Pathological mechanisms responsible for deafness in Pou3f4-deficient mouse, an animal model for human DFNX2 (DFN3)

Abstract

Pou3f4, encoding a POU domain transcription factor, is the major gene responsible for DFNX2 (DFN3), an X-chromosome linked nonsyndromic deafness in human. C3HeB/FeJ-Pou3f4del-J/J (The Jackson Laboratory, Bar Harbor, ME, USA) mice carrying spontaneous deletion in the Pou3f4 locus was used as an animal model for DFNX2 (DFN3). Analysis of the cochlea from Pou3f4del-J mice showed that the lack of Pou3f4 causes abnormalities in the temporal bone and otic fibrocytes similar to the phenotypes in human X-linked nonsyndromic hearing loss DFNX2 (DFN3). To better understand the role of Pou3f4 in cochlear structure of mouse inner ear during development, I analysed the cochlea of Pou3f4del-J mice inner ears by immunohistochemistry, in situ hybridization, HE, and alizarin red staining. Mid-modiolar sections of 3 week-old mutant mice showed severe defects of the modiolus and lateral wall. The immunohistochemistry and in situ hybridization results suggested that all types of otic fibrocytes were affected by Pou3f4 deficiency. A portion of mesenchymal cells in the lateral wall that were fated to go through mesenchymal-epithelial transition to form the basal cells of the stria vascularis underwent normal condensation and localization process in mutant mice, although the timing was slightly delayed. Therefore, unlike the otic fibrocytes, stria vascularis completed its differentiation with a slight delay, yet the expression of Kir4.1 in the strial intermediate cells that required for EP generation, was lost afterwards.Affymetrix mouse array was performed with wild type and Pou3f4del-J inner ears at P0. Among the many genes investigated, only 5 genes were shown > 2 fold downregulation. Carbonic anhydrase3 (Car3), a cytoplasmic isozyme of carbonic anhydrase family, which has a low activity for CO2 hydration but functions as an oxyradical scavenger to protect cells from oxidative stress, was identified from the 5 downregulated genes in Pou3f4del-J inner ear. Thus, a severe decreased expression of Car3 in otic fibrocytes was corresponded with hyperpigmentation in the melanocytes (intermediate cells) of the stria vascularis in Pou3f4del-J inner ear, suggestive of free radical involvement in the spiral ligament defects. However, Car3 transcript was not directly regulated by Pou3f4.The in vitro studies suggested that overexpression of E-cadherin led to inhibition of osteoblast differentiation, supporting the hypothesis - the loss of modiolar bone may be dependent on the ectopic expression of E-cadherin in the modiolus region in Pou3f4-deficient cochlea. In situ hybridization was performed on otic capsule surrounding membranous labyrinth by using specific markers for every stage of bone development. The results demonstrated that cartilage formation of otic capsule may be processed almost normally, yet hypertrophic chondrogenesis and bone maturation procedure may be happened abnormally in Pou3f4-deficient cochlea. Taken together, the results highlight the pathological mechanisms of the defects in various tissues of cochlea in inner ear resulted from Pou3f4 deficiency, which may contribute to understand the role of Pou3f4 in mouse cochlea.