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March5 Governs the Convergence and Extension Movement for Organization of the Telencephalon and Diencephalon in Zebrafish Embryos

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March5 Governs the Convergence and Extension Movement for Organization of the Telencephalon and Diencephalon in Zebrafish Embryos Molecules and Cells march5 Governs the Convergence and Extension Movement for Organization of the Telencephalon and Diencephalon in Zebrafish Embryos 1 1 1 2 3 1, Jangham Jung , Issac Choi , Hyunju Ro , Tae-Lin Huh , Joonho Choe , and Myungchull Rhee * 1Department of Life Science, BK21 Plus Program, Graduate School, Chungnam National University, Daejeon 34134, Korea, 2School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea, 3Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea *Correspondence: [email protected] https://doi.org/10.14348/molcells.2019.0210 www.molcells.org MARCH5 is a RING finger E3 ligase involved in mitochondrial INTRODUCTION integrity, cellular protein homeostasis, and the regulation of mitochondrial fusion and fission. To determine the function Membrane-associated RING-CH protein 5 (MARCH5) is an of MARCH5 during development, we assessed transcript E3 ubiquitin ligase located in the mitochondrial outer mem- expression in zebrafish embryos. We found that march5 brane (with the E3 ligase domain facing the cytoplasm) and transcripts were of maternal origin and evenly distributed at is involved in a variety of mitochondrial and cellular processes the 1­cell stage, except for the mid­blastula transition, with (for review; Nagashima et al., 2014). For example, MARCH5 expression predominantly in the developing central nervous ubiquitylates the mitochondrial outer membrane proteins system at later stages of embryogenesis. Overexpression of involved in mitochondrial fusion such as mitofusin 1 (Mfn1), march5 impaired convergent extension movement during which regulates mitochondrial docking and fusion, and gastrulation, resulting in reduced patterning along the Mfn2, which stabilizes the interactions between mitochon- dorsoventral axis and alterations in the ventral cell types. dria. Increased expression of Mfn1 in MARCH5-depleted cells Overexpression and knockdown of march5 disrupted enhances mitochondrial fusion but interferes with fission the organization of the developing telencephalon and (Chen et al., 2003; Park and Cho, 2012; Park et al., 2010). diencephalon. Lastly, we found that the transcription of Ubiquitylation of Mfn2 generates a mitochondrion-associated march5 was tightly regulated by the transcriptional regulators endoplasmic reticulum membrane domain for Mfn2 oligo- CHOP, C/EBPα, Staf, Znf143a, and Znf76. These results merization, enabling Mfn2 proteins to interact and transfer demonstrate the essential role of March5 in the development Ca2+ from the endoplasmic reticulum to the mitochondrion of zebrafish embryos. (De Brito and Scorrano, 2008; Sugiura et al., 2013; Szabadkai et al., 2006). Keywords: convergence and extension movement, dience- Another target of MARCH5, cytoplasmic dynamin-relat- phalon, March5/MITOL, telencephalon, ubiquitin proteasome ed protein 1 (Drp1), regulates mitochondrial fission. Drp1 system transiently interacts with tetratricopeptide repeats in the outer-membrane-associated protein Fis1 via cytosolic adap- tor proteins Mdv1 and Caf4 (Lackner et al., 2009; Mears et Received 19 September, 2019; revised 25 November, 2019; accepted 4 December, 2019; published online 7 January, 2020 eISSN: 0219-1032 ©The Korean Society for Molecular and Cellular Biology. All rights reserved. cc This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. 76 Mol. Cells 2020; 43(1): 76-85 march5 Regulation of Brain Development in Zebrafish Jangham Jung et al. al., 2011) while MARCH5 counteracts mitochondrial fission the manufacturer’s instructions. mRNAs were dissolved in nu- by marking Drp1 and Fis1 for degradation (Nakamura et al., clease-free water and diluted in 0.5% phenol red solution for 2006; Yonashiro et al., 2006). MARCH5 also negatively reg- microinjection via a Picopump microinjection device (World ulates mitochondrial fission via stress-induced degradation of Precision Instruments, USA). mitochondrial dynamics protein 49 kDa (MiD49) (Cherok et al., 2017; Xu et al., 2016). MiD49 is located in the mitochon- Whole­mount in situ hybridization (WISH) drial outer membrane and recruits Drp1 to the mitochondrial Embryos were fixed in 4% paraformaldehyde (PFA) over- surface rather than the peroxisomal surface; thus, MiD49 night, and dehydrated in 100% methanol. Embryos after is thought to facilitate Drp1-directed mitochondrial fission 24 h post-fertilization (hpf) were digested with 10 µg/ml (Palmer et al., 2011; Zhao et al., 2011). protease K (Thermo Scientific, USA). WISH was performed MARCH5 marks mitophagy receptor FUN14 domain-con- with minor modifications as described in (Kumar et al., 2019; taining protein 1 for degradation in response to hypoxia (Wu Thisse et al., 1993). Antisense probes of march5 were synthe- et al., 2017). MARCH5 also regulates mitochondrial transport sized from a specific region within the ORF. Antisense probes via the degradation of abnormal proteins, such as microtu- of march5 were synthesized using the DIG RNA Labeling Kit bule-associated protein 1B, which interfere with dynein mo- (SP6/T7) (Roche, USA). tor function and block the transport of mitochondria along axonal microtubules (Yonashiro et al., 2012). Cell culture Despite the many reports describing the various molecular HEK293T (human embryonic kidney 293T) cells were ob- functions of MARCH5 in mammalian cells, its embryological tained from KCLB (Korean Cell Line Bank, Korea) for the functions have yet to be described. Therefore, we conduct- Dual-Luciferases assay. HEK 293T cells were cultured in Dul- ed whole-mount in situ hybridization (WISH) and induced becco’s modified Eagle medium (DMEM; Welgene, Korea) ectopic expression and knockdown of march5 in zebrafish containing 10% fetal bovine serum (FBS; Welgene) and 1% embryos to investigate the role of MARCH5 in vertebrate em- Antibiotic-Antimycotic solution (Gibco, USA). bryogenesis. Luciferase activity assay MATERIALS AND METHODS Cells were harvested and lysates were collected 24 h post transfection. Firefly and Renilla luciferase activities were mea- Zebrafish care and embryos sured using a Dual-Luciferase Reporter Assay System (Prome- Wild-type (WT) zebrafish were obtained from Korea Ze- ga, USA) according to the manufacturer’s instructions. Rela- brafish Organogenesis Mutant Bank (ZOMB) and grown at tive luciferase activities are the ratios of the activity of firefly 28.5˚C. Embryos were obtained through natural spawning luciferase to that of the Renilla luciferase control. and raised, and staged as described previously (Kimmel et al., 1995; Westerfield, 2000). Embryonic pigmentation was Reverse transcription­quantitative polymerase chain reac­ blocked by treating the embryos with 0.002% phenylth- tion (RT­qPCR) iourea after onset of somitogenesis. Total RNA from WT and march5 morpholino injected zebraf- ish embryos at 4.7 hpf was extracted by RNAiso Plus reagent Sequence analysis (TaKaRa Bio, Japan), according to the manufacturer’s proto- March5 sequence similarity searches to identify homologous col. The PrimeScript First strand cDNA synthesis kit (TaKaRa sequences were performed as described previously (Kim et Bio) was applied for cDNA generation. qPCR was performed al., 2008) and phylogenic analysis of March5 was conducted using SYBR-Green qPCR mix (TaKaRa Bio) in a Thermal Cycler at http://www.treefam.org. Dice Real-Time system TP950 1 Set (TaKaRa Bio). Each assay was performed in triplicate. The specificity of the amplified Molecular cloning products was determined by corresponding melting curve Total RNA was isolated from the embryos at various stages analysis. Primers used for RT-qPCR are listed in Supplementa- using the easy BLUE total RNA extraction Kit (iNtRON Bio, ry Table S1. Korea) according to the manufacturer’s guidelines. cDNA was synthesized using Superscript III reverse transcriptase Statistical analysis (Invitrogen, USA) as described in (Kumar et al., 2017). For All data are presented as mean ± SD. Statistically significant overexpression studies, the open reading frame (ORF) of differences between the two groups were determined using march5 was subcloned into the pcGlobin2 vector between the two-tailed Student’s t-test. One-way ANOVA was used the restriction sites for Xho1 and EcoR1 (Ro et al., 2004). for comparisons among multiple groups. P < 0.05 was con- sidered to indicate a statistically significant difference. Data Morpholino, in vitro transcription, and microinjections analysis were carried out using SPSS 17.0 (IBM, USA). Splicing-blocking morpholinos (E1/I1: 5’TTTGTTTCTTTCACT- TACCTGTCCACG3’) were purchased from Gene-Tools (USA), and dissolved in water. march5-specific morpholinos (0.8 to 1 ng) or control morpholinos were injected into zebrafish embryos at the 1-cell stage. march5 mRNA was synthesized using the Ambion mMESSAGE mMACHINE kit according to Mol. Cells 2020; 43(1): 76-85 77 march5 Regulation of Brain Development in Zebrafish Jangham Jung et al. RESULTS Thus, march5 likely contributes to the development of these regions. march5 is maternally expressed and restricted to the optic vesicles, telencephalon, midbrain, and hindbrain in em­ Ectopic expression and knockdown of march5 cause de­ bryos at 18 hpf velopmental defects in the brain and eyes and dorsoven­ To investigate the contribution of march5 to vertebrate tralization
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