XB-ART-45533Dev Biol 2012 May 01;3651:196-207. doi: 10.1016/j.ydbio.2012.02.029.
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Early neural crest induction requires an initial inhibition of Wnt signals.
Neural crest (NC) induction is a long process that continues through gastrula and neurula stages. In order to reveal additional stages of NC induction we performed a series of explants where different known inducing tissues were taken along with the prospective NC. Interestingly the dorso-lateral marginal zone (DLMZ) is only able to promote the expression of a subset of neural plate border (NPB) makers without the presence of specific NC markers. We then analysed the temporal requirement for BMP and Wnt signals for the NPB genes Hairy2a and Dlx5, compared to the expression of neural plate (NP) and NC genes. Although the NP is sensitive to BMP levels at early gastrula stages, Hairy2a/Dlx5 expression is unaffected. Later, the NP becomes insensitive to BMP levels at late gastrulation when NC markers require an inhibition. The NP requires an inhibition of Wnt signals prior to gastrulation, but becomes insensitive during early gastrula stages when Hairy2a/Dlx5 requires an inhibition of Wnt signalling. An increase in Wnt signalling is then important for the switch from NPB to NC at late gastrula stages. In addition to revealing an additional distinct signalling event in NC induction, this work emphasizes the importance of integrating both timing and levels of signalling activity during the patterning of complex tissues such as the vertebrate ectoderm.
PubMed ID: 22394485
PMC ID: PMC3657187
Article link: Dev Biol
Species referenced: Xenopus
Genes referenced: bmp4 dkk1 dlx5 hes4 msx1 nog npb pax3 snai1 snai2 sox2 sox3 zic3
Phenotypes: Xla Wt + Hsa.BMP4 (fig.4.d)
Article Images: [+] show captions
|Fig. 1. The DLMZ is able to promote expression of neural plate border (NPB) makers in absence of neural crest (NC). Explants of different sizes were taken at stage 10 then cultured for 30 h. A. Prospective neural crest/neural plate (NC/NP) tissue alone, together with dorso-lateral marginal zone (DLMZ; B) or with DLMZ and epidermis (EP; C). D. DLMZ alone. Markers examined: the neural crest markers Pax3 (i)and Snail2 (ii), the neural plate marker Sox2 (iii), the neural plate border markers Hairy2a (iv), Dlx5 (v), Msx1 (vi) and Zic3 (vii). Also the epidermal marker Keratin (viii). When NC/NP is taken alone, only epidermis is formed (A). In NC/NP/DLMZ explants: only NP and NPB markers are expressed (Biii–viii), but not Pax3 (Bi) or Snail2 (Bii). With the addition of the epidermis (EP), all markers are present (C). None of the ectodermal markers tested are expressed in the DLMZ alone (D). E. Explants of NC/NP/DLMZ and FDX-labelled epidermis (EP) were cultured for 30 h either separately or as conjugates. As expected, no Snail2 expression is observed either in the NC/NP/DLMZ explants (Ei; 0/15) or in the epidermis explants (Eii; 0/15) when cultured alone. However Snail2 expression (purple) is seen in conjugates (Eiii; 12/15). Epidermis has been stained cyan for FDX label, note that Snail2 is induced within NC/NP/DLMZ and not in the epidermal region (Eiii).|
|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).|
|Fig. 3. An inhibition of Wnt signalling is required for neural plate border specification. A–F. Explants of prospective neural crest and neural plate with adjacent dorso-lateral mesoderm (NC/NP/DLMZ explants) were taken at stage 10, cultured for 27 h and then analysed for the expression of the neural plate border (NPB) markers Dlx5 and Hairy2a and the neural plate (NP) marker Sox2. For each condition the % of explants expressing each marker is shown in the graphs (C–F). B. Control explants cultured with beads soaked in 0.1% BSA (B). C. Explants cultured with Dkk1 protein soaked beads maintained expression of NP and NPB markers. D. Prior to taking explants, embryos were injected at the two-cell stage with β-cateninGR mRNA and explants were cultured in presence or absence (not shown) of dexamethasone. Acitivating β-catenin leads to a loss of NPB border markers, but no change in Sox2. E. Explants cultured in the presence of Noggin protein had no affect on NPB markers, and a slight increase in Sox2. F. Explants cultured with BMP4 protein had no affect on NPB markers, though Sox2 was abolished.|
|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.|
|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|
|Fig. 6. A dynamic modulation of Wnt levels accompanies the transition from neural plate border to neural crest specification. A, B. Measurements of canonical Wnt activity were measured using a TOPflash luciferase reporter assay. Measurements normalised both protein concentration to control for differences in explant size and displayed as a percentage of stage 10 prospective neural crest (NC) activity. Measurements of Wnt activity made from NC explants at different stages (A) or in explants of NC, DLMZ or EP and measured immediately (B). C. Measurements of cultured explants of NC/NP together with either the dorso-lateral marginal zone (DLMZ) or the DLMZ together with epidermis. D–F. Explants of st11.5 prospective NC were taken from embryos previously injected with a dexamethosone (DEX) inducible beta-catenin construct. No Snail2 expression seen the absence of DEX (0% of cases, n = 21; E), though is expressed upon DEX addition (75% of cases, n = 20; F). G–I. To have an inhibition of Wnt signalling during gastrulation, but a later activation of the signalling pathway during neurulation we first injected 2-cell stage embryos with the inducible construct β-cateninGR. Grafts of Dkk1 soaked beads were then added to the embryos at stage 10, which resulted in an inhibition of Snail2 expression at stage 13 (data not shown, 75%, n = 12). Experimental design (G). In the absence of dexamethasone, this inhibition is still observed at stage 17 (H; 66%, n = 21). However activation of β-cateninGR during the maintenance stage rescues this inhibition (I; 65% of embryos rescued, n = 17). All embryos shown in dorsal view with anterior to the left.|
|Fig. 7. A three-step model for NC induction. Initially Wnt signals need to be repressed for the early neural plate border markers Hairy2a and Dlx5. Subsequently, an activation of Wnt together with intermediate BMP signals is required for NC specification. Finally, an activation of both BMP and Wnt is required for NC maintenance (see text for details).|
References [+] :
Aybar, Snail precedes slug in the genetic cascade required for the specification and migration of the Xenopus neural crest. 2002, Pubmed, Xenbase