The Pax2 gene is expressed in the developing otocyst, kidney, and midbrain-hindbrain boundary. We generated Pax2-Cre transgenic lines by modification of a Pax2 bacterial artificial chromosome (BAC). In one Pax2-Cre line, Cre mRNA starts to be expressed in the otic placode at the late presomite stage. R26R reporter mouse analysis revealed that the Cre expression is sufficient to delete the loxP-flanked sequences in most of the cells in the inner ear. Reporter-positive cells are also detected in other Pax2-expressing tissues such as midbrain, cerebellum, olfactory bulb, and kidney, suggesting that these cells are the descendants of Pax2-expressing cells in these tissues and that Pax2-Cre transgenic mice can delete genes efficiently in these tissues.

Summary The organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Here we show that Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and that – unlike other Hes/Hey factors – Hey2 expression is Notch-independent. Hey2 is activated by FGF and blocks hair cell differentiation, while mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. We speculate that co-option of FGF signaling to render Hey2 Notch-independent, also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear.

The otic placode, the anlagen of the inner ear, develops from an ectodermal field characterized by expression of the transcription factor Pax2. Previous fate mapping studies suggest that these Pax2 + cells will give rise to both otic placode tissue and epidermis, but the signals that divide the Pax2 + field into placodal and epidermal territories are unknown. We report that Wnt signaling is normally activated in a subset of Pax2 + cells, and that conditional inactivation of ␤-catenin in these cells causes an expansion of epidermal markers at the expense of the otic placode. Conversely, conditional activation of ␤-catenin in Pax2 + cells causes an expansion of the otic placode at the expense of epidermis, and the resulting otic tissue expresses exclusively dorsal otocyst markers. Together, these results suggest that Wnt signaling acts instructively to direct Pax2 + cells to an otic placodal, rather than an epidermal, fate and promotes dorsal cell identities in the otocyst.

The human serine/threonine kinase, mammalian STE20-like kinase (MST), is considerably homologous to the budding yeast kinases, SPS1 and STE20, throughout their kinase domains. The cellular function and physiological activation mechanism of MST is unknown except for the proteolytic cleavage-induced activation in apoptosis. In this study, we show that MST1 and MST2 are direct substrates of caspase-3 both in vivo and in vitro. cDNA cloning of MST homologues in mouse and nematode shows that caspase-cleaved sequences are evolutionarily conserved. Human MST1 has two caspasecleavable sites, which generate biochemically distinct catalytic fragments. Staurosporine activates MST either caspase-dependently or independently, whereas Fas ligation activates it only caspase-dependently. Immunohistochemical analysis reveals that MST is localized in the cytoplasm. During Fas-mediated apoptosis, cleaved MST translocates into the nucleus before nuclear fragmentation is initiated, suggesting it functions in the nucleus. Transiently expressed MST1 induces striking morphological changes characteristic of apoptosis in both nucleus and cytoplasm, which is independent of caspase activation. Furthermore, when stably expressed in HeLa cells, MST highly sensitizes the cells to death receptor-mediated apoptosis by accelerating caspase-3 activation. These findings suggest that MST1 and MST2 play a role in apoptosis both upstream and downstream of caspase activation.

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.