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DVDY_22620_sm_suppfig1.tif3737KSupporting Figure 1 Schematic of MYC-MAX-MNT/MXD/MGA and possible MYC pathway with N-Myc CKO affect. MAX forms a heterodimer with MYC but is also capable of forming a heterodimer with one of three antagonists, MGA, MNT, or MXD. Either the MYC-MAX or MNT/MGA/MXD (MMM)-MAX pairs will bind to the E-box sequence on target genes. Along with accessory binding partners, MYC-MAX will allow for the transcription of target genes responsible for leading to proliferation. Contrarily, MMM-MAX will inhibit the transcription of these target genes, leading to differentiation. Target genes of MYC-MAX include CYCLINs, IDs, E2F, and various microRNAs. Both the CYCLINs and IDs remove the pRB from E2F through phosphorylation and allow E2F to bind to S-phase promoting genes and allow subsequent proliferation. IDs are also able to bind bHLH transcription factors but since they lack a basic motif, are unable to bind E-Boxes, inhibiting differentiation. In the absence of IDs or CYCLINs, bHLH transcription factors are free to bind to E-proteins and the E-box, promoting differentiation (A). In WT controls, upon activation of NOTCH1 by JAG1, the Notch IntraCellular Domain (NICD) is cleaved by γ-SECRETASE. NICD translocates to the nucleus (shown by gray dotted arrow) to form a complex with RBPJ to positively regulate the transcription of the Hes family of bHLH transcription factors (Doetzlhofer et al., 2009). MYC is regulated by a number of pathways, including the Wnt/β-CATENIN pathway, TGF-β or BMP/SMAD, Fgf/Erk1-2, and the Shh/Smo pathways. Once activated, MYC interacts with partner proteins (A) to regulate a number of downstream effectors, including the CYCLINs, E2F, IDs, CDKIs, and various microRNAs. This regulation ultimately leads to the inhibition of activator bHLH transcription factors from binding to the E-proteins, which then allows the HES to occupy the E-proteins. It also leads to the removal of pRB from E2F and allows E2F to bind S-phase promoting genes. When E2F binds with pRB, proliferation cannot commence and the cell enters a state of quiescence where differentiation may occur (B). To the right, a WT control cartoon shows the phenotype of a WT inner ear (B′). In the absence of Myc, Myc downstream effectors have a dosage-related decrease and growth is reduced while onset of differentiation may be accelerated (C). To the right are the resulting phenotypes of knocking out N-Myc in the inner ear using both Pax2-Cre (C′) and Foxg1-Cre (C′′). Asterisks mark genes whose phenotypes have similar characteristics as seen in N-Myc CKO and are qualified in Table 3. The larger circle represents the cell as a whole while the smaller circle is the nucleus. Arrows indicate upregulation while blunted lines represent blocking. Dashed lines represent a reduction to endogenous protein levels. Red indicates pathways favoring differentiation, blue indicates pathways favoring proliferation. Modified after Fritzsch et al. (2006). Note: given the conservation and functional redundancy of the Mycs from yeast to man, it is reasonable to assume that the possible interactions for a Myc revealed in the literature apply to the ear.
DVDY_22620_sm_suppmovie1.avi5047KSupp movie 1 Three-dimensional view of WT cochlea. After 3D reconstruction of TSLIM images using Amira software, we were able to rotate the 6-week WT littermate to our N-Myc CKO. Translucent structures are the three scalae while the basilar membrane, organ of Corti, and Rosenthal′s canal are opaque.
DVDY_22620_sm_suppmovie2.avi4723KSupp movie 2 Three-dimensional view of N-Myc CKO cochlea. 3D reconstruction and animation allows for a unique perspective to analyze mutants with grossly abnormal phenotypes. Provided here is a 3D reconstruction and video of our 6-week N-Myc CKO. Translucent structures are the three scalae while the basilar membrane, organ of Corti, and Rosenthal′s canal are opaque.

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