PMC

Reactivation of GFP expression by exogenous Gal4-VP16. (A) Mock-injected larvae from c269 GFP^off intercrosses show no GFP labeling (n = 100). (B) GFP^off siblings injected with ∼25 pg of plasmid DNA encoding the EF1α ubiquitous promoter driving Gal4-VP16 expression are extensively labeled with GFP (98%; n = 60). (C) DNA from injected or mock-injected larvae was subjected to bisulfite sequencing, and the methylation status of each CG site was reported as a percentage of the total sites tested for 10 clones. Methylated sites are solid circles. Data are representative of two biological replicates.

Variegated expression of UAS-regulated GFP in zebrafish larvae. (A) Representative larva carrying unique insertions of the gene/enhancer trap vector designated by c numbers. For a given insertion, sibling F₂ larvae express gfp either in a pattern similar to the F₁ generation (GFP^high, left panels) or in a smaller subset of cells within this pattern (GFP^low, right panels). Larvae were imaged at 3 dpf. (B) Fluorescent labeling of the forebrain, midbrain, and hindbrain (r5–6, arrowhead) of c269 GFP^high larvae at 2 dpf. (C) GFP^low c269 sibling larvae show significantly fewer GFP-labeled cells, which are restricted to within the c269 GFP^high pattern of expression. (D) Corresponding images of GFP (top) and mCherry (bottom) fluorescence in c269 GFP^high, GFP^low, and GFP^off larvae carrying a newly integrated UAS:mCherry transgene at 2 dpf. Irrespective of the extent of GFP expression, mCherry labeling recapitulates the complete c269 GFP^high expression pattern.

Distinct regions of the brain show differential silencing and reactivation. Corresponding images of GFP fluorescence (A, D, G, and J), mCherry fluorescence (B, E, H, and K), and an overlay of mCherry, GFP, and differential interference contrast (C, F, I, and L) from 3 dpf larvae. (A–C) c269 GFP^off larvae that are wild type or heterozygous for the dnmt1^s872 mutation do not show GFP labeling. (D–F) c269 GFP^off larvae that are homozygous dnmt1^s872 mutants show GFP labeling in the midbrain and forebrain. Despite transactivation of mCherry, GFP labeling is not observed in r5–6 (arrow in E and F). (G–I) The homozygous dnmt1^s872 mutant with gfp reactivation in a small number of bilateral cells in r5–6. The insert in I is an enlargement of reactivated cells indicated by the arrowhead. (J–L) Larvae from a c269 GFP^high adult mated to wild type. Labeling of GFP is present in the forebrain and midbrain, but not in r5–6 mCherry-positive cells.

Methylation of the multicopy UAS is correlated with decreased GFP labeling. (A) Genomic DNA from pooled GFP^high or GFP^off larvae was digested with SacI to produce an 840-bp SacI fragment encompassing the 14× UAS upstream of gfp. The 840-bp SacI fragment has increased sensitivity to HpaII cleavage in GFP^high larvae compared to GFP^off larvae. Transactivation of a 14× UAS:mCherry transgene was used to identify GFP^off larvae that inherited the c269 transgene. The 14× UAS probe also recognizes the 14× UAS regulating mCherry, although SacI cleavage releases a larger fragment (2.2 kb). This fragment, containing the 14× UAS upstream of mCherry and the mCherry coding sequence, is largely digested, indicating that it is unmethylated in GFP^high and GFP^off samples. (B) DNA from heads and bodies of individual GFP^high or GFP^off larvae was subjected to bisulfite sequencing. The methylation status of each CG site is reported as a percentage of total sites tested for 10 clones from the head and 10 clones from the body of each individual. Methylated sites are solid circles; horizontal bars indicate pairs of CpG dinucleotides within individual Gal4-binding sites. The final 8 CpG sites reside within the minimal promoter sequence.