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First published online 3 May 2006
doi: 10.1242/dev.02380

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Drosophila Plexin B is a Sema-2a receptor required for axon guidance

¹ Howard Hughes Medical Institute, Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, 1001 PCTB, 725 North Wolfe Street, Baltimore, MD 21205, USA.
² Center for Basic Neuroscience and Department of Pharmacology, The University of Texas Southwestern Medical Center, NA4.301/5323 Harry Hines Boulevard, Dallas, TX 75390, USA.

View larger version (66K): [in a new window]   Fig. 1. Drosophila Plexin A and Plexin B share similar embryonic expression patterns. (A) A scale representation of the PlexB gene. ATG and the green arrow indicate the position of the open reading frame start codon. The SUPor-P element used for LOF analyses (KG00878) is inserted 25 animo acids downstream of the PlexB start codon and is denoted by the red line and triangle. (B-E) Whole-mount wild-type stage 15-16 embryos hybridized with digoxigenin-labeled cRNA probes specific for the intracellular regions of plexB (B,C) and plexA (D,E). Both plexin transcripts show robust expression within the ventral nerve cord (VNC). Scale bar in B: 60 µm for B-E.

View larger version (111K): [in a new window]   Fig. 2. ISNb motor axon pathfinding defects in plexB mutants resemble plexA mutants. (A-F) Filleted preparations of late stage 16 embryos stained with the anti-Fasciclin II monoclonal antibody to reveal motor axons of the ISNb. Arrows and open arrows indicate proper and absent innervation, respectively, by axons of the ISNb. Anterior, left; dorsal, up. (A) In a wild-type embryo, axons within the ISNb innervate the ventral longitudinal muscles 12, 13, 6 and 7 (arrows). (B) ISNb motor axons fail to defasciculate in plexADf(4)C3 mutants and often do not innervate their proper muscle targets (open arrows). (C) plexBKG00878 mutant ISNb motor axon pathways, like plexADf(4)C3 mutants, also fail to reach their proper muscle targets (open arrows). (D) Neuronal expression of plexB in a plexBKG00878 mutant background restores proper neuromuscular connectivity (arrows). (E) Neuronal plexB expression in a plexADf(4)C3 mutant background completely fails to rescue the plexADf(4)C3 mutant phenotype (open arrows). (F) Neuronal plexA expression in a plexBKG00878 mutant background partially rescues the plexBKG00878 mutant phenotype (absent, left, and normal, right, innervation are shown). (G) Schematics of two adjacent hemisegments illustrating ISNb phenotypes observed in wild type (left), plexA mutants (middle) and plexB mutants (right). Scale bar in A: 10 µm for A-F.

View larger version (112K): [in a new window]   Fig. 3. SNa motor axon pathfinding defects in plexB mutants resemble plexA mutants and display novel guidance errors. (A-F) Filleted preparations of late stage 16 embryos stained with the anti-Fasciclin II monoclonal antibody to reveal motor axons of the SNa. Anterior, left; dorsal, up. (A) In wild-type embryos, the dorsal branch of the SNa projects correctly between muscles 22 and 23 before defasciculating and innervating muscle 24 (arrowhead). (B) In plexADf(4)C3 mutants, the dorsal-most projecting axon of the SNa fails to reach its proper target, muscle 24 (open arrowhead). (C') plexBKG00878 mutant SNa pathways show defects in pathfinding choice. This mutant SNa axon bundle projects incorrectly between muscles 21 and 22 (open arrow), and then extends toward its proper target, muscle 24. The dashed line indicates the path that this bundle of SNa axons normally follows. (C'') plexBKG00878 mutants, like plexADf(4)C3 mutants, also show a lack of muscle 24 innervation (open arrowhead). (D) Neuronal expression of plexB in a plexBKG00878 mutant background restores proper neuromuscular connectivity (arrowheads). (E) Neuronal plexB expression in a plexADf(4)C3 mutant background fails to rescue the plexADf(4)C3 mutant phenotype (open arrowheads). (F) Neuronal expression of plexA in a plexBKG00878 mutant background fails to rescue the plexBKG00878 mutant pathfinding and innervation defects (open arrow and open arrowhead, respectively). (G) Schematics of two adjacent hemisegments illustrating SNa phenotypes observed in wild type (left), plexA mutants (middle) and plexB mutants (right). Scale bar in A: 10 µm for A-F.

View larger version (99K): [in a new window]   Fig. 4. plexB mutants display a novel CNS axon bundling defect. (A-F) Filleted preparations of late stage 16 embryos stained with the anti-Fasciclin II monoclonal antibody to reveal three fascicles of longitudinally projecting axon bundles on both sides of the midline (dashed line in A) within the ventral nerve cord. Anterior, left. (A) In a wild-type embryo the three bundles of axons on either side of the midline are tightly fasciculated (arrowheads). (B) The outermost bundle of axons in plexADf(4)C3 mutants is disrupted at several places along the embryo (open arrows). (C) The medial bundle of Fasciclin II-positive axons in plexBKG00878 mutants is split apart and appears as two separate fascicles (open arrowheads). (D) Neuronal expression of plexB in a plexBKG00878 mutant background restores proper bundling of the medial fascicle (arrowheads). (E) Neuronal plexB expression in a plexADf(4)C3 mutant background fails to rescue the plexADf(4)C3 mutant phenotype (open arrows). (F) Neuronal plexA expression in a plexBKG00878 mutant background fails to rescue the plexBKG00878 mutant phenotype (open arrowheads). (G) Schematics illustrating CNS phenotypes observed in wild type (left), plexA mutants (middle) and plexB mutants (right). Scale bar in A: 10 µm for A-F.

View larger version (91K): [in a new window]   Fig. 5. PlexB genetically interacts with MICAL and forms a complex with PlexA. (A,B) Filleted preparations of late stage 16 embryos stained with the anti-Fasciclin II monoclonal antibody to reveal motor axons of the ISNb. (A) ISNb axons of plexBKG00878 heterozygous embryos defasciculate properly and resemble wild-type embryos. (B) ISNb axons in embryos double heterozygous for both plexB and MICAL (Df(3R)swp2MICAL/+; plexBKG00878/+) often fail to defasciculate properly and do not innervate their proper muscle targets (open arrows). (C) A chart outlining the results from yeast two-hybrid interaction assays with cytoplasmic regions of PlexA and PlexB. Numbers indicate the amino acids of PlexA or PlexB that comprise each fragment. A positive interaction is indicated by a plus sign (+). The C2 region of the PlexA cytoplasmic domain (animo acids 1702-1945) and the C1 region of the PlexB cytoplasmic domain (amino acids 1402-1784) support growth and reporter gene transcription when grown on selective plates, whereas all other combinations between PlexA and PlexB do not. (D) Lysates from embryos expressing Myc-PlexB, with or without HA-PlexA, were immunoprecipitated using anti-HA antibodies and blotted with anti-HA or anti-Myc antibodies to detect the presence of HA-PlexA or Myc-PlexB, respectively. HA-PlexA immunoprecipitates from embryo lysates also contain Myc-PlexB. Scale bar in A: 10 µm for A-B.

View larger version (98K): [in a new window]   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-1aEC-AP, or AP-Sema-1bEC 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-1aEC-AP and AP-Sema-1bEC (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 plexBKG00878 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.