Under Arrest: The Embryo in Diapause

Embryonic diapause is the reversible arrest in development of mammalian embryos at the blastocyst stage. In this issue of Developmental Cell, Hussein et al., 2020Hussein A.M. Wang Y. Mathieu J. Margaretha L. Chaozhong S. Jones D.C. Cavanaugh C. Miklas J.W. Mahen E. Showalger M.R. et al.Metabolic control over mTOR dependent diapause-like state.Dev. Cell. 2020; 52 (this issue): 236-250Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar reveal that alternative splicing of Lkb1 is essential for diapause to persist and find the elevation of glycolytic and lipolytic pathways that were previously considered dormant. Embryonic diapause is the reversible arrest in development of mammalian embryos at the blastocyst stage. In this issue of Developmental Cell, Hussein et al., 2020Hussein A.M. Wang Y. Mathieu J. Margaretha L. Chaozhong S. Jones D.C. Cavanaugh C. Miklas J.W. Mahen E. Showalger M.R. et al.Metabolic control over mTOR dependent diapause-like state.Dev. Cell. 2020; 52 (this issue): 236-250Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar reveal that alternative splicing of Lkb1 is essential for diapause to persist and find the elevation of glycolytic and lipolytic pathways that were previously considered dormant. Embryos from more than 130 species of mammals have the capacity to undergo a developmental arrest at the blastocyst state, with the teleological significance of ensuring the best conditions for the survival of the offspring (Fenelon et al., 2014Fenelon J.C. Banerjee A. Murphy B.D. Embryonic diapause: development on hold.Int. J. Dev. Biol. 2014; 58: 163-174Crossref PubMed Scopus (83) Google Scholar). This arrest, known as embryonic diapause, was first uncovered in rodent species well over a century ago (Fenelon and Renfree, 2018Fenelon J.C. Renfree M.B. The history of the discovery of embryonic diapause in mammals.Biol. Reprod. 2018; 99: 242-251Crossref PubMed Scopus (22) Google Scholar). In the usual course of events, mouse embryos hatch from their zonae pellucidae and initiate implantation late on the fourth day following mating (Figure 1). This event is triggered by a brief ovarian spike of estrogen secretion (Fenelon et al., 2014Fenelon J.C. Banerjee A. Murphy B.D. Embryonic diapause: development on hold.Int. J. Dev. Biol. 2014; 58: 163-174Crossref PubMed Scopus (83) Google Scholar). Under conditions of metabolic stress in rodents, most commonly attributable to suckling by the previous litter, this signal is absent. The embryos nonetheless hatch, but they cease development. This condition is reversible and can be recapitulated by any one of a number of strategies that abrogate the secretion of the key estrogen signal. Much has been learned about the process of mouse implantation from this model. Further, mouse embryos in diapause have provided another important scientific contribution, in that pluripotent stem cells were first established from the inner cell mass (ICM) of the embryo in arrest (Evans and Kaufman, 1981Evans M.J. Kaufman M.H. Establishment in culture of pluripotential cells from mouse embryos.Nature. 1981; 292: 154-156Crossref PubMed Scopus (6429) Google Scholar). A recent study demonstrated that reversible diapause could be induced in mouse blastocysts by partial inhibition of mechanistic target of rapamycin (mTOR) (Bulut-Karslioglu et al., 2016Bulut-Karslioglu A. Biechele S. Jin H. Macrae T.A. Hejna M. Gertsenstein M. Song J.S. Ramalho-Santos M. Inhibition of mTOR induces a paused pluripotent state.Nature. 2016; 540: 119-123Crossref PubMed Scopus (124) Google Scholar). A more recent study of transcriptomic and proteomic analysis comparing whole embryos in diapause with the activated embryos (exiting from diapause) revealed upregulation of both mTOR expression and activity (He et al., 2019He B. Zhang H. Wang J. Liu M. Sun Y. Guo C. Lu J. Wang H. Kong S. Blastocyst activation engenders transcriptome reprogram affecting X-chromosome reactivation and inflammatory trigger of implantation.Proc. Natl. Acad. Sci. USA. 2019; 116: 16621-16630Crossref PubMed Scopus (20) Google Scholar), indicating that quiescence in this signaling pathway is a major element of diapause. In this issue of Developmental Cell, Hussein et al., 2020Hussein A.M. Wang Y. Mathieu J. Margaretha L. Chaozhong S. Jones D.C. Cavanaugh C. Miklas J.W. Mahen E. Showalger M.R. et al.Metabolic control over mTOR dependent diapause-like state.Dev. Cell. 2020; 52 (this issue): 236-250Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar extend these findings to provide a comprehensive understanding of both the signals upstream of mTOR inhibition and the cellular processes downstream implicated in the reversible arrest in embryo development. In their investigation, Hussein et al., 2020Hussein A.M. Wang Y. Mathieu J. Margaretha L. Chaozhong S. Jones D.C. Cavanaugh C. Miklas J.W. Mahen E. Showalger M.R. et al.Metabolic control over mTOR dependent diapause-like state.Dev. Cell. 2020; 52 (this issue): 236-250Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar compared the transcriptomes of the ICM of the preimplantation blastocyst and the epiblast during early implantation with the embryo in diapause. By this means, they defined gene ontology pathways that were up- or downregulated specifically during the diapause condition. Standouts in this analysis were the genes in the glycolytic and pyruvate pathways and those associated with cholesterol metabolism. Together these findings indicate that a unique metabolic profile characterizes diapause. In further analysis, these authors showed that either mTOR inhibition or starvation of naive mouse embryonic stem cells (mESC) resulted in a transcriptome that recapitulated, in many respects, the diapause cellular phenotype. A key difference in the transcriptomes among the developmental isotypes (epiblast, preimplantation, and diapause ICM) was the presence of a number of alternatively spliced isoforms, suggesting that this process may contribute to the diapause condition. Indeed, one of the splice variants identified was in the Lkb1 gene, resulting a long and short form of this key metabolic regulator. An essential finding was that only the long form, with a C-terminal regulatory domain, was present in the ICM cells derived from diapause embryos. Experiments in which mESC lines were modified to express the Lkb1 short form revealed that deletion of the C-terminal regulatory domain rendered Lkb1 constitutively active. Further, forced expression of only the long form of Lkb1 in mESCs (i.e., absence of the short form) induced a state highly reminiscent of diapause. The sum of the evidence indicates that the absence of the shortened, constitutively active form provokes the diapause arrest. The downstream target of Lkb1 is AMPK, a major player in cellular energy homeostasis, and AMPK is an upstream regulator of the mTOR cascade (Jacinto, 2019Jacinto E. Amplifying mTORC2 signals through AMPK during energetic stress.Sci. Signal. 2019; 12: eaax5855Crossref PubMed Scopus (6) Google Scholar). Together, these observations have established a chain of signals essential for continued development. The authors postulate that the short LKB1 form and its constitutive activity disappear at diapause (Hussein et al., 2020Hussein A.M. Wang Y. Mathieu J. Margaretha L. Chaozhong S. Jones D.C. Cavanaugh C. Miklas J.W. Mahen E. Showalger M.R. et al.Metabolic control over mTOR dependent diapause-like state.Dev. Cell. 2020; 52 (this issue): 236-250Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Whether this is a direct cause or a consequence of diapause is unclear, but these findings serve to further define the enigmatic mechanisms that regulate the reversible developmental arrest of the mouse embryo. The cells of the ICM are dormant in diapause and in starvation models employed by the authors (Hussein et al., 2020Hussein A.M. Wang Y. Mathieu J. Margaretha L. Chaozhong S. Jones D.C. Cavanaugh C. Miklas J.W. Mahen E. Showalger M.R. et al.Metabolic control over mTOR dependent diapause-like state.Dev. Cell. 2020; 52 (this issue): 236-250Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Early investigations of the diapause state in rodent embryos indicated dormancy in the form of a depressed metabolic rate and infrequent proliferation (Fenelon et al., 2014Fenelon J.C. Banerjee A. Murphy B.D. Embryonic diapause: development on hold.Int. J. Dev. Biol. 2014; 58: 163-174Crossref PubMed Scopus (83) Google Scholar). In addition, as the mTOR signaling system integrates upstream metabolic pathways, acting as a sensor for cell-nutrient availability, and regulates proliferation, the expected response to its inhibition would be reduced metabolism. It was somewhat surprising that Hussein et al. found that the pathways that had most significant upregulation during diapause were those related to glycolysis. Starvation and treatment of mESCs with a pan-mTOR inhibitor produced analogous elevations in the glycolytic products. Moreover, metabolomic analyses of whole embryos collected in diapause demonstrated not a decrease but rather an overall increase in lipolysis, accompanied by an increase in the content of both saturated and unsaturated fatty acids. This was attributed to the formation and accumulation of phospholipids. Explanation of the significance of increased lipid and carbohydrate metabolism in the context of diapause begs further investigation. Another regulatory mechanism revealed by global analysis was related to amino acid transport. The glutamine transporter Slc38i was elevated in the ICM from diapause embryos. Pharmacological or siRNA interference with SLC38A transporters prevented diapause. This was interpreted to be due to inhibitory effects of high intracellular glutamine concentration on activation of the mTOR signaling pathway. Some caveats to the conclusions can be recognized. The mouse embryo in diapause has two cell compartments, the trophoblast and the ICM, with the former comprising two-thirds of the total cell number (Houghton, 2006Houghton F.D. Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst.Differentiation. 2006; 74: 11-18Crossref PubMed Scopus (155) Google Scholar). The trophoblast has a significantly different metabolic profile (Houghton, 2006Houghton F.D. Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst.Differentiation. 2006; 74: 11-18Crossref PubMed Scopus (155) Google Scholar), and autophagy is more frequent in trophoblast cells compared to the ICM (Lee et al., 2011Lee J.E. Oh H.A. Song H. Jun J.H. Roh C.R. Xie H. Dey S.K. Lim H.J. Autophagy regulates embryonic survival during delayed implantation.Endocrinology. 2011; 152: 2067-2075Crossref PubMed Scopus (71) Google Scholar). Additionally, there is evidence for communication between the two compartments in the mouse embryo (Desrochers et al., 2016Desrochers L.M. Bordeleau F. Reinhart-King C.A. Cerione R.A. Antonyak M.A. Microvesicles provide a mechanism for intercellular communication by embryonic stem cells during embryo implantation.Nat. Commun. 2016; 7: 11958Crossref PubMed Scopus (132) Google Scholar). Influences other than those visited on the ICM could also be important to regulation of diapause. The mouse model of diapause is perhaps the best studied, but it is important to note that there are many other species that display this trait, and there are significant differences in the manner that the embryo manifests diapause among species (Fenelon et al., 2014Fenelon J.C. Banerjee A. Murphy B.D. Embryonic diapause: development on hold.Int. J. Dev. Biol. 2014; 58: 163-174Crossref PubMed Scopus (83) Google Scholar). Nonetheless, the findings of Hussein et al., 2020Hussein A.M. Wang Y. Mathieu J. Margaretha L. Chaozhong S. Jones D.C. Cavanaugh C. Miklas J.W. Mahen E. Showalger M.R. et al.Metabolic control over mTOR dependent diapause-like state.Dev. Cell. 2020; 52 (this issue): 236-250Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar provide valuable new avenues of exploration into the perplexing trait of mammalian embryonic diapause. Metabolic Control over mTOR-Dependent Diapause-like StateHussein et al.Developmental CellJanuary 27, 2020In BriefHussein et al. report that, during starvation, mTOR is repressed through LKB1-AMPK, inducing a reversible metabolically active but epigenetically silenced embryonic diapause-like state that upregulates expression of the glutamine transporters SLC38A1/2. These transporters are required for the H4K16ac-negative, diapause state. Full-Text PDF Open Archive