Cxcr4 promotes Cyclin D1 expression through ERK signaling during DFC proliferation. (A) ERK1/2 phosphorylation levels were dramatically decreased in cxcr4aum20 mutants. WT and cxcr4a-deficient Tg(sox17:GFP) embryos were harvested at the 75% epiboly stage and subjected to immunostaining for p-ERK1/2 (red) and GFP (green). All embryos are shown in dorsal views with anterior to the top. Scale bar, 20 μm. (B–E) caMEK mRNA overexpression in DFCs rescued L–R patterning defects in cxcr4aum20 mutants. Different types of heart looping and liver laterality at 48 hpf in cxcr4aum20 mutants following midblastula injection of different caMEK mRNA doses were visualized by cmlc2 and hhex expression (B and C). The ratios are shown in (D) and (E). Underlying data can be found in  S1 Data. (F) Cxcr4a-deficient Tg(sox17:GFP) embryos were injected with 60 pg caMEK mRNA at the 256-cell stage and then harvested at the 75% epiboly stage for fluorescence in situ hybridization experiments with cyclin D1 (red) and GFP (green) probes. Dorsal views with anterior to the left. Scale bar, 20 μm. (G–H) WT embryos were treated with 0.5 μM PD0332991 or 0.2 μM CY202 from the shield stage to bud stage and then analyzed for L–R patterning defects at 48 hpf by in situ hybridizations with cmlc2 and hhex probes. The proportion of treated embryos exhibiting each type of heart looping and liver laterality is shown in (G) and (H). Underlying data can be found in  S1 Data. (I–J) Reintroduction of cyclin D1 into DFCs relieves DFC proliferation defects in cxcr4aum20 mutants. Cxcr4a-deficient Tg(sox17:GFP) embryos were injected with or without 300 pg cyclin D1 mRNA at the 256-cell stage, followed by coimmunostaining with anti-BrdU (red) and anti-GFP (green) antibodies at the 75% epiboly stage. Representative images are shown in (I), and the percentage of BrdU-positive DFCs is indicated in (J). Scale bar, 20 μm. Student t test, ***P < 0.001. Underlying data can be found in  S1 Data. (K–M) The reduced ratios of embryos affected in KV size (K) and cmlc2 (L) or hhex (M) expression show that DFC-specific overexpression of cyclin D1 rescued the defects of KV formation (K) and L–R patterning (L and M) in cxcr4a mutants. Underlying data can be found in  S1 Data. BrdU, bromodeoxyuridine; caMEK, constitutively activated version of MEK; cmlc2, cardiac myosin light chain 2; DFC, dorsal forerunner cell; ERK, extracellular regulated MAP kinase; GFP, green fluorescent protein; hhex, hematopoietically expressed homeobox; hpf, hours postfertilization; KV, Kupffer’s vesicle; L–R, left–right; ns, no significant difference; p-ERK1/2, phosphorylated ERK1/2; sox, SRY-box transcription factor; Tg, transgene; WT, wild-type

irx2a acts upstream of etv5a. WISH at the 28 ss revealed decreased etv5a expression in the pronephros of irx2a morphants. Black bars denote the etv5a expression domain. Scale bar is 50 μm. (B) Quantification of etv5a length in μm at the 28 ss. Each dot represents one nephron and data is presented +/− SEM. Significance was evaluated with an unpaired student’s T-test. (C) The irx2a domain is unchanged in etv5a morphants at the 28 ss. Solid black bars denote strong irx2a expression and the dashed black lines denote faint expression in the pronephros. (D) Quantification of irx2a length (μm) at the 28 ss. Each dot represents one nephron and data is presented +/− SEM. An unpaired student’s T-test was used to determine significance. WISH – whole mount in situ hybridization, ss – somite stage.

Chronic ethanol treatment induced hepatic injury, inflammation in the liver and elevation of serum glucose and triglycerides. Adult zebrafish were exposed to 0.2% ethanol for 4 weeks. (A) Hematoxylin and eosin (H&E) staining and Picrosirius Red staining in the liver of untreated and 0.2% ethanol treated adult zebrafish (top and middle). Control livers showed normal hepatocytes (n = 8/10) and ethanol treated siblings showed hepatic injury (n = 9/10). Control livers showed thin layer of picrosirius red positive staining surrounding blood vessels (n = 6/6), ethanol exposed group showed intense staining of fibrotic tissue in some cases (n = 2/5) Scale bar = 50 μm (top, bottom), 25 μm (middle). (B) Alanine aminotransferase assay from blood samples of control, 0.2% ethanol exposed zebrafish. (C) Relative mRNA expression of genes associated with inflammation and liver injury. RNAs extracted from three to five siblings were used for analysis. (D) Quantification of serum glucose in control and ethanol treated zebrafish. (E) Quantification of serum triglycerides in control and ethanol treated zebrafish. Error bars indicate standard deviation of the mean. * p < 0.05, ** p < 0.005.

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).  

Whole mount in situ hybridization demonstrates reduction in four key brain transcription factors after Apex1 knockdown in both wild-type and p53 mutant embryos with rescue by co-injection of mRNA for human APEX1.Whole mount in situ hybridization shows aberrant distribution of critical brain markers after Apex1 knockdown. Rescue was achieved by co-injection of transcript for human APEX1. Similar results were obtained in p53 mutant embryos in which Apex1 was knocked down. Whole mount in situ hybridization was performed to examine fezf2, otx2, egr2a, and pax2a expression after knockdown of Apex1 in wild-type and p53 mutant embryos. Expression of each transcription factor decreased, and distribution was altered in both Apex1 MO injected wild-type and p53 mutant embryos, but was rescued by co-injection with human APEX1 capped mRNA. Note the small heads and eyes in Apex1 knockdown embryos. Hindbrain neurons (HBN) indicated by pax2a expression were no longer visible in Apex1 MO injected embryos (pax2a panel). Alteration in distribution or amount of signals is marked with arrows or brackets. KD knock down, WT wild-type, Res Apex1MO + human Apex1 rescue, p53m p53 mutant embryos, FB forebrain, MB midbrain, r5 hindbrain rhombomere 5, OS optic stalk, MHB midbrain-hindbrain boundary, OV otic vesicle. Whole mount in situ hybridization was performed with 20 embryos/group. All embryos are shown with anterior to the left

Depletion of Bnc2 Causes Pericardial Effusion, Hydrocephalus, Glomerular Cysts, and Distal Pronephric-Outlet Obstruction in Zebrafish (A) Immunoblot analysis shows a protein decrease in bnc2 MO-injected zfl for Bnc2-202 (130 kDa, C7DZJ6 according to UniProt ID) but not for Bnc2-201 (110 kDa, F1R42 according to UniProt ID) at 2 days dpf, which is as predicted for the specific MO target side. UI = uninjected. (B) H&E staining of a sagittal section of a MO-injected zfl (lateral view, head to the left) shows (PE), hydrocephalus (HY), abnormal body curvature and a “vesicle” due to an outlet obstruction of the pronephric ducts (arrowhead in enlargement in B) at 56 hpf. (C) Zfl injected with bnc2 MO frequently develop a distal-outlet obstruction at 33 hpf; this obstruction is highlighted by an arrowhead in the enlargement of (C). (D) Whole-mount ISH with a pax2a probe relates the constituent parts of the bnc2 MO-induced pronephric-outlet obstruction to distal parts of the pronephric ducts and the cloacal region (black arrow). (E) ISH with a bnc2 probe in bnc2 MO-injected embryos shows bnc2 mRNA expression in relevant tissues forming a pronephric-outlet obstruction (black arrow). (F and G) Zfl injected with bnc2 MO develop glomerular cysts (white arrowheads in G) and dilatation of the pronephric ducts (white asterisk in G). Images from in vivo observation through fluorescence microscopy (dorsal view) in Tg(wt1b:GFP) were taken at 54 hpf in zfl injected with control (Ctrl) MO (F) and bnc2 MO (G). Tg = transgenic zebrafish line. (H) The graph shows 100% of all at 1 dpf surviving zfl of the five different cohorts: UI, Ctrl MO, bnc2 MO, bnc2 MO + WT human RNA, and bnc2 MO + mutated human RNA (transcript ENST00000380672) bearing the p.His888Arg variant (for absolute numbers, see Figure 3H). A distal “vesicle” due to an outlet obstruction (phenotype) of the pronephric ducts can be seen in 21% of bnc2MO-injected zfl compared to 0% of zfl with a “vesicle” in both control groups (p < 0.05, unpaired t test). 6% of zfl injected with bnc2 MO + WT mRNA develop a “vesicle,” and so do 16% of zfl injected with bnc2 MO + p.H888R. Data are presented as means with standard error of the mean (SEM). (I) Quantification of glomerular cyst rates (phenotype) in Tg(wt1b:GFP) zfl at 2 dpf depicts significantly (p < 0.05, unpaired t test) clear glomerular cysts in 57% of bnc2 MO-injected zfl. 26% of zfl injected with bnc2 MO + WT RNA develop pronephric cysts; 51% of those injected with bnc2 MO + p.His888Arg RNA develop pronephric cysts. Data are presented as means with SEM. (J) Quantification of death rates at up to 3 dpf shows an increased mortality up to 64% in bnc2 MO-injected zfl compared to zfl injected with UI (7%) and Ctrl MO (6%). With bnc2 MO injected, zfl show significantly (p < 0.0001, Mantel-Cox test) reduced survival. Co-injection of bnc2 MO + human WT RNA results in a mortality of 69%. Aggravated mortality up to 81% can be detected in the zfl injected with bnc2 MO + p.His888Arg RNA. Scale bars represent 200 μm (B), 50 μm (magnification in B), 500 μm (C), 100 μm (magnification in C), and 100 μm (D–G). ∗∗p < 0.005, ∗∗∗ p < 0.0005.

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.

irx2a genetic mutants do not have a renal or eye phenotype. (A) Schematic of the irx2a transcript and location of the irx2asa10716 mutation, where grey is non-coding and black represents coding sequence. The forward arrow demarcates the ATG start site. (B) WISH analysis at the 28 ss shows odf3b and slc12a3 expression in irx2asa10716+/+, irx2asa10716+/−, and irx2asa10716−/− embryos. The black lines highlight the DL marker slc12a3. An enlarged dorsal view of the odf3b+ MCCs for each embryo is shown in the inset. Scale bars are 50 μm. (C) Gel image of the restriction digest product for each genotype. Wild-type bands are marked with a blue star, and mutant bands with a red star. (D) Quantification of odf3b+ cells at the 28 ss. Each dot represents one pronephros. (E) Quantification of the slc12a3 length (μm) at the 28 ss. Each dot represents one nephron. (F) Quantification of eye area (μm2). Each dot represents one eye. For all graphs, data is presented +/− SEM and statistical significance was determined by ANOVA. WISH – whole mount in situ hybridization, ss – somite stage, DL – distal late, MCC – multiciliated cell.

pen/lgl2 mutant distal retina displays no significant abnormalities in polarity or cellular morphology.Immunostaining of transverse retinal sections at 5 dpf. (A,C,E,G,I,K,M,O) Sibling and (B,D,F,H,J,L,N,P) mutant fish. (A–B′) Zpr1, (C–D′) WGA, (E–F′) Zpr2, (G–H′) Crb2a, (I–J′) aPKC, (K–L′) Moe, (M–N′) ZO-1 and (O–P′) N-cadherin immunostaining of distal retina. PRC, photoreceptor layer; INL, inner nuclear layer; OS, outer segment; OPL, outer plexiform layer; RPE, retinal pigment layer; OLM, outer limiting membrane. Scale bar: 10 µm.