p38 (Mapk14/11) occupies a regulatory node governing entry into primitive endoderm differentiation during preimplantation mouse embryo development

Abstract

During mouse preimplantation embryo development, the classically described second cell-fate decision involves the specification and segregation, in blastocyst inner cell mass (ICM), of primitive endoderm (PrE) from pluripotent epiblast (EPI). The active role of fibroblast growth factor (Fgf) signalling during PrE differentiation, particularly in the context of Erk1/2 pathway activation, is well described. However, we report that p38 family mitogen-activated protein kinases (namely p38α/Mapk14 and p38β/Mapk11; referred to as p38-Mapk14/11) also participate in PrE formation. Specifically, functional p38-Mapk14/11 are required, during early-blastocyst maturation, to assist uncommitted ICM cells, expressing both EPI and earlier PrE markers, to fully commit to PrE differentiation. Moreover, functional activation of p38-Mapk14/11 is, as reported for Erk1/2, under the control of Fgf-receptor signalling, plus active Tak1 kinase (involved in non-canonical bone morphogenetic protein (Bmp)-receptor-mediated PrE differentiation). However, we demonstrate that the critical window of p38-Mapk14/11 activation precedes the E3.75 timepoint (defined by the initiation of the classical ‘salt and pepper’ expression pattern of mutually exclusive EPI and PrE markers), whereas appropriate lineage maturation is still achievable when Erk1/2 activity (via Mek1/2 inhibition) is limited to a period after E3.75. We propose that active p38-Mapk14/11 act as enablers, and Erk1/2 as drivers, of PrE differentiation during ICM lineage specification and segregation.

p38-mitogen activated kinases mediate a developmental regulatory response to amino acid depletion and associated oxidative stress in mouse blastocyst embryos

“…Chemical inhibition of p38-MAPKs was carried out using SB220025 (Calbiochem® cat. # 559396; dissolved in dimethyl sulfoxide/DMSO) at 20μM working concentration in the respective culture medium, as described previously (Thamodaran and Bruce, 2016). DMSO (Sigma-Aldrich® cat.…”

“…The third lineage, represented by the pluripotent epiblast (EPI), residing deep within the ICM, serves as a progenitor pool for all subsequent tissues of the foetus; extensively reviewed (Frum and Ralston, 2015;Chazaud and Yamanaka, 2016;Rossant, 2016)). We have previously reported a role for p38-mitogen activated kinases α/β(herein referred to as p38-MAPK), employing pharmacological inhibition, in regulating primitive endoderm (PrE) differentiation within mouse blastocyst inner cell mass (ICM)(Thamodaran and Bruce, 2016). p38-MAPK was found to act during the early stages of ICM maturation (the period between E3.5-E4.5), downstream of fibroblast growth factor (FGF) signalling, permitting PrE progenitors to resolve their uncommitted fate (as is characteristic of the majority of nascent ICM cells at E3.5 (Chazaud et al, 2006)) and thus, functionally diverge and segregate from the EPI lineage (Thamodaran and Bruce, 2016).…”

“…We have previously reported a role for p38-mitogen activated kinases α/β(herein referred to as p38-MAPK), employing pharmacological inhibition, in regulating primitive endoderm (PrE) differentiation within mouse blastocyst inner cell mass (ICM)(Thamodaran and Bruce, 2016). p38-MAPK was found to act during the early stages of ICM maturation (the period between E3.5-E4.5), downstream of fibroblast growth factor (FGF) signalling, permitting PrE progenitors to resolve their uncommitted fate (as is characteristic of the majority of nascent ICM cells at E3.5 (Chazaud et al, 2006)) and thus, functionally diverge and segregate from the EPI lineage (Thamodaran and Bruce, 2016). Although, p38-MAPK (and their related paralogs, p38γ/δ) belong to the wider family of serine-threonine and tyrosine kinases, regulating a wide variety of cellular functions (Cargnello and Roux, 2011), they differ from the other family members, such as extra-cellular regulated kinases [ e.g.…”

“…Although, p38-MAPK (and their related paralogs, p38γ/δ) belong to the wider family of serine-threonine and tyrosine kinases, regulating a wide variety of cellular functions (Cargnello and Roux, 2011), they differ from the other family members, such as extra-cellular regulated kinases [ e.g. ERK1/2 – themselves implicated in FGF-mediated PrE differentiation at a developmental point succeeding that identified for p38-MAPK (Nichols et al, 2009;Yamanaka et al, 2010;Frankenberg et al, 2011;Kang et al, 2013;Thamodaran and Bruce, 2016)] in that they are classically known to be activated by extracellular stress stimuli; for example pro-inflammatory cytokines, U.V. radiation and physical stress, rather than liganded growth-factor associated receptor tyrosine kinases (Remy et al, 2010).…”

“…Accordingly, we have examined our previously identified p38-MAPK inhibition induced defective PrE phenotypes, themselves associated with reduced blastocyst cell number (Thamodaran and Bruce, 2016), to ascertain if p38-MAPK not only regulates PrE differentiation per se , but also perform a dual regulative/homeostatic role in mediating preimplantation mouse embryo/blastocyst development in response to limited amino acid (AA) availability, as could be inferred from earlier studies (Kwong et al, 2000;Kwong et al, 2006;Sun et al, 2014;Fleming et al, 2015;Sun et al, 2015;Fleming et al, 2018) addressing DOHaD.…”