DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 4159
Development 137, 4159-4169 (2010) doi:10.1242/dev.056630 © 2010. Published by The Company of Biologists Ltd Initiation of trophectoderm lineage specification in mouse embryos is independent of Cdx2 Guangming Wu1, Luca Gentile1, Takuya Fuchikami2, Julien Sutter1, Katherina Psathaki1, Telma C. Esteves1, Marcos J. Araúzo-Bravo1, Claudia Ortmeier1, Gaby Verberk1, Kuniya Abe2 and Hans R. Schöler1,3,*
SUMMARY The separation of the first two lineages – trophectoderm (TE) and inner cell mass (ICM) – is a crucial event in the development of the early embryo. The ICM, which constitutes the pluripotent founder cell population, develops into the embryo proper, whereas the TE, which comprises the surrounding outer layer, supports the development of the ICM before and after implantation. Cdx2, the first transcription factor expressed specifically in the developing TE, is crucial for the differentiation of cells into the TE, as lack of zygotic Cdx2 expression leads to a failure of embryos to hatch and implant into the uterus. However, speculation exists as to whether maternal Cdx2 is required for initiation of TE lineage separation. Here, we show that effective elimination of both maternal and zygotic Cdx2 transcripts by an RNA interference approach resulted in failure of embryo hatching and implantation, but the developing blastocysts exhibited normal gross morphology, indicating that TE differentiation had been initiated. Expression of keratin 8, a marker for differentiated TE, further confirmed the identity of the TE lineage in Cdx2-deficient embryos. However, these embryos exhibited low mitochondrial activity and abnormal ultrastructure, indicating that Cdx2 plays a key role in the regulation of TE function. Furthermore, we found that embryonic compaction does not act as a ‘switch’ regulator to turn on Cdx2 expression. Our results clearly demonstrate that neither maternal nor zygotic Cdx2 transcripts direct the initiation of ICM/TE lineage separation.
KEY WORDS: Cdx2, Trophectoderm, Lineage specification, Preimplantation mouse embryos
INTRODUCTION and in establishing anteroposterior patterning of the intestine The first visible cell lineage split in the development of the (Silberg et al., 2000). Dysregulation of Cdx2 expression has been mammalian embryo occurs during blastulation. The cells of the found in intestinal metaplasia and carcinomas (da Costa et al., inner part of the blastocyst, called the inner cell mass (ICM), are 1999; Bai et al., 2002; Eda et al., 2003). In colon cancer cells, for pluripotent and eventually give rise to the embryo proper and to the example, oncogenic ras downregulates Cdx2 expression by extra-embryonic tissues. By contrast, the cells of the outer layer activating protein kinase C (PKC) pathway signaling and by differentiate into an epithelium, called the trophectoderm (TE), reducing activity of the Cdx2 promoter AP-1 site through changes which subsequently develops into the placenta. To date, a full in the relative expression of c-June/c-Fos (Lorentz et al., 1999); by understanding has been lacking of the molecular mechanisms that contrast, in the embryo, ras-MAPK signaling activates Cdx2 underlie the fate decision for the initial totipotent cells of the expression (Lu et al., 2008). Most importantly, one study found that embryo to become either the TE or ICM lineage. although Cdx2 expression appears to be required for TE cell fate Cdx2 is a class I homeobox transcription factor that belongs to specification in the early mouse embryo, Cdx2 zygotic mutants can the caudal-related homeobox gene family, which comprises still initiate TE differentiation and blastulation (Strumpf et al., mammalian homologs of Drosophila caudal (James et al., 1994; 2005). Similarly, another study found that Cdx2-deficient embryos, Suh et al., 1994). Cdx family members function as upstream generated from Cdx2 short hairpin (sh) RNA-expressing somatic positive regulators of Hox genes (Lorentz et al., 1997), which are cells using nuclear transfer, failed to implant into the uterus, just purported to confer positional identity to cells along the like Cdx2–/– embryos (Meissner and Jaenisch, 2006). However, anteroposterior body axis and are sequentially activated in a neither study eliminated maternal Cdx2 expression. More recent temporal and spatial manner (Davidson et al., 2003). Loss of Cdx studies have found that Tead4 acts upstream of Cdx2 and is function has been shown to be associated with inactivation of Hox essential for TE specification and blastocyst formation (Yagi et al., gene expression (Davidson et al., 2003). Cdx1 and Cdx2, which are 2007; Nishioka et al., 2008), although a subsequent study regulated by p38 MAPK (Mapk14) (Houde et al., 2001), are contradicted these findings by reporting that maternal Cdx2 is involved in defining the anteroposterior body axis responsible for compaction and TE lineage initiation (Jedrusik et (Chawengsaksophak et al., 1997; Chawengsaksophak et al., 2004) al., 2010). Therefore, although it is well established that Cdx2 is indispensable for the maintenance and proper functioning of the TE lineage, its role in the initiation of TE lineage specification remains obscure. It is therefore imperative to study the full effect of Cdx2 1 Department of Cell and Developmental Biology, Max Planck Institute for Molecular expression in early mammalian embryonic development by Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany. 2BioResource Center, RIKEN Tsukuba Institute, Ibaraki 305-0074, Japan. 3University of Münster, Medical eliminating both maternal and zygotic Cdx2 transcripts. Faculty, Domagkstr. 3, 48149 Münster, Germany. In the present study, we microinjected a robust Cdx2 small interfering (si) RNA duplex into zygotes and metaphase II (MII) *Author for correspondence ([email protected]) oocytes and determined the effects of eliminating both maternal
Accepted 11 October 2010 and zygotic expression of Cdx2 on the initiation of ICM/TE lineage DEVELOPMENT 4160 RESEARCH ARTICLE Development 137 (24) separation and cellular differentiation. As Cdx2 acts downstream amplification were custom designed: PF, 5Ј-AACCAGTGGTTGAATA - of Tead4, we also targeted Tead4 with siRNA to further confirm CTAGCAATG-3Ј; PR, 5Ј-CTGCAATGGATGCTGGGATACT-3Ј; and these results. The findings of this study help to better define the probe, 5Ј-6FAM-TTCAGAAGGGCTCAGCAC-MGB-3Ј. Three to six roles played by Cdx2 during early mammalian development. biological replicates were used and each sample was run with three technical replicates; negative controls lacked reverse transcriptase or MATERIALS AND METHODS template. The cycle threshold (CT) values were collected using Applied Embryo culture and microinjection of siCdx2 duplex Biosystems SDS v2.0 software and transferred to a Microsoft Excel Fertilized oocytes were collected from the oviducts of primed B6C3F1 spreadsheet for further relative quantification analysis using the DDCT female mice after mating with CD1 male mice 18 hours post-hCG in M2 method (User Bulletin #2, ABI Prism 7700 Sequence Detection System, medium. Oocytes were cultured in KSOMAA (potassium simplex optimized 1997). Some of the data were presented using the percentage of peak method, which is an adaptation of the C method (Wang et al., 2004). medium plus 19 natural amino acids) (Ho et al., 1995) at 37°C and 5% CO2 DD T in air until microinjection. For MII oocyte microinjection, mature oocytes Immunofluorescent staining of embryos were collected from the oviducts of primed B6C3F1 female mice 14 hours Immunocytochemical staining was performed as described previously with post-hCG in M2 medium, injected with siCdx2, and then fertilized in vitro minor modifications (Strumpf et al., 2005). Briefly, samples were fixed in in modified KSOM (Summers et al., 2000) with epididymal spermatozoa 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X- from adult OG2 male mice. 100 (Sigma-Aldrich, St Louis, MO, USA) for 1 hour, and blocked with 3% We used the online tool BLOCK-iT RNAi Designer to select specific BSA in PBS for 1 hour. Control and Cdx2-deficient embryos were target sequences for siRNA (https://rnaidesigner.invitrogen.com/ processed and examined in parallel. Samples were incubated with the rnaiexpress/). The lyophilized siRNA duplexes (Invitrogen, Karlsruhe, following antibodies: monoclonal mouse anti-Cdx2 IgG (1:100; BioGenex, Germany) were resuspended in 1 ml DEPC-treated water according to the San Ramon, CA, USA), rabbit polyclonal anti-Cdx2 [1:2000; gift from manufacturer’s instructions and stored in single-use aliquots at –20°C. We Felix Beck (Victoria, Australia) and Alexey Tomilin, (Freiburg, Germany)], tested three regular oligonucleotides and three Stealth RNAi rabbit anti-Nanog IgG (1:500; Cosmo Bio Company, Tokyo, Japan), oligonucleotides containing the coding region of Cdx2 (target sequence 5Ј- monoclonal mouse anti-Oct4 IgG (1:200; SC-5279; Santa Cruz Biotech., GCAGTCCCTAGGAAGCCAA-3Ј) in zygotes and selected the most Santa Cruz, CA, USA), rabbit anti-Oct4 IgG [1:2500; generated in our effective duplex (siRNA3) for use in all our experiments. Unless otherwise laboratory (Palmieri et al., 1994)], rat anti-E-cadherin IgG (1:100; specified, a scrambled siRNA duplex was used as control (target sequence ECCD-2; Calbiochem, Darmstadt, Germany), Troma-1 (1:100; DSHB, 5Ј-GCACCCGATAAGCGGTCAA-3Ј). Iowa City, IA, USA) or rabbit anti-ISP1 IgG [1:100; kindly provided by siRNAs were microinjected using an Eppendorf FemtoJet microinjector Dr D. Rancourt (O’Sullivan et al., 2001)], all at 4°C overnight, or with and Narishige micromanipulators. Microinjection pipettes were pulled with rabbit anti-ZO-1 IgG (1:150; Zytomed, Berlin, Germany) at 37°C for a Sutter P-97 pipette puller. siRNA solution (5 l of 8 M) was loaded into m m 1 hour. the pipette and ~2 pl was injected into the cytoplasm of each oocyte. A Embryo samples were then washed several times with PBS and stained relatively consistent amount was carefully injected each time. Specifically, with Alexa 488-conjugated goat anti-rabbit IgG, Alexa 488-conjugated from 73 separate experiments, 3894 of 4093 zygotes (95.1%) were goat anti-mouse IgG (Invitrogen), Cy3-conjugated goat anti-rat IgG, or successfully injected with Cdx2 siRNA, in comparison with 3846 of 4065 Cy3-conjugated goat anti-mouse IgG (Jackson Laboratory, Bar Harbor, zygotes (94.6%) successfully injected with scrambled siRNA. ME, USA) at 1:400 dilution for 1 hour at room temperature. After further In addition, from 18 separate experiments, 345 of 1202 MII oocytes washes and counterstaining with 5 mM DRAQ5 (Biostatus, Shepshed, UK) successfully injected with Cdx2 siRNA were fertilized in vitro, in for 30 minutes, samples were examined under a laser-scanning confocal comparison with 207 of 743 MII oocytes successfully injected with microscope (UltraVIEW; PerkinElmer Life Sciences, Jügesheim, scrambled siRNA and fertilized in vitro and with 327 of 415 uninjected Germany) with 488, 568 and 647 nm lasers. ECCD-2 immunostaining was MII oocytes that were fertilized in vitro. Unsuccessfully injected oocytes quantified with ImageJ 1.42 software (http://rsbweb.nih.gov/ij/ or lysed oocytes were excluded from all subsequent experiments. download.html). Troma-1 antibody and DRAQ5 staining were used to Following microinjection, oocytes were washed and cultured in determine the ICM:TE cell number ratio by comparing confocal images of KSOMAA at 37°C and 5% CO in air and evaluated for cleavage twice 2 neighboring sections using a 5 m scanning distance. daily. The implantation capability of early-stage embryos was evaluated by m transfer of embryonic day (E) 3.5 mouse embryos into the uterus of a 2.5 Transmission electron microscopy days post-coitum (dpc) pseudopregnant CD1 female mouse. Cells were fixed in 2.5% glutaraldehyde (Merck, Darmstadt, Germany) in Animal care was in accordance with the institutional guidelines of the 0.1 M sodium cacodylate buffer (pH 7.4), post-fixed in 1% aqueous osmium Max Planck Institute. tetroxide, dehydrated in ethanol and embedded in Epon 812 (Fluka, Buchs, Switzerland). Ultrathin sections (50 nm) were prepared with an EM UC6 RNA extraction, cDNA synthesis and real-time reverse ultramicrotome (Leica Microsystems, Wetzlar, Germany), stained with 1% transcription PCR uranyl acetate and then 3% lead citrate, and subsequently examined under a For real-time analysis of gene expression, embryos were harvested in RLT Zeiss EM 109 electron microscope (Carl Zeiss, Oberkochen, Germany). buffer (Qiagen, Hilden, Germany) at different stages of development and processed as previously described (Boiani et al., 2005). Briefly, total RNA Measurement of mitochondrial membrane potential and ATP was extracted from individual blastocysts using the MicroRNeasy Kit content (Qiagen) and cDNA synthesis was performed with the High-Capacity Mitochondrial membrane potential was measured by staining with 5,5Ј,6,6Ј- cDNA Archive Kit (Applied BioSystems, Darmstadt, Germany) according tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazolyl carbocyanide iodide (JC-1; to the manufacturer’s instructions. Molecular Probes, Eugene, OR, USA) (Cossarizza et al., 1994). Cells were Real-time PCR was carried out on the ABI PRISM 7900HT Sequence incubated in KSOMAA containing 5.0 mg/ml JC-1 for 15 minutes at 37°C in Detection System (Applied BioSystems) using TaqMan probes (Applied the dark. Cells were washed with KSOMAA, excited at 488 nm, and then Biosystems) for the housekeeping gene Hprt1 (Mm00446968_m1) as a observed at either 510 nm (green mitochondria) or 590 nm (red-to-orange control and the following genes of interest: Cdx2 (Mm00432449_m1), mitochondria) with the confocal imaging system. The ATP content of each Eomes (Mm01351984_m1), Efs (Mm00468700_m1), Atpaf1 embryo was measured as previously described (Boiani et al., 2004). (Mm00619286_g1), Atp1b1 (Mm00437612_m1), Akt3 (Mm00442194_m1), Fgfr2 (Mm00438941_m1), Rab13 Induction and inhibition of embryonic compaction (Mm00503311_m1), Cdh1 (Mm00486906_m1), Hand1 To induce compaction, 4-cell stage embryos were transferred into (Mm00433931_m1), Grn (Mm00433848_m1), Oct4 (Mm00658129_gH), KSOMAA containing 200 mM DiC8 (Sigma-Aldrich) and incubated
Gata6 (Mm00802636_m1), Sox17 (Mm00488363_m1). Oligos for Nanog overnight at 37°C. To prevent compaction, precompacted 8-cell stage DEVELOPMENT Cdx2 in trophectoderm specification RESEARCH ARTICLE 4161 embryos were cultured in KSOMAA containing 200 mg/ml anti-E-cadherin formation (see Table S1 in the supplementary material). As shown antibody (ECCD-1; Calbiochem). Embryo samples were collected after 18 in our time-lapse observations (see Fig. S2D in the supplementary and 24 hours of treatment and subjected to immunocytochemical analysis material), compaction and the onset of blastocyst formation were for Cdx2 expression. unaffected in siCdx2-treated embryos. However, most likely owing Measurement of DNA fragmentation by TUNEL assay to a defect in tight junctions, Cdx2-deficient blastocysts could not The degree of apoptosis in embryo samples was determined using terminal initially maintain the integrity of the blastocyst cavity, collapsed deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end three times and were delayed by ~7.5 hours in starting cavity labeling (TUNEL) with an in situ cell death detection kit (DeadEnd expansion, but were of similar size and gross morphology to Fluorimetric TUNEL System; Promega, Madison, WI, USA) according to siControl embryos at 4.0 dpc. Then, after 5-8 pulses of collapse and the manufacturer’s recommendations. Cells were then viewed/counted expansion, all Cdx2-deficient embryos eventually collapsed in the under a confocal microscope. zonae pellucidae between 5.5 and 6.0 dpc, rather than hatching like Statistical analysis control blastocysts. The Jarque-Bera test was performed on the ICM:TE cell number ratio and To exclude the possibility that any residual maternal Cdx2 apoptotic cell numbers, revealing that the cell number did not follow a protein could have driven TE lineage separation and blastocyst normal, or Gaussian, distribution with a significance level 0.05. formation in siCdx2-treated embryos, we co-injected siCdx2 with Therefore, the Wilcoxon rank-sum test was performed as an appropriate a rabbit polyclonal Cdx2 antibody at 100-fold higher concentration non-parametric statistical test with significance level 0.05 to compare than the effective concentration for immunostaining, but still differences in cell number. observed no phenotype prior to the blastocyst stage (see Table S2 in the supplementary material). RESULTS In addition, an increase in the ICM:TE cell number ratio was Maternal and zygotic expression of Cdx2 found (0.58 versus 0.37; siControl, n40; siCdx2, n41; We examined Cdx2 mRNA and protein levels in developing early P1.05ϫ10–5), but the total number of cells in Cdx2-deficient E4.0 mouse embryos by quantitative real-time PCR (qRT-PCR) and blastocysts was not different from that of control blastocysts (66.3 immunocytochemistry. Maternal Cdx2 mRNA was readily versus 66.9; P0.64; Fig. 1H). TUNEL analysis revealed the detectable in oocytes (CT value of 36.5 from pools of ten MII presence of slightly more apoptotic cells within both the inner (5.4 oocytes) and 2-cell stage embryos, but levels were reduced by 73% versus 2.7; P7.35ϫ10–5) and outer (2.9 versus 1.8; P3.48ϫ10–4; in 4-cell stage embryos and increased by 23-fold upon Cdx2 siControl, n19; siCdx2, n13) cells of Cdx2-deficient blastocysts activation in 8- to 16-cell stage embryos (Fig. 1A). As previously (Fig. 1I). However, transfer of Cdx2-deficient blastocysts into the reported (Niwa, H. et al., 2005; Strumpf et al., 2005; Dietrich and uterus of pseudopregnant foster mice did not lead to any Hiiragi, 2007; Yagi et al., 2007), Cdx2 protein levels could only be implantation (0 implanted out of 109 transferred in the siCdx2- detected as a very weak nuclear signal in 8-cell stage embryos (Fig. treated group versus 60 out of 108 in the siControl, from three 1C); however, a stronger Cdx2 protein signal was detected in some separate experiments), constituting another specific phenotype of blastomeres during the next cell cycle, gradually extending to all the zygotic Cdx2–/– embryos. outer blastomeres (see Fig. S1 in the supplementary material). This Gene expression analysis by qRT-PCR revealed that knockdown expression profile provides evidence for the presence of maternal of maternal Cdx2 led to a 77.5% reduction in the transcript levels Cdx2 transcripts in early mouse embryos and indicates that of the TE-associated gene Eomes in 8-cell stage embryos, whereas activation of zygotic Cdx2 expression is concomitant with the first a reduction in the Fgfr2 transcript and an increase in the transcripts lineage differentiation event during the asymmetric fourth division of the pluripotency gene Oct4 (Pou5f1 – Mouse Genome after compaction in 8-cell stage mouse embryos (Johnson and Informatics) became significant only after blastulation (Fig. 2A,B; McConnell, 2004). Fig. 4; see Fig. S3A in the supplementary material). Notably, Hand1, which is expressed in the TE and regulates trophoblast cell Effective reduction in maternal and zygotic Cdx2 differentiation into trophoblast giant cells (Cross et al., 1995), and by RNAi has profound effects on TE cell Grn, which is an important factor for blastocyst hatching, adhesion differentiation, function and embryo and outgrowth (Qin et al., 2005), were overexpressed in Cdx2- developmental potential deficient blastocysts (Fig. 4). Of the six Cdx2-specific siRNA duplexes tested, siRNA3 was To determine why the normally shaped Cdx2-deficient selected for subsequent experiments owing to its unique sequence blastocysts failed to hatch from the zonae pellucidae, we performed and high efficiency (see Fig. S2A in the supplementary material); gene expression studies with qRT-PCR or immunocytochemistry siRNA3 is hereafter referred to as siCdx2. A scrambled siRNA on E4.0 Cdx2-deficient blastocysts. We found that Grn (an duplex (siControl) with the same composition as siCdx2 but no autocrine growth factor) and ISP1 (a tryptase; Prss28 – Mouse specific target was used as control. A single injection of siCdx2 Genome Informatics), which have been reported to play crucial into MII oocytes or zygotes was found to reduce Cdx2 mRNA roles in the hatching process (O’Sullivan et al., 2001; Qin et al., levels by at least 95%, as determined by qRT-PCR (Fig. 1B). 2005), were not reduced in Cdx2-deficient blastocysts (Fig. 4; see Furthermore, siCdx2 injection also led to an efficient reduction in Fig. S3B in the supplementary material). However, further Cdx2 protein to undetectable levels in each of the 8-cell (siControl, examination revealed the cause of this hatching failure. n26; siCdx2, n28), morula (siControl, n26; siCdx2, n25) and Immunocytochemical analysis of embryos using the tight junction- blastocyst stage (siControl, n71; siCdx2, n67) embryos, as associated protein zona occludens 1 (ZO-1; Tjp1 – Mouse Genome determined by three independent immunocytochemistry Informatics) showed that the apical zonular structure of the tight experiments (Fig. 1C,D; see Fig. S2B in the supplementary junctions was discontinuous in siCdx2-treated embryos (Fig. 1F), material) and a western blot with replicates (see Fig. S2C in the indicating compromised intercellular sealing and epithelial supplementary material). The robust elimination of both maternal integrity. This specific phenotype was previously observed in –/– and zygotic Cdx2 expression did not affect the rate of blastocyst experiments with mouse Cdx2 embryos (Strumpf et al., 2005). DEVELOPMENT 4162 RESEARCH ARTICLE Development 137 (24)
Fig. 1. Efficient reduction of Cdx2 mRNA and protein levels in different preimplantation stage mouse embryos by siCdx2 results in phenotypes similar to Cdx2 knockout. (A)Relative Cdx2 mRNA expression levels as a percentage of peak expression in mouse embryos at different preimplantation stages, as assessed by qRT-PCR. The maternal mRNA decreased at the 4-cell stage, followed by upregulation at the 8-cell stage due to the zygotic activation of Cdx2. Embryo collection times (post-hCG): 2-cell stage, 40 hours; 4-cell stage, 52 hours; 8-cell stage, 62 hours; 16-cell stage, 72 hours; morula, 85 hours; early blastocyst, 96 hours; late blastocyst, 115 hours. (B) A comparison of Cdx2 mRNA levels in siControl- and siCdx2- treated zygotes at different preimplantation stages demonstrated robust downregulation of Cdx2 by siCdx2 treatment. KD, knockdown. (C)Confocal images of 8-cell stage embryos immunolabeled with anti-Cdx2 monoclonal antibody illustrate the reduction of Cdx2 protein (green) by siCdx2 treatment. Red, nuclear counterstaining with DRAQ5. (D)Confocal images of blastocyst stage embryos immunolabeled with anti-Cdx2 (green) and anti- Oct4 (red) antibodies. Following siCdx2 treatment, Cdx2 was eliminated, but Oct4 was ectopically expressed in the trophectoderm (TE). (E)DIC images of E4.0 and E6.0 embryos. All siCdx2-treated embryos failed to hatch. Arrows indicate empty zonae pellucidae left by hatched control embryos. Scale bar: 100mm. (F)Confocal images of ZO-1 (green) and E-cadherin (red) immunohistochemistry show normal E-cadherin distribution, which marks the lateral-basal cell boundary, but disoriented cell polarity of the TE, as indicated by the presence of ZO-1 at both the apical and basal sides (arrowhead and inset). (G)Defective tight junction (arrowhead) in the TE of a Cdx2-deficient blastocyst as shown by electron microscopy. Scale bar: 0.2 mM. (H) Effect of Cdx2 depletion on TE and inner cell mass (ICM) cell numbers. The number of ICM cells was significantly increased (P<0.01), but the total cell number remained unchanged (P0.64) upon Cdx2 depletion. (I)Quantitation of apoptotic cells shows an increase in apoptotic activity in a Cdx2- deficient E4.0 blastocyst,. MII, metaphase II oocyte; ZP, zona pellucida. Error bars indicate standard deviation.
Moreover, we also observed the presence of the zonular form of apicobasal polarity. Examination of Cdx2-deficient blastocysts ZO-1 at both the apical and basal sides of the epithelium, under the electron microscope (Fig. 1G) showed that the tight
suggesting that some TE cells exhibited disorientation in cellular junction structure was actually missing, although ZO-1 protein was DEVELOPMENT Cdx2 in trophectoderm specification RESEARCH ARTICLE 4163
Fig. 2. Significant effect of Cdx2 reduction on gene expression at various developmental stages but no blockage of expression of the differentiated TE marker Krt8. (A)Relative gene expression levels as a percentage of peak expression of Cdx2, Eomes, Fgfr2 and Oct4 in mouse preimplantation embryos after siControl and siCdx2 injection into zygotes. The downstream transcription factor Eomes was reduced to a basal level. Fgfr2 reduction is shown at the early and expanded blastocyst stages. Oct4 expression was increased ~2-fold in both the early and expanded blastocyst stages (P<0.0001). Error bars indicate standard deviation. (B) Ectopic expression of Nanog in the TE of Cdx2-deficient blastocysts after microinjection of siCdx2 at the zygote stage. The embryonic stem cell marker Nanog was ectopically expressed in TE, as shown in this z-stack confocal image of an immunostained siCdx2-treated blastocyst. (C)Sectional confocal image showing the presence of Krt8 (as detected by the Troma- 1 antibody) in an interrupted pattern in Cdx2 knockdown blastocysts.
detected by immunocytochemistry. However, the ultrastructure of presence of a functionally defective TE (Fig. 3A). This finding was lateral-basal adherens junctions was present as in control embryos corroborated by the underexpression of genes involved in both ATP (Fig. 1G). Immunocytochemical analysis of embryos revealed a synthesis (Atpaf1) and consumption (Atp1b1) (Fig. 4A,B) in Cdx2- stronger intensity of E-cadherin, a major component of adherens deficient embryos, as determined by qRT-PCR. However, an junctions, at the basolateral border of TE cells in Cdx2-deficient increase in cytoplasmic ATP content was found in blastocyst stage embryos than in control embryos (Fig. 1F; P<0.01), without an embryos that had Cdx2 knockdown-induced mitochondrial increase in mRNA levels (Fig. 4). Therefore, although adherens dysfunction, as compared with wild-type blastocysts (0.41 versus junctions could compensate for the lack of tight junctions and 0.17 pmol per embryo; Fig. 3B). support delayed blastocoele formation and expansion in the mutated embryos, they failed to maintain intercellular sealing for siCdx2 microinjection into MII oocytes produces a the more demanding process of hatching. phenotype identical to that of zygotes Finally, to further confirm the identity of the outer cells, we Our results showed that microinjection of siCdx2 into MII oocytes performed immunocytochemical analysis of blastocysts for keratin also led to an efficient reduction in Cdx2 mRNA expression levels 8 (Krt8; also known as EndoA), a widely used marker for (Fig. 4A), with no protein detectable in blastocyst stage embryos differentiated TE, with the monoclonal antibody Troma-1 (Brulet et (siControl, n61; siCdx2, n52) by immunostaining. Changes in al., 1980; Ralston and Rossant, 2008). We found that Krt8 was the gene expression profile at E4.0 were similar to those observed present in the outer cells of Cdx2-deficient blastocysts, albeit with an after injection of siCdx2 into zygotes (Fig. 4B), as determined by uneven distribution, clearly showing that establishment of the TE qRT-PCR. Injection of siCdx2 into MII oocytes led to ectopic lineage was independent of Cdx2 at the molecular level (Fig. 2C). expression of Oct4 and Nanog proteins in the outer cells of Cdx2- Our time-lapse analysis revealed that Cdx2-deficient embryos deficient blastocysts (see Fig. S3C in the supplementary material), compacted and initiated cavitation at the correct time and only failed comparable to that observed after siCdx2 injection into zygotes to hatch afterwards. In addition, the zonular form of ZO-1 was found (Fig. 1D; Fig. 2B). Injection of siCdx2 at an earlier time point to be present at the apical sides of the outer cells and certain TE- resulted in the same phenotype and gene expression changes as at associated genes (Hand1 and Grn) were actually overexpressed in the zygote stage, further supporting the conclusion that the Cdx2-depleted embryos. Taking all these data together, we conclude initiation of compaction and separation of ICM/TE in Cdx2- that elimination of both maternal and zygotic Cdx2 expression did deficient embryos was not due to Cdx2 protein produced from not block the initiation of ICM/TE lineage separation, but it did maternal mRNA before the mRNA was knocked down. impact the continuation of TE differentiation and function. Embryonic compaction does not lead to activation Effect of Cdx2 knockdown on mitochondrial of Cdx2 expression activity To evaluate the effect of embryonic compaction on the activation Upregulation of mitochondrial activity is a feature of TE of Cdx2 expression, we used two complementary approaches. First, differentiation. The JC-1 assay was performed to explore the we induced compaction in 4-cell stage embryos with the natural impact of Cdx2 expression on mitochondrial function and revealed PKC agonist DiC8 (Pauken and Capco, 1999). Embryos at this a significant reduction in mitochondrial activity at the apical cortex stage of normal development do not initiate compaction. However,
of 8-cell embryos and TE cells of blastocysts, indicating the upon treatment with DiC8, 4-cell stage embryos rapidly initiated DEVELOPMENT 4164 RESEARCH ARTICLE Development 137 (24)
Fig. 3. Significant effect of Cdx2 knockdown on mitochondrial activity. (A)Confocal images showing mitochondrial activity as assessed by JC-1 staining at the 8-cell, morula and blastocyst stages. Fig. 4. Relative gene expression levels in E4.0 blastocysts after siCdx2 treatment significantly reduced mitochondrial activity, as microinjection of siCdx2 into MII oocytes and zygotes. Elimination indicated by the accumulation of the red aggregated form of JC-1 in of Cdx2 RNA by microinjection of siCdx2 into mouse (A) MII oocytes the mitochondria (appears yellow, as all mitochondria were stained and (B) zygotes had similar effects on gene expression levels, as with the green form of JC-1). (B)Measurement of ATP content in assessed by qRT-PCR. Cdx2 knockdown reduced expression of genes individual embryos, showing a significant increase in ATP from 0.17 crucial in TE lineage differentiation, with overexpression of pmol to 0.41 pmol in Cdx2-deficient blastocysts. Error bars indicate pluripotency-related genes. Error bars indicate standard deviation. standard deviation. compaction, as seen by the flattening and adherence of 2007; Nishioka et al., 2008), these two duplexes were found to blastomeres, which became well compacted within 30 minutes effectively prevent embryo cavitation and clear separation of the (Fig. 5A). Cdx2 expression could not be detected after 18 hours of ICM and TE (Fig. 6B), but had no effect upon compaction at 2.5 treatment by immunocytochemistry (Fig. 5B; DiC8, n21; Control, dpc (see Fig. S4 in the supplementary material), just as with Cdx2 n19). Using the second approach, we blocked the compaction of knockdown embryos. Significant underexpression of TE marker 8-cell stage embryos (Fig. 5C) with ECCD-1, an antibody directed genes (Cdx2, Eomes, Fgfr2 and Atp1b1), but not pluripotency- against the extracellular domain of E-cadherin (Yoshida-Noro et related genes (Oct4, Nanog and Sox2), was observed by qRT-PCR al., 1984), and examined Cdx2 expression after 24 hours in 12- to upon Tead4 knockdown (Fig. 6C). 16-cell stage embryos (Fig. 5D; ECCD-1, n20; Control, n21). Although there was no compaction, Cdx2 expression could still be DISCUSSION detected in 12-cell stage embryos. These results clearly In the present study, we defined the expression profile of Cdx2 in demonstrate that the compaction process does not lead to activation mouse oocytes and embryos by the sensitive and specific technique of Cdx2 expression in mouse embryos. of qRT-PCR and systematically investigated the impact of knocking down both maternal and zygotic mRNA levels by an Reduction in Tead4, which is upstream of Cdx2, siRNA approach on the development of preimplantation embryos. prevents the cavitation and separation of the ICM The consistency in gene expression profile and phenotype of the and TE but does not prevent compaction siCdx2-treated embryos with those of the zygotic Cdx2-null We tested the efficiency of four Tead4-specific siRNA duplexes to embryos (Strumpf et al., 2005), such as the failure of embryos to knock down Tead4 mRNA expression levels by the same hatch from the zonae pellucidae and implant into the uterus, the microinjection approach that we used for Cdx2 knockdown. underexpression of Eomes, and the ectopic expression of Oct4 and Injected oocytes were cultured for 72 hours to reach the Nanog in the TE, demonstrates the efficacy and specificity of our morula/blastocyst stage and collected individually or in groups for siCdx2 treatment. qRT-PCR. Two siRNA duplexes were found to result in a ~90% Developmental biologists have sought for years to understand knockdown efficiency of Tead4 (siTead4A and siTead4B; Fig. 6A). how mammalian embryos establish their developmental pattern and
Consistent with the zygotic Tead4 knockout phenotype (Yagi et al., initiate their first differentiation event – the separation of the TE DEVELOPMENT Cdx2 in trophectoderm specification RESEARCH ARTICLE 4165
responsible for the initiation of TE lineage differentiation. However, the same group later proposed that Cdx2 does not lead the TE transcription factor hierarchy (Rossant and Tam, 2009), as a loss-of-function Cdx2 mutation had no effect on the initiation of blastocyst formation (Strumpf et al., 2005), and that Cdx2-mutant cells were not excluded from the TE layer in chimeric blastocysts (Ralston and Rossant, 2008). These cells, however, do not undergo further trophoblast differentiation and thus are not functional (Strumpf et al., 2005). In the present study, we used an siRNA approach to robustly knock down both maternal and zygotic Cdx2 transcripts, and are the first group to provide unequivocal and direct evidence that maternal Cdx2 is not a determinant of TE lineage commitment. In these maternal and zygotic Cdx2-deficient embryos, compaction and cavitation initiation were not interrupted, as clearly shown by our time-lapse observations (see Fig. S2D in the supplementary material), even though the morula-to-blastocyst transformation was delayed as a consequence of a defect in ZO-1/tight junctions (Wang et al., 2008). The outer cells of Cdx2-deficient blastocysts expressed the TE marker Krt8, confirming their TE identity. However, disorientation of the TE, as indicated by the presence of linear ZO-1 at the base of the epithelium, also highlights the importance of Cdx2 in cell polarization at the preimplantation stages. An increased ICM:TE cell number ratio might reflect compromised TE proliferation and function in regulating the number of ICM cells. These embryos subsequently failed to hatch, implant into the uterus or survive, indicating that TE lineage separation is independent of Cdx2 but that TE function supporting embryogenesis is completely dependent on Cdx2. A recent report from M. Zernicka-Goetz’s group (Jedrusik et al., 2010) has provided results that are contradictory to those of the present study. In that study, the authors achieved a much lower knockdown efficiency of maternal Cdx2 transcripts, of ~80-85% before the 8-cell stage, with Cdx2 protein levels still detectable in some of the treated embryos, but they reported a more severe Fig. 5. Neither induction nor blockage of embryonic compaction Cdx2 phenotype and claimed that the maternal pool of Cdx2 mRNA affects the activation of expression. (A)Premature played a crucial role in compaction and polarization at the 8- to 16- compaction induced within 30 minutes of treatment with the PKC agonist DiC8 (right). Normal 4-cell stage mouse embryos are shown on cell stages of embryonic development. In our experiments, we the left, with obvious, individual blastomeres. (B)Cdx2 expression (red) achieved a knockdown efficiency of maternal Cdx2 transcripts of could not be detected by immunocytochemistry 18 hours after DiC8 greater than 99.0%, without any detectable Cdx2 proteins, and yet treatment. Nuclei are blue (DAPI) and Oct4 (a positive control for 88.4% of these embryos compacted and developed to the blastocyst transcriptional activity of the treated embryos) green. (C)Compaction stage, with the same morphology and developmental rate as those was inhibited by anti-E-cadherin (ECCD-1) antibody treatment for 24 injected with scrambled siRNA (87%) or non-injected embryos hours (right). Normally compacted 14-cell stage embryos are shown on (85.3%) (see Table S1 in the supplementary material). The failure the left. (D)Cdx2 was detected (arrowhead) in uncompacted 12-cell of such embryos to hatch and implant and the formation of stage embryos. Heavy cytoplasmic background (especially around the embryoid body (EB)-like structures on mouse embryonic cell surface) was due to cross-reaction of ECCD-1 (rat IgG monoclonal), fibroblasts (MEFs) (Wu et al., 2010) were reported to be which was used at a very high concentration (200mg/ml) to block –/– embryo compaction, with the Cdx2 antibody (mouse IgG monoclonal). phenotypes specific to zygotic Cdx2 embryos by Rossant’s group (Strumpf et al., 2005), yet we did not observe any significant effect on compaction and blastocyst formation. In the study by M. Zernicka-Goetz’s group, heterogeneous from the ICM (Hiiragi et al., 2006). Cdx2, the first TE marker gene phenotypes were observed as embryos arrested at various stages of to be expressed in mouse embryos, was reported in a study by development. Furthermore, their Cdx2-deficient embryos not only Strumpf and colleagues to be required for correct TE fate totally lacked expression of TE-associated genes, such as Krt8, specification (Strumpf et al., 2005). The zygotic Cdx2–/– embryos Eomes, Ecad (Cdh1), aPKC (Prkcz), Par1 (Mark1) and Par3 failed to downregulate Oct4 and Nanog levels in the outer cells of (Pard3), but also that of the pluripotent marker gene Nanog, which the blastocyst, resulting in loss of epithelial integrity and in a contradicts the findings of other publications, as a reciprocal subsequent failure to hatch and implant. However, these Cdx2-null relationship between Nanog and Cdx2 has already been clearly embryos were still capable of forming a TE, as evidenced by established (Chen et al., 2009) and overexpression of Nanog has cavitation and blastocyst formation, a finding that contradicted the been observed in zygotic Cdx2 knockout experiments (Strumpf et main conclusion of that study and the authors hypothesized that the al., 2005) as well as in the present study. In support of our –/–
expression of maternal Cdx2 in these Cdx2 embryos might be observations, an independent group has reported that following DEVELOPMENT 4166 RESEARCH ARTICLE Development 137 (24)
Fig. 6. Effective knockdown of Tead4 in preimplantation mouse embryos by microinjection of siRNA into zygotes reproduces the Tead4–/– embryo phenotype. (A)Tead4 knockdown efficiency (%) determined by qRT-PCR in a single E4.5 mouse blastocyst in comparison to the scrambled siRNA control. Error bars indicate standard deviation. (B) Failure of blastulation 72 hours (E3.5) after microinjection of siTead4A and siTead4B into mouse zygotes. In this particular experiment, 25 zygotes per group were successfully injected. Note that the phenotype is consistent and highly reproducible within the siTead4A and siTead4B groups (except for one embryo in each group that was blocked at the 2-cell stage), but was variable in groups with lower Tead4 knockdown efficiency. (C)Tead4 knockdown reduced the expression of genes crucial in TE lineage differentiation but did not reduce the expression of pluripotency-related genes. Shown are qRT-PCR results from pools of six embryos, with biological and technical triplicates. injection of Cdx2-specific siRNA at the 1-cell stage, Cdx2-deficient (Ralston and Rossant, 2008). Of note, an increase in ATP content embryos do not exhibit developmental arrest prior to the blastocyst was consistently found in Cdx2-deficient embryos, in duplicate stage and do overexpress the pluripotent gene Oct4 at the blastocyst experiments with triplicate tests, which was probably due to stage (Wang et al., 2010). In a broader sense, research on primates underexpression of the ATP-dependent Na+ pump of the TE, a has also indicated that CDX2 plays an essential role in functional main consumer of ATP at this developmental stage (Houghton, TE formation and that the ICM lineage of CDX2-depleted embryos 2006). These data indicate that deficiency in Cdx2 has profound supports the isolation of functional embryonic stem cells effects on mitochondrial activity and that Cdx2 is indeed required (Sritanaudomchai et al., 2009), which is consistent with our for proper functioning of the TE. Further study is needed to observations in the mouse. determine precisely how Cdx2 regulates mitochondrial activity and In the present study, we observed an increase in mitochondrial ATP levels in the embryo. activity concomitant with TE differentiation. This is consistent with Observations from classical embryonic experiments have the involvement of Na+/K+-ATPase in mouse embryos preparing suggested that ICM/TE lineage separation is initiated by for cavitation and production of nascent blastocoele fluid (Wiley, asymmetric cell divisions that act to establish the inner and outer 1984) and with the stage-specific translocation of active cell populations following compaction and polarization in 8-cell mitochondria during early porcine embryo development (Sun et al., stage mouse embryos (Johnson and Rossant, 1981). During 2001). Interestingly, TE cells have been reported to produce and compaction, changes at the cellular level include enhanced cell consume most of the ATP of the blastocyst (Houghton, 2006). adhesion between blastomeres resulting in the formation of a Functional analysis with JC-1 staining showed reduced activity in smooth ball of blastomeres, and the establishment of cell the mitochondria of Cdx2-deficient embryos. Further gene polarity, as demonstrated by the formation of apical and expression analysis demonstrated that the reduction in basolateral surfaces on the blastomeres (Hyafil et al., 1980; Butz mitochondrial activity was mediated by the underexpression of and Larue, 1995; Ohsugi et al., 1996). At the next cell division genes involved in both ATP synthesis and consumption. Krt8 has after compaction, the developmental fate of the embryo becomes been shown to modulate the shape, distribution and function of restricted, as outer blastomeres become destined to form the first hepatocyte mitochondria and to protect cells from apoptosis (Tao epithelial layer, the TE, and inner blastomeres are pluripotent et al., 2009), suggesting that some of the phenotypes of Cdx2- and form the ICM (Tarkowski and Wroblewska, 1967; Johnson deficient embryos might be mediated by the reduced and uneven and Rossant, 1981). Par3, Par6 and aPKC are components of an Krt8 expression pattern that we observed. Reduction in Krt8 apical polarity complex that has been shown to influence –/– expression has also been observed in zygotic Cdx2 embryos TE/ICM fate choice (Plusa et al., 2005). In contrast to the two DEVELOPMENT Cdx2 in trophectoderm specification RESEARCH ARTICLE 4167
siRNA approach that we used to knock down Cdx2. After specifically and efficiently knocking down Tead4 transcripts, we found that none of the treated embryos was capable of initiating cavitation, consistent with the results of the two previous Tead4 knockout studies. In these Tead4–/– embryos, the maternal pool of Cdx2, which was not at all affected, did not sustain the embryos through the early stages of development to support initiation of TE separation in the positive-feedback loop manner proposed by M. Zernicka-Goetz’s group. Our Tead4 knockdown results provided another solid piece of evidence to support the validity of our Cdx2 knockdown approach and confirmed that maternal Cdx2 does not lead the cascade that regulates TE lineage specification. Notably, Nishioka et al. (Nishioka et al., 2008) performed a trophoblast outgrowth assay and showed that all the TE-specific genes examined, including Cdx2, Eomes, Fgfr2 (Arman et al., Fig. 7. Postulated TE differentiation regulatory network. Cdx2 is 1998), integrin alpha 7 (Itga7) (Klaffky et al., 2001), placental not required for the TE lineage specification but it is critical for further differentiation of TE. Interactions among the processes of compaction cadherin (Cdh3) (Niwa, T. et al., 2005) and the giant-cell-specific and cellular polarization could affect the distribution of Cdx2 along the genes Hand1 (Riley et al., 1998) and Prl3d1 (Faria et al., 1991), –/– inside/outside axis and influence tight junction formation and were not expressed in Tead4 embryos corresponding to the late localization. These interactions could therefore affect ICM/TE lineage blastocyst stage. Similarly, expression of Hand1 and Prl3d1 was allocation. SCMC, subcortical maternal complex. detected in Cdx2+/+ and Cdx2+/– embryos but not in Cdx2–/– embryos after 72 hours of culture of 3.5 dpc embryos (Strumpf et al., 2005). On the contrary, another Tead4 knockout study (Yagi et studies by M. Zernicka-Goetz’s group (Jedrusik et al., 2008; al., 2007) concluded that Tead4 was required for expression of Jedrusik et al., 2010), other studies have shown that Cdx2 some, but not all, TE-specific genes, as expression of Fgfr2 was appears to act downstream of the first lineage decision in the not affected in Tead4–/– embryos. Our qRT-PCR analysis (Fig. 6C) early mouse embryo – i.e. following cell polarization – to showed significant downregulation of TE-associated genes (Cdx2, promote TE cell fate determination in a cell-autonomous manner, Eomes, Atp1b1 and Fgfr2) and significant overexpression of suggesting that processes influencing lineage allocation or Hand1. These results, together with previous observations that morphogenesis might regulate Cdx2 expression along the Tead4–/– embryos weakly and briefly express Cdx2 between the 8- inside/outside axis of the embryo (Ralston and Rossant, 2008). and 18-cell stages, and that they appear to have normal adherens However, it is not yet clear whether compaction induces Cdx2 junctions, cell polarities and JNK and p38 MAPK signaling expression. It is known that compaction can be induced (Nishioka et al., 2008), lead us to propose the existence of prematurely in 4-cell stage embryos by PKC activators (Winkel additional, Cdx2- and Tead4-independent mechanisms in TE et al., 1990; Ohsugi et al., 1993; Pauken and Capco, 1999). With lineage specification, which might include the subcortical maternal this approach, we showed that induction of compaction does not complex (SCMC) and its component Floped (also known as activate Cdx2 expression. Moreover, blocking compaction with Moep19 or Ooep) in the mouse oocyte cortex (Herr et al., 2008; Li ECCD-1, a monoclonal antibody that specifically neutralizes E- et al., 2008). Although the SCMC does not appear to be sufficient cadherin-mediated adhesion (Johnson et al., 1986) and induces for establishing the TE lineage, the persistence of the SCMC in the disorientation of cell polarity in mouse early embryos (Fleming outer cells of E3.5 Tead4-null embryos indicates that it might be et al., 1989), does not prevent Cdx2 expression. These results present in early mouse embryos as an intrinsic pre-patterning factor suggest that Cdx2 expression and compaction are two parallel related to blastomere polarity and TE specification (Li et al., 2008). processes in regulating TE differentiation. This conclusion is As illustrated in Fig. 7, based on the results of this study we consistent with a very recent report from Rossant’s group that confirm the findings of recent studies and clarify conflicting studies demonstrated that the epithelial integrity mediated by E-cadherin by concluding that although Cdx2 plays a crucial role in the is not required for Cdx2 expression (Stephenson et al., 2010). maintenance of TE differentiation and function, neither maternal We next sought to confirm that even though we effectively nor zygotic Cdx2 expression is essential for initiating the knocked down maternal Cdx2 transcripts and attempted to block segregation of the ICM and TE lineages in the early stages of the function of any residual proteins with a Cdx2 antibody, we still could not confer an essential role for maternal Cdx2 in embryo mouse embryonic development. compaction and initiation of the TE lineage, contrary to M. Acknowledgements Zernicka-Goetz’s group (Jedrusik et al., 2010). Here, we add We thank Jeanine Mueller-Keuker, Margit Preusser and Susanne Koelsch for another piece of evidence to support our conclusions. Tead4 has assistance in preparing the manuscript and Dr D. Rancourt for providing the been reported by two studies to act upstream of Cdx2 and to be ISP1 antibody. The content of this manuscript is solely the responsibility of the essential for TE specification (Yagi et al., 2007; Nishioka et al., authors and does not necessarily represent the official views of the Eunice –/– Kennedy Shriver National Institute of Child Health & Human Development or 2008). In these two studies, zygotic Tead4 embryos were found the National Institutes of Health. This research was supported by the Max to be devoid of the TE lineage, and expression of Tead4 was Planck Society and grant NIH R01HD059946-01 from the Eunice Kennedy activated at the 2-cell stage and was postulated to subsequently Shriver National Institute of Child Health & Human Development. This work trigger differentiation of totipotent blastomeres into trophoblast by was also supported in part by DFG grant Germ Cell Potential DFG FOR 1041 the differential activation of the protein through the Hippo SCHO 340/7-1. Deposited in PMC for release after 12 months. signaling pathway (Nishioka et al., 2008; Nishioka et al., 2009). In Competing interests statement
the present study, we attempted to knock down Tead4 by the same The authors declare no competing financial interests. DEVELOPMENT 4168 RESEARCH ARTICLE Development 137 (24)
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