Abstract
Engrailed-2 (En-2), a homeodomain transcription factor, is expressed in a caudal-to-rostral gradient in the developing midbrain, where it has an instructive role in patterning the optic tectum—the target of topographic retinal input1,2. In addition to its well-known role in regulating gene expression through its DNA-binding domain, En-2 may also have a role in cell–cell communication, as suggested by the presence of other domains involved in nuclear export, secretion and internalization3. Consistent with this possibility, here we report that an external gradient of En-2 protein strongly repels growth cones of Xenopus axons originating from the temporal retina and, conversely, attracts nasal axons. Fluorescently tagged En-2 accumulates inside growth cones within minutes of exposure, and a mutant form of the protein that cannot enter cells fails to elicit axon turning. Once internalized, En-2 stimulates the rapid phosphorylation of proteins involved in translation initiation and triggers the local synthesis of new proteins. Furthermore, the turning responses of both nasal and temporal growth cones in the presence of En-2 are blocked by inhibitors of protein synthesis. The differential guidance of nasal and temporal axons reported here suggests that En-2 may participate directly in topographic map formation in the vertebrate visual system.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Retaux, S. & Harris, W. A. Engrailed and retinotectal topography. Trends Neurosci. 19, 542–546 (1996)
Itasaki, N. & Nakamura, H. A role for gradient en expression in positional specification on the optic tectum. Neuron 16, 55–62 (1996)
Prochiantz, A. & Joliot, A. Can transcription factors function as cell–cell signalling molecules? Nature Rev. Mol. Cell Biol. 4, 814–819 (2003)
Sperry, R. W. Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl Acad. Sci. USA 50, 703–710 (1963)
Cheng, H. J., Nakamoto, M., Bergemann, A. D. & Flanagan, J. G. Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cell 82, 371–381 (1995)
Drescher, U. et al. 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 (1995)
Shigetani, Y., Funahashi, J. I. & Nakamura, H. En-2 regulates the expression of the ligands for Eph type tyrosine kinases in chick embryonic tectum. Neurosci. Res. 27, 211–217 (1997)
Logan, C. et al. Rostral optic tectum acquires caudal characteristics following ectopic engrailed expression. Curr. Biol. 6, 1006–1014 (1996)
Feldheim, D. A. et al. Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping. Neuron 25, 563–574 (2000)
Frisen, J., Holmberg, J. & Barbacid, M. Ephrins and their Eph receptors: multitalented directors of embryonic development. EMBO J. 18, 5159–5165 (1999)
Lohof, A. M., Quillan, M., Dan, Y. & Poo, M. M. Asymmetric modulation of cytosolic cAMP activity induces growth cone turning. J. Neurosci. 12, 1253–1261 (1992)
de la Torre, J. R. et al. Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC. Neuron 19, 1211–1224 (1997)
Derossi, D., Joliot, A. H., Chassaing, G. & Prochiantz, A. The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269, 10444–10450 (1994)
Joliot, A. et al. Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein. Curr. Biol. 8, 856–863 (1998)
Topisirovic, I. et al. The proline-rich homeodomain protein, PRH, is a tissue-specific inhibitor of eIF4E-dependent cyclin D1 mRNA transport and growth. EMBO J. 22, 689–703 (2003)
Topisirovic, I. et al. Eukaryotic translation initiation factor 4E activity is modulated by HOXA9 at multiple levels. Mol. Cell. Biol. 25, 1100–1112 (2005)
Campbell, D. S. & Holt, C. E. Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron 32, 1013–1026 (2001)
Nedelec, S. et al. 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 (2004)
Foucher, I., Montesinos, M. L., Volovitch, M., Prochiantz, A. & Trembleau, A. Joint regulation of the MAP1B promoter by HNF3β/Foxa2 and Engrailed is the result of a highly conserved mechanism for direct interaction of homeoproteins and Fox transcription factors. Development 130, 1867–1876 (2003)
Hay, N. & Sonenberg, N. Upstream and downstream of mTOR. Genes Dev. 18, 1926–1945 (2004)
Gingras, A. C., Raught, B. & Sonenberg, N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68, 913–963 (1999)
Kablar, B. et al. Xotx genes in the developing brain of Xenopus laevis. Mech. Dev. 55, 145–158 (1996)
Friedman, G. C. & O'Leary, D. D. Retroviral misexpression of engrailed genes in the chick optic tectum perturbs the topographic targeting of retinal axons. J. Neurosci. 16, 5498–5509 (1996)
Charron, F. & Tessier-Lavigne, M. Novel brain wiring functions for classical morphogens: a role as graded positional cues in axon guidance. Development 132, 2251–2262 (2005)
Macdonald, R. et al. Regulatory gene expression boundaries demarcate sites of neuronal differentiation in the embryonic zebrafish forebrain. Neuron 13, 1039–1053 (1994)
Cohen, J., Burne, J. F., McKinlay, C. & Winter, J. The role of laminin and the laminin/fibronectin receptor complex in the outgrowth of retinal ganglion cell axons. Dev. Biol. 122, 407–418 (1987)
Montesinos, M. L. et al. The neuronal microtubule-associated protein 1B is under homeoprotein transcriptional control. J. Neurosci. 21, 3350–3359 (2001)
Acknowledgements
We thank all of our colleagues for discussions, and A. Dwivedy and E. Ipendey for technical assistance. This work was supported by the Wellcome Trust, UK (to C.H. and W.H.), the Human Frontier Sciences Program and the European Commission (to A.P.). Author Contributions I.B. did the turning assays and internalization experiments in Figs 1, 2, 3a and 3c, and in the Supplementary Figs. C.W. did the quantitative immunofluorescence work in Fig. 4, and trained I.B. in turning assays. M.P. did the 3H-leucine experiments in Fig. 3b. M.V. provided all the constructs and proteins. A.P. proposed the hypothesis that En-2 guides retinal axons. A.P. and A.T. initiated the collaboration and discussed experiments. W.H. and C.H. wrote the manuscript and discussed the experiments.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
Supplementary information
Supplementary Figures
Supplementary Figures 1–4 show that En-2 induces opposite turning behaviour in nasal and temporal axons in ‘blind’ experiments and does not alter their extension rate. Photomicrographs show growth cones severed from their cell bodies exhibit differential turning in an En-2 gradient. Otx2 only elicits weakly attractive turning responses. (DOC 526 kb)
Rights and permissions
About this article
Cite this article
Brunet, I., Weinl, C., Piper, M. et al. The transcription factor Engrailed-2 guides retinal axons. Nature 438, 94–98 (2005). https://doi.org/10.1038/nature04110
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nature04110
