Fig. 3

Knockdown of nrarp-a and nrarp-b in Zebrafish Leads to Abnormal Intersegmental Vessel Formation

(A–D) Tg(fli1:EGFP)^y1 zebrafish were injected with 10 ng control (A), nrarp-a (B), nrarp-b (C), or 5 ng each of nrarp-a and -b (D) morpholinos and analyzed at ∼56 hpf. (B′–D′) nrarp-a and -b morphants show defective ISVs. Arrows indicate disconnected vessels; arrowheads indicate ISVs with delayed dorsal migration; yellow arrowheads show misdirected ISV formation. Anterior of embryos is to the left. DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel; PAV, parachordal vessel; DA, dorsal aorta; PCV, posterior cardinal vein.

(E–H) Whole-mount collagen XII (red) staining in 48 hpf zebrafish Tg(fli1:EGFP)^y1 embryos. Normal myotome boundaries are observed in nrarp-a, -b, and double nrarp-a and –b morphants (F–H). Arrows indicate disconnected vessels. ^* in (G) indicates missing ISV at the myotome boundary.

(I–K) Confocal time-lapse stills of control (I) and nrarp-a (J) or nrarp-b (K) morphants between 48 and 56 hpf. The ISVs of control morphants remained connected to the DA and DLAV (I). Note that the middle and right ISVs of the control morphant is out of focus. ISVs in nrarp-a and -b morphants regress over time (J and K). In (J), red arrows indicate poor connection between vessels and yellow arrow points to a regressing vessel. In (K), red arrow points to regressing vessel along the path of DLAV formation; yellow arrow indicates detachment of ISV from DA.

(L) Quantification of vessel disconnection. Graph shows the percentage of ISVs with disconnection between the ISV and DA, within the ISV, or between the ISV and DLAV. Ten embryos (8 ISVs/embryo) were analyzed for each morphant.