Identification of a Rudimentary Neural Crest in a Non-Vertebrate Chordate

LETTER doi:10.1038/nature11589

Identification of a rudimentary neural crest in a non-vertebrate chordate

Philip Barron Abitua1, Eileen Wagner1, Ignacio A. Navarrete1 & Michael Levine1

Neural crest arises at the neural plate border, expresses a core set as a positional cue to trigger differentiation of the posterior a10.97 of regulatory genes and produces a diverse array of cell types, melanocyte. including ectomesenchyme derivatives that elaborate the verte- Wnt signalling was selectively perturbed in the a9.49 lineage by brate head1,2. The evolution of neural crest has been proposed to the targeted misexpression of Wnt7, stable b-catenin and dominant- be a key event leading to the appearance of new cell types that negative Tcf (DTcf, which lacks the b-catenin amino-terminal binding fostered the transition from filter feeding to active predation in domain) using the Mitf enhancer (Fig. 1c–f). A bc-crystallin reporter ancestral vertebrates3. However, the origin of neural crest remains was used to distinguish the melanocytes, as it is expressed in the otolith controversial, as homologous cell types have not been unambigu- but not the ocellus (Fig. 1c). Both pigmented precursors were converted ously identified in non-vertebrate chordates1,4. Here we show that the tunicate Ciona intestinalis possesses a cephalic melanocyte Wnt7 ISH lineage (a9.49) similar to neural crest that can be reprogrammed a a9.38 a9.34 a9.50 Mitf into migrating ‘ectomesenchyme’ by the targeted misexpression of IV a9.37 Twist (also known as twist-like 2). Our results suggest that the III a9.33 a9.49 neural crest melanocyte regulatory network pre-dated the diver- b9.38 A9.14 gence of tunicates and vertebrates. We propose that the co-option II A9.16 A9.30 of mesenchyme determinants, such as Twist, into the neural plate b9.37 A9.13 A9.15 ectoderm was crucial to the emergence of the vertebrate ‘new I A9.29 head’3. A9.32

Whole-genome phylogenetic analyses place the tunicates as the true ZicL Msx ZicL, Msx, Mitf, Tyr and Tyrp1 b 5 sister clade to vertebrates , and consequently they are well suited for Mitf>lacZ Mitf>Wnt7 investigating the evolutionary origins of the neural crest. In a previous βγ-Cry>GFP βγ-Cry>GFP report on the mangrove tunicate, a migratory cell population origin- ating from the vicinity of the neural tube was likened to the neural crest6. However, subsequent studies of eleven additional tunicates pro- vided unequivocal evidence that these cells arise from the mesoderm cd flanking the neural tube7. It was then suggested that a mesoderm- Mitf>Stable β-catenin Mitf>ΔTcf derived mesenchyme lineage (A7.6) in Ciona possessed some of the βγ-Cry>GFP βγ-Cry>GFP properties of the neural crest8, although these cells do not arise from the neural plate border and lack expression of key neural crest regulatory genes. We present evidence that the bilateral a9.49 pigment cell lineage of Ciona embryos represents a rudimentary neural crest. It arises at the ef Zicl>Ets–VP16, Mitf>lacZ Zicl>Ets–VP16, Mitf>Wnt7 neural plate border and expresses several neural plate border genes, as βγ-Cry>GFP βγ-Cry>GFP well as a number of neural crest specification genes, including Id, Snail, Ets and FoxD8–13 (Fig. 1a and Supplementary Fig. 1). In vertebrates, MITF (microphthalmia-associated transcription factor) directly activates several target genes required for melanogenesis of neural- crest-derived melanocytes, including TYR and TYRP1 (ref. 14). In g h tunicates, Mitf is expressed in the a9.49 lineage15, which can be labelled Figure 1 | Wnt signalling promotes ocellus formation. a, Schematic of . by electroporation of a reporter, Mitf lacZ (Mitf regulatory sequence neural plate at gastrulation, indicating the lineage-specific expression of driving the expression of lacZ) (Fig. 1b). The posterior daughters of the enhancers in this study. b, Tailbud embryo electroporated with Mitf.lacZ, lineage (a10.97) intercalate at the dorsal midline and form the gravity- detected with an antibody (green) and hybridized with a Wnt7 probe (red). ISH, sensing otolith and melanocyte of the light-detecting ocellus (Fig. 1c)16. in situ hybridization. c–f, Larvae electroporated with bc-crystallin.GFP (bc- We sought to understand the basis for the differential specification of cry.GFP) marks the otolith and anterior palps. c, Larva co-electroporated with these pigmented cells. Mitf.lacZ (166 of 196 had an otolith and ocellus). d, Larva co-electroporated Wnt signalling has a conserved role in neural crest induction, and with Mitf.Wnt7 (172 of 205 had two ocelli). e, Larva co-electroporated with . b promotes melanocyte formation from cephalic neural crest in zebra- Mitf stable -catenin (189 of 205 had two ocelli). f, Larva co-electroporated 17 with Mitf.DTcf (dominant-negative Tcf) (100 of 205 had two otoliths). fish . Both a10.97 cells express Tcf/Lef, the transcriptional effector of g, h, Larvae electroporated with Zicl.Ets–VP16 and bc-crystallin.GFP. 13 Wnt signalling , thus Wnt may also have a role in Ciona melanogenesis. g, Larva co-electroporated with Mitf.lacZ (75 of 100 had extra otoliths). We found that Wnt7 is expressed along the dorsal midline just posterior h, Larva co-electroporated with Mitf.Wnt7 (only 11 of 100 had extra otoliths). to the presumptive ocellus (Fig. 1b), suggesting that it might serve Scale bars, 50 mm.

1Center for Integrative Genomics, Division of Genetics, Genomics and Development, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

104 | NATURE | VOL 492 | 6 DECEMBER 2012 ©2012 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH to ocelli after misexpression of Wnt7 in the a9.49 lineage (Wnt7 is not expression in the presumptive ocellus (Fig. 2c), and is dependent on normally expressed in this lineage) (Fig. 1d). A similar transforma- Wnt signalling, as expression is lost in the presence of DTcf (Fig. 2d). tion was observed after targeted expression of a stabilized form of To investigate the role of FoxD in melanogenesis, we expressed b-catenin,thecoactivatorofTcf (Fig. 1e). In contrast, misexpression variants of FoxD in the midline of the CNS, including the a9.49 line- of a dominant-negative form of Tcf (Mitf.DTcf) produced the recipro- age, using 59 regulatory sequences from the Msx gene (Fig. 1a and cal transformation: both a10.97 melanocytes differentiated into otoliths Supplementary Fig. 3). Targeted expression of either full-length and expressed the bc-crystallin reporter (Fig. 1f). FoxD or the N-terminal third of FoxD (non-DNA binding) abolished Supernumerary otoliths were induced by the expression of a con- expression of the Mitf.GFP reporter gene (Fig. 2e, f and Supplemen- stitutively active form of the transcription factor for MAPK signalling, tary Fig. 3). However, misexpression of a constitutive repressor form of Ets1/2 (also known as Ets/pointed2) (Fig. 1g and Supplementary Fig. 2). FoxD (DNA-binding domain fused to a WRPW repressor motif) had Co-electroporation of Mitf.Wnt7 transformed these extra otoliths into little effect on Mitf expression (Supplementary Fig. 3). These results ocelli (Fig. 1h). These results suggest that Wnt7 signalling is crucial in suggest that FoxD represses Mitf independent of its DNA-binding transforming otoliths to ocelli; it specifies the ocellus and suppresses the domain, which is consistent with its mode of regulation in avian development of the otolith. To determine the underlying mechanism we embryos, in which Foxd3-mediated repression of Mitf is thought to sought to identify neural crest specification genes that are selectively occur through the sequestration of the transcriptional activator Pax3 activated in the presumptive ocellus in response to Wnt7 signalling. (ref. 18). In vertebrates, Foxd3 has been shown to repress melanogenesis of Our results suggest a simple gene regulatory network (Wnt7 signal- neural crest cells through downregulation of Mitf14,18.InCiona, FoxD ling activates FoxD, which inhibits Mitf expression) for the differential is activated directly by the accumulation of nuclear b-catenin in the specification of the otolith and ocellus in the Ciona tadpole (Sup- early embryo, indicating a potential link between Wnt signalling and plementary Fig. 4). Both a10.97 cells express Mitf before neurulation FoxD expression19. We found that FoxD is selectively expressed in the and during the convergence of the two cells along the dorsal midline of presumptive ocellus, (Fig. 2a, b) adjacent to the site of Wnt7 expression the anterior neural tube. Subsequently, the posterior a10.97 cell re- in the dorsal midline (Fig. 1b). A FoxD enhancer recapitulates this ceives a localized Wnt7 signal and activates FoxD, which attenuates Mitf leading to diminished pigmentation in the ocellus. Mitf expression is sustained in the anterior a10.97 cell, which forms the densely pigmented otolith. a Ot Oc b a/a10.97 Notably, zebrafish uses a very similar mechanism to specify neural crest-derived pigmented melanophores and iridophores, which derive a/a10.98

FoxD ISH Tyrp1 FoxD ISH Tyrp1 c d Twist> GFP H2B Twist ISH cd

Oc Ot

Mitf>lacZ Mitf>ΔTcf Mitf>lacZ Mitf>Twist FoxD>mCherry FoxD>mCherry Mitf>GFP H2B Mitf>GFP H2B Mitf>GFP Mitf>GFP ef ef

Msx>lacZ Msx>FoxD N terminus Msx>H2B Msx>H2B Mitf>GFP Mitf>GFP Mitf>lacZ Mitf>Twist Tyr>mCherry H2B Tyr>mCherry H2B Figure 2 | FoxD represses Mitf in the ocellus. a, Tailbud embryo electroporated with Tyrp1.lacZ detected with an antibody (green) marking the Figure 3 | Twist reprograms the a9.49 lineage. a, Neurula hybridized with a precursors of the otolith and ocellus, and hybridized with a FoxD probe (red). Twist probe. b, Tailbud embryo during mesenchyme migration (arrows) co- b, FoxD is expressed in the posterior a10.97 cell. c, d, Tailbud embryos electroporated with Twist.GFP and Twist.H2B mCherry. c, d, Larvae electroporated with FoxD.mCherry and Mitf.GFP. Arrowheads mark the electroporated with Mitf.GFP and Mitf.H2B mCherry. Insets show lineage presumptive ocellus. c, Co-electroporated with Mitf.lacZ (126 of 180 marked with Tyr.mCherry and Tyr.H2B YFP (yellow fluorescent protein); expressed mCherry). d, Co-electroporated with Mitf.DTcf (only 30 of 180 both the Tyr and Mitf reporters label the same a9.49 cells. c, Co-electroporated expressed mCherry). e, f, Tailbuds electroporated with Msx.histone H2B with Mitf.lacZ. Oc, ocellus; Ot, otolith. d, Co-electroporated with Mitf.Twist. mCherry and Mitf.GFP. Arrowhead shows GFP expression in a9.49 Arrowheads indicate ectopic position of a9.49 derivatives. e, f, Juveniles derivatives. e, Co-electroporated with Msx.lacZ. f, Co-electroporated with electroporated with Tyr.mCherry and Tyr.H2B YFP. Arrows identify the Msx.FoxD N terminus. Scale bars, 50 mm(a, c–f); 25 mm(b). Oc, ocellus; position of a9.49 derivatives. e, Juvenile co-electroporated with Mitf.lacZ. Ot, otolith. f, Co-electroporated with Mitf.Twist. Scale bars, 50 mm.

6 DECEMBER 2012 | VOL 492 | NATURE | 105 ©2012 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER from a common bipotent Mitf1 progenitor14. The conservation of this mesenchyme (Fig. 3a). It is not expressed in any region of the neural network strengthens the argument that the a9.49 lineage of Ciona plate, including the a9.49 lineage. Twist-expressing mesoderm under- represents a rudimentary neural crest. However, the a9.49 lineage goes long-range migration (Fig. 3b) and produces a number of diverse lacks some of the defining properties of cephalic neural crest, such tissues in juveniles and adults, including body-wall muscles, tunic cells as long-range migration and the potential to form ectomesenchyme (which populate the protective covering of the adult) and blood cells25. derivatives. The migration and differentiation of these mesoderm tissues are inhi- We therefore sought to identify vertebrate neural crest determinants bited when Twist expression is reduced25. that are not expressed in the Ciona a9.49 lineage. In vertebrates, the To determine whether ectomesenchyme could be formed in Ciona, craniofacial mesenchyme is derived from primary mesoderm and the we misexpressed Twist in the a9.49 lineage using the Mitf enhancer ectomesenchyme arising from cephalic neural crest2,20. Both sources of (Fig. 3c, d). The manipulated cells exhibit a mesenchymal phenotype, cranial mesenchyme express the conserved mesodermal determinant including protrusive activity, proliferation and long-range migration, Twist21 and produce diverse cranial tissues including muscle, cartilage which was not observed by the misexpression of other related genes and bone. In tetrapods, it seems that only the cephalic neural crest (Supplementary Video 1 and Supplementary Fig. 6). Moreover, mis- expresses Twist and produces ectomesenchyme21–23, whereas trunk expression of Twist in the notochord and motor ganglion causes some neural crest lacks Twist expression and generates non-ectomesenchymal disruptions in terminal differentiation, but does not transform these derivatives (for example, neurons, glia and melanocytes)2. Disruption of tissues into mesenchyme (Supplementary Fig. 7). The reprogrammed Twist activity causes severe cephalic neural crest phenotypes, including a9.49 cells exhibit expression of mesenchyme genes, including Erg defects in cell migration and survival, as well as morphological defects of (Supplementary Fig. 8), which is expressed in the ectomesenchyme the skull vault and heart21,22,24. of mouse embryos26. The affected lineage was visualized in juveniles There are three Twist-related genes in Ciona. In this study we using reporters for Tyr, a gene that is activated by Mitf in melanocytes14 focused on the gene most similar to Twist1 in vertebrates (Supplemen- (Fig. 3e, f). Normally the a9.49 derivatives are located solely in an tary Fig. 5). In Ciona, Twist is expressed solely in mesoderm-derived anterior region of the CNS (Fig. 3e). In contrast, embryos expressing

abcMitf>Twist Kaede Kaede

Merge

d Mitf>Twist Kaede ef Kaede UV treated

Merge g 0′ 14.13′ 28.27′ 42.40′ 56.53′

Mitf>Twist

Figure 4 | Lineage tracing of reprogrammed a9.49 cells. a–f, Ciona f, UV-treated embryo results in a juvenile with green and red ectopic cells electroporated with Mitf.Twist and Tyr.Kaede. a, b, Non-ultraviolet (UV)- (arrows). g, Time-lapse frames of Supplementary Video 2 (frames were taken treated tailbud embryo shows only green fluorescence. c, Embryo never from the times indicated) shows the migration of reprogrammed a9.49 cell exposed to UV results in a juvenile with only green ectopic cells (arrows). labelled with Tyr.mCherry (arrowhead). The white cells correspond to d, e, UV-treated tailbud embryos show green and red fluorescence, respectively. endogenous tunic cells. Scale bars, 50 mm.

Mitf.Twist result in juveniles with ectopic a9.49 cells (Fig. 3f). The 7. Jeffery, W. R. Ascidian neural crest-like cells: phylogenetic distribution, relationship to larval complexity, and pigment cell fate. J. Exp. Zool. B 306B, reprogrammed cells seem to produce mesodermal derivatives, such as 470–480 (2006). tunic cells, based on their location and distinct, rounded morphology 8. Jeffery, W. R. et al. Trunk lateral cells are neural crest-like cells in the ascidian Ciona (Supplementary Fig. 9). intestinalis: Insights into the ancestry and evolution of the neural crest. Dev. Biol. 324, 152–160 (2008). Additional evidence for the reprogramming of the a9.49 lineage was 9. Tassy, O. et al. The ANISEED database: digital representation, formalization, and obtained with Kaede, a photoconvertible fluorescent protein that was elucidation of a chordate developmental program. Genome Res. 20, 1459–1468 previously used in Ciona to trace the formation of the CNS27. Embryos (2010). . . 10. Russo, M. T. et al. Regulatory elements controlling Ci-msxb tissue-specific were co-electroporated with Mitf Twist and Tyr Kaede, which expression during Ciona intestinalis embryonic development. Dev. Biol. 267, mediate expression in the a9.49 lineage (Fig. 4a). Tailbud embryos that 517–528 (2004). were not exposed to ultraviolet light show no red fluorescence (Fig. 4b) 11. Imai, K. S., Levine, M., Satoh, N. & Satou, Y. Regulatory blueprint for a chordate and result in juveniles that have only green a9.49 descendants (Fig. 4c). embryo. Science 312, 1183–1187 (2006). 12. Wada, H. & Makabe, K. Genome duplications of early vertebrates as a possible In contrast, tailbud embryos treated with ultraviolet light (Fig. 4d, e) chronicle of the evolutionary history of the neural crest. Int. J. Biol. Sci. 2, 133–141 develop into juveniles that display red cells throughout the body (2006). (Fig. 4f). Control juveniles lacking Mitf.Twist exhibit the expected 13. Squarzoni, P., Parveen, F., Zanetti, L., Ristoratore, F. & Spagnuolo, A. FGF/MAPK/Ets signaling renders pigment cell precursors competent to respond to Wnt signal by expression solely in the CNS (Supplementary Fig. 10). Finally, time- directly controlling Ci-Tcf transcription. Development 138, 1421–1432 (2011). lapse microscopy was used to examine the Mitf.Twist expressing cells 14. Curran, K. et al. Interplay between Foxd3 and Mitf regulates cell fate plasticity in the in juveniles. Some of these cells migrate like the normal tunic cells zebrafish neural crest. Dev. Biol. 344, 107–118 (2010). 15. Yajima, I. et al. Cloning and functional analysis of ascidian Mitf in vivo:insightsinto derived from the mesenchyme (Fig. 4g and Supplementary Video 2). the origin of vertebrate pigment cells. Mech. Dev. 120, 1489–1504 (2003). Thus, the misexpression of Twist is sufficient to reprogram the a9.49 16. Nishida, H. & Satoh, N. Determination and regulation in the pigment cell lineage of lineage into migrating mesenchymal cells, reminiscent of vertebrate the ascidian embryo. Dev. Biol. 132, 355–367 (1989). ectomesenchyme. 17. Dorsky, R. I., Moon, R. T. & Raible, D. W. Control of neural crest cell fate by the Wnt signalling pathway. Nature 396, 370–373 (1998). The mesenchymal properties of neural crest were proposed to be the 18. Thomas, A. J. & Erickson, C. A. FOXD3 regulates the lineage switch between neural last features to appear during its evolution3,28. Our studies of the non- crest-derived glial cells and pigment cells by repressing MITF through a non- vertebrate chordate Ciona intestinalis support this hypothesis. We canonical mechanism. Development 136, 1849–1858 (2009). 19. Imai, K. S., Satoh, N. & Satou, Y. An essential role of a FoxD gene in notochord propose that cephalic neural crest arose from the co-option of one induction in Ciona embryos. Development 129, 3441–3453 (2002). or more mesenchyme determinants (for example, Twist) in a rudi- 20. Yoshida, T., Vivatbutsiri, P., Morriss-Kay, G., Saga, Y. & Iseki, S. Cell lineage in mentary neural crest cell type. Thus, this enigmatic cell population mammalian craniofacial mesenchyme. Mech. Dev. 125, 797–808 (2008). 21. Bildsoe, H. et al. Requirement for Twist1 in frontonasal and skull vault development should not be considered a vertebrate innovation but an elaboration of in the mouse embryo. Dev. Biol. 331, 176–188 (2009). an ancestral chordate gene network. 22. Soo, K. et al. Twist function is required for the morphogenesis of the cephalic neural Note added in proof: After this paper was accepted for publication, our tube and the differentiation of the cranial neural crest cells in the mouse embryo. Dev. Biol. 247, 251–270 (2002). time-lapse imaging revealed that the a10.97 cells undergo an additional 23. Hopwood, N. D., Pluck, A. & Gurdon, J. B. A Xenopus mRNA related to Drosophila divisionjust before their intercalation at the dorsal midline. This finding twist is expressed in response to induction in the mesoderm and the neural crest. does not alter any of the preceding conclusions. Cell 59, 893–903 (1989). 24. Vincentz, J. W. et al. An absence of Twist1 results in aberrant cardiac neural crest morphogenesis. Dev. Biol. 320, 131–139 (2008). METHODS SUMMARY 25. Tokuoka, M., Satoh, N. & Satou, Y. A bHLH transcription factor gene, Twist-like 1, is Ciona intestinalis transgenesis, and protein–RNA double-labelling assays, were essential for the formation of mesodermal tissues of Ciona juveniles. Dev. Biol. 288, performed as described previously29. All enhancer and misexpression sequences 387–396 (2005). were cloned into a pCESA vector using the primer pairs shown in Supplementary 26. Vlaeminck-Guillem, V. et al. The Ets family member Erg gene is expressed in mesodermal tissues and neural crests at fundamental steps during mouse Table 1. The ZicL, Msx, FoxD, Tyr, Tyrp1, Brachyury and Dmbx enhancers have 10,13,19,29,30 embryogenesis. Mech. Dev. 91, 331–335 (2000). been described previously . Photoconversion of Kaede was achieved by 27. Horie, T. et al. Ependymal cells of chordate larvae are stem-like cells that form the treating tailbud embryos with ultraviolet light using a fluorescence stereomicro- adult nervous system. Nature 469, 525–528 (2011). scope. All images were generated on a Zeiss Axio Imager A2 or a Zeiss LSM 700 28. Shimeld, S. M. & Holland, P. W. H. Vertebrate innovations. Proc. Natl Acad. Sci. USA microscope. 97, 4449–4452 (2000). 29. Shi, W. & Levine, M. Ephrin signaling establishes asymmetric cell fates in an Full Methods and any associated references are available in the online version of endomesoderm lineage of the Ciona embryo. Development 135, 931–940 (2008). the paper. 30. Stolfi, A. & Levine, M. Neuronal subtype specification in the spinal cord of a protovertebrate. Development 138, 995–1004 (2011). Received 19 March; accepted 13 September 2012. Supplementary Information is available in the online version of the paper. Published online 7 November 2012. Acknowledgements We thank A. Stolfi for his continued support and guidance, 1. Bronner, M. E. & Le Douarin, N. M. Evolution and development of the neural crest: Y. Satou for isolating the Twist enhancer, N. Ellis for cloning Dmbx.Twist and B. Gainous An overview. Dev. Biol. 366, 2–9 (2012). for critical reading of the manuscript. P.B.A is supported by a graduate fellowship from 2. Le Douarin, N. M. et al. Neural crest cell plasticity and its limits. Development 131, the US National Science Foundation. This work was supported by a grant from the US 4637–4650 (2004). National Institutes of Health (NS 076542). 3. Gans, C. & Northcutt, R. G. Neural crest and the origin of vertebrates: a new head. Author Contributions P.B.A. designed and performed most experiments in Science 220, 268–273 (1983). consultation with M.L. E.W. isolated the cis-regulatory element for the bc-crystallin 4. Yu, J. K., Meulemans, D., McKeown, S. J. & Bronner-Fraser, M. Insights from the reporter and made the stable b-catenin transgene. I.A.N. examined Mech2 and Erg amphioxus genome on the origin of vertebrate neural crest. Genome Res. 18, expression in wild-type and reprogrammed tailbud embryos. P.B.A., M.L. and E.W. 1127–1132 (2008). wrote the manuscript. 5. Delsuc, F., Brinkmann, H., Chourrout, D. & Philippe, H. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439, Author Information Reprints and permissions information is available at 965–968 (2006). www.nature.com/reprints. The authors declare no competing financial interests. 6. Jeffery, W. R., Strickler, A. G. & Yamamoto, Y. Migratory neural crest-like cells form Readers are welcome to comment on the online version of the paper. Correspondence body pigmentation in a urochordate embryo. Nature 431, 696–699 (2004). and requests for materials should be addressed to M.L. ([email protected]).

METHODS sequence was cloned 59 of a basal friend of GATA (Zfpm1) promoter. The lacZ Embryo preparation and imaging. Ciona intestinalis adults were obtained, in coding sequence of the pCESA vector was replaced with UNC-76–GFP, UNC-76– vitro fertilized and electroporated for transient transgenesis as described29. For mCherry, H2B–mCherry, enhanced GFP or Kaede. The ZicL, Msx, FoxD, Tyr, 10,13,19,29,30 each electroporation, typically 70 mg of DNA was resuspended in 100 ml buffer. Tyrp1 Brachyury and Dmbx enhancers have been previously described .A Embryos were fixed at the appropriate developmental stage for 15 min in 4% for- similar cloning strategy was used to create misexpression vectors using Not1 or maldehyde. The tissue was then cleared in a series of washes of 0.01% Triton-X in EcoR1 sites for control group A basic helix–loop–helix genes, Wnt7, FoxD and PBS. Actin was stained overnight with Alexa-647-conjugated phalloidin at a dilution FoxD N-terminal, or NotI or BlpI sites for stabilized b-catenin and Twist of 1:500. Samples were mountedin 50% glycerol in PBS with2% DABCO compound (Supplementary Table 1). The FoxD–DBD–WRPW coding sequence was made for microscopy. Differential interference-contrast microscopy was used to obtain by amplifying the DBD (DNA-binding domain) of FoxD (Supplementary Table transmitted light micrographs with a Zeiss Axio Imager A2 using the 340 EC Plan 1), which was subcloned into a pCESA vector containing an HA–NLS peptide and Neofluar objective. Confocal images were acquired on a Zeiss LSM 700 microscope a WRPW repressor motif using NheI or SpeI sites. Additional coding sequences using a plan-apochromat 320 or 340 objective. Confocal stacks contained approxi- for Ets–VP16, Ets–WRPW and DTcf were subcloned from existing expression 13 mately 50 optical slices at a thickness of 1 to 2 mm each. Images were rendered in vectors . three dimensions using Volocity 6 with the three-dimension opacity visualization In situ hybridization and immunohistochemistry. The double-fluorescent in tool. For time-lapse microscopy, larvae and juveniles were anaesthetized in artificial situ hybridizations and immunohistochemistry were performed as described30 sea water supplemented with 0.04% tricaine mesylate in a glass-bottom dish. Time- using linearized complementary DNA clones for Wnt7 (cilv33g04), FoxD lapse images were taken on a Zeiss LSM 700 microscope at intervals of 3 to 4 min. (citb8o13), Erg (cilv04i11), Mech2 (cicl04m09), and Twist (cicl20p07) from the Molecular cloning. The University of California Santa Cruz Genome Browser cDNA library of N. Satoh. Gateway facilitated the identification of conserved non-coding sequences between Kaede lineage tracing. Embryos electroporated with Tyr.Kaede and co-electro- Ciona intestinalis and Ciona savignyi. Primers (Supplementary Table 1) were used porated with Mitf.lacZ or Mitf.Twist were developed in the dark until the late to PCR (polymerase chain reaction) amplify these putative enhancer sequences tailbud stage. Tailbuds expressing Kaede were then photoconverted with UV light which were cloned into a pCESA vector using either AscI or NotI restriction sites using the DAPI filter on a Zeiss Stereo Lumar.V12 for 3 min. Embryos were for Prop1, Twist and bc-crystallin, or AscI and XhoI for Mitf.TheMitf enhancer continuously reared in the dark from tailbud stages and prepared for imaging.