FIGURE

Fig. 5

ID
ZDB-FIG-150924-9
Publication
Wu et al., 2015 - Histone Demethylases KDM4A and KDM4C Regulate Differentiation of Embryonic Stem Cells to Endothelial Cells
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Fig. 5

KDM4A and KDM4C Are Required for Capillary Tube Formation and Vasculogenesis In Vivo

(A) mESCs were transfected with scrambled, Kdm4a, or Kdm4c siRNA and underwent differentiation protocol. Unsorted differentiated cells were plated on Matrigel, and tube formation was evaluated by phase-contrast microscopy. Knockdown of either Kdm4a or Kdm4c prevented tube formation. Values are mean ± SD (n = 3 independent experiments). Bar graphs of tube lengths in indicated groups (p < 0.05 by Student’s t test).

(B) Unsorted differentiated cells treated as in (A) were suspended in Matrigel containing VEGF and injected subcutaneous into nude mice. Matrigel plugs were harvested on day 7 and assessed by H&E staining. Bar graphs show vessel area. Immunostaining was performed using anti-CD31 and anti-VE-cadherin antibodies. Values are mean ± SD (n = 3 independent experiments). Bar graphs show CD31 and VE-cadherin signal intensity (p < 0.05 by Student’s t test).

(C) Targeting either Kdm4a or Kdm4c with MOs induced significant defects in vasculogenesis in zebrafish embryos, with defects being more prominent in the Kdm4a-MO group. In each case, fertilized zebrafish eggs were injected with 2-ng Control-MO, Kdm4a-MO, or Kdm4c-MO, and the embryos were observed at 24 and 48 hpf. Kdm4a morphants displayed only residual inter-somite vessels (ISV) derived from DA and complete lack of DLAV, whereas Kdm4c-MO showed a less severe phenotype with reduced number of DLAV at 24 hpf. Kdm4a morphants did not survive beyond 36 hpf. In contrast, although the response was delayed in Kdm4c morphants, they developed an apparently normal vasculature at 48 hpf similar to control embryos. Bar graphs show blood vessel area and the length of inter-somite vessels at 24 and 48 hpf (p < 0.05 by ANOVA).

Expression Data

Expression Detail
Antibody Labeling
Phenotype Data
Fish:
Knockdown Reagents:
Observed In:
Stage Range: Prim-5 to Long-pec

Phenotype Detail
Acknowledgments
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