JCS038232 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The stimulation of dendrite growth by Sema3A depends on integrin activation and is mediated by Nrp-1 and A-type plexins. (A) Rat hippocampal neurons were plated on fibronectin and incubated with control medium (−Sema3A) or medium containing Sema3A (+Sema3A) at 3 d.i.v. for 16-20 hours. After fixation, neurons were stained with an anti-MAP2 antibody to visualize dendrites. (B) The percentage of dendrites with a specific length is shown (n=75 neurons, 3 experiments). (C) Neurons were transfected at 2 d.i.v. with expression vectors for dominant-negative mutants (dn) of Nrp-1, plexin-A1 or plexin-B1 and incubated at 3 d.i.v. for 16-20 hours with control medium (−) or medium containing Sema3A (+) as indicated. The length of the dendrites was determined after staining the neurons with an anti-MAP2 antibody (means ± s.e.m.; n=30-50 neurons; 3 experiments; *P<0.001 compared to control medium without Sema3A).

• Supplemental Figure S2 -

  Fig. S2. Growth-cone collapse is independent of integrin activation. (A,B) Hippocampal neurons were plated on fibronectin. At 3 d.i.v., control medium (−) or medium containing Sema3A (+) was added for 1 hour. (A) Phase contrast images of representative neurons and their axonal growth cones are shown. (B) Control medium, Sema3A, 1mM MnCl₂ or 200µM RGD were added to the cultures as indicated. The percentage of collapsed axonal growth cones is shown (means ± s.e.m.; n=50 neurons, 3 experiments; *P<0.001 compared to control medium without Sema3A).

• Supplemental Figure S3 -

  Fig. S3. Expression of β1 integrins in hippocampal neurons. (A) Mouse hippocampal neurons were stained at 5 d.i.v. with the Tau-1 (blue) or an anti-MAP2 (blue) antibody and an anti-β1-integrin antibody (red). (B) Neurons from Itgb1^flox/flox embryos were cultured on fibronectin and transfected at 3 d.i.v. with a vector for EGFP-Cre (pBS505, green) and stained at 5 d.i.v. with an anti-MAP2 (blue) and an anti-β1-integrin antibody. After expression of Cre, the expression of β1 integrins is lost in transfected neurons. Scale bars: 50 µm (A) and 10 µm (higher magnification in A and B).

• Supplemental Figure S4 -

  Fig. S4. Inactivation of Itgb1 does not affect axon length. Neurons from Itgb1^flox/flox embryos were transfected at 3 d.i.v. with vectors for mCherry and EGFP or EGFP-Cre (pBS505) to visualize neuronal morphology and analyzed at 5 d.i.v. by measuring the length of axons (means ± s.e.m.; n=25 neurons, 3 experiments).

• Supplemental Figure S5 -

  Fig. S5. Sema3A stimulates cGMP production in dendrites and axons. (A) Hippocampal neurons were cultured on fibronectin and incubated with control medium (control) or medium containing Sema3A (Sema3A) including the volume marker CMFDA (green) at 3 d.i.v. for 30 minutes. Neurons were stained with an anti-cGMP (red) and the Tau-1 antibody (blue). The marked axonal and dendritic growth cones are shown at a higher magnification. (B) The immunofluorescence signal for cGMP was measured in the growth cones of dendrites and axons and normalized to the signal for CMFDA (volume marker) to determine the relative level of cGMP in arbitrary units (a.u.) (means ± s.e.m.; 25 neurons, 3 experiments; * p<0.001). Scale bar: 20 µm.

• Supplemental Figure S6 -

  Fig. S6. Sema3A increases FAK phosphorylation in dendrites. Hippocampal neurons from rat embryos were cultured on fibronectin and incubated with control medium (control) or medium containing Sema3A (Sema3A) at 3 d.i.v. for 30 minutes and stained with anti-pFAK (397) and anti-MAP2 (dendritic marker) antibodies. Scale bar: 10 µm.

• Supplemental Figure S7 -

  Fig. S7. FAK is not required for the collapse of axonal growth cones by Sema3A. Neurons from Fak^flox/flox embryos were cultured on PO, transfected at 3 d.i.v. with vectors for mCherry (red) and EGFP or EGFP-Cre (green), incubated 48 hours after the transfection with control medium (control) or medium containing Sema3A for 1 hour, and stained with an anti-MAP2 antibody (blue). A higher magnification of the marked axonal growth cones is shown. The axonal growth cones that are formed by hippocampal neurons from mouse embryos have a less elaborate structure compared to that of rat neurons. Scale bars: 100 µm and 10 µm (higher magnification).

• Supplemental Figure S8 -

  Fig. S8. Inactive FAK-R454 blocks the collapse of axonal growth cones by Sema3A. (A) Neurons from wild-type embryos were cultured on fibronectin, transfected at 1 d.i.v. with vectors for EGFP (green) or EGFP and FAK-R454, incubated 48 hours after the transfection with control medium (control) or medium containing Sema3A for 1 hour, and stained with rhodamine-phalloidin (red), which stains F-actin in growth cones and non-neuronal cells. A higher magnification of the marked axonal growth cones is shown. Scale bars: 12 µm. (B) The percentage of collapsed axonal growth cones after addition of control medium (−) or Sema3A (+) is shown means ± s.e.m.; n=109−178 neurons, 3 experiments; *P<0.001 compared to control (GFP, − Sema3A).