Transdifferentiation from cornea to lens in Xenopus laevis depends on BMP signalling and involves upregulation of Wnt signalling
© Day and Beck; licensee BioMed Central Ltd. 2011
Received: 24 March 2011
Accepted: 6 September 2011
Published: 6 September 2011
Surgical removal of the lens from larval Xenopus laevis results in a rapid transdifferention of central corneal cells to form a new lens. The trigger for this process is understood to be an induction event arising from the unprecedented exposure of the cornea to the vitreous humour that occurs following lens removal. The molecular identity of this trigger is unknown.
Here, we have used a functional transgenic approach to show that BMP signalling is required for lens regeneration and a microarray approach to identify genes that are upregulated specifically during this process. Analysis of the array data strongly implicates Wnt signalling and the Pitx family of transcription factors in the process of cornea to lens transdifferentiation. Our analysis also captured several genes associated with congenital cataract in humans. Pluripotency genes, in contrast, were not upregulated, supporting the idea that corneal cells transdifferentiate without returning to a stem cell state. Several genes from the array were expressed in the forming lens during embryogenesis. One of these, Nipsnap1, is a known direct target of BMP signalling.
Our results strongly implicate the developmental Wnt and BMP signalling pathways in the process of cornea to lens transdifferentiation (CLT) in Xenopus, and suggest direct transdifferentiation between these two anterior eye tissues.
Urodele amphibians, for example the axolotl, are well known for their incredible ability to regenerate appendages, such as the limb. However, axolotls are unable to regenerate the lens of the eye following its removal (lentectomy). In contrast, the anuran amphibian Xenopus laevis, in which limb regeneration is subject to an ontogenic decline leading up to metamorphosis, is able to regenerate a new lens from the overlying central corneal cells (for review see [1, 2]). This process was first described by Freeman in 1963, and involves a transdifferentiation of one cell type (corneal epithelium) to another (lens) . It differs from the better-known Wolffian regeneration in adult newts, where a new lens is formed from cells of the pigmented dorsal iris epithelium and is known as cornea to lens transdifferentiation, or CLT .
The trigger for CLT in vivo is exposure of the outer corneal cells to an unidentified factor present in the vitreous of the eye, most likely originating from the neural retina [4, 5]. In vitro, epithelial cells from any location within the lentogenic area, a region extending twice the diameter of the eye  can respond to the vitreous factor and initiate CLT, whereas cells outside this region are refractory to the trigger [6–8]. The limitation of lens forming ability to the lentogenic area correlates with Pax6 expression, and ectopic Pax6 in flank epidermis can confer competence to undergo CLT . As with other cases of regeneration in Xenopus, there is an ontogenic decline in the ability to initiate CLT in vivo , however, this is thought to arise due to a mechanical barrier formed by the healing of the inner cornea rather than a loss of competence . Interestingly, the close relative Xenopus tropicalis, which exhibits more rapid healing of the inner cornea following lentectomy, fails to initiate CLT in vivo although reciprocal transplants show that the central corneal cells of X. tropicalis can respond to the vitreous factor .
Fifty years on from the discovery of CLT, we still know little of the molecular mechanisms that drive the process. While it is generally believed that transdifferentiation occurs directly and not via proliferation of stem cells , a direct demonstration of this is lacking. Jon Henry and colleagues have shown that several transcription factors known to be fundamental to lens development are re-expressed during the process of CLT (Pax6, Prox1, Otx2 and Sox3), suggesting that similar regulation of gene expression drives differentiation during both development and regeneration of the lens [13, 14]. Previous EST analysis of corneal cells undergoing CLT has identified several hundred transcripts from a library constructed from corneal tissue at 1-4 days after lens removal [14, 15].
Despite the identification of multiple candidate pathways from these expression studies, functional analysis of potential transdifferentiation factors has so far been lacking. A single in vitro study demonstrated the ability of acidic fibroblast growth factor (aFGF) to induce lens fibre formation in cultured outer corneas, although morphological organisation of the fibres does not occur . In the current study, we have used a transgenic line of Xenopus laevis to reveal a need for functional BMP signalling during the process of CLT along with a microarray strategy to identify genes and pathways that are likely to be specifically involved in the process of transdifferentiation. The microarray strategy differs from previous library based approaches in that we can specifically compare expression in wounded, non-regenerating corneas to that in corneal tissue undergoing CLT, with the aim of identifying genes associated with the regenerative process. Analysis of this microarray data indicates an important role for wingless/int1 (Wnt) pathway signalling in CLT and suggests that, as with tail and limb regeneration in Xenopus [17–19], several morphogens may be acting to trigger the regenerative process of CLT. We have identified several new candidates for CLT, some of which are also involved in lens formation during development, and many of which are associated with lens pathology, particularly cataract development.
Lens regeneration is dependent on BMP signalling
In WT eyes fixed three days post lentectomy, in 5/10 samples, the transdifferenting cornea had reached early Freeman stage 3, and a cluster of aggregated cells is beginning to invade the vitreous (Figure 2F). A further 2/10 had reached mid to late Freeman stage 3 and no CLT was observed in the remaining 3 samples. At this stage the connection to the cornea is very clear. By five days, the aggregate has detached from the cornea and become a lens vesicle and primary lens fibres have begun to form (Figure 2G, Freeman stage 4). By ten days, primary and secondary lens fibres have formed and the lens appears essentially complete (Figure 2H, Freeman stage 5). The cornea has returned to its original state and is once more composed of squamous epithelial cells.
Microarray analysis of CLT I: Pattern matching based on crystallin expression (CRY list)
Crystallins used in the pattern match are shown in Figure 3B. Three further crystallin genes, Cryba2, Cryaa and a gene most similar to Crybb3, were detected by the match. Prox1, a known eye development gene, was also captured. The Wnt family member Wnt7a appeared twice (due to the presence of multiple probe sets captured by the filter) in the top 50, along with the Wnt receptor Frz7, which occurred a total of four times in the CRY list. Seven of the top 50 ranked genes could not be assigned to a protein family and are annotated as transcribed. Interestingly, two homologues of a CUG triplet repeat RNA binding protein, Cugbp1, were also ranked highly in the match. These proteins, also known as EDEN-BP, are members of the Bruno family and are involved in degradation of mRNA through binding to instability sites .
Top 10 statistically over represented GO categories in the CRY* list
nuclear migration along microtubule/during conjugation with cellular fusion
multicellular organismal development
establishment of wing hair orientation
pyrimidine nucleotide biosynthetic process
R3/R4 cell fate commitment
eye development (sensu Vertebrata)
frizzled signaling pathway
Q-rtPCR validation of 11 genes from the CRY list
Rank in array
Microarray analysis of CLT II: Identification of regeneration associated genes (RAG)
Also included in the RAG list were the matrix metalloproteinases Mmp11 and Mmp14, transcription factors Pitx2, Pitx2a, Six1, Pax6, and the signalling proteins Fgf8b and Wnt6, which both appear twice. Tcf4, which encodes a bHLH transcription factor unrelated to Wnt signalling, appears three times on the list and a closely related gene that we have annotated as Tcf4b (similar) appears twice.
Top 10 statistically over represented GO categories in the RAG* list.
Wnt receptor signaling pathway
patterning of blood vessels
chromatin assembly or disassembly
metal ion transport
tetrahydrobiopterin biosynthetic process
perception of mechanical stimulus
determination of left/right symmetry
iron ion transport
induction of positive chemotaxis
Q-rtPCR validation of 13 genes from the RAG list.
Rank in array
Pluripotency genes are not upregulated in 3 day corneas undergoing CLT
BMP signalling is essential for CLT
BMP signalling has not been previously linked to transdifferentiation of the cornea into lens. However, Faber et al demonstrated that BMP signalling is required for mammalian primary lens cell differentiation both in vivo and in vitro . Furthermore, BMP4 is essential for lens development in mice and acts upstream of the transcription factor and early lens placode marker Sox2 [29, 30], which in turn directly regulates crystallins . Finally, BMP7 is also expressed in the developing lens and regulates expression of Pax6, which also regulates crystallins [29, 30].
We have previously used the transgenic line N1, in which noggin overexpression is controlled by the inducible Hsp70 heat shock promoter, to demonstrate a requirement for functional BMP signalling in limb and tail regeneration in Xenopus laevis [17, 20]. Both tail and limb regeneration involve epimorphic type regeneration, the regrowth of a patterned organ comprised of multiple tissues. Here, we use the same line to show that a distinct type of regenerative process, that of transdifferentiation of cornea to lens, also depends on active BMP signalling. Noggin overexpression, which blocks BMP signalling, appeared to have no effect on the early corneal thickening and cell shape changes associated with wound healing. However, prolonged inhibition of BMP signalling prevented the subsequent progression of cells to transdifferentiating aggregates that eventually form the new lens. Instead, the extra cells seem to become hypertrophic and die. Cellular hypertrophy was also observed in the AEC of poorly regenerating Xenopus limb buds , suggesting that BMP may be a survival signal for cells during regeneration.
In support of this, we have shown that increased expression of Nipsnap1, a known direct BMP target gene , is associated with CLT. The expression of Nipsnap1 in developing lens suggests that it may act downstream of BMPs in specifying the lens fate. In another study of Xenopus CLT, Malloch et al detected transcripts from a gene annotated as similar to BMP5 . CLT in Xenopus differs from the better studied Wolffian regeneration of the lens in newts, which takes place via dedifferentiation of the pigmented epithelial cells (PECs) of the dorsal iris rather than transdifferentiation from the more closely related cornea . Despite this difference, similar signals may be involved in the process. In a recent study, a large number of ESTs were generated from Cynops pyrrhogaster dorsal iris PECs undergoing dedifferentiation after lentectomy . In this dataset, crystallins were not identified, suggesting that the dedifferentiated PECs had not yet begun to transdifferentiate into lens cells. Maki and colleagues detected multiple members of the BMP and TGFβ growth factor pathways but report that components of the Wnt, Fgf and hedgehog signalling pathways were not detected. In newts, however, BMP inhibition leads to enhanced regeneration, in conflict with our observations in Xenopus. Functional evidence showed that inhibition of BMP induces formation of a lens from the ventral iris, which does not normally regenerate . This difference in the role of BMP may reflect inherent differences between Wolffian regeneration in newts and CLT in Xenopus, possibly due to the requirement for dedifferentiation in the newt.
Wnt signalling pathway components are upregulated during lens regeneration
Wnt/β-catenin signalling (via the canonical pathway) is known to be important in driving lens cell differentiation in mammals, with several Wnt ligands, along with their receptors (Frizzled family) and the Lrp5/6 co-receptors expressed during development of the lens [36–38]. Reporter strains in mice have shown a period of Tcf/Lef activity in the lens epithelium as it develops [39, 40], and it is thought that canonical Wnt signalling is required for initial formation of the lens epithelium. In contrast, the Wnt/PCP pathway is thought to play a role in the later lens fibre differentiation from the lens epithelium . Regulation of Wnt signalling via inhibition by secreted frizzled related proteins (Sfrp) also occurs in mammalian lens development . Sfrp2 expression under a lens specific crystallin promotor in mice led to cataract formation .
Here, we present evidence that the developmental role of canonical wnt signalling in mammalian anterior eye formation is recapitulated in lens regeneration from the cornea in Xenopus. Gene ontology analysis of our RAG list demonstrated that regeneration of the lens in Xenopus is accompanied by significantly increased expression of several components of the Wnt signalling pathway. Although the gene ontology for the CRY list suggested involvement of Wnt/PCP pathway, this result is entirely due to the expression of a single Wnt receptor, Frz7, and there is therefore insufficient evidence to implicate Wnt/PCP rather than canonical Wnt signalling, in CLT. The antagonist Sfrp2 was also recovered from the CRY list and was expressed at 1.5 fold higher levels in regenerating CLT corneas than in sham operated controls.
Wnt signalling components were also recovered from an EST collection generated from tissue undergoing the early stages of CLT. Wnt7b and two genes related to the Wnt modulator Sfrp5 were identified by Malloch and colleagues . Furthermore, Wolffian regeneration of the newt lens, while occurring from the neural crest derived pigmented epithelial cells of the dorsal iris rather than the cornea, may well employ some of the same mechanisms (Reviewed in ). Experiments have demonstrated that if Wnt is made available, the ventral iris can also regenerate a lens .
Pitxgenes, which may be Wnt targets, are upregulated during lens regeneration
Three members of the bicoid related homeobox transcription factor Pitx were captured in the RAG list: Pitx2, Pitx2a and Pitx1. A fourth, Pitx3, was captured in the CRY list and was expressed in lens tissue as well as 1.4 × higher in corneas undergoing CLT than in control corneas. Pitx factors have not previously been implicated in vertebrate regeneration although a recent report identified a role in asexual reproduction/regeneration of the ascidian Botryllus schlosseri . Mutations in Pitx genes in humans are known to cause eye developmental defects, particularly affecting the anterior eye structures: cornea, iris and lens. Pitx2 mutations cause Axenfeld-Rieger syndrome type 1, a congenital malformation syndrome affecting the anterior eye [46, 47] iridogoniodysgenesis type 2 (iris hypoplasia) , Peters' anomaly (defective cornea)  and ring dermoid of the cornea . Mutations in the related gene Pitx3 are known to cause congenital cataracts . Pitx2 is induced by the canonical Wnt signalling pathway directly via Lef1 . Pitx genes may therefore act downstream of Wnt signalling in lens regeneration in Xenopus.
Lens crystallins are markers of differentiation and are expressed during CLT
Eleven probesets representing seven UniGenes belonging to the lens crystallin family were used to search for genes with similar patterns of expression across the nine datasets. Searching our microarray data for similarly expressed genes uncovered three more crystallins: Cryaa, cryba2 and crybb3 (similar). Of these, only cryba2 showed a significantly higher expression in corneas undergoing CLT than in sham-operated corneas, i.e. a regenerative response. The expression of three selected crystallin genes was observed at different times during the process of embryonic lens formation (Figure 4). The α-crystallin cryaa was expressed late, between lens stage 4 and 5 according to McDevitt and Brahma . The β-crystallin Cryba1 was the earliest of the three to be expressed in the lens placode, beginning at lens stage 1-2 and the γ-crystallin crygb was expressed slightly later at lens stage 3-4. This pattern of developmental expression is somewhat reflected in the corneas undergoing CLT, with α-crystallins unchanged between regenerating and sham operated corneas and all identified β and γ-crystallins on the array being upregulated during CLT. Therefore we believe we have captured corneas in the act of transdifferentiating, just as the first crystallins become expressed. Furthermore, we observe a correlation in timing of α, β and γ-crystallin expression that reflects that seen during lens development. However, others have reported that the timing of crystallin expression differs between regeneration and development .
Genes associated with congenital cataract formation have similar expression profiles to crystallins in our microarray data
Formation of congenital cataract often results from mutation of genes involved in the formation of the anterior eye, which includes the lens. Many cases of congenital cataract result from mutations in the crystallin genes discussed above. Our search for regeneration specific gene upregulation revealed changes in several other genes known to be involved the formation of congenital cataracts. One such gene, Pitx3, is discussed earlier. The b-Zip transcription factor L-maf ranks 31st in the CRY list, and is 2-fold upregulated in corneas undergoing CLT relative to sham operated controls (Figure 3). L-maf is expressed in the lens placode and later in fibre cells in Xenopus, and directly activates the expression of several lens crystallins . Mutations in a human homologue of this gene cause a type of congenital cataract called CCA4 (OMIM#610202) .
Lens intrinsic membrane protein 2 (Lim2) is ranked 17th on the CRY list and a potential homologue, Lim2 similar, is ranked 13th. Lim2, but not its homolgue, was upregulated in CLT. Lim2 protein is very abundant in the human lens, and mutations in Lim2, also known as MP19, are associated with congenital or early onset cataract . The forkhead transcription factor Lens1 (FoxE3) was ranked 218th in the CRY list and expression was 1.6 fold higher in CLT corneas than in sham operated controls. FoxE3 is associated with ocular dysgenesis and cataracts in humans (OMIM#601094).
Cugbp1 is an RNA binding protein targeting CUG repeats. Xenopus laevis appears to have two homolgues of this gene, Cugbp1b was ranked 8th in the CRY list and expression was 2.4 fold higher in CLT corneas relative to controls, and Cugbp1a was ranked 14th with a 1.5 fold higher relative expression. While Cugbp1 is not directly linked to the formation of cataracts, expansion of CTG repeats in the 3'UTR of the human DMPK gene cause myotonic dystrophy, a form of adult muscular dystrophy that is accompanied by cataract formation . Here, we have shown that Cugbp1b is expressed specifically in the forming lens and is upregulated during CLT, suggesting it could be involved in the pathology of myotonic dystrophy. However, the Cugbp1 duplication appears to be unique to Xenopus laevis and an eye developmental role has not yet been described for these CUG binding proteins in other vertebrates.
Genes associated with pluripotency are not upregulated in three day CLT tissue but chromatin modification may occur
Recent transcriptome analysis of Wolffian regeneration of the newt lens identified several genes associated with reprogramming, such as histone deacetylases and the oncogene c-myc, highly suggestive of dedifferentiation [34, 59]. Furthermore, expression of a subset of pluripotency associated genes (Klf4, Sox2 and c-myc) was found to be increased during newt lens and limb regeneration . In contrast, recent evidence has shown that pluripotency genes were not up regulated during zebrafish fin regeneration, although reduction of either Oct4 or Sox2 activity prevented fins from regenerating . Our microarray data, while limited to only one stage of the process, suggests that CLT occurs without dedifferentiation in Xenopus. Thirteen genes associated with pluripotency were present on the GeneChip array. However, with the exception of Sox2, none of these were up regulated during CLT (Figure 7). Sox2 was expressed at highest levels in the lens samples but was not significantly elevated in corneas undergoing CLT compared to sham operated corneas. Sox2 then may be involved in lens differentiation but does not seem to be indicative of dedifferentiation in this case. Similarly, Sox2 expression was found in limbs and tails before amputation . Another pluripotency associated gene, Fut6, was significantly upregulated in sham operated corneas when compared to corneas undergoing CLT (3.7x), with no expression in lens. Christen et al recently showed the same result in Xenopus limb blastema vs. pseudoblastema, with what they call Fut1 (the same gene) being upregulated regardless of regenerative success . While limited to a single timepoint in the regenerative process, our results show no evidence for pluripotency gene up regulation during CLT and we therefore suggest that returning to a pluripotent state is not part of the CLT process, unlike the lens regeneration from newt PECs. We do note, however, that genes associated with chromatin assembly and disassembly were statistically overrepresented in the RAG list, suggesting that epigenetic changes may be taking place during CLT.
We have shown a functional role for BMP signalling in the process of lens regeneration from the cornea (CLT) in Xenopus laevis tapoles, and identified a new role for Nipsnap1, a of BMP signalling, in the process of lens development. Furthermore, we present strong evidence for the involvement of Wnt signalling and Pitx transcription factors in CLT. Our microarray analysis has identified many genes that are involved in lens pathology, in particular the development of congenital cataract. Finally, we have shown that although there may be alterations to chromatin, there is no evidence for a return to pluripotency, or dedifferentiation, seen in 3 day corneas undergoing CLT.
The N1 stable line of transgenic Xenopus has been previously described . Briefly, the animals contain a transgene (Hsp70:Noggin; γ-Crystallin:GFP) comprised of two linked parts, the first containing X. laevis Noggin coding sequence under the control of the Hsp70 promoter, and the second the green fluorescent protein (GFP) coding sequence under the control of the lens specific promoter γ-crystallin. The line is derived from a single insertion founder made by sperm nuclear injection using the method of  Kroll and Amaya 1996 modified as in Beck et al (2003) . All animal experiments were subject to New Zealand's animal welfare standards for vertebrates and were reviewed by the University of Otago Animal Ethics Committee (AEC). The AEC approved all experiments under protocols AEC78/06 and AEC78/09.
Stage 50-51 tadpoles were anaesthetised in MS222 (1/4000 w/v) in 0.1 × MMR, then placed on their left sides on damp paper towels for surgery. The outer cornea was first snipped with Vannas iridectomy scissors from the posterior side and cut dorsally and ventrally before lifting up as a flap. The inner cornea was ruptured and the lens gently removed using forceps, and the flap of cornea and epidermis was replaced gently over the eye. Animals were allowed to recover in 0.1 × MMR overnight before returning to a marine biotech aquarium and fed as normal. For sham-operated animals, the flap of outer cornea/epidermis was raised as for lentectomy but then replaced without further intervention.
Tadpoles were fixed overnight in cold ethanol/glycine fixative (70% ethanol, 15 mM glycine pH 2.0) at -20°C, dehydrated in methanol and embedded in paraffin wax. 5 μm sections were cut using a Leica microtome and stained with haematoxylin and eosin. At least five animals were sectioned for each timepoint and condition reported.
St. 50-51 tadpoles were either subjected to lentectomy or sham operated as described above. After three days, corneas (containing some epidermis) from lentectomised eyes (R) or sham operated eyes (S) were dissected from tadpoles under anaesthesia in MS222 using Vannas iridectomy scissors and fine forceps and transferred to small pieces of dry Whatman 3 M filter paper, cut with a hole punch, in groups of 8-10. The paper discs were immediately transferred to RNA later in a 1.5 ml centrifuge tube and stored on ice until RNA isolation. Groups of 8-10 dissected lenses (L) were placed directly in RNAlater (Qiagen). Three biological replicate samples were prepared in each case and RNA was isolated using an RNaqueous micro kit (Ambion) with brief manual homogenisation in buffer before removing the paper discs manually. An amplification step (in vitro transcription) was performed using 50 ng of starting material and the samples were labelled with Biotin using a Nugen Ovation kit according to the manufacturers instructions. Three biological replicate pools for each treatment (L, R and S) were hybridised to Xenopus laevis GeneChips (Affymetrix, version 1) and washed using protocol WS2v4_450 before scanning on a 7G Plus GeneChip Scanner 3000 (Affymetrix). Data was normalised for the nine samples using an RMA algorithm with the software GenePattern. Heatmaps were prepared in Microsoft Excel using a custom macro to colour cells according to their values.
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (GEO)  and are accessible through GEO Series accession number GSE28014.
Annotation and gene naming
Annotation was done manually by searching NCBI UniGene with the accession number of the source sequence used to design the Affymetrix probesets. Gene nomenclature follows that used by UniGene: where this differs from common usage, it has been highlighted in the text where practical. Genes that could not be annotated confidently are marked as transcribed. Genes with high similarity to a known gene are marked (similar) or (sim).
Gene ontology analysis
Xenopus TC (Tentative Consensus) numbers and Gene Ontology (GO) assignments for biological function were obtained for the Affymetrix Xenopus laevis GeneChip using Resourcerer v13.0 , December 2006 release. Lists of TC numbers were generated for both CRY and RAG lists and duplicate TC numbers (arising when the GeneChip contained multiple probe sets for one gene) removed using the online BAR duplicate remover tool . Genemerge v1.2  was used to determine GO groups that were statistically over-represented in either list compared to the genes represented on the array.
Primers for quantitative real time PCR were designed, where possible, to different exons, to avoid amplification of genomic DNA. The NCBI program Spidey  was used to predict intron-exon boundaries by comparing X. laevis cDNA sequence to X. tropicalis genomic and transcript sequence from the Joint Genome Institute . Primer sequences, annealing temperatures and product sizes can be found in additional file 3.
Cloning and ISH
Primer pairs for amplification of cDNA can be found in additional file 3.
Crystallins were amplified from reverse transcribed st 50 lens RNA and all other cDNAs from reverse transcribed RNA isolated from mixed embryo stages (13, 19, 33 and 37), using Mango TAQ (Bioline). PCR products were cloned directly into T-tailed PCRIIscript vector using a TOPO kit (Invitrogen) and transformed into chemically competent TOP10 E.coli (Invitrogen). Insertions were verified by DNA sequencing, performed by Otago University's Gene Analysis Service. Digoxygenin labelled ribonucleotide probes were made by linearising plasmids with XhoI and transcribing using SP6 polymerase labelled with digoxigenin-UTP labelling mix (Roche). DNase I (Invitrogen) was used to remove templates following transcription and the probes were precipitated with 2.5 M LiCl. Whole-mount in situ hybridisation of albino embryos and tadpoles was performed as described previously . Proteinase K treatment was 10 μg/ml for 10 minutes for embryos.
The authors acknowledge the assistance of Ellen Hansen with preparation of histological sections, James McEwan for Prox1 and Sox2 in situ hybridisation, and Amy Armstrong for care of the frog colony at Otago.
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