Specific developmental defects of Dnmt2 morphants. (A) Gross morphology of Dnmt2 morphants at 80 hpf, displaying pericardial edema, a primitive yolk, a thin trunk, and deformed eyes, brain, somites, and tail. Bar, 0.5 mm. (B–D) Dnmt2 morphants display tissue-specific differentiation defects. Whole-mount in situ of embryos at 80 hpf. Rows display expression of mRNA encoding l-fabp or transferrin-α (A), gata6 or foxa3 (B), and i-fabp or insulin (C). Columns display control morphants, Dnmt2 morphants, or Dnmt2 morphants coinjected with mRNA encoding Dnmt2^WT or Dnmt2^C82S. Dnmt2 morphants lose the expression of terminal differentiation marker of liver l-fabp and transferrin-α, whereas they retain expression of markers of liver precursors (gata6 and foxa3) and markers of terminal differentiation of intestine (i-fabp) and endocrine pancreas (insulin). Arrows denote the cells expressing the respective marker.

Dnmt2 morphants display a developmental delay in the eye. (A) Cross-sections of wild-type and Dnmt2 morphant eyes at 80 hpf stained with toluidine blue. Note the presence of retinal pigmented epithelium only in the dorsal side (red arrow) and duplication of a portion of ventral retina (yellow arrow). (B) Whole-mount in situ for pcna, vsx2, neurod, crx, zash1a, and gso1 in embryos injected with control morpholino or Dnmt2 morpholino alone, or coinjected with V5-Dnmt2^WT. Expression of all markers except zash1a (which was examined at 48 hpf) was analyzed at 80 hpf. Note the continued expression of markers of progenitors (pcna and vsx2), and the loss of expression of markers of specified precursors (neurod, crx, and zash1a).

Cytoplasmic localization of Dnmt2 rescues Dnmt2 morphant phenotypes. (A) Confocal images of GFP and TOPRO-3 fluorescence in ZF4 and RKO cells transfected with empty vector, or vectors containing tagged Dnmt2^WT, NLS-Dnmt2, mNLS-Dnmt2, or NES-Dnmt2. (B) Whole-mount in situ of embryos at 80 hpf, stained for expression of l-fabp and vsx2. Columns depict embryos from control morphants, Dnmt2 morphants, or Dnmt2 morphants coinjected with NLS-Dnmt2, mNLS-Dnmt2, or NES-Dnmt2.

Zebrafish Dnmt2 is an RNA methyltransferase. Total RNA was purified from wild-type and Dnmt2 morphant zebrafish embryos at 72 hpf and subjected to a H³-labeled methyltransfer reaction using immunoprecipitated wild-type or catalytically inactive (C82S) zebrafish Dnmt2. After the reaction, recovered RNA was separated on a denaturing UREA-PAGE (see Supplementary Fig. 9). The band corresponding to the size of tRNA^Asp (∼80 bp) (see Supplementary Fig. 8) was excised and H³ incorporation was measured by scintillation counting. The bar graph depicts the fold increase in H³ counts per minute from the band containing tRNA^Asp (band 5, ∼80 bases) (Supplementary Fig. 8) from Dnmt2 morphants compared with wild-type zebrafish embryos. The two assays at the left utilized wild-type zebrafish Dnmt2 protein, whereas the two at the right utilized the catalytically inactive (C82S) enzyme. Assays represent three biological replicates; error bars are ±1 SD.