News and Views
Taking striping up a notch Makoto Goda, Robert N. Kelsh and Hisashi Hashimoto email: [email protected]
Color patterns in animal skins are of the utmost importance for the survival and breeding success of many species. Consequently, understanding the molecular basis for pigment pattern formation has been a priority within the pigmentation field. The reaction-diffusion system, which was originally proposed by A. Turing in 1952, is one of the most studied theoretical models capable of generating spatially periodic patterns. Kondo and Asai provided experimental evidence for the existence of a Turing-type mechanism underlying the skin pattern changes of growing marine angelfish, Pomacanthus imperator (Kondo and Miura, 2010). A combination of short- and long-range signals is integral to Turing-type mechanisms, but the same mathematics underlies a whole variety of biochemical and cellular mechanisms (Kelsh, 2004), making identification of the cellular and molecular players in periodic patterning mechanisms crucial. For these purposes, major progress has been made since employing the zebrafish (Danio rerio), allowing investigation of both the molecular genetics and cell biology of adult pigment pattern formation. Adult zebrafish have a longitudinally striped pattern, and mutants with perturbed stripe formation provide sources for identifying key genes involved in pigment pattern formation. Genetic studies to date have not produced compelling evidence for a diffusible substance driving a reactiondiffusion system, although the identification of mutations in connexin41.8, a component of gap junctions, underlying the spotted leopard phenotype gave some hope (Kondo and Miura, 2010). In contrast, the inwardly rectifying potassium channel 7.1 (Kir7.1) gene is responsible for the wider melanophore stripes in jaguar/obelix mutants (Kondo and Miura, 2010). The same group then
Coverage on: Hamada H, Watanabe M, Lau HE, Nishida T, Hasegawa T, Parichy DM, Kondo S. (2014) Involvement of Delta/Notch signaling in zebrafish adult pigment stripe patterning. Development 141(2):318–24. . doi: 10.1111/pcmr.12288
688
showed using live cell imaging techniques that direct contact of xanthophores and melanophores induced membrane depolarization and repulsive migration of melanophores in a Kir7.1dependent manner (Inaba et al., 2012), suggesting involvement of Kir7.1 in a short-range signal. Laser ablation of pigment cells in vivo identified a crucial role for a cellular interaction network between melanophores and xanthophores (Kondo and Miura, 2010), suggesting that interactions at both short and long ranges act as a substitute for diffusing mor“goes a long phogens proposed in the way toward original Turing convincingly model. identifying the In this context, molecular the study by mechanism of Hamada et al. long-range provides further signaling as strong evidence involving Delta– for a cell–cell Notch interaction netsignaling.” work underlying zebrafish stripe formation and maintenance and furthermore To identify candidate mediators between xanthophores and melanophores, they carried out microarray analysis and searched for genes expressed more abundantly in xanthophores than in melanophores and which encoded candidate signaling molecules. For functional assay of these candidates, they took a transgenic approach, expressing them ectopically in melanophores under an mitf promoter. They found a proportion of adult transgenic founder (transient or mosaic) fish exhibited altered pattern only when carrying deltaC, which encodes a membranebound ligand of Notch signaling. Their RT-PCR analysis further highlighted Delta ligand expression in xanthophores and Notch receptor in melanophores, leading to the suggestion that Delta– Notch signals mediate xanthophore– melanophore interactions. Hamada et al. next aimed to test whether melanophore survival depends on the Delta–Notch signal. To block the signal, they used DAPT, an inhibitor of proteolytic cleavage of the Notch
intracellular domain (NICD) fragment of the Notch receptor. In Delta–Notch signaling, a membrane-bound Delta ligand on one cell binds to the transmembrane Notch receptor on a neighboring cell, which in turn induces cleavage and release of the NICD fragment. DAPT treatment reduced the number of melanophores in the melanophore stripes, whereas it did not affect the number of xanthophores, implying loss of Notch signaling results in melanophore death. To test this possibility, they generated stable transgenic lines in which Notch signaling was stimulated or constitutively activated in melanophores. In Tg(mitfa: deltaC) fish, melanophores express both DeltaC as well as the endogenous Notch, so that a Notch signal is expected to be exchanged among melanophores independent of xanthophores. The authors show that the stripes of melanophores in this transgenic fish are wider than those of wild type, consistent with enhanced melanophore survival due to local Notch signal activation within the stripes. As exposure of this fish to DAPT reduces the width of the melanophore stripes and the number of melanophores, the effect of ectopic Notch expression in melanophores depends on signal transduction through cleavage of Notch. In Tg(mitfa:NICD1a), Notch signaling pathway should be constitutively and autonomously activated in melanophores, and as predicted this results in wider melanophore stripes. However, in contrast with those of Tg (mitfa: deltaC) fish, the melanophores of Tg(mitfa: NICD1a) fish are resistant to DAPT. The results shown by Hamada et al. strongly indicate that xanthophores promote melanophore survival through a Delta–Notch signaling mechanism, but it is still unclear why melanophores only partially undergo cell death although all melanophores were exposed to DAPT treatment and presumably were deprived of Notch signaling. A similar phenomenon was observed in zebrafish csf1ra mutant, which has no xanthophores and decreased melanophores. These findings consistently suggest that only a proportion of melanophores
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
News and Views depends on xanthophores and thus on Delta–Notch signal for survival. Finally, the authors demonstrate elegantly that stripe melanophores are in distant contact with xanthophores. They demonstrate by two distinct methods that long, fine processes project from melanophores toward xanthophores. These presumed intercellular connections provide a cellular mechanism for transmission of a survival signal directly from xanthophores to melanophores. In conclusion, Hamada et al. have shown that xanthophores are a distant (long range) source of Delta–Notch signal for melanophore survival, although the authors also speculate that involvement of the Delta–Notch signaling mediated by xanthophores is only a part of a complex mechanism for melanophore survival. Together with previous work, this study argues for the intriguing conclusion that the underlying basis for the Turing-type mechanism of zebrafish pigment pattern formation does not require physical diffusion of molecules, but instead results from direct cell–cell contacts mediating both short- and longrange signaling. Direct contact resulting in cell depolarization acts as a shortrange signal; Kir7.1 is likely involved in this. Similarly, long-range signaling is
Delta–Notch dependent; long projections (cytonemes; Muller et al., 2013) of melanophores allow them to sense Delta expression in distant xanthophores. Further studies are desired to test whether Notch receptor proteins are localized at the tip of projection of melanophores and whether Delta ligand binding to Notch receptors occurs at the contact sites between melanophores and xanthophores. One final consideration concerns the recent discovery of a role in pigment pattern formation for the third pigment cell type in zebrafish, iridophores. Two recent studies have shown that three mutants, rose, shady, and transparent, lacking iridophores have altered pigment patterns on the skin of the trunk, strongly suggesting a role for this third pigment cell type in adult pigment pattern formation (Frohnhofer et al., 2013). However, it is noteworthy that whereas mutants such as panther, leopard, and jaguar perturb the stripe pattern in both of the trunk and fins, in the iridophoreless mutants, stripe formation is not affected in the anal and tail fins. Thus, stripe formation in both the trunk and the fins likely shares an underlying mechanism, but that in the trunk iridophores are additionally involved. Thus,
further consideration needs to be given to how iridophores interact with melanophores and xanthophores if we are to fully understand how the zebrafish gets its stripes.
References Frohnhofer, H.G., Krauss, J., Maischein, H.M., and Nusslein-Volhard, C. (2013). Iridophores and their interactions with other chromatophores are required for stripe formation in zebrafish. Development 140, 2997–3007. Inaba, M., Yamanaka, H., and Kondo, S. (2012). Pigment pattern formation by contact-dependent depolarization. Science 335, 677. Kelsh, R.N. (2004). Genetics and evolution of pigment patterns in fish. Pigment Cell Res. 17, 326–336. Kondo, S., and Miura, T. (2010). Reactiondiffusion model as a framework for understanding biological pattern formation. Science 329, 1616–1620. Muller, P., Rogers, K.W., Yu, S.R., Brand, M., and Schier, A.F. (2013). Morphogen transport. Development 140, 1621– 1638.
Ctr1-ing BRAF signaling with copper Stuart G. Jarrett and John A. D’Orazio e-mail: [email protected]
The V600E gain-of-function mutation in the BRAF serine/threonine kinase is a driver mutation in roughly 50% of human melanomas, in nearly all hairy cell leukemias and in certain subsets of colorectal, ovarian, lung, breast, and papillary thyroid cancers. BRAFV600E is generated as a result of a 1799T>A missense change in exon 15 of the BRAF gene, causing valine to be replaced by a phosphomimetic moiety (glutamic acid) at the 600th residue of the mature protein. This amino acid substitution
Coverage on: Brady, D.C., et al. (2014). Copper is required for oncogenic BRAF signaling and tumorigenesis. Nature, doi:10.1038/nature13180.
endows BRAFV600E with exaggerated and unregulated signaling, resulting in continuous activation of the MAPK signaling cascade on which melanogenesis seems to depend. Normally positioned downstream of G-proteins in the RAS family, BRAF promotes MEK1/2 activation which in turn leads to ERK1/2 signaling and induction of transcription factors and other cellular mediators that promote cell growth and survival. In their recent Nature paper, Donita Brady and Christopher Counter of Duke University and their colleagues report a novel insight into how aberrant BRAF signaling drives melanoma. They found that BRAFV600E-driven melanomas require adequate cellular transport of copper through copper transporter 1 (Ctr1), the major copper transporter in the extracellular membrane (Ohrvik and
Thiele, 2014), for sustained oncogenesis. Furthermore, they provide evidence in a mouse model that systemic copper chelation interferes with BRAFV600Emediated oncogenesis, introducing an unexpected and potentially feasible therapeutic approach targeting BRAFV600Edriven cancers (Figure 1). Building on their prior discovery that copper interacts with MEK1 to enhance phosphorylation of ERK1/2 (Turski et al., 2012), Brady et al. reported that interfering with MEK1–copper interactions decreased BRAFV600E-mediated MAPK signaling and reduced tumor aggressiveness in melanomas driven by the BRAFV600E mutation. They found that BRAFV600E-mediated ERK phosphorylation and cell growth were blunted in the absence of Ctr1 expression, rescuable only when copper transport-proficient
doi: 10.1111/pcmr.12265
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
689
Taking striping up a notch Makoto Goda, Robert N. Kelsh and Hisashi Hashimoto email: [email protected]
Color patterns in animal skins are of the utmost importance for the survival and breeding success of many species. Consequently, understanding the molecular basis for pigment pattern formation has been a priority within the pigmentation field. The reaction-diffusion system, which was originally proposed by A. Turing in 1952, is one of the most studied theoretical models capable of generating spatially periodic patterns. Kondo and Asai provided experimental evidence for the existence of a Turing-type mechanism underlying the skin pattern changes of growing marine angelfish, Pomacanthus imperator (Kondo and Miura, 2010). A combination of short- and long-range signals is integral to Turing-type mechanisms, but the same mathematics underlies a whole variety of biochemical and cellular mechanisms (Kelsh, 2004), making identification of the cellular and molecular players in periodic patterning mechanisms crucial. For these purposes, major progress has been made since employing the zebrafish (Danio rerio), allowing investigation of both the molecular genetics and cell biology of adult pigment pattern formation. Adult zebrafish have a longitudinally striped pattern, and mutants with perturbed stripe formation provide sources for identifying key genes involved in pigment pattern formation. Genetic studies to date have not produced compelling evidence for a diffusible substance driving a reactiondiffusion system, although the identification of mutations in connexin41.8, a component of gap junctions, underlying the spotted leopard phenotype gave some hope (Kondo and Miura, 2010). In contrast, the inwardly rectifying potassium channel 7.1 (Kir7.1) gene is responsible for the wider melanophore stripes in jaguar/obelix mutants (Kondo and Miura, 2010). The same group then
Coverage on: Hamada H, Watanabe M, Lau HE, Nishida T, Hasegawa T, Parichy DM, Kondo S. (2014) Involvement of Delta/Notch signaling in zebrafish adult pigment stripe patterning. Development 141(2):318–24. . doi: 10.1111/pcmr.12288
688
showed using live cell imaging techniques that direct contact of xanthophores and melanophores induced membrane depolarization and repulsive migration of melanophores in a Kir7.1dependent manner (Inaba et al., 2012), suggesting involvement of Kir7.1 in a short-range signal. Laser ablation of pigment cells in vivo identified a crucial role for a cellular interaction network between melanophores and xanthophores (Kondo and Miura, 2010), suggesting that interactions at both short and long ranges act as a substitute for diffusing mor“goes a long phogens proposed in the way toward original Turing convincingly model. identifying the In this context, molecular the study by mechanism of Hamada et al. long-range provides further signaling as strong evidence involving Delta– for a cell–cell Notch interaction netsignaling.” work underlying zebrafish stripe formation and maintenance and furthermore To identify candidate mediators between xanthophores and melanophores, they carried out microarray analysis and searched for genes expressed more abundantly in xanthophores than in melanophores and which encoded candidate signaling molecules. For functional assay of these candidates, they took a transgenic approach, expressing them ectopically in melanophores under an mitf promoter. They found a proportion of adult transgenic founder (transient or mosaic) fish exhibited altered pattern only when carrying deltaC, which encodes a membranebound ligand of Notch signaling. Their RT-PCR analysis further highlighted Delta ligand expression in xanthophores and Notch receptor in melanophores, leading to the suggestion that Delta– Notch signals mediate xanthophore– melanophore interactions. Hamada et al. next aimed to test whether melanophore survival depends on the Delta–Notch signal. To block the signal, they used DAPT, an inhibitor of proteolytic cleavage of the Notch
intracellular domain (NICD) fragment of the Notch receptor. In Delta–Notch signaling, a membrane-bound Delta ligand on one cell binds to the transmembrane Notch receptor on a neighboring cell, which in turn induces cleavage and release of the NICD fragment. DAPT treatment reduced the number of melanophores in the melanophore stripes, whereas it did not affect the number of xanthophores, implying loss of Notch signaling results in melanophore death. To test this possibility, they generated stable transgenic lines in which Notch signaling was stimulated or constitutively activated in melanophores. In Tg(mitfa: deltaC) fish, melanophores express both DeltaC as well as the endogenous Notch, so that a Notch signal is expected to be exchanged among melanophores independent of xanthophores. The authors show that the stripes of melanophores in this transgenic fish are wider than those of wild type, consistent with enhanced melanophore survival due to local Notch signal activation within the stripes. As exposure of this fish to DAPT reduces the width of the melanophore stripes and the number of melanophores, the effect of ectopic Notch expression in melanophores depends on signal transduction through cleavage of Notch. In Tg(mitfa:NICD1a), Notch signaling pathway should be constitutively and autonomously activated in melanophores, and as predicted this results in wider melanophore stripes. However, in contrast with those of Tg (mitfa: deltaC) fish, the melanophores of Tg(mitfa: NICD1a) fish are resistant to DAPT. The results shown by Hamada et al. strongly indicate that xanthophores promote melanophore survival through a Delta–Notch signaling mechanism, but it is still unclear why melanophores only partially undergo cell death although all melanophores were exposed to DAPT treatment and presumably were deprived of Notch signaling. A similar phenomenon was observed in zebrafish csf1ra mutant, which has no xanthophores and decreased melanophores. These findings consistently suggest that only a proportion of melanophores
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
News and Views depends on xanthophores and thus on Delta–Notch signal for survival. Finally, the authors demonstrate elegantly that stripe melanophores are in distant contact with xanthophores. They demonstrate by two distinct methods that long, fine processes project from melanophores toward xanthophores. These presumed intercellular connections provide a cellular mechanism for transmission of a survival signal directly from xanthophores to melanophores. In conclusion, Hamada et al. have shown that xanthophores are a distant (long range) source of Delta–Notch signal for melanophore survival, although the authors also speculate that involvement of the Delta–Notch signaling mediated by xanthophores is only a part of a complex mechanism for melanophore survival. Together with previous work, this study argues for the intriguing conclusion that the underlying basis for the Turing-type mechanism of zebrafish pigment pattern formation does not require physical diffusion of molecules, but instead results from direct cell–cell contacts mediating both short- and longrange signaling. Direct contact resulting in cell depolarization acts as a shortrange signal; Kir7.1 is likely involved in this. Similarly, long-range signaling is
Delta–Notch dependent; long projections (cytonemes; Muller et al., 2013) of melanophores allow them to sense Delta expression in distant xanthophores. Further studies are desired to test whether Notch receptor proteins are localized at the tip of projection of melanophores and whether Delta ligand binding to Notch receptors occurs at the contact sites between melanophores and xanthophores. One final consideration concerns the recent discovery of a role in pigment pattern formation for the third pigment cell type in zebrafish, iridophores. Two recent studies have shown that three mutants, rose, shady, and transparent, lacking iridophores have altered pigment patterns on the skin of the trunk, strongly suggesting a role for this third pigment cell type in adult pigment pattern formation (Frohnhofer et al., 2013). However, it is noteworthy that whereas mutants such as panther, leopard, and jaguar perturb the stripe pattern in both of the trunk and fins, in the iridophoreless mutants, stripe formation is not affected in the anal and tail fins. Thus, stripe formation in both the trunk and the fins likely shares an underlying mechanism, but that in the trunk iridophores are additionally involved. Thus,
further consideration needs to be given to how iridophores interact with melanophores and xanthophores if we are to fully understand how the zebrafish gets its stripes.
References Frohnhofer, H.G., Krauss, J., Maischein, H.M., and Nusslein-Volhard, C. (2013). Iridophores and their interactions with other chromatophores are required for stripe formation in zebrafish. Development 140, 2997–3007. Inaba, M., Yamanaka, H., and Kondo, S. (2012). Pigment pattern formation by contact-dependent depolarization. Science 335, 677. Kelsh, R.N. (2004). Genetics and evolution of pigment patterns in fish. Pigment Cell Res. 17, 326–336. Kondo, S., and Miura, T. (2010). Reactiondiffusion model as a framework for understanding biological pattern formation. Science 329, 1616–1620. Muller, P., Rogers, K.W., Yu, S.R., Brand, M., and Schier, A.F. (2013). Morphogen transport. Development 140, 1621– 1638.
Ctr1-ing BRAF signaling with copper Stuart G. Jarrett and John A. D’Orazio e-mail: [email protected]
The V600E gain-of-function mutation in the BRAF serine/threonine kinase is a driver mutation in roughly 50% of human melanomas, in nearly all hairy cell leukemias and in certain subsets of colorectal, ovarian, lung, breast, and papillary thyroid cancers. BRAFV600E is generated as a result of a 1799T>A missense change in exon 15 of the BRAF gene, causing valine to be replaced by a phosphomimetic moiety (glutamic acid) at the 600th residue of the mature protein. This amino acid substitution
Coverage on: Brady, D.C., et al. (2014). Copper is required for oncogenic BRAF signaling and tumorigenesis. Nature, doi:10.1038/nature13180.
endows BRAFV600E with exaggerated and unregulated signaling, resulting in continuous activation of the MAPK signaling cascade on which melanogenesis seems to depend. Normally positioned downstream of G-proteins in the RAS family, BRAF promotes MEK1/2 activation which in turn leads to ERK1/2 signaling and induction of transcription factors and other cellular mediators that promote cell growth and survival. In their recent Nature paper, Donita Brady and Christopher Counter of Duke University and their colleagues report a novel insight into how aberrant BRAF signaling drives melanoma. They found that BRAFV600E-driven melanomas require adequate cellular transport of copper through copper transporter 1 (Ctr1), the major copper transporter in the extracellular membrane (Ohrvik and
Thiele, 2014), for sustained oncogenesis. Furthermore, they provide evidence in a mouse model that systemic copper chelation interferes with BRAFV600Emediated oncogenesis, introducing an unexpected and potentially feasible therapeutic approach targeting BRAFV600Edriven cancers (Figure 1). Building on their prior discovery that copper interacts with MEK1 to enhance phosphorylation of ERK1/2 (Turski et al., 2012), Brady et al. reported that interfering with MEK1–copper interactions decreased BRAFV600E-mediated MAPK signaling and reduced tumor aggressiveness in melanomas driven by the BRAFV600E mutation. They found that BRAFV600E-mediated ERK phosphorylation and cell growth were blunted in the absence of Ctr1 expression, rescuable only when copper transport-proficient
doi: 10.1111/pcmr.12265
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
689
