Retinal Regeneration in the Adult Zebrafish Following Intraocular Ouabain Injection

Doctoral Dissertation
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Abstract

We examined the regenerative capacity of the adult zebrafish retina by intravitreal injection of a low ouabain concentration to rapidly damage the ganglion cell layer (GCL) and inner nuclear layer (INL), with minimal photoreceptor cell damage. By 24 hours post-ouabain injection, maximal numbers of TUNEL-positive cells were detected in the INL and GCL, with low numbers of TUNEL-positive cells in the outer nuclear layer (ONL). Immunolabeling revealed nearly 85% of the HuC/D-positive amacrine and ganglion cells were lost by 7 dpi (days post-ouabain injection). This ganglion cell loss was consistent with the small, but statistically significant, decrease in the optic nerve diameter. The regeneration response began within 1 dpi with increased PCNA expression in both the INL and GCL. By 3 dpi, PCNA expression is largely restricted to the MÌller glia. By 5 dpi, most of the PCNA expression was localized to neuronal progenitors expressing the olig2:egfp transgene rather than the MÌller glia. By 7 dpi, the neuronal progenitors began committing to the ganglion cell fate based on the coexpression of the atoh7:EGFP transgene and the zn5 antigen. The regeneration of ganglion and amacrine cells continued until 60 dpi, when they reached 75% of their uninjected control number. This demonstrates that inner retinal damage, without extensive photoreceptor damage, is sufficient to induce a regeneration response that is marked by the MÌller glial cells re-entering the cell cycle to produce neuronal progenitor cells that regenerate INL and ganglion cells in the zebrafish retina. Similar to light-induced retinal regeneration in the adult zebrafish, ouabain-induced damage triggers the regeneration of cell type(s) in a precisely regulated manner which includes, high levels of retinal cell proliferation, progenitor cell migration, and a commitment of the progenitors to differentiate into specific cell types (Vihtelic and Hyde, 2000; Kassen et al., 2007; Fimbel et al., 2007). Therefore, the characterization of the cellular response to the ouabain-induced damage provides the foundation for examining the global transcriptional changes associated with retinal neurogenesis. To identify gene expression changes during the ouabain-induced regeneration response, a microarray analysis was performed at four time points during the ouabain-induced damage time course, which included a time point during INL cell death (24 hrs), a time point during maximal proliferation of MÌller glia (55 hrs), a time point during progenitor cell migration (96 hrs), and a 140 hr time point, which corresponds to the initial stages of ganglion cell differentiation. Mean expression values for every gene were determined relative to the control (3 h saline injected retina) and statistical analysis by one-way ANOVA identified 2,843 genes exhibiting significant changes in gene expression during the time course. Subsequently, genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for several selected genes, which provided the basis for comparisons with the purpose of identifying candidate genes that play critical roles during these selected biological events

Attributes

Attribute NameValues
URN
  • etd-11212007-123954

Author Shane Michael Fimbel
Advisor Dr. David Hyde
Contributor Dr. David Hyde, Committee Chair
Contributor Dr. Lei Li, Committee Member
Contributor Dr. Michael Ferdig, Committee Member
Contributor Dr. Edward Hinchcliffe, Committee Member
Degree Level Doctoral Dissertation
Degree Discipline Biological Sciences
Degree Name PhD
Defense Date
  • 2007-10-31

Submission Date 2007-11-21
Country
  • United States of America

Subject
  • ouabain

  • zebrafish

  • retina

  • regeneration

Publisher
  • University of Notre Dame

Language
  • English

Record Visibility Public
Content License
  • All rights reserved

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