Abstract

   The plasticity of human retinal pigment epithelium (RPE) has been
   observed during proliferative vitreoretinopathy, a defective repair
   process during which injured RPE gives rise to fibrosis. In contrast,
   following injury, the RPE of the embryonic chicken can be reprogrammed
   to regenerate neural retina in a fibroblast growth factor 2
   (FGF2)-dependent manner. To better explore the mechanisms underlying
   embryonic RPE reprogramming, we used laser capture microdissection to
   isolate RNA from (1) intact RPE, (2) transiently reprogrammed RPE
   (t-rRPE) 6 h post-retinectomy, and (3) reprogrammed RPE (rRPE) 6 h
   post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the
   acute repression of genes related to cell cycle progression in the
   injured t-rRPE, as well as up-regulation of genes associated with
   injury. In contrast, the rRPE was strongly enriched for
   mitogen-activated protein kinase (MAPK)-responsive genes and retina
   development factors, confirming that FGF2 and the downstream MAPK
   cascade are the main drivers of embryonic RPE reprogramming. Clustering
   and pathway enrichment analysis was used to create an integrated
   network of the core processes associated with RPE reprogramming,
   including key terms pertaining to injury response, migration, actin
   dynamics, and cell cycle progression. Finally, we employed gene set
   enrichment analysis to suggest a previously uncovered role for
   epithelial-mesenchymal transition (EMT) machinery in the initiation of
   embryonic chick RPE reprogramming. The EMT program is accompanied by
   extensive, coordinated regulation of extracellular matrix (ECM)
   associated factors, and these observations together suggest an early
   role for ECM and EMT-like dynamics during reprogramming. Our study
   provides for the first time an in-depth transcriptomic analysis of
   embryonic RPE reprogramming and will prove useful in guiding future
   efforts to understand proliferative disorders of the RPE and to promote
   retinal regeneration.

   Keywords: retina, RPE, reprogramming, EMT, RNA-seq, LCM

1. Introduction

   The retinal pigment epithelium (RPE) is a monolayer of pigmented cells
   located between the neural retina and choriocapillaris and constitutes
   the outer blood-retinal barrier. As such, the RPE plays an important
   role in visual disorders and has received special attention for its
   regenerative potential following retinal cell loss
   [[38]1,[39]2,[40]3,[41]4,[42]5]. In humans, RPE plasticity has been
   observed during pathological conditions such as proliferative
   vitreoretinopathy (PVR), a defective repair process that leads to
   visual impairment [[43]6]. The pathophysiology of PVR is a
   multifactorial process in which RPE cells dedifferentiate, shed
   epithelial characteristics, and acquire a mesenchymal phenotype in a
   process known as epithelial-mesenchymal transition (EMT) [[44]7]. PVR
   can be incited as a repair mechanism following acute trauma, such as
   retinal detachment, but instead of regenerating lost retina, the RPE
   attempts wound repair via scar formation, ultimately resulting in
   visual impairment [[45]6].

   Mammalian RPE can generate neuronal or retinal cells under certain
   conditions. Between embryonic days 12 and 13, rodent RPE can be
   reprogrammed in vitro to form neural retina when treated with
   fibroblast growth factor 2 (FGF2) [[46]8,[47]9]. Similarly, human fetal
   RPE cells are able to proliferate and generate RPE stem-like cells that
   can be differentiated into RPE cells, rod photoreceptors, and
   mesenchymal lineages [[48]10]. Moreover, RPE from adult humans can be
   reprogrammed with small molecules in vitro to form neuron-like cells
   that synthesize dopamine [[49]11]. Together, the observations that
   human RPE responds to injury by undergoing EMT, as well as the capacity
   for fetal RPE cells to differentiate to retinal neurons, suggest that
   mature mammalian RPE may retain some capacity for producing neural
   retina. However, whether there is a specific combination of factors and
   conditions that can efficiently reprogram mature human RPE to retinal
   neurons remains unknown.

   The limited plasticity observed in mammals stands in contrast to
   organisms such as urodele amphibians and the chick embryo (Gallus
   gallus), which have the ability to fully regenerate their retina after
   damage [[50]4,[51]12,[52]13,[53]14]. The embryonic chick can regenerate
   its retina between embryonic day (E) 4–4.5 (Hamburger and Hamilton (H &
   H) stages 23–25) by reprogramming RPE cells into retinal progenitors
   that are capable of generating all major retinal cell types
   [[54]4,[55]12,[56]15,[57]16,[58]17]. RPE reprogramming in the chick has
   been characterized at the molecular level in 3 distinct phases
   [[59]18]. Phase I of RPE reprogramming can be incited by surgical
   retina removal (retinectomy) alone and occurs within 6 h of injury.
   During this phase, RPE cells dedifferentiate and transiently reprogram
   by up-regulating pluripotency-associated factors and eye field
   transcription factors, while simultaneously repressing RPE identity
   genes. However, RPE reprogramming stalls in Phase I unless an exogenous
   reprogramming-inducing factor, such as FGF2, is introduced at the time
   of retinectomy. In the presence of FGF2, these transient changes in
   gene expression are augmented and sustained, committing the RPE to
   reprogram. Importantly, we have previously shown that the RPE remains
   non-proliferative during Phase I, and only in the presence of FGF2 is
   the RPE able to proceed to a proliferative stage in Phase II [[60]18].
   During this phase, the onset of proliferation can be observed by 24 h
   post-retinectomy (PR), and a new neuroepithelium containing a pool of
   retinal progenitor cells forms within 3 days PR. Finally, during Phase
   III, the differentiation of retinal cells takes place within 7 days PR
   [[61]15,[62]18].

   A number of different factors and pathways have been implicated in
   chick RPE reprogramming, but the central mechanisms underlying this
   process remain unclear. For instance, FGF2 induces RPE reprogramming
   through activation of the mitogen-activated protein kinase (MAPK)
   pathway, leading to the induction of the eye field transcription factor
   PAX6 and suppression of the RPE marker MITF [[63]15,[64]19]. Moreover,
   the induction of ectopic SHH signaling in the RPE can inhibit
   reprogramming by protecting the RPE identity [[65]15]. Inhibition of
   β-catenin/LEF/TCF transcriptional activity is sufficient to induce RPE
   reprogramming, even in the absence of FGF2, demonstrating that a
   decrease in canonical WNT signaling occurs during reprogramming
   [[66]20,[67]21]. RPE cells can be reprogrammed in vitro at later stages
   (HH Stage 28–31) by using combined treatment with FGF2 and TGFβ
   inhibitors, suggesting that signaling by TGFβ family members also exert
   an active role in promoting RPE differentiation [[68]9,[69]22]. More
   recently, we demonstrated that the epigenetic landscape of RPE is
   dynamic during reprogramming, including histone modifications
   (H3K27me3/H3K4me3) and changes in DNA methylation profile, and that
   overexpression of DNA demethylation enzyme TET3 is sufficient to
   reprogram RPE [[70]23]. Despite efforts to characterize the various
   pathways which govern RPE identity, there has not been a study to date
   characterizing the global changes in gene expression, which occur
   during embryonic RPE reprogramming.

   The intimacy of the RPE with surrounding tissues such as extraocular
   mesenchyme and retina has impeded efforts to profile the in-situ
   transcriptome associated with RPE reprogramming. Compounding this
   problem, we previously demonstrated that retinectomy leads to rapid
   changes in the transcriptome and epigenetic profile of the injured RPE
   cells, underscoring the need for tissue isolation techniques that do
   not introduce injury signals that may confound the analysis
   [[71]18,[72]23]. In order to avoid the use of dissociation protocols
   that could mask crucial processes, such as those influenced by cell-ECM
   interactions, we performed Laser Capture Microdissection followed by
   bulk RNA-sequencing (LCM-RNAseq). LCM-RNAseq allows for the capture of
   a precise and pure population of RPE cells without the disruption of
   the local tissue microenvironment, preserving the native transcript
   profile. In this study, we employed LCM-RNAseq to analyze the global
   transcriptome associated with early (Phase I) RPE reprogramming. We
   investigated the intact RPE as well as the transiently reprogrammed RPE
   (t-rRPE), which represents RPE 6 h following retinectomy, and
   reprogrammed RPE (rRPE), which represents RPE 6 h following
   retinectomy, additionally treated with FGF2. We identified a number of
   genes in the t-rRPE which have been previously associated with retinal
   injury, as well as novel processes associated with transient
   reprogramming, such as the induction of neural development genes and
   the suppression of proliferation-associated transcripts. In the rRPE,
   we identified a number of FGF-responsive genes and transcription
   factors which drive RPE reprogramming, as well as uncover previously
   unknown roles for EMT and extracellular matrix (ECM) remodeling in
   facilitating the early transition from RPE to neural retina progenitor.

2. Materials and Methods

2.1. Chick Embryos and Surgical Procedures

   White Leghorn chicken eggs from Michigan State University were
   incubated in a humidified rotating incubator at 38 °C. At embryonic day
   4 (H&H Stage 24), retinectomies were performed as previously described
   [[73]15], and FGF2-soaked beads were introduced to the eye cup of
   embryos in the rRPE condition. Embryos were collected at 6 h PR and
   processed for LCM collection.

2.2. Laser Capture Microdissection (LCM)

   LCM collection was performed as previously described [[74]18,[75]24].
   Briefly, embryos were collected and infiltrated with a sucrose gradient
   at 4 °C followed by 2:1 25% sucrose: OCT compound (Sakura Finetek,
   Torrance, CA, USA) for 1 h and snap-frozen in dry ice and methylbutane.
   Cryosections (12 μm) were collected on PEN Membrane Frame Slides
   (ThermoFisher Scientific, Waltham, MA, USA) followed by fixation in 70%
   ethanol at −20 °C for 30 sec. Slides were rehydrated in 4 °C
   DEPC-treated water for 30 s, stained with he-matoxylin (Millipore
   Sigma, Burlington, MA, USA) for 10 s, and dehydrated in a 70%, 95%, and
   100% ethanol gradient for 30 s each. LCM was performed using a Veritas
   Laser Capture Microdissection system and software to collect RNA
   samples for library prepara-tion as described previously [[76]18]. RPE
   was isolated from sections using IR capture and UV dissection on
   CapSure HS LCM Caps (ThermoFisher Scientific, Waltham, MA, USA), and
   total RNA extraction was performed using PicoPure RNA Isolation Kit
   (Applied Biosys-tems, Waltham, MA, USA), including a treatment with
   DNase I (RNase-Free DNase Set, Qiagen, Hilden, Germany). The quality
   and quantity of RNA were determined using Ag-ilent RNA 6000 pico kit
   (Thermo Fisher, Waltham, MA, USA) on the Agilent 2100 Bioana-lyzer.

2.3. mRNA Enrichment and RNA Sequencing Library Preparation

   Total RNA was processed for library construction by Cofactor Genomics
   (St. Louis, MO, USA) according to the following procedure. Briefly,
   total RNA was reverse-transcribed using an Oligo(dT) primer, and
   limited cDNA amplification was performed using the SMARTer Ultra Low
   Input RNA Kit for Sequencing—v4 (Takara Bio, Shiga, Japan). The
   resulting full-length cDNA was fragmented and tagged, followed by
   limited PCR enrichment to generate the final cDNA sequencing library
   (Nextera® XT DNA Library Prep, Illumina, San Diego, CA, USA). Libraries
   were sequenced as single-end 75 base pair reads on the Illumina
   NextSeq500 per the manufacturer’s instructions.

2.4. Mapping of RNA-Seq Reads to the Chicken Genome

   A total of 44–68 million single-end 75 bp reads were processed for each
   of 9 RNA-seq samples. Read quality was assessed using FastQC v0.11.8
   and MultiQC v1.8 [[77]25,[78]26]. Reads were quality trimmed and
   adapter sequences removed using Trim Galore v0.6.1 and Cutadapt v1.18
   with the parameters: --stringency 3 --length 36 [[79]27,[80]28].
   Trimmed reads were aligned to Gallus gallus genome assembly GRCg6a
   using STAR v2.7.3, and splice sites were incorporated from Ensembl
   annotation 98 [[81]29,[82]30]. Aligned reads were sorted with Samtools
   v1.9 [[83]31]. Quantification of transcripts was performed with
   Stringtie v2.0.4 using parameters and -A -C -B -e for unstranded
   libraries, and counts were summarized at the gene level [[84]32].

2.5. Data Filtration and Differential Expression Testing

   The resulting gene count matrix from Stringtie was passed to DESeq2
   v1.22.2 for differential expression testing [[85]33]. Low coverage
   genes were discarded from analysis (combined counts <20), and apeglm
   shrinkage of log2 fold change estimates were applied during
   differential expression testing [[86]34]. Differentially expressed
   genes (DEGs) were defined as |log2(fold change)| ≥1 and adjusted
   p-value ≤ 0.05. All expression values are displayed in DESeq2
   normalized counts or transcripts per million (TPM), as specified in the
   figures. The PCA analysis was performed on the DESeq2 normalized gene
   count matrix. Genome browser visualizations were rendered with the
   Integrative Genomics Viewer, using bam files normalized to TPM with the
   bamCoverage function of deepTools2.0 [[87]35,[88]36].

2.6. Clustering and Pathway Enrichment Analysis

   Genes meeting the DEG criteria were clustered using affinity
   propagation clustering with the R package APCluster [[89]37,[90]38].
   DEGs associated with pairwise comparisons or identified clusters were
   analyzed with g:Profiler for pathway enrichment analysis [[91]39].
   Enrichment results with adjusted p ≤ 0.05 and less than 2000 total term
   genes were included in the analysis. Gene Set Enrichment Analysis was
   performed using GSEA v4.0.3 [[92]40,[93]41]. EMT genes were obtained
   from dbEMT 2.0, a curated database of EMT-associated genes [[94]42].
   Chicken orthologs were identified using BioMart, yielding a final list
   of 839 EMT-associated genes. ECM-associated terms were obtained from
   the matrisome gene sets, and Gallus gallus Ensembl IDs were obtained
   for available orthologs using BioMart [[95]43]. Figures were generated
   in the R environment, as well as using Cytoscape v3.7.2
   [[96]44,[97]45].

3. Results

3.1. LCM-RNAseq of the Early Reprogrammed RPE

   In order to analyze changes in the transcriptome during the early
   stages (Phase I) of RPE reprogramming, we performed LCM-RNAseq. For the
   analysis, we considered that the RPE was in close proximity with other
   tissues, such as the developing retina and extraocular mesenchyme.
   Additionally, previous studies have demonstrated the importance of the
   ECM during retina development and regeneration, as well as interactions
   between the RPE and Bruch’s membrane which influence RPE behavior and
   differentiation [[98]3,[99]6,[100]46,[101]47]. Therefore, to avoid
   disruptive tissue isolation protocols that may mask crucial processes,
   as well as to isolate a precise and pure population of RPE cells, we
   performed laser capture microdissection followed by RNA-seq
   (LCM-RNAseq) ([102]Figure 1a) [[103]48]. RPE cells from 12 µm-serial
   tissue sections (~n = 30) of 9 embryos (3 for each condition) were
   obtained from: (1) un-injured RPE at embryonic day 4 of development;
   (2) transiently reprogrammed RPE (t-rRPE) 6 h PR; and (3) reprogrammed
   RPE (rRPE) 6 h PR, with the addition of an FGF2-soaked bead immediately
   after retinectomy ([104]Figure 1a,b). For rRPE, only RPE in the
   immediate proximity of an FGF2-soaked bead was targeted for laser
   capture. The 6 h PR time point encompassed Phase I of chick RPE
   reprogramming, representing an acute response to retinal injury during
   which t-rRPE and rRPE remain non-proliferative (BrdU-negative,
   p27Kip1-positive), as we have previously shown [[105]18].

Figure 1.

   [106]Figure 1
   [107]Open in a new tab

   RNA-seq experimental design, quality control, and data are summarized.
   (a) RPE was isolated using LCM. (b) RNA-seq was performed on intact
   RPE, transiently reprogrammed RPE (t-rRPE) 6h post-retinectomy (PR),
   and reprogrammed RPE (rRPE) 6h PR in the presence of an FGF2-soaked
   bead. (c,d) Snapshot of Integrative Genomics Viewer genome browser of
   RPE-specific gene MITF and retinal progenitor gene VSX2, respectively.
   The scale bar below shows the genomic distance in base pairs. Labels:
   CM: Ciliary Margin; L: Lens; RPE: Retinal pigment epithelium; *:
   FGF2-soaked bead.

   Total RNA extraction yielded ~10 ng of RNA for each sample and was used
   to generate libraries for low-input poly-A enrichment RNA sequencing.
   Bulk RNA-seq resulted in 44.5−67.5 million high-quality reads per
   sample with a genomic alignment rate of 91.5–94% ([108]Figure S1). A
   three-dimensional representation of Principal Component Analysis (PCA)
   using normalized gene expression counts showed the samples forming
   three distinct clusters ([109]Figure S2a). In addition, unsupervised
   clustering of total normalized gene expression confirmed congruence
   among biological replicates ([110]Figure S2b). The genomic distribution
   of reads among the conditions was similar, with the majority of reads
   (64−69%) mapped to annotated genic regions of the Gallus gallus genome
   ([111]Figure S2c). An additional 22−26% of reads aligned to unannotated
   genomic regions, with another 3.52−3.95% of reads multi-mapping,
   1.7−1.8% aligned to ambiguous features, and only 3.0−3.8% unaligned
   ([112]Figures S1c and S2c). Following TPM normalization, housekeeping
   genes GAPDH and SDHA showed uniform coverage and consistent expression
   across samples, reflecting unbiased amplification between libraries and
   suitable post-sequencing normalization measures ([113]Figure S2d;
   Supplementary Files S1–S3). Consistent with previous results, we
   observed the down-regulation of RPE marker MITF in the t-rRPE and rRPE,
   as well as the up-regulation of retinal progenitor genes ASCL1 and VSX2
   in the t-rRPE and rRPE, although VSX2 was only modestly increased in
   the t-rRPE (log2[fold change] = 0.78, adjusted p = 0.08) ([114]Figure
   1c,d and [115]Figure S2d; Supplementary Files S1 and S2) [[116]18].
   Changes in expression of cell identity markers MITF, ASCL1, and VSX2 in
   the t-rRPE, as well as the close spatial proximity of t-rRPE and rRPE
   in the PCA, lend further support for a model of RPE reprogramming which
   is partially initiated by retinectomy, even in the absence of exogenous
   FGF2 ([117]Figure 1c,d and [118]Figure S2a,d).

   We next turned our attention to differential expression testing in
   order to identify genes regulated during early RPE reprogramming. To
   conduct differential expression testing, we used the R package DESeq2
   and defined differentially expressed genes (DEGs) by the cutoff
   criteria of |Log2(Fold Change) | ≥1 and adjusted p-value ≤0.05
   [[119]33,[120]34]. To summarize each of the pairwise comparisons, we
   constructed MA plots displaying each gene’s log fold change plotted
   against average expression, revealing no readily apparent biases in log
   fold change estimates and highlighting the distribution of genes with
   an adjusted p-value ≤0.05 in red ([121]Figure S2e). Using this method,
   we identified in the t-rRPE and rRPE, 296 and 363 DEGs, relative to
   intact RPE, respectively; another 127 DEGs were identified between
   t-rRPE and rRPE. These DEGs were used for further analysis in the
   subsequent sections ([122]Supplementary Files S1–S3).

3.2. Injury-Responsive Genes Initiate Reprogramming and Suppress
Proliferation in the t-rRPE

   Of the 296 DEGs discovered in t-rRPE relative to intact RPE, we
   identified 175 up- and 121 down-regulated genes, which are summarized
   in a volcano plot with DEGs marked in red ([123]Figure S3a). A complete
   list of these injury-responsive DEGs is also provided
   ([124]Supplementary File S1; Table S1). To visualize the top DEGs, we
   display heatmaps of the 25 down- and up-regulated genes, which
   exhibited the strongest evidence for differential expression, ranked by
   adjusted p-value ([125]Figure 2a). Interestingly, many of these genes
   have been previously associated with eye development, injury response,
   and retinogenesis across broad contexts. We briefly discuss below
   notable genes, which warrant further consideration in connection to RPE
   reprogramming.

Figure 2.

   [126]Figure 2
   [127]Open in a new tab

   Injury-responsive DEGs control cell cycle dynamics and initiate
   reprogramming in the early t-rRPE. (a) Top 25 up- and down-regulated
   genes, as ranked by adjusted p-value, are displayed in heatmaps. Values
   are row-normalized. (b) Pathway enrichment analysis of up-regulated
   genes. (c) Down-regulated genes were used for pathway enrichment
   analysis, using the same criteria as in (b).

   Among the top 25 up-regulated genes, we identified STRA6, which encodes
   a protein associated with retinol metabolism in the RPE, as well as
   lens-associated genes such as the crystallins CRYAA and CRYAB, the
   filensin BFSP1, and the avian D-crystallin ASL1 ([128]Figure 2a)
   [[129]49,[130]50,[131]51,[132]52,[133]53,[134]54]. Alpha crystallins
   CRYAA and CRYAB serve a dual role as heat shock proteins with
   anti-apoptotic effects and have been observed to be stress-responsive
   in human RPE in-vitro [[135]55,[136]56]. These findings may be
   reconciled with observations that RPE can form lentoid structures when
   maintained in culture conditions and may reflect a propensity for RPE
   to express genes associated with lens identity when placed in ectopic
   or otherwise stressful environments
   [[137]57,[138]58,[139]59,[140]60,[141]61]. Similarly, we identified as
   a top up-regulated DEG GADD45G, which serves as a DNA repair factor
   that can promote cell cycle arrest under stressful conditions
   [[142]62]. The dual-specificity phosphatase (DUSP) CDC14A which not
   only counteracts MAPK signaling but also plays a direct role in DNA
   repair and exit from mitosis, further displayed potent up-regulation
   [[143]63,[144]64,[145]65]. Together, these results highlight a number
   of stress-responsive genes which may play determining roles in stalling
   cell cycle progression within the early t-rRPE, and potentially serve
   inhibitory roles to retina regeneration in the absence of FGF2.

   In addition to the above injury-associated factors, we identified a
   number of up-regulated genes, which are involved in the formation of
   neural retina, lending further support to a model of RPE reprogramming,
   which is initiated by retinectomy alone. Among the top DEGs is ASCL1
   ([146]Figure 2a and [147]Figure S2d), encoding a transcription factor
   necessary for Müller glia reprogramming in mice and zebrafish; it has
   previously been demonstrated that the overexpression of ASCL1 alone can
   reprogram chick RPE cell cultures to retinal neurons
   [[148]66,[149]67,[150]68,[151]69,[152]70,[153]71,[154]72]. Moreover,
   the transcription factor-encoding gene STAT3 was also induced in the
   t-rRPE after injury and has been shown to play an important role in
   neuroprotection and retina regeneration in mice, zebrafish, and birds
   ([155]Supplementary File S1)
   [[156]68,[157]73,[158]74,[159]75,[160]76,[161]77,[162]78,[163]79]. It
   is possible that Müller glia and RPE reprogramming may share
   commonalities via ASCL1 or STAT3 regulatory mechanisms
   [[164]75,[165]79]. AXIN2, a negative regulator of canonical WNT/TCF, is
   also up-regulated, further emphasizing the importance of decreasing WNT
   signaling for the initiation of RPE reprogramming ([166]Supplementary
   File S1) [[167]21,[168]80,[169]81,[170]82,[171]83,[172]84]. Similarly,
   we identified the up-regulated gene Neuromedin-U (NMU), a neuropeptide,
   which is able to activate the extracellular signal-regulated kinase
   (ERK) pathway, as well as the transcription cofactors BTG1 and BTG2,
   which are involved in neural precursor differentiation and the
   suppression of proliferation ([173]Supplementary File S1; [174]Figure
   2a) [[175]85,[176]86,[177]87]. Together, these observations highlight
   key shifts in known modulators of RPE and retinal identity immediately
   following retinectomy.

   In order to glean more comprehensive insight into the biological
   functions regulated in the t-rRPE, we performed pathway enrichment
   analysis on the identified DEGs ([178]Figure 2b,c). As expected, we
   found terms associated with lens and eye development to be highly
   enriched among our up-regulated genes (including BFSP1, ASL1, ASL2,
   CRYBA4, CRYAA, CRYBA2, CRYAB, NTRK3, and STRA6) ([179]Figure 2b).
   Interestingly, our up-regulated dataset was also associated with axon
   injury and regeneration, including the genes KIAA0319, TNC, JUN, and
   NREP, highlighting potential similarities between the injured RPE and
   neural injuries in other contexts ([180]Figure 2b; [181]Supplementary
   File S4) [[182]88,[183]89,[184]90,[185]91]. Arginine biosynthetic
   process is also highly enriched and could likely reflect the role of
   ASL family proteins as crystallins during avian lens development.
   Intrigued by these findings, we further examined our gene list using
   the Enrichr tool and the Jensen DISEASES database, and found high
   combined enrichment scores of our DEG set with a number of eye
   pathologies, including retinal detachment ([186]Figure S3b)
   [[187]92,[188]93,[189]94]. The extensive overlap between DEGs in the
   injured t-rRPE and genes associated with eye pathologies highlights the
   conserved and vital functions of many of the identified DEGs in
   maintaining eye physiology.

   On the other hand, analysis of the 121 down-regulated DEGs bolsters the
   indication that cell proliferation is suppressed in the t-RPE. Several
   of the top down-regulated DEGs are important regulators of cell cycle
   progression, including PCNA, E2F1, MCM2, CDK6, and RRM2 ([190]Figure
   2a). The broad suppression of cell cycle-related functions was further
   emphasized in the pathway enrichment analysis, which includes terms
   largely related to DNA replication and cell migration, as well as
   related terms such as cell cycle DNA replication, mitotic DNA
   replication, and pre-replicative complex assembly ([191]Figure 2c,
   [192]Supplementary File S4). It is possible that the observed
   suppression of cell cycle-related transcripts plays a determining role
   in stalling the t-rRPE in a non-proliferative state and could prevent
   the t-rRPE from progressing past Phase I of reprogramming in the
   absence of FGF2. Interestingly, enrichment analysis suggested that
   genes associated with cell migration and motility were also
   down-regulated in the t-rRPE, including the semaphorin SEMA3G, as well
   as many structural components, including fibronectin FN1, collagen
   COL1A1, and cadherin CDH5 ([193]Figure 2a,c, [194]Supplementary File
   S4). Together, these results highlight a previously undescribed network
   of gene regulation that is incited by retinectomy, and reflects a
   dynamic regulation of RPE cell behavior that extends to proliferation,
   migration, and retinal identity.

3.3. Transcriptome Changes in the Early rRPE reflect MAPK Signaling Activity
and AP-1 Transcription Factors

   We next sought to evaluate transcriptional changes, which occur during
   early reprogramming in response to combined injury and FGF2 treatment.
   Using the same parameters described above, we identified 100 down- and
   263 up-regulated genes in the rRPE relative to the intact RPE
   ([195]Figure S3c; Supplementary File S2; Table S2). To this end, we
   further displayed the top 25 down- and up-regulated genes, as ranked by
   adjusted p-value, in row-normalized heatmaps ([196]Figure 3a).
   Interestingly, we observed up-regulation of both FGFR1 and FGFR4 in the
   rRPE ([197]Figure 3a; [198]Supplementary File S2). We have previously
   shown that FGF2 acts through FGF receptors to stimulate the MAPK
   cascade, establishing that the FGF-FGFR-MEK-ERK pathway is necessary
   for the RPE reprogramming process [[199]19]. In the same study, it was
   shown that blocking FGFR1/2 signaling during reprogramming leads to the
   reduced presence of PAX6, and also results in reduced proliferation of
   the rRPE at 24 h PR. The evidence presented here also implies that
   FGFR1 is regulated at the transcript level during reprogramming.
   Notably, we also identified FGFR3 as a top down-regulated gene, further
   underscoring that FGF2 signaling entails the regulation of FGF receptor
   expression ([200]Figure 3a).

Figure 3.

   [201]Figure 3
   [202]Open in a new tab

   FGF2-responsive genes reveal a unique transcript profile in rRPE. (a)
   Top 25 up- and down-regulated genes are displayed in heatmaps, as
   ranked by adjusted p-value. Values are row-normalized. (b) Pathway
   enrichment analysis of the 263 up-regulated genes. (c) Summary of
   pathway enrichment results for the 100 down-regulated genes, created
   with the same criteria as in (b).

   Surprisingly, among our top up-regulated genes were four prominent
   members of the AP-1 transcription factor complex, FOS, JUN, ATF3, and
   MAFA ([203]Figure 3a; [204]Supplementary File S2). The expression of
   AP-1 subunits has been shown to be MAPK-inducible, and together can
   regulate a broad range of cellular functions ranging from proliferation
   to differentiation in a context-dependent manner [[205]95]. AP-1
   transcription factor activity is influenced by the available protein
   subunits, binding site accessibility, and the present intracellular
   environment. Future studies may clarify a role for AP-1 transcription
   factors during RPE reprogramming. A number of other up-regulated genes
   were identified in the rRPE, which were not observed following
   retinectomy alone, such as tyrosine kinase signaling antagonists SPRY1
   and SPRY4, which control a wide range of biological functions such as
   metabolism and differentiation ([206]Supplementary File S2) [[207]96].
   Two DUSP family members were also up-regulated, DUSP1 and DUSP5, which
   have the capacity to regulate the activity and strength of MAPK
   signaling ([208]Supplementary File S2) [[209]97]. Interestingly, two
   members of the secreted ligands of TGF-β family were up-regulated, BMP2
   and BMP4, which is notable given the crucial role of BMP signaling in
   controlling the specification and identity of the RPE and neural retina
   ([210]Supplementary File S2) [[211]84,[212]98,[213]99,[214]100].
   Importantly, SMAD6, an inhibitory signal transducer of BMP and TGF-β
   signaling, was also up-regulated ([215]Supplementary File S2)
   [[216]101]. These observations suggest that the correct balance of BMP
   signaling is important for the specification of RPE and neural retina,
   and the mechanisms of action of BMPs might differ between development,
   injury response, and reprogramming contexts.

   In agreement with our previous study, we observed increased expression
   of GADD45Β in the rRPE, and it is possible that GADD45B regulates DNA
   methylation to facilitate the epigenetic reprogramming of RPE
   ([217]Supplementary File S2) [[218]23]. As noted prior, and in
   agreement with previous studies, the neural retinal progenitor marker
   VSX2 was identified as one of the top up-regulated DEGs in rRPE
   ([219]Figure 3a) [[220]18]. Of note, interleukin 6 signal transducer
   (IL6ST) was also up-regulated, which is associated with the signal
   transduction of several cytokines, including IL6 ([221]Supplementary
   File S2). In this regard, we previously demonstrated that complement
   component C3a is sufficient to induce retina regeneration through
   activation of progenitor cells located in the ciliary margin via STAT3
   activation, which in turn activates inflammation-responsive factors
   such as IL6 [[222]76]. It is possible that IL6 acts in a paracrine mode
   to contribute to RPE reprogramming. Similar to the t-rRPE, STAT3
   remained up-regulated in the rRPE, and although STAT3 activity is
   largely regulated at the protein level, further studies are necessary
   to explore its function in this context ([223]Supplementary Files S2
   and S3).

   As expected, pathway enrichment analysis identified a diverse set of
   overrepresented terms among the up-regulated genes in the rRPE,
   especially those related to phosphate metabolism and the MAPK cascade.
   Notable terms include regulation of MAPK cascade, regulation of the
   ERK1 and ERK2 cascade, and negative regulation of phosphate metabolic
   processes, which includes genes such as SPRY4, PTPRO, ATF3, SOCS3,
   DUSP1, EZR, SPRY1, DUSP5, and PTN. These observations further point
   toward activation of the MAPK cascade as a main driver of RPE cell
   proliferation and reprogramming. Interestingly, the rRPE was enriched
   for up-regulated genes associated with cell migration, cell motility,
   and cell population proliferation, indicating a trend toward the
   partial recovery of these processes with the addition of FGF2
   ([224]Figure 3b), as opposed to the t-rRPE, which displayed
   down-regulated genes associated with cell migration, motility, and
   proliferation ([225]Figure 2c). Given this pattern, it is likely that
   the associated genes play indispensable roles in initiating a
   regenerative response in the injured RPE. The complete list of enriched
   pathways and their associated gene sets are included in
   [226]Supplementary File S4.

   In contrast to the up-regulated gene set, MITF was among the top 25
   down-regulated genes in the rRPE, reflecting the loss of RPE identity
   that is a defining feature of reprogramming ([227]Figure 1c and
   [228]Figure 3a). Similar to the t-rRPE, the structural genes
   fibronectin FN1 and CDH5 remained down-regulated in the rRPE. A
   substantial number of transcription factors were among the top 25
   down-regulated genes, including ID2, HEY1, PITX1, ETS1, and BARX2,
   providing further evidence of a changing regulatory landscape that
   accompanies early RPE reprogramming ([229]Figure 3a). Pathway
   enrichment analysis of the down-regulated genes in the rRPE reflected
   processes associated with structural, metabolic, and transcriptional
   changes. A number of terms related to extracellular space were
   identified, as well as a significant enrichment of genes associated
   with primary metabolic processes. Another important term associated
   with cell structure, metalloendopeptidase inhibitor activity, was also
   enriched, including the down-regulated genes TIMP3 and RARRES1. Other
   down-regulated terms include regulation of nitrogen compound metabolic
   process and transcription by RNA polymerase II, which reflects changes
   in metabolism occurring during early embryonic RPE reprogramming
   ([230]Figure 3c, [231]Supplementary File S4).

3.4. Gene Clusters Summarize the Transcriptional Dynamics of RPE
Reprogramming and Associated Functions

   We next sought to more precisely define the networks of gene expression
   that are regulated during RPE reprogramming, as we considered that a
   number of DEGs could demonstrate regulatory patterns that are not
   readily captured by pairwise analysis. To address this problem, we
   turned to affinity propagation clustering, a method of organizing DEGs
   into discrete groups based on similarities in expression pattern
   [[232]37,[233]38]. After compiling a list of all non-redundant DEGs
   across conditions (n = 592), we used affinity propagation clustering to
   organize the DEGs into five clusters, with each cluster resembling a
   distinct, archetypal pattern of gene expression ([234]Figure 4a and
   [235]Figure S4a; Supplementary File S5). In addition, we analyzed each
   of these clusters using pathway enrichment analysis and integrated the
   results into a unified network ([236]Figure S4b). These five clusters
   are particularly revealing in expounding upon the gene networks which
   differentiate the non-regenerative response of the t-rRPE from the
   rRPE’s regenerative outcome, and they lend insights to the mechanisms
   by which FGF2 signaling commits injured RPE to reprogram.

Figure 4.

   [237]Figure 4
   [238]Open in a new tab

   Clustering reveals gene expression dynamics during RPE reprogramming.
   (a) Affinity propagation clustering of 592 non-redundant DEGs
   delineated five gene clusters adhering to unique expression patterns.
   The heatmap is row-normalized and notable genes associated with retinal
   development and regeneration are annotated on the right. (b) Bubble
   chart displays key terms associated with each DEG cluster. The size of
   each bubble represents the number of DEGs identified for each term; the
   color of each bubble is associated with the cluster number from (a).

   Clusters 1 and 2 contain DEGs with expression patterns exclusively
   regulated in the rRPE, suggesting that the expression of these genes is
   directly or indirectly dependent on the presence of FGF2 ([239]Figure
   4a and [240]Figure S4a). Cluster 1 and 2 exhibit inverse patterns, with
   cluster 1 genes showing markedly elevated expression in the rRPE, and
   cluster 2 genes being distinctively down-regulated in the rRPE. Given
   these patterns, we anticipated these clusters to be of importance in
   effectuating processes downstream of FGF2 signaling, with genes in
   cluster 1 acting as determining factors associated with neural retina
   and genes in cluster 2 of importance in maintaining RPE identity. In
   line with these expectations, cluster 1 contains SIX6, an eye field
   transcription factor required for neural retina development, while
   cluster 2 contains the transcription factor OTX2, which is associated
   with early RPE identity and is acutely down-regulated in amphibian RPE
   during retina regeneration [[241]102].

   We previously demonstrated that MAPK signaling is the downstream
   effector of FGF2 signaling in rRPE, and accordingly, pathway enrichment
   analysis of cluster 1 revealed MAPK signaling to be a highly enriched
   term ([242]Figure 4b; [243]Supplementary File S5) [[244]19]. Cluster 1
   encapsulates a number of MAPK signaling antagonists, such as DUSP1,
   DUSP7, RASA2, SPRY1, and SPRY2, as well as the phosphatase PTPRO
   ([245]Figure 4a; [246]Supplementary File S5). The pathway enrichment
   term Tissue Development is also highly enriched in cluster 1, which
   included genes belonging to a number of prominent developmental
   signaling pathways, including FGFR1, as well as TGFB3 and the
   downstream negative regulator SMAD6. Similarly, cluster 1 is also
   strongly enriched for genes associated with actin filaments and
   epithelial polarization, such as the cytoskeletal elements ACTN1 and
   EZR, the myosin MYO1E, cytoskeleton regulator LIMA1, and
   actin-interacting proteins FSCN1 and SLC9A3R1 ([247]Figure 4;
   [248]Supplementary File S5). When considering cluster 2, the role of
   FGF2 in inciting actin rearrangements is reinforced, which includes the
   actin-associated genes CGNL1, RHOF, and FMN1 ([249]Figure 4;
   [250]Supplementary File S5) [[251]103,[252]104,[253]105]. Cluster 2
   further suggest shifts in extracellular matrix composition and
   inflammation, containing collagen COL8A1, fibronectin FN1, fibrillin
   FBN1, interleukins IL16 and IL18, as well as complement receptor C1R
   ([254]Figure 4a; [255]Supplementary File S5). Despite these
   observations, pathway enrichment analysis of cluster 2 did not return
   any significantly overrepresented pathways at the stringent cut-off of
   adjusted p ≤0.05. However, given the known functions of these genes and
   their altered expression in the rRPE, it is likely they play
   significant roles in modulating injury response and priming the RPE for
   the formation of retinal progenitor cells.

   The largest number of DEGs are found in cluster 3 (n = 202). These
   genes adhere to an expression pattern following modest up-regulation in
   the t-rRPE (i.e., in response to injury), and further up-regulation in
   the rRPE, suggesting that exogenous FGF2 treatment enhances, but is not
   solely responsible for, these genes’ expression. Similar to cluster 1,
   cluster 3 is highly enriched for MAPK-associated genes, demonstrating a
   significant enrichment of genes associated with the MAPK cascade and
   regulation of phosphorylation ([256]Figure 4b). Genes associated with
   these processes included SPRY4, ATF3, JUN, DUSP5, BMP2, FGFR4, STAT3,
   and MAP3K4 ([257]Figure 4a; [258]Supplementary File S5), which are all
   directly a part of the MAPK signal transduction cascade or have been
   shown to be regulated by this pathway. Although we observe significant
   elevation of these genes’ expression in the t-rRPE and rRPE, many of
   these genes are also regulated at the protein level, and their activity
   is likely only partially explained by their transcriptional regulation.
   The observation that these genes are modestly elevated in the t-rRPE
   suggests that they may play a role in transient reprogramming or in
   priming the injured RPE toward neural retina production. This may
   additionally indicate that these genes are also regulated by some
   pathway other than FGF2 signaling, especially one that leads to a burst
   in MAPK activity in the t-rRPE. It is unclear if the mechanism
   regulating these genes is unrelated to FGF2, or if this could be caused
   by endogenous FGFs, albeit at a lower concentration, which was
   insufficient to induce committed reprogramming in the t-rRPE.
   Surprisingly, similar to cluster 1, we also identified enrichment of
   the term cell migration in cluster 3, including genes such as CXCL14,
   CCL4, CCL19, VEGFD, and TGFB2, further reinforcing that the RPE is
   primed for cell migration within the first 6 h of reprogramming
   ([259]Figure 4b; [260]Supplementary File S5).

   Genes in clusters 4 and 5 adhere to inverse expression patterns and
   encompass genes with markedly reduced expression in the t-rRPE (cluster
   4), and conversely, genes with elevated expression in the t-rRPE
   (cluster 5). These patterns suggest that regulation of these gene
   clusters is unique to an injured transcriptome and can be reversed with
   the addition of FGF2, and thus are likely to be associated with the
   injured RPE state. In general, there was more variability in gene
   expression in clusters 4 and 5, and the relative enrichment was
   somewhat less pronounced than that observed in clusters 1–3
   ([261]Figure 4a and [262]Figure S4a). As expected, cluster 4 is
   overwhelmingly associated with genes involved with cell proliferation
   and DNA metabolism, including factors such as MCM3, MCM4, MCM5, MCM10,
   ORC6, PCNA, and CDK6, indicating that upon injury, the t-rRPE represses
   cell cycle-related factors, a trend that is partially reversed in the
   rRPE. In this regard, metabolic activity and cell cycle have been
   linked with reprogramming and differentiation, and it may be possible
   that a non-proliferative state may be a prerequisite to provide a
   window for RPE reprogramming [[263]106,[264]107,[265]108]. Cluster 4
   also contained a number of genes whose expression remains reduced in
   both the t-rRPE and rRPE, notably HEY1, MITF, and PITX1. On the other
   hand, cluster 5 contains a number of t-rRPE-enriched genes, which
   increase in expression with retinal injury alone, but which were also
   largely reduced in the rRPE. Terms associated with cluster 5 include
   structural constituents of eye lens, argininosuccinate lyase activity,
   and amino acid metabolism ([266]Figure 4b, [267]Supplementary File S5).
   These terms can be explained by the elevation of lens-related
   crystallins, such as CRYAA, CRYAB, CRYBA2, ASL1, and ASL2 in the
   t-rRPE. However, the function of crystallin genes in RPE is still yet
   to be determined, and it is still unclear if the crystallins are acting
   in accordance with their roles as heat shock proteins as a response to
   retinal injury.

3.5. RPE Reprogramming Exhibits Qualities of an Epithelial-Mesenchymal
Transition and Is Accompanied by Extensive Extracellular Matrix Remodeling

   PVR occurs when human RPE responds to injury signals by undergoing EMT
   and forming fibrous epiretinal membranes. Known modulators of EMT are
   thought to play an inciting role in this pathology, as elevated TGFβ
   levels have been directly implicated in PVR incidence both in-vitro and
   in-vivo, and injuries to epithelial integrity are also thought to be an
   inciting factor
   [[268]7,[269]109,[270]110,[271]111,[272]112,[273]113,[274]114]. PVR
   certainly highlights the cellular plasticity of RPE, but it is unclear
   why the mammalian healing response is limited to fibrotic wound
   closure. Interestingly, knockdown of the eye field transcription factor
   PAX6 in the newt RPE is sufficient to initiate a PVR-like fibrotic
   response, suggesting that there may also be a role for the regulation
   of EMT mechanisms in regenerative systems [[275]3,[276]115]. Given
   these observations, we were intrigued by our finding that TGFB2 is
   up-regulated in the t-rRPE and rRPE, leading us to speculate a possible
   role for EMT in initiating embryonic RPE reprogramming
   ([277]Supplementary Files S1 and S2). To test this hypothesis, we
   performed gene set enrichment analysis (GSEA) using dbEMT 2.0, a
   curated compendium of EMT-associated genes, from which we identified
   839 chicken orthologs [[278]40,[279]42]. Surprisingly, GSEA revealed a
   significant enrichment of the EMT-associated genes expressed in the
   regenerative rRPE relative to the non-regenerative t-rRPE ([280]Figure
   5a,b). To better explain this observation, we performed GSEA on the
   remaining pairwise comparisons, which revealed an overall trend in
   which EMT-related genes were modestly depleted in the t-rRPE following
   injury, and conversely, were highly enriched in the rRPE in relation to
   RPE ([281]Supplementary Figure S5). Together, these results suggest a
   robust ability for the RPE to modulate the transcription of
   EMT-associated factors. This observation supports a model in which
   injury alone leads to the suppression of EMT transcripts, a trend,
   which is reversed in the presence of FGF2. This pattern was especially
   evident in the genes most dramatically repressed in the t-rRPE and
   elevated in the rRPE, such as SNAI1, TGFBR2, ELK3, SMAD3, and TGFB3
   ([282]Figure 5b). It is possible that EMT is in part a shared mechanism
   for initiating both RPE fibrosis and regeneration, with differing
   context-dependent outcomes.

Figure 5.

   [283]Figure 5
   [284]Open in a new tab

   Epithelial-mesenchymal transition genes are highly enriched in the rRPE
   transcriptome, and extracellular matrix remodeling emerges as a
   defining feature of reprogramming. (a) Gene set enrichment analysis
   (GSEA) was conducted using the dbEMT 2.0 database and revealed
   significant enrichment (false discovery rate [FDR] = 0.005) of
   EMT-associated genes in the rRPE relative to t-rRPE. The GSEA report
   displays genes associated with the term as black bars, and the running
   enrichment score is shown above in green. (b) Leading edge genes
   identified in (a) are displayed in a row-normalized heatmap to
   visualize changes in expression. (c) GSEA using the Matrisome database
   revealed Basement membrane-associated genes were significantly enriched
   in the intact RPE relative to the other combined conditions (t-rRPE and
   rRPE). Conversely, the Matrisome database terms ECM regulators (d) and
   Secreted factors (e) were significantly enriched in rRPE relative to
   the other conditions (RPE and t-rRPE), suggesting that extensive
   remodeling of the extracellular matrix, including degradation of the
   basement membrane, occurs in the presence of FGF2.

   We next sought to identify changes in ECM structure that accompany the
   EMT response and may further reflect a change in cell identity. To
   accomplish this, we performed GSEA using the matrisome, a collection of
   gene sets encoding ECM components and regulators [[285]43]. RPE
   behavior is heavily influenced by interactions with the basement
   membrane, and separation from the basement membrane is a hallmark of
   EMT [[286]47,[287]116]. GSEA revealed that genes associated with the
   basement membrane are enriched in the intact RPE relative to both
   t-rRPE and rRPE, suggesting that the initiation of RPE reprogramming
   entails the dissolution of the basement membrane following injury
   ([288]Figure 5c and [289]Figure S6a). Notably, we observed a general
   depletion of basement membrane components during reprogramming,
   including collagens COL6A3, COL4A1, and COL4A6, laminins LAMA2, LAMA4,
   LAMB2, and LAMC1, netrins NTN3 and NTNG1, and nidogens NID1 and NID2.

   Concomitantly, GSEA also revealed that within the rRPE there is a
   general shift toward the elevated expression of ECM remodeling
   components and secreted factors, emphasizing that reprogrammed RPE
   adopts an expression profile, which exerts an active role in reshaping
   ECM and the local tissue microenvironment. The matrisome category of
   ECM Regulators was significantly enriched in the rRPE (FDR = 0.006),
   which notably includes genes such as the matrix metalloproteinases MMP9
   and MMP28, lysyl oxidase-like genes LOXL1 and LOXL2, the lysyl
   hydroxylase PLOD1, and metalloendopeptidase genes ADAMTS9 and ADAMTS15
   ([290]Figure 5d and [291]Figure S6b). Similarly, there was a high
   enrichment of secreted factors in the rRPE (FDR = 0.006), reflecting a
   group of genes that are likely to be broadly involved in reshaping the
   local ocular environment and potentiating cellular cues ([292]Figure
   5e). Genes in this category include CXCL14, HGF, NGF, BMP2, WNT6,
   HBEGF, and TGFB3, among others ([293]Figure S6c). Taken all together,
   these observations suggest that a major feature of RPE reprogramming
   includes the repression of genes associated with the RPE basement
   membrane, with a simultaneous shift toward the production of remodeling
   factors and secreted proteins. These results are consistent with a
   model in which RPE cells in Phase I begin to dedifferentiate, dampen
   expression of an epithelial gene program, prime themselves to
   dissociate from the basement membrane, and actively secrete factors
   that influence and reshape their immediate environment ([294]Figure 6,
   [295]Table 1). However, the extent to which the early mechanisms of RPE
   reprogramming overlap with EMT regulation during PVR are yet unclear,
   and it is also unknown how embryonic RPE ultimately averts fibrosis.

Figure 6.

   [296]Figure 6
   [297]Open in a new tab

   Schematic representation of the changes in gene expression. Major
   findings from the study are summarized in the diagram and table below,
   including the key biological processes associated with changes in gene
   expression in t-rRPE and rRPE. Example genes are listed for each
   category.

Table 1.

   Associated functions in the t-rRPE and rRPE.
   t-rRPE rRPE
   Reduced basement membrane synthesis
   COL6A3, COL4A1, LAMA2 Reduced basement membrane synthesis
   COL6A3, COL4A1, LAMA2
   Reduction in EMT machinery
   SNAI1, E2F1, TGFBR2, TGFB3 Activation of EMT machinery
   SNAI1, E2F1, TGFBR2, TGFB3
   Transient reprogramming
   ASCL1 Retinal progenitor profile
   VSX2, ASCL1, SIX6
   Suppression of proliferation
   MCM2/4/5/10, CDK6 Production of secreted factors
   PDGFD, CXCL14, WNT6, NGF, HGF
   Injury and damage-response
   CRYAA, CRYAB, CRYBA2, GADD45G ECM remodeling
   ADAMTS9, MMP9, LOXL2, PLOD1
   --- MAPK transcript profile
   SPRY1/2, DUSP1/7
   --- AP-1 transcription factor expression
   FOS, JUN, ATF3, MAFA
   [298]Open in a new tab

4. Discussion

   The loss of retinal neurons is irreversible in adult mammals. Following
   retinal injury, the reparative contribution from mammalian RPE is
   limited to fibrotic wound closure, which can be readily observed as
   PVR-derived membranes formed following retinal detachment. An
   attractive approach to combating retinal cell loss would entail
   redirecting this fibrotic response toward a regenerative outcome,
   similar to the spontaneous regeneration of urodele amphibians or the
   FGF2-dependent processes during embryonic chick reprogramming. Here, we
   have for the first time conducted a study to uncover the global
   transcriptional changes associated with early embryonic RPE
   reprogramming, representing a crucial step in understanding the
   molecular events that make tissue reprogramming possible. The evidence
   presented here supports a model in which retinectomy alone initiates a
   partial reprogramming response, yet this response is not sufficiently
   robust to sustain committed reprogramming in the absence of exogenously
   supplied FGF2.

   There are potential therapeutic implications for a model in which RPE
   transiently reprograms following injury alone, and future studies on
   this topic will help elucidate the mechanisms which set apart the
   injury response observed during embryonic stages from the fibrotic
   outcomes following adult injury. In the RPE of spontaneously
   regenerating organisms, such as the newt, retinectomy alone is
   sufficient to induce a gene expression profile characteristic of
   retinal progenitors, including pluripotency factors and eye field
   transcription factors [[299]117,[300]118,[301]119]. In previous
   studies, we demonstrated that this behavior partially holds true in the
   embryonic chick system but is also in part FGF2-dependent [[302]18].
   Here, we reaffirm many of these observations and extend the regulatory
   network to include other factors characteristic of neural injury and
   development. Interestingly, the current study further suggests an EMT
   program is repressed in t-rRPE and robustly activated in rRPE,
   including prominent regulators of EMT such as TGFBR2, TGFB3, and SNAI1
   [[303]116]. Numerous studies have implicated TGFB signaling and EMT in
   the pathogenesis of PVR, and future studies which compare the EMT
   profile of embryonic and adult models of RPE injury will lend insight
   to mechanisms underlying the differing outcomes
   [[304]110,[305]112,[306]113,[307]114,[308]120].

   Accompanying the EMT program, we observed extensive regulation of ECM
   factors during embryonic reprogramming. The changes in ECM-associated
   gene expression can largely be characterized by repression of basement
   membrane-associated genes in reprogrammed conditions and an increased
   expression of remodeling factors in the rRPE. Extensive remodeling of
   the ECM is required during EMT to facilitate the separation of
   epithelial cells from the basement membrane. Interestingly, there is
   evidence to suggest a role for cell-ECM interactions in controlling the
   cell cycle reentry of amphibian RPE. For example, MMPs, which serve
   roles in the degradation of various ECM components, are up-regulated in
   regenerating Xenopus RPE and are required for RPE transdifferentiation
   in-vitro [[309]121]. Given the importance of these interactions, we
   chose to employ LCM in this study, as it is highly desirable to protect
   the intact RPE from injury signals while still isolating the cell
   population with high precision. However, this technique is not without
   limitations and can suffer from reduced, degraded RNA yields, requiring
   relatively increased numbers of PCR cycles during library preparation,
   and ultimately may result in lowered sensitivity to DEG identification.
   Thus, the targets identified herein via LCM-RNAseq may serve as a
   non-exhaustive repertoire of genes regulated during early RPE
   reprogramming.

5. Conclusions

   We present a comprehensive and in-depth analysis of the gene regulation
   which occurs during early embryonic RPE reprogramming, facilitated by
   the use of LCM to capture the native transcript profile. These findings
   shed light on the biological processes governing the transient,
   injury-induced reprogramming of the t-rRPE, and committed, FGF2-induced
   reprogramming of the rRPE. Gene-level analysis points toward extensive
   regulation of pathways and functions known to be essential to
   RPE-derived retinal regeneration, including cell proliferation, injury
   response, MAPK activity, and transcription factor dynamics.
   Importantly, we observe extensive regulation of EMT-associated genes
   and ECM remodeling factors in the t-rRPE and rRPE, suggesting that
   RPE-to-neural retina reprogramming may be facilitated by an EMT program
   that has been associated with fibrosis in other contexts ([310]Figure
   6, [311]Table 1). In conclusion, the genes discussed in this study
   implicate a diverse set of processes with putative roles in governing
   embryonic RPE reprogramming and will serve as an important reference
   for future studies which may further explore the latent plasticity of
   the RPE.

Acknowledgments