Cancer Letters, 173, 83C91. stalk cells or spores. The fates of these cells can be predicted in the vegetative phase: Cells expressing higher and lower levels of differentiate into stalk cells and spores, respectively. However, is merely a marker gene and changes in its expression do not influence the cell fate, and determinant factors remain unknown. In this study, we analyzed cell fate determinants in the stalk\destined and spore\destined cells that were sorted based on expression. Luciferase assay demonstrated higher levels of intracellular ATP in the stalk\destined cells than in the spore\destined cells. Live\cell observation during development using ATP sensor probes revealed that cells with higher Tropisetron HCL ATP levels differentiated into stalk cells. Furthermore, reducing the ATP level by treating with an inhibitor of ATP production changed the differentiation fates of the stalk\destined cells to spores. These results suggest that intracellular ATP levels influence cell fates in differentiation. differentiation. 1.?INTRODUCTION Multicellular organisms consist of a variety of differentiated cells, and their differentiation processes must be tightly regulated to ensure their proper functions; errors that occur during the differentiation process may induce fatal defects in organisms. Thus, understanding the regulatory mechanisms that determine cell lineages is a fundamental question for the fields of biology and medicine (Avior, Sagi, & Benvenisty, 2016; Castanon & Gonzlez\Gaitn, 2011; Zakrzewski, Dobrzynski, Szymonowicz, & Rybak, 2019). At the beginning of the cell differentiation process, different cell types appear stochastically within a genetically identical cell population, which is known as the salt and pepper model and has been observed in various organisms, such as nematode worms, flies and mice (Chazaud, Yamanaka, Pawson, & Rossant, 2006; Miller, Seymour, King, & Herman, 2008; Schnabel et al., 2006). In such stochastic differentiation, non\genetic cellular heterogeneity, which arises from fluctuations of intrinsic and extrinsic factors, appears to be a key factor in the determination of cell fates. Intercellular variations in gene expression, metabolism and responses to cellular signals have been proposed to be intrinsic factors that affect cell fate (Elowitz, Levine, Siggia, & Swain, 2002; Evers et al., 2019; Raser & OShea, 2004; Yamanaka & Blau, 2010). Among these potential factors, metabolism appears to play a significant role in cell fate decisions. Increasing evidence has indicated that the activity levels of the mitochondria, important organelles associated with metabolism, play a role in the differentiation of human cells (Buck et al., 2016; Khacho et al., 2016). However, the critical factors that determine cell fate remain unknown. The cellular slime mold is an amoebozoa and represents a good model Tropisetron HCL organism for studying relationships between cellular heterogeneity and cell differentiation during the development of multicellular organisms. Amoeboid cells continue to proliferate under nutrient\rich conditions (vegetative phase). Upon starvation, amoeboid cells initiate the process of multicellular development, differentiating into 2 major cell types: stalk cells and spore cells (Figure?1a). In the early stages of development, amoeboid cells move collectively toward extracellular cAMP oscillations, originated from an aggregation center, to form a multicellular mound. Cells that enter the mound phase begin to differentiate into stalk or spore progenitor cells, called prestalk and prespore cells, respectively, which arise stochastically in a salt and pepper fashion. Prestalk cells are sorted to the top Tropisetron HCL side of the mound, forming the tip region, which later forms the anterior region of the migrating body (slug), whereas prespore cells constitute the posterior region of the slug. In the process of fruiting body formation, prestalk cells differentiate CHK1 into stalk cells, penetrating into the prespore region of the slug. Spore cells generating progenies are moved to the top of the fruiting body through the support of stalk cells (Maeda, Inouye, & Takeuchi, 1997). Open in a separate window Figure Tropisetron HCL 1 Gene expression analysis of stalk\destined and spore\destined cells. (a) Schematic illustration of the developmental process for cells are predetermined in the vegetative phase, prior to multicellular formation, and can be predicted by expression levels of the gene (Kuwana, Senoo, Sawai, & Fukuzawa, 2016). Vegetative cells expressing high levels of are destined to differentiate into spore cells (designated as spore\destined cells, hereafter). However, is merely a marker gene, and changes in its expression levels do not influence the cell fates (Kuwana et al., 2016). The identities of the factors that drive differentiation of stalk\destined cells (expressing high levels) and spore\destined cells (expressing low levels) remain unknown. To address this question, we first carried out RNA sequencing (RNA\seq) analyses to examine differences in gene expression levels between stalk\destined and spore\destined cells sorted by flow cytometry based on expression levels. The subsequent RNA\seq analysis and luciferase assay suggested that differences in ATP levels may be a factor.