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First published online 20 March 2008
doi: 10.1242/dev.015412

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Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells

¹ Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK.
² Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
³ MRC Centre for Developmental Neurobiology, King's College London, London SE1 1UL, UK.

View larger version (61K): [in this window] [in a new window]   Fig. 1. Expression pattern of Sema3a, Sema3f and Nrp1 during cranial neural crest migration. (A,B) Sox10 expression in the trigeminal and hyoid neural crest streams at 8.25 and 8.5 dpc (10- and 12-somite stage, respectively); the hyoid stream is indicated by the black arrowheads. (C,D) Nrp1 expression in the hyoid stream (black arrowheads), in the distal pharyngeal arch mesenchyme (open arrowheads), and in areas where major vessels form (asterisks). (E,F) At 8.25 dpc, Sema3a was expressed in a stripe between the trigeminal and hyoid neural crest streams (arrow in E). Sema3f was expressed in r3 and r5 (arrows in F). (G,H) Dorsal view of the hindbrain at 8.25 dpc, when it has not yet closed. Sema3a was expressed only weakly in the hindbrain, but prominently in the domain just outside r3 (arrows in G), corresponding to the stripe indicated with an arrow in E. Sema3F was expressed strongly in r3 (arrow in H). (J-L) Coronal sections at r4 level through an 8.75 dpc mouse embryo subjected to in situ hybridisation for Nrp1 (J) and immunolabelling of the neural crest marker p75 (K). Both markers detected the same population of neural crest cells (black arrowheads in overlay, L). Open arrowheads (J,L) indicate weak Nrp1 expression in the p75-negative distal mesenchyme. (I) Summary of the expression patterns for Nrp1, Sema3a and Sema3f during neural crest migration on embryonic day 8. ov, otic vesicle; e, eye anlage; ncc, neural crest cell; pa1 and pa2, pharyngeal arches 1 and 2. Scale bars: 250 µm in A for A-H, in J for J-L.

View larger version (84K): [in this window] [in a new window]   Fig. 2. SEMA3A/NRP1 signalling guides cranial neural crest migration. (A-F) Wholemount in situ hybridisation of 9.5 dpc (21- to 23-somite stage) mouse embryos with a Sox10 probe revealed a neural crest-free zone beneath r3 (bracket) in wild-type embryos; two examples are shown (A,D). Loss of NRP1 (B,E) or SEMA3A (C,F) caused neural crest cell invasion into the territory adjacent to r3 (arrowheads) in all mutants examined (two examples are shown for each mutation). Invading neural crest cells appeared to emerge from the hyoid (h) neural crest stream (arrowhead in C) or formed bridges between the trigeminal (t) and hyoid neural crest streams (arrowheads in B,E,F). Ectopically migrating neural crest cells were usually found at the level of the dorsal neural tube, but in some mutants an additional smaller ventral stream formed (lower arrowhead in B,F). In some cases, trigeminal neural crest cells invaded the territory adjacent to r3 (arrow in E). (G-O) Coronal sections through the wild-type embryo shown in D, the Nrp1-null mutant shown in E, and the Sema3a-null mutant shown in F at r2 (G-I), r3 (J-L) and r4 (M-O) level; the planes of sectioning are indicated with dotted white lines in the insets (miniaturised images of the embryo) in D-F. Arrowheads in K,L,I indicate ectopic neural crest cells in the cranial mesenchyme adjacent to r3. t, trigeminal neural crest cells; h, hyoid neural crest cells. ov, otic vesicle; e, eye anlage; ncc, neural crest cell; pa1 and pa2, pharyngeal arches 1 and 2. Scale bars: 250 µm in A for A-F, in G for G-O.

View larger version (91K): [in this window] [in a new window]   Fig. 3. NRP1 is required for cranial neural crest migration in a cell-autonomous fashion. (A-I) Double-label immunofluorescence analysis of 9.5 dpc mouse embryos (21-23 somites) with the neural crest cell marker p75 (red, A-C) and the blood vessel marker endomucin (green, D-F); (G-I) merged images. (A,D,G) In wild type, neural crest cells avoided the head mesenchyme at the level of r3 (bracket in A), but migrated ventrally in close proximity to the anterior cardinal vein (arrows in D). Vascular patterning in the head appeared disorganised in Nrp1-null mutants (E) compared with wild-type littermates (D). In Nrp1-null mutants, ectopically migrating hyoid neural crest cells travelled rostrally along the anterior cardinal vein (arrowhead in B,H). A coronal section at r3 level through the Nrp1-null mutant shown in B,E,H revealed that ectopic neural crest cells (arrowhead in C) migrated just dorsally of the anterior cardinal vein (arrow in F,I); the plane of sectioning is indicated with a dotted white line in the inset in H. (J-L) Wholemount in situ hybridisation with a Sox10 probe at 9.5 dpc (21-23 somites) revealed a neural crest-free zone (bracket in J,K) in control embryos and in embryos lacking NRP1 in CRE-expressing blood vessel endothelium (Tie2 promoter; K). By contrast, loss of NRP1 from CRE-expressing cranial neural crest (Wnt1 promoter; L) caused neural crest invasion into the mesenchymal territory adjacent to r3 and bridging between the trigeminal and hyoid neural crest streams (arrowhead in L). t, trigeminal neural crest cells; h, hyoid neural crest cells; ov, otic vesicle; e, eye anlage; pa1 and pa2, pharyngeal arches 1 and 2. Scale bars: 250 µm in A for A,B,D,E,G,H and in J for J-L; 200 µm in C for C,F,I.

View larger version (97K): [in this window] [in a new window]   Fig. 4. ERBB4 does not control Sema3A expression. The expression of Sema3a in control (A,C) and Erbb4-null mutant (B,D) littermates was monitored by wholemount in situ hybridisation at 8.0 dpc (A,B) and 8.5 dpc (C,D). At 8.5 dpc, expression was prominent in a dorsoventral stripe that initiates below r3 (arrow in C,D). Scale bar: 100 µm.

View larger version (138K): [in this window] [in a new window]   Fig. 5. NRP1 and NRP2 cooperate to guide cranial neural crest cells. (A-C) Wholemount in situ hybridisation of 9.5 dpc mouse embryos (20-somite stage) with a Sox10 probe revealed a neural crest-free zone (bracket) between the trigeminal and hyoid neural crest streams in wild type (A), but not in stage-matched Nrp2-null mutants (arrowhead in B). Loss of semaphorin signalling through both NRP1 and NRP2 caused more extensive invasion of neural crest cells into the territory that normally separates the trigeminal and hyoid neural crest streams (arrowheads in C). (D-F) Coronal sections through the double-null mutant shown in C at r2 (D), r3 (E) and r4 (F) level; the three planes of sectioning are indicated with dotted white lines in the inset in C. Arrowheads in E indicate dorsal and ventral ectopic neural crest cell streams in the cranial mesenchyme at the r3 level. t, trigeminal neural crest cells; h, hyoid neural crest cells; ov, otic vesicle; pa1 and pa2, pharyngeal arches 1 and 2. Scale bars: 250 µm in A for A-C, in D for D-F.

View larger version (132K): [in this window] [in a new window]   Fig. 6. Neuropilin mutants contain ectopic neurons and extend misprojecting axons. Wholemount immunochemistry at 10.5 dpc for HUC/D-positive neuronal cell bodies (green) and neurofilament-containing axons (red) in wild-type mouse embryos (A,D,G,J), in Nrp1-null mutants (B,E,H,K), and in Nrp2-null mutants with reduced semaphorin signalling though NRP1 (C,F,I,L). Defasciculated axon bundles (white arrows) extended from the trigeminal (Vg) and facioacoustic (VII/VIIIg) ganglia in Nrp1-null (B) and in compound mutants (C). The wavy arrow in A indicates the greater superficial petrosal nerve. Ectopic axon tracts extended between the trigeminal (Vg) and facioacoustic (VII/VIIIg) ganglia in single and compound mutants (boxed area in B and C, respectively; shown at higher magnification in D-F and as single labels in G-I and J-L, respectively). Note absence of neurons in the area between the trigeminal and facioacoustic ganglia in wild-type embryos (bracket in D), and presence of ectopic neurons between the trigeminal and facioacoustic ganglia in single and compound mutants (arrowheads in H and circled in I). Misprojecting axons extend from ectopic neurons (arrowheads in K, circled in L). Vg, VIIg, VIIIg, cranial ganglia; e, eye anlage; pa1 and pa2, pharyngeal arches 1 and 2. Scale bars: 200 µm in A for A-C; 50 µm in D for D-L.

View larger version (47K): [in this window] [in a new window]   Fig. 7. SEMA3/NRP control axon guidance during cranial gangliogenesis. (A-C) Wholemount immunolabelling of neurofilament-containing axons in stage-matched 11.5 dpc mouse embryos shows that the trigeminal ganglion (Vg) and facioacoustic ganglion complex (VIIg and VIIIg) are well separated in the wild type (red bracket, A), but not in mutants lacking NRP2 (B); note the presence of ectopic projections that appear to extend from the trigeminal ganglion towards the geniculate ganglion (arrow, B). The trigeminal and facioacoustic ganglia appeared completely fused in mutants lacking semaphorin signalling through both NRP1 and NRP2 (outlined in red, C). Scale bar: 250 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 8. Ectopic neurons in Nrp1 mutants have placodal identity. In situ hybridisation of Nrp1-null mutants and heterozygous control littermates at 10.5 dpc with probes that detect (A,B) all cranial sensory neurons (Isl1) or (C-H) subpopulations of placodal neurons (Phox2b, Ngn1 and Brn3b). Ectopic neurons could be clearly identified by in situ hybridisation (arrowhead in B). At 10.5 dpc, Phox2b marked neurons from the geniculate placode (gp, C), whereas Ngn1 was expressed by neurons derived from the trigeminal neural crest stream and trigeminal placode (tnc/p, E). However, neither marker labelled the ectopic neurons in any of the Nrp1-null mutants examined (compare B with D,F). Brn3b labelled the same population of trigeminal neurons as Ngn1 and, in addition, neurons derived from the otic placode (otp, G); this marker was expressed by the ectopic neurons (arrowhead in H). Scale bar: 100 µm. (I) Working model for the multiple roles of semaphorin signalling in cranial sensory neuron patterning. By acting on neuropilin/plexin receptors, gradients formed by the class 3 semaphorins SEMA3A and SEMA3F synergise to repel cranial neural crest cells and the axonal growth cones of sensory neurons. Neural crest cells in turn determine the position of cell bodies from placodal sensory neurons through an unknown molecular interaction (bi-directional arrow).

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