, 2002, Iwamasa et al , 1999, Kania and Jessell, 2003, Luria et a

, 2002, Iwamasa et al., 1999, Kania and Jessell, 2003, Luria et al., 2008 and Marquardt et al., 2005). We considered the possibility that in order to permit the selection of LMC axon trajectory with high fidelity, the function of Eph receptors

expressed in LMC neurons might be modulated by coexpressed ephrins. We focused on the time of LMC axon growth into the limb mesenchyme, between Hamburger-Hamilton stage (HH st.) 25 and 27 in chick and between the embryonic day (e) 10.5 and e11.5 in mouse (Hamburger and Hamilton, 1951, Kania et al., 2000 and Tosney and Landmesser, 1985). We determined the levels of total ligand-unbound RO4929097 Eph receptors (∑EphFREE) using ephrin-A5-Fc and ephrin-B2-Fc protein overlay and found that ∑EphBFREE levels are higher in medial LMC neurons when compared with

lateral LMC neurons, while ∑EphAFREE levels are higher in lateral LMC neurons when compared with medial LMC neurons in tissue sections (Figures 1B–1E and 1U; p < 0.001; quantification details in Table S2) and cultured neurons (Figures 1P, 1Q, and 1U; p < 0.001) in spite of the presence of EphA and EphB proteins in both LMC divisions. To determine if some of the Eph receptors expressed in LMC neurons were present on the cell surface, we overlaid live explanted ventral spinal check details cord neurons with an anti-EphA3 antiserum followed by transcriptional identity assignment. We detected surface EphA3 in both medial and lateral LMC neurons

and their axons, at apparently similar expression levels (Figure 1T; Figure S1). We next surveyed the levels of ∑ephrinFREE by Eph-Fc overlay, as well as ephrin protein and mRNA expression profile in LMC neurons (Imondi et al., 2000, Iwamasa et al., 1999, Luria et al., 2008 and Marquardt et al., 2005). We observed Megestrol Acetate that ∑ephrin-BFREE and ∑ephrin-AFREE levels were high in medial and lateral LMC neurons, respectively, in both, tissue sections (Figures 1F and 1G; p < 0.001) and cultured neurons (Figures 1R and 1S; p < 0.001). We found ephrin-B2 mRNA in lateral LMC neurons at a much higher level when compared with medial LMC neurons ( Figures 1C and 1H; p < 0.001). In parallel, relative to lateral LMC neurons, ephrin-A5 mRNA and protein was found to be highly enriched in medial LMC neurons ( Figures 1B and 1I; p < 0.001), with higher levels of ephrin-A5 protein found in axons in the ventral limb nerves ( Figures 1J–1M; p < 0.001). We also detected ephrin-A5 expression in lateral LMC neurons and dorsal limb nerve axons as previously shown ( Marquardt et al., 2005), but at considerably lower levels relative to medial LMC neurons and ventral limb nerve axons ( Figures 1N and 1O; p < 0.001).

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