BMP signaling is necessary for patterning the sensory and nonsensory regions of the developing mammalian cochlea

Abstract

The mammalian inner ear detects sound with the organ of Corti, an intricately patterned region of the cochlea in which one row of inner hair cells and three rows of outer hair cells are surrounded by specialized supporting cells. The organ of Corti derives from a prosensory domain that runs the length of the cochlear duct and is bounded by two nonsensory domains, Kölliker's organ on the neural side and the outer sulcus on the abneural side. Although much progress has been made in identifying the signals regulating organ of Corti induction and differentiation, less is known about the mechanisms that establish sensory and nonsensory territories in the cochlear duct. Here, we show that a gradient of bone morphogenetic protein (BMP) signaling is established in the abneural-neural axis of the cochlea. Analysis of compound mutants of Alk3/6 type I BMP receptors shows that BMP signaling is necessary for specification of the prosensory domain destined to form the organ of Corti. Reduction of BMP signaling in Alk3/6 compound mutants eliminates both the future outer sulcus and the prosensory domain, with all cells expressing markers of Kölliker's organ. BMP4 upregulates markers of the future outer sulcus and downregulates marker genes of Kölliker's organ in cochlear organ cultures in a dose-dependent manner. Our results suggest BMP signaling is required for patterning sensory and nonsensory tissue in the mammalian cochlea.

Figure 1.

Dynamic expression of molecular markers during mouse cochlear prosensory formation. A–G, Sections from E11.5 to E13.5 cochlear duct showing expression of SOX2 (A), JAG1 (B), P27^KIP1 (green in B at E13.5), Fgf10 (C), Lfng (D), Bmp4 (E), phospho-SMAD1/5/8 (F), and Id2 (G). The approximate region of the prosensory domain at E13.5 is marked by brackets. H, Schematic drawing summarizing the changes in expression of molecular markers that regionalize the E13.5 cochlear epithelium. The arrow in E indicates the region of Bmp4 expression. Note that the epithelium of the Bmp4⁺ domain at E13.5 is significantly thinner than other domains as indicated by the bars in E. Scale bars, 100 μm.

Figure 2.

BMP signaling is necessary for prosensory specification in the mouse cochlea. A–E, Sections of the cochlear duct (mid-turn) from E13.5 embryos. Left, Control littermate. Right, Alk3-CKO; Alk6^+/− compound mutant. BMP signaling activity is almost completely reduced in mutant embryos, as revealed by phosphorylated-SMAD1/5/8 (A) and the BMP4-responsive gene Id2 (B). Markers of the prosensory domain are absent in mutant embryos: P27^KIP1 (C), BrdU (D), and Hey2 (E). The approximate location of the prosensory domain in control embryos is marked by brackets. Scale bar, 100 μm.

Figure 3.

Markers of Kölliker's organ expand at the expense of the prosensory domain and the outer sulcus in Alk3-CKO; Alk6^+/− compound mutants. A–D, Sections of the cochlear duct (mid-turn) at E13.5. Left, Control littermate. Right, Alk3-CKO; Alk6^+/− compound mutant. Markers of Kölliker's organ such as Fgf10 (A), JAG1 (B), and Lfng (C) are expressed throughout the dorsal cochlear duct of compound mutants, whereas Bmp4 (D), a marker of outer sulcus, is absent. The approximate location of the prosensory domain is marked by brackets. E, A schematic drawing representing the phenotype of the Alk3-CKO; Alk6^+/− compound mutant. In the Alk3-CKO; Alk6^+/− compound mutant, Kölliker's organ domain (red) expands at the expense of the prosensory domain (green) and the outer sulcus domain (blue). F, G, Sections of the cochlear duct (mid-turn) at E15.5. F, The arrow indicates apoptotic cells marked by activated CASPASE3 (green). Nuclei are labeled with DAPI (blue). G, P27^KIP1 (green) and SOX2 (magenta) staining, showing an absence of the p27^kip1+ domain in the compound mutants. Scale bars, 100 μm.

Figure 4.

BMP4 represses Kölliker's organ gene expression and activates outer sulcus genes in a dose-dependent manner. Quantitative PCR analysis of 2 d cultures of E11.5 cochlear explants with genes downregulated (A) and upregulated (C) by increasing concentrations of BMP4 protein (n = 4, ±SEM). B, Whole-mount in situ hybridization of explants with Fgf10 and Id2 probes shows that Fgf10 is downregulated by BMP4, whereas Id2 is upregulated. D, Quantitative PCR analysis of 4 d cultures of E11.5 cochlear explants with outer sulcus marker genes (n = 4, ±SEM). E, Four day culture of cochlear explants from Math1–GFP mice at the presence or in the absence of BMP4 protein in culture medium. The number of GFP⁺ cells per explant is quantified and shown in the graph (n > 20, ±SEM).

Figure 5.

The gross morphology of the inner ear in BMP receptor mutants. A, Whole-mount in situ hybridization of the E12.5 inner ear, showing the expression of Fgf10, Sox2, and Bmp4. ac, Anterior crista; lc, lateral crista; pc, posterior crista; ed, endolymphatic duct; u, utricle; s, saccule; c, cochlea. B, Paint-fills of the E13.5 inner ears of control and Alk3-CKO; Alk6^+/− mutants. The paint often fails to fill the middle of the semicircular canals of Alk3-CKO; Alk6^+/− (arrowheads). The cochlear duct is severely shortened and widened in the Alk3-CKO; Alk6^+/− mutants (asterisk). C, Vestibular sensory hair cell development revealed by Myosin VI staining (green) appears grossly normal in Alk3-CKO; Alk6^+/− mutants at E15.5. Nuclear staining by DAPI is indicated in magenta. Scale bars, 100 μm.