September 15, 2007;
Neural crests are actively precluded from the anterior neural fold by a novel inhibitory mechanism dependent on Dickkopf1 secreted by the prechordal mesoderm.
It is known the interactions between the neural plate
generate neural crest
), but it is unknown why the NC
develops only at the lateral
border of the neural plate
and not in the anterior
fold. Using grafting experiments we show that there is a previously unidentified mechanism that precludes NC
from the anterior
region. We identify prechordal mesoderm
as the tissue
that inhibits NC
in the anterior
territory and show that the Wnt/beta-catenin antagonist Dkk1
, secreted by this tissue
, is sufficient to mimic this NC
inhibition. We show that Dkk1
is required for preventing the formation of NC
in the anterior
neural folds as loss-of-function experiments using a Dkk1
blocking antibody in Xenopus as well as the analysis of Dkk1
-null mouse embryos transform the anterior neural fold
. This can be mimicked by Wnt/beta-catenin signaling activation without affecting the anterior posterior
patterning of the neural plate
, or placodal specification. Finally, we show that the NC
cells induced at the anterior neural fold
are able to migrate and differentiate as normal NC
. These results demonstrate that anterior
regions of the embryo
because of a mechanism, conserved from fish to mammals, that suppresses Wnt/beta-catenin signaling via Dkk1
[+] show captions
Fig. 1. Alternative models for the absence of NC at the ANF. (A, B) Current models. (A) The NC inductive signals (neural plate/epidermis interaction) are present only where the NC is induced and absent from the ANF. (B) Posteriorizing signals required to induce NC do not reach the ANF. (C) Proposed model: the NC inductive signals are present along the entire border of the neural plate and the NC is induced along the entire neural plate border, but at a later stage a specific NC inhibitory signal is produced at the ANF that restricts NC development to the lateral sides of the neural plate border. (D) Expression of Snai1 at stage 11, showing expression at the ANF (arrow). (E) Expression of Snail1 at stage 13, showing absence of expression at the ANF (arrow). (F) Expression of Pax3 at stage 11, showing expression at the ANF (arrow). (G) Expression of Pax3 at stage 13, showing absence of expression at the ANF (arrow).
Fig. 2. The NC is inhibited at the ANF by the PM. (A–G) Inhibition of NC induction at the ANF. (A) Embryos were injected at the 1-cell stage with FLDX, at stages 14–18 the NC were dissected and grafted into the ANF, or lateral epidermis of a stage 13/14 control embryos, or cultured in vitro until stage 18/19, when the expression of Snail2 was analyzed. (B) NC dissected at stage 15 and cultured in vitro until stage 18 showing expression of Snail2 (n = 60, 100% of expression). (C) Anterior view of an embryo containing a graft of stage 15 NC as described. Dotted line shows the green color of the FLDx graft. (D) Same embryo as in C, showing the absence of Snail2 in the graft (dotted line). (E) Lateral view of an embryo in which NC taken from a stage 15 embryo was grafted in lateral epidermis, showing expression of Snail2 in the graft. Approximately 50 embryos were analyzed for each case. (F–K) PM inhibits NC induction. (F) Anterior ectoderm (AE, green square) or prechordal mesoderm (PM, yellow circle) was dissected from a stage 15 embryo and grafted next to the NC from a control embryo, after culture until stage 18, the expression of Snail2 was analyzed. (G) No effect on Snail2 expression was observed with the AE graft. Asterisk: graft, n = 24, 100% of expression. (H) Inhibition of Snail2 expression with PM graft. Asterisk: graft, n = 35; 78% of grafted embryos exhibited inhibition of Snail2 expression. (I) PM and NC were dissected from stage 15 embryos, conjugated in vitro and the expression of Snail2 was analyzed at the equivalent of stage 18. (J) NC cultured in vitro showing normal expression of Snail2 (arrow); n = 23; 100% of expression. (K) Conjugate of NC and PM showing inhibition of Snail2 expression; n = 38, 80% of conjugates with inhibition of Snail2 expression.
Fig. 3. Assay to identify NC inhibitors in vitro. (A) Embryos were injected at the 1-cell stage with mRNA coding for the tested molecules, at stage 9 animal caps were dissected and conjugated with NC taken from stages 13–17 control embryo; the expression of Snail2 was analyzed. (B) Stage 15 NC conjugated with control animal caps shows normal Snail2 expression; n = 67, 100% of expression. (C) Stage 15 NC conjugated with AC expressing Dkk1 shows absence of Snail2 expression. (D) Summary of expression of Snail2 in grafts into the ANF of NC taken from embryos at different stages (as described in Fig. 2A; black bars) and Dkk1 expressing animal caps (as described in panel A; light gray bars). Note a similar trend on NC inhibition in the graft and in the Dkk1 conjugates. Approximately 50 embryos were analyzed for each stage. (E) Beads soaked with BSA or Dkk1 protein were grafted next to the NC of a stage 15 embryo. (F) Control bead soaked with BSA (arrow) shows no effect on Snail2 expression; n = 20, 100% of expression. (G) Bead soaked with Dkk1 shows inhibition of Snail2 expression; n = 14, 92% of inhibition. (H–M) Analysis of Pax3 and Dkk1 expression. (H) Expression of Pax3 in a stage 11 embryo. (I) Expression of Dkk1 in stage 11 embryo. (J) Overlapping of pictures shown in panels I and J after artificial color change. (K) Expression of Pax3 in a stage 13 embryo. (L) Expression of Dkk1 in stage 13 embryo. (M) Overlapping of pictures shown in panels K and L after artificial color change. Note the correlation between Dkk1 expression and Pax3 restriction.
Fig. 4. Dkk1 is essential for NC inhibition at the ANF. (A–D) Effect on Xenopus. Embryos are shown in dorso-anterior view. (A) Embryo injected at the blastula stage with an anti-prolactin antibody as control, it shows absence of Snail2 expression at the ANF (arrow); n = 40, 0% of expression at the ANF. (B) Embryo injected at the blastula stage with anti-Dkk1 antibody showing Snail2 expression at the ANF (arrow); n = 86, 60% of expression. (C) Control NC/PM conjugates (0% of Snail2 expression, n = 7). (D) NC/PM conjugates cultured with the anti-Dkk1 antibody (75% Snail2 of expression, n = 8). (E, F) Effect on mouse. (E) Anterior view of wild type mouse embryo (E9.5) showing normal expression of Sox10, and no expression at the ANF (arrow). Inset: lateral view of the same embryo. (F) Anterior view of Dkk1 mutant mouse embryo (E9.5) showing expression of Sox10 at the ANF (arrow). Inset: lateral view of the same embryo; n = 12, 83% of embryos showing expression of Sox10 at the ANF. Note that the expression of Sox10 is normal in the midbrain, hindbrain and trunk NC, suggesting more NC rather than a truncation.
Fig. 5. Activation of Wnt signaling leads to transformation of ANF into NC. (A–F) Effect on Xenopus. Embryos are shown in dorso-anterior view. (A) Diagram showing where the injections were performed. (B) Control embryo showing normal Snail2 expression. (C, D) Embryos injected with 2 ng of Wnt8 mRNA and FLDx into A1 blastomere of a 32-cell stage embryo. Green cells in panel D correspond to the injected cells, which are indicated by the dotted line in panels C and D. Note expression of Snail2 in all the ANF (arrows and arrowheads); n = 50, 55% of embryos showing ectopic Snail2 expression. (E, F) Embryos injected with 130 pg of β-catenin-GR mRNA and FLDx into A1 blastomere of a 32-cell stage embryo, induced at stage 10.5. Green cells in F correspond to the injected cells which are indicated by the dotted line in E. Note expression of Snail2 at the ANF but this time restricted to the injected cells (arrow); n = 67, 56% of embryos showing ectopic Snail2 expression. (G, H) Zebrafish embryos showing expression of Foxd3. (G) Control embryo. (H) Embryo injected with 0.01 pmol of Tcf3a MO.
Fig. 6. Temporal analysis of transformation of ANF into NC. (A) Representative embryo showing transformation phenotype monitored by the expression of the NC-specific gene Snail2. Note the transformation of the ANF into NC with almost no effect in the medio-lateral axis. (B) Representative embryo showing the expansion phenotype monitored by the expression of Snail2. Note that the expansion of the NC is as much in the anterior–posterior axis as it is in the medio-lateral axis. (C) Diagram to show the experimental design used in panels D and E. Embryos were injected with different doses of β-catenin-GR mRNA at the 32-cell stage and treated with dexamethasone at different stages (green bar), the expression of NC markers was analyzed at stage 16. (D) Analysis of the ANF transformation phenotype and NC expansion phenotype (inset) after injection of 100 pg of β-catenin-GR and activation at different stages. Note that the highest ANF transformation is reached with activation at stage 10–11 while the highest NC expansion phenotype is reached with activation at stage 9. (E) Analysis of the ANF transformation phenotype and NC expansion phenotype (inset) after injection of different doses of β-catenin and activation at stage 11. Note that the higher doses of β-catenin can still induce NC expansion at later stages (inset).
Fig. 7. Analysis of neural plate and placodal markers in embryos with NC at the ANF. (A–D, I–L) Xenopus embryos were injected with 130 pg of β-catenin mRNA in A1 blastomere at 32-cell stage, treated with dexamethasone at stage 10.5, and the expression of different markers was analyzed at stage 16. All embryos are shown in anterior views; injected side is to the right and recognized by FLDx or β-gal staining (blue color). (A) Snail2/Krox20. Note expression of Snail2 (52%, n = 50) at the ANF and normal expression of Krox20 (white arrowheads, 95%, n = 40). (B) Otx2. Note no effect on Otx2 in the injected side (23%, n = 45). (C) Bf1. No effect on Bf1 at the injected side (12%, n = 41). (D) Cpl1. No effect on Cpl1 at the injected side (17%, n = 42). (E, F) Zebrafish embryos. (E) Wild type embryos showing expression of FoxD3. (F) Embryo injected with 0.01 pmol of Tcf3a MO. Note the expression of FoxD3 at the ANF (100%, n = 50). (G, H) Sibling embryos of panels E and F, respectively analyzed for Rxr3 expression. Note the normal expression of Rxr3 in wild type and injected embryo (75%, n = 40). (I–L) Placodal markers. White lines indicate the distance between the neural grove or neural plate and the placodal markers. (I) Six1 expression. Note the anterior–ventral shift in the expression of Six1 at the injected side (47%, n = 48). (J, K) Sox3 expression. Note the shift in the preplacodal domain of Sox3 expression (45%, n = 45), while the neural plate domain is not affected. (L) Dkk1 expression; note similar expression in the injected and uninjected side (99%, n = 40). (M) Summary of anterior markers. NP: neural plate; NC: neural crest; TL: telencephalon; PL: placodal field. (N) BrdU staining. Left: control side; right: injected side. Bar indicates the thickness of the ectoderm which is higher in the injected side; black arrowhead shows the ANF where there is a particularly higher number of stained nuclei in the injected side. (O) Summary of BrdU staining. BrdU-positive nuclei in the injected ectoderm compared with the ones on the contralateral uninjected region.