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doi: 10.1242/10.1242/dev.00327

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Molecular analysis of axon repulsion by the notochord

Christopher N. G. Anderson*, Kunimasa Ohta, Marie M. Quick, Angeleen Fleming, Roger Keynes and David Tannahill{dagger}

Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK
* Present address: Centre for Cell and Molecular Dynamics, Department of Anatomy and Developmental Biology, University College London, 5th Floor Rockefeller Building, Gower Street, London WC1E 6BT, UK



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  		Fig. 1. (A) Surround repulsion of peripheral DRG axons in the chick embryo trunk. Schematic diagram through the trunk in transverse section showing tissues that are sources of secreted repulsive signals (red arrows). These guidance cues operate in concert to constrain DRG axon paths in a typical bipolar trajectory (black arrows). (B) Expression of SEMA3A in the chick embryo notochord. Following whole-mount in situ hybridisation on stage 19 embryos, transverse sections (30 µm) were cut and mounted. The section shown is through the brachial region and SEMA3A expression is seen in the notochord and dermamyotome. Note that the neural tube split open during the hybridisation procedure. dm, dermamyotome; drg, dorsal root ganglion; fp, floor plate of neural tube; nt, neural tube; n, notochord; sc, sclerotome. Scale bar: ~100 µm.

 


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  		Fig. 2. Growth of different axon populations in response to the notochord in collagen gel co-cultures. (A-E) Dark-field photomicrographs of representative co-cultures. (A,B) Stage 36 (St36) DRG explants (n=90 and 36, respectively); (C,D) Stage 26-27 (St26) DRG explants (n=55 and 34, respectively); (E) Stage 36 SCG explants (n=28). Explants were cultured with NGF (A,C,E) or with NT3 (B,D). (F) Histogram showing the mean repulsion score±s.e.m. for neural explants co-cultured with stage 17-21 notochord. Asterisk indicates no significant repulsion. Scale bar: ~100 µm.

 


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  		Fig. 3. Growth of different axon populations in response to SEMA3A-expressing COS cells in collagen gel co-cultures. (A) Left, stage 36 (St36) DRG; right, stage 26-27 (St26) DRG; (B) Left, stage 36 DRG; right, stage 36 SCG. Scale bar: ~100 µm.

 


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  		Fig. 4. Effects of soluble neuropilin/Fc reagent in collagen gel co-culture experiments. (A-D) Stage 36 DRG explants cultured with COS cell aggregates transfected with vector (VEC) alone (A) (n=25) or SEMA3A (SEMA3A) (B-D). (B) Repulsion by SEMA3A-expressing cells in the absence of neuropilin/Fc (n=14). (C) Neuropilin 1/Fc at 10 µg/ml [NP1(10)] reduces repulsion by SEMA3A (n=13); whereas (D) neuropilin 2/Fc at 10 µg/ml [NP2(10)] has little or no effect on SEMA3A-induced repulsion (n=8). (E) Histogram showing the mean repulsion score±s.e.m. Neuropilin 1 or 2/Fc does not affect axon outgrowth in co-cultures of notochord and vector-expressing cells (n=12 and 7, respectively). (F,G) Stage 36 DRG explants co-cultured with stage 17-21 notochords in the presence of (F) 3 µg/ml neuropilin 1 [NP1(3)] or (G) 10 µg/ml neuropilin 1/Fc [NP1(10)]; (n=12 for both). (H,I) Stage 36 DRG explants co-cultured with stage 17-21 notochords in the presence of (H) 3 µg/ml neuropilin 2 [NP2(3)] or (I) 10 µg/ml neuropilin 2/Fc [NP2(10)]; (n=12 and 16, respectively). (J) Histogram showing the mean repulsion score±s.e.m. (n=25 for control cultures in the absence of neuropilin/Fc). Asterisks indicate no significant repulsion, or a significant reduction in repulsion, compared to positive controls (SEMA). Scale bar: ~100 µm.

 


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  		Fig. 5. Effects of soluble neuropilin/Fc reagents on repulsion by the notochord of stage 26-27 DRG and stage 36 SCG axons in collagen gel co-cultures. (A-C) Stage 26-27 DRG explants cultured with (A) 10 µg/ml neuropilin 1/Fc (NP1), (B) 10 µg/ml neuropilin 2/Fc (NP2) and (C) 5 µg/ml each of neuropilin 1 and 2/Fc (NP1/2); (n=27, 20 and 11, respectively). (D-F) As for A-C, but with stage 36 SCG explants (n=24, 16 and 10, respectively). (G) Histogram showing the mean repulsion score±s.e.m. (n=24 and 27 for DRG and SCG explants, respectively, in the absence of neuropilin/Fc reagents. The neuropilin/Fc reagent dose in µg/ml is indicated in brackets). Asterisks indicate a significant reduction in repulsion compared to controls. Scale bar: ~100 µm.

 


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  		Fig. 6. Expression of SEMA3C and neuropilin 2 in the developing chick embryo trunk. (A,C) Lateral views of wholemount in situ hybridisation for SEMA3C at stage 18 (A) and for neuropilin 2 at stage 20 (C). Somites anterior to the wing bud are shown (anterior is up, dorsal is right). SEMA3C expression in (A) is seen in the notochord (white arrows) and posterior halves of consecutive sclerotomes (white arrowheads). Neuropilin 2 expression in (C) is seen in the DRGs within the anterior half-sclerotomes (white arrowheads); black arrowheads indicate somite boundaries. Scale bars: ~100 µm. (B,D) Transverse sections (30 µm) through the trunk of stage 19 embryos after whole-mount in situ hybridisation. (B) SEMA3C expression at the level of a posterior half-sclerotome. Expression is seen in the notochord, posterior half-sclerotome and ventral half of the neural tube. (D) Neuropilin 2 expression at the level of the anterior half-sclerotome. Expression is seen in the anterior half sclerotome in the region of DRG formation, and in the ventral neural tube. Scale bar: ~100 µm. (E) Lateral view of whole-mount in situ hybridisation for neuropilin 2 at stage 24. Expression is seen in the anterior half-sclerotomes of recently formed somites (arrow) and in the DRGs forming more anteriorly (arrowheads). Scale bar: ~1 mm. (F) Whole-mount in situ hybridisation for neuropilin 2 on a stage 26 embryo trunk. After in situ hybridisation, the trunk was hemisected and viewed from the neural tube side to help visualise expression. Weak neuropilin 2 expression is seen in the DRGs (arrows) and axons leaving the DRGs (arrowheads). Scale bar: ~100 µm. as, anterior half-sclerotome; drg, dorsal root ganglion; nt, neural tube; n, notochord; ps, posterior half-sclerotome.

 


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  		Fig. 7. SEMA3C repels SCG but not early DRG axons in collagen gel co-cultures. (A,B) Stage 35 SCG explants cultured with COS cell aggregates transfected with (A) vector (VEC) alone (n=31) or (B) SEMA3C (SEMA3C) (n=24). (C,D) Stage 26-27 DRG explants cultured with COS cell aggregates transfected with (C) vector (VEC) alone (n=22) or (D) SEMA3C (SEMA3C) (n=18). (E) Histogram showing the mean repulsion score±s.e.m. Scores were assessed in cultures in which the explants lay between 150-450 µm of the COS cell aggregate. Asterisks indicate no significant repulsion. Scale bar: ~100 µm.

 


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  		Fig. 8. Axon repulsion of other neuronal populations by the notochord. (A) Axons from stage 26-29 retinal explants are repelled by the notochord (n=52), but axons from stage 36 olfactory bulb explants are not (B) (n=20). (A,B) Co-cultures viewed by phase-contrast microscopy. Scale bar: ~100 µm. (C) Histogram showing the mean repulsion score±s.e.m. Soluble neuropilins do not reduce the repulsion of retinal axons by the notochord; n=11 for both neuropilin 1/Fc (NP1) and neuropilin 2/Fc (NP2), respectively. Asterisk indicates no significant repulsion.

 

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