culture-adult-transgenic-zebrafish-retinal-explants-for-live-cell

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

University of Notre Dame

Acoustic overstimulation and ototoxic drugs can cause permanent hearing loss as a result of the damage and death of cochlear hair cells. Relatively little is known about the signaling pathways triggered by such trauma, although a significant role has been described for the c-Jun N-terminal kinase [one of the mitogen-activated protein kinases (MAPKs)] pathway. We investigated the role of another MAPK family, the extracellularly regulated kinases 1 and 2 (ERK1/2) during hair cell damage in neonatal cochlear explants. Within minutes of subjecting explants to mechanical damage, ERK1/2 were transiently activated in Deiters' and phalangeal cells but not in hair cells. The activation of ERK1/2 spread along the length of the cochlea, reaching its peak 5-10 min after damage onset. Release of extracellular ATP and the presence of functional connexin proteins were critical for the activation and spread of ERK1/2. Damage elicited an intercellular Ca(2+) wave in the hair cell region in the first seconds after damage. In the absence of Ca(2+) influx, the intercellular Ca(2+) wave and the magnitude and spread of ERK1/2 activation were reduced. Treatment with the aminoglycoside neomycin produced a similar pattern of ERK1/2 activation in supporting cells surrounding pyknotic hair cells. When ERK1/2 activation was prevented, there was a reduction in the number of pyknotic hair cells. Thus, activation of ERK1/2 in cochlear supporting cells in vitro is a common damage signaling mechanism that acts to promote hair cell death, indicating a direct role for supporting cells in regulating hair cell death.

Damage-induced activation of ERK1/2 in cochlear supporting cells is a hair cell death-promoting signal that depends on extracellular ATP and calcium.

Intercellular Ca(2+) waves can coordinate the action of large numbers of cells over significant distances. Recent work in many different systems has indicated that the release of ATP is fundamental for the propagation of most Ca(2+) waves. In the organ of hearing, the cochlea, ATP release is involved in critical signalling events during tissue maturation. ATP-dependent signalling is also implicated in the normal hearing process and in sensing cochlear damage. Here, we show that two distinct Ca(2+) waves are triggered during damage to cochlear explants. Both Ca(2+) waves are elicited by extracellular ATP acting on P2 receptors, but they differ in their source of Ca(2+), their velocity, their extent of spread and the cell type through which they propagate. A slower Ca(2+) wave (14 mum/s) communicates between Deiters' cells and is mediated by P2Y receptors and Ca(2+) release from IP(3)-sensitive stores. In contrast, a faster Ca(2+) wave (41 mum/s) propagates through sensory hair cells and is mediated by Ca(2+) influx from the external environment. Using inhibitors and selective agonists of P2 receptors, we suggest that the faster Ca(2+) wave is mediated by P2X(4) receptors. Thus, in complex tissues, the expression of different receptors determines the propagation of distinct intercellular communication signals.

Damage-induced cell-cell communication in different cochlear cell types via two distinct ATP-dependent Ca waves.

Retinal damage in teleosts, unlike mammals, induces robust Müller glia-mediated regeneration of lost neurons. We examined whether Notch signaling regulates Müller glia proliferation in the adult zebrafish retina and demonstrated that Notch signaling maintains Müller glia in a quiescent state in the undamaged retina. Repressing Notch signaling, through injection of the γ-secretase inhibitor RO4929097, stimulates a subset of Müller glia to reenter the cell cycle without retinal damage. This RO4929097-induced Müller glia proliferation is mediated by repressing Notch signaling because inducible expression of the Notch Intracellular Domain (NICD) can reverse the effect. This RO4929097-induced proliferation requires Ascl1a expression and Jak1-mediated Stat3 phosphorylation/activation, analogous to the light-damaged retina. Moreover, coinjecting RO4929097 and TNFα, a previously identified damage signal, induced the majority of Müller glia to reenter the cell cycle and produced proliferating neuronal progenitor cells that committed to a neuronal lineage in the undamaged retina. This demonstrates that repressing Notch signaling and activating TNFα signaling are sufficient to induce Müller glia proliferation that generates neuronal progenitor cells that differentiate into retinal neurons, mimicking the responses observed in the regenerating retina.

Repressing notch signaling and expressing TNFα are sufficient to mimic retinal regeneration by inducing Müller glial proliferation to generate committed progenitor cells.

In the adult zebrafish, death of retinal neurons stimulates Müller glia to re-enter the cell cycle to produce neuronal progenitor cells (NPCs) that undergo further cell divisions and differentiate to replace lost neurons in the correct spatial locations. Understanding the mechanisms regulating retinal regeneration will ultimately provide avenues to overcome vision loss in human. Recently, the observation of interkinetic nuclear migration (INM) of Müller glia in the regenerating zebrafish retina resulted in the inclusion of an additional complex step to the regeneration process. The pathways regulating INM and its function in the regenerating retina have not been well studied. Here, we summarize the evidence for INM in the regenerating retina and review mechanisms that control INM during neuro-epithelial development in the context of pathways known to be critical during retinal regeneration.

Interkinetic Nuclear Migration in the Regenerating Retina.

Loss of retinal neurons in adult zebrafish (Danio rerio) induces a robust regenerative response mediated by the reentry of the resident Müller glia into the cell cycle. Upon initiating Müller glia proliferation, their nuclei migrate along the apicobasal axis of the retina in phase with the cell cycle in a process termed interkinetic nuclear migration (INM). We examined the mechanisms governing this cellular process and explored its function in regenerating the adult zebrafish retina. Live-cell imaging revealed that the majority of Müller glia nuclei migrated to the outer nuclear layer (ONL) to divide. These Müller glia formed prominent actin filaments at the rear of nuclei that had migrated to the ONL. Inhibiting actin filament formation or Rho-associated coiled-coil kinase (Rock) activity, which is necessary for phosphorylation of myosin light chain and actin myosin-mediated contraction, disrupted INM with increased numbers of mitotic nuclei remaining in the basal inner nuclear layer, the region where Müller glia typically reside. Double knockdown of Rho-associated coiled-coil kinase 2a (Rock2a) and Rho-associated coiled-coil kinase 2b (Rock2b) similarly disrupted INM and reduced Müller glial cell cycle reentry. In contrast, Rock inhibition immediately before the onset of INM did not affect Müller glia proliferation, but subsequently reduced neuronal progenitor cell proliferation due to early cell cycle exit. Long-term, Rock inhibition increased the generation of mislocalized ganglion/amacrine cells at the expense of rod and cone photoreceptors. In summary, INM is driven by an actin-myosin-mediated process controlled by Rock2a and Rock2b activity, which is required for sufficient proliferation and regeneration of photoreceptors after light damage.

Article	Year
Damage-induced activation of ERK1/2 in cochlear supporting cells is a hair cell death-promoting signal that depends on extracellular ATP and calcium. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 18463245	2008
Damage-induced cell-cell communication in different cochlear cell types via two distinct ATP-dependent Ca waves. Purinergic signalling\| PubMed ID: 20806011	2010
Repressing notch signaling and expressing TNFα are sufficient to mimic retinal regeneration by inducing Müller glial proliferation to generate committed progenitor cells. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 25339752	2014
Interkinetic Nuclear Migration in the Regenerating Retina. Advances in experimental medicine and biology\| PubMed ID: 26427463	2016
Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 26609156	2015

Manuela Lahne

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