A novel retinal degeneration locus identified by linkage and comparative mapping of canine early retinal degeneration.

Early retinal degeneration (erd) is an early onset progressive retinal atrophy, a hereditary canine retinal disease phenotypically similar to human retinitis pigmentosa (RP). In previous efforts to identify the erd locus, canine homologs of genes causally associated with RP in humans, such as opsin (RHO), the beta-subunit gene for cyclic GMP phosphodiesterase (PDE6B), and RDS/peripherin, were excluded. A genome-wide screen was undertaken on canine families segregating the erd disease. Analysis of over 150 canine-specific markers has localized erd to a single linkage group comprising two previously identified canine linkage groups, 20 and 26, corresponding to canine radiation hybrid groups RH.34-a and RH.40-a. Multipoint analysis places erd in the interval between marker FH2289 (distance 23.6 cM) and FH2407 (5.9 cM) with a lod score of 12.23. Although the erd linkage group has not been assigned to an identified canine chromosome, conserved synteny of this linkage group with human 12p13-q13 suggests several candidates for erd and identifies a novel retinal degeneration locus. The rapid progress now occurring in canine genetics will expedite identification of the genes and molecular mechanisms underlying the inherited traits and diseases that make the dog a unique asset for study of mammalian traits.     

Identical mutation in a novel retinal gene causes progressive rod-cone degeneration in dogs and retinitis pigmentosa in humans

Progressive rod-cone degeneration (prcd) is a late-onset, autosomal recessive photoreceptor degeneration of dogs and a homolog for some forms of human retinitis pigmentosa (RP). Previously, the disease-relevant interval was reduced to a 106-kb region on CFA9, and a common phenotype-specific haplotype was identified in all affected dogs from several different breeds and breed varieties. Screening of a canine retinal EST library identified partial cDNAs for novel candidate genes in the disease-relevant interval. The complete cDNA of one of these, PRCD, was cloned in dog, human, and mouse. The gene codes for a 54-amino-acid (aa) protein in dog and human and a 53-aa protein in the mouse; the first 24 aa, coded for by exon 1, are highly conserved in 14 vertebrate species. A homozygous mutation (TGC --> TAC) in the second codon shows complete concordance with the disorder in 18 different dog breeds/breed varieties tested. The same homozygous mutation was identified in a human patient from Bangladesh with autosomal recessive RP. Expression studies support the predominant expression of this gene in the retina, with equal expression in the retinal pigment epithelium, photoreceptor, and ganglion cell layers. This study provides strong evidence that a mutation in the novel gene PRCD is the cause of autosomal recessive retinal degeneration in both dogs and humans.     

Photoreceptor cell death, proliferation and formation of hybrid rod/S-cone photoreceptors in the degenerating STK38L mutant retina

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene.     

Altered miRNA expression in canine retinas during normal development and in models of retinal degeneration

Background

Although more than 246 loci/genes are associated with inherited retinal diseases, the mechanistic events that link genetic mutations to photoreceptor cell death are poorly understood. miRNAs play a relevant role during retinal development and disease. Thus, as a first step in characterizing miRNA involvement during disease expression and progression, we examined miRNAs expression changes in normal retinal development and in four canine models of retinal degenerative disease.

Results

The initial microarray analysis showed that 50 miRNAs were differentially expressed (DE) early (3 vs. 7 wks) in normal retina development, while only 2 were DE between 7 and 16 wks, when the dog retina is fully mature. miRNA expression profiles were similar between dogs affected with xlpra2, an early-onset retinal disease caused by a microdeletion in RPGRORF15, and normal dogs early in development (3 wks) and at the peak of photoreceptor death (7 wks), when only 2 miRNAs were DE. However, the expression varied much more markedly during the chronic cell death stage at 16 wks (118 up-/55 down-regulated miRNAs). Functional analyses indicated that these DE miRNAs are associated with an increased inflammatory response, as well as cell death/survival. qRT-PCR of selected apoptosis-related miRNAs (“apoptomirs”) confirmed the microarray results in xlpra2, and extended the analysis to the early-onset retinal diseases rcd1 (PDE6B-mutation) and erd (STK38L-mutation), as well as the slowly progressing prcd (PRCD-mutation). The results showed up-regulation of anti-apoptotic (miR-9, -19a, -20, -21, -29b, -146a, -155, -221) and down-regulation of pro-apoptotic (miR-122, -129) apoptomirs in the early-onset diseases and, with few exceptions, also in the prcd-mutants.

Conclusions

Our results suggest that apoptomirs might be expressed by diseased retinas in an attempt to counteract the degenerative process. The pattern of expression in diseased retinas mirrored the morphology and cell death kinetics previously described for these diseases. This study suggests that common miRNA regulatory mechanisms may be involved in retinal degeneration processes and provides attractive opportunities for the development of novel miRNA-based therapies to delay the progression of the degenerative process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-172) contains supplementary material, which is available to authorized users.     

Distribution of protein kinase C immunoreactivity in rat retina

Summary A polyclonal antiserum to protein kinase C has been used to study the distribution of the enzyme antigenic sites in rat retina. The results indicate that the kinase is concentrated in photoreceptor outer segments as well as in the outer and inner plexiform layers. In identified components of retinal neuronal circuits, the kinase immunoreactivity is present in photoreceptor presynaptic terminals, in bipolar cell dendrites and axons, and probably in bipolar cell presynaptic terminals impinging on retinal ganglion cell dendrites. Thus, protein kinase C is positioned to play a role in specialized compartments of photoreceptor membrane and at both pre- and postsynaptic levels in the function of retinal neuronal circuits. Label in the nucleus is observed in retinal ganglion cells, but not bipolar or horizontal cells and probably not in amacrine cells. A role for protein kinase C in neuronal function at the level of the cell nucleus is therefore not likely to be universal, but to be determined by the particular properties of individual neuronal types.     

Function for Hedgehog genes in zebrafish retinal development

The hedgehog (hh) genes encode secreted signaling proteins that have important developmental functions in vertebrates and invertebrates. In Drosophila, expression of hh coordinates retinal development by propagating a wave of photoreceptor differentiation across the eye primordium. Here we report that two vertebrate hh genes, sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), may perform similar functions in the developing zebrafish. Both shh and twhh are expressed in the embryonic zebrafish retinal pigmented epithelium (RPE), initially in a discrete ventral patch which then expands outward in advance of an expanding wave of photoreceptor recruitment in the subjacent neural retina. A gene encoding a receptor for the hedgehog protein, ptc-2, is expressed by retinal neuroepithelial cells. Injection of a cocktail of antisense (alphashh/alphatwhh) oligonucleotides reduces expression of both hh genes in the RPE and slows or arrests the progression of rod and cone photoreceptor differentiation. Zebrafish strains known to have mutations in Hh signaling pathway genes similarly exhibit retardation of photoreceptor differentiation. We propose that hedgehog genes may play a role in propagating photoreceptor differentiation across the developing eye of the zebrafish.     

pipsqueak encodes a novel nuclear protein required downstream of seven-up for the development of photoreceptors R3 and R4.

Photoreceptor induction in the Drosophila eye is mediated by activation of the Ras signal transduction cascade. Although this process is well understood, little is known about how the diversity of photoreceptor subtypes is generated. The pipsqueak (psq) gene is expressed at high levels in the R3/R4 precursors during eye development and this expression depends on seven-up (svp) gene function. Moreover, strong psq alleles are dominant suppressors of a svp-induced cone cell transformation phenotype. Although the gene was previously identified and described as a member of the maternal posterior group of genes, the strong semilethal alleles isolated here demonstrate a specific requirement for psq function downstream of svp for the development of photoreceptors R3/R4. The gene has three independent 5' ends and codes for several nuclear protein isoforms, some containing the POZ domain which has been implicated in protein-protein interactions. Interestingly, all viable alleles with a maternal posterior group phenotype cluster around one specific 5' exon, while all semilethal alleles have lesions which map to a different alternative 5' exon.     

Roles of cell-intrinsic and microenvironmental factors in photoreceptor cell differentiation

Photoreceptor differentiation requires the coordinated expression of numerous genes. It is unknown whether those genes share common regulatory mechanisms or are independently regulated by distinct mechanisms. To distinguish between these scenarios, we have used in situ hybridization, RT-PCR, and real-time PCR to analyze the expression of visual pigments and other photoreceptor-specific genes during chick embryo retinal development in ovo, as well as in retinal cell cultures treated with molecules that regulate the expression of particular visual pigments. In ovo, onset of gene expression was asynchronous, becoming detectable at the time of photoreceptor generation (ED 5-8) for some photoreceptor genes, but only around the time of outer segment formation (ED 14-16) for others. Treatment of retinal cell cultures with activin, staurosporine, or CNTF selectively induced or down-regulated specific visual pigment genes, but many cognate rod- or cone-specific genes were not affected by the treatments. These results indicate that many photoreceptor genes are independently regulated during development, are consistent with the existence of at least two distinct stages of gene expression during photoreceptor differentiation, suggest that intrinsic, coordinated regulation of a cascade of gene expression triggered by a commitment to the photoreceptor fate is not a general mechanism of photoreceptor differentiation, and imply that using a single photoreceptor-specific "marker" as a proxy to identify photoreceptor cell fate is problematic.     

Genome-wide association and linkage analyses localize a progressive retinal atrophy locus in Persian cats

Hereditary eye diseases of animals serve as excellent models of human ocular disorders and assist in the development of gene and drug therapies for inherited forms of blindness. Several primary hereditary eye conditions affecting various ocular tissues and having different rates of progression have been documented in domestic cats. Gene therapy for canine retinopathies has been successful, thus the cat could be a gene therapy candidate for other forms of retinal degenerations. The current study investigates a hereditary, autosomal recessive, retinal degeneration specific to Persian cats. A multi-generational pedigree segregating for this progressive retinal atrophy was genotyped using a 63 K SNP array and analyzed via genome-wide linkage and association methods. A multi-point parametric linkage analysis localized the blindness phenotype to a ~1.75 Mb region with significant LOD scores (Z ≈ 14, θ = 0.00) on cat chromosome E1. Genome-wide TDT, sib-TDT, and case–control analyses also consistently supported significant association within the same region on chromosome E1, which is homologous to human chromosome 17. Using haplotype analysis, a ~1.3 Mb region was identified as highly associated for progressive retinal atrophy in Persian cats. Several candidate genes within the region are reasonable candidates as a potential causative gene and should be considered for molecular analyses.     

The CNTF/LIF signaling pathway regulates developmental programmed cell death and differentiation of rod precursor cells in the mouse retina in vivo

Natural cell death is critical for normal development of the nervous system, but the extracellular regulators of developmental cell death remain poorly characterized. Here, we studied the role of the CNTF/LIF signaling pathway during mouse retinal development in vivo. We show that exposure to CNTF during neonatal retinal development in vivo retards rhodopsin expression and results in an important and specific deficit in photoreceptor cells. Detailed analysis revealed that exposure to CNTF during retinal development causes a sharp increase in cell death of postmitotic rod precursor cells. Importantly, we show that blocking the CNTF/LIF signaling pathway during mouse retinal development in vivo results in a significant reduction of naturally occurring cell death. Using retroviral lineage analysis, we demonstrate that exposure to CNTF causes a specific reduction of clones containing only rods without affecting other clone types, whereas blocking the CNTF/LIF receptor complex causes a specific increase of clones containing only rods. In addition, we show that stimulation of the CNTF/LIF pathway positively regulates the expression of the neuronal and endothelial nitric oxide synthase (NOS) genes, and blocking nitric oxide production by pre-treatment with a NOS inhibitor abolishes CNTF-induced cell death. Taken together, these results indicate that the CNTF/LIF signaling pathway acts via regulation of nitric oxide production to modulate developmental programmed cell death of postmitotic rod precursor cells.     

PKG activity causes photoreceptor cell death in two retinitis pigmentosa models

Photoreceptor degeneration in retinitis pigmentosa is one of the leading causes of hereditary blindness in the developed world. Although causative genetic mutations have been elucidated in many cases, the underlying neuronal degeneration mechanisms are still unknown. Here, we show that activation of cGMP-dependent protein kinase (PKG) hallmarks photoreceptor degeneration in rd1 and rd2 human homologous mouse models. When induced in wild-type retinae, PKG activity was both necessary and sufficient to trigger cGMP-mediated photoreceptor cell death. Target-specific, pharmacological inhibition of PKG activity in both rd1 and rd2 retinae strongly reduced photoreceptor cell death in organotypic retinal explants. Likewise, inhibition of PKG in vivo, using three different application paradigms, resulted in robust photoreceptor protection in the rd1 retina. These findings suggest a pivotal role for PKG activity in cGMP-mediated photoreceptor degeneration mechanisms and highlight the importance of PKG as a novel target for the pharmacological intervention in RP.     

Identification and Characterization of Autosomal Genes That Interact with Glass in the Developing Drosophila Eye

The glass gene encodes a zinc finger, DNA-binding protein that is required for photoreceptor cell development in Drosophila melanogaster. In the developing compound eye, glass function is regulated at two points: (1) the protein is expressed in all cells' nuclei posterior to the morphogenetic furrow and (2) the ability of the Glass protein to regulate downstream genes is largely limited to the developing photoreceptor cells. We conducted a series of genetic screens for autosomal dominant second-site modifiers of the weak allele glass(3), to discover genes with products that may regulate glass function at either of these levels. Seventy-six dominant enhancer mutations were recovered (and no dominant suppressors). Most of these dominant mutations are in essential genes and are associated with recessive lethality. We have assigned these mutations to 23 complementation groups that include multiple alleles of Star and hedgehog as well as single alleles of Delta, roughened eye, glass and hairy. Mutations in 18 of the complementation groups are embryonic lethals, and of these, 13 show abnormal adult retinal phenotypes in homozygous clones, usually with altered numbers of photoreceptor cells in some of the ommatidia.     

Midkine-a Protein Localization in the Developing and Adult Retina of the Zebrafish and Its Function During Photoreceptor Regeneration

Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation.