The neural crest is a stem population critical for development of the vertebrate craniofacial skeleton and peripheral ganglia. Neural crest cells originate along the border between the neural plate and epidermis, migrate extensively and generate numerous derivatives, including neurons and glia of the peripheral nervous system, melanocytes, bone and cartilage of the head skeleton. Impaired neural crest development is associated with human defects, including cleft palate. Classically, the neural crest has been thought to form by interactions at the border between neural and non-neural ectoderm or mesoderm, and defined factors such as bone morphogenetic proteins (BMPs) and Wnt proteins have been postulated as neural crest-inducers. Although competence to induce crest cells declines after stage 10 (ref. 14), little is known about when neural crest induction begins in vivo. Here we report that neural crest induction is underway during gastrulation and well before proper neural plate appearance. We show that a restricted region of chick epiblast (stage 3-4) is specified to generate neural crest cells when explanted under non-inducing conditions. This region expresses the transcription factor Pax7 by stage 4 + and later contributes to neural folds and migrating neural crest. In chicken embryos, Pax7 is required for neural crest formation in vivo, because blocking its translation inhibits expression of the neural crest markers Slug, Sox9, Sox10 and HNK-1. Our results indicate that neural crest specification initiates earlier than previously assumed, independently of mesodermal and neural tissues, and that Pax7 has a crucial function during neural crest development.

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 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.

Sensorineural hearing loss is most commonly caused by the death of hair cells in the organ of Corti, and once lost, mammalian hair cells do not regenerate. In contrast, other vertebrates such as birds can regenerate hair cells by stimulating division and differentiation of neighboring supporting cells. We currently know little of the genetic networks which become active in supporting cells when hair cells die and that are activated in experimental models of hair cell regeneration. Several studies have shown that neonatal mammalian cochlear supporting cells are able to trans-differentiate into hair cells when cultured in conditions in which the Notch signaling pathway is blocked. We now show that the ability of cochlear supporting cells to trans-differentiate declines precipitously after birth, such that supporting cells from six-day-old mouse cochlea are entirely unresponsive to a blockade of the Notch pathway. We show that this trend is seen regardless of whether the Notch pathway is blocked with gamma secretase inhibitors, or by antibodies against the Notch1 receptor, suggesting that the action of gamma secretase inhibitors on neonatal supporting cells is likely to be by inhibiting Notch receptor cleavage. The loss of responsiveness to inhibition of the Notch pathway in the first postnatal week is due in part to a down-regulation of Notch receptors and ligands, and we show that this down-regulation persists in the adult animal, even under conditions of noise damage. Our data suggest that the Notch pathway is used to establish the repeating pattern of hair cells and supporting cells in the organ of Corti, but is not required to maintain this cellular mosaic once the production of hair cells and supporting cells is completed. Our results have implications for the proposed used of Notch pathway inhibitors in hearing restoration therapies.