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Page 104 Perkins. J Transl Genet Genom 2022;6:95-110 https://dx.doi.org/10.20517/jtgg.2021.47

[144]
in mice [141-143] , rats , and frogs [145-148] . These models exhibit progressive rod degeneration and have proven
invaluable toward understanding the mechanisms of degeneration caused by different pathogenic mutations
in the rhodopsin gene. To determine if a zebrafish model of RP could be utilized to study the molecular
signals stimulating regeneration, a construct was made that contained a 1.8 kb fragment of the zebrafish
rhodopsin promoter to drive expression of a mouse rhodopsin carrying the P23H mutation fused to a C-
[149]
terminal Flag tag . The P23H mutation was chosen as it is the most frequent rhodopsin mutation to cause
adRP and was the first mutation identified in humans . The resulting stable transgenic line,
[150]
Tg(rho:Mmu.Rho_P23H-FLAG)uth4Tg expressed the P23H-rhodopsin-FLAG beginning at 3 dpf. The
mutant rhodopsin protein was mislocalized throughout the cell body and synapses in larval animals. Rod
outer segments were shorter in larval stages but cones appeared normal. In adult transgenic animals, very
few rods were observed and the P23H-rhodopsin-FLAG protein was significantly mislocalized in the
remaining rods. Endogenous wild-type rhodopsin was also mislocalized, indicating that expression of the
P23H-rhodopsin had deleterious effects on trafficking of outer segment proteins. Adult transgenic animals
had a 3-fold reduction in the number of nuclei in the ONL, indicating degeneration of rods. Interestingly,
cone inner segments and outer segments were shorter in transgenic animals compared to adults, although
the total number of Zpr1+ cones was unchanged. These results suggested that transgenic expression of a
P23H-rhodopsin mutant causes secondary damage to cones. This differs from the rhodopsin (rh1-1)
knockout zebrafish mutants, which had normal cone morphology . At both 4 mpf and 6 mpf, it was
[42]
noticed that the number of proliferating cells (PCNA+) was considerably higher in the ONL of transgenic
animals compared to wild-type. There was no signs of proliferation within the INL. Cell proliferation was
confirmed by BrdU labeling and many of the BrdU+ cells also expressed rhodopsin, indicating that the
newly post-mitotic cells were rods. Together, these results demonstrate that rod degeneration caused by
expression of a P23H-rhodopsin transgene can initiate a regeneration response from rod precursors but not
from Müller glia.

PERSPECTIVES
The mutants and transgenic models described herein represent a variety of human retinal dystrophy
conditions, ranging from ciliopathies to RP to cone-rod dystrophy. As zebrafish also possesses the capacity
to regenerate, it remains unclear why the response to progressive degeneration differs so greatly from acute
damage. Inflammation is believed to be critical for the initial reprogramming of Müller glia , but excess
[151]
inflammation compromises survival of regenerated photoreceptors . Whether chronic inflammation may
[152]
limit Müller glia reprogramming remains unanswered. It is also known that Notch signaling suppresses
regeneration [153,154] . Following acute injury, expression of notch3 is significantly downregulated prior to
Müller glia proliferation. Understanding potential differences in the transcriptional responses of Müller
glia and microglia to disease and injury will hopefully help answer why zebrafish do not regenerate in
[155]
[156]
these IRD models.
CONCLUSION
The zebrafish represents an ideal genetic model to study the pathology of retinal degeneration in a cone-rich
retina. The development of new genetic tools for genome editing allow investigators to target candidate
genes of interest. Indeed, many of the models described herein were generated by genome editing
technologies. The power of unbiased forward genetic screens thrust zebrafish into the scientific mainstream
by uncovering genes essential for vertebrate development and function. In the future, more sophisticated or
sensitive forward genetic screening approaches could be utilized to identify adult-onset phenotypes in a
space- and cost-efficient manner that will reveal even more genes and pathways critical for the health and
survival of photoreceptors.