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Fig. 2. NPB specification occurs earlier than Snail2. A, B. Stage 11.5 embryos were injected with small quantities of the lipophilic marker DiI. Groups of cells were marked and their resulting contribution determine at stage 28. The key shows which colours were used in the map to represent each tissue (A). Circles were drawn around each labelled area and mapped onto stage 11.5 (B). Dorsal is to the right, animal to the top. White arrow indicates dorsal blastopore lip. C–E. Expression of Hairy2a (C) and Dlx5 (D) overlaps with the prospective NC, adjacent to the neural plate (NP) marker Sox3 (E). F–I. At stage 13 the neural crest marker Snail2 (NC; F) overlaps with the neural plate border (NPB) markers Hairy2a (G) and Dlx5 (H) adjacent to the NP marker Sox3 (I). J–P. Explants of prospective neural crest cells at stage 10 (K–M) or stage 11.5 (N–P) and cultured until sibling embryos were stage 20 and analyzed for Snail2 (K,N), Dlx5 (L,O) or Hairy2a (M,P). R–Z. Explants of dorso-lateral marginal zone (DLMZ), prospective neural crest and neural plate (NP) and epidermis (EP) were taken together and cultured for various time-points. Expression Snail2 (Q,U,X), Dlx5 (S,V,Y) and the neural plate marker Sox2 (T,W,Z) were analysed by in situ hybridisation. Note that after 23 h only Sox2 is induced (T), then after 27 h both Dlx5 and Sox2 are present (V,W). After 30 h all markers are expressed (X–Z). |
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Fig. 4. The neural plate (NP) and neural plate border (NPB) respond to BMP signals in successive time windows. A. Experimental design; Embryos were manipulated (red arrow) at the beginning of gastrulation and the affect on neural plate border (hairy2a/dlx5) and neural plate (sox2) markers was assessed. B–C. A bead soaked in Noggin results in lateral shift of Dlx5 (C) compared to control side (B) when fixed at stage 12. D–E. A bead soaked in BMP4 leads to a shift of Hairy2a towards the midline (D; 71%, n = 17) with a corresponding reduction in the Sox2 expression (E; K; 81%, n = 11). F–G. A bead soaked in Noggin leads to a lateral shift in Hairy2a but without affecting the thickness of the NPB (F; 69%, n = 13) (F) expression with a corresponding expansion of Sox2 (G; 65%, n = 32 G). H–K. Stage 12 explants of both NP and epidermis (EP) were taken, conjugated and cultured until sibling embryos were at stage 20. I. When co-cultured with a PBS bead, Snail2 expression is induced (70% of cases, n = 20). This is inhibited when co-cultures with either a BMP4 (J; 0% with expression, n = 15) or Noggin (K; 20% with expression, n = 10) soaked bead. L–M stage 12 explants continue to express Sox2 in the presence of either a PBS (L; 100% of cases, n = 7) or BMP4 (M; 100% of cases, n = 8) soaked bead. Black lines indicate distances from midline to the NPB. Black arrowheads indicate position of bead. |
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Display additional annotations [+]
| Gene |
Clone |
Species |
Stages |
Anatomy |
|
sox2
|
|
laevis
|
NF stage 15
|
neuroectoderm
,
pre-chordal neural plate
,
chordal neural plate
,
neural plate
|
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sox3
|
|
laevis
|
NF stage 12
to
NF stage 18
|
pre-chordal neural plate
,
chordal neural plate
,
neural plate
,
anterior neural fold
|
|
sox3
|
|
laevis
|
NF stage 16
|
pre-chordal neural plate
,
chordal neural plate
,
neural plate
,
anterior neural fold
|
|
hes4
|
|
laevis
|
NF stage 12
|
neural groove
,
pre-chordal neural plate border
,
chordal neural plate border
,
anterior neural ridge
|
|
hes4
|
|
laevis
|
NF stage 15
|
neural plate border
,
posterior placodal area
|
|
| |
Fig. 5. The neural plate (NP) and neural plate border (NPB) respond to Wnt signals in successive time windows. A–M. Embryos were injected at the 64-cell stage with the inducible beta-catenin contruct β-cateninGR and fixed at stage 12 to determine the specific affect on either NP or NPB markers. B–C. No affect on Dlx5 is seen in the absence of dexamethasone (0% of embryos, n = 9). D–E. Dlx5 is inhibited within injected cells when the construct is activated at stage 10 (70% with inhibition, n = 10). H–I. Inhibition of Sox2 is observed within injected cells when the construct is activated immediately after injection (75% of embryos with inhibition, n = 16). J–K. Little affect is observed upon Sox3 activation at stage 10 (14% of embryos with slight inhibition, n = 14). L–M. No affect is observed in the absence of dexamethasone 0% of embryos affected, n = 22). F–O. Injection of β-cateninGR at the 32-cell stages leads to a loss of both Hairy2a (F; 50% of embryos affected, n = 8) and Dlx5 (G; 65%, n = 23) at stage 15. No affect was observed in the absence of dexamethasone (data not shown, 0%, n = 24). No inhibition of Sox2 was observed either in the presence (G; 0%, n = 38) or absence (data not shown, 0% n = 36) or dexamethasone. P–V. Dkk1/PBS or PBS soaked beads were grafted next to the prospective neural crest at stage 10 and embryos were fixed at stage 15. No expansion of the neural plate was detected either with PBS beads (Q; 0%, n = 24) or with Dkk1 (R; 0%, n = 12). No effect was observed with BSA soaked beads on Hairy2a (S; 0%, n = 26), an expansion was observed with Dkk1 (T; 63%, n = 36). An expansion of Dlx5 was also observed with Dkk1 beads (V; 75% of cases, n = 8) compared to control side (U). Black arrows indicate areas affected, black circles indicate position of grafted bead |
