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First published online 1 February 2006
doi: 10.1242/dev.02271

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Wnt signals mediate a fate decision between otic placode and epidermis

¹ Gonda Department of Cell and Molecular Biology, House Ear Institute, 2100 West 3rd Street, Los Angeles CA 90057, USA.
² Department of Obstetrics and Gynecology, McGill University, Montreal, Canada.
³ Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

View larger version (65K): [in a new window]   Fig. 1. Detection of Wnt signaling in Pax2+ ectoderm with TCF/Lef reporter mice. (A,B) Comparison of Pax2 expression, Wnt8a expression and TCF/Lef reporter ß-galactosidase activity at E8.0 and E8.25. Significant reporter expression is observed in presumptive otic ectoderm after the onset of Pax2 expression at E8.25 but not at E8.0 (arrowheads). Wnt8a expression is observed in rhombomere 4 at both E8.0 and E8.25. (C) A whole-mount E8.5 TCF/Lef reporter embryo stained for ß-galactosidase activity, with sections through the anterior and posterior regions of the presumptive otic placode. The lower panels show a similar embryo immunostained for Pax2 (red) and ß-galactosidase (green) protein. The bracket shows the extent of the Pax2 domain, and the arrowhead shows Pax2+, ß-galactosidase- ectoderm. The arrow indicates migrating neural crest cells. (D) Sections through the otic cup and otocyst of E8.75 and E9.5 reporter embryos. The white arrowhead and black bars indicate ß-galactosidase- regions of placodal epithelium. The black arrowhead indicates ß-galactosidase+ vestibuloacoustic ganglion cells migrating from the otocyst, and black arrows indicate ß-galactosidase+ migrating neural crest cells. n, neural tube. Scale bars: 100 µm.

View larger version (78K): [in a new window]   Fig. 2. Conditional inactivation of ß-catenin in Pax2+ cells promotes epidermal fates at the expense of the otic placode. (A) Cre-loxP reporter analysis of Pax2-Cre mice (left) and whole-mount images of the ß-catenin CKO embryo at E10.5 from lateral (top) and dorsal (bottom) aspects. Cre-loxP reporter signal (green) is detected in the Pax2+ ectoderm from E8.5. By E8.75, reporter signal is detected both in the thickened otic placode and in the ectoderm (brackets) lateral to the otic placode. DAPI staining is shown in magenta. By E10.5, reporter signal (blue) is detected in virtually the entire otic vesicle (arrowheads). In E10.5 ß-catenin CKO embryos, the mid-hindbrain region is entirely missing (asterisk) and the size of the otic vesicle (broken line) is significantly smaller than controls. (B) Whole-mount in situ hybridization of otic, epidermal and hindbrain markers in ß-catenin CKO embryos. Pax2 and Pax8 are downregulated in presumptive otic ectoderm (arrowheads) and in the mid-hindbrain boundary of CKO embryos (asterisks) at E8.5. The epidermal marker Foxi2 indicates the size of the otic placode as a white patch (dashed oval). In E8.5 CKO embryos the Pax2/Pax8-expressing domain is smaller and the Foxi2+ ectodermal domain is expanded. At E8.75, Krox20 marks rhombomeres (r) 3 and 5 in the CKO and control. A small otic cup (dashed oval) is formed adjacent to r5. The posterior end of the otic cup in CKO is the same level as that in control. Dlx5 expression is greatly diminished in CKO embryos, but remains strongly expressed in the rim of the otic cup in control embryos. Scale bars: 100 µm in fluorescent images.

View larger version (70K): [in a new window]   Fig. 3. Apoptosis and proliferation in ß-catenin CKO embryos. (A) Whole-mount anti activated caspase 3 staining in E8.5 embryos. Increased numbers of activated caspase 3 cells detected mainly in the anterior (A) region of the otic ectoderm in CKO (arrowhead). Brackets indicate the position of the contralateral otic placode. Broken lines show the first (1p) and second (2p) branchial pouches. Sections of anterior and posterior parts of the otic region are shown stained with activated caspase 3 (green), DAPI (magenta) and processed with a probe to Foxi2. Apoptotic cells are observed in unthickened, Foxi2+ ectoderm (arrowheads) in CKO embryos but are not seen in controls. No capase 3 cells can be seen in the thickened, Foxi2- otic placode (brackets). (B) Average number of activated caspase 3-expressing cells per section in the otic region (indicated as rectangles in A). (C) Dividing cells (green, arrowheads) marked by phospho-Histone H3 (pH3) at E8.75 co-stained with ß-catenin (red) and DAPI (blue). (D) Average number of the pH3-positive cells per section in the otic cup at E8.75 shows a reduction in dividing cells in the CKO otic cup. Numbers are normalized to the length of the apical surface of the placode (see Materials and methods). Scale bars: 100 µm.

View larger version (81K): [in a new window]   Fig. 4. Cadherin-mediated adhesion in ß-catenin CKO embryos. (A) Double immunolabeling of ß-catenin (ß-cat) and E-cadherin (E-cad), -catenin (-cat) or -catenin (-cat) at E8.75 and E9.0. Brackets indicate ß-catenin-negative regions of ectoderm in CKO embryos. (B) Double immunolabeling of E10.5 embryos with ß-catenin and either E-cadherin or -catenin. Dashed ovals indicate the outline of ß-catenin-negative otocysts. Both E-cadherin and -catenin are expressed in the ß-catenin-negative cysts. (C) Neurogenesis in ß-catenin CKO embryos at E10.5. A Neurod1-positive ganglion can be seen delaminated from the small CKO otocyst (arrowhead). This ganglion is a mixture of ß-catenin-positive (red, white arrow) and negative (dashed line) cells. ß-Catenin-negative cells are still ßIII tubulin (TuJ1, green) positive. t, trigeminal; g, geniculate; cv, cochleovestibular; p, petrosal and n, nodose ganglia. Scale bars: 100 µm.

View larger version (56K): [in a new window]   Fig. 5. Conditional activation of ß-catenin in Pax2+ cells expands the otic placode at the expense of epidermis. (A) Whole-mount in situ hybridization of otic and epidermal markers in ß-catenin cAct embryos. Expression of the early otic markers Pax2, Pax8 and Dlx5 are upregulated in ectoderm lateral to the otic cup (arrows) at E8.75, while the epidermal marker Foxi2 is downregulated at E9.5. The broken line indicates the outline of the Foxi2- placodal domain. Markers of the mid-hindbrain boundary (asterisk) are also expanded in cAct embryos. (B) Expansion of the placodal domain without an increase in cell division in cAct embryos. The ectoderm lateral to the otic cup is thickened from E8.75 in cAct embryos and ß-catenin protein (red) is expressed strongly in the placodal epithelium. By E9.5, the placodal epithelium fails to close in cAct embryos, forming a grossly enlarged placode extending ventrally to the level of the pharynx, with accumulated ß-catenin and ectopic invaginating regions (asterisk). (C) The average number of pH3-positive cells is not significantly increased in cAct placodal epithelium. Numbers are normalized to the length of the apical surface of the placode (see Materials and methods). Scale bars: 100 µm.

View larger version (78K): [in a new window]   Fig. 6. Activation of ß-catenin promotes dorsal otocyst cell fates. (A) Whole-mount in situ hybridization of dorsal otocyst markers in cAct embryos at E9.5. Dlx5, Gbx2 and Msx1 are upregulated in the placodal epithelium, although Dlx5 and Msx1 are downregulated in the most deeply invaginated part of the placode, closest to the ventral midline (asterisks). Borders of the Dlx5 and Gbx2 expressions in control embryos are indicated with bars. Msx1 expression is just starting to appear in the control endolymphatic bud at E9.5 (arrowhead). (B) Pax2 expression in cAct is downregulated at E9.5, while Pax8 is expanded. A dorsal marker, Hmx3, is not expanded. (C) Expression of neurogenic and non-neurogenic markers. Neurod1 and Lfng (anteroventral markers) and Tbx1 (posteroventral marker) are not expressed in cAct embryos. Broken lines represent the malformed otocyst and open rim. Anterior (A) and posterior (P) orientation is indicated with arrows.

View larger version (21K): [in a new window]   Fig. 7. Proposed model for the role of Wnt signaling in mediating the otic placode-epidermis fate decision and dorsal otocyst cell fates. (A) Summary of the ß-catenin loss-of-function (CKO) and gain-of-function (cAct) phenotypes. Control: Wnt-TCF/Lef is activated (magenta) in Pax2+ surface ectoderm by Wnt (blue) from hindbrain (HB) to delineate the size of the otic placode (dark blue). Pax2+ cells that do not receive Wnt signals differentiate as epidermis (gray). In CKO, in the absence of Wnt signaling, epidermis (gray) is expanded at the expense of the otic placode. In cAct, stabilized ß-catenin activates Wnt-responsive genes in a wide region of the surface ectoderm, thus expanding the otic placode at the expense of epidermis. Ovals in the top panels represent the otic placode. A, anterior; P, posterior. Lower panels represent a transverse section at the level shown in the top panels as a broken line. The strength of the Wnt-mediated response is shown as graph. (B) A model of the induction and polarization of the otic placode. E8.0: Pax2 in the surface ectoderm (SE) is induced by Fgf signaling from underlying tissues and/or the hindbrain. E8.5: Wnt signaling from rhombomere (r) 4 directs the medial-most region of the Pax2+ ectoderm to an otic placode fate and activates expression of dorsal otic markers such as Dlx5. Cells that do not receive Wnt signaling in the Pax2+ ectoderm are directed to an epidermal fate and express epidermal-specific genes such as Foxi2. E8.75+: As the otic placode invaginates, signals from the ventral midline (orange) such as Shh activate expression of ventral otocyst genes and repress dorsal cell fates (Riccomagno et al., 2002; Riccomagno et al., 2005).