Limits...
Changes in the regulation of the Notch signaling pathway are temporally correlated with regenerative failure in the mouse cochlea.

Maass JC, Gu R, Basch ML, Waldhaus J, Lopez EM, Xia A, Oghalai JS, Heller S, Groves AK - Front Cell Neurosci (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Baylor College of Medicine Houston, TX, USA ; Department of Otolaryngology, Hospital Clínico Universidad de Chile Santiago, Chile ; Interdisciplinary Program of Physiology and Biophysics, ICBM Universidad de Chile Santiago, Chile ; Department of Otolaryngology, Clínica Alemana de Santiago, Facultad de Medicina Clínica Alemana-Universidad del Desarrollo Santiago, Chile.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Notch pathway components are down-regulated during the first postnatal week. In situ hybridization of Notch pathway genes in the cochlea at 0, 3 and 6 postnatal days (P0, P3 and P6). Left panels: Whole mount in situs of cochlear explants, with the samples curved clockwise from apex to base. Scale = 200 μm. Right panels: In situ hybridization of frozen sections; a: apex region, b: basal region. Scale 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4379755&req=5

Figure 4: Notch pathway components are down-regulated during the first postnatal week. In situ hybridization of Notch pathway genes in the cochlea at 0, 3 and 6 postnatal days (P0, P3 and P6). Left panels: Whole mount in situs of cochlear explants, with the samples curved clockwise from apex to base. Scale = 200 μm. Right panels: In situ hybridization of frozen sections; a: apex region, b: basal region. Scale 50 μm.

Mentions: The preceding results suggest that the Notch pathway is deployed to stabilize supporting cell fate of neonatal cochlear supporting cells, but that inhibition of the Notch pathway has no effect on supporting cell fate even a few days later. To determine out if this change in the response of supporting cells to Notch inhibition was related to changes in the endogenous activity of the Notch pathway, we examined the expression of mRNA for Notch receptors (Notch1 and Notch3), ligands (Dll1, Jag1 and Jag2) and downstream effectors of Notch signaling (Hey1, Hey2, HeyL and Hes5) in the cochlea from P0 to P6 by in situ hybridization on whole mount cochleas, sectioned cochleas and by Q-PCR of cochlear tissue (Figures 4, 5A). In general, all components of the Notch pathway evinced a down-regulation between P0 and P6 starting at the base and proceeding down to the apex. Specifically, Notch1 and Notch3 were expressed throughout the supporting cell layer and into Kölliker’s organ and the outer sulcus, and both receptors showed a basal-apical down-regulation between P0 and P6. Jag2 and Dll1 were both down-regulated in hair cells between P0 and P6, along with the hair cell marker Atoh1. Hey2 and Hes5 were down-regulated from pillar cells and Deiters’ cells respectively in a basal-apical gradient, whereas Hey1, HeyL and Jag1 were expressed in all supporting cells and cells of Kölliker’s organ, and down-regulated again in a basal-apical direction. The speed of down-regulation varied considerably from gene to gene—for example, Dll1 was down-regulated in hair cells more quickly than Jag2, and Hes5 was down-regulated much more quickly in supporting cells than Hey1. We also saw a general trend towards down-regulation of each gene by Q-PCR (Figure 5A), although the degree of down-regulation measured by this method was somewhat blunted as a result of including the entire basal-apical extent of the cochlear duct in each sample. To confirm that activation of the Notch1 receptor was also decreasing between P0 and P6, we immunostained cochlear sections with antibodies to the Notch1 intracellular domain (Notch1-ICD) which is released and localized to the nucleus after Notch activation (Figure 5B). We observed Notch1-ICD staining in Deiters’ cells at P0, but could not detect staining in the supporting cells at later stages.


Changes in the regulation of the Notch signaling pathway are temporally correlated with regenerative failure in the mouse cochlea.

Maass JC, Gu R, Basch ML, Waldhaus J, Lopez EM, Xia A, Oghalai JS, Heller S, Groves AK - Front Cell Neurosci (2015)

Notch pathway components are down-regulated during the first postnatal week. In situ hybridization of Notch pathway genes in the cochlea at 0, 3 and 6 postnatal days (P0, P3 and P6). Left panels: Whole mount in situs of cochlear explants, with the samples curved clockwise from apex to base. Scale = 200 μm. Right panels: In situ hybridization of frozen sections; a: apex region, b: basal region. Scale 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379755&req=5

Figure 4: Notch pathway components are down-regulated during the first postnatal week. In situ hybridization of Notch pathway genes in the cochlea at 0, 3 and 6 postnatal days (P0, P3 and P6). Left panels: Whole mount in situs of cochlear explants, with the samples curved clockwise from apex to base. Scale = 200 μm. Right panels: In situ hybridization of frozen sections; a: apex region, b: basal region. Scale 50 μm.
Mentions: The preceding results suggest that the Notch pathway is deployed to stabilize supporting cell fate of neonatal cochlear supporting cells, but that inhibition of the Notch pathway has no effect on supporting cell fate even a few days later. To determine out if this change in the response of supporting cells to Notch inhibition was related to changes in the endogenous activity of the Notch pathway, we examined the expression of mRNA for Notch receptors (Notch1 and Notch3), ligands (Dll1, Jag1 and Jag2) and downstream effectors of Notch signaling (Hey1, Hey2, HeyL and Hes5) in the cochlea from P0 to P6 by in situ hybridization on whole mount cochleas, sectioned cochleas and by Q-PCR of cochlear tissue (Figures 4, 5A). In general, all components of the Notch pathway evinced a down-regulation between P0 and P6 starting at the base and proceeding down to the apex. Specifically, Notch1 and Notch3 were expressed throughout the supporting cell layer and into Kölliker’s organ and the outer sulcus, and both receptors showed a basal-apical down-regulation between P0 and P6. Jag2 and Dll1 were both down-regulated in hair cells between P0 and P6, along with the hair cell marker Atoh1. Hey2 and Hes5 were down-regulated from pillar cells and Deiters’ cells respectively in a basal-apical gradient, whereas Hey1, HeyL and Jag1 were expressed in all supporting cells and cells of Kölliker’s organ, and down-regulated again in a basal-apical direction. The speed of down-regulation varied considerably from gene to gene—for example, Dll1 was down-regulated in hair cells more quickly than Jag2, and Hes5 was down-regulated much more quickly in supporting cells than Hey1. We also saw a general trend towards down-regulation of each gene by Q-PCR (Figure 5A), although the degree of down-regulation measured by this method was somewhat blunted as a result of including the entire basal-apical extent of the cochlear duct in each sample. To confirm that activation of the Notch1 receptor was also decreasing between P0 and P6, we immunostained cochlear sections with antibodies to the Notch1 intracellular domain (Notch1-ICD) which is released and localized to the nucleus after Notch activation (Figure 5B). We observed Notch1-ICD staining in Deiters’ cells at P0, but could not detect staining in the supporting cells at later stages.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Baylor College of Medicine Houston, TX, USA ; Department of Otolaryngology, Hospital Clínico Universidad de Chile Santiago, Chile ; Interdisciplinary Program of Physiology and Biophysics, ICBM Universidad de Chile Santiago, Chile ; Department of Otolaryngology, Clínica Alemana de Santiago, Facultad de Medicina Clínica Alemana-Universidad del Desarrollo Santiago, Chile.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus