Page 57 - Read Online

Page 102 Perkins. J Transl Genet Genom 2022;6:95-110 https://dx.doi.org/10.20517/jtgg.2021.47

[120]
connecting cilium , while some isoforms localize to the outer segments and other subcellular structures,
[121]
including the nucleus . RPGRIP1 is proposed to anchor RPGR at the connecting cilium where they likely
[122]
function within the ciliary transition zone to regulate protein trafficking . A zebrafish rpgrip1 mutant was
made by ENU mutagenesis . The zebrafish rpgrip1 contains 1342 amino acids and the mutation
[123]
introduced a stop codon at amino acid 736 (Q736X). At 7 dpf, rod outer segments failed to form in rpgrip1
mutants, although cones were unaffected and the disk membranes appeared normal. Rhodopsin was
mislocalized throughout the inner segments of the rpgrip1 mutant rods. Rods degenerated rapidly and only
a few rod nuclei were present by 3 mpf. By 6 mpf, cone degeneration was apparent. Rod degeneration
preceded the loss of cones, which is consistent with an RP model with rod-cone dystrophy rather than the
cone-rod dystrophy seen in humans. By 13 mpf, both rods and cones had degenerated and few
photoreceptor nuclei could be found within the presumptive ONL. Cell proliferation was not reported for
the rpgrip1 mutant so future studies may explore whether rod precursors attempt to regenerate dying rods
and whether the rapid degeneration triggers a response from Müller cells.

RHODOPSIN
Rhodopsin is the visual pigment of rod photoreceptors that absorbs photons of light to mediate vision.
Rhodopsin is a proto-typical G-protein coupled receptor that binds the light-absorbing chromophore 11-
[124]
cis-retinal within the transmembrane region of the protein . Mutations in the RHODOPSIN (RHO) gene
are responsible for approximately 18%-26% of all adRP cases, which is far more than any other gene [125-127] .
Zebrafish possess two genes that encode rhodopsin, the rh1-1 and rh1-2 genes [128,129] . The protein product of
the rh1-1 gene shares strong homology with other vertebrate RHO proteins and the pattern of expression is
consistent with function as the rod opsin gene. Multiple mutations in the zebrafish rhodopsin gene (rh1-1)
[42]
fl6
were generated by CRISPR/Cas9 mutagenesis . The rho allele encoded an N-terminal nonsense mutation
at amino acid 17 (T17*), which resulted in rod degeneration in homozygous animals as early as 5 dpf.
Heterozygous animals for the rho allele did not exhibit a phenotype, suggesting this mutation caused
fl6
recessive rod degeneration. Two other N-terminal mutations, the rho and rho alleles, encoded in-frame
fl7
fl10
fl7
deletions that disrupted a highly conserved glycosylation sequence. Heterozygous larvae of both the rho
fl10
and rho alleles exhibited rapid rod degeneration by 6 dpf, consistent with a dominant rod degeneration.
fl8
fl9
The rho allele encodes an in-frame deletion in the C-terminus of the protein, while the rho allele results
in a nonsense mutation at amino acid 347 (S347*). Heterozygous larvae of both alleles show loss of rods in
the central retina. In adults, few rod photoreceptors were present and the rod outer segments were missing
in heterozygous animals. These new zebrafish mutants will serve as useful models of both adRP while the
rho allele could serve as model for autosomal recessive RP. Interestingly, the zebrafish cone photoreceptors
fl6
were unaffected by the loss of rods. This differs from humans with RP-causing mutations in RHO, where
rod degeneration results in the indirect death of cones. Future work may uncover novel mechanisms that
permit cone survival in the absence of rods and reveal potential targets for therapies to preserve cones in
patients with RP.

Transgenic zebrafish models of retinal degeneration
Tg(Xla.Rho:GAP-CFP)q13Tg
The first report of a model with rod-specific degeneration described a transgenic line of zebrafish that
expresses a membrane-targeted cyan fluorescent protein (mCFP) using a 5.5 kb promoter sequence from
the Xenopus laevis rhodopsin gene [130,131] . The mCFP protein included an N-terminal palmitoylation signal
sequences from neuromodulin (GAP-43), which targets proteins to the plasma membrane . This was
[132]
originally named the Tg(XOPS:mCFP) transgenic line . The Fadool lab had previously generated a
[130]
transgenic line of zebrafish that expressed eGFP from the Xenopus rhodopsin promoter . This
[133]
Tg(XOPS:eGFP) line specifically labeled rods with eGFP and did not result in any deleterious effects on rod
photoreceptors . In the Tg(XOPS:mCFP) line, however, rods exhibited an abnormal morphology shortly
[133]