FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2753476076> ?p ?o ?g. }

• W2753476076 endingPage "2997" @default.
• W2753476076 startingPage "2987" @default.
• W2753476076 abstract "Article7 September 2017free access Source DataTransparent process Epigenetic regulation of left–right asymmetry by DNA methylation Lu Wang State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Science, Beijing, China Search for more papers by this author Zhibin Liu State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Science, Beijing, China Search for more papers by this author Hao Lin MOE Key Laboratory of Protein Sciences, Tsinghua University School of Life Sciences, Beijing, China Search for more papers by this author Dongyuan Ma State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Science, Beijing, China Search for more papers by this author Qinghua Tao MOE Key Laboratory of Protein Sciences, Tsinghua University School of Life Sciences, Beijing, China Search for more papers by this author Feng Liu Corresponding Author [email protected] orcid.org/0000-0003-3228-0943 State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Science, Beijing, China Search for more papers by this author Lu Wang State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Science, Beijing, China Search for more papers by this author Zhibin Liu State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Science, Beijing, China Search for more papers by this author Hao Lin MOE Key Laboratory of Protein Sciences, Tsinghua University School of Life Sciences, Beijing, China Search for more papers by this author Dongyuan Ma State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Science, Beijing, China Search for more papers by this author Qinghua Tao MOE Key Laboratory of Protein Sciences, Tsinghua University School of Life Sciences, Beijing, China Search for more papers by this author Feng Liu Corresponding Author [email protected] orcid.org/0000-0003-3228-0943 State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China University of Chinese Academy of Science, Beijing, China Search for more papers by this author Author Information Lu Wang1,2, Zhibin Liu1,2, Hao Lin3, Dongyuan Ma1,2, Qinghua Tao3 and Feng Liu *,1,2 1State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 2University of Chinese Academy of Science, Beijing, China 3MOE Key Laboratory of Protein Sciences, Tsinghua University School of Life Sciences, Beijing, China *Corresponding author. Tel: +86 10 64807307; E-mail: [email protected] EMBO J (2017)36:2987-2997https://doi.org/10.15252/embj.201796580 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract DNA methylation is a major epigenetic modification; however, the precise role of DNA methylation in vertebrate development is still not fully understood. Here, we show that DNA methylation is essential for the establishment of the left–right (LR) asymmetric body plan during vertebrate embryogenesis. Perturbation of DNA methylation by depletion of DNA methyltransferase 1 (dnmt1) or dnmt3bb.1 in zebrafish embryos leads to defects in dorsal forerunner cell (DFC) specification or collective migration, laterality organ malformation, and disruption of LR patterning. Knockdown of dnmt1 in Xenopus embryos also causes similar defects. Mechanistically, loss of dnmt1 function induces hypomethylation of the lefty2 gene enhancer and promotes lefty2 expression, which consequently represses Nodal signaling in zebrafish embryos. We also show that Dnmt3bb.1 regulates collective DFC migration through cadherin 1 (Cdh1). Taken together, our data uncover dynamic DNA methylation as an epigenetic mechanism to control LR determination during early embryogenesis in vertebrates. Synopsis During vertebrate development, asymmetric positioning of organs is regulated by a laterality organizer, which is specified by Nodal signaling. Here, DNA methylation via Dnmt1 and Dnmt3bb.1 is shown to be essential for determination of left–right asymmetry and lateral organizer formation in zebrafish. DNA methylation controls specification and clustering of the laterality organizer progenitors during early embryogenesis. Dnmt1 deficiency induces hypo-methylation of the lefty2 enhancer. Upon dnmt1 knockdown, increased Lefty2 expression represses Nodal signaling. Dnmt3bb.1 regulates collective laterality organizer progenitor cell migration by promoting Cdh1-mediated cell adhesion. Introduction DNA methylation is one of the major epigenetic modifications in vertebrates, which involves adding a methyl group to the 5th carbon of cytosine to form 5-methylcytosine (5mC). 5mC is established and maintained by DNA methyltransferases (DNMT), which are classified into two groups, that is, maintenance DNMTs (Dnmt1) and “de novo” DNMTs (Dnmt3a and Dnmt3b) (Smith & Meissner, 2013). Genomewide 5mC mapping has implicated the role of DNA methylation during early embryogenesis in that DNMTs maintain the existing DNA methylation pattern to stabilize the genome integrity. In vertebrates, after fertilization, the embryo shows a relatively stable (in zebrafish) or low level (in mice) of DNA methylation before early cleavage stages, and then, the overall DNA methylation level gradually increases until gastrulation (Jiang et al, 2013; Potok et al, 2013; Wang et al, 2014). Various development processes including gastrulation and organogenesis occur after the cleavage stages (Kimelman, 2006). However, our knowledge on how DNA methylation regulates early embryonic development is still limited. The body pattern determination is initiated during gastrulation, followed by organogenesis. The organs such as the heart, pancreas, liver, and intestines are then asymmetrically positioned within the body cavity. This left–right (LR) asymmetry is first established by symmetry breaking, and then followed by laterality organizer formation, that is, the node in mammals and the Kupffer's vesicle (KV) in zebrafish (Blum et al, 2014). The progenitor cells of KV are dorsal forerunner cells (DFCs), which arise from a subset of dorsal surface epithelial (DSE) cells at the sphere stage. The DFC cluster first appears adjacent to the embryonic shield, then migrates to the vegetal pole, and forms a rosette-shaped structure, finally differentiates into ciliated epithelial cells of KV (Oteiza et al, 2008). Cilia in laterality organ generate directional fluid flow (i.e., Nodal flow), which thereby leads to asymmetric expression of genes such as Nodal gene (e.g., spaw in fish) and its targets (Matsui & Bessho, 2012; Blum et al, 2014). This asymmetric expression (usually left side-specific expression of Nodal genes) directs internal organ primordium to position asymmetrically in later stages of development (Okada et al, 2005). Several signaling pathways, including Nodal, have been demonstrated to be critical in determining LR asymmetry during vertebrate embryogenesis (Burdine & Schier, 2000; Tanaka et al, 2005; Matsui & Bessho, 2012). However, very little is known about the role of epigenetic regulation in LR determination. In this study, we demonstrate a critical role of DNA methylation in LR asymmetry in vertebrates including zebrafish and Xenopus. We show that loss of dnmt1 or dnmt3bb.1 disrupts laterality of organs including heart, pancreas, and liver. Mechanistically, hypomethylation of the lefty2 gene enhancer caused by loss of dnmt1 can promote lefty2 expression, which in turn inhibits Nodal signaling, therefore leading to impaired DFC specification and loss of LR asymmetry. In addition, Dnmt3bb.1 is required for cadherin 1 (cdh1)-mediated DFC clustering to ensure proper LR determination. Our finding unravels a new layer of regulation involving dynamic DNA methylation in embryonic development in vertebrates. Results DNA methylation signal is enriched in the LR organizer To explore a potential role of DNA methylation in gastrulation, we first knocked down dnmt1 by injecting dnmt1 antisense MOs into zebrafish embryos to reduce the global DNA methylation level as reported previously (Rai et al, 2006), and then performed MeDIP-seq and RNA-seq in control and dnmt1-deficient embryos at early and late gastrulation stages, respectively. The overall methylation level in dnmt1-deficient embryos was much lower than that in control embryos (Fig 1A and Appendix Fig S1). Consistently, differential methylation analysis showed that dnmt1-deficient embryos had significantly more hypomethylated peaks (n = 1,715) than hypermethylated peaks (n = 284) (Fig 1B). Gene ontology (GO) analysis of genes with differentially methylated peaks in dnmt1-deficient embryos revealed an enrichment of genes that are implicated in developmental processes, such as embryo development, pattern specification process, and cell fate commitment (Fig 1C). Notably, genes involved in determination of bilateral symmetry and LR asymmetry were also enriched (Table EV1), indicating a potential role of DNA methylation in this process. Consistently, GO analysis of RNA-seq data showed that developmental process terms were highly enriched among the differentially expressed genes in dnmt1 morphants, such as determination of bilateral symmetry and pattern specification (Fig EV1A and B). To determine whether DNA methylation is directly involved in LR asymmetry determination, we examined the 5mC level in wild-type embryos using immunofluorescence analysis. 5mC was readily detectable in sox17+ DFCs, the progenitor of KV, suggesting potential involvement of DNA methylation in LR determination (Fig 1D). We then investigated which DNMTs are likely to control this process. In zebrafish, there are eight DNMTs, including maintenance DNMT (Dnmt1), “de novo” DNMTs (Dnmt3a/b), and Dnmt2 (Campos et al, 2012). We examined the expression of Dnmt family genes in sox17+ DFCs and found that dnmt1, dnmt3bb.2, dnmt3bb.1, and dnmt3bb.3 were expressed at higher level in DFCs (Fig EV1C). Double fluorescence in situ analysis with the DFC marker, sox17, further showed that dnmt1 and dnmt3bb.1 were specifically enriched in KV and DFCs, respectively (Fig EV1D). Taken together, these data suggest that DNA methylation is possibly involved in LR asymmetry. Figure 1. DNA methylation signal is enriched in Left–Right organizer Circos representation of genomewide DNA methylation level in control and dnmt1 morphants. Each chromosome is marked with different color. The central and outer circles indicate the methylation level in control and dnmt1-deficient embryos, respectively. The middle circle displays distribution of DMRs. Decreased and upregulated DMRs in dnmt1-deficient embryos are indicated in green and red, respectively. P < 0.05, FDR q < 0.05. Differentially methylated peaks in control and dnmt1 morphants. P < 0.05, FDR q < 0.05. Gene ontology analysis of genes with DMRs in dnmt1 morphants. Immunofluorescence of 5mC at 60% epi stage in Tg(sox17:eGFP) embryos. Scale bar, 20 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. DNA methyltransferases expressed in LR organizer Differentially expressed genes in dnmt1 morphants (P < 0.05). Representative GO terms enriched in differentially expressed genes in dnmt1 morphants. Expression level of DNA methyltransferases in DFCs. Error bars, mean ± SD, n = 3 technical replicates. Student's t-test. Double FISH revealed that dnmt1 and dnmt3bb.1 are co-expressed with sox17 in KV and DFC region. Scale bar, 20 μm. Download figure Download PowerPoint DNA methylation is required for LR determination To investigate whether DNA methylation can modulate LR patterning directly, we injected dnmt1 MO or dnmt3bb.1 MO into the yolk at the 512-cell stage as demonstrated previously (Amack & Yost, 2004), to block translation of endogenous dnmt1 or dnmt3bb.1 specifically in DFCs. The efficacy of the dnmt1 translation-blocking MO (atgMO) or dnmt3bb.1 MO (atgMO) was validated using Western blotting or co-injection with an EGFP plasmid reporter (due to lack of Dnmt3b-specific antibody in zebrafish) (Fig EV2A and Appendix Fig S2). Knockdown of dnmt1 or dnmt3bb.1 did not cause any defects during early gastrulation (data not shown). Then, we examined the knockdown effects on organ laterality. During zebrafish embryogenesis, the initially midline-positioned heart tube undergoes a leftward jogging at 28 h post-fertilization (hpf) (Stainier, 2001). In dnmt1-deficient embryos, we observed abnormal cardiac jogging, including rightward and middling looping by detecting nkx2.5 expression and visualizing heart position in cmlc2:GFP transgenic embryos at 30 hpf (Fig 2A). To test the specificity of dnmt1 MO, we generated dnmt1 mis-mRNA (with the mutated atgMO target sequence without changing amino acid coding) and co-injected it with dnmt1 MO into the 1-cell stage embryos. Western blotting results showed that dnmt1 mis-mRNA can efficiently restore Dnmt1 expression in dnmt1 morphants (Fig EV2A). As expected, overexpression of dnmt1 mis-mRNA restored the randomized organ laterality in dnmt1 morphants (Fig EV2B). Furthermore, a dnmt1 splice-blocking MO was also used, and its specificity was validated by Western blotting (Fig EV2C). The abnormal cardiac jogging was also found in embryos injected with dnmt1 splice MO (Fig EV2D), supporting that the disrupted organ laterality was caused by dnmt1 knockdown specifically. To further confirm these results, we outcrossed the dnmt1 mutant (dnmt1s872) with Tg(fabp10:dsRed, ela3l:GFP)gz12; Tg(ins:dsRed)m1081 to examine the organ laterality (Anderson et al, 2009). Because of the maternal expression of dnmt1, the mutant appeared morphologically normal in comparison with the wild type and heterozygotes (Fig EV2E). Therefore, a low dose of dnmt1 MO was injected into 1-cell stage embryos including homozygous mutants, wild-type and heterozygous siblings to block the maternal expression of dnmt1. The results showed that a low dose of MO injection failed to affect organ laterality in wild type and heterozygotes, while the injected homozygous mutant exhibited randomized liver and pancreas (Fig EV2E). Collectively, these data support a critical role of dnmt1 in LR asymmetry. Click here to expand this figure. Figure EV2. Deficiency of dnmt1 or dnmt3bb.1 causes defects of organ patterning and DFC development Protein level of Dnmt1 in the control, dnmt1 morphants, dnmt1 MO and mis-mRNA-co-injected embryos at 75% epi stage by Western blotting. Representative images showing heart asymmetry labeled by nkx2.5 at 30 hpf (left panel) with quantification (right panel). Western blotting analysis of Dnmt1 in the control and dnmt1 splice MO-injected embryos at 75% epi stage. Representative images showing heart asymmetry labeled by nkx2.5 at 30 hpf (left panel) with quantification (right panel). Representative images showing pattern of liver and pancreas in Tg(fabp10:dsRed; ela3l:GFP); Tg(ins:dsRED) embryos at 4 dpf (left panel) with quantification (right panel). Scale bar, 100 μm. Representative images showing nkx2.5 expression at 30 hpf in control, dnmt3bb.1 morphants and dnmt3bb.1 mis-mRNA-rescued embryos (left panel) with quantification (right panel). Generation of dnmt3bb.1 mutant using the CRISPR/Cas9 technique. Representative images showing nkx2.5 expression at 30 hpf in control, dnmt3bb.1 mutants (left panel) with quantification (right panel). The expression of foxj1a and sox17 in DFCs at 75% epi stage in control and embryos injected with dnmt1 splice MO. The expression of sox17 in DFCs at 75% epi stage was reduced in dnmt1 homozygous mutant injected with a lose-dose dnmt1 MO. Download figure Download PowerPoint Figure 2. Defects in LR determination in Dnmt morphants A–C. Representative images showing nkx2.5 expression in the heart at 30 hpf, foxa3 expression in visceral organs at 46 hpf or heart looping at 30 hpf using Tg(cmlc2: GFP) embryos (bottom panel) in control and DFC-specific-deficient embryos. Statistical analysis is shown on the right with the total observed number of embryos indicated above each bar. Effects of DFC-specific dnmt1 (A), dnmt3bb.1 (B), and dnmt1 + dnmt3bb.1 (C) knockdown on organ laterality. Scale bar, 100 μm. Dashed lines denote the embryonic midline. Download figure Download PowerPoint Interestingly, dnmt3bb.1 MODFC embryos also showed abnormal laterality based on examination of the cardiac marker nkx2.5 at 30 hpf and the liver/pancreas marker, foxa3 at 46 hpf (Fig 2B), and the modified dnmt3bb.1 mRNA (dnmt3bb.1 mis-mRNA) injection can partially restore this abnormal laterality (Fig EV2F). To further confirm these results, we next generated a dnmt3bb.1 null mutant with an 11-bp insertion in the third exon (236 bp after the start codon) using the CRISPR/Cas9 technique (Fig EV2G). We found that the cardiac jogging was also disturbed in dnmt3bb.1 mutant (Fig EV2H), indicating an important role of dnmt3bb.1 in LR development. Since both dnmt1 and dnmt3bb.1 are required for LR patterning, we next investigated whether the LR defects would be more severe in double-knockdown embryos. Examination of nkx2.5 at 30 hpf indeed revealed more severe abnormal laterality (37 out of 56 embryos) in embryos co-injected with dnmt1 and dnmt3bb.1 MOs (Fig 2C), compared to individual morphants (Fig 2A and B). Together, these results demonstrate that DNA methylation is required for organ laterality in zebrafish. Dnmt1 modulates DFC specification and KV formation Previous studies in vertebrates including zebrafish, Xenopus, chicken and mice have demonstrated that organ laterality is regulated by asymmetric expression of LR genes (Hamada et al, 2002; Long et al, 2003). We thus analyzed the early asymmetric marker, southpaw (spaw, the Nodal-related gene in zebrafish), and its downstream target gene lefty2. Normally, spaw is expressed in the left lateral plate mesoderm (LPM), while lefty2 is expressed in the anterior LPM at late somitogenesis and later on in the heart primordium on the left side of embryo. Knockdown of dnmt1 resulted in the randomized expression pattern of spaw and lefty2 (Appendix Fig S3). Given that the asymmetric gene expression is induced by the laterality organizer, we then examined the LR organizer, KV, including organizer formation and ciliogenesis at earlier stages. Live imaging of Tg(sox17:eGFP) embryos and immunofluorescence staining using anti-acetylated tubulin showed that dnmt1 deficiency led to the disrupted lumen formation as well as the decrease in the number and length of primary cilia in the KV (Fig 3A and B). Ciliated epithelial cells of the KV generate the leftward Nodal flow, which is required for LR patterning (Hamada et al, 2002). We found that directional KV fluid flow was also disrupted in dnmt1 morphants (Fig 3C and Movies EV1 and EV2), indicating malformed KV function. Together, these results indicate the requirement of Dnmt1 for KV formation and ciliogenesis. Figure 3. Dnmt1 modulates KV formation and DFC specification Live imaging of KV in Tg(sox17:eGFP) embryo. Scale bar, 100 μm. Confocal images of cilia immunostained with Ac-tubulin and cell membrane labeled with exogenous CAAX-mCherry in KV region (dashed circles). Right panel shows quantification analysis of cilia number and length. Scale bar, 20 μm. KV fluid flow in control embryos and dnmt1 morphants with bead tracks. Yellow circles denote KV region. Scale bar, 50 μm. Expression of foxj1a and sox17 in DFCs in control- and dnmt1-deficient embryos. DFCs were visualized at shield and 60% epi stage in Tg(sox17:eGFP) embryos (left panel). The right panel shows quantification analysis of cell number. Scale bar, 20 μm. Injection of dnmt1 mis-mRNA restored the reduced expression of sox17 and foxj1a in dnmt1 morphants. Visualization of DFCs at 75% epi stage in Tg(sox17:eGFP) embryos showing that overexpression of dnmt1 mis-mRNA restored the number of DFCs in dnmt1 morphants (left panel) with quantification (right panel). Scale bar, 20 μm. Data information: (B, E and G) Error bars, mean ± SD, n ≥ 5 embryos per experiment and n ≥ 2 technical replicates. *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test. (D and F) Numbers indicate the number of embryos with the respective phenotype/total number of embryos analyzed in each experiment. Download figure Download PowerPoint To investigate whether DNA methylation affects LR patterning through regulation of DFC specification, we examined the expression of early markers specific for DFC fate specification (sox17) and differentiation (foxj1a), respectively. The expression of these two genes was decreased in dnmt1 MODFC embryos and in dnmt1 splice MO-injected embryos (Figs 3D and EV2I). Furthermore, the number of sox17:eGFP-labeled DFCs at shield and 60% epiboly (epi) stages was significantly reduced in dnmt1 morphants (Fig 3E), indicating that both DFC specification and the cell number were affected by dnmt1 knockdown. TUNEL assay showed more apoptosis in the DFCs of dnmt1 morphants, compared to controls, whereas there was no discernable difference on the cell proliferation in DFCs (Appendix Fig S4A and B). In addition, overexpression of dnmt1 mis-mRNA efficiently rescued the reduced expression of sox17 and foxj1a as well as the number of DFCs at shield stage in dnmt1 MO-injected embryos (Fig 3F and G). Given there were no obvious differences in the expression of sox17 in DFCs among embryos from dnmt1 heterozygous carrier fish, we then used a low dose of dnmt1 MO injection. As a result, we found a significantly reduced sox17 expression in homozygous mutant but not in wild-type sibling or heterozygous embryos (Fig EV2J). Collectively, these data suggest that dnmt1 affects LR asymmetry through modulating DFC specification and KV formation. Next, to determine whether regulation of LR asymmetry by dnmt1 is conserved across vertebrates, we used Xenopus, a well-established model for LR asymmetry studies (Blum et al, 2009). We injected 15–30 ng of xdnmt1 MO (Dunican et al, 2008) into one dorsal blastomere of Xenopus embryos at the 4-cell stage and validated its efficiency using Western blotting (Fig EV3A). Examination of the heart and gut looping at the tadpole stage showed that about 60% of xdnmt1 MO-injected embryos (27 out of 47 embryos) exhibited heterotaxia or situs inversus, suggesting a conserved and gene-specific effect of dnmt1 on the LR patterning in Xenopus (Fig EV3B and C). Accordingly, we examined the cilia in gastrocoel roof plate (GRP), the LR organizer of Xenopus, by anti-acetylated tubulin staining. The number of acetylated tubulin-positive cilia was decreased on the side that received the xdnmt1 MO injection, but not on the non-injected side that served as control (Fig EV3D). Collectively, these results demonstrate that DNA methylation is a conserved regulatory mechanism during LR asymmetry determination in vertebrates. Click here to expand this figure. Figure EV3. Knockdown of dnmt1 in Xenopus disrupts left–right asymmetry and ciliogenesis A. Western blotting analysis showing the protein level of DNMT1 in the control and xdnmt1 morphants at stage 45. B, C. Representative imaging showing heart and gut looping in Xenopus embryos at stage 45 (B) with quantification (C). (B) The white and red dashed lines mark heart and gut, respectively. D. Visualization of cilia in GRP using anti-acetylated tubulin immunofluorescence with gastrocoel roof cells labeled by Alexa488-phalloidin staining, showing that the cilia length and number were decreased on the side that received the xdnmt1 MO injection, but not on the uninjected side. Scale bar, 20 μm. Source data are available online for this figure. Download figure Download PowerPoint Lefty2 mediates DFC specification downstream of Dnmt1 Our results above reveal an epigenetic regulation of DFC formation by DNA methylation. Numerous studies have demonstrated that the Nodal signaling pathway plays crucial roles in the process of DFC formation, whereas the number of DFCs increases or decreases in response to the enhanced or reduced Nodal signaling, respectively (Schier & Talbot, 2005). As a repressor of Nodal signaling, the elevated lefty2 expression leads to reduced number of DFCs (Choi et al, 2007; Oteiza et al, 2008). Since the morphological patterning defects of DFCs observed in dnmt1-deficient embryos resemble those of lefty2-mRNA-injected embryos (Oteiza et al, 2008), we next analyzed lefty2 expression in dnmt1-deficient embryos to test whether dnmt1 affects DFC formation through altering lefty2 expression. Whole-mount in situ hybridization (WISH) analysis confirmed that the expression of lefty2 was markedly increased at 60% epi and 75% epi stages in dnmt1-deficient embryos (Fig EV4A). Similarly, qPCR analysis also confirmed a significant increase of lefty2 expression upon dnmt1 knockdown in manually dissected sox17:eGFP+ DFCs (Figs 4A and EV4B). Double fluorescence in situ hybridization using Tg(sox17:eGFP) showed that lefty2 expression in DFCs was obviously increased upon dnmt1 deficiency (Fig 4B). We also evaluated the expression of Nodal target genes in DFCs. The qPCR analysis showed that the mRNA levels of these Nodal-regulated genes (chd, ntl, dkk1b, dusp6, gata5, id3, pitx2a, and fgf8) were decreased in dnmt1 morphants (Fig 4C). To test whether overexpression of lefty2 alone can mimic the defects of DFC specification observed in dnmt1 morphants, we injected lefty2-mRNA into sox17:eGFP transgenic embryos at 1-cell stage. As expected, overexpression of lefty2 decreased the number of DFCs in a dose-dependent manner (Fig EV4C). Click here to expand this figure. Figure EV4. lefty2 deficiency rescues DFC specification defects through restoration of Nodal signaling The expression of lefty2 at 60% epi and 75% epi stage in dnmt1-deficient embryos was upregulated, compared to control embryos. Schematic diagram showing the experimental procedure for the isolation of GFP-positive DFCs from sox17:GFP transgenic zebrafish and using DNA and RNA extracted from these DFCs for bisulfite PCR or qPCR. lefty2 mRNA overexpression decreased the number of sox17+ DFCs at 75% epi stage. Scale bar, 20 μm. The reduced expression of foxj1a and sox17 at 60% epi in dnmt1 morphants was restored by lefty2-MO co-injection. lefty2 MO partially rescued the reduced expression of lefty1, gata6 and ephrinb2b in DFCs of dnmt1 morphants. The dotted black outlines denote DFC region. qPCR analysis of Nodal target genes in control embryos, dnmt1 morphants, and dnmt1 morphants co-injected with lefty2 MO. Representative images showing heart pattern labeled by cmlc2 at 30 hpf (upper panel) with quantification (lower panel). Data information: Error bars, mean ± SD, n ≥ 5 embryos per experiment, n ≥ 2 technical replicates (C) and n = 3 technical replicates (F). *P < 0.05, **P < 0.01, ***P < 0.001. Student's t-test. (A, D and E) Numbers indicate the number of embryos with the respective phenotype/total number of embryos analyzed in each experiment. Download figure Download PowerPoint Figure 4. Lefty2 mediates Dnmt1 regulation of DFC specification qPCR analysis of lefty2 expression in DFCs in control and dnmt1 MO-injected embryos. FISH analysis using Tg(sox17:eGFP) showing that lefty2 expression in DFCs was increased upon dnmt1 deficiency. Scale bar, 20 μm. qPCR analysis of Nodal target genes (chd, ntl, dkk1b, dusp6, gata5, id3, pitx2a, fgf8) in DFCs. Visualization of sox17+ DFCs in control embryos, dnmt1 MODFC- and dnmt1 + lefty2 MODFC-injected embryos (left panel) with quantification (right panel). Scale bar, 20 μm. Representative heart looping pattern in Tg(cmlc2:GFP) embryos at 30 hpf (left panel) and statistical analysis is shown on the right with the total observed number of embryos indicated above each bar. Scale bar, 100 μm. Data information: Error bars, mean ± SD, n = 3 technical replicates (A and C), n = 5 embryos per experiment and n ≥ 2 technical replicates (D). *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test. Download figure Download PowerPoint Next, we tested whether decreasing lefty2 expression would rescue the LR defects in dnmt1 morphants. Single lefty2 MO injection into wild-type embryos did not result in any laterality defects; however, co-injection of dnmt1 and lefty2 MOs led to restoration of sox17 and foxj1a expression and the number of DFCs (Figs 4D and EV4D). A significant restoration of Nodal target gene expression was also observed in co-injected embryos (Fig EV4E and F). Moreover, the proportion of embryos with normal heart looping was increased, compared to dnmt1 morphants and dnmt1DFC-injected embryos (Figs 4E and EV4G). Taken together, these results demonstrate that Dnmt1 regulates LR asymmetry through Lefty2-mediated DFC specification. Dnmt1 represses the expression of lefty2 through DNA methylation To verify whether lefty2 expression alteration in dnmt1 morphants was directly regulated by DNA methylation. Gene-specific inspection of MeDIP data revealed a region with decreased DNA methylation at the lefty2 enhancer in dnmt1-deficient embryos (Fig" @default.
• W2753476076 created "2017-09-15" @default.
• W2753476076 creator A5021602083 @default.
• W2753476076 creator A5029484872 @default.
• W2753476076 creator A5045468337 @default.
• W2753476076 creator A5049171847 @default.
• W2753476076 creator A5060470951 @default.
• W2753476076 creator A5073270106 @default.
• W2753476076 date "2017-09-07" @default.
• W2753476076 modified "2023-10-12" @default.
• W2753476076 title "Epigenetic regulation of left–right asymmetry by <scp>DNA</scp> methylation" @default.
• W2753476076 cites W1559457086 @default.
• W2753476076 cites W1911369697 @default.
• W2753476076 cites W1966871815 @default.
• W2753476076 cites W1967546149 @default.
• W2753476076 cites W1969212379 @default.
• W2753476076 cites W1980398966 @default.
• W2753476076 cites W1981197560 @default.
• W2753476076 cites W1982215720 @default.
• W2753476076 cites W1987356909 @default.
• W2753476076 cites W1987557775 @default.
• W2753476076 cites W1995542274 @default.
• W2753476076 cites W2005756834 @default.
• W2753476076 cites W2020088788 @default.
• W2753476076 cites W2023856799 @default.
• W2753476076 cites W2029689141 @default.
• W2753476076 cites W2034793355 @default.
• W2753476076 cites W2040063035 @default.
• W2753476076 cites W2041884054 @default.
• W2753476076 cites W2046243315 @default.
• W2753476076 cites W2049365982 @default.
• W2753476076 cites W2059813768 @default.
• W2753476076 cites W2064107554 @default.
• W2753476076 cites W2066473933 @default.
• W2753476076 cites W2073264990 @default.
• W2753476076 cites W2076812266 @default.
• W2753476076 cites W2077429965 @default.
• W2753476076 cites W2083904901 @default.
• W2753476076 cites W2088352274 @default.
• W2753476076 cites W2089096062 @default.
• W2753476076 cites W2093543973 @default.
• W2753476076 cites W2094370232 @default.
• W2753476076 cites W2097851896 @default.
• W2753476076 cites W2099057248 @default.
• W2753476076 cites W2099635668 @default.
• W2753476076 cites W2110061827 @default.
• W2753476076 cites W2125349610 @default.
• W2753476076 cites W2136905998 @default.
• W2753476076 cites W2139122477 @default.
• W2753476076 cites W2142535592 @default.
• W2753476076 cites W2145055902 @default.
• W2753476076 cites W2152274458 @default.
• W2753476076 cites W2159869422 @default.
• W2753476076 cites W2164550220 @default.
• W2753476076 cites W2165603299 @default.
• W2753476076 cites W2166901270 @default.
• W2753476076 cites W2167773905 @default.
• W2753476076 cites W2412536215 @default.
• W2753476076 cites W2532812479 @default.
• W2753476076 cites W2587996389 @default.
• W2753476076 doi "https://doi.org/10.15252/embj.201796580" @default.
• W2753476076 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5641680" @default.
• W2753476076 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28882847" @default.
• W2753476076 hasPublicationYear "2017" @default.
• W2753476076 type Work @default.
• W2753476076 sameAs 2753476076 @default.
• W2753476076 citedByCount "21" @default.
• W2753476076 countsByYear W27534760762018 @default.
• W2753476076 countsByYear W27534760762019 @default.
• W2753476076 countsByYear W27534760762020 @default.
• W2753476076 countsByYear W27534760762021 @default.
• W2753476076 countsByYear W27534760762022 @default.
• W2753476076 countsByYear W27534760762023 @default.
• W2753476076 crossrefType "journal-article" @default.
• W2753476076 hasAuthorship W2753476076A5021602083 @default.
• W2753476076 hasAuthorship W2753476076A5029484872 @default.
• W2753476076 hasAuthorship W2753476076A5045468337 @default.
• W2753476076 hasAuthorship W2753476076A5049171847 @default.
• W2753476076 hasAuthorship W2753476076A5060470951 @default.
• W2753476076 hasAuthorship W2753476076A5073270106 @default.
• W2753476076 hasBestOaLocation W27534760761 @default.
• W2753476076 hasConcept C104317684 @default.
• W2753476076 hasConcept C150194340 @default.
• W2753476076 hasConcept C153911025 @default.
• W2753476076 hasConcept C190727270 @default.
• W2753476076 hasConcept C33288867 @default.
• W2753476076 hasConcept C41091548 @default.
• W2753476076 hasConcept C54355233 @default.
• W2753476076 hasConcept C552990157 @default.
• W2753476076 hasConcept C86803240 @default.
• W2753476076 hasConcept C95444343 @default.
• W2753476076 hasConceptScore W2753476076C104317684 @default.
• W2753476076 hasConceptScore W2753476076C150194340 @default.
• W2753476076 hasConceptScore W2753476076C153911025 @default.
• W2753476076 hasConceptScore W2753476076C190727270 @default.
• W2753476076 hasConceptScore W2753476076C33288867 @default.
• W2753476076 hasConceptScore W2753476076C41091548 @default.
• W2753476076 hasConceptScore W2753476076C54355233 @default.

• next