Deletion of p66Shc Dysregulates ERK and STAT3 Activity in Mouse Embryonic Stem Cells,Enhancing Their Naive-Like Self-Renewal in the Presence of Leukemia Inhibitory Factor

Abstract

The ShcA adapter protein is necessary for early embryonic development. The role of ShcA in development is primarily attributed to its 52 and 46 kDa isoforms that transduce receptor tyrosine kinase signaling through the extracellular signal regulated kinase (ERK). During embryogenesis, ERK acts as the primary signaling effector, driving fate acquisition and germ layer specification. P66Shc, the largest of the ShcA isoforms, has been observed to antagonize ERK in several contexts; however, its role during embryonic development remains poorly understood. We hypothesized that p66Shc could act as a negative regulator of ERK activity during embryonic development, antagonizing early lineage commitment. To explore the role of p66Shc in stem cell self-renewal and differentiation, we created a p66Shc knockout murine embryonic stem cell (mESC) line. Deletion of p66Shc enhanced basal ERK activity, but surprisingly, instead of inducing mESC differentiation, loss of p66Shc enhanced the expression of core and naive pluripotency markers. Using pharmacologic inhibitors to interrogate potential signaling mechanisms, we discovered that p66Shc deletion permits the self-renewal of naive mESCs in the absence of conventional growth factors, by increasing their responsiveness to leukemia inhibitory factor (LIF). We discovered that loss of p66Shc enhanced not only increased ERK phosphorylation but also increased phosphorylation of Signal transducer and activator of transcription in mESCs, which may be acting to stabilize their naive-like identity, desensitizing them to ERK-mediated differentiation cues. These findings identify p66Shc as a regulator of both LIF-mediated ESC pluripotency and of signaling cascades that initiate postimplantation embryonic development and ESC commitment.

Introduction

Embryonic development and cellular differentiation rely on the accurate transduction of extrinsic cues to mediate transcriptional programs. These complex cellular events are characterized by just a handful of intracellular cascades that elicit a broad range of cellular responses through regulation of signal timing, magnitude, and localization [1]. Signaling is achieved, in part, through adaptor proteins that possess two or more distinct protein binding domains which facilitate interactions between proteins [2].

ShcA (Src homology domain containing transforming protein 1) belongs to a family of adaptor proteins that regulate pathways responsible for cell proliferation and differentiation. ShcA plays a key role in facilitating the rat sarcoma/mitogen activated protein kinase (RAS/MAPK) cascade [3 –6], which mediates many stages of embryonic development, particularly the early acquisition of cell identity [7,8]. The ShcA locus encodes three isoforms with molecular weights of 46, 52, and 66 kDa (p46Shc, p52Shc, and p66Shc, respectively), which are largely conserved across vertebrates [5,9,10].

Despite their structural similarity, the ShcA isoforms have functionally distinct roles in signal transduction. The p52/46 kDa isoforms predominantly transduce mitogenic signaling by binding to Grb2, facilitating its interaction with Son of Sevenless, which subsequently activates the MAPK cascade [10 –12]. In contrast, p66Shc antagonizes mitogenic signaling and promotes oxidative stress [13 –15]. Although p66Shc binds to Grb2 [16], this interaction does not necessarily lead to the activation of ERK1/2. Expression of p66Shc is correlated with the inhibition of ERK1/2 signaling in some cell lineages, including fibroblasts [16], T-cells [17], and CHOK-1 cells [18], while in other cell types, p66Shc expression is associated with the activation of ERK1/2 [19 –21]. These conflicting findings suggest that p66Shc regulates ERK1/2 signaling in a cell-context dependent manner.

Several studies have implicated p66Shc in the regulation of cell fate: kidney development in rats [22], morphogenesis of fetal mouse lungs [23], and B cell maturation [24]. We previously demonstrated that p66Shc transcript abundance alters the expression of lineage-associated transcription factors (TFs) and the timing of blastocyst cavitation [25,26]. Collectively, these studies implicate p66Shc in a wide variety of developmental roles; however, the extent and mechanism by which p66Shc affects cell fate have not been established.

To explore the involvement of p66Shc at the earliest stage of lineage commitment, we employed the use of mouse embryonic stem cells (mESCs). In vitro, mESCs can be maintained in a state transcriptionally similar to the inner-cell mass of the preimplantation blastocyst, referred to as naive-like pluripotency, and are capable of recapitulating the early events of epiblast maturation [27].

The propagation of naive-like mESCs can be achieved by the addition of growth factors and pathway inhibitors, to reinforce self-renewal while preventing differentiation. Current approaches commonly involve modulating key pathways: (1) blocking extracellular signal regulated kinase (ERK) activity, traditionally through the inhibition of mitogen-activated protein kinase (MEK), the kinase responsible for ERK1/2(T202/Y204) phosphorylation, (2) inhibition of glycogen synthase kinase-3 beta (GSK3β), the kinase responsible for the phosphorylation of β-catenin(S33/S37/T41), targeting β-catenin for proteasomal degradation, and (3) enhancing STAT3 activity, through supplementation with exogenous LIF which stimulates Janus kinase (JAK) the enzyme responsible for the phosphorylation of STAT3(Y705). When combined, the in vitro supplementation of LIF together with pharmacologic inhibitors of MEK and GSK3β is commonly referred to as 2i/LIF media [28].

The relevance of these pathways to the acquisition of cell fate is evident from the effect of their targeted disruption on mouse embryogenesis. Germline deletion of ERK2 results in developmental abnormalities before E5.5 and embryonic lethality by E8.5, with failure of trophoblast maturation and endoderm specification [29]. Deletion of β-catenin prevents mesoderm formation, causing embryonic lethality at the gastrulation stage E6.5-E9.5 [30]. Targeted disruption of STAT3 similarly prevented mesoderm formation, resulting in embryonic lethality by E7.0 [31].

In vitro, the imbalance of these signals can induce precocious differentiation, loss of ESC self-renewal, or impair lineage commitment. Notably, ERK and STAT3 have been shown to have opposing roles at this stage of naive-like identity. ERK is generally considered essential for early ESC differentiation, driving commitment toward primed-like identity, which is representative of postimplantation epiblast cells [8,32 –35]. STAT3 activity, conversely, opposes ESC differentiation and its forced activation can durably suppress ESC differentiation [36 –38].

Phosphorylation of STAT3 at Y705 is primarily mediated by JAK, which is critical for its nuclear translocation and transcriptional activity. However, STAT3 activity can also be modulated by phosphorylation at S727. Although the regulation of S727 phosphorylation and its contribution to STAT3 transcriptional activity remain controversial [39 –42], preventing STAT3(S727) phosphorylation, by replacement of serine residue with alanine, has been reported to reduce the expression of core pluripotency TF Nanog in mESCs [43].

Embryogenesis relies on coordinated timing and activity of these critical signal effectors, and consequently, disrupting their upstream regulators can also have dramatic effects across multiple stages of embryogenesis. The targeted disruption of the upstream activators of ERK can lead to delayed ESC differentiation and embryonic lethality early to mid-gestation [44 –47]. Similarly, germline deletion of ShcA in mice causes profound cardiovascular defects and results in embryonically lethal by E11.5 [12].

We hypothesized that p66Shc could act as a negative regulator of ERK activity in mESCs, to block lineage commitment and promote the retention of naive-like pluripotency. To explore this hypothesis, we examined the differentiation capacity of p66Shc knockout (KO) mESCs in vitro and the effect of p66Shc deletion on the critical signaling effectors involved in pluripotency. Consistent with our hypothesis, the deletion of p66Shc enhanced ERK phosphorylation but, surprisingly, also simultaneously reinforced mESC naive-like identity. We discovered that loss of p66Shc enhanced not only ERK phosphorylation but also STAT3(S727) phosphorylation. We propose that p66Shc intersects with both ERK1/2 and STAT3 pathways during mESC commitment, thereby sensitizing mESCs to differentiation by suppressing STAT3(S727) phosphorylation, despite simultaneously inhibiting ERK1/2.

Materials and Methods

Generating p66Shc KO mESCs

R1 wild type (WT) mESCs (provided by Janet Rossant, Sick Kids Hospital, Canada) were targeted with CRISPR/Cas9 to induce a knockout of p66Shc. Guide RNAs were designed to target the p66Shc-specific promoter and the 5′ region of exon 2, which encodes for the CH2 region unique to p66Shc. Guide RNA sequences, 5′-TCGGGGTCTACCCCTCCGG-3′ and 5′-ATATATCTGTAGGTCCGAG-3′, were synthesized by Life Technologies and were introduced into the pSpCas9(BB)-2A-Puro (PX459, No. 48139; Addgene [48]) plasmid following digestion with the restriction enzyme Bbs1. The plasmid was transfected into the R1 mESC line using Lipofectamine 3000 (Life Technologies) according to manufacturer's guidelines. Transfected cells were selected following exposure to puromycin, and single-cell clones were picked and expanded upon mouse embryonic fibroblasts (MEFs) and feeder-free conditions (2i/LIF).

Genomic DNA of clonal lines was isolated using GenElute™ Mammalian Genomic DNA Miniprep (Sigma), amplified using LA Taq DNA polymerase (Takara Bio, USA), and screened by polymerase chain reaction (PCR) product size using agarose gel electrophoresis. Genomic PCR products were extracted, purified, and sequenced at the London Regional Genomics Center (Robarts Research Institute, London, Canada). P66Shc deletion was further confirmed by immunoblotting with a ShcA specific antibody (610879; BD Biosciences), and densitometric analysis was performed to identify lines with loss of p66Shc expression but unmodified levels of p52/46Shc.

Cell culture

R1 WT and p66Shc KO mouse embryonic stem and p66Shc knockout clonal lines were maintained on a mouse embryonic fibroblast (MEF) feeder layer and in Dulbecco's modified Eagle's medium (DMEM) containing 15% ESC qualified fetal bovine serum (Life Technologies), 0.1% 2-Mercaptoethanol (Millipore Sigma), 1 × GlutaMAX (ThermoFisher), and 1000 U/mL mouse Leukemia Inhibitory Factor (mLIF; Millipore Sigma) in a 37°C, 5% carbon dioxide incubator. Cells were dissociated using 0.025% Trypsin-EDTA (ThermoFisher) at 37°C and passaged onto MEF-coated cell culture plates (Millipore Sigma).

For feeder-free conditions, R1 WT and p66Shc KO mESCs were plated on 0.2% wt/vol porcine gelatin coated plates (Millipore Sigma) and cultured with 2i/LIF media comprising: N2B27 media [KO 50% DMEM F12 medium (Life Technologies), 50% Neurobasal Medium (Life Technologies), 1 × GlutaMAX (ThermoFisher), 0.1% vol 2-Mercaptoethanol (Millipore Sigma), 0.5 × N₂ Supplement (ThermoFisher), 0.5 × B27 Supplement without vitamin A (ThermoFisher)], 3 μM CHIR99021 (Reagents Direct), 1 μM PD0325901 (Reagents Direct), and 1000 U/mL mLIF (Millipore Sigma). Cells were passaged using StemPro™ Accutase™ Cell Dissociation Reagent (Life Technologies), according to the manufacturer's instructions, onto 0.2% wt/vol porcine gelatin coated plates (Millipore Sigma). Under all conditions, cells were passaged —three to four times post-thaw before experimentation.

The cytotoxicity of 2i was quantified using the trypan blue exclusion assay during the transition of mESCs to feeder-free conditions. In brief, mESCs maintained on MEFs were dissociated using 0.025% Trypsin-EDTA and separated from MEF feeder layer by allowing the MEFs to readhere to the culture plates. The mESCs were passaged onto 0.2% gelatin coated plates in N2B27 media containing LIF/2i and serum with a seeding density of 40,000 cells/cm². The cells were then passaged every 48 h and plated with N2B27 media containing LIF/2i. For assessment of cytotoxicity, media was removed during passaging and sampled along with the dissociated cell suspension to calculate the net concentration of live and dead cells. Cells were counted in duplicate using a hemocytometer following the addition of 0.4% Trypan Blue (Gibco, ThermoFisher).

For colony forming assays, dissociated cells were seeded in 6-well plates, 600 cells/well on 0.2% gelatin, with a minimum of three technical and biological replicates. Colonies were counted after 4 days.

Differentiation of mESCs

Mouse (m)ESCs were differentiated using a monolayer differentiation protocol established for 2i culture systems, with minor modifications [28]. Briefly, mESCs were dissociated using StemPro Accutase Cell Dissociation Reagent (Life Technologies) and then seeded on gelatin-coated tissue culture plates at 10⁴ cells/cm² in N2B27 or N2B27 supplemented with LIF. JAK inhibitor treatments used a final concentration of 0.5 μM Ruxolitinib (Cayman Chemical Company Culture). Medium was changed daily.

Immunocytochemistry and microscopy

Cells were seeded in eight-well plastic chamber slides (Ibidi) coated with gelatin. Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min and washed with cold phosphate-buffered saline (PBS). Cells were then permeabilized with 0.1% Triton X (Millipore Sigma) in PBS for 30 min then incubated with blocking solution containing 3wt/vol% bovine serum albumin (BSA; ThermoFisher) and 0.1% Tween 20 in PBS (PBS-T). Following blocking, cells were incubated with primary antibodies diluted in PBST containing 1% BSA overnight at 4°C. A list of the primary antibodies used is shown in Supplementary Table S1. Cells were washed with PBS, counterstained with NucBlue (ThermoFisher, Invitrogen) according to manufacturer instructions, and then mounted using ProLong Gold™ (ThermoFisher, Invitrogen).

Immunofluorescence (IF) images were obtained using a LSM800 laser-scanning confocal microscope (Zeiss) and Zen imaging software (Zeiss). Laser settings were unchanged when imaging the same primary antibodies across conditions. Each condition was examined with three biological replicates. Phase contrast images were acquired using Leica DMI 6000 microscope (Leica Microsystems) and Application Suite X software (Leica Microsystems).

Immunoblotting

Cells were lysed and scraped into radioimmunoprecipitation assay (RIPA) buffer containing 1 × phosphatase inhibitor cocktail 2 (Millipore Sigma) and protease inhibitor cocktail 1 (Millipore Sigma) and stored at −80°C until processing. Proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 4%–12% gradient gels (NuPage™, Life Technologies). Membranes were treated with 0.1 wt/vol% Ponceau S for 15 min, washed in methanol, and then imaged using a ChemiDoc MP Imaging system (Bio-Rad). A list of the primary antibodies used for immunoblotting is shown in Supplementary Table S2. Protein bands were visualized using enhanced chemiluminescence (Forte ECL, Millipore Sigma) and imaged using a ChemiDoc MP Imaging System (Bio-Rad). Densitometric analysis of Western blots was performed using ImageLab (Bio-Rad).

RNA extraction and quantitative real time PCR

Cells were scraped and lysed in TRIzol reagent (ThermoFisher, Invitrogen), then stored at −20°C until RNA extraction. RNA was extracted into chloroform then isolated using the RNeasy Mini Kit (Qiagen), according to manufacturer's instructions. Genomic DNA was digested using the RNase-Free DNase Set (Qiagen), and cDNAs were generated using SuperScript III Reverse Transcriptase (ThermoFisher, Invitrogen). Quantitative real time PCR (RT-qPCR) was performed using a SensiFast™ SYBR No-ROX Kit (FroggaBio), and amplification was assessed using a real-time CFX384 thermal cycler (Bio-Rad). Relative transcript abundance was determined by the $Δ Δ C t$ method, normalized to Gapdh, Ppia, and Tbp housekeeping genes. Primer sequences are shown in Supplementary Table S3.

Teratomas

Experimental animals

Male and female immunodeficient mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl /SzJ, Stock No.: 005557) were ordered from The Jackson Laboratory (Bar Harbor, ME, USA) and kept in the Robarts Research Institute's mouse barrier facility (University of Western Ontario, London, Ontario, Canada). Mice were housed in Thoren vent rack cages (Thoren caging systems, Inc., PA, USA), in rooms kept at 23–26°C with a 35%–60% relative humidity, 12-h light/12-h dark cycle, and provided with constant food (Teklad 2920X irradiated soy protein-free diet) and water access. Cages were changed once per week in a biosafety cabinet, and mice were checked daily for overall health. All mice were handled according to the Western University Animal Care Committee guidelines approved by the Canadian Council on Animal Care (UWO animal utilization protocol 2022-072).

Stem cell injections

WT and p66Shc KO mESCs were cultured and passaged as described above. Cells were counted using a hemocytometer, and one million cells were collected, spun down, resuspended in 50 μL aliquots of 100 × Geltrex (Life Technologies catalog No. A1413302), and placed on ice. Mice were anesthetized using isoflurane (5% induction, 2% maintenance) through inhalation and injected with the 50 μL cell/geltrex mixture subcutaneously in the dorsal region.

Teratoma collection and characterization

All mice were euthanized using isoflurane and cervical dislocation once WT teratomas reached 1.5 cm in length (∼21 days postinjection). Teratomas were frozen on dry ice, then placed in a cryo-block, covered in optimal cutting temperature. Compound (Fisher catalog No. 23-730-571) and snap frozen in liquid nitrogen-chilled isopentane, and stored at −80°C for future cryo-sectioning and slide preparation at the Robarts Research Institute's Molecular Pathology Core Facility (University of Western Ontario). Excised tissue underwent immersion fixation for 24 h in formalin followed by paraffin embedding and sectioning using routine histological methods. Representative sections of each teratoma were stained with Hematoxylin & Eosin (H&E) for microscopic examination. Germ layers were identified from the combined study of individual cell morphology and cellular arrangement observed in the H&E-stained histological sections by histopathologist Dr. Patti Kaiser, without a priori knowledge of experimental conditions. For individual cell morphology, cell size, shape, and borders (indistinct vs. distinct) were considered. In addition, cytologic and nuclear cellular features were assessed to identify cell type. Cellular arrangements were assessed by cell density, pattern, and stroma. Microscopy was performed using an optical microscope (Olympus CX33) equipped with a camera (Lumenera INFINITY1-1M). Digitally recorded images' histologic measurements were conducted using Infinity Analyze software.

Chimera and embryo culture

To identify contribution of p66Shc KO mESCs to blastocysts, we genetically modified WT and p66Shc KO mESC lines with a mCherry fluorescent reporter and injected the cells into eight-cell mouse embryos. In brief, WT and p66Shc KO mESCs were transfected with H2B promoter-driven mCherry with a PiggyBac™ vector using Lipofectamine 3000 according to manufacturer's protocols. Transfected mESCs were selected by puromycin (2 μg/mL) over 72 h. Mouse embryos were harvested from oviducts 2 days after detection of mating plug and incubated in KSOMaa Evolve (Zenith Biotech) at 37°C. Noncompacted eight-cell embryos were selected and incubated for 30 min before injection. Using a micromanipulator-assisted injection pipette (CellTram, Eppendorf) and inverted microscope, ∼8–10 cells of either WT or p66Shc KO mCherry-mESCs were injected into the perivitelline space under the zona pellucida. A total of 10 embryos were injected with mESCs (5WT, 5KO) and then cultured in a hypoxic incubator for 24 h before fixation.

For extended culture, a similar approach was used, with modification, to remove the zona pellucida. The zona pellucida was removed at E3.5 by first washing each embryo in Acid Tyrodes droplets (30 μL, 1 min), transferring the embryo to a neutralizing M2 medium, then transferring them to M11-6 medium (Cell Guidance Systems) in eight-well culture slides (Ibidi, μ-plates). After 2 days the medium was changed to M12-6 (Cell Guidance Systems). Embryos were cultured for a total of 5 days in a hypoxic incubator at 37°C, before fixation with 4% paraformaldehyde.

Statistical analysis

Statistical tests were performed in Prism 8 (GraphPad Inc.) using Student's t-test (equal variance, unpaired, two-tailed) and one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference test to correct for multiple comparisons. Two-way ANOVAs were used for transcriptional analysis across multiple experimental conditions and cell types, and Dunnett's test was used when all comparisons were made to a single control. Experiments were performed with a minimum of three biological replicates.

Results

Deletion of p66Shc in mESCs increases basal ERK activity, Stat3(S727) phosphorylation, and enhances expression of pluripotency TF Nanog in serum and LIF containing media

ShcA has pleiotropic roles in RTK signal transduction, but is most notably involved in promoting ERK phosphorylation [5]. To explore the potential involvement of p66Shc in mESCs, we assessed the effect of p66Shc deletion on the core effectors of signaling pathways that govern ESC self-renewal and pluripotency: ERK, Stat3, and β-catenin.

p66Shc-null (p66Shc KO) mESCs were generated from the R1 mESC line using CRISPR-Cas9 nickase-mediated editing, targeting the p66Shc promoter and a part of p66Shc exon-specific region of the ShcA locus (Fig. 1A) [49]. Immunoblot analysis using a SHCA specific antibody revealed that the p66Shc isoform was absent but the 52 and 46 kDa isoforms were unaffected by genomic editing of the R1 mESC line (Fig. 1B).

FIG. 1.

P66ShcKO mESCs have enhanced P-ERK and P-STAT3(S727) and have a more naive-like pluripotency state in serum/LIF-containing media. (A) Structure of the mouse ShcA locus indicating the region targeted by CRISPR-Cas9, the p66Shc specific promoter, highlighted in red, and the start sites of the three SHCA isoforms. (B) Representative immunoblot using a ShcA antibody, illustrating the absence of p66Shc protein in the p66ShcKO mESCs. (C) Representative immunoblots of the pluripotency signaling effectors and their phosphorylation status in WT and p66ShcKO mESCs, and (D) densitometric analysis of their phosphorylation, normalized to total abundance for each effector. (E) Phase contrast images of WT and p66ShcKO mESCs grown in serum/LIF on MEFs, scalebars = 100 μm. (F) Quantitative RT-PCR analysis of pluripotent TF expression associated with naive and primed-like pluripotency states of WT and p66ShcKO mESCs. (G) Representative immunoblots of core pluripotency TFs in WT and p66ShcKO mESCs, and (H) their densitometric analysis relative to β-Actin. All data include n = 3 biological replicates from protein samples collected 3–4 days after cell seeding. Error bars of all data represent ± SEM. Statistic analysis was performed using a two-way ANOVA with Tukey's multiple comparisons, where significance is represented by *P < 0.05, **P < 0.01, ***P < 0.005. ANOVA, analysis of variance; RT-PCR, real time polymerase chain reaction; SEM, standard error of the mean; TF, transcription factor; WT, wild type.

Deletion of p66Shc significantly increased ERK1/2(T202/Y204) phosphorylation, without influencing levels of WNT pathway effector, β-catenin, or its GSK3β mediated phosphorylation (Fig. 1C, D). We also observed a significant increase in Stat3(S727) phosphorylation, while no significant alterations in Stat3(Y705) phosphorylation were observed (Fig. 1C).

Despite increased ERK phosphorylation, the p66Shc KO mESCs did not exhibit evidence of early commitment. P66Shc KO mESCs maintained tight colony boundaries and limited cell spreading (Fig. 1E), consistent with the morphology of naive mESCs. We observed alterations in naive-like marker expression, with a significant decrease in Rex1, but also a contradictory increase in Nanog. In addition, the p66Shc KO mESCs had reduced expression of the primed transcript FGF5 and a collective decrease across the remaining primed markers, although individually, the changes in each were not significant (Fig. 1F). We observed negligible changes in core pluripotency factors Oct3/4 and Sox2 and a significant increase in the levels of Nanog (Fig. 1G, H).

Collectively, these findings did not support our prediction that the loss of p66Shc would impair mESC self-renewal and pluripotency. Unexpectedly, p66Shc deletion appeared to stabilize naive TF circuitry, despite increasing ERK activity.

MEK and GSK3β inhibition abrogates differences in P-ERK and P-STAT3 signaling and Nanog expression between WT and p66Shc KO mESCs

Media supplemented with serum and MEFs contains a collection of cytokines that contribute to RTK signaling and that support mESC naive pluripotency, despite high levels of ERK activity. To eliminate the contributions of serum and MEFs to extrinsic signaling, we transitioned the WT and p66Shc KO mESCs to a defined media containing LIF, MEK inhibitor PD0325901, and GSK3β inhibitor CHIR99021, commonly referred to as 2i/LIF conditions. This media promotes naive-like pluripotency and Nanog expression by inhibiting ERK phosphorylation and the degradation of β-catenin [8,28,50]. These conditions would bypass upstream adaptor-control over ERK and WNT signaling, allowing us to better assess whether the loss of p66Shc was contributing to Nanog expression through alterations in canonical pluripotency signaling cascades.

Transitioning mESCs to 2i/LIF media required several passages before the mESCs adapted to the new conditions (Fig. 2A). The initial addition of 2i appeared to stress both WT and p66Shc KO mESCs, with significantly higher levels of cell death in WT mESCs (Fig. 2B). Following this transition, both WT and p66Shc KO mESCs could be maintained in tight naive-like colonies, without evidence of cell death or differentiation (Fig. 2C).

FIG. 2.

The addition of MEK and GSK3β inhibitors, PD0325901 and CHIR99021, abrogates P-ERK and P-STAT3(S727) differences between WT and p66ShcKO mESCs, and increases expression of Nanog in WT mESCs. (A) The protocol used for transitioning mESCs from serum and LIF containing media to 2i/LIF media under feed-free conditions. Cells were exposed to 2i for 24 h during the first passage, then allowed to acclimate to 2i/LIF media for three passages. (B) The number of dead cells during transition of mESCs to 2i media was tested by trypan blue exclusion assay and found to be significantly greater in WT relative to p66ShcKO mESCs during the first day of transition (P0) but not in subsequent passages (P1 and P2). (C) After three passages in 2i/LIF media, WT and p66ShcKO mESCs exhibited similar colony morphologies, tightly packed with well-defined borders, consistent with typical naive mESCs. (D) Representative immunoblots and (E) densitometric analysis for levels of p52/46 ShcA and signaling proteins (ERK, β-catenin, STAT3) associated with pluripotency and their respective key phosphorylation states following the transition of WT and p66ShcKO mESCs to 2i/LIF media, normalized to β-Actin and signal effector total abundance. (F) Representative immunoblots and (G) densitometric analysis for the change in core pluripotency factors (Nanog, Oct3/4, and Sox2) following the transition of WT and p66ShcKO mESCs to 2i/LIF media, normalized to β-Actin. Proteins were collected from n = 3 separate passages of mESCs, independently treated with 2i for four passages, after establishing morphological uniformity across mESC colonies. Statistical analysis was performed to compare WT and p66ShcKO mESCs and the effect of treatment using a two-way ANOVA with Tukey's multiple comparisons, where significance is represented by *P < 0.05, **P < 0.01, ***P < 0.005.

The presence of 2i abrogated all differences between WT and p66Shc KO phenotypes that we previously observed in serum/LIF media. The addition of 2i inhibited ERK activity and significantly enhanced levels of β-catenin in both WT and p66Shc KO mESCs (Fig. 2D, E). Notably, 2i also influenced STAT3 activity, abrogating the differences in STAT3(S727) phosphorylation, despite the inhibitors not being directly involved in canonical STAT3 signaling cascades. 2i also abrogated differences in Nanog expression by increasing levels of Nanog in the WT mESCs, without significantly altering levels of Nanog in the p66Shc KO mESCs (Fig. 2F, G).

Deletion of p66Shc permits the self-renewal of pluripotent mESCs in the absence of serum, 2i, or MEFs

2i appeared to abrogate intracellular signaling and pluripotency TF differences between WT and p66Shc mESCs; however, it remained unclear which aspect of the serum/LIF/MEF signaling environment was mediating these differences. It was also unclear whether p66Shc was directly driving these differences or whether they were due to indirect adaptations within the p66Shc KO mESCs compensating for the absence of p66Shc.

To clarify the conditions necessary to reassert these differences, we assessed early commitment of WT and p66Shc KO mESCs in response to withdrawal of one or more factors from the 2i/LIF media. Initial commitment, following the withdrawal of one or more components (MEKi, GSK3βi, and/or LIF) from the 2i/LIF media, was assessed by changes in mESC morphology over 2 days. Across most conditions, WT and p66Shc KO mESCs were morphologically indistinguishable (data not shown). Withdrawal of any single factor was insufficient to provoke noticeable morphological changes, while withdrawal of all three factors induced rapid cell separation and spreading in both the WT and p66Shc KO mESC lines. However, one condition, the withdrawal of 2i with maintenance of LIF provoked rapid morphological changes in the WT but not the p66Shc KO mESCs (Fig. 3A).

FIG. 3.

p66ShcKO mESCs maintain naive-like pluripotency in LIF containing media, despite withdrawal of 2i. (A) Phase contrast images of WT and p66ShcKO mESCs grown in N2B27 + LIF media, indicating the number of days following replacement of media without 2i, scale bars = 100 μm. (B) Effect of 2i withdrawal and serial passaging on self-renewal was assessed by colony formation per 600 cells seeded (C) Expression of core and (D) naive-like mESC pluripotency markers in WT and p66ShcKO mESCs following the withdrawal of 2i, by qRT-PCR. (E) Representative immunoblots and (F) densitometric analysis of core pluripotency factors (Nanog, Oct3/4, and Sox2) in WT and p66ShcKO mESCs following the withdrawal of 2i. (G) Immunofluorescent staining of Nanog, Oct3/4, and E-cadherin in WT and p66ShcKO mESCs in 2i/LIF media and after 6 days, or 5 passages (p5) of incubation in -2i + LIF media, scale bars = 20 μm. Statistical analyses were performed for comparisons between WT and p66ShcKO at each condition, using Student's t-tests, where significance is represented by *P < 0.05, **P < 0.01, ***P < 0.005. LIF, leukemia inhibitory factor.

The effect of p66Shc deletion on mESC self-renewal was assessed by their capacity for colony formation. Although WT and p66Shc KO mESCs had similar colony formation efficiency in 2i/LIF, only the p66Shc KO mESCs maintained efficient colony formation in the absence of 2i (Fig. 3B). Moreover, colony formation efficiency decreased with repeat passaging of the WT mESCs in the absence of 2i. However, LIF alone was able to support p66Shc KO mESC self-renewal across multiple passages without loss of efficiency. The addition of higher doses of LIF (from 10³ to 10⁴ U/mL) also failed to support the colony formation in the WT mESCs (data not shown).

To assess whether the observed morphological changes might be rationalized by changes in mESC identity, we assessed levels of pluripotency associated transcripts over 6 days. Withdrawal of 2i decreased the expression of core and naive pluripotency associated transcripts in the WT mESCs (Fig. 3C, D), concomitant with a depletion of Nanog and SOX2 protein levels (Fig. 3E, F). However, p66Shc KO mESCs were insensitive to the loss of 2i and maintained expression of both core and naive pluripotency transcripts after 6 days. Nanog protein levels decreased in the absence of 2i but remained significantly elevated relative to the WT mESCs. Levels of pluripotency markers in the p66Shc KO mESCs were retained even after repeat passaging in the absence of 2i, for a minimum of five passages (p5).

Immunofluorescence microscopy analysis corroborated these results, revealing decreased Nanog immunostaining within the WT mESCs and a loss of E-cadherin at the intercellular boundaries following withdrawal of 2i (Fig. 3G). Conversely, the p66Shc KO mESCs maintained stable Nanog levels within nuclei and E-cadherin at the cellular interfaces within colonies.

P66Shc regulates the timing and levels of ERK and STAT3 phosphorylation during mESC commitment in LIF-containing media

To examine the underlying signaling mechanism mediating the retention of naive pluripotency of the p66Shc KO mESCs, we examined the phosphorylation of STAT3 and ERK following the withdrawal of 2i, during the early stages of naive mESC commitment.

Over 6 days of differentiation, we observed a significant increase in levels of p66Shc in the WT mESCs (Fig. 4A, B), concomitant with a decrease in STAT3(Y705) and STAT3(S727) phosphorylation. The p66Shc KO mESCs, however, retained both P-STAT3(Y705) and P-STAT3(S727) across the period of 2i withdrawal.

FIG. 4.

p66ShcKO mESCs exhibit elevated ERK activity relative to WT mESCs, in response to LIF-JAK extrinsic signaling. Effect of 2i withdrawal on activity of pluripotency-associated signaling effectors in WT and p66ShcKO mESCs over a 6-day period, and after multiple passages (p5) in p66ShcKO mESCs: (A) Representative immunoblots and (B) densitometric analysis of immunoblots quantifying the phosphorylation status of ERK and STAT3, relative to their total respective levels, and levels of ShcA isoforms and β-catenin relative to total protein. (C) Representative immunoblots and (D) densitometric analysis for P-STAT3 and P-ERK of mESCs treated with 2 μM RUX (Ruxolitinib, JAK1/2 inhibitor) and PD (PD0325901, MEK inhibitor) for 1 h, to determine the potential involvement of p66Shc in LIF signaling. Statistical analyses were performed for comparisons between WT and p66ShcKO at each condition, using Student's t-tests, where significance is represented by *P < 0.05,**P < 0.01, ***P < 0.005.

The absence of p66Shc altered the timing of ERK phosphorylation following 2i withdrawal. Compared to the WT mESCs, which showed peak ERK phosphorylation at 2 days, ERK activity in the p66Shc KO mESCs was highest after 6 days. In contrast with STAT3 and ERK, the absence of p66Shc did not significantly modulate levels of β-catenin.

These findings indicate that p66Shc KO mESCs have altered patterns of ERK and STAT3 activity concomitant with the retention of their naive pluripotency in the presence of LIF. To explore the role of p66Shc in LIF signal transduction, the mESCs were starved of LIF for 4 h, then stimulated with LIF in conjunction with the targeted inhibition of either JAK1/2, using Ruxolitinib, ERK, using PD, or in the presence of serum for 1 h (Fig. 4C, D).

LIF appeared to enhance the P-STAT3(S727) phosphorylation of both WT and p66Shc KO mESCs. However, in contrast with STAT3(Y705) phosphorylation, JAK inhibition only partially inhibited the STAT3(S727) phosphorylation. This suggests that Stat3(S727) is, in part, mediated by non-LIF and non-JAK dependent cascades. Deletion of p66Shc did not significantly affect STAT3(Y705) phosphorylation; however, it significantly enhanced STAT3(S727) phosphorylation in response to LIF stimulation. Notably, the difference in STAT3(S727) phosphorylation could be abrogated by MEK inhibition and the addition of serum, but not by JAK inhibition. The deletion of p66Shc significantly enhanced ERK phosphorylation, both in response to LIF and its absence. Again, this difference could be abrogated by MEK inhibition or by the addition of serum, but not by JAK inhibition.

Collectively, these findings suggest that p66Shc antagonizes ERK and STAT3(S727) phosphorylation in naive mESCs. Once the constraints on signaling imposed by 2i were removed, the p66Shc KO mESCs responded by phosphorylating ERK and STAT3(S727), which could be durably sustained in LIF containing media. Thus, the regulatory effect of p66Shc on STAT3 phosphorylation appears to be ERK dependent and JAK independent.

P66Shc KO mESCs incorporate into the inner cell mass of chimeric embryos and contribute to all somatic germ layers but form small and immature teratomas

Elevated ERK activity has been shown to preferentially select for “primed” ERK-dependent epiblast-like ESC populations in LIF/serum, while STAT3 is necessary for the maintenance of naive inner cell mass (ICM)-like lineages [32,37,51,52]. Unlike naive mESCs, primed ESCs are incapable of contributing to the ICM when injected into preimplantation embryos [53,54]. Having observed both elevated ERK phosphorylation, but also increased levels of naive-associated transcripts in the p66Shc KO, we were interested in how these contradictory phenotypes would influence the ability of p66Shc KO mESCs to contribute to preimplantation embryos.

To assess the effect of p66Shc deletion on preimplantation development, we generated mCherry-reporter WT and p66Shc KO mESC lines. Between 8 and 10 labeled cells were injected into each mouse embryo at the 8-cell stage. After 1 day in vitro, both WT and p66Shc KO mESC chimeric embryos developed into the mid-blastocyst stage, similar to E3.5. Like WT chimeras, the donor p66Shc KO mESCs were restricted to the ICM and dispersed in a mosaic among the host's ICM cells (Fig. 5A). The WT and p66Shc KO cells predominantly expressed NANOG, a marker indicative of epiblast identity. We did not observe expression of GATA4, a primitive endoderm marker, in either the WT or p66Shc KO donor cells.

FIG. 5.

p66ShcKO mESCs contribute to the inner cell mass of blastocysts and contribute to all germ layers in teratomas, but teratoma-derived tissues remain immature. (A) mCherry-reporter WT and p66ShcKO mESCs injected into mouse preimplantation blastocysts, with cell identity assessed by immunofluorescence: primitive endoderm (GATA4) and inner cell mass (NANOG), scale bars = 50 μm. (B) Representative photos of WT and p66ShcKO mESC derived teratomas, and (C) their mass, n = 5 biological replicates, statistical analysis performed by Student's t-test, with error bars representing mean standard deviation and significance represented by *P < 0.05, **P < 0.01, ***P < 0.005. (D) Sections of the WT and p66ShcKO teratomas containing discreet structures. Hatched boxes denote enlarged images below: ectoderm (i), mesoderm (ii), endoderm (iii), and undifferentiated cells (iv). H&E stained; scale bars represent 1,000 and 100 μm for the low and high-magnification images, respectively. (E) Representative sections of WT and p66ShcKO teratomas, showing differences in tissue maturity, with arrows indicating discreet structures and asterisks indicating areas of undifferentiated neoplasm. H&E stained; scale bars represent 100 μm. (F) Qualitative assessment of proportion of teratoma area containing mature structures relative to undifferentiated tissue. H&E, hematoxylin and eosin.

The WT and p66Shc KO chimeras were also grown in extended embryo culture conditions to allow development to the egg-cylinder stage (Supplementary Fig. S1). The cells were stained with SOX17 and OCT3/4 to distinguish between their contribution to visceral endoderm and epiblast lineages, respectively. At this stage of development, we did not observe any distinct differences in their potential. Both WT and KO donor cells expressed OCT3/4, but not SOX17, suggesting that neither WT nor p66Shc KO donor cells contributed to visceral endoderm.

To explore the differentiation potential of the p66Shc KO mESCs, we used the teratoma assay [55]. Transplanted tumors were observed in nearly all mice, aside from one mouse injected with the p66Shc KO mESCs. WT derived tumors were all encapsulated masses with prominent solid components, while the p66Shc KO derived tumors were poorly defined soft tissue clumps, with significantly smaller mass than the WT derived tumors (Fig. 5B, C).

Histological analysis identified each of the three germ layers (ectoderm, mesoderm, and endoderm) in both the WT and p66Shc KO derived teratomas (Fig. 5D); however, these structures appeared less defined in the p66Shc KO tumors. WT teratomas consisted primarily of well-defined structures; however, the p66Shc KO teratomas were composed almost entirely of undifferentiated neoplasm (Fig. 5E, F).

Together, these data indicate that the p66Shc KO mESCs are pluripotent, with a naive-like identity, capable of differentiating to each germ layer; however, they have an attenuated maturation rate relative to WT mESCs.

The withdrawal of LIF or inhibition of JAK signaling restores p66Shc KO mESC differentiation competency

Although we had observed significant differences in intracellular activity of P-ERK and P-STAT3(S727) in p66Shc KO mESCs, these signaling differences did not appear directly mediated by canonical LIF-LIFR-JAK extrinsic cascades.

The reduced tissue complexity and size of the p66Shc KO derived teratomas appeared to suggest developmental impairments of the p66Shc KO mESCs, but it remained unclear whether these impairments were restricted to naive-commitment and whether LIF signaling was necessary for their enhanced naive-like self-renewal.

To assess early lineage commitment in the absence of LIF, we withdrew both 2i and LIF and monitored changes in expression of naive-associated genes. The withdrawal of 2i and LIF caused rapid morphological changes (Fig. 6A), concomitant with a loss of naive marker expression in both WT and p66Shc KO mESCs (Fig. 6B). Differences between WT and p66Shc KO were negligible across many of the markers assessed, including Nanog.

FIG. 6.

LIF-JAK signaling is necessary for enhanced naive-like pluripotency in p66ShcKO mESCs. (A) Phase contrast images of WT and p66ShcKO mESCs following withdrawal of 2i and LIF for 3 days, scale bars = 100 μm. (B) Effect of 2i withdrawal on expression of TFs associated with naive pluripotency in WT and p66ShcKO mESCs after 2 days, quantified by qRT-PCR. Statistical analysis was performed for comparison of +2i+LIF and -2i-LIF conditions within each cell type, with significance displayed above each bar, and comparisons were made between WT and KO within each treatment, with significance displayed below each bar, using Student's t-tests. (C) Phase contrast images of p66ShcKO mESCs adapted to N2B27 + LIF media, without 2i, MEFs, or serum, following the withdrawal of LIF or inhibition of JAK1/2 with 0.5 μM Ruxolitinib, scale bars = 100 μm. (D) Effect of LIF withdrawal or treatment with 0.5 μM Ruxolitinib on expression of core and naive pluripotency transcription factors. Statistical analyses were performed for comparisons between LIF-Serum condition and each treatment, using a two-way ANOVA. Significance is represented by *P < 0.05, **P < 0.01, ***P < 0.005.

To explore whether canonical LIF-JAK-STAT signaling was mediating enhanced LIF permissiveness in p66Shc KO mESCs, we attempted to induce differentiation of p66Shc KO mESCs that we had adapted to LIF-only media, by the targeted inhibition of components of the JAK-STAT, MAPK, Phosphoinositide-3-kinase (PI3K), and BMP-SMAD pathways (data not shown). Notably, only the withdrawal of LIF or the presence of JAK/STAT3 inhibitors could recapitulate the morphological changes of mESC commitment (Fig. 6C). Concomitant with changes in morphology, LIF withdrawal induced the loss of both core and naive pluripotency markers from the LIF-only p66Shc KO mESCs (Fig. 6D). Moreover, persistent JAK1/2 inhibition could forcibly induce loss of naive marker expression and partially recapitulate the effect of LIF withdrawal (Fig. 6D).

Discussion

In this study, we show that the deletion of p66Shc from mESCs is sufficient to permit the maintenance of their naive pluripotency in minimal media containing LIF, without serum, supplemental growth factors, or pharmacologic inhibitors. Surprisingly, this effect was associated with a loss of sensitivity to P-ERK mediated differentiation cues in the p66Shc KO mESCs. This is the first study to identify the involvement of p66Shc in the negative regulation of ERK activity in ESCs and to implicate p66Shc in the negative regulation of LIF-permissive naive-like pluripotency.

Initially, we hypothesized that p66Shc would be essential to suppress epiblast commitment by negatively regulating ERK activity. Consequently, we predicted that p66Shc deletion from mESCs would increase ERK phosphorylation and drive differentiation. Contrary to our expectation, we discovered that p66Shc is neither essential for the self-renewal of naive mESCs nor their incorporation into the ICM of preimplantation embryos. Instead, we observed that the deletion of p66Shc increased the expression of naive-associated pluripotency genes and promoted the retention of Nanog. This was a surprising finding, considering that ERK is a negative regulator of Nanog and an established driver of epiblast and mESC commitment [28,33,34,56].

Notably, aberrant retention of naive pluripotency in p66Shc KO mESCs depended on the presence of LIF. LIF is conventionally added to mESCs in vitro to reinforce self-renewal and naive pluripotency through the downstream phosphorylation of STAT3(Y705) [36,51,57]. It has been previously shown that P-ERK mediated differentiation cues are blocked by JAK/STAT3 activity [38]. In this study, we suspect that the deletion of p66Shc is conferring P-ERK insensitivity due to elevated P-STAT3(S727). P-STAT3(S727) has been previously shown to enhance the transcriptional activity of P-STAT3(Y705) [39,40] and facilitate the maintenance of mESC naive pluripotency [43]. This interpretation is supported by enhanced expression of STAT3 target genes, klf4 and tfcp2l1, in the p66Shc KO mESCs and their sensitivity to both JAK inhibition and LIF withdrawal.

How might p66Shc inhibit ERK and STAT3(S727) phosphorylation in mESCs? Canonically, SHC adaptor proteins transduce RTK signaling and promote ERK phosphorylation by first binding to the intracellular motifs of activated RTKs, then binding to GRB2 to facilitate GRB2-RAS interactions [58 –60]. We observed that deletion of p66Shc enhanced ERK phosphorylation in the presence of LIF, consequently, one possibility is that p66Shc is antagonizing LIFR/gp130 signaling cascade. This is supported by previous findings suggesting that p66Shc can directly interact with gp130 and JAK2, in response to IL6 stimulation [61]. Alternatively, p66Shc could be directly binding to ERK to inhibit its activity, similar to what has been reported in cancer cell lines in the absence of EGF [62]. Given that SHC adaptors lack affinity for traditional LIFR/gp130 binding motifs [63], it is unlikely that p66Shc is directly participating in the regulation of LIFR/gp130 signaling.

We speculate that it is more likely that p66Shc is indirectly influencing ERK and STAT3 phosphorylation by competitively inhibiting other adaptors, most notably, SHP2 which directly participates in LIF-ERK cascades. In contrast with the other SHC isoforms, p66Shc may bind to RTKs and form stable complexes with GRB2, without activating ERK [17,18,64,65]. Analogous findings have been reported for the closely related SHCD adaptor, which has been observed to suppress ERK signaling in response to neurotrophic RTK activation by binding to and competitively inhibiting GRB2 [66]. The binding of p66Shc to a limited pool of GRB2 could competitively inhibit a wide range of GRB2 interactions and prevent ERK phosphorylation, both in response to LIF and in the absence of LIF (Fig. 7).

FIG. 7.

Proposed model for p66Shc's involvement in ERK and STAT3 signaling in mESCs in LIF-media. 1. P66Shc attenuates ERK activity by directly binding to the intracellular domains of RTKs with SHC binding motifs (EGFR, FGFR, IGFR, TRKA/Bs, IL-2/3, OCMR), competitively inhibiting the ERK-promoting SHC proteins: p52/46ShcA and the ShcB, ShcC, and ShcD families. 2. Binding of LIF to the LIFR/GP130 heterodimer activates both the JAK-STAT and SHP2-MAPK cascades. P66Shc competitively inhibits SHP2-ERK in LIF signaling by binding to and sequestering GRB2. P66Shc's inhibition of ERK impairs ERK-dependent phosphorylation of P-STAT3(S727), inhibiting STAT3 activity without influencing STAT3(Y705) phosphorylation. P66Shc may also participate in the negative regulation of P-STAT3(S727) in response to other kinases (PKC $δ,$ MAPKs, etc.), through mechanisms yet to be identified. Despite inhibiting ERK activity, p66Shc has the cumulative effect of destabilizing naive pluripotency circuit, due to its simultaneous involvement in the negative regulation of STAT3 activity, through the inhibition of STAT3(S727) phosphorylation.

Several studies have suggested that STAT3(S727) phosphorylation is mediated by ERK [67]. Consistent with this, enhanced P-STAT3(S727) levels in p66Shc KO mESCs coincided with elevated P-ERK. However, we also observed that ERK inhibition simultaneously promoted STAT3(S727) phosphorylation in the WT mESCs. To date, there is limited information surrounding the regulation of P-STAT3(S727); however, our findings suggest that p66Shc may antagonize STAT3 phosphorylation in mESCs in a P-ERK dependent manner. These findings warrant further investigation, which may offer insight into regulatory crosstalk between STAT3 and MAPK signaling cascades and the contradictory reports regarding P-STAT3(S727) regulation [68].

Given the dynamic nature of differentiation, and the limited number of timepoints assessed in this study, it's possible that the observed alterations in ERK and STAT3 activity are not directly responsible for the naive-like retention in the p66Shc KO mESCs. Future approaches could use phospho-proteomic screens with high temporal resolution, to closely establish the causal links between each phosphorylation event and the acquisition of cell fate. In addition, we did not assess how the deletion of p66Shc influenced mitochondrial function. P66Shc may be contributing to differentiation through its putative role in the production of reactive oxygen species, which may influence commitment through a mitochondrial-specific mechanism.

Derivation of naive-like mESCs, using conventional serum and LIF media, is restricted to certain mouse strains. Similarly, the ESCs derived from many other species, including humans, typically lack naive-like features and more closely resemble the developmentally primed epiblast stem cells of postimplantation embryos [53]. A central feature that distinguishes naive and primed ESCs is their responsiveness to LIF. Previous studies have shown that reinforcement of STAT3 activity in non-LIF-permissive mESCs, and even primed hESCs, reprograms these cells to a LIF-dependent naive-like state [69,70]. Based on these findings, studies have theorized that the function of LIF to maintain a naive-like pluripotency is conserved across species and that LIF permissiveness in vitro is determined by intracellular regulators that modulate the relative contribution of ERK and STAT3 activity [57].

Our findings implicate p66Shc's participation in the signaling cascades that mediate mESC commitment and may offer additional insight into the molecular basis of LIF permissiveness and the regulatory mechanisms contributing to preimplantation pluripotency status.

Footnotes

Acknowledgments

The authors thank the members of the Cumming and Betts labs for their valuable contributions to ideas and analysis, Jonathan Teichroeb and Courtney Brooks for their assistance in generating the p66Shc KO mESCs. The authors also acknowledge funding from the Western Strategic Support for CIHR Success Program and the Natural Sciences and Engineering Research Council of Canada (NSERC). A.P acknowledges support from the Ontario Graduate Scholarship.

Data Availability

Data available on request.

The data underlying this article will be shared on reasonable request to the corresponding author.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This research was funded by Discovery Grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada, Seed and Bridge grants from Western Strategic Support and an internal research grant from the Children's Health Research Institute (CHRI). AMP was supported by an Ontario Graduate Scholarship (OGS).

Supplementary Material

Supplementary Figure S1

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

References

Perrimon

, Pitsouli

and Shilo.

B-Z

(2012) Signaling mechanisms controlling cell fate and embryonic patterning. Cold Spring Harb Perspect Biol, 4:a005975.

Pawson

and Scott

. (1997). Signaling through scaffold, anchoring, and adaptor proteins. Science, 278:2075–2080.

Ahmed SBM and Prigent

. (2017). Insights into the Shc family of adaptor proteins. J Mol Signal, 12:2.

Hardy

, Li

, Wang

, Sedy

, Fawcett

, Frank

, Kucera

and Pawson

. (2007). Combinatorial ShcA docking interactions support diversity in tissue morphogenesis. Science, 317:251–256.

Pelicci

, Lanfrancone

, Grignani

, McGlade

, Cavallo

, Forni

, Nicoletti

, Grignani

, Pawson

and Giuseppe Pelicci

. (1992). A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell, 70:93–104.

Ravichandran KS. (2001). Signaling via Shc family adapter proteins. Oncogene, 20:6322–6330.

Corson

, Yamanaka

, Lai

K-MV

and Rossant

. (2003). Spatial and temporal patterns of ERK signaling during mouse embryogenesis. Development, 130:4527–4537.

Nichols

, Silva

, Roode

and Smith

. (2009). Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo. Development, 136:3215–3222.

Pelicci

, Dente

, De Giuseppe

, Verducci-Galletti

, Giuli

, Mele

, Vetriani

, Giorgio

, Pandolfi

, Cesareni

and Pelicci PG

. (1996). A family of Shc related proteins with conserved PTB, CH1 and SH2 regions. Oncogene, 13:633–641.

10.

Luzi

, Confalonieri

, Di Fiore

and Pelicci

. (2000). Evolution of Shc functions from nematode to human. Curr Opin Genet Dev, 10:668–674.

11.

Sakaguchi

, Okabayashi

, Kido

, Kimura

, Matsumura

, Inushima

and Kasuga

. (1998). Shc phosphotyrosine-binding domain dominantly interacts with epidermal growth factor receptors and mediates Ras activation in intact cells. Mol Endocrinol Baltim Md, 12:536–543.

12.

Lai K-MV and Pawson

. (2000). The ShcA phosphotyrosine docking protein sensitizes cardiovascular signaling in the mouse embryo. Genes Dev, 14:1132–1145.

13.

Migliaccio

, Giorgio

, Mele

, Pelicci

, Reboldi

, Pandolfi

, Lanfrancone

and Pelicci

. (1999). The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature, 402:309–313.

14.

Giorgio

, Migliaccio

, Orsini

, Paolucci

, Moroni

, Contursi

, Pelliccia

, Luzi

, Minucci

, Marcaccio

, Pinton

, Rizzuto

, Bernardi

, Paolucci

and Pelicci

. (2005). Electron transfer between cytochrome c and p66shc generates reactive oxygen species that trigger mitochondrial apoptosis. Cell, 122:221–233.

15.

Pinton

, Rimessi

, Marchi

, Orsini

, Migliaccio

, Giorgio

, Contursi

, Minucci

, Mantovani

, Wieckowski

, Sal

, Pelicci

and Rizzuto

. (2007) Protein kinase C ß and prolyl isomerase 1 regulate mitochondrial effects of the life-span determinant p66Shc. Science, 315:659–663.

16.

Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor–MAP kinase–fos signalling pathway. (1997). EMBO J, 16:706–716.

17.

Pacini

, Pellegrini

, Migliaccio

, Patrussi

, Ulivieri

, Ventura

, Carraro

, Naldini

, Lanfrancone

, Pelicci

and Baldari

. (2004). p66SHC promotes apoptosis and antagonizes mitogenic signaling in T cells. Mol Cell Biol, 24:1747–1757.

18.

Okada

, Kao

, Ceresa

, Blaikie

, Margolis

and Pessin

. (1997). The 66-kDa Shc isoform is a negative regulator of the epidermal growth factor-stimulated mitogen-activated protein kinase pathway*. J Biol Chem, 272:28042–28049.

19.

Jackson

, Yoneda

, Clark

and Yee

. (2000). Elevated levels of p66 Shc are found in breast cancer cell lines and primary tumors with high metastatic potential. Clin Cancer Res Off J Am Assoc Cancer Res, 6:1135–1139.

20.

Veeramani

, Igawa

, Yuan

T-C

, Lin

F-F

, Lee

M-S

, Lin

, Johansson

and Lin

M-F

. (2005). Expression of p66(Shc) protein correlates with proliferation of human prostate cancer cells. Oncogene, 24:7203–7212.

21.

Perrini

, Tortosa

, Natalicchio

, Pacelli

, Cignarelli

, Palmieri

, Caccioppoli

, De Stefano

, Porro

, Leonardini

, Ficarella

, De Fazio

, Cocco

, Puglisi

, Laviola

, Palasciano

and Giorgino

. (2015). The p66Shc protein controls redox signaling and oxidation-dependent DNA damage in human liver cells. Am J Physiol Gastrointest Liver Physiol, 309:G826–840.

22.

Miller

, Palygin

, El-Meanawy

, Mattson

, Geurts

, Staruschenko

and Sorokin

. (2021). p66Shc-mediated hydrogen peroxide production impairs nephrogenesis causing reduction of number of glomeruli. Life Sci, 279:119661.

23.

Lee

, Smith

, Banerjee

, Li

, Minoo

, Volpe

and Nielsen

. (2014). The p66Shc adapter protein regulates the morphogenesis and epithelial maturation of fetal mouse lungs. Am J Physiol-Lung Cell Mol Physiol, 306:L316–L325.

24.

Onnis

, Cianfanelli

, Cassioli

, Samardzic

, Pelicci

, Cecconi

and Baldari

. (2018). The pro-oxidant adaptor p66SHC promotes B cell mitophagy by disrupting mitochondrial integrity and recruiting LC3-II. Autophagy, 14:2117–2138.

25.

Edwards

, Watson

and Betts

. (2016). P66Shc, a key regulator of metabolism and mitochondrial ROS production, is dysregulated by mouse embryo culture. Mol Hum Reprod, 22:634–647.

26.

Edwards

, Watson

and Betts

. (2018). Knockdown of p66Shc alters lineage-associated transcription factor expression in mouse blastocysts. Stem Cells Dev, 27:1479–1493.

27.

Nichols

and Smith

. (2009). Naive and primed pluripotent states. Cell Stem Cell, 4:487–492.

28.

Ying

Q-L

, Wray

, Nichols

, Batlle-Morera

, Doble

, Woodgett

, Cohen

and Smith

. (2008). The ground state of embryonic stem cell self-renewal. Nature, 453:519–523.

29.

Saba-El-Leil

, Vella

FDJ

, Vernay

, Voisin

, Chen

, Labrecque

, Ang

S-L

and Meloche

. (2003). An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development. EMBO Rep, 4:964–968.

30.

Haegel

, Larue

, Ohsugi

, Fedorov

, Herrenknecht

and Kemler

. (1995). Lack of beta-catenin affects mouse development at gastrulation. Development, 121:3529–3537.

31.

Takeda

, Noguchi

, Shi

, Tanaka

, Matsumoto

, Yoshida

, Kishimoto

and Akira

. (1997). Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci U S A, 94:3801–3804.

32.

Lanner

and Rossant

. (2010). The role of FGF/Erk signaling in pluripotent cells. Development, 137:3351–3360.

33.

Stavridis

, Lunn

, Collins

and Storey

. (2007). A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification. Development, 134:2889–2894.

34.

Kunath

, Saba-El-Leil

, Almousailleakh

, Wray

, Meloche

and Smith

. (2007). FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development, 134:2895–2902.

35.

Hamilton

and Brickman

. (2014). Erk signaling suppresses embryonic stem cell self-renewal to specify endoderm. Cell Rep, 9:2056–2070.

36.

Niwa

, Burdon

, Chambers

and Smith

. (1998). Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev, 12:2048–2060.

37.

, Ueda

, Messerschmidt

, Lorthongpanich

, Zhou

, Feng

, Guo

, Lin

, Hossain

, Zhang

, Moh

, Wu

, Robson

, Ng

, Poellinger

, Knowles

, Solter

and Fu

X-Y

. (2013). A genetic and developmental pathway from STAT3 to the OCT4–NANOG circuit is essential for maintenance of ICM lineages in vivo. Genes Dev, 27:1378–1390.

38.

van Oosten

, Costa

, Smith

and Silva

JCR

. (2012). JAK/STAT3 signalling is sufficient and dominant over antagonistic cues for the establishment of naive pluripotency. Nat Commun, 3:817.

39.

Wen

, Zhong

and Darnell

. (1995). Maximal activation of transcription by statl and stat3 requires both tyrosine and serine phosphorylation. Cell, 82:241–250.

40.

Zhang

, Blenis

, Li

H-C

, Schindler

and Chen-Kiang

. (1995). Requirement of serine phosphorylation for formation of STAT-promoter complexes. Science, 267:1990–1994.

41.

Wakahara

, Kunimoto

, Tanino

, Kojima

, Inoue

, Shintaku

and Nakajima

. (2012). Phospho-Ser727 of STAT3 regulates STAT3 activity by enhancing dephosphorylation of phospho-Tyr705 largely through TC45. Genes Cells Devoted Mol Cell Mech, 17:132–145.

42.

Tkach

, Rosemblit

, Rivas

, Proietti Anastasi

, Díaz Flaqué

, Mercogliano

, Beguelin

, Maronna

, Guzman

, Gercovich

, Gil Deza

, Elizalde

and Schillaci

. (2013). p42/p44 MAPK-mediated Stat3 Ser727 phosphorylation is required for progestin-induced full activation of Stat3 and breast cancer growth. Endocr Relat Cancer, 20:197–212.

43.

Huang

, Yan

, Ye

, Tong

and Ying

Q-L

. (2014). STAT3 Phosphorylation at tyrosine 705 and serine 727 differentially regulates mouse ESC fates. Stem Cells, 32:1149–1160.

44.

C-K

, Yu

W-M

, Azzarelli

and Feng

G-S

. (1999). Genetic evidence that Shp-2 tyrosine phosphatase is a signal enhancer of the epidermal growth factor receptor in mammals. Proc Natl Acad Sci, 96:8528–8533.

45.

Cheng

, Saxton

, Sakai

, Kulkarni

, Mbamalu

, Vogel

, Tortorice

, Cardiff

, Cross

, Muller

and Pawson

. (1998). Mammalian Grb2 regulates multiple steps in embryonic development and malignant transformation. Cell, 95:793–803.

46.

Giroux

, Tremblay

, Bernard

, Cardin-Girard

, Aubry

, Larouche

, Rousseau

, Huot

, Landry

, Jeannotte

and Charron

. (1999). Embryonic death of Mek1-deficient mice reveals a role for this kinase in angiogenesis in the labyrinthine region of the placenta. Curr Biol CB, 9:369–372.

47.

Goldin

and Papaioannou

. (2003). Paracrine action of FGF4 during periimplantation development maintains trophectoderm and primitive endoderm. Genesis, 36:40–47.

48.

Ran

, Hsu

, Wright

, Agarwala

, Scott

and Zhang

. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 8:2281–2308.

49.

Ventura

, Luzi

, Pacini

, Baldari

and Pelicci

. (2002). The p66Shc longevity gene is silenced through epigenetic modifications of an alternative promoter*. J Biol Chem, 277:22370–22376.

50.

Silva

, Barrandon

, Nichols

, Kawaguchi

, Theunissen

and Smith

. (2008). Promotion of reprogramming to ground state pluripotency by signal inhibition. PLOS Biol, 6:e253.

51.

Boeuf

, Hauss

, Graeve

, Baran

and Kedinger

. (1997). Leukemia inhibitory factor–dependent transcriptional activation in embryonic stem cells. J Cell Biol, 138:1207–1217.

52.

Hamilton

, Mosesson

, Monteiro

, Emdal

, Knudsen

, Francavilla

, Barkai

, Olsen

and Brickman

. (2019). Dynamic lineage priming is driven via direct enhancer regulation by ERK. Nature, 575:355–360.

53.

Brons

IGM

, Smithers

, Trotter

MWB

, Rugg-Gunn

, Sun

, Chuva de Sousa Lopes

, Howlett

, Clarkson

, Ahrlund-Richter

, Pedersen

and Vallier

. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature, 448:191–195.

54.

Tesar

, Chenoweth

, Brook

, Davies

, Evans

, Mack

, Gardner

, McKay

RDG

. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature, 448:196–199.

55.

Evans

and Kaufman

. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature, 292:154–156.

56.

Kim

S-H

, Kim

, Cho

Y-Y

, Yao

, Kim

, Jeong

C-H

, Yu

, Bae

, Cho

, Jung

, Lee

, Chen

, Kim

, Bode

and Dong

. (2014). ERK1 phosphorylates Nanog to regulate protein stability and stem cell self-renewal. Stem Cell Res, 13:1–11.

57.

Ohtsuka

, Nakai-Futatsugi

and Niwa

. (2015). LIF signal in mouse embryonic stem cells. JAK-STAT, 4:e1086520.

58.

van der Geer

, Wiley

, Gish

and Pawson

. (1996). The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein–protein interactions. Curr Biol, 6:1435–1444.

59.

Ravichandran

, Zhou

, Pratt

, Harlan

, Walk

, Fesik

and Burakoff

. (1997). Evidence for a requirement for both phospholipid and phosphotyrosine binding via the Shc phosphotyrosine-binding domain in vivo. Mol Cell Biol, 17:5540–5549.

60.

Kolch

. (2005). Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat Rev Mol Cell Biol, 6:827–837.

61.

Giordano

, Falco

, Chiari

, Quinto

, Pelicci

, Bartholomew

, Delmastro

, Gadina

and Scala

. (1997). Shc mediates IL-6 signaling by interacting with gp130 and Jak2 kinase. J Immunol, 158:4097–4103.

62.

Suen

, Lin

C-C

, George

, Melo

, Biggs

, Ahmed

, Drake

, Arur

, Arold

and Ladbury

. (2013). Interaction with Shc prevents aberrant Erk activation in the absence of extracellular stimuli. Nat Struct Mol Biol, 20:620–627.

63.

Hermanns

, Radtke

, Schaper

, Heinrich

and Behrmann

. (2000). Non-redundant signal transduction of interleukin-6-type cytokines: the adapter protein Shc is specifically recruited to the oncostatin m receptor*. J Biol Chem, 275:40742–40748.

64.

Natalicchio

, Laviola

, Tullio

, Renna

, Montrone

, Perrini

, Valenti

, Procino

, Svelto

and Giorgino

. (2004). Role of the p66Shc isoform in insulin-like growth factor I receptor signaling through MEK/Erk and regulation of actin cytoskeleton in rat myoblasts*. J Biol Chem, 279:43900–43909.

65.

Migliaccio

, Mele

, Salcini

, Pelicci

, Lai

, Superti-Furga

, Pawson

, Di Fiore

, Lanfrancone

and Pelicci

. (1997). Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway. EMBO J, 16:706–716.

66.

Wills

MKB

, Keyvani Chahi

, Lau

, Tilak

, Guild

, New

, Lu

, Jacquet

, Meakin

, Bisson

and Jones

. (2017). Signaling adaptor ShcD suppresses extracellular signal-regulated kinase (Erk) phosphorylation distal to the Ret and Trk neurotrophic receptors. J Biol Chem, 292:5748–5759.

67.

Gough

, Koetz

and Levy

. (2013). The MEK-ERK pathway is necessary for serine phosphorylation of mitochondrial STAT3 and Ras-mediated transformation. PLoS One, 8:e83395.

68.

Tesoriere

, Dinarello

and Argenton

. (2021). The roles of post-translational modifications in STAT3 biological activities and functions. Biomedicines, 9:956.

69.

Yang

, van Oosten

, Theunissen

, Guo

, Silva

JCR

and Smith

. (2010). Stat3 activation is limiting for reprogramming to ground state pluripotency. Cell Stem Cell, 7:319–328.

70.

Chen

, Aksoy

, Gonnot

, Osteil

, Aubry

, Hamela

, Rognard

, Hochard

, Voisin

, Fontaine

, Mure

, Afanassieff

, Cleroux

, Guibert

, Chen

, Vallot

, Acloque

, Genthon

, Donnadieu

, De Vos

, Sanlaville

, Guérin

J-F

, Weber

, Stanton

, Rougeulle

, Pain

, Bourillot

P-Y

, Savatier

. (2015). Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency. Nat Commun, 6:7095.

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