Expression profiling of the RPE in zebrafish smarca4 mutant revealed altered signals that potentially affect RPE and retinal differentiation

Fish maintenance and embryo collection

The following zebrafish lines were used in this study: wild-type (WT) AB and smarca4^a50/+ (yng), which was originally generated from the AB line [21,22]. The WT parents were the genotyped WT siblings of the smarca4^a50/+, and are isogenic other than the smarca4^a50 allele. The adult breeders were maintained according to standard procedures [28]. Embryo collection, staging, and incubation were performed according to standard procedures. The embryos used for an individual experiment were collected from the same parents and spawned within a 15 min interval. All protocols were approved by the Purdue Animal Care and Use Committee.

Microdissection of zebrafish retina and RPE–attached retinas

Microdissection of retinas and RPE-attached retinas from WT and smarca4 ^{a50/ a50} embryos at 52 h post-fertilization (hpf) was conducted as described [9]. WT retinas (WR52) and WT RPE-attached retinas (WRR52) were originally collected and reported in [8], smarca4/yng retinas (YR52) were originally collected and reported in [23], and smarca4 RPE-attached retinas (YRR52) were collected in this study. Three biologic replicates were collected for each condition, and the number of tissues in each replicate is shown in Table 1.

Total RNA extraction and microarray analysis

Total RNA extraction was performed with an optimized procedure that combined TRIzol (Life Technologies, Grand Island, NY) and column-based purification (Qiagen, Valencia, CA) [8,29]. The yield (Table 1) and the quality of the purified total RNAs were evaluated with NanoDrop spectrophotometry (Thermo Scientific, Wilmington, DE) and Bioanalyzer electrophoresis (Agilent Technologies, Santa Clara, CA), respectively. Total RNAs were amplified and labeled using a two-cycle target-labeling protocol (Affymetrix, Santa Clara, CA), and hybridized to GeneChip Zebrafish Whole Genome arrays (Affymetrix). The input RNA amounts are specified in Table 1. Hybridization, washing, and scanning were performed according to the manufacturer’s standard procedure.

Histology

Histological analysis on 1 μm plastic sections was conducted as previously described [8] and imaged with a SPOT-RT3 color slider camera (Diagnostic Instruments, Sterling Heights, MI) mounted on a BX51 fluorescence compound microscope (Olympus, Center Valley, PA). To analyze the ultrastructural changes in the RPE, transmission electron microscopy (TEM) was used. The samples were fixed and processed as previously described [30]. Ultrathin sections (100 nm) through the optic nerve region were collected for TEM analysis with a Philips CM-10 transmission electron microscope (FEI Company, Hillsboro, OR). The resulting images were merged in Adobe Photoshop CS6 (Adobe, San Jose, CA).

In situ hybridization

In situ hybridization was conducted as described [24]. The following riboprobes were used in this study: dopachrome tautomerase (dct; GenBank accession number: NM_131555) and retinal pigment epithelium-specific protein 65a (rpe65a; NM_200751). To prepare the probes for these genes, a fragment of each gene was amplified from a cDNA library prepared as described [29]. The primer sequences are as follows: dct-1F: 5′-ACT TCT TCG TCT GGC AGC AT-3′, dct-1R: 5′-CGG CTT ATC ATA TCC CTC CA-3′, rpe65–1F: 5′- GCT TCG AGT CGG ATG AAG AG-3′ and rpe65–1R: 5′-CAG GGA CGA AAT GGT TGA GT-3′. The resulting PCR fragment was cloned into the pGEM-T easy vector (Promega, Madison, WI) for propagation. The riboprobes were synthesized according to standard procedures [31].

Morphometric measurements and general statistical analysis

Melanosome and RPE parameters were measured and extracted from the images with i-Solution 10.1 (IMT i-Solution, Burlington, Canada). The morphology of the melanosomes was measured by area and roundness. The latter parameter is defined as 4A/f2π (A = area, f – max Feret diameter (the longest diameter along the region-of-interest boundary). Thus, 1 = round, 0 = elongated). Standard error propagation was used to combine the measurement errors of the variables. All standard descriptive statistics and data analyses were performed in the R statistical environment version 2.15.2. Melanosome numbers, area and roundness, as well as RPE area were analyzed with standard ANOVA. The melanosomes along the apical/basal axis was demarcated by the center of the RPE nuclei and their number counted. The resulting data were analyzed with logistic regression. An alpha level of 0.05 was used for all general statistical tests.

Microarray data analysis

The analysis conducted in this study used the WR52, WRR52, YR52, and YRR52 data as described, as well as the whole embryo data obtained from WT (WA52) and smarca4 (YA52) previously collected [23]. The microarray data were deposited at the Gene Expression Omnibus (GEO) under the accession number (GSE50241).

Probe-level analysis and retinal pigment epithelium expression value estimation

The probe-level data of these sample groups were background-adjusted, normalized, and summarized with a robust multiarray average (RMA) algorithm [32] implemented in the affy library of the Bionconductor [33] in R statistical environment version 2.15.2, using default parameters. RPE expression values were estimated from the comparison between the retinal samples and the RPE-attached retinal samples of the same genotype based on a method we previously developed [8]. First, the RMA-normalized expression values in RPE-attached retinal sample were adjusted according to the yield and number of tissues with the following equations:

A. WRR52

adjExprWRR52_ij=ExprWRR52_ij x (mean(yieldWRR52_j/tissueNoWRR52_j)/mean(yieldWR52_j/tissueNoWR52_j))

B. WR52

ExprWR52_ij (i.e., no adjustment)

C. YRR52

adjExprYRR52_ij=ExprYRR52_ij x (mean(yieldYRR52_j/tissueNoYRR52_j)/mean(yieldYR52_j/tissueNoYR52_j))

D. YR52

ExprYR52_ij (i.e., no adjustment)

where Expr is the expression value of a gene i in replicate j, adjExpr is the adjusted expression value of a gene i in replicate j, yield is the total RNA yield of replicate j, and tissueNo is the number of tissue used in replicate j; i=1,…,n; j=1,2,3.

Then, the RPE expression in WT and smarca4 was estimated as follows:

RPEWT52=adjExprWRR52_ij – ExprWR52_ij

RPEyng52=adjExprYRR52_ij – ExprYR52_ij

Factorial analysis and gene selection

A factorial analysis [23] was conducted using the adjusted RPE expression values, as well as the retinal and whole-embryo values. The overall design is a 3×2 model (Figure 1A) that determines the effect of tissue type (T) and mutation (M) on the expression level. The corresponding levels of each factor are listed below:

Mutation (M) – two levels: WT and smarca4

Tissue (T) – three levels: whole embryo, retina, and RPE

For any gene g, its expression (y_g) in the six experimental conditions in Figure 1A was modeled with the following equations:

WT_embryo: y_g = μ (Eq. 2.1)

WT_retina: y_g = μ + T_R (Eq. 2.2)

WT_RPE: y_g = μ + T_RPE (Eq. 2.3)

smarca4_embryo: y_g = μ + M (Eq. 2.4)

smarca4_retina: y_g = μ + M + T_R + M*T_R (Eq. 2.5)

smarca4_RPE: y_g = μ + M + T_RPE + M*T_RPE (Eq. 2.6)

The candidate genes for a specific biologic question were selected by building contrast with the coefficients in Equations 2. In other words, two conditions were statistically compared to identify differentially expressed genes among the conditions (see [23] for further discussion and examples). Depending on whether the two-way interaction term in the Equations 2 was significant or not, the resulting contrasts had to be built differently. In other words, there were first-order (two-way interaction term is not significant) and second-order models (two-way interaction term is significant). Multiple hypothesis testing was corrected by calculating the false discovery rate (FDR). A gene was inferred as differentially expressed when the contrast in consideration had a q value less than 0.001 unless specified otherwise. By combining multiple contrasts, specific rules were built to select 1) smarca4-regulated RPE genes, 2) smarca4-regulated retinal genes, and 3) smarca4-regulated RPE genes that are not differentially expressed in the retina (see the corresponding Result sections for the list of contrasts used). A schematic diagram that shows the relationship between these selections and the resulting candidate genes is shown in Figure 1B. General gene annotations were adopted from an annotation file from Affymetrix (Zebrafish annotation release 30). The selected RPE genes were also analyzed with the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.7 [34,35] using default parameters. Further grouping was aided by the fold change of a comparison. If an estimated expression value was less than zero, indicating the gene was not expressed in the condition, the value was artificially set as one.

Differentiation of smarca4 RPE is abnormal

One distinctive feature of smarca4 mutant was the lower level of pigmentation in pigment cells, including the RPE. The smarca4 RPE cells developed pigmentation at around 24 hpf, a stage when the WT RPE cells began to differentiate and form pigment. However, the pigmentation level was always less intense in smarca4. By 52 hpf, the whole eye was covered with RPE and appeared dark in the WT (Figure 2A,E), while the smarca4 RPE cells were noticeably less pigmented, particularly on the ventral side (Figure 2B,F, red arrow). At this stage, there is differentiation of several retinal cell types in the WT eye, and retinal lamination is present [30,36,37]. In contrast, the smarca4 retina at 52 hpf was dystrophic and would gradually decrease [21-23] (Figure 2F). As a result, the smarca4 eyes were noticeably different from the WT eyes at 72 hpf (Figure 2C,D,G,H). Although the smarca4 RPE was more pigmented at this stage, the RPE cells were still abnormal with holes detected in the RPE layer (Figure 2G,H, red arrow).

To analyze the differentiation problem of the smarca4 RPE, we first conducted an in situ hybridization analysis at 52 hpf with dct and rpe65a, a pigment cell and an RPE-specific marker, respectively. Dct is a key enzyme in the melanogenesis pathway melanin synthesis [38], which gives the pigment cells the distinctive black color. The dct signal generally covered the eye surface of the WT (Figure 2I,K) and smarca4 embryos (Figure 2J,L), suggesting that the smarca4 RPE cells were still committed to the pigment cell lineage. However, the smarca4 staining was irregular, which indicates abnormal RPE differentiation. Indeed, this was supported by the staining of rpe65a, an RPE-specific enzyme that mediates a critical step of isomerization of all-trans-retinol to 11-cis-retinal in the visual cycle [39]. In WT embryos, the staining was intense in the anterior region of the eye and highlighted the RPE cell shape clearly (Figure 2M,O), while in the smarca4 embryos, the staining was irregular (Figure 2N,P), and the extent of staining was far less comprehensive than that observed with dct expression (Figure 2J,O).

The differentiation defects of the smarca4 RPE were further analyzed with TEM at 52 hpf. The smarca4 RPE had fewer melanosomes compared with WT (Figure 3A), which is consistent with the pigmentation defects observed in the semithin sections (Figure 2F,H). In addition, there appeared to be fewer melanosomes on the basal side of the cell (red arrow). To quantify these changes, three independent transverse sections of WT and smarca4 eyes were collected and several RPE attributes measured. First, the number of melanosomes per RPE cell area was analyzed (Figure 3B and Table 2). The RPE on the dorsal and ventral sides of the optic nerve (ON) was analyzed separately. A two-way ANOVA indicated that there were fewer melanosomes/RPE cell area in smarca4 (p value=1.43e-4) and in the RPE ventral to the ON in both genotypes (p value=4.91e-4; see detailed descriptions of the ANOVA output in Appendix 1). In addition, the total RPE area in the smarca4 embryos was not smaller than that in the WT embryos (WT: 1176.36±58.24 μm⁻²; smarca4:1078.35±133.05 μm⁻²; Mann–Whitney test; p value=0.7). Together, these results suggest that there was a general decrease in the melanosome number at this stage. Second, the distribution of the melanosomes in the RPE was evaluated by calculating the apical/basal ratio, using the center of the flattened nuclei to demarcate the apical and basal sides of the RPE cells (Figure 3C and Table 2). A logistic regression analysis of the counts demonstrates that the melanosomes in the smarca4 RPE had an apical bias (p value=3.47e-12) and that there was a general apical bias in the ventral RPE compared with the dorsal RPE in both genotypes (p value=6.15e-07; Appendix 2).

Melanosomes mature through distinctive phases from the circular immature type to the ellipsoidal mature type [40]. To assess the maturity of the melanosomes, their roundness (1: circular; 0: elongated; Figure 3D and Table 2) and area (Figure 3E and Table 2) were separately evaluated with two-way ANOVA. The analysis shows that the roundness of the melanosomes was not affected by genotype (p value=0.67; Appendix 3). There was also no difference in melanosome roundness between the dorsal and ventral RPE (p value=0.07). These observations suggest that the melanosome maturity, as assessed by shape, was similar between the smarca4 and WT genotypes. However, there was a small but statistically significant decrease in the melanosome area in smarca4 RPE (p value=1.96e-7), and in the ventral RPE for both genotypes (p value=2.37e-6; Appendix 4). In addition, the decrease in melanosome size in smarca4 RPE was less drastic in the ventral side (p value=0.0066). Together, these histological data suggest that smarca4 RPE cells were formed, but the differentiation was abnormal at 52 hpf. This observation is consistent with the overall impairment of melanogenesis observed in the smarca4 mutants.

Microarray analysis of smarca4 retina and RPE

The differentiation defects in the smarca4 RPE suggest that the absence of smarca4 disrupted the regulation and expression of genes that were key to normal RPE differentiation. Furthermore, terminal differentiation was also impaired in the smarca4 retina, resulting in abnormal retinal lamination (Figure 2F) [21-23]. Since the RPE and the retina interact closely during development, we were interested in elucidating whether the retinal phenotype might be secondary to disruption of normal signals between the developing RPE and retina. To this end, we extended a factorial analysis framework [23] that was previously used to study smarca4 retinal differentiation. An overview is described (also see the Methods section for the details of the statistical framework).

First, the RPE expression values were estimated by comparing the retinas and the RPE-attached retinas [8]. Then, a 3×2 factorial design (Figure 1A) was used to analyze the expression values of three kinds of tissue (T: whole embryo, retina, and RPE) in two mutation backgrounds (M: WT and smarca4). The expression value of a gene in the individual conditions (y_g) was deconstructed into coefficients that represented the contribution to the expression level by the presence of that factor (Figure 1A, equations under the condition name). Using these coefficients, contrasts were built to identify candidate genes that fulfilled specific criteria. The false discovery rate (FDR) q value cutoff for a contrast was 0.001. Several contrasts were ultimately combined to select genes that were regulated by smarca4 in the retina and the RPE (Figure 1B) and will be discussed below. In addition, a fold-change between smarca4 and WT was calculated to aid ranking of the resulting gene list.

Smarca4-regulated RPE genes

The contrasts selected genes that were differentially expressed in the WT or smarca4 RPE and at the same time were regulated by smarca4, either directly or indirectly (Figure 1B, black circle; Table 3). A total of 591 genes were selected (Appendix 5). Among them, 432 and 162 genes were under- and overexpressed in the smarca4 RPE when compared with the WT RPE, respectively. Seven genes had negative RPE expression value in WT and smarca4 and were not biologically meaningful. DAVID analysis of these genes revealed two functional annotation groups that were highly enriched (Appendix 6; Benjamini-adjusted p value=2.7e-2). These groups include two gene ontology (GO) terms for cellular components: non-membrane-bounded organelle (32 genes/6.1% of total annotated genes) and intracellular non-membrane-bounded organelle (32 genes/6.1% of total annotated genes). These terms refer to organized structures that are not bound by a lipid bilayer membrane including the cytoskeleton, suggesting that the cytoskeletal dynamics of the smarca4 RPE were impaired. Further selection of these RPE candidate genes is described in the third subsection.

Smarca4-regulated retinal genes

The contrasts selected genes that were differentially expressed in the WT or smarca4 retina and at the same time regulated either directly or indirectly by smarca4 (Figure 1B, dark blue circle; Table 4). A total of 412 genes were selected (Appendix 7). Among them, 252 and 160 genes were under- and overexpressed in the smarca4 RPE when compared with the WT RPE, respectively. Many gene candidates were previously identified from a microarray analysis of the smarca4 retinas [23] and were validated in subsequent follow-up experiments [24,25,27]. Furthermore, we identified the smarca4 gene itself as under-expressed in the smarca4 retina, which validated the array experiment and our statistical selection. This is because the null-mutation was expected to result in nonsense-mediated decay of the mRNA and reduce its gene expression. DAVID analysis of these genes revealed several similar functional annotation groups that were enriched (Appendix 8; Benjamini-adjusted p value <0.05; see the table for the gene count and percentage of total annotated genes). For example, eight terms are related to cytoskeletal dynamics, nine terms are related to the cell cycle, and four terms are related to neuron/eye morphogenesis.

Comparing the final list in groups (i) and (ii) showed that 51 genes were commonly expressed in both groups (Figure 1B, the white region between the black and blue circles), while 540 and 361 genes were uniquely expressed in (i) and (ii), respectively. Thus, a total of 952 genes were identified. Among them, 293 (30.78%) had no annotation.

Smarca4-regulated RPE genes that were not differentially expressed in the retina

We theorized that paracrine signaling genes would likely be differentially expressed in one of the two interacting cell-types (i.e., the retina and the RPE), either as secreted ligands or the intracellular signal transducers. To identify these candidate genes in smarca4 RPE, additional selection criteria were imposed on the group (i; Table 5). In particular, these genes should not be differentially expressed in WT or smarca4 retinas. In other words, we selected for genes with RPE-specific differential expression. Specific contrasts were first built to identify retinal-specific genes in WT and smarca4 retinas with a relatively inclusive cutoff (q value <0.05; Figure 1B, light blue circle). Then, these genes were removed from the candidate gene group (i) to select for the genes that were regulated by smarca4 in the RPE but were not differentially expressed in the retina.

A total of 39 genes were selected based on these criteria (Appendix 9). Among them, 31 were under-expressed in the smarca4 RPE, while the remaining eight were overexpressed. Based on their annotations and previous investigations in humans [41-44], mice [44-46], chicks [10], and zebrafish [8], we found that many of the known genes are expressed in the RPE and potentially play an important role in RPE signaling and physiology (Appendix 9; supporting evidence columns). For example, previous studies had detected expression of 31 out of 39 (79.5%) genes in the RPE. Among these data, the zebrafish data represent carefully selected RPE genes that were consistently expressed in our previous investigation [8] (Appendix 9). Even if the zebrafish data are excluded from this calculation, studies in the other systems still support the expression of 22 out of 39 (56.4%) genes in RPE. Therefore, these selected Smarca4-regulated RPE genes represent a group of highly validated RPE-specific genes. DAVID analysis of these genes also revealed two enriched functional annotation groups with more than two genes in each group (Appendix 10; p value<0.05). These functional annotation groups include the focal adhesion pathway (four genes; 12.5% of total annotated genes) and the melanogenesis pathway (three genes; 9.375% of total annotated genes).