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First published online 1 September 2005
doi: 10.1242/dev.02009

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Specification and maintenance of the spinal cord stem zone

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

View larger version (93K): [in a new window]   Fig. 1. Comparison of early neural, mesodermal and stem zone-specific gene expression adjacent to the primitive streak during body axis extension. Expression of Sox3, Sox2, bra and Fgf8 at HH3-3+ (A-D'), HH4 (E-H') and, additionally, Sax1 and Hoxb8 at HH6-7 (M-N'), HH8+-9 (S-T') and HH10+ (U-Z'). Levels of transverse sections are indicated with a black bar and epiblast adjacent to the primitive streak lies between arrowheads. Arrowhead in Z indicates somite 5. Diagrams show the positions of the stem zone from HH stages 4-10.

View larger version (58K): [in a new window]   Fig. 2. Timing and tissue interactions underlying stem zone specification. 3c epiblast explants (A) do not express Sax1 (B) or Hoxb8 (C), but do express bra (D) and Sox2 (E). 4c explants (F) express Sax1 (G), Hoxb8 (H), bra (I) and Sox2 (J). 4a and 4b explants each include a region medial (a) or lateral (b) to 4c (K). 4a explants express Sax1 (L), Hoxb8 (M), bra (N) and Sox2 (O). 4b explants do not express Sax1 (P), Hoxb8 (Q) or bra (R), but are Sox2 positive (S). All explants assessed after 24 hours. (T) Explants 3c, 4b, 7CPM (caudal paraxial mesoderm) and combination experiments (a,b,c and d, respectively). Stage 7 CPM induces expression of Sax1 (U) and Hoxb8 (V) in 3c explants. In 4b explants, the CPM induces Sax1 (W) but not bra (X) after 24 hours. Expression of Sax1 (Y) and bra (Z) is also undetected after 8-12 hours. Scale bar: 100 µm.

View larger version (92K): [in a new window]   Fig. 3. FGF signalling is insufficient for stem zone specification. FGF8 soaked bead in extra-embryonic epiblast at HH3 (A). bra is induced by FGF8 within 4 hours (B) (high magnification in C), but stem zone gene Sax1 (D) is only detected after 10 hours. 4b explants do not express Sax1 following exposure to 200 ng/ml FGF4 (E,F) or to 250 ng/ml FGF8 (G,H), but can express bra following exposure to 200 ng/ml FGF4 (I,J) or to 250 ng/ml FGF8 (K,L). Scale bar: 100 µm.

View larger version (66K): [in a new window]   Fig. 4. FGF signalling is required for stem zone specification. 3c and stage 7 CPM explant combination experiments (A). The ability of 7CPM to induce Sax1 (B,C) and Hoxb8 (D,E) is inhibited by exposure to SU5402 but not DMSO. Electroporation scheme at HH4 (F). Mis-expression of GFP only does not alter Sax1 expression (G,H), but that of DnFGFR1 inhibits Sax1 onset (red arrowheads, I-J'), while not reducing Sox2 (K-L').

View larger version (79K): [in a new window]   Fig. 5. FGF/MAPK signalling is required for stem zone maintenance. Electroporation scheme at HH10 (A). Mis-expression of GFP only does not reduce Sax1 expression (B-C'), while dnFGFR1 inhibits Sax1 (D-E'), but not Sox2 (F-G'). SU5402, but not DMSO, leads to loss Sprouty2 (H,I) and Sax1 (J,K), but not Sox2 expression (L,M). Similarly, PD184352, but not DMSO, reduces Sprouty2 (N,O) and Sax1 (P,Q), but does not attenuate Sox2 expression (R,S).

View larger version (65K): [in a new window]   Fig. 6. Stem zone gene expression in FGF- and retinoid-deficient embryos. SU5402, but not DMSO, leads to loss of Hoxb8 expression (A,B). Vitamin A-deficient (VAD) quails commence expression of Sax1 (C,C') and Hoxb8 (D,D') in the stem zone as in normal quail embryos.

View larger version (29K): [in a new window]   Fig. 7. Steps in stem zone specification and maintenance. At HH4, an overlap between pan-neural (Sox2) and early mesodermal gene (bra) expression is observed in epiblast cells close to the anterior primitive streak. Underlying paraxial mesoderm begins to express Raldh2, while Fgf8 expression is restricted to the primitive streak. From HH7, stem zone-specific gene expression (Sax1) commences in epiblast cells close to the primitive streak. Sax1 onset requires signals from the paraxial mesoderm and an intact FGF pathway, which works at least in part by inhibiting the retinoid pathway (see text). At this time, Raldh2 is rostrally restricted by FGF8 in newly emerged paraxial mesoderm (Diez del Corral et al., 2003). Retinoic acid can also attenuate Fgf8 levels (Diez del Corral et al., 2003) and so a fine balance between FGF and retinoid signalling is required for formation of the stem zone. Sax1 induction can take place without prior creation of an ambivalent mesodermal/neural cell state. At HH10, Sax1 and Fgf8 expression are also present in the transition zone and Sax1 is maintained by FGF/MAPK signalling. There is a persisting overlap of neural and mesodermal genes in the stem zone, which may be the location of bipotent stem cells.