Fgf-dependent otic induction requires competence provided by Foxi1 and Dlx3b

Abstract

Background: The inner ear arises from a specialized set of cells, the otic placode, that forms at the lateral edge of the neural plate adjacent to the hindbrain. Previous studies indicated that fibroblast growth factors (Fgfs) are required for otic induction; in zebrafish, loss of both Fgf3 and Fgf8 results in total ablation of otic tissue. Furthermore, gain-of-function studies suggested that Fgf signaling is not only necessary but also sufficient for otic induction, although the amount of induced ectopic otic tissue reported after misexpression of fgf3 or fgf8 varies among different studies. We previously suggested that Foxi1 and Dlx3b may provide competence to form the ear because loss of both foxi1 and dlx3b results in ablation of all otic tissue even in the presence of a fully functional Fgf signaling pathway.

Results: Using a transgenic line that allows us to misexpress fgf8 under the control of the zebrafish temperature-inducible hsp70 promoter, we readdressed the role of Fgf signaling and otic competence during placode induction. We find that misexpression of fgf8 fails to induce formation of ectopic otic vesicles outside of the endogenous ear field and has different consequences depending upon the developmental stage. Overexpression of fgf8 from 1-cell to midgastrula stages leads to formation of no or small otic vesicles, respectively. Overexpression of fgf8 at these stages never leads to ectopic expression of foxi1 or dlx3b, contrary to previous studies that indicated that foxi1 is activated by Fgf signaling. Consistent with our results we find that pharmacological inhibition of Fgf signaling has no effect on foxi1 or dlx3b expression, but instead, Bmp signaling activates foxi1, directly and dlx3b, indirectly. In contrast to early activation of fgf8, fgf8 overexpression at the end of gastrulation, when otic induction begins, leads to much larger otic vesicles. We further show that application of a low dose of retinoic acid that does not perturb patterning of the anterior neural plate leads to expansion of foxi1 and to a massive Fgf-dependent otic induction.

Conclusion: These results provide further support for the hypothesis that Foxi1 and Dlx3b provide competence for cells to respond to Fgf and form an otic placode.

Figure 1

Otic vesicle size is affected by the overexpression of fgf8 depending on the developmental stage. (A, B) Overexpression of fgf8 by mRNA injections at the 1-cell stage completely ablates all indications of otic fate (27/40 embryos, B) in comparison to wild-type vesicles (A). (C-E) Misexpression of fgf8 at 30% epiboly (28/28 embryos, C), shield stage (27/28 embryos, D) and 75% epiboly (29/29 embryos, E) results in smaller otic vesicles. (F-H) Otic vesicles are increased in size if fgf8 is misexpressed at the tailbud (26/29 embryos, F) or the 4-somite stage (24/28 embryos, G) whereas at the 8-somite stage no change in vesicle size is observed (26/26 embryos, H). Lateral views of otic vesicles highlighted with starmaker at 24h with anterior to the left and dorsal towards the top. Scale bar: 30 μm.

Figure 2

Ectopic Fgf-signaling represses foxi1 and dlx3b before and during gastrulation. (A, B, F, G) Expression of foxi1 and dlx3b is absent after overexpression of fgf8 by mRNA injections at the 1-cell stage in comparison to wild-type embryos. (C, D, H, I) Misexpression of fgf8 at 30% epiboly or shield stages results in smaller foxi1 and dlx3b expression domains. (E, J) Loss of Fgf-signaling after pharmacological inhibition with SU5402 from late blastula stages until the end of gastrulation has no effect on the ventral expression of foxi1 and dlx3b. Lateral views at the end of gastrulation with dorsal to the right and anterior towards the top. Scale bar: 100 μm.

Figure 3

Bmp-signaling is required for foxi1 and dlx3b expression during gastrulation. (A-C, F-H) Expression of foxi1 and dlx3b is strongly reduced in bmp7 mutants or embryos injected with Vox and Vent morpholinos (MOs) in comparison to wild-type embryos. (D, E, I, J) Overexpression of bmp2b by mRNA injections at the 1-cell stage leads to an expansion of both foxi1 and dlx3b expression domains at the expense of anterior neural plate. (A-D, F-I) Lateral views at the end of gastrulation with dorsal to the right and anterior upward; (E, J) dorsal views of embryos shown in D and I with anterior towards the top. Scale bar: 100 μm

Figure 4

foxi1 and vent but not dlx3b are direct targets of Bmp-signaling during gastrulation. (A, B, E, F, I, J) Expression of foxi1, dlx3b and vent is expanded after Bmp2a protein injection at late blastula stages in comparison to wild-type embryos. (C, D, G, H, K, L) Pharmacological inhibition of protein synthesis with cycloheximide (CHX) at late blastula stages blocks all indications of foxi1, dlx3b and vent expression (C, G, K), whereas foxi1 and vent but not dlx3b expression is restored in CHX-treated embryos after Bmp2a protein injection (D, H, L). Lateral views at the end of gastrulation with dorsal to the right and anterior towards the top (note that CHX treatment blocks epiboly movements). Scale bar: 90 μm.

Figure 5

Ubiquitous fgf8 expression at late gastrulation stages leads to ectopic otic induction within the preotic field. (A, G) Ectopic fgf8 expression can be detected throughout the embryo after a 30 minute heat shock in transgenic hsp:fgf8 embryos in comparison to non-transgenic embryos. (B, C, H, I) Within 2 hours, expression of the two Fgf reporter genes, erm and pea3, is upregulated in cells of the transgenic embryos whereas non-transgenic siblings are unaffected. (D-F, J-L) After misexpression of fgf8 at late gastrulation stages, expression of foxi1 is reduced, whereas the preotic expression domains of pax8 and pax2a are enlarged. Dorsal views of 2–5-somite stage embryos with anterior towards the top. mhb, midbrain-hindbrain border; po, preotic region; p, pronephros. Scale bar: 100 μm for A-C, G-I; 40 μm for D-F, J-L.

Figure 6

Ectopic otic induction after fgf8 expression at late gastrulation stages leads to formation of larger but correctly patterned placodes and vesicles. (A, E) Otic placodes labeled by cldna expression are increased in size in transgenic hsp:fgf8 embryos in comparison to non-transgenic embryos following a heat shock at late gastrulation stages. (B-D, F-H) The enlarged otic vesicles of transgenic embryos show no apparent patterning defects in comparison to non-transgenic siblings assessed by morphology and marker gene expression, including otx1 and pax2a. (A, E) Dorsal views of 12-somite stage embryos with anterior towards the top; (B-D, F-H) lateral views of otic vesicles at 24h with anterior to the left and dorsal towards the top. o, otolith. Scale bar: 50 μm for A, E; 30 μm for B-D, F-H.

Figure 7

Ectopic otic induction after fgf8 expression at late gastrulation stages requires both, Foxi1 and Dlx3b. (A-D) Inactivation of Dlx3b or Foxi1 in wild-type embryos by morpholino injection (MO) leads to a reduction of ear size in comparison to wild-type embryos, and combined loss of Dlx3b and Foxi1 results in loss of all indications of otic specification. (E-H) Ear size reduction by depletion of Dlx3b or Foxi1 but not combined loss of Dlx3b and Foxi1, can be partially rescued in transgenic hsp:fgf8 embryos heat shocked at late gastrulation stages. Lateral views of otic vesicles hybridized with starmaker at 24h with anterior to the left and dorsal towards the top. Scale bar: 40 μm.

Figure 8

Ectopic foxi1 expression after treatment with retinoic acid (RA) results in ectopic Fgf-dependent otic induction. (A, B, E, F) In comparison to wild-type control embryos treated with DMSO, embryos treated with 20nM RA show ectopic pax8 and foxi1 expression surrounding the anterior neural plate border without affecting the neural expression of otx2. (C, G) In fgf3, fgf8 double mutants, pax8 is completely abolished in the control embryos, whereas RA-treated double mutant embryos show weak anterior expression of pax8. (D, H) In foxi1 mutants treated with DMSO, pax8 expression can not be detected in the preotic region; but in foxi1 mutants treated with RA, residual anterior pax8 expression is present. Dorsal views of 1–3-somite stage embryos with anterior towards the top. Scale bar: 40 μm.

Figure 9

Model summarizing the early events upstream of otic induction. During gastrulation, Fgf signaling antagonizes Bmp signaling that activates the competence factors Foxi1 and Dlx3b-4b (green). Foxi1 is activated by BMP signaling in a direct manner whereas Dlx3b-4b requires an additional factor X. It is unknown whether Fgf signaling inhibits Bmp signaling at a transcriptional or translational level (?). The proper balance of Bmp and Fgf signaling during gastrulation promotes the positioning and size of the preotic region leading to the induction of otic fate as indicated by Pax8 and Pax2a expression (blue).