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Brn3a is a transcriptional regulator of soma size, target field innervation and axon pathfinding of inner ear sensory neurons

Eric J. Huang1,*, Wei Liu2, Bernd Fritzsch3, Lynne M. Bianchi4, Louis F. Reichardt1,{ddagger} and Mengqing Xiang2,{ddagger}

1 Program in Neuroscience, Department of Physiology, and Howard Hughes Medical Institute, University of California, San Francisco, CA 94143, USA
2 Graduate Program in Molecular Genetics and Microbiology, Center for Advanced Biotechnology and Medicine, and Department of Pediatrics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
3 Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, USA
4 Neuroscience Program, Oberlin College, Oberlin, OH, USA
* Present address: Pathology Service, VAMC and Department of Pathology, University of California, San Francisco, CA 94121, USA



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  		Fig. 1. Expression of Brn3a and Brn3b during development of the mouse facial-stato-acoustic ganglion. (A-J) Whole-mount (A,B) and inner ear sections (C-J) from embryos at the indicated stages were immunostained with anti-Brn3a (A,C,E,G,I) and anti-Brn3b (B,D,F,H,J) antibodies. Prominent Brn3a expression is initially found in the facial-stato-acoustic ganglion (FSAG) by E9-E9.5, and persists at later embryonic stages in all the ganglia derived from it, including the vestibulocochlear ganglion (VCG), spiral ganglion (SG), vestibular ganglion (VG) and geniculate ganglion (GG). Brn3b exhibits a much more delayed temporal expression pattern than Brn3a with no expression in the FSAG at E9-E10.5, weak expression in some cells of the VCG and GG by E12.5-E14.5, and prominent expression in the SG, VG and GG starting only at E16.5. The location of the otic vesicle (OV) is indicated in A-D. (K) Schematic illustrating the derivation of inner ear and gustatory sensory ganglia during embryogenesis, and temporal expression patterns of Brn3a and Brn3b in these ganglia and their primordia. Scale bar: 25 µm in A-F,I,J; 100 µm in G,H.

 


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  		Fig. 2. Neuronal loss, neuron size reduction and migration defects in the spiral, vestibular and geniculate ganglia of Brn3a-/- mice. (A,B) Geniculate ganglion sections from P0 Brn3a+/+ (A) and Brn3a-/- (B) mice were stained with Cresyl Violet. Compared with the wild type, fewer neurons are present in the mutant geniculate ganglion and they appear to be reduced in size. (C,D) Inner ear sections from P0 Brn3a+/+ (C) and Brn3a-/- (D) neonates were labeled with Cresyl Violet. Spiral ganglion (SG) neurons are clustered in the modiolus of the wild-type cochlea (C), whereas they are widely scattered in the mutant (D). (E) Quantitation of neuron number in developing spiral (SG), vestibular (VG) and geniculate (GG) ganglia of wild-type, Brn3a-/- and Brn3b-/- mice. Each histogram represents the mean±s.d. for three to four ganglia. Wild type includes both Brn3a+/+ and Brn3a+/- ganglia, which are indistinguishable in neuron number. (F-H) Distributions of cross-sectional areas of neuronal profiles in Cresyl Violet-labeled vestibular ganglion (F), spiral ganglion (G), and geniculate ganglion (H) from P0 wild type and Brn3a-/- mice. Cross-sectional areas were measured for neuron profiles using NIH Image and the data were plotted using Microsoft Excel. For all three ganglia, the mutant shows a much narrower distribution with the peak substantially shifted to the left compared to the wild type. Scale bar: 25 µm in A,B; 100 µm in C,D.

 


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  		Fig. 3. Reduction of TrkC, parvalbumin and Brn3b expression in the Brn3a-/- spiral ganglion. (A-F) Compared with the wild-type ganglia (A,C), TrkC immunoreactivity is greatly reduced in the E15.5 (B) and E18.5 (D) mutant spiral ganglia. By contrast, a similar level of TrkB immunoreactivity is present in E18.5 spiral ganglion between wild-type (E) and Brn3a mutant (F). Arrows point to radial fibers. (G,H) Parvalbumin immunoreactivity shows substantial decrease in the mutant spiral and vestibular ganglia and their nerve fibers. (I-L) In contrast to the robust expression of Brn3b in wild-type ganglia, the intensity of Brn3b expression and the number of neurons positive for Brn3b is dramatically diminished in E18.5 mutant embryos. Cri, crista; Sac, saccule; SG, spiral ganglion; Utr, utricule; VG, vestibular ganglion. Scale bar: 25 µm in A-D,K,L; 50 µm in E,F; 100 µm in G-J.

 


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  		Fig. 4. Afferent and efferent projections to the cochlea in wild type, Brn3a-/- and TrkC-/- mice. (A-C) Afferent projection to the basal turn of cochlea at P0 is demonstrated by labeling the fibers with DiI. This labeling highlights the modiolus (M), spiral ganglion (SG) and inner ear hair cells (HC). In contrast to the densely organized afferent fibers in the wild-type cochlea (A), there is an overall reduction in fiber density in the basal turn of cochlea in Brn3a-/- mice, including an almost complete absence of the hook region of the ganglion in the basal turn (double arrowheads) and gapping between fiber bundles (B). These abnormalities in the Brn3a-/- cochlea are similar to those present in TrkC-/- mice (C). (D-G) The acetylated tubulin antibody labels both afferent (arrows) and efferent (arrowheads) fibers of the cochlea. In contrast to the orderly arrangement of fibers in wild-type mice (D), there is disordered outgrowth of efferent fibers in the basal turn (BT), middle turn (MT) and apical turn (AT) of the Brn3a-/- cochlea (E-G). Scale bar: 200 µm in A-C; 100 µm in D-G.

 


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  		Fig. 5. Abnormal afferent fiber projections in the semicircular canals of Brn3a-/- mice. (A,B) DiI-labeled flat mount of E13.5 inner ears shows a dramatic reduction in fiber projection to the posterior vertical canal (pvc) and the cochlea in Brn3a-/- embryos. Although slightly reduced, the innervation to the anterior vertical canal (avc), horizontal canal (hc) and utricle (u) in Brn3a-/- mice is comparable with that in the wild type. (C,D) In contrast to the dense innervation in the wild type, the posterior vertical canal of Brn3a-/- mice shows essentially no innervation at P0. Scale bars: 200 µm.

 


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  		Fig. 6. Axon misrouting in the Brn3a-/- vestibular system. Inner ear sections from E16.5 (A-E) and E18.5 (F) Brn3a+/+ (A) and Brn3a-/- (B-F) embryos were immunostained with anti-NF 150 (A-C) and anti-p75NTR (D,E) antibodies with counterstaining by Hematoxylin, or labeled by silver impregnation (F). Although in the wild type (A), vestibular ganglion (VG)-derived nerve fibers immunoreactive for NF 150 or p75NTR invariantly follow pathways leading to innervation of only sensory epithelia of the saccule (Sac), utricule (Utr) and crista (Cri), they often form aberrant branches in the mutant that project into the outside of the inner ear (B-D), or into the cochlea (Co) (E). Tracing nerve fiber trajectories by silver impregnation reveals similar anomalous fiber branches that penetrate the temporal bone and project away from the inner ear (F). Arrows indicate abnormal nerve fiber branches. Scale bar: 50 µm in C,F; 100 µm in A,B,D,E.

 


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  		Fig. 7. Expression of EphB1 in the nervous system of Brn3a mutant mice. Sections of E16.5 wild-type (A-C) and Brn3a-/- (D-F) embryos show no difference in the expression of EphB1. Neurons in the spiral ganglion, trigeminal ganglion and inferior olivary nucleus, which are affected by Brn3a mutation, express similar level of EphB1 protein in both wild type and Brn3a mutants. Co, cochlea; ION, inferior olivary nucleus; SG, spiral ganglion; TG, trigeminal ganglion. Scale bar: 100 µm in A,D; 50µm in B,C,E,F.

 


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  		Fig. 8. Schematics illustrating inner ear defects and genetic interactions revealed by analysis of Brn3a mutant mice. (A) Inner ear defects in Brn3a-/- mice. In the mutant, the spiral ganglion (SG) loses ~30% of neurons by P0, whereas the vestibular ganglion (VG) contains normal number of neurons. Substantial reduction in soma size of neurons is seen in both Brn3a-/- SG and VG. In addition, the mutant SG neurons are defective in migration and thus do not become clustered. In the mutant cochlea (Co), there is overall decrease in afferent fiber density in the basal turn with a nearly complete loss of innervation at the base (broken green arrow). Moreover, efferent innervation displays profound pathfinding defects throughout the entire cochlea. In the mutant vestibular system, the saccule (S), utricle (U), and anterior vertical (AVC) and horizontal (HC) canals are all well innervated by afferent fibers. However, the posterior vertical canal (PVC) lacks afferent innervation (broken pink arrow). (B) Genetic interactions between Brn3a and other transcriptional regulators during sensory gangliogenesis. Brn3a is required for differentiation and survival of sensory neurons in the trigeminal (TG), spiral, vestibular, geniculate (GG) and dorsal root (DRG) ganglia. For fate commitment of neuron progenitors, the TG, SG and VG require Ngn1; the GG requires Ngn2; and the DRG requires both Ngn1 and Ngn2. Therefore, Ngn1 and Ngn2 genetically act upstream of Brn3a. Brn3a regulates expression of TrkA, TrkB, TrkC, Brn3b and Brn3c in TG and DRG, and that of parvalbumin (Parv) and Brn3b in SG and VG. In addition, it controls Brn3b expression in GG, and TrkC expression in SG. During GG development, Phox2a also genetically acts downstream of Ngn2 as it is only required for differentiation and survival of GG neurons. Phox2a has been shown to control expression of c-Ret and dopamine-ß-hydroxylase (DBH) in GG.

 

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