ZFIN Image: Chen et al., 2017, Fig. 5

Image

Figure Caption

Fig. 5

Cilia Are Required to Drive vMC Recruitment

(A) Pharmacological impairment of primary cilia formation negatively regulates vMC recruitment. Confocal images of partial z-projections of the trunk region (somite 8–14) of Tg(kdrl:egfp)^s843;Tg(acta2:mCherry)^uto5 embryos at 80 hpf (merged and single channels) after disrupting cilia formation by CBD treatment. Compared to controls, CBD-treated embryos show reduced vMC coverage around the DA (arrows). Scatter plots show the quantification of vMC number per 500 μm of the DA at 80 hpf. n = 39 and 53 embryos. Data are represented as mean ± SD. Stars represent the results of unpaired t tests of mean difference = 0 (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001). g, gut.

(B) Endothelial-specific genetic inhibition of ift88 gene expression impairs vMC coverage. Confocal images of partial z-projections of the trunk region (somite 8–14) of Tg(kdrl:egfp)^s843Tg(acta2:mCherry)^uto5 embryos at 80 hpf (merged and single channels) injected with the integratable CRISPR vector for endothelial-specific ift88 gene targeting. Cmlc2:GFP-negative embryos were used a negative control (ctrl). Scatter plots show the quantification of vMC number per 500 μm of the DA at 80 hpf. n = 20 and 20 embryos. Data are represented as mean ± SD. Stars represent the results of unpaired t tests of mean difference = 0 (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001).

(C) Inhibition of cilia function by CBD treatment impairs Notch signaling in living zebrafish blood vessels. qPCR analysis of Notch target genes in DMSO and 1 and 2 μM CBD-treated embryos. qPCR analysis of Notch target gene expression in zebrafish treated with different concentrations of CBD at 48 hpf. A zebrafish trunk was used for RNA extraction and qPCR analyses. Notch1b, dll4, and Jagged1b are the most affected Notch targets by the lack of cilia at this stage of development (48 hpf). Data are represented as mean ± SD. Stars represent the results of two-way ANOVA. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.