The auditory sensory epithelium (organ of Corti), where sound waves are converted to electrical signals, comprises a highly ordered array of sensory receptor (hair) cells and nonsensory supporting cells. Here, we report that Sprouty2, which encodes a negative regulator of signaling via receptor tyrosine kinases, is required for normal hearing in mice, and that lack of SPRY2 results in dramatic perturbations in organ of Corti cytoarchitecture: instead of two pillar cells, there are three, resulting in the formation of an ectopic tunnel of Corti. We demonstrate that these effects are due to a postnatal cell fate transformation of a Deiters' cell into a pillar cell. Both this cell fate change and hearing loss can be partially rescued by reducing Fgf8 gene dosage in Spry2 null mutant mice. Our results provide evidence that antagonism of FGF signaling by SPRY2 is essential for establishing the cytoarchitecture of the organ of Corti and for hearing.

Multiple signaling molecules, including Fibroblast Growth Factor (FGF) and Wnt, induce two patches of ectoderm on either side of the hindbrain to form the progenitor cell population for the inner ear, or otic placode. Here we report that in Spry1, Spry2 compound mutant embryos (Spry1−/−; Spry2−/− embryos), the otic placode is increased in size. We demonstrate that the otic placode is larger due to the recruitment of cells, normally destined to become cranial epidermis, into the otic domain. The enlargement of the otic placode observed in Spry1−/−; Spry2−/− embryos is preceded by an expansion of a Wnt8a expression domain in the adjacent hindbrain. We demonstrate that both the enlargement of the otic placode and the expansion of the Wnt8a expression domain can be rescued in Spry1−/−; Spry2−/− embryos by reducing the gene dosage of Fgf10. Our results define a FGF-responsive window during which cells can be continually recruited into the otic domain and uncover SPRY regulation of the size of a putative Wnt inductive center.

Sprouty (SPRY) appears to act as a tumor suppressor in cancer, whereas we demonstrated that SPRY2 functions as a putative oncogene in colorectal cancer (CRC) [Oncogene, 2010, 29: 5241–5253]. We investigated the mechanisms by which SPRY regulates epithelial-mesenchymal transition (EMT) in CRC. SPRY1 and SPRY2 mRNA transcripts were significantly upregulated in human CRC. Suppression of SPRY2 repressed AKT2 and EMT-inducing transcription factors and significantly increased E-cadherin expression. Concurrent Downregulation of SPRY1 and SPRY2 also increased E-cadherin and suppressed mesenchymal markers in colon cancer cells. An inverse expression pattern between AKT2 and E-cadherin was established in a human CRC tissue micro array. SPRY2 negatively regulated miR-194-5p that interacts with AKT2 3′UTR. Mir-194 mimics increased E-cadherin expression and suppressed cancer cell migration and invasion. By confocal microscopy we demonstrated redistribution of E-cadherin to plasma membrane in colon cancer cells transfected with miR-194. Spry1−/− and Spry2−/− double mutant mouse embryonic fibroblasts (MEFs) exhibited decreased cell migration, while acquiring several epithelial markers. In CRC, SPRY drive EMT and may serve as a biomarker of poor prognosis.

BackgroundIn multiple vertebrate organisms, including chick, Xenopus, and zebrafish, Fibroblast Growth Factor (FGF) and Wnt signaling cooperate during formation of the otic placode. However, in the mouse, although FGF signaling induces Wnt8a expression during induction of the otic placode, it is unclear whether these two signaling pathways functionally cooperate. Sprouty (Spry) genes encode intracellular antagonists of receptor tyrosine kinase signaling, including FGF signaling. We previously demonstrated that the Sprouty1 (Spry1) and Sprouty2 (Spry2) genes antagonize FGF signaling during induction of the otic placode. Here, we investigate cross talk between FGF/SPRY and Wnt signaling during otic placode induction and assess whether these two signaling pathways functionally cooperate during early inner ear development in the mouse.MethodsEmbryos were generated carrying combinations of a Spry1 null allele, Spry2 null allele, β-catenin null allele, or a Wnt reporter transgene. Otic phenotypes were assessed by in situ hybridization, semi-quantitative reverse transcriptase PCR, immunohistochemistry, and morphometric analysis of sectioned tissue.ResultsComparison of Spry1, Spry2, and Wnt reporter expression in pre-otic and otic placode cells indicates that FGF signaling precedes and is active in more cells than Wnt signaling. We provide in vivo evidence that FGF signaling activates the Wnt signaling pathway upstream of TCF/Lef transcriptional activation. FGF regulation of Wnt signaling is functional, since early inner ear defects in Spry1 and Spry2 compound mutant embryos can be genetically rescued by reducing the activity of the Wnt signaling pathway. Interestingly, we find that although the entire otic placode increases in size in Spry1 and Spry2 compound mutant embryos, the size of the Wnt-reporter-positive domain does not increase to the same extent as the Wnt-reporter-negative domain.ConclusionsThis study provides genetic evidence that FGF and Wnt signaling cooperate during early inner ear development in the mouse. Furthermore, our data suggest that although specification of the otic placode may be globally regulated by FGF signaling, otic specification of cells in which both FGF and Wnt signaling are active may be more tightly regulated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0083-8) contains supplementary material, which is available to authorized users.

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in (1,2)).Visualization of this cellular mosaic often requires that the organ of Corti is cross-sectioned. In particular, the nonsensory pillar and Deiters' cells, whose nuclei are located basally with respect to the hair cells, cannot be visualized without cross-sectioning the organ of Corti. However, the delicate cytoarchitecture of the mammalian organ of Corti, including the fine cytoplasmic processes of the pillar and Deiters' cells, is difficult to preserve by routine histological procedures such as paraffin and cryo-sectioning, which are compatible with standard immunohistochemical staining techniques. Here I describe a simple and robust procedure consisting of vibratome sectioning of the cochlea, immunohistochemical staining of these vibratome sections in whole mount, followed by confocal microscopy. This procedure has been used widely for immunhistochemical analysis of multiple organs, including the mouse limb bud, zebrafish gut, liver, pancreas, and heart (see (3-6) for selected examples). In addition, this procedure was sucessful for both imaging and quantitificaton of pillar cell number in mutant and control organs of Corti in both embryos and adult mice (7). This method, however, is currently not widely used to examine the mammalian organ of Corti. The potential for this procedure to both provide enhanced preservation of the fine cytoarchitecture of the adult organ of Corti and allow for quantification of various cell types is described.