Ectopic Bergmann glial cells (BGs) in reln mutants. (A–H) Aberrant localization of BGs in reln mutants. Sagittal section of adult (96 dpf) SAGFF(LF)251A; UAS:GFP brains, which express GFP in BGs, harboring WT (control, n= 5) or homozygous reln mutant (relnΔ7/Δ7, n= 5) alleles were stained with anti-Pvalb7 (magenta) and anti-GFP (green) antibodies. (B-D, F–H) High magnification images of the boxes in A and E. (I) Ratio of the BG (GFP+) area in the GCL to the total BG area in controls and relnΔ7/Δ7mutants. Fluorescence images were captured of every fourth section (14 total sections in each fish). The GFP+ area in the Pvalb7-negative GCL layer was measured and divided by the total GFP+ area in the cerebellum. *p< 0.05 (Mann-Whitney test). Scale bars: 300 μm in A (applies to A and E); 50 μm in B (applies to B-D and F–H).

Defects in the migration and polarity of PCs in reln mutants. Sagittal sections of the brain from WT (A-C, n= 4) and relnΔ7/Δ7 (D-H, n= 4) 15-dpf larvae, and from WT (I, n= 3) and relnΔ7/Δ7 (J, n= 3) 30-dpf fish, were stained with anti-Pvalb7 (magenta) and anti-Vglut1 (green) antibodies, and Hoechst (cyan). Typical cerebellum images are shown. The ventral limit of the cerebellum is indicated by a dotted line. (Ba, Ca, Da, Ea, Fa, Ga, Ha) High magnification images of the PCs marked by “a” in B–H. The Pvalb7 and Vglut1-double positive region marks the ML. Migrating PCs were detected in the GCL in WT, and they extended a neurite (primary dendrite) toward the pial side (Ba, Ca, n= 4). Many of the migrating cells extended one or multiple neurites in aberrant directions in the relnΔ7/Δ7mutants (Ea, Fa, Ga, Ha, n= 4). At 30 dpf, most of the PCs had reached the PCL in WT (I), whereas many ectopic PCs were detected in the GCL in relnΔ7/Δ7 mutants (J, n= 3). The abbreviations are described in the legend for Fig. 1. Scale bars: 20 μm in A (applies to A-H); 100 μm in I (applies to I-J).

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.  

Tracing liver tumor cells during tumor regression in adult fish. (A) Flowchart of fish treatment and sample collection. CreER and CreER/xmrk adult fish (4 mpf) were treated with Dox for 6 weeks to induce HCC. 4-OHT was added at 5 wpi (weeks post-induction) for 3 days to cause loxPrecombination for marking the tumor cell lineage with RFP expression. Fish were collected at 6 wpi, 1 wpr (weeks post-regression), 2 wpr and 4 wpr for various assays. (B) Fluorescent protein expression and histological analyses of livers. Rows 1 and 2 are representative images of gross observation of GFP and RFP in the livers under a stereomicroscope. Row 3 shows representative confocal images of the fresh liver tissue immersed in phosphate buffer to show the re-establishment of the two-cell hepatic plate structure from tumor-reverted hepatocytes during tumor regression. Row 4 shows representative images of fixed liver sections for GFP expression, RFP expression (stained with anti-RFP antibody) and nuclear staining by DAPI for quantification of cell number. Row 5 shows representative images of H&E staining. Black arrows, large and irregular nuclei; yellow arrows, prominent nucleoli. (C) Quantification of percentages of GFP+ hepatocytes and RFP+ hepatocytes in total hepatocytes based on liver sections from panel B row 3 (n=10). (D) Quantification of 2D liver size based on the left lateral view of dissected fish as shown in row 2 of panel B (n=10/group). *P<0.05.

Identification and expression of zebrafish shootin genes. (a) Schematic representation of zebrafish shootin1, shootin2, shootin3 and mouse shootin1 protein structures. The top box for mouse shootin1 indicates the structural domains described previously26, and the bottom three boxes for zebrafish shootin1, shootin2 and shootin3 indicate the corresponding structural domains. CC1-3, coiled-coil domains 1–3; PR: proline-rich domain. (b) RT-PCR analysis of shootin1, shootin2 and shootin3transcripts. Elongation factor 1a (EF1a) was used as a control. Developmental stages are denoted in hours post-fertilization (hpf). RT-PCR products produced in the presence (RT+) or absence (RT−) of reverse transcriptase were electrophoresed using a 3% agarose gel. (c–e) Whole-mount in situ hybridization of shootin1 (c), shootin2 (d) and shootin3 (e) in zebrafish embryos at 36 hpf. Arrowheads and arrows indicate neuromasts and PLLP, respectively. Enlarged images indicate expression of shootin1and shootin3 in the last deposited neuromasts (c’ and e’) and the PLLP (c” and e”). Scale bars: 200 μm for whole body images and 25 μm for enlarged images.

Development of the microglia population in larval zebrafish. (a) Representative confocal images are shown to illustrate zebrafish larval head development from 1 to 7 days postfertilization (dpf) and microglial cell distribution throughout the developing brain. Upper panels correspond to a brightfield transmission image and lower panels represent the maximum intensity projection of 4C4+ microglia (magenta) at each developmental stage. Microglia start colonizing the brain (dotted line) at 3 dpf whereas signal can be detected in the retina from 1 dpf onwards. Scale bar represents 50 μm. (b) Upper panels show 4C4 antibody immunohistochemistry, lower panels show segmented images of microglia morphology at 3, 5, and 7 dpf using the Imaris surface tool. Microglia morphology changes from amoeboid (1 dpf) to ramified (7 dpf) with an intermediate feature at 5 dpf. Scale bar represents 10 μm. (c) Schematic representation of the protocol used to isolate 4C4+ microglia from larval zebrafish brains at 3, 5, and 7 dpf. All images represent maximum intensity projections of confocal stacks. Images were captured using a Zeiss LSM710 confocal microscope with a 20×/NA 0.8 objective [Color figure can be viewed at http://wileyonlinelibrary.com]

RNA in situ hybridization of slc2a3a in the adult brain. Pictures (A–R) show cross sections of an adult zebrafish brain (80 μm) from anterior to posterior as indicated in the scheme. (B’–R’) are high magnifications of boxed areas in (B–R). Detailed descriptions can be found in the text, for abbreviations see Table 1. Scale bar in (A), 100 μm and pertains to (A–R); scale bars in (B’–R’), 20 μm.

Localization of Reln protein in the tectum and the cerebellum. Medial and lateral sagittal sections of the brain from 30-dpf (A–P) and 90-dpf (Q–T) WT and relnΔ7/Δ7 mutant zebrafish were stained with anti-Reln (magenta), anti-Vglut1 (green), and Hoechst (cyan). Three fish for 30-dpf and one fish for 90-dpf WT or relnΔ7/Δ7 mutant fish were analyzed. Typical images are shown. (E-H, M-P) High magnification images of the boxes in C, D, K, and L. In WT, Reln protein was detected strongly in the SM and relatively weakly in the SO in the TeO. Reln was also detected strongly in the ML in the Cb. Weak Reln signals were also detected in the GCs in the TL and the GCL. These signals were absent in relnΔ7/Δ7mutants. CC, crista cerebellaris. The other abbreviations are described in the legend for Fig. 1. Scale bars: 100 μm in A (applies to A-D, I-L); 50 μm in E (applies to E-H, M-P); 200 μm in Q (applies to Q-T).

Heterogeneous Wnt reporter expression and β-catenin cytoplasmic localization in adult β-catenin-driven HCC. (A) Stacked bar graph showing analysis of Wnt reporter (7xTCF-Xla.Siam:mCherry) expression in liver tissue of Tg(fabp10a:flox-pt-β-cat) (HepABC) HCC and zebrafish livers of sibling controls lacking this transgene (NonTg). Wnt reporter expression was scored as: A, absent (no mCherry expression); L, low (mCherry expression in less than 10% of cells); or H, high (mCherry expression in greater than 10% of cells). P-values derived from Fisher's exact test comparing samples with (low or high) and without (absent) Wnt reporter expression. Graph shows data from one experiment. (B,C) Stacked bar graphs showing quantification of β-catenin localization by immunofluorescence staining performed on paraffin-embedded sections (B) or cryosections (C) in HepABC HCC and NonTg zebrafish livers. Samples were scored based on amount of cytoplasmic staining: 0, no cytoplasmic staining; 1+, focal (<10%) weak to moderate cytoplasmic staining; 2+, focal strong cytoplasmic staining or patchy (10–50%) weak to moderate cytoplasmic staining; 3+, diffuse (>50%) cytoplasmic staining. P-values derived from Fisher's exact test comparing samples with membrane staining only (0+) to those with cytoplasmic staining (1+ to 3+). Each experiment was performed once. (D) Stacked bar graph showing quantification of β-catenin localization in 6-mpf CreLox zebrafish with HCC and control siblings without HCC lacking either the Cre driver or lox-switch transgene [Tg(fabp10a:flox-pt-β-cat) and Tg(fabp10a:CreERT2), control]. Numbers above x axis indicate the sample size for each group. P-values determined by Fisher's exact test. Experiment was performed once. (E) Representative β-catenin and Wnt reporter images of cryosections from a HepABC liver diagnosed as HCC (top panels) and a NonTg liver diagnosed as no/minimal changes (bottom panels). Arrow indicates a cell with cytoplasmic β-catenin localization; white arrowheads indicate Wnt reporter expression. Scale bars: 30 µm. Insets contain 5× magnified images of regions of tissue in smaller boxes for each image. (F) Representative immunofluorescence images of β-catenin staining. Control zebrafish showed membrane staining only, whereas CreLox zebrafish with HCC showed varying degrees of cytoplasmic staining. Scale bars: 50 µm. In each image, large inset box is 5× magnification of small box.