|Home | About | Journals | Submit | Contact Us | Français|
During preimplantation development, the embryo must establish totipotency and enact the earliest differentiation choices, processes that involve extensive chromatin modification. To identify novel developmental regulators, we screened for genes that are preferentially transcribed in the pluripotent inner cell mass (ICM) of the mouse blastocyst. Genes that encode chromatin remodeling factors were prominently represented in the ICM, including Chd1l, a member of the Snf2 gene family. Chd1l is developmentally regulated and expressed in embryonic stem (ES) cells, but its role in development has not been investigated. Here we show that inhibiting Chd1l protein production by microinjection of antisense morpholinos causes arrest prior to the blastocyst stage. Despite this important function in vivo, Chd1l is non-essential for cultured ES cell survival, pluripotency, or differentiation, suggesting that Chd1l is vital for events in embryos that are distinct from events in ES cells. Our data reveal a novel role for the chromatin remodeling factor Chd1l in the earliest cell divisions of mammalian development.
The first differentiation decision in the mammalian embryo is made prior to the blastocyst stage, when blastomeres must commit to becoming either part of the trophectoderm (TE) or the inner cell mass (ICM) (Rossant and Tam, 2009). Cells of the ICM possess the property of pluripotency and will contribute to the many tissues of the embryo, whereas the TE will give rise to extra-embryonic material (Rossant and Tam, 2009). We reasoned that factors compartmentalized in the ICM could be novel developmental regulators of pluripotency or early differentiation. To identify candidate preimplantation regulators, we performed an expression analysis on purified ICM and whole blastocysts and identified genes enriched in the ICM. Gene ontology clustering revealed a large group of chromatin regulatory enzymes.
The high degree of chromatin organization within the nucleus is oppressive to transcription and other processes that require DNA accessibility (Knezetic and Luse, 1986; Lorch et al., 1987). Chromatin remodeling factors (CRFs) utilize energy to alter nucleosome positioning and contain a core SNF2-like ATPase/helicase domain responsible for enzymatic activity (Hirschhorn et al., 1992; Flaus et al., 2006). CRFs participate in key chromatin-dependent processes including transcriptional activation and repression, histone exchange, cell cycling, DNA repair, and many others (Hirschhorn et al., 1992; Cairns, 2005; Morrison et al., 2004; Fyodorov and Kadonaga, 2001). CRFs assemble into multi-subunit complexes, and their functions depend in part on the composition of the complexes (Flaus and Owen-Hughes, 2004; Wang et al., 1996a; Wang et al., 1996b).
During the development of the zygote and the preimplantation embryo, chromatin undergoes profound changes that allow the parental genomes to achieve a state of totipotency and that are necessary for normal development. Despite successful reprogramming of somatic cells, reprogramming in the embryo remains largely enigmatic (Takahashi and Yamanaka, 2006; Okita et al., 2007; Wernig et al., 2007; Yu et al., 2007; Niemann et al., 2008). Relatively few factors involved in preimplantation development have been identified because early phenotypes of homozygous mutants are often masked by maternally provided transcripts and proteins. Therefore, techniques aimed at early development will likely be fruitful in discovering additional chromatin modifiers that are essential in the preimplantation embryo.
Among the genes identified in our screen was Chd1l, encoding a largely unexplored CRF of the Snf2-like family. Its compartmentalization in the ICM, expression in ES cells, and temporal regulation prior to the blastocyst stage (Wang et al., 2004) led us to hypothesize that Chd1l is a chromatin enzyme critical for early development. The protein has a Snf2-like ATPase domain but lacks any of the signature domains of the four classic Snf2 subclasses. Instead, Chd1l contains a unique C-terminal “macro” domain and therefore defines a distinct subclass (Yan et al., 2002; Mohrmann and Verrijzer, 2005). The macro domain binds poly(ADP-ribose), or PAR, a post-translational modification added to nuclear acceptor proteins. The nucleosome remodeling activity of Chd1l is dependent on PAR synthesis, indicating that the PAR-binding macro domain is central to its function as a chromatin remodeler (Ahel et al., 2009; Gottschalk et al., 2009).
Chd1l is involved in the DNA damage response. Chd1l localization to sites of induced DNA damage is dependent on a functional macro domain, and dissociation from sites of damage is dependent on a functional ATPase domain (Ahel et al., 2009; Gottschalk et al., 2009). Recent studies have also implicated Chd1l as an oncogene. The majority of hepatocellular carcinomas in humans are associated with genomic amplification of a region that includes Chd1l, and its overexpression in liver cell lines and mouse models is tumorigenic (Chen et al., 2009; Ma et al., 2008). While evidence is accumulating for a role for Chd1l as an oncogene and in DNA repair, its importance during development has not been examined.
We find that Chd1l is expressed in cultured embryonic stem (ES) cells, which are derived from the ICM and share the ability to differentiate into the three major germ layers. Our data show that Chd1l is not required for ES cell viability, pluripotency or differentiation. Using a morpholino (MO) knockdown approach in the zygote-stage embryo, we show that Chd1l is required for the very earliest stages of development.
The decision to become inner cell mass (ICM) or trophectoderm (TE) is the first lineage commitment a totipotent blastomere must make. The ICM retains pluripotency, the ability to give rise to the three primary germ layers, whereas the TE will give rise to extra-embryonic tissue. We reasoned that mRNAs enriched in the ICM would encode proteins that contribute to the development of the blastocyst and/or the establishment of pluripotency. An alternative model would be that repression of these mRNAs in the TE marks an important step in the differentiation of TE and that continued expression in the ICM restricts TE differentiation. To screen for ICM-enriched mRNAs, we purified the ICM by immunosurgery (Solter and Knowles, 1975), taking advantage of the structural organization of the blastocyst (Fig. 1A). Outer TE cells of the blastocyst were labeled with IgG by incubation with rabbit anti-mouse serum and specifically lysed by the complement cascade, leaving behind purified ICMs. RNA extracted from the ICM was compared with total blastocyst RNA using genome-wide expression analysis.
Transcripts encoding Oct4 and Nanog, factors known to be critical for pluripotency, were enriched in the ICM 1.9- and 2.4-fold, respectively, providing proof of sound methodology (Fig. 1B). In addition, mRNAs encoding Cdx2 and Eomes, markers of extra-embryonic material, were under-represented 4.5-fold and 2.4-fold, respectively, in ICM compared to the whole blastocyst (Fig. 1B). Ubiquitously expressed transcripts encoding β-actin and β-tubulin demonstrate roughly equivalent levels in ICM and whole blastocyst (Fig. 1B). Clustering of genes whose transcripts are enriched in ICM revealed three major GO-term classes: cell signaling molecules, transcription factors, and chromatin-modifying enzymes. Some of the chromatin factors identified have known enzymatic activity and/or developmental roles, including the DNA methyltransferases, the polycomb group proteins, and the Snf2 family of chromatin remodeling enzymes (Fig. 1C).
Chromatin remodeling factors are often found in large, multi-subunit complexes (Wang et al., 1996a). Subtle changes in the composition of a complex can have dramatic effects on its function, and on the differentiation status of a cell (Ho and Crabtree, 2010; Lessard et al., 2007; Ho et al., 2009). Enrichment (or repression) of one or more subunits of a complex is one way in which the composition of a complex can be regulated (Peng et al., 2009). In general, our data support a model in which, compared to the trophectoderm, the ICM is characterized as having a chromatin state with tight transcriptional control and an abundance of chromatin proteins that mediate transcription and differentiation.
Among the Snf2 family of chromatin enzymes whose mRNAs were enriched in the ICM was the CRF called Chd1l. Its enrichment score of 4.28-fold was higher than that of the “master regulator” of pluripotency, Oct4 (1.8-fold) (Fig. 2A). The Snf2 family of chromatin remodeling factors has powerful and diverse roles in development and transcriptional regulation (Ho and Crabtree, 2010; Eisen et al., 1995), and Chd1l is a member of this family by virtue of the split DNA-dependent ATPase/helicase domain (Flaus et al., 2006). Chd1l is the only member of the Snf2 family that contains a poly(ADP-ribosyl)ation binding macro domain (Fig. 2B) (Yan et al., 2002). Chd1l protein expression was confirmed in ES cells using a Chd1l-specific antibody (Fig. 3B).
Our lab previously reported genome-wide gene expression profiles during preimplantation development from the zygote through the blastocyst stage (Wang et al., 2004). In these studies, Chd1l expression was found to increase through the first several cell divisions of development, peaking at the late morula stage (Fig. 2C). Upon formation of the blastocyst, total Chd1l expression decreases slightly; our ICM data indicate it then becomes preferentially expressed in the ICM. Compartmentalization in the ICM, expression in ES cells, and developmental regulation support a potential role for Chd1l in pluripotency and during early embryogenesis. We chose to investigate the developmental function of this novel CRF of the Snf2 family (Fig. 2D).
Mouse ES cells are derived from the ICM of blastocyst stage embryos and maintain the property of pluripotency indefinitely. Because Chd1l mRNA is enriched in the ICM and abundant in ES cells, we asked whether Chd1l is essential for ES cell survival and pluripotency. To knock down Chd1l in ES cells, we introduced shRNA-encoding sequence into the EBRTcH3 ES cell line (Masui et al., 2005). These cells are engineered to allow stable, Cre-mediated integration and inducible transgene expression under the control of a CMV promoter (Tet-Off) (Fig. 3A). First, we created a control ES cell line, NS-shRNA EBRTcH3, by integrating DNA encoding shRNA that does not target any transcript in the mouse genome (“Non-Silencing”). Transcription of the shRNA from the CMV promoter was confirmed by observing robust Venus reporter gene expression 24 hours after inducing expression by Tetracycline withdrawal (“Tet-Off” induction). We created the Chd1l-shRNA EBRTcH3 ES cell line by integration of a sequence encoding shRNA that targets the Chd1l transcript. To assess knockdown efficiency of Chd1l protein, we blotted ES cell lysates using an α-Chd1l antibody generated in our laboratory, which detects a band at ~100 kD corresponding to Chd1l. Chd1l protein was consistently and reproducibly reduced to nearly undetectable levels in Chd1l-shRNA EBRTcH3 cells 48 hours after tetracycline withdrawal (Fig. 3B). In contrast, NS-shRNA ES cells induced to express NS-shRNA for 48 hours had normal levels of Chd1l. These data confirm that induction of Chd1l shRNA by removal of Tetracycline from the Chd1l-shRNA EBRTcH3 ES cell line is a robust system in which to knock down Chd1l. Chd1l-shRNA ES cells with reduced Chd1l had normal levels of Oct4 expression (Fig. 3B), no obvious abnormalities in ES cell morphology or colony formation (Fig. 3C), and normal proliferation over a period of eight days, or ~10 doublings (Fig. 3D). Our results are consistent with a recent RNAi screen performed in ES cells in which Chd1l was included among the chromatin factors screened, but was not identified as necessary for ES cell proliferation or for expression of a pluripotency reporter gene (Fazzio et al., 2008). However, no validation of Chd1l knockdown was provided in that study, and no further investigation of more inconspicuous phenotypes was attempted. We therefore proceeded to investigate gene expression profiles and differentiation in ES cells in which Chd1l has been knocked down.
A primary function of the SNF2 family of DNA-dependent ATPases is transcriptional regulation (Fry and Peterson, 2001; Flaus and Owen-Hughes, 2001). The four major subfamilies, SWI/SNF, CHD, ISWI, and INO80 all regulate gene expression during development (Ho and Crabtree, 2010). Chd1l contains a seven-motif, DNA-dependent ATPase module that defines the SNF2 family of chromatin remodeling factors as well as a macro domain that recognizes PAR-modified nuclear proteins, including PAR-modified histones. Chd1l might also regulate transcription, and ES cells lacking Chd1l could have transcription changes even in the absence of obvious morphological changes. We took a whole-genome approach and obtained the expression profiles of induced (−Tet) EBRTcH3 ES cells expressing Chd1l-shRNA or NS-shRNA and uninduced (+Tet) ES cells that did not express shRNA. Expression indices confirm the reduction of Chd1l in ES cells expressing Chd1l-shRNA (Fig. 3E). Only a small number of other transcripts that changed more than 1.4-fold between induced ES cells expressing Chd1l-shRNA and uninduced ES cells (~30), and these transcripts were also differentially expressed between induced ES cells expressing NS-shRNA and uninduced ES cells, indicating the expression changes were a byproduct of inducing shRNA expression. We found no statistically significant changes in expression of pluripotency markers, differentiation markers, or cell cycling genes (Fig. 3E). Our data suggest that Chd1l does not regulate transcription in ES cells.
Like the ICM, ES cells are capable of differentiating into the three germ layers. While ES cells maintain this property indefinitely in vitro, the ICM is only transiently pluripotent as cells rapidly differentiate during embryogenesis. In the absence of the pluripotency cytokine LIF, ES cells can be grown into embryoid bodies (EBs), differentiating cellular aggregates that mimic in vivo post-implantation development. We reasoned that Chd1l may regulate gene expression in differentiating cells, when new gene expression patterns are being established. To ask whether Chd1l is required for the formation of the germ layers, Chd1l was reduced in Chd1l-shRNA ES cells, which were then differentiated into EBs. We measured the expression of a panel of lineage markers by q-rtPCR over time. For comparison, we measured gene expression in EBs made from induced and uninduced NS-shRNA ES cells. Quantitative rt-PCR confirmed knockdown of Chd1l mRNA in induced Chd1l-shRNA EBs but not in induced NS-shRNA EBs over nine days of differentiation (Fig. 3F). The lineage markers included genes associated with the establishment of endoderm (Sox17, AFP, Gata4), mesoderm (Lhx1), and ectoderm (Fgf5, Otx2), as well as pluripotency (Oct4) and extra-embryonic (Eomes) tissues. Under differentiating conditions, EBs expressing Chd1l-shRNA reduced Oct4 expression in a manner similar to EBs expressing NT-shRNA (Fig. 3F). Expression of markers for all three germ layers was induced in a temporally appropriate manner (Fig. 3F). Our results indicate Chd1l does not control gene expression in pluripotent ES cells or in differentiating embryoid bodies under normal culture conditions.
Next, we addressed the question of whether Chd1l plays a role in development prior to differentiation of the ICM. The preimplantation embryo can be cultured in vitro through the blastocyst and hatching stages. We took a rapid knockdown approach, utilizing synthetic antisense oligos called morpholinos (MOs) that inhibit translation and splicing machinery. This approach has been used extensively to study the early development of diverse organisms (Gore et al., 2005; Imai et al., 2006; Sumanas and Larson, 2002; Yamada et al., 2003). In the mouse preimplantation embryo, MOs have been employed to show that Oct4 has a critical role prior to the blastocyst stage (Foygel et al., 2008). MOs are stable oligos and effectively reduce production of specific proteins in the preimplantation embryo, with minimal toxicity or off-target effects because they function through steric hindrance rather than through activation of the RNAi pathway (Foygel et al., 2008).
Splice-blocking MOs were designed to target Chd1l pre-mRNA. The predicted splice mutants produce truncated proteins due to stop codons within the intron (Fig. 4A). Chd1l MO-1 was microinjected into the cytoplasm of one-cell stage mouse embryos collected from superovulated and mated females. To confirm that MO-1 was functioning as predicted, we used microfluidic q-rtPCR on RNA collected from single MO-injected and control embryos. We used a TaqMan primer-probe assay that targeted the junction between exons 2 and 3 (ex2–3). This junction would be present in the wild-type Chd1l transcript but absent if the MO blocks its targeted splicing event. Microfluidic qPCR confirmed abrogation of the wild-type transcript. Ct values showed that amplification of the ex2–3 splice junction was efficient in control embryos but nearly absent in injected embryos (a difference in Ct values of ~20, reflecting >99% reduction of transcripts containing the normal splice junction in injected embryos) (Fig. 4B,C). Wild-type transcripts were similarly abrogated by injection of a second splice-blocking MO (MO-2), which targeted the ex4–5 splice junction (a difference in Ct values of ~10, reflecting a >99% reduction of normal transcripts) (Fig. 4C). Changes in Oct4 gene expression in embryos injected with Chd1l MO were not statistically significant (α=0.05).
Using either MO, the altered splicing would lead to the introduction of a stop codon within the intron. The effect of impaired splicing on the transcript as a whole is unknown, and whether or not nonsense mediated decay (NMD) will be initiated cannot be predicted. The Chd1l transcript is not significantly reduced in embryos injected with MO-1 compared to uninjected embryos, as demonstrated by the lack of any significant change in amplification of the 3′ sequences (ex20–21). This suggests that the transcript is stable despite abrogation of splicing between exons 2 and 3. On the other hand, the Chd1l transcript (ex20–21) is somewhat reduced in embryos injected with MO-2, indicating that the transcript resulting from the abrogation of splicing between exons 4 and 5 may be more unstable or may trigger NMD. Regardless of transcript stability, the only protein products translated would be truncated near the N-terminus and devoid of any of the known functional protein domains.
To ask whether Chd1l is required during early development we microinjected the zygote-stage embryo with MO-1 targeting Chd1l and observed embryos for a period of four days. MO-injected embryos did not reach the blastocyst stage and instead arrested at the compaction stage (Fig. 5A,B). Cells of arrested embryos do not fragment and instead appear morphologically normal. An arrest prior to blastocyst formation is consistent with the peak in Chd1l expression at the late morula stage and enrichment in the ICM. In contrast, the majority of embryos microinjected with MO targeting the Snf2l transcript, encoding another Snf2-like chromatin remodeling factor, reached the blastocyst stage (Fig. 5A,B). This result demonstrates that embryonic arrest is not a general effect of microinjection of a CRF MO. To further test our finding we microinjected MO-2 targeting ex4–5 splice junction. These embryos also arrested prior to the blastocyst stage (Fig. 5A,B). The precise timing of the arrest varied between the MOs, perhaps due to different binding affinities of the MO sequences.
To confirm that the embryonic arrest phenotype is a result of disrupting Chd1l protein production, mRNA encoding Chd1l was co-injected along with Chd1l MO. We reasoned that embryos arrested at an earlier stage would be more able to progress to later developmental stages with addition of mRNA than embryos arrested at later stages, so we used MO-2 for co-injection. MO-2 targets a splicing junction and therefore could not target the injected mRNA, which was synthesized from cDNA lacking intron sequences. Embryos injected with MO-2 alone did not progress to the morula stage, nor did embryos co-injected with MO-2 plus GFP mRNA. About 50% of the embryos co-injected with MO-2 plus Chd1l mRNA progressed to the morula stage or further (Fig. 5C). Mitigation of the developmental arrest phenotype by Chd1l mRNA confirmed that loss of Chd1l was responsible for the embryonic arrest.
Chromatin remodeling activities are abundant in preimplantation embryos and in ES cells, and many of these activities are geared toward initiating pluripotent transcriptional competence and ensuring that differentiation programs are locked in epigenetically (Corry et al., 2009; Albert and Peters, 2009). Although Chd1l is part of the Snf2 family of DNA-dependent ATPases (Flaus et al., 2006), many of which are potent transcriptional regulators, Chd1l itself does not seem to regulate gene expression, at least in ES cells. It remains a formal possibility that Chd1l regulates gene expression in the preimplantation embryo, where a prominent developmental arrest phenotype was observed, but subsequently ceases to have gene regulation activity in ES cells. Despite being abundantly expressed, Chd1l is not required for normal proliferation, pluripotency, or differentiation of ES cells. Chd1l might have no function at all in ES cells, its function may be masked by the function of a redundant ES cell protein, its function may only become apparent when some trigger or insult presents itself, or the ES cell protein may represent a store of protein that will be important later in development. Chd1l is likely function differently in the transiently pluripotent ICM than in ES cells that have undergone artificial epigenetic changes to adapt to immortal growth in culture.
The critical developmental role of Chd1l in preimplantation embryos may stem from the ADP-ribose-binding macro module that distinguishes Chd1l from the other members of its family (Ahel et al., 2009; Gottschalk et al., 2009; Karras et al., 2005). ADP-ribose is a post-translational modification that is added to acceptor proteins by several enzymes including the PARPs, which catalyze poly-ADP-ribosylation, and the sirtuins, which catalyze mono-ADP-ribosylation in addition to histone deacetylation (Landry et al., 2000; Imai et al., 2000; Frye, 1999). Chd1l may bind to ADP-ribose catalyzed by these enzymes and act as an effector protein for sirtuin- and/or PARP-mediated processes. The sirtuins have diverse cellular roles, including gene regulation and DNA repair, and have been shown to be critical during embryogenesis (Wang et al., 2006; Rine and Herskowitz, 1987; McBurney et al., 2003; Mao et al., 2011). However, mono-ADP-ribosylation by the sirtuins appears to be weak compared to histone deacetylation, and the physiological significance this activity remains somewhat controversial (Du et al., 2009).
Poly-ADP-ribose (PAR) is synthesized by the PARP family of PAR polymerases and has important roles in diverse chromatin-dependent processes. Evidence is accumulating for the importance of PAR regulation in the embryo. Double knockout of PAR polymerases Parp-1 and the partially redundant Parp-2 in mice is embryonic lethal at the onset of gastrulation (Ménissier de Murcia et al., 2003), whereas knockout of the non-redundant PAR depolymerase PARG is lethal at E3.5 (Koh et al., 2004). These data suggest that PAR levels are tightly regulated in the embryo and that fluctuations are highly deleterious. Chd1l contains a macro module responsible for binding PAR. Thus Chd1l could contribute to PAR regulation, and the Chd1l embryonic arrest phenotype may be due to aberrant PAR levels or downstream PAR signaling.
Two independent groups recently demonstrated the ability of Chd1l to respond to DNA damage by interacting with PAR (Ahel et al., 2009; Gottschalk et al., 2009). DNA damage is one of the most prominent triggers of PAR modification and signaling. Parp-1 is activated by DNA damage and synthesizes PAR onto itself in an auto-modification reaction, which initiates DNA repair mechanisms (Berger, 1985; Benjamin and Gill, 1980). Blocking Parp-1 activity with specific inhibitors, or through null mutations, results in cellular hypersensitivity to DNA damaging agents and defects in DNA repair (Herceg and Wang, 2001). Repair of damaged DNA is critical in the early embryo. Damage to DNA occurs frequently as a result of normal cellular metabolism, and the repair of resulting errors is critical in the early embryo as it must maintain genomic integrity for the future organism. Consistent with this requirement, genes involved in all of the major DNA repair pathways are expressed in the preimplantation embryo (Jaroudi and SenGupta, 2007). Therefore, because Chd1l responds to DNA damage through its interaction with PAR, and because DNA repair is crucial during embryogenesis, defects in DNA repair could underlie the Chd1l early embryonic arrest.
Large number of double-stranded break repair proteins are embryonic lethal when deleted (Hakem, 2008). Double-stranded breaks are the most toxic form of DNA damage and can be repaired through either non-homologous end-joining (NHEJ) or homologous recombination (HR). NHEJ can function throughout the cell cycle, whereas HR is restricted to S/G2 phase (Rothkamm et al., 2003). In the zygote, repair of the paternal genome is especially crucial because double-stranded breaks and other errors are introduced during spermatogenesis, and the extreme chromatin compaction of the sperm is inhibitory to repair (Generoso et al., 1979; Matsuda et al., 1985). The zygote spends ~20 hours in G1 prior to the first cell division, and much of the paternal DNA is repaired through NHEJ (Fiorenza et al., 2001; Hagmann et al., 1996; Hagmann et al., 1998; Lee et al., 1997). The ability of Chd1l to function in NHEJ is suggested by its PARP-dependent association with a major NHEJ component, DNA-PKcs, upon induced DNA damage (Ahel et al., 2009).
An intriguing question is why is Chd1l essential in the earliest stages of embryogenesis but not in ES cells? In contrast to the zygote, ES cells have rapid cell cycles with abbreviated G1 and G2 phases and rely heavily on HR to repair lesions during S phase (Savatier et al., 2002). Therefore, one explanation for why reduced Chd1l causes preimplantation arrest but is dispensable for ES cells is that Chd1l plays a role in NHEJ, which is acutely essential during early embryogenesis but not in ES cells.
In summary, numerous mRNAs encoding chromatin remodeling enzymes are enriched in the ICM, which will differentiate into all the cellular lineages of the adult organism. Chd1l, a candidate regulator of pluripotency studied here, is essential for preimplantation embryonic development even prior to the formation of the ICM. Despite its requirement for the earliest cell divisions in the embryo and its expression in ES cells, Chd1l appears to be dispensable for ES cell viability, pluripotency, differentiation, and gene expression. The function of Chd1l could be in PAR signaling via the PAR-binding macro domain and in downstream DNA repair. Recent studies have demonstrated a role for Chd1l as a DNA damage response protein that interacts with members of the NHEJ pathway in a PARP-dependent manner. Impaired NHEJ repair could explain why Chd1l deficiency results in developmental arrest of preimplantation embryos that rely heavily on NHEJ but causes no detectable abnormalities in ES cells. The differential requirement of Chd1l in the preimplantation embryo and in ES cells exemplifies the limitations of extrapolating conclusions from experiments using in vitro-derived ES cells and highlights the importance of studying early development directly in mouse embryos.
E3.5 blastocysts were collected from timed-pregnant mothers and washed in M2 medium. The zona pellucida was removed by incubation in Acid Tyrode solution for 3 minutes. Outer TE cells were labeled with IgGs by incubation with 10% rabbit anti-mouse serum for 60 minutes. Embryos were washed three times in M2 medium, and then TE cells were lysed through the complement cascade by incubation with 30% guinea pig complement for 15–30 minutes, or until lysis was visible. Remaining ICMs were washed three times in M2 medium with a fine pipette to remove residual TE cells. Total RNA was extracted from purified ICMs and whole blastocysts with Trizol reagent. Purified RNA was amplified using an Affymetrix kit, labeled, and hybridized to one Affymetrix mouse 430 2.0 Expression Array per sample. Chip analyses were performed with Dchip, a model-based method for expression analysis (http://www.dchip.org). Normalization of data was performed by the Invariant Set Normalization method (Li and Wong, 2001).
The EBRTcH3 cell line contains a cassette acceptor utilizing loxP and loxPV sites at the Rosa locus to allow efficient and directional integration of a transgene by Cre-mediated recombination. ShRNA-mir cDNAs were subcloned from pGIPZ vectors (OpenBiosystems, Chd1l shRNA Oligo ID: V2LMM_18041 and “non-silencing” shRNA-mir) into the pPthC exchange vector for recombination into the EBRTcH3 ES cell line. The parental EBRTcH3 ES cells and the pPthC exchange vector were gifts from the lab of Dr Hitoshi Niwa of Japan.
The exchange vector containing the shRNA-mir sequence was cotransfected with a Cre expression plasmid using lipofectamine. Transfected cells were plated at single-cell density and cultured with Puromycin (1.5 µg/ml) to select for successful recombinants and with Tetracycline (1.0 µg/ml) to repress transgene expression. Clones were confirmed by PCR genotyping of the 5′ and 3′ recombination sites. To induce shRNA expression, the derived ES cell lines were cultured without Tetracycline but with high Puromycin (7.5 µg/ml). Control, uninduced ES cells were cultured in high Tetracycline (1.5 µg/ml) and high Puromycin (7.5 µg/ml).
Total RNA was extracted from Chd1l-shRNA and NS-shRNA ES cells three days after inducing the expression of Chd1l-shRNA or NS-shRNA by Tetracycline removal (7.5 µg/ml Puromycin), and from uninduced Chd1l-shRNA and NS-shRNA ES cells that do not express shRNA (7.5 µg/ml Puromycin, 1.5 µg/ml Tetracycline). Three different Chd1l-shRNA EBRTcH3 clones and one NS-shRNA EBRTcH3 clone were used. RNA was amplified from the eight samples, labeled using an Affymetrix kit, and hybridized to mouse 430 2.0 Expression Arrays. Fold changes in expression indices were calculated for shRNA-induced ES cells versus shRNA-uninduced ES cells. The statistical significance of fold-changes between Chd1-shRNA induced and uninduced samples was determined using a paired t-test, a minimum fold change of 1.4, and a delta value of 1.9 (SAM Analysis) (Tusher et al., 2001).
Expression of shRNA was induced by Tetracycline withdrawal in Chd1l-shRNA and NS-shRNA EBRTcH3 ES cells for three days prior to differentiation into embryoid bodies (EBs) to ensure complete Chd1l knockdown. RNA was collected at Day 0 of differentiation from induced and uninduced Chd1l-shRNA and NS-shRNA EBRTcH3 ES cells. ES cells were suspended at a density of 2×104 cells/ml of medium without LIF, and EBs were made using hanging droplets of 500 cells in 25 µl. After two days, embryoid bodies were collected into 10-cm Ultralow Attachment plates (Corning) and cultured for an additional seven days in the absence of LIF. RNA was collected every three days after LIF removal. cDNA was synthesized from each sample and subjected to qPCR. Relative quantities for each cell line were calculated using Gapdh as the internal control and shRNA-uninduced, Day 0 samples as references.
TaqMan gene expression assays used in these experiments were predesigned and ordered from Applied Biosystems: Gapdh: Mm99999915_g1; Oct4: Mm00658129_gH; Gata4: Mm00484689_m1; Eomes: Mm01351988_m1; Sox17: Mm00488363_m1; Fgf5: Mm00438919_m1; AFP: Mm01351348_g1; LhX1: Mm00521776_m1; Otx1: Mm00446859_m1; Chd1l: Mm00471579_m1.
Three to five-week-old wild-type F1 (C57BL6×DBA/2) females (Charles Rivers) were superovulated by intraperitoneal injections of 5 IU of pregnant mare's serum gonadotropin (Sigma) followed by 5 IU of human chorion gonadotropin (Sigma) 48 hours later and mated with wild-type males. Mice were sacrificed by cervical dislocation 17 hours after hCG injection, and 1-cell embryos were dissected and released from oviducts. Cumulus cells were removed using hyaluronidase digestion, and single-cell zygotes at the two-pronuclei stage were recovered and immediately micro-injected cytoplasmicly with 5–10 pL of 0.6 mM antisense morpholino. Prior to injection, the MO was heated at 65° for 15 minutes to remove any secondary structure.
Preimplantation embryos were cultured in vitro in 20 µl droplets of Quinn's Advantage Cleavage Medium (Sage) supplemented with 10% SPS serum and covered with mineral oil. Dishes were placed in a desiccator filled with mixed gas (90% nitrogen, 5% oxygen, 5% carbon dioxide) in a 37° incubator. Embryos were observed every 24 hours for a period of four days, about the time of hatching.
Morpholinos were obtained from GeneTools: Chd1l MO-1: tcattccacagcagatacCTGGCAG (in2-EX2); Chd1l MO-2: ttggagagaagcagagggctaCCTC (in4-EX4); Snf2l: tgctgtttaccaccttacCAAGGGC (in2-EX2).
Single embryos were collected 48 hours after injection and lysed by one freeze thaw cycle. cDNA was synthesized using the CellsDirect One-Step rtPCR kit (Invitrogen) and subjected to 18 rounds of gene-specific amplification using TaqMan primer/probe assays (Applied Biosystems). TaqMan primer/probe assays and cDNA from single embryos were loaded onto a Fluidigm 48.48 microfluidic array for qPCR analysis using a Biomark thermalcycler.
TaqMan gene expression assays used in these experiments were predesigned and ordered from Applied Biosystems: Gapdh: Mm99999915_g1; Chd1l Ex2–3: Mm00471561_m1; Chd1l Ex4–5: Mm01257091_m1; Chd1l Ex20–21: Mm00471578_m1.
A hydrophilic sequence of 122 aa corresponding to amino acid numbers 557–678 of the Chd1l protein was selected for the antigenic region. The antigen was produced as a TrpE fusion protein from the pATH11 vector in BL21 E. coli, solubilized, and subjected to SDS-PAGE. The gel slice was excised and submitted to Josman, LLC for injection into two rabbits and two rats. The antiserum was affinity purified using a GST-Chd1l-bound Sepharose column and eluted with low pH buffer. Both the rabbit and the rat antibodies recognize a ~100 kD band on Western blot. Rabbit α-Chd1l antibody was used in all experiments reported here.
The animal experiments were performed in the labs of M.W.M.Y. and M.P.S. under ethical protocols approved by the Stanford Institutional Animal Care and Use Committee.
M.P.S. is an Investigator of the Howard Hughes Medical Institute. M.W.M.Y. and D.L. are supported by NIH (HD057970, HD00849, HD01249). We thank S. Quake for the use of the BiomarkH (Fluidigm Corp.) for microfluidic qPCR experiments.
Competing interests: The authors have no competing interests to declare.