Motor axon exit from the mammalian spinal cord is controlled by the homeodomain protein Nkx2.9 via Robo-Slit signaling

DEV072256 Supplementary Material

Files in this Data Supplement:

  • Supplemental Table S1 -
  • Supplemental Figure S1 -

    Fig. S1. vMN axons properly exit the spinal cord in Nkx2.9−/− embryos. (A,B) Cryosections derived from E11.5 WT (A) and Nkx2.9−/− (B) embryos were colabeled with anti-BEN and anti-L1. L1-labeled vMN axons extend through ventral roots (arrowhead) in both WT and Nkx2.9−/− embryos (A,B). Scale bar: 100 µm.

  • Supplemental Figure S2 -

    Fig. S2. Nkx2.9-lacZ-expressing cells likely give rise to SACMNs. (A-C) Cryosection derived from an E9.5 Nkx2.9-lacZ+/− embryo was colabeled with anti-β-galactosidase (A) and a riboprobe specific for Nkx2.9 mRNA (B). Nkx2.9+ progenitors express β-galactosidase (B, arrow). (D) β-galactosidase enzymatic staining of a cryosection derived from an E10.5 Nkx2.9-lacZ−/− mouse reveals β-galactosidase-expressing SACMNs (arrows). (E-X) E10.5 Nkx2.9-lacZ+/− (E-H,K-N,Q-U) and Nkx2.9-lacZ−/− (I,J,O,P,V-X) mice were colabeled with anti-β-galactosidase and antibodies specific for BEN, Islet1 or Phox2b. Nkx2.9-lacZ-expressing SACMNs (G,I,M,O,S,V, solid arrow) express BEN (E-J), Islet1 (K-P) and Phox2b (Q-X). H, J, N, P, T, U, W and X are magnified views of boxed areas in G, I, M, O, S (top), S (bottom), V (top) and V (bottom), respectively. Scale bars: 50 µm in C for A-C, in D for D-G,I,K-M,O,Q-S,V; 20 µm in J for H,J, in X for N,P,T,U,W,X.

  • Supplemental Figure S3 -

    Fig. S3. Misexpression of Nkx2.9 in the embryonic chick spinal cord does not alter Robo2 mRNA levels. (A,B) Transverse cryosections derived from the cervical spinal cord of E4 chick embryos that had been unilaterally electroporated with pMESmNkx2.9 (4 µg/µl) at E2 were subjected to in situ hybridization for mouse (m) Nkx2.9 (A, arrow) and chick (c) Robo2 (B, arrowhead) mRNA. Forced expression of Nkx2.9 (left side) does not alter Robo2 mRNA levels (B). Scale bar: 50 µm.

  • Supplemental Figure S4 -

    Fig. S4. Misexpression of Slit1 in the embryonic chick spinal cord does not perturb SACMN axon pathfinding. (A-C) Transverse cryosections derived from the cervical spinal cord of E4 chick embryos that had been electroporated with Slit1-LRR (0.5 µg/µl) at E2 were labeled with anti-GFP (A) and anti-SC1 (B). Forced expression of Slit1 (right side) does not perturb the pathfinding/exit of anti-SC1-labeled SACMN axons (B,C, arrows). Scale bar: 50 µm.