Fig. 5

Fig. 5

Rescue of the tmem2^hu5935 Mutant Phenotype by Degrading HA Requires the Vegf Pathway

(A) Whole-mount pErk immunofluorescence staining (white) in sibling and tmem2^hu5935 mutants on the Tg(kdrl:EGFP) background (green) at 22 hpf, injected just 3 hr prior with dextran. Scale bar, 10 μm.

(B) Graph depicting the significant reduction in fluorescence intensity of pErk-positive endothelial cells in mutants (n = 43) versus sibling embryos (n = 56) following dextran injection.

(C) Whole-mount pErk immunofluorescence staining in sibling and mutant embryos 3 hr after HA'se injection. Scale bar, 10 μm.

(D) Scatterplots showing no significant difference in fluorescence intensity of pErk-positive endothelial cells in mutants (n = 90) versus sibling embryos following HA'se injection (n = 89).

(E) Untreated vegfaa morphants (n = 8) had fewer ISVs compared with uninjected controls (n = 10) and this reduction in sprouting was not significantly different following HA'se (n = 8), o-HA (n = 11) or HA'se inact treatment (n = 9).

(F) Likewise, untreated kdr/kdrl double morphants (n = 25) had no detectable ISVs compared with normal uninjected controls (n = 11), and this loss of sprouting was not significantly different following HA'se (n = 17), o-HA (n = 20) or HA'se inact treatment (n = 20).

(G) Design of rescue experiments for low and high dose SU5416 treatments shown in (H) and (I).

(H) Scatterplot for DMSO-treated embryos injected with HA'se (n = 27), o-HA (n = 29), or HA'se inact (n = 23) showing no difference from uninjected (n = 8) embryos. Low-dose (0.8 μM) SU5416 mildly but significantly impaired ISV sprouting in uninjected embryos (n = 21). SU5416-treated embryos injected with HA'se (n = 40) or HA'se inact (n = 46) were not significantly different from uninjected but o-HA injected embryos (n = 53) were significantly rescued (p < 0.0001).

(I) At a higher dose (1 μM SU5416), DMSO treatment again had no effect on embryos not injected (n = 8), injected with HA'se (n = 12), o-HA (n = 16), or HA'se inact (n = 11). At the higher dose, all treatments (uninjected [n = 13], HA'se [n = 12], o-HA [n = 18] and HA'se inact [n = 11]) were significantly different from DMSO with no rescue in any of these conditions observed. For all scatterplots uninjected (white circles), HA'se injected (yellow squares), o-HA injected (gray triangles), HA'se inact (red diamonds). ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001. Error bars represent ±SD.