ZFIN Figure: De Angelis et al., 2017, 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.

Expression Data

Gene:	EGFP
Antibody:	Ab9-mapk
Fish:	s843Tg cemip2^{hu5935/hu5935}; s843Tg
Anatomical Term:	dorsal aorta endothelial cell
Stage:	26+ somites

Expression Detail

Antibody Labeling

Phenotype Data

Fish:	WT WT + MO2-vegfaa s843Tg WT + MO1-kdr + MO4-kdrl
Condition:	chemical treatment by environment: semaxanib
Knockdown Reagents:	MO1-kdr MO2-vegfaa MO4-kdrl
Observed In:	dorsal aorta endothelial cell intersegmental vessel
Stage Range:	26+ somites to Prim-25

Phenotype Detail

Acknowledgments

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Reprinted from Developmental Cell, 40, De Angelis, J.E., Lagendijk, A.K., Chen, H., Tromp, A., Bower, N.I., Tunny, K.A., Brooks, A.J., Bakkers, J., Francois, M., Yap, A.S., Simons, C., Wicking, C., Hogan, B.M., Smith, K.A., Tmem2 Regulates Embryonic Vegf Signaling by Controlling Hyaluronic Acid Turnover, 123-136, Copyright (2017) with permission from Elsevier. Full text @ Dev. Cell