FIGURE 3.

c-Abl1 interacts with Abi1 but does not affect eye development. A, whole mount in situ hybridization of stage 34 embryos using a c-abl1 antisense probe, showing expression in eye, among other tissues. ba, branchial arches; ov, otic vesicle; fb, forebrain; p, pronephros. B, Xenopus embryos were injected with HA-tagged abi1 RNA (500 pg) and/or FLAG-tagged c-abl1b RNA (1 ng). Extracts were co-immunoprecipitated (IP) with anti-FLAG or -HA antibodies and probed with anti-FLAG, -HA, or -Tyr(P) (p-Tyr) antibodies as indicated. Abi1 is detected in the c-Abl1b immune complexes and vice versa, and Abi1 is phosphorylated in the presence of Abl1b. C, top, Western blot of Xenopus oocyte lysates probed with anti-FLAG antibody, showing that c-Abl1b MO (10 ng) effectively blocks translation of exogenous c-Abl1b-FLAG protein (10 ng). Center, Western blot to show effect of c-Abl1b MO on endogenous protein. Xenopus embryos were injected into both blastomeres at the two-cell stage with 10–40 ng of c-Abl1b MO or 40 ng of STD MO. Embryos were analyzed at stage 12.5, using a c-Abl1 antibody. Bottom, 10 ng of c-Abl1b MO or STD MO was co-injected with GFP mRNA into the D1.1.1 blastomere at the 32-cell stage, and eye development was analyzed at stage 37–38. The fluorescent cells containing c-Abl1b MO can enter the eye field, and the eye forms normally. D, top, embryos were injected into the D1.1.1 blastomere at the 32-cell stage with GFP mRNA alone or with c-abl1a (K270R) or c-abl1b (K290R) RNAs, and eye development was analyzed at stage 37. The fluorescent progeny cells can enter the eye field, and the eye forms normally. Bottom, Western blot performed on siblings of the embryos shown above, showing that the dominant-negative c-Abl1 proteins are expressed above endogenous c-Abl1 levels.