# Knockdown of Zebrafish Blood Vessel Epicardial Substance Results in Incomplete Retinal Lamination

Authors: Yu-Ching Wu; Ruei-Feng Chen; Chia-Yang Liu; Fung Rong Hu; Chang-Jen Huang; I-Jong Wang
Journal: The Scientific World Journal
 (2014)

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## Abstract

Cell polarity during eye development determines the normal retinal lamination and differentiation of photoreceptor cells in the retina. In vertebrates, blood vessel epicardial substance (Bves) is known to play an important role in the formation and maintenance of the tight junctions essential for epithelial cell polarity. In the current study, we generated a transgenic zebrafish Bves (zbves) promoter-EGFP zebrafish line to investigate the expression pattern of Bves in the retina and to study the role of zbves in retinal lamination. Immunostaining with different specific antibodies from retinal cells and transmission electron microscopy were used to identify the morphological defects in normal and Bves knockdown zebrafish. In normal zebrafish, Bves is located at the apical junctions of embryonic retinal neuroepithelia during retinogenesis; later, it is strongly expressed around inner plexiform layer (IPL) and retinal pigment epithelium (RPE). In contrast, a loss of normal retinal lamination and cellular polarity was found with undifferentiated photoreceptor cells in Bves knockdown zebrafish. Herein, our results indicated that disruption of Bves will result in a loss of normal retinal lamination.


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## Body

## 1. Introduction

The vertebrate retina can be used as a model to study cell patterning and cell fate determination within the central nervous system, from which the retina is derived; moreover, the retina is easily observed and accessible during development [1]. The neural retina in vertebrates differentiates between a sheet of multipotent and proliferating neuroepithelial cells, undergoing a dramatic morphogenetic change that depends on a proper epithelial polarity and integrity to reshape the original cell layers during retinogenesis [2]. In the early stage, progenitor cells at the ventricular margin of the neural retina undergo mitosis, are divided into six classes of cells, photoreceptors, horizontal cells, bipolar cells, amacrine, ganglion cells, and Müller glia, and extend their neurites, leading to a laminar pattern of the retina [3].The photoreceptor cells have both neuronal and epithelial properties [4]. Therefore, cell polarity is a major feature of vertebrate photoreceptors, each of which is further subdivided into four parts in the developed retina: an outer segment (OS), an inner segment (IS), a cell body (CB), and a synaptic terminus (ST) [4]. The differentiation of retinal pigment epithelium (RPE) is related to the development of photoreceptors [5, 6]. Several junctional complexes, including adherens junctions and tight junctions, participate in this process [6]. Coordinating with RPE, these tight connections in photoreceptors are able to prevent certain substances in choroid vessels from entering the retinal tissue [6, 7]. Here, the establishment and regulation of junctional components are indispensable for the function and the integrity of RPE and photoreceptor cells in retinogenesis. Most importantly, the molecular mechanism controlling cell polarity formation in the retinal photoreceptor cells is interesting and important in retinal development using an appropriate model. For example, several studies have shown that several mutation loci in zebrafish that encode proteins required for apicobasal polarity, such as mosaic eyes (moe), oko meduzy (ome), nagie oko (nok), and heart and soul (has), showed disruption of retinal lamination [8–11]. These studies also demonstrated that the zebrafish is a good animal model to study the effect of junctional complexes on retinal development.Thebves (blood vessel/epicardial substance) gene encodes a membrane protein [12, 13], and its protein has aPopeye domainbelonging to thePopeye domain—containing (Popdc) family. Bves is widely expressed in many epithelial tissues, including the eye [12, 14–18] and plays a role in cell adhesion, epithelial integrity, and cell motility [19]. During early eye development, Bves is initially localized at the apical-lateral position of epithelial precursors; later on, it is expressed in the retina, lens, and cornea [20].In vitro knockdown experiments in a cultured corneal cell line demonstrated that Bves may affect epithelial cell movement during corneal reepithelialization [15]. Our previous study further elucidated that Bves might regulate the formation of a polarized epithelial sheet through the association with the polarity protein, aPKC (atypical protein kinase c) [21]. Combined with its expression pattern in the eye and its role in cell junctions, we believe that the expressions of Bves in epithelial adhesion and movement are crucial for eye development. Although we speculate that Bves should play a physiological role during eye development, little is known about the role of Bves in retinal lamination and photoreceptor differentiation. In this study, we generated a transgenic fish line in whichzbves promoter, driven EGFP, could be visualized to localize its expression pattern during eye development. We further used a knockdown technique to study the expression of Bves during retinal lamination and photoreceptor differentiation.


## 2. Materials and Methods

### 2.1. Transgenic Zebrafish Line

The Tg(zbves :EGFP) transgenic zebrafish line was established using the generated construct T2 K-zbves-p (Figure 1(a)), containing two transposon sequences and the 6-kb DNA of the zebrafishzbves promoter—conjugated EGFP sequence. This transposon-donor plasmid and transposase mRNAs were coinjected into fertilized eggs and the transgenic fish line was created. F1 embryos exhibiting EGFP expression at normal temperatures (~28°C) were raised and F3 embryos were used for observation in this study.Figure 1
ZBves expression in the eyes of transgenic line. The Tol2 transposon system was established to produce a Tg(zbves :EGFP) transgenic zebrafish line. Thezbves promoter sequences were cloned in the Tol2-containing vector (a). The F3 transgenic embryos were observed using a confocal microscope to investigate the expression pattern of Bves. Before 18 hpf, the GFP signal (transgene) mainly appeared on the surface of whole organism and eye (b)–(d). At 18 hpf, Bves expression initially spread into the retina from the border between the cornea and retina (e), (e′). At 24 hpf, zBves was expressed in whole retina of the zebrafish eye (f), (f’). At 96 hpf, zBves was localized around IPL and RPE (g), (g′), and (g′′). Antibody staining with Bves was preformed in 24-hpf, 96-hpf, and adult fish (h), (i), and (i′). The lateral side of the eye with phalloidin staining (red) is shown in (e′), (f′), (g′), and (g′′). Scale bar, 50 μm.


### 2.2. Morpholino Knockdown

The following antisense morpholino oligonucleotides (MOs) were purchased from Gene Tools (Philomath, OR), azbves translation blocking morpholino (ATGmo; 5′-GATGTTGTGTTGGACATTCTGAGGC-3′), and azbves splice inhibition morpholino (splice mo; 5′-AGAGCAGCCTGAAAGACAATAAAGA-3′). Zebrafish embryos at the 1- or 2-cell stages were chosen for injection. We chose 2 ng of ATGmo and 4 ng of splice mo in subsequent experiments. These concentrations resulted in 90% survival from injected embryos with more than 50% exhibiting a phenotype [21].


### 2.3. Immunofluorescence Staining

For immunofluorescence detection of proteins on cryosections of embryos, staged embryos were fixed in 4% paraformaldehyde at 4°C overnight and then transferred to 30% sucrose. After overnight immersion in sucrose, embryos were embedded within OCT (Tissue-Tek) and stored at –80°C. 20μm cryosections were cut and loaded on glass slides. Then these samples were washed in Triton X-100 for 30 min at room temperature and then blocked in 5% BSA at 4°C overnight. The embryos were incubated with the following primary antibodies in blocking buffer at 4°C overnight: affinity purified anti-zBves (1 : 500) [21], anti-ZO-1 (1 : 200, Zymed, S. San Francisco, CA), anti-PKCζ (1 : 200, Santa Cruz Biotechnology, Santa Cruz, CA), zn8 (1 : 50, Zebrafish International Resource Center, Eugene, OR), anticarbonic anhydrase II (CA; 1 : 100, Abcam, Cambridge, England), antityrosine hydroxylase (TH; 1 : 100, Millipore, Temecula, CA), anti-CtBP (1 : 200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Crx (1 : 200, Santa Cruz Biotechnology, Santa Cruz, CA), and zpr-1 (1 : 400, Zebrafish International Resource Center, Eugene, OR). Embryos were washed in PT/1% BSA, and then appropriate secondary antibodies (i.e., Alexa 488 1 : 200, Invitrogen, Carlsbad, CA) were added for 2 h at room temperature. Samples were washed and images were acquired using confocal microscopy (LSM510 Meta, Zeiss, Thornwood, NY).


### 2.4. Electron Microscopy

At 96 hpf, the controls and thezbves ATGmo-injected embryos were immersed in a fixative solution containing 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h at room temperature. After a wash in 0.1 M cacodylate buffer, the samples were postfixed in 0.2% osmium tetroxide in 0.1 M cacodylate buffer for 16–18 h. The samples were then rinsed in the buffer, dehydrated in a graded series of ethanol, and embedded in Epon 812; 1 m sections were then observed after toluidine blue staining. Thin sections were contrasted with uranyl acetate and lead citrate. Grids were observed by the transmission electron microscopy (TEM; Hitachi H-7500, Tokyo, Japan).


## 2.1. Transgenic Zebrafish Line

The Tg(zbves :EGFP) transgenic zebrafish line was established using the generated construct T2 K-zbves-p (Figure 1(a)), containing two transposon sequences and the 6-kb DNA of the zebrafishzbves promoter—conjugated EGFP sequence. This transposon-donor plasmid and transposase mRNAs were coinjected into fertilized eggs and the transgenic fish line was created. F1 embryos exhibiting EGFP expression at normal temperatures (~28°C) were raised and F3 embryos were used for observation in this study.Figure 1
ZBves expression in the eyes of transgenic line. The Tol2 transposon system was established to produce a Tg(zbves :EGFP) transgenic zebrafish line. Thezbves promoter sequences were cloned in the Tol2-containing vector (a). The F3 transgenic embryos were observed using a confocal microscope to investigate the expression pattern of Bves. Before 18 hpf, the GFP signal (transgene) mainly appeared on the surface of whole organism and eye (b)–(d). At 18 hpf, Bves expression initially spread into the retina from the border between the cornea and retina (e), (e′). At 24 hpf, zBves was expressed in whole retina of the zebrafish eye (f), (f’). At 96 hpf, zBves was localized around IPL and RPE (g), (g′), and (g′′). Antibody staining with Bves was preformed in 24-hpf, 96-hpf, and adult fish (h), (i), and (i′). The lateral side of the eye with phalloidin staining (red) is shown in (e′), (f′), (g′), and (g′′). Scale bar, 50 μm.


## 2.2. Morpholino Knockdown

The following antisense morpholino oligonucleotides (MOs) were purchased from Gene Tools (Philomath, OR), azbves translation blocking morpholino (ATGmo; 5′-GATGTTGTGTTGGACATTCTGAGGC-3′), and azbves splice inhibition morpholino (splice mo; 5′-AGAGCAGCCTGAAAGACAATAAAGA-3′). Zebrafish embryos at the 1- or 2-cell stages were chosen for injection. We chose 2 ng of ATGmo and 4 ng of splice mo in subsequent experiments. These concentrations resulted in 90% survival from injected embryos with more than 50% exhibiting a phenotype [21].


## 2.3. Immunofluorescence Staining

For immunofluorescence detection of proteins on cryosections of embryos, staged embryos were fixed in 4% paraformaldehyde at 4°C overnight and then transferred to 30% sucrose. After overnight immersion in sucrose, embryos were embedded within OCT (Tissue-Tek) and stored at –80°C. 20μm cryosections were cut and loaded on glass slides. Then these samples were washed in Triton X-100 for 30 min at room temperature and then blocked in 5% BSA at 4°C overnight. The embryos were incubated with the following primary antibodies in blocking buffer at 4°C overnight: affinity purified anti-zBves (1 : 500) [21], anti-ZO-1 (1 : 200, Zymed, S. San Francisco, CA), anti-PKCζ (1 : 200, Santa Cruz Biotechnology, Santa Cruz, CA), zn8 (1 : 50, Zebrafish International Resource Center, Eugene, OR), anticarbonic anhydrase II (CA; 1 : 100, Abcam, Cambridge, England), antityrosine hydroxylase (TH; 1 : 100, Millipore, Temecula, CA), anti-CtBP (1 : 200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Crx (1 : 200, Santa Cruz Biotechnology, Santa Cruz, CA), and zpr-1 (1 : 400, Zebrafish International Resource Center, Eugene, OR). Embryos were washed in PT/1% BSA, and then appropriate secondary antibodies (i.e., Alexa 488 1 : 200, Invitrogen, Carlsbad, CA) were added for 2 h at room temperature. Samples were washed and images were acquired using confocal microscopy (LSM510 Meta, Zeiss, Thornwood, NY).


## 2.4. Electron Microscopy

At 96 hpf, the controls and thezbves ATGmo-injected embryos were immersed in a fixative solution containing 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h at room temperature. After a wash in 0.1 M cacodylate buffer, the samples were postfixed in 0.2% osmium tetroxide in 0.1 M cacodylate buffer for 16–18 h. The samples were then rinsed in the buffer, dehydrated in a graded series of ethanol, and embedded in Epon 812; 1 m sections were then observed after toluidine blue staining. Thin sections were contrasted with uranyl acetate and lead citrate. Grids were observed by the transmission electron microscopy (TEM; Hitachi H-7500, Tokyo, Japan).


## 3. Results

### 3.1. Spatiotemporal Expression of zBves in Zebrafish Eye during Development

To examine the Bves expression pattern, a transgenic zebrafish line—Tg(zbves:EGFP)—was generated using a transposon system, in which Tol2 vector was used as previously described (Figure 1(a)) [22]. The fluorescent signal of F3 transgenic zebrafish embryos appears at 4 hpf and is highly expressed in the cells of yolk syncytial layer (YSL) (Figure 1(b)). Then, the fluorescence spreads to the envelope layer (EVL) at 8 hpf (Figure 1(c)). Before forming the optic vesicle at 12 hpf, the eye field begins to evaginate and the fluorescence significantly appears in the epidermis layer (Figure 1(d)). At 18 hpf, when the optic cup is formed, the fluorescent signals concentrate at the anterior margin of retina in the eye field (Figures 1(e), and 1(e′)). At 24 hpf, it can be spread to whole eye tissue (Figures 1(f), and 1(f′)). At 96 hpf, Bves expressed cells were localized around inner plexiform layer (IPL) and RPE (Figures 1(g), 1(g′), and 1(g′′)).We used an anti-zBves antibody to confirm the distribution and location of the Bves protein in 24 hpf, 96 hpf, and adult fish. Compared with the fluorescent signals in the transgenic embryos, the expression pattern of Bves protein is compatible with the fluorescent pattern in transgenic zebrafish (Figures1(f′), 1(g′), 1(g′′), 1(h), 1(i), and 1(i′)). At 24 hpf, the Bves protein is localized at the apical region of the retinal epithelium (Figure 2(i)). Compared with the adherens junction-associated actin bundles colocalized with phalloidin, high level Bves expression is at apical sites of retina epithelium (Figure 2(i)).Figure 2
Zbves morphants exhibited defects in eye size and lamination. The morphological observations of wild-type and ATGmo- and splice mo-injected embryos are shown. Compared with wild-type (a), the eyes formed abnormally and resulted in microphthalmos after knockdown of zBves (b), (c). Statistical analyses of the length of whole eye and lens in both groups were performed (n
=
11,t-test for two groups, P
<
0.01) (d). HE staining in paraffin sections of wild-type and morphant embryos revealed the eye without a laminated retina (e), (f). The same patterns were observed in the semithin Epon sections with toluidine blue staining (g), (h). Scale bar, 50 μm. Immunostaining with anti-zBves antibody (green) and phalloidin (red) was performed in the retinal neuroepithelia of wild-type (i) and zBves MO-treated embryos (j) at 24 hpf and laminated retinas of untreated (k) and zBves MO-treated embryos (l) at 96 hpf. ln, lens. Scale bar, 10 μm.


### 3.2. Bves Knockdown Results in Microphthalmos with Retinal Lamination Defect

To examine the physiological function of Bves in eye development, two kinds of morpholino oligonucleotides (MOs)—ATGmo and splicing mo—were used to disrupt the production of the zBves protein. In our previous study, zBves knockdown embryos exhibited epidermal defects. In addition, a dramatically small eye field could be observed in morphants (Figure2). Compared with the untreated embryos, MO-injected embryos have significantly small eyes (Figures 2(a)–2(c)). The thicknesses of lenses and the lengths of whole eyes were measured and their differences were found to be statistically different at 1, 2, and 3 dpf, respectively (Figure 2(d)).To further compare the microstructural differences between untreated and zBves knockdown embryos, we found that the morphants had a microphthalmos without a laminated retina through hematoxylin and toluidine blue staining (Figures2(e)–2(h)). Additionally, there was a space between the retina and the RPE, indicating subretinal fluid retention (Figures 2(g) and 2(h)). This indicates that zBves disruption might break the RPE barrier in retina and implies that Bves might play an important role in the precise formation of retinal layer structure during retinogenesis. The expressions of actin and Bves were examined in morphants and revealed an unclosed apical pattern of phalloidin-stained adherens junction-associated actin bundles (Figure 2(j)). This result implies that Bves is involved in the generation or maintenance of epithelial cell polarity like that in the epithelia of other tissues [21, 23]. Furthermore, we examined the expression of Bves after retinal lamination. Interestingly, at 96 hpf, Bves was found in the cell membranes of all epithelial layers of the eye; however, the retina exhibited the strongest expression. In the retina, Bves was expressed near RPE barrier and around the inner plexiform layer (Figure 2(k)), which is composed of the amacrine cells and the ganglion cells. Furthermore, the bundles of actin in zBves knockdown retina were dramatically disrupted (Figure 2(l)). These suggest that Bves might play a role in cell-cell junctional maintenance, especially at the apical side of the RPE membrane after eye formation, as described previously [21].


### 3.3. Incomplete Photoreceptor Formation in Bves Morphants

Becausehas encodes atypical protein kinase C, which requires Bves to regulate cell junction, and the zebrafishhas mutant exhibits loss of normal retinal lamination [24], we further investigated whether Bves is required for retinal lamination. The retinae of morphants were stained with different retinal cell-specific antibodies to identify the differentiation of retinal cells in lamination defect during eye development. Our results showed that ganglion cells, Müller glia, and interplexiform cells were present in both morphants (Figures 3(e)–3(g)); however, most of them were mislocalized and unlike those of the normal retina (Figures 3(a)–3(c)). Interestingly, immunostaining with the zpr-1 antibody revealed that photoreceptors are not present at 96 hpf after Bves knockdown (asterisks in Figure 5(h)) whereas they exist in wild-type embryos (arrows in Figure 3(d)). To confirm these results, cone-rod homebox-containing gene (Crx) in relation with eye development was further observed in 72-hpf retina (Figure 3(i)). Surprisingly, Crx protein did not disappear in all mutant retinae. 37.5% of mutant retina expressed Crx protein (Figure 3(j)) but 62.5% of them did not. These results indeed demonstrate that Bves is required for proper retinal lamination, especially for photoreceptors.Figure 3
Disruption of zBves affected the dysregulated patterns in the retina. The frozen sections and immunofluorescence staining with different marker antibodies—zn8 for ganglion cells (a), (e), anti-CAII for Müller glia (b), (f), anti-TH for interplexiform neurons (c), (g), and zpr-1 for photoreceptors (d), (h) were performed in eyes of 96 hpf wild-type and zBves MO-injected embryos. Immunofluorescence staining with Crx (i), (j) antibody in the frozen sections was performed in 72 hpf embryos. 37.5% (6/16) of mutant retina showed Crx protein. The ultrastructures of the retina in wild-type (m), (m′) and zBves MO-injected embryos (n), (n′) were analyzed by TEM. Double staining with CtBP and zpr-1 was adopted in frozen crytosections (k), (l). 33.3% (4/12) of mutant retina showed CtBP protein without zpr-1 expression (l). Asterisks indicate the outer segment. Black arrows indicate the inner segment. N, nucleus; S, Scale bar in immunostaining photographs, 50μm. Scale bar in TEM picture, 1000 nm.To examine the morphology of photoreceptor and to confirm the result of zpr-1 and Crx staining, the ultrastructures and CtBP (synaptic ribbon) expression of the photoreceptors were analyzed by TEM and immunostaining. Compared with the microstructure of a normal retina (Figures3(m), and 3(m′)),zbves morphants were found to lack outer segments under transmission electronic microscopy (Figures 3(n), and 3(n′)). 33.3% of mutant retina had CtBP expression (Figure 3(l)) and 66.6% of them did not show CtBP protein, whereas all wild-type retinae showed CtBP protein (Figure 3(k)). These results indicate that zBves knockdown results in immature and even undifferentiated photoreceptors.


### 3.4. Bves is Essential for the Formation of Cell Polarity between Retina Neuron and RPE to Assist in the Differentiation of Photoreceptor Cells

As Bves is localized in adherens junctions, we examined whether the expression patterns of ZO-1 (zona occludens protein 1), aPKC, andβ-catenin, which are initially associated with adherens junctions in the RPE, are altered after Bves knockdown. The strongest expressions of ZO-1, aPKC, and β-catenin are normally observed at the apical surface of the photoreceptor cells and the RPE in a uniformly honeycomb-like pattern (Figures 4(a), 4(e), and 4(i)). In Bves knockdown embryos, however, the patterns of ZO-1, aPKC, and β-catenin were found to be discontinuous along the apical surface of photoreceptor cells and formed into rosette structures (Figures 4(b), 4(c), 4(f), 4(g), 4(j), and 4(k)). This defect could be rescued by coinjection withzbves mRNA in morphants (Figures 4(d), 4(h), and 4(l)). These results indicate that Bves is crucial for the proper localization of adherens junction proteins along the apical border of the photoreceptor cells and RPE.Adherens junctions were not formed in the outer limiting membrane ofzbves morphants. Immunostaining of adherens junction-related molecules-ZO-1 (a)–(d), aPKC (e)–(h), and β-catenin (i)–(l)—was carried out in the retinas of wild-type and zBves MO-injected embryos andzbvesmRNA rescued morphants at 72 hpf. In wild-type embryos, β-catenin obviously appeared in the apical site of photoreceptors (i), whereas it disappeared inzbve morphants (j), (k). The defect in this protein among morphants could be rescued by coinjection withzbve mRNA. Arrows indicate the apical edge. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)Figure 5
Illustration of Bves effect on zebrafish retinogenesis. RGC, retinal ganglion cells; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Red spot indicates tight junction.


## 3.1. Spatiotemporal Expression of zBves in Zebrafish Eye during Development

To examine the Bves expression pattern, a transgenic zebrafish line—Tg(zbves:EGFP)—was generated using a transposon system, in which Tol2 vector was used as previously described (Figure 1(a)) [22]. The fluorescent signal of F3 transgenic zebrafish embryos appears at 4 hpf and is highly expressed in the cells of yolk syncytial layer (YSL) (Figure 1(b)). Then, the fluorescence spreads to the envelope layer (EVL) at 8 hpf (Figure 1(c)). Before forming the optic vesicle at 12 hpf, the eye field begins to evaginate and the fluorescence significantly appears in the epidermis layer (Figure 1(d)). At 18 hpf, when the optic cup is formed, the fluorescent signals concentrate at the anterior margin of retina in the eye field (Figures 1(e), and 1(e′)). At 24 hpf, it can be spread to whole eye tissue (Figures 1(f), and 1(f′)). At 96 hpf, Bves expressed cells were localized around inner plexiform layer (IPL) and RPE (Figures 1(g), 1(g′), and 1(g′′)).We used an anti-zBves antibody to confirm the distribution and location of the Bves protein in 24 hpf, 96 hpf, and adult fish. Compared with the fluorescent signals in the transgenic embryos, the expression pattern of Bves protein is compatible with the fluorescent pattern in transgenic zebrafish (Figures1(f′), 1(g′), 1(g′′), 1(h), 1(i), and 1(i′)). At 24 hpf, the Bves protein is localized at the apical region of the retinal epithelium (Figure 2(i)). Compared with the adherens junction-associated actin bundles colocalized with phalloidin, high level Bves expression is at apical sites of retina epithelium (Figure 2(i)).Figure 2
Zbves morphants exhibited defects in eye size and lamination. The morphological observations of wild-type and ATGmo- and splice mo-injected embryos are shown. Compared with wild-type (a), the eyes formed abnormally and resulted in microphthalmos after knockdown of zBves (b), (c). Statistical analyses of the length of whole eye and lens in both groups were performed (n
=
11,t-test for two groups, P
<
0.01) (d). HE staining in paraffin sections of wild-type and morphant embryos revealed the eye without a laminated retina (e), (f). The same patterns were observed in the semithin Epon sections with toluidine blue staining (g), (h). Scale bar, 50 μm. Immunostaining with anti-zBves antibody (green) and phalloidin (red) was performed in the retinal neuroepithelia of wild-type (i) and zBves MO-treated embryos (j) at 24 hpf and laminated retinas of untreated (k) and zBves MO-treated embryos (l) at 96 hpf. ln, lens. Scale bar, 10 μm.


## 3.2. Bves Knockdown Results in Microphthalmos with Retinal Lamination Defect

To examine the physiological function of Bves in eye development, two kinds of morpholino oligonucleotides (MOs)—ATGmo and splicing mo—were used to disrupt the production of the zBves protein. In our previous study, zBves knockdown embryos exhibited epidermal defects. In addition, a dramatically small eye field could be observed in morphants (Figure2). Compared with the untreated embryos, MO-injected embryos have significantly small eyes (Figures 2(a)–2(c)). The thicknesses of lenses and the lengths of whole eyes were measured and their differences were found to be statistically different at 1, 2, and 3 dpf, respectively (Figure 2(d)).To further compare the microstructural differences between untreated and zBves knockdown embryos, we found that the morphants had a microphthalmos without a laminated retina through hematoxylin and toluidine blue staining (Figures2(e)–2(h)). Additionally, there was a space between the retina and the RPE, indicating subretinal fluid retention (Figures 2(g) and 2(h)). This indicates that zBves disruption might break the RPE barrier in retina and implies that Bves might play an important role in the precise formation of retinal layer structure during retinogenesis. The expressions of actin and Bves were examined in morphants and revealed an unclosed apical pattern of phalloidin-stained adherens junction-associated actin bundles (Figure 2(j)). This result implies that Bves is involved in the generation or maintenance of epithelial cell polarity like that in the epithelia of other tissues [21, 23]. Furthermore, we examined the expression of Bves after retinal lamination. Interestingly, at 96 hpf, Bves was found in the cell membranes of all epithelial layers of the eye; however, the retina exhibited the strongest expression. In the retina, Bves was expressed near RPE barrier and around the inner plexiform layer (Figure 2(k)), which is composed of the amacrine cells and the ganglion cells. Furthermore, the bundles of actin in zBves knockdown retina were dramatically disrupted (Figure 2(l)). These suggest that Bves might play a role in cell-cell junctional maintenance, especially at the apical side of the RPE membrane after eye formation, as described previously [21].


## 3.3. Incomplete Photoreceptor Formation in Bves Morphants

Becausehas encodes atypical protein kinase C, which requires Bves to regulate cell junction, and the zebrafishhas mutant exhibits loss of normal retinal lamination [24], we further investigated whether Bves is required for retinal lamination. The retinae of morphants were stained with different retinal cell-specific antibodies to identify the differentiation of retinal cells in lamination defect during eye development. Our results showed that ganglion cells, Müller glia, and interplexiform cells were present in both morphants (Figures 3(e)–3(g)); however, most of them were mislocalized and unlike those of the normal retina (Figures 3(a)–3(c)). Interestingly, immunostaining with the zpr-1 antibody revealed that photoreceptors are not present at 96 hpf after Bves knockdown (asterisks in Figure 5(h)) whereas they exist in wild-type embryos (arrows in Figure 3(d)). To confirm these results, cone-rod homebox-containing gene (Crx) in relation with eye development was further observed in 72-hpf retina (Figure 3(i)). Surprisingly, Crx protein did not disappear in all mutant retinae. 37.5% of mutant retina expressed Crx protein (Figure 3(j)) but 62.5% of them did not. These results indeed demonstrate that Bves is required for proper retinal lamination, especially for photoreceptors.Figure 3
Disruption of zBves affected the dysregulated patterns in the retina. The frozen sections and immunofluorescence staining with different marker antibodies—zn8 for ganglion cells (a), (e), anti-CAII for Müller glia (b), (f), anti-TH for interplexiform neurons (c), (g), and zpr-1 for photoreceptors (d), (h) were performed in eyes of 96 hpf wild-type and zBves MO-injected embryos. Immunofluorescence staining with Crx (i), (j) antibody in the frozen sections was performed in 72 hpf embryos. 37.5% (6/16) of mutant retina showed Crx protein. The ultrastructures of the retina in wild-type (m), (m′) and zBves MO-injected embryos (n), (n′) were analyzed by TEM. Double staining with CtBP and zpr-1 was adopted in frozen crytosections (k), (l). 33.3% (4/12) of mutant retina showed CtBP protein without zpr-1 expression (l). Asterisks indicate the outer segment. Black arrows indicate the inner segment. N, nucleus; S, Scale bar in immunostaining photographs, 50μm. Scale bar in TEM picture, 1000 nm.To examine the morphology of photoreceptor and to confirm the result of zpr-1 and Crx staining, the ultrastructures and CtBP (synaptic ribbon) expression of the photoreceptors were analyzed by TEM and immunostaining. Compared with the microstructure of a normal retina (Figures3(m), and 3(m′)),zbves morphants were found to lack outer segments under transmission electronic microscopy (Figures 3(n), and 3(n′)). 33.3% of mutant retina had CtBP expression (Figure 3(l)) and 66.6% of them did not show CtBP protein, whereas all wild-type retinae showed CtBP protein (Figure 3(k)). These results indicate that zBves knockdown results in immature and even undifferentiated photoreceptors.


## 3.4. Bves is Essential for the Formation of Cell Polarity between Retina Neuron and RPE to Assist in the Differentiation of Photoreceptor Cells

As Bves is localized in adherens junctions, we examined whether the expression patterns of ZO-1 (zona occludens protein 1), aPKC, andβ-catenin, which are initially associated with adherens junctions in the RPE, are altered after Bves knockdown. The strongest expressions of ZO-1, aPKC, and β-catenin are normally observed at the apical surface of the photoreceptor cells and the RPE in a uniformly honeycomb-like pattern (Figures 4(a), 4(e), and 4(i)). In Bves knockdown embryos, however, the patterns of ZO-1, aPKC, and β-catenin were found to be discontinuous along the apical surface of photoreceptor cells and formed into rosette structures (Figures 4(b), 4(c), 4(f), 4(g), 4(j), and 4(k)). This defect could be rescued by coinjection withzbves mRNA in morphants (Figures 4(d), 4(h), and 4(l)). These results indicate that Bves is crucial for the proper localization of adherens junction proteins along the apical border of the photoreceptor cells and RPE.Adherens junctions were not formed in the outer limiting membrane ofzbves morphants. Immunostaining of adherens junction-related molecules-ZO-1 (a)–(d), aPKC (e)–(h), and β-catenin (i)–(l)—was carried out in the retinas of wild-type and zBves MO-injected embryos andzbvesmRNA rescued morphants at 72 hpf. In wild-type embryos, β-catenin obviously appeared in the apical site of photoreceptors (i), whereas it disappeared inzbve morphants (j), (k). The defect in this protein among morphants could be rescued by coinjection withzbve mRNA. Arrows indicate the apical edge. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
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(l)Figure 5
Illustration of Bves effect on zebrafish retinogenesis. RGC, retinal ganglion cells; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Red spot indicates tight junction.


## 4. Discussion

During eye development, the formation of different ocular layers relies on the tight junctions to anchor the changing tissue shape and cellular polarities [25], and the function of Bves in tight junctions has also been confirmed in this process and described in many studies [21, 23, 26]. In our study, we found that Bves can be expressed earlier in single-layer epithelia of the EVL and later expressed in the ocular surface epithelium, lens, and retina of the eye (Figure 1). At 24 hpf, Bves spreads throughout the eye tissue and locates at cell plasma membranes (Figure 2(i)). At that time, neuroepithelia in eyes are already migrating to the eye cup; these then proliferate or differentiate according to the signals from their niches [27]. Therefore, according to the expression pattern, we consider that Bves might play a role in assisting and regulating the epithelial cell shape and polarity during early eye development.Interestingly, Bves is strongly expressed in the apical site of neuroepithelia and the retina (Figure2(k)), which contact with RPE before closure of the subretinal space and form an outer limiting membrane and a retina-blood barrier [28]. Our recentin vivostudies have shown that Bves can regulate paracellular permeability in epithelia [21]. Thus, it is reasonable to speculate that Bves may be involved in the regulation of the blood-retina barrier and the formation of the outer limiting membrane. At 4 dpf,zbves morphants show fluid accumulation between the RPE and the retina (Figure 2(h)). We further reason that this fluid accumulation results from the disruption of the blood-retina barrier.After Bves knockdown, the eye becomes smaller and loses its normal arrangement as in microphthalmia (Figures2(b) and 2(c)). Many transcription and regulatory factors, such as Pax6, Crx, Otx2, MITF, Sox2, and shh, are involved in eye development, and the mutation of these factors will also induce microphthalmia [29]. Although the cause of small eye in these mutants is still unclear [29], these studies showed the importance of the lens in regulating eye growth, in which ablation of the lens or mutation of genes vital for lens development leads to microphthalmia [30]. In our study, we also showed poorly arranged lenticular fibers in the lens at 3 dpf (Figure 2(i)). Therefore, it is possible to speculate that the microphthalmia could result from the disruption of lenticular formation inzbves morphants and it will be of interest to investigate the possible causes of microphthalmia in future study. In addition, we find that Crx protein expressions in zBves mutants are disordered and even disappeared (Figures 3(i), and 3(j)). These imply that defects in lamination and photoreceptor development in zBves knockdown retinae might result from abnormal transcriptions factor expressions in early stage which arises from the zBves knockdown and disruption of cell polarity.The outer blood-retinal barrier lies between RPE and the outer surface of the photoreceptor layer [6]. To form this barrier, a belt of cell junctions around the apical domain of the photoreceptor should be formed to support the cell polarity of photoreceptors and to form a barrier between the choroid and the retina [6]. Studies onDrosophila have indicated that genetic regulators of photoreceptor polarity are similar to the genes regulating cell polarity in other epithelial tissue [31–33]. The analysis of the vertebrate eye also lends credence to the idea that the regulators of photoreceptor polarity are related to epithelial polarity pathways [34, 35]. Our results confirm this notion and indicate that Bves is also crucial for the proper localization of adherens junction proteins along the apical border of the photoreceptor cells and RPE.The most important thing in our study is thatzbves morphants lost their retinal lamination. In neural tissue, a proper differentiation of apical and basal domains in neuroepithelial cells determines cell fate and lamination during development [1]. Because Bves is located in the apical regions in the retina and plays a role in cell polarity, we think that Bves knockdown would disrupt normal cell polarity during retinogenesis. Furthermore, immunostaining with specific retinal cell makers showed that retinal cell differentiation inzbves morphants was not affected, except in the photoreceptor cells (Figure 3(h)), most of which revealed an undifferentiated pattern without a normal outer segment formation (Figures 3(n) and 3(n′)). Koike  et al. indicated that polarized photoreceptors could assist progenitors to anchor at the apical edge of the retina, which led to the formation of a correct laminar retina [36]. Therefore, we suggest that Bves is involved in retinal lamination through polarized photoreceptors. Furthermore, the bundles of junction proteins between retina and RPE in Bves knockdown embryos revealed a disordered and a discontinuous arrangement (Figures 4(b), 4(c), 4(f), 4(g), 4(j), and 4(k)), andzbves mRNA could rescue these defects (Figures 4(d), 4(h), and 4(l)). Taking them all together, our results suggest that Bves can regulate photoreceptor differentiation in retinal lamination (Figure 5).



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*Source: 803718-2014-03-06.xml*