Conceived and designed the experiments: SJK. Performed the experiments: MK JR KAI. Analyzed the data: MK CP FE CMW SJK. Contributed reagents/materials/analysis tools: CP FE CMW. Wrote the paper: MK SJK.
The authors have declared that no competing interests exist.
In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that
We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic
We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages.
Blastocyst formation coincides with demarcation of the first two lineages in the mammalian preimplantation embryo: the ICM that gives rise to the embryo proper, extraembryonic endoderm and mesoderm, and the trophectoderm (TE) that generates the placenta
Homozygous
Temporal and spatial expression of Sox2 protein was examined in preimplantation mouse embryos by immunofluorescence. Sox2 protein was found in oocytes and 2-cell stage embryos; fluorescence intensity increased from the 4-8-cell stage to the morula stage, peaking at the blastocyst, where it was present in both TE and ICM. Sox2 protein was located in nuclei in cleavage stage embryos, although nuclear staining at the 2-cell stage was weak and cytoplasmic staining was observed. However in some 4-cell embryos, Sox2 was localized in nuclei in all blastomeres, while in others, nuclear localization was observed in half of the blastomeres, while still in others Sox2 staining was detected only in the cytoplasm and not in nuclei (
Expression of Sox2 was detected at all stages (oocyte to blastocyst) of mouse preimplantation development by immunocytochemistry and RT-PCR. (
(
In order to rule out that the effects observed were not specific to the cross used (
To determine whether developmental arrest resulted from loss of Sox2 protein, we assessed protein expression by immunofluorescence with three different Sox2 antibodies (
(
The
(
Due to the similarity of sequence in the HMG domain (which
The cell-permeant Sox2 protein was added 24 h after RNAi in the
In order to investigate the
After reversal of
(
Cell number per embryo in
We found that Sox2 is expressed throughout preimplantation mouse development with cytoplasmic or nuclear Sox2 protein detected from the unfertilized oocyte to the blastocyst stage. This finding is partially in agreement with Avilion et al.
The aim of our study was to define the effect of knocking down
After rescue experiments, using Sox2 protein fused with a TAT protein transduction domain
Loss of
In our study, the pluripotency marker proteins Oct4 and Nanog remained unchanged despite the lack of Sox2. This is surprising taking into account their interconnected regulation in ES cells. A similar maintenance of pluripotency markers was also observed in the absence of Tead4
Fgf4 is expressed at the blastocyst stage
Recent findings in mES cells show an inverse relationship between Sox2 and TE associated markers, as ES cells can differentiate into trophectoderm in the absence of Sox2
Our results lead us to conclude that Sox2 plays two distinctively different roles at two different early developmental stages, namely the morula and the ICM-derived ES cells and early epiblast
In summary, after knocking down
This study was approved by the Ethics Review Committee of the University of Manchester and the UK Home Office (Licence Numbers 40/1939 and 40/2669).
Outbred female
On day 2 female mice were killed by cervical dislocation, oviducts were dissected and transferred into M2 medium (Sigma) with 4 mg/ml bovine serum albumin (BSA, ICN Biomedical) before flushing. Embryos were flushed using sterile M2/BSA and a 34-Gauge blunt-ended stainless steel needle (Coopers Needleworks) attached to a 1 ml syringe.
Embryos were washed and cultured in a 30 µl drop of KSOM (EmbryoMax, Chemicon), under embryo-tested mineral oil (Sigma) at 37°C with 5% CO2 as described previously
Specific oligonucleotide RNAi duplexes (Eurogentec) to
The 5′-end of each sense oligo was conjugated to rhodamine. 30 µl of each sense and antisense oligo from the stock solutions (100 µM) were mixed and 15 µl of annealing buffer (50 mM Tris/pH 7.5, 100 mM NaCl, Eurogentec) was added. Oligos were annealed by heating to 95°C for 2 minutes and then allowed to cool for 1 hour at 4°C. The annealed duplex was diluted 1∶20 in 4-(2-hydroxyethyl)-1 piperazine ethanesulfonic acid (HEPES)-buffered saline (HBS) supplemented with 4 mg/ml BSA to give a final concentration of 2 µM (individual
Flushed 2-cell embryos were washed in HBS/BSA, transferred into the electroporation microchamber slides (VWR) in a minimal volume of medium and electroporated using an ECM®830 electroporator (VWR). Thirty embryos per group in 60 µl of each annealed siRNA (50 µM) were loaded onto the electroporation slides. The embryos were electroporated based on an assessment of several voltages and times, the optimum conditions being those used by Grabarek et al.
Glycerol stocks of purified recombinant Sox2-TAT fusion protein were generated as described previously
Embryos were fixed in 4% paraformaldehyde (PFA, Sigma), washed through PBS-Tween with 4 mg/ml BSA (PBS/BSA), permeabilised with 1% Triton X-100 (Sigma) and washed through PBS/BSA. In order to decrease background staining, embryos were incubated for 10 min at room temperature in a solution of 2.6 mg/ml NH4Cl (Sigma) in PBS, and subsequently permeabilised and washed as above. They were then immersed in 1∶20 normal goat serum (NGS, Sigma) or normal donkey serum (NDS, Sigma) before incubation in the appropriate dilution of the primary antibody in PBS/BSA at 4°C overnight. They were then washed and transferred into the appropriate secondary antibody (Alexa-Fluor, Molecular Probes) at 1∶200 dilution in PBS/BSA and incubated for 1 hour in the dark at room temperature. After washing, embryos were mounted in DAPI Vectashield mountant (Vector Laboratories) and aspirated by capillary action into 0.2 µM diameter microcapillaries (Camlab), which were sealed in both ends. Primary antibodies were as follows: Sox2 mouse monoclonal (1∶50, R&D), Sox2 rabbit polyclonal (1∶500, Abcam), Sox2 rabbit polyclonal (1∶100, Chemicon), Oct4 mouse monoclonal (1∶250, BD Biosciences), Nanog goat IgG (1∶10, R&D systems), Yap rabbit polyclonal (1∶25, Cell Signaling), Cdx2 rabbit polyclonal (1∶500, raised in rabbits to an N-terminal KLH-linked peptide of mouse Cdx2 by Dr J. Collins, University of Southampton), Cdx2 mouse monoclonal IgG (1∶400, Biogenex, gift from Dr J. Draper, Toronto Hospital for Sick Children, Toronto), Eomesodermin rabbit polyclonal (1∶100, Orbigen), Fgfr2 mouse IgG (1∶100, Santa Cruz Biotechnology), Fgf4 goat polyclonal (1∶50, Santa Cruz Biotechnology), Occludin rabbit polyclonal (1∶100, Zymed), ZO1 mouse monoclonal IgG (1∶100, Zymed), Desmoplakin mouse monoclonal IgG (1∶10, gift from Prof. D. Garrod, University of Manchester), E-cadherin rat monoclonal IgG (1∶500, Sigma), Gata4 goat IgG (1∶200, Santa Cruz Biotechnology), Gata6 goat IgG (1∶200, R&D). Irrelevant antibodies of the same species/isotype as the primary antibodies replaced the primary antibodies, as negative controls: rabbit IgG (Vector laboratories); mouse IgG (Serotec); goat IgG (Santa Cruz Biotechnology); rat IgG (Santa Cruz Biotechnology).
Embryos were viewed under a multi-photon scanning laser confocal microscope (MRC 1024, BioRad) or a Leica (TCS Sp2 AOBS) inverted confocal microscope at the Bioimaging Facility, University of Manchester. A Z-series of 2.5 µM optical sections was collected and analysed using Confocal Assistant software (version 4.02) or LCS Lite software.
In order to assess the lethality of the
Control and
Apoptotic indices were calculated as number of apoptotic cells divided by total number of cells (DAPI stained nuclei) per embryo. Cell number per embryo was calculated by counting DAPI stained nuclei in confocal images produced by a compressed Z-series of 2.5 µM optical sections for each embryo by means of Confocal Assistant software (version 4.02).
SPSS software (version 11.5) was used to perform Independent Samples T-test for the apoptosis assessment. GraphPad Prism software (version 4) was used for the chi-square tests to monitor distribution of embryos among preimplantation developmental stages.
RNA was extracted from groups of 30 embryos by Dynabeads mRNA Direct Micro Kit (Dynal). cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) according to manufacturer's guidelines. The primers used (5′-3′) were:
Forty-cycle PCR amplification was performed using thermal cycler Mastercycler Gradient (Eppendorf). Amplicons, each amplified from 50 ng cDNA, were run by electrophoresis alongside a 100 bp DNA ladder (Invitrogen) or Hyperladder IV (Bioline) on 2% agarose gels, at 100 V and 150 mA for 1 hour. Mouse genomic DNA (Bioline) was used as positive control.
PCR product bands of the correct size were confirmed to contain the expected gene product by DNA sequencing. Bands were excised from the agarose gels and DNA extracted and purified using QIAquick Gel-Extraction-Kit (QIAGEN) which were then sequenced (in the Sequencing Unit) using an ABI Prism 377 sequencer (Applied Biosystems). Chromas software was used to read the base compositions, which were copied into the NCBI BLAST database (
Validation of Sox2 antibody specificity was performed by Western blotting using 2×107 mouse ES cells (line R1)/ml of lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 0.02% Na azide, 1% Triton X-100) with 1 protease inhibitor cocktail tablet (Complete Mini, Roche)/10 ml lysis buffer. A protein assay was performed using the bicinchoninic acid (BCA) Kit (Pierce), following the Microplate procedure, according to manufacturers' instructions. Twenty five µl lysates samples (50 µg of protein) were resolved by a 4–12% NuPAGE Bis-Tris gel according to manufacturer's recommendations (Invitrogen). 10 µl of BioRad kaleidoscope Multi-Coloured Standard markers were run in parallel at 200 V, 110 mA for 50 minutes. Samples were transferred to a PVDF membrane (Amersham Biosciences), which was blocked in 5% w/v dried milk powder (Marvel) in 0.1% PBS-Tween, incubated with Sox2 antibody (1∶1000, Abcam) in 5% w/v dried milk powder (Marvel) at 4°C overnight and then with a peroxidase-labelled anti-rabbit IgG (1∶3000, DAKO) in 5% w/v dried milk powder (Marvel) for 1 hour. The enhanced chemiluminescence (ECL) detection system (Amersham Biosciences) was used to visualize the protein bands.
(A) Sox2 antibody positive controls: pluripotent mES cells (Sox2-ES) stained for Sox2 (Abcam) in nuclei with some cytoplasmic staining, as well as neurally differentiated E14 cells (Sox2-N) with mainly nuclear Sox2 protein localisation and negative immunological controls (rabbit IgG isotype control and 2° Ab only control) for Sox2 embryo staining. Bars: 100 μm. (B) Western blot to validate specificity of the Sox2 (Abcam) antibody; 50μg mES protein loaded. Lane 1: 2o antibody only; 2: Sox2 (37kDa).
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Development of MF1xMF1 embryos after RNAi. Sox2-duplex-1-siRNA embryos (hatched, N = 30) were compared with incubator-control (grey, N = 20), FFL-siRNA (black, N = 27) and bench-control embryos (white, N = 22). On day 5, while control embryos formed blastocysts from 80% to 92.6%, only 3.3% of the Sox2-siRNA embryos formed blastocysts, with 96.7% arresting at the morula stage. On day 5, chi-square tests revealed significant differences (p<0.0001) between the % of Sox2-siRNA morulae and all control morulae, as well as between the % of Sox2-siRNA blastocysts and all control blastocysts.
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Immunostaining of day 4 and day 5 incubator ctrl (untreated) and Sox2-duplex-3-siRNA embryos, with different Sox2 (Chemicon) and Cdx2 (Biogenex) antibodies than the ones presented in
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RT-PCR for Sox2, Fgfr2, Fgf4, Cdx2, Eomes, Tead4, Oct4, Nanog, Sox1, Sox3, Sox14, Sox15, Sox21 and Beta-actin (40 cycles) on day 4: incubator-control embryos (lanes 1 and 3); Sox2-duplex-1-siRNA embryos (lane 2); Sox2-duplex-3-siRNA embryos (lane 4). In the absence of Sox2 transcripts after siRNA, a clear reduction of Fgfr2, Ffg4, Cdx2, Eomes and Tead4 transcripts in Sox2-siRNA embryos was observed. Oct4 and Nanog transcripts were unaffected in Sox2 knock-down morulae compared to incubator-control morulae. Sox1, Sox3, Sox14, Sox15 and Sox21 transcripts were not expressed in any of the control or Sox2 knock-down morulae. Beta actin transcripts were detected in all embryos.
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Immunofluorescence confirmation of transferred Sox2 after initiation of the rescue experiment using the Sox2-TAT protein. Sox2 siRNA R embryos were immunostained with Sox2 6h, 12h and 18h after the first addition of Sox2-TAT protein, to assess Sox2 protein recovery efficiency. Gradual expression of Sox2 protein was confirmed, with signs of possible endocytic uptake of the protein, as indicated by patchy expression of Sox2.
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Immunological controls of embryo staining presented in
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We thank Robert Fernandez and Jane Kott at the University of Manchester Bioimaging Facility for help with confocal microscopy, and Dr Fozia Chaudry for help with optimization of the siRNA electroporation parameters. We also thank Professor Daniel Brison and Dr Nicoletta Bobola (University of Manchester, UK) for useful comments on the manuscript, Dr Jenny Nichols (University of Cambridge, UK) for the Gata4 and Gata6 antibodies, Dr Jane Collins (University of Southampton, UK), as well as Dr Jon Draper (Toronto Hospital for Sick Children, Canada), for the Cdx2 antibodies.