–Q’) Expression of mRNA for fgf genes in embryos treated with 100 μM cyclopamine (cyc) from the 10-somite stage (14 hpf) until 22.5 hpf. Controls in the left-hand panels of each pair of images were treated with vehicle (ethanol) only. Numbers of embryos with the phenotype shown for individual treatments are indicated in the dorsal view panels. There was little change to the otic expression patterns of fgf3 or fgf8a at 22.5 hpf (8.5 hours post treatment) (G–J’), but note the loss of fgf3 expression ventral to the ear (H’; asterisk). Expression of fgf10a in the otic vesicle was strengthened after inhibition of Hh signalling in about 50% of treated embryos (L,L’). At 36 hpf (8.5 hpt + 13.5 h wash), ectopic expression of both fgf3 and fgf8a appeared in a new posteromedial domain in the ears of cyclopamine-treated embryos (M-P’; arrowheads). (Q,Q’) Expression of fgf8a in the posterior of the otic vesicle at 48 hpf (8.5 hpt + 25.5 h wash). Ectopic expression has strengthened (arrowhead) and medial epithelium is thinner than normal (brackets). Dorsal views of the left ear, with anterior to the top. Scale bar in A, 50 μm (applies to A–L); scale bar in A’, 50 μm (applies to A’–L’); scale bar in M, 50 μm (applies to M–P); scale bar in M’, 50 μm (applies to M’–P’); scale bar in Q, 20 μm (applies to Q,Q’).

Forward genetic screen reveals tfap2a is necessary for nephrogenesis in the developing zebrafish pronephros. (A) Zebrafish pronephros schematic. P, podocytes; N, neck; PCT, proximal convoluted tubule; PST, proximal straight tubule; DE, distal early; CS, corpuscle of Stannius; DL, distal late; CD, collecting duct. (B) Whole-mount in situ hybridization at 24 hpf. Scale bar: 70 µm. (C) SNPtrack from whole-genome sequencing at chromosome 24, with G>A tfap2a mutation in trm−/−. Exon diagram: tfap2a spliceoforms (pink, cyan and orange); black asterisks indicate alternative start sites; location of MO (blue) and tfap2am819 lesion (red); conserved nucleotides (green), mutant nucleotides (red) and primer locations (purple). (D) RT-PCR with mutant bands 1-4 (green) and table of predicted consequences from sequence analysis. TAD, transcriptional activation domain; DBD, DNA-binding domain. (E) Whole-mount in situ hybridization with pharyngeal arches (white outlines) and DE (black box) indicated. Scale bars: 70 µm (left) and 35 µm (right). (F) trm mutants exhibit abnormal craniofacial cartilage (black arrowheads) and pericardial edema (blue arrowheads). Scale bar: 200 µm. (G) Alcian Blue staining, with gaping jaw (black arrowhead); black dotted lines trace Meckel's cartilage. Scale bar: 100 µm. (H) Immunofluorescence of Tfap2a (green) and Slc12a1/2 (magenta). White dots show the pronephros border. Scale bars: 50 µm (top), 10 µm (bottom).

Adult mutants have normal retinal structure, but mir-183/96/182lri78 survival is depressed. (a,b) Survey of retinal anatomy in adult fish at (a) 12 m or (b) 6 m. Markers used here are equivalent to those used for imaging of larval fish, except that we also examined PCNA and Gfap staining to look for evidence of cell proliferation and gliosis, respectively. (c) Quantification of PCNA labeling in double mutants. PCNA + cells were counted across a 10μm thick retinal cross-section at the level of the optic nerve. Proliferating cells in the optic nerve head and in the ciliary marginal zone were excluded. ns = not significant (ANOVA). For all data points, n = 3. (d) Survival curve of fish from incrosses of WT and mir-183/96/182lri78 heterozygous parents. Each of the two cohorts began the study with a population of 51 fry. (e) Histological analysis of a surviving 15wk mir-183/96/182lri78 homozygote, showing normal retinal anatomy. Arrows in (a), (b), and (e) denote PCNA+ nuclei.  

Thalidomide Retards Brain Development and Causes Microcephaly (A) Head morphology of 72-hpf zebrafish that were grown in the presence of 0.1% DMSO (left), 200 μM thalidomide (middle), or 400 μM thalidomide (right). Thin vertical lines indicate the distance between the rostral-most tip of olfactory bulbs and the temporal-most edge of eyes. Otic vesicles (arrowheads) and pectoral fins (arrows) are also indicated. (B) A schematic diagram depicting body length (black arrow), eye diameter (blue arrow) and head thickness (red arrow) that were used to determine ratios. (C and D) The ratios of eye diameter (blue arrow) in (C) and head thickness (red arrow) in (D) to body length (black arrow) of zebrafish that were grown with or without 400 µM thalidomide were determined and are shown as means ± SEM (n = 20 per group). (E) Primary neurons of 24-hpf embryos that were grown in the presence of 0.1% DMSO or 200 or 400 μM thalidomide. Where indicated, capped mRNA encoding crbnWT or crbnYW/AA was microinjected at the 1-cell stage before thalidomide treatment. Bright-field (BF) (upper panels) and fluorescence (lower panels) images of embryos immunostained with acetylated α-tubulin antibody are shown. Tel, telencephalon; SOC, supraoptic commissure; TPOC, tract of postoptic commissure; PC, posterior commissure. (F) Fluorescence intensity of acetylated α-tubulin-positive neural clusters in telencephalon and TPOC. Fluorescence intensities of the regions indicated with rectangles in (E) were measured and normalized to the intensity of DMSO-treated embryos and are shown as means ± SD (n = 15 per group). (G) Percent incidence of head phenotype (n = 100 per group in a single trial). The head sizes of 72-hpf embryos were classified based on the head-to-body ratio using the following criteria: ≥13%, normal; <13%, small. Scale bar, 50 μm. *p < 0.05, ***p < 0.001.