A Cxcl12-Cxcr4 Chemokine Signaling Pathway Defines

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1 Supplemental Data A Cxcl12-Cxcr4 Chemokine Signaling Pathway Defines the Initial Trajectory of Mammalian Motor Axons Ivo Lieberam, Dritan Agalliu, Takashi Nagasawa, Johan Ericson, and Thomas M. Jessell Figure S1. Expression of Cxcr4 by Trigeminal but Not Other dmns in the Hindbrain (A C) Expression of Cxcr4 (A), Hb9 (B) and Isl1 (C) by trigeminal motor neurons. Only a subset of cells that express Isl1 also express Cxcr4. (E G) Expression of Cxcr4, Hb9 and Isl1 in rhombomere 5. Cxcr4 (E) and Hb9 (F) are expressed in the abducens nucleus (nvi). The facial nucleus (nvii, G) only expresses Isl1 but not Cxcr4 and Hb9. (I K) In rhombomere 4, as in rhombomere 5, the facial nucleus (nvii) only expresses Isl1 but not Cxcr4 and Hb9. (M O). Expression of Cxcr4 (M), Hb9 (N) and Isl1 (O) mrnas in the anterior part of rhombomere 7 (r7), where the vagus nucleus (nx) is located. Cxcr4 and Hb9 mrnas are absent from cells expressing Isl1. (D, H, L, and P) Expression of Cxcl12 in the mesenchyme surrounding different rhombomeres in the hindbrain. At more rostral levels, two additional motor neuron classes (oculomotor (niii) and trochlear (niv)) are generated near the boundary of the hindbrain and midbrain. The progenitor domains for these two motor nuclei differ from those of motor neurons found at more caudal levels of the neuraxis, and their initial axonal trajectories also diverge dramatically; oculomotor neurons send axons via a ventral exit point, whereas trochlear motor neurons send axons dorsally to reach the midline of the neural tube. We find that oculomotor, but not trochlear, motor neurons express Cxcr4.

2

3 Figure S2. Neurofilament Labels Motor Axons but Not Medial Processes (A and B). Expression of β3-tubulin and Isl1/2 in spinal cords of Cxcr4 heterozygous (A) and mutant (B) mice at e9.5. β3-tubulin labels both medial processes and axons. (C and D) Expression of neurofilament and Isl1/2 in spinal cords of Cxcr4 heterozygous (C) and mutant (D) mice at e9.5. Neurofilament labels motor axons but not medial processes.

4 Figure S3. The Transcriptional Identity of vmns Remains Unchanged in Cxcr4 Mutants (A) Diagram summarizing the progenitor domains and transcription factors expressed by two subtypes of motor neurons. Progenitor cells within the pmn domain (red box) express Pax6, followed by Lhx3/4 that transiently persist in mature neurons. Motor neurons generated from the pmn domain acquire vmn identity. The p3 precursors (blue box) expressing Nkx2 generate dmn motor neurons in the hindbrain, marked by Phox2a/b. In Pax6 mutants (B), the pmn domain is converted into a p3 domain and vmns are converted to dmns. (C E) In situ hybridization with Cxcr4 (C), Hb9 (D) and Isl1 (E) at the posterior r7 level in Pax6 heterozygous mice (e9.5). Cxcr4, Hb9 and Isl1 are expressed in the hypoglossal nucleus (nxii). The X/XI nuclei are marked with Isl1. (F H). Expression of Cxcr4 (F), Hb9 (G) and Isl1 (H) at r7 level in Pax6 mutants (e9.5). No Cxcr4 and Hb9 expression is detected in any region of r7. (I N) Expression of Lhx3 (I and L) Isl1/2 (J and M) and merge panels (K and N) in Cxcr4 heterozygous and Cxcr4 mutant spinal cords (e10.0).

5 Figure S4. Increase in Spinal Accessory Nerve Size in Cxcr4 Mutants Expression of neurofilament and egfp in the spinal accessory nerve (SAN) at cervical (C1-C2) level of the spinal cord in wild-type (A), Cxcr4 (B) and Cxcl12 (C) mutants. In mutants, the SAN cross-section area is increased (D) due to a major contribution of egfp+ vmn axons to the nerve (n=2 embryos, 4 sections per embryo; mean ± SEM).

6 Figure S5. Aberrant dmn-like Trajectory of Hindbrain vmn Axons in Cxcr4 and Cxcl12 Mutant Mice (A I) Expression of egfp and Isl1/2 in r5 (nvi; A C) and r7 (nxii; D I) in controls, Cxcr4 and Cxcl12 mutant embryos (e11.5). In both mutants, a few cranial egfp+ vmn axons project dorsally within the neural epithelium (B, C, E, and F; white arrows). Some egfp+ axons exit dorsally in r7 and enter the vagus nerve in Cxcr4 and Cxcl12 mutants (H and I) but not in controls (G).

7 Figure S6. Normal Trajectory of dmn Axons within the CNS in Cxcr4 Mutants (A and B) The initial trajectory of the trigeminal motor nerve in Cxcr4 heterozygous and mutant mice (e10.0) is normal. dmn axons are marked with LacZ by means of the Nkx6.2::τlacZ marker allele. Their trajectory within the neural epithelium and their exit points are identical in controls and Cxcr4 mutants. (C and D) The initial trajectory of the vagus motor nerve in Cxcr4 heterozygous and mutant mice (e10.0) is normal. dmn axons are marked by LacZ. (E and F) Whole-mount X-Gal staining of hindbrain and cranial nerves from Cxcr4 heterozygous and mutant mice (e11.5) carrying the Nkx6.2::τlacZ marker allele. Most cranial dmn nerves are normal in Cxcr4 mutants, with the exception of the trigeminal motor nerve (F; red arrows).

8 Figure S7. Motor Axons Exhibit Variable Growth Cones (A D) Staining of Hb9::eGFP motor axons with phalloidin after a 20h culture period. Motor neurons were scored as growth cone negative if their axons exhibited shaft-like growth cones (A and B) and growth cone positive if they had intermediate or large fan-like growth cones (C and D).

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