Intermediate filaments of zebrafish retinal and optic nerve astrocytes and Müller glia: differential distribution of cytokeratin and GFAP
© Koke et al; licensee BioMed Central Ltd. 2010
Received: 5 December 2009
Accepted: 1 March 2010
Published: 1 March 2010
Optic nerve regeneration (ONR) following injury is a model for central nervous system regeneration. In zebrafish, ONR is rapid - neurites cross the lesion and enter the optic tectum within 7 days; in mammals regeneration does not take place unless astrocytic reactivity is suppressed. Glial fibrillary acidic protein (GFAP) is used as a marker for retinal and optic nerve astrocytes in both fish and mammals, even though it has long been known that astrocytes of optic nerves in many fish, including zebrafish, express cytokeratins and not GFAP. We used immunofluorescence to localize GFAP and cytokeratin in wild-type zebrafish and transgenic zebrafish expressing green fluorescent protein (GFP) under control of a GFAP promoter to determine the pattern of expression of intermediate filaments in retina and optic nerve.
GFAP labeling and GFAP gene expression as indicated by GFP fluorescence was found only in the Müller glial cells of the retina. Within Müller cells, GFP fluorescence filled the entire cell while GFAP labelling was more restricted in distribution. No GFAP expression was observed in optic nerves. Cytokeratin labeling of astrocytes was observed throughout the optic nerve and less intensely in cells in the retinal inner plexiform layer. The retinal inner limiting membrane was strongly labeled by anti-cytokeratin.
Studies of astrocyte function during ONR in zebrafish cannot solely rely on GFAP as an astrocyte marker or indicator of reactivity. Future studies of ONR in zebrafish should include evaluation of changes in cytokeratin expression and localization in the optic nerve.
Because of the accessibility of the optic nerve, optic nerve regeneration (ONR) is often used for studies of central nervous system regeneration. In fish, typified by zebrafish, regeneration of the optic nerve after injury by crushing or transectioning is rapid with new neurites crossing the lesion and entering the optic tectum in as few as 7 days . In mammals, typified by mice, regeneration does not take place in the absence of specific molecular interventions and suppression of astrocyte reactivity in the optic nerve [2, 3] (for a recent review, see ).
As part of an ongoing study of ONR in zebrafish , we examined intermediate filament (IF) expression of astrocytes in the zebrafish retina and optic nerve. Many previous studies have used the type III IF glial fibrillary acidic protein (GFAP) as a marker for retinal and optic nerve astrocytes, both in fish and mammals, even though it has been known for some time that astrocytes of optic nerves in many fish, including zebrafish, express cytokeratins rather than GFAP [6, 7]. A possible exception are astrocytes of goldfish optic nerve, which, as reported by Nona et al, appear GFAP positive both before and after optic nerve injury.
Results and Discussion
The rapid regeneration of optic nerve in zebrafish as compared non-regeneration in mammals (who express GFAP in their optic nerves) suggests that GFAP itself is non-permissive to axonal regeneration. In mammals, reactive astrogliosis that includes upregulation of GFAP and vimentin provides a neuroprotective effect, particularly in a stroke model. Li et al.  found transection of the middle cerebral artery in Gfap(-/-) Vim(-/-) mice generated an infarct that was 210% to 350% larger than in wild type mice. They also report that Gfap(-/-) Vim(-/-) mice show attenuated reactive gliosis and improved post-traumatic regeneration as compared to wild type. In goldfish, Nona et al.  reported the presence of GFAP-positive astrocytes 7 days following an optic nerve crush injury on both proximal and distal to the lesion site; however, the injury site itself remained GFAP-negative, and astrocytes were excluded until after axonal regeneration was complete. Thus one could speculate that the absence of GFAP expression in fish optic nerve contributes to an environment that is permissive to nerve regeneration, but there seems to be no evidence that cytokeratins promote regeneration. In the present study, we found no evidence of increased cytokeratin expression in the injured optic nerve as compared to the uninjured. This result is consistent with previous studies of cytokeratin expression during optic nerve regeneration by Fuchs et al. (1994) in goldfish, where no changes in mRNA expression for the goldfish optic nerve cytokeratins GK48 and GK49 were found 10 days post-injury.
On the basis of these results, it appears that if Müller glial cells can be considered astrocytes, zebrafish have two populations of astrocytes in their retina, the GFAP expressing Müller cells, and the cytokeratin expressing reticular astrocytes that appear to extend into the retina from the optic nerve, forming the inner limiting membrane and contributing to the bundled nerve fiber and inner plexiform layers. According to Watanabe and Raff , a similar situation exists in mammalian retina with respect to non-Müller astrocytes entering the retina from the optic nerve along retinal vasculature, and in the mature retina, locating near the retinal vasculature and nerve fiber layer (although mammalian astrocytes do express GFAP and not cytokeratin). Because of the apparent absence of GFAP expression by any cell type in the zebrafish optic nerve - either injured or uninjured - studies of the role astrocytes may play during ONR in zebrafish cannot rely on GFAP as an marker for astrocytes or an indicator of reactivity. Future studies of ONR in zebrafish should include evaluation of changes in cytokeratin expression and localization in the optic nerve.
The authors acknowledge the contributions of undergraduate Kyle Henry, who performed the anti-GFAP labeling. This work was made possible by NSF grants IOB-0615762 to DMG and DBI-0821252 to JRK and DMG.
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