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Fig. 6. PlexB is a Sema-2a receptor. (A-C) Cultured S2R+ cells transfected with cDNAs for either PlexB or GFP to which Sema-2a-AP, Sema-1a^EC-AP, or AP-Sema-1b^EC was added. (A) AP-tagged Sema-2a binds to the surface of S2R+ cells transfected with PlexB. Sema-2a-AP does not bind to S2R+ cells transfected with GFP (inset). (B) Sema-1a^EC-AP and AP-Sema-1b^EC (inset) do not bind to S2R+ cells transfected with PlexB. (C) Sema-2a-AP binds to PlexB in the presence of 1 mM and 10 mM (inset) EDTA. (D) Scatchard analysis of Sema-2a-AP binding to PlexB-expressing S2R+ cells. Data points were normalized to Sema-2a-AP binding to GFP-expressing cS2R+ cells. (E-G) Filleted preparations of late stage 16 embryos stained with the anti-Fasciclin II monoclonal antibody to reveal the transverse nerve (TN). (E) In a wild-type embryo, in the vicinity of the ventral longitudinal muscle field two axonal projections come together to form the TN (arrowheads). (F) Expression of Sema-2a in all muscles inhibits the TN from forming properly. Axons from the TN motoneuron and LBD neuron, which normally fasciculate and form the TN, stall and are unable to complete the nerve (open arrowheads). (G) Using the plexB^KG00878 mutant allele to remove one copy of plexB from this Sema-2a GOF background restores the formation of the TN (arrowheads). (H) Schematics of two adjacent hemisegments illustrating the TN phenotypes observed in wild type (left), Sema-2a overexpressing embryos (middle), and Sema-2a overexpressing embryos with one copy of plexB removed (right). Scale bars: in A, 50 µm for A-C; in E, 10 µm for E-G.

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