Development Of The Vertebrate Retina Biology Essay

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The vertebrate retina is a multilayered neural tissue and is derived from the inner surface of the optic vesicle via cell migration and cell differentiation. In the early stages of embryonic eye development, the anterior neural tube evaginates to form paired optic vesicles, in the region of the diencephalon (forebrain) (Chow and Lang, 2001). When the optic vesicles contact the ectoderm, inductive events take place to cause the epithelium to form a lens placode (Grainger et al, 1997). The lens placode fold inwards and ultimately becomes the lens. During these events, the optic vesicle invaginates and forms a bilayered cup, the optic cup. Cells within the inner layer of the optic cup undergo proliferation to form a dense neuroblastic layer, containing undifferentiated multipotential retinal progenitor cells (RPCs). When lineages of these cells were analysed using retroviral infection they were found to have an ability to generate variety of ganglion cells, glia, interneurons and light sensitive photoreceptor neurons (Capko, 1993; Turner and Cepko, 1987). Collectively, the cells constitute the multi-layered neural retina. The neural retina is specialised for the reception and transduction of light energy from visual image of the environment, and for the generation and integration of the neural responses.

In most vertebrate tissues including fish, frogs and birds, the RPCs are organised into a zone of stem cells called the ciliary or circumferential marginal zone (CMZ), located at the transition between the neural retina and ciliary epithelium of marginal retina. However, no evidence for RPCs organisation in CMZ in mammals has been found yet (Close et al., 2005; Moshiri and Reh, 2004; Kanika et al., 2007). An interesting fact is that the proliferation and differentiation of the RPCs are precisely regulated, and cells located in the periphery of the neural retina proliferate for longer period than the ones in the central region, as found from the classical autoradiography studies (Kubo et al, 2005).

The retinal ganglion cells are neurons whose axons send electrical impulses to the brain. The axons of the retinal ganglion cells meet at the base of the eye and travel down the optic stalk, which is then known as the optic nerve.

The outer layer of the optic cup produces melanin pigment that differentiates into a mono-layered retinal pigmented epithelium (RPE). The RPE lines the back of the neural retina, and pigmented layer of the ciliary epithelium. In other words, it lies in direct apposition with the apical surface of the RPCs. RPE is thought to be involved in nutrient exchange, phagocytosis of photoreceptor discs after shedding, and absorption of stray light.

The different cell fates (formation of neural retina and RPE) are not immediately fixed, and apparently there is a period of development during which the fates can be reversed. Experimental studies in mammals and chicks show that cells fate is restricted either to the neural retinal cell or RPE, shortly after the formation of the optic cup. In contrast, the cell fate in the amphibians is unfixed during development and the RPE have a potential to regenerate neural retina after damage (Fuhrmann et al, 2000).

Figure 1. D (A) The optic vesicle contacts the overlying ectoderm, inducing a lens placode. (B) As the optic vesicle invaginated the overlying ectoderm differentiates into lens cells (C) The optic vesicle becomes the pigmented and neural retina, as the lens is internalised.

Structure of the retina

The retina is highly accessible light sensory region and covers the interior of the eye. As a consequence of its embryological origin, the adult vertebrate retina has a relatively simple laminated structure comparable to that of the cerebral cortex of the brain (Ramon y Cajol 1892; Rodieck, 1998). One cannot disregard that retina is formed as a projection of the neuroectoderm, which develops into the central nervous system (CNS), therefore retina is always considered as part of the CNS. Due to its simplicity, retina serves as an excellent model to study the molecular mechanisms of the CNS, such as cell fate specification and synapse formation (Purves et al, 2004).

All known vertebrate retinas are organised according to the same basic plan. Figure 2 is a diagram of the typical cell types of retinal neurones and organisation of retinal layers. The retina consists of at least five major types of retinal neurons (photoreceptor, bipolar, horizontal, amacrine, and ganglion cells) and non-neuronal glial cells (Muller cells). The constituent cells are arranged in a penta-laminar array; three nuclear layers [outer nuclear layer (ONL), inner nucleur layer (INL) and ganglion cell layer (GCL)] and two synaptic layers [outer plexiform layer (OPL) and inner plexiform layer (IPL)] (Cajal, S.R, 1972).

Light, entering the eye, passes through the retina and is captured by photoreceptor cells [rod (R) and cone (C)], whose somata are located at the ONL in a layer of photoreceptor cell (RCL). The photoreceptor cells absorb light energy, convert this into electrical responses (phototransduction), and transmit them as visual information to second-order neurons, such as bipolar cella (B) and horizontal cells (H). The OPL is the first synaptic zone of the retina where photoreceptor, bipolar and horizontal cells are connected. The bipolar cells receive input from photoreceptor cells and transmit electrical signals to third-order neurons, such as amacrine cells (A) and rerinal ganglion cells (RGC). The INL contains somata of horizontal, bipolar, and amarcine cells. The IPL is the second synaptic zone of the retina where bipolar, amacrine and ganglion cells interact. Ganglion cells whose somata located in the GCL encode electrical signals into action potentials, and transmit them to the brain via the axons, which are bundled together in the optic nerve. When the sensory signals are passed back from the brain to the RGCs it has to channel through two 'check posts', the first involves amacrine cells (AC) which are found in between the BPs and RGCs, and the second is horizontal cell (HC), orientated between BPs and photoreceptors (Trevor et al, 2008). Horizontal cells extend processes widely in the OPL, and mediate lateral interactions within the first synaptic zone whereas amacrine cells, like horizontal cells, extend processes widely in the IPL, and mediate lateral interaction within it. The predominant type of glial cell in the vertebrate retina is called the Müller cell. These cells extend vertically through the retina from the distal margin of the ONL to the inner margin of the retina.

Fig 2. Shows a highly specialised structure of the retina (Junqueira & Carneiro, 2005).

Figure 2. The adult vertebrate retina. In this representation, light enters from the top of the diagram (i.e., at the vitreal border). The main cell types are rods (R), cones (C), horizontal cells (H), bipolar cells (B), amacrine cells (A), and ganglion cells (G). At the bottom of the figure is the RPE. Its finger-like cellular processes interact with the photoreceptors.

The entire cell-population in the development of retina is produced during a short time span and although these cells are born in sequential order there is a considerable overlap in the cell lineage. According to Young (1985), the sequential order of generation of these cells begin with retinal ganglion cells, cone photoreceptor cells, horizontal cells and 50% of the amacrine cells in the first wave of differentiation, then followed by formation of rod photoreceptors, bipolar cells, müller glia and the rest of amacrine cells. It is believed that the mechanism of intrinsic and extrinsic patterning is reflected by the chronological control of retinal cell type development. So can retinal cells regenerate? Well, unlike fish and amphibians, the damage to the retina causes irreversible visual impairment in mammals, due to its lack of regenerative potential (Raymond and Hitchcock, 2000).

Wnt signaling pathway

Wnts are family of 19 cysteine-rich glycoproteins, which build-up in the extracellular matrix to activate pathways in adjacent cells. The activation of pathways is triggered when Wnt ligands bind and activate an appropriate receptor that belong to a family of frizzled receptors, a member of seven transmembrane domain proteins of which there are 10 members. Previous studies have shown that there is a requirement of co-receptor, lipoprotein receptor-related proteins 5 and 6 (LRP5/6), to activate Wnt signaling pathways, and blockage of such pathways occurs when LRP5/6 is bound to a secreted inhibitory protein, Dickkopf-1 (Zerlin et al, 2008). So far, Wnt signaling has been studied from a variety of organisms, including mouse, fly, zebrafish, xenopus laevis, and using mammalian cultured cells.

The Wnt signaling pathway is an important intracellular communication pathway which is involved in many cell biological and developmental processes during embryogenesis and adult tissues, ranging from body axis determination and axonal outgrowth to cellular proliferation and differentiation. (Moon et al. 2004; Wodarz et al 1998).

Figure 3. Canonical Wnt signaling pathway. (A) Binding of Wnt ligands to Frizzled and LRP5/6 activates Dsh, leading to inhibition of GSK3β and APC, which causes translocation of β-catenin to the nucleus by stabilising it. β-catenin then interacts with TCF/LEF, and activates the transcription of the Wnt target genes. (B) When Wnt ligand is not present, phosphorylation of β-catenin via Gsk3β occurs, followed by β-catenin degradation via proteosome. Inhibition of transcription of Wnt target protein is a result of interactions between TCF/LEF and co-repressors, CtBp and Groucho.

There are several different signaling cascades activated by Wnts, including the β-catenin-dependent pathway (canonical Wnt pathway), the planar cell polarity pathway, the Wnt/Ca2- pathway, and a pathway that regulates spindle orientation and asymmetric cell division. Of all these, the canonical Wnt pathway is the best understood in mammalian tissues. β-Catenin is an important component of cell adhesion through its association with E-cadherin, and β-catenin at the plasma membrane and it is also an essential mediator of canonical Wnt signaling.

The canonical Wnt signaling pathway is initiated by the binding of Wnt ligand to two receptor molecules, Frizzled receptors (G-protein-coupled receptors) and low-density lipoprotein receptor-related proteins (LRP), at the plasma membrane, inducing tyrosine phosphorylation and activation of dishevelled (Dsh), which then leads to the inhibition of a complex containing glycogen synthase kinase 3β (GSK3β), axin, and adenomatous polyposis coli (APC) (Figure. 3a). This ultimately stabilise β-catenin protein which is translocated into the nucleus where it interacts with transcription factors such as the lymphoid enhancer binding factor 1 (LEF1) or the T cell-specific transcription factor (TCF), converting them from repressors to activators, and initiating transcription of Wnt target genes. In the absence of Wnt ligands, the central mediator protein, β-catenin is recruited by the cytoplasmic APC-Axin destruction complex. β-catenin is then phophorylated by the associated kinases casein kinase 1α (CKIα) (not shown in the diagram) and glycogen synthase kinase 3β (GSK3β) in the NH2-terminal degradation box, resulting in ubiquination and rapid degradation of β-catenin mediated by the proteosome (Figure.3b).

Alteration of Wnt signaling has been a contributing factor in several ocular diseases and malignancies of the eye. Despite increasing recognition that the Wnt pathway regulates vertebrate eye development, the expression and function of Wnt signaling in retina is not fully understood. In this report we will focus mainly on some of the key Wnt players that have been a topic of interest in current research.

Wnt2b and its role in retinogenesis

Wnt-2b is a member of the Wnt signaling molecule family, which activates the Wnt/β-catenin signaling cascade. It was initially designated as Wnt-13 due to genetic similarity between human Wnt-13 gene and Wnt-2 (Katoh et al, 1996).

In mice and chicken, Wnt2b mRNA is highly expressed in the anterior rim of the optic vesicles, neighbouring the retinal progenitor cells (RPC) and also in the RPE, overlying the ciliary epithelium (Zakin et al, 1998; Jasoni et al, 1999; Lie et al, 2003; Cho & Cepko 2006). Moreover, comprehensive analyses on the expression patterns of Wnt family members have shown that several Wnt signaling family members including the Wnt2b and Frizzled genes are expressed in the peripheral retina (Jin et al. 2002; Fuhrmann et al. 2003; Liu et al. 2003).

Canonical Wnt signaling is occurring at high rate at the site of undifferentiated RPC. Previous studies has shown that in the frog eye, the canonical Wnt signaling pathway has driven progenitor proliferation and induction of proneural gene expression (Van Raay et al., 2005). Also, inhibition of the Wnt signaling pathway by Lef1, a downstream effector of canonical Wnt signaling, stopped the proliferation of the cells and induced premature neuronal differentiation in the marginal retina (Kubo et al., 2003). In contrary, an increased Wnt signaling in the chicken eye at the optic vesicle stage, by the Wnt2b expression has shown to inhibit the differentiation of the RPCs that are derived from the central retina (Cho and Cepko, 2006; Kubo et al., 2003).

To find out whether Wnt signaling promotes proliferation of RPCs, the retinal cells proliferation in an explant was assessed, in the presence or absence of constitutively active, CA-β-catenin. Retinas were electroporated with a control GFP plasmid with a the ubiquitous CAG promoter (pCAG:GFP) only, or with a mixture of plasmids encoding CA-β-catenin and GFP (pCAG:CA-β-catenin+pCAG:GFP). The expression of CA-β-catenin in embryonic day 5.5 explants showed a decrease in mitotic RPCs compared with that of the control cells (GFP-expressing non-electroporated cells), from 25.2±2.4% to 10.7±3.5%.

Figure 4. Retina progenitor cells proliferation in explants. Retinal explants were electroporated with pCAG:CA-β-catenin or pCAG-GFP which were then left to culture for about 18 hours followed by their exposure to radiolabelled thymidine [3H]thymidine for 6hrs. Then autoradiography was performed to determine the percentage of 3H]thymidine+ cells. The bars represent the percentage of cells labelled with [3H]thymidine among the GFP+ population. Error bars represent the standard deviation (SD).

But evidence suggests that reduced proliferation of RPCs upon Wnt signal activation could be the result of inhibition of the cell cycle in RPCs. A cell fate changes from highly mitotic retina to that of less mitotic peripheral tissue, which could lead to a reduction in cell proliferation (Beebe, 1986; Kubota et al., 2004), therefore the validity of the results is questionable at this time. The research led by Cho and his collegues also proposed that constituvely active CA-β-catenin or Wnt2b interferes with the maintenance of retinal progenitor identity and leads to the conversion of retinal cells into the peripheral fates of ciliary body/iris.

In situ hybridisation shows dynamic expression pattern for Wnt2b means it is likely to be involved in several aspects of eye development. Furthermore, the role of Wnt2b was shown in the formation of laminae in the retina (Nakagawa et al., 2003).

These studies indicate that Wnt2b could be responsible for the maintenance of RPCs in the marginal retina but the precise molecular mechanism by which Wnt2b maintains progenitor cells in the undifferentiated state remains an unknown mystery.

Wnt3a

Wnt3a are expressed within the CNS, mainly along the dorsal midline from the diencephalon to the spinal code (Roelink and Nusse 1991; McMahon et al. 1992; Parr et al. 1993). In previous years, Wnt3a has been reported to be linked with self-duplication of HSCs (Reya et al, 2003).

A more recent study by Inoue and colleagues demonstrated the role of Wnt3a in promoting the proliferation of explanted adult retinal progenitor cells via canonical Wnt signaling pathway. It was found that the number of pigmented sphere colonies (PSCs) derived from the Ciliary margin (CM) was increased in the presence of Wnt3a, in vitro (Inoue et al. 2006). Also, Wnt3a treated sphere cells were able to generate secondary spheres and differentiate to express different retinal cell-specific markers,

syntaxin, rhodopsin, and Pax6, under culture conditions that promoted retina cell differentiation (Table 5). Therefore the sphere cells from CM possessed multineage potential and self-renewal character of stem cells.

Table extracted from Inoue et al, 2007

Table 5. Expansion of sphere cells via Wnt3a or GSK3 Inhibitor, SB216763. The column shows the mean percentage ± standard deviation of cell types in the number of nuclei. Sphere colonies were grown in the medium of either Wnt3a or SB216763 for 5 days. Each sphere colony was then plated and cultured under condition that promoted the differentiation of retinal cells, for 21 days. The derivative of sphere cells were found to express retinal cell-specific markers, such as rhodopsin as rod photoreceptors, Gln synthetase as Müller glia or Pax6 and syntaxin as amacrine cells.

It was claimed that canonical Wnt signaling pathway was linked to the proliferation of Wnt3a on pigmented sphere colonies and the synergistic effect of Wnt3a and fgf2 aided to the proliferation effect (Inoue et al. 2006). Initially, when PSCs were cultured under conditions that promoted the differentiation of retinal cells, some of the cells expressed the neurofilament and pan-neuronal marker microtubule-associated protein 2 (MAP2), whereas other cells were found to express astrocytic marker, glial fibrillary acidic protein (GFAP). The cells which differentiation later contained a small population of nestinpositive cells that remained concentrated to the centres of the colonies (Tropepe et al. 2000), in addition to many markers that are usually seen in differentiated retinal cells, rod photoreceptors, bipolar cells, retinal ganglion cells, and Müller glia. Thus, the cells comprising PSCs have an ability to differentiate into a variety of retinal cell types.

The progenitor cells in the ciliary body epithelium were found to respond to the growth factor treatment and injury by means of extensive proliferation in vivo, implying that they are able to be activated endogenously (Ahmad et al. 2004). Although PSC cells are proliferative, self-renewable and multipotent, more research is still needed to determine the precise indentifcation of retinal stem cells in the ciliary body.

Müller glia

A study by Osakada and collegues have found that Wnt3a has an effect of inducing Muller glia cells to proliferate to and differentiate into retinal cells (Oskada et al, 2007). The idea was brought by a previous study in which the potential of avian retina to regenerate after neurotoxic damage was tested (Fischer & Reh 2001). When cells death was induced by inducing toxic levels of NMDA, large number of mitotically active cells were present in the retina, which could adapt one of the three different fates; less than 4% of Muller glia-derived cells differentiated into retinal neurones that expressed calretinin, about 20% differentiated into GS expressing muller glial cells, and the remaining stayed in undifferentiated (Fischer & Reh 2003).

Taking factors into account such as risks associated with invasive surgery, and the number of newly generated neurons, an alternative was considered by examining the effect of Wnt signaling in the process of regeneration after retinal injury in respect to intrinsic progenitor cells.

The retinal explants were isolated from adult rats and were then used as an in-vitro injury model. Muller glia cells were seen to proliferate in the damaged retinal explant, and re-entered the cell cycle to a certain extent in response to injury. After culturing these explants in the presence of Wnt3a, an increased proliferation of muller glia derived retinal precursor cells was noticed after damage (Figure 6). In addition, Wnt3a treatment promoted nuclear translocation of β-catenin only in the damaged retina, although β-catenin was observed in both the plasma memberane and the cytoplasm of damaged and non-damaged retina (Osakada et al. 2007). The activation of Wnt signaling by an inhibitor of glycogen syntase kinase (GSK)-3β, which destabilises and phosphorylates β-catenin, triggered neural reegeneration of the retina. On the other hand, inhibition of Wnt signaling by a negative modulator Dkk-1, attenuated the regeneration of retina. When Wnt3 treatment was given to the damaged retinal explants, followed by incubation in a medium in the absence of Wnt3a, the Müller glia-derived retinal progenitors moved into the outer nuclear (ONL) where they differentiated into rod photoreceptor cells. The findings therefore explain that in the adult retina, regeneration of retinal neurons after injury is regulated by Wnt/β-catenin signaling pathway.

Figure 6. Retinal explants from adult rats were cultured in the absence (C) or presence (D) of Wnt3a for 4 days. C, BrdU positive (proliferative) cells (green) are observed in the damaged retina. A Few brdU-labeled cells (green) in the inner nuclear layer (INL) express GS (a marker. D, Wnt3a increases the number of BrdU-positive cells (green) positive for GS (red) in the damaged retina.

The findings implicate that the molecules which activate Wnt/β-catenin signaling may have a therapeutic importance in promoting regeneration of neurons in the mammalian CNS. These moecules may be applied for genetic eye condition retinistis pigmentosa, vascular occlusion (reduced blood flow to the retina), and acute retinal damage.

Wnt4

Wnt4 are expressed in dorsal regions of the neural tube (Daneman et al, 2008). Early studies show their involvement in sex determination (Vainio et al, 1999; Jordan et al, 2001). However, recent studies by Brunken and collegues have found that Wnt Inhibitory Factor-1 (WIF-1) ligand, Wnt4 is involved in retinal development. The WIF-1, Wnt4 receptor, fdz4, and LRP6, a Wnt4 coreceptor, were all found to be expressed in rod photoreceptor morphogenesis. It was claimed that WIF-1 and Wnts act together to modulate the rod photoreceptor genesis. Also that the Wif-1 and Wnt4 are co-expresssed in retinal development and that WIF-1 binds to and inhibit Wnt4 in the extracellular matrix, which would reduce the level of Wnt4 available for the activation of Wnt receptors including LRP6 and fzd6, leading to lowered activation of Wnt signaling through fzd4 and LRP6.

The co-expression of Wnt4 and wif-1 was proved by the use of antibodies directed against Wnt4 and Wif-1, suggesting that both these proteins are present in the IPM near the inner segments (IS) of photoreceptors (Fig.7a). Wnt4 and Wif-1 antisera was used to demonstrate the presence of Wnt/Wif-1 complex in the retina. this was done by homogenating the tissues, initially, and then lysates were immunoprecepitated with an anti-Wnt4 antiserum, an anti-WIF-1 antiserum, or an anti-fzd4 antiserum, then protein transfer blot was used to detect the presence of Wnt4 and WIF-1 (Fig.7b). Wnt4 was found to be precipitated by anti-Wnt4 and by anti-wif-1 but not by another primary antibody such as anti-β-galactosidase. Also, wif-1 was precipitated by using anti-Wnt4. The presence of Wnt/wif4 complex is clearly noticeable by the coprecipitation of Wnt4 and wif-1 by an antibody against either component.

Fig. 7. Wnt4 and WIF-1 are coexpressed and are bound to each other. (A) Simultaneous double immunohistochemical analysis of adult mouse retina displayed Wnt4 (red) and WIF-1 (green), coexpressed in the outer retina (yellow), particularly in the region of the photoreceptor inner segments (IS). WIF-1 is expressed in the OPL, portions of the INL, and in blood vessels (bv). (B) Co-immunoprecipitation of a retinal extract demonstrates a WIF-1/Wnt4 complex in the retina. Immunoprecipitations performed with antibodies to Wnt4 (anti-Wnt4) and WIF-1 (anti-WIF-1) both contain Wnt4. Immunoprecipitations with anti-WIF-1 contain Wnt4. Immunoprecipitation performed with antibodies to h-galactosidase (anti-h gal) do not contain Wnt4. Fzd4 is present in immunoprecipitations performed with antibodies to fzd4 (anti-fzd4), but not with immunoprecipitations performed with antibodies to WIF-1.

In vitro retinal development method with two approaches was used to study the function of the WIF-1 and Wnt4 in retinogenesis. Initially, disscociated retinal cells from newborn rats were cultured on glass coverslips, a technique which promotes the development of rod photoreceptor development (Hunter and Brunken, 1997), and analysed the activity of WIF-1 in this system. Due to the fact that the time course of photoreceptor differentiation goes along with the changes in protein distribution of WIF-1 and Wnt4, the effect of WIF-1 on rod photoreceptor differentiation could be studied in vitro. The production of rod photoreceptors, as assessed by rhodopsin production was found to be inhibited in dose-dependent manner when retinal cells were cultured in the presence of WIF-1 protein (Fig.8a). In contrast, the endogenous WIF-1 activity was inhibited with the addition of anti-WIF-1 antiserum and increased rod photoreceptor production of about 125% was noticeable (Fig.8b).

Fig. 8. the production of presumed photoreceptor was enhanced by Wnt4 and inhibited by WIF-1. (A). number of cells which express rhodopsin (rods) were inhibited by synthetic WIF-1, in dissociated culture in a dose-dependant manner. (B) the number of rods increased in dissociated culture by using an anti-serum against WIF-1. The number of rod cells production in dissociated culture was increased using a conditioned medium containing Wnt4, but not with Wnt14.

In a second approach, developing cells from similarly dissociated mouse retinae were cocultured with Wnt-producing chicken fibroblasts. When the culture medium was supplied with exogenously secreted Wnt4, it triggered the production of rod photoreceptors, which means Wnt4 is a capable modulator in the development of retina (Fig. 8c). When Wnt14, another Wnt molecule, was secreted exogenously into the medium no sign of photoreceptor production was observed.

These in vitro approaches jointly demonstrate that exogenous WIF-1 and Wnt4 have negative and positive effects, respectively, on rod photoreceptor differentiation. These data further support a modulatory interaction between the two molecules that affects rod photoreceptor development.

Wnt9a/Wnt14

Wnt14, also known as Wnt9a, share 64% of amino acids with the shark Wnt9 (Bergstein et al.,1997). Wnt9 gene of shark is homologues to the Wnt9a of a mouse and the level of expression of Wnt9 was found to be different between embryonic days 3 (ED7), ED11 and ED15, implying a possible role of Wnt9/14 in the development of the retina. Research has found that human Wnt14 is located on chromosome 1q42 (Katoh et al, 2001).

In the process of postnatal retinogenesis, there are various steps including migration, differentiation and proliferation and are all programmed into the RPC, in order to get a fully functional retina (Snow and Robson, 1994). Cell death is also considered to be an important process in the development of retina which is suggested to be by means of programmed cell death or apoptosis (Frade et al, 1997).

The formation of RGCs from progenitor cells occur around embryonic days 3 (ED3). RGCs thereafter proliferate between ED8 and ED11, followed by their reduction in numbers from ED11 to ED14. According to Snow and colleagues (1994), about 1/3rd of progenitor cells are directed to undergo apoptosis between ED11 and ED16 (Snow and Robson, 1994). In a recent study, Mizukami and colleagues found that a change in the expression of Wnt14 was linked with downregulation of retina at ED15, suggesting its possible role in retinogenesis.

The effect of over-expression of Wnt14 on the survival of R28 cells was determined. R28 are a precursor cell line with an ability to differentiate into RGCs. They express both glial and the neuronal cell markers (Seigel et al.,1996).

R28 cells were induced to cell death by the serum deprivation time-course dependent technique, in which the R28 cells were initially transfected with either Wnt14 expression or with the control vector. The number of surviving colonies was then measured after serum deprivation for 0, 12, 24, 48, and 96 h. After serum deprivation of 96 hours, the mean number of Wnt14 expressed R28 surviving colonies decreased from 329, at the start, to 66 after 96 hours, whereas the respective figures for control colonies were 350 at 0 hours and 43 after 96 hours. The efficiency of transfecting R28 cells with Wnt14 was found to be 30%, which means that only 30% of the cells could be transfected with Wnt14. By taking this into consideration the results show clearly that the death of R28 cells was inhibited by over-expression of Wnt14 (Fig.9a).

Later, a colony formation assay was carried out by exposing L-glutamate to induce cell death, which is a commonly used method for the induction of retinal cell death (Levinthal and DeFranco,2005). The R28 were over-expressed with Wnt14 and were then exposed to different concentrations of L-glutamate as 0,10, 20, and 40 mM for 12 hours. In the absence of L-glutamate, the number of R28 colonies was found to be 1176 and that of control cells was 1109.3. In the presence of 40mM exposure of L-glutamate, the number of Wnt14 over-expressed R28 cell colonies was 581, which was much higher than that in the control value of 362.7. The results further demonstrate that the L-glutamate induced death of R28 cells was inhibited by an over-expression of Wnt14 (Fig. 9b).

Fig. 9. Effect of Wnt14 over-expression on R28 cell death after serum deprivation and glutamate treatment. R28 cell, transfected with Wnt14-pCMV (black bars) or empty pCMV-vector (gray bars), was serum deprived. The cell death was time-dependent (A) and glutamate dose-dependent (B). After incubation for five days with medium containing 10% serum, the number of surviving colonies is shown to determine the viability. Error bars indicate the standard deviations in the numbers of colony in three dishes.

The study was extended further to find out the possible pathway involved in inhibiting the cell death of R28 cells via Wnt14. The caspase cascade is the well understood signaling pathway for apoptosis, and caspase-3 is essential for its activation. Such activation can be detected by using anti-cleaved caspase-3 antibody by immunohistochemitry (Yin and Thummel, 2004).

The caspase-3 was activated by serum deprivation (a trigger for cell death) which was detected by immunostaining R28 transfected cells (Fig.10a). It was then found that the expression of activated caspase-3 (cleaved caspase-3) was reduced from 56.9% in the control to 26.5% after Wnt14 transfection (Fig.10b and 10c). Thus, Wnt14 transfection reduced the numbers of cells whose caspase-3 was activated by serum deprivation.

Fig. 10. Showing the effect of Wnt14 over expression on the activity of caspase-3. A. R28 cells were transfected using HA-tagged Wnt14-pCMV (middle image) or using pCMV-vector using serum deprivation (left image), for 24 hours. The activation of caspase-3 was assessed by immunostaining the cells. rabit anti-cleaved caspase-3 antibody was used to detect the cleaved and activated caspase-3. Anti-cleaved caspase-3 antibody and anti-HA antibody was used to co-immunostain the cells. Red-stain was used to see the cleaved caspase-3 immunoreactive cells and HA-positive cells were seen by green stain. Yellow stained cells are shown with a co-expression of cleaved caspase-3 and HA-tagged Wnt14 (right image). Serum-deprived and Wnt14-pCMV transfected cells are also stained with secondary antibody only as negative control.

The question remains unclear is that if over-expression of Wnt14 inhibited the death of glutamate treated R28 cells then why most of the surviving colonies were dead (mean surviving R28 colonies decreased from 1174 to 581). One possible explanation is that death was induced by the invasive experimental procedure, in which the cell culture was too long and confluent cell density with no passage of cells.

Wnt3a prevented neurotoxin-induced cell death by the reduction of caspase-3 activation(Chaco´n et al. 2008). This experiment has similar finding to the role of Wnt3a as inactivation of caspase-3 is associated with Wnt14. An assumption can be based that Wnt14 may bind with a similar receptor as Wnt3a. Further experiment in this field can elucidate canonical signaling pathway and possibly confirm the finding of this experiment.

Future indications

Previous models of the patterning of the eye recognized the early

division of the OV into domains that would give rise to the outer OC

and inner OC, and thus the RPE and retina, respectively (Chow and

Lang, 2001; Graw, 2003; Martinez-Morales et al., 2004). We are

now suggesting that there is a third domain of the OV, determined

by high Wnt signaling, which will give rise to the peripheral OC, and

thus the ciliary body and iris. Expression of Wnt2b/Lef1/Fz4 and

canonical Wnt reporter activity had been previously observed at the

OC stages, but not at the OV stage (Jasoni et al., 1999; Kubo et al.,

2003; Liu et al., 2003). The sensitive reporter SuperTopAP allowed

lens determination. Dev. Genet. 20, 246-257.

Chow, R.L., Lang, R.A., 2001. Early eye development in vertebrates. Annu. Rev. Cell Dev.

Biol. 17, 255-296.

Grainger, R.M., Mannion, J.E., Cook Jr., T.L., Zygar, C.A.,1997. Defining intermediate stages

in cell determination: acquisition of a lens-forming bias in head ectoderm during

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