Zebra fish have emerged as a powerful genetic model system for identifying and mapping signaling pathways critical to embryological development (1
). In addition, zebra fish harbor orthologs of each of the mammalian DNA methyltransferases, including the founding member, DNMT1 (28
). By convention, we present zebra fish genes in lowercase and human genes in uppercase. To evaluate the role of the Dnmt1 protein in zebra fish embryonic development, we designed and injected a translation-blocking antisense morpholino, which efficiently reduced Dnmt1 protein levels (Fig. ). At an injection amount of 4 ng, approximately 40% of the embryos died within 24 hpf, an effect that was reminiscent of DNMT1 knockout in mice (23
). With the remaining viable embryos, we determined global DNA methylation levels by using a mass spectrometry-based assay (45
) and observed an approximate 40% decrease in the global levels of cytosine DNA methylation, with mdC/dG contents of 7.15% ± 0.06% (average ± standard deviation [SD]) and 4.48% ± 0.35% (average ± SD) for the wild type and Dnmt1 morphants, respectively. The viable morphants retained an overall normal appearance but displayed developmental defects, including curled tails, pericardial edema (Fig. ), and the absence of the P1 (mandibular) and P2 (hyoid) segments in the jaw (Fig. ).
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) (more ...)
At 72 hpf, dnmt1
is expressed in developing gut, eye, and hindbrain (Fig. ) (53
). This led us to examine possible defects in these organs. We observed near-normal expression levels of markers of the primordial gut foxa3
), and hnf1β
) within Dnmt1 morphant embryos (Fig. ), thereby confirming formation and specification of the early gut tube. However, cross-sections of the morphant embryo guts revealed failed epithelial differentiation, as evidenced by lack of established columnar cells throughout the intestine (Fig. ). This was paralleled by absence of intestinal fatty acid binding protein (fabp2
) expression, a marker of terminally differentiated enterocytes (33
), in 97% of Dnmt1 morphants (Fig. ). The penetrance statistics of all morphant phenotypes, as well as all other statistical analyses, are provided in Table . To determine whether lack of intestinal differentiation resulted from specific knockdown of Dnmt1 activity, we coinjected a plasmid encoding V5-tagged zebra fish Dnmt1 or mRNA encoding human DNMT1 along with the Dnmt1 morpholino and confirmed expression by Western analysis (Fig. ). These coinjections effectively rescued fabp2
expression to near-normal levels in more than 80% of the embryos (Fig. and Table ). In contrast, coinjection of a catalytically inactive derivative of zebra fish (Dnmt1C1109S
) or human (DNMT1C1226S
) origin failed to rescue fabp2
expression (Fig. and Table ). Taken together, these data indicate that zebra fish intestinal differentiation requires the catalytic activity of Dnmt1 and that human DNMT1 activity can substitute for that of zebra fish Dnmt1.
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. (more ...)
Organ-specific defects extended to the pancreas, which failed to undergo exocrine differentiation, as indicated by lack of trypsin
) (Fig. and Table ). Remarkably, differentiation of endocrine pancreas and liver appeared unaffected in Dnmt1 morphants, as assessed by the expression levels of insulin (Fig. ) (2
) and liver fatty acid binding protein (fabp10
) (Fig. ) (5
), respectively. Again, injection of wild-type but not catalytically inactive Dnmt1 (or human DNMT1) restored trypsin expression in 72% and 71% of the morphant embryos, respectively (Fig. and Table ). As the exocrine pancreas (2
), endocrine pancreas, and liver (8
) emerge from the same primordial precursors, these findings imply highly specific, temporally regulated roles for Dnmt1 within the developing gut.
Considering that dnmt1
is strongly expressed within the developing eye (Fig. ), it was surprising that the morphological appearance of the eye remained relatively normal in Dnmt1 morphants (Fig. ). Indeed, markers of retinal development otx2
) and otx5
) were similarly expressed in both control and Dnmt1 morphants at 96 hpf (Fig. ). However, examination of cross-sections of the Dnmt1 morphant eyes revealed a profound disorganization of all retinal layers (Fig. ) as well as failed establishment of the dorsal retinal pigmented epithelium (RPE). In keeping with this, expression of interphotoreceptor retinoic binding protein (irbp
), which marks terminal differentiation of the RPE and photoreceptor cell layers (49
), was lost from the eyes of Dnmt1 morphants (Fig. and Table ). Consistent with observations of the intestine and exocrine pancreas, irbp
expression and organization of retinal cell layers were restored in greater than 70% of the Dnmt1 morphants upon coinjection of wild-type but not catalytically inactive zebra fish Dnmt1 or human DNMT1 (Fig. and Table ).
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. (more ...)
A number of investigations support functional and physical interactions between DNA methyltransferases and H3K9 methyltransferases (9
). Consistent with these studies, we observed a significant reduction in the global H3K9me3 levels in Dnmt1 morphants (Fig. ). Since SUV39H1 proteins establish H3K9 methylation in heterochromatic regions (39
), we next investigated the in vivo functions of Suv39h1 in zebra fish embryonic development. Using database search tools, we identified a zebra fish Suv39h1 ortholog that shares 63% identity with both human and murine SUV39H1 and contains all of the essential functional domains (data not shown). We also determined its developmental expression profile by whole-mount in situ hybridization and RT-PCR (Fig. ), which demonstrated that suv39h1
expression in zebra fish overlapped with the tissue expression profile of dnmt1
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 (more ...)
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 (more ...)
To compare the consequences of Suv39h1 loss with those of Dnmt1 loss, we knocked down suv39h1
mRNA transcript levels by using a splice-blocking morpholino directed against the exon 2/intron 2 boundary and verified incomplete splicing by RT-PCR (Fig. ). To confirm knockdown of Suv39h1, we analyzed H3K9me3 levels in Suv39h1 morphants by Western blotting and noticed a significant reduction, a reduction that was greater than that observed for Dnmt1 morphants (Fig. ). Since knockout of H3K9 methyltransferase leads to reduction in global DNA methylation levels in Neurospora crassa
) and Arabidopsis thaliana
), we analyzed global levels of 5-methyl-2′-deoxycytidine in genomic DNA from 72-hpf Suv39h1 morphant embryos. We were unable to detect any change in global cytosine methylation levels within the Suv39h1 morphant embryos (for wild-type embryos, the mdC/dG ratio was 7.15% ± 0.06% [average ± SD], whereas for Suv39h1 morphants, it was 7.21% ± 0.46% [average ± SD]), despite robust reductions in the H3K9me3 levels. Although this method does not assess locus-specific DNA methylation changes, these data show that in zebra fish significant reduction in H3K9 methylation does not appreciably reduce DNA methylation.
Suv39h1 morphants did not show a significantly increased rate of death at 24 hpf (~10% in Suv39h1 morphants, compared to ~5% in control-injected embryos). However, they displayed overall morphological defects that were strikingly similar to those of Dnmt1 morphants, including pericardial edema and curled tails (Fig. ). However, unlike the jaws of Dnmt1 morphants, the jaws of Suv39h1 morphants developed normally (data not shown), suggesting that there are both unique and overlapping development requirements for Dnmt1 and Suv39h1. We tested this concept by examining expression of terminal differentiation markers of the eye and gut that were absent in Dnmt1 morphants. Similarly to Dnmt1 morphants, Suv39h1 morphants lacked expression of irbp in the retina (Fig. and Table ). This defect was rescued when embryos were coinjected with the Suv39h1 morpholino and a plasmid containing V5-tagged, wild-type zebra fish Suv39h1, thereby confirming specific knockdown of suv39h1 (Fig. and Table ). Similarities between Dnmt1 and Suv39h1 morphants extended to the gut: Suv39h1 morphants expressed the primordial gut tube markers foxa3, gata6, and hnf1β (Table and data not shown) but did not express fabp2 within the intestinal epithelial cell layer (Fig. ). As with irbp, coinjection of suv39h1 rescued fabp2 expression (Fig. and Table ). Dnmt1 and Suv39h1 morphants also shared loss of trypsin and retention of insulin and fabp10 (Fig. and Table ). The above-described findings indicate that Dnmt1 and Suv39h1 cooperate to promote tissue-specific differentiation.
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 (more ...)
Reduction of global H3K9me3 and CpG methylation levels in Dnmt1 morphants and no change in DNA methylation levels in Suv39h1 morphants suggest that H3K9me3 levels might be dependent upon CpG methylation levels. Also, phenotypic similarities between Dnmt1 and Suv39h1 morphants suggest that maintaining H3K9me3 levels might be important for terminal differentiation programs. Hence, we tested whether increasing H3K9me3 levels in Dnmt1 morphants by overexpression of suv39h1 would rescue the terminal differentiation defects. Strikingly, injection of suv39h1 mRNA (100 pg) or a plasmid containing V5-tagged, wild-type zebra fish Suv39h1 (12 pg) rescued the expression of fabp2, trypsin, and irbp in Dnmt1 morphants (Fig. ). In contrast, injection of a catalytically inactive Suv39h1 derivative (suv39h1H323K) failed to rescue the phenotypic defects (Fig. ). Furthermore, overexpression of wild-type suv39h1, but not of suv39h1H323K, increased H3K9me3 levels in Dnmt1 morphants (Fig. ). As a control, expression of wild-type suv39h1 and suv39h1H323K constructs was confirmed by Western blotting using V5 antibody (Fig. ). Taken together, these data suggest that reduced levels of H3K9 methylation account for the terminal differentiation defects present in Dnmt1 and Suv39h1 morphants.
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 (suv39h1WT) or (more ...)