Figure 5.

Vessel Sprouting Is Compromised by Nrp1 Knockdown or Cdc42 Inhibition in Zebrafish

(A) Alignment of the nrp1a/b MO nucleotide target sequence with the 5′ UTR immediately upstream of the start codon (underlined) in nrp1a and nrp1b; asterisks indicate identical nucleotides.

(B) Alignment of the peptide sequence recognized by anti-NRP1 antibody with the cytoplasmic tail of human and mouse NRP1 as well as zebrafish Nrp1a and Nrp1b; asterisks indicate identical amino acid residues, and the colon indicates a conservative substitution.

(C and D) Protein lysates from control and nrp1a/b and nrp1b MO-injected 32-hpf fish embryos were used for Nrp1 and Akt immunoblotting after electrophoresis using a 10% gel (C) or 4%–12% gradient gel (D).

(E–L) Confocal z stacks of trunks from 32 hpf Tg(fli1a:EGFP)^y5 zebrafish and corresponding quantitation of the indicated vascular parameters for controls versus nrp1a/b MO (E–H) and vehicle versus ML141 treatment (I–L); scale bar, 200 μm. A kinked tail caused by Nrp1 knockdown is also seen after Cdc42 inhibition (red arrows). Boxed areas are shown at higher magnification adjacent to each corresponding panel to illustrate delayed sprout extension; scale bar, 25 μm. Δ indicates impaired sprout invasion into the dorsal trunk. The number of all ISV sprouts (F and J), full-length ISV sprouts (G and K), or ISV sprouts that have fused laterally (H and L) is shown as mean ± SD (n ≥ 4 fish for each treatment condition). Asterisks indicate p values for nrp1a/b MO or ML141 relative to controls ^∗∗p < 0.01, ^∗∗∗p < 0.001; hashtags indicate p values for 75 μM relative to 25 μM ML141 #p < 0.05, ###p < 0.001.