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First published online 14 September 2005
doi: 10.1242/dev.02038

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Slit-mediated repulsion is a key regulator of motor axon pathfinding in the hindbrain

MRC Centre for Developmental Neurobiology, 4th Floor New Hunt's House, King's College, Guy's Campus, London SE1 1UL, UK

View larger version (92K): [in a new window]   Fig. 1. Expression patterns of Slit1 and Slit2 and Robo1 and Robo2 in the rat embryo. (A) Diagram showing organisation of cranial motoneurons in E12 rat hindbrain; somatic motor (SM) neurons are on the left (red), BM/VM neurons are on the right (blue). Nuclei: III, oculomotor; IV, trochlear; V, trigeminal; VI, abducens; VII, facial; IX, glossopharyngeal; X/XI, vagus and cranial accessory; XII, hypoglossal (inner ear efferent neurons at r4 level omitted). (B,C,H-J) Flat-mount rat hindbrains in situ hybridised with Slit or Robo probes as indicated. Asterisk in H indicates the floor plate. (D-G,K-N) Vibratome sections of E12 rat embryos labelled with fluorescein dextran axon tracer then in situ hybridised; BM/VM projection labelled in D,F,K,M; SM projection labelled in E,G,L,N. Insets in K and M show in situ hybridisation without axon tracing. The axial levels of the vibratome sections are D rhombomere 2(r2); E, r5; F, r2; G, r8; K, r3; L, r7; M, r6; N, r6. (O-R) Peroxidase-stained sections of a rat E12 hindbrain with adjacent sections stained with neurofilament (NF) or anti-Robo1+2 antibodies as labelled. (O,P) r2/3 level; (Q,R) r8. HB or H, hindbrain; MB or M, midbrain; FP, floor plate; r, rhombomere; RL, rhombic lip; SM, somatic motoneurons; TMA, trigeminal motor axons; TG, trigeminal ganglion; VMA, vagus motor axons; HMA, hypoglossal motor axons. Scale bar: 500 µm in B,C,H,I,J,Q,R; 200 µm in D-G,K-N; 300 µm in O,P; 800 µm in insets.

View larger version (43K): [in a new window]   Fig. 2. Responses of motor axons to Slit proteins in vitro. (A) Dissection of tissues for use in collagen gel co-cultures (see Materials and methods). (B,C,E) Trigeminal (r2+3) level explants co-cultured with cells transfected as indicated. (D) Histogram showing quantitation (mean % pixels on facing side of explant are on the y axis) of outgrowth from explants at r2-8 levels in the presence of transfected cell clusters; n>30 for each condition. Asterisks represent significant differences from the controls. (F-M) Semi-quantitative data. A shift to negative explant net scores indicates inhibition of outgrowth (see Materials and methods); inhibition was significant when P<0.1 in all cases, and significant when P<0.05 in G,H,I,K,M; n>25 for each condition. Scale bar: 400 µm.

View larger version (92K): [in a new window]   Fig. 3. Responsiveness of motoneuron subpopulations to Slit proteins in vitro. (A,D,G) Immunostained trochlear axons from explants co-cultured with transfected cell clusters. (B) Histogram showing mean angle difference representing trochlear axon bundle chemorepulsion. Significant differences from controls represented by asterisks (P<0.05); n>20 in each condition. (C,F,I) Spinal cord explants growing in the presence of transfected cell clusters. (E,H) Histograms representing quantitation of mean % of axons facing cluster (y-axis) for retrogradely labelled dorsally projecting (BM/VM) axons (E) or ventrally projecting (SM) axons (H). Asterisks indicate significant deviations from the control values (P<0.05); n>10 for BM/VM labelled explants, n>25 for SM labelled explants. (J-L) Explants placed in apposition to Slit1-transfected cell clusters. (M-O) Explants facing mock-transfected control cell clusters. (J,M) Ventrally projecting abducens axons. (K,N) Dorsally projecting glossopharyngeal axons. (L,O) Dorsally projecting hypoglossal/cranial accessory axons. Scale bars: 400 µm in I for A,C,D,F,G,I; 400 µm in O for J-O.

View larger version (142K): [in a new window]   Fig. 4. DiI labelling of motor axon pathways in the hindbrains of E11.5 mouse embryos mutant for Slit proteins or Robo proteins. All panels show flat-mount hindbrains with DiI labelling of dorsally projecting BM/VM neurons. (A,E) DiI labelling is bilateral; (B-D,F-H) labelling is unilateral (right-hand side). Genotypes as labelled. Rhombomeres are numbered, white arrowheads indicate floor-plate borders and red arrows indicate axon guidance errors. (A,B,F,I) Floor plate is to left of the white arrow. (H) Basal plate with floor plate towards the left (asterisk). (J) Centre of floor plate. IEE, inner ear efferent axons. Scale bar: 200 µm in A,G; 400 µm in B-F,H.

View larger version (138K): [in a new window]   Fig. 5. Effects of ectopic expression of Slit1 in chick hindbrains. All panels show flat-mount chick hindbrains electroporated at stage 10-11, fixed at stage 17-19 and immunostained using anti-SC1 antibodies to show motor axon pathways (green). (A) A control GFP construct has been electroporated (red); (B-F) a hSlit1-myc construct has been electroporated (red). Rhombomere levels are numbered on the non-electroporated (right-hand) side. Exit points are shown by an asterisk in some cases. White arrowheads show regions in which axons exit at the incorrect exit point (B,C), stall (D) or overshoot the exit point and form tangles (E,F). Scale bar: 150 µm.

View larger version (104K): [in a new window]   Fig. 6. Effects of expression of dominant-negative Robo1 and Robo2 constructs in chick hindbrains. (A-J) Flat-mount chick hindbrains that have been electroporated with plasmids encoding myristylated GFP (myr-GFP), dominant-negative Robo1-GFP (Robo1-GFP) or Robo2-GFP (Robo2-GFP) as labelled. Hindbrains were fixed at stage 17-19 and immunostained with anti-Islet 1/2 antibodies to detect motoneuron cell bodies (red), while GFP expression is shown in green. (F,I-N) Preparations additionally immunostained using anti-neurofilament antibodies (blue) to reveal all axon tracts. (G-J) Asterisks show borders of floor plate. (K-M) Transverse cryostat sections of embryos electroporated with plasmids labelled as above, with insets showing higher magnification. Immunostaining is as above. Yellow and white arrows show dorsal and ventral exit points, respectively. Both dorsal and ventral GFP-labelled motor axons exit in the control whereas only ventral axon projections are present in the dominant-negative Robo-electroporated embryos. (L) The trigeminal ganglion is immunostained in red (Islet 1/2). Scale bar: 100 µm in A-F; 15 µm in G-I; 50 µm in K,L,N; 30 µm in M; 20 µm in insets.

View larger version (41K): [in a new window]   Fig. 7. The role of Slit-Robo signalling in motor axon pathfinding in the hindbrain. (A-D) Schematic diagrams of the vertebrate embryo hindbrain at stage 19 chick (A-C) or E11.5 mouse (D) is presented. Rhombomere levels are indicated (r) and the midbrain (MB)-hindbrain (HB) boundary. BM/VM neurons are shown in blue with corresponding exit points (blue ellipses) while SM neurons are shown in red. (A) Composite patterns of Slit and Robo expression (see key) in regions relevant to this study. (B) BM/VM axon pathway errors (thicker blue axons) in Slit1-electroporated sides of chick hindbrains (left-hand side) compared with non-electroporated or GFP-electroporated (right-hand side). Axon stalling and inappropriate, caudal projection of motor axons is shown. (C) Behaviour of BM/VM neurons electroporated with Robo dominant-negative GFP constructs (left-hand side; green axons), compared with myr-GFP electroporated axons (right-hand side; green axons). Stalling, misprojections and ectopic midline crossing is shown. (D) Behaviour of BM/VM axons in Robo2 mutant mice at E11.5 (left-hand side) compared with wild-type embryos (right-hand side). Ectopic midline crossing, longitudinal extension and looping are shown. Purple axons in D represent IEE axons which also show pathfinding errors. (E) Schematic of two rhombomeres with DiI labelling of the sensory ganglion outside the hindbrain, leading to labelling of motor axons and cell bodies within the hindbrain (see Materials and methods).