Figure 2. Zebrafish gprc5b morphant embryos exhibit decreased lymphatic vessel formation. (A) Confocal images of Tg(fli1a: EGFP); Tg(kdrl: Hsa.HRASmCherry) control (uninjected) and 1 ng of gprc5ba morpholino-injected larvae at 72 h post-fertilization (hpf). The boxes in the above panels are enlarged in the bottom panels, respectively. White arrows point to EGFP-positive TD in the trunk region. Red arrows point to defects in TD formation in the trunk region. Anterior to the left, dorsal to the top. DA, dorsal aorta; PCV, posterior cardinal vein; TD, thoracic duct. (B) Quantification of EGFP-positive/kdrl-negative thoracic duct in the trunk (six somites). Uninjected embryos (N = 10) and gprc5ba MO-injected larvae (N = 13), from three independent clutches.

Figure 3. RNA sequencing analysis of slc38a9 mutant zebrafish. (A) Principal component analysis (PCA) plot of four wild type and slc38a9 mutant RNA-seq datasets. Principal component 1 (PC1), principal component 2 (PC2), and principal component 3 (PC3) were used for analysis. (B) Volcano plot of differential expression analysis of slc38a9 mutant and control larvae showing the relationship between −log10(padj) and log2FoldChanges. The red dot shows upregulated genes, the green dot shows downregulated genes, and the blue dot shows an unchanged gene. (C) Gene ontology (GO) analysis of differentially expressed genes in biological processes (red column), cellular component (green column), and molecular function (blue column). (D) The directed acyclic graph (DAG) of biological process. The lower terms are more specific, while the upper terms are more general. (E) Average heart rate of 60 hpf WT and slc38a9 mutant zebrafish larvae. Data shown are mean ± SEM, n = 10. * p < 0.05. (F) Correlation between qRT-PCR and RNA-seq results for select DEGs. The fold change values derived from the RNA-seq analysis of DEGs are compared with those obtained by qRT-PCR determined by 2−ΔΔCT. The reference line indicates the expected linear relationship.

Whole mount in situ hybridization detection of marker genes in Adar morpholino knockdown and adar mRNA overexpression. a–f pax6 expression demarcates anterior brain structures including dorsal retina (R), forebrain (FB) as well as hindbrain (HB) and anterior spinal cord. g–l tbx2b expression marking diencephalon (DE), R, epiphysis (EP), trigeminal ganglion (TG), and otic vesicle (OV). m–o expression of tbxta marks the notochord. p–r shhb expression indicates floor plate (FP) and midbrain-hindbrain boundary (MHB). The experiment was performed three times on embryos from three different mating pairs with similar results.

Inhibition of cingulin b affects the normal deposition of neuromasts in zebrafish. (A–E) In transgenic cldnb:lynGFP embryos, the neuromasts of PLL are labeled with green fluorescence. At 48 hpf, the deposition of neuromasts in zebrafish is shown in the control group (A), cingulin b knockdown group (B,C), cingulin b-MO + p53 group (D), and cingulin b-MO + cingulin b mRNA group (E), respectively. (F) The number of PLL neuromasts in controls (n = 235), cingulin b knockdown (2 ng or 3 ng) group (n = 86 and 264, respectively), cingulin b-MO (3 ng) + p53 group (n = 81), and cingulin b-MO (3 ng) + mRNA embryos (n = 180) at 48 hpf. The number of neuromasts decreased dose-dependently after knockdown of cingulin b(A–C,F). The decrease in the number of neuromasts is also confirmed when co-injecting with cingulin b-MO and p53(C,D,F). Combined injection of cingulin b-MO and cingulin b mRNA can partially rescue the decrease in the number of neuromasts caused by cingulin b-MO (E,F). Red arrowheads mark the neuromasts in the trunk, and white arrowheads mark the terminal neuromasts of the PLL system (A–E). Scale bars represent 100 μm. Data are shown in mean ± SEM. *Stands by the comparison with the control group: ***p < 0.0001. #Stands by the comparison between cingulin b-MO group and cingulin b-MO + cingulin b mRNA group: ####p < 0.0001. &Stands by the comparison between 2 ng cingulin b-MO group and 3 ng cingulin b-MO group: &⁣&⁣&⁣&p < 0.0001. ns means no significance. (G,H) The number of eya1 labeled neuromasts is markedly reduced after knocking down of cingulin b(G)n = 21 compared to the Control-MOs (H)n = 14. The black arrows in G and H indicate the neuromasts. Scale bars in panel (G,H) mark 50 μm.