Determining integration time of the Tol2 transposon following injection in 1–2 cell embryos.

(A) A plasmid construct containing the ubiquitously expressed Xenopus ef1α promoter driving GFP (Xef1α>GFP) and flanked by Tol2 transposon arms was co-injected with transposase mRNA into 1–2 cell embryos. (B) Following injection, typical mosaic Xef1α>GFP expression at 2 days. Note that a small minority of cells, in a wide variety of tissues, is labeled throughout the embryo. (C) Model relating the size of the germ cell precursor (GCP) population at the time of integration of the Xef1α>GFP transposon to transmission rates from F0 founders to their F1 progeny. (D) F0 founders grouped by the developmental stages at which integration took place, as inferred from the number of calculated GCPs. Number of GCPs based on previous counts of vasa mRNA expressing cells at various stages of development [30], [31]. Note that the majority of integration events occur after the mid blastula transition and prior to the end of the shield stage (59% unadjusted, 75% adjusted).

Three classes of melanocyte clones are revealed from clonal analysis.

(A) Plasmid construct containing the melanocyte specific Takifugu rubripes Tyrosinase related protein 1 promoter driving GFP (fTyrp1>GFP) and flanked by Tol2 transposon arms was co-injected with transposase mRNA into 1–2 cell embryos. (B) Experimental protocol used for clonal analysis of melanocyte lineages. Following transposon injection, ontogenetic melanocytes are allowed to develop normally through 3 dpf, and then screened for GFP+ melanocytes. 4-HA treatment between 3 and 5 dpf was then used to ablate melanocytes. Drug was then washed out to allow melanocyte regeneration from MSCs. At 8 dpf fish are again screened for the presence of GFP in regeneration melanocytes. Vertical arrows indicate time of scoring for labeled melanocytes. (C–E) Representative pictures of fish showing ontogenetic melanocytes prior to ablation (C and D) and regeneration melanocytes following ablation and regeneration (C′ and E). Note that the same fish is shown in C and C′ as well as D and D′. Arrows point to GFP expressing ontogenetic and regeneration melanocytes. Fish with GFP labeled melanocytes can be divided in 3 clone classes: Class 1, with both ontogenetic and regeneration melanocytes labeled, indicating integration in a bipotent melanogenic precursor; Class 2, with only ontogenetic melanocytes labeled, indicating integration in a restricted direct-developing precursor; and Class 3, with only regeneration melanocytes labeled, indicating integration in a restricted MSC precursor.