October 1, 2009;
The posteriorizing gene Gbx2 is a direct target of Wnt signalling and the earliest factor in neural crest induction.
Wnt signalling is required for neural crest
) induction; however, the direct targets of the Wnt pathway during NC
induction remain unknown. We show here that the homeobox gene Gbx2
is essential in this process and is directly activated by Wnt/beta-catenin signalling. By ChIP and transgenesis analysis we show that the Gbx2
regulatory elements that drive expression in the NC
respond directly to Wnt/beta-catenin signalling. Gbx2
has previously been implicated in posteriorization of the neural plate
. Here we unveil a new role for this gene in neural fold
patterning. Loss-of-function experiments using antisense morpholinos against Gbx2
and expand the preplacodal domain, whereas Gbx2
overexpression leads to transformation of the preplacodal domain into NC
cells. We show that the NC
specifier activity of Gbx2
is dependent on the interaction with Zic1
and the inhibition of preplacodal genes such as Six1
. In addition, we demonstrate that Gbx2
is upstream of the neural fold
. Our results place Gbx2
as the earliest factor in the NC
genetic cascade being directly regulated by the inductive molecules, and support the notion that posteriorization of the neural folds is an essential step in NC
specification. We propose a new genetic cascade that operates in the distinction between anterior
placodal and NC
[+] show captions
Fig. 1. Gbx2 is expressed in posterior ectoderm that includes the prospective neural crest. (A-C) Hypothesis of neural crest (NC) induction by the posteriorizing activity of Gbx2. (D-S) In situ hybridization at the indicated stages for the indicated genes. (D-G) Dorsal view, anterior to the top. (H-K) Transverse sections. (L-O) Lateral view, anterior to the left. (P-R) Dorsal view, anterior to the top. Arrowhead, NC; arrow, gap in Gbx2 expression. (S) Detail of the neural fold region in a lateral view, anterior to the top, midline to the right. (T) Summary of Gbx2 and Snail2 expression at stage 16. Anterior to the top, midline to the right. Different tones of purple denote different levels of Gbx2 expression. Blue, NC.
Fig. 2. Gbx2 is required for NC induction. Embryos were injected in animal blastomeres at the eight-cell stage with the indicated MO, and the expression of Snail2 was analysed between stages 12 and 13. In all the images, in situ hybridizations are shown in dorsal view with anterior to the top and the inset corresponds to the overlay of in situ hybridization and fluorescence to show the injected side to the right. (A) Control MO (20 ng). (B) Gbx2 splicing MO (20 ng). (C) Gbx2 translational MO (16 ng). (D) Gbx2 mRNA (1 ng). (E) Gbx2 translational MO (16 ng) and Gbx2EnR-GR (1 ng). Dexamethasone was added at stage 10. (F) Gbx2EnR-GR (1 ng). Dexamethasone was added at stage 10. (G) Gbx2 translational MO (16 ng) and seven-mismatch (7mismatch) Gbx2 mRNA (1 ng). (H) 7mismatch Gbx2 mRNA (1 ng). (I) Summary of rescue experiment showing percentage of embryos with Snail2 inhibition. ** P<0.001. (J) Efficiency of splicing MO. RT-PCR of embryos injected with 20 ng of control MO or 20 ng of Gbx2 splicing MO. Gbx2 and ODC were analysed. ODC, loading control. (K-N) Targeted injection of Gbx2 translational MO. A1, A3 or A4 blastomeres were injected with Gbx2 MO to target neural plate, NC or epidermis, respectively. (K) NC injection. (L) Neural plate injection. (M) Epidermis injection. (N) Summary of targeted injection, showing percentage of Snail2 inhibition after injecting in prospective NC, neural plate or epidermis. A minimum of 30 embryos was analysed in each experiment. E, epidermis; NP, neural plate.
Fig. 5. NC induction by Wnt signalling is Gbx2 dependent. Embryos were injected in animal blastomeres at the eight-cell stage as indicated. The expression of Snail2, Pax3 and Msx1 was analysed at stage 12. (A-C) β-catenin-GR (1 ng) and induced at stage 10 with DEX. Between 75 and 86% of NC expansion; n=153 embryos. (D-F)β -catenin-GR (1 ng), induced at stage 10 with DEX and 16 ng of Gbx2 MO. NC expansion was reduced to less than 2%; n=174. (G-I) 1 ng of Dsh dominant-negative DD1. Between 78 and 82% of inhibition of NC genes; n=124. (J-L) Dsh dominant-negative DD1 (1 ng) and 1 ng of Gbx2 mRNA. NC inhibition was reduced to less than 2%; n=120.
Fig. 6. Early requirement of Gbx2 for NC induction. (A-F) In situ hybridization for Gbx2, Pax3 and Msx1 at the indicated stages. Note that only Gbx2 is observed at stage 11. (G-J) Gbx2 MO was injected at the eight-cell stage and the expression of Pax3 and Msx1 was analysed at the indicated stages. Asterisks indicate the injected side (visualized in the inset). Note almost complete inhibition of Pax3 and Msx1 at stage 11.5 (43% of total and 31% of partial inhibition; n=83), and partial inhibition at stage 14 (70% of partial inhibition; n=110). (K) Percentage of embryos with defects in Snail2 expression after activation of GbxEnR-GR with dexamethasone at the indicated stages (s).
Fig. 7. Gbx2 is upstream of Pax3 and Msx1 in the NC genetic cascade. Embryos were injected as indicated and the expression of the indicated genes was analysed at stage 12. (A-F) Gbx2 MO alone (16 ng) (A,D) or co-injected with 1 ng of Pax3 mRNA (B,E) or 1 ng of Msx1 mRNA (C,F). Eighty-one to 83% of NC inhibition by Gbx2 MO (n=178) was rescued to less than 1% of inhibition by co-injection with Pax3 (n=78) or Msx1 (n=69) mRNA. (G-I) Pax3 MO alone (20 ng) (G) or co-injected with 1 ng of Gbx mRNA (H,I). Seventy-eight percent (n=90) of NC inhibition by Pax3 MO was not rescued by Gbx2 MRNA (75-79% inhibition; n=189). (J-L) Msx1 dominant-negative HD-Msx1 alone (1 ng) (J) or co-injected with 1 ng of Gbx2 mRNA (K,L). Sixty-eight percent (n=89) of NC inhibition by HD-Msx1 alone was not rescued by Gbx2 mRNA (73-77% of inhibition; n=124).
Fig. 8. Gbx2 is required for the posteriorization of neural folds. Embryo injections and expression of indicated genes were analysed at stage 12. (A,B) Snail2. (C,D) FoxD3. (E,F) Six1. Arrows, posterior end of Six1 expression. (G) RT-PCR of animal caps analysing Six1 and FoxD3 expression. (H,I) Cpl1. (J,K) En2. (L,M) Otx2. (N) Sox2. (O) Keratin. (P,Q) Summary phenotypes produced by Gbx2 MO (P) and Gbx2 mRNA (Q). Anterior part of the embryo is represented, with left side as control and right-hand side as that injected. Percentages of phenotypes are shown in Fig. S1 in the supplementary material. A minimal of 35 embryos was analysed in each experiment. AC, animal cap; E, whole embryo; Gb, 1 ng of Gbx2 mRNA; ODC, loading control; tBR, 2 ng of dominant-negative of BMP4 receptor.