Expression of cingulin b is detected during the early development of zebrafish. (A,B)In situ hybridization staining of cingulin b at 3.7 hpf (n = 13) from the lateral view (A) and the top view (B). (C)Cingulin b is expressed in the whole somite at 14 hpf from the lateral view (n = 14). (D–E) The expression of cingulin b is focused on the neuromasts of the posterior lateral line system at 48 hpf (n = 11). Scale bars mark 50 μm in panel (A–D). The black arrows in D indicate neuromasts, and the white dotted lines labeled neuromast in D is magnified in panel (E).

The efficacy of cingulin b-MO. (A–D) The expression of cingulin b is significantly reduced in the cingulin b-MO morphants (C), n = 8 compared to that in the Control-MO embryos (A)n = 5. (E) Quantitative analysis on the level of cingulin b between Control-MO and cingulin b-MO groups (n = 8 in each group). Data are shown in mean ± SEM, **p < 0.01. Scale bars in panel (A,C) mark 50 μm. The black arrows in panel (A) indicate the neuromasts, and the white dotted lines labeled neuromasts in panel (A,C) are magnified in panel (B,D), respectively.

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.

The proliferative cells in the PLL primordium are severely decreased while downregulation of cingulin b. (A–F) Representative images of BrdU positive proliferating cells and DAPI labeled nuclei in the controls (A,C,E) and cingulin b-deficient embryos (B,D,F) at 36 hpf. Red arrows indicate the rosette-shaped cell clusters in the primordium (A). Scale bars mark the 10 μm scale. (G) The quantitative analysis of BrdU index in control (n = 16) and cingulin b-MO embryos (n = 18). Data are shown in mean ± SEM. ****p < 0.0001.

Knockdown of cingulin b reduces the number of HCs and cell proliferation in the neuromasts of zebrafish at 72 hpf. (A–H) The immunochemical staining of PLL neuromasts in the Control-MO group (n = 19) and cingulin b-MO group (n = 15). DAPI (green) labels nuclei (A,E) and BrdU (red) labels proliferative cells in the neuromast (B,F). In transgenic Tg (brn3c: mGFP)^s356t lines, the membrane of HCs in PLL neuromasts are labeled with green fluorescence (GFP) (C,G). (I) The average number of hair cells per neuromast is significantly reduced after inhibition of cingulin b. (J) BrdU index in the neuromasts is also severely downregulated in the cingulin b-MO injected embryos compared to the controls. Scale bars mark 10 μm (A–H). Data are shown in mean ± SEM, and ****p < 0.0001. (K) The differentiation of HCs indicated by atoh1 ISH staining is inhibited after injection with cingulin b-MO. Scale bars in panel (K) mark 30 μm.

KEGG enrichment analysis screens out top 13 pathways which are highly differentiated expressed between controls and cingulin b-MO mutants. The analysis is conducted from three independent experiments in different groups (n = 30 embryos in each group), and p value <0.05 is considered as remarkable difference.

The key KEGG pathways: MAPK signaling pathway and cellular senescence signaling pathway. The red nodes represent upregulated DEGs in cingulin b-MO mutants, the green marked node represents downregulated DEGs in cingulin b-MO mutants, and the blue marked node represents overlapping targets between Control-MO and cingulin b-MO embryos. The analysis is conducted from three independent experiments in different groups, and each group has 30 embryos.

Heatmap analysis of MAPK signaling pathway and cellular senescence signaling pathway in comparison between Control-MO embryos and cingulin b-MO morphants. The red indicates upregulated DEGs and the blue indicates downregulated DEGs. The analysis is conducted from three independent experiments in different groups, and each group has 30 embryos.

The relative mRNA levels of the indicated genes from MAPK and cellular senescence signaling pathways were normalized to the GAPDH level as determined by qRT-PCR. The results are recorded as mean ± SEM from three independent experiments (n = 8 embryos in each group). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.