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Keywords:

  • Retina;
  • Multipotency;
  • Xenopus;
  • Transcription factors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusion
  7. Disclosure of Potential Conflicts of Interest
  8. References
  9. Supporting Information

The molecular mechanisms underlying the acquisition of retinal precursor identity are scarcely defined. Although the homeobox gene Rx1 (also known as Rax) plays a major role in specifying retinal precursors and maintaining their multipotent state, the involved mechanisms remain to be largely deciphered. Here, following a highthroughput screen for genes regulated by Rx1, we found that this transcription factor specifies the fate of retinal progenitors by repressing genes normally activated in adjacent ectodermal territories. Unexpectedly, we also observed that Rx1, mainly through the activation of the transcriptional repressors TLE2 and Hes4, is necessary and sufficient to inhibit endomesodermal gene expression in retinal precursors of the eye field. In particular, Rx1 knockdown leads retinogenic blastomeres to adopt an endomesodermal fate, indicating a previously undescribed function for Rx1 in preventing the expression of endomesoderm determinants known to inhibit retinal fate. Altogether these data suggest that an essential requirement to establish a retinal precursor identity is the active inhibition of pathways leading to alternative fates. Stem Cells 2013;31:2842–2847


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusion
  7. Disclosure of Potential Conflicts of Interest
  8. References
  9. Supporting Information

All retinal cell types of the vertebrate eye derive from multipotent retinal precursors, which are first specified at early neurula stage in an anterior region of the neuroectoderm called the eye field (reviewed in reference 1). Identifying the molecular mechanisms underlying the generation of retinal precursors and their multipotency represents a primary goal not only for developmental neurobiology but also to define efficient protocols for cell reprogramming aimed at generating retinal cells in vitro. A major role in defining retinal identity is played by Rx1 (also known as Rax), a homeobox gene required for retina development and expressed in retinal precursors of the eye field and in retinal stem cells of the amphibian ciliary marginal zone (CMZ; 2–6). Notably, Rx1 was shown to control proliferation and multipotency of retinal progenitors [7, 8], to trigger generation of Müller glia, a cell type sharing many similarities with retinal progenitors and retinal stem cells [9-11], and to induce a retinal fate in embryonic stem cells [12]. Despite the crucial role played by Rx1, the genetic program regulated by this transcription factor remains to be largely deciphered. Here, we show that a prerequisite for Rx1 to maintain retinal progenitor specification is the repression of alternative fates, in particular by preventing expression of endomesoderm determinants known to inhibit retinal fate [13].

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusion
  7. Disclosure of Potential Conflicts of Interest
  8. References
  9. Supporting Information

Microinjection and in situ hybridizations were performed as previously described [5, 14, 15]. Microarray results are available from the GEO database (Accession Number GSE35918). More details are described in Supporting Information.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusion
  7. Disclosure of Potential Conflicts of Interest
  8. References
  9. Supporting Information

Rx1 Represses Genes Expressed in Territories Surrounding the Eye Field

To gain insight into the molecular pathways controlled by Rx1, we compared the transcriptome of Xenopus laevis embryos overexpressing Rx1 to the transcriptome of embryos injected with MoRx1, an antisense morpholino oligonucleotides targeting Rx1 (Supporting Information Table S1; see Supporting Information Material and Methods for details). A main feature emerging from this analysis is that Rx1 appears to repress genes expressed in territories surrounding the retinal precursors of the eye field. One of the primarily affected genes is Crx-b (previously named Xotx5b; [16]), which displays an expression domain complementary to that of Rx1, including presumptive cement gland, forebrain, and midbrain (Fig. 1A–1H). Accordingly, Rx1 overexpression represses Crx-b in all these territories (Fig. 1M), while Rx1 knockdown causes Crx-b upregulation in the eye field (Figs. 1N, 4K). A similar repressive effect could be potentially exerted by Rx1 also in the peripheral mature retina, where Crx-b is excluded from the CMZ, which expresses Rx1 (Fig. 1D, 1H). Concomitantly, we found that Rx1 activates TLE2, a corepressor of the groucho family, and, as we previously described [5], one of its putative interactors, the transcriptional repressor Hes4 (previously named hairy2). TLE2 expression correlates well with both early and late Rx1 expression sites, including the CMZ (Fig. 1I–1L), and functional experiments indicate that Rx1 is necessary and sufficient for TLE2 activation (Fig. 1O, 1P). Hes4, which similarly to Rx1 maintains retinal precursors in a proliferative state, is coexpressed with Rx1 at early neurula, in the retinal pigmented epithelium and in the CMZ [5, 7, 19, 20]. We found that Rx1GR, an Rx1 hormone-inducible protein [8], is able to activate the expression of TLE2, but not of Hes4, in pluripotent ectodermal explants (animal caps) even in the absence of protein synthesis, thus suggesting that TLE2 is a direct target of Rx1 (Fig. 1Q).

image

Figure 1. Rx1 is necessary and sufficient to activate TLE2 and repress Crx-b. (A-L): Expression of Rx1, Crx-b, and TLE2 at early neurula (A, E, I), midneurula (B, F, J), late neurula (C, G, K), and in the mature retina of early tadpole (D, H, L). In D, H, and L black and white arrowheads indicate presence or absence of CMZ expression, respectively. (M-P): Embryos injected unilaterally with Rx1 (M, O) or MoRx1 (P), or bilaterally with MoRx1 (N) and analyzed at early neurula stage. Embryos are shown in frontal views with dorsal to the top. In O, ectopic TLE2 expression is indicated by an arrowhead, while increased expression of TLE2 within its normal expression domain is indicated by an arrow. Scale bars = 400 µm (A-C, E-G, I-K, M-P) or 50 µm (D, H, L). (Q): Relative quantification by real-time quantitative RT-PCR (RT-qPCR) of TLE2 and Hes4 expression in animal caps injected with Rx1GR in the presence (+) or absence (−) of cycloheximide (C) or dexamethasone (D) and cultured to stage 11. Expression levels were normalized by setting the expression level of the condition −C +D to 100. As already described in other cases, cycloheximide treatment may lead to transcription activation probably due to a block in the protein synthesis of transcriptional repressors that normally maintain silent the analyzed genes in animal caps [17, 18]. Error bars indicate SEM. *, p < .05 (Student's t test). n, number of analyzed embryos.

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Rx1 Inhibits the Expression of Endomesodermal Genes in the Eye Field

Unexpectedly, microarray data indicated that Rx1 appears to repress also endomesodermal genes such as Xenf, Goosecoid (Gsc), VegT, Cerberus (Cer), Eomesodermin (Eomes), and Sox17β Supporting Information Table S1. Although these genes are typically expressed in the endomesoderm, low expression may be also present in dorso-animal regions, which were targeted by Rx1 or MoRx1 injections in microarray experiments. This has been actually documented for VegT, which forms a concentration gradient from vegetal to animal poles [21, 22]. To better test the ability of Rx1 to repress endomesodermal genes, we analyzed embryos misexpressing Rx1 in dorsal endomesoderm. RT-qPCR and in situ hybridization indicate that Rx1 is indeed sufficient to repress all the analyzed genes (Fig. 2A, 2B). To understand whether Rx1 could also antagonize the endomesodermal inducing activity of VegT, we analyzed the effects of these genes in animal caps. We found that Rx1 strongly represses VegT-mediated activation of Xenf, but not of Sox17β (Fig. 2C), suggesting that the inhibition of Sox17β observed in Rx1 injected embryos (Fig. 2B) is likely to be mainly a consequence of the repression of VegT.

image

Figure 2. Rx1 misexpression inhibits the expression of endomesodermal genes. (A, B): Expression of endomesodermal genes in early neurulae injected vegetally with Rx1 and analyzed by RT-qPCR (A) and in situ hybridization (B, lateral views of bisected embryos, dorsal to the top, and anterior to the left). Black arrowheads indicate expression sites in control embryos, except in Goosecoid where they delimit the extent of the expression domain. White arrowheads indicate reduced expression in corresponding sites of Rx1 injected embryos. (C): Animal caps injected with VegT and green fluorescent protein (GFP) (VegT) or VegT, Rx1, and GFP (VegT+Rx1) and analyzed for the expression of Xenf and Sox17β. Percentages of affected caps are shown in Supporting Information Table S2A. Error bars indicate SEM. *, p < .05 (Student's t test). n, number of analyzed embryos. Scale bars = 400 µm (B) or 600 µm (C).

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To assess the requirement for Rx1 in repressing endomesodermal genes, we analyzed MoRx1 injected embryos. While RT-qPCR shows a significant upregulation for all the analyzed endomesodermal genes (Fig. 3A), sections of hybridized embryos reveal increased expression levels and/or expansion of their expression domains in the endomesodermal layer but no ectopic activation in the neural plate (Fig. 3B). As Rx1 does not appear to repress endomesodermal gene expression in a non-cell-autonomous manner (Supporting Information Fig. S1), a possible interpretation of these data is that an impairment of Rx1 activity could lead presumptive retinal progenitors to adopt an endomesodermal fate. This is indeed what happens when VegT, a gene activated in Rx1 knockdown, is misexpressed in retinogenic blastomeres [13]. To this aim, we injected the retinogenic blastomere D1.1 at 16-cell stage with MoRx1 and green fluorescent protein (GFP) and followed the fate of the cell progeny at tadpole stage comparing it with the fate of D1.1 blastomere injected with GFP alone. To analyze the retinal contribution of the MoRx1 injected blastomere, we focused on microphthalmic, rather than anophthalmic, larvae, which were obtained by decreasing the dose of injected MoRx1. Each organ was scored based on the extent of the population of GFP-positive cells [23]. Fate mapping analysis shows that the D1.1 lineage contributes significantly more frequently to the head endoderm in microphthalmic tadpoles than in control embryos (Fig. 3C–3G). Interestingly, Rx1 knockdown leads to a significant decrease in the number of embryos displaying the largest extent of labeled cells in retina as well as in brain (Fig. 3C, class 1), which also expresses Rx1 in the early presumptive telencephalon, the pineal organ, and ventral diencephalon [2, 3, 20]. These effects can be explained in part as the result of a reduced proliferation of these territories [5]. However, as no other significant variation between control and MoRx1 injected embryos is observed in other tissues, these data suggest that the increased contribution to anterior endoderm of D1.1 blastomere in Rx1 knockdown embryos appears to be at the expense of retina and brain. The close vicinity of the anterior neural plate and the underlying anterior endomesoderm at the end of gastrulation, when Rx1 is first activated [24], and during neurulation is likely to facilitate migration of reprogrammed MoRx1 injected cells from the neuroectoderm to the endomesoderm. Thus, the increased levels of endomesodermal markers, as well as the expansion of their expression domains, observed in MoRx1 injected embryos (Fig. 3A, 3B) can be interpreted as the consequence of an increased number of endomesodermal cells. Previous loss of function studies on Rx genes performed both in Xenopus and in other animal models did not report these effects [3, 5, 20, 25]. This could be due to the more visible, and possibly predominant, effect of Rx1 on cell proliferation, or to the lack of an extended early cell fate analysis, although, in some cases, the existence of species-specific differences cannot be ruled out. However, some studies [26, 27] have indicated that the inactivation of Rx genes can lead to a fate switch within the neuroectoderm, supporting the idea that Rx1 may act by repressing alternative fates.

image

Figure 3. Rx1 knockdown leads retinal progenitors to adopt an endomesodermal fate. (A, B): Expression of endomesodermal genes in early neurulae injected with MoRx1 in dorsal ectoderm as determined by RT-qPCR (A) and in situ hybridization (B). (B): Midsagittal sections of paraffin-embedded embryos, anterior to the left, dorsal to the top. Anterior-dorsal sectors are shown, except for VegT where a posterior detail is shown. Arrowheads indicate the extent of Cerberus, Goosecoid, and Sox17β expression in control and MoRx1 injected embryos. (C): Fate mapping of D1.1 blastomere in control (C) and MoRx1 injected (M) embryos. The graph shows the percentages of embryos displaying green fluorescent protein (GFP)-labeled cells in specific organs. Based on the classes defined by Moody and Kline [23], each column of the graph is subdivided in three fractions (class 1, 2, 3) indicating an estimate of the sizes of the GFP-positive clones populating each organ. (D-G): Examples of cryosections at the level of the retina (D, E) and anterior endoderm (F, G) showing the different contribution of D1.1 progeny in control (D, F) and MoRx1 injected embryos (E, G). Structures labeled in MoRx1 injected or control embryos are indicated: r, retina; b, brain; e, epidermis; g, gut. n, number of analyzed embryos. Error bars indicate SEM. *, p < .05; **, p < .01 (Student's t test). n, number of analyzed embryos. The white asterisks in C refer to a significant decrease of retina and brain class 1 observed in MoRx1 injected embryos compared to class 1 of control embryos. Scale bars = 200 µm (B) or 100 µm (D--G). Abbreviations: A. END., anterior endoderm; A.P. MES., axial and paraxial mesoderm; C. GL., cement gland; EPID., epidermis; H. MES., head mesoderm; P. END., posterior endoderm.

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image

Figure 4. TLE2 and Hes4 mediate the repressive effects of Rx1. (A, B, F, G): Crx-b expression in embryos injected in dorsal-animal blastomeres with TLE2(T) and Hes4(H) (A), Rx1 (B), MoRx1(M), TLE2, and Hes4 (F), and Rx1, VP16TLE2 (dnT: dominant negative TLE2), and Hes4VP16-GR (dnH: dominant negative Hes4) (G); frontal views, dorsal to the top. (C, H): Eomesodermin expression in bisected neurulae injected vegetally with nuclear lacZ alone (C), or TLE2 and Hes4 (H). (D, E, I, J): Eomesodermin expression in bisected neurulae injected in dorsal-animal blastomeres with MoRx1 (D), nuclear lacZ alone (E), MoRx1, TLE2, and Hes4 (I) or dnT and dnH (J). (C-E, H-J): Lateral view, anterior to the left and dorsal to the top. n, number of analyzed embryos. Percentage of affected embryos for rescue experiments is shown in Supporting Information Table S2B, S2C. Scale bar = 400 µm (A-J). (K): Relative quantification by RT-qPCR of Eomesodermin and Crx-b in embryos injected with either green fluorescent protein (GFP) or MoRx1, or coinjected with either TLE2 and Hes4, or MoRx1, TLE2, and Hes4. Error bars indicate SEM. *, p < .05; **, p < .01 (Student's t test). No significant difference is observed between samples injected with GFP alone and MoRx1+Tle2+Hes4.

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TLE2 and Hes4 Mediate the Repressive Activity of Rx1

To further characterize the mechanisms of Rx1 repressive activity, we tested whether TLE2 together with its putative interactor Hes4 [28] could mediate this effect. We observed that, similarly to Rx1, coinjection of TLE2 and Hes4 represses Crx-b in dorsal-animal injection (Fig. 4A, 4K) and Eomes when injected vegetally (Fig. 4C, 4H, 4K), consistently with the described ability of Hes4 to repress anterior endodermal genes [29]. Moreover, coinjection of TLE2 and Hes4 effectively rescues an almost normal expression of Crx-b and Eomes in MoRx1 injected embryos (Fig. 4D, 4F, 4I, 4K, Supporting Information Table S2B). Conversely, blocking TLE2 and Hes4 activity in the neural ectoderm, by the use of the dominant negative constructs VP16TLE2 and Hes4VP16-GR ([19]; Supporting Information Fig. S2), is sufficient to phenocopy the upregulation of Eomesodermin described for MoRx1 injected embryos (Fig. 4E, 4J). Furthermore, we found that coexpression of VP16TLE2 and Hes4VP16-GR in the dorsal ectoderm efficiently rescues the repressive effects of Rx1 on Crx-b (Fig. 4B, 4G, Supporting Information Table S2C). However, no rescue on the Rx1-mediated repression of Eomesodermin is observed in vegetal injections (Supporting Information Table S2C), suggesting that when Rx1 is ectopically expressed in the endomesoderm it may repress Eomesodermin independently of TLE2 and Hes4. Altogether these data suggest that in the neural plate TLE2 and Hes4 are the main mediators of Rx1 repressive activity.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusion
  7. Disclosure of Potential Conflicts of Interest
  8. References
  9. Supporting Information

Our data indicate a role for Rx1 in inhibiting gene programs leading to cell fates typical of territories surrounding the eye field, including the underlying endomesoderm. Because of the key function played by Rx1 in retinal precursors, this is likely to be an essential element to generate and maintain a retinal multipotent identity that may prove crucial in efficiently directing embryonic and adult cells toward a retinal fate.

Acknowledgments

We are grateful to Fondazione Fiorgen onlus and Alessandro Provenzani for the use of microarray scanner and to Naoto Ueno, Morgane Locker and Muriel Perron for generously providing plasmids. We thank Robert Vignali and Federico Cremisi for helpful discussion and Giuseppe Lupo for critical reading of a previous version of the manuscript. We also gratefully acknowledge Marzia Fabbri, Donatella De Matienzo and Elena Landi for technical assistance, and Salvatore Di Maria for frog care. This work was supported by Cofinanziamento PRIN-Universita' di Pisa (prot. 2007TEFM7M). G.G. is currently affiliated with the Scuola Superiore S. Anna, The BioRobotics Institute, Translational Neural Engineering Group, Viale Rinaldo Piaggio 34, 56025 Pontedera (Pisa), Italy. D.B. is currently affiliated with the Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Cambridge, U.K. S.M. is currently affiliated with the Laboratorio di Scienze Mediche, Istituto di Scienze della Vita, Scuola Superiore Sant'Anna, Pisa 56127, Italy.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusion
  7. Disclosure of Potential Conflicts of Interest
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusion
  7. Disclosure of Potential Conflicts of Interest
  8. References
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
stem1530-sup-0001-suppfig1.tif8172KSupporting Information Figure 1.
stem1530-sup-0002-suppfig2.tif3847KSupporting Information Figure 2.
stem1530-sup-0003-supptab1.docx70KSupporting Information Table 1.
stem1530-sup-0004-supptab2.docx23KSupporting Information Table 2.
stem1530-sup-0005-supptab3.docx70KSupporting Information Table 3.
stem1530-sup-0006-suppfigcap.docx118KSupporting Information Figure Caption
stem1530-sup-0007-suppinfo.docx124KSupporting Information

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