Because we are interested in identifying the transcriptional regulatory networks that regulate photoreceptor regeneration in zebrafish, we chose to study further three transcription factors, with previously described roles in neurogenesis, that demonstrated increased expression in XOPS-mCFP retinas. These include the zebrafish homologues of
insm1, a zinc-finger transcription factor associated with neuroendocrine development and neurogenesis
27,42 ;
sox11, a transcription factor involved in the regulation of embryonic development and neural cell fate determination
19,23 ; and c-
myb, a transcription factor best known for its role in regulating hematopoiesis but which has also been linked to maintenance of neural stem cells.
15 In situ hybridization analysis demonstrated that in wild-type zebrafish all three genes are expressed in the CMZ, suggesting that they are involved in persistent neurogenesis in the adult retina. Furthermore, expression of all three genes was localized to the base of the ONL in the XOPS-mCFP retinas, where mitotic rod progenitor cells and postmitotic rod precursors have been shown to reside.
6,9 Double in situ hybridization analysis indicated that some
sox11b +,
insm1a +, and c-
myb + cells co-localized with the proliferation marker PCNA, although this result is not conclusive because of the technical limitations of the colorimetric in situ assay. In single in situ hybridization experiments,
sox11b and
insm1a demonstrated the most widespread expression in the ONL of XOPS-mCFP retinas. This may suggest that they are expressed in both mitotic and postmitotic rod progenitors. Indeed, previous studies have suggested a role for
insm1 and
sox11 in regulating progenitor cell cycle exit and the establishment of neuronal identity, respectively
43 –45 ; therefore, it is possible that they act in a similar fashion to regulate rod photoreceptor regeneration. Interestingly, both c-
myb and to some extent
insm1a displayed an increase in expression in cells that had a Müller glial morphology in XOPS-mCFP retinas. At this point the functional significance of this expression pattern is unclear and must be confirmed using Müller-glial–specific markers in adult retinal sections (although we did observe co-localization of c-
myb-GFP with the Müller glial antibody Zrf-1 at 5 dpf). We have demonstrated that Müller glia do not proliferate or show signs of reactive gliosis in XOPS-mCFP retinas.
6,9 The increase in expression of c-
myb and
insm1a in a subset of Müller glia may be associated with a proliferation-independent signaling pathway in response to chronic rod degeneration. Alternatively, we cannot rule out the possibility that c-
myb and
insm1a are expressed in a subset of Müller glia proliferating at a much slower rate than is observed in response to acute retinal damage, below the threshold of detection in our assays.