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Adult subsynaptic myonuclei beneath the neuromuscular junction (NMJ) express specific genes, the products of which coordinately function in the maintenance of the pre\u2010 and post\u2010synaptic regions. However, the gene expression profiles that promote the NMJ formation during embryogenesis remain largely unexplored. We performed single\u2010nucleus RNA sequencing (snRNA\u2010seq) analysis of embryonic and neonatal mouse diaphragms, and found that each myonucleus had a distinct transcriptome pattern during the NMJ formation. Among the previously reported NMJ\u2010constituting genes,\n                    <jats:italic>Dok7<\/jats:italic>\n                    ,\n                    <jats:italic>Chrna1<\/jats:italic>\n                    , and\n                    <jats:italic>Chrnd<\/jats:italic>\n                    are specifically expressed in subsynaptic myonuclei at E18.5. In the E18.5 diaphragm, ca. 10.7% of the myonuclei express genes for the NMJ formation (\n                    <jats:italic>Dok7<\/jats:italic>\n                    ,\n                    <jats:italic>Chrna1<\/jats:italic>\n                    , and\n                    <jats:italic>Chrnd<\/jats:italic>\n                    ) together with four representative \u03b2\u2010catenin regulators (\n                    <jats:italic>Amotl2<\/jats:italic>\n                    ,\n                    <jats:italic>Ptprk, Fam53b<\/jats:italic>\n                    , and\n                    <jats:italic>Tcf7l2<\/jats:italic>\n                    ). Additionally, the temporal gene expression patterns of these seven genes are synchronized in differentiating C2C12 myoblasts. Amotl2 and Ptprk are expressed in the sarcoplasm, where \u03b2\u2010catenin serves as a structural protein to organize the membrane\u2010anchored NMJ structure. In contrast, Fam53b and Tcf7l2 are expressed in the myonucleus, where \u03b2\u2010catenin serves as a transcriptional coactivator in Wnt\/\u03b2\u2010catenin signaling at the NMJ. In C2C12 myotubes, knockdown of\n                    <jats:italic>Amotl2<\/jats:italic>\n                    or\n                    <jats:italic>Ptprk<\/jats:italic>\n                    markedly, and that of\n                    <jats:italic>Fam53b<\/jats:italic>\n                    and\n                    <jats:italic>Tcf7l2<\/jats:italic>\n                    less efficiently, impair the clustering of acetylcholine receptors. In contrast, knockdown of\n                    <jats:italic>Fam53b<\/jats:italic>\n                    and\n                    <jats:italic>Tcf7l2<\/jats:italic>\n                    , but not of\n                    <jats:italic>Amotl2<\/jats:italic>\n                    or\n                    <jats:italic>Ptprk<\/jats:italic>\n                    , impairs the gene expression of\n                    <jats:italic>Slit2<\/jats:italic>\n                    encoding an axonal attractant for motor neurons, which is required for the maturation of motor nerve terminal. Thus, Amotl2 and Ptprk exert different roles at the NM compared to Fam53b and Tcf7l2. Additionally, Wnt ligands originating from the spinal motor neurons and the perichondrium\/chondrocyte are likely to work remotely on the subsynaptic nuclei and the myotendinous junctional nuclei, respectively. We conclude that snRNA\u2010seq analysis of embryonic\/neonatal diaphragms reveal a novel coordinated expression profile especially in the Wnt\/\u03b2\u2010catenin signaling that regulate the formation of the embryonic NMJ.\n                    <jats:boxed-text content-type=\"graphic\" position=\"anchor\">\n                      <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" mimetype=\"image\/png\" position=\"anchor\" specific-use=\"enlarged-web-image\" xlink:href=\"graphic\/jnc16013-fig-0004-m.png\">\n                        <jats:alt-text>image<\/jats:alt-text>\n                      <\/jats:graphic>\n                    <\/jats:boxed-text>\n                  <\/jats:p>","DOI":"10.1111\/jnc.16013","type":"journal-article","created":{"date-parts":[[2023,11,24]],"date-time":"2023-11-24T19:14:07Z","timestamp":1700853247000},"page":"342-354","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Transcriptome profile of subsynaptic myonuclei at the neuromuscular junction in embryogenesis"],"prefix":"10.1111","volume":"168","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1987-458X","authenticated-orcid":false,"given":"Bisei","family":"Ohkawara","sequence":"first","affiliation":[{"name":"Division of Neurogenetics, Center for Neurological Diseases and Cancer Nagoya University Graduate School of Medicine  Nagoya Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masaomi","family":"Kurokawa","sequence":"additional","affiliation":[{"name":"Department of Informatics National Institute of Genetics  Shizuoka Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akinori","family":"Kanai","sequence":"additional","affiliation":[{"name":"Department of Computational Biology and Medical Sciences Graduate School of Frontier Sciences, The University of Tokyo  Chiba Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kiyomi","family":"Imamura","sequence":"additional","affiliation":[{"name":"Department of Computational Biology and Medical Sciences Graduate School of Frontier Sciences, The University of Tokyo  Chiba Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guiying","family":"Chen","sequence":"additional","affiliation":[{"name":"Division of Neurogenetics, Center for Neurological Diseases and Cancer Nagoya University Graduate School of Medicine  Nagoya Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruchen","family":"Zhang","sequence":"additional","affiliation":[{"name":"Division of Neurogenetics, Center for Neurological Diseases and Cancer Nagoya University Graduate School of Medicine  Nagoya Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akio","family":"Masuda","sequence":"additional","affiliation":[{"name":"Division of Neurogenetics, Center for Neurological Diseases and Cancer Nagoya University Graduate School of Medicine  Nagoya Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koichi","family":"Higashi","sequence":"additional","affiliation":[{"name":"Department of Informatics National Institute of Genetics  Shizuoka Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Mori","sequence":"additional","affiliation":[{"name":"Department of Informatics National Institute of Genetics  Shizuoka Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yutaka","family":"Suzuki","sequence":"additional","affiliation":[{"name":"Department of Computational Biology and Medical Sciences Graduate School of Frontier Sciences, The University of Tokyo  Chiba Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ken","family":"Kurokawa","sequence":"additional","affiliation":[{"name":"Department of Informatics National Institute of Genetics  Shizuoka Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kinji","family":"Ohno","sequence":"additional","affiliation":[{"name":"Division of Neurogenetics, Center for Neurological Diseases and Cancer Nagoya University Graduate School of Medicine  Nagoya Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,11,23]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1177\/1535370217749494"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1002\/dneu.22190"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.stem.2015.11.002"},{"issue":"826","key":"e_1_2_10_5_1","article-title":"SnapShot: Neuromuscular junction","volume":"144","author":"Burden S. 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