The Vegf pathway functions downstream of tbx16 in HSC specification. Whole-mount in situ hybridization of embryos between 36 and 40 hpf (c-myb expression) or approximately 28 hpf (vegf, flk1, or efrinb2a). Red arrowheads represent arterial expression of HSC markers; green arrowheads, intersomitic vessels. Lateral views, anterior left; low magnification (original magnification ×10) and higher magnification (original magnification ×40) of the trunk region. (A,B) Injection of approximately 40 pg vegf₁₂₁ RNA into wild-type embryos expands c-myb–positive HSCs. (C,D) vegf expression in wild-type (C) and tbx16^−/− mutants (D). (E-J) Uninjected (E,G,I) or vegf injected (F,H,J) tbx16 mutant embryos showing substantial rescue of c-myb, flk1, and efrinb2a in the trunk.