title: Controlled Cre/loxP site-specific recombination in the developing brain in medaka fish, Oryzias latipes.
abstract: BACKGROUND
  Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.
  
  
  RESULTS
  To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0-1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2-3 dpf embyos compared with 0-1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.
  
  
  CONCLUSIONS
  We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan ; National Institute for Basic Biology, Myodaiji, Okazaki, Aichi, Japan.
authors.forename: Teruhiro
authors.initials: T
authors.lastname: Okuyama
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Isoe
authors.forename: Masahito
authors.initials: M
authors.lastname: Hoki
authors.forename: Yuji
authors.initials: Y
authors.lastname: Suehiro
authors.forename: Genki
authors.initials: G
authors.lastname: Yamagishi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Naruse
authors.forename: Masato
authors.initials: M
authors.lastname: Kinoshita
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kamei
authors.forename: Atushi
authors.initials: A
authors.lastname: Shimizu
authors.forename: Takeo
authors.initials: T
authors.lastname: Kubo
authors.forename: Hideaki
authors.initials: H
authors.lastname: Takeuchi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001921:Brain / Q000166:cytology / Q000196:embryology*; D002454:Cell Differentiation / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000166:cytology; D047108:Embryonic Development / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D006358:Hot Temperature; D007259:Infrared Rays; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D007834:Lasers; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism; D009875:Operator Regions, Genetic / Q000235:genetics; D009990:Oryzias / Q000196:embryology* / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013269:Stochastic Processes
chemical_list: D012333:RNA, Messenger; C045073:Cre recombinase; D019426:Integrases