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Syne-1 and Syne-2 Play Crucial Roles in Myonuclear Anchorage and Motor Neuron Innervation

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Syne-1 and Syne-2 Play Crucial Roles in Myonuclear Anchorage and Motor Neuron Innervation RESEARCH ARTICLE 901 Development 134, 901-908 (2007) doi:10.1242/dev.02783 Syne-1 and Syne-2 play crucial roles in myonuclear anchorage and motor neuron innervation Xiaochang Zhang1, Rener Xu1,*, Binggen Zhu1, Xiujuan Yang2, Xu Ding1, Shumin Duan2, Tian Xu1,3, Yuan Zhuang1,4 and Min Han1,5 Proper nuclear positioning is important to cell function in many biological processes during animal development. In certain cells, the KASH-domain-containing proteins have been shown to be associated with the nuclear envelope, and to be involved in both nuclear anchorage and migration. We investigated the mechanism and function of nuclear anchorage in skeletal muscle cells by generating mice with single and double-disruption of the KASH-domain-containing genes Syne1 (also known as Syne-1) and Syne2 (also known as Syne-2). We showed that the deletion of the KASH domain of Syne-1 abolished the formation of clusters of synaptic nuclei and disrupted the organization of non-synaptic nuclei in skeletal muscle. Further analysis indicated that the loss of synaptic nuclei in Syne-1 KASH-knockout mice significantly affected the innervation sites and caused longer motor nerve branches. Although disruption of neither Syne-1 nor Syne-2 affected viability or fertility, Syne-1; Syne-2 double-knockout mice died of respiratory failure within 20 minutes of birth. These results suggest that the KASH-domain-containing proteins Syne-1 and Syne-2 play crucial roles in anchoring both synaptic and non-synaptic myonuclei that are important for proper motor neuron innervation and respiration. KEY WORDS: Synaptic nuclei, Neuromuscular junction, Neonatal lethality, Nuclear envelope, KASH, SUN domain, Nesprin, ANC-1, MSP-300, Mouse INTRODUCTION Thomson, 1982; Horvitz and Sulston, 1980; Malone et al., 2003; The proper positioning of nuclei in cells is crucial for many biological Starr and Han, 2002; Starr et al., 2001). In Drosophila, the KASH- processes, including fertilization, cell division, cell migration and domain protein Klarsicht has been shown to be important for nuclear other cell functions. Nuclear migration and anchorage have been migration during eye development and for the movement of lipid extensively studied, and both the microtubule and the actin droplets (Mosley-Bishop et al., 1999; Welte et al., 1998). The cytoskeleton systems have been found to play important roles in these Drosophila MSP-300, a homologue of the worm ANC-1 protein in processes (Morris, 2003; Starr and Han, 2003). In recent years, genetic overall structure (Starr and Han, 2002; Volk, 1992; Zhang et al., analyses in several model organisms have shown that the 2002), was also shown to be associated with the nuclear envelope Klarsicht/ANC-1/Syne homologue (KASH)-domain-containing and to play a crucial role in anchoring nurse-cell nuclei during proteins that are associated with the nuclear envelope play important oogenesis (Yu et al., 2006). roles in nuclear positioning during various cellular and developmental Three KASH-domain-containing proteins have been discovered processes (Grady et al., 2005; Malone et al., 2003; Mosley-Bishop et in mammals, namely Syne-1 (also known as Syne1, Myne1, al., 1999; Starr and Han, 2002; Starr et al., 2001; Yu et al., 2006). Nesprin-1, Enaptin165), Syne-2 (also known as Syne2 and Nesprin- The KASH domain is a conserved protein motif of approximately 2; and as NUANCE in humans) and Nesprin-3 (Apel et al., 2000; 60 amino acids that is located at the C-terminus of KASH-family Gough et al., 2003; Mislow et al., 2002; Padmakumar et al., 2004; proteins (Starr and Fischer, 2005). KASH domains have been shown Wilhelmsen et al., 2005; Zhang et al., 2001; Zhen et al., 2002). to bind to nuclear envelope and are likely to be responsible for the Nesprin-3 is a much smaller protein compared to the other two Syne association of the nuclear envelope with KASH proteins (Fischer et proteins. Syne-1 and Syne-2 are orthologs of ANC-1 and MSP-300: al., 2004; Grady et al., 2005; Malone et al., 2003; Starr and Han, all four proteins are very large (>6000 amino acids) and contain 2002; Wilhelmsen et al., 2005; Yu et al., 2006; Zhang et al., 2001; actin-binding domains at their N-terminus, a large middle part and Zhen et al., 2002). In C. elegans, the KASH-domain proteins UNC- a KASH domain at their C-terminus (Starr and Fischer, 2005). The 83, ANC-1 and ZYG-12 have been shown to be associated with the KASH domains of Syne proteins have been shown to target proteins nuclear envelope and to play roles in nuclear migration, nuclear to the nuclear envelope (Apel et al., 2000; Grady et al., 2005; Zhang anchorage of syncytial cells and association of centrosomes with the et al., 2001; Zhen et al., 2002). The structural similarities of Syne nuclear envelope during cell division, respectively (Hedgecock and proteins to ANC-1 and MSP-300 suggest that they may also be involved in nuclear anchorage during important cellular and developmental processes. 1Institute of Developmental Biology and Molecular Medicine, School of Life Science, 2 Syne proteins have been implicated in playing important roles Fudan University, Shanghai, 200433, China. Institute of Neuroscience and Key T Laboratory of Neurobiology, Shanghai Institutes for Biological Sciences, Chinese in nuclear positioning in multinucleated skeletal muscle cells. Academy of Sciences, Shanghai, 200031, China. 3Howard Hughes Medical Institute During early development of the skeletal muscle, hundreds of and Department of Genetics, Yale University School of Medicine, New Haven, CT myoblasts fuse together to form multinucleated myotubes, and 06520, USA. 4Department of Immunology, Duke University Medical Center, Durham, NC 27706, USA. 5Howard Hughes Medical Institute and Department of MCDB, nuclei undergo migration (Englander and Rubin, 1987). Later on, University of Colorado, Boulder, CO 80309, USA. each myotube matures into a large syncytial muscle fiber and nuclei are stably anchored at the periphery of each individual cell *Author for correspondence (e-mail: [email protected]) (Bruusgaard et al., 2003). Noticeably, except for a 3-8 nuclei Accepted 14 December 2006 (synaptic nuclei) cluster under the neuromuscular junction (NMJ), DEVELOPMEN 902 RESEARCH ARTICLE Development 134 (5) myonuclei distribute evenly in muscle fibers (Sanes and protein in skeletal muscle cells. We aim to understand: (1) Are Lichtman, 1999). The evenly spaced localization pattern of non- KASH-domain-containing isoforms of Syne-1 and/or Syne-2 synaptic nuclei is speculated to result from nuclei repelling each essential for the anchorage of synaptic nuclei in skeletal muscle other to minimize the transport distance (Bruusgaard et al., 2003). cells? (2) Do these proteins play a role in anchoring non-synaptic However, the underlying mechanism responsible for nuclear nuclei? (3) Do these two homologous proteins function redundantly? anchorage remains unknown. In addition, synaptic nuclei have and (4) What are the physiological consequences of eliminating the long been proposed to be transcriptionally specialized and activities of either or both proteins? essential in maintaining the postsynaptic components of the NMJ (Sanes and Lichtman, 2001; Schaeffer et al., 2001), but the MATERIALS AND METHODS Syne-1 Syne-2 anchoring mechanism for those nuclei has also been obscure. Generation of and single and double-knockout Both Syne-1 and Syne-2 have been found to be expressed at high mice The gene-targeting vectors for the Syne-1 and Syne-2 KASH domain were levels in the skeletal muscle (Apel et al., 2000; Zhang et al., 2005; constructed from 129 genomic DNA fragments screened out of a BAC Zhang et al., 2001). More recently, a direct involvement of Syne library (Invitrogen). For the Syne-1 construct, the HindIII fragment in Fig. proteins in nuclear positioning has been indicated in the study of 1A was first cloned into pBluescript. The AvrII-HindIII and HindIII-BamHI transgenic mice expressing a dominant-negative form of Syne-1 fragments were then cloned into pPNT at the NotI-XhoI and BamHI sites, (Grady et al., 2005). This study showed that the ectopic respectively. For Syne-2, the HindIII-BamHI fragment was blunt-end ligated expression of the dominant-negative form of Syne-1 disrupted the into the XhoI site of pPNT, while the SpeI-EcoRI genomic fragment was positioning of synaptic nuclei but had no effect on the even cloned into pPNT at XbaI-EcoRI. Each vector was linearized with NotI and spacing of non-synaptic nuclei. However, it remains to be electroporated into embryonic stem (ES) cells. After double selections, determined to what degree this dominant-negative effect reflects positive clones were screened by PCR and Southern blot, which was the function of Syne proteins and which Syne protein is directly followed by injection to obtain chimeric mice. Chimeras and their positive progenies were backcrossed with C57/B6J mice. involved in myonuclear positioning during muscle development. Genomic DNA was extracted and genotyped using a three-primer PCR. To thoroughly understand the cellular and physiological functions Primers for Syne-1 genotyping were prcmy016, prcmy017 and prcmy018 of Syne-1 and Syne-2, we generated mice deficient in the KASH- (sequence details of all primers mentioned in this paper are available upon domain-containing isoforms of both genes. In addition, we also request). Primers for Syne-2 genotyping were prcmy019, prcmy020 and generated transgenic mice that overexpress the Syne-2 KASH prcmy021. Fig. 1. The generation of Syne-1 and Syne-2 KASH-domain-deletion mice. (A,B) Schematic representation of the knockout strategies for Syne-1 and Syne-2. Exons are labeled and coding regions are indicated by black boxes. In the targeting vector, the last exon of Syne-1 (the last two T exons of Syne-2) was replaced by a neomycin-resistance expression cassette (neo). A HSV-TK cassette was linked to the 5Ј end (3Ј end for Syne-2) for negative selection.
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