FGF8 signaling regulates growth of midbrain dopaminergic axons by inducing semaphorin 3F

Abstract

Accumulating evidence indicates that signaling centers controlling the dorsoventral (DV) polarization of the neural tube, the roof plate and the floor plate, play crucial roles in axon guidance along the DV axis. However, the role of signaling centers regulating the rostrocaudal (RC) polarization of the neural tube in axon guidance along the RC axis remains unknown. Here, we show that a signaling center located at the midbrain-hindbrain boundary (MHB) regulates the rostrally directed growth of axons from midbrain dopaminergic neurons (mDANs). We found that beads soaked with fibroblast growth factor 8 (FGF8), a signaling molecule that mediates patterning activities of the MHB, repelled mDAN axons that extended through the diencephalon. This repulsion may be mediated by semaphorin 3F (sema3F) because (1) FGF8-soaked beads induced an increase in expression of sema3F, (2) sema3F expression in the midbrain was essentially abolished by the application of an FGF receptor tyrosine kinase inhibitor, and (3) mDAN axonal growth was also inhibited by sema3F. Furthermore, mDAN axons expressed a sema3F receptor, neuropilin-2 (nrp2), and the removal of nrp-2 by gene targeting caused caudal growth of mDAN axons. These results indicate that the MHB signaling center regulates the growth polarity of mDAN axons along the RC axis by inducing sema3F.

Figure 1.

Ectopically expressed FGF8 disorganizes the rostrally directed growth of mDAN axons. A, Schematic diagram showing the position of FGF8-bead implantation. FGF8-beads are embedded into the mDAN axonal pathway in the diencephalon. PC, Posterior commissure. B, C, mDAN axonal growth in an experimental (B) and a control (C) brain 2 d after bead placement. TH-positive axons were deflected dorsally in an FGF8-bead implanted brain. In contrast, in a BSA-bead-implanted brain, TH-positive axons grew rostrally ignoring the bead. White arrowheads show the position of beads, which were removed during the fixation procedure. D, Dorsal; R, rostral. D, A higher-magnification image of the boxed area in B. A caudally deflected mDAN axon is labeled by arrowheads. Growth cone-like structures were observed at the tip of axons (arrows). Scale bars: B, C, 200 μm; D, 50 μm.

Figure 2.

Expression pattern of sema3F and its induction by FGF8-beads in whole-embryo culture. A, The distribution of sema3F transcripts in rat midbrain at E13.5. B, sema3F expression in a midbrain preparation 2 d after an FGF8-bead (arrowhead) implantation. Sema3F transcripts were induced in the region just ventral to the bead (arrow). C, The distribution of nrp2 mRNAs in an E13.5 rat midbrain. Nrp2 mRNAs were observed in the rostral region of the ventral most midbrain (arrowhead). nrp2 was also detected in the IIIn and IVn. D, An E13.5 rat midbrain hemisphere immunostained for TH. mDAN axons appear to avoid the sema3F-expressing region (compare A, D). PC, Posterior commissure; Zli, zona limitans intrathalamica; IIIn, oculomotor nucleus; IVn, trochlear nucleus. Scale bars: A, C, D, 300 μm; B, 375 μm.

Figure 3.

FGF signaling is required for sema3F expression in the midbrain. A–D, Whole-mount in situ hybridization with Fgf8 (A, C) and sema3F (B, D) antisense riboprobes in E10.0 (A, B) and E10.5 (C, D) rat embryos. Expression of Fgf8 temporally preceded sema3F at the MHB. E, F, The distribution of sema3F (blue) and otx2 (brown) in a DMSO-treated (E) and a SU5402-treated (F) embryo. The colocalization of sema3F (blue) and otx2 (brown) was observed in a control embryo (E). In contrast, sema3F mRNAs in the otx2-positive region was nearly undetectable in a SU5402-treated embryo (F). sema3F expressions in the hindbrain were not affected by the application of SU5402. Scale bars: 200 μm.

Figure 4.

Nrp2 is expressed on a subset of mDAN axons. A, A midbrain coronal section of E12.5 nrp2^+/lacZ midbrain immunostained for TH (green) and β-gal (magenta). B–D, Higher-magnification views of the boxed area in A. B, C, TH (B) and β-gal (C) expression. D, A merged image of B and C. β-gal immunoreactivity was observed in almost all TH-positive cells (arrowheads). E, An E13.5 rat midbrain/diencephalon parasagittal section immunostained for TH (green) and nrp2 (magenta). F–K, High-power views of the boxed areas (f–h, i–k) in E. F, G, I, J, TH (F, I) and nrp2 (G, J) expression. H, A merged view of F and G. K, A merged image of I and J. The colocalization of TH and nrp2 was observed in the rostroventral region (F–H) but not in the dorsal region (I–K). Scale bar: A, E, 100 μm; B–D, F–H, I–K, 20 μm.

Figure 5.

Sema3F inhibits the mDAN neurite outgrowth in vitro. A, B, VRM explants from E13.5 rat embryos were cocultured with aggregates of COS-7 cells transfected with either the pCAGGs-myc (A) or the pCAGGs-sema3F-myc (B) vector in collagen gels. After 2 d, the explants were fixed and stained with an anti-TH antibody. Neurite outgrowth from VRM explants, which is almost symmetric in a control coculture (A), appears to be inhibited by sema3F (B). C, Scheme of the method used to quantify neurite outgrowth. Scale bar, 250 μm.

Figure 6.

A subset of mDAN axons deflect in the caudal direction in nrp2-knock-out mice. A, B, TH immunostaining in coronal sections of wild-type (A) and nrp2-knock-out (B) mice at E14.5. Aberrant TH-positive fibers invaded the rostral hindbrain in an nrp2-knock-out mouse (B, arrowheads). C, Schematic representation showing the level of coronal sections in A and B. Note that the RC axis curves in the midbrain. PC, Posterior commissure; Tel, telencephalon. D, E, TH immunostaining in parasagittal sections of nrp2⁺⁺ (D) and nrp2^lacZ/lacZ (E) brains at P0. TH-positive fibers derived from the ventral midbrain, all of which were directed rostrally in a wild-type mouse (D), were deflected caudally in an nrp2-knock-out mouse (E, arrowheads). F–H, Aberrant TH-positive fibers (magenta) immunostained for DAT (green). F, G, TH (F) and DAT (G) expression. H, A merged view of F and G. DAT was expressed on an aberrant TH-positive fiber (arrowheads). I–K, Aberrant mDAN axons stained with antibodies against TH (green) and β-gal (magenta). I, J, TH (I) and β-gal (J) expression. K, A merged view of I and J. β-gal was expressed on aberrant mDAN axons (arrowheads). Scale bars: A, B, D, E, 200 μm; F–K, 10 μm.

Figure 7.

Dorsal spread of mDAN axons in nrp2-knock-out mice. A, B, TH immunostaining in flat-mount brain preparations of wild-type (A) and nrp2^lacZ/lacZ (B) mice at E12.5. TH-positive fibers, which are relatively tightly fasciculated in a wild type (A), were defasciculated, with some deviating from their normal pathway in an nrp2-knock-out mouse (B, arrows). Scale bar, 200 μm. C, Schematic diagram showing the trajectory of mDAN axons in nrp2-knock-out mice. In nrp2-knock-out mice, the trajectories of nrp2-positive mDAN axons were disrupted: a subset of axons grow caudally (1) and another part of them deflect dorsally (2). The remaining part projects normally (3). PC, Posterior commissure.

Figure 8.

Summary diagram of the present study and model diagram of the regulation of axonal growth polarity by signaling centers. A, Summary diagram showing the regulation of growth polarity of mDAN axons by FGF8 signaling. In early stages, MHB-derived FGF8 induces the sema3F expression at the MHB. Later, the axons of differentiated mDANs are guided rostrally by sema3F. Sema3F also provides the dorsal nonpermissive/repulsive territory to mDAN axons, defining the dorsal border of these axons. B, Model diagram showing the roles of signaling centers in axon guidance along the DV and RC axes. Along the DV axis, the growth polarity of axons is regulated by the signaling centers controlling the DV polarization of the neural tube. In an analogous way, the signaling centers controlling the RC polarization of the neural tube are also involved in the growth polarity of axons along the RC axis.