DEV01067 Supplemental Figures

Files in this Data Supplement:

• Figure S1 -

  Fig. S1. Local suppression of NeuroD by Tbx1 in the otocyst epithelium. Selected serial (7 mm) sections of whole-mount hybridized otocysts arrayed by AP level (% otocyst), genotype, and probe. In A-E arrowheads mark the ventral or medial border of Tbx1 expression and black arrows indicate the delaminated neural progenitors apposed to Tbx1-negative epithelium (compare with delaminated NeuroD-positive cells in F-I). Red arrows mark the medial extent of NeuroD signal in F-I. (K-T) Increased (cyan asterisks) or ectopic (red asterisks) NeuroD expression is present in loss-of-function mutants. (P-T) Ectopic epibranchial ganglion signal (ep) is present in null homozygotes.

• Figure S2 -

  Fig. S2. Tbx1/TBX1 gene dose affects the position of NeuroD gene expression borders. Spatial mapping of expression domain borders by genotype at the developmental stages (E9.5 and E10) modeled in Fig. 4A. y-axis represents relative position along the otocyst AP axis. x-axis represents developmental time (ss, somite stage) and, within each stage, the otocyst medial-lateral axis as defined by points L (lateral), VL (ventrolateral), and VM (ventromedial). These points were determined throughout transverse serial section sets as shown in the schematic at bottom left (angles L,O,V=90°; VL,O,V=45°; VM,O,V=22.5°; line dv was positioned along the longest dorsal-ventral length of the otocyst). Sections were 7 mm thick, which constitutes 3-4% of the average otocyst AP length, depending on stage. Each plotted point represents the mean and standard deviation of an expression border coordinate; NeuroD domains lie anterior to the plotted points while the Tbx1 domain lies posterior. Sample sizes per genotype per stage range between 10 and 12 otocysts from a minimum of two litters. P values for specified comparisons (two-tailed t-tests) are shown below the graph. The transgene (Tg) causes significant (asterisks) anterior displacement of NeuroD border coordinates, while heterozygosity results in significant posterior displacement in the ventral otocyst at E10. ns, non-significant. Wild-type NeuroD and Tbx1 border positions are classified as overlapping (o) or non-overlapping (no) if P<0.01 and abutting (a) if P>0.01. These domains overlap only in the ventral otocyst at E9.5.

• Figure S3 -

  Fig. S3. Histodifferentiation of the VIIIth ganglion rudiment at E10.5. (A) Transverse 7 mm section through a wild-type E10.5 embryo, anterior to the otocyst which was reacted with mAb4D5 (anti-islet1/2), reveals histodifferentiation of three ganglion rudiment subdivisions. Axes in B apply. Labeled cells ventral and lateral to white arrows derive from branchial arch ectoderm (black arrow) and constitute the facial (f, VIIth) ganglion rudiment. Lateral (L) and ventromedial (VM) subdivisions constitute the VIIIth ganglion rudiment. Asterisk denotes VIIIth ganglion rudiment central projection. hb, hindbrain. Scale bar: 30 mm. (B) Topographic projection of the E10.5 VIIIth ganglion rudiment and otocyst (light gray) in anterior view, obtained from serial sections prepared as in A. Lateral (red) and ventromedial (cyan) subdivisions are each continuous with a discrete region of delaminating islet1/2-positive cells (arrows). (C) Histograms show distributions of mAb4D5-positive nuclear areas for lateral (red) and ventromedial (cyan) VIIIth ganglion subdivisions in wild-type (6 ears) and Tbx1^–/– embryos (3 ears). n indicates total number of cells sampled per class. Nuclei of each subdivision were sampled exhaustively from alternating (Tbx^+/+) or every fourth (Tbx^–/–) section. Means and standard deviations (microns²) are shown and differences between subdivisions are significant for both genotypes (P<0.0001).