Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

C C D

RPE

POS

PIS

ONL

OPL

INL

IPL

GCL

Figure 29.1. Histology of control and lasered mouse retinae at 3 days and 90 days post-argon laser photocoagulation. A, Control retina and B, lasered retina at 3 days post-treatment. C, Control retina and D, lasered retina at 90 days post-treatment. C, choroid; RPE, retinal pigment epithelium; POS, photoreceptor outer segments; PIS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Magnification 40¥. Scale bars represent 10 mm.

Late changes in the retina following laser photocoagulation include further extension of glial processes and denser areas of glial scars. The outer nuclear layer reduces in thickness over time (Figure 29.1D) and the number of apoptotic cells present in the laser lesion decreases. Interestingly, photoreceptors adjacent to laser lesions appear to have increased survival compared to photoreceptors in normal retinae as shown by an increase in basic fibroblast growth factor (bFGF)-immuno-reactive cells in this area (Xiao et al., 1998, 1999). The mechanism contributing to the increased survival of these photoreceptors may be due to the increase in bFGF, suppressing apoptosis in these cells.

The benefits of laser photocoagulation therapy are not without risks. Potentially dangerous complications such as haemorrhaging, corneal burns and the development of cataracts are risks in laser therapy. Ultimately, understanding the underlying molecular mechanisms which produce the beneficial effects of laser photocoagulation could lead to the development of non-destructive treatments, thereby circumventing these potential hazards.

3.2. Changes in Gene Expression

3.2.1. Early Changes

High oxygen demand in the diabetic retina is thought to play a role in the initiation and progression of microvascular changes. The development of areas of hypoxia stimulates the up-regulation of angiogenic factors such as vascular endothelial growth factor (VEGF) and angiopoietins, potentiating neovascularisation (Oh et al., 1999, Park et al., 2003, Yancopoulos et al., 2000). Laser therapy decreases the oxygen demand of the tissue by localised destruction of the photoreceptors and the consequential development of glial scars facilitates the diffusion of oxygen through the retina. It is also thought to diminish the stimulation of angiogenesis by photocoagulation of normal and abnormal vessels and/or stimulate the expression of anti-angiogenic factors.

Many studies have been conducted to examine the effects of laser photocoagulation on specific factors in the rat and mouse. These studies clearly demonstrated that laser photocoagulation does not only destroy oxygen-demanding photoreceptor cells within the laser lesions, but does have a very important and significant effect on the expression of genes within the retina. These include bFGF/FGF2, epithelial growth factor (EGF), transforming growth factor alpha and beta (TGFa, TGFb), insulin growth factor I (IGF-I), glial fibrillary acidic protein (GFAP), platelet derived growth factor (PDGF) and VEGF among others (Humphrey et al., 1997; Xiao et al., 1999). However, these factors are mainly associated with wound healing, an early response to the treatment.

With the advent of array-based gene expression studies, we can now examine the entire gene expression profile of any given tissue, identifying many novel associations and functions for both known and unknown genes. A previous study in our laboratory aimed to identify those genes that were affected by laser photocoagulation (Wilson et al., 2003). This study demonstrated the effect on gene expression of a normal mouse eye three days postargon laser photocoagulation. Angiogenic factors such as FGF14 and FGF16 were found to be down-regulated whilst angiotensin II type 2 receptor, a potent inhibitor of VEGF and VEGF-induced angiogenesis, was significantly up-regulated. As expected, proteins implicated in tissue remodelling and wound healing were also differentially expressed.

3.2.2. Late Changes

Few studies have followed the expression of factors past the initial wound healing response. Xiao et al. (1998) demonstrated a sustained increase (up to 180 days) in bFGF immuno-reactive photoreceptor cells adjacent to the laser lesions. GFAP-positive glial cells were also evident for more than 30 days post-treatment. Zhang et al. (1993) reported that RPE cells became aFGF and bFGF-positive while losing their CRALBP-immuno- reactivity for up to 80 days post laser treatment. These studies demonstrated some of the changes that occurred following laser photocoagulation and indicated that cells within these damaged areas can change the expression of factors controlling angiogenesis.

We sought to extend our earlier study to measure the changes in gene expression after laser photocoagulation long-term. The focus was on identifying genes that had a known functional relationship with angiogenesis. Ultimately, this study aimed to identify novel targets for diabetic retinopathy therapy whereby a directed change in expression would lead to a reduction in neovascularisation without the need to use lasers. Changes in gene expres-

sion at 90 days post-laser photocoagulation in the normal mouse were measured by microarray analysis and further examined by real-time PCR (Binz et al., 2005). At 90 days post laser photocoagulation, 107 genes were identified as differentially expressed compared to unlasered controls. Of these 107 genes, 34 had previously been identified as differentially expressed at three days post-treatment. Therefore, these genes demonstrated a true longterm change in expression due to laser photocoagulation.

Inducers of angiogenesis such as VEGF, PDGF, or bFGF were not differentially expressed, indicating that beneficial effects of laser photocoagulation do not stem from longterm changes to this pathway. However, this study identified genes previously associated with cytoskeletal and structural remodelling as differentially expressed at 90 days posttreatment and the level of protein measured corroborated this large increase. Interestingly, some of these genes were not differentially expressed at three days, suggesting there was a late stage of remodelling in the retina following initial wound healing and scar formation. Alternatively, these gene products could be functioning via a novel mechanism contributing to the beneficial effects of laser photocoagulation.

4. CONCLUSIONS

The goal of prevention and treatment of diabetic retinopathy requires the knowledge of factors and events that reduce or prevent neovascularisation. One approach to achieve this goal is to identify genes differentially expressed following successful laser photocoagulation. With the identification of genes initially affected by laser treatment and those whose expression remains changed long-term, we can now apply this knowledge to the diabetic retina. Ultimately, this will enable the development of therapeutic targets for long-term protection and prevention of vision impairment caused by chronic conditions such as diabetes.

5. ACKNOWLEDGEMENTS

We thank the Foundation for Fighting Blindness for C.E. Graham’s Young Investigator Award to attend the RD2004 Conference. The authors gratefully acknowledge financial support from the Juvenile Diabetes Research Foundation International, the Australian National Health and Medical Research Council and Westpac Foundation. This work is part of the research effort of the Diabetic Retinopathy Consortium, Perth, Western Australia.

6. REFERENCES

Binz, N., Graham, C. E., Simpson, K., Lai, Y. K. Y., Shen, W.-Y., Lai, C.-M., Speed, T. P. and Rakoczy, P. E., 2005, Long-term effect of therapeutic laser photocoagulation on gene expression in the eye, EMBO:Submitted.

Humphrey, M. F., Chu, Y., Mann, K. and Rakoczy, P., 1997, Retinal GFAP and bFGF expression after multiple argon laser photocoagulation injuries assessed by both immunoreactivity and mRNA levels, Exp Eye Res. 64:361-9.

Lewis, G. P., Erickson, P. A., Guerin, C. J., Anderson, D. H. and Fisher, S. K., 1992, Basic fibroblast growth factor: a potential regulator of proliferation and intermediate filament expression in the retina, J Neurosci. 12:396878.

Mainster, M. A., 1999, Decreasing retinal photocoagulation damage: principles and techniques, Semin Ophthalmol. 14:200-9.

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Oh, H., Takagi, H., Suzuma, K., Otani, A., Matsumura, M. and Honda, Y., 1999, Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells, J Biol Chem. 274:15732-9.

Park, Y. S., Kim, N. H. and Jo, I., 2003, Hypoxia and vascular endothelial growth factor acutely up-regulate angiopoietin-1 and Tie2 mRNA in bovine retinal pericytes, Microvasc Res. 65:125-31.

Petrovic, V. and Bhisitkul, R. B., 1999, Lasers and diabetic retinopathy: the art of gentle destruction, Diabetes Technol Ther. 1:177-87.

Porta, M. and Allione, A., 2004, Current approaches and perspectives in the medical treatment of diabetic retinopathy, Pharmacology & Therapeutics. 103:167-77.

Roider, J., El Hifnawi, E. S. and Birngruber, R., 1998, Bubble formation as primary interaction mechanism in retinal laser exposure with 200-ns laser pulses, Lasers Surg Med. 22:240-8.

Wilson, A. S., Hobbs, B. G., Shen, W.-Y., Speed, T. P., Schmidt, U., Begley, C. G. and Rakoczy, P. E., 2003, Argon Laser Photocoagulation-Induced Modification of Gene Expression in the Retina, Invest Ophthalmol Vis Sci. 44:1426-34.

Xiao, M., McLeod, D., Cranley, J., Williams, G. and Boulton, M., 1999, Growth factor staining patterns in the pig retina following retinal laser photocoagulation, Br J Ophthalmol. 83:728-36.

Xiao, M., Sastry, S. M., Li, Z. Y., Possin, D. E., Chang, J. H., Klock, I. B. and Milam, A. H., 1998, Effects of retinal laser photocoagulation on photoreceptor basic fibroblast growth factor and survival, Invest Ophthalmol Vis Sci. 39:618-30.

Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J. and Holash, J., 2000, Vascular-specific growth factors and blood vessel formation, Nature. 407:242-8.

Zhang, N. L., Samadani, E. E. and Frank, R. N., 1993, Mitogenesis and retinal pigment epithelial cell antigen expression in the rat after krypton laser photocoagulation, Invest Ophthalmol Vis Sci. 34:2412-24.

Figure 30.1. (a) Retinal histology of transverse cryosections of 5 dpf zebrafish stained with methylene blue and Azure II. ON, optic nerve; RPE, retinal pigment epithelium; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (b), (c) Merged fluorescent imaging of GFP in GFP-transgenic zebrafish retinal cryosection, and immunohistochemistry with anti-rhodopsin antibody visualised with Cy3-conjugated secondary antibody.

post-fertilisation (hpf ), the retina is differentiated at 72 hpf and there is a measurable visual response at 120 hpf. This rapid development makes zebrafish suitable for the study of all stages of retinal development. Ex utero development and larval transparency means that gene expression patterns may be observed in wholemount zebrafish larvae. Established behavioural tests exist for fast and accurate assay of visual transduction in zebrafish (Brockerhoff et al., 1995; Neuhauss et al., 1999). Electroretinography (ERG) though a more invasive and more time-consuming assay enables detailed characterisation of visual function (Brockerhoff et al., 1995).

Zebrafish are a cost-effective model organism. Adult zebrafish, being ~3 cm in length, can be maintained at high densities in multiple tanks, which allows for the establishment of a large experimental population. This population can produce large numbers of offspring, since a given mating pair can routinely produce batches of 150 eggs every 3-4 days. Zebrafish are amenable to transgenesis, whereby foreign DNA sequences injected into developing embryos are incorporated into the genome. The first transgenic zebrafish were generated in 1988, demonstrating the inheritance of an antibiotic cassette (Stuart et al., 1988). Transgenic zebrafish lines are now routinely used to study diverse aspects of zebrafish development and physiology.

2. GENERATION OF TRANSGENIC ZEBRAFISH

Transgenic zebrafish are generated by injecting transgenes into newly fertilised zebrafish eggs. These embryos are raised to adulthood (~3 months) and founder zebrafish that transmit the transgene through the germline are identified by PCR and/or reporter gene expression.

Zebrafish routinely mate at daybreak, which is artificially simulated in laboratory conditions by the onset of a light-dark cycle. The evening before embryo injection, male and female adults are placed in mating tanks. These tanks have a gridded insert to aid collection of the eggs, and an optional barrier insert to separate male and female zebrafish. This

Figure 30.2. Schematic showing the three modes of delivery of a transgene for germline integration. (a) linearised DNA constructs are injected alone. (b) DNA constructs are packaged into retrovirus for infection. (c) DNA constructs are flanked by transposon inverted repeats and co-injected with in vitro transcribed transposase mRNA. The schematic shows the major components of the microinjection apparatus. Transgenes are represented by black bars, and inverted repeats by black triangles.

insert enables the investigator to delay matings so that newly fertilised eggs can be collected at several intervals. DNA is delivered for transgenesis using fine glass needles that can pass easily through an egg’s chorion and yolk into the animal pole. These needles are filled with a DNA and a tracer dye, usually phenol red. To optimise transgene integration, eggs are injected at the 1- or 2-cell stage. The eggs are immobilised in an agarose trough, a micromanipulator is used to guide the needle into the animal pole, and a microinjector used to inject the DNA solution. Between 50 and 70% of injected eggs routinely survive the procedure.

DNA injected for transgenesis can integrate into the genome by random insertion, by transposase-mediated insertion, or by retroviral insertion (Fig. 30.2). During random integration, injected DNA assembles into concatemers consisting of many head-to-tail copies of the construct. The zebrafish’s DNA repair machinery targets the free double-stranded ends and incorporates the concatemers into the genome, usually, at a single, random point. The transgene can integrate into native enhancer or repressor regions, which can distort the levels and patterns of transcription driven by the promoter. The major disadvantage to this method is the low efficiency of transgene integration. It is advantageous, however, since it can be performed without labour-intensive cloning, as even PCR amplicons can be injected. Retroviral systems can also generate transgenic zebrafish (Gaiano et al., 1996). The transgenic DNA is packaged by a viral packaging line, and delivered using a separate viral vehicle. Integration is brought about by infection of the zebrafish cells with the transgenic DNA. The disadvantages to this method are that the generation of high-titre viral stocks can be technically difficult and requires specialised safety procedures. The advantages are that the retrovirus inserts the transgene into the genome with high efficiency.

Most recently, technologies have been developed to generate transgenic zebrafish by transposition. In this procedure the transgene is flanked with inverted repeats from the Tol2

transposable element from the medaka zebrafish, Oryzias latipes (Kawakami et al., 1998). The construct is injected as a circular plasmid along with in vitro-synthesised transposase mRNA. The zebrafish’s ribosomes produce active transposase, which enzymatically integrates the transgene into multiple sites of the genome. The initial cloning steps can be problematic, since the inverted-repeat regions are prone to random rearrangements during bacterial passage, and the transposase mRNA must be synthesised and protected from degradation. However, the Tol2 transposase based system is highly efficient, with transgenes integrating in greater than 50% of injected zebrafish (Kawakami et al., 2000).

3. TRANSGENIC ZEBRAFISH TECHNOLOGY APPLIED TO THE RETINA

There are several transgenic zebrafish lines and tissue/cell-specific promoters available to study the retina. These lines/promoters can be used to study temporal and spatial gene regulation, to characterise gene function by overexpression and to report on retinal cell/tissue integrity in genetic and pharmacological screens.

Transgenic zebrafish have been successfully applied to characterise the temporal and spatial regulation of genes in vivo. The patterning and timing of the expression of a gene can be characterised by fusing reporters (e.g. EGFP) to putative promoter regions and generating transgenic zebrafish transmitting this construct. Expression of fluorescent transgenes can be analysed in anaesthetised zebrafish by fluorescence microscopy. Phenyl-thio-urea (0.003%) added to embryo media prevents pigment formation, facilitating the analysis of fluorophores in transparent larvae up to 8 days post-fertilisation. Apart from generating stable transgenic lines, reporter constructs can also be used to test promoter activity using transient expression assays. By analysing approximately one hundred zebrafish injected with constructs containing promoter deletions/mutations, it is possible to quickly locate cis-acting enhancer or repressor elements that confer tissueor temporal-specific expression (Luo et al., 2004).

The line Tg(1.2ZOP-EGFP) expresses enhanced GFP (EGFP) under the control of 1.2 kb of promoter sequence from the zebrafish rod opsin gene (Kennedy et al., 2001). EGFP is expressed exclusively in rod photoreceptors, and exhibits a similar developmental expression profile to the rod opsin protein. Similarly, the line Tg(ZUV-GFP) expresses GFP under the control of a UV opsin promoter, which directs expression solely to UVcones (Luo et al., 2004). This line was used to analyse proximal and distal elements of the UV opsin promoter, and showed that a UV opsin promoter element could direct ectopic expression of a chimeric rhodopsin promoter to UV cones. Promoters from other species can also be used to study the zebrafish retina, such as a Xenopus laevis rod opsin promoter (Perkins et al., 2002).

Another application of transgenic zebrafish technology is enhancer trapping. This process takes advantage of the random nature of transgene integration. Constructs consisting of GFP coupled to a minimal promoter are injected into zebrafish embryos. Integration of the transgene near strong cis-acting elements can result in zebrafish expressing EGFP in spatial/temporal expression dictated by the cis-element and easily visualised in the transparent embryos by fluorescent microscopy.

Expression of GFP in nascent cells can be used to plot the migration of cells during development of primordial structures. Constructs highlighting the migration of presumptive cells within the eye can give insights into signalling pathways. A wave of differentiation

generating ganglion and amacrine cells in the retina during neurogenesis has been imaged using GFP under the regulation of a sonic hedgehog promoter (Shkumatava et al., 2004).

Promoters that direct expression of transgenes in the retina can also be used to characterise gene function. Expression of foreign genes in zebrafish allows novel genes to be characterised by analyzing phenotypes associated with overexpression of that gene. Transgenic zebrafish technologies can also “rescue” mutant/disease phenotypes. The lakritz mutant, which has a mutation in ath5, an eye-specific transcription factor, causes elimination of the ganglion cell layer (Kay et al., 2001). Injection of a plasmid carrying the unmutated gene under control of its native promoter rescues the wild type phenotype, confirming that the mutant phenotype is caused by the mutation. Similarly, a nonsense mutation in the gene for the rx3 transcription factor causes an eyeless phenotype, which can be rescued by the injection of constructs carrying the wild type gene (Kennedy et al., 2004).

The low efficiency of transgenesis by random integration previously impeded the generation of transgenic lines. To overcome this, existing lines often took advantage of dual expression of both an effector transgene and a reporter gene for easy screening. Transgenic lines exist that express both EGFP and protein kinase A (PKA), or EGFP and glycogen synthase kinase-3b (GSK-3b), under the control of a retinal ganglion cell (RGC) promoter (Yoshida and Mishina, 2003). These lines allow characterisation of the promoter, clarification of the roles of PKA and GSK-3b, and efficient screening owing to the concomitant expression of the reporter gene. Likewise, transgenic expression of a Gap43-GFP fusion protein allows visualisation of dynamic behaviour of GFP-labeled amacrine cell neurites in vivo from the earliest stages of neurite outgrowth (Kay et al., 2004). The recent emergence of an efficient transposase-mediated system for transgenesis eliminates the need for a reporter to be co-injected with the effector transgene.

4. LIMITATIONS AND FUTURE DEVELOPMENTS

Transgenic zebrafish technology has advanced such that transgenic zebrafish can now be routinely generated in-house. Transgenic zebrafish will provide powerful tools for functional genomics, for modelling human diseases and for drug discovery. However, a number of limitations exist, particularly the current inability to apply homologous recombination to generate targeted knockouts/knockins in zebrafish.

Standard approaches to understand gene function include analysing “gain-of function” and “loss-of function” phenotypes. In zebrafish the most common method to overexpress a gene (gain-of function) is by microinjection of embryos with in vitro-synthesised mRNA. However, as the mRNA is translated throughout the developing larvae, this assay may be misinformative. Also, it is difficult to limit the temporal onset of expression of injected mRNAs, which are typically degraded by 5 dpf. Thus, it is desirable to express a transgene under regulation of a promoter that drives specific temporal and spatial expression patterns. Though a few promoters have been identified that direct specific patterns of expression in the zebrafish retina, there is a need to identify specific promoters for other cell types so that these can be comprehensively studied. In addition, there is a need for inducible expression of transgenes in the retina. A heat-shock promoter can induce selective expression when coupled with highly localised laser treatment (Halloran et al., 2000). In the future transgenic lines generated from retinal-specific promoters coupled with the Tet ON/OFF system will provide greater flexibility for inducible expression of transgenes in the zebrafish retina.

Analysis of phenotypes arising from the specific reduction of gene expression (loss-of- function) is a key method to understanding gene function. In zebrafish, loss-of-function phenotypes have been generated by morpholino “knockdown” or by mutagenesis screening (Warren and Fishman, 1998; Nasevicius and Ekker, 2000). However, neither of these approaches are designed to target selective and complete eliminate of the expression of a specific gene. The gold standard of “loss-of-function” analyses, i.e. targeted knockout by homologous recombination, is currently not feasible in zebrafish. Though cells displaying embryonic stem cell properties and of producing zebrafish germ-line chimeras from embryo cell cultures have been reported the long-term culture of zebrafish embryonic stem cells required for selection of homologous recombinants has been elusive though concerted efforts to overcome this hurdle continue ((Ma et al., 2001; Fan et al., 2004). Once this is achieved other variations including the Cre/lox binary system for conditional knockouts will become feasible. As a result, targeted knockout lines of zebrafish cannot be generated, currently. The ability to make directed knockout lines will facilitate reverse genetic studies accelerating the characterisation of genes whose function is either partially or wholly unknown.

Currently available transgenic technologies in zebrafish now enable the generation of transgenic models of human retinopathies, especially to study the approximately 50 genes associated with dominant forms of inherited human blindness (http://www.sph.uth.tmc.edu/Retnet/). Currently, promoters exist for expressing transgenes in rod photoreceptors, cone photoreceptors, or in ganglion cells, and many more promoters for other retinal cells will become available in the future. Such zebrafish models will aid in understanding the molecular pathogenesis of disease. In addition, it is envisaged that zebrafish models of human retinopathies can be used in pharmacological screens to identify drugs that modulate the disease. The features of small size, rapid development, larval transparency and large offspring numbers that are inherent to zebrafish are also beneficial to pharmacological screens.

5. ACKNOWLEDGEMENTS

The authors wish to thank Susan Brockerhoff, Tom Vihtelic, and David Hyde for providing photographic figures.

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