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Slit proteins are not dominant chemorepellents for olfactory tract and spinal motor axons

¹ MRC Centre for Developmental Neurobiology, New Hunt’s House, Guy’s Hospital, King’s College London, London Bridge, London SE1 1UL, UK
² The 2^nd Research Deapartment, Central Technology Laboratory, Asahi Kasei Corporation, 2-1 Samejima, Fuji, Shizuoka, Japan 416-8501

View larger version (39K): [in a new window]   Fig. 1. Expression and processing of c-myc-tagged human Slit proteins and human Robo1/Fc chimera in CHOP cells. (A) Immunoprecipitation of c-myc-tagged human Slit1, Slit2 or Slit3 from conditioned medium was carried out with monoclonal antibody 9E10. The proteins were separated by SDS-PAGE and following transfer to nitrocellulose membranes, the blots were probed with monoclonal antibody 9E10. (B) Immunoprecipitation of human Robo1/Fc chimera from conditioned medium was carried out with Protein G-Sepharose followed by SDS-PAGE and western blotting with an antibody to human immunoglobulin. Molecular mass markers: myosin (220 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa), lysozyme (14.3 kDa).

View larger version (145K): [in a new window]   Fig. 2. Chemorepulsion of olfactory tract axons caused by Slit2 but not by the septum is inhibited in the presence of soluble Robo/Fc. E14.5-15 olfactory bulb explants were co-cultured with either aggregates of Slit2-expressing cells (A-C), E14. 5-15 septal explants (D-E) or alone (G-I) in collagen gels in the absence (A,D,G) or presence of Robo1/Fc conditioned medium (B,E,H) or Robo2/Fc conditioned medium (C,F,I). The cultures were incubated at 37°C for 24-48 hours and examined by phase contrast microscopy for axonal outgrowth of olfactory tract axons.

View larger version (141K): [in a new window]   Fig. 3. Chemorepulsion of olfactory tract axons caused by Slit1 or Slit3 is inhibited in the presence of soluble Robo1/Fc. E14.5-15 olfactory bulb explants were co-cultured with aggregates of cells expressing either Slit1 (A,B) or Slit3 (C,D) in collagen gels in the absence (A,C) or presence (B,D) of Robo1/Fc conditioned medium. The cultures were incubated at 37°C for 24-48 hours and examined by phase contrast microscopy for chemorepulsion of olfactory tract axons.

View larger version (123K): [in a new window]   Fig. 4. Chemorepulsion of spinal motor axons caused by floor plate-derived chemorepulsive activity is not inhibited in the presence of soluble Robo1/Fc. E12 basal plate explants were co-cultured with E12 floor plate explants in collagen gels in the absence (A) or presence (B) of Robo1/Fc conditioned medium. The cultures were incubated at 37°C for 24-48 hours and examined by phase contrast microscopy for chemorepulsion of spinal motor axons growing out of basal plate explants.