vdINVP grew in a VEGF-dependent manner.

(A) Experimental design to examine effects by sFLT-1 or VEGF. cDNAs were electroporated unilaterally into the neural tube at E2 (HH14), and manipulated embryos were infused with highlighter ink at E5/HH26, flowed by flat-mounted preparation. (B) Control electroporation with EGFP. (C) sFLT-1 was co-electroporated with EGFP. vdINVP was excluded from the sFLT-1/EGFP positive area. (D) The inhibitory action by sFLT-1 was also observed in conventional transverse sections (arrowhead). sFLT-1-overexpression did not cause gross effect at least on the progenitor zone. (E) VEGF co-electroporated with DsRed caused ectopic vascularization extending to VEGF-electroporated sites (arrows). Such hypervascularization (arrow) was also confirmed in a transverse view processed for 70 μm thick (Z-stack) by confocal microscopy, where growing plexus could be seen as continuous tissue (similar to Fig. 2 A, B, E) (F). (G-J) Similar manipulations with four different cDNAs as indicated (flat-mounted preparation). pBI-Wnt7a-EGFP/pCAGGS-tTA, pBI-SFRP2-EGFP/pCAGGS-tTA, pCMS-Noggin-EGFP, and pCMS-BMP4-EGFP, were tested. No gross effects were appreciated on the vdINVP patterning. Scale bars: 100 μm for (D, F), 200 μm for (B, C, E, G–J).