Biomedical subjects

Marcelo N Rivolta

Publications and source records attributed to Marcelo N Rivolta.

7 recordsLinked to original sources

PubMed2006

Generation of inner ear cell types from embryonic stem cells.

The senses of hearing and balance are mediated by hair cells located in the cochlea and in the vestibular organs of the vertebrate inner ear. Loss of hair cells and other cell types of the inner ear results in hearing and balance disorders that substantially diminish the quality of life. The irreversibility of hearing loss in mammals is caused by the inability of the cochlea to replace lost hair cells. No drugs are available that stimulate inner ear cell regeneration. We describe here protocols to generate inner ear progenitor cells from murine ES cells and to differentiate these progenitors into hair cells and potentially into other inner ear cell types. In addition, we provide a modification of the protocol describing culture conditions in which human ES cells express a similar set of inner ear markers. Inner ear progenitor cells, generated from ES cells, may be used for the development of cell replacement therapy for the diseased inner ear, for high-throughput drug screening, and for the study of inner ear development.

Animals↗

PubMed2004

GATA3 and NeuroD distinguish auditory and vestibular neurons during development of the mammalian inner ear.

The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expressed GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD and that migrate from the antero-ventral, otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3.

Animals↗

PubMed2002

Cell lines in inner ear research.

Cell lines have provided important experimental tools that have enhanced our understanding of neural and sensory function. They are particularly valuable in inner ear research because the auditory and vestibular systems are small, complex, and encased in several layers of bone. Organotypic cultures provide an invaluable experimental resource but require repeated microdissection and culture, and remain complex in terms of cell types and states of differentiation. A number of laboratories have established cell lines that offer a range of potential applications to hearing research. This review describes the advances that have already been made with these lines and the potential applications that they offer in the future. The majority of the cell lines are immortalized with a conditionally expressed, temperature sensitive variant of the SV40 tumor antigen. We discuss the value of these cells in developmental studies.

Animals↗

PubMed2002

E-cadherin and the differentiation of mammalian vestibular hair cells.

E-cadherin is expressed in vestibular, mechanosensory epithelia during early embryonic development. During late embryonic and neonatal stages it is expressed in supporting cells but down-regulated in differentiating sensory hair cells. We used a conditionally immortal cell line (UB/UE-1) from the neonatal mouse utricle to test the hypothesis that constitutive expression of E-cadherin inhibits the progression of hair cell differentiation. Under differentiating culture conditions, transfected E-cadherin inhibited expression of the cytoskeletal protein myosin VIIa and functional expression of both acetylcholine receptors and potassium channels, which are normally expressed by neonatal hair cells. However, it had no effect on the expression of the transcription factor Brn3c or the cytoskeletal protein fimbrin, which are also expressed by neonatal hair cells. The number of adherens junctions increased significantly under differentiating conditions but there was no detectable change in formation of tight junctions or gap junctions. However, E-cadherin expression led to density-dependent cell death under differentiating conditions. We have shown that E-cadherin is expressed in vestibular supporting cells, which form the basis of the sensory epithelium, but that constitutive expression inhibits the full differentiation of hair cells. Down-regulation of E-cadherin is thus likely to be a key element in the regeneration of hair cells.

Acetylcholine↗

PubMed2002

Asymmetric segregation of mitochondria and mortalin correlates with the multi-lineage potential of inner ear sensory cell progenitors in vitro.

The sensory epithelia of the inner ear include hair cells and supporting cells that share a common precursor. One possible mechanism involved in the genesis of these cell types is through asymmetric cell division. In this work we have studied asymmetric division of inner ear sensory cell progenitors in vitro in an attempt to understand how the different cell phenotypes are generated. In the search for molecules that will segregate asymmetrically we have found that mitochondria in general, and a mitochondrial protein named mortalin in particular, are asymmetrically segregated during certain cell divisions. In one conditionally immortal cell line (UB/OC-1), which represents a population of committed hair cell precursors, mortalin is uniformly distributed in the cytoplasm and shared equally between sibling cells during division. In another cell line (UB/UE-1), which represents a bipotent, vestibular supporting cell that can produce both neonatal hair cells as well as supporting cells, mortalin segregates asymmetrically. In UB/UE-1, approximately 12% of the cells display an asymmetric distribution of mortalin and mitochondria. The proportion of asymmetric cells increases immediately after the release of the immortalizing gene and before the onset of differentiation. The asymmetric segregation of mortalin in the bipotent cell line and its uniform distribution in a committed, lineage-restricted cell line raises the possibility that it may play a role in cell fate determination.

Animals↗

PubMed2002

Expression of the transcription factors GATA3 and Pax2 during development of the mammalian inner ear.

The transcription factors GATA3 and Pax2 are expressed throughout development of the mouse inner ear. We have used antibodies to study their temporal and spatial expression patterns from embryonic days E8-E16.5. The two factors show reciprocal relationships in the regional patterning of the early otocyst and cellular patterning within the sensory epithelia. GATA3 is expressed in the whole otic placode at E8. In the otocyst at E9.5-10.5, the distribution is lateral and complementary to the medial expression pattern of Pax2. Only Pax2 is expressed in the endolymphatic duct, but both factors are expressed in the cochlea. At E11.5-13.5, GATA3 is expressed strongly in the cochlea, but in the dorsal, vestibular region it is downregulated. In all sensory epithelia, downregulation coincides with sensory innervation. Pax2 is expressed in all sensory and some nonsensory epithelia, but within sensory epithelia at E16.5 it is restricted to hair cells. GATA3 is expressed throughout key periods of cell proliferation, fate determination, and differentiation and is not specifically associated with any of these processes. Expression persists most strongly in the main components of the developing auditory system. These include the auditory sensory epithelium, the afferent and efferent nerves, and the mesenchymal and ectodermal cells in regions that form key parts of the middle and outer ear. GATA3 is thus expressed in functionally distinct groups of cells that integrate to form a complete sensory system. The results suggest that both factors may be involved in tissue compartmentalisation, morphogenesis, and cell signalling.

Aging↗

PubMed2002

Transcript profiling of functionally related groups of genes during conditional differentiation of a mammalian cochlear hair cell line.

We have used Affymetrix high-density gene arrays to generate a temporal profile of gene expression during differentiation of UB/OC-1, a conditionally immortal cell line derived from the mouse cochlea. Gene expression was assessed daily for 14 days under differentiating conditions. The experiment was replicated in two separate populations of cells. Profiles for selected genes were correlated with those obtained by RT-PCR, TaqMan analysis, immunoblotting, and immunofluorescence. The results suggest that UB/OC-1 is derived from a population of nonsensory epithelial cells in the greater epithelial ridge that have the potential to differentiate into a hair-cell-like phenotype, without the intervention of Math1. Elements of the Notch signaling cascade were identified, including the receptor Notch3, with a transient up-regulation that suggests a role in hair cell differentiation. Several genes showed a profile similar to Notch3, including the transcriptional co-repressor Groucho1. UB/OC-1 also expressed Me1, a putative partner of Math1 that may confer competence to differentiate into hair cells. Cluster analysis revealed expression profiles for neural guidance genes associated with Gata3. The temporal dimension of this analysis provides a powerful tool to study genetic mechanisms that underlie the conversion of nonsensory epithelial cells into hair cells.

Animals↗