Current Biology Vol 15 No 1 R14 Dispatches Notochord Morphogenesis: A Prickly Subject for Ascidians Orchestrated cell movements marshalled by proper cell polarity in the developing body axes are fundamental to the elongation of the notochord during chordate embryogenesis. A recent study shows that, in ascidians, the planar cell polarity gene prickle regulates sequential establishment of cell polarity during two phases of notochord morphogenesis. Masazumi Tada How cell polarity is established and propagated within a large population of cells is a key issue to understanding the coordinated and directional tissue movements that shape the body axes of animal embryos. One such process is convergent extension, best described in the presumptive notochord of the Xenopus embryo, by which medio-laterally aligned cells converge on the dorsal midline and intercalate between one another, narrowing the dorsal tissue medially and simultaneously extending it along the anterior–posterior axis of the developing body [1]. This fundamental process appears to be conserved in other chordates, including zebrafish and ascidians [2,3]. Recently, a molecular picture of the pathways involved in the regulation of convergent extension has emerged. Components of the so-called planar cell polarity pathway, homologs of which are known to play an essential role in the establishment of cell polarity within the plane of epithelial tissues in Drosophila, have been found to be key regulators of this critical process [4]. Indeed, the vertebrate planar cell polarity pathway has also been implicated in the regulation of a variety of other coordinated morphogenetic movements, such as neural tube closure, cochlear hair cell orientation and neuronal migration [5,6]. In ascidian embryos, a mere 40 notochord cells are specified before convergent extension takes place by rearranging a four by ten sheet of cells into a forty by one stack of cells, thereby elongating the notochord along the anterior–posterior axis. Later, individual notochord cells extend along their anterior–posterior axis as the tail elongates. Cellular mechanisms underlying convergent extension in ascidians are similar to those in Xenopus in that medio-lateral cell intercalation is thought to be a cellautonomous driving force of this process [3]. As reported in this issue of Current Biology, by examining this beautifully simple system, Jiang and colleagues [7] have identified the aimless locus, which encodes a homolog of the core planar cell polarity protein Prickle, as a key regulator of notochord morphogenesis in the ascidian Ciona savignyi. Notochord cells are specified normally in larvae homozygous for the aimless mutation, but fail to undergo the normal movements of convergent extension. A shorter and thicker notochord is generated, reminiscent of those found in mutant zebrafish or Xenopus embryos defective for planar cell polarity components such as Prickle Strabismus or Dishevelled [5,6,8]. This aimless mutant phenotype is reminiscent of that seen in Ciona embryos expressing a dominant-negative form of Dishevelled that specifically disrupts its function in planar cell polarity [9]. The defective convergent extension phenotype is consistent with expression of prickle in the notochord. In aimless/prickle embryos, the notochord cell behavior that underlies medio-lateral intercalation is disrupted, such that the orientation of lamellipodia-like outgrowth is randomised rather than having a medio-lateral biased orientation as in wild-type embryos. How do planar cell polarity proteins establish and maintain cell polarity with respect to the axis of the developing embryo? This is best understood in the case of the Drosophila wing epithelium. Initially, all the components are colocalised symmetrically at the cell membrane. The establishment of cell polarity crucially involves feedback amplification in which proximally localised Prickle and Strabismus suppress Frizzled and Dishevelled on the proximal side, thereby facilitating the accumulation of Frizzled and Dishevelled on the distal side (Figure 1A) [4,10]. This localised Frizzled/Dishevelled activity leads to distal outgrowth of hairs. In vertebrates, however, there is no consensus as to where planar cell polarity proteins are localised with respect to the axis of alignment of polarised cells. In Xenopus dorsal marginal explants undergoing convergent extension, Dishevelled is variously reported as localised to the membrane uniformly [11] or in a medio-lateral biased manner [12]. Colocalisation or differential localisation of Dishevelled and other planar cell polarity components has not been yet reported in cells undergoing cell intercalations, though Dishevelled and Prickle colocalise to the membrane in the presence of Fz7 in static Xenopus animal pole cells [13,14], while Prickle inhibits Fz7-mediated membrane localisation of Dishevelled in static zebrafish animal pole cells [15]. In ascidian notochord cells actively undergoing convergent Dispatch R15 extension, Dishevelled colocalises with Prickle to the membrane uniformly without any bias along the medio-lateral axis (Figure 1B). This membrane localisation of Dishevelled is dependent on Prickle activity, as Dishevelled is no longer localised to the membrane in the aimless/prickle mutant. It will be intriguing to see if the ability of Prickle to localise to the membrane is also dependent on Dishevelled, as an interdependency between Prickle and Dishevelled is observed in the initial localisation of planar cell polarity proteins in establishing cell polarity in the Drosophila wing [10]. Interestingly, both Prickle and Dishevelled disappear from the cell membranes in contact with the notochord–somite boundary (Figure 1B). This asymmetric localisation of the two proteins at the notochord–somite boundary suggests the presence of a cue that influences the establishment of cell polarity within the notochord, consistent with the observations of Munro and Odell [3]. What molecular mechanism might underlie this event? One obvious candidate is the secreted signaling molecule Wnt5, one of the so-called non-canonical Wnts. Wnt5 is secreted by the adjacent muscle cells in Ciona [16] and might help establish cell polarity in notochord cells. This would be consistent with the defective convergent extension phenotype of pipetail/wnt5 mutant zebrafish [8]. Another candidate, possibly expressed in the somites but not in the notochord and implicated in convergent extension, is paraxial protocadherin (papc). In embryos with compromised Papc activity, convergence is impaired but extension in the notochord is relatively normal, and Papc appears to act in part together with the Fz7-mediated planar cell polarity signal by activating the RhoA small GTPase [17]. Importantly, protocadherins such as Dachsous and Fat are also implicated in propagation of the Frizzled-mediated signal in Drosophila planar cell polarity [4]. A P Pk D Dsh Stbm Fz B A P Somites L M WT L Somites Somites aim Somites C P A Somites WT Somites Somites aim Somites Current Biology Figure 1. Sub-cellular localisation of planar cell polarity proteins during establishment of polarity in the Drosophila wing and ascidian notochord. (A) During establishment of planar cell polarity in the Drosophila wing, Prickle (Pk) and Strabismus (Stbm) are localised to the proximal side of the cell, while Frizzled (Fz) and Dishevelled (Dsh) are to the distal side. (B) During notochord morphogenesis in ascidians, Prickle and Dishevelled co-localise uniformly at the cell membranes, apart from at the notochord–somite boundary in wild-type embryos. Dishevelled is no longer localised to the cell membranes in aimless/prickle mutant embryos, which consequently develop a shorter and thicker notochord. (C) As the nuclei position posteriorly within the extending notochord cells, Prickle and Strabismus colocalise to the anterior edge of the cells, whereas Dishevelled is laterally localised to the notochord–somite boundary in wild embryos. Note that the localisation of Dishevelled and Strabismus in aimless/prickle embryos, in which the position of nuclei is randomised, is not reported. Following convergent extension, individual notochord cells elongate along the anterior–posterior axis and Prickle and Strabismus become localised to the anterior edge of cells, while Dishevelled is found laterally at the notochord–somite boundary (Figure 1C). Correlated with the anterior localisation of Prickle, nuclei within the notochord become localised to the posterior edge of the cells. In aimless/prickle mutant embryos, the position of nuclei in notochord cells is randomised with respect to their anterior–posterior axis, suggesting that Prickle might establish or maintain anterior–posterior polarity in the later aspects of notochord morphogenesis. The biological significance of this process is yet to be clarified, but it will be interesting to examine the position of nuclei, as well as the anterior localisation of Prickle, during this stage when Dishevelled activity is compromised. One planar cell Current Biology Vol 15 No 1 R16 polarity-dependent process occurring during zebrafish gastrulation is directional cell division aligned along the anterior–posterior axis, which contributes in part to the elongation of the body axis [18]. William Smith’s group [19] has previously reported that the chobi mutant Ciona embryos show a phenotype reminiscent of aimless/prickle mutant embryos. Further identification of Ciona mutants that exhibit a shorter tail phenotype with properly differentiated notochord could uncover novel members of the planar cell polarity pathway, as has been done for gastrulation defects in zebrafish and for neural tube defects in mice. Considering that the extent of genome redundancy in ascidians is similar to that in Drosophila with respect to planar cell polarity genes [20], this approach might be more successful in these species than in other vertebrates. 4. 5. 6. 7. 8. 9. 10. 11. References 1. Keller, R., Davidson, L., Edlund, A., Elul, T., Ezin, M., Shook, D., and Skoglund, P. (2000). Mechanisms of convergence and extension by cell intercalation. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355, 897–922. 2. Warga, R.M., and Kimmel, C.B. (1990). Cell movements during epiboly and gastrulation in zebrafish. Development 108, 569–580. 3. Munro, E.M., and Odell, G.M. (2002). Polarized basolateral cell motility 12. 13. underlies invagination and convergent extension of the ascidian notochord. Development 129, 13–24. Strutt, D. (2003). Frizzled signalling and cell polarisation in Drosophila and vertebrates. Development 130, 4501–4513. Wallingford, J.B., Fraser, S.E., and Harland, R.M. (2002). Convergent extension: the molecular control of polarized cell movement during embryonic development. Dev. Cell 2, 695–706. Veeman, M.T., Axelrod, J.D., and Moon, R.T. (2003). A second canon. Functions and mechanisms of beta-cateninindependent Wnt signaling. Dev. Cell 5, 367–377. Jiang, D., Munro, E.M., and Smith, W.C. (2005). Ascidian prickle regulates both mediolateral and anterior-posterior cell polarity of notchord cells. Curr. Biol. 15, this issue. Heisenberg, C.P., and Tada, M. (2002). Zebrafish gastrulation movements: bridging cell and developmental biology. Semin. Cell Dev. Biol. 13, 471–479. Keys, D.N., Levine, M., Harland, R.M., and Wallingford, J.B. (2002). Control of intercalation is cell-autonomous in the notochord of Ciona intestinalis. Dev. Biol. 246, 329–340. Tree, D.R., Shulman, J.M., Rousset, R., Scott, M.P., Gubb, D., and Axelrod, J.D. (2002). Prickle mediates feedback amplification to generate asymmetric planar cell polarity signaling. Cell 109, 371–381. Wallingford, J.B., Rowning, B.A., Vogeli, K.M., Rothbacher, U., Fraser, S.E., and Harland, R.M. (2000). Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405, 81–85. Kinoshita, N., Iioka, H., Miyakoshi, A., and Ueno, N. (2003). PKC delta is essential for Dishevelled function in a noncanonical Wnt pathway that regulates Xenopus convergent extension movements. Genes Dev. 17, 1663–1676. Veeman, M.T., Slusarski, D.C., Kaykas, A., Louie, S.H., and Moon, R.T. (2003). Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr. Biol. 13, 680–685. Circadian Biology: Fibroblast Clocks Keep Ticking Real-time cellular imaging of gene expression has revealed that fibroblasts contain a robust, self-sustained and cell-autonomous circadian oscillator, with a range of properties that both overlap and contrast with those of the neural clock of the suprachiasmatic nuclei. Michael H. Hastings Circadian clocks enable organisms to define biological day and night, synchronising daily rhythms of metabolism and behaviour to the demands and opportunities of the world [1]. Hence, clocks confer selective advantage and are hard-wired into our make-up. Our most obvious circadian rhythm is that of sleep and wakefulness, but they range from mucosal cell division through hormonal profiles to susceptibility to cardiac arrest. The circadian mantra used to be easy; “there is but one true clock and it sits in the brain”. By using real-time cellular imaging, two papers [2,3] have revealed a new truth:, “clocks are all over the body and all are equal, but some are more equal than others”. After early skepticism that circadian rhythms are artefactual, 14. 15. 16. 17. 18. 19. 20. Takeuchi, M., Nakabayashi, J., Sakaguchi, T., Yamamoto, T.S., Takahashi, H., Takeda, H., and Ueno, N. (2003). The prickle-related gene in vertebrates is essential for gastrulation cell movements. Curr. Biol. 13, 674–679. Carreira-Barbosa, F., Concha, M.L., Takeuchi, M., Ueno, N., Wilson, S.W., and Tada, M. (2003). Prickle1 regulates cell movements during gastrulation and neuronal migration in zebrafish. Development 130, 4037–4046. Imai, K.S., Hino, K., Yagi, K., Satoh, N., and Satou, Y. (2004). Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks. Development 131, 4047–4058. Unterseher, F., Hefele, J.A., Giehl, K., De Robertis, E.M., Wedlich, D., and Schambony, A. (2004). Paraxial protocadherin coordinates cell polarity during convergent extension via Rho A and JNK. EMBO J. 23, 3259–3269. Gong, Y., Mo, C., and Fraser, S.E. (2004). Planar cell polarity signalling controls cell division orientation during zebrafish gastrulation. Nature 430, 689–693. Nakatani, Y., Moody, R., and Smith, W.C. (1999). Mutations affecting tail and notochord development in the ascidian Ciona savignyi. Development 126, 3293–3301. Hotta, K., Takahashi, H., Ueno, N., and Gojobori, T. (2003). A genome-wide survey of the genes for planar polarity signaling or convergent extensionrelated genes in Ciona intestinalis and phylogenetic comparisons of evolutionary conserved signaling components. Gene 317, 165–185. Department of Anatomy and Developmental Biology, University College London, Gower St, London WC1E 6BT, UK. E-mail: m.tada@ucl.ac.uk DOI: 10.1016/j.cub.2005.36.25 the field was boosted in the 1980s with the identification of the hypothalamic suprachiasmatic nuclei (SCN) as the body clock controlling the sleep/wake and endocrine cycles [4]. Further respectability came in the nineties with the discovery of genes encoding the SCN clockwork [5]. Driven by complexes of the transcription factors CLOCK and BMAL, the ‘clock genes’ Period and Cryptochrome encode transcriptional inhibitors that oppose CLOCK/BMAL activity, thereby closing a negative feedback loop that oscillates with an approximately daily period. Electrophysiological recordings of dispersed cultures revealed that the clockwork is cell-autonomous, its activity within single SCN neurons indicating that there must