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Abnormal Mesenchymal Differentiation in the Superior Semicircular Canal of Brn4/Pou3f4 Knockout Mice

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Abnormal Mesenchymal Differentiation in the Superior Semicircular Canal of Brn4/Pou3f4 Knockout Mice ORIGINAL ARTICLE 2 Abnormal Mesenchymal Differentiation in the Superior Semicircular Canal of Brn4/Pou3f4 Knockout Mice Steven E. Sobol, MD, MSc; Xiuyin Teng, MSc; E. Bryan Crenshaw III, PhD Objective: To examine the developmental time course of the SSCC in Brn4 knockout mice was initially detect- of the mutant phenotype and cellular mechanisms that able at P14. Interestingly, the mutant SSCC is indistin- result in malformations of the superior semicircular ca- guishable from control mice at earlier neonatal time points. nal (SSCC) in Brn4 knockout mice. Mutations in the Brn4/ In mutant neonates, there is persistence of immature wo- Pou3f4 gene result in characteristic inner ear abnormali- ven bone with high cellularity surrounding the perilym- ties in mutant mouse pedigrees, and the findings in these phatic space of the SSCC. These findings are not pres- mice are similar to those in human X-linked deafness type ent in control animal specimens, which demonstrate III. appropriate lamellar bony architecture. Design: Mutant and control mice were killed at vari- Conclusions: In Brn4 knockout mice, constriction of the ous neonatal time points to assess the development of SSCC with narrowing of the bony labyrinth develops in the SSCC. Measurements of SSCC diameter were made the postnatal period at approximately P14. The persis- on paint-perfused specimens at postnatal day (P) 0, P7, tence of immature bone in affected mice indicates that P10, and P14. Histologic evaluation of the SSCC was signaling abnormalities disrupt normal mesenchymal dif- made on hematoxylin-eosin–stained sections at P10. ferentiation in the SSCC. Results: A dysmorphic constriction of the superior arc Arch Otolaryngol Head Neck Surg. 2005;131:41-45 ORMAL INNER EAR DEVEL- have revealed multiple defects in affected opment results from a individuals, including stapes footplate fixa- complex series of mor- tion, dilatation of the internal auditory phogenetic changes that canal (IAC), reduced diameter of the semi- occur in response to in- circular canals, and varying degrees of coch- teractions between epithelial and mesen- lear dysplasia.5,7 High-resolution com- N 1 Author Affiliations: Division chymal embryonic tissues. The molecu- puted tomography has demonstrated that of Pediatric Otolaryngology and lar signaling events underlying normal there is a reduction of bone thickness at the Mammalian Neurogenetics inner ear development have yet to be fully cochlear modiolus.8,9 Group, The Center for elucidated. The recent development of X-linked deafness type 3 results from Childhood Communication, genetic approaches provides the means to mutations in the POU3F4 gene,10,11 a mem- The Children’s Hospital of Philadelphia (Drs Sobol and study the underlying pathogenesis of he- ber of the POU domain family of tran- Crenshaw and Ms Teng); and reditary auditory dysfunction in animal scription factors. POU domain genes play Department of models of human congenital malforma- critical roles in the development of the cen- Otorhinolaryngology–Head and tions. tral nervous system and the inner ear as Neck Surgery, University of X-linked deafness type 3 is the most well as other organ systems.12-22 Linkage Pennsylvania School of common type of X-linked hearing loss in analysis has localized the POU3F4 gene to Medicine (Drs Sobol and humans, accounting for 0.8% of all cases the Xq13-21.1 region.23,24 Crenshaw), Philadelphia. of hereditary hearing loss2,3 and character- The Brn4 gene (also called Pou3f4)is Dr Sobol is currently with ized by early-onset mixed hearing loss with the murine ortholog of the human POU3F4 the Department of a high incidence of perilymphatic gusher gene. This gene is expressed early in the Otolaryngology–Head and Neck 4,5 Surgery, Emory University during stapedectomy. Many patients with development of the nervous system and is School of Medicine, X-linked deafness type 3 display abnor- the only gene in the POU domain family Atlanta, Ga. mal vestibular responses on electronystag- expressed in the mesenchyme of the de- Financial Disclosure: None. mographic studies.5,6 Radiologic studies veloping inner ear.25-29 The gene has been (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 131, JAN 2005 WWW.ARCHOTO.COM 41 ©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 localized by immunocytochemical analysis to the mes- (P) 0 (9 controls, 8 mutants), P7 (9 controls, 12 mutants), P10 enchymal cells of the developing otic capsule and plays (5 controls, 20 mutants), and P14 (14 controls, 10 mutants). a role in the regulation of normal mesenchymal remod- These whole-mount preparations were obtained by bisecting eling, epithelial-mesenchymal interactions, and mesen- the head in the sagittal plane, dissecting out the temporal bone, chymal-mesenchymal interactions, which are vital for nor- and placing the specimens in Bodian fixative (75% ethanol, 15% 30,31 water, 5% formalin, and 5% glacial acetic acid) for 24 to 48 hours. mal labyrinthine development. Preparations were then dehydrated through a graded series of Mutations in Brn4 result in a number of characteristic ethanols and cleared in methyl salicylate. Inner ears were per- behavioral abnormalities in mutant mouse pedigrees, in- fused through the oval window with a suspension of 1% gloss cluding hearing loss, vertical head bobbing, changes in gait, paint in methyl salicylate using a 33 gauge standard Hamilton and reduced whisker mobility.30-32 A number of studies have needle. These paint fills visualized the bony labyrinth within evaluated auditory function as well as middle and inner ear the temporal bones of these mice. structure in Brn4 knockout mice. Evaluation of the audi- tory brainstem response in mutant mice has demon- HISTOLOGIC ANALYSIS strated severe hearing loss and evidence of cochlear dys- function.31 Abnormal fibrocyte structure within the spiral Anesthetized mutant and control animals (6 of each) were fixed ligament of Brn4 knockout mice has been hypothesized to by cardiac perfusion using 4% paraformaldehyde in phosphate- result in an abnormal endolymphatic potential, which may buffered saline on P10. Preparations were obtained by bisect- explain these auditory brainstem response results. Knock- ing the head in the sagittal plane, dissecting out the temporal out alleles of the Brn4 gene have demonstrated multiple bone, and placing the specimens in 4% paraformaldehyde for malformations of tissues derived from the otic mesen- overnight immersion fixation. Specimens were washed 3 times using phosphate-buffered chyme, including hypoplasia of the temporal bone, en- saline and decalcified in 0.1M EDTA for 2 to 3 days. After de- larged internal auditory meatus, dysplastic fibrocytes, and 30,31 calcification, temporal bone specimens were serially dehy- malformed stapes. Other morphologic abnormalities drated in alcohol, embedded in paraffin, and sectioned (8 µm) identified in knockout mice have included cochlear hypo- in the coronal plane (cross section through the SSCC) in prepa- plasia, a reduction in the number of cochlear turns, and ration for staining with hematoxylin-eosin. endolymphatic hydrops. Vestibular abnormalities of Brn4 knockout mice give DATA ANALYSIS rise to the vertical head-bobbing phenotype detected in the mutant mice. The vertical nature of the aberrant head Measurements of SSCC diameter were made on paint- motion prompted evaluation of the superior semicircu- perfused specimens and reported in micrometers as lar canal (SSCC, which lies in the vertical plane) by paint mean±standard error of the mean (SEM). On sections stained perfusion in studies by Phippard et al.30,32 Findings in- with hematoxylin-eosin, the narrowest point of the SSCC peri- cluded a constriction of the bony labyrinth surrounding lymphatic space was identified for each sample. To account for the canal with a reduction in the thickness of the bone the natural variability that may exist between specimens, a fixed in this area in adult mutant mice. This was not observed point anterior to the common crus that could be consistently in the posterior semicircular canal and was only rarely located was identified in all samples. Multiple cross-sectional area measurements were made at these points on the hema- observed in the horizontal canal. toxylin-eosin–stained sections by tracing and analysis using the The first objective of the present study was to delin- National Institutes of Health Image J program (version 1.32; eate the timing of the development of the SSCC constric- available at http://rsb.info.nih.gov/ij/. The mean cross- tion by analyzing the canal diameter of Brn4 knockout sectional area of the narrowest and fixed points was reported and control mice at different postnatal time points us- in square micrometers ± SEM. ing paint perfusion analyses. The second goal was to in- Statistical analysis was performed using a 2-tailed t test with vestigate the histologic findings within the mesenchy- PϽ.01 used for statistical significance. All animal protocols used mal compartment of mutant and control animal SSCCs during this research were approved by the institutional ani- at a time point preceding the constriction. We postu- mal care and use committee at The Children’s Hospital of Phila- lated that the vestibular abnormalities observed in Brn4 delphia in accordance with regulations established by the Na- tional Institutes of Health. knockout mice were the result of aberrant mesenchy- mal differentiation in the developing SSCC. RESULTS METHODS SSCC DIAMETER Mouse pedigrees containing a null mutation of the Brn4 gene have been described.30 CD1 and wild-type littermate mice served Although there was a trend of decreasing SSCC diam- as controls. Normal and Brn4 knockout mice were killed at sev- eter in Brn4 knockout mice with increasing develop- eral defined postnatal time points to assess the development mental age, no statistically significant difference was of the SSCC. found between mutant mice and controls at P0, P7, and P10. The mean±SEM diameters of the SSCC in P0, P7, PAINT PERFUSION ANALYSIS and P10 mutant mice were 194.8±4.2 µm, 149.5±10.3 µm, and 126.1±8.3 µm, respectively.
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