NEWS AND VIEWS
Pigment Cell Melanoma Res.
New functions for old genes: Pax6 and Mitf in eye pigment biogenesis Florencia Cavodeassi and Paola Bovolenta e-mail: [email protected]
Melanin synthesis constitutes a fundamental aspect of the terminal differentiation program of pigment cells. In vertebrates, there are two major pigment cell types: melanocytes, derived from the neural crest and cells of the pigmented layer of the retina, known as retinal pigmented epithelium (RPE), which are generated from neuroepithelial cells of the optic primordium. Despite their distinct origin, these cells share a conserved battery of genes devoted to control pigment biogenesis, which, in turn, is activated by different isoforms of the microphthalmia transcription factor (MITF) (reviewed in Martinez-Morales et al., 2004). The RPE is essential for the homeostasis of the retina and failure in its development or physiology leads to severe retinal degeneration. RPE specification occurs at very early stages of embryonic development when the optic primordium becomes subdivided into optic stalk, neural retina, and RPE (Figure 1A). This regionalization occurs under the control of well-organized gene regulatory networks (Beccari et al., 2013), which, in the RPE, involve the activity of transcription factors of the Otx and Pax families, the positive input of the Wnt/ßcatenin pathway and the strong induction of Mitf (reviewed in Martinez-Morales et al., 2004). In particular, studies devoted to understand the role of Pax genes during optic vesicle specification have shown that Pax2 and Pax6 have redundant roles in optic vesicle regionalization: in the absence of both genes, Mitf expression fails to be activated and the presumptive RPE develops as neural retina-like tissue.
Coverage on: Raviv S, Bharti K, RencusLazar S, Cohen-Tayar Y, Schyr R, Evantal N, Meshorer N, Zilberberg A, Idelson M, Reubinoff B, Grebe R, Rosin-Arbesfeld R, Lauderdale J, Lutty G, Arnheiter H, and Ashery-Padan R. (2014) PAX6 regulates melanogenesis in the retinal pigmented epithelium through feed-forward regulatory interactions with MITF. Plos Genetics 10(5):e1004360. doi: 10. 1371/journal.pgen.1004360. doi: 10.1111/pcmr.12308
Likely, Pax6 has a more prominent role in this process because Pax2 is soon downregulated from the presumptive RPE, whereas Pax6 expression is maintained. Furthermore, loss of a single Pax6 allele in a Mitf null background is sufficient to enhance the acquisition of a neural retina fate by the RPE (Bharti et al., 2012), suggesting that Pax6 is a main regulator of RPE formation. Pax6 is considered as one of the eye ‘kernel’ genes, in other words as a gene that acts at the top of a gene regulatory network that imparts the fate of a given tissue or organ (Beccari et al., 2013). Indeed, Pax6 is expressed in the entire eye primordium and loss of its function leads to severe eye defects across evolution. Its expression, however, is not restricted to the earliest stages of eye formation and is thereafter recurrently required to promote neural retina and lens development, raising the question of how a single transcription factor controls the specification and differentiation of the different tissues in which it is expressed. A very recent study from the laboratory of Ruth Ashery-Padan provides an answer to this question, focusing on the yet unexplored role of Pax6 after the RPE is specified and its differentiation begins. The authors show that Pax6 is once again recruited during eye development, this time to activate a specific aspect of the terminal differentiation of the RPE: the formation of melanin pigment. This Pax6 function involves a well-orchestrated interaction with MITF, considered a master regulator of melanocyte formation. To reach this conclusion, the authors generated a Pax6 conditional mouse mutant, which specifically removes full-length Pax6 from the RPE but only after its specification (Pax6loxP/loxP; DctCre or Pax6RPE mutants). The RPE of the resulting mutants maintained its fate with typical monolayer morphology, normal levels of junctional proteins and expression of RPE markers such as Otx2 and Sox9 but presented a remarkable reduction in pigmentation. This suggested that, once established, Pax6 is no longer needed to maintain RPE specification but is required for promoting its melanogenic program. To verify this possibility, the authors com-
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
pared the transcriptional profile of Pax6-deficient and wild-type RPE cells. This analysis revealed an enrichment of melanogenic genes among those significantly downregulated in the mutant RPE. Notably, seven of the ten melanogenesis-related genes found downregulated in Pax6-RPE mutants were well-known direct targets of MITF. Nonetheless, of the three Mitf isoforms expressed in the RPE, only D-Mitf, which seems to contribute minimally to pigment regulation, was strongly downregulated, whereas the other two Mitf isoforms, A-Mitf and H-Mitf, were slightly increased, with a global 1.45-fold reduction of Mitf levels as compared to wild type. This modest change unlikely explained the loss of pigmentation seen in Pax6-RPE mutants. Thus, the authors reasoned that Pax6, besides controlling Mitf expression, was likely a direct regulator of the melanogenesis program. In keeping with this hypothesis, Raviv et al. (2014) first demonstrated that Pax6 synergizes with MITF to transactivate the D-Mitf promoter with a mechanism that depends upon binding of the paired domain of Pax6 to the DNA but involves no direct MITF-DNA interaction. Thereafter, they analyzed the transactivation activity of Pax6 on the promoter of three pigmentation genes (Tyrp1, Tyr, and Mlana). Pax6 by itself was unable to activate the transcription of any of these genes but, when coexpressed with MITF, had a strongly synergistic effect on the transactivation of both Tyrp1 and Tyr promoters. The authors went on to show that Pax6 and MITF physically interact with a paired domain independent mechanism and that this interaction is critical. Indeed, transactivation by the Pax6/MITF complex requires MITF but not Pax6 binding to the melanogenic loci. Intriguingly, the presence of the Pax6 paired domain, which can interact with specific DNA sequences, is necessary to assure
“Pax6 is no longer needed to maintain RPE specification but is required for promoting its melanogenic program.”
1
News and Views
A
B
C
Figure 1. (A) Schematic representation of the optic primordium. (B) Diagram of Pax6/ Mitf interactions controlling retinogenesis versus RPE formation. Blue lines indicate the interactions involved in retinogenesis, whereas red lines highlight those required for RPE differentiation. The thicker red line indicates a synergistic effect between Pax6 and Mitf in Mitf activation. (C) Cartoon highlighting the genetic network underlying melanogenesis in the RPE and the melanocytes. Note that a conserved network composed of Wnt/ßcatenin signalling and transcription factors of the Pax and Mitf families operates in both cell types. The additional participation of Otx2 in the RPE and Sox10 in the melanocytes as well as different co-transcriptional interactions diversify the melanogenic cascade in these two cell types (see text for further explanations).
efficiency although the authors did not explore why this is so. In summary, Raviv et al. (2014) uncover a new function for Pax6 in the RPE and show an interesting functional dependence between Pax6 and MITF: Pax6 induces the expression of melanogenic genes only in the presence of MITF, whereas MITF regulates the expression of its D isoform with a Pax6-dependent mechanism (Figure 1B). An interaction between Pax6 and MITF had been previously reported and proposed to be important for RPE specification. At early optic vesicle stages, the Pax6/MITF complex prevents the prospective RPE from acquiring a neural retina fate by suppressing the expression of retinogenic genes, which are otherwise activated when Pax6 acts in conjunction with other proretinal factors, including Fgfs and Wnt signalling inhibitors (Bharti et al., 2012). By directly controlling Mitf expression, Pax6 later on presets the conditions (expression of Mitf), which enable its function as a regulator of melanogenesis. Such a feed forward loop locks the RPE fate into a stable state, providing further evidence to explain how Pax6 promotes a contextspecific outcome in each of the tissues in
2
which it is expressed. This concept may have wider implications for the evolution of animal body plan. A similar network of genes, including Pax6, specifies visual structures across phyla. The most primitive of these structures likely required a small battery of genes. As eye complexity increased, so it did the underlying gene regulatory network. The co-option of genes from the original core network into more specialized functions seems thus to be a parsimonious solution to drive the differentiation of increasingly complex structures. Viewed from a different angle, the findings of Raviv et al. might also imply that the architecture of the gene regulatory networks that control embryonic development does not follow a strict hierarchy. Regulatory genes at the core of the network set up the developmental programs devoted to tissue specification, by regulating the expression of differentiation and effector genes. These, in turn, provide the architectural and functional features characteristic of each tissue (reviewed in Beccari et al., 2013). In the RPE, such a strict hierarchy seems to have been loosen up, because Pax6, which acts as an eye ‘kernel’ gene, is repurposed for a very specialized function, the control of melanogenesis during terminal tissue differentiation. Notably, in the Pax6-RPE mutants, defects are restricted to the lack of pigmentation, whereas other RPE features, such as the cuboidal epithelial morphology or the specialized intercellular junctions, are preserved, indicating that different genetic modules control different aspects of RPE differentiation and function. This independent regulation, often controlled by transcriptional complexes, may ensure robustness to the system, minimizing deleterious effects of gene mutations. In this respect, recent studies have shown that Otx2, besides controlling genes of the melanogenesis pathway (reviewed in MartinezMorales et al., 2004), activates retinol metabolism (Housset et al., 2013). Furthermore, Sox9 and Otx2 synergize to activate the expression of visual cycle genes (Masuda et al., 2014), whereas a complex formed by Sox9, Otx2, and MITF critically drives the RPE expression of the BEST1 gene, encoding a basolateral membrane protein associated with macular dystrophies. These specific functions of Otx2 and Sox9 in RPE cells together with the results of Raviv et al. bring in a different but connected issue: the use of similar sets of transcription factors in different tissues. In fact, these observations provide further support to the idea that the partial conservation of the melanogenic program in melanocytes and RPE cells
likely represent the co-option of an efficient gene regulatory network. This network might have evolved in the context of eye function and have been thereafter adapted in other cell types as the melanocytes, for example by recruiting different cofactors. Indeed, although the Wnt/ßcatenin pathway together with members of the Sox, Pax, and Mitf families all contribute to specification and differentiation of both RPE and melanocytes (Figure 1C), their functional relationships are largely different. Raviv et al. demonstrate that Pax6 teams up with MITF to control melanogenesis, whereas the cognate Pax3 has little control on the expression of melanogenic genes in neural crest-derived melanocytes. Rather, MITF synergize with both Sox10 and the Wnt/ßcatenin pathway effector LEF-1 to promote melanogenesis in this lineage. This synergism seems to require physical interaction between MITF and LEF-1/Sox10 and their cooperative binding to the promoter of melanogenic genes, in a mechanism comparable to that described by Raviv et al. in the RPE. By contrast, there is no evidence, at least so far, which supports a role of Sox9 – the RPE Sox family member – in the regulation of the melanogenic cascade in the RPE. This may represent an additional diversification in the control of the melanogenic cascades between RPE and melanocytes.
References Beccari, L., Marco-Ferreres, R., and Bovolenta, P. (2013). The logic of gene regulatory networks in early vertebrate forebrain patterning. Mech. Dev. 130, 95–111. Bharti, K., Gasper, M., Ou, J., Brucato, M., Clore-Gronenborn, K., Pickel, J., and Arnheiter, H. (2012). A regulatory loop involving PAX6, MITF, and WNT signalling controls retinal pigment epithelium development. PLoS Genet. 8, e1002757. Housset, M., Samuel, A., Ettaiche, M., Bemelmans, A., Beby, F., Billon, N., and Lamonerie, T. (2013). Loss of Otx2 in the adult retina disrupts retinal pigment epithelium function, causing photoreceptor degeneration. J. Neurosci. 33, 9890–9904. Martinez-Morales, J.R., Rodrigo, I., and Bovolenta, P. (2004). Eye development: a view from the retina pigmented epithelium. BioEssays 26, 766–777. Masuda, T., Wahlin, K., Wan, J. et al. (2014). Transcription factor SOX9 plays a key role in the regulation of visual cycle gene expression in the retinal pigment epithelium. J. Biol. Chem. 289, 12908–12921.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Pigment Cell Melanoma Res.
New functions for old genes: Pax6 and Mitf in eye pigment biogenesis Florencia Cavodeassi and Paola Bovolenta e-mail: [email protected]
Melanin synthesis constitutes a fundamental aspect of the terminal differentiation program of pigment cells. In vertebrates, there are two major pigment cell types: melanocytes, derived from the neural crest and cells of the pigmented layer of the retina, known as retinal pigmented epithelium (RPE), which are generated from neuroepithelial cells of the optic primordium. Despite their distinct origin, these cells share a conserved battery of genes devoted to control pigment biogenesis, which, in turn, is activated by different isoforms of the microphthalmia transcription factor (MITF) (reviewed in Martinez-Morales et al., 2004). The RPE is essential for the homeostasis of the retina and failure in its development or physiology leads to severe retinal degeneration. RPE specification occurs at very early stages of embryonic development when the optic primordium becomes subdivided into optic stalk, neural retina, and RPE (Figure 1A). This regionalization occurs under the control of well-organized gene regulatory networks (Beccari et al., 2013), which, in the RPE, involve the activity of transcription factors of the Otx and Pax families, the positive input of the Wnt/ßcatenin pathway and the strong induction of Mitf (reviewed in Martinez-Morales et al., 2004). In particular, studies devoted to understand the role of Pax genes during optic vesicle specification have shown that Pax2 and Pax6 have redundant roles in optic vesicle regionalization: in the absence of both genes, Mitf expression fails to be activated and the presumptive RPE develops as neural retina-like tissue.
Coverage on: Raviv S, Bharti K, RencusLazar S, Cohen-Tayar Y, Schyr R, Evantal N, Meshorer N, Zilberberg A, Idelson M, Reubinoff B, Grebe R, Rosin-Arbesfeld R, Lauderdale J, Lutty G, Arnheiter H, and Ashery-Padan R. (2014) PAX6 regulates melanogenesis in the retinal pigmented epithelium through feed-forward regulatory interactions with MITF. Plos Genetics 10(5):e1004360. doi: 10. 1371/journal.pgen.1004360. doi: 10.1111/pcmr.12308
Likely, Pax6 has a more prominent role in this process because Pax2 is soon downregulated from the presumptive RPE, whereas Pax6 expression is maintained. Furthermore, loss of a single Pax6 allele in a Mitf null background is sufficient to enhance the acquisition of a neural retina fate by the RPE (Bharti et al., 2012), suggesting that Pax6 is a main regulator of RPE formation. Pax6 is considered as one of the eye ‘kernel’ genes, in other words as a gene that acts at the top of a gene regulatory network that imparts the fate of a given tissue or organ (Beccari et al., 2013). Indeed, Pax6 is expressed in the entire eye primordium and loss of its function leads to severe eye defects across evolution. Its expression, however, is not restricted to the earliest stages of eye formation and is thereafter recurrently required to promote neural retina and lens development, raising the question of how a single transcription factor controls the specification and differentiation of the different tissues in which it is expressed. A very recent study from the laboratory of Ruth Ashery-Padan provides an answer to this question, focusing on the yet unexplored role of Pax6 after the RPE is specified and its differentiation begins. The authors show that Pax6 is once again recruited during eye development, this time to activate a specific aspect of the terminal differentiation of the RPE: the formation of melanin pigment. This Pax6 function involves a well-orchestrated interaction with MITF, considered a master regulator of melanocyte formation. To reach this conclusion, the authors generated a Pax6 conditional mouse mutant, which specifically removes full-length Pax6 from the RPE but only after its specification (Pax6loxP/loxP; DctCre or Pax6RPE mutants). The RPE of the resulting mutants maintained its fate with typical monolayer morphology, normal levels of junctional proteins and expression of RPE markers such as Otx2 and Sox9 but presented a remarkable reduction in pigmentation. This suggested that, once established, Pax6 is no longer needed to maintain RPE specification but is required for promoting its melanogenic program. To verify this possibility, the authors com-
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
pared the transcriptional profile of Pax6-deficient and wild-type RPE cells. This analysis revealed an enrichment of melanogenic genes among those significantly downregulated in the mutant RPE. Notably, seven of the ten melanogenesis-related genes found downregulated in Pax6-RPE mutants were well-known direct targets of MITF. Nonetheless, of the three Mitf isoforms expressed in the RPE, only D-Mitf, which seems to contribute minimally to pigment regulation, was strongly downregulated, whereas the other two Mitf isoforms, A-Mitf and H-Mitf, were slightly increased, with a global 1.45-fold reduction of Mitf levels as compared to wild type. This modest change unlikely explained the loss of pigmentation seen in Pax6-RPE mutants. Thus, the authors reasoned that Pax6, besides controlling Mitf expression, was likely a direct regulator of the melanogenesis program. In keeping with this hypothesis, Raviv et al. (2014) first demonstrated that Pax6 synergizes with MITF to transactivate the D-Mitf promoter with a mechanism that depends upon binding of the paired domain of Pax6 to the DNA but involves no direct MITF-DNA interaction. Thereafter, they analyzed the transactivation activity of Pax6 on the promoter of three pigmentation genes (Tyrp1, Tyr, and Mlana). Pax6 by itself was unable to activate the transcription of any of these genes but, when coexpressed with MITF, had a strongly synergistic effect on the transactivation of both Tyrp1 and Tyr promoters. The authors went on to show that Pax6 and MITF physically interact with a paired domain independent mechanism and that this interaction is critical. Indeed, transactivation by the Pax6/MITF complex requires MITF but not Pax6 binding to the melanogenic loci. Intriguingly, the presence of the Pax6 paired domain, which can interact with specific DNA sequences, is necessary to assure
“Pax6 is no longer needed to maintain RPE specification but is required for promoting its melanogenic program.”
1
News and Views
A
B
C
Figure 1. (A) Schematic representation of the optic primordium. (B) Diagram of Pax6/ Mitf interactions controlling retinogenesis versus RPE formation. Blue lines indicate the interactions involved in retinogenesis, whereas red lines highlight those required for RPE differentiation. The thicker red line indicates a synergistic effect between Pax6 and Mitf in Mitf activation. (C) Cartoon highlighting the genetic network underlying melanogenesis in the RPE and the melanocytes. Note that a conserved network composed of Wnt/ßcatenin signalling and transcription factors of the Pax and Mitf families operates in both cell types. The additional participation of Otx2 in the RPE and Sox10 in the melanocytes as well as different co-transcriptional interactions diversify the melanogenic cascade in these two cell types (see text for further explanations).
efficiency although the authors did not explore why this is so. In summary, Raviv et al. (2014) uncover a new function for Pax6 in the RPE and show an interesting functional dependence between Pax6 and MITF: Pax6 induces the expression of melanogenic genes only in the presence of MITF, whereas MITF regulates the expression of its D isoform with a Pax6-dependent mechanism (Figure 1B). An interaction between Pax6 and MITF had been previously reported and proposed to be important for RPE specification. At early optic vesicle stages, the Pax6/MITF complex prevents the prospective RPE from acquiring a neural retina fate by suppressing the expression of retinogenic genes, which are otherwise activated when Pax6 acts in conjunction with other proretinal factors, including Fgfs and Wnt signalling inhibitors (Bharti et al., 2012). By directly controlling Mitf expression, Pax6 later on presets the conditions (expression of Mitf), which enable its function as a regulator of melanogenesis. Such a feed forward loop locks the RPE fate into a stable state, providing further evidence to explain how Pax6 promotes a contextspecific outcome in each of the tissues in
2
which it is expressed. This concept may have wider implications for the evolution of animal body plan. A similar network of genes, including Pax6, specifies visual structures across phyla. The most primitive of these structures likely required a small battery of genes. As eye complexity increased, so it did the underlying gene regulatory network. The co-option of genes from the original core network into more specialized functions seems thus to be a parsimonious solution to drive the differentiation of increasingly complex structures. Viewed from a different angle, the findings of Raviv et al. might also imply that the architecture of the gene regulatory networks that control embryonic development does not follow a strict hierarchy. Regulatory genes at the core of the network set up the developmental programs devoted to tissue specification, by regulating the expression of differentiation and effector genes. These, in turn, provide the architectural and functional features characteristic of each tissue (reviewed in Beccari et al., 2013). In the RPE, such a strict hierarchy seems to have been loosen up, because Pax6, which acts as an eye ‘kernel’ gene, is repurposed for a very specialized function, the control of melanogenesis during terminal tissue differentiation. Notably, in the Pax6-RPE mutants, defects are restricted to the lack of pigmentation, whereas other RPE features, such as the cuboidal epithelial morphology or the specialized intercellular junctions, are preserved, indicating that different genetic modules control different aspects of RPE differentiation and function. This independent regulation, often controlled by transcriptional complexes, may ensure robustness to the system, minimizing deleterious effects of gene mutations. In this respect, recent studies have shown that Otx2, besides controlling genes of the melanogenesis pathway (reviewed in MartinezMorales et al., 2004), activates retinol metabolism (Housset et al., 2013). Furthermore, Sox9 and Otx2 synergize to activate the expression of visual cycle genes (Masuda et al., 2014), whereas a complex formed by Sox9, Otx2, and MITF critically drives the RPE expression of the BEST1 gene, encoding a basolateral membrane protein associated with macular dystrophies. These specific functions of Otx2 and Sox9 in RPE cells together with the results of Raviv et al. bring in a different but connected issue: the use of similar sets of transcription factors in different tissues. In fact, these observations provide further support to the idea that the partial conservation of the melanogenic program in melanocytes and RPE cells
likely represent the co-option of an efficient gene regulatory network. This network might have evolved in the context of eye function and have been thereafter adapted in other cell types as the melanocytes, for example by recruiting different cofactors. Indeed, although the Wnt/ßcatenin pathway together with members of the Sox, Pax, and Mitf families all contribute to specification and differentiation of both RPE and melanocytes (Figure 1C), their functional relationships are largely different. Raviv et al. demonstrate that Pax6 teams up with MITF to control melanogenesis, whereas the cognate Pax3 has little control on the expression of melanogenic genes in neural crest-derived melanocytes. Rather, MITF synergize with both Sox10 and the Wnt/ßcatenin pathway effector LEF-1 to promote melanogenesis in this lineage. This synergism seems to require physical interaction between MITF and LEF-1/Sox10 and their cooperative binding to the promoter of melanogenic genes, in a mechanism comparable to that described by Raviv et al. in the RPE. By contrast, there is no evidence, at least so far, which supports a role of Sox9 – the RPE Sox family member – in the regulation of the melanogenic cascade in the RPE. This may represent an additional diversification in the control of the melanogenic cascades between RPE and melanocytes.
References Beccari, L., Marco-Ferreres, R., and Bovolenta, P. (2013). The logic of gene regulatory networks in early vertebrate forebrain patterning. Mech. Dev. 130, 95–111. Bharti, K., Gasper, M., Ou, J., Brucato, M., Clore-Gronenborn, K., Pickel, J., and Arnheiter, H. (2012). A regulatory loop involving PAX6, MITF, and WNT signalling controls retinal pigment epithelium development. PLoS Genet. 8, e1002757. Housset, M., Samuel, A., Ettaiche, M., Bemelmans, A., Beby, F., Billon, N., and Lamonerie, T. (2013). Loss of Otx2 in the adult retina disrupts retinal pigment epithelium function, causing photoreceptor degeneration. J. Neurosci. 33, 9890–9904. Martinez-Morales, J.R., Rodrigo, I., and Bovolenta, P. (2004). Eye development: a view from the retina pigmented epithelium. BioEssays 26, 766–777. Masuda, T., Wahlin, K., Wan, J. et al. (2014). Transcription factor SOX9 plays a key role in the regulation of visual cycle gene expression in the retinal pigment epithelium. J. Biol. Chem. 289, 12908–12921.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
