- Email: [email protected]
Engrailed: Complexity and economy of a multi-functional transcription factor
FEBS Letters 580 (2006) 2531–2533
Minireview
Engrailed: Complexity and economy of a multi-functional transcription factor Richard Morgan* Postgraduate Medical School, University of Surrey, Guildford, Surrey GU2 7XH, UK Received 14 February 2006; revised 12 April 2006; accepted 17 April 2006 Available online 27 April 2006 Edited by Ivan Sadowski
Abstract Engrailed is a homeodomain-containing transcription factor with numerous, overlapping roles in neural development. Its multifunctional nature depends upon an extremely diverse group of molecular functions including the ability to regulate both transcription and translation, and to be released from and internalized by cells. Recent findings have shown how some of these functions relate to specific roles in development and disease. Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Engrailed; Transcription; Development; Neural; Cancer
1. Introduction Engrailed (En) was first characterized as a Drosophila mutation that results in a failure of the border that normally divides the posterior and anterior wing compartments, resulting in the homeotic transformation of the former into the latter [1]. The cloning and subsequent characterization of the Drosophila Engrailed gene revealed that it belongs to the ever expanding family of homeodomain-containing transcription factors that play key roles in development. Homologues of Engrailed are present in numerous animal groups including annelids [2], mollusks [3], insects [4], echinoderms [5], chordates [6], and vertebrates [7]. The latter have two Engrailed genes, En-1 and En-2, which are largely functionally equivalent [8,9], although En-1 has some specific functions during limb development.
2. Engrailed proteins have complex structures and are multifunctional Despite a very high degree of functional conservation, the En homologues have a relatively limited degree of sequence conservation at the protein level. The highest level of conservation is within the DNA-binding domain, where it averages 90% [10]. Comparing the sequences of En genes from different phyla * Address: Postgraduate Medical School, University of Surrey, Daphne Jackson Road, Guildford GU2 7WG, United Kingdom. Fax: +44 1483 688558. E-mail address: [email protected] (R. Morgan).
have revealed four further regions of similarity [10], and together these are termed En homology regions (EHs), and are designated numbers 1–5, where EH4 is the homeodomain (Fig. 1). EH1 and EH5 mediate transcriptional repression, the former by recruiting the co-repressor groucho [11], whilst EH2 and EH3 bind PBX, a second homeodomain-containing transcription factor that modifies the DNA binding affinity and specificity of En [12,13]. In addition to transcriptional regulation, En proteins have a number of other, apparently unrelated properties that are functionally important. The first of these is the ability to be secreted from cells, and to be internalized by others [14,15]. The exact mechanistic basis for this phenomenon is unknown, although it is clear that it depends upon a conserved nuclear export sequence located in the homeodomain [16] (Fig. 1). A proportion of cytoplasmic En protein associates with vesicles, an interaction also mediated by homeodomain sequences [17], and is subsequently secreted from the cell by an unconventional and as yet uncharacterized pathway [15]. The mechanism for the internalization of En is likewise unconventional, occurring both at 4 and 37 °C and not requiring a specific chiral receptor [18,19]. As with secretion, internalization is also dependant upon sequences within the homeodomain [20]. More recent advances have revealed that secretion is negatively regulated by the CK2 kinase that targets a serine rich site on the C-terminal end of the En protein [21]. Another noteworthy property of En protein is its ability to bind directly to the eukaryotic translation initiation factor 4E (eIF4E), through a sequence located on the N-terminal side [22] (Fig. 1). The high affinity and specificity of this interaction suggests that En may have a regulatory role in translation, as well as transcription.
3. En and neural development En has multiple regulatory roles at different stages of development, involving transcriptional and translational regulation, and secretion and internalization. The earliest of these roles is the determination of the midbrain/hindbrain (MH) border, an important organizing center during brain development. The alar (dorsal) cells of this region express high levels of both En-1 and En-2, and continue to do so even when transplanted to other areas of the developing brain that do not usually express En [23,24]. Furthermore, these cells then promote the expression of En in their new neighbors, which in turn take on the characteristics of the transplanted tissue. This inductive
0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.04.053
2532
R. Morgan / FEBS Letters 580 (2006) 2531–2533
Fig. 1. Functional domains within the En protein.
capacity is phylogenetically conserved as the equivalent embryonic tissue from mice has the same effect when transplanted into chick embryos [24,25]. Also conserved is at least one of the transcriptional targets that En-1 and En-2 regulate – the secreted peptide FGF8, which in turn can induce the expression of the En genes [26–29]. One of the earliest neuronal subtypes that arise from the MH is the mesencephalic dopaminergic (mesDA) neurons. Despite the large deletion of midbrain and hindbrain structures in En-1 / and En-2 / double mutants, mesDA neurons are still specified but undergo apoptosis shortly afterwards [30– 32]. Their continued survival in wild type embryos depends on En expression [30,32].
4. En and axonal guidance Axonal guidance is key to developing the neural network, and here too En plays an important role. Then guidance of axons depends upon the interaction between receptor proteins in the axon and neighboring cells. Many of these receptors and ligands have now been characterized, but the classic example is that of the EphrinA ligand and its receptor, EphA. EphrinA is expressed in a caudal (low) -to-rostral (high) gradient in the tectum, and repels retinal axons expressing high levels of EphA receptors [33,34]. These axons thus map to the rostral tectum and avoid the caudal tectum where EphrinA is most densely expressed. En-2 is expressed in a similar caudal-to-rostral gradient within the tectum, where it promotes EphrinA transcription [35,36] and thus has an important role in the establishment of the EphrinA gradient.
Intriguingly, recent results by Christine Holt’s and Alain Prochiantz’s groups have shown that an external gradient of En-2 protein can also act directly on axons after being internalized by these cells [37]. In this study, the authors show that En2 protein can repel cultured Xenopus axons originating from the temporal retina whilst attracting nasal axons (Fig. 2). In both cases it does not seem to be the transcriptional activity of En-2 that is important, but rather its ability to modify translation.
5. En in the adult Developmental genes often have adult functions, and frequently have a role in disease states. This is certainly true of the closely related HOX gene family [38], but relatively little is known about En function in the adult. Within the nervous system, the dopanergic neurons that depend on En-2 for survival during development continue to express high levels of En-2 in the adult, indicating that En-2 may continue to allow the survival of these cells [39]. In addition, En-2 is known to be expressed in a number of cancers, and more recently has been shown to be a potential oncogene in breast cancer [40]. In this study, non-tumorogenic murine mammary cell lines were forced to express En-2 and subsequently exhibited a number of malignant characteristics including a reduction in cell cycling time, a loss of cell to cell contact and a failure to differentiate in response to lactogenic hormones. In addition, RNAi interference studies suggested that En-2 expression is required for the maintenance of the transformed phenotype in a human breast cancer cell line.
6. Conclusions En is a transcription factor with exceptional functional complexity, as it not only modulates transcription, but also translation, and in addition seems to be capable of acting as a morphogen in its own right. It is this functional diversity that may underlie the diverse set of roles that En has during development. There are undoubtedly further functions of En yet to be elucidated, especially with regards to its expression in adult tissues and in cancer. References Fig. 2. An external gradient of En-2 protein repels Xenopus axons that originate in the temporal retina, but attracts nasal axons (after Ref. [37]). Adapted by permission from Macmillan Publishers Ltd. Nature 438, 94–98, copyright 2005.
[1] Garcia-Bellido, A. and Santamaria, P. (1972) Developmental analysis of the wing disc in the mutant Engrailed of Drosophila melanogaster. Genetics 72, 87–104.
R. Morgan / FEBS Letters 580 (2006) 2531–2533 [2] Wedeen, C.J. and Weisblat, D.A. (1991) Segmental expression of an Engrailed-class gene during early development and neurogenesis in an annelid. Development 113, 805–814. [3] Wanninger, A. and Haszprunar, G. (2001) The expression of an Engrailed protein during embryonic shell formation of the tuskshell, Antalis entalis (Mollusca, Scaphopoda). Evol. Dev. 3, 312– 321. [4] Fjose, A., McGinnis, W.J. and Gehring, W.J. (1985) Isolation of a homoeo box-containing gene from the Engrailed region of Drosophila and the spatial distribution of its transcripts. Nature 313, 284–289. [5] Lowe, C.J. and Wray, G.A. (1997) Radical alterations in the roles of homeobox genes during echinoderm evolution. Nature 389, 718–721. [6] Holland, L.Z., Kene, M., Williams, N.A. and Holland, N.D. (1997) Sequence and embryonic expression of the amphioxus Engrailed gene (AmphiEn): the metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila. Development 124, 1723–1732. [7] Joyner, A.L., Kornberg, T., Coleman, K.G., Cox, D.R. and Martin, G.R. (1985) Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila Engrailed gene. Cell 43, 29–37. [8] Hanks, M., Wurst, W., Anson-Cartwright, L., Auerbach, A.B. and Joyner, A.L. (1995) Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2. Science 269, 679–682. [9] Hanks, M.C., Loomis, C.A., Harris, E., Tong, C.X., AnsonCartwright, L., Auerbach, A. and Joyner, A. (1998) Drosophila engrailed can substitute for mouse Engrailed1 function in midhindbrain, but not limb development. Development 125, 4521– 4530. [10] Logan, C., Hanks, M.C., Noble-Topham, S., Nallainathan, D., Provart, N.J. and Joyner, A.L. (1992) Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions. Develop. Genet. 13, 345–358. [11] Tolkunova, E.N., Fujioka, M., Kobayashi, M., Deka, D. and Jaynes, J.B. (1998) Two distinct types of repression domain in engrailed: one interacts with the groucho corepressor and is preferentially active on integrated target genes. Mol. Cell Biol. 18, 2804–2814. [12] Peltenburg, L.T. and Murre, C. (1997) Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins. Development 124, 1089–1098. [13] Dijk, M.A. van and Murre, C. (1994) Extradenticle raises the DNA binding specificity of homeotic selector gene products. Cell 78, 617–624. [14] Cosgaya, J.M., Aranda, A., Cruces, J. and Martin-Blanco, E. (1998) Neuronal differentiation of PC12 cells induced by engrailed homeodomain is DNA-binding specific and independent of MAP kinases. J. Cell Sci. 111 (Pt 16), 2377–2384. [15] Joliot, A., Maizel, A., Rosenberg, D., Trembleau, A., Dupas, S., Volovitch, M. and Prochiantz, A. (1998) Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein. Curr. Biol. 8, 856–863. [16] Maizel, A., Bensaude, O., Prochiantz, A. and Joliot, A. (1999) A short region of its homeodomain is necessary for engrailed nuclear export and secretion. Development 126, 3183–3190. [17] Joliot, A., Trembleau, A., Raposo, G., Calvet, S., Volovitch, M. and Prochiantz, A. (1997) Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties. Development 124, 1865–1875. [18] Chatelin, L., Volovitch, M., Joliot, A.H., Perez, F. and Prochiantz, A. (1996) Transcription factor hoxa-5 is taken up by cells in culture and conveyed to their nuclei. Mech. Dev. 55, 111–117. [19] Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G. and Prochiantz, A. (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271, 18188–18193. [20] Derossi, D., Joliot, A.H., Chassaing, G. and Prochiantz, A. (1994) The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269, 10444–10450.
2533 [21] Maizel, A., Tassetto, M., Filhol, O., Cochet, C., Prochiantz, A. and Joliot, A. (2002) Engrailed homeoprotein secretion is a regulated process. Development 129, 3545–3553. [22] Nedelec, S., Foucher, I., Brunet, I., Bouillot, C., Prochiantz, A. and Trembleau, A. (2004) Emx2 homeodomain transcription factor interacts with eukaryotic translation initiation factor 4E (eIF4E) in the axons of olfactory sensory neurons. Proc. Natl. Acad. Sci. USA 101, 10815–10820. [23] Martinez, S. and Alvarado-Mallart, R.M. (1990) Expression of the homeobox Chick-en gene in chick/quail chimeras with inverted mes-metencephalic grafts. Develop. Biol. 139, 432– 436. [24] Martinez, S., Wassef, M. and Alvarado-Mallart, R.M. (1991) Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en. Neuron 6, 971–981. [25] Bally-Cuif, L., Alvarado-Mallart, R.M., Darnell, D.K. and Wassef, M. (1992) Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon. Development 115, 999–1009. [26] Liu, A. and Joyner, A.L. (2001) EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region. Development 128, 181–191. [27] Shamim, H., Mahmood, R., Logan, C., Doherty, P., Lumsden, A. and Mason, I. (1999) Sequential roles for Fgf4, En-1 and Fgf8 in specification and regionalisation of the midbrain. Development 126, 945–959. [28] Ristoratore, F., Carl, M., Deschet, K., Richard-Parpaillon, L., Boujard, D., Wittbrodt, J., Chourrout, D., Bourrat, F. and Joly, J.S. (1999) The midbrain–hindbrain boundary genetic cascade is activated ectopically in the diencephalon in response to the widespread expression of one of its components, the medaka gene Ol-eng2. Development 126, 3769–3779. [29] Gemel, J., Jacobsen, C. and MacArthur, C.A. (1999) Fibroblast growth factor-8 expression is regulated by intronic Engrailed and Pbx1-binding sites. J. Biol. Chem. 274, 6020–6026. [30] Simonn, H.H., Saueressig, H., Wurst, W., Goulding, M.D. and O’Leary, D.D. (2001) Fate of midbrain dopaminergic neurons controlled by the engrailed genes. J. Neurosci. 21, 3126–3134. [31] Simon, H.H., Bhatt, L., Gherbassi, D., Sgado, P. and Alberi, L. (2003) Midbrain dopaminergic neurons: determination of their developmental fate by transcription factors. Ann. N.Y. Acad. Sci. 991, 36–47. [32] Alberi, L., Sgado, P. and Simon, H.H. (2004) Engrailed genes are cell-autonomously required to prevent apoptosis in mesencephalic dopaminergic neurons. Development 131, 3229–3236. [33] Cheng, H.J., Nakamoto, M., Bergemann, A.D. and Flanagan, J.G. (1995) Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cell 82, 371–381. [34] Drescher, U., Kremoser, C., Handwerker, C., Loschinger, J., Noda, M. and Bonhoeffer, F. (1995) In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases. Cell 82, 359–370. [35] Shigetani, Y., Funahashi, J.I. and Nakamura, H. (1997) En-2 regulates the expression of the ligands for Eph type tyrosine kinases in chick embryonic tectum. Neurosci. Res. 27, 211–217. [36] Logan, C., Wizenmann, A., Drescher, U., Monschau, B., Bonhoeffer, F. and Lumsden, A. (1996) Rostral optic tectum acquires caudal characteristics following ectopic Engrailed expression. Curr. Biol. 6, 1006–1014. [37] Brunet, I., Weinl, C., Piper, M., Trembleau, A., Volovitch, M., Harris, W., Prochiantz, A. and Holt, C. (2005) The transcription factor Engrailed-2 guides retinal axons. Nature 438, 94–98. [38] Morgan, R. (2006) Hox genes: a continuation of embryonic patterning? Trends. Genet. 22, 67–69. [39] Bannon, M.J., Pruetz, B., Barfield, E. and Schmidt, C.J. (2004) Transcription factors specifying dopamine phenotype are decreased in cocaine users. Neuroreport 15, 401–404. [40] Martin, N.L., Saba-El-Leil, M.K., Sadekova, S., Meloche, S. and Sauvageau, G. (2005) EN-2 is a candidate oncogene in human breast cancer. Oncogene 24, 6890–6901.
Minireview
Engrailed: Complexity and economy of a multi-functional transcription factor Richard Morgan* Postgraduate Medical School, University of Surrey, Guildford, Surrey GU2 7XH, UK Received 14 February 2006; revised 12 April 2006; accepted 17 April 2006 Available online 27 April 2006 Edited by Ivan Sadowski
Abstract Engrailed is a homeodomain-containing transcription factor with numerous, overlapping roles in neural development. Its multifunctional nature depends upon an extremely diverse group of molecular functions including the ability to regulate both transcription and translation, and to be released from and internalized by cells. Recent findings have shown how some of these functions relate to specific roles in development and disease. Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Engrailed; Transcription; Development; Neural; Cancer
1. Introduction Engrailed (En) was first characterized as a Drosophila mutation that results in a failure of the border that normally divides the posterior and anterior wing compartments, resulting in the homeotic transformation of the former into the latter [1]. The cloning and subsequent characterization of the Drosophila Engrailed gene revealed that it belongs to the ever expanding family of homeodomain-containing transcription factors that play key roles in development. Homologues of Engrailed are present in numerous animal groups including annelids [2], mollusks [3], insects [4], echinoderms [5], chordates [6], and vertebrates [7]. The latter have two Engrailed genes, En-1 and En-2, which are largely functionally equivalent [8,9], although En-1 has some specific functions during limb development.
2. Engrailed proteins have complex structures and are multifunctional Despite a very high degree of functional conservation, the En homologues have a relatively limited degree of sequence conservation at the protein level. The highest level of conservation is within the DNA-binding domain, where it averages 90% [10]. Comparing the sequences of En genes from different phyla * Address: Postgraduate Medical School, University of Surrey, Daphne Jackson Road, Guildford GU2 7WG, United Kingdom. Fax: +44 1483 688558. E-mail address: [email protected] (R. Morgan).
have revealed four further regions of similarity [10], and together these are termed En homology regions (EHs), and are designated numbers 1–5, where EH4 is the homeodomain (Fig. 1). EH1 and EH5 mediate transcriptional repression, the former by recruiting the co-repressor groucho [11], whilst EH2 and EH3 bind PBX, a second homeodomain-containing transcription factor that modifies the DNA binding affinity and specificity of En [12,13]. In addition to transcriptional regulation, En proteins have a number of other, apparently unrelated properties that are functionally important. The first of these is the ability to be secreted from cells, and to be internalized by others [14,15]. The exact mechanistic basis for this phenomenon is unknown, although it is clear that it depends upon a conserved nuclear export sequence located in the homeodomain [16] (Fig. 1). A proportion of cytoplasmic En protein associates with vesicles, an interaction also mediated by homeodomain sequences [17], and is subsequently secreted from the cell by an unconventional and as yet uncharacterized pathway [15]. The mechanism for the internalization of En is likewise unconventional, occurring both at 4 and 37 °C and not requiring a specific chiral receptor [18,19]. As with secretion, internalization is also dependant upon sequences within the homeodomain [20]. More recent advances have revealed that secretion is negatively regulated by the CK2 kinase that targets a serine rich site on the C-terminal end of the En protein [21]. Another noteworthy property of En protein is its ability to bind directly to the eukaryotic translation initiation factor 4E (eIF4E), through a sequence located on the N-terminal side [22] (Fig. 1). The high affinity and specificity of this interaction suggests that En may have a regulatory role in translation, as well as transcription.
3. En and neural development En has multiple regulatory roles at different stages of development, involving transcriptional and translational regulation, and secretion and internalization. The earliest of these roles is the determination of the midbrain/hindbrain (MH) border, an important organizing center during brain development. The alar (dorsal) cells of this region express high levels of both En-1 and En-2, and continue to do so even when transplanted to other areas of the developing brain that do not usually express En [23,24]. Furthermore, these cells then promote the expression of En in their new neighbors, which in turn take on the characteristics of the transplanted tissue. This inductive
0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.04.053
2532
R. Morgan / FEBS Letters 580 (2006) 2531–2533
Fig. 1. Functional domains within the En protein.
capacity is phylogenetically conserved as the equivalent embryonic tissue from mice has the same effect when transplanted into chick embryos [24,25]. Also conserved is at least one of the transcriptional targets that En-1 and En-2 regulate – the secreted peptide FGF8, which in turn can induce the expression of the En genes [26–29]. One of the earliest neuronal subtypes that arise from the MH is the mesencephalic dopaminergic (mesDA) neurons. Despite the large deletion of midbrain and hindbrain structures in En-1 / and En-2 / double mutants, mesDA neurons are still specified but undergo apoptosis shortly afterwards [30– 32]. Their continued survival in wild type embryos depends on En expression [30,32].
4. En and axonal guidance Axonal guidance is key to developing the neural network, and here too En plays an important role. Then guidance of axons depends upon the interaction between receptor proteins in the axon and neighboring cells. Many of these receptors and ligands have now been characterized, but the classic example is that of the EphrinA ligand and its receptor, EphA. EphrinA is expressed in a caudal (low) -to-rostral (high) gradient in the tectum, and repels retinal axons expressing high levels of EphA receptors [33,34]. These axons thus map to the rostral tectum and avoid the caudal tectum where EphrinA is most densely expressed. En-2 is expressed in a similar caudal-to-rostral gradient within the tectum, where it promotes EphrinA transcription [35,36] and thus has an important role in the establishment of the EphrinA gradient.
Intriguingly, recent results by Christine Holt’s and Alain Prochiantz’s groups have shown that an external gradient of En-2 protein can also act directly on axons after being internalized by these cells [37]. In this study, the authors show that En2 protein can repel cultured Xenopus axons originating from the temporal retina whilst attracting nasal axons (Fig. 2). In both cases it does not seem to be the transcriptional activity of En-2 that is important, but rather its ability to modify translation.
5. En in the adult Developmental genes often have adult functions, and frequently have a role in disease states. This is certainly true of the closely related HOX gene family [38], but relatively little is known about En function in the adult. Within the nervous system, the dopanergic neurons that depend on En-2 for survival during development continue to express high levels of En-2 in the adult, indicating that En-2 may continue to allow the survival of these cells [39]. In addition, En-2 is known to be expressed in a number of cancers, and more recently has been shown to be a potential oncogene in breast cancer [40]. In this study, non-tumorogenic murine mammary cell lines were forced to express En-2 and subsequently exhibited a number of malignant characteristics including a reduction in cell cycling time, a loss of cell to cell contact and a failure to differentiate in response to lactogenic hormones. In addition, RNAi interference studies suggested that En-2 expression is required for the maintenance of the transformed phenotype in a human breast cancer cell line.
6. Conclusions En is a transcription factor with exceptional functional complexity, as it not only modulates transcription, but also translation, and in addition seems to be capable of acting as a morphogen in its own right. It is this functional diversity that may underlie the diverse set of roles that En has during development. There are undoubtedly further functions of En yet to be elucidated, especially with regards to its expression in adult tissues and in cancer. References Fig. 2. An external gradient of En-2 protein repels Xenopus axons that originate in the temporal retina, but attracts nasal axons (after Ref. [37]). Adapted by permission from Macmillan Publishers Ltd. Nature 438, 94–98, copyright 2005.
[1] Garcia-Bellido, A. and Santamaria, P. (1972) Developmental analysis of the wing disc in the mutant Engrailed of Drosophila melanogaster. Genetics 72, 87–104.
R. Morgan / FEBS Letters 580 (2006) 2531–2533 [2] Wedeen, C.J. and Weisblat, D.A. (1991) Segmental expression of an Engrailed-class gene during early development and neurogenesis in an annelid. Development 113, 805–814. [3] Wanninger, A. and Haszprunar, G. (2001) The expression of an Engrailed protein during embryonic shell formation of the tuskshell, Antalis entalis (Mollusca, Scaphopoda). Evol. Dev. 3, 312– 321. [4] Fjose, A., McGinnis, W.J. and Gehring, W.J. (1985) Isolation of a homoeo box-containing gene from the Engrailed region of Drosophila and the spatial distribution of its transcripts. Nature 313, 284–289. [5] Lowe, C.J. and Wray, G.A. (1997) Radical alterations in the roles of homeobox genes during echinoderm evolution. Nature 389, 718–721. [6] Holland, L.Z., Kene, M., Williams, N.A. and Holland, N.D. (1997) Sequence and embryonic expression of the amphioxus Engrailed gene (AmphiEn): the metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila. Development 124, 1723–1732. [7] Joyner, A.L., Kornberg, T., Coleman, K.G., Cox, D.R. and Martin, G.R. (1985) Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila Engrailed gene. Cell 43, 29–37. [8] Hanks, M., Wurst, W., Anson-Cartwright, L., Auerbach, A.B. and Joyner, A.L. (1995) Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2. Science 269, 679–682. [9] Hanks, M.C., Loomis, C.A., Harris, E., Tong, C.X., AnsonCartwright, L., Auerbach, A. and Joyner, A. (1998) Drosophila engrailed can substitute for mouse Engrailed1 function in midhindbrain, but not limb development. Development 125, 4521– 4530. [10] Logan, C., Hanks, M.C., Noble-Topham, S., Nallainathan, D., Provart, N.J. and Joyner, A.L. (1992) Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions. Develop. Genet. 13, 345–358. [11] Tolkunova, E.N., Fujioka, M., Kobayashi, M., Deka, D. and Jaynes, J.B. (1998) Two distinct types of repression domain in engrailed: one interacts with the groucho corepressor and is preferentially active on integrated target genes. Mol. Cell Biol. 18, 2804–2814. [12] Peltenburg, L.T. and Murre, C. (1997) Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins. Development 124, 1089–1098. [13] Dijk, M.A. van and Murre, C. (1994) Extradenticle raises the DNA binding specificity of homeotic selector gene products. Cell 78, 617–624. [14] Cosgaya, J.M., Aranda, A., Cruces, J. and Martin-Blanco, E. (1998) Neuronal differentiation of PC12 cells induced by engrailed homeodomain is DNA-binding specific and independent of MAP kinases. J. Cell Sci. 111 (Pt 16), 2377–2384. [15] Joliot, A., Maizel, A., Rosenberg, D., Trembleau, A., Dupas, S., Volovitch, M. and Prochiantz, A. (1998) Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein. Curr. Biol. 8, 856–863. [16] Maizel, A., Bensaude, O., Prochiantz, A. and Joliot, A. (1999) A short region of its homeodomain is necessary for engrailed nuclear export and secretion. Development 126, 3183–3190. [17] Joliot, A., Trembleau, A., Raposo, G., Calvet, S., Volovitch, M. and Prochiantz, A. (1997) Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties. Development 124, 1865–1875. [18] Chatelin, L., Volovitch, M., Joliot, A.H., Perez, F. and Prochiantz, A. (1996) Transcription factor hoxa-5 is taken up by cells in culture and conveyed to their nuclei. Mech. Dev. 55, 111–117. [19] Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G. and Prochiantz, A. (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271, 18188–18193. [20] Derossi, D., Joliot, A.H., Chassaing, G. and Prochiantz, A. (1994) The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269, 10444–10450.
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