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First published online 21 March 2007
doi: 10.1242/dev.02835

Development 134, 1679-1689 (2007)
Published by The Company of Biologists 2007
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Clathrin-mediated endocytic signals are required for the regeneration of, as well as homeostasis in, the planarian CNS

Takeshi Inoue1,2, Tetsutaro Hayashi1,*, Katsuaki Takechi1,{dagger} and Kiyokazu Agata1,3,{ddagger}

1 Group for Evolutionary Regeneration Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
2 KAN Research Institute, Inc., Kobe MI R&D Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
3 Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.

{ddagger} Author for correspondence (e-mail: agata{at}mdb.biophys.kyoto-u.ac.jp)

Accepted 13 February 2007

Planarians have a well-organized central nervous system (CNS), including a brain, and can regenerate the CNS from almost any portion of the body using pluripotent stem cells. In this study, to identify genes required for CNS regeneration, genes expressed in the regenerating CNS were systematically cloned and subjected to functional analysis. RNA interference (RNAi) of the planarian clathrin heavy chain (DjCHC) gene prevented CNS regeneration in the intermediate stage of regeneration prior to neural circuit formation. To analyze DjCHC gene function at the cellular level, we developed a functional analysis method using primary cultures of planarian neurons purified by fluorescence-activated cell sorting (FACS) after RNAi treatment. Using this method, we showed that the DjCHC gene was not essential for neural differentiation, but was required for neurite extension and maintenance, and that DjCHC-RNAi-treated neurons entered a TUNEL-positive apoptotic state. DjCHC-RNAi-treated uncut planarians showed brain atrophy, and the DjCHC-RNAi planarian phenotype was mimicked by RNAi-treated planarians of the mu-2 (µ2) gene, which is involved in endocytosis, but not the mu-1 (µ1) gene, which is involved in exocytosis. Thus, clathrin-mediated endocytic signals may be required for not only maintenance of neurons after synaptic formation, but also axonal extension at the early stage of neural differentiation.

Key words: Planarian, Regeneration, Central nervous system (CNS), Clathrin heavy chain, Endocytosis, AP-2, Neuronal homeostasis, Dugesia japonica


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