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Additional Supporting Information may be found in the online version of this article.

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DVDY_22308_sm_SuppFigS1.tif18565KSupporting Figure 1. β-catenin gain-of-function (BcatGOF) mutants generated using Pax2-Cre have a more severe otic vesicle (OV) phenotype as compared to Foxg1-Cre. A,B: Lateral views of RNA in situ hybridization to Tbx1 at embryonic day (E) 10.5 in BcatGOF mutants generated using Pax2-Cre (A) as compared to Foxg1-Cre (B). Pax2-Cre BcatGOF mutants have fusion of the 1st (I) and 2nd (II) pharyngeal arches as can be detected by continuous Tbx1 expression in the pharyngeal mesoderm (black arrow). In addition, the OV (red dotted outline) is highly dysmorphic due to early activation of β-catenin in the otic placode, and has failed to undergo complete closure of the otic cup by E10.5 (white dotted outline). In comparison, Foxg1-Cre BcatGOF mutants have a fully developed OV (red dotted outline) by E10.25 despite a slight delay in otic cup closure at E9.5 (see Figure 3B, asterisk).
DVDY_22308_sm_SuppFigS2.tif7173KSupporting Figure 2. Whole-mount RNA in situ hybridization for downstream targets of canonical Wnt signaling. Lateral views of mRNA expression as detected by whole-mount RNA in situ hybridization on control (left) and β-catenin gain-of-function (BcatGOF) mutant (right) littermates at embryonic day (E) 10.5. Bmp4, Dlx5, Sp5, and Msx1 are all known downstream targets of Wnt signaling and exhibit ectopic expression in the forebrain and pharyngeal region of BcatGOF mutants, consistent with the activation domain of Foxg1-Cre. Bmp4 is also ectopically expressed in the otic vesicle (OV), while Sp5 does not appear to be altered in this tissue. Dlx5 and Msx1 have increased expression in the dorsal OV, but do not undergo complete dorsalization.
DVDY_22308_sm_SuppFigS3.tif15237KSupporting Figure 3. Abnormalities in olfactory placode development in β-catenin gain-of-function (BcatGOF) mutants. High magnification lateral views of embryos with whole-mount RNA in situ hybridization using probes to NeuroD, Eya1, and Six1. All three genes are no longer expressed in the olfactory epithelium of BcatGOF mutants at embryonic day (E) 9.5 compared with controls. Because it is possible that there may be loss of tissue in this region in BcatGOF mutants, we analyzed cryosections of the embryos to determine if the olfactory placode was present. In the control, there is a clear thickening of the ectoderm (black bracket) that is expressing NeuroD (epithelium is defined between solid and dotted red lines). Development of the olfactory placode fails in BcatGOF mutants where a thickening of the ectoderm cannot be detected.

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