Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve

Abstract

Developing neurons accurately position their somata within the neural tube to make contact with appropriate neighbors and project axons to their preferred targets. Taking advantage of a collection of genetically engineered mouse mutants, we now demonstrate that the behavior of somata and axons of the facial nerve is regulated independently by two secreted ligands for the transmembrane receptor neuropilin 1 (Nrp1), the semaphorin Sema3A and the VEGF164 isoform of Vascular Endothelial Growth Factor. Although Sema3A is known to control the guidance of facial nerve axons, we now show that it is not required for the pathfinding of their somata. Vice versa, we find that VEGF164 is not required for axon guidance of facial motor neurons, but is essential for the correct migration of their somata. These observations demonstrate, for the first time, that VEGF contributes to neuronal patterning in vivo, and that different compartments of one cell can be co-ordinately patterned by structurally distinct ligands for a shared receptor.

Figure 1.

Nrp1 is required for the correct pathfinding of facial branchiomotor somata. (A) Schematic representation of the spatial relationship of trigeminal, facial, abducens, and glossopharyngeal motor neurons in a flat-mounted mouse hindbrain. The axon exit points for trigeminal and facial branchiomotor neurons (hatched circles) and the floorplate (gray) are indicated. The cross-sections on the right-hand side show the position of facial branchiomotor somata on the ventricular (v) side in r4 (top section) and on the pial (p) side in r6 (bottom section). (B,C) The migration of Isl1-positive facial branchiomotor somata (VIIm) from r4 to r6 was observed on the ventricular side in the presence (B) or absence (C) of Nrp1 at 12.5 dpc. Wild-type somata stayed loosely associated to form one continuous stream, but mutant somata separated into several distinct streams (arrows and arrowheads indicate separation in r4 and r5/r6, respectively). (D) In the presence of Nrp1, many facial somata have reached the pial side of the hindbrain by 12.5 dpc and formed facial motor nuclei (VIIn). (E) In the absence of Nrp1, many mutant somata emerged on the pial side in an ectopic anterior location (star) instead of their normal location (Δ). Panel D shows one side of a wild-type hindbrain, and panel E shows the opposite side of a stage-matched mutant hindbrain to highlight differences in the position of facial branchiomotor neurons relative to the trigeminal nucleus (Vn). (F) Few facial branchiomotor somata were normally present on the ventricular side at 13.5 dpc (bracket). (G) In contrast, many mutant somata were still migrating on the ventricular side at 13.5 dpc (arrowheads). (I)On the pial side, some mutant somata had contributed to normally positioned nuclei (VIIn), while others had formed an ectopic nucleus (star). One side of a wild-type hindbrain (F,G) and the opposite side of a stage-matched mutant hindbrain (H,I) are shown. (J,K) By 14.5 dpc, all facial branchiomotor somata have integrated into nuclei on the pial side, which in wild types appeared round (J), but in mutant littermates were usually dumbbell-shaped, with an ectopic anterior component (star; K). Bars, 250 μm (one for B,C; one for D,E; one for F–I; and one for J,K). The midline is indicated with an asterisk.

Figure 2.

Soma migration of facial branchiomotor neurons does not rely on peripheral axons or normal vessel networks. (A–D) Wild-type hindbrains (11.5 dpc) were dissected free from peripheral axons and mesenchyme and fixed (0 h) or cultured for 2 d (2d) under serum-free conditions. In such explants, the somata of Isl1-positive facial branchiomotor neurons (VIIm, indicated with an arrowhead) traveled from the ventricular (v) side to form facial motor nuclei (VIIn) in a posterior position on the pial (p) side. Facial branchiomotor neurons were not seen prior to explanting on the pial side (Δ; B), or on the ventricular side after 2 d in culture (C). (E–H) PECAM-positive vessel sprouts had entered the hindbrain from the pial side (F) and formed an extensive vessel network on the ventricular side (E) at the time of explanting (0 h). After 2 d in culture, vessel segments had severely degenerated on the ventricular side (G) and completely degenerated on the pial side (H). (I–L) When 11.5-dpc littermate hindbrains containing (I) or lacking (K) Nrp1 were explanted, facial motor nuclei formed on the pial side in wild-type (VIIn; J) and mutant (VIIn; L) explants, but mutant explants also formed an ectopic anterior nucleus (star; L). As soma migration was delayed in explants lacking Nrp1, facial nuclei were never completely assembled after 2 d in culture, and they were therefore smaller than those in wild-type explants. (M,N) When a conditionally targeted Nrp1 allele was removed from vascular endothelial cells (EC) with Cre recombinase under the control of the Tie2 promoter, the migration of facial branchiomotor somata was not impaired. One side of a control hindbrain (M) and the opposite side of a stage-matched mutant hindbrain (N) are shown next to each other to highlight the similar position of the facial (VIIn) relative to the trigeminal (Vn) nuclei. (O) Nrp1 contains CUB domains essential for binding the Sema domain of class 3 semaphorins and CFV/VIII domains for binding VEGF164. Bars, 250 μm (one for A–L; one for M,N). The midline is indicated with an asterisk.

Figure 3.

Sema3A and Sema3C are expressed in the hindbrain, but are not required for the pathfinding of facial branchiomotor somata. (A,B) In situ hybridization (ISH) shows that at 12.5 dpc, the Isl1-positive somata of facial branchiomotor neurons were migrating caudally (VIIm) on the ventricular side (A) and began to condense into the paired facial motor nuclei (VIIn) on the pial side (B) of the hindbrain. (C–F) In stage-matched hindbrains, Sema3A (C) and Sema3C (E) were expressed on the ventricular side in the area where facial somata migrate (arrowheads in C,E) and in neighboring regions (arrows in C,E). Sema3A (D) and Sema3C (F) were down-regulated on the pial side (cf. positions of Δ in D,F and VIIn in B). (G–I) By 13.5 dpc, most facial somata were located within their nuclei (VIIn) on the pial side (cf. the size of facial motor nuclei in B and G). Loss of Sema3A (H) or Sema3C (I) did not impair the formation or positioning of facial motor nuclei (cf. the relative positions of VIIn and Vn). Bars: A–F, 250 μm; G–I, 200 μm. The midline is indicated with an asterisk.

Figure 4.

Semaphorin signaling through Nrp1 is not required for the migration of facial branchiomotor somata. (A–F) Loss of both Sema3A and Sema3C did not impair the pathfinding of Isl1-positive facial somata on the ventricular side (cf. A and D), nor the positioning of facial motor nuclei on the pial side at 12.5 dpc (cf. B and E) or 13.5 dpc (cf. C and F). (G–L) Mutation of the Sema-binding domain of Nrp1 did not impair pathfinding of Isl1-positive facial somata on the ventricular side (cf. G and J), or the positioning of facial motor nuclei on the pial side at 12.5 dpc (cf. H and K) or 13.5 dpc (cf. I and L). Bars, 250 μm (one for A,B,D,E; one for G,H,J,K); 200 μm (one for C,F; one for I,L). The midline is indicated with an asterisk.

Figure 5.

VEGF controls the pathfinding of facial branchiomotor somata. (A,B) Whole-mount in situ hybridization at 12.5 dpc shows expression of the VEGF-A gene (B) in the area were the Isl1-positive somata of facial branchiomotor neurons assemble into facial motor nuclei (VIIn) on the pial side of the hindbrain (A). (C–F) Expression of a VEGF-A LacZ reporter at 12.5 (C–E) and 13.5 dpc (F). (C) At 12.5 dpc on the pial side, VEGF-A LacZ expression was prominent in the area of facial motor nucleus assembly (VIIn). (D) At 12.5 dpc on the ventricular side, VEGF-A LacZ expression was prominent near the midline (arrow), in a more dorsally located pair of stripes (wavy arrow), and in the area where the hypoglossal nuclei form (XIIn). (E) A higher magnification of the boxed area in C. (F) At 13.5 dpc, expression was elevated in the area of facial motor nucleus assembly and in an adjacent stripe (open arrowhead). (G–N) The migration of the Isl1-positive somata of facial branchiomotor neurons (arrowheads) was observed in the presence (wt/wt or wt/120) or absence (120/120) of VEGF164. In the absence of VEGF164, facial branchiomotor neurons formed nuclei in anterior positions, which appeared elongated or dumbbell shaped (stars; J,L,N). One side of a control hindbrain and the opposite side of a stage-matched mutant hindbrain are shown next to each other in G–L to highlight the position of the facial branchiomotor neurons relative to the hypoglossal (XIIn; G,H) or trigeminal (Vn; I–L) nuclei (cf. the length of the square brackets in G–J controls and mutants). In some mutants, somata emerged in an abnormally large area on the pial side and could be identified as scattered Isl1-positive cells (cf. boxed areas in K and L). (O,P) A comparison of the facial motor nuclei (VIIn) formed in littermate hindbrains capable of expressing all VEGF isoforms (wt/wt; O) or VEGF164 only (164/164; P) showed that VEGF164 was sufficient to drive normal soma migration. (Q–U) Hindbrain tissue (11.25 dpc) expressing VEGF164 only was fixed (0 h; Q) or cultured for 2 d in the presence of function-blocking antibodies for VEGF (αVEGF; R,S) to ablate VEGF164 function. When VEGF164 function was blocked, somata continued to migrate caudally out of r4 on the ventricular side, and a proportion of somata dived through the basal plate in the expected position (open arrowhead; R) to form facial motor nuclei on the pial side (VIIn; S). However, ectopically migrating somata were seen on the ventricular side in the r5 territory (arrowheads in R). (T,U) When heparin beads (b) were implanted into normal hindbrain tissue, somata did not respond to control beads (T), but moved toward beads coated with VEGF164 (U); accordingly, somata were positioned more posteriorly on the side containing VEGF beads relative to the untreated hindbrain side (double bracket in U). Bars, 250 μm (one for A–D; one for E,F one for G–J; one for K,L; on for M–P; one for Q–U). The midline is indicated with an asterisk; in K and L the midline is located outside the panel, but its position is indicated with an open arrowhead.

Figure 6.

VEGF164 is not required for the target finding of facial nerve axons. (A–D) Neurofilament staining of cranial nerve axons in 11.5-dpc embryos. Facial nerve axons extend normally into the branchial arches in the presence (A,C) or absence (D) of VEGF164, but they defasciculate in mutants lacking Nrp1 (B). The three major branches of the lower facial nerve (VII) are indicated with arrows. The position of the maxillary (Vmx), mandibular (Vmd), and ophthalmic (Vop) branches of the trigeminal nerve, the vestibulocochelar nerve (VIII) and the trigeminal (Vg) and facial (VIIg) ganglia are indicated in A. In B, defasciculation of the facial nerve is highlighted with a circle, and an abnormally positioned branch of the facial nerve is indicated with an open arrowhead. The trigeminal nerve branches are also defasciculated. Bar, 250 μm. (E) Working model for the control of axon and soma guidance in facial branchiomotor neurons by Nrp1 ligands. Sema3A binds to Nrp1-containing receptors in the growth cone to control axon guidance in the periphery, while VEGF164 binds to Nrp1-containing receptors on the cell body in the hindbrain to control soma migration. The most prominent site of VEGF164 expression on the pial hindbrain side is shown in green to indicate its likely role as an attractive signal for migrating somata. Sema3A is shown in red to indicate its role as an axon repellent in the periphery. Nrp1 associates with plexins to transmit semaphorin signals in the growth cone, but the signaling coreceptor in the soma is not known.