BACKGROUND
photoreceptor  cells are specialized retinal neurons that efficiently capture light and transduce it into a neural signal.
their intricate and highly specific structure is dependent on the expression of multiple genes, including those involved in pr specification, differentiation and maintenance  <cit> .
indeed, ~ <dig> genes and loci are involved in retinal degeneration in man , and a lower but still substantial number of genes in animals  <cit> .
while mutations in pr-specific or enriched genes are common, and cause a broad spectrum of inherited retinal diseases , an understanding of the mechanistic links between mutation and disease is limited.
moreover, while apoptotic cell death is the final common pathway in most retinal degenerative diseases  <cit> , the rate of degeneration and cell death pathway utilized varies in a disease, mutation and species-specific manner, demonstrating a high level of complexity .
identifying the principal molecular players and providing an in-depth knowledge of the mechanisms that induce and regulate the underlying pr cell death will provide essential insight into disease progression.
this will help to develop pharmacological agents and specific therapies that provide pr protection in the hopes of treating inherited retinopathies.
while stimulating the proliferation of diseased yet functional prs to maintain the pr layer would likely be therapeutically beneficial in some retinal degenerative diseases, the proliferative potential of pr cells is controversial, and not well understood.
prs are terminally differentiated, and undergo their final cell division just prior to cell fate specification; compensatory neurogenesis mechanisms do not exist to replace all dying cells in the normal retina  <cit> .
indeed, it was originally postulated that new prs are generated in naturally occurring retinal diseases, however this hypothesis has been disproved  <cit> .
furthermore, a recent comprehensive analysis of different rodent retinal mutants, along with a light-induced retinal degeneration model, clearly demonstrated that reactivation of the expression of cell cycle genes did not correlate with pr cell division as determined by ethynyl deoxyuridine incorporation and phospho histone h <dig>  labeling.
instead, this process was essential to promote the cell death pathways  <cit> .
in contrast, under some specific circumstances  <cit> , a limited number of prs and other neurons can be generated from presumably terminally differentiated müller cells that dedifferentiate, proliferate and express neuronal progenitor markers in the adult rat, mouse as well as chicken and fish .
in contrast to normal prs and other disease models, we recently reported that a mutation in the ndr family kinase stk38l  found in the erd canine model results in a period of sustained pr proliferation, and that newly generated cells are unique hybrid rod/s-cones  <cit> , and did not originate from resident retinal stem cells.
the stk38l mutation eliminates the binding sites for regulatory proteins s100b and mob, and part of the n-terminal regulatory region that is highly conserved in all ndr subclass of agc protein kinases  <cit> .
ndr kinases, including lats <dig>  interact with the hippo pathway through mob <dig> binding to regulate aspects of cell growth, metabolism, proliferation and survival  <cit> .
thus, we hypothesize that terminally differentiated normal prs are kept from dividing by ndr2-mob <dig> interaction, and removing this control in mutants allows the cell to re-enter the cell cycle and divide  <cit> .
in the present study, we examined whether pr proliferation may also occur in other early-onset inherited retinal diseases to determine if common molecular pathways were involved.
in addition to erd, where no equivalent disease has been reported in man  <cit> , two other early onset canine diseases with comparable cell death kinetics and histopathology were examined: x-linked progressive retinal atrophy  <dig>  and rod cone dysplasia  <dig> , which are caused, respectively, by mutations in rpgrorf <dig>  <cit>  and pde6b  <cit> .
both diseases bear mutations in genes that cause human inherited blindness, and the disease phenotypes are similar and comparable.
in all three diseases, the early and rapid degeneration of the prs makes the disease course predictable and highly suitable for comparative studies of the involved events.
however, the exact mechanisms by which mutations in these genes drive the degeneration events are currently unknown.
to this end, we examined the retinal and retinal pigment epithelium  expression of selected genes and proteins that are involved in cell cycle regulation, or belong to the ndr protein-kinase family and the hippo pathway  <cit> ;  <cit> .
notably, our results indicate that pr proliferation also occurred in xlpra <dig> and rcd <dig>  but that formation of hybrid rod/s-cones is unique to erd.
furthermore, we demonstrate a concurrent dysregulation of critical cell cycle genes that were differentially expressed  in all three diseases, while hippo pathway genes were more specifically altered in erd.
RESULTS
morphology of early-onset canine retinal degeneration models
we initially characterized the retinal morphology of the  <dig> early-onset disease models that generally have a similar pattern of pr development and degeneration .
although overall retinal development is initially normal , there were differences in the subsequent rates and kinetics of pr degeneration; retinal degeneration started at different ages and occurred more rapidly in rcd <dig>  where rod pr development was abnormal, and outer segments were sparse, failed to elongate, and inner segments were short already at  <dig> wks.
the disease is slightly more delayed in xlpra <dig>  while erd showed preservation of the onl thickness until at least  <dig>  wks.fig.
<dig> age-dependent structural changes in normal and mutant retinas.
disease occurs earlier and progresses more rapidly in rcd <dig>  while it is slightly delayed in xlpra <dig>  the outer nuclear layer  in erd is preserved during the time course of the study.
scale bar: 20 μm; rpe = retinal pigment epithelium, pr = photoreceptors, onl = outer nuclear layer, opl = outer plexiform layer, inl = inner nuclear layer, ipl = inner plexiform layer, gcl = ganglion cell layer
photoreceptor cell proliferation in mutant retinas
to determine if pr proliferation was exclusive to erd-mutants, we used phh <dig> and pcna labeling to examine pr mitosis in the onl of additional early-onset disease models.
phh <dig> is a specific marker for mitotic cells in the late g <dig> and m-phases  <cit> , while pcna labels both cells undergoing proliferation and dna repair  <cit> .
the number of labeled cells/ <dig> million μm <dig> of onl was analyzed at different time points between  <dig> and  <dig> wks.
the results showed similar trends for both phh <dig> and pcna labeling in the different models and in normals , although the number of pcna-positive cells was lower than the number of phh3-positive cells at every time point examined.
in addition to labeling different phases of the cell cycle, the lower pcna results suggest that there is limited ongoing dna damage and repair.
minimal numbers of phh3-positive cells were found in normal retinas after  <dig> wks of age; these were located adjacent to the external limiting membrane and limited to the retinal periphery.fig.
<dig> photoreceptor cell proliferation in the outer nuclear layer of normal and mutant retinas.
a phh3-labeling was used to identify mitotic cells, and b pcna-labeling to identify cells undergoing proliferation and dna repair.
normal retinas exhibited essentially no mitotic cells after  <dig> wks of age, while they were present in the onl of the  <dig> diseased retinas.
mitotic cells were continually present throughout the first 14– <dig> wks of life in all  <dig> diseases, with the highest numbers observed in erd-mutants.
the pcna data for erd has been published previously .
data points are expressed as mean of triplicates ± sd.
note the difference in the scale of the y-axes between erd  and normal, xlpra <dig> and rcd <dig>
as previously shown with pcna labeling , phh3-labeled mitotic prs were found at ≥ <dig>  wks in erd, and these levels were sustained, ranging from >50– <dig> cells/ <dig> million μm <dig> of onl.
in contrast, pr proliferation peaked at  <dig> wks of age in rcd <dig> and at  <dig> wks in xlpra <dig>  these time points precede  or approximately coincide  the peaks of cell death for each disease .
however, regardless of the disease, the number of phh3-labeled cells in mutants far exceeded the numbers in normals, and was sustained for the time period analyzed.
for example, at ~ <dig> wks of age the mutants had 7– <dig> times the number of dividing cells than controls, and these increased to 12– <dig> times at ~ <dig> wks.
we examined the association between cells undergoing mitosis , and those in mitotic checkpoint arrest  to exclude the possibility that entry into the late g2-m phase of the cell cycle preceded apoptosis.
to this end, we compared control  and mutant retinas at time points with high rates of pr mitosis in mutants: xlpra2 =  <dig> wks, rcd1 =  <dig> wks, erd =  <dig>  wks.
in the control, the phh <dig> labeled onl nuclei also labeled with mad2; overall, the number of labeled cells with each antibody was low, and the phh3/mad <dig> ratio per  <dig> million μm <dig> of onl was  <dig>  in contrast, the phh3/mad <dig> ratio in the mutants was much higher , and smaller number of nuclei/ <dig> million μm <dig> of onl co-expressed both proteins .
this indicates that pr mitosis is occurring in onl, and the dividing cells contribute to maintenance of the onl.
to control for the possibility that the same pr cells were not undergoing concurrent apoptosis and mitotic division, we performed dual-labeling with tunel and phh <dig> at the ages that correspond to the peaks of cell death for each disease  <cit> .
the results indicate that with few exceptions the majority of cells were positive for either tunel or phh <dig>  but not for both, an indication that different cell populations in the onl were undergoing either apoptosis or mitosis .
co-localization of tunel and phh <dig> labeling was rare and limited to few cells  in the erd model.
these results establish that proliferating and degenerating cells in the onl are different.fig.
<dig> phh <dig> labeling identifies mitotic cells in the outer nuclear layer.
a combined phh <dig>  and tunel  labeling in normal , xlpra <dig> , rcd <dig>  and erd  retinas at the peak of cell death for each disease  <cit> .
there are no tunel or phh <dig> labeled cells in the normal retina.
in the mutants, some of the red  and green  labeled nuclei in onl are identified.
note that in erd , two cells are undergoing combined proliferation and apoptosis .
b cd <dig> labeling  shows microglial cells and processes distributed primarily in the opl and ipl, with increased expression in xlpra <dig> , rcd <dig>  and erd  compared to normal .
phh <dig> labeling in the mutants shows mitotic cells in onl .
the two dividing cells in the opl  are of unknown origin and are not counted in the analysis.
c glutamine synthetase  labeling in normal , and dual phh <dig> and gs labeling in xlpra <dig> , rcd <dig>  and erd .
dividing cells in onl  are not labeled with gs.
note that in xlpra <dig> and rcd <dig> there are phh3-positive nuclei in the inner border of the inl .
d confocal microscopy image of dual-immunolabeling of retinal sections with phh <dig>  and rod opsin  in the onl of xlpra <dig> , rcd <dig> , and erd .
the delocalized rod opsin associated with the cell membrane forms a green halo around the labeled nuclei.
rpe = retinal pigment epithelium, onl = outer nuclear layer, inl = inner nuclear layer, ipl = inner plexiform layer, nfl = nerve fiber layer.
scale bar: a-c = 40 μm; d = 5 μm
mitotic cells in the onl are rods
we used confocal microscopy in sections double-labeled with phh <dig> and rod opsin to determine if the proliferating cells in the onl were rods.
as rod opsin delocalizes to the onl in the early stages of disease  <cit> , it serves as a specific marker for rods.
confocal microscopy imaging clearly demonstrated co-localization between phh <dig> and rod opsin, and revealed an opsin-positive cytoplasmic halo surrounding the phh3-positive cells in the onl .
these results indicate that in the three diseases the proliferating cells in the onl are rods.
in contrast, phh <dig> labeling of putative cones, i.e.
the larger nuclei located immediately vitreal to the external limiting membrane, was not detected.
mitotic cells in the onl are neither microglia nor müller cells
to determine if microglia or müller cells contributed to the pool of proliferating onl cells observed with ihc, we undertook dual-labeling with phh <dig> and markers specific for microglia  and müller cells ;  <cit> ).
cd18-labeled microglia normally reside in the inner and outer plexiform layers , and retinal diseases, in general, can result in microglial activation.
however, neither microglia  nor müller cells  appeared to contribute to the phh <dig> positive onl cell pool.
it is important to note, however, that robust phh <dig> labeling of gs positive müller cells in the vitreal border of the inner nuclear layer  was present in some areas of the rcd <dig> retina, and, to a lesser extent in xlpra <dig> ; however there was no indication that these dividing müller cells migrated to the onl.
retinal gene expression
age-dependent changes in normal and mutant retinas – intra-group comparisons
to properly interpret gene expression changes that occur with aging in both normals and mutants, we first performed an intra-group comparison that examined expression at time points when pr and retinal development is occurring , when they have just reached structural maturation  and when the retina is fully mature   <cit>  .
overall, the major changes in gene expression occurred between  <dig> vs.  <dig>  and  <dig> vs.  <dig> wks, where many genes showed significant down-regulation compared to the previous time point within each group, suggesting that the majority of molecular events and changes take place when retinal development is still ongoing before reaching maturation .
with the exception of nr2e <dig>  rb <dig>  e2f <dig>  and cdk <dig>  the expression profiles were not shared between normal and mutant retinas.
in the case of cdk <dig>  expression was markedly up-regulated at  <dig> and  <dig> wks in normal, in rcd <dig> at  <dig> wks, and xlpra <dig> at  <dig> vs.  <dig> wks.
the expression of some cyclins decreased in normal retinas over time, as expected by the lack of cell division after two wks of age.
however, this decrease was not accompanied by decreases in cyclin-dependent kinases, cell cycle phosphatases, or cyclin-dependent kinase inhibitors.
in contrast, cyclin-dependent kinases, cdk <dig>   <dig> and  <dig>  decreased in expression in rcd <dig> at  <dig> compared to  <dig> wks; the former time point is characterized by a very high rate of apoptotic pr cell death  <cit> .
the expression of the transcription regulators e2f <dig> and rb was lower at all time points examined in xlpra <dig> in comparison to  <dig> wks of age.
two genes, cdk25b and cdkn1b were specifically up-regulated in erd at 8– <dig> and  <dig> – <dig>  wks in comparison to  <dig>  wks of age.
notably, there were no changes in expression with age in members of the ndr kinases or hippo signaling pathway.
details of these results are presented in additional file  <dig>  all together, these results show that expression changes in the selected genes parallel the structural development of the dog retina in normal and diseased retinas.
disease associated changes in retinal gene expression
to determine the changes in expression as a result of disease, we compared the mutant dogs to age-matched normal controls at different ages.
concurrent with pr cell degeneration, there is decreased expression of genes associated with pr function and structure .
grk <dig> decreases first in rcd <dig> at  <dig> wks, and at  <dig> wks in xlpra <dig>  by  <dig> and  <dig> – <dig>  wks, rcvrn is decreased in all  <dig> diseases.
consistent with incomplete outer segment  formation prior to early pr degeneration in rcd <dig> dogs, there is decreased expression of rds at  <dig> wks and subsequent time points.
nrl and nr2e <dig>  genes associated with pr development and cell fate specification  <cit> , are down-regulated in rcd <dig> at  <dig>   <dig> , and  <dig> wks, and in xlpra <dig> also at  <dig> wks.
nrl, the upstream regulator of nr2e <dig>  is down-regulated at all ages examined in erd, and may account for the formation of hybrid rod/s-cones in the retina following pr cell proliferation  <cit>  .table  <dig> qrt-pcr retinal gene expression results in xlpra <dig>  rcd <dig>  and erd mutants compared to normal retinas
cell cycle: cyclins
ccna1
ccna2
ccnb1
ccnd1
ccnd3
ccne1
cell cycle: cyclin-dependent kinases
cdk1
cdk2
cdk4
cdk6
cell division cycle phosphatases
cdc25a
cdc25b
cdc25c
cyclin-dependent kinase inhibitors
cdkn1a
cdkn1b
cdkn2a
cell cycle transcription regulators
bmi1
e2f1
rb1
hippo signaling/ndr kinases
lats1
lats2
mob1a
ndr1
eye development regulation via proliferation/apoptosis
pax6
pcna
pr function
grk1
rbp3
rcvrn
pr development/structure
crb1
nrl
nr2e3
rds
statistically significant  differences in retinal gene expression are reported in comparison to normals at different ages .
the examined genes are reported in alphabetical order within functional groups.
the complete list of genes tested is available as additional file  <dig>  n.s = not statistically significant; + = up-regulated and - = down-regulated compared to normal
fig.
<dig> retinal mrna expression changes in xlpra <dig>  rcd <dig>  and erd-mutants compared to normal between  <dig> and  <dig> wks of age.
a photoreceptor function; b photoreceptor development/structure; c cell cycle transcription regulators; d cyclin-dependent kinases; e cyclins; f hippo signaling/ndr kinases.
an asterisk indicates statistically significant differences  in expression.
data points are reported as mean +/− sd
three cell cycle transcription regulators, bmi <dig>  e2f <dig> and rb <dig>  were included in the analysis.
while bmi <dig> showed no changes associated with disease, both e2f <dig> and rb <dig> expression was up-regulated in xlpra <dig> and rcd <dig> at  <dig> wks, early in the degenerative process, and e2f <dig> at the  <dig> wk time point.
in contrast, there were marked changes in expression of cyclin dependent kinases and cyclins with disease .
cdk <dig> was down-regulated in all diseases beginning at  <dig> wks in rcd <dig>  cdks  <dig> , <dig> were up-regulated at  <dig> and  <dig> wks in rcd <dig>  and cdk <dig> at the same time points in xlpra <dig>  up-regulation of ccna <dig> and ccne <dig> occurred in the  <dig> mutants at  <dig> wks, and  <dig> wks ; similar up-regulation was found for ccna <dig> at  <dig> wks.
lastly, ccnb <dig>  d <dig> and d <dig> were up-regulated at  <dig> wks in rcd <dig>  and ccnb <dig> and d <dig> in xlpra <dig> .
these results indicate that changes in the expression of some cyclin dependent kinases and cyclins occur at specific disease stages, and, in some cases, the expression patterns are shared among the  <dig> diseases.
disease-associated changes in gene expression for members of the ndr kinases and hippo pathway were only found in erd at the  <dig> – <dig>  wks time point.
there was up-regulation of lats <dig>  mob1a and ndr <dig> .
there were no expression differences in stk38l , the gene mutated in the disease when primers located 3′ to the deletion were used.
disease-associated changes in rpe gene expression
because of the close rpe-pr physical proximity, shared functional interactions, and the rpe’s role in immune modulation .
even though there was no evidence of rpe cell mitosis based on phh <dig> labeling, we did find that at  <dig> wks there was a general up-regulation of cdk <dig>  ccnd <dig>  and ccne <dig> in all three diseases.
these expression commonalities suggest that similar mechanisms related to the cell cycle pathway might be activated and preserve the integrity in both mutant prs and rpes.
as well, expression of the cell cycle transcription regulator e2f <dig> was increased at  <dig> wks in rcd <dig>  in general, de genes in retina and rpe samples were not the same and the ages of dysregulation differed.
in contrast, as with the retinal analyses, expression changes in ndr kinases and the hippo signaling pathway were only found in erd , although not at the same ages and lats <dig> was specifically de only in rpe.
these results indicate specific and likely independent differences in the expression of cell cycle genes in the  <dig> different cell types, and do not suggest a key role for rpe cells in retinal degeneration.fig.
<dig> rpe mrna expression changes in xlpra <dig>  rcd <dig>  and erd-mutants compared to normal at  <dig>  and  <dig>  wks of age.
at  <dig> wks, there were no differentially expressed genes that reached statistical significance.
an asterisk indicates statistically significant differences  in expression.
bars indicate mean +/− sd of triplicate samples
protein expression and disease
western blot analyses
to complement the mrna expression studies, we examined a selected number of proteins encoded by genes relevant to the cell cycle and pr cell fate specification, and which were differentially expressed.
in addition to nrl, this included transcription regulators , the cyclin dependent kinase cdk <dig>  cyclins , and the lats <dig> and mob1a members of the ndr kinase/ hippo signaling pathway .
the number of genes and time points tested was limited by availability of samples, i.e.
one sample/disease/time point, and the lack of specificity to canine tissues for several of the antibodies tested .fig.
<dig> western blot analysis of normal, xlpra <dig>  rcd <dig>  and erd retinas.
a blots exposed on autoradiograph films; b li-cor immunoblotting system.
the band intensity normalized to the actin density of the normal control  was set to one, and fold changes were calculated and shown below each of the blots
relative to normal retina, nrl is down-regulated at  <dig> ,  <dig>  and  <dig> wks in rcd <dig>  and at  <dig>  wks in erd, which was the only time point examined by western blot analysis for the latter disease.
this result is not unexpected, as the transcription factor nrl is a critical intrinsic regulator of pr development and function, and regulates the expression of several rod-specific genes, including rho, that, particularly in rcd <dig>  is also down-regulated early in the disease process.
the transcription regulators prb and e2f <dig> showed similar expression patterns with low levels in normal retinas, and higher levels in the  <dig> disease models.
cdk <dig> was lower in normals and xlpra <dig> at  <dig> wks, while ccnd <dig> and ccne <dig> levels were higher in all diseases.
the levels of ccna <dig> in the 7/ <dig>  wks diseased retinas were markedly higher.
lastly, we found that expression of mob1a was specifically elevated in erd, but lats <dig> was increased in rcd <dig> and erd at ~ <dig> wks.
in general, there was good agreement between the qrt-pcr and western analyses, which indicate a dysregulation of the expression of some cell cycle proteins in the canine models studied.
immunohistochemistry
we used ihc to localize the expression of selected proteins, and to qualitatively assess their expression levels.
while expression of crx, a pr-specific transcription factor that plays a role in the differentiation of pr cells, was low in all samples, regardless of disease status, the expression of nrl differed in normal and mutants.
normal retinas showed labeling in the cone outer segments and basal row of onl nuclei.
similar os labeling was found in xlpra <dig> and rcd <dig>  but with a larger area of the onl occupied by labeled nuclei.
in contrast, the erd retina, composed primarily of hybrid rod/s-cones, had a paucity of nrl-labeled cone outer segments, further underlying this particular feature of this disease model.
on the other hand, pax <dig>  a transcription factor present in developing eyes, primarily labeled müller cell nuclei in normal and mutants, but not prs .fig.
<dig> immunolabeling in normal and mutant retinas for retinal transcription factors.
a crx; b nrl; c pax <dig>  samples were from normal , xlpra <dig> , rcd <dig>  and erd .
in normal, xlpra <dig> and rcd <dig>  nrl is expressed in cone outer segments  and in the basal aspect of the outer nuclear layer; the rcd <dig> retina has few rods, and almost no rod outer segments remain so nrl distinctly labels the surviving cone outer segments.
pr = photoreceptor, onl = outer nuclear layer, inl = inner nuclear layer, ipl = inner plexiform layer, nfl = nerve fiber layer.
scale bar = 40 μm
due to lack of specificity, we were unable to assess immunolabeling with cell cycle specific antibodies against prb <dig>  cdk <dig>  cdk <dig>  and ccna <dig> .
antibodies against e2f <dig> showed robust labeling localized to the rpe, ipl and especially the opl.
mutant retinas, particularly at younger ages, showed more intense rpe labeling .
of the  <dig> cyclins analyzed, ccnd <dig> primarily showed intense labeling over the rpe layer, and, with the exception of erd, was more intense than in controls.
in most cases, the labeling was cytoplasmic as well as nuclear .
in contrast, ccne <dig> labeled horizontal processes in the opl equally in normals and mutants .fig.
<dig> immunolabeling in normal and mutant retinas for selected cell cycle proteins.
paired sections from normal, xlpra <dig>  rcd <dig>  erd retina taken at two ages were labeled with antibodies against: a e2f <dig>  b ccnd <dig>  c ccne <dig>  more intense labeling of the rpe layer is present in mutants compared with normals with antibodies against e2f <dig> and ccnd <dig>  note that in the  <dig> wk xlpra <dig> retina , the rpe layer has separated from the section during immunolabeling and is not included in the image.
rpe = retinal pigment epithelium, onl = outer nuclear layer, opl = outer plexiform layer, inl = inner nuclear layer, ipl = inner plexiform layer, nfl = nerve fiber layer.
scale bar = 40 μm
immunolocalization of mob1a was comparable in normal and mutants, and in xlpra <dig> and rcd <dig> for lats <dig> .
in erd, however, more diffuse labeling of the pr layer was evident with lats <dig>  with os- <dig>  a monoclonal antibody that specifically labels s-cones  <cit> , we found labeling of a distinct population of s-cone in normal, xlpra <dig> and rcd <dig> .
in erd, however, the os- <dig> antibody resulted in uniform labeling of the entire pr layer .
the merged os- <dig> and lats <dig> images showed no co-localization in normal , but complete co-localization in the pr layer of erd .
these results confirm a previous study which described that newly generated prs in erd are hybrid rod/s-cones  <cit> , and indicates that in erd the lats <dig> protein is now expressed in these newly formed cells.fig.
<dig> lats <dig>  mob1a, os- <dig> immunolabeling of normal and mutant retinas.
a lats <dig> labels the inl and gcl/nfl in normal retina.
in mutants, there is additional faint labeling of the photoreceptor and rpe layers.
b low levels of mob1a labeling are present in the normal retina, and increase in mutants in the same layers which express lats <dig>  c the os- <dig> antibody labels a subpopulation of cones  in normal, xlpra <dig> and rcd <dig> .
dual labeling with os- <dig> and lats <dig> antibodies in normal shows labeling in different retinal domains .
in erd, os- <dig> labels the inner and outer segments of the entire photoreceptor population , and colocalizes with lats <dig> .
rpe = retinal pigment epithelium, onl = outer nuclear layer, inl = inner nuclear layer, nfl = nerve fiber layer.
scale bar = 40 μm for
discussion
cell division in normal canine retina ends before  <dig> wks of age, and prs mature in the first 5– <dig> wks after birth  <cit> .
pr proliferation beyond  <dig> wks occurs infrequently, sporadically, and only in the periphery, adjacent to the ora serrata.
in contrast, mitotic prs are observed in one inherited retinal disease, erd, in which concurrent proliferation and apoptosis of different pr cell populations occurs, and is followed by formation of hybrid rod/s-cones.
the cell proliferation and death kinetics are balanced, albeit for a finite period, as the onl maintains normal thickness until  <dig> wks of age.
thereafter, the balance tilts towards cell death and the onl progressively thins  <cit> .
to determine if aberrant pr proliferation is exclusive to erd, we assessed two other phenotypically comparable diseases, xlpra <dig> and rcd <dig>  and used erd as a mutant control.
these diseases are caused by mutations in distinct genes that impair the early phases of pr development, and, generally, have similar structural features and disease time course with rcd <dig> being faster and more aggressive, followed by xlpra <dig> and erd  <cit> .
using phh <dig> as a specific marker of the m-phase of the cell cycle, we identified mitotic cells in the onl in all three diseases, with the greatest number observed in erd.
pcna labeling largely recapitulated trends in phh <dig> labeling over time, and the highest numbers of pcna-positive cells were identified in erd.
confocal microscopy demonstrated conclusively the co-localization of phh <dig> with the rod-specific protein rho, and confirmed that cells undergoing proliferation in all  <dig> diseases were rod prs.
the higher overall numbers of phh3-positive than pcna-positive cells suggests that the majority of proliferating cells in the onl are in the m-phase of the cell cycle for all  <dig> diseases.
in xlpra <dig> and particularly in rcd <dig>  there is marked pr proliferation that is many times higher than that found in normals.
in both, proliferation is sustained for the  <dig> wk period of observation.
the magnitude of pr proliferation in the two diseases parallels cell death kinetics based on tunel labeling, which is greater in rcd <dig>  balance between cell proliferation and death in these two diseases does not occur, and the onl progressively thins beginning at early time points  <cit> .
the overall lack of co-localization of tunel- and phh3-labeled pr cells indicated that different cells undergo proliferation and apoptosis, and that the two events are not linked in a common pathway.
furthermore, the large differences between the number of cells labeled with phh <dig> and mad <dig>  and the smaller number of cells that co-express both markers in the mutant retinas emphasizes that the cells entering the m-phase of the cell cycle do not undergo mitotic checkpoint arrest, but continue to divide, thus contributing new pr cells to the onl.
in contrast, the control retina showed only a very small number of phh <dig> positive cells, and all of these co-expressed mad <dig>
in rd <dig> mice, proliferating cells in the onl have been reported to be microglia in origin, based on f4/ <dig> immunostaining  <cit> .
in contrast, although the mutant canine retinas showed an increased numbers of cd18-positive microglial cells and processes, these did not co-localize with phh <dig> labeling.
in addition, gs-positive müller cell labeling did not co-localize with the dividing pr cells.
while the methods used cannot exclude completely a contribution by microglia and/or müller cells to the dividing cell population in the onl, the results indicate that such a contribution, if present, is minor.
it is possible that some of the phh3-labeled müller cells in the vitreal border on the inl in xlpra <dig> and rcd <dig> , proliferated and migrated into the onl.
however, prior studies have shown that pax <dig> positive müller glia can express cell cycle markers, but not proliferate  <cit> .
all together, our results show that in the onl of the  <dig> canine diseases there are distinct subsets of pr cells one that is dying, while the other is dividing.
a recent study showed that in several rodent models of retinal degeneration there is a reactivation of cell cycle genes before the onset of apoptosis  <cit> .
these findings were in accord with previous studies of neurodegenerative diseases, e.g.
alzheimer disease, parkinson’s disease and others, where aberrant reentry of susceptible neuronal populations into the cell cycle is associated with the death of post-mitotic, terminally differentiated neurons .
in the retina of the rodent models, nuclear expression of cdk <dig>  cdk <dig>  and cdk <dig>  as well as phosphorylation of rb precede pr death.
furthermore, deleting e2f <dig> in the rd <dig> model resulted in a transient neuroprotection, whereas deletion of bmi <dig>  the upstream activator of e2f <dig> and cdks, resulted in a marked delay in pr loss.
at least in the rodent retinas, re-activation of these cell cycle proteins resulted in pr cell death, but not pr mitosis  <cit> .
unlike these rodent models, pr proliferation is readily observed in  <dig> early-onset canine models of retinal degeneration .
to begin to assess the mechanistic basis for pr proliferation, we first examined selected genes involved in different phases of the cell cycle.
we find that at  <dig> and  <dig> wks, the first and last time points examined, mrna expression of rb <dig> and e2f <dig> are significantly up-regulated in xlpra <dig> and rcd <dig>  and protein levels of prb <dig> and e2f <dig> were increased in all  <dig> diseases.
expressions of cdk <dig> , <dig> also are up-regulated at the same time points, particularly in rcd <dig>  but cdk <dig> expression is down-regulated.
indeed, western analysis of cdk <dig> showed increased expression at  <dig> wks in mutants.
furthermore, mrna expression changes in cyclins were quite uniform across all diseases; ccna <dig> and ccne <dig> were up-regulated at  <dig> wks, while increased expression of the other cyclins was found at the  <dig> wk time point.
these data were confirmed by western analysis, showing comparable results at the mrna and protein levels.
these results indicate that in the mutant canine retinas there is a dysregulation of cell cycle gene expression in comparison to the normal retina.
even though the  <dig> diseases investigated are primary pr degenerations, we also examined the rpe expression changes of selected genes, to determine potential differences or commonalities.
we find altered mrna expression patterns in the rpe at time points when this cell layer is not affected.
at  <dig> wks of age, de genes were primarily up-regulated, and included cdk <dig>  ccna <dig>  and ccne <dig>  which showed increased expression levels in the three diseases.
at 7 weeks, on the other hand, statistically higher mrna levels were observed mainly in erd, and included ndr <dig> and lats <dig>  while, as expected, stk38l expression of exon  <dig>  deleted in the disease, was absent.
other than ccne <dig>  there was poor correlation between the retina and rpe mrna expression findings at comparable ages.
these results are not unexpected given that the prs are affected by the disease.
indeed, changes in the rpe occur in response to pr degeneration or alternatively could be due to changes in contaminating tissue, including choroid, microglia/macrophages and white blood cells trapped in the highly vascular choroid.
however, in contrast to the retina, the observed differences in rpe cell cycle gene expression in the  <dig> diseases suggests an activation of pathways that lead to the long-term survival.
this hypothesis warrants further studies that could also be performed on primary rpe cell cultures.
while the mrna and protein expression studies indicate activation of a subset of cell cycle genes in proliferating pr, some of the ihc results are more difficult to reconcile with these findings given the apparent lack of specificity of several of the antibodies.
in tissue sections, we could not assess expression of prb <dig>  cdk <dig>  cdk <dig>  ccna <dig>  ccnb <dig>  and ccnd <dig>  moreover, in none of the mutant tissues that showed phh3-labeled proliferating prs did we find labeling with e2f <dig>  ccnd <dig> or ccne <dig> in the onl, the expected site of these cellular events.
obviously, both qrt-pcr and western analyses include the entire neuroretina, while proteins with ihc were more precisely localized to specific cellular layers, but quantification of labeling intensity is qualitative.
these data suggest that the cells expressing different levels of cell cycle-related transcripts may not be the ones that are dividing, thus are not prs.
the specific analysis of these questions will require single cell analysis of pr populations.
in contrast to the retina, ihc of the rpe clearly showed expression of several of the cell cycle gene products, e.g.
e2f <dig> and ccnd <dig>  labeling intensity was greater in the mutants vs. controls, and there was a general increase in label intensity in the older affected animals of each genotype.
this evidence appears to confirm an activation of specific cell cycle related pathways in rpe that might promote survival of these cells.
most notably, we find unique changes in the expression of ndr kinases and hippo signaling genes in erd-mutants, a disease caused by a mutation in the ndr kinase stk38l .
we found an up-regulation of the ndr kinases and hippo signaling pathway members mob1a, lats <dig>  and ndr <dig>  lats <dig> is a tumor suppressor gene expressed in vertebrate retina  <cit>  and stimulates the kinase activity of stk38l upon mob1a binding and subsequent activation after association with s100b  <cit> .
the increased expression of lats <dig> in erd may be an attempt to suppress the aberrant pr proliferation or to induce apoptosis, as several studies have reported this particular feature of this gene .
lats <dig> and os- <dig> co-localization in the pr inner and outer segments was observed with ihc exclusively in erd, and confirms the previous finding that aberrant rod/s-cone hybrids are generated following pr proliferation in erd-mutants  <cit> .
finding marked down-regulation of expression of nrl in erd is also consonant with its known function in pr cell fate specification  <cit> .
nrl knockout mouse retinas are almost entirely composed of s-cone prs.
given that the proliferating population of prs in the erd retina resulted in l/m-cone and hybrid rod/s-cones, the decreased expression of nrl is not surprising.
the hippo pathway controls organ size via the regulation of apoptosis and proliferation.
it interacts in cell cycle control by cdk <dig> phosphorylation of yap, a downstream hippo pathway effector  <cit> , and cyclin e is a known hippo pathway target gene  <cit> .
indeed, the hippo pathway, via mob1a activation of lats <dig> tumor suppressor can negatively regulate cell proliferation by modulating cyclin a activity  <cit> .
however, as we found that expression changes in cdk <dig>  ccne <dig>  ccna <dig>  and ccna <dig> were not specific to erd, but were comparable also in xlpra <dig> and rcd <dig>  we expect that the observed retinal changes in these genes did not result from the altered hippo signaling pathway.
CONCLUSIONS
in summary, our results show that in early phases of three retinal degenerative diseases in dogs, phh <dig> labeling is present in the outer nuclear layer, an indication that a subset of pr cells have entered the m-phase of the cell cycle.
these cells are of rod origin, and there is no evidence that the proliferating cells are microglia or müller cells.
in erd, the proliferating cells transiently maintain the integrity of the onl in spite of high rates of concurrent apoptotic cell death in cells that are not proliferating .
similar but lower levels of pr proliferation occur in xlpra <dig> and rcd <dig>  but the high rate of apoptotic cell death in comparison to pr proliferation result in a more rapid loss of onl in the early stages of disease.
associated with these events is a dysregulation of cell cycle genes that appears to occur in a different subset of retinal cells, that might play a primary or bystander role in the degenerative process.

 photoreceptor cells are specialized retinal neurons that efficiently capture light and transduce it into a neural signal. their intricate and highly specific structure is dependent on the expression of multiple genes, including those involved in pr specification, differentiation and maintenance. indeed, ~ genes and loci are involved in retinal degeneration in man, and a lower but still substantial number of genes in animals. while mutations in pr-specific or enriched genes are common, and cause a broad spectrum of inherited retinal diseases, an understanding of the mechanistic links between mutation and disease is limited. moreover, while apoptotic cell death is the final common pathway in most retinal degenerative diseases, the rate of degeneration and cell death pathway utilized varies in a disease, mutation and species-specific manner, demonstrating a high level of complexity. identifying the principal molecular players and providing an in-depth knowledge of the mechanisms that induce and regulate the underlying pr cell death will provide essential insight into disease progression. this will help to develop pharmacological agents and specific therapies that provide pr protection in the hopes of treating inherited retinopathies. while stimulating the proliferation of diseased yet functional prs to maintain the pr layer would likely be therapeutically beneficial in some retinal degenerative diseases, the proliferative potential of pr cells is controversial, and not well understood. prs are terminally differentiated, and undergo their final cell division just prior to cell fate specification; compensatory neurogenesis mechanisms do not exist to replace all dying cells in the normal retina. indeed, it was originally postulated that new prs are generated in naturally occurring retinal diseases, however this hypothesis has been disproved. furthermore, a recent comprehensive analysis of different rodent retinal mutants, along with a light-induced retinal degeneration model, clearly demonstrated that reactivation of the expression of cell cycle genes did not correlate with pr cell division as determined by ethynyl deoxyuridine incorporation and phospho histone h labeling. instead, this process was essential to promote the cell death pathways. in contrast, under some specific circumstances, a limited number of prs and other neurons can be generated from presumably terminally differentiated müller cells that dedifferentiate, proliferate and express neuronal progenitor markers in the adult rat, mouse as well as chicken and fish. in contrast to normal prs and other disease models, we recently reported that a mutation in the ndr family kinase stk38l found in the erd canine model results in a period of sustained pr proliferation, and that newly generated cells are unique hybrid rod/s-cones, and did not originate from resident retinal stem cells. the stk38l mutation eliminates the binding sites for regulatory proteins s100b and mob, and part of the n-terminal regulatory region that is highly conserved in all ndr subclass of agc protein kinases. ndr kinases, including lats interact with the hippo pathway through mob binding to regulate aspects of cell growth, metabolism, proliferation and survival. thus, we hypothesize that terminally differentiated normal prs are kept from dividing by ndr2-mob interaction, and removing this control in mutants allows the cell to re-enter the cell cycle and divide. in the present study, we examined whether pr proliferation may also occur in other early-onset inherited retinal diseases to determine if common molecular pathways were involved. in addition to erd, where no equivalent disease has been reported in man, two other early onset canine diseases with comparable cell death kinetics and histopathology were examined: x-linked progressive retinal atrophy and rod cone dysplasia, which are caused, respectively, by mutations in rpgrorf and pde6b. both diseases bear mutations in genes that cause human inherited blindness, and the disease phenotypes are similar and comparable. in all three diseases, the early and rapid degeneration of the prs makes the disease course predictable and highly suitable for comparative studies of the involved events. however, the exact mechanisms by which mutations in these genes drive the degeneration events are currently unknown. to this end, we examined the retinal and retinal pigment epithelium expression of selected genes and proteins that are involved in cell cycle regulation, or belong to the ndr protein-kinase family and the hippo pathway;. notably, our results indicate that pr proliferation also occurred in xlpra and rcd but that formation of hybrid rod/s-cones is unique to erd. furthermore, we demonstrate a concurrent dysregulation of critical cell cycle genes that were differentially expressed in all three diseases, while hippo pathway genes were more specifically altered in erd.  morphology of early-onset canine retinal degeneration models we initially characterized the retinal morphology of the early-onset disease models that generally have a similar pattern of pr development and degeneration. although overall retinal development is initially normal, there were differences in the subsequent rates and kinetics of pr degeneration; retinal degeneration started at different ages and occurred more rapidly in rcd where rod pr development was abnormal, and outer segments were sparse, failed to elongate, and inner segments were short already at wks. the disease is slightly more delayed in xlpra while erd showed preservation of the onl thickness until at least wks.fig. age-dependent structural changes in normal and mutant retinas. disease occurs earlier and progresses more rapidly in rcd while it is slightly delayed in xlpra the outer nuclear layer in erd is preserved during the time course of the study. scale bar: 20 μm; rpe = retinal pigment epithelium, pr = photoreceptors, onl = outer nuclear layer, opl = outer plexiform layer, inl = inner nuclear layer, ipl = inner plexiform layer, gcl = ganglion cell layer photoreceptor cell proliferation in mutant retinas to determine if pr proliferation was exclusive to erd-mutants, we used phh and pcna labeling to examine pr mitosis in the onl of additional early-onset disease models. phh is a specific marker for mitotic cells in the late g and m-phases, while pcna labels both cells undergoing proliferation and dna repair. the number of labeled cells/ million μm of onl was analyzed at different time points between and wks. the results showed similar trends for both phh and pcna labeling in the different models and in normals, although the number of pcna-positive cells was lower than the number of phh3-positive cells at every time point examined. in addition to labeling different phases of the cell cycle, the lower pcna results suggest that there is limited ongoing dna damage and repair. minimal numbers of phh3-positive cells were found in normal retinas after wks of age; these were located adjacent to the external limiting membrane and limited to the retinal periphery.fig. photoreceptor cell proliferation in the outer nuclear layer of normal and mutant retinas. a phh3-labeling was used to identify mitotic cells, and b pcna-labeling to identify cells undergoing proliferation and dna repair. normal retinas exhibited essentially no mitotic cells after wks of age, while they were present in the onl of the diseased retinas. mitotic cells were continually present throughout the first 14– wks of life in all diseases, with the highest numbers observed in erd-mutants. the pcna data for erd has been published previously. data points are expressed as mean of triplicates ± sd. note the difference in the scale of the y-axes between erd and normal, xlpra and rcd as previously shown with pcna labeling, phh3-labeled mitotic prs were found at ≥ wks in erd, and these levels were sustained, ranging from >50– cells/ million μm of onl. in contrast, pr proliferation peaked at wks of age in rcd and at wks in xlpra these time points precede or approximately coincide the peaks of cell death for each disease. however, regardless of the disease, the number of phh3-labeled cells in mutants far exceeded the numbers in normals, and was sustained for the time period analyzed. for example, at ~ wks of age the mutants had 7– times the number of dividing cells than controls, and these increased to 12– times at ~ wks. we examined the association between cells undergoing mitosis, and those in mitotic checkpoint arrest to exclude the possibility that entry into the late g2-m phase of the cell cycle preceded apoptosis. to this end, we compared control and mutant retinas at time points with high rates of pr mitosis in mutants: xlpra2 =  wks, rcd1 =  wks, erd =  wks. in the control, the phh labeled onl nuclei also labeled with mad2; overall, the number of labeled cells with each antibody was low, and the phh3/mad ratio per million μm of onl was in contrast, the phh3/mad ratio in the mutants was much higher, and smaller number of nuclei/ million μm of onl co-expressed both proteins. this indicates that pr mitosis is occurring in onl, and the dividing cells contribute to maintenance of the onl. to control for the possibility that the same pr cells were not undergoing concurrent apoptosis and mitotic division, we performed dual-labeling with tunel and phh at the ages that correspond to the peaks of cell death for each disease. the results indicate that with few exceptions the majority of cells were positive for either tunel or phh but not for both, an indication that different cell populations in the onl were undergoing either apoptosis or mitosis. co-localization of tunel and phh labeling was rare and limited to few cells in the erd model. these results establish that proliferating and degenerating cells in the onl are different.fig. phh labeling identifies mitotic cells in the outer nuclear layer. a combined phh and tunel labeling in normal, xlpra, rcd and erd retinas at the peak of cell death for each disease. there are no tunel or phh labeled cells in the normal retina. in the mutants, some of the red and green labeled nuclei in onl are identified. note that in erd, two cells are undergoing combined proliferation and apoptosis. b cd labeling shows microglial cells and processes distributed primarily in the opl and ipl, with increased expression in xlpra, rcd and erd compared to normal. phh labeling in the mutants shows mitotic cells in onl. the two dividing cells in the opl are of unknown origin and are not counted in the analysis. c glutamine synthetase labeling in normal, and dual phh and gs labeling in xlpra, rcd and erd. dividing cells in onl are not labeled with gs. note that in xlpra and rcd there are phh3-positive nuclei in the inner border of the inl. d confocal microscopy image of dual-immunolabeling of retinal sections with phh and rod opsin in the onl of xlpra, rcd, and erd. the delocalized rod opsin associated with the cell membrane forms a green halo around the labeled nuclei. rpe = retinal pigment epithelium, onl = outer nuclear layer, inl = inner nuclear layer, ipl = inner plexiform layer, nfl = nerve fiber layer. scale bar: a-c = 40 μm; d = 5 μm mitotic cells in the onl are rods we used confocal microscopy in sections double-labeled with phh and rod opsin to determine if the proliferating cells in the onl were rods. as rod opsin delocalizes to the onl in the early stages of disease, it serves as a specific marker for rods. confocal microscopy imaging clearly demonstrated co-localization between phh and rod opsin, and revealed an opsin-positive cytoplasmic halo surrounding the phh3-positive cells in the onl. these results indicate that in the three diseases the proliferating cells in the onl are rods. in contrast, phh labeling of putative cones, i.e. the larger nuclei located immediately vitreal to the external limiting membrane, was not detected. mitotic cells in the onl are neither microglia nor müller cells to determine if microglia or müller cells contributed to the pool of proliferating onl cells observed with ihc, we undertook dual-labeling with phh and markers specific for microglia and müller cells; ). cd18-labeled microglia normally reside in the inner and outer plexiform layers, and retinal diseases, in general, can result in microglial activation. however, neither microglia nor müller cells appeared to contribute to the phh positive onl cell pool. it is important to note, however, that robust phh labeling of gs positive müller cells in the vitreal border of the inner nuclear layer was present in some areas of the rcd retina, and, to a lesser extent in xlpra; however there was no indication that these dividing müller cells migrated to the onl. retinal gene expression age-dependent changes in normal and mutant retinas – intra-group comparisons to properly interpret gene expression changes that occur with aging in both normals and mutants, we first performed an intra-group comparison that examined expression at time points when pr and retinal development is occurring, when they have just reached structural maturation and when the retina is fully mature. overall, the major changes in gene expression occurred between vs. and vs. wks, where many genes showed significant down-regulation compared to the previous time point within each group, suggesting that the majority of molecular events and changes take place when retinal development is still ongoing before reaching maturation. with the exception of nr2e rb e2f and cdk the expression profiles were not shared between normal and mutant retinas. in the case of cdk expression was markedly up-regulated at and wks in normal, in rcd at wks, and xlpra at vs. wks. the expression of some cyclins decreased in normal retinas over time, as expected by the lack of cell division after two wks of age. however, this decrease was not accompanied by decreases in cyclin-dependent kinases, cell cycle phosphatases, or cyclin-dependent kinase inhibitors. in contrast, cyclin-dependent kinases, cdk and decreased in expression in rcd at compared to wks; the former time point is characterized by a very high rate of apoptotic pr cell death. the expression of the transcription regulators e2f and rb was lower at all time points examined in xlpra in comparison to wks of age. two genes, cdk25b and cdkn1b were specifically up-regulated in erd at 8– and – wks in comparison to wks of age. notably, there were no changes in expression with age in members of the ndr kinases or hippo signaling pathway. details of these results are presented in additional file all together, these results show that expression changes in the selected genes parallel the structural development of the dog retina in normal and diseased retinas. disease associated changes in retinal gene expression to determine the changes in expression as a result of disease, we compared the mutant dogs to age-matched normal controls at different ages. concurrent with pr cell degeneration, there is decreased expression of genes associated with pr function and structure. grk decreases first in rcd at wks, and at wks in xlpra by and – wks, rcvrn is decreased in all diseases. consistent with incomplete outer segment formation prior to early pr degeneration in rcd dogs, there is decreased expression of rds at wks and subsequent time points. nrl and nr2e genes associated with pr development and cell fate specification, are down-regulated in rcd at, and wks, and in xlpra also at wks. nrl, the upstream regulator of nr2e is down-regulated at all ages examined in erd, and may account for the formation of hybrid rod/s-cones in the retina following pr cell proliferation.table qrt-pcr retinal gene expression results in xlpra rcd and erd mutants compared to normal retinas cell cycle: cyclins ccna1 ccna2 ccnb1 ccnd1 ccnd3 ccne1 cell cycle: cyclin-dependent kinases cdk1 cdk2 cdk4 cdk6 cell division cycle phosphatases cdc25a cdc25b cdc25c cyclin-dependent kinase inhibitors cdkn1a cdkn1b cdkn2a cell cycle transcription regulators bmi1 e2f1 rb1 hippo signaling/ndr kinases lats1 lats2 mob1a ndr1 eye development regulation via proliferation/apoptosis pax6 pcna pr function grk1 rbp3 rcvrn pr development/structure crb1 nrl nr2e3 rds statistically significant differences in retinal gene expression are reported in comparison to normals at different ages. the examined genes are reported in alphabetical order within functional groups. the complete list of genes tested is available as additional file n.s = not statistically significant; + = up-regulated and - = down-regulated compared to normal fig. retinal mrna expression changes in xlpra rcd and erd-mutants compared to normal between and wks of age. a photoreceptor function; b photoreceptor development/structure; c cell cycle transcription regulators; d cyclin-dependent kinases; e cyclins; f hippo signaling/ndr kinases. an asterisk indicates statistically significant differences in expression. data points are reported as mean +/− sd three cell cycle transcription regulators, bmi e2f and rb were included in the analysis. while bmi showed no changes associated with disease, both e2f and rb expression was up-regulated in xlpra and rcd at wks, early in the degenerative process, and e2f at the wk time point. in contrast, there were marked changes in expression of cyclin dependent kinases and cyclins with disease. cdk was down-regulated in all diseases beginning at wks in rcd cdks, were up-regulated at and wks in rcd and cdk at the same time points in xlpra up-regulation of ccna and ccne occurred in the mutants at wks, and wks; similar up-regulation was found for ccna at wks. lastly, ccnb d and d were up-regulated at wks in rcd and ccnb and d in xlpra. these results indicate that changes in the expression of some cyclin dependent kinases and cyclins occur at specific disease stages, and, in some cases, the expression patterns are shared among the diseases. disease-associated changes in gene expression for members of the ndr kinases and hippo pathway were only found in erd at the – wks time point. there was up-regulation of lats mob1a and ndr. there were no expression differences in stk38l, the gene mutated in the disease when primers located 3′ to the deletion were used. disease-associated changes in rpe gene expression because of the close rpe-pr physical proximity, shared functional interactions, and the rpe’s role in immune modulation. even though there was no evidence of rpe cell mitosis based on phh labeling, we did find that at wks there was a general up-regulation of cdk ccnd and ccne in all three diseases. these expression commonalities suggest that similar mechanisms related to the cell cycle pathway might be activated and preserve the integrity in both mutant prs and rpes. as well, expression of the cell cycle transcription regulator e2f was increased at wks in rcd in general, de genes in retina and rpe samples were not the same and the ages of dysregulation differed. in contrast, as with the retinal analyses, expression changes in ndr kinases and the hippo signaling pathway were only found in erd, although not at the same ages and lats was specifically de only in rpe. these results indicate specific and likely independent differences in the expression of cell cycle genes in the different cell types, and do not suggest a key role for rpe cells in retinal degeneration.fig. rpe mrna expression changes in xlpra rcd and erd-mutants compared to normal at and wks of age. at wks, there were no differentially expressed genes that reached statistical significance. an asterisk indicates statistically significant differences in expression. bars indicate mean +/− sd of triplicate samples protein expression and disease western blot analyses to complement the mrna expression studies, we examined a selected number of proteins encoded by genes relevant to the cell cycle and pr cell fate specification, and which were differentially expressed. in addition to nrl, this included transcription regulators, the cyclin dependent kinase cdk cyclins, and the lats and mob1a members of the ndr kinase/ hippo signaling pathway. the number of genes and time points tested was limited by availability of samples, i.e. one sample/disease/time point, and the lack of specificity to canine tissues for several of the antibodies tested.fig. western blot analysis of normal, xlpra rcd and erd retinas. a blots exposed on autoradiograph films; b li-cor immunoblotting system. the band intensity normalized to the actin density of the normal control was set to one, and fold changes were calculated and shown below each of the blots relative to normal retina, nrl is down-regulated at, and wks in rcd and at wks in erd, which was the only time point examined by western blot analysis for the latter disease. this result is not unexpected, as the transcription factor nrl is a critical intrinsic regulator of pr development and function, and regulates the expression of several rod-specific genes, including rho, that, particularly in rcd is also down-regulated early in the disease process. the transcription regulators prb and e2f showed similar expression patterns with low levels in normal retinas, and higher levels in the disease models. cdk was lower in normals and xlpra at wks, while ccnd and ccne levels were higher in all diseases. the levels of ccna in the 7/ wks diseased retinas were markedly higher. lastly, we found that expression of mob1a was specifically elevated in erd, but lats was increased in rcd and erd at ~ wks. in general, there was good agreement between the qrt-pcr and western analyses, which indicate a dysregulation of the expression of some cell cycle proteins in the canine models studied. immunohistochemistry we used ihc to localize the expression of selected proteins, and to qualitatively assess their expression levels. while expression of crx, a pr-specific transcription factor that plays a role in the differentiation of pr cells, was low in all samples, regardless of disease status, the expression of nrl differed in normal and mutants. normal retinas showed labeling in the cone outer segments and basal row of onl nuclei. similar os labeling was found in xlpra and rcd but with a larger area of the onl occupied by labeled nuclei. in contrast, the erd retina, composed primarily of hybrid rod/s-cones, had a paucity of nrl-labeled cone outer segments, further underlying this particular feature of this disease model. on the other hand, pax a transcription factor present in developing eyes, primarily labeled müller cell nuclei in normal and mutants, but not prs.fig. immunolabeling in normal and mutant retinas for retinal transcription factors. a crx; b nrl; c pax samples were from normal, xlpra, rcd and erd. in normal, xlpra and rcd nrl is expressed in cone outer segments and in the basal aspect of the outer nuclear layer; the rcd retina has few rods, and almost no rod outer segments remain so nrl distinctly labels the surviving cone outer segments. pr = photoreceptor, onl = outer nuclear layer, inl = inner nuclear layer, ipl = inner plexiform layer, nfl = nerve fiber layer. scale bar = 40 μm due to lack of specificity, we were unable to assess immunolabeling with cell cycle specific antibodies against prb cdk cdk and ccna. antibodies against e2f showed robust labeling localized to the rpe, ipl and especially the opl. mutant retinas, particularly at younger ages, showed more intense rpe labeling. of the cyclins analyzed, ccnd primarily showed intense labeling over the rpe layer, and, with the exception of erd, was more intense than in controls. in most cases, the labeling was cytoplasmic as well as nuclear. in contrast, ccne labeled horizontal processes in the opl equally in normals and mutants.fig. immunolabeling in normal and mutant retinas for selected cell cycle proteins. paired sections from normal, xlpra rcd erd retina taken at two ages were labeled with antibodies against: a e2f b ccnd c ccne more intense labeling of the rpe layer is present in mutants compared with normals with antibodies against e2f and ccnd note that in the wk xlpra retina, the rpe layer has separated from the section during immunolabeling and is not included in the image. rpe = retinal pigment epithelium, onl = outer nuclear layer, opl = outer plexiform layer, inl = inner nuclear layer, ipl = inner plexiform layer, nfl = nerve fiber layer. scale bar = 40 μm immunolocalization of mob1a was comparable in normal and mutants, and in xlpra and rcd for lats. in erd, however, more diffuse labeling of the pr layer was evident with lats with os- a monoclonal antibody that specifically labels s-cones, we found labeling of a distinct population of s-cone in normal, xlpra and rcd. in erd, however, the os- antibody resulted in uniform labeling of the entire pr layer. the merged os- and lats images showed no co-localization in normal, but complete co-localization in the pr layer of erd. these results confirm a previous study which described that newly generated prs in erd are hybrid rod/s-cones, and indicates that in erd the lats protein is now expressed in these newly formed cells.fig. lats mob1a, os- immunolabeling of normal and mutant retinas. a lats labels the inl and gcl/nfl in normal retina. in mutants, there is additional faint labeling of the photoreceptor and rpe layers. b low levels of mob1a labeling are present in the normal retina, and increase in mutants in the same layers which express lats c the os- antibody labels a subpopulation of cones in normal, xlpra and rcd. dual labeling with os- and lats antibodies in normal shows labeling in different retinal domains. in erd, os- labels the inner and outer segments of the entire photoreceptor population, and colocalizes with lats. rpe = retinal pigment epithelium, onl = outer nuclear layer, inl = inner nuclear layer, nfl = nerve fiber layer. scale bar = 40 μm for discussion cell division in normal canine retina ends before wks of age, and prs mature in the first 5– wks after birth. pr proliferation beyond wks occurs infrequently, sporadically, and only in the periphery, adjacent to the ora serrata. in contrast, mitotic prs are observed in one inherited retinal disease, erd, in which concurrent proliferation and apoptosis of different pr cell populations occurs, and is followed by formation of hybrid rod/s-cones. the cell proliferation and death kinetics are balanced, albeit for a finite period, as the onl maintains normal thickness until wks of age. thereafter, the balance tilts towards cell death and the onl progressively thins. to determine if aberrant pr proliferation is exclusive to erd, we assessed two other phenotypically comparable diseases, xlpra and rcd and used erd as a mutant control. these diseases are caused by mutations in distinct genes that impair the early phases of pr development, and, generally, have similar structural features and disease time course with rcd being faster and more aggressive, followed by xlpra and erd. using phh as a specific marker of the m-phase of the cell cycle, we identified mitotic cells in the onl in all three diseases, with the greatest number observed in erd. pcna labeling largely recapitulated trends in phh labeling over time, and the highest numbers of pcna-positive cells were identified in erd. confocal microscopy demonstrated conclusively the co-localization of phh with the rod-specific protein rho, and confirmed that cells undergoing proliferation in all diseases were rod prs. the higher overall numbers of phh3-positive than pcna-positive cells suggests that the majority of proliferating cells in the onl are in the m-phase of the cell cycle for all diseases. in xlpra and particularly in rcd there is marked pr proliferation that is many times higher than that found in normals. in both, proliferation is sustained for the wk period of observation. the magnitude of pr proliferation in the two diseases parallels cell death kinetics based on tunel labeling, which is greater in rcd balance between cell proliferation and death in these two diseases does not occur, and the onl progressively thins beginning at early time points. the overall lack of co-localization of tunel- and phh3-labeled pr cells indicated that different cells undergo proliferation and apoptosis, and that the two events are not linked in a common pathway. furthermore, the large differences between the number of cells labeled with phh and mad and the smaller number of cells that co-express both markers in the mutant retinas emphasizes that the cells entering the m-phase of the cell cycle do not undergo mitotic checkpoint arrest, but continue to divide, thus contributing new pr cells to the onl. in contrast, the control retina showed only a very small number of phh positive cells, and all of these co-expressed mad in rd mice, proliferating cells in the onl have been reported to be microglia in origin, based on f4/ immunostaining. in contrast, although the mutant canine retinas showed an increased numbers of cd18-positive microglial cells and processes, these did not co-localize with phh labeling. in addition, gs-positive müller cell labeling did not co-localize with the dividing pr cells. while the methods used cannot exclude completely a contribution by microglia and/or müller cells to the dividing cell population in the onl, the results indicate that such a contribution, if present, is minor. it is possible that some of the phh3-labeled müller cells in the vitreal border on the inl in xlpra and rcd, proliferated and migrated into the onl. however, prior studies have shown that pax positive müller glia can express cell cycle markers, but not proliferate. all together, our results show that in the onl of the canine diseases there are distinct subsets of pr cells one that is dying, while the other is dividing. a recent study showed that in several rodent models of retinal degeneration there is a reactivation of cell cycle genes before the onset of apoptosis. these findings were in accord with previous studies of neurodegenerative diseases, e.g. alzheimer disease, parkinson’s disease and others, where aberrant reentry of susceptible neuronal populations into the cell cycle is associated with the death of post-mitotic, terminally differentiated neurons. in the retina of the rodent models, nuclear expression of cdk cdk and cdk as well as phosphorylation of rb precede pr death. furthermore, deleting e2f in the rd model resulted in a transient neuroprotection, whereas deletion of bmi the upstream activator of e2f and cdks, resulted in a marked delay in pr loss. at least in the rodent retinas, re-activation of these cell cycle proteins resulted in pr cell death, but not pr mitosis. unlike these rodent models, pr proliferation is readily observed in early-onset canine models of retinal degeneration. to begin to assess the mechanistic basis for pr proliferation, we first examined selected genes involved in different phases of the cell cycle. we find that at and wks, the first and last time points examined, mrna expression of rb and e2f are significantly up-regulated in xlpra and rcd and protein levels of prb and e2f were increased in all diseases. expressions of cdk, also are up-regulated at the same time points, particularly in rcd but cdk expression is down-regulated. indeed, western analysis of cdk showed increased expression at wks in mutants. furthermore, mrna expression changes in cyclins were quite uniform across all diseases; ccna and ccne were up-regulated at wks, while increased expression of the other cyclins was found at the wk time point. these data were confirmed by western analysis, showing comparable results at the mrna and protein levels. these results indicate that in the mutant canine retinas there is a dysregulation of cell cycle gene expression in comparison to the normal retina. even though the diseases investigated are primary pr degenerations, we also examined the rpe expression changes of selected genes, to determine potential differences or commonalities. we find altered mrna expression patterns in the rpe at time points when this cell layer is not affected. at wks of age, de genes were primarily up-regulated, and included cdk ccna and ccne which showed increased expression levels in the three diseases. at 7 weeks, on the other hand, statistically higher mrna levels were observed mainly in erd, and included ndr and lats while, as expected, stk38l expression of exon deleted in the disease, was absent. other than ccne there was poor correlation between the retina and rpe mrna expression findings at comparable ages. these results are not unexpected given that the prs are affected by the disease. indeed, changes in the rpe occur in response to pr degeneration or alternatively could be due to changes in contaminating tissue, including choroid, microglia/macrophages and white blood cells trapped in the highly vascular choroid. however, in contrast to the retina, the observed differences in rpe cell cycle gene expression in the diseases suggests an activation of pathways that lead to the long-term survival. this hypothesis warrants further studies that could also be performed on primary rpe cell cultures. while the mrna and protein expression studies indicate activation of a subset of cell cycle genes in proliferating pr, some of the ihc results are more difficult to reconcile with these findings given the apparent lack of specificity of several of the antibodies. in tissue sections, we could not assess expression of prb cdk cdk ccna ccnb and ccnd moreover, in none of the mutant tissues that showed phh3-labeled proliferating prs did we find labeling with e2f ccnd or ccne in the onl, the expected site of these cellular events. obviously, both qrt-pcr and western analyses include the entire neuroretina, while proteins with ihc were more precisely localized to specific cellular layers, but quantification of labeling intensity is qualitative. these data suggest that the cells expressing different levels of cell cycle-related transcripts may not be the ones that are dividing, thus are not prs. the specific analysis of these questions will require single cell analysis of pr populations. in contrast to the retina, ihc of the rpe clearly showed expression of several of the cell cycle gene products, e.g. e2f and ccnd labeling intensity was greater in the mutants vs. controls, and there was a general increase in label intensity in the older affected animals of each genotype. this evidence appears to confirm an activation of specific cell cycle related pathways in rpe that might promote survival of these cells. most notably, we find unique changes in the expression of ndr kinases and hippo signaling genes in erd-mutants, a disease caused by a mutation in the ndr kinase stk38l. we found an up-regulation of the ndr kinases and hippo signaling pathway members mob1a, lats and ndr lats is a tumor suppressor gene expressed in vertebrate retina and stimulates the kinase activity of stk38l upon mob1a binding and subsequent activation after association with s100b. the increased expression of lats in erd may be an attempt to suppress the aberrant pr proliferation or to induce apoptosis, as several studies have reported this particular feature of this gene. lats and os- co-localization in the pr inner and outer segments was observed with ihc exclusively in erd, and confirms the previous finding that aberrant rod/s-cone hybrids are generated following pr proliferation in erd-mutants. finding marked down-regulation of expression of nrl in erd is also consonant with its known function in pr cell fate specification. nrl knockout mouse retinas are almost entirely composed of s-cone prs. given that the proliferating population of prs in the erd retina resulted in l/m-cone and hybrid rod/s-cones, the decreased expression of nrl is not surprising. the hippo pathway controls organ size via the regulation of apoptosis and proliferation. it interacts in cell cycle control by cdk phosphorylation of yap, a downstream hippo pathway effector, and cyclin e is a known hippo pathway target gene. indeed, the hippo pathway, via mob1a activation of lats tumor suppressor can negatively regulate cell proliferation by modulating cyclin a activity. however, as we found that expression changes in cdk ccne ccna and ccna were not specific to erd, but were comparable also in xlpra and rcd we expect that the observed retinal changes in these genes did not result from the altered hippo signaling pathway.  in summary, our results show that in early phases of three retinal degenerative diseases in dogs, phh labeling is present in the outer nuclear layer, an indication that a subset of pr cells have entered the m-phase of the cell cycle. these cells are of rod origin, and there is no evidence that the proliferating cells are microglia or müller cells. in erd, the proliferating cells transiently maintain the integrity of the onl in spite of high rates of concurrent apoptotic cell death in cells that are not proliferating. similar but lower levels of pr proliferation occur in xlpra and rcd but the high rate of apoptotic cell death in comparison to pr proliferation result in a more rapid loss of onl in the early stages of disease. associated with these events is a dysregulation of cell cycle genes that appears to occur in a different subset of retinal cells, that might play a primary or bystander role in the degenerative process. 
mitotic terminally differentiated photoreceptors are observed in early retinal degeneration, an inherited canine retinal disease driven by mutations in the ndr kinase stk38l. unique to erd, however, are mutation-specific changes in the expression of ndr kinases and hippo signaling members with increased expression of mob and lats in the newly generated hybrid rod/s-cones. changes in cyclin expression differed across all genes, diseases and time points analyzed, although ccna and ccne expression increased with age in the three models suggesting that there is a dysregulation of cell cycle gene expression in all three diseases. proliferating cells are rod prs, and not microglia or müller cells. our data raise the intriguing possibility that terminally differentiated normal prs are kept from dividing by ndr2-mob interaction. expression of the cell cycle related genes rb and e2f as well as cdk, was up-regulated, but changes were mutation-specific. canine modelscell cycleerdhippo pathwayphotoreceptor degenerationphotoreceptor mitosisrcd1xlpra2http://dx.doi.org/ /100000002national institutes of health ey06855aguirre gustavo d. http://dx.doi.org/ /100000002national institutes of healthey017549p ey001583aguirre gustavo d. http://dx.doi.org/ /100001116foundation fighting blindnessnih-merial fellowship and 2012alcon research institute awardvan sloun fundhope for visionissue-copyright-statement© the author 2016
 mitotic terminally differentiated photoreceptors are observed in early retinal degeneration, an inherited canine retinal disease driven by mutations in the ndr kinase stk38l.  we demonstrate that a similar proliferative response, but of lower magnitude, occurs in two other early onset disease models, x-linked progressive retinal atrophy and rod cone dysplasia. proliferating cells are rod prs, and not microglia or müller cells. expression of the cell cycle related genes rb and e2f as well as cdk, was up-regulated, but changes were mutation-specific. changes in cyclin expression differed across all genes, diseases and time points analyzed, although ccna and ccne expression increased with age in the three models suggesting that there is a dysregulation of cell cycle gene expression in all three diseases. unique to erd, however, are mutation-specific changes in the expression of ndr kinases and hippo signaling members with increased expression of mob and lats in the newly generated hybrid rod/s-cones.  our data raise the intriguing possibility that terminally differentiated normal prs are kept from dividing by ndr2-mob interaction. furthermore, they provide the framework for the selection of candidate genes for further investigation as potential targets of therapy. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords canine modelscell cycleerdhippo pathwayphotoreceptor degenerationphotoreceptor mitosisrcd1xlpra2http://dx.doi.org/ /100000002national institutes of health ey06855aguirre gustavo d. http://dx.doi.org/ /100000002national institutes of healthey017549p ey001583aguirre gustavo d. http://dx.doi.org/ /100001116foundation fighting blindnessnih-merial fellowship and 2012alcon research institute awardvan sloun fundhope for visionissue-copyright-statement© the author 2016 