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Onset of neuronal differentiation is regulated by paraxial mesoderm and requires attenuation of FGF signalling

Ruth Diez del Corral1, Dorette N. Breitkreuz2 and Kate G. Storey1,*

1 Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dow Street, Dundee, DD1 5EH, UK
2 Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK



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  		Fig. 1. Spatial and temporal separation of the events of neurogenesis during the generation of the spinal cord (D,E,G,I,M-P are tissue sections). (A,B) Summary of the gene expression domains in the developing embryo (medio-lateral extent of expression is not indicated). (A) Stage 7+ embryo, CNP, caudal neural plate; PS, primitive streak; S, first formed somite; PSM, presomitic mesoderm; dashed lines, underlying tissues. (B) Stage 8 embryo. PNT, preneural tube; NT, neural tube. (C-E) The first neurons appear opposite recently formed somites (arrowhead) as revealed by NeuroM expression in a stage 8– embryo. (F-I) NeuroM expression extends caudally within the neural tube and appears adjacent to the most recently formed somites (arrowheads) in stage HH8+ (F,G) and stage HH9 (H,I) embryos. (J-L) Sax1 expression in the regressing CNP and PNT prefigures neurogenesis and is down regulated in the neural tube at about the level of the most recently formed somite (arrowheads). (M-P) NeuroM-expressing cells (blue, arrowheads) do not incorporate BrdU (FITC/green) in stage 12 neural tube. Double exposures (M,O), bright field (N), FITC only (P). Scale bars, (C,F,H,J,K,L) 200 µm; (D) 100 µm; (E,G,I) 50 µm; (M) 20 µm; (N,O,P) 10 µm.

 


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  		Fig. 2. Somites elicit neuronal differentiation in CNP explants. (B-H,J,L,O-Q are tissue sections.) (A) Diagram of CNP explant in a stage 7+ embryo. (B-H) CNP explants continue to express Sox2 (B), cash4 (C), Sax1 (D) bra (E) and homogenous Delta 1 (F), but do not express NeuroM (G) or contain cells with neurofilament (NF)-positive fine processes (H). (I-L) CNP explants cultured with differentiating PSM contain isolated Delta 1-positive cells (I,J) and NeuroM-expressing cells (K,L), and this presomitic mesoderm differentiates into paraxis-expressing tissue (M) (note, NeuroM-positive cells in K,L appear at one end of the explant and are likely to be underlain by paraxis-positive cells, as seen in M). (N,O) CNP explants cultured with somites also express NeuroM and (P) contain cells with neurofilament-positive fine processes (arrowhead). (Q) cash4 is down regulated in CNP explants cultured with differentiating PSM. (R) CNP explants cultured alone express NeuroM after 48 hours in culture and (S) in some cases contain paraxis-positive cell. Scale bars, (B-H,J,L,O,P) 50 µm; (I,K,M,N,R,S) 100 µm; (Q) 100 µm.

 


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  		Fig. 3. Removal of the presomitic mesoderm elicits premature neuronal differentiation and the down regulation of CNP-specific genes. (C,E,H,J,L,N,P,R are transverse sections.) (A) Diagram illustrating removal of PSM at stage 7+ and the embryo 6 hours later. (B,C) Depletion of NeuroM-expressing cells in neural tube developing following PSM removal. S1, most recently formed somite on un-operated side. (D,E) Sox2 expression is unaffected by PSM removal. (F) Diagram of the removal of somites at stage 8/8+ and the embryo 6 hours later. (G,H) Depletion of NeuroM-expressing cells following somite removal. S, first somite formed after removal on operated side. (I,J) Sox2 and (K,L) Delta 1 are unaffected by somite removal. (M,N) NeuroM expression extends into the PNT in the absence of PSM. (O,P) Removal of PSM also down regulates cash4 and (Q,R) Sax1 in the PNT. Tissue between arrowheads in B,G,M,O and Q are regions of ectopic (M) or absent gene expression (B,G,O,Q). Scale bars: (B,D,G,I,K,M,O,Q) 100 µm; (C,E,H,J,L,N,P,R) 100 µm.

 


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  		Fig. 4. FGF signalling upregulates CNP-specific genes and represses neuronal differentiation in vivo. (A) Diagram showing position of bead beneath stage 7+ CNP. (B,C) FGF4 beads placed beneath the CNP maintain ectopic expression of cash4 (B) and Sax1 (C), but control PBS beads (D,E) do not alter gene expression. (F,G) FGF8 beads inhibit expression of NeuroM (F), while PBS beads do not (G). Scale bar: (B-G) 200 µm.

 


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  		Fig. 5. FGF signalling represses neuronal differentiation and somites repress Fgf8, but attenuation of this pathway is not sufficient to elicit neuronal differentiation. (A) CNP explants cultured with PSM express NeuroM after 24 hours, but (B) only a few NeuroM-positive cells appear in the presence of FGF4. (C) CNP explants contain NeuroM-positive cells after 48 hours, but (D) fail to express this gene with FGF4. (E) Diagram to show the position of PNT and NT explants from a stage HH8 embryo. (F) PNT explants cultured for 24 hours contain NeuroM-positive cells but FGF4 treatment (G) inhibits NeuroM expression. (H) PNT explants cultured for 24 hours do not contain any bra-expressing cells but maintain some expression in the presence of FGF4 (I). (J) NT explants contain NeuroM +ve cells, while (K) FGF4 inhibits NeuroM expression. (L) CNP explants retain Fgf8 after 24 hours, but (M) Fgf8 is repressed when cultured with somites. (N) CNP explants cultured for 24 hours express bra but treatment with SU5402 down regulates bra (O) and maintains expression of Sox2 (P). (Q) CNP explants do not contain NeuroM-positive cells after 24 hours even following treatment with SU5402 (R). Scale bar: 100 µm.

 


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  		Fig. 6. Model for the regulation of neuronal differentiation by paraxial mesoderm and levels of FGF signalling. (A) Tissue level regulation of neuronal differentiation. The neural tube in contact with somites contains NeuroM-expressing cells (purple dots) while the CNP/PNT underlain by the PSM does not express this neuronal marker and expresses caudal neural genes. Somites provide signals that repress caudal neural genes and activate neuronal differentiation, whereas the PSM provides signals that maintain caudal neural gene expression and prevent neuronal differentiation. Removal experiments indicate that signals from other tissues in the embryo must also influence the appearance of neurons. *Induction of neurons by other tissues at the level of the CNP/PNT may also be a consequence of the down-regulation of caudal neural genes. (B) FGF signalling and the regulation of spinal cord differentiation. FGF8 and FGF4 are present in the primitive streak and FGF8 is also present in the PSM and the CNP/PNT where these signals promote bra and caudal neural genes and prevent the appearance of NeuroM-positive cells. Somites repress Fgf8 expression in CNP/PNT. Attenuation of FGF signalling down regulates the early mesodermal gene bra but does not elicit neuronal differentiation (NeuroM); somites must therefore also interact with additional pathways to promote NeuroM either by inducing an activator (dashed line) and/or repressing a repressor (see text). (C) Fgf8 expression in stage 9– embryo, showing transcripts in the CNP and PNT, aligned with B. Scale bar: 100 µm.

 

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