December 1, 2008;
Hindbrain-derived Wnt and Fgf signals cooperate to specify the otic placode in Xenopus.
Induction of the otic placode
, the rudiment of the inner ear
, is believed to depend on signals derived from surrounding tissues, the head mesoderm
and the prospective hindbrain
. Here we report the first attempt to define the specific contribution of the neuroectoderm
to this inductive process in Xenopus. To this end we tested the ability of segments of the neural plate
(NP), isolated from different axial levels, to induce the otic marker Pax8
when recombined with blastula stage
animal caps. We found that one single domain of the NP, corresponding to the prospective anterior hindbrain
, had Pax8
-inducing activity in this assay. Surprisingly, more than half of these recombinants formed otic vesicle
-like structures. Lineage tracing experiments indicate that these vesicle-like structures are entirely derived from the animal cap
and express several pan-otic markers. Pax8
activation in these recombinants requires active Fgf and canonical Wnt signaling, as interference with either pathway blocks Pax8
induction. Furthermore, we demonstrate that Fgf and canonical Wnt signaling cooperate to activate Pax8
expression in isolated animal caps. We propose that in the absence of mesoderm
cues the combined activity of hindbrain
-derived Wnt and Fgf signals specifies the otic placode
in Xenopus, and promotes its morphogenesis into an otocyst
[+] show captions
Fig. 4. Ablation of the NP2 domain reduces the otic expression of Pax8 in stage 23/24 embryos. Otx2 (forebrain and eyes) and Krox20 (rhombomeres 3 and 5, arrowheads) (A, B) and Pax8 (C, D) otic expression (arrows) in control embryos at stage 23/24. (E–H) Unilateral ablation of NP2 domain results in reduced Pax8 expression on the manipulated side (G–H; arrows). Sibling embryos after NP2 domain removal (E, F) show reduced Krox20 expression in the hindbrain (residual rhombomere 5 expression can be detected in these embryos; arrowhead) without affecting Krox20 expression domain in the neural crest (nc). Dorsal (A, C, E, G) and lateral (B, D, F, H) views, anterior to left.
Fig. 7. Developmental expression of putative hindbrain-derived otic-inducing factors. (A) Fgf3, Fgf8, Wnt1 and Wnt8 are expressed in a region of NP corresponding to NP2 domain at the early neurula stage; dorsal views, anterior to top. (B) Pax8 otic expression at stage 15 (black arrow). (C) Double in situ hybridization showing the relationship of expression of Pax8 (black arrows; green staining) and Fgf3, Fgf8, Wnt1 and Wnt8 (white arrows; purple staining) at the neurula stage. Lateral views, anterior to the left. At this stage Wnt8 is also expressed outside of the hindbrain in a region of the ectoderm presumably corresponding to the neural crest. (D) Real-Time RT-PCR analysis of Fgf3, Fgf8, Wnt1 and Wnt8 expression in various segments of NP (NP1, NP2 and NP3) shortly after dissection. All four ligands are enriched in NP2 domain. WNP; whole NP.
Fig. 8. Wnt and Fgf signaling are required for otic placode specification. (A) Embryos injected with different combinations of Fgf3 (F3MO), Fgf8 (F8MO), Wnt1 (W1MO) and Wnt8 (W8MO), morpholino antisense oligonucleotides show reduction of Pax8 and Sox9 otic expression at the neurula stage (stage 15). The affected otic domain is indicated by an arrow on the injected side. In these embryos two morpholinos were co-injected at the 4-cell stage to target the NP. For the same embryos the control and injected sides are shown for comparison. Lateral views. For the control panels anterior to right. For the injected panels anterior to left. (B) Quantification of the in situ hybridization results. The number on the top of each bar indicates the number of embryo analyzed.