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Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development
E XP ER I ME NTAL C E LL RE S E ARCH
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Research Article
Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development Zenzele T.V. Sillaa,1, Jerolen Naidooa,1, Susan H. Kidsonb, Paula Sommera,n a
School of Life Sciences, University of KwaZulu-Natal Westville Campus, Durban 4001, South Africa Department of Human Biology, University of Cape Town Medical School, Anzio Road, Observatory, South Africa
b
article information
abstract
Article Chronology:
Correct formation of the corneal endothelium is essential for continued development of the
Received 28 October 2013
anterior segment of the eye. Corneal endothelial development is initiated at E12 when precursor
Received in revised form
peri-ocular mesenchyme cells migrate into the space between the lens and the presumptive
14 January 2014
corneal epithelium and begin to respond to signals from the lens, undergoing a mesenchymal to
Accepted 16 January 2014
epithelial transition (MET) that is complete by E15.5. To study the initiation of MET, peri-ocular mesenchyme cell lines were derived from E12.5 and E13.5 murine embryos. These cells expressed
Keywords:
key transcription factors, Foxc1 and Pitx2, as well as Slug and Tsc22, genes involved in MET. We
Peri-ocular mesenchyme
have shown that all these genes must be down-regulated by E13.5 for differentiation to proceed.
Corneal endothelial development
Lens-derived signals play a role in this down-regulation with Tgfβ2 specifically down-regulating
Lens
Foxc1 and Pitx2. Over-expression and knock-down of Foxc1 significantly and similarly affected the
Foxc1
expression of Pitx2, Tsc22 and Slug while Foxc1 was shown to play a role in mediating the lens
Pitx2
effects on Slug. Thus, for the progression of initial corneal endothelial development, the key transcription factors, Foxc1 and Pitx2, as well as genes involved in MET, Slug and Tsc-22, must be down-regulated, a process driven by the lens and Foxc1. & 2014 Published by Elsevier Inc.
Introduction A fully functioning cornea is essential for visual acuity. In adults, the cornea is composed of several layers: the outer epithelium which rests on Bowman's layer; the stroma which is responsible for transparency; Dua's layer; Descemet's membrane and the final, innermost layer, the corneal endothelium [23]. Physiologically, the corneal endothelium is the most important layer of the cornea, forming a barrier between the corneal stroma and the aqueous humour of the anterior chamber. The mature corneal endothelium pumps fluid from the stroma and is thus responsible
for maintaining the relatively acellular stroma in a hydrated, yet still transparent state [23]. Importantly, failure of correct endothelial development leads to abnormal development of the anterior segment of the eye [24]. Eye development is initiated by the formation of the optic pits from the neural ectoderm. By embryonic day (E) 9, the optic pits deepen to form the optic vesicles which extend laterally to meet the outer surface ectoderm of the developing head. Contact of the optic vesicle with the surface ectoderm results in the induction of the lens placode from an area of competent surface ectoderm. At E10.5, the lens placode invaginates to form the lens pit which, by
n
Corresponding author. E-mail address: [email protected] (P. Sommer). 1 These authors contributed equally to this work.
0014-4827/$ - see front matter & 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.yexcr.2014.01.016
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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E11, then separates from the overlying surface ectoderm to form the lens vesicle. Importantly, from E12, peri-ocular mesenchyme cells, composed of predominantly neural crest but also mesoderm mesenchyme, migrate into the space between the developing lens and the now continuous surface ectoderm which will mature into the corneal epithelium [15]. From E12, the peri-ocular mesenchyme cells respond to cues from the lens initiating the development of the corneal endothelium by a process of mesenchyme to epithelial transition. The maturing corneal endothelium physically dissociates from the anterior lens between E13.5 and E14.5, resulting in a space between the lens and the corneal endothelium that will become the anterior chamber, filled with aqueous humour. By E15, the maturing corneal endothelium consists of a monolayer of cells, characterised by tight junctions creating a tight barrier of cells capable of actively pumping fluid [13,18]. The molecular cues necessary for the differentiation of the corneal endothelium into an organised monolayer are not well understood. The lens plays an essential role in the development of the corneal endothelium as removal of the lens in chick embryos [3] or ablation of the lens in mice [31] inhibits corneal endothelial and stroma formation. Not only is the presence of the lens essential, but correct and precise instructions from the lens are also necessary for proper corneal endothelial development as in mice, transgenic for lens-specific TGFα or EGF [24] or mice overexpressing lens-specific TGFβ1 [12], the corneal endothelium failed to differentiate correctly. The lens is thus thought to act as a major signalling centre, secreting molecules that pass through the anterior chamber and direct the induction of the corneal endothelium and stroma [15]. Despite the important role of the lens in corneal development, there is little information on the molecules produced and secreted by the lens. To date, the only well described molecule known to be secreted by the lens, with secretion peaking between E13.5 and E15, is TGFβ2 [16]. The importance of Tgfβ2 secretion by the lens is evident in Tgfβ2 / [25] and in Tgfβr2 / [16] mice where the corneal endothelium fails to form, preventing separation of the lens and cornea. A recent microarray study revealed that E12.5 lens epithelial cells produce the BMP antagonist, follistatin [32]. BMPs play a crucial role in eye development [10]. Follistatin is able to inhibit BMP activity, thus playing a crucial role in embryonic patterning. Two known key genes essential for corneal endothelial development are the transcription factors, Foxc1 and Pitx2. Foxc1 belongs to the Fox family of genes, characterised by a conserved forkhead domain of 11 amino acids, many of which play important roles in neural crest differentiation [20]. The paired homeodomain transcription factor, Pitx2, is essential for normal ocular development, encoding three alternatively spliced transcripts in mice, Pitx2A, 2B and 2C. Foxc1 and Pitx2 are both highly, but variably, co-expressed in the peri-ocular mesenchyme from E11.5 [4,15]. Expression of both of these transcription factors in periocular mesenchyme cells is essential for corneal endothelial development as the lack of either one prevents corneal endothelial development and subsequent formation of the anterior chamber ([14,17]). Heterozygous mutations in FOXC1 or PITX2 are directly associated with Axenfeld–Rieger Syndrome, a broad spectrum of clinical anterior segment disorders resulting from impaired differentiation of the peri-ocular mesenchyme [8]. Tsc22 (Tgfβ1i4) is also a transcriptional regulator that has previously been shown to be expressed in peri-ocular mesenchyme
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cells and is a downstream target of Foxc1 [27]. Originally described as an early response gene of TGFβ1, it is highly expressed during development, including in neural crest derived cells. Tsc22 is upregulated at sites of epithelial to mesenchymal interactions and plays a role in differentiation [7,9,29]. Slug (Snai2) is a zinc finger transcription factor that is known to play a key role in epithelial to mesenchymal transitions (EMT) and the converse, mesenchyme to epithelial transitions (MET). It is expressed in the developing mouse eye [26]. Peri-ocular mesenchyme cells migrate into the space between the lens and the surface ectoderm at E12 where they respond to cues from the lens and begin to differentiate into the corneal endothelium. To study these initial steps in corneal endothelial development, we created two cell lines representing peri-ocular mesenchyme at E12.5 and E13.5. We analysed the expression of the two essential transcription factors, Foxc1 and Pitx2, as well as two other transcriptional regulators known to be involved in MET, Tsc22 and Slug, at these two developmental time points. We determined the effects of the lens after exposure of the cells to chick lenses and recombinant Tgfβ2 and follistatin and mapped out their dependency on Foxc1 expression.
Materials and methods Derivation and maintenance of cell lines Peri-ocular mesenchyme cell lines derived from the anterior region of the eyes of E12.5 mouse embryos were immortalised as described [27]. In addition, an immortalised peri-ocular mesenchyme cell line at E13.5 was derived using similar methods (Ethics approval number 019/13/Animal). The cell lines were maintained in DMEM (PAA) supplemented with 10% FBS (Invitrogen) and 0.5 mg/mL penicillin streptomycin (Invitrogen). Cell counts were performed using trypan blue exclusion and metabolic assays were performed using the MTS assay (Promega). All experiments were performed on low and similar passage cells.
Lens exposures, transfections and treatments For the lens exposures, whole chick lenses were dissected at E6 and E8. Ten lenses were added to peri-ocular mesenchyme cells cultured in 60 mm dishes (Corning) containing 4 mL of culture medium for a period of 24 h. Boiled lenses, in which the lens epithelium was dissected away, were used for control experiments. For over-expression of Foxc1, cells were transfected with pFoxc1-eGFP or peGFP-N1 using FuGene 6 (Roche) for 24 h. For Foxc1 knock-down, five putative shRNAs to Foxc1 were chosen from the MIT/Harvard Broad Institute's murine shRNA library database (http://www.broad.mit.edu/rnai/trc/lib). Primers, containing a 21 bp sequence corresponding to the 50 end of the human U6 promoter; 22 bp sequence corresponding to the sense and antisense sequence of the potential shRNA separated by 9 bp loop sequence and 6 bp termination sequence, were designed and, along with a forward primer designed to amplify the 50 end of the human U6 promoter, used to amplify human genomic DNA [5]. The resulting amplicons therefore contained the full-length U6 promoter and the shRNA sequence. The amplicons were cloned into pGEM-T easy (Promega) and transiently transfected into peri-ocular mesenchyme cells to determine knock-down
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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efficiency. Two of the five clones demonstrated a knock-down efficiency of 498% by qPCR and western blot analysis. For knockdown and lens treatment, cells were transfected with the shRNA targeting Foxc1 or scrambled shRNA (Santa Cruz) for 16 h and subsequently exposed to the lenses for 24 h. Where applicable, cells were treated with 30 ng/ml recombinant human Tgfβ2 (Prepotech) or 0.5 mg/ml recombinant mouse follistatin (R & D Systems).
Conventional and quantitative real time PCR (qPCR) RNA was extracted from the cells using the RNeasy mini kit (QIAGEN) and cDNA synthesised using the SuperScripts VILO™ cDNA Synthesis Kit (Invitrogen). Conventional PCR was performed in a Bio-Rad MyCycler using 95 1C for 2 min; 35 cycles of 95 1C for 30 s, 60 1C for 1 min and 72 1C for 1 min using the primers listed below. Real-time quantitative PCR was performed in a Bio-Rad miniopticon thermal cycler using SYBR green jumpstart Taq ready mix (Sigma-Aldrich) under the following conditions: an initial denaturation step of 94 1C for 2 min; followed by 40 cycles of 94 1C for 15 s, 60 1C for 30 s, 72 1C for 1 min, a single plate read step; and an extra 72 1C extension step for 10 min. The melting curve analysis ranged from 50 1C to 95 1C with a 0.5 1C increment and a plate read at every 1 1C increment after a 5 s hold. Results were analysed using the 2 ΔΔCT method relative to the geometric means of the expression levels of two reference genes, Rps12 and Hprt [21,30]. Primer sequences are as follows: Rps12F (50 -ggaaggcatagctgctggaggt-30 ), Rps12R (50 cgatgacatccttggcctgag-30 ) (364 bp); HprtF (50 -gtcccagcgtcgtgattagcgat-30 ), HprtR (50 -gggccacaatgtgatggcctcc-30 ) (206 bp); Foxc1F(50 -tcgctttcctgctcattcgtc-30 ), Foxc1R (50 -tgcagaaaacgctgtagggg-30 ) (559 bp); Pitx2F (50 -agctgtgcaagaatggcttt-30 ), Pitx2R (50 -caccatgctggacgacatac-30 ) (232 bp); Tsc22F (50 -gtagaccagtggcgatggat-30 ), Tsc22R (50 -tccagctgggagtttttctc-30 ) (256 bp); Tgfβ2F (50 -cttcaccacaaagacaggaacct-30 ), Tgfβ2R (50 -tgccatcaatacctgcaaatct-30 ) (136 bp); Tgfβr1F (50 -gcttcatttagtgccacacc-30 ), Tgfβr1R (50 -acagatggcagagctgtgag-30 ) (490 bp); Tgfβr2F (50 -ttaacagtTgfβr2R (50 -agacttcatgcggcttctcacaga-30 ) gatgtcatggccagcg-30 ), 0 (156 bp); SlugF (5 -aagaagcccaactacagcga-30 ), SlugR (50 -gcttttccccagtgtgagtt-30 ) (595 bp). The data were compared using an unpaired Student's t-test. Analyses were run using SPSS.
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well as the two reference genes, Rps12 and Hprt (Fig. 1a and b). Although the cell lines exhibit similar morphology and express similar transcripts, their growth rates are considerably different with the E12.5 peri-ocular mesenchyme cells proliferating more rapidly than the E13.5 cells (Fig. 1c). The differences between the two cell lines are highlighted by an analysis of the expression levels of the transcription factors. Foxc1, Pitx2, Tsc22 and Slug are all expressed at both E12.5 and E13.5 however; the expression of all of these genes is significantly down-regulated by E13.5 (Fig. 1d). These data suggest that, in order for the E12.5 cells to progress to the more differentiated state at E13.5, Foxc1, Pitx2, Tsc22 and Slug expression must persist, but be down-regulated.
The lens plays a role in regulating expression of key genes associated with corneal endothelial differentiation As many studies have shown that the lens plays an important role in corneal endothelial development, E12.5 and E13.5 peri-ocular
Western Blots Proteins were extracted from wild-type cells and cells transfected for 24 h with shRNA targeted to Foxc1, pFoxc1-eGFP or scrambled shRNA (Santa Cruz) by lysing cells with RIPA buffer containing protease and phosphatase inhibitors (Sigma). Proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrances and probed with antibodies to Foxc1 and GAPDH (Cell Signaling Technology).
Results Peri-ocular mesenchyme cell lines are appropriate model cell lines to study corneal endothelial differentiation Both E12.5 and E13.5 peri-ocular mesenchyme cell lines were shown to express the transcription factors Foxc1, Pitx2, Tsc22, Slug/ Snai2; the growth factor Tgfβ2 and its receptors, Tgfβr1 and 2; as
Fig. 1 – The peri-ocular mesenchymal cell lines are an appropriate model to study the onset of corneal endothelial differentiation: Conventional PCR revealed that both E12.5 (A) and E13.5 (B) peri-ocular mesenchyme cell lines express Rps12, Hprt, Foxc1, Pitx2, Tsc22, Tgfb2, Tgbr1, Tgfbr2 and Slug transcripts. (C) E12.5 peri-ocular mesenchyme cells of a similar passage proliferate more rapidly over a 4 day period than E13.5 cells as determined by cell counts and MTS assay (n ¼3, po0.05). (D) Foxc1, Pitx2, Tsc22 and Slug are expressed at lower levels relative to the expression of two reference genes, Rps12 and Hprt, in E13.5 cells compared to E12.5 cells as determined by qPCR (n¼ 3, po0.05).
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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mesenchyme cells were exposed to chick lenses dissected at E6 and E8 as chick lenses at these stages have previously been shown to induce gene expression in mouse optic cups [29]. Exposure of E12.5 peri-ocular mesenchyme cells to E6 lenses for 24 h resulted in at least a 50% down-regulation of Foxc1, Pitx2, Tsc22 and Slug (Fig. 2a). While all genes remain expressed at E13.5, only Foxc1 and Slug are down-regulated by exposure to E8 lenses. Pitx2 expression is increased by 50% while Tsc22 expression is unaffected (Fig. 2a), suggesting that E13.5 cells are still competent to
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respond to lens-derived signals but respond in a different manner than the less differentiated E12.5 cells. To determine the possible nature of the lens-derived signal, the cells were exposed to recombinant Tgfβ2 protein and the expression levels of the transcription factors analysed by qPCR. A time and dose exposure to Tgfβ2 was performed (Supplementary data Figs. 1 and 2). Exposure of the E12.5 peri-ocular mesenchyme cells to 30 ng/ml Tgfβ2 for 24 h resulted in a significant 50% decrease in Foxc1 expression suggesting that secreted Tgfβ2 plays a role in down-regulating Foxc1 expression by E13.5. Pitx2 expression was dramatically reduced after 24 h of Tgfβ2 exposure suggesting that Tgfβ2 is the major possible lens-secreted molecule regulating Pitx2 expression (Fig. 2b). However, Tgfβ2 exposure had no discernible effect on Tsc22 and Slug at E12.5 suggesting that other signals from the lens at E12.5 are largely responsible for the down-regulation of these genes (Fig. 2b). Boiled lenses with the epithelium removed were used as controls and showed no effects on gene expression. At E13.5, Tgfβ2 effects on the expression levels of Foxc1 or Pitx2 were abolished but exposure significantly down-regulated Slug expression. As Slug is down-regulated by lens exposure at E13.5 (Fig. 2a), it is possible that Tgfβ2 may be responsible for this down-regulation (Fig. 2b). Tsc22 expression was slightly, but significantly, reduced compared to the control. Taken together, Tgfβ2, possibly secreted by the lens may play a role in downregulating Foxc1 and Pitx2 expression at E12.5 and Slug expression at E13.5 but does not largely affect Tsc22 or Slug expression at E12.5 or Foxc1 or Pitx2 expression at E13.5. It is clear, therefore, that there are other possible lens-secreted factors that play a role in regulating these genes. As follistatin is produced by E12.5 lens epithelial cells, we exposed E12.5 cells to 0.5 mg/ml follistatin. This exposure had little effect on Foxc1, Pitx2 or Tsc22 expression but, in contrast to whole lens effects, significantly increased Slug expression (Fig. 2c). As there is little information on follistatin concentrations, we determined the effects of various concentrations of follistatin on Foxc1 expression, none of which showed any effect (Supplementary data Fig. 3).
The role of Foxc1 in the regulation of Pitx2, Tsc22 and Slug
Fig. 2 – The lens regulates the expression of key genes in the onset of corneal endothelial differentiation: (A) exposure of E12.5 cells to E6 lenses for 24 h down-regulates the expression of all tested genes. At E13.5, exposure to E8 lenses causes continued Foxc1 and Slug down-regulation and upregulation of Pitx2 while Tsc22 is unaffected by lens exposure. (B) Exposure of E12.5 cells to Tgfβ2 (30 ng/ml) for 24 h resulted in reduced expression of Foxc1 and Pitx2 while at E13.5, Tsc22 and Slug expression was reduced. (C) Exposure of E12.5 cells to 0.5 lg/ml follistatin for 24 h resulted in an increase in Slug expression. For all experiments, gene expression was measured by qPCR relative to the two reference genes (n¼ 3, po0.05). Boiled lenses with the epithelia removed were used as controls.
Western blot analysis showed that the shRNA targeted to Foxc1 mediated a 490% knock-down of Foxc1 transcripts while transfection with pFoxc1-eGFP induced more than 100-fold overexpression of Foxc1. Transfection with a scrambled shRNA control vector had no effect on Foxc1 protein levels (Fig. 3a). At E12.5, both over-expression and knock-down of Foxc1 had major and, interestingly, similar effects on the expression levels of all genes, decreasing Pitx2 expression by 99% and Slug by 60% and increasing Tsc22 expression levels by 2.5–3-fold (Fig. 3b). The effects of varying Foxc1 levels on Pitx2 expression remain at E13.5 but the effects on Tsc22 are abolished (Fig. 3c). At 13.5, Foxc1 knock-down does not affect Slug expression but Foxc1 over-expression significantly induces Slug expression (Fig. 3c).
The role of Foxc1 in interpreting lens-derived signals. It is therefore clear that Foxc1 plays some role in regulating the expression levels of Pitx2, Tsc22 and Slug at E12.5. The expression levels of all of these genes are also affected by exposure to lenses at E12.5. To determine whether Foxc1 plays a role in mediating
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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This suggests that lens-derived signals down-regulate Tsc22 expression independent of the effects of Foxc1. In the absence of Foxc1, Tgfβ2 and follistatin, which normally do not affect Tsc22 expression, act to significantly reduce Tsc22 expression (Fig. 4). Foxc1 positively regulates Slug expression while signals from the lens reduce Slug expression. In the absence of Foxc1 and the presence of the lens, Slug expression levels increase significantly compared to the effects of Foxc1 knock-down and lens exposure. These data suggest that Foxc1 does play a role in mediating the correct lens-derived effects on Slug expression. Slug expression is normally unresponsive to Tgfβ2 signalling but, as with Tsc22, in the absence of Foxc1, exposure to Tgfβ2 reduces Slug expression. This suggests that the Foxc1 mediation of lens-derived signals on Slug expression is independent of Tgfβ2. It also suggests that Foxc1 expression acts in some way to block Tgfβ2 signalling effects on Tsc22 and Slug (Fig. 4). Follistatin exposure significantly increases Slug expression which is further increased in the absence of Foxc1. As lens signals act to down-regulate Slug expression, it may be that Foxc1 acts to block these follistatin effects.
Discussion
Fig. 3 – Foxc1 plays a role in regulating genes expressed in periocular mesenchyme cells: (A) Foxc1 expression was determined by western blotting in wild-type cells; cells transfected with an shRNA directed to knock-down Foxc1 expression; cells overexpressing Foxc1 by transfection with pFoxc1-eGFP and cells transfected with a scrambled shRNA control. (B) Both Foxc1 knock-down and over-expression mediate similar effects on the target genes at E12.5. (C) At E13.5, Pitx2 expression remains down-regulated by both Foxc1 knock-down and overexpression while only Slug expression is upregulated in response to Foxc1 over-expression. Relative gene expression was measured by qPCR relative to the two reference genes (n¼ 3, po0.05). Transfection with a scrambled shRNA control had no effect on gene expression compared to the control.
the effects of lens-derived signals, Foxc1 expression was knockeddown in E12.5 peri-ocular mesenchyme cells and the cells were subsequently exposed to lenses, Tgfβ2 or follistatin for 24 h. We have shown that, at E12.5, Foxc1 positively regulates Pitx2 expression while the lens acts to inhibit its expression. In the absence of Foxc1 but the presence of the lens, Pitx2 expression is significantly less than when exposed to the lens alone but not significantly different from Pitx2 expression levels in Foxc1 knockdown cells. This suggests that the presence of Foxc1 has a greater effect on Pitx2 expression than lens-derived signals. Pitx2 expression is normally inhibited by Tgfβ2 and this is unaffected by the absence of Foxc1. Follistatin has no effect on Pitx2 expression but, in the absence of Foxc1, acts to downregulate Pitx2 expression (Fig. 4). Under normal conditions, Foxc1 negatively regulates Tsc22 expression [27] and signals from the lens decrease Tsc22 expression. In the absence of Foxc1, but the presence of the lens, Tsc22 expression is reduced compared to the effect of the knock-down alone but levels do not differ from those exposed to lenses alone.
During normal murine eye development, from E12, peri-ocular mesenchyme cells migrate into the space created by the separation of the lens from the surface ectoderm. These cells undergo a process of mesenchymal to epithelial transition, differentiating to form the mature corneal endothelium. The maturing corneal endothelium dissociates from the anterior lens between E13.5 and E14.5, forming a tight monolayer of cells by E15. In order to understand the cues that drive the initial differentiation process of the corneal endothelium, we created peri-ocular mesenchyme cells lines from E12.5 and E13.5 embryos. These cell lines were shown to be appropriate model cell lines for understanding this process as they express the key genes known to be associated with corneal endothelial development. Moreover, the cell lines differ in their proliferation rates with E12.5 cells proliferating more rapidly than E13.5 cells, consistent with the E13.5 cells representative of a more differentiated state of the E12.5 cells. We showed that, in order for differentiation to proceed from E12.5 to E13.5, Foxc1, Pitx2, Tsc22 and Slug expression must persist, but be down-regulated. The reduction in Foxc1 expression at E13.5 has been previously reported [17] while down-regulation of Slug, a mesenchymal marker, is necessary for mesenchyme to epithelial transition to occur [11]. To determine whether signals from the lens are responsible for down-regulation, the cells were exposed to chick lenses for 24 h. Ectopic chick lenses have previously been shown to induce distinct expression patterns of key genes in eye development suggesting that an evolutionarily conserved signal/s from the lens controls differentiation. In these experiments, Tsc22 expression in the optic cup margins of E10.5– E13.5 murine embryos was induced by E4–E8 ectopic chick lenses [29]. In our model, we showed that exposure of E12.5 cells to lenses for 24 h reduced Foxc1, Pitx2, Tsc22 and Slug expression by 50%. As these genes are normally down-regulated by E13.5, it suggests that the lens plays a major role in their down-regulation. Exposure of E13.5 cells to lenses for 24 h revealed that lensderived effects on Foxc1 and Slug are robust and are maintained for a further 24 h. Tsc22 expression is unaffected while Pitx2 expression is increased. Thus, in this model, signal/s from the lens
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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Fig. 4 – The role of Foxc1 in interpreting lens-derived signals: The effect of lens, Tgfβ2 and Follistatin exposure on Pitx2, Tsc22 and Slug expression in E12.5 cells in which Foxc1 expression has been knocked-down measured by qPCR relative to the two reference genes (n¼3, po0.05). Some of the data are repeated for ease of comparison.
play a role in driving differentiation from E12.5 and E13.5, variably affecting the expression of Foxc1 and Pitx2 and the two drivers of MET, Tsc22 and Slug. Tgfβ2 is the most well described lens-secreted molecule and it has been shown to play an important role in corneal endothelial development. The peri-ocular mesenchyme cells at E12.5 and E13.5 express Tgfβr1 and 2 receptors and are thus able to respond to Tgfβ2. Moreover, the peri-ocular mesenchyme cells themselves produce Tgfβ2 (Figs. 1a and b) suggesting a possible autocrine function. In E13.5 Tgfβr2 / mice, Foxc1 expression in the periocular mesenchyme was dramatically reduced compared to wildtype mice and both Foxc1 and Pitx2 expression absent at E15 [16]. This suggests that Tgfβ2 signalling, paracrine or possible autocrine, is important for the expression of these transcription factors in a temporal-specific manner [16]. In line with these data and in this model, exposure of E12.5 cells to Tgfβ2 was shown to down-regulate Foxc1 and very significantly, Pitx2 expression, indicating that Tgfβ2 may play a role in Foxc1 down-regulation but appears to be the major possible lens-derived signalling molecule affecting Pitx2 expression at this time point. Exposure of E13.5 cells to Tgfβ2 for 24 h significantly reduced Slug expression suggesting that Tgfβ2 may be largely responsible for downregulating Slug expression at this stage. In a study designed to understand the role EMT may play during the development of anterior polar cataracts in the lens, lens epithelial cells exposed to TGFβ induced Slug expression by binding to the Sp1 site in the Slug promoter, thus driving EMT [6]. Thus, at least in these cell lines, in EMT, Tgfβ signalling induces Slug while in MET, Tgfβ2 represses Slug. While Tgfβ2 down-regulates Foxc1 and Pitx2 expression at E12.5 and Slug expression at E13.5, it has little or no effect, on Tsc22 expression at E12.5 and E13.5 suggesting that other lens-derived factors affect its expression at these developmental time points. We thus investigated the effects of the BMP antagonist, follistatin, shown to be expressed by lens epithelial cells at E12.5. Follistatin had no effect on Foxc1, Pitx2 or Tsc22 but significantly enhanced the expression of Slug, in contrast to the inhibitory effect of whole lens. BMP4 and 7 have been shown to induce Slug expression during EMT in neural crest formation [22] thus presumably inhibition of BMP signalling by follistatin would inhibit Slug expression. However, in this model, where the cells
are beginning to undergo MET, inhibition of BMP signalling by follistatin, results in increased Slug expression. The reason for this is unknown as Slug expression must be down-regulated for MET to occur [11] and exposure of whole lens (presumably the source of any follistatin) results, as expected, in Slug down-regulation. It is possible that the effects seen are due to follistatin concentration as a previously published concentration (0.5 mg/ml) was used to perform the experiments [2]. Nevertheless, these data show that Slug expression can be regulated by follistatin. Foxc1 expression is essential for normal corneal endothelial development. We have previously shown that Tsc22 is negatively regulated by Foxc1 at E12.5 [27]. Here, we show that Foxc1 positively regulates, either directly or indirectly, the expression of Pitx2 and Slug at E12.5. Furthermore, knock-down and overexpression of Foxc1 has a dramatic and similar effect on Pitx2, Tsc22 and Slug expression. These data are consistent with reports that precise levels of Foxc1 are required for correct differentiation of the peri-ocular mesenchyme and that both mutations in FOXC1 or duplication of FOXC1 can result in abnormal anterior segment development and ARS [28]. Foxc1 and Pitx2 have been shown to physically interact where the homeodomain of Pitx2A binds to the C-terminal transactivation domain of Foxc1, resulting in impairment of Foxc1 activation of target genes [4]. Here, we show that, when Foxc1 expression is knocked-down, Pitx2 expression is also reduced. The transcriptional regulator protein PRKC apoptosis Wilm's tumour 1 regulator (PAWR) was previously shown to modulate the activities of Foxc1 and Pitx2 where, in the absence of Foxc1, PAWR reduced Pitx2 activity [1]. Foxc1 control over Pitx2 expression persisted at E13.5 while effects on Tsc22 were abolished. Interestingly, at E13.5, Foxc1 over-expression induced Slug expression by two-fold. Increased expression of Foxc1 through chromosomal duplication causes iris hypoplasia and can lead to glaucoma [19]. The significant upregulation in Slug expression following overexpression of Foxc1 may partially prevent proper MET, impairing development and may thus be a contributing factor to the disease. We have therefore shown that lens-derived signals at E12.5 play a role in down-regulating the expression of Foxc1 and Pitx2, transcription factors whose expression is essential for normal corneal endothelial development, Slug, a key regulator of EMT and MET and Tsc22, a regulator expressed highly at areas of EMT. This down-regulation appears necessary for continued differentiation.
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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Our results suggest that possible lens-derived Tgfβ2 plays a role in down-regulating Foxc1 and Pitx2 expression at E12.5 and Slug at E13.5. Although Foxc1 and Pitx2 interact and Foxc1 can regulate Pitx2 expression, Foxc1 does not play a role in Tgfβ2-mediated down-regulation of Pitx2. Similarly, lens-mediated down-regulation of Tsc22 appears unaffected by Foxc1 and does not appear to be mediated by Tgfβ2 and follistatin suggesting that other lensderived factors force its down-regulation. However, although Tgfβ2 and follistatin have no effect on Tsc22 expression, in the absence of Foxc1, their expression is dramatically reduced suggesting that Foxc1 acts in some way to mediate the effects of these two molecules on Tsc22 expression. This, however, has little effect when compared to whole lens exposure where other factors down-regulate Tsc22 expression. Foxc1 does play a role in mediating the effects of whole lenses on Slug expression. The down-regulatory effects of the lens do not appear to be mediated by Tgfβ2 (which has no effect) or follistatin (which acts to increase Slug expression), suggesting that, as with Tsc22, other lensderived factors play a greater role in regulating Slug expression. Foxc1 may act to inhibit the positive effects of follistatin and may, as with Tsc22 and Pitx2, play a role in mediating Tgfβ2 effects. Our data reveal that Foxc1 does not play a role in translating lens-derived effects on Tsc22 or Pitx2 expression but that Foxc1 may play a role in correctly mediating the lens-derived signal/s negative effects on Slug. In addition, Foxc1 may act in some way to block Tgfβ2 signalling and to mediate follistatin effects on Tsc22 and Slug expression. These data provide an indication of how Foxc1 expression is important for MET in corneal endothelial development. Taken together, in this model we show that lensderived factors and key transcription factors interact to drive the initial process of MET in corneal endothelial development.
Acknowledgments This study was supported by a grant from the Medical Research Council of South Africa. The authors would like to thank Dr. Marco Weinberg (University of the Witwatersrand) for his help with the shRNA design and Y.Liu, J. Huang and D.Beebe (Washington University in St. Louis) for supplying the array data on enriched E12.5 lens epithelial transcripts.
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2014.01.016.
references [1] M. Acharya, L. Huang, V.C. Fleisch, W.T. Allison, M.A. Walter, A complex regulatory network of transcription factors critical for ocular development and disease, Hum. Mol. Genet. 20 (2011) 1610–1624. [2] D. Beebe, C. Garcia, X. Wang, R. Rajagopal, M. Feldmeier, J.Y. Kim, A. Chytil, H. Moses, R. Ashery-Padan, M. Rauchman, Contributions by members of the TGFbeta superfamily to lens development, Int. J. Dev. Biol. 48 (2004) 845–856. [3] D.C. Beebe, J.M. Coats, The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye, Dev. Biol. 220 (2000) 424–431.
] (]]]]) ]]]–]]]
7
[4] F.B. Berry, M.A. Lines, J.M. Oas, T. Footz, D.A. Underhill, P.J. Gage, M.A. Walter, Functional interactions between FOXC1 and PITX2 underlie the sensitivity to FOXC1 gene dose in Axenfeld–Rieger syndrome and anterior segment dysgenesis, Hum. Mol. Genet. 15 (2006) 905–919. [5] D. Castanotto, H. Li, J.J. Rossi, Functional siRNA expression from transfected PCR products, RNA 8 (2002) 1454–1460. [6] J. Choi, S.Y. Park, C.K. Joo, Transforming growth factor-beta1 represses E-cadherin production via slug expression in lens epithelial cells, Invest. Ophthalmol. Vis. Sci. 48 (2007) 2708–2718. [7] S.J. Choi, J.H. Moon, Y.W. Ahn, J.H. Ahn, D.U. Kim, T.H. Han, Tsc-22 enhances TGF-beta signaling by associating with Smad4 and induces erythroid cell differentiation, Mol. Cell. Biochem. 271 (2005) 23–28. [8] A. Cvekl, E.R. Tamm, Anterior eye development and ocular mesenchyme: new insights from mouse models and human diseases, Bioessays 26 (2004) 374–386. [9] C.E. Dohrmann, M. Belaoussoff, L.A. Raftery, Dynamic expression of TSC-22 at sites of epithelial–mesenchymal interactions during mouse development, Mech. Dev. 84 (1999) 147–151. [10] A.T. Dudley, K.M. Lyons, E.J. Robertson, A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye, Genes Dev. 9 (1995) 2795–2807. [11] N. Fenouille, M. Tichet, M. Dufies, A. Pottier, A. Mogha, J.K. Soo, S. Rocchi, A. Mallavialle, M.D. Galibert, A. Khammari, J.P. Lacour, R. Ballotti, M. Deckert, S. Tartare-Deckert, The epithelial–mesenchymal transition (EMT) regulatory factor SLUG (SNAI2) is a downstream target of SPARC and AKT in promoting melanoma cell invasion, PLoS One 7 (2012) e40378. [12] C. Flugel-Koch, A. Ohlmann, J. Piatigorsky, E.R. Tamm, Disruption of anterior segment development by TGF-beta1 overexpression in the eyes of transgenic mice, Dev. Dyn. 225 (2002) 111–125. [13] P.J. Gage, A.L. Zacharias, Signaling “cross-talk” is integrated by transcription factors in the development of the anterior segment in the eye, Dev. Dyn. 238 (2009) 2149–2162. [14] P.J. Gage, H. Suh, S.A. Camper, Dosage requirement of Pitx2 for development of multiple organs, Development 126 (1999) 4643–4651. [15] P.J. Gage, W. Rhoades, S.K. Prucka, T. Hjalt, Fate maps of neural crest and mesoderm in the mammalian eye, Invest. Ophthalmol. Vis. Sci. 46 (2005) 4200–4208. [16] L.M. Ittner, H. Wurdak, K. Schwerdtfeger, T. Kunz, F. Ille, P. Leveen, T.A. Hjalt, U. Suter, S. Karlsson, F. Hafezi, W. Born, L. Sommer, Compound developmental eye disorders following inactivation of TGFbeta signaling in neural-crest stem cells, J. Biol. 4 (2005) 11. [17] S.H. Kidson, T. Kume, K. Deng, V. Winfrey, B.L. Hogan, The forkhead/winged-helix gene, Mf1, is necessary for the normal development of the cornea and formation of the anterior chamber in the mouse eye, Dev. Biol. 211 (1999) 306–322. [18] T. Langenberg, A. Kahana, J.A. Wszalek, M.C. Halloran, The eye organizes neural crest cell migration, Dev. Dyn. 237 (2008) 1645–1652. [19] O.J. Lehmann, N.D. Ebenezer, T. Jordan, M. Fox, L. Ocaka, A. Payne, B.P. Leroy, B.J. Clark, R.A. Hitchings, S. Povey, P.T. Khaw, S.S. Bhattacharya, Chromosomal duplication involving the forkhead transcription factor gene FOXC1 causes iris hypoplasia and glaucoma, Am. J. Hum. Genet. 67 (2000) 1129–1135. [20] O.J. Lehmann, J.C. Sowden, P. Carlsson, T. Jordan, S.S. Bhattacharya, Fox's in development and disease, Trends Genet. 19 (2003) 339–344. [21] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method, Methods 25 (2001) 402–408. [22] S. Ohta, G.C. Schoenwolf, G. Yamada, The cessation of gastrulation: BMP signaling and EMT during and at the end of gastrulation, Cell Adhes. Migr. 4 (2010) 440–446.
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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[23] G.S. Peh, R.W. Beuerman, A. Colman, D.T. Tan, J.S. Mehta, Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview, Transplantation 91 (2011) 811–819. [24] L.W. Reneker, D.W. Silversides, L. Xu, P.A. Overbeek, Formation of corneal endothelium is essential for anterior segment development – a transgenic mouse model of anterior segment dysgenesis, Development 127 (2000) 533–542. [25] S. Saika, S. Saika, C.Y. Liu, M. Azhar, L.P. Sanford, T. Doetschman, R.L. Gendron, C.W. Kao, W.W. Kao, TGFbeta2 in corneal morphogenesis during mouse embryonic development, Dev. Biol. 240 (2001) 419–432. [26] M. Sefton, S. Sanchez, M.A. Nieto, Conserved and divergent roles for members of the Snail family of transcription factors in the chick and mouse embryo, Development 125 (1998) 3111–3121. [27] P. Sommer, H.R. Napier, B.L. Hogan, S.H. Kidson, Identification of Tgf beta1i4 as a downstream target of Foxc1, Dev. Growth Differ. 48 (2006) 297–308.
] (]]]]) ]]]–]]]
[28] M.H. Strungaru, I. Dinu, M.A. Walter, Genotype-phenotype correlations in Axenfeld–Rieger malformation and glaucoma patients with FOXC1 and PITX2 mutations, Invest. Ophthalmol. Vis. Sci. 48 (2007) 228–237. [29] C.J. Thut, R.B. Rountree, M. Hwa, D.M. Kingsley, A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens, Dev. Biol. 231 (2001) 63–76. [30] J. Vandesompele, K. De Preter, F. Pattyn, B. Poppe, N. Van Roy, A. De Paepe, F. Speleman, Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes, Genome Biol. 3 (2002) (RESEARCH0034). [31] Y. Zhang, P.A. Overbeek, V. Govindarajan, Perinatal ablation of the mouse lens causes multiple anterior chamber defects, Mol. Vis. 13 (2007) 2289–2300. [32] Y. Lui, J. Huang, D. Beebe, unpublished data.
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development Zenzele T.V. Sillaa,1, Jerolen Naidooa,1, Susan H. Kidsonb, Paula Sommera,n a
School of Life Sciences, University of KwaZulu-Natal Westville Campus, Durban 4001, South Africa Department of Human Biology, University of Cape Town Medical School, Anzio Road, Observatory, South Africa
b
article information
abstract
Article Chronology:
Correct formation of the corneal endothelium is essential for continued development of the
Received 28 October 2013
anterior segment of the eye. Corneal endothelial development is initiated at E12 when precursor
Received in revised form
peri-ocular mesenchyme cells migrate into the space between the lens and the presumptive
14 January 2014
corneal epithelium and begin to respond to signals from the lens, undergoing a mesenchymal to
Accepted 16 January 2014
epithelial transition (MET) that is complete by E15.5. To study the initiation of MET, peri-ocular mesenchyme cell lines were derived from E12.5 and E13.5 murine embryos. These cells expressed
Keywords:
key transcription factors, Foxc1 and Pitx2, as well as Slug and Tsc22, genes involved in MET. We
Peri-ocular mesenchyme
have shown that all these genes must be down-regulated by E13.5 for differentiation to proceed.
Corneal endothelial development
Lens-derived signals play a role in this down-regulation with Tgfβ2 specifically down-regulating
Lens
Foxc1 and Pitx2. Over-expression and knock-down of Foxc1 significantly and similarly affected the
Foxc1
expression of Pitx2, Tsc22 and Slug while Foxc1 was shown to play a role in mediating the lens
Pitx2
effects on Slug. Thus, for the progression of initial corneal endothelial development, the key transcription factors, Foxc1 and Pitx2, as well as genes involved in MET, Slug and Tsc-22, must be down-regulated, a process driven by the lens and Foxc1. & 2014 Published by Elsevier Inc.
Introduction A fully functioning cornea is essential for visual acuity. In adults, the cornea is composed of several layers: the outer epithelium which rests on Bowman's layer; the stroma which is responsible for transparency; Dua's layer; Descemet's membrane and the final, innermost layer, the corneal endothelium [23]. Physiologically, the corneal endothelium is the most important layer of the cornea, forming a barrier between the corneal stroma and the aqueous humour of the anterior chamber. The mature corneal endothelium pumps fluid from the stroma and is thus responsible
for maintaining the relatively acellular stroma in a hydrated, yet still transparent state [23]. Importantly, failure of correct endothelial development leads to abnormal development of the anterior segment of the eye [24]. Eye development is initiated by the formation of the optic pits from the neural ectoderm. By embryonic day (E) 9, the optic pits deepen to form the optic vesicles which extend laterally to meet the outer surface ectoderm of the developing head. Contact of the optic vesicle with the surface ectoderm results in the induction of the lens placode from an area of competent surface ectoderm. At E10.5, the lens placode invaginates to form the lens pit which, by
n
Corresponding author. E-mail address: [email protected] (P. Sommer). 1 These authors contributed equally to this work.
0014-4827/$ - see front matter & 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.yexcr.2014.01.016
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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E11, then separates from the overlying surface ectoderm to form the lens vesicle. Importantly, from E12, peri-ocular mesenchyme cells, composed of predominantly neural crest but also mesoderm mesenchyme, migrate into the space between the developing lens and the now continuous surface ectoderm which will mature into the corneal epithelium [15]. From E12, the peri-ocular mesenchyme cells respond to cues from the lens initiating the development of the corneal endothelium by a process of mesenchyme to epithelial transition. The maturing corneal endothelium physically dissociates from the anterior lens between E13.5 and E14.5, resulting in a space between the lens and the corneal endothelium that will become the anterior chamber, filled with aqueous humour. By E15, the maturing corneal endothelium consists of a monolayer of cells, characterised by tight junctions creating a tight barrier of cells capable of actively pumping fluid [13,18]. The molecular cues necessary for the differentiation of the corneal endothelium into an organised monolayer are not well understood. The lens plays an essential role in the development of the corneal endothelium as removal of the lens in chick embryos [3] or ablation of the lens in mice [31] inhibits corneal endothelial and stroma formation. Not only is the presence of the lens essential, but correct and precise instructions from the lens are also necessary for proper corneal endothelial development as in mice, transgenic for lens-specific TGFα or EGF [24] or mice overexpressing lens-specific TGFβ1 [12], the corneal endothelium failed to differentiate correctly. The lens is thus thought to act as a major signalling centre, secreting molecules that pass through the anterior chamber and direct the induction of the corneal endothelium and stroma [15]. Despite the important role of the lens in corneal development, there is little information on the molecules produced and secreted by the lens. To date, the only well described molecule known to be secreted by the lens, with secretion peaking between E13.5 and E15, is TGFβ2 [16]. The importance of Tgfβ2 secretion by the lens is evident in Tgfβ2 / [25] and in Tgfβr2 / [16] mice where the corneal endothelium fails to form, preventing separation of the lens and cornea. A recent microarray study revealed that E12.5 lens epithelial cells produce the BMP antagonist, follistatin [32]. BMPs play a crucial role in eye development [10]. Follistatin is able to inhibit BMP activity, thus playing a crucial role in embryonic patterning. Two known key genes essential for corneal endothelial development are the transcription factors, Foxc1 and Pitx2. Foxc1 belongs to the Fox family of genes, characterised by a conserved forkhead domain of 11 amino acids, many of which play important roles in neural crest differentiation [20]. The paired homeodomain transcription factor, Pitx2, is essential for normal ocular development, encoding three alternatively spliced transcripts in mice, Pitx2A, 2B and 2C. Foxc1 and Pitx2 are both highly, but variably, co-expressed in the peri-ocular mesenchyme from E11.5 [4,15]. Expression of both of these transcription factors in periocular mesenchyme cells is essential for corneal endothelial development as the lack of either one prevents corneal endothelial development and subsequent formation of the anterior chamber ([14,17]). Heterozygous mutations in FOXC1 or PITX2 are directly associated with Axenfeld–Rieger Syndrome, a broad spectrum of clinical anterior segment disorders resulting from impaired differentiation of the peri-ocular mesenchyme [8]. Tsc22 (Tgfβ1i4) is also a transcriptional regulator that has previously been shown to be expressed in peri-ocular mesenchyme
] (]]]]) ]]]–]]]
cells and is a downstream target of Foxc1 [27]. Originally described as an early response gene of TGFβ1, it is highly expressed during development, including in neural crest derived cells. Tsc22 is upregulated at sites of epithelial to mesenchymal interactions and plays a role in differentiation [7,9,29]. Slug (Snai2) is a zinc finger transcription factor that is known to play a key role in epithelial to mesenchymal transitions (EMT) and the converse, mesenchyme to epithelial transitions (MET). It is expressed in the developing mouse eye [26]. Peri-ocular mesenchyme cells migrate into the space between the lens and the surface ectoderm at E12 where they respond to cues from the lens and begin to differentiate into the corneal endothelium. To study these initial steps in corneal endothelial development, we created two cell lines representing peri-ocular mesenchyme at E12.5 and E13.5. We analysed the expression of the two essential transcription factors, Foxc1 and Pitx2, as well as two other transcriptional regulators known to be involved in MET, Tsc22 and Slug, at these two developmental time points. We determined the effects of the lens after exposure of the cells to chick lenses and recombinant Tgfβ2 and follistatin and mapped out their dependency on Foxc1 expression.
Materials and methods Derivation and maintenance of cell lines Peri-ocular mesenchyme cell lines derived from the anterior region of the eyes of E12.5 mouse embryos were immortalised as described [27]. In addition, an immortalised peri-ocular mesenchyme cell line at E13.5 was derived using similar methods (Ethics approval number 019/13/Animal). The cell lines were maintained in DMEM (PAA) supplemented with 10% FBS (Invitrogen) and 0.5 mg/mL penicillin streptomycin (Invitrogen). Cell counts were performed using trypan blue exclusion and metabolic assays were performed using the MTS assay (Promega). All experiments were performed on low and similar passage cells.
Lens exposures, transfections and treatments For the lens exposures, whole chick lenses were dissected at E6 and E8. Ten lenses were added to peri-ocular mesenchyme cells cultured in 60 mm dishes (Corning) containing 4 mL of culture medium for a period of 24 h. Boiled lenses, in which the lens epithelium was dissected away, were used for control experiments. For over-expression of Foxc1, cells were transfected with pFoxc1-eGFP or peGFP-N1 using FuGene 6 (Roche) for 24 h. For Foxc1 knock-down, five putative shRNAs to Foxc1 were chosen from the MIT/Harvard Broad Institute's murine shRNA library database (http://www.broad.mit.edu/rnai/trc/lib). Primers, containing a 21 bp sequence corresponding to the 50 end of the human U6 promoter; 22 bp sequence corresponding to the sense and antisense sequence of the potential shRNA separated by 9 bp loop sequence and 6 bp termination sequence, were designed and, along with a forward primer designed to amplify the 50 end of the human U6 promoter, used to amplify human genomic DNA [5]. The resulting amplicons therefore contained the full-length U6 promoter and the shRNA sequence. The amplicons were cloned into pGEM-T easy (Promega) and transiently transfected into peri-ocular mesenchyme cells to determine knock-down
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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efficiency. Two of the five clones demonstrated a knock-down efficiency of 498% by qPCR and western blot analysis. For knockdown and lens treatment, cells were transfected with the shRNA targeting Foxc1 or scrambled shRNA (Santa Cruz) for 16 h and subsequently exposed to the lenses for 24 h. Where applicable, cells were treated with 30 ng/ml recombinant human Tgfβ2 (Prepotech) or 0.5 mg/ml recombinant mouse follistatin (R & D Systems).
Conventional and quantitative real time PCR (qPCR) RNA was extracted from the cells using the RNeasy mini kit (QIAGEN) and cDNA synthesised using the SuperScripts VILO™ cDNA Synthesis Kit (Invitrogen). Conventional PCR was performed in a Bio-Rad MyCycler using 95 1C for 2 min; 35 cycles of 95 1C for 30 s, 60 1C for 1 min and 72 1C for 1 min using the primers listed below. Real-time quantitative PCR was performed in a Bio-Rad miniopticon thermal cycler using SYBR green jumpstart Taq ready mix (Sigma-Aldrich) under the following conditions: an initial denaturation step of 94 1C for 2 min; followed by 40 cycles of 94 1C for 15 s, 60 1C for 30 s, 72 1C for 1 min, a single plate read step; and an extra 72 1C extension step for 10 min. The melting curve analysis ranged from 50 1C to 95 1C with a 0.5 1C increment and a plate read at every 1 1C increment after a 5 s hold. Results were analysed using the 2 ΔΔCT method relative to the geometric means of the expression levels of two reference genes, Rps12 and Hprt [21,30]. Primer sequences are as follows: Rps12F (50 -ggaaggcatagctgctggaggt-30 ), Rps12R (50 cgatgacatccttggcctgag-30 ) (364 bp); HprtF (50 -gtcccagcgtcgtgattagcgat-30 ), HprtR (50 -gggccacaatgtgatggcctcc-30 ) (206 bp); Foxc1F(50 -tcgctttcctgctcattcgtc-30 ), Foxc1R (50 -tgcagaaaacgctgtagggg-30 ) (559 bp); Pitx2F (50 -agctgtgcaagaatggcttt-30 ), Pitx2R (50 -caccatgctggacgacatac-30 ) (232 bp); Tsc22F (50 -gtagaccagtggcgatggat-30 ), Tsc22R (50 -tccagctgggagtttttctc-30 ) (256 bp); Tgfβ2F (50 -cttcaccacaaagacaggaacct-30 ), Tgfβ2R (50 -tgccatcaatacctgcaaatct-30 ) (136 bp); Tgfβr1F (50 -gcttcatttagtgccacacc-30 ), Tgfβr1R (50 -acagatggcagagctgtgag-30 ) (490 bp); Tgfβr2F (50 -ttaacagtTgfβr2R (50 -agacttcatgcggcttctcacaga-30 ) gatgtcatggccagcg-30 ), 0 (156 bp); SlugF (5 -aagaagcccaactacagcga-30 ), SlugR (50 -gcttttccccagtgtgagtt-30 ) (595 bp). The data were compared using an unpaired Student's t-test. Analyses were run using SPSS.
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well as the two reference genes, Rps12 and Hprt (Fig. 1a and b). Although the cell lines exhibit similar morphology and express similar transcripts, their growth rates are considerably different with the E12.5 peri-ocular mesenchyme cells proliferating more rapidly than the E13.5 cells (Fig. 1c). The differences between the two cell lines are highlighted by an analysis of the expression levels of the transcription factors. Foxc1, Pitx2, Tsc22 and Slug are all expressed at both E12.5 and E13.5 however; the expression of all of these genes is significantly down-regulated by E13.5 (Fig. 1d). These data suggest that, in order for the E12.5 cells to progress to the more differentiated state at E13.5, Foxc1, Pitx2, Tsc22 and Slug expression must persist, but be down-regulated.
The lens plays a role in regulating expression of key genes associated with corneal endothelial differentiation As many studies have shown that the lens plays an important role in corneal endothelial development, E12.5 and E13.5 peri-ocular
Western Blots Proteins were extracted from wild-type cells and cells transfected for 24 h with shRNA targeted to Foxc1, pFoxc1-eGFP or scrambled shRNA (Santa Cruz) by lysing cells with RIPA buffer containing protease and phosphatase inhibitors (Sigma). Proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrances and probed with antibodies to Foxc1 and GAPDH (Cell Signaling Technology).
Results Peri-ocular mesenchyme cell lines are appropriate model cell lines to study corneal endothelial differentiation Both E12.5 and E13.5 peri-ocular mesenchyme cell lines were shown to express the transcription factors Foxc1, Pitx2, Tsc22, Slug/ Snai2; the growth factor Tgfβ2 and its receptors, Tgfβr1 and 2; as
Fig. 1 – The peri-ocular mesenchymal cell lines are an appropriate model to study the onset of corneal endothelial differentiation: Conventional PCR revealed that both E12.5 (A) and E13.5 (B) peri-ocular mesenchyme cell lines express Rps12, Hprt, Foxc1, Pitx2, Tsc22, Tgfb2, Tgbr1, Tgfbr2 and Slug transcripts. (C) E12.5 peri-ocular mesenchyme cells of a similar passage proliferate more rapidly over a 4 day period than E13.5 cells as determined by cell counts and MTS assay (n ¼3, po0.05). (D) Foxc1, Pitx2, Tsc22 and Slug are expressed at lower levels relative to the expression of two reference genes, Rps12 and Hprt, in E13.5 cells compared to E12.5 cells as determined by qPCR (n¼ 3, po0.05).
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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mesenchyme cells were exposed to chick lenses dissected at E6 and E8 as chick lenses at these stages have previously been shown to induce gene expression in mouse optic cups [29]. Exposure of E12.5 peri-ocular mesenchyme cells to E6 lenses for 24 h resulted in at least a 50% down-regulation of Foxc1, Pitx2, Tsc22 and Slug (Fig. 2a). While all genes remain expressed at E13.5, only Foxc1 and Slug are down-regulated by exposure to E8 lenses. Pitx2 expression is increased by 50% while Tsc22 expression is unaffected (Fig. 2a), suggesting that E13.5 cells are still competent to
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respond to lens-derived signals but respond in a different manner than the less differentiated E12.5 cells. To determine the possible nature of the lens-derived signal, the cells were exposed to recombinant Tgfβ2 protein and the expression levels of the transcription factors analysed by qPCR. A time and dose exposure to Tgfβ2 was performed (Supplementary data Figs. 1 and 2). Exposure of the E12.5 peri-ocular mesenchyme cells to 30 ng/ml Tgfβ2 for 24 h resulted in a significant 50% decrease in Foxc1 expression suggesting that secreted Tgfβ2 plays a role in down-regulating Foxc1 expression by E13.5. Pitx2 expression was dramatically reduced after 24 h of Tgfβ2 exposure suggesting that Tgfβ2 is the major possible lens-secreted molecule regulating Pitx2 expression (Fig. 2b). However, Tgfβ2 exposure had no discernible effect on Tsc22 and Slug at E12.5 suggesting that other signals from the lens at E12.5 are largely responsible for the down-regulation of these genes (Fig. 2b). Boiled lenses with the epithelium removed were used as controls and showed no effects on gene expression. At E13.5, Tgfβ2 effects on the expression levels of Foxc1 or Pitx2 were abolished but exposure significantly down-regulated Slug expression. As Slug is down-regulated by lens exposure at E13.5 (Fig. 2a), it is possible that Tgfβ2 may be responsible for this down-regulation (Fig. 2b). Tsc22 expression was slightly, but significantly, reduced compared to the control. Taken together, Tgfβ2, possibly secreted by the lens may play a role in downregulating Foxc1 and Pitx2 expression at E12.5 and Slug expression at E13.5 but does not largely affect Tsc22 or Slug expression at E12.5 or Foxc1 or Pitx2 expression at E13.5. It is clear, therefore, that there are other possible lens-secreted factors that play a role in regulating these genes. As follistatin is produced by E12.5 lens epithelial cells, we exposed E12.5 cells to 0.5 mg/ml follistatin. This exposure had little effect on Foxc1, Pitx2 or Tsc22 expression but, in contrast to whole lens effects, significantly increased Slug expression (Fig. 2c). As there is little information on follistatin concentrations, we determined the effects of various concentrations of follistatin on Foxc1 expression, none of which showed any effect (Supplementary data Fig. 3).
The role of Foxc1 in the regulation of Pitx2, Tsc22 and Slug
Fig. 2 – The lens regulates the expression of key genes in the onset of corneal endothelial differentiation: (A) exposure of E12.5 cells to E6 lenses for 24 h down-regulates the expression of all tested genes. At E13.5, exposure to E8 lenses causes continued Foxc1 and Slug down-regulation and upregulation of Pitx2 while Tsc22 is unaffected by lens exposure. (B) Exposure of E12.5 cells to Tgfβ2 (30 ng/ml) for 24 h resulted in reduced expression of Foxc1 and Pitx2 while at E13.5, Tsc22 and Slug expression was reduced. (C) Exposure of E12.5 cells to 0.5 lg/ml follistatin for 24 h resulted in an increase in Slug expression. For all experiments, gene expression was measured by qPCR relative to the two reference genes (n¼ 3, po0.05). Boiled lenses with the epithelia removed were used as controls.
Western blot analysis showed that the shRNA targeted to Foxc1 mediated a 490% knock-down of Foxc1 transcripts while transfection with pFoxc1-eGFP induced more than 100-fold overexpression of Foxc1. Transfection with a scrambled shRNA control vector had no effect on Foxc1 protein levels (Fig. 3a). At E12.5, both over-expression and knock-down of Foxc1 had major and, interestingly, similar effects on the expression levels of all genes, decreasing Pitx2 expression by 99% and Slug by 60% and increasing Tsc22 expression levels by 2.5–3-fold (Fig. 3b). The effects of varying Foxc1 levels on Pitx2 expression remain at E13.5 but the effects on Tsc22 are abolished (Fig. 3c). At 13.5, Foxc1 knock-down does not affect Slug expression but Foxc1 over-expression significantly induces Slug expression (Fig. 3c).
The role of Foxc1 in interpreting lens-derived signals. It is therefore clear that Foxc1 plays some role in regulating the expression levels of Pitx2, Tsc22 and Slug at E12.5. The expression levels of all of these genes are also affected by exposure to lenses at E12.5. To determine whether Foxc1 plays a role in mediating
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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This suggests that lens-derived signals down-regulate Tsc22 expression independent of the effects of Foxc1. In the absence of Foxc1, Tgfβ2 and follistatin, which normally do not affect Tsc22 expression, act to significantly reduce Tsc22 expression (Fig. 4). Foxc1 positively regulates Slug expression while signals from the lens reduce Slug expression. In the absence of Foxc1 and the presence of the lens, Slug expression levels increase significantly compared to the effects of Foxc1 knock-down and lens exposure. These data suggest that Foxc1 does play a role in mediating the correct lens-derived effects on Slug expression. Slug expression is normally unresponsive to Tgfβ2 signalling but, as with Tsc22, in the absence of Foxc1, exposure to Tgfβ2 reduces Slug expression. This suggests that the Foxc1 mediation of lens-derived signals on Slug expression is independent of Tgfβ2. It also suggests that Foxc1 expression acts in some way to block Tgfβ2 signalling effects on Tsc22 and Slug (Fig. 4). Follistatin exposure significantly increases Slug expression which is further increased in the absence of Foxc1. As lens signals act to down-regulate Slug expression, it may be that Foxc1 acts to block these follistatin effects.
Discussion
Fig. 3 – Foxc1 plays a role in regulating genes expressed in periocular mesenchyme cells: (A) Foxc1 expression was determined by western blotting in wild-type cells; cells transfected with an shRNA directed to knock-down Foxc1 expression; cells overexpressing Foxc1 by transfection with pFoxc1-eGFP and cells transfected with a scrambled shRNA control. (B) Both Foxc1 knock-down and over-expression mediate similar effects on the target genes at E12.5. (C) At E13.5, Pitx2 expression remains down-regulated by both Foxc1 knock-down and overexpression while only Slug expression is upregulated in response to Foxc1 over-expression. Relative gene expression was measured by qPCR relative to the two reference genes (n¼ 3, po0.05). Transfection with a scrambled shRNA control had no effect on gene expression compared to the control.
the effects of lens-derived signals, Foxc1 expression was knockeddown in E12.5 peri-ocular mesenchyme cells and the cells were subsequently exposed to lenses, Tgfβ2 or follistatin for 24 h. We have shown that, at E12.5, Foxc1 positively regulates Pitx2 expression while the lens acts to inhibit its expression. In the absence of Foxc1 but the presence of the lens, Pitx2 expression is significantly less than when exposed to the lens alone but not significantly different from Pitx2 expression levels in Foxc1 knockdown cells. This suggests that the presence of Foxc1 has a greater effect on Pitx2 expression than lens-derived signals. Pitx2 expression is normally inhibited by Tgfβ2 and this is unaffected by the absence of Foxc1. Follistatin has no effect on Pitx2 expression but, in the absence of Foxc1, acts to downregulate Pitx2 expression (Fig. 4). Under normal conditions, Foxc1 negatively regulates Tsc22 expression [27] and signals from the lens decrease Tsc22 expression. In the absence of Foxc1, but the presence of the lens, Tsc22 expression is reduced compared to the effect of the knock-down alone but levels do not differ from those exposed to lenses alone.
During normal murine eye development, from E12, peri-ocular mesenchyme cells migrate into the space created by the separation of the lens from the surface ectoderm. These cells undergo a process of mesenchymal to epithelial transition, differentiating to form the mature corneal endothelium. The maturing corneal endothelium dissociates from the anterior lens between E13.5 and E14.5, forming a tight monolayer of cells by E15. In order to understand the cues that drive the initial differentiation process of the corneal endothelium, we created peri-ocular mesenchyme cells lines from E12.5 and E13.5 embryos. These cell lines were shown to be appropriate model cell lines for understanding this process as they express the key genes known to be associated with corneal endothelial development. Moreover, the cell lines differ in their proliferation rates with E12.5 cells proliferating more rapidly than E13.5 cells, consistent with the E13.5 cells representative of a more differentiated state of the E12.5 cells. We showed that, in order for differentiation to proceed from E12.5 to E13.5, Foxc1, Pitx2, Tsc22 and Slug expression must persist, but be down-regulated. The reduction in Foxc1 expression at E13.5 has been previously reported [17] while down-regulation of Slug, a mesenchymal marker, is necessary for mesenchyme to epithelial transition to occur [11]. To determine whether signals from the lens are responsible for down-regulation, the cells were exposed to chick lenses for 24 h. Ectopic chick lenses have previously been shown to induce distinct expression patterns of key genes in eye development suggesting that an evolutionarily conserved signal/s from the lens controls differentiation. In these experiments, Tsc22 expression in the optic cup margins of E10.5– E13.5 murine embryos was induced by E4–E8 ectopic chick lenses [29]. In our model, we showed that exposure of E12.5 cells to lenses for 24 h reduced Foxc1, Pitx2, Tsc22 and Slug expression by 50%. As these genes are normally down-regulated by E13.5, it suggests that the lens plays a major role in their down-regulation. Exposure of E13.5 cells to lenses for 24 h revealed that lensderived effects on Foxc1 and Slug are robust and are maintained for a further 24 h. Tsc22 expression is unaffected while Pitx2 expression is increased. Thus, in this model, signal/s from the lens
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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Fig. 4 – The role of Foxc1 in interpreting lens-derived signals: The effect of lens, Tgfβ2 and Follistatin exposure on Pitx2, Tsc22 and Slug expression in E12.5 cells in which Foxc1 expression has been knocked-down measured by qPCR relative to the two reference genes (n¼3, po0.05). Some of the data are repeated for ease of comparison.
play a role in driving differentiation from E12.5 and E13.5, variably affecting the expression of Foxc1 and Pitx2 and the two drivers of MET, Tsc22 and Slug. Tgfβ2 is the most well described lens-secreted molecule and it has been shown to play an important role in corneal endothelial development. The peri-ocular mesenchyme cells at E12.5 and E13.5 express Tgfβr1 and 2 receptors and are thus able to respond to Tgfβ2. Moreover, the peri-ocular mesenchyme cells themselves produce Tgfβ2 (Figs. 1a and b) suggesting a possible autocrine function. In E13.5 Tgfβr2 / mice, Foxc1 expression in the periocular mesenchyme was dramatically reduced compared to wildtype mice and both Foxc1 and Pitx2 expression absent at E15 [16]. This suggests that Tgfβ2 signalling, paracrine or possible autocrine, is important for the expression of these transcription factors in a temporal-specific manner [16]. In line with these data and in this model, exposure of E12.5 cells to Tgfβ2 was shown to down-regulate Foxc1 and very significantly, Pitx2 expression, indicating that Tgfβ2 may play a role in Foxc1 down-regulation but appears to be the major possible lens-derived signalling molecule affecting Pitx2 expression at this time point. Exposure of E13.5 cells to Tgfβ2 for 24 h significantly reduced Slug expression suggesting that Tgfβ2 may be largely responsible for downregulating Slug expression at this stage. In a study designed to understand the role EMT may play during the development of anterior polar cataracts in the lens, lens epithelial cells exposed to TGFβ induced Slug expression by binding to the Sp1 site in the Slug promoter, thus driving EMT [6]. Thus, at least in these cell lines, in EMT, Tgfβ signalling induces Slug while in MET, Tgfβ2 represses Slug. While Tgfβ2 down-regulates Foxc1 and Pitx2 expression at E12.5 and Slug expression at E13.5, it has little or no effect, on Tsc22 expression at E12.5 and E13.5 suggesting that other lens-derived factors affect its expression at these developmental time points. We thus investigated the effects of the BMP antagonist, follistatin, shown to be expressed by lens epithelial cells at E12.5. Follistatin had no effect on Foxc1, Pitx2 or Tsc22 but significantly enhanced the expression of Slug, in contrast to the inhibitory effect of whole lens. BMP4 and 7 have been shown to induce Slug expression during EMT in neural crest formation [22] thus presumably inhibition of BMP signalling by follistatin would inhibit Slug expression. However, in this model, where the cells
are beginning to undergo MET, inhibition of BMP signalling by follistatin, results in increased Slug expression. The reason for this is unknown as Slug expression must be down-regulated for MET to occur [11] and exposure of whole lens (presumably the source of any follistatin) results, as expected, in Slug down-regulation. It is possible that the effects seen are due to follistatin concentration as a previously published concentration (0.5 mg/ml) was used to perform the experiments [2]. Nevertheless, these data show that Slug expression can be regulated by follistatin. Foxc1 expression is essential for normal corneal endothelial development. We have previously shown that Tsc22 is negatively regulated by Foxc1 at E12.5 [27]. Here, we show that Foxc1 positively regulates, either directly or indirectly, the expression of Pitx2 and Slug at E12.5. Furthermore, knock-down and overexpression of Foxc1 has a dramatic and similar effect on Pitx2, Tsc22 and Slug expression. These data are consistent with reports that precise levels of Foxc1 are required for correct differentiation of the peri-ocular mesenchyme and that both mutations in FOXC1 or duplication of FOXC1 can result in abnormal anterior segment development and ARS [28]. Foxc1 and Pitx2 have been shown to physically interact where the homeodomain of Pitx2A binds to the C-terminal transactivation domain of Foxc1, resulting in impairment of Foxc1 activation of target genes [4]. Here, we show that, when Foxc1 expression is knocked-down, Pitx2 expression is also reduced. The transcriptional regulator protein PRKC apoptosis Wilm's tumour 1 regulator (PAWR) was previously shown to modulate the activities of Foxc1 and Pitx2 where, in the absence of Foxc1, PAWR reduced Pitx2 activity [1]. Foxc1 control over Pitx2 expression persisted at E13.5 while effects on Tsc22 were abolished. Interestingly, at E13.5, Foxc1 over-expression induced Slug expression by two-fold. Increased expression of Foxc1 through chromosomal duplication causes iris hypoplasia and can lead to glaucoma [19]. The significant upregulation in Slug expression following overexpression of Foxc1 may partially prevent proper MET, impairing development and may thus be a contributing factor to the disease. We have therefore shown that lens-derived signals at E12.5 play a role in down-regulating the expression of Foxc1 and Pitx2, transcription factors whose expression is essential for normal corneal endothelial development, Slug, a key regulator of EMT and MET and Tsc22, a regulator expressed highly at areas of EMT. This down-regulation appears necessary for continued differentiation.
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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Our results suggest that possible lens-derived Tgfβ2 plays a role in down-regulating Foxc1 and Pitx2 expression at E12.5 and Slug at E13.5. Although Foxc1 and Pitx2 interact and Foxc1 can regulate Pitx2 expression, Foxc1 does not play a role in Tgfβ2-mediated down-regulation of Pitx2. Similarly, lens-mediated down-regulation of Tsc22 appears unaffected by Foxc1 and does not appear to be mediated by Tgfβ2 and follistatin suggesting that other lensderived factors force its down-regulation. However, although Tgfβ2 and follistatin have no effect on Tsc22 expression, in the absence of Foxc1, their expression is dramatically reduced suggesting that Foxc1 acts in some way to mediate the effects of these two molecules on Tsc22 expression. This, however, has little effect when compared to whole lens exposure where other factors down-regulate Tsc22 expression. Foxc1 does play a role in mediating the effects of whole lenses on Slug expression. The down-regulatory effects of the lens do not appear to be mediated by Tgfβ2 (which has no effect) or follistatin (which acts to increase Slug expression), suggesting that, as with Tsc22, other lensderived factors play a greater role in regulating Slug expression. Foxc1 may act to inhibit the positive effects of follistatin and may, as with Tsc22 and Pitx2, play a role in mediating Tgfβ2 effects. Our data reveal that Foxc1 does not play a role in translating lens-derived effects on Tsc22 or Pitx2 expression but that Foxc1 may play a role in correctly mediating the lens-derived signal/s negative effects on Slug. In addition, Foxc1 may act in some way to block Tgfβ2 signalling and to mediate follistatin effects on Tsc22 and Slug expression. These data provide an indication of how Foxc1 expression is important for MET in corneal endothelial development. Taken together, in this model we show that lensderived factors and key transcription factors interact to drive the initial process of MET in corneal endothelial development.
Acknowledgments This study was supported by a grant from the Medical Research Council of South Africa. The authors would like to thank Dr. Marco Weinberg (University of the Witwatersrand) for his help with the shRNA design and Y.Liu, J. Huang and D.Beebe (Washington University in St. Louis) for supplying the array data on enriched E12.5 lens epithelial transcripts.
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2014.01.016.
references [1] M. Acharya, L. Huang, V.C. Fleisch, W.T. Allison, M.A. Walter, A complex regulatory network of transcription factors critical for ocular development and disease, Hum. Mol. Genet. 20 (2011) 1610–1624. [2] D. Beebe, C. Garcia, X. Wang, R. Rajagopal, M. Feldmeier, J.Y. Kim, A. Chytil, H. Moses, R. Ashery-Padan, M. Rauchman, Contributions by members of the TGFbeta superfamily to lens development, Int. J. Dev. Biol. 48 (2004) 845–856. [3] D.C. Beebe, J.M. Coats, The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye, Dev. Biol. 220 (2000) 424–431.
] (]]]]) ]]]–]]]
7
[4] F.B. Berry, M.A. Lines, J.M. Oas, T. Footz, D.A. Underhill, P.J. Gage, M.A. Walter, Functional interactions between FOXC1 and PITX2 underlie the sensitivity to FOXC1 gene dose in Axenfeld–Rieger syndrome and anterior segment dysgenesis, Hum. Mol. Genet. 15 (2006) 905–919. [5] D. Castanotto, H. Li, J.J. Rossi, Functional siRNA expression from transfected PCR products, RNA 8 (2002) 1454–1460. [6] J. Choi, S.Y. Park, C.K. Joo, Transforming growth factor-beta1 represses E-cadherin production via slug expression in lens epithelial cells, Invest. Ophthalmol. Vis. Sci. 48 (2007) 2708–2718. [7] S.J. Choi, J.H. Moon, Y.W. Ahn, J.H. Ahn, D.U. Kim, T.H. Han, Tsc-22 enhances TGF-beta signaling by associating with Smad4 and induces erythroid cell differentiation, Mol. Cell. Biochem. 271 (2005) 23–28. [8] A. Cvekl, E.R. Tamm, Anterior eye development and ocular mesenchyme: new insights from mouse models and human diseases, Bioessays 26 (2004) 374–386. [9] C.E. Dohrmann, M. Belaoussoff, L.A. Raftery, Dynamic expression of TSC-22 at sites of epithelial–mesenchymal interactions during mouse development, Mech. Dev. 84 (1999) 147–151. [10] A.T. Dudley, K.M. Lyons, E.J. Robertson, A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye, Genes Dev. 9 (1995) 2795–2807. [11] N. Fenouille, M. Tichet, M. Dufies, A. Pottier, A. Mogha, J.K. Soo, S. Rocchi, A. Mallavialle, M.D. Galibert, A. Khammari, J.P. Lacour, R. Ballotti, M. Deckert, S. Tartare-Deckert, The epithelial–mesenchymal transition (EMT) regulatory factor SLUG (SNAI2) is a downstream target of SPARC and AKT in promoting melanoma cell invasion, PLoS One 7 (2012) e40378. [12] C. Flugel-Koch, A. Ohlmann, J. Piatigorsky, E.R. Tamm, Disruption of anterior segment development by TGF-beta1 overexpression in the eyes of transgenic mice, Dev. Dyn. 225 (2002) 111–125. [13] P.J. Gage, A.L. Zacharias, Signaling “cross-talk” is integrated by transcription factors in the development of the anterior segment in the eye, Dev. Dyn. 238 (2009) 2149–2162. [14] P.J. Gage, H. Suh, S.A. Camper, Dosage requirement of Pitx2 for development of multiple organs, Development 126 (1999) 4643–4651. [15] P.J. Gage, W. Rhoades, S.K. Prucka, T. Hjalt, Fate maps of neural crest and mesoderm in the mammalian eye, Invest. Ophthalmol. Vis. Sci. 46 (2005) 4200–4208. [16] L.M. Ittner, H. Wurdak, K. Schwerdtfeger, T. Kunz, F. Ille, P. Leveen, T.A. Hjalt, U. Suter, S. Karlsson, F. Hafezi, W. Born, L. Sommer, Compound developmental eye disorders following inactivation of TGFbeta signaling in neural-crest stem cells, J. Biol. 4 (2005) 11. [17] S.H. Kidson, T. Kume, K. Deng, V. Winfrey, B.L. Hogan, The forkhead/winged-helix gene, Mf1, is necessary for the normal development of the cornea and formation of the anterior chamber in the mouse eye, Dev. Biol. 211 (1999) 306–322. [18] T. Langenberg, A. Kahana, J.A. Wszalek, M.C. Halloran, The eye organizes neural crest cell migration, Dev. Dyn. 237 (2008) 1645–1652. [19] O.J. Lehmann, N.D. Ebenezer, T. Jordan, M. Fox, L. Ocaka, A. Payne, B.P. Leroy, B.J. Clark, R.A. Hitchings, S. Povey, P.T. Khaw, S.S. Bhattacharya, Chromosomal duplication involving the forkhead transcription factor gene FOXC1 causes iris hypoplasia and glaucoma, Am. J. Hum. Genet. 67 (2000) 1129–1135. [20] O.J. Lehmann, J.C. Sowden, P. Carlsson, T. Jordan, S.S. Bhattacharya, Fox's in development and disease, Trends Genet. 19 (2003) 339–344. [21] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method, Methods 25 (2001) 402–408. [22] S. Ohta, G.C. Schoenwolf, G. Yamada, The cessation of gastrulation: BMP signaling and EMT during and at the end of gastrulation, Cell Adhes. Migr. 4 (2010) 440–446.
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016
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E XP E RI ME N TAL CE L L R ES E ARC H
[23] G.S. Peh, R.W. Beuerman, A. Colman, D.T. Tan, J.S. Mehta, Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview, Transplantation 91 (2011) 811–819. [24] L.W. Reneker, D.W. Silversides, L. Xu, P.A. Overbeek, Formation of corneal endothelium is essential for anterior segment development – a transgenic mouse model of anterior segment dysgenesis, Development 127 (2000) 533–542. [25] S. Saika, S. Saika, C.Y. Liu, M. Azhar, L.P. Sanford, T. Doetschman, R.L. Gendron, C.W. Kao, W.W. Kao, TGFbeta2 in corneal morphogenesis during mouse embryonic development, Dev. Biol. 240 (2001) 419–432. [26] M. Sefton, S. Sanchez, M.A. Nieto, Conserved and divergent roles for members of the Snail family of transcription factors in the chick and mouse embryo, Development 125 (1998) 3111–3121. [27] P. Sommer, H.R. Napier, B.L. Hogan, S.H. Kidson, Identification of Tgf beta1i4 as a downstream target of Foxc1, Dev. Growth Differ. 48 (2006) 297–308.
] (]]]]) ]]]–]]]
[28] M.H. Strungaru, I. Dinu, M.A. Walter, Genotype-phenotype correlations in Axenfeld–Rieger malformation and glaucoma patients with FOXC1 and PITX2 mutations, Invest. Ophthalmol. Vis. Sci. 48 (2007) 228–237. [29] C.J. Thut, R.B. Rountree, M. Hwa, D.M. Kingsley, A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens, Dev. Biol. 231 (2001) 63–76. [30] J. Vandesompele, K. De Preter, F. Pattyn, B. Poppe, N. Van Roy, A. De Paepe, F. Speleman, Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes, Genome Biol. 3 (2002) (RESEARCH0034). [31] Y. Zhang, P.A. Overbeek, V. Govindarajan, Perinatal ablation of the mouse lens causes multiple anterior chamber defects, Mol. Vis. 13 (2007) 2289–2300. [32] Y. Lui, J. Huang, D. Beebe, unpublished data.
Please cite this article as: Z.T.V. Silla, et al., Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.01.016