Scleraxis is Required for Differentiation of the Stapedius and Tensor Tympani Tendons of the Middle Ear

Abstract

Scleraxis (Scx) is a basic helix-loop-helix transcription factor expressed in tendon and ligament progenitor cells and the differentiated cells within these connective tissues in the axial and appendicular skeleton. Unexpectedly, we found expression of the Scx transgenic reporter mouse, Scx-GFP, in interdental cells, sensory hair cells, and cochlear supporting cells at embryonic day 18.5 (E18.5). We evaluated Scx-null mice to gain insight into the function of Scx in the inner ear. Paradoxical hearing loss was detected in Scx-nulls, with 50% of the mutants presenting elevated auditory thresholds. However, Scx-null mice have no obvious, gross alterations in cochlear morphology or cellular patterning. Moreover, we show that the elevated auditory thresholds correlate with middle ear infection. Laser interferometric measurement of sound-induced malleal movements in the infected Scx-nulls demonstrates increased impedance of the middle ear that accounts for the hearing loss observed. The vertebrate middle ear transmits vibrations of the tympanic membrane to the cochlea. The tensor tympani and stapedius muscles insert into the malleus and stapes via distinct tendons and mediate the middle ear muscle reflex that in part protects the inner ear from noise-induced damage. Nothing, however, is known about the development and function of these tendons. Scx is expressed in tendon progenitors at E14.5 and differentiated tenocytes of the stapedius and tensor tympani tendons at E16.5-18.5. Scx-nulls have dramatically shorter stapedius and tensor tympani tendons with altered extracellular matrix consistent with abnormal differentiation in which condensed tendon progenitors are inefficiently incorporated into the elongating tendons. Scx-GFP is the first transgenic reporter that identifies middle ear tendon lineages from the time of their formation through complete tendon maturation. Scx-null is the first genetically defined mouse model for abnormal middle ear tendon differentiation. Scx mouse models will facilitate studies of tendon and muscle formation and function in the middle ear.

Regulation of fibronectin gene expression in cardiac fibroblasts by scleraxis

“…Scleraxis, a basic helix-loop-helix (bHLH) transcription factor, regulates expression of the Col1a2/COL1A2 gene in cardiac fibroblasts, and is significantly up-regulated in the post-infarct scar in a rodent model of MI (Bagchi and Czubryt 2012;Espira et al 2009). Scleraxis has been implicated in aiding wound healing, as well as in the development of the auditory system and periodontal ligaments Mann et al 2013;Wang et al 2011). A recent report from our group demonstrated for the first time that scleraxis controls the expression of multiple ECM genes at the basal level in the myocardium, and is a critical mediator of fibroblast-to-myofibroblast phenotype conversion by direct regulation of key genes involved in the process such as α-smooth muscle actin (Bagchi et al 2016a).…”

Expression and Function of Scleraxis in the Developing Auditory System

“…Resultantly, GFP expression is driven by Scx regulatory sequences in a pattern that closely ressembles that of endogenous Scx expression [29]. In addition, the ability of Scx-GFP to accurately convey activity of the Scx promoter has been confirmed by in situ hybridization [13]. Figure 1 shows cross sections through the cochlear duct at embryonic day 13.5 (E13.5), a time point prior to hair cell and supporting cell differentiation, and at E15.5 when cells in the cochlea are post mitotic and cellular differentiation has begun [30], [31], [32].…”

“…Scx is a twist-like bHLH transcription factor that plays an important role in the development of somites and chondrocyte cell lineages [9], [10], force transmitting tendons of the musculo-skeletal system [11], [12], tendons of the middle ear [13] and numerous mesenchymal cell masses [10]. Despite a predominant expression in developing tissues, there are also reports of Scx transcripts in adult tissues such as the tongue, diaphragm, limb and cartilage of the bronchi [10].…”

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

“…. The stereofluorescence dissecting microscope is a powerful tool to visualize structures in evaluating reporter mice expressing fluorescent proteins in the middle or inner ear 33 . In addition, various in vivo or ex vivo fluorescence labeling methods and whole mount immunofluorescence detection could be undertaken.…”

“…Finally, despite their small size, structures associated with ossicles such as muscles 33 and joints 34,35 are accessible.…”