The ENU-induced cetus mutation reveals an essential role of the DNA helicase DDX11 for mesoderm development during early mouse embryogenesis

Authors


Abstract

Background: DDX11 is a DNA helicase of the conserved FANCJ/RAD3/XPD family involved in maintaining genome stability. Studies in yeast and humans have shown requirements for DDX11 in sister chromatid cohesion and DNA repair. In mouse, loss of Ddx11 results in embryonic lethality. However, the developmental defects of Ddx11 mutants are poorly understood. Results: We describe the characterization and positional cloning of cetus, a mouse ENU-induced mutation in Ddx11. We demonstrate that cetus causes a nonconservative amino acid change in DDX11 motif V and that this mutation is a null allele of Ddx11. cetus mutant embryos failed to thrive beyond embryonic day 8.5 and displayed placental defects similar to those described in Ddx11 null embryos. Additionally, our characterization of Ddx11cetus mutants identified embryonic phenotypes that had not been previously reported in Ddx11KO embryos, including loss of somitic mesoderm, an open kinked neural tube and abnormal heart looping. We show that loss of Ddx11 causes widespread apoptosis from early embryonic stages and that loss of Ddx11 disrupts somitic mesoderm more dramatically than other embryonic cells. Conclusions: Our results identify novel roles of Ddx11 during embryo morphogenesis and demonstrate that the activity of its motif V is essential for DDX11 function. Developmental Dynamics 241:1249–1259, 2012. © 2012 Wiley Periodicals, Inc.

INTRODUCTION

Genome integrity is preserved by mechanisms that ensure accurate DNA replication, proper segregation of the duplicated material into daughter cells, and repair of DNA damage (Luijsterburg and van Attikum,2011; Mannini and Musio,2011). These mechanisms include a series of cell cycle checkpoint controls that safeguard genome stability by inducing cell cycle arrest and/or apoptosis (Rieder,2011). DEAD/H box polypeptide 11 (DDX11), also known as CHLR1, is a helicase of the SF2 superfamily essential for genome integrity (Gerring et al.,1990; Spencer et al.,1990; Parish et al.,2006; Inoue et al.,2007; van der Lelij et al.,2010). First identified in genetic screens for chromosome transmission fidelity in S. cerevisiae, chl1 yeast mutants show a high rate of chromosome missegregation and G2/M cell cycle arrest (Liras et al.,1978; Gerring et al.,1990). In mammals, loss of Ddx11 also causes defects characteristic of genomic instability such as chromosome misalignment during mitosis, G2/M cell cycle arrest, aneuploidy, and increased apoptosis (Parish et al.,2006; Inoue et al.,2007).

Loss of genome integrity in yeast chl1 mutants, as well as human and mouse cells lacking Ddx11, has been attributed to a precocious separation of sister-chromatids during mitosis (Petronczki,2004; Skibbens,2004; Inoue et al.,2007; van der Lelij et al.,2010). Consistent with a role in sister-chromatid cohesion, DDX11 has been shown to physically interact with structural components of the cohesin complex, a ring-shaped multiprotein complex that is loaded onto DNA before replication and encircles the replicated chromosomes after passage of the replication fork (Parish et al.,2006; Uhlmann,2009; Sherwood et al.,2010). Chl1p/DDX11 has also been shown to bind factors that participate in the establishment of cohesion, including Ctf4, Ctf7, Ctf18-RFC and Swi1/TIMELESS (Skibbens,2004; Farina et al.,2008; Leman et al.,2010), and a functional role for some of these complexes has been supported by genetic interactions in yeast (Petronczki,2004; Skibbens,2004). These findings, together with the fact that only ectopic expression of CHL1 at S phase can restore the defects of chl1 yeast mutants (Petronczki,2004), have led to the conclusion that Chl1p/DDX11 is required during the S phase for the establishment of sister-chromatid cohesion, rather than for its maintenance through the rest of the cell cycle (Sherwood et al.,2010; reviewed in Uhlmann,2009).

In addition to its well described roles in the establishment of sister-chromatid cohesion, Chl1p/DDX11 has been suggested to maintain genome stability by participating in DNA repair. Although expression of Ddx11 is not up-regulated in response to UV-induced DNA damage (Amann et al.,1997), evidence for a role of Chl1p/DDX11 in DNA repair comes from the findings that loss of CHL1/DDX11 in yeast and humans renders cells hypersensitive to DNA damaging agents (Chang et al.,2002; Laha et al.,2006; Pan et al.,2006; Ogiwara et al.,2007; van der Lelij et al.,2010). Nonetheless, because sister-chromatid cohesion is a prerequisite for efficient double-stranded DNA break repair (Ström et al.,2004,2007; Unal et al.,2007), the prevailing view is that Chl1p/DDX11 has a an indirect role in DNA repair as a cohesion establishment factor, rather than a direct function in repairing DNA damage.

The importance of DDX11 in maintaining genome stability has been emphasized by a recent study showing that loss of DDX11 function in humans is responsible for Warsaw Breakage syndrome, an inherited disorder characterized by growth retardation, defects in DNA repair, and abnormal sister-chromatid cohesion (van der Lelij et al.,2010). Additionally, a targeted null allele of Ddx11 causes early embryonic lethality in mice (Inoue et al.,2007). Analysis of mouse mutants, as well as studies in cell culture, have indicated that loss of Ddx11 causes G2/M cell cycle arrest and apoptosis (Inoue et al.,2007). Ddx11 null mutants have growth retardation as early as embryonic day (E) 7.5, sparse cellularity at somite stages, and fail to survive beyond E10.5 (Inoue et al.,2007). The embryonic lethality of Ddx11KO embryos was attributed to a failure to develop a robust placenta (Inoue et al.,2007). However, the embryonic defects of Ddx11KO mutants have not been extensively characterized.

Here, we describe the phenotypic characterization and positional cloning of cetus, a mouse N-ethyl-N-nitrosourea (ENU) -induced mutation. We determined that cetus causes a mutation in DDX11 helicase motif V and demonstrate genetically that this mutation is a null allele of Ddx11. Similar to Ddx11KO embryos, chorioallantoic fusion failed in cetus mutants, preventing the development of the placenta. However, we found that cetus embryos also contained severe morphological defects in somitic mesoderm, neural tube, and heart. We show that loss of Ddx11 causes widespread apoptosis from early gastrula stages and that a significant percentage of Ddx11cetus and Ddx11KO embryos die at this developmental stage. In Ddx11cetus and Ddx11KO embryos with late embryonic lethality, loss of Ddx11 disrupts somitic mesoderm more dramatically than other embryonic cell types, suggesting that Ddx11 is differentially required in embryonic tissues during early development. Together, our results define novel roles of DDX11 during early mouse embryogenesis and demonstrate that its motif V is essential for DDX11 function.

RESULTS

The cetus Mutation Disrupts Embryonic Development

The cetus mutation was isolated in an ENU-mutagenesis screen to identify recessive mutations that disrupt embryonic morphogenesis (Garcia-Garcia et al.,2005). Embryos homozygous for the cetus mutation arrested before E8.5 with severe developmental defects (Fig. 1). Most cetus mutants had a morphology typical of an E8.5 embryo, but were significantly smaller and lacked morphologically distinguishable somites (Fig. 1A,C,E). In some cases, cetus mutants contained blisters at both sides of the midline (Fig. 1C,D). Additionally, the neural tube epithelia of cetus embryos remained open and was frequently deformed, appearing wavy and/or kinked along the anterior–posterior axis (Fig. 1E,F). All cetus embryos that survived until late E8.5 also failed to undergo cardiac looping, retaining a straight heart tube (Fig. 2A,B). By E10.5, cetus mutant embryos were resorbed and only residual embryonic tissue could be observed inside the decidua.

Figure 1.

cetus mutants have severe embryonic defects. A–F: Whole-mount (A,C,E) and transverse sections (B,D,F) of E8.5 wild-type (A,B) and cetus mutant embryos (C–F). Arrowhead in C,D point to blisters (bl), areas devoid of cells that were confined dorsally by surface ectoderm (sect), ventrally by definitive endoderm (end) and laterally by lateral plate mesoderm (LPM). Cell debris was frequently observed in the blisters of cetus mutants. Arrowhead in F points to the flank of a cetus mutant lacking somites (som). Scale bars = 200 μm.

Figure 2.

cetus mutants have heart defects and abnormally localized cells in the amniotic cavity. A,B: Frontal views of wild-type (A) and cetus mutant (B) embryos at embryonic day (E) 8.5 showing the heart area. Dashed lines in A,B represent the direction of heart looping. In the cetus mutant, the heart remains unlooped as a straight tube and no chambers are discernible (B). C,E,G: Whole-mount in situ hybridization for Mox1 expression on wild-type embryos and cetus mutants, with late (E) and early (G) developmental arrest, dissected at E8.5. D,F,H: Transverse sections of embryos in C, E, and G, respectively. Dashed lines in D and F mark the amnion (amn). Arrowheads point to abnormally localized cells in the amniotic cavity of cetus mutants. Bracket in E highlights the absence of Mox1 expression. Scale bars in A (for pictures in A,B) and C (for pictures in C, E, G) represent 200 μm. Scale bars in D (for pictures in D and F) and in H represent 100 μm.

Analysis of transverse embryonic sections corroborated the lack of somitic mesoderm cells in cetus mutants. In cetus embryos containing blisters, somites were substituted by an area devoid of cells which was confined dorsally by the surface ectoderm, ventrally by the definitive endoderm and laterally by the lateral plate mesoderm (Fig. 1D). In cetus mutants without blisters, somitic mesoderm cells were also missing, but the surface ectoderm was in direct apposition to the endoderm and lateral plate mesoderm (Fig. 1F).

In addition to embryos with E8.5 developmental arrest, we found that some cetus mutants arrested at E7.5 with a morphology most similar to that of gastrula stage embryos (Fig. 2G,H). The penetrance of this early lethality was dependent on the genetic background. Hence, in a congenic C3H/FeJ background, 46.9% (n = 23/49) of cetus mutants arrested at E7.5, but in a CAST/Ei mixed background only 11.1% (n = 10/90) of cetus embryos had an early lethality. Although early-arrest cetus mutants retained the characteristic cylindrical shape of E7.5 embryos, they contained cells abnormally localized in the amniotic cavity (Fig. 2G,H). We also found cells accumulated in the amniotic cavity of cetus mutants with late developmental arrest at E8.5 (Fig. 2E,F). Some of these cells were pyknotic when visualized with DAPI staining (Fig. 6B, inset), suggesting that the phenotypes of cetus mutants could originate as a consequence of cell apoptosis. Despite the fact that the penetrance of the early and late phenotypes was influenced by the genetic background, embryos with early lethality had similar developmental defects regardless of the genetic background in which they were raised, as did embryos with late lethality.

cetus Is a Mutation in the DEAD/H-Box Helicase DDX11

Using microsatellite-based linkage analysis, the cetus mutation was mapped to a 1.1 Mb interval on chromosome 17 (Fig. 3A). This region contained 11 candidate genes, all of which were sequenced in wild-type and cetus embryonic samples. In cetus mutants, this analysis identified a single T to C mutation in the DEAD/H-box helicase Ddx11 (Fig. 3B). This T-C mutation created a BseRI restriction site polymorphism that was used to confirm linkage to cetus mutant embryos and heterozygote cetus carrier animals (Fig. 3C).

Figure 3.

cetus positional cloning revealed a mutation in Ddx11. A: cetus was mapped to a 1.1 Mb interval on chromosome 17 containing 11 genes (red line). Numbers to the right of the chromosome indicate the number of independent recombinants separating the mutation from the corresponding simple sequence length polymorphism (SSLP) markers indicated to the left. For linkage analysis, a total of 2,018 chromosomes were analyzed. B: Chromatograms showing Ddx11 cDNA sequence from wild-type and cetus embryos. Nucleotide 2228 of Ddx11 open reading frame (highlighted in yellow) was mutated in cetus, producing a L743P amino acid substitution. C: The cetus mutation creates a BseRI restriction site polymorphism that was used to confirm linkage. A 1,817 bp polymerase chain reaction-amplified DNA (odd lanes) was digested with BseRI (even lanes), producing fragments of 1,272 bp, 335 bp, and 210 bp for the wild-type allele and fragments of 1,482 bp and 335 bp for the cetus allele. The empty arrowhead points to the absence of the 210 bp product in homozygote cetus embryos. The solid arrowhead points to the 1,482 bp product, present in all embryos carrying the cetus mutation.

The cetus mutation is predicted to produce a nonconservative Leu to Pro amino acid substitution in DDX11 motif V, a highly conserved motif in helicases of the FANCJ/RAD3/XPD family (Fig. 4A; Wu et al.,2008; Fairman-Williams et al.,2010). To confirm whether the cetus mutation in motif V disrupts the activity of DDX11, we performed genetic complementation analysis with a null allele of Ddx11 (Ddx11KO; Inoue et al.,2007). We found that Ddx11cetus/KO heterozygous embryos arrested between E7.5 and E8.5 with similar defects to those we previously observed in cetus embryos, including small size, absence of somitic mesoderm and kinked neural tube (Fig. 4D). The same phenotypes were also observed in Ddx11KO embryos (Figs. 4E, 5). Altogether, these results demonstrate that cetus is a null allele of Ddx11 and that its motif V is absolutely required for DDX11 function.

Figure 4.

The cetus mutation in DDX11 motif V completely disrupts DDX11 function. A: Domain structure of mDDX11 showing the seven characteristic motifs of DNA helicases. B: The sequence comparison indicates the amino acid conservation of motif V among members of the FANCJ/RAD3/XPD family and RECQL, a DNA helicase of the SF2 superfamily. Gray boxes indicate areas of high conservation across different phyla. The red box highlights the Leu residue disrupted in cetus mutants, which is conserved in most, but not all FANCJ/RAD3/XPD family members. Amino acids in red font indicate residues mutated in human patients with xeroderma pigmentosum, Cockayne syndrome, or trichothiodystrophy (Fan et al.,2008). C–E: The cetus mutation did not complement a null (KO) allele of Ddx11, as judged from the similarities between the phenotypes of cetus (C), cetus/KO (D), and KO (E) embryos dissected at embryonic day (E) 9. Brackets highlight the absence of somites (som). Scale bar = 200 μm.

Figure 5.

Embryonic defects in Ddx11KO embryos. A–C: Whole-mount wild-type (A) and Ddx11KO embryos with late (B) and early (C) developmental arrest when dissected at embryonic day (E) 8.5. D,E: Whole-mount in situ hybridization showing Mox1 expression in E8.5 wild-type (D) and Ddx11KO (E) embryos. Bracket in B highlights the lack of somitic mesoderm. Arrowhead in C points to abnormal cells in the amniotic cavity of an early-arrest Ddx11KO mutant. som, somites. Scale bars = 200 μm.

The cetus Mutation Causes Widespread Apoptosis from Early Embryonic Stages

The presence of pyknotic cells in the amniotic cavity of cetus mutants with both early and late embryonic lethality suggested that apoptosis might contribute to the embryonic phenotypes of cetus embryos (Fig. 2E–H). To test this hypothesis, we analyzed cetus mutants using TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling) staining, which detects the DNA fragmentation characteristic of the late stages of the apoptosis process (Gavrieli et al.,1992). To quantitatively compare the levels of apoptosis in cetus embryos, we also performed TUNEL staining on Ddx11KO mutants, which were previously described to have increased levels of cell death due to genome instability (Inoue et al.,2007). We found that E7.5 Ddx11cetus and Ddx11KO mutants contained a dramatic increase in the number of TUNEL-positive cells as compared with wild-type littermates (Fig. 6A–D). Detailed quantification of the number of TUNEL-positive cells indicated that the levels of apoptosis in Ddx11cetus embryos were similar to those of null Ddx11 mutants (Fig. 6D), providing further evidence that the cetus mutation in motif V causes defects similar to those caused by complete loss of DDX11 function.

Figure 6.

Loss of Ddx11 causes widespread apoptosis at early embryonic stages, but does not affect the proliferative index of the embryo. A–C: TUNEL (green; terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling) and DAPI (blue; 4′,6-diamidine-2-phenylidole-dihydrochloride) staining in embryonic day (E) 7.5 wild-type (A), Ddx11cetus (B), and Ddx11KO (C) embryos. D: Quantification of TUNEL-positive cells in Ddx11cetus mutants raised in a C3H/FeJ background revealed a dramatic increase in apoptosis as compared to wild-type littermates (n = 12 sections, 3 embryos for wild-type and Ddx11cetus; n = 7 sections, 3 embryos for Ddx11KO). Ddx11cetus embryos raised in a CAST/Ei background showed a similar increase in widespread apoptosis (not shown). E–G: Phospho-histone-3 (red) and DAPI (blue) staining in E7.5 wild-type (E), Ddx11cetus (F), and Ddx11KO (G) embryos. H: Quantification of phospho-histone-3-positive cells failed to reveal statistically significant differences between wild-type and Ddx11 mutants (n = 9 sections, 3 embryos for wild-type; n = 12 sections, 3 embryos for Ddx11cetus; n = 3 sections, 3 embryos for Ddx11KO). Insets in B, C, and F are higher magnifications of the embryos in the corresponding panels. Discontinuous lines indicate the border between the embryonic epithelia and the amniotic cavity. Arrowheads in B,C insets point to pyknotic cells with highly condensed chromatin. Arrowheads in F point to phospho-histone-3-positive cells in the amniotic cavity. Error bars indicate standard deviation. Scale bars = 50 μm.

Apoptotic cells in Ddx11cetus and Ddx11KO embryos did not seem to localize to any specific cell population, but rather where evenly distributed in the epithelia, mesoderm, and endoderm (Fig. 6B,C). Of interest, most of the cells that accumulated in the amniotic cavity of Ddx11cetus and Ddx11KO mutants were not TUNEL positive (Fig. 6B,C, insets). We hypothesize that these cells might have recently entered the apoptosis process but do not yet have substantial DNA fragmentation. Because loss of Ddx11 has been described to cause G2/M arrest (Inoue et al.,2007), one possibility is that disruption of cell cycle progression in Ddx11 mutants leads first to a loss of adhesiveness of the apically localized mitotic epithelial cells, then to apoptosis, once cells are in the amniotic cavity. Consistent with this hypothesis, we found that some of the cells in the amniotic cavity of Ddx11 mutants contained high levels of phospho-histone 3 (Fig. 6F, inset), a marker of chromosome condensation during mitosis (Hendzel et al.,1997). These observations, together with the fact that the proliferative index of embryonic cells was not affected in Ddx11 mutants (Fig. 6E–H), suggest that widespread apoptosis, and not lack of proliferation, contributes to the smaller size and developmental defects of Ddx11 mutant embryos from very early embryonic stages.

Loss of Ddx11 Function Causes Depletion of Somitic Mesoderm, But Does Not Severely Disrupt Other Mesenchymal Tissues

The previous phenotypic characterization of Ddx11KO mutants reported by Inoue et al. (2007) determined that embryonic lethality in Ddx11KO embryos is a consequence of their failure to establish chorioallantoic fusion, a critical step in the formation of the placenta (Watson and Cross,2005). Our analysis of Ddx11cetus mutants revealed defects in chorioallantoic fusion and placental development similar to those previously described for the Ddx11 null allele. We found that Ddx11cetus embryos expressed Tbx4, a marker of allantoic mesoderm (Chapman et al.,1996), but the allantois in Ddx11cetus mutants was rudimentary (Fig. 7B,D) and chorioallantoic fusion failed in all the Ddx11cetus embryos analyzed.

Figure 7.

cetus mutants have severe embryonic defects. A–J: Whole-mount in situ hybridizations with Tbx4 (A,B), Mox1 (C,D), Foxf1 (E,F), brachyury (G,H), and Sox2 (I,J) probes on embryonic day (E) 8.5 wild-type (A,C,E,G,I) and cetus mutant (B,D,F,H,J) embryos. Arrowhead in D points to the ball-like allantois of a cetus mutant. Empty arrowhead in D points to vestigial expression of Mox1 in the anterior part of a cetus embryo. This phenotype was only observed in a few cases, most cetus mutants completely lacked Mox1 expression as shown in Figure 2. Bracket in D highlights the lack of somites (som). al, allantois. LPM, lateral plate mesoderm. not, notochord (axial mesoderm). ps, primitive streak. NE, neuroepithelia. Scale bars = 200 μm.

In addition to allantoic defects, our phenotypic characterization of Ddx11cetus mutants revealed severe morphological abnormalities that were not previously described in Ddx11 null embryos. Ddx11cetus mutants lacked somites and, in some cases, had blisters at the locations where the somites should have been present (Fig. 1). Analysis of markers of somitic mesoderm such as Mox1 (Candia et al.,1992) confirmed that most Ddx11cetus embryos were devoid of somitic mesoderm (Fig. 2C–H). However, we observed small bilateral patches of Mox1 expression in a small percentage of Ddx11cetus mutants (Fig. 7C,D). These small patches of Mox1-expressing cells were always localized to the anterior embryonic flanks and suggested that in some cases somitogenesis is initiated in Ddx11cetus mutants. Analysis of transverse sections revealed that Mox1-expressing cells in Ddx11cetus embryos were sparse and that they failed to organize into the spherical structures characteristic of epithelial somites (Fig. 8A,B). The fact that some embryos showed residual expression of Mox1, suggested that somitic mesoderm cells can be specified in Ddx11cetus mutants, but the sparse cellularity and presence of cell debris in the flanks and blisters of Ddx11cetus embryos (Figs. 1D,F, 8B,D) suggests that these cells die either before or shortly after they are committed to become somitic mesoderm.

Figure 8.

Analysis of mesodermal markers in Ddx11cetus embryos. A–F: Transverse sections of embryonic day (E) 8.5 wild-type (A,C,E) and cetus mutant (B,D,F) embryos analyzed for expression of Mox1 (A,B), Foxf1 (C,D), and brachyury (E,F). Sections correspond to the respective embryos shown in Figure 7. NT, neural tube. som, somite. LPM, lateral plate mesoderm. not, notochord (axial mesoderm). Scale bar = 100 μm.

To determine whether other types of mesoderm lineages are affected in Ddx11cetus embryos, we analyzed the expression of molecular markers of the lateral plate mesoderm (LPM) and the notochord. We found that the lateral plate mesoderm marker Foxf1 (Mahlapuu et al.,2001), was normally expressed in Ddx11cetus embryos, as shown by whole-mount in situ hybridization (Figs. 7E,F, 2C–F) and analysis of transverse sections (Fig. 8C,D). Likewise, expression of brachyury, a marker of the primitive streak and axial mesoderm (Wilkinson et al.,1990), was normal in Ddx11cetus mutants (Figs. 7G,H, 8E,F). These results contrast with the severe effects of loss of Ddx11 on somitic mesoderm. Therefore, our data indicate that Ddx11 is differentially required by embryonic cells during development.

Analysis of Sox2, a marker of neuroepithelia (Collignon et al.,1996), confirmed our previous observation that the neural tube of Ddx11cetus mutants was open and wavy (Figs. 7I,J, 1C–F). Because Ddx11cetus embryos looked underdeveloped as compared to wild-type littermates, the open neural tube of Ddx11cetus mutants could be a consequence of early developmental arrest. However, mouse mutants that disrupt mesoderm development including fibronectin, alpha5-integrin, and lulu mutants, display an open and wavy neuroepithelia similar to that observed in Ddx11cetus embryos (George et al.,1993; Yang et al.,1993; Lee et al.,2007). Therefore, some of the neuroepithelial defects of Ddx11cetus mutants might originate from the lack of somitic mesoderm.

Morphological characterization and analysis with molecular markers revealed that the phenotypes we identified in Ddx11cetus embryos were also present in mutants for the previously characterized Ddx11 null allele, including lack of somitic mesoderm, presence of blisters, and defects in neuroepithelia (Figs. 4B–E, 5). Therefore, our results demonstrate that loss of Ddx11 does not only disrupt placental development, but also causes severe malformations in embryonic tissues, especially in the somitic mesoderm.

Ddx11 Is Expressed Ubiquitously During Early Embryogenesis

Because our phenotypic characterization of Ddx11cetus and Ddx11KO embryos indicates that Ddx11 disrupts somitic mesoderm cells more dramatically than other embryonic cell types, we aimed to determine whether this specificity could be attributed to a higher expression level of Ddx11 in the somitic mesoderm, as compared to other embryonic tissues. To this end, we analyzed Ddx11 expression pattern in embryos using in situ hybridization. Consistent with the role of DDX11 in the development of the placenta, we found that Ddx11 was expressed at high levels in the placenta and allantois (Fig. 9A). Ddx11 was also expressed in other extraembryonic tissues such as the visceral endoderm layer of the yolk sac, albeit at lower levels (ys, Fig. 9C). In the embryo, we found that Ddx11 was widely expressed in all embryonic tissues at both E8.5 and E9.5 embryos (Fig. 9). The widespread expression of Ddx11 does not support differential gene expression as the cause of the drastic defects of Ddx11 mutants in somitic mesoderm. Therefore, our data suggest that other mechanisms might explain the differential requirements for Ddx11 in embryonic tissues during embryogenesis.

Figure 9.

Expression of Ddx11 at embryonic day (E) 8.5 and E9.5. A,B: Whole-mount in situ hybridizations with a Ddx11 probe in wild-type E8.5 (A) and E9.5 (B) embryos. The discontinuous lines in A,B indicate the section planes shown in C,D, respectively. emb, embryo; pl, placenta; al, allantois; ys, yolk sac; ht, heart; me, mesoderm; NT, neural tube; lmb, limb. Scale bars = 200 μm in A (applies to A,B) 50 μm in C (applies to C,D).

DISCUSSION

Ddx11 Is Required for Morphogenesis of the Early Mouse Embryo

Previous studies have demonstrated that Ddx11 is essential for maintaining genome integrity and that complete loss of Ddx11 function leads to embryonic lethality in mice (Gerring et al.,1990; Skibbens,2004; Inoue et al.,2007; Wu et al.,2012). However, the embryonic phenotypes of Ddx11 had been poorly characterized. The analysis of Ddx11cetus and Ddx11KO mutants presented here revealed requirements for DDX11 during early embryogenesis that had not been previously reported. We found that loss of Ddx11 caused severe defects in somitic mesoderm, neuroepithelia, and heart development. Additionally, we found that Ddx11 is required during gastrulation, as judged from the significant percentage of Ddx11cetus mutants that arrested at E7.5. We show that the cetus mutation disrupts DDX11 motif V and demonstrate genetically that this mutation creates a null allele of Ddx11. Therefore, the phenotypic analysis presented here expands the current understanding of the roles of Ddx11 during embryonic development.

Loss of Ddx11 Causes Embryonic Lethality Due to Genome Instability

The embryonic lethality of Ddx11KO mutants was previously attributed to defects in chorioallantoic fusion, which prevent the formation of a functional placenta and the supply of nutrients to the embryo (Inoue et al.,2007). Although our results also indicate that placental defects might contribute to the embryonic lethality of Ddx11 mutants, our phenotypic characterization of Ddx11cetus and Ddx11KO embryos showed that a significant percentage of the mutants have developmental arrest and lethality at E7.5, when embryonic survival does not depend on a functional placenta.

We observed that Ddx11cetus and Ddx11KO embryos have widespread apoptosis from very early embryonic stages and that embryonic cells were abnormally found in the amniotic cavity at E7.5. Although only a small fraction of the cells in the amniotic cavity of Ddx11cetus embryos were positive with TUNEL staining, other cells were pyknotic, a sign of chromatin condensation characteristic of early apoptosis (Kerr et al.,1972). Additionally, we found that a fraction of the cells in the amniotic cavity of Ddx11cetus mutants were positive for the mitotic marker phospho-histone 3. It has been previously shown that the presence of cells in the amniotic cavity of gastrula stage embryos is linked to conditions that disrupt mitosis (Poelmann,1980). Because loss of Ddx11 has been described to cause G2/M cell cycle arrest (Inoue et al.,2007), we hypothesize that phospho-histone-3-positive cells in Ddx11cetus embryos correspond to cells that have become arrested in mitosis and expelled from the embryonic epithelia into the amniotic cavity. Furthermore, we show that the proliferative index of Ddx11 mutants is not affected at E7.5. Together, these observations indicate that loss of Ddx11 triggers cell cycle arrest and apoptosis during embryogenesis. Given the roles of DDX11 in sister-chromatid cohesion and DNA repair, our results strongly suggest that the embryonic lethality of Ddx11 mutants originates from genomic instability and cell death from early embryonic stages.

Ddx11 Is Differentially Required in Embryonic Tissues During Early Embryogenesis

We show that Ddx11cetus and Ddx11KO embryos with late embryonic lethality lack discernible somites. In some Ddx11cetus mutants, the lack of somitic mesoderm gives the embryos a slim appearance, while in other cases Ddx11cetus embryos have blisters filled with cell debris in the places that should have been occupied by somitic mesoderm. Most Ddx11cetus mutants lack Mox1 expression, but some Ddx11cetus embryos have residual expression of Mox1 in the anterior somitic mesoderm. Therefore, these results suggest that somitic mesoderm cells die either before or shortly after they are committed to become somites. In contrast, other types of mesoderm, such as the notochord or the lateral plate mesoderm, are relatively unaffected in Ddx11cetus mutants. The fact that loss of Ddx11 affects somitic mesoderm cells more dramatically than other embryonic cell types indicates that Ddx11 is differentially required in embryonic tissues during development.

We show that expression of Ddx11 is not enriched in somitic mesoderm but rather ubiquitously expressed in all embryonic tissues. Therefore, factors other than differential gene expression must explain the specific effects of Ddx11 in the somitic mesoderm. One possibility is that differences in the developmental mechanisms that specify the somitic mesoderm, as compared to other mesoderm lineages, might explain the differential requirement of Ddx11 in embryonic tissues. For instance, it is possible that somitic mesoderm originates from a very limited number of precursor cells as compared to other types of mesoderm. If this were the case, widespread apoptosis might be more likely to deplete completely the somitic mesoderm as compared to other tissues. Another possibility is that intrinsic characteristics of somitic mesoderm cells, such as small differences in the length of the cell cycle and the mitotic index, might be responsible for the differential requirements of Ddx11 during embryogenesis. Variations in the rates of proliferation during embryonic development are well described in the literature. In mouse embryos, early cleavages after fertilization take place every 10–14 hr (Sawicki et al.,1978; Bolton et al.,1984), but postimplantation development is marked by a dramatic increase in proliferation and cell cycle progression, which peaks at gastrulation (E7.5) with cells dividing every 7 hr (Solter et al.,1971) and then declines by the neurula stage (E9), with cells dividing each 9 hr (Kauffman,1968). The unusually rapid cell cycle progression in mammalian gastrula stage embryos is achieved by a shortening of G1, S, and G2 phases (Mac Auley et al.,1993; Power and Tam,1993). This high proliferative index and shortening of cell cycle has been described to impair the ability of embryonic cells to repair DNA damage, as evidenced by the hypersensitivity of gastrula stage embryos to ionizing radiation (Heyer et al.,2000). Therefore, it is possible that differences in the proliferative index of somitic mesoderm with respect to other embryonic cell types could make cells more sensitive to the genomic instability produced by lack of Ddx11. In this respect, genome instability caused by lack of Hus1, a component of the RAD9/RAD1/HUS1 checkpoint complex, causes defects in somitic mesoderm similar to those we observed in Ddx11 mutants (Weiss et al.,2000). Similar phenotypes have also been described in mouse mutants for the tumor suppressor genes Brca2 and Palb2 (Suzuki et al.,1997; Rantakari et al.,2010). These observations, together with our phenotypic analysis of Ddx11 embryos, suggest that intrinsic properties of the somitic mesoderm, such as its particular proliferative index, might make this tissue specifically sensitive to DNA damage.

Helicase Motif V Is Required for DDX11 Activity

DDX11 contains the seven motifs characteristic of DNA helicases (Wu et al.,2008; Fairman-Williams et al.,2010). The positional cloning of cetus revealed a Leu to Pro mutation in amino acid 743 of DDX11, a residue that maps to motif V and is conserved among members of the FANCJ/RAD3/XPD DNA helicase family (Fairman-Williams et al.,2010). Human DDX11 has been shown to function as an ATP-dependent helicase that unwinds DNA duplex substrates (Hirota and Lahti,2000; Farina et al.,2008; Wu et al.,2008,2012). Mutations in the motif V of other SF1 and SF2 DNA helicases have been shown to disrupt DNA-binding and ATPase activities (Moolenaar et al.,1994; Hsu et al.,1995; Graves-Woodward et al.,1997; Hall and Matson,1999; Korolev et al.,1997; Tuteja and Tuteja,2004) and to cause human disease (Fig. 4A; Fan et al.,2008). These previous findings, together with the fact that Leu to Pro substitutions cause dramatic changes in protein structure, suggest that the cetus mutation in motif V of DDX11 affects the catalytic activities of this DNA helicase, ultimately disrupting its roles maintaining genome stability. We show that the cetus mutation did not complement a null allele of Ddx11 and that Ddx11cetus mutants have phenotypes that are indistinguishable from those of Ddx11cetus/KO and Ddx11KO embryos. Hence, our experiments provide genetic evidence that motif V is essential for DDX11 function.

In conclusion, the phenotypic characterization and positional cloning of the Ddx11cetus allele presented here reveals novel requirements for Ddx11 during embryonic development. We show that some Ddx11 mutants die at the gastrula stage embryo, likely from a dramatic increase in apoptosis, and that in embryos with late lethality, loss of Ddx11 disrupts somitic mesoderm more drastically than other embryonic cell types. These studies expand our knowledge about the effects of genome instability on embryonic development and define an essential role of the DNA helicase motif V for DDX11 function.

EXPERIMENTAL PROCEDURES

Mouse Strains

cetus was characterized on CAST/Ei and congenic C3H/FeJ Mus musculus strain backgrounds. The simple sequence length polymorphism (SSLP) markers D17CU33 and D17CU40 (CAST/Ei background) or D17CU35 and D17CU39 (C3H/FeJ background) were used for genotyping. Ddx11KO mice were obtained from Dr Jill Lahti (Inoue et al.,2007) and were raised on CAST/Ei and C3H/FeJ genetic backgrounds. Experiments involving mice were approved by the institutional IACUC and done in accordance with regulations.

Positional Cloning of cetus

The cetus mutation was generated by ENU-mutagenesis as described previously (Garcia-Garcia et al.,2005). To facilitate positional cloning, the cetus mutation was created on a C57BL6/J background and outcrossed to C3H/FeJ and CAST/Ei for linkage analysis. The cetus mutation was mapped to a 1.1 Mb region of chromosome 17 using a collection of SSLPs from public databases or identified by us from genome sequence data with the Etandem EMBOSS software (http://bioweb2. pasteur.fr/docs/EMBOSS/etandem.html). Primer sequences for these novel SSLP markers are specified below. cDNAs of all candidate genes in the cetus interval (E13009J12Rik, VapA, Txndc2, Rab31, Ppp4r1, Ralbp1, Twsg1, Ankrd12, Ndufv2, Orf19, and Ddx11) were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR; Superscript One-Step RT-PCR, Invitrogen) using RNA from E8.5 cetus and C57BL/6J (control) embryos. RNAs were extracted using RNA STAT-60 (Tel-Test). Amplification products were sequenced at the Cornell University Life Sciences Core Laboratory Center (CLC). Linkage to the BseRI RFLP was confirmed in a collection of two heterozygote cetus animals and eight homozygote cetus embryos.

Primers for SSLP Analysis

The following pairs of forward (F) and reverse (R) primers were used for detection of SSLP on chromosome 17:

  • D17CU33-F ACACAGGAGAACG GAATGGA

  • D17CU33-RTTTTTAGGTGAGTTAGCCCAACA

  • D17CU35-FTGCGTTCAGCTTAC AGAGGA

  • D17CU35-RCAATCATTTCTATCAGTTGACATGG

  • D17CU39-FGCCACTCCATTACCACACG

  • D17CU39-RGGATTCAGGGGAGTCCATTT

  • D17CU40-FCTCCCTCTGCTTCCA AAGTG

  • D17CU40-RAATGCAGCTCCAGG ACATCT

  • D17CU44-FTGTACAGCATTTGG CAGAGC

  • D17CU44-RAAGCACAGAGGATG CCACTC

  • D17CU45-FGGTGTGAGTCAGCG GAAAAG

  • D17CU45-RGTCCCCAAAGACGG AGATTA

Embryo Analysis

The analysis of cetus mutant embryos comprised the dissection of 150 cetus mutants raised in a C3H/FeJ background (49 of those were congenic) and 90 cetus embryos raised in a CAST/Ei background. Representative examples of embryos with early or late lethality mutants are shown in the figures as indicated. Embryos shown were raised in a CAST/Ei background for Figures 1A–F, 2B, 4C, 5B,C,E, 7D,H,J, 8B, and 8F; in a C3H/FeJ background for Figures 2E–H, 6B,F, 7B,F, and 8D; and in a hybrid genetic background for Figures 4D,E, 6C, and 6G. Embryos were dissected in phosphate buffered saline containing 0.4% bovine serum albumin at different stages as indicated. Whole-mount RNA in situ hybridization was performed as previously described (Belo et al., 1997; Shibata and Garcia-Garcia, 2011). Whole-mount embryos were imaged in methanol. For analysis of sections, embryos were embedded, then cryosectioned at 10 μm. All comparisons of wild-type and mutant embryos are at the same magnification unless otherwise noted. For phospho-histone-H3 immunohistochemistry and TUNEL, E7.5 embryos were cryosectioned (8–10 μm) as previously described (Garcia-Garcia and Anderson, 2003). Anti-phospho-histone H3 (Ser10; Upstate) was used at 1/250. TUNEL was performed using the ApopTag Detection Kit (Chemicon). As positive controls for TUNEL, sections were treated with DNase I. For E7.5 embryos, extra-embryonic tissue was removed before embedding and used for genotyping.

Acknowledgements

We thank Joe Peters, Qiaojuan Shi, Maria Spies, and members of the laboratory for helpful discussions and for comments on the manuscript; Drs. Jill Lahti, Eishi Noguchi, John Schimenti, and Holger Sondermann for mice and reagents. Cornell's CARE staff for mice husbandry and care.

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