ETV4 and ETV5 Orchestrate FGF-Mediated Lineage Specification and Epiblast Maturation during Early Mouse Development

Cell fate decisions in early mammalian embryos are tightly regulated processes crucial for proper development. While FGF signaling plays key roles in early embryo patterning, its downstream effectors remain poorly understood. Our study demonstrates that the transcription factors Etv4 and Etv5 are critical mediators of FGF signaling in cell lineage specification and maturation in mouse embryos. We show that loss of Etv5 compromises primitive endoderm formation at pre-implantation stages. Furthermore, Etv4/5 deficiency delays naïve pluripotency exit and epiblast maturation, leading to elevated NANOG and reduced OTX2 expression within the blastocyst epiblast. As a consequence of delayed pluripotency progression, Etv4/5 deficient embryos exhibit anterior visceral endoderm migration defects post-implantation, a process essential for coordinated embryonic patterning and gastrulation initiation. Our results demonstrate the successive roles of these FGF signaling effectors in early lineage specification and embryonic body plan establishment, providing new insights into the molecular control of mammalian development.


Introduction
FGF signalling is essential for mouse pre-implantation development where it acts in multiple successive steps.At the blastocyst stage, within the inner cell mass FGF drives the specification of primitive endoderm progenitors, an extra-embryonic endoderm lineage that later forms the yolk sac.Culture in exogenous FGF ligands directs all inner cell mass progenitors to differentiate to primitive endoderm (Nichols et al., 2009;Yamanaka et al., 2010).Conversly, inactivation of FGF signalling through genetic or biochemical means generates embryos with an inner cell mass containing all epiblast cells (Chazaud et al., 2006;Kang et al., 2017;Kang et al., 2013;Krawchuk et al., 2013;Molotkov et al., 2017;Nichols et al., 2009;Yamanaka et al., 2010).However, these epiblast cells have sustained elevated NANOG levels, indicating an inability to exit naïve pluripotency (Kang et al., 2017;Kang et al., 2013;Molotkov et al., 2017;Nichols et al., 2009).FGF signalling is well known to regulate naive pluripotency exit and later germ-layer differentiation in vivo (Lanner and Rossant, 2010;Nichols et al., 2009), and in mouse embryonic stem cells (Kunath et al., 2007;Ying et al., 2008).However, the direct targets and molecular mechanism of how FGF/ERK signalling executes cell fate decisions is poorly understood.
Etv4 and Etv5, are ETS transcription factors, and downstream transcriptional activators of the FGF signaling pathway during embryonic development, with cooperative roles in the morphogenesis of the lung, limb bud and kidney (Herriges et al., 2015;Zhang et al., 2009).
Single-cell transcriptomics of mouse (Ohnishi et al., 2014) and human (Blakeley et al., 2015) blastocysts revealed expression of Etv4 (epiblast and primitive endoderm) and Etv5 (inner cell mass progenitors and epiblast), which are downregulated in Fgfr1 mutant mouse blastocysts 3 (Kang et al., 2017), suggesting these ETVs may govern the transcriptional output downstream of FGF signaling in the inner cell mass.However, despite being implicated in regulation of the pluripotent state in vitro (Akagi et al., 2015;Kalkan et al., 2019;Zhang et al., 2018), the role of these transcription factors during epiblast development in vivo has not been assessed.
Here, we investigated the role of Etv4 and Etv5 as potential downstream effectors of FGF signalling in the establishment and maturation of the epiblast lineage.Analysing compound mutant mouse embryos, we find that loss of Etv5 compromises the formation of the primitive endoderm at pre-implantation stages.While, at peri-implantation the loss of Etv4/5 causes a delay in the progression of pluripotency and epiblast maturation, leading to developmental delay and AVE migration defects at early post-implantation stages.Together, our work sheds light on the successive roles of FGF signalling effectors in mediating inner cell mass fate decisions, and later epiblast maturation required for establishing the embryonic body plan at gastrulation.

Etv4 and Etv5 expression during mouse embryonic development
Given the role of PEA3 family members in the regulation of the pluripotent state in stem cells, we hypothesised that PEA3 family member ETS transcription factors were involved in the establishment of the epiblast lineage.We first characterised where ETV transcripts were expressed during early mouse embryo development using our scRNA-seq dataset from mouse pre-to early post-implantation embryo development (Nowotschin et al., 2019).Etv4 transcripts were expressed in late-blastocyst stage primitive endoderm (PrE) and epiblast (Epi), whereas, Etv5 transcripts were expressed in early-blastocyst stage inner cell mass (ICM) progenitors, and later in epiblast cells (Fig. 1A).
Next, we assessed protein expression at pre-implantation stages.We were unable to detect the ETV4 protein at pre-implantation stages, likely due to the lack of a good commercially available antibody, however ETV5 protein was readily detectable.We performed immunofluorescence staining for ETV5, NANOG, GATA6, and quantified nuclear fluorescence intensity to categorise cell lineages (Supplemental Fig. 1A,B), as described previously (Saiz et al., 2016).In early blastocyst (32-64 cells) stage embryos, ETV5 protein was not expressed.At mid-blastocyst (64-128 cells) stage, low levels of ETV5 protein were detected in uncommitted ICM progenitor cells (NANOG+GATA6+), and at higher levels in epiblast cells (NANOG+GATA6-) (Fig. 1B,C).The highest levels of ETV5 protein were detected in the epiblast cells (NANOG+GATA6-or NANOG-GATA6-) of late blastocyst stage embryos (Fig. 1B,C).ETV5 protein was not present in the primitive endoderm (NANOG-GATA6+) or outer trophectoderm cells.
Further to this, we assessed early post-implantation embryos for ETV5 protein expression.
We performed immunofluorescence analysis for ETV5, GATA6 (visceral endoderm marker), and SOX2 (epiblast and extra-embryonic ectoderm marker).At the egg cylinder stage (E5.5),ETV5 protein is lowly expressed within the nuclei of the epiblast and in the distalmost part of the extra-embryonic ectoderm adjacent to the epiblast, and absent in the visceral endoderm (Supplementary Fig. 1C).At mid-gastrulation stage (E6.5),ETV5 was expressed heterogeneously in the epiblast and in the nascent mesoderm (Supplementary Fig. 1C).
Taken together, the transcript and protein profiling reveal that Etv5 is first expressed in ICM progenitors, and then specifically upregulated in epiblast cells, where expression is maintained in these pluripotent cells through peri-and post-implantation stages.

Loss of Etv5 compromises the formation of primitive endoderm
To determine if ETVs might act as downstream effectors of FGF signalling in preimplantation development, we next wanted to assess the role ETVs in blastocyst formation.
Therefore, we analysed cell lineages upon loss of one, or both, Etv4 (Livet et al., 2002) and Etv5 (Zhang et al., 2009) genes .We collected embryos from Etv4 +/-;Etv5 +/-inter-cross matings at early to late blastocyst stage, and assessed the number of cells in each lineage by quantitative immunofluorescence staining for CDX2 (trophectoderm), NANOG (epiblast) and GATA6 (PrE, Supplemental Fig. 2A) as described earlier.Uncommitted ICM progenitor cells are specified asynchronously from early blastocyst stage to epiblast and primitive endoerm, so that by the late-blastocyst (>128 cells) stage there is a stereotyped proportion of 40% epiblast cells and 60% primitive endoderm within the inner cell mass (Saiz et al., 2016).
The reduction in the proportion of primitive endoderm cells in Etv5 null embryos was driven by both an increase in epiblast cell numbers and a decrease in primitive endoderm cell numbers, without changes in total cell counts (Supplemental Fig. 2C).The compensatory increase in epiblast versus primitive endoerm cell numbers in Etv5 null embryos suggested that uncommitted cells were preferentially specified to epiblast.While the similarity in the number of DP cells in single Etv5 -/-and Etv4 -/-, and double Etv4 -/-;Etv5 -/-knockout embryos, and wild-type embryos indicated timely specification of uncommitted progenitors for all embryo genotypes (Fig. 2C).These data, combined with the comparable number of ICM cells between all genotypes (Supplemental Fig. 2D), argue against a delay in specification, or the selective loss of primitive endoerm cells.
Although epiblast specification did not appear to be negatively affected in any of the mutant embryos, we wanted to determine if the epiblast lineage was being properly maintained after specification.Declining NANOG levels are indicative of the exit of naïve pluripotency as the epiblast undergoes maturation prior to implantation, and is dependent on FGF signalling (Kang et al., 2017;Molotkov et al., 2017;Nichols et al., 2009).NANOG levels were elevated in Etv4 -/-and Etv5 -/-single knockout embryos, when compared with wild-type embryos at the late-blastocyst stage (Fig. 2D).This unphysiologically elevated level of NANOG was further compounded in double knockout embryos (Fig. 2D), with NANOG levels significantly higher in Etv4 +/-;Etv5 -/-and Etv4 -/-;Etv5 -/-compared to wild-type embryos, suggesting that both ETV factors play complementary and synergistic roles in the maturation of the epiblast lineage.
All together, these data suggest Etv5, but not Etv4, is required for balancing the specification of uncommitted ICM cells towards primitive endoerm and away from epiblast cells, to achieve the robust and stereotyped tissue proportions of the blastocyst.

Mechanism of Etv5 action on inner cell mass cell fate decision
Given the phenotypic similarity of Etv5 mutants to mutants with reduced FGF signalling activity (e.g.Fgf4 +/-and Fgfr1 -/-embryos), we reasoned that FGF signalling may be disrupted or dampened in Etv5 mutant embryos.Etv5 is expressed at intermediate levels in uncommitted ICM cells, and upregulated in epiblast cells, mirroring the expression of Fgf4 (Fig. 1, Supplemental Fig. 1B, (Nowotschin et al., 2019).Hypothesising that Etv5 may regulate Fgf4 transcription, we analysed ChIP-seq data of ETV5 binding (Kalkan et al., 2019) in mouse embryonic stem cells (mESC) cultured in 2i (naïve pluripotent conditions), and 16h after transfer to N2B27 (to induce transition out of the naïve pluripotent state).ETV5, a transcriptional activator, is enriched at the upstream promoter region of Fgf4 in naïve and transitioning mESCs (Fig. 3A), suggesting direct regulation in epiblast cells in vivo as well.
To test this hypothesis, we analysed Fgf4 expression after loss of Etv5 in embryos.
Quantitative real-time PCR (qRT-PCR) of E4.5 whole blastocysts demonstrated that Fgf4 transcripts are elevated in Etv5 -/-embryos compared with wild-type (Fig. 3B, fold change = 1.59,P = 0.01).Therefore, in embryos, Etv5 is dispensable for Fgf4 expression, which is sustained in Etv5 -/-embryos with higher Fgf4 expression correlating with increased number of epiblast cells.Thus, loss of primitive endoderm in the Etv5 mutants is not caused by noncell autonomous effect of limited FGF4 availability.
Given the dampened response to FGF signalling in the Etv5 -/-embryos, we attempted to rescue the reduction in primitive endoderm numbers with excess exogenous FGF ligand.
We treated wild-type, Etv5 +/-, and Etv5 -/-embryos with a saturating dose of FGF4 (1μg/ml) and Heparin (1μg/ml) from E2.5 (8-16cell stage) for 48 hours.After FGF treatment, Etv5 -/- embryos consisted of GATA6+NANOG-primitive endoderm cells throughout their ICMs, similar to wild-type and heterozygote embryos (Fig. 3C,D).These experiments demonstrate that high, non-physiological levels of exogenous FGF4 can rescue the specification of the primitive endoerm in Etv5 -/-mutants.In conclusion, these experiments show that while primitive endoderm specification can be rescued with exogenous FGF4, under physiological conditions, Etv5 plays a crucial cell-autonomous role in tuning sensitivity of ICM cells to the FGF4 signal during primitive endoderm specification.

Loss of Etv4/5 causes a delay in the progression of pluripotency
We hypothesised that ETV factors may play a role in epiblast maturation, as suggested by the elevated NANOG levels observed in mutant embryos (Fig. 2D).To probe the role of Etv4 and Etv5 in pluripotency exit further, we assessed additional markers of pluripotency at the late blastocyst stage.Embryos from Etv4 +/-;Etv5 +/-inter-crosses were stained for a core pluripotency marker (SOX2), naïve marker (KLF4), and formative/primed marker (OTX2) (Fig. 4A).While SOX2 and KLF4 in the epiblast were expressed at similar levels across all genotypes (Supplemental Fig. 4A, B), epiblast OTX2 expression was significantly reduced in embryos lacking Etv5 (Fig. 4B, C).Analysis of published Etv5 chromatin binding in mESC (Kalkan et al., 2019) shows strong binding at a downstream enhancer region (Fig. 4D).
Together, these data indicate that during pre-implantation development Etv5 is required for timely exit of pluripotency, likely through direct regulation of Otx2, and indirect regulation of Nanog by an as yet unknown mechanism.
We then assessed pluripotency at early post-implantation stages at E5.5.In wild-type, Etv4, and Etv5 null mutants embryos, the epiblast cavitated and retained expression of the core pluripotency factor SOX2 (Supplemental Fig. 4C).Interestingly, OTX2 was expressed in both the visceral endoderm and the epiblast at this stage, indicating that ETV factors are not necessary for upregulation of primed pluripotency factors at early post-implantation stage.
We then tested the requirements of Etv4 and Etv5 for pluripotency in ESCs.We differentiated double knockout Etv4 -/-;Etv5 -/-ESCs (Lu et al., 2009) to epiblast-like cells (EpiLC) representative of the early post-implantation epiblast, which required exogenous FGF2 and Activin.After two days of EpiLC differentiation, both wild-type and double knockout cells maintained core pluripotency transcription factors (OCT4, SOX2), downregulated naïve markers (NANOG, KLF4) and upregulated primed markers (Supplemental Fig. 4D,E).Indicating, that loss of Etv4/5 is not sufficient to completely block naïve pluripotency exit, in agreement with in vivo early post-implantation embryo data (Supplemental Fig. 4C).However, given the failure in downregulation of NANOG and upregulation of OTX2 at pre-implantation stages, ETVs likely control the timely exit of naïve pluripotency in the embryo, this delay has been similarly shown in ESCs (Kalkan et al., 2019).

Compound Etv4/5 mutants display developmental delay and anterior visceral endoderm migration defects
We next looked at later embryonic time-points to determine if the delayed naïve pluripotency exit impacted later stages of development.At E6.5, prior to gastrulation, wild-type and Etv4 mutant embryos expressed high levels of OTX2 in the anterior visceral endoderm (AVE), and NANOG expression was confined to the proximal posterior region of the epiblast, marking the primitive streak (Fig. 5A).In Etv5 null, and compound mutants, the distal visceral endoderm (DVE) marked by high OTX2 failed to migrate anteriorly (Fig. 5A, left panel arrow heads).In single Etv5 mutants, this resulted in a failure to properly position the anterior-posterior axis, as evidenced by the ~45 degree rotation of the NANOG expression domain, marking the primitive streak.Double knockout embryos at E6.5 were overall smaller compared to E5.5 wild-type embryos.The extra-embryonic ectoderm region was noticeably reduced in size, with a disordered morphology of the visceral endoderm epithelium (Fig. 5A, right panel arrowhead).NANOG expression was elevated throughout the epiblast, indicating that either the entire epiblast had failed to exit naïve pluripotency, or the presence of an expanded primitive streak region.
To determine if mutant embryos were specifying the AVE correctly and able to initiate gastrulation, we stained for an AVE marker, CER1, and mesoderm marker, T (Brachyury).In wild-type and Etv4 null embryos, CER1 was localised to the anterior region of the embryo, extending from the embryonic-extraembryonic junction to the distal tip (Fig. 5B), while T was localised to the posterior epiblast of marking the nascent mesoderm.However, in single Etv5 and double Etv4;Etv5 homozygous mutant embryos, while CER1 was expressed, indicating that the AVE had been specified, the migration of the AVE appeared to be disrupted, delayed (Fig. 5B, arrowheads), or arrested at the distal tip (Supplemental Fig. 5A).The AVE, as evidenced by CER1 expression could sometimes be seen extending beyond the embryonic-extraembryonic junction, indicting an over migration of the AVE.The CER1 domain size was noticeably reduced relative to the size of the embryos.Again, abnormal thickening, and disordered DVE/AVE epithelial morphology was observed in these mutants (Fig. 5B arrowhead).Finally, mutants were devoid of T+ cells, indicating a failure in gastrulation and/or specification of mesoderm cell types.
The AVE migration phenotype and reduced embryo size was more severe and pronounced in compound mutant embryos Etv4 +/-;Etv5 -/-and Etv4 -/-;Etv5 -/-when compared with single homozygous Etv4 +/+ ;Etv5 -/-embryos.So, while Etv4 single mutants did not have an overt phenotype, partial or complete loss of Etv4 did increase the severity of the Etv5 phenotype, suggesting that these factors may partially compensate, or have overlapping roles in the early post-implantation epiblast.
By late-gastrulation (E7.5)Etv5 and double homozygous null embryos were severely developmentally retarded with a failure in gastrulation, as indicated by the absence of the primitive streak marker, T (Supplemental Fig. 5B).Epiblast morphology was grossly abnormal, with ruffles and folds to the epithelium.As a consequence, Etv5 homozygous mutant pups were not recovered from Etv4;Etv5 heterozygous intercrosses, and were drastically under-represented in Etv5 heterozygous intercrosses (Supplemental Fig. 5C,D).
Etv4 -/-;Etv5 +/-pups were also not recovered, further suggesting a combinatorial role for ETV factors during embryonic development.All together, these data demonstrate that embryos of Etv4;Etv5 allelic series have increasingly severe phenotypes with loss of ETV gene dosage at early postimplantation stages exhibiting developmental delay and AVE migration defects which compromise embryonic development.

Discussion
Here, we have shown that Etv5 -/-blastocysts have a reduction in primitive endoderm specification.This phenotype bears a striking similarity to that of Fgfr1 -/-and Fgf4 +/-embryos (Brewer et al., 2015;Kang et al., 2017;Kang et al., 2013).Given that Fgf4 transcripts are not diminished in Etv5 mutants, the phenocopying of low dosage FGF mutants cannot be due to a decreased ligand availably.Our findings support the hypothesis that Etv5 relays FGF signalling activity in uncommitted ICM cells, as in other developmental contexts (Herriges et al., 2015;Zhang et al., 2009).The direct involvement of Etv5 in initiating the primitive endoderm program is further supported by its ability to upregulate multiple endoderm genes (including Sox7 and Sox17) when overexpressed in mouse ESCs (Correa-Cerro et al., 2011).However, FGF hyperstimulation can rescue primitive endoderm cell numbers in Etv5-deficient embryos, suggesting that under these non-physiological conditions, related ETS family members such as Etv1 and Etv4 may compensate for loss of Etv5 function.
Etv4 and Etv5, have been implicated in regulating pluripotency in vitro.For example, Etv5 was shown to promote MET during iPSC reprogramming (Zhang et al., 2018).In addition, these ETV factors appear to regulate ESC proliferation, but there is conflicting evidence as to whether they promote or repress epiblast-like fate during differentiation (Akagi et al., 2015;Zhang et al., 2018).Triple knock-out of Etv5, Rbpj and Tcf3 in ESC can maintain naïve pluripotent state in absence of 2i (Kalkan et al., 2019), implicating these factors in dissolution of the naïve pluripotency transcription factor network.Given that ETV5 does not directly bind to the Nanog locus in ESC (Kalkan et al., 2019), it seems likely that this regulation by ETV factors in vivo is indirect.Instead, ETVs may activate the primed pluripotency network, which then, in turn, repress the naïve state.Consistent with such a model, the primed pluripotency marker, OTX2, fails to be upregulated in Etv5 mutant blastocysts.ETV5 has been shown to directly bind an Otx2 enhancer in mESCs (Kalkan et al., 2019), suggesting it is a direct target in vivo.As Otx2 and Nanog are antagonistic (Acampora et al., 2017;Acampora et al., 2016), the role of ETV in epiblast cells may be to turn on Otx2, and consequently reduce Nanog levels thereby promoting the exit from naïve pluripotency.
OTX2, a marker of primed pluripotency, is normally expressed in the epiblast from late blastocyst stage (E4.5) through early post-implantation.In Etv5 -/-embryos however, OTX2 is absent at E4.5 but eventually expressed by E5.5, indicating a delayed exit from naïve pluripotency.This delay is consistent with our observation of elevated NANOG levels in Etv5 mutant blastocysts.Fruthermore, this is further supported by time-course analysis of Etv5 -/- ESC differentiation showing delayed naïve pluripotency exit (Kalkan et al., 2019).Together, these findings highlight the role for the ETVs in regulating the robustness and timely exit of naïve pluripotency.Timely, Nodal-dependent maturation of the epiblast at early post-implantation stages is required for the co-ordinated and spatial patterning of the embryo and initiation of gastrulation (Huang et al., 2017;Zang et al., 2022).Given that Etv4 and Etv5 are not expressed in the visceral endoderm, we hypothesize that the delayed epiblast maturation observed in Etv5 and Etv4;Etv5 compound mutants leads to the later AVE development defects.In the Etv5 and Etv4;Etv5 compound mutants there is a reduction in the number and migration CER1 expressing cells, with disordered discontinuous AVE and DVE populations, and ectopic protrusions.These phenotypes are reminiscent of mutants affecting AVE development, including mutants in Nodal signalling pathway components (Stower and Srinivas, 2014).The most severely affected Etv4 -/-;Etv5 -/-embryos either fail to migrate the AVE/DVE, or the AVE over-migrates beyond the embryonic-extraembryonic boundary, and exhibit a disordered multi-layered epithelium, reminiscent of the two classes of Lefty1 mutants (a Nodal antagonist) (Trichas et al., 2011).Our research suggests that FGF, in conjunction with Nodal, plays a role in epiblast development and its interaction with the visceral endoderm, which is crucial for establishing the anterior-posterior axis.
All together, our results demonstrate the successive roles of ETS factors Etv4 and Etv5 as FGF signaling effectors in early lineage specification and embryonic body plan establishment, increasing our understanding of the molecular mechanisms of mammalian development.

Immunofluorescence
Pre-implantation embryo immunofluorescence was carried out as previously described (Saiz et al., 2016).Briefly, embryos were fixed for 10 min at room temperature in 4% PFA.Fixed blastocysts were washed in PBX; 0.1% Triton X-100 (Sigma-Aldrich) in PBS, permeabilised for 5 min in a solution of 0.5% Triton X-100, 100 mM glycine in PBS and then washed in PBX for 5 min.Embryos were blocked in blocking buffer: 2% horse serum in PBS, for 40 min at room temperature, followed by incubation overnight at 4°C with primary antibodies diluted in blocking buffer (see antibody list below).The next day, embryos were washed three times in PBX, incubated in blocking buffer for 40 min at room temperature before a 1 h incubation with secondary antibodies at 4°C.Embryos were then washed in PBX and incubated in 5 μg/ml Hoechst in PBS for at least 30 min prior to imaging.
For post-implantation stages, embryos were fixed for 30 min at room temperature in 4% PFA.Then, fixed embryos were washed in PBX, permeabilised in 0.5% Triton-X in PBS for 30 min and then washed three times in PBX.Embryos were then incubated in blocking buffer, 5% donkey serum and 0.2% BSA in PBX for 2hrs at RT, followed by incubation overnight at 4°C with primary antibodies diluted in blocking buffer.The next day, embryos were washed in PBX, followed by a second blocking step for at least 2hrs at room temperate, and incubation with secondary antibodies in blocking buffer overnight at 4 °C.
After antibody staiing, embryos were washed in PBX, and incubated with 5 μg/ml Hoechst in PBX for a minimum of 2 hours to visualise DNA prior to imaging.

FGF embryo treatment
Embryos for this study were obtained from natural matings between Etv5 male and female heterozygotes.The sex of embryos was not determined.E2.5 morulae were flushed from oviducts with flushing holding medium (FHM, Millipore) as described (Behringer et al., 2014).Embryos within litters were randomly assigned in even sized groups for control and exogenous FGF treatment.Control: KSOM (MR-121-D, Sigma) and FGF stimulation: 1μg/ml FGF4 (R&D Systems) and 1μg/ml Heparin (Sigma) in KSOM.Medium was equilibrated 30 mins prior to culture to reach the correct temperature and pH.Embryos were cultured in groups in droplets of medium in 35mm dishes (approximately 1μl/embryo) overlaid with mineral oil (Sigma), for 48 hours in total in a humidified incubator at 37°C with 5% CO2.After 24 hours (E3.5) the zona pellucidae were removed by brief incubation in acid Tyrode's solution (Sigma), washed three times in their respective culture medium, and then cultured in fresh droplets of medium.Embryos were assay for lineage markers by immunofluorescence at the end of 48hr culture.
Single-embryo qPCR E4.5 stage embryos from Etv5 intercrosses were prepared for genotyping and qPCR as previously described (Kang et al., 2017;Morgani et al., 2018), using CellsDirect One-Step kit in accordance with the manufacturer's instructions.Blastocyst were first washed in PBS, then incubated in 0.5% trypsin for 3 mins at 37°C.Using a glass capillary, a small number of mural TE cells were removed for PCR genotyping, then each blastocyst was added to 5 µl of 2 × Reaction Mix (Invitrogen, CellsDirect One-Step qRT-PCR Kit) and snap-frozen on dry ice and stored at -80°C until processing.
For cDNA and target specific pre-amplification 5μl of a reverse transcription/pre-amp mix was added to each blastocyst lysate.For each sample this comprised of 0.2μl SuperScript II RT/Platinum Taq mix (Invitrogen), 2.5μl TaqMan assay (pooled assay mix with a concentration of 0.2x for each probe, detailed in supp.Table X), 2.3μl RNase-free H2O.To perform combined cell lysis, cDNA synthesis and pre-amplification of specific targets, samples were incubated at 50 °C for 20 min, 95 °C for 2 min, followed by 18 cycles of 95 °C for 15 sec then 60 °C for 4 min, in a T100 Thermal Cycler (Bio-Rad).
Blastocyst cDNA was diluted 1 in 5 by adding 40μl H20 to the 10μl cDNA to a total of 50μl.
To assay the amount of mRNA, each qPCR reaction was set up in duplicate in 96-well plates (Applied Biosystems 4306737) overlaid with MicroAmp clear adhesive film (Applied Biosystems 4306311).For each qPCR reaction the mix was as follows: 7.5μl TaqMan Universal PCR Master Mix, 0.75μl TaqMan Assay, 5.25μl RNase free H2O.1.5μl diluted sample cDNA was then added to each reaction, to a total of 15μl.Real-time PCR was then carried out in a QuantStudio 7 Flex System (Applied Biosystems).Gene expression was calculated as 2 ΔΔCt .Target gene (FGF) expression was calibrated to the arithmetic mean of the wild-type samples, and normalised to two reference genes' expression within each sample, Gapdh and Actb, using the geometric mean (Vandesompele et al., 2002).
award from NYSTEM (C32599GG).Work in the laboratory of KKN was supported by the Wellcome (221856/Z/20/Z) and the Wellcome Human Developmental Biology Initiative (215116/Z/18/Z).Work in the laboratory of K.K.N. is also supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001120), the UK Medical Research Council (FC001120) and the Wellcome (FC001120).