Knockdown of ephrin-A5 expression by 40% does not affect motor axon growth or migration into the chick hindlimb

Abstract

Bidirectional signaling between Eph receptor tyrosine kinases and their cell-surface protein signals, the ephrins, comprises one mechanism for guiding motor axons to their proper targets. During projection of motor axons from the lateral motor column (LMC) motor neurons of the spinal cord to the hindlimb muscles in chick embryos, ephrin-A5 has been shown to be expressed in the LMC motor axons until they reach the base of the limb bud and initiate sorting into their presumptive dorsal and ventral nerve trunks, at which point expression is extinguished. We tested the hypothesis that this dynamic pattern of ephrin-A5 expression in LMC motor axons is important for the growth and guidance of the axons to, and into, the hindlimb by knocking down endogenous ephrin-A5 expression in the motor neurons and their axons. No perturbation of LMC motor axon projections was observed in response to this treatment, suggesting that ephrin-A5 is not needed for LMC motor axon growth or guidance.

Keywords: chick; in ovo electroporation; motor neurons.

Figure 1

Expression of EphA4, ephrin-A5, and ephrin-A2 during motor axon projections to the chick hindlimb.

Figure 2

Knockdown of ephrin-A5 expression by 236 shRNA. Chick embryos were co-transfected at stage 15/16 with pCAX alone (A,B), pCAX and 236 shRNA, against ephrin-A5 (C,D), or pCAX and 236 M shRNA, a mutated form of 236 (E,F). Vibratome sections of embryos fixed at stage 21 were stained with antibody against ephrin-A5 and examined for GFP expression (A,C,E) or antibody staining (B,D,F).

Figure 3

Effect of ephrin-A5 shRNA on LMC axon growth and migration. Embryos were co-transfected at stage 15/16 with HB9 and 236 M (control; A–F) or HB9 and 236 (ephrin-A5 shRNA; G–L) and fixed at stage 26. Vibratome sections were stained with anti-neurofilament antibody (NF) and examined for GFP fluorescence (A,C,E;G,I,K) and antibody staining (B,D,F;H,J,L). Note: motor axons have extended into the limb at stage 26 in A,B but are omitted here.

Figure 4

Effect of ephrin-A5 shRNA on islet-1 (isl-1) expression. Embryos were co-transfected at stage 15/16 with HB9 and 236 M (Control; A,B) or with HB9 and 236 shRNA (against ephrin-A5; C,D) and fixed at stage 26. Vibratome sections were stained with antibody against isl-1 and examined for GFP fluorescence (A,C) and antibody staining (B, D).