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


Neural crest arises at the neural plate border, expresses a core set of regulatory genes and produces a diverse array of cell types, including ectomesenchyme derivatives that elaborate the vertebrate head1,2. The evolution of neural crest has been proposed to be a key event leading to the appearance of new cell types that fostered the transition from filter feeding to active predation in ancestral vertebrates3. However, the origin of neural crest remains controversial, as homologous cell types have not been unambiguously identified in non-vertebrate chordates1,4. Here we show that the tunicate Ciona intestinalis possesses a cephalic melanocyte lineage (a9.49) similar to neural crest that can be reprogrammed into migrating ‘ectomesenchyme’ by the targeted misexpression of Twist (also known as twist-like 2). Our results suggest that the neural crest melanocyte regulatory network pre-dated the divergence of tunicates and vertebrates. We propose that the co-option of mesenchyme determinants, such as Twist, into the neural plate ectoderm was crucial to the emergence of the vertebrate ‘new head’3.

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Figure 1: Wnt signalling promotes ocellus formation.
Figure 2: FoxD represses Mitf in the ocellus.
Figure 3: Twist reprograms the a9.49 lineage.
Figure 4: Lineage tracing of reprogrammed a9.49 cells.


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We thank A. Stolfi for his continued support and guidance, Y. Satou for isolating the Twist enhancer, N. Ellis for cloning Dmbx>Twist and B. Gainous for critical reading of the manuscript. P.B.A is supported by a graduate fellowship from the US National Science Foundation. This work was supported by a grant from the US National Institutes of Health (NS 076542).

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Authors and Affiliations



P.B.A. designed and performed most experiments in consultation with M.L. E.W. isolated the cis-regulatory element for the βγ-crystallin reporter and made the stable β-catenin transgene. I.A.N. examined Mech2 and Erg expression in wild-type and reprogrammed tailbud embryos. P.B.A., M.L. and E.W. wrote the manuscript.

Corresponding author

Correspondence to Michael Levine.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10 and Supplementary Table 1. (PDF 5463 kb)

Lateral view of a Mitf>Twist expressing larva labeled with Tyr>mCherry

The time-lapse covers a period of about 168 minutes, during which time the protrusive behavior of the a9.49 lineage is seen. The a9.49 lineage misexpressing Twist commonly forms filopodia, which is indicative of cellular reprogramming to mesenchymal fate. (MOV 2329 kb)

Lateral view of a Mitf>Twist expressing early juvenile labeled with Tyr>mCherry

The lateral view of a Mitf>Twist expressing early juvenile labeled with Tyr>mCherry (grey is UV autofluorescence). The time-lapse covers about 6 hours of development, during which time the ectopic a9.49 lineage can be seen migrating around the periphery like the endogenous migrating tunic cells (autofluorescence). The movie loops twice and during the second time the migrating descendants of a9.49 are labeled with asterisks. Other Tyr>mCherry that have been incorporated into the mesenchyme remain stationary. (MOV 19826 kb)

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Abitua, P., Wagner, E., Navarrete, I. et al. Identification of a rudimentary neural crest in a non-vertebrate chordate. Nature 492, 104–107 (2012).

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