Germ cell regeneration-mediated, enhanced mutagenesis in the ascidian Ciona intestinalis reveals flexible germ cell formation from different somatic cells.
The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona. This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona.
Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka 415-0025, Japan.
Keita
K
Yoshida
Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka 415-0025, Japan.
Akiko
A
Hozumi
Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka 415-0025, Japan.
Nicholas
N
Treen
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
Tetsushi
T
Sakuma
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
Takashi
T
Yamamoto
Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Japan.
Maki
M
Shirae-Kurabayashi
Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka 415-0025, Japan. Electronic address: sasakura@shimoda.tsukuba.ac.jp.
Yasunori
Y
Sasakura
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002938:Ciona intestinalis / Q000166:cytology* / Q000235:genetics*; D018274:Electroporation; D055786:Gene Knockout Techniques; D016678:Genome; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000196:embryology; D008297:Male; D008675:Metamorphosis, Biological; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D059645:Mutation Rate; D009928:Organ Specificity; D010063:Ovum / Q000166:cytology; D012038:Regeneration; D013094:Spermatozoa / Q000166:cytology; D013623:Tail; D000069896:Transcription Activator-Like Effector Nucleases / Q000378:metabolism
D018398:Homeodomain Proteins; D000069896:Transcription Activator-Like Effector Nucleases
Ascidian; Ciona; Mutagenesis; Primordial germ cell; Regeneration; TALEN