Abstract
The temporal bone encases conductive and sensorineural elements of the ear. Mutations of POU3F4 are associated with unique temporal bone abnormalities and X-linked mixed deafness (DFNX2/DFN3). However, the target genes and developmental processes controlled by POU3F4 transcription factor activity have remained largely uncharacterized. Ephrin-B2 (Efnb2) is a signaling molecule with well-documented effects on cell adhesion, proliferation, and migration. Our analyses of targeted mouse mutants revealed that Efnb2 loss-of-function phenocopies temporal bone abnormalities of Pou3f4 hemizygous null neonates: qualitatively identical malformations of the stapes, styloid process, internal auditory canal, and cochlear capsule were present in both mutants. Using failed/insufficient separation of the stapes and styloid process as a quantitative trait, we found that single gene Efnb2 loss-of-function and compound Pou3f4/Efnb2 loss-of-function caused a more severe phenotype than single gene Pou3f4 loss-of-function. Pou3f4 and Efnb2 gene expression domains overlapped at the site of impending stapes-styloid process separation and at subcapsular mesenchyme surrounding the cochlea; at both these sites, Efnb2 expression was attenuated in Pou3f4 hemizygous null mutants relative to control. Results of immunoprecipitation experiments using chromatin isolated from nascent middle ear mesenchyme supported the hypothesis of a physical association between Pou3f4 and specific non-coding sequence of Efnb2. We propose that Efnb2 is a target of Pou3f4 transcription factor activity and an effector of mesenchymal patterning during temporal bone development.
Highlights
Paired temporal bones at the sides and base of the skull house conductive and sensori-neural components of the peripheral auditory system [1]
Our results indicate that Efnb2 lies downstream of Pou3f4 in a genetic pathway governing embryonic-stage development of the mouse temporal bone
Previous work demonstrates that Pou3f4 transcript and gene product are expressed in proximal branchial mesenchyme and in mesenchyme surrounding the nascent cochlea as early as E10.5 [31]
Summary
Paired temporal bones at the sides and base of the skull house conductive and sensori-neural components of the peripheral auditory system [1]. The tympanic part (middle ear) is air-filled and contains structures that conduct sound energy to the cochlea: the tympanic membrane, auditory ossicles (malleus, incus, stapes), muscles, and ligaments (Fig. 1A). The petrous portion of the temporal bone (bony capsule) encases inner ear sensory end organs and neuronal ganglia within a labyrinthine space, and forms canals for passage of cranial nerves and major vessels. It protects the auditory-vestibular system and contributes to the architecture of partitioned, fluid-filled spaces that comprise the inner ear. The auditory ossicles and petrous portion form by endochondral ossification, and in mice, these structures are cartilaginous at the time of birth. The stapes is of mixed origin [5]; head and crura derive exclusively from neural crest, and the footplate is a mixed derivative of neural crest and head mesoderm (Fig. 1B)
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