Figure 7: Overexpression of Nrp1 sensitizes proprioceptive axons to Sema3A.

(a) General scheme illustrating the generation of neuronal aggregates. DRGs are dissociated, electroporated with plasmids encoding the desired receptor, and incubated overnight in a ‘hanging drop’. The newly formed neuronal aggregates are tested the following day in collagen assay. (b,c) Neuronal aggregates, overexpressing either Nrp1 or L1-CAM, Flag tagged at their (extracellular) N-termini, were prepared from E14 rat DRGs. The two types of aggregates were embedded in collagen next to Sema3A-secreting aggregate of COS-1 cells, as indicated. NT3 was added to the cultures to facilitate growth of proprioceptive axons. After incubation, cultures were fixed and stained for tubulin βIII (c, green) to visualize all the axons. The subpopulations of axons overexpressing Nrp1 (left aggregate) or L1-CAM (right aggregate) were visualized using anti-Flag staining (b, red). Arrowheads indicate representative proximal axons (positive for tubulin βIII) that are clearly negative for Flag-tagged Nrp1. (d) P/D ratios calculated for axons overexpressing Nrp1 or L1-CAM (Flag positive axons). n=number of neuronal aggregates measured. Bars represent mean±s.e.m.