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substantial support for the possibility that the LIP activity in question is associated with preparation for eye movement. The alternative interpretation offered by Janssen and Shadlen [4], “the intention-related signals seen in our experiments could underlie a shift in spatial attention that is not in competition with an eye movement plan” [4], is not obviously compelling. However, a related scenario in which these cells code for intentionality regarding a response, without being involved in a specific motor plan or tied to a specific motor affector, should also be considered. The likelihood that LIP neurons do not actually measure time, and that their temporally sensitive responding codes instead for eye movement, do not render these findings uninteresting to the field of time measurement. The parietal cortex is frequently activated in neuroimaging studies of timing — for example, see [8,12], but see also review [11] — and damage to this region [13], as well as temporary disruption via transcranial magnetic stimulation, have both been shown to cause temporal deficits. A number of authors have speculated about the role of parietal cortex in temporal processing, with some papers [8,14] suggesting an attentional function, while another[15] suggests a system for calculating magnitude. Until now, little has been known about how these involvements could be manifest at the neural level. The demonstration by Janssen and Shadlen [4] that individual LIP neurons can respond to visuotemporal information confirms that temporal information is available to the parietal cortex. These findings also provide novel and welcome insight into what this area may be doing in time measurement tasks, suggesting that the role of parietal cortex in multisensory integration and the planning of action extends to the modality of time. Thus, by exploring temporal processing in the parietal cortex with single unit recording, Shadlen’s group has taken a useful step towards describing the type of temporal

processes performed in that region. Their work is also novel because it illustrates the potential of single unit recording as a tool for discrimination between the different forms of temporal processing: perception, memory, preparation for movement, and so on. Similar work in other structures associated with timing may lead to even greater insights. Thus, Shadlen and colleagues may not have found the holy grail of timing research, but they have certainly discovered a treasure trove of information which will undoubtedly lead to a better understanding of this system. And really, its about time. References 1. Gibbon, J. (1977). Scalar expectancy theory and Weber’s law in animal timing. Psychol. Rev. 84, 279–325. 2. Staddon, J.E., and Higa, J.J. (1999). Time and memory: towards a pacemaker-free theory of interval timing. J. Exp. Anal. Behav. 71, 215–251. 3. Grossberg, S., and Schmajuk, N.A. (1989). Neural dynamics of adaptive timing and temporal discrimination during associative learning. Neural Netw. 2, 79–102. 4. Janssen, P., and Shadlen, M.N. (2005). A representation of the hazard rate of elapsed time in macaque area LIP. Nat. Neurosci. 8, 234–241. 5. Leon, M.I., and Shadlen, M.N. (2003). Representation of time by neurons in the posterior parietal cortex of the macaque. Neuron 38, 317–327. 6. Walsh, V. (2003). Time: the back-door of perception. Trends Cogn. Sci. 7, 335–338. 7. Ivry, R.B., and Spencer, R.M. (2004). The neural representation of time. Curr. Opin. Neurobiol. 14, 225–232.

8. Rao, S.M., Mayer, A.R., and Harrington, D.L. (2001). The evolution of brain activation during temporal processing. Nat. Neurosci. 4, 317–323. 9. Macar, F., Vidal, F., and Casini, L. (1999). The supplementary motor area in motor and sensory timing: evidence from slow brain potential changes. Exp. Brain Res. 125, 271–280. 10. Onoe, H., Komori, M., Onoe, K., Takechi, H., Tsukada, H., and Watanabe, Y. (2001). Networks recruited for time perception: a monkey positron emission tomography (PET) study. Neuroimage 13, 37–45. 11. Lewis, P.A., and Miall, R.C. (2003). Distinct systems for automatic and cognitively controlled time measurement: evidence from neuroimaging. Curr. Opin. Neurobiol. 13, 250–255. 12. Coull, J.T., Vidal, F., Nazarian, B., and Macar, F. (2004). Functional anatomy of the attentional modulation of time estimation. Science 303, 1506–1508. 13. Harrington, D.L., Haaland, K.Y., and Knight, R.T. (1998). Cortical networks underlying mechanisms of time perception. J. Neurosci. 18, 1085–1095. 14. Macar, F., Lejeune, H., Bonnet, M., Ferrara, A., Pouthas, V., Vidal, F., and Maquet, P. (2002). Activation of the supplementary motor area and of attentional networks during temporal processing. Exp. Brain Res. 142, 475–485. 15. Walsh, V. (2003). A theory of magnitude: common cortical metrics of time, space and quantity. Trends Cogn. Sci. 7, 483–488. 1Institute

of Science and Culture, 187 The Terrace, Wellington, New Zealand. E-mail: [email protected] 2Institute of Cognitive Neuroscience & Department of Psychology, University College London, 17 Queen Square, London WC1N 3AR, UK.

DOI: 10.1016/j.cub.2005.05.008

Axis Formation: Redundancy Rules The role of BMP antagonists in the Spemann-Mangold organizer of vertebrate embryos is a controversial issue. A study using combined knock down of multiple antagonists finally reveals dramatic effects. Christof Niehrs The Spemann-Mangold organizer of vertebrate embryos plays a paramount role during embryogenesis by releasing a cocktail of molecules that induce the embryonic axes and various cell fates. Bone morphogenetic protein (BMP) antagonists are an important class of such inducers, as was discovered in Xenopus, where their over-expression has dramatic effects, such as inducing

a secondary embryonic axis. By contrast, studies in higher vertebrates — such as chicken and mouse — have yielded less impressive results and have led to a controversy over how important BMP antagonists really are and what their precise role is. In a bold approach, Khoka et al. [1] have now knocked down in parallel three BMP antagonists in Xenopus embryos and observe dramatic effects on embryonic axis formation.

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BMP Antagonists and Embryonic Axis Formation The Spemann-Mangold organizer is of central importance for the establishment of the vertebrate body axes during early embryogenesis. In amphibian embryos, a grafted organizer — also known as the dorsal blastopore lip — induces secondary embryonic axes and thus results in a twinned embryo. Specifically, the organizer is thought to dorsalize ventral mesoderm, to induce neural cell fates within the ectoderm and to promote gastrulation movements [2,3]. It is now widely accepted that one main function of the organizer is to secrete molecules that antagonise three classes of growth factors — BMPs, Wnts and Nodals — which inhibit all or some organizer cell fates. Typically, these antagonists bind directly to the growth factors, thereby inactivating them and protecting the organizer from signaling molecules that would inhibit its function. The expression domains of these growth factors and their antagonists create gradients of signaling activity, which pattern the axes of the early embryo in a combinatorial fashion. Combinatorial signaling by these growth factor gradients accounts for regionally specific induction of different organizers, known as head, trunk and tail organizers [4]. BMPs are members of the TGFβ superfamily and they were the first growth factors whose negative effect on the Spemann organizer was discovered and for which antagonists were identified. Overexpression of BMP antagonists on the ventral side of Xenopus embryos recapitulates axis formation with the induction of a secondary embryonic axis or twinned embryo in amphibians, zebrafish and chick. Furthermore, in naïve Xenopus animal caps BMP inhibition leads to the induction of neural tissue. This led to the proposal of the ‘default model’ of neural induction according to which ectoderm forms neural tissue by default, unless it is exposed to BMPs, which cause the formation of epidermal tissue [5]. However, the

importance of BMP antagonists was questioned, as — in particular in chick embryos — they failed to induce neural tissue and only mildly expanded the neural plate. This is in contrast to the function of FGF, which readily induces neural tissue in chick and represents an evolutionarily conserved neural inducer [6]. Furthermore, mutants of BMP antagonists in zebrafish and mouse show only mild axial or head defects and thus failed to support a major role of BMP antagonists in axis formation or neural induction [3]. Functional Redundancy of BMP Antagonists The lack of major axial defects in mutants raises the possibility that BMP antagonists are only involved in fine tuning the patterning of neural and organizer tissue. However, another possibility is that there is functional redundancy among BMP antagonists, as there is a whole zoo of anti-BMPs expressed in the Spemann organizer — including noggin, chordin, follistatin, Xnr3 and cerberus. Recent studies from the Harland and De Robertis laboratories [1,7] indicate that the latter is true. Oelgeschläger et al. [7] have used sensitized assays in Xenopus to show that morpholino antisense oligonucleotide (MO) knockdown of chordin completely blocks dorsal mesoderm formation in activin treated animal caps as well as inhibiting neural induction by a transplanted organizer. Even more impressively, Khokha et al. [1] used MOs in Xenopus to generate a triple knock down of follistatin, chordin and noggin (FCN). These embryos fail to gastrulate, and show a dramatic loss of dorsal structures and neural tissue. By contrast, when only one or two of the antagonists are blocked, no or only mild effects are observed. In an elegant control experiment, Khoka et al. [1] showed that knockdown of BMP4 partially rescues the effect of the triple knockdown. These data clearly indicate a requirement for BMP antagonists in early axis formation. However, it is not clear

whether the failure in FCN embryos to form a neural plate is due to a direct role of BMP antagonists as neural inducers. An indirect effect cannot be ruled out because in FCN embryos dorsal mesoderm, which has the capacity to induce neural tissue, does not form. Moreover, convergent extension movements are inhibited in these embryos and therefore presumptive neural plate cells may remain stuck under the influence of cells that suppress neural fate. However, an elegant study of Kuroda et al. [8] supports a direct requirement for BMP antagonists in neural induction. In addition to being continuously expressed in the organizer, BMP antagonists are also transiently expressed outside the organizer and prior to its formation in the so-called ‘blastula chordin and noggin expressing center’. This region is fated to become neural tissue. Importantly, both chordin or noggin MOs block its ability to differentiate into neural tissue, suggesting a direct role for these BMP antagonists in neural induction. The studies by the Harland and De Robertis [1,7] groups strongly suggest that the negative or minor results with BMP antagonists obtained in other model organisms are due to lack of appropriate assays as well as to incomplete knockouts of BMP antagonists. Functional redundancy among BMP antagonists in mouse was already noted in the case of noggin and chordin [9]; double mutant mice lose head structures due to anterior gastrula defects, while in single mutants the anterior gastrula is normal. Triple mutants including additional BMP antagonists, e.g. cerberus, are of course painful to generate in mice, and an alternative may be to ask what happens to embryos in which all BMP signaling is blocked, e.g. by ectopic expression of BMP antagonists. Are such embryos neuralized? Do they form ectopic, supernumary organizers? What is the effect of the knockdown of multiple BMP antagonists in zebrafish and chick? From an evolutionary point

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of view, the redundancy of BMP antagonists raises the question of why Nature chose to have so many growth factor antagonists in vertebrates, while Drosophila can do without most of them. FGF is now recognized as an evolutionarily conserved neural inducer in ascidians, fish, Xenopus and chick [10,11]. However, one of its main functions is to block the BMP signaling cascade, as in embryos inhibited for FGF signaling, the BMP antagonist Noggin can rescue neural cell fate [12]. This effect of FGF may not only involve regulation of BMP gene expression but also direct inhibition of Smads, which mediate BMP signaling intracellularly. Activation of the FGF receptor activates MAP kinase to phosphorylate Smad1 in the linker region, thus inhibiting Smad1 transcriptional activity [13]. This again highlights the importance of blocking BMP signaling during neural induction.

However, in light of the crucial role of FGF as an instructive neural inducer, the ‘default model’ of neural induction appears to be a misnomer. References 1. Khokha, M.K., Yeh, J., Grammer, T.C., and Harland, R.M. (2005). Depletion of three BMP antagonists from Spemann’s organizer leads to a catastrophic loss of dorsal structures. Dev. Cell 8, 401–411. 2. Harland, R.M., and Gerhart, J. (1997). Formation and function of Spemann’s organizer. Annu. Rev. Cell Dev. Biol. 13, 611–667. 3. De Robertis, E.M., and Kuroda, H. (2004). Dorsal-ventral patterning and neural induction in Xenopus embryos. Annu. Rev. Cell Dev. Biol. 20, 285–308. 4. Niehrs, C. (2004). Regionally specific induction by the Spemann-Mangold organizer. Nat. Rev. Genet. 5, 425–434. 5. Weinstein, D.C., and Hemmati-Brivanlou, A. (1999). Neural induction. Annu. Rev. Cell Dev. Biol. 15, 411–433. 6. Stern, C.D. (2002). Induction and initial patterning of the nervous system - the chick embryo enters the scene. Curr. Opin. Genet. Dev. 12, 447–451. 7. Oelgeschläger, M., Kuroda, H., Reversade, B., and Robertis, E.M.D. (2003). Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos. Dev. Cell 4, 219–230. 8. Kuroda, H., Wessely, O., and De Robertis, E.M. (2004). Neural induction in

Xenopus: requirement for ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin, and Cerberus. PLoS Biol. 2, E92. 9. Bachiller, D., Klingensmith, J., Kemp, C., Belo, J.A., Anderson, R.M., May, S.R., McMahon, J.A., McMahon, A.P., Harland, R.M., Rossant, J., et al. (2000). The organizer factors Chordin and Noggin are required for mouse forebrain development. Nature 403, 658–661. 10. Wilson, S.I., and Edlund, T. (2001). Neural induction: toward a unifying mechanism. Nat. Neurosci. 4 (Suppl), 1161–1168. 11. Böttcher, R.T., and Niehrs, C. (2004). Fibroblast growth factor signalling during early vertebrate development. Endocr. Rev. 26, 63–77. 12. Wilson, S.I., Rydstrom, A., Trimborn, T., Willert, K., Nusse, R., Jessell, T.M., and Edlund, T. (2001). The status of Wnt signalling regulates neural and epidermal fates in the chick embryo. Nature 411, 325–330. 13. Pera, E.M., Ikeda, A., Eivers, E., and De Robertis, E.M. (2003). Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction. Genes Dev. 17, 3023–3028.

Division of Molecular Embryology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail: [email protected] DOI: 10.1016/j.cub.2005.05.002