Changes in the Expression of Mitochondrial Morphology-Related

1 Changes in the expression of mitochondrial morphology-related

2 genes during the differentiation of murine embryonic stem cells

3 Jeong Eon Lee1,a, Bong Jong Seo1,a, Min Ji Han1, Yean Ju Hong1, Kwonho Hong1, Hyuk 4 Song1, Jeong Woong Lee2, and Jeong Tae Do1,*

6 1Department of Stem Cell and Regenerative Biotechnology, KU Institute of Science and 7 Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of 8 Korea

9 2Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and 10 Biotechnology, Daejeon 305-806, Republic of Korea

11 a These authors contributed equally to this work.

12 *Correspondence should be addressed to Jeong Tae Do, Ph.D. 13 E-mail: [email protected]

14 Tel: 82-2-450-3673

15 Fax: 82-2-355-1044 16 Running title: Mitochondrion-related genes and cellular differentiation

18 Abstract

19 During embryonic development, cells undergo changes in gene expression, signaling 20 pathway activation/inactivation, metabolism, and intracellular organelle structures, which are 21 mediated by mitochondria. Mitochondria continuously switch their morphology between 22 elongated tubular and fragmented globular via mitochondrial fusion and fission. 23 Mitochondrial fusion is mediated by proteins encoded by Mfn1, Mfn2, and Opa1, whereas 24 mitochondrial fission is mediated by proteins encoded by Fis1 and Dmn1L. Here, we 25 investigated the expression patterns of mitochondria-related genes during the differentiation 26 of mouse embryonic stem cells (ESCs) in response to leukemia inhibitory factor (LIF) 27 withdrawal. The expression of Mfn2 and Dnm1L was, as expected, increased and decreased, 28 respectively. By comparing gene expression and mitochondrial morphology, we proposed an 29 index that could precisely represent mitochondrial changes during the differentiation of 30 pluripotent stem cells by analyzing the expression ratios of three fusion- and two fission- 31 related genes. Surprisingly, increased Mfn2/Dnm1L ratio was correlated with elongation of 32 mitochondria during the differentiation of ESCs. Moreover, application of this index to other 33 specialized cell types revealed that neural stems cells (NSCs) and mouse embryonic 34 fibroblasts (MEFs) showed increased Mfn2/Dnm1L ratio compared to ESCs. Thus, we 35 suggest that the Mfn2/Dnm1L ratio could reflect changes in mitochondrial morphology 36 according to the extent of differentiation.

37 Keywords: pluripotent stem cells, differentiation, mitochondria, mitochondrial morphology, 38 mitochondrial dynamics

40 Introduction 41 During embryonic development, cells undergo various changes in gene expression 42 (Carter et al., 2003; Hamatani et al., 2004) and signaling pathways (Wang et al., 2004). 43 Metabolism and intracellular organelle structures are also altered during development and 44 differentiation (Folmes et al., 2012; Leese, 1995; Van Blerkom, 2004). Specifically, the 45 organellar changes are observed during the regaining of pluripotency (also known as 46 reprogramming) (Choi et al., 2015). For example, Folmes et al. showed that the globular 47 shape of mitochondria progressively changed to elongated during embryonic development 48 from zygote to somite embryo. Accordingly, metabolic features such as pyruvate oxidation, 49 glucose oxidation, glycolysis, and the pentose phosphate pathway (PPP) were also changed 50 dynamically (Folmes et al., 2012). These features return to the developmental early-stage 51 status during the reprogramming process (Panopoulos et al., 2012). Some of the most 52 dramatic changes in cells during development and differentiation occur in the mitochondria, 53 which play essential roles in cellular processes, including energy metabolism (Seo et al., 54 2018), apoptosis (Suen et al., 2008), aging (Bratic and Larsson, 2013), reactive oxygen 55 species production, calcium homeostasis, and differentiation (Wanet et al., 2015). 56 Mitochondria continuously change their morphology through fusion and fission in 57 response to cellular requirements; this process is called mitochondrial dynamics or 58 mitochondrial biogenesis (Chan, 2006; Chen and Chan, 2004; Cho et al., 2006; Westermann, 59 2012). In pre-implantation embryonic and pluripotent stem cells, immature mitochondria 60 characterized by a small and globular shape with poorly developed cristae are observed (Cho 61 et al., 2006; Chung et al., 2007; St John et al., 2005). Cells with immature mitochondria show 62 low oxygen consumption and high levels of glycolytic enzymes (Kondoh et al., 2007). Thus, 63 undifferentiated embryonic stem cells (ESCs) also exhibit low levels of ATP production, 64 modest levels of antioxidant enzymes, and poor oxidant capacity (Cho et al., 2006; Chung et 65 al., 2007; Kondoh et al., 2007). Upon differentiation of ESCs, mitochondria in these cells 66 become elongated, showing developed cristae and dense matrices (Lonergan et al., 2007); this 67 results in high oxygen consumption and ATP production for more efficient cellular activity 68 (Cho et al., 2006; Chung et al., 2007; St John et al., 2005). 69 In mammals, mitochondrial morphology switches between elongated tubular and 70 fragmented globular by fusion and fission, respectively (Okamoto and Shaw, 2005;

71 Westermann, 2010). Mitochondrial fusion is mediated by the dynamin family GTPases, such 72 as mitofusin (MFN) 1, MFN2, and optic atrophy 1 (OPA1) (Chen et al., 2003; Delettre et al., 73 2000; Santel and Fuller, 2001). Although the exact fusion mechanism has yet to be defined, 74 MFN1 and MFN2 form a dimer that inserts itself into the mitochondrial outer membrane, 75 whereas OPA1 is located in the mitochondrial inner membrane (Meeusen et al., 2006; Song et 76 al., 2009). MFN1, MFN2, and OPA1 contain a GTPase domain, hydrophobic heptad repeat 77 (HR) domain, and transmembrane domain (Ishihara et al., 2006; Santel and Fuller, 2001). 78 MFN1 and MFN2 play similar roles in mitochondrial fusion and thus can functionally replace 79 each other and form homotypic or heterotypic dimers (Chen et al., 2003; Shirihai et al., 2015). 80 In contrast, the major proteins related to mitochondrial fission are FIS1 (James et al., 81 2003; Yoon et al., 2003; Zhang and Chan, 2007) and dynamin-related protein 1 (DNM1L, 82 also called DRP1) (Legesse-Miller et al., 2003; Otsuga et al., 1998; Smirnova et al., 1998). 83 DNM1L is mainly located in the cytosol and recruited to the outer membrane of the 84 mitochondria where it induces fission (Shirihai et al., 2015). FIS1 is located in the outer 85 mitochondrial membrane and is closely related to DNM1L (James et al., 2003; Yoon et al., 86 2003). DNM1L can interact with other mitochondrial fission proteins, including 87 mitochondrial fission factor (MFF) and FIS1. Interestingly, a recent study suggested that 88 DNM1L can interact with the fusion protein MFN and facilitate MFN-mediated fusion 89 (Shirihai et al., 2015). Although many studies have evaluated the fusion and fission of 90 mitochondria, the mechanisms and signaling pathways that determine mitochondrial 91 dynamics are still unclear. Activation of the mammalian target of rapamycin (mTOR) 92 pathway by the withdrawal of LIF induced mouse ESC differentiation with suppression of 93 pluripotent genes such as Klf4, Oct4, and Nanog (Cherepkova et al., 2016). When pluripotent 94 stem cells differentiate, they require more energy to meet the demands of their newly 95 acquired functions (Folmes et al., 2012) 96 Accordingly, mitochondria undergo dynamic remodeling during differentiation, and 97 thus, mitochondrial morphology and metabolism are changed. Therefore, we hypothesized 98 that expression of mitochondrial fusion- and fission-related genes may be changed toward a 99 certain direction during the spontaneous differentiation of ESCs. Here, we quantified the 100 expression levels of the fusion-related genes Mfn1, Mfn2, and Opa1 and the fission-related 101 genes Fis1 and Dnm1L during the differentiation of murine ESCs. Here, we investigated

102 these genes to determine whether they could be used as an index of the extent of 103 differentiation and changes in mitochondrial morphology. 104

105 Materials and Methods 106 All methods used in this study were carried out in accordance with animal care and 107 use guidelines, and all experimental protocols were approved by the Institutional Animal 108 Care and Use Committee of Konkuk University. 109 110 Cell cultures 111 Mouse ESCs (E14tg2a) were purchased from the American Type Culture Collection 112 (ATCC, Manassas, VA, USA) and cultured on culture dishes layered with inactivated MEFs 113 in an ESC medium, consisting of Dulbecco’s modified Eagle’s medium (DMEM; Gibco) 114 supplemented with 15% fetal bovine serum (FBS; HyClone), 1× 115 Penicillin/Streptomycin/Glutamine (P/S/G; Gibco), 0.1 mM non-essential amino acids 116 (NEAAs; Gibco), 1 mM β-mercaptoethanol (Gibco) with 1000 U/mL leukemia inhibitory 117 factor (ESGRO, Chemicon International). MEFs were cultured in culture dishes coated with 118 0.15% porcine gelatin (Sigma, St. Louis, MO, USA) in MEF medium consisting of DMEM 119 (Gibco) supplemented with 15% FBS (HyClone), 1× P/S/G (Gibco), 0.1 mM NEAAs (Gibco), 120 and 1 mM β-mercaptoethanol (Gibco). NSCs were cultured in culture dishes coated with 0.15% 121 porcine gelatin (Sigma) in NS medium consisting of DMEM:Nutrient Mixture F-12 (Gibco), 122 0.5 mg/ml bovine serum albumin (BSA; Sigma) 1% N2 supplement (Gibco), 1x NEAAs 123 (Gibco), 1x P/S/G (Gibco), 10 ng/mL basic fibroblast growth factor (bFGF; R&D systems), 124 and 10 ng/mL epidermal growth factor (EGF; Gibco). All cell lines were incubated at 37 oC

125 in an atmosphere of 5% CO2 and maintained on tissue culture dishes (Corning, Amsterdam, 126 The Netherlands). 127 128 In vitro differentiation 129 In the pre-plating process, ESCs cultured with feeder cells were dissociated by 130 trypsin-EDTA (0.25%) (Gibco) and transferred to a 0.15% gelatin-coated dish and incubated o 131 at 37 C in an atmosphere of 5% CO2 for 2 h to remove the feeder cells. Because the feeder 132 cells attached to the gelatin-coated dish much earlier than ESCs, supernatant of the culture 133 mostly contain ESCs without contamination of feeder cells. Supernatant was transferred 134 another gelatin-coated dish and used as the differentiation experiment. For in vitro 135 differentiation, 1 × 105 ESCs were seeded in 100-mm cell culture dishes coated with 0.15%

136 porcine gelatin (Sigma) with MEF medium. The medium was refreshed every day for 15 days 137 of differentiation. The cells were collected by scraping for experimental analysis. 138 139 RNA isolation and qRT-PCR 140 Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) 141 according to the manufacturer’s protocol. The cDNA was then synthesized from 1 mg total 142 RNA using SuperScript III Reverse Transcriptase (Invitrogen) and oligo(dT)20-primer 143 (Invitrogen) according to the manufacturer’s instructions. qPCR was performed in duplicate 144 with Power SYBR Green Master Mix (Takara, Shiga, Japan), and results were analyzed on a 145 Roche LightCycler 5480 (Roche). Thermal cycling was carried out via 45 cycles of 10 s at 95 146 oC, 10 s at 60 oC, and 20 s at 72 oC. The primers for qRT-PCR are shown in Table 1. 147 148 Western blot analysis 149 Total cells were lysed using RIPA buffer (Thermo Fisher) according to the 150 manufacturer’s instructions. Cell lysates (20 μg protein) were separated on NuPAGE 4–12% 151 Bis-Tris Gels (Invitrogen) and transferred to polyvinyl difluoride membranes. The 152 membranes were blocked using a blocking solution containing 5% skim milk powder and 153 0.05% Tween 20 in phosphate-buffered saline (PBS). The primary antibodies used in this 154 study were as follows: anti-OCT4 (rabbit, 1:1000; Santa Cruz Biotechnology, Santa Cruz, 155 CA, USA), anti-Nanog (rabbit, 1:1000; Abcam, Cambridge, UK), anti-MFN2 (mouse, 1:1000; 156 Abcam), anti-DRP1 (rabbit, 1:1000; Millipore), anti-MFN1 (mouse, 1:1000; Abcam), anti- 157 FIS1 (mouse, 1:500; Abcam), and anti-β-actin (mouse, 1:10000; Sigma). The membranes 158 were incubated with these antibodies overnight at 4°C. Secondary antibodies were conjugated 159 with anti-mouse IgG-peroxidase (1:10000; Sigma), anti-goat IgG-horseradish peroxidase 160 (HRP), and anti-rabbit IgG-HRP (1:10000; Santa Cruz Biotechnology) and the membranes 161 were incubated with these antibodies for 90 min at room temperature. 162 Antigens were detected using Pierce ECL Western Blotting chemiluminescent 163 substrate (Thermo Fisher), according to the manufacturer’s instructions. Blots were then 164 exposed to X-ray film for development and stripped for reuse of the membranes. Anti-β-actin 165 antibody (mouse, 1:10000; Sigma) was used as a control. Densitometry of the bands for the 166 proteins and their loading controls was performed using Image J 1.43 (NIH) software.

167 168 Immunocytochemistry 169 For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 20 min at 170 room temperature. The cells were washed with PBS and then treated with PBS containing 3% 171 bovine serum albumin and 0.03% Triton X-100 for 30 min at room temperature. The cells 172 were then incubated with the following primary antibodies: anti-OCT4 (1:500; Santa Cruz 173 Biotechnology), anti-Nanog (1:200; Abcam), anti-β-tubulin (TUJ1; 1:500; R&D), anti- 174 SMA (1:200; Abcam), anti-SOX17 (1:300; R&D), and anti-TO20 (1:200; Santa Cruz 175 Biotechnology). Fluorescently labeled (Alexa Fluor 488 or 647; Abcam) secondary 176 antibodies were used according to the manufacturer’s specifications. Images for anti-TOM20 177 staining were obtained with a confocal microscope (Zeiss). 178 179 Electron microscopy 180 For transmission electron microscope (TEM) experiments, the samples were fixed in 181 4% paraformaldehyde (Sigma) and 2.5% glutaraldehyde (Sigma) in 0.1M phosphate 182 (Sigma) buffer for 24 h. After washing in 0.1M phosphate buffer, the samples were post- 183 fixed for 1h in 1% osmium tetroxide (Sigma) prepared in the same buffer. The samples 184 were dehydrated with a graded series of ethyl alcohol concentrations, embedded in Epon 812, 185 and polymerized at 60°C for 3 days. Ultrathin sections (60–70nm) were obtained using an 186 ultramicrotome (Leica Ultracut UCT), collected on grids (200 mesh), and examined under a 187 TEM (JEM 1010) operating at 60kV, and images were recorded by a charge-coupled device 188 camera (SC1000; Gatan). 189 190 Mitochondrial length analysis 191 The images from electron microscopy were analyzed and measured by the Image J 192 1.43 (NIH) software for calculating the maximum (Max)/minimum/(Min) ratio of 193 mitochondrial length. At least over fifty mitochondria were measured and analyzed per 194 sample to obtain data. 195 196 Statistical analysis 197 All experiments were performed in triplicate, and data are presented as means ±

198 standard error of mean (SEM). Differences were assessed using unpaired or paired t-tests, and 199 differences with p-values of less than 0.05 were considered significant. 200

201 Results

202 Changes in pluripotency- and tissue-specific markers during the differentiation of mouse 203 ESCs 204 To examine changes in gene expression during the differentiation of ESCs, we 205 randomly differentiated mouse ESCs by the withdrawal of LIF without feeder cells for 0, 3, 6, 206 9, 12, and 15 days (Fig. 1a). Dome-like colonies of undifferentiated ESCs became flat and 207 showed changes in morphology. First, we checked whether ESCs were properly differentiated 208 in vitro after 15 days in differentiation medium. Immunocytochemistry analysis showed that 209 OCT4 and NANOG, which were expressed in undifferentiated ESCs, were silenced at day 15 210 after differentiation. Differentiation markers, neuron-specific class III β-tubulin (TUJ1), 211 smooth muscle actin (SMA), and SRY-box 17 (SOX17) for ectodermal, mesodermal, and 212 endodermal cells, respectively, were not detectable in undifferentiated ESCs at day 0, but 213 were detected after differentiation at day 15, indicating that ESCs lost pluripotency and were 214 differentiated into various cell types, including all three germ layers (Fig. 1b). 215 Next, we evaluated the expression levels of pluripotency and differentiation markers 216 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis (Fig. 1c, 217 d). The expression levels of the pluripotency markers Oct4 and Nanog were gradually 218 decreased as ESCs were differentiated (Fig. 1c). In contrast, early mesoderm marker T (also 219 known as Brachyury) expression was increased until day 6 post-differentiation and then 220 downregulated afterward (Fig. 1d), indicating that early mesoderm cells appeared at day 6 221 after differentiation and then differentiated further. Taken together, our findings demonstrated 222 that ESCs differentiated gradually and lost pluripotency over 15 days upon LIF withdrawal 223 from the ESC medium. 224 225 Changes in mitochondrial morphology during the differentiation of mouse ESCs 226 Mitochondrial morphology was expected to change from fragmented to elongated 227 shapes during the differentiation of ESCs. Thus, we examined mitochondrial morphology by 228 immunostaining using antibodies targeting translocase of outer membrane 20 (TOM20), 229 which is in the outer mitochondrial membrane. In ESCs, small mitochondria (green dots in 230 Fig. 2a) were evenly distributed in the cytoplasm, indicating that the fragmented form of 231 mitochondria was the major form in ESCs. However, green dots became larger beginning on 10

232 day 6 after differentiation of ESCs (Fig. 2a). This observation was also accurately confirmed 233 by electron microscopic analysis (Fig. 2b). Undifferentiated ESCs had primarily globular 234 mitochondria with immature cristae, and this morphology was maintained until day 3 of 235 differentiation. From day 6 to 15, mitochondria were gradually elongated and showed mature 236 cristae (Fig. 2b). We measured the Max and Min axes of mitochondria to quantify 237 mitochondrial length (Fig. 2c-d). The Max/Min ratios of mitochondria was 1.51, 1.66, 3.65, 238 3.92, 4.30, and 6.87 on days 0, 3, 9, 12, and 15, respectively (Fig. 2e). These data clearly 239 demonstrated that the globular and immature mitochondria in ESCs became elongated and 240 showed developed mature cristae after the spontaneous differentiation. 241 242 Changes in mitochondrial morphology-related genes during the differentiation of mouse 243 ESCs 244 Because mitochondria were elongated during the differentiation of ESCs, we 245 predicted that fusion-related genes would be up-regulated, and fission-related genes would be 246 down-regulated during this differentiation. Thus, we examined mitochondrial morphology- 247 related genes by qRT-PCR analysis. Unexpectedly, the fusion-related gene Mfn1 was 248 progressively downregulated, whereas Mfn2 and Opa1 expression patterns fluctuated at day 3 249 post-differentiation (Fig. 3a). Next, we evaluated the expression of two fission-related genes, 250 Dnm1L and Fis1 (Fig. 3b); consistent with the reduced mitochondrial fission, which resulted 251 in enhanced mitochondrial elongation, the expression of Dnm1L decreased as mouse ESCs 252 underwent spontaneous differentiation (Fig. 3b). However, Fis1 expression did not decrease 253 but rather increased after slight downregulation at the beginning of differentiation (Fig. 3b). 254 This result supported previous findings that FIS1 is less related to mitochondrial fission than 255 DNM1L and functions to recruit DNM1L to the mitochondria (Loson et al., 2013; Zhang et 256 al., 2016). Collectively, our results showed that Mfn2 and Dmn1L mRNA expression levels 257 reflected mitochondrial elongation with ESC differentiation. 258 Accordingly, we next investigated the levels of MFN2 and DNM1L proteins by 259 western blotting in ESCs (day 0) and in differentiated cells on days 3, 6, 9, 12, and 15 (Fig. 260 3d). As a control for ESC spontaneous differentiation, the expression of the pluripotency 261 marker OCT4 gradually decreased during differentiation and was almost undetectable at day 262 15. MFN2 was expressed at a low level in ESCs but showed a dramatic increase in

263 expression upon spontaneous differentiation. Furthermore, MFN2 protein levels showed a 264 fluctuating expression pattern during differentiation, similar to the qRT-PCR data (Fig. 3a and 265 3d). DNM1L protein levels decreased gradually during the differentiation of ESCs, similar to 266 the result of qRT-PCR analysis (Fig. 3b and 3d). Thus, we observed similar changes in the 267 mRNA and protein levels for these two targets. 268 269 Establishment of indexes representing the extent of differentiation 270 Given that many genes involved in mitochondrial fusion and fission are not 271 correlated with mitochondrial morphology, we next attempted to find an index to represent 272 mitochondrial morphology. Based on the qRT-PCR data, we analyzed the ratios between three 273 fusion- and two fission-related genes during the differentiation of ESCs. A total of 6 274 combinatorial ratios for three fusion- and two fission-related genes were analyzed (Fig. 3c). 275 One of these, the Mfn2/Dnm1l ratio, was interesting because it was similar to the 276 mitochondrial length Max/Min ratio during the differentiation of ESCs (Fig. 2d and 3c in red 277 square). Next, we confirmed the protein expression level in the MFN2/DNM1L ratio (Fig. 3e). 278 Indeed, the MFN2/DNM1L ratio was also similar to the mRNA expression and mitochondrial 279 length Max/Min ratio. Thus, we concluded that the mitochondrial changes during ESC 280 spontaneous differentiation could be reflected by the progressive increase in the Mfn2/Dnm1l 281 ratio. 282 283 Mfn2/Dnm1L index represented mitochondrial morphology according to the extent of 284 differentiation 285 To investigate whether this index was applicable to other cell types, we compared 286 this ratio among ESCs, neural stem cells (NSCs) and mouse embryonic fibroblasts (MEFs). 287 We chose these cell types because ESCs are undifferentiated, NSCs are less specialized, and 288 MEFs are differentiated. Only ESCs express high levels of Oct4 (Fig. 4b). Although the 289 mRNA expression level of the mitochondrial fusion-related gene Mfn2 was slightly changed 290 in these cell types, the mRNA expression level of the mitochondrial fission-related gene 291 Dnm1l was gradually decreased in NSCs and MEFs (Fig. 4c, 4d), consistent with the 292 observed changes in mitochondrial morphology (Fig. 4a). Next, we applied the Mfn2/Dnm1l 293 ratio to these cell types, with adjustment to a ratio of 1.0 for ESCs. Interestingly, the adjusted

294 Mfn2/Dnm1L ratios were 1.0, 2.97, and 3.86 in ESCs, NSCs, and MEFs, respectively. Next, 295 we also confirmed MFN2 and DNM1L protein expression (Fig. 4f). As expected, the DNM1L 296 protein expression levels were decreased in NSCs and MEFs, compared with those in ESCs. 297 Interestingly, the MFN2 protein expression levels were gradually increased in NSCs and 298 MEFs. The MFN2/DNM1L protein expression ratios were 0.16, 2.94, and 4.61 in ESCs, 299 NSCs, and MEFs, respectively. Thus, we concluded that the Mfn2/Dnm1l ratio at the mRNA 300 and protein expression levels corresponded to the length of mitochondria (Fig. 4e-g).

301 Discussion 302 In this study, we investigated the dynamics of mitochondrial morphology-related 303 genes, which are responsible for mitochondrial fusion and fission, during the differentiation 304 of mouse ESCs. Given that differentiated cells contain elongated mitochondria and ESCs 305 contain globular mitochondria (Seo et al., 2018), we expected to observe increases in the 306 expression of mitochondrial fusion-related genes (Mfn1, Mfn2, and Opa1) and decreases in 307 the expression of fission-related genes (Fis1 and Dnm1L). 308 This is because several reports have shown that the overexpression of fusion-related 309 genes such as Mfn1/Mfn2 (Chen et al., 2003) and Opa1 (Frezza et al., 2007) induces 310 mitochondrial elongation in MEFs. In this context, the overexpression of fission-related 311 genes such as Drp1 (Yamamori et al., 2015) and Fis1 (Otera et al., 2010) induces 312 mitochondrial fragmentation in MEFs and HeLa cells, respectively. However, there were only 313 minor changes in the expression levels of Mfn1 and Opa1 during differentiation, and changes 314 in the expression of Fis1 were opposite to the expected results. Actually, FIS1 has been 315 reported to have a weaker effect on mitochondrial fission than DNM1L in MEFs but not in 316 HeLa (Loson et al., 2013; Otera et al., 2010; Yamamori et al., 2015) possibly due to the 317 different mechanisms of mitochondrial morphology regulation in humans and mice. Fig. 3a 318 shows that the expression pattern of Mfn2 better reflects mitochondrial morphology than that 319 of Mfn1 or Opa1 during ESC spontaneous differentiation. A loss-of-function experiment in a 320 previous study showed that Mfn1 or Mfn2 deficiency results in fragmented mitochondrial 321 morphology and loss of Mfn2 has more dramatic effects than loss of Mfn1 (Chen et al., 2003). 322 Moreover, OPA1, processed for mitochondrial fusion by protease isoenzymes and OMA1, 323 might not reflect the morphology of mitochondria (Ehses et al., 2009). This is because 324 various proteins affect the processing of OPA1 protein for its function in the mitochondrial 325 fusion. Only Mfn2 and Dnm1L expression patterns were, as expected, increased and 326 decreased, respectively. 327 Next, we aimed to identify a special index that gradually increased during ESC 328 spontaneous differentiation. We found that a gradual increase in the Mfn2/Dnm1L ratio was 329 closely related to mitochondrial elongation during the elapsed time after the differentiation of 330 ESCs. Furthermore, we found that this index could be applied to other cell types, including 331 NSCs and MEFs. More specialized cells showed higher Mfn2/Dnm1L ratios than ESCs.

332 There were some limitations to our research. Firstly, technologies such as High- 333 Content Imaging and Machine Learning were recently developed for the analysis of the 334 shapes of mitochondria (Leonard et al., 2015). This technical method allows the analysis of 335 the mitochondria by further subdividing the shape of the mitochondria e.g., fragmented, rods, 336 networks, and large/round. However, the functions of the genes that affect the dynamic of 337 mitochondria have yet to be identified and therefore cannot explain how mitochondria form a 338 network (Seo et al., 2018). Therefore, we focused on classifying the mitochondria in just two 339 categories, i.e., fragmented and elongated. Second, some cell types at final stages of 340 differentiation such as erythropoietic cells (Jensen et al., 2019; Liu et al., 2017) and 341 hepatocytes (Das et al., 2012) have fragmented mitochondria. If ESCs were differentiated by 342 LIF-withdrawal, various cell types will be observed among differentiated cell population 343 (Cherepkova et al., 2016; Duval et al., 2000), including erythropoietic cells and hepatocytes. 344 The fragmentation of mitochondria related to the specific role of cells cannot be limited to 345 gene expression, which controls the dynamic of mitochondria. Thus, the tendency of gene 346 expression patterns can only be interpreted as the indexes that predict the degree of 347 differentiation and shape of mitochondria, because of the random differentiation process (i.e., 348 no lineage-specific differentiation). Thus, our findings suggested that this ratio could also be 349 used as an index for mitochondrial morphology during differentiation. 350 351

352 Acknowledgments

353 This research was supported by the Basic Science Research Program through the National 354 Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future 355 Planning of the Republic of Korea (grant nos. 2016M3A9B6946835 and 2015R15A1009701).

356

357 Author contribution statement

358 JEL, KH, BJS, and JTD wrote the main manuscript text and designed the study. JEL, BJS, 359 and YJH, MJH performed experiments and analyzed the data. JEL, BJS, HS, and JWL 360 performed data analysis. All authors reviewed the manuscript.

361

362 Ethical standards

363 All methods used in this study were carried out in accordance with animal care and use 364 guidelines, and all experimental protocols were approved by the Institutional Animal Care 365 and Use Committee of Konkuk University. 366

367 Conflict of interest 368 The authors declare that they have no conflict of interest 369

370

371 References

372 Bratic, A. and Larsson, N. G. (2013). The role of mitochondria in aging. J Clin Invest 123, 373 951-7. 374 Carter, M. G., Hamatani, T., Sharov, A. A., Carmack, C. E., Qian, Y., Aiba, K., Ko, N. T., 375 Dudekula, D. B., Brzoska, P. M., Hwang, S. S. et al. (2003). In situ-synthesized novel microarray 376 optimized for mouse stem cell and early developmental expression profiling. Genome Res 13, 1011- 377 21. 378 Chan, D. C. (2006). Mitochondrial fusion and fission in mammals. Annu Rev Cell Dev Biol 22, 379 79-99. 380 Chen, H. and Chan, D. C. (2004). Mitochondrial dynamics in mammals. Curr Top Dev Biol 381 59, 119-44. 382 Chen, H., Detmer, S. A., Ewald, A. J., Griffin, E. E., Fraser, S. E. and Chan, D. C. (2003). 383 Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic 384 development. J Cell Biol 160, 189-200. 385 Cherepkova, M. Y., Sineva, G. S. and Pospelov, V. A. (2016). Leukemia inhibitory factor (LIF) 386 withdrawal activates mTOR signaling pathway in mouse embryonic stem cells through the 387 MEK/ERK/TSC2 pathway. Cell Death Dis 7, e2050. 388 Cho, Y. M., Kwon, S., Pak, Y. K., Seol, H. W., Choi, Y. M., Park, D. J., Park, K. S. and Lee, 389 H. K. (2006). Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the 390 spontaneous differentiation of human embryonic stem cells. Biochem Biophys Res Commun 348, 391 1472-8. 392 Choi, H. W., Kim, J. H., Chung, M. K., Hong, Y. J., Jang, H. S., Seo, B. J., Jung, T. H., Kim, J. 393 S., Chung, H. M., Byun, S. J. et al. (2015). Mitochondrial and metabolic remodeling during 394 reprogramming and differentiation of the reprogrammed cells. Stem Cells Dev 24, 1366-73. 395 Chung, S., Dzeja, P. P., Faustino, R. S., Perez-Terzic, C., Behfar, A. and Terzic, A. (2007). 396 Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells. Nat Clin 397 Pract Cardiovasc Med 4 Suppl 1, S60-7. 398 Das, S., Hajnóczky, N., Antony, A. N., Csordás, G., Gaspers, L. D., Clemens, D. L., Hoek, J. 399 B. and Hajnóczky, G. (2012). Mitochondrial morphology and dynamics in hepatocytes from normal 400 and ethanol-fed rats. Pflügers Archiv-European Journal of Physiology 464, 101-109. 401 Delettre, C., Lenaers, G., Griffoin, J. M., Gigarel, N., Lorenzo, C., Belenguer, P., Pelloquin, L., 402 Grosgeorge, J., Turc-Carel, C., Perret, E. et al. (2000). Nuclear gene OPA1, encoding a mitochondrial 403 dynamin-related protein, is mutated in dominant optic atrophy. Nat Genet 26, 207-10. 404 Duval, D., Reinhardt, B., Kedinger, C. and Boeuf, H. (2000). Role of suppressors of cytokine 405 signaling (Socs) in leukemia inhibitory factor (LIF)-dependent embryonic stem cell survival. The 406 FASEB Journal 14, 1577-1584.

407 Ehses, S., Raschke, I., Mancuso, G., Bernacchia, A., Geimer, S., Tondera, D., Martinou, J.- 408 C., Westermann, B., Rugarli, E. I. and Langer, T. (2009). Regulation of OPA1 processing and 409 mitochondrial fusion by m-AAA protease isoenzymes and OMA1. J Cell Biol 187, 1023-1036. 410 Folmes, C. D., Dzeja, P. P., Nelson, T. J. and Terzic, A. (2012). Metabolic plasticity in stem 411 cell homeostasis and differentiation. Cell Stem Cell 11, 596-606. 412 Frezza, C., Cipolat, S. and Scorrano, L. (2007). Measuring mitochondrial shape changes and 413 their consequences on mitochondrial involvement during apoptosis. Methods Mol Biol 372, 405-20. 414 Hamatani, T., Carter, M. G., Sharov, A. A. and Ko, M. S. (2004). Dynamics of global gene 415 expression changes during mouse preimplantation development. Dev Cell 6, 117-31. 416 Ishihara, N., Fujita, Y., Oka, T. and Mihara, K. (2006). Regulation of mitochondrial 417 morphology through proteolytic cleavage of OPA1. EMBO J 25, 2966-77. 418 James, D. I., Parone, P. A., Mattenberger, Y. and Martinou, J. C. (2003). hFis1, a novel 419 component of the mammalian mitochondrial fission machinery. J Biol Chem 278, 36373-9. 420 Jensen, E. L., Gonzalez-Ibanez, A. M., Mendoza, P., Ruiz, L. M., Riedel, C. A., Simon, F., 421 Schuringa, J. J. and Elorza, A. A. (2019). Copper deficiency-induced anemia is caused by a 422 mitochondrial metabolic reprograming in erythropoietic cells. Metallomics 11, 282-290. 423 Kondoh, H., Lleonart, M. E., Nakashima, Y., Yokode, M., Tanaka, M., Bernard, D., Gil, J. and 424 Beach, D. (2007). A high glycolytic flux supports the proliferative potential of murine embryonic stem 425 cells. Antioxid Redox Signal 9, 293-9. 426 Leese, H. J. (1995). Metabolic control during preimplantation mammalian development. Hum 427 Reprod Update 1, 63-72. 428 Legesse-Miller, A., Massol, R. H. and Kirchhausen, T. (2003). Constriction and Dnm1p 429 recruitment are distinct processes in mitochondrial fission. Mol Biol Cell 14, 1953-63. 430 Leonard, A. P., Cameron, R. B., Speiser, J. L., Wolf, B. J., Peterson, Y. K., Schnellmann, R. 431 G., Beeson, C. C. and Rohrer, B. (2015). Quantitative analysis of mitochondrial morphology and 432 membrane potential in living cells using high-content imaging, machine learning, and morphological 433 binning. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1853, 348-360. 434 Liu, X., Zhang, Y., Ni, M., Cao, H., Signer, R. A., Li, D., Li, M., Gu, Z., Hu, Z. and Dickerson, 435 K. E. (2017). Regulation of mitochondrial biogenesis in erythropoiesis by mTORC1-mediated protein 436 translation. Nat Cell Biol 19, 626. 437 Lonergan, T., Bavister, B. and Brenner, C. (2007). Mitochondria in stem cells. Mitochondrion 438 7, 289-96. 439 Loson, O. C., Song, Z., Chen, H. and Chan, D. C. (2013). Fis1, Mff, MiD49, and MiD51 440 mediate Drp1 recruitment in mitochondrial fission. Mol Biol Cell 24, 659-67. 441 Meeusen, S., DeVay, R., Block, J., Cassidy-Stone, A., Wayson, S., McCaffery, J. M. and 442 Nunnari, J. (2006). Mitochondrial inner-membrane fusion and crista maintenance requires the 443 dynamin-related GTPase Mgm1. Cell 127, 383-95. 18

444 Okamoto, K. and Shaw, J. M. (2005). Mitochondrial morphology and dynamics in yeast and 445 multicellular eukaryotes. Annu Rev Genet 39, 503-36. 446 Otera, H., Wang, C., Cleland, M. M., Setoguchi, K., Yokota, S., Youle, R. J. and Mihara, K. 447 (2010). Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in 448 mammalian cells. J Cell Biol 191, 1141-1158. 449 Otsuga, D., Keegan, B. R., Brisch, E., Thatcher, J. W., Hermann, G. J., Bleazard, W. and 450 Shaw, J. M. (1998). The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in 451 yeast. J Cell Biol 143, 333-49. 452 Panopoulos, A. D., Yanes, O., Ruiz, S., Kida, Y. S., Diep, D., Tautenhahn, R., Herrerias, A., 453 Batchelder, E. M., Plongthongkum, N., Lutz, M. et al. (2012). The metabolome of induced pluripotent 454 stem cells reveals metabolic changes occurring in somatic cell reprogramming. Cell Res 22, 168-77. 455 Santel, A. and Fuller, M. T. (2001). Control of mitochondrial morphology by a human 456 mitofusin. J Cell Sci 114, 867-74. 457 Seo, B., Yoon, S. and Do, J. (2018). Mitochondrial Dynamics in Stem Cells and 458 Differentiation. Int J Mol Sci 19, 3893. 459 Shirihai, O. S., Song, M. and Dorn, G. W., 2nd. (2015). How mitochondrial dynamism 460 orchestrates mitophagy. Circ Res 116, 1835-49. 461 Smirnova, E., Shurland, D. L., Ryazantsev, S. N. and van der Bliek, A. M. (1998). A human 462 dynamin-related protein controls the distribution of mitochondria. J Cell Biol 143, 351-8. 463 Song, Z., Ghochani, M., McCaffery, J. M., Frey, T. G. and Chan, D. C. (2009). Mitofusins and 464 OPA1 mediate sequential steps in mitochondrial membrane fusion. Mol Biol Cell 20, 3525-32. 465 St John, J. C., Ramalho-Santos, J., Gray, H. L., Petrosko, P., Rawe, V. Y., Navara, C. S., 466 Simerly, C. R. and Schatten, G. P. (2005). The ex pression of mitochondrial DNA transcription factors 467 during early cardiomyocyte in vitro differentiation from human embryonic stem cells. Cloning Stem 468 Cells 7, 141-53. 469 Suen, D. F., Norris, K. L. and Youle, R. J. (2008). Mitochondrial dynamics and apoptosis. 470 Genes Dev 22, 1577-90. 471 Van Blerkom, J. (2004). Mitochondria in human oogenesis and preimplantation 472 embryogenesis: engines of metabolism, ionic regulation and developmental competence. 473 Reproduction 128, 269-80. 474 Wanet, A., Arnould, T., Najimi, M. and Renard, P. (2015). Connecting Mitochondria, 475 Metabolism, and Stem Cell Fate. Stem Cells Dev 24, 1957-71. 476 Wang, Q. T., Piotrowska, K., Ciemerych, M. A., Milenkovic, L., Scott, M. P., Davis, R. W. and 477 Zernicka-Goetz, M. (2004). A genome-wide study of gene activity reveals developmental signaling 478 pathways in the preimplantation mouse embryo. Dev Cell 6, 133-44. 479 Westermann, B. (2010). Mitochondrial fusion and fission in cell life and death. Nat Rev Mol 480 Cell Biol 11, 872-84. 19

481 Westermann, B. (2012). Bioenergetic role of mitochondrial fusion and fission. Biochim 482 Biophys Acta 1817, 1833-8. 483 Yamamori, T., Ike, S., Bo, T., Sasagawa, T., Sakai, Y., Suzuki, M., Yamamoto, K., Nagane, 484 M., Yasui, H. and Inanami, O. (2015). Inhibition of the mitochondrial fission protein dynamin-related 485 protein 1 (Drp1) impairs mitochondrial fission and mitotic catastrophe after x-irradiation. Mol Biol Cell 486 26, 4607-17. 487 Yoon, Y., Krueger, E. W., Oswald, B. J. and McNiven, M. A. (2003). The mitochondrial 488 protein hFis1 regulates mitochondrial fission in mammalian cells through an interaction with the 489 dynamin-like protein DLP1. Mol Cell Biol 23, 5409-20. 490 Zhang, Y. and Chan, D. C. (2007). Structural basis for recruitment of mitochondrial fission 491 complexes by Fis1. Proc Natl Acad Sci U S A 104, 18526-30. 492 Zhang, Z., Liu, L., Wu, S. and Xing, D. (2016). Drp1, Mff, Fis1, and MiD51 are coordinated to 493 mediate mitochondrial fission during UV irradiation-induced apoptosis. FASEB J 30, 466-76.

494

495

496

497 Table 1. Primer sets used for quantitative RT-PCR

Gene Forward Reverse Mfn1 5’-CATGGGCATCATGGTTGTTGGG-3’ 5’-TCTCCACTGCTCGGGTGTAG-3’ Mfn2 5’-CAAGTGTCCGCTCCTGAAGG-3’ 5’-GAACCTCCTTGGCAGACACG-3’ Opa1 5’-CAAGCATTACAGGAAGGTGTCAGAC-3’ 5’-CACTGAGAGTCACCTTCACTGG-3’ Fis1 5’-CATCGTGCTGCTGGAGAGC-3’ 5’-GCAGAGAGCAGGTGAGGCTG-3’ Dnm1L 5’-GGAGTTGAAGCAGAAGAATGGGG-3’ 5’-CAGTGACGGCGAGGATAATGG-3’ Oct4 5’-GATGCTGTGAGCCAAGGCAAG-3’ 5’-GGCTCCTGATCAACAGCATCAC-3’ Nanog 5’-CTTTCACCTATTAAGGTGCTTGC-3’ 5’-TGGCATCGGTTCATCATGCTAC-3’ T 5’-CCGGTGCTGAAGGTAAATGT-3’ 5’-CCTCCATTGAGCTTGTTGGT-3’ ACTB 5’-C G C CAT G GAT G ACG ATAT C G-3’ 5’-CGAAGCCGGCTTTGCACATG-3’

498

499 Figure Legends 500 Fig. 1 In vitro differentiation and characterization of ESCs. (a) Phase-contrast images of 501 ESCs and differentiating cells on days 0, 3, 6, 9, 12, and 15. Scale bars = 200 μm. (b) 502 Immunofluorescence images of the pluripotency markers OCT4 and NANOG and 503 differentiation markers for ectodermal (TUJ1), mesodermal (SMA), and endodermal (SOX17) 504 cells in undifferentiated ESCs and ESC-derived differentiated cells on days 0 and 15. Nuclear 505 was counterstained with DAPI. Scale bars = 100 μm. (c-d) Quantitative RT-PCR analysis of 506 ESCs and differentiating cells on days 0, 3, 6, 9, 12, and 15 (D0, D3, D6, D9, D12, and D15). 507 Data are presented as mean ± SEM for n = 3 independent experiments. (c) Pluripotent marker 508 Oct4 and Nanog expression on day 0 to 15 (d) Differentiation marker T expression from day 509 0 to 15. t-test: *p < 0.05, **p < 0.01, and ***p < 0.001. 510 511 Fig. 2 Changes in mitochondrial morphology during the differentiation of ESCs. (a) 512 Analysis of mitochondrial morphology by staining of mitochondria using anti-TOM20 513 antibodies on days 0, 3, 6, 9, 12, and 15 after differentiation of ESCs. Green dots represent 514 mitochondria. Nuclear was counterstained with DAPI. Scale bars = 10 μm. (b) Electron 515 microscopic images of mitochondria in undifferentiated ESCs and differentiated cells from 516 ESCs on days 0, 3, 6, 9, 12 and 15. Yellow dotted lines represent mitochondrial morphologies. 517 Scale bars = 0.5 μm. (c) Measurement of the maximum (Max) and minimum (Min) axes of 518 mitochondria. (d) The mitochondrial length (nm) in ESCs on differentiating days 0, 3, 6, 9, 519 12 and 15. (e) The Max/Min ratios in ESCs and differentiating cells on days 0, 3, 6, 9, 12, and 520 15. Data are presented as mean ± SEM for n = 30 independent experiments. t-test: *p < 0.05, 521 **p < 0.01, and ***p < 0.001. 522 523 Fig. 3 Changes in the expression levels of mitochondrial fusion- and fission-related genes 524 and in indexes from combinations of fusion- and fission-related genes. Quantitative RT- 525 PCR analysis of (a) mitochondrial fusion-related genes (Mfn1, Mfn2, and Opa1) and (b) 526 mitochondrial fission-related genes (Fis1 and Dnm1L) on days 0, 3, 6, 9, 12, and 15 after 527 differentiation of ESCs. Gene expression levels were normalized to the expression of Actb. (c) 528 Six combinatorial ratios from the analysis of gene expression levels between three fusion- 529 and two fission genes. (d) Western blotting for the expression of OCT4, MFN2, and DNM1L.

530 β-actin was used as a control for other proteins. (e) MFN2/DNM1L protein ratios were 531 gradually increased according to the elapsed time after ESC differentiation. All data are 532 presented as mean ± SEM for n = 3 independent experiments. t-test: *p < 0.05, **p < 0.01, 533 and ***p < 0.001.

534 Fig. 4 Mfn2/Dnm1L ratios in ESCs, NSCs, and MEFs. (a) Electron microscopic images of 535 mitochondria in undifferentiated ESCs, NSCs, and MEFs. Yellow dotted lines represent 536 mitochondrial morphologies. Scale bars = 0.5 μm. Quantitative RT-PCR analysis of (b) 537 pluripotency-, (c) mitochondrial fusion-, and (d) mitochondrial fission-related genes in ESCs, 538 NSCs, and MEFs. Gene expression levels were normalized to the expression of Actb. (e) 539 Mfn2/Dnm1L gene expression ratios in ESCs, NSCs, and MEFs. (f) Western blotting for the 540 expression of DNM1L and MFN2 in ESCs, NSCs, and MEFs. β-actin was used as a control 541 for other proteins. (g) MFN2/DNM1L protein ratios in ESCs, NSCs, and MEFs. All data are 542 presented as mean ± SEM for n = 3 independent experiments. t-test: *p < 0.05, **p < 0.01, 543 and ***p < 0.001.

bioRxiv preprint doi: https://doi.org/10.1101/644906; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/644906; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/644906; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/644906; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.