Figure 2: Nrp1 is developmentally reduced from the growth cones of proprioceptive axons in a metalloprotease-dependent manner.

DRGs were dissected from E13, E14 and E15 rat embryos, and cultured in the presence of the MP inhibitor TAPI-1 (20 μM), or DMSO (carrier). Either NT3 or NGF was added to the cultures to facilitate growth of proprioceptive (a and Supplementary Fig. 1), or cutaneous axons (Supplementary Fig. 1), respectively. After fixation, cultures were surface stained (without permeabilization) against Nrp1 (red) or TAG-1 (green in Supplementary Fig. 1). Relative fluorescence of proprioceptive and cutaneous axonal growth cones at the different developmental stages was measured for both Nrp1 (b) and TAG-1 (d) staining. Mean fluorescence values±s.e.m. for cultures supplemented with TAPI-1 or DMSO are presented in red and blue, respectively. All results were normalized relative to the E13/DMSO value, which was set to 1. For each developmental stage, the percent change in growth cone fluorescence of TAPI-1-supplemented cultures relative to DMSO-supplemented cultures is shown in green (c,e); n (number of growth cones quantified) >1,200 for all conditions. Results for each condition were collected from 3–4 separate experiments. Mann–Whitney U-test. *P<0.05, **P<0.01, ***P<0.001. Scale bars, 25 μm. MP, metalloproteases; a.u., arbitrary units.