Epha3/4pMNΔflox embryos displayed severely defective formation of epaxial sensory pathways (compare Figures 3A–3C and 3D–3F, see also Figures S3G and S3J). At the same time, hypaxial sensory pathways remained unaffected in these mutants ( Figures S3I and 3L). Both the frequency and pattern of these epaxial selleck screening library sensory projection defects were virtually indistinguishable from those observed in Epha3/4null embryos ( Figure 3G). Because in Epha3/4null embryos no alterations in DRG sensory neuron numbers were detected we could rule out that the selective epaxial projection defects in these mutants were caused by loss of a subset of sensory neurons ( Figures

S3N). Moreover, in both Epha3/4pMNΔflox and Epha3/4null embryos the absence of epaxial sensory projections was further accompanied by a consistent increase in diameter of the hypaxial nerves ( Figure 3H and Figures 3I–3L). This suggested that sensory projections that failed to extend epaxially instead grew hypaxially in these mutants ( Figures 3M–3N). We next tested whether these sensory projection defects were accompanied BIBW2992 order by similar defects in epaxial motor projections. Neither Epha3/4pMNΔflox nor Epha3/4null embryos showed absence of epaxial motor projections, thus ruling out that the failure to form epaxial sensory projections was due to the loss of epaxial motor projections (compare Figures 3J and 3L; see also Figures S3H and S3K). We next asked whether loss of epaxial sensory

projections in these mutants could have been caused by hypaxial

misrouting of epaxial motor axons. We tested this by retrogradely tracing of hypaxially projecting motor neurons by focal injection of fluorescent cholera toxin B (CtxB) into hypaxial intercostal nerves. In both control and Epha3/4null animals this effectively labeled hypaxial motor neurons residing within the lateral division of the medial motor column (MMCl) ( Figures S3O–S3Q and S3R–S3T). At the same time, neither in control nor in Epha3/4null embryos were epaxial motor neurons in the medial MMC (MMCm) labeled by hypaxial CtxB injection ( Figures S3O–S3U). Thus, removal of motor axonal EphA3/4 selectively disrupts epaxial sensory projections, without resulting in the hypaxial misrouting of epaxial motor axons ( Figures 3M–3N). In addition to the sensory projection Oxymatrine defects, both Epha3/4null and Epha3/4pMNΔflox mutants display misrouting of epaxial motor axons into DRGs due to loss of repulsive EphA3/4 signaling in motor growth cones (data not shown) ( Gallarda et al., 2008). We therefore asked whether the requirements of EphA3/4 for determining epaxial sensory projections could be uncoupled from their actions in repelling motor growth cones from DRGs. To address this, we tested how sensory projections would develop upon eliminating EphA3/4 repulsive intracellular signaling, while retaining the ability of motor axonal EphAs to engage their putative interaction partners on sensory axons.