ZFIN Figure: Wang et al., 2023, Fig. 8

Fig. 8

Vascular patterning mediated by the interaction between stap2b and the Jak-Stat3 pathway. (Aa?Ac) Bright field images of wild-type, stap2bATG morphants and embryos treated with the STAT3-specific inhibitor stattic at 30 hpf. (Ad?Af) At 30 hpf, compared with the effect in the wild-type control (Ad), treatment of embryos with stattic caused a growth defect of ISVs similar to that of stap2b morphants (hollow arrowheads in Ae and Af). (B) Quantification of the percentage of completed ISVs in stap2b morphant and stattic-treated embryos was reduced approximately 60?65% compared to wt control. (C) A Western blot analysis showed reduced phosphorylation of Stat3 in stap2b morphants, and ?-actin was a loading control. The densitometry results are normalized and shown aside. (D?G) Double knockdown of stap2b and stat3 enhanced vascular defects during zebrafish development. (Da?Dd) At 30 hpf, the wild-type control showed normal growth of ISVs and CVP formation (arrowheads in Da, arrows in De). Double knockdown of stap2b and stat3 enhanced vascular defects in ISVs (hollow arrowheads in Dd), and fewer sprouting or loop structures in the CVP (arrows in Dh) compared to lower- dosage of stap2b (Db and Df, 0.85 ng) or stat3 (Dc and De, 0.85 ng) morpholino knockdown. (E) Quantification of the percentage of completed ISVs is approximately 95%, 38%, 39% and 8% in the wt control, stap2bMO, stat3MO and double-knockdown morphants (n = 20 in wt, n = 17 in stap2b MO, n = 16 in stat3 MO and n = 16 in stap2b + stat3 MOs) at 30 hpf. (F) Double knockdown of stap2b and stat3 caused the shorter average length of ISVs than in stap2bMO and in stat3MO. The average length in wt control, stap2bMO, stat3MO, and double-knockdown morphants was 113.1 � 4.9 ?m, 90.7 � 8.1 ?m, 92.9 � 6.8 ?m, and 68.5 � 5.2 ?m (n = 30 in wt, stap2b MO, stat3 MO and in stap2b + stat3 MOs) at 30 hpf. (G) Quantification of the number of loop structures in the CVP was approximately 28, 11, 12 and 8 in wt control, stap2bMO, stat3MO and double-knockdown morphants (n = 22 in wt, n = 21 in stap2b MO, n = 18 in stat3 MO and n = 18 in stap2b + stat3 MOs). (Ha?Hc) At 30hpf, compared to DMSO control in WT, treatment with JAK-specific inhibitor AG490 caused a growth defect in the ISVs (hollow arrowheads in Hc), similar to the vessel defects in stattic-treated embryos (hollow arrowheads in Hb). While chemical treatments in stap2b mutant (stap2b?3), stattic-treated stap2b mutants (He) showed the enhanced impairment of ISV growth compared to the vessel defect in DMSO-treated stap2b mutant (Hd) or stattic-treated (Hb) wild-type embryos. Similarly, AG490-treated stap2b mutants (Hf) enhanced the defect of ISV growth compared to DMSO-treated stap2b mutant (Hd) or AG490-treated wild-type embryos (Hc). (I) The percentage of completed ISVs in static or AG490-treated WT embryos or stap2b knockout embryos was quantified. Data are represented as means � SD. ***Refers to p < .001, **refers to p < .01, and *refers to p < .05 by an unpaired Student's t-test. Scale bars are 200 ?m for Aa?Ac, and 100 ?m for Ad?Af, Da?Dh, Hd?Hf.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ FASEB J.