Abstract
During embryonic development, cells undergo changes in gene expression, signaling pathway activation/inactivation, metabolism, and intracellular organelle structures, which are mediated by mitochondria. Mitochondria continuously switch their morphology between elongated tubular and fragmented globular via mitochondrial fusion and fission. Mitochondrial fusion is mediated by proteins encoded by Mfn1, Mfn2, and Opa1, whereas mitochondrial fission is mediated by proteins encoded by Fis1 and Dnm1L. Here, we investigated the expression patterns of mitochondria-related genes during the differentiation of mouse embryonic stem cells (ESCs). Pluripotent ESCs maintain stemness in the presence of leukemia inhibitory factor (LIF) via the JAK-STAT3 pathway but lose pluripotency and differentiate in response to the withdrawal of LIF. We analyzed the expression levels of mitochondrial fusion- and fission-related genes during the differentiation of ESCs. We hypothesized that mitochondrial fusion genes would be overexpressed while the fission genes would be downregulated during the differentiation of ESCs. Though the mitochondria exhibited an elongated morphology in ESCs differentiating in response to LIF withdrawal, only the expression of Mfn2 was increased and that of Dnm1L was decreased as expected, the other exceptions being Mfn1, Opa1, and Fis1. Next, by comparing gene expression and mitochondrial morphology, we proposed an index that could precisely represent mitochondrial changes during the differentiation of pluripotent stem cells by analyzing the expression ratios of three fusion- and two fission-related genes. Surprisingly, increased Mfn2/Dnm1L ratio was correlated with elongation of mitochondria during the differentiation of ESCs. Moreover, application of this index to other specialized cell types revealed that neural stems cells (NSCs) and mouse embryonic fibroblasts (MEFs) showed increased Mfn2/Dnm1L ratio compared to ESCs. Thus, we suggest that the Mfn2/Dnm1L ratio could reflect changes in mitochondrial morphology according to the extent of differentiation.
Highlights
During embryonic development, cells undergo various changes in gene expression [1, 2] and signaling pathways [3]
neural stems cells (NSCs) were cultured in culture dishes coated with 0.15% porcine gelatin (Sigma) in NS medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) : Nutrient Mixture F-12 (Gibco), 0.5 mg/mL bovine serum albumin (BSA; Sigma), 1% N2 supplement (Gibco), 1x nonessential amino acids (NEAAs) (Gibco), 1x P/S/G (Gibco), 10 ng/mL basic fibroblast growth factor, and 10 ng/mL epidermal growth factor (EGF; Gibco)
We investigated the mitochondrial biogenesis by immunostaining using antibodies targeting translocase of the outer membrane 20 (TOM20), which is in the outer mitochondrial membrane during the differentiation of embryonic stem cells (ESCs)
Summary
Cells undergo various changes in gene expression [1, 2] and signaling pathways [3]. Folmes et al showed that the globular shape of mitochondria progressively changed to elongated during embryonic development from zygote to somite embryo. Metabolic features such as pyruvate oxidation, glucose oxidation, glycolysis, and the pentose phosphate pathway (PPP) were changed dynamically [6]. These features return to the developmental early-stage status during the reprogramming process [8].
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