1 Centre for Genome Research, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JQ, UK
2 Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1, Honjo, Kumamoto, 860-0811, Japan
*Author for correspondence (e-mail: jenny.nichols{at}ed.ac.uk)
Accepted April 9, 2001
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SUMMARY |
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Key words: Pluripotency, Blastocyst, Diapause, Epiblast, Self-renewal, Stem cell, Mouse
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INTRODUCTION |
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In the absence of LIF, ES cells differentiate and pluripotency cannot be maintained. LIF acts by engaging a heterodimeric cell surface receptor complex comprising the LIF receptor subunit (LIFR) (Gearing et al., 1991) and glycoprotein 130 (gp130; also known as Il6st) (Davis et al., 1993). A family of related cytokines, including cardiotrophin 1, oncostatin M and ciliary neurotrophic factor, that interact with the LIFR/gp130 complex can substitute for LIF and support ES cell self-renewal (Conover et al., 1993; Pennica et al., 1995; Rose et al., 1994). Alternatively, the combination of interleukin 6 and soluble interleukin 6 receptor activates gp130 homodimers and can be used to derive and maintain ES cells without involvement of LIFR (Nichols et al., 1994; Yoshida et al., 1994).
LIF, LIFR and gp130 mRNAs are all expressed in the mouse blastocyst, and moreover in a reciprocal pattern between the trophectoderm and the inner cell mass (ICM) (Nichols et al., 1996). This is suggestive of a paracrine interaction whereby cytokine production by the trophectoderm would act to sustain embryonic pluripotency. However, embryos lacking LIF, LIFR or gp130 develop normally, at least until mid-gestation. Lif mutants are viable into adulthood, although homozygous females are sterile due to a failure of the uterus to support implantation (Stewart et al., 1992). Lifr-/- embryos die around the time of birth with severe deficits in motor neuron and glial cell populations (Li et al., 1995; Ware et al., 1995). Gp130 mutants exhibit defects in placental, cardiac and haematopoietic development in addition to the nervous system and die between 12 and 18 days post coitum (d.p.c.), depending on genetic background (Nakashima et al., 1999; Yoshida et al., 1996). No evidence for early embryo loss has been reported for any of these mutations. In contrast to the dependency of ES cells on gp130 signalling, therefore, the epiblast in vivo does not appear to rely on this pathway in the course of unperturbed development.
Embryogenesis in mice can be arrested temporarily at the blastocyst stage. This phenomenon of diapause has evolved in certain mammals to overcome sub-optimal conditions for reproduction associated with climate or demands on maternal nutrients due to the presence of a suckling litter (Mantalenakis and Ketchel, 1966; Yoshinaga and Adams, 1966). Under such conditions embryos develop to the hatched blastocyst stage, but then cease development and remain unimplanted in the uterus. In the mouse this state of diapause may persist for several weeks throughout which the blastocysts remain capable of resuming development once restored to an oestrogen-rich environment. Diapause can be induced experimentally by removal of the ovaries after fertilisation. Following ovariectomy embryos progress through blastocyst formation, hatch from the zona pellucida, and then arrest. When transferred to a primed recipient such embryos can implant and develop normally. This alteration of the normal schedule of development necessitates extended maintenance of the epiblast. Interestingly, ES cells were first established from embryos in diapause (Evans and Kaufman, 1981) and this generally appears to facilitate their derivation (Brook and Gardner, 1997).
We have examined Lifr and gp130 mutant embryos for their ability to support maintenance of epiblast cells during embryonic diapause. Whereas normal embryos can produce foetuses after several weeks in diapause, we find that this capacity is completely lost in the mutants. These findings establish a cryptic but critical role for gp130 receptor signalling in the mouse epiblast in vivo and thus provide an explanation for the responsiveness of ES cells to this pathway.
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MATERIALS AND METHODS |
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Delaying implantation of embryos and analysis of developmental potential
Embryonic diapause was induced by surgical removal of the ovaries of pregnant female mice at 2.5 d.p.c. and subcutaneous administration of 0.5 mg of Depo provera. Diapause commences 2 days later, just before the normal time for implantation (4.5 d.p.c.). The ability of delayed blastocysts to resume development was investigated by flushing embryos from the uterus at the designated time and transferring them to pseudopregnant recipients at 2.5 d.p.c. Dissections were performed 7 days later (9.5 d.p.c. recipient age). Intact embryos were examined by light microscopy and genotyped by Southern blotting, as described previously (Li et al., 1995; Yoshida et al., 1996). All subsequent genotyping on embryos, ICMs and trophectoderm lysates was performed using polymerase chain reaction (PCR).
PCR genotyping
Gp130 genotypes were determined by PCR using an oligonucleotide located at the gp130 initiation codon 5'-AGATGTCAGCACCAAGGATTTGGCTA-3', in combination with oligonucleotides located in the second intron of gp130 5'-CCCCAACCTTAACATTATGGAGGTAG-3' or in the tk promoter of tk neo 5'-CCGACTGCATCTGCGTGTTCGAATT-3'. This generated products of 300 base pairs (bp) for wild-type and 450 bp for mutant allelles respectively. Embryos or trophectoderm debris were lysed as previously described (Nichols et al., 1998). Amplification was carried out on 1-7 µl of DNA for 35 cycles (following 95°C hot start for 10 minutes) of 94°C, 15 seconds; 60°C, 12 seconds; 72°C, 60 seconds, with a final extension at 72°C for 10 minutes. Reaction products were resolved by agarose gel electrophoresis.
Immunosurgery
Immunosurgical isolation of internal cell populations (Solter and Knowles, 1975) was carried out on freshly flushed delayed blastocysts. Whole anti-mouse antiserum was used at 20% (v/v) in phosphate-buffered medium 1 (PB1; Whittingham and Wales, 1969). Embryos were incubated in this at 37°C for 2-3 hours, rinsed in PB1 containing 10% foetal calf serum (FCS), then exposed to 20% rat serum (non-heat inactivated as a source of complement) in PB1 at 37°C for 15 minutes. Embryos were then transferred individually to drops of Glasgow modified Eagles medium (GMEM) with 10% FCS under mineral oil for a further hour. Trophectodermal debris was then separated and transferred into individual tubes containing 10 µl of lysis buffer for PCR genotyping.
SSEA-1 immunofluorescence
Freshly isolated ICMs were rinsed in PB1 with 0.15% BSA and incubated in anti-SSEA-1 (stage-specific embryonic antigen-1) supernatant (1/1000 dilution) at 4°C for 45 minutes. After rinsing in PB1 with BSA they were transferred to FITC-conjugated rabbit anti-mouse IgM (1/10 dilution) for 30 minutes at 4°C. They were then rinsed and observed using a fluorescence microscope. ICMs were then processed separately for genotyping by PCR. The anti-SSEA-1 monoclonal antibody developed by Solter and Knowles was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242 (Solter and Knowles, 1978).
Outgrowth of isolated ICMs in culture
Following immunosurgery residual inside cell clumps were transferred to individual gelatinised wells in 4-well plates containing GMEM + 20% FCS. Outgrowths were monitored daily and cultures were fixed for in situ hybridisation analysis after 5 days in culture.
In situ hybridisation
In situ hybridisation was carried out according to the protocol described by Rosen and Beddington (Rosen and Beddington, 1993) adapted for use on cell cultures. The digoxigenin-labelled antisense riboprobe corresponds to nucleotides 7-1013 of SPARC (secreted acidic cysteine rich glycoprotein; GenBank accession number 04017).
ICM cell counts
Cell counts were facilitated by disaggregating freshly isolated ICMs into single cells or doublets. Five minutes incubation in 0.5% pronase in PBS was followed by at least 20 minutes in Ca2+- and Mg2+-free KSOM (Bhatnagar et al., 1995) until the cells could be separated easily.
Detection of dead cells
Hoechst staining was used to identify condensed or fragmented nuclei, indicative of dead cells. Blastocysts undergoing diapause were flushed from uteri, rinsed in PBS, then incubated in 0.05 mM Hoechst in absolute ethanol at 4°C for at least an hour. Before observation they were rinsed in absolute ethanol for 30 minutes, then placed individually into drops of glycerol on a glass slide, each covered with a fragment of siliconised coverslip for fluorescence microscopy. After observation and scoring for dead cells each delayed blastocyst was rinsed in PBS and placed in 10 µl of PCR buffer for genotyping. In subsequent experiments fragmented DNA was detected by TdT-mediated dUTP nick end labelling (TUNEL) according to the manufacturers protocol (Roche Molecular Biochemicals).
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RESULTS |
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Gp130-/- embryos are unable to resume embryogenesis after diapause
The observation that mutant foetuses could not be recovered from recipient females after uterine transfer of delayed blastocysts could be due to embryo loss during diapause or to inability to reinitiate development. The incidence of empty decidua after transfer suggested that mutant embryos had implanted in the host uterus but failed to develop subsequently (Fig. 1). To confirm this, embryos were flushed from pregnant females of gp130 heterozygous intercross matings after 6 days in diapause and genotyped by PCR. The predicted Mendelian ratio for the three genotypes was obtained (15 +/+, 24 +/-, 13 -/-), establishing that gp130-/- blastocysts do persist during diapause. Gp130 signalling therefore plays a specific role in maintaining the developmental potential of the embryo during implantation delay.
Pluripotent cells are absent in gp130-/- blastocysts after diapause
Internal cell populations were isolated immunosurgically (Solter and Knowles, 1975) from blastocysts generated by heterozygous intercross matings after 6 days in diapause. Expression of the epiblast-specific marker SSEA-1 (Solter and Knowles, 1978) was examined by whole-mount immunofluorescence staining, followed by PCR genotyping. Whereas ICMs from wild-type or heterozygous embryos exhibited a distinct region of fluorescence representing the epiblast component of the embryo (Fig. 2A,B), gp130-/- ICMs showed no immunoreactivity (5/5; Table 1). This implies that although gp130-/- embryos can survive diapause, the epiblast compartment is not maintained.
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DISCUSSION |
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The loss of epiblast identity in gp130-/- embryos after 6 days in delay was indicated by the lack of SSEA-1 immunostaining on freshly isolated ICMs (Fig. 2A,B). These ICMs produced only parietal endoderm in culture (Fig. 2D), whereas ICMs from wild-type or heterozygous blastocysts exhibited a prominent mass of epiblast and outgrew both visceral and parietal endoderm (Fig. 2C). Formation of visceral endoderm requires sustained contact with the epiblast (Hogan and Tilly, 1981). Therefore the absence of visceral endoderm in outgrowths from gp130-/- ICMs provides further evidence for the lack of a functional epiblast.
The reduction in the number of ICM cells in gp130-/- embryos during delayed implantation (Fig. 3) implicates cell death rather than inappropriate differentiation as the likely mechanism for loss of the epiblast in gp130-/- embryos during diapause. The elevated numbers of dead cells detected in the gp130-/- embryos compared with their wild-type and heterozygous littermates substantiates this interpretation (Fig. 5). The detection of a number of dead cells in wild-type and heterozygous delayed blastocysts indicates that the cell death observed during normal blastocyst development (Copp, 1978; El-Shershaby and Hinchliffe, 1974) is also ongoing during diapause, albeit at a lower level. Overall there is a modest increase in ICM cell numbers in normal embryos during diapause, as previously suggested by Evans and Kaufman (Evans and Kaufman, 1981). It is striking that we have never observed mutant embryos with more than four dead or dying cells at any one time. Thus the loss of epiblast does not appear to occur simultaneously, but rather to be progressive. Trophectoderm cells in the blastocyst have been shown to exhibit phagocytic activity (Rassoulzadegan et al., 2000). It is possible that dead cell debris is rapidly removed by the trophectoderm and degraded so that only nascent dead cells are detectable, and a cumulative increase in the number of dead cells therefore is not observed. TUNEL-positive cells appearing in the trophectoderm region of delayed blastocysts may also be attributable to trophectoderm phagocytic activity.
There are two possible reasons why epiblast cells might enter into apoptosis in the absence of gp130 signalling. One possibility is that gp130 provides a direct cell survival signal. STAT3, which is activated downstream of gp130, has been shown to have anti-apoptotic activity in some circumstances (Hirano et al., 2000). However, although gp130 signalling can enhance ES cell viability under sub-optimal conditions, its main function in these cells in vitro is to suppress differentiation (Smith, 2001). Therefore an alternative explanation for the diapause phenotype could be that, in the absence of gp130, epiblast cells may begin to differentiate inappropriately and that this subsequently results in apoptosis.
During unperturbed development gp130 and Lifr are expressed in the ICM of the blastocyst, and this expression is maintained during diapause (Nichols et al., 1996, and unpublished data). As shown in Fig. 1, some Lifr-/- embryos are able to resume development after 6 days in delay, although embryogenesis may be compromised. After 12 days of diapause, however, no Lifr-/- embryos were found following transfer, although wild-type or heterozygous embryos were still viable. These data indicate that epiblast maintenance during delayed implantation shows a more acute dependence on gp130 than LIFR. This implies that the action of LIF or related cytokines that signal through the LIFR/gp130 heterodimeric complex, are augmented, at least for a limited period, by a cytokine acting through a separate gp130 receptor complex that does not include LIFR. Interleukin 6 (IL6), which acts via gp130 homodimers is a candidate, since IL6 and IL6R mRNAs have been detected in blastocysts (Murray et al., 1990; Rothstein et al., 1992). We have previously shown that IL6/sIL6R
can support both the derivation and propagation of ES cells with similar efficiency to LIF (Nichols et al., 1994; Yoshida et al., 1994).
Diapause is observed in many different mammals. The hormonal mechanisms, growth characteristics, stimuli (lactational, seasonal or nutritional) and potential duration are sufficiently diverse to imply that this phenomenon has been adopted independently during evolution (Renfree and Shaw, 2000). A speculative possibility is that there may be a relationship between the persistence of gp130/LIFR expression and the distinct duration of diapause seen in different species. The finding that gp130 signalling serves a highly evolved adaptive function is consistent with the proposal that an alternative pathway plays the primary role in supporting normal transient propagation of the epiblast (Dani et al., 1998; Lake et al., 2000; Rathjen et al., 1999). It is noteworthy that mouse epiblast cells appear more amenable to conversion into ES cells if they have been subjected to diapause (Brook and Gardner, 1997; Robertson, 1987). This may be related to the activation of dependency on gp130 signalling that could prime the cells for continued self-renewal. However, ES cell derivation is a multifactorial process (Smith, 2001) and imposition of diapause alone is not sufficient in non-permissive mouse strains or other rodents such as the rat.
The results presented here may have implications for attempts to derive and propagate ES cell equivalents from mammals that do not undergo diapause. Primate blastocyst-derived stem cells have been reported not to respond to gp130 cytokines and to be more difficult to propagate than mouse ES cells (Reubinoff et al., 2000; Thomson et al., 1998). However, the generality of these observations is not certain (Schuldiner et al., 2000). It is worth noting that LIFR and gp130 mRNAs have been detected in blastocysts of various species including humans (Geisterfer and Gauldie, 1996; Nichols et al., 1996; Sharkey et al., 1995). Therefore, the expression of these components has been conserved, but whether this extends to complete or only partial functionality remains to be determined.
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ACKNOWLEDGMENTS |
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