DEV009118 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Ngn1-CreER and Ngn1-GFP BAC transgenic lines mimic neurogenic gene expression. (A-H) Comparison of Cre mRNA (in Ngn1-CreER embryos), GFP protein (in Ngn1-GFP embryos) and endogenous Ngn1 and NeuroD mRNA expression at otocyst and labyrinth stages. Arrows indicate the lateral (left) and medial (right) extent of otocyst domains. Brackets highlight the later utricular domains. (I) GFP reports on Ngn1-CreER-mediated recombination (Ngn1-CreER;Z/EG double-transgenic embryos) in VIIIth ganglion rudiment neurons (green), double labeled with an antibody against islet 1 protein (red). GFP is distributed throughout the VIIIth ganglion rudiment. VIIth (facial) and VIIIth ganglion rudiments are distinguished by a dotted line. (J) GFP reports directly on Ngn1 promoter activity in the VIIIth ganglion rudiment of Ngn1-GFP BAC transgenic embryos, double labeled with an antibody against islet 1 protein (red). GFP signal is absent from the most mature ‘core’ regions of the ganglion; these neurons have downregulated Ngn1 promoter activity.

• Supplemental Figure S2 -

  Fig. S2. Reduction of Ngn1 promoter activity in the saccular rudiment over time. The bar chart compares spatial distributions of all saccular Ngn1-derivatives from two groups of embryos with different tamoxifen administration start times. Once begun, tamoxifen was administered twice daily to all litters until E13.5 and all embryos were sacrificed at E14.5. The saccule is binned into three sectors along its anteroposterior axis. Distributions are based on 327 labeled cells from four ears for the E8.5 start group and 87 labeled cells from ten ears for the E12.5 start group. Shown at the bottom are representative sections for each of the three saccular regions from an embryo with an E8.5 tamoxifen start.

• Supplemental Figure S3 -

  Fig. S3. Excess neurogenesis in the Math1<^b>⁻<^/b>^/<^b>⁻ embryonic ear. Sections highlighting the ear epithelium (A-D) and VIIIth ganglion (E-H) at various levels, hybridized with a NeuroD RNA probe. Arrow in A indicates the normal NeuroD signal at E15.5. Asterisk in B highlights excessive delamination in the mutant. White dots in E and F outline the most mature part of the vestibular ganglion, which no longer expresses NeuroD at the stage shown. Epithelia are outlined in white. lc, lateral crista; mu, macula of the utricle; sacc, saccule; coch, cochlea; ivg, inferior vestibular ganglion; sg, spiral ganglion. Scale bar: 100 μm in A-D; 50 μm in E,F.

• Supplemental Figure S4 -

  Fig. S4. Sensory epithelial phenotypes in Ngn1 mutant embryos. (A) Wild-type and Ngn1^−/− saccule (inset) at E14.5, hybridized with an RNA probe for Math1 and shown to scale. (B,C) Sections through the E12.5 saccular rudiment of wild-type (B) and Ngn1^−/− (C) littermates, reacted for TUNEL. Arrowheads in B highlight the normal level of TUNEL in this part of the wild-type ear. (D-F) Myosin VIIa immunohistochemistry reveals differences in density and shape of embryonic hair cells in the utricular maculae of wild-type (D), Ngn1^+/− (E) and Ngn1^−/− (F) littermates at E14.5.