Zebra fish Dnmt1 and Suv39h1 regulate organ-specific terminal differentiation during development

Abstract

DNA methylation and histone methylation are two key epigenetic modifications that help govern heterochromatin dynamics. The roles for these chromatin-modifying activities in directing tissue-specific development remain largely unknown. To address this issue, we examined the roles of DNA methyltransferase 1 (Dnmt1) and the H3K9 histone methyltransferase Suv39h1 in zebra fish development. Knockdown of Dnmt1 in zebra fish embryos caused defects in terminal differentiation of the intestine, exocrine pancreas, and retina. Interestingly, not all tissues required Dnmt1, as differentiation of the liver and endocrine pancreas appeared normal. Proper differentiation depended on Dnmt1 catalytic activity, as Dnmt1 morphants could be rescued by active zebra fish or human DNMT1 but not by catalytically inactive derivatives. Dnmt1 morphants exhibited dramatic reductions of both genomic cytosine methylation and genome-wide H3K9 trimethyl levels, leading us to investigate the overlap of in vivo functions of Dnmt1 and Suv39h1. Embryos lacking Suv39h1 had organ-specific terminal differentiation defects that produced largely phenocopies of Dnmt1 morphants but retained wild-type levels of DNA methylation. Remarkably, suv39h1 overexpression rescued markers of terminal differentiation in Dnmt1 morphants. Our results suggest that Dnmt1 activity helps direct histone methylation by Suv39h1 and that, together, Dnmt1 and Suv39h1 help guide the terminal differentiation of particular tissues.

FIG. 1.

Dnmt1 knockdown in zebra fish. (A) Western analysis of Dnmt1 protein levels in embryos injected with a Dnmt1-directed morpholino (0, 2, 4, or 8 ng). Levels of histone H3 served to normalize Dnmt1 expression levels, as indicated by percent wild-type (WT) levels. (B) Nomarski images of 96-hpf embryos injected with either control or Dnmt1 morpholino. Bar = 0.5 mm. (C) Alcian blue staining showing loss of branchial arches P1 (mandibular) and P2 (hyoid) in Dnmt1 morphants. (D) Whole-mount in situ hybridization of 72-hpf wild-type embryos with dnmt1 shows dnmt1 expression in the retina and gut (indicated by arrows). (E) Early gut tube specification was assessed in control or Dnmt1 morphant embryos (96 hpf) by whole-mount in situ hybridization for expression of gata6, foxa3, and hnf1β (indicated by arrows). (F) Histological sectioning shows loss of columnar cells throughout the intestine of Dnmt1 morphants.

FIG. 2.

Intestinal and pancreatic differentiation defects in Dnmt1 morphants. (A) Whole-mount in situ hybridization assessing levels of fabp2 and trypsin (arrows) in Dnmt1 morphants shows failed differentiation of intestinal epithelial cells and exocrine pancreas. Injection of a plasmid encoding V5-tagged, wild-type Dnmt1 (dnmt1^WT) or human DNMT1^WT RNA rescues fabp2 and trypsin expression, but use of V5-tagged, catalytically inactive Dnmt1 (dnmt1^C1109S) or human DNMT1^C1226S does not. (B) Whole-mount in situ hybridization assessing levels of insulin and fabp10 (arrows) in Dnmt1 morphants. Levels of insulin and fabp10 are unchanged, indicating apparent normal development of the endocrine pancreas and liver, respectively. (C) Western analysis confirming equal expression of wild-type or catalytically inactive Dnmt1 within the injected embryos. Vinculin (Vin) served as a loading control. (D) Western analysis showing equal expression of DNMT1^WT and DNMT1^C1226S RNA in injected embryos. HEK 293 extract was used as a positive control, and histone H3 was used as a loading control.

FIG. 3.

Retinal differentiation defects in Dnmt1 morphants. (A) Control or Dnmt1 morphant embryos (96 hpf) were subjected to whole-mount in situ hybridization for otx5 and otx2. (B) TOPRO-1 staining of histological cross-sections within the Dnmt1 morphant retinas. Arrows indicate the dorsal loss of RPE. Injection of a plasmid encoding V5-tagged, wild-type Dnmt1 (dnmt1^WT) rescues the RPE and retinal layering. (C) Whole-mount in situ hybridization of irbp expression within the retina of Dnmt1 morphants. Injection of a plasmid encoding V5-tagged, wild-type Dnmt1 (dnmt1^WT) rescues irbp expression, but use of V5-tagged, catalytically inactive Dnmt1 (dnmt1^C1109S) does not.

FIG. 4.

Reduced levels of H3K9me3 in Dnmt1 and Suv39h1 morphant embryos. (A) Western analysis of H3K9me3 levels in embryos injected with a Dnmt1-directed morpholino. The amount of protein loaded in each lane is indicated at top. Analysis of histone H3 loading served to normalize levels of H3K9me3 for morphants relative to levels for the wild type (WT). Percent control levels are indicated at bottom. (B) cDNA was prepared at 96 hpf from embryos injected with increasing amounts of Suv39h1 morpholino (0, 4, 8, and 12 ng); RT-PCR using primers flanking the exon 2/intron 2 boundary of suv39h1 (against which the morpholino was targeted) showed that the level of spliced suv39h1 transcripts was reduced with increasing amounts of Suv39h1 splice-blocking morpholino. β-actin was used as a loading control. Percent control levels are indicated at bottom. (C) H3K9me3 levels in Suv39h1 morphants.

FIG. 5.

Expression pattern of suv39h1 during embryonic development. (A) Whole-mount in situ hybridization for suv39h1 shows expression of suv39h1 during early embryonic development. suv39h1 expression is ubiquitous until early somatic stages, after which it becomes restricted to the head of the embryo. After 48 hpf, the expression reduces drastically. Very low levels of expression can be seen in the pancreas starting at 72 hpf. dpf, days postfertilization. (B) RT-PCR analysis shows suv39h1 expression in adult tissues of brain, eyes, fins, gut, skeletal muscle, and skin.

FIG. 6.

Suv39h1 morphants produce phenocopies of Dnmt1 morphants. (A) Nomarski image of 96-hpf embryos injected with either control morpholino or Suv39h1 morpholino. Bar = 0.5 mm. (B) Whole-mount in situ hybridization for irbp, fabp2, trypsin, insulin, and fabp10 (arrows) in an Suv39h1 morphant in comparison to a control embryo indicates failed terminal differentiation of retina, exocrine pancreas, and intestine. irbp, fabp2, and trypsin (arrows) expression levels were rescued in Suv39h1 morphants by coinjection of V5-tagged, wild-type suv39h1 (suv39h1^WT). As seen with Dnmt1 morphants, Suv39h1 morphants continue to express insulin and fabp.

FIG. 7.

Suv39h1 overexpression rescues Dnmt1 morphants. (A) Whole-mount in situ hybridization for irbp, fabp2, and trypsin (arrows) in embryos injected with Dnmt1 morpholino alone or coinjected with plasmid encoding V5-tagged, wild-type Suv39h1 (suv39h1^WT) or catalytic mutant Suv39h1 (suv39h1^H323K) indicates that Dnmt1 morphants can be rescued by overexpression of wild-type Suv39h1 but not catalytically inactive Suv39h1. (B) Western analysis showing expression of V5-tagged suv39h1^WT and suv39h1^H323K in embryos injected with these constructs along with Dnmt1 morpholino. Vinculin is used as a loading control. (C) Western analysis of H3K9me3 levels in embryos injected with Dnmt1 morpholino alone or coinjected with suv39h1^WT or suv39h1^H323K. Analysis of histone H3 loading served to normalize levels of H3K9me3.