QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 17 March 2004
doi: 10.1242/dev.01067

This Article Summary Full Text Full Text (PDF) Supplemental Figures Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Raft, S. Articles by Morrow, B. E. Search for Related Content PubMed PubMed Citation Articles by Raft, S. Articles by Morrow, B. E.

Suppression of neural fate and control of inner ear morphogenesis by Tbx1

Steven Raft^1,*, Sonja Nowotschin², Jun Liao² and Bernice E. Morrow^2,

¹ Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
² Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

View larger version (91K): [in a new window]   Fig. 1. Tbx1/TBX1 suppresses expression of neural bHLH genes in the otic placode. (A-F) Lateral surface views of whole mount hybridized embryos. Arrowheads mark the anterior and posterior poles of the invaginating otic placode. asterisk, facial ganglion progenitors; m, Tbx1-positive mesenchyme. ss, somite stage. (G,H,J-L) Transverse sections (7 µm) through the otic epithelium of whole-mount hybridized embryos. Arrowheads in G and H mark limits of detectable signal; Tg, transgenic. Scale bars in A for A-F and in G for G-L. (I) Quantification of otic epithelial Ngn1 hybridization signals (perinuclear rings) by serial section analyses for various genotypes at two stages near the onset of neurogenesis (16-17 somites, 19-21 somites; n=8-10 epithelia/genotype/stage. *P<0.01).

View larger version (88K): [in a new window]   Fig. 2. Tbx1/TBX1 suppresses NeuroD expression in the otocyst. Lateral surface views of whole-mount hybridized embryos arrayed by stage, genotype, and hybridization probe. All transgenic (Tg) embryos shown are of Tg+/+ genotype. Mottled otocyst NeuroD signals are epithelial; dense signals apposed to the otocyst indicate the VIIIth ganglion. Asterisks mark the facial ganglion. Arrows in G,H,I mark heightened/ectopic NeuroD expression in Tbx1+/– otocysts. p, petrosal ganglion. m, Tbx1-positive mesenchyme. Scale bar: 100 µm applies to all panels.

View larger version (51K): [in a new window]   Fig. 4. Tbx1 determines the position of neural bHLH gene expression domain borders. (A) Expression domain morphologies (colored regions) revealed by 3-D reconstruction of serial section sets. Otocysts are shown in ventral view and rendered transparently to visualize dorsal Tbx1 expression. Models are normalized for AP length across stages. (B) 3-D reconstructions of the otocyst showing the Ngn1 expression domain (green) at E10 for each genotype.

View larger version (97K): [in a new window]   Fig. 3. Tbx1 patterns Lfng and Bmp4 otocyst expression. Lateral surface views of whole mount hybridized embryos arrayed by stage and genotype. Arrowheads denote position of the Bmp4 anterior stripe. Black arrows highlight the position of posterior Bmp4 expression. White arrows mark the anterior region of Lfng/neural bHLH gene expression. Brackets in D and H mark the extent of diffuse anterodorsolateral Bmp4 expression in Tbx1–/– otocysts. All images are shown to scale.

View larger version (60K): [in a new window]   Fig. 5. Ectopic neurogenesis correlates with an expansion of VIIIth ganglion rudiment form. (A) Mean occurrence of epithelial NeuroD signals (perinuclear rings) by stage and otocyst region for each of the phenotypes examined, obtained by analyses of serial 7 µm thick transverse sections. Between four and eight right otocysts per genotype per stage were analyzed. Data were normalized to account for differences with wild type in average number of sections when this exceeded 5% (normalization factors: E9.5 Tg, 1.08; E9.5 Tbx1–/–, 1.09; E10 Tbx1–/–, 1.22; E11 Tbx1–/–, 1.53; E11 Tbx1+/–, 1.09). Transgenic data are for Tg+/+, except at E10 (Tg+/–). Values are plotted on a log scale, and non-overlapping error bars represent significant differences with P<0.003. At later stages these regions are distinguished as shown in A'. (A') Tracings of E11 wild-type sections at 20 and 32% otocyst (with anterior pole set to zero) showing spatial distributions of NeuroD-positive lateral (red in B) and ventral (cyan in B) cells. a, anterior cardinal vein. Lateral and ventral regions at stages E9.5 and E10 are distinguished by line VL in Fig. S2, http://dev.biologists.org/supplemental/. (B) 3-D reconstructions of E10.5 otocysts and ganglia (red, lateral subdivision; cyan, ventromedial subdivision), obtained by analyses of sections reacted with mAb4D5 (anti-islet1/2). Brackets demarcate regions of delamination in wild type. Otocysts are rendered transparently. ed, endolymphatic duct outgrowth. Scale bar: 100 µm. (C) Representative section of E10.5 Tbx1–/– posterior otocyst reacted with mAb4D5. Arrows highlight sites of cell delamination into the lateral (red) and ventromedial (cyan) pools. epibr., epibranchial ganglion. Scale bar: 60 µm. (D) Lateral (red arrows) and ventromedial (cyan arrows) mAb4D5-positive nuclei in the wild-type E10.5 ganglion. Scale bar: 10 µm. (E) Section through the posterior pole of the E10.5 Tbx1–/– otocyst shows delaminating mAb4D5-positive cells.

View larger version (131K): [in a new window]   Fig. 8. Tbx1–/– inner ear and VIIIth ganglion morphology at E13.5. (A-D,F,G,J,K) Transverse sections through wild-type and Tbx1–/– ears/ganglia at E13.5, reacted with mAb2H3 (anti-neurofilament) and counter-stained with Cresyl Violet. Axes in F apply to all except G. Red arrows in A and C indicate regions of vestibular (vest) neuronal cytology and cyan arrows in C auditory (aud) neuronal cytology. The distinguishing cytological features (nuclear size and basophilia) from a mutant posterior ganglion are shown at high magnification in G, with vestibular neuronal cytology shown at right. White asterisks in A and B indicate a branch of the facial nerve. Section shown in F passes through a Tbx1–/– posterior compound ganglion and epithelial posterior pole. Pvest, presumptive vestibular ganglion; Paud, presumptive auditory ganglion. Brackets and arrowheads in J and K indicate cellular stratification. Scale bars: in D, 60 µm for A-D; 20 µm for J,K. (E) 3-D reconstructions of serial sections reacted as described above. Presumptive vestibular (red) and auditory (cyan) ganglion tissue is shown in relation to the inner ear epithelium. c, cochlea; sa, superior ampulla; la, lateral ampulla; pa, posterior ampulla; sc, superior canal; lc, lateral canal; pc, posterior canal; ed/es, endolymphatic duct/sac; u, utricle; s, saccule; lt, lateral tube; ms, medial sac; dp, dorsal projection. (H) Double immunolabeling with mAb2H3 and anti-MATH1 shows neurites (in green, arrowheads) enveloping the base of a MATH1-positive cell. Dotted line indicates the basement membrane. (I) Anteroventrolateral view of the Tbx1–/– inner ear; the region of stratification and innervation is mapped in brown. Approximate section levels are indicated. (L) MATH1-positive nuclei occupy an apical position in the epithelium. Dashed line indicates the lumen, which is collapsed. Dotted line indicates the basement membrane.

View larger version (68K): [in a new window]   Fig. 6. Tbx1 is a determinant of otocyst anterior-posterior patterning. (A) 7 µm transverse section through the wild-type anterior otocyst shows a lateral to medial gradient of Lfng signal, decreasing medially. Arrow marks the medial extent of detactable signal. Axes are as shown in I. (B) Section, prepared as in A, through the posterior Tbx1–/– otocyst. (C-H) Lateral surface views of whole-mount hybridized embryos. Arrowhead in C marks posteroventral Lfng signal. Arrowheads in G and H indicate borders of midbrain Otx1 signal. Arrows indicate the otocyst. (I,J) 40 µm transverse vibratome sections through the anterior otocyst. Arrows indicate common dorsal borders of Pax2 and Gata3 signal; asterisk, low intensity ventromedial Gata3 signal.

View larger version (157K): [in a new window]   Fig. 7. Otocyst Bmp4 expression is sensitive to Tbx1 gene copy number. (A-F) 30 µm transverse cryosections of Bmp4 hybridized embryos at the level of the anterior stripe (arrows in B). Asterisk in C indicates the absence of a definitive anterior stripe in E11 Tbx1+/– otocysts. Arrowheads in F indicate mesenchymal Bmp4 signal of low intensity compared to wild type.

View larger version (60K): [in a new window]   Fig. 9. Summary. (A) Gene expression and predicted fates of the E10.5 otocyst lateral wall. Specification of neural and sensory organ fates appears to involve both reciprocal compartmentalization (Tbx1/Bmp4/Otx1 vs. bHLH gene expression domain) and change in competence of a pluripotent progenitor field over time (bHLH gene/Lfng domain). (B) Neural and particular sensory organ fates may be specified in parallel through opposing actions of Tbx1 on gene expression. Question marks indicate potential relationships that are unsubstantiated by genetic analyses.