RESUMEN
This study is the first to demonstrate that macrophage migration inhibitory factor (MIF), an immune system 'inflammatory' cytokine that is released by the developing otocyst, plays a role in regulating early innervation of the mouse and chick inner ear. We demonstrate that MIF is a major bioactive component of the previously uncharacterized otocyst-derived factor, which directs initial neurite outgrowth from the statoacoustic ganglion (SAG) to the developing inner ear. Recombinant MIF acts as a neurotrophin in promoting both SAG directional neurite outgrowth and neuronal survival and is expressed in both the developing and mature inner ear of chick and mouse. A MIF receptor, CD74, is found on both embryonic SAG neurons and adult mouse spiral ganglion neurons. Mif knockout mice are hearing impaired and demonstrate altered innervation to the organ of Corti, as well as fewer sensory hair cells. Furthermore, mouse embryonic stem cells become neuron-like when exposed to picomolar levels of MIF, suggesting the general importance of this cytokine in neural development.
Asunto(s)
Oído Interno/embriología , Oxidorreductasas Intramoleculares/fisiología , Factores Inhibidores de la Migración de Macrófagos/fisiología , Factores de Crecimiento Nervioso/fisiología , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Oído Interno/efectos de los fármacos , Oído Interno/crecimiento & desarrollo , Oído Interno/metabolismo , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Oxidorreductasas Intramoleculares/farmacología , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Factores Inhibidores de la Migración de Macrófagos/farmacología , Ratones , Ratones Noqueados , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Órgano Espiral/embriología , Órgano Espiral/crecimiento & desarrollo , Órgano Espiral/metabolismo , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/crecimiento & desarrollo , Ganglio Espiral de la Cóclea/metabolismoRESUMEN
Inner ear sensory hair cells (HCs), supporting cells (SCs), and sensory neurons (SNs) are hypothesized to develop from common progenitors in the early embryonic otocyst. Because little is known about the molecular signals that control this lineage specification, we derived a model system of early otic development: conditionally immortalized otocyst (IMO) cell lines from the embryonic day 9.5 Immortomouse. This age is the earliest stage at which the otocyst can easily be separated from surrounding mesenchymal, nervous system, and epithelial cells. At 9.5 days post coitum, there are still pluripotent cells in the otocyst, allowing for the eventual identification of both SN and HC precursors--and possibly an elusive inner ear stem cell. Cell lines derived from primitive precursor cells can also be used as blank canvases for transfections of genes that can affect lineage decisions as the cells differentiate. It is important, therefore, to characterize the "baseline state" of these cell lines in as much detail as possible. We characterized seven representative "precursor-like" IMO cell populations and the uncloned IMO cells, before cell sorting, at the molecular level by polymerase chain reaction (PCR) and immunocytochemistry (IHC), and one line (IMO-2B1) in detail by real-time quantitative PCR and IHC. Many of the phenotypic markers characteristic of differentiated HCs or SCs were detected in IMO-2B1 proliferating cells, as well as during differentiation for up to 30 days in culture. These IMO cell lines represent a unique model system for studying early stages of inner ear development and determining the consequences of affecting key molecular events in their differentiation.