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First published online 21 May 2008
doi: 10.1242/dev.017905

Development 135, 2251-2261 (2008)
Published by The Company of Biologists 2008
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Notch signaling augments the canonical Wnt pathway to specify the size of the otic placode

Chathurani S. Jayasena1,*, Takahiro Ohyama1, Neil Segil2 and Andrew K. Groves2,{dagger},{ddagger}

1 Gonda Department of Cell and Molecular Biology, House Ear Institute, 2100 West 3rd Street, Los Angeles, CA 90057, USA.
2 Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.


Figure 1
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  		Fig. 1. Expression profile of Notch pathway genes during otic placode development. (A) Schematic views of embryos during otic placode development. The dotted line indicates the approximate level of sections in C. (B) Summary showing the onset of Notch1 (blue), jagged 1 (Jag1; brown), delta-like 1 (Dll1; green) and Hes1 (yellow-orange) expression with respect to Pax2 expression (pink-red) and Wnt signaling (purple). Preceding neurogenesis, Dll1 expression is high in the pre-otic field but progressively becomes weaker in the placode. During neurogenesis, Dll1 localizes to neuroblasts. (C) Sections comparing the expression of Wnt6, Notch1, Jag1, Dll1 and Hes1 with respect to Pax2 (red) and the Wnt reporter (blue). Arrowheads mark the pre-otic field. Brackets mark the extent of the otic placode. (D) Top row, Jag1 expression in the anterior and posterior pre-otic field; bottom row, consecutive serial sections through the pre-otic field immunostained with anti-Pax2 and anti-Jag1 antibodies, respectively. Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. The canonical Wnt pathway positively regulates components of the Notch pathway in the otic placode. (A) Jag1, Notch1, and Hes1 domains are ectopically expanded in cAct embryos (bracket). Insets show corresponding whole mounts. Arrowheads indicate normal (top row) or ectopic (bottom row) expression; dotted outline demarcates the otic area. (B) Jag1, Notch1 and Hes1 domains are reduced in β-cat-CKO embryos. (Top panels) Anti-Jag1 (red) and β-catenin (β-cat, green) co-immunostaining. Bracket indicates β-catenin-; Jag1- cells. Inset shows Jag1 expression at E9.5 in whole mount. (Lower panels) Sections of Notch1- and Hes1-hybridized embryos. Scale bar: 50 µm.

 

Figure 3
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  		Fig. 3. Some, but not all otic markers are expanded at the expense of epidermis in conditionally activated Notch1 (cN1ICD) embryos. (A-C') Expanded otic placode in cN1ICD embryos hybridized with probes for Pax8 (A,A'), Pax2 (B,B') and the epidermal marker Foxi2 (C,C'). Arrowheads indicate ectopic expression. Dotted outline in C indicates the invaginating otic cup (controls) or expanded otic region (cN1ICD mutants). (A'-C') Corresponding transverse sections. Brackets indicate the lateral ectopic placode region. Scale bars: 100 µm. (D) Hmx3, Sox9 and Gbx2 otic markers are not expanded in cN1ICD mutants. (E) In β-cat-CKO; cN1ICD mutants, Pax8 (top row) and Jag1 (green, bottom row) are expanded at the expense of Foxi2 (middle row). (Bottom row) β-catenin (red) and Jag1 protein expression in Foxi2 hybridized embryos. Insets show whole mounts. Scale bar: 100 µm.

 

Figure 4
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  		Fig. 4. Domains of Pax2 and Pax8 are reduced in Notch1 mutants. (A,B) Dorsal and lateral whole-mount views of Pax2 (A) and Pax8 (B) expression. Anterior and posterior sections through control and Notch1 mutant placodes are also shown. Brackets indicate otic expression. e, epibranchial placode; h, hyoid arch. Scale bars: 50 µm.

 

Figure 5
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  		Fig. 5. The otic placode is significantly reduced in Notch1 mutants. (A,B) Foxi2 expression at 10-11ss (A) and at 12-13ss (B) in whole mounts and sections. Dotted outline/brackets indicate the thickened otic placode region that does not express Foxi2. (A) Corresponding sections showing Foxi2 transcript (blue) and Pax2 protein expression (brown). (C,D) Quantitative comparison of mediolateral placode length in control and Notch1 mutant embryos at 9-11ss (C) and 12-13ss (D; refer to Materials and methods). *P<0.05; **P<0.005. (E) Quantification of average cell density in control and Notch1 mutants. Placode n values are given in parentheses. Error bars indicate s.e.m. (F) Activated caspase 3 expression (red), indicating a lack of apoptotic cells within the Notch1 mutant otic placode at 10-11ss. Brackets indicate the thickened placode. Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. Notch1 signaling augments Wnt signaling in the otic placode. (A) Wnt reporter and Dlx5 mRNA expression is increased in conditionally overexpressing Notch1-ICD (cN1ICD) embryos in whole mounts and corresponding mid-placode transverse sections. Dotted outline demarcates the otic cup; red arrowhead indicates the mediolateral otic region. (B) Wnt reporter expression in transverse sections of control and cN1ICD; Wnt reporter embryos. Wnt reporter mice were co-immunostained with anti-β-galactosidase (β-gal; red) and anti-GFP (green) antibodies. Note that only the medial part of the expanded placode expresses the Wnt reporter. The inset shows anti-β-galactosidase staining in a normal Wnt reporter mouse. Brackets in A and B indicate that the ectopic lateral placode region is negative for Wnt reporter and Dlx5. (C) Wnt reporter and Dlx5 expression is diminished in Notch1 mutants relative to in controls. Brackets indicate the otic placode. (D) A comparison of Pax8 and Foxi2 expression in Notch1 mutant, cAct and Notch1; cAct double mutant littermates. Arrowheads indicate otic expression. Scale bars: 50 µm.

 

Figure 7
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  		Fig. 7. Model of how Wnt and Notch pathways interact to regulate the size of the otic placode. (A) The generation of the otic placode can be divided into three stages. Pax2 in the pre-otic field is induced by FGFs (arrows). A gradient of Wnts (light blue) determines the size of the otic field: above a certain threshold, Wnts drive cells towards an otic fate (dark blue), and, below the threshold, cranial epidermis is formed (Foxi2) (Ohyama et al., 2006Go). Notch1 signaling is superimposed on the Wnt gradient (pink-blue) and acts to augment the otic fate imposed by Wnts. NE, neuroectoderm; SE, surface ectoderm. (B) The Wnt pathway is the primary signal (denoted by bold lettering) that controls otic fate (blue region) by positively regulating (green arrows) the expression of Dlx5, Sox9, Gbx2, Pax2, Pax8 and components of the Notch1 pathway, such as Notch1 and Hes1 (Figs 1, 2). Jag1 expression is initiated by Wnts (striped green arrow; see Fig. 2). Notch1 acts to: (1) augment Wnt and Notch1 activity within otic cells (pink arrow; plus sign); and (2) co-operate with Wnt to negatively regulate Foxi2 (red) and positively regulate Pax8 (dark green), and to maintain a thickened otic placode. (C) A model summarizing the various otic placode phenotypes observed in this study. A gradient of Wnt activity emanating from the midline is established across the mediolateral axis of the pre-otic field. Cells exposed to a certain threshold of Wnt signals express Jag1 and differentiate as otic placode (blue). Below this threshold, cells differentiate as epidermis (gray). Jag1-Notch1 signaling augments Wnt signals in the medial region of the otic placode, whereas more lateral regions are not exposed to Notch1 signals and Wnt signaling is not augmented. In the absence of Notch1 (brown line), the gradient of Wnt signaling becomes weaker, resulting in a smaller placode and more epidermis. When Notch1 is activated in the pre-otic field (green line), the Wnt gradient is augmented further. Some Wnt-dependent markers (Dlx5) are expressed only in the expanded Wnt domain, whereas markers such as Pax8 are expressed throughout the pre-otic field (marked as Pax8+ placode). When β-catenin is activated in the entire pre-otic field (purple line), all cells differentiate as otic placode (Ohyama et al., 2006Go).

 

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