Together, these observations strongly suggest that in LMC neurons, endogenous ephrins modulate in cis the ability of Eph receptors to bind and signal in response to ephrins in trans. Since LMC axons are tightly fasciculated as they form limb nerves, we considered the possibility that axon-axon interactions might contribute to ephrin modulation of Eph signaling in LMC neurons and affect axon trajectory choice.

We thus analyzed ephrin Vorinostat manufacturer stripe preference of LMC neurites in low-density cultures, such that axon-axon interactions are virtually absent. Lumbar HH st. 25/26 neurons were dissociated, cultured for 18–24 hr, and the LMC divisional identity assigned to individual neurites by examining nuclear Foxp1 and Isl1 expression (Figure S7). Cultured neurons expressing both Foxp1 and Isl1 were classified as medial LMC neurons, and those expressing Foxp1 but not Isl1 were classified as lateral LMC neurons (Figure 5). Dissociated LMC neurons responded to ephrins: lateral LMC neurite P[ephrin-A5/Fc] but PFI-2 research buy not P[ephrin-B2/Fc] was significantly lower

than P[Fc/Fc] of controls (Figure S7, p < 0.01 and p = 0.07, respectively). On the other hand, medial LMC neurite P[ephrin-B2/Fc] but not P[ephrin-A5/Fc] was significantly lower than P[Fc/Fc] of control neurites (Figure S7; p < 0.01 and p = 0.925, respectively), arguing that ephrin:Eph forward signaling can guide LMC axons in the absence of axon-axon interactions. We next challenged dissociated [eA5]siRNA and GFP or GFP-expressing LMC neurons with ephrin-A5-Fc/Fc. [eA5]siRNA and GFP-expressing medial LMC neurites were found over ephrin-A5 stripes less frequently when compared with controls ( Figures 5A and 5B; p < 0.01). We next challenged dissociated

eA5::GFP-electroporated LMC neurons with ephrin-A5-Fc/Fc stripes and found that their P[ephrin-A5/Fc] was similar to GFP controls ( Figures 5A and 5C; p = 0.425). In contrast, eA5::GFP-expressing lateral LMC neurites had a decreased sensitivity to ephrin-A5 stripes while loss of ephrin-A5 function Carnitine dehydrogenase had no effect, when compared with GFP-expressing controls ( Figures 5D–5F; p < 0.01). Thus, the ability of ephrin-A to modulate EphA function in LMC neurons in the absence of axon-axon interactions strongly suggests that it is a cell-autonomous process. Cis-attenuation of Eph function by ephrins in LMC neurons apparently contradicts the in vitro evidence implying that in LMC neurons, ephrin-As can mediate attractive EphA:ephrin-A reverse signaling by binding to EphAs in trans ( Marquardt et al., 2005). To better understand the relation between ephrin-mediated reverse signaling in response to exogenous Ephs and ephrin-mediated cis-attenuation of Eph signaling, we challenged LMC explants, as above, with EphA2-Fc/Fc or EphB1-Fc/Fc stripes.