Essential Role of POU–Domain Factor Brn-3C in Auditory and Vestibular Hair Cell Development (Inner Ear Development)

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9445–9450, August 1997 Neurobiology Essential role of POU–domain factor Brn-3c in auditory and vestibular hair cell development (inner ear development) MENGQING XIANG*†,LIN GAN†‡,DAQING LI†§,ZHI-YONG CHEN*, LIJUAN ZHOU¶i,BERT W. O’MALLEY,JR.§, WILLIAM KLEIN‡**, AND JEREMY NATHANS¶i**†† *Center for Advanced Biotechnology and Medicine, Department of Pediatrics, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, Piscataway, NJ 08854; ‡Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030; Departments of §Otolaryngology, Head and Neck Surgery, ¶Molecular Biology and Genetics, ††Neuroscience and Ophthalmology, iHoward Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205 Contributed by Jeremy Nathans, June 11, 1997 ABSTRACT The Brn-3 subfamily of POU–domain tran- Unc-86 loss-of-function mutations affect some of these cells by scription factor genes consists of three highly homologous causing a daughter cell to assume the fate of its mother or by members—Brn-3a, Brn-3b, and Brn-3c—that are expressed in altering cell phenotypes postmitotically (21–23). In mammals, sensory neurons and in a small number of brainstem nuclei. the three highly homologous class IV POU–domain genes, This paper describes the role of Brn-3c in auditory and Brn-3a, Brn-3b, and Brn-3c (also referred to as Brn-3.0, vestibular system development. In the inner ear, the Brn-3c Brn-3.2, and Brn-3.1, respectively), are expressed in distinct but protein is found only in auditory and vestibular hair cells, and overlapping patterns in the developing and adult brainstem, the Brn-3a and Brn-3b proteins are found only in subsets of retina, and dorsal root and trigeminal ganglia (16, 18–20, 24, spiral and vestibular ganglion neurons. Mice carrying a 25). targeted deletion of the Brn-3c gene are deaf and have Targeted mutations in Brn-3a and Brn-3b have revealed a impaired balance. These defects reflect a complete loss of high degree of functional specificity in vivo. Targeted deletion auditory and vestibular hair cells during the late embryonic of the Brn-3a gene results in a decreased number of neurons and early postnatal period and a secondary loss of spiral and in the trigeminal ganglia, altered gene expression in the dorsal vestibular ganglion neurons. Together with earlier work dem- root and trigeminal ganglia, and defects in brainstem nuclei onstrating a loss of trigeminal ganglion neurons and retinal involved in sensory processing and motor control (26, 27). ganglion cells in mice carrying targeted disruptions in the Targeted deletion of the Brn-3b gene results in a selective Brn-3a and Brn-3b genes, respectively, the Brn-3c phenotype absence of '70% of retinal ganglion cells with little or no reported here demonstrates that each of the Brn-3 genes plays effect on other neurons (28, 29). In this paper we show that distinctive roles in the somatosensory, visual, and auditoryy Brn-3c is expressed in auditory and vestibular hair cells and vestibular systems. that targeted deletion of Brn-3c leads to defective inner ear function secondary to a complete absence of sensory hair cells. A number of transcription factors have been implicated in Results similar to those described here have been reported by decisions related to neuronal vs. nonneuronal cell fate, re- Erkman et al. (29). gional specification in the nervous system, or determination of the terminally differentiated phenotype. For example, bHLH MATERIALS AND METHODS factors such as neuroD and the achaete–scute family control neural vs. ectodermal cell fates (1, 2), Hox genes control Targeted Deletion of Brn-3c. To generate the gene targeting regional specification along the neuraxis (3), and several construct, the 4.8-kb BamHI–EcoRI fragment containing the POU–domain genes act at late stages to control the survival Brn-3c gene was cloned into the BamHI site of pMCTK (L.G., and final differentiated phenotype of particular neuronal unpublished work). Subsequently, the 1.8-kb XhoI–XhoI frag- subtypes (4). The POU–domain family was intially defined by ment containing the first exon and the majority of the second the mammalian pituitary-specific transcription factor Pit-1y exon, and encompassing most of the coding region of Brn-3c, GHF-1, the octamer binding proteins Oct-1 and Oct-2, and the was replaced with a 1.6-kb XhoI–XhoI segment containing the Caenorhabditis elegans gene Unc-86 (5). Genetic studies in PGKneo cassette. The targeting vector was linearized at a mice and humans indicate that many POU–domain genes unique NotI site and 25 mg of linearized DNA was electropo- 7 function in the terminal stages of central nervous system rated into 10 AB1 embryonic stem (ES) cells that were then development. SCIPyTst-1yOct-6 controls the differentiation of selected for resistance to G418 and 1-(2-deoxy-2-fluoro-b-D- Schwann cells (6–8), Pit-1yGHF-1 is required for the normal arabinofuranosyl)-5-iodouracil (FIAU). Among the 288 ES development of the anterior pituitary (9), Brn-4yRHS2y colonies resistant to G418 and FIAU, 7 contained the expected POU3F4 is required for the normal development of the middle homologous recombination event, as indicated by Southern ear (10), and Brn-2 is required for the specification of subsets blot analysis using flanking probes. Two targeted ES cell clones of neurons in the hypothalamus (11, 12). were injected into C57BLy6J blastocysts to generate chimaeric The class IV POU–domain group is defined by the Unc-86 mice, and the chimaeric mice were bred to C57BLy6J mice to gene (13), the Drosophila I-POU gene (14, 15), and the three produce heterozygotes and homozygotes for further analysis. vertebrate Brn-3 genes (16–20). The Unc-86 protein is found Behavioral Testing. Hearing was tested by observing the exclusively within a subset of neurons and neuroblasts, and startle response. Mice were placed individually in a small The publication costs of this article were defrayed in part by page charge Abbreviations: ES, embryonic stem; E, embryonic day; SPL, sound pressure level; DAPI, 49,6-diamidino-2-phenylindole; P, postembry- payment. This article must therefore be hereby marked ‘‘advertisement’’ in onic day. accordance with 18 U.S.C. §1734 solely to indicate this fact. †M.X., L.G., and D.L. contributed equally to this work and should be © 1997 by The National Academy of Sciences 0027-8424y97y949445-6$2.00y0 considered co-first authors. PNAS is available online at http:yywww.pnas.org. **To whom reprint requests should be addressed. 9445 Downloaded by guest on September 28, 2021 9446 Neurobiology: Xiang et al. Proc. Natl. Acad. Sci. USA 94 (1997) Plexiglas restrainer in a sound-insulated room. At 10-sec cochlea, stained with 49,6-diamidino-2-phenylindole (DAPI), intervals, each animal was exposed five times to a sharp sound and mounted in glycerin. Immunostaining of the developing produced by striking a metal rod. Ten Brn-3c (1y1), 10 Brn-3c auditory and vestibular system was performed on animals after (1y2), and 11 Brn-3c (2y2) animals were tested. Balance was up to 5 days of postnatal development without decalcification tested by placing mice individually on a soft rubber-coated as described (20). horizontal drum 6 cm in diameter and 5.5 cm deep, positioned 15 cm above a cushioned pad. In 1 set of 10 trials the drum was stationary, and in a second set of 10 trials the drum was rotated RESULTS by a variable speed motor at 7 rpm. In each trial the mouse was Defective Hearing and Balance in Mice Homozygous for a observed for 60 sec after it was placed on the drum and the Targeted Deletion of the Brn-3c Gene. To assess the role of time elapsed until it fell from the drum was recorded. Seven Brn-3c in vivo, a targeted deletion of this gene was constructed Brn-3c (1y1), 7 Brn-3c (1y2), and 7 Brn-3c (2y2) animals in ES cells and introduced via blastocyst fusion into the mouse were tested. germ line. Brn-3c heterozygotes [(1y2)] are indistinguishable Auditory Brainstem Responses. Auditory-evoked brainstem from the wild-type [(1y1)] in viability, fertility, and readily responses were recorded using a Nicolet Compact Four (Nico- observable behaviors. They are also indistinguishable by all of let). Mice were anesthetized with Avertin by intraperitoneal the histologic criteria described below. Brn-3c homozygotes injection, and electrodes were placed at the vertex (active), [(2y2)] have normal viability, but are 10–20% smaller than bilaterally in the neighborhood of the postauricular bullae wild type, have low fertility, and spend much of their time (reference), and in the forehead (ground). The acoustic stim- running in circles. We think that the low weight and fertility of ulus consisted of a click of approximately 0.1 msec duration, Brn-3c (2y2) mice may be secondary to the circling behavior presented at a rate of 11.4 per second. Responses were and the resulting higher energy expenditure. averaged over 1,000 stimuli. Auditory thresholds were deter- Circling behavior has been described for a number of mouse mined by visual inspection of response traces obtained at lines with inner ear defects (34). As a first step in examining stimulus intervals of 5 dB. Absolute stimulus intensities were inner ear function, we determined whether a sharp sound calibrated to obtain the sound pressure level (SPL). could elicit a startle response in Brn-3c (1y1), (1y2), and Semi-Thin Plastic Sections. Deeply anesthetized mice were (2y2) mice. Brn-3c (1y1) and (1y2) mice show a robust perfused transcardially with PBS, 2% paraformaldehyde, 2% startle response (10 mice for each genotype), whereas Brn-3c gluteraldehyde, 1 mM CaCl2, 1 mM MgCl2.

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