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Supplemental Figure 1: Nrp1/Plexin-A complexes do not mediate Sema3Ainduced FAK recruitment
(A) Sema3A does not induce FAK recruitment to Nrp1/Plex-A2 and Nrp1/Plex-A3
complexes. (B) In Nrp1/Plex-A4-expressing cells immunoprecipitation of either FAK
or Plex-A4 did not reveal Plex-A4/FAK interaction, both in cont and Sema3A-treated
condition. LpIP: Lysate post IP. (D) Immunodetection of L1 forms mutated on serine
residues, L1S1194L and L1S1224L expressed in COS7 cells showing that L1 proteins are
present at the cell surface. The ser mutations do not prevent L1/Nrp1 coprecipitation.
Supplemental Figure 2: FAK and L1 signaling are required for the guidance of
cortical axons in slice overlay assays.
Schematic drawing and microphotographs of the assay to illustrate the quantification
of axon trajectory of cortical neurons grown on top of cortical slices. Axons are
normally directed away from the pial surface by the secretion of Sema3A. As shown
by the histogram, over-expression of L1S1194 and FAFKD strongly alter the trajectory
taken by axons from neurons over-expressing L1WT and FAKWT.
Supplemental figure 3: Defects of cortical projections in horizontal sections
from mice lacking Plexin-A3, Plexin-A4 or L1.
(A) Schematic diagram of horizontal brain sections of neonatal brain showing the
position of the corpus callosum and the internal capsule. (B) Immunolabelling of
horizontal brain sections illustrating the reduced density of Nrp1-expressing axons in
the intermediate zone (black arrows) and extending from lateral cortical regions in the
internal capsule (black asterisks) of Plexin-A3/A4 -/-, Plexin-A3- and Plexin-A4-/mice, compared with wild-types. Note that the decrease of Nrp1-expresising axons
found in the double mutant is fully reproduced in the simple Plexin-A4 null mutant
while is also present but less pronounced in the simple Plexin-A3 null mutant. The
reduction of fibers in the internal capsule can also be visualized by antibodies to L1.
(C) Immunodetection of Nrp1 in horizontal brain sections illustrating the sharp
reduction of Nrp1+ axons in the internal capsule of mice lacking L1, compared to
wild-type mice. DCC labeling shows abnormal fasciculation of axons still present in
the internal capsule of mice lacking L1. Scale bar: 1 mm. CP: cortical plate; FT: fiber
tract; IC: internal capsule. (C) DCC labelling of callosal axons shows that the corpus
callosum is formed normally in Plexin-A3/A4 mice.
Supplemental Figure 4: Antero-posterior organization of Nrp1+ axons in the
corpus callosum of Plexin-A3, Plexin-A4 and L1 knockout mice.
Nrp1+ axons are positioned at the anterior pole of the corpus callosum, as illustrated
by these horizontal sections immunolabelled with antibodies to Nrp1. This
organization is maintained in mice lacking Plexin-A3 and Plexin-A4. In contrast in
mice lacking L1, sections were found for which Nrp1+ axons were no longer present.
Scale bar: 1 mm. CC: Corpus callosum