Figure 2.

Motor neuron subtype-specific, protein synthesis-independent response to ephrin-A5. A, RALDH2 is expressed by ESC-derived LMC motor neurons [(Foxp1⁺; Hb9⁺), arrow] and not by other motor neuron subtypes [(Foxp1⁻; Hb9⁺), arrowhead]. Scale bar, 10 μm. B, Immunostaining on mouse embryo section (E12.5) for RALDH2 (red) shows that the enzyme specifically labels LMC axons projecting toward the forelimb but is not present in other motor branches. SC: Spinal cord; Limb Br.: limb axonal branch (LMC axons); Axial Br.: axial axonal branch. Scale bar, 100 μm. C, RALDH2 is observed in a subset of axons growing from ESC-derived brachiothoracic motor neurons plated on laminin substrate. Scale bar: 15 μm. D, ephrin-A5 at 50 ng/ml induces preferentially the collapse of non-LMC motor axons (HB9-GFP⁺/RALDH2⁻ axons) compared with LMC axons (HB9-GFP⁺/RALDH2⁺ axons) (n = 3), while 300 ng/ml leads to similar collapse of both populations. GC, Growth cone. Scale bar, 15 μm. E, Dose response of brachiothoracic ES-MNs to ephrin-A5 in absence or presence of cycloheximide (CHX). All significantly active concentrations of ephrin-A5 (starting at 30 ng/ml; n = 3) induce collapse in a protein synthesis-independent manner. Two-way ANOVA. n.s., No significance. Error bar indicates SD.