Patterns of Gene Expression Associated with <i>Pten</i> Deficiency in the Developing Inner Ear

<div><p>In inner ear development, phosphatase and tensin homolog (PTEN) is necessary for neuronal maintenance, such as neuronal survival and accurate nerve innervations of hair cells. We previously reported that <i>Pten</i> conditional knockout (cKO) mice exhibited disorganized fasciculus with neuronal apoptosis in spiral ganglion neurons (SGNs). To better understand the genes and signaling networks related to auditory neuron maintenance, we compared the profiles of differentially expressed genes (DEGs) using microarray analysis of the inner ear in E14.5 <i>Pten</i> cKO and wild-type mice. We identified 46 statistically significant transcripts using significance analysis of microarrays, with the false-discovery rate set at 0%. Among the DEGs, expression levels of candidate genes and expression domains were validated by quantitative real-time RT-PCR and <i>in situ</i> hybridization, respectively. Ingenuity pathway analysis using DEGs identified significant signaling networks associated with apoptosis, cellular movement, and axon guidance (i.e., secreted phosphoprotein 1 (<i>Spp1</i>)-mediated cellular movement and regulator of G-protein signaling 4 (<i>Rgs4</i>)-mediated axon guidance). This result was consistent with the phenotypic defects of SGNs in <i>Pten</i> cKO mice (e.g., neuronal apoptosis, abnormal migration, and irregular nerve fiber patterns of SGNs). From this study, we suggest two key regulatory signaling networks mediated by <i>Spp1</i> and <i>Rgs4</i>, which may play potential roles in neuronal differentiation of developing auditory neurons.</p></div

Microarray analysis identifies novel <i>Pten</i> targets.

<p>Heat maps for relative gene expression of interest (FDR = 0) obtained from three microarrays comparing <i>Pten</i> cKO to wild-type embryos. Green and red indicate decreased and increased expression, respectively, in <i>Pten</i> cKO mice.</p

Cochlear innervation defects in <i>Pten-</i>deficient mice.

<p>(A, B) Patterns of cochlear innervation were assessed by NeuroVue-tracing at E18.5. (A) In <i>Pax2^Cre/+</i>;<i>Pten^loxP/loxP</i> mice, nerve innervation was evident in the apical and basal turns of the cochlea in wild-type (a, c) and <i>Pten</i> cKO (b, d) mice. <i>Pten</i>-deficient inner ears displayed sparse radial fibers, a disorganized pattern, and loss of spiral ganglia in the cochlea (arrowheads in b, d). Abnormal innervation was distributed evenly throughout the cochlea. M, modiolus; R, radial fibers; SG, spiral ganglion. Scale bar: 100 µm. (B) <i>Neurog1^Cre/+</i>;<i>Pten^loxP/loxP</i> mice were administered tamoxifen as an IP injection to perform tamoxifen-inducible deletion of <i>Pten</i> between E8.5 and E11.5. Neuronal abnormalities were seen in <i>Pax2^Cre/+</i>;<i>Pten^loxP/loxP</i> mice and similar innervation defects in <i>Neurog1^Cre/+</i>;<i>Pten^loxP/loxP</i> mice revealed spacing or several gathered radial fibers and a disorganized pattern in the innervation of the cochlea (arrowheads in b, d). The innervation defects were distributed evenly throughout the cochlea. M, modiolus; R, radial fibers; SG, spiral ganglion. Scale bar: 100 µm. (C, D) Neuronal loss in the spiral ganglion in <i>Pax2^Cre/+</i>;<i>Pten^loxP/loxP</i> mice at E18.5. (C) Neurofilament immunoreactivity (arrows) and (D) the number of the spiral ganglia were significantly decreased in <i>Pten</i> cKO mice compared to wild-type mice at E18.5 (3 cochleae, <i>P</i><0.01). M, modiolus; R, radial fibers; SG, spiral ganglion. Scale bar: 100 µm.</p

Genes selected for validation of microarray data by qRT-PCR.

<p>Genes selected for validation of microarray data by qRT-PCR.</p

Akt and GSK3β phosphorylation in <i>Pten</i>-deficient mice.

<p>(A) Phosphorylation of Akt and GSK3β was measured by immunofluorescence staining using anti-phospho-Akt (Ser473) (pAkt) and anti-phospho-GSK3β (Ser9) (pGSK3β) antibodies at E13.5. Compared to wild-type spiral ganglia, Akt phosphorylation (green) increased substantially in <i>Pten</i>-deficient mice (a–f). GSK3β phosphorylation (green) was also highly expressed in the spiral ganglia of <i>Pten</i>-deficient mice compared to that in wild-type mice (g–l). In the absence of <i>Pten</i>, all pAkt and pGSK3β cells were maintained in Islet-1 positive cells (red) (a–l). Wild-type mice showed well-organized patterns of Islet-1-positive cells (a–c), whereas <i>Pten</i>-deficient mice showed a slightly scattered form of spiral ganglia (d–f). Cleaved caspase-3-positive apoptotic neurons (red) were sometimes co-localized with TrkC-positive cells (green) (arrowheads in r). DAPI-stained nuclei (blue) are seen in all images. Scale bar: 100 µm. (B) Western blotting analysis of Pten, pAkt, Akt, pGSK3β, GSK3β, and β-actin expression in the inner ear at E14.5. Proteins were extracted from four pooled inner ears at E14.5. (C) The relative intensity of each phospho-protein was normalized to the total level of the same protein. Levels of both pAkt (a) and pGSK3β (b) were significantly increased in <i>Pten</i> cKO mice (4 cochleae, <i>P<</i>0.05).</p

Functional network analysis associated with <i>Pten</i>-deficient inner ear.

<p>Network analysis using the Ingenuity Pathway Analysis (IPA) software was conducted using selected genes that were differentially expressed and their close relationships. IPA results show two core networks consisted of <i>Spp1</i>-(red line) and <i>Rgs4</i>-associated interactions (blue line). Genes that were differentially expressed are indicated in pink, and predicted interacting genes (not contained in the microarray data) are indicated in white. Axon guidance signaling pathway-related genes are outlined in magenta. Molecular interactions between connected genes represent direct (solid line) or indirect (dotted line) functional relationships based on the IPA database. Green indicates negative fold changes, while red denotes positive fold changes, according to color intensity.</p

Differentially expressed genes in wild-type and <i>Pten</i> cKO mice at E14.5.

<p>Differentially expressed genes in wild-type and <i>Pten</i> cKO mice at E14.5.</p

Pten expression patterns during inner ear development.

<p>(A, B) Pten expression in the inner ear was examined by immunofluorescence at E10.5, E11.5, E12.5, E14.5, and E16.5. (A) Pten protein (green) was first detected between E10.5 and E11.5 in the cytoplasm of the cochleovestibular ganglion (CVG) complex (white outlines in a–l), and mainly overlapped with TrkC-positive neural precursor cells (red) rather than co-localizing with NeuroD (red) (arrows in a–l). From E12.5 to E14.5, Pten (green) was clearly expressed in TrkC-positive cells (red) in cochlear ganglion neurons, which were defined by Tuj1-positive ganglia (red) (m–x). DAPI-stained nuclei (blue) are seen in all images. CVG, cochleovestibular ganglion; D, dorsal; GG, geniculate ganglion; M, medial; OV, otic vesicle; SA, saccule. Scale bars: 100 µm. (B) At E16.5, Pten-immunopositive signals (arrowheads in b) were observed in the MyoVIIa-positive hair cells (green) (a–c) but not in the Prox1-positive supporting cells (green) (d–f). Expression of Pten was detected in the Hensen’s and Claudius’ cells (arrows in h), Tuj1-positive neurons (green), but not in p75^NTR-positive pillar cells (green) (g-i). DAPI-stained nuclei (blue) are seen in all images. DC, Deiter’s cell; GER, greater epithelial ridge; H&C, Hensen’s and Claudius’ cells; HCs, hair cells; IH, inner hair cell; IP, inner pillar cell; KO, Kölliker’s organ; LER, lesser epithelial ridge; OH, outer hair cell; OP, outer pillar cell; SG, spiral ganglion. Scale bars: 100 µm.</p

Conditional Deletion of <em>Pten</em> Leads to Defects in Nerve Innervation and Neuronal Survival in Inner Ear Development

<div><p>All cellular phenomena and developmental events, including inner ear development, are modulated through harmonized signaling networks. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor, is a major signaling component involved in cross talk with key regulators of development; <em>i</em>.<em>e</em>., Wnt, Notch, and bone morphogenetic proteins. Although <em>Pten</em> function has been studied in various systems, its role in inner ear development is poorly understood. Here, we used inner ear-specific <em>Pten</em> conditional knockout mice and examined the characteristics of the inner ear. In a detailed analysis of the phenotype, reduced cochlear turning and widened epithelia were observed. Phalloidin staining of sensory epithelium revealed that hair cell patterns were disturbed; <em>i</em>.<em>e</em>., additional rows of hair cells were discovered. The neural abnormality revealed a reduction in and disorganization of nerve fibers, including apoptosis at the neural precursor stage. <em>Pten</em> deficiency induced increased phosphorylation of Akt at Ser473. The elevation of inhibitory <b><em>g</em></b>lycogen synthase kinase 3β Ser9 phosphorylation (pGSK3β) was sustained until the neuronal differentiation stage at embryonic day 14.5, instead of pGSK3β downregulation. This is the first report on the influence of Pten/Akt/GSK3β signaling on the development of spiral ganglia. These results suggest that <em>Pten</em> is required for the maintenance of neuroblast number, neural precursors, and differentiation in the inner ear.</p> </div

Inner ear phenotypes in <i>Pten</i> conditional knockout (cKO) mice.

<p>(A) Paint-filled inner ears of <i>Pten</i> cKO embryos at E14.5 shown in a lateral view. <i>Pten</i>-deficient inner ears showed abnormal morphology in the vestibule, including a thickened semicircular canal and canal pouches with a widened endolymphatic duct and common cruses. There was a grossly widened morphology with a coarse pattern in the cochlea. aa, anterior ampulla; asc, anterior semicircular canal; ed/s, endolymphatic duct and sac; la, lateral ampulla; oc, organ of Corti; pa, posterior ampulla; psc, posterior semicircular canal; s, saccule; u, utricle; A, anterior; D, dorsal. Scale bar: 50 µm. (B) At E17.5, the <i>Pten</i> deletion-induced morphology became more evident compared to that in the wild type (black double arrow in a, b). D, dorsal; P, posterior. Scale bar: 100 µm. (C) The morphological pattern of the epithelium was analyzed by whole-mount phalloidin with p75^NTR immunofluorescence. <i>Pten</i>-deficient mice showed additional rows of outer and inner hair cells, whereas wild-type mice showed three rows of outer hair cells and one row of inner hair cells at E18.5 (arrowheads in b). p75^NTR-positive pillar cells were irregular and widened compared to those in wild-type mice (arrow in b). IH, inner hair cell; OH, outer hair cell. Scale bar: 100 µm.</p