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ZFIN ID: ZDB-FIG-170302-14
Chen et al., 2017 - Cilia Control Vascular Mural Cell Recruitment in Vertebrates. Cell Reports   18:1033-1047 Full text @ Cell Rep.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagents:
Anatomical Terms:
Stage Range: Long-pec to Protruding-mouth
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage Range: Long-pec to Protruding-mouth

Fig. 4

Notch Signaling Acts Downstream of Blood Flow to Recruit vMCs

(A) Shear-stress-mediated Notch activation in arterial-fated endothelial cells. Western blot analyses show increased levels of cleaved intracellular Notch (NICD) after 4 hr of LSS, whereas total Notch1 is unchanged. β-actin has been used as loading control. Histograms show the quantification of the amount of cleaved Notch1 (NICD)/over total Notch1 upon shear stress in endothelial cells.

(B) Shear stress induces Notch signaling in ECs. Effects of shear stress on the expression of different Notch target genes in human endothelial cell lines after 24 hr of LSS. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are represented as mean ± SD. Stars represent the results of two-way ANOVA.

(C) Loss of Notch signaling by blockade of blood flow in living zebrafish vessels. qPCR analyses of different Notch target genes are downregulated in ECs sorted from Tg(kdrl:egfp)s843 injected with control or gata1 MO at 48 hpf. ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Data are represented as mean ± SD. Stars represent the results of two-way ANOVA.

(D) Arterial Notch activation restores vMC recruitment in the absence of blood flow in zebrafish vessels. Confocal images of Tagln staining (green) on section of TgBAC(dll4:GAL4FF;UAS:RFP)hu10049Tg(UAS:NICD)kca3 injected with tnnt2 or control morpholino. Expression of NICD in arterial ECs (dll4:RFP) rescue vMC recruitment (arrow) in the absence of blood flow (gata1 morphants). All the embryos analyzed (n = 15 in both conditions) show the indicated phenotype. Blue, nuclei. Scale bar, 25 μm.

(E) Blood flow can promote Notch signaling and vMC coverage in arterial vessels in the absence of dll4. Injection of dll4 morpholino induces a reduction of EGFP-positive Se vessels in Tg(tp1:egfp)um14 embryos. The few Se vessels that are positive for Notch activity and vMC coverage are the ones with circulation (arrow). A total number of 23 controls and 30 morphants were analyzed. Scale bar, 50 μm.

(F) Notch signaling is impaired after dll4 inactivation (KD). Histograms show the percentage of tp1:GFP+ Se vessels in 500 μm of DA, which are reduced in dll4 MO (n = 22/155 Se) compared to controls (n = 85/128 Se). The coverage of tp1+ vessels by vMCs is not altered in dll4 MO (n = 9/22) compared to controls (55/85), showing that flow can still recruit vMCs. A total number of 23 controls and 30 morphants were analyzed. 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).

Arrows indicate vessels with blood flow in dll4 morphants.

See also Figures S5 and S6.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP um14Tg ; uto5Tg control Protruding-mouth artery endothelial cell IFL
Protruding-mouth dorsal aorta IFL
Protruding-mouth intersegmental artery IFL
um14Tg ; uto5Tg + MO2-dll4 control Protruding-mouth artery endothelial cell IFL
Protruding-mouth dorsal aorta IFL
Protruding-mouth intersegmental artery IFL
her9 s843Tg + MO1-gata1a control Long-pec endothelial cell RTPCR
hey1 s843Tg + MO1-gata1a control Long-pec endothelial cell RTPCR
hey2 s843Tg + MO1-gata1a control Long-pec endothelial cell RTPCR
jag1b s843Tg + MO1-gata1a control Long-pec endothelial cell RTPCR
mCherry um14Tg ; uto5Tg control Protruding-mouth dorsal aorta vascular associated smooth muscle cell IFL
Protruding-mouth intersegmental artery vascular associated smooth muscle cell IFL
um14Tg ; uto5Tg + MO2-dll4 control Protruding-mouth dorsal aorta vascular associated smooth muscle cell IFL
Protruding-mouth intersegmental artery vascular associated smooth muscle cell IFL
notch1b s843Tg + MO1-gata1a control Long-pec endothelial cell RTPCR
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
hu10049Tg; kca3Tg + MO1-tnnt2a control Protruding-mouth blood circulation decreased rate, abnormal
Protruding-mouth dorsal aorta vascular associated smooth muscle cell amount, ameliorated
s843Tg + MO1-gata1a control Long-pec endothelial cell notch1b expression decreased amount, abnormal
Long-pec endothelial cell jag1b expression decreased amount, abnormal
Long-pec endothelial cell hey1 expression decreased amount, abnormal
Long-pec endothelial cell hey2 expression decreased amount, abnormal
Long-pec endothelial cell her9 expression decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Cell Reports for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Cell Rep.