Ontogeny of mdDA axon projections in the mouse forebrain. Sagittal sections are immunostained for TH (green) to visualize dopaminergic axons and counterstained with either PI (red) or fluorescent Nissl (blue). A, B, At E11.5, TH-positive neurons emerge within the ventral midbrain lining the mesencephalic flexure (mf) and project neurites dorsally (arrowheads). C, By E13.5, the MFB has formed and takes a ventrolateral course in the diencephalon. The dotted line indicates rostral (diencephalic) boundary of mdDA system. D, At E14.5, the MFB curves dorsally (bent arrow) and reaches the striatum. E represents a higher magnification of the boxed region in D.E, A subset of TH-positive axons diverges from the main MFB trajectory and extends rostrally. F, At E16.5, axons in the MFB are fasciculated into thick axon bundles. G, TH-positive axons curve dorsally just caudal of the OB to enter the cortical SP (arrow). C, Caudal; Ctx, cortex; D, dorsal; Di, diencephalon; DT, dorsal thalamus; LGE, lateral ganglionic eminence; Mes, mesencephalon; MGE, medial ganglionic eminence; MZ, marginal zone; Ol, olfactory epithelium; R, rostral; RP, Rathke's pouch; SC, superior colliculus; Str, striatum; Tel, telencephalon; V, ventral; VT, ventral thalamus.

mdDA neurons in the rostral mVTA project axons to the mPFC during development. A, Schematic showing in gray the derivation of the mPFC sections shown in B–F. B–D, Sagittal sections are immunostained for TH (green) to visualize mdDA axons and counterstained with fluorescent Nissl (blue). B, TH-positive axons start to enter the embryonic cortex around E15.5 and travel tangentially within the SP. In addition, some TH-positive puncta can be observed within the MZ (arrowheads). C, At E16.5, fibers appear within the SP. However, the CP is still mostly devoid of TH-positive axons. D, By E18.5, axons extend from the SP to the CP following a radial trajectory (arrows). E, F, To identify the origin of mesoprefrontal mdDA projections during development, DiI tracer was microinjected in the medial aspect of the VTA at different developmental time points (for an overview of all injection sites, see supplemental Fig. S1, available at www.jneurosci.org as supplemental material). Coronal sections are counterstained with Hoechst (blue) and show DiI in red. E, F, After injections in the rostral mVTA, but not in more lateral, middle, or caudal aspects of the mVTA, DiI-labeled axons were found in the mPFC. At E16.5, DiI-positive axons derived from rostral mVTA enter the mPFC through the SP (E) and start to innervate the CP around E18.5 (F, arrow). IZ, Intermediate zone; VZ, ventricular zone. Scale bar: B-F, 100 μm.

The mPFC releases diffusible molecules that exert neuronal subset-specific directional effects on mVTA axon outgrowth. A, Schematic representation of the coculture system used to identify axon guidance activities between cortex and mVTA. Explants of E14.5 embryos were taken from the ventral midbrain along the rostrocaudal axis (mVTA), the mPFC, and somatosensory (S1) cortex. Explants were analyzed after 56–58h in culture using immunohistochemistry for TH and βIII-tubulin to visualize dopaminergic (green) and all (red) neurites, respectively. Rostral (R), middle (M), or caudal (C) parts of mVTA explants were cocultured with mPFC (n = 8, 10, and 9, respectively) and S1 (n = 8). B–E, J, Cocultures of rostral mVTA and mPFC show attraction of TH-positive axons. F–J, In contrast, TH-positive axons extending from the caudal mVTA are strongly repelled. The boxed areas in C and G are shown in D, E, H, and I. J, Quantification of the length of TH-positive neurites in the proximal and distal quadrants of the coculture assays as shown in B–I. Graph shows average P/D ratio ± SEM. TH-positive axon outgrowth from the mVTA in the presence of mPFC is shown separately for the R, M, and C parts of the mVTA. For S1 P/D ratios for R, M, and C explants are pooled. For each experimental condition, at least three independent experiments were performed. **p < 0.01; ***p < 0.001, one-way ANOVA. Ctx, Cortex; dist, distal; D, dorsal; IF, interfascicular nucleus; L, lateral; prox, proximal; SN, substantia nigra; Str, striatum. Scale bar, 250 μm.

Neuropilin-2 mediates the attractive effect of mPFC on mdDA rostral mVTA axons. A–C′, In situ hybridization shows expression of npn-1 (B) and npn-2 (C) within the TH-positive (A) mdDA system (A′–C′). The boxes in A–C are enlarged in A′–C′. Note the expression of npn-2 (C′) in specific neuronal subsets. D, E, Immunostaining of coronal sections for TH (green) and Npn-2 (red) counterstained with fluorescent Nissl (blue) shows Npn-2-positive cells (D, arrowheads) within the TH-positive rostral mVTA. D′, Enlargement of the area indicated by top arrowhead in D shows colocalization of a subset of TH-positive cells with Npn-2 (arrowheads). E, Npn-2 (red) is present in the TH-positive MFB. F, Npn-1 (red) is present in cells of the IZ and CP. G, Axons within the SP of the mPFC are Npn-2-positive (boxed area enlarged on the right, arrowheads) and colocalize with TH (H, arrowheads). I, Representative examples of cocultures of rostral mVTA and mPFC explants in the presence of IgG-Fc, Npn-1-Fc, or Npn-2-Fc immunostained for TH (green). The arrowheads indicate the growth cones (leading edge) of some of the longest TH-positive neurites. J, Quantification of the length of TH-positive neurites in the proximal and distal quadrants of rostral mVTA/mPFC cocultures in the presence of IgG-Fc (n = 13), Npn-1-Fc (n = 12), or Npn-2-Fc (n = 9). Graph shows average P/D ratio ± SEM. Three independent blocking experiments were performed. *p < 0.05; **p < 0.01, one-way ANOVA (compared with control IgG-Fc). Note the ability of Npn-2-Fc to block the positive effect of mPFC on rostral mVTA axons. aca, Anterior commissure; CP, cortical plate; CPu, caudate–putamen; D, dorsal; fr, fasciculus retroflexus; IZ, intermediate zone; ls, lateral septum; mf, mesencephalic flexure; MZ, marginal zone; PZ, proliferative zone; V, ventral.

Expression of Sema3s in the mPFC. A, RT-PCR showing the expression of sema3A, 3B, 3C, 3D, 3E, 3F, and 3G in mouse head and mPFC at E14.5. Only sema3A, 3C, and 3F can be found in the mPFC (arrows). Splice forms of sema3F are indicated by arrowheads (Kusy et al., 2003). Actin serves as a control. B–G, In situ hybridization on coronal sections shows sema3C and sema3F expression in the E14.5 (B, E) and E18.5 (C–F) mPFC. Sema3C is expressed within the SVZ and IZ and weakly within the upper layers of the CP, whereas sema3F is almost exclusively expressed in the CP.

Sema3F is a bifunctional axon guidance cue for rostral mVTA axons. A, TH immunostaining (green) of E14.5 rostral mVTA explants (proximal quadrants are shown; arrowheads indicate the leading edge of a set of axons) cultured adjacent to HEK293 cells (dotted line) secreting either control protein (n = 8), Sema3C (n = 10), or Sema3F (n = 11). B, Quantification of the length of TH-positive neurites in the proximal and distal quadrants of the culture assays shown in A. Graph shows average P/D ratio ± SEM. Note that TH-positive rostral mVTA axons are attracted by Sema3F (P/D ratio, 1.33; p < 0.05) but not by Sema3C (P/D ratio, 1.01) compared with control. This attraction is blocked by Npn-2-Fc (n = 9; P/D ratio, 1.07; p < 0.05). Interestingly, TH-positive axons extending from caudal mVTA explants are repelled by Sema3F (n = 12; P/D ratio, 0.56; p < 0.001). C, TH immunostaining (green) of E12.5 rostral mVTA explants cultured adjacent to HEK293 cells (dotted line) secreting either control protein (n = 12) or Sema3F (n = 11). Axons extending from E12.5 rostral mVTA explants are repelled by Sema3F (P/D ratio, 0.52; p < 0.001). For each experimental condition, at least three independent experiments were performed. *p < 0.05; ***p < 0.001, one-way ANOVA. div, Days in vitro.

sema3F^−/− mice display defects in the rostral growth of mdDA axons in the forebrain. A, Schematic showing the derivation of the sections shown in B and C in gray. The developing MFB is shown in red. B, C, Sagittal sections of E13.5 wt (n = 3) (B) and sema3F^−/− (n = 2) (C) mice are immunostained for TH (green) to visualize mdDA axons and counterstained with fluorescent Nissl (blue). The rostral extent of the mdDA neuron pool is indicated by a dotted line. B, At E13.5, TH-positive axons project into the diencephalon and grow beyond the LH (arrowhead). C, In sema3F^−/− mice, the MFB is defasciculated and spreads out over a larger area along the dorsoventral axis. In addition, in sema3F^−/− mice, TH-positive axons accumulate at the level of the LH and fail to extend further rostrally.

Abnormal channeling, fasciculation, rostral growth, and EC crossing of mdDA projections in sema3F- and npn-2-deficient mice. A, Schematic showing in gray the derivation of the sections in B–M and R–U. The developing mesoprefrontal projection is shown in red. B–M, R–U, Sagittal sections at the level of the MFB (B–I), diencephalon (J–M), and EC (R–U) of E18.5 wt (n = 5) (B, F, J, R), sema3F^−/− (n = 4) (C, G, K, S), npn-2^−/− (n = 4) (D, H, L, T), and npn-2^−/−;TH-Cre (n = 3) (E, I, M, U) mice. Sections are immunostained for TH (green) to visualize mdDA axons and counterstained with fluorescent Nissl (blue). The boxed areas in B–E are enlarged in F–I. N–Q, Schematic representations of the development of the mesoprefrontal projection (in red) of wild-type (N), sema3F^−/− (O), npn-2^−/− (P), and npn-2^−/−;TH-Cre (Q) mice with an enlargement at the level of the MFB (dark red within circle). B, F, In wild-type mice, the compact MFB (boundaries indicated by dotted lines) projects into the diencephalon with individual TH axons fasciculated into thick bundles. The double-arrowed line in B indicates location of line used to quantify MFB width and axon fascicle number. C, G, In sema3F^−/− mutants, the MFB occupies a larger dorsoventral area and individual TH-positive axons are severely defasciculated. In npn-2^−/− (D, H) and npn-2^−/−;TH-Cre mice (E, I), the MFB is broader and TH-positive axons are defasciculated. In sema3F^−/− mutants (K), but not in wild-type (J), npn-2^−/− (L), and npn-2^−/−;TH-Cre mice (M), many axons in the diencephalon are displaced ventrally and appear to stall in the LH region. R–U, A subset of axons innervates the mPFC by crossing the EC (R). Although this innervation appears normal in sema3F^−/− mice (S), TH-positive axons excessively cross the EC in npn-2^−/− and npn-2^−/−;TH-Cre mice (arrowheads). Str, Striatum.

sema3F- and npn-2-deficient mice display defects in intracortical targeting of mesoprefrontal axons. A–D, Sagittal sections of E18.5 mPFC in wild-type (A), sema3F^−/− (B), npn-2^−/− (C), and npn-2^−/−;TH-Cre mice (D) immunostained for TH. The arrows indicate direction of growth of TH-positive axons in the CP. B–H, Camera lucida drawings of three consecutive sections within the mPFC of wild-type (E), sema3F^−/− (F), npn-2^−/− (G), and npn-2^−/−;TH-Cre mice (H). The insets show axon orientation plots of randomly selected axons within the CP [dotted line indicates trajectory perpendicular to the pial surface (0°)]. B, F, Inset, Overall innervation of the mPFC in sema3F^−/− mice is reduced and axons ectopically innervate deeper cortical areas. Furthermore, axons within the CP display a random orientation. C, G, Inset, In npn-2^−/− mutants, overall mPFC innervation is increased, mdDA axon growth in the CP is randomized, and many axons form club-like structures (arrowheads). D, H, Inset, In npn-2^−/−;TH-Cre mice, overall TH-positive innervation is increased and ectopic innervation of deeper cortical areas is prominent. TH-positive axon growth in the CP is randomized. I, Quantification of total TH axon density at E18.5 in the mPFC in arbitrary units (a.u.) confirms the qualitative observations shown in A–H. Graph shows average density ± SEM. There is a significant decrease of TH-positive wiring in the mPFC of sema3F^−/− animals (p < 0.001) and an increase in npn-2^−/− and npn-2^−/−;TH-Cre animals (p < 0.01 and p < 0.05, respectively) compared with wild type. J, Quantification of the distribution of TH-positive axon density in 10 bins dividing the mPFC as indicated in A. Graph shows average density per bin ± SEM. Note that innervation of the mPFC in sema3^−/− and npn-2^−/−;TH-Cre mice is shifted toward the lower bins. K, Axon orientation histogram. Quantification of the orientation of mesoprefrontal axons in the CP. 0° represents a trajectory perpendicular to the pial surface. Note that mesoprefrontal axon growth in the CP of the mPFC in all mutant mouse lines is random, whereas in wild-type mice most axons are biased toward a radial trajectory. White, Wild type; dark gray, sema3F^−/−; light gray, npn-2^−/−; intermediate gray, npn-2^−/−;TH-Cre. *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA. CP, Cortical plate; IZ, intermediate zone; MZ, marginal zone; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone.

Proposed model for the role of Sema3F and Npn-2 during mesoprefrontal pathway development. (1) Surround repulsion by Sema3F expressed around the (presumptive) MFB trajectory (blue) induces channeling and fasciculation of mesoprefrontal axons (in red) in the MFB through Npn-2. (2) A temporally expanding high-caudal to low-rostral repulsive Sema3F gradient (blue) is required to push mesoprefrontal axons into a rostral direction in the forebrain. This effect does not require Npn-2. (3) Mesoprefrontal axons arrive in the SP around E15, and after a waiting period of ∼2d innervate the overlaying CP. As they develop, mesoprefrontal axons change their responsiveness to Sema3F from repulsion to attraction. Attraction by Sema3F-Npn-2 is necessary to orient mesoprefrontal axons in the CP toward the pial surface. (4) A small subset of TH-labeled axons traverses the striatum and crosses the developing EC to innervate the overlaying mPFC. Access of axons to the mPFC via the EC is controlled by Npn-2 in combination with a repulsive ligand other than Sema3F, presumably Sema3C (green gradient), which is expressed in deeper cortical layers of the mPFC. IZ, Intermediate zone; MZ, marginal zone; PZ, proliferative zone.