{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:17:27Z","timestamp":1760059047024,"version":"build-2065373602"},"reference-count":38,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2025,5,17]],"date-time":"2025-05-17T00:00:00Z","timestamp":1747440000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key Research and Development Program of China","award":["2022YFE0199800"],"award-info":[{"award-number":["2022YFE0199800"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cleft palate is one of the most common congenital abnormalities and one of the main symptoms of Stickler syndrome. Secondary palate development is a complex multi-step process that involves raising the palatal frame from a vertical to a horizontal position. Lysyl oxidase-like 3 (LOXL3), a member of the lysyl oxidase family responsible for the crosslinking in collagen, is also one of the mutated genes detected in Stickler syndrome. Loss of Loxl3 causes delayed palatal shelf elevation, which in turn resulted in cleft palate. However, the precise mechanisms of palatal shelf delayed elevation remain unclear. In this study, we deeply investigated the mechanism of Loxl3 induced delayed elevation in palatal shelves. We found that Loxl3 deficiency caused reduced cell proliferation in both medial and posterior palatal mesenchyme through BrdU labeling and Western blot analysis (p &lt; 0.05, p &lt; 0.01), decreased migration of palatal mesenchymal cells through cell scratch assay (p &lt; 0.05), and decreased expression of genes associated with proliferation through Western blot analysis (p &lt; 0.05, p &lt; 0.01) at E14. We found that the specific deletion of Loxl3 in the palatal mesenchyme resulted in delayed elevation but normal fusion of palatal shelves, also reduced cell proliferation and collagen fibers deposition in medial palatal mesenchyme through BrdU labeling and histological analysis (p &lt; 0.05, p &lt; 0.01). Thus, our data suggest that Loxl3 regulates cell proliferation and collagen fibers deposition in the palatal mesenchyme, thus controlling palatal shelf elevation.<\/jats:p>","DOI":"10.3390\/ijms26104815","type":"journal-article","created":{"date-parts":[[2025,5,19]],"date-time":"2025-05-19T07:34:03Z","timestamp":1747640043000},"page":"4815","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Loxl3 Affects Palatal Shelf Elevation by Regulating Cell Proliferation and Collagen Deposition"],"prefix":"10.3390","volume":"26","author":[{"given":"Ziyi","family":"Liu","sequence":"first","affiliation":[{"name":"School of Life Science, Shandong University, Qingdao 266237, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fan","family":"Mo","sequence":"additional","affiliation":[{"name":"School of Life Science, Shandong University, Qingdao 266237, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinyu","family":"Dong","sequence":"additional","affiliation":[{"name":"School of Life Science, Shandong University, Qingdao 266237, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ge","family":"Chen","sequence":"additional","affiliation":[{"name":"School of Life Science, Shandong University, Qingdao 266237, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiangang","family":"Gao","sequence":"additional","affiliation":[{"name":"School of Life Science, Shandong University, Qingdao 266237, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6231-0301","authenticated-orcid":false,"given":"Jian","family":"Zhang","sequence":"additional","affiliation":[{"name":"School of Life Science, Shandong University, Qingdao 266237, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1038\/nrg2933","article-title":"Cleft lip and palate: Understanding genetic and environmental influences","volume":"12","author":"Dixon","year":"2011","journal-title":"Nat. Rev. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1016\/j.clp.2018.07.006","article-title":"Cleft Lip and Palate","volume":"45","author":"Worley","year":"2018","journal-title":"Clin. Perinatol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1242\/dev.067082","article-title":"Palatogenesis: Morphogenetic and molecular mechanisms of secondary palate development","volume":"139","author":"Bush","year":"2012","journal-title":"Development"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1465","DOI":"10.1093\/bib\/bbz082","article-title":"Critical microRNAs and regulatory motifs in cleft palate identified by a conserved miRNA-TF-gene network approach in humans and mice","volume":"21","author":"Li","year":"2020","journal-title":"Brief. Bioinform."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1002\/wsbm.1265","article-title":"Signaling networks in palate development","volume":"6","author":"Lane","year":"2014","journal-title":"Wiley Interdiscip. Rev. Syst. Biol. Med."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Nixon, T.R.W., Richards, A.J., Martin, H., Alexander, P., and Snead, M.P. (2022). Autosomal Recessive Stickler Syndrome. Genes, 13.","DOI":"10.3390\/genes13071135"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1007\/s00439-015-1531-z","article-title":"LOXL3, encoding lysyl oxidase-like 3, is mutated in a family with autosomal recessive Stickler syndrome","volume":"134","author":"Alzahrani","year":"2015","journal-title":"Hum. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1111\/cge.13465","article-title":"LOXL3 novel mutation causing a rare form of autosomal recessive Stickler syndrome","volume":"95","author":"Chan","year":"2019","journal-title":"Clin. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"660","DOI":"10.1002\/jcb.10413","article-title":"Lysyl oxidase: Properties, specificity, and biological roles inside and outside of the cell","volume":"88","author":"Kagan","year":"2003","journal-title":"J. Cell Biochem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"6174","DOI":"10.1093\/hmg\/ddv333","article-title":"Loss of lysyl oxidase-like 3 causes cleft palate and spinal deformity in mice","volume":"24","author":"Zhang","year":"2015","journal-title":"Hum. Mol. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"959","DOI":"10.1177\/0022034519851786","article-title":"Regulatory Mechanisms of Soft Palate Development and Malformations","volume":"98","author":"Li","year":"2019","journal-title":"J. Dent. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"4135","DOI":"10.1242\/dev.129.17.4135","article-title":"Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis","volume":"129","author":"Zhang","year":"2002","journal-title":"Development"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5269","DOI":"10.1242\/dev.00708","article-title":"Conditional inactivation of Tgfbr2 in cranial neural crest causes cleft palate and calvaria defects","volume":"130","author":"Ito","year":"2003","journal-title":"Development"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1543","DOI":"10.1002\/ar.24263","article-title":"Extracellular Matrix in Secondary Palate Development","volume":"303","author":"Logan","year":"2020","journal-title":"Anat. Rec."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1080\/15476278.2020.1735239","article-title":"Extracellular Matrix Remodeling During Palate Development","volume":"16","author":"Wang","year":"2020","journal-title":"Organogenesis"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1242\/dev.103.Supplement.41","article-title":"Palate development","volume":"103","author":"Ferguson","year":"1988","journal-title":"Development"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4709","DOI":"10.1242\/dev.099028","article-title":"Pax9 regulates a molecular network involving Bmp4, Fgf10, Shh signaling and the Osr2 transcription factor to control palate morphogenesis","volume":"140","author":"Zhou","year":"2013","journal-title":"Development"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1016\/j.ydbio.2016.10.001","article-title":"Bmp4-Msx1 signaling and Osr2 control tooth organogenesis through antagonistic regulation of secreted Wnt antagonists","volume":"420","author":"Jia","year":"2016","journal-title":"Dev. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1451","DOI":"10.1177\/0022034517719749","article-title":"Identification of Osr2 Transcriptional Target Genes in Palate Development","volume":"96","author":"Fu","year":"2017","journal-title":"J. Dent. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"504","DOI":"10.2174\/1566524014666140414204829","article-title":"Fgf10: A paracrine-signaling molecule in development, disease, and regenerative medicine","volume":"14","author":"Itoh","year":"2014","journal-title":"Curr. Mol. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3207","DOI":"10.1242\/dev.01175","article-title":"Odd-skipped related 2 (Osr2) encodes a key intrinsic regulator of secondary palate growth and morphogenesis","volume":"131","author":"Lan","year":"2004","journal-title":"Development"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Liu, Z., Bai, X., Wan, P., Mo, F., Chen, G., Zhang, J., and Gao, J. (2021). Targeted Deletion of Loxl3 by Col2a1-Cre Leads to Progressive Hearing Loss. Front. Cell Dev. Biol., 9.","DOI":"10.3389\/fcell.2021.683495"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1038\/nn.2467","article-title":"A robust and high-throughput Cre reporting and characterization system for the whole mouse brain","volume":"13","author":"Madisen","year":"2010","journal-title":"Nat. Neurosci."},{"key":"ref_24","unstructured":"Adam, M.P., Mirzaa, G.M., Pagon, R.A., Wallace, S.E., Bean, L.J.H., Gripp, K.W., and Amemiya, A. (2000). Stickler Syndrome. GeneReviews(\u00ae), University of Washington."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"520","DOI":"10.1016\/j.ydbio.2010.12.028","article-title":"Bmpr1a signaling plays critical roles in palatal shelf growth and palatal bone formation","volume":"350","author":"Baek","year":"2011","journal-title":"Dev. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4029","DOI":"10.1242\/dev.050591","article-title":"Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation","volume":"137","author":"Enomoto","year":"2010","journal-title":"Development"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Yoshioka, H., Jun, G., Suzuki, A., and Iwata, J. (2021). Dexamethasone Suppresses Palatal Cell Proliferation through miR-130a-3p. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222212453"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1159\/000337543","article-title":"Hedgehog signalling in development of the secondary palate","volume":"16","author":"Cobourne","year":"2012","journal-title":"Front. Oral. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1692","DOI":"10.1172\/JCI20384","article-title":"Disruption of Fgf10\/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate","volume":"113","author":"Rice","year":"2004","journal-title":"J. Clin. Investig."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1242\/dev.028167","article-title":"Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth","volume":"136","author":"Lan","year":"2009","journal-title":"Development"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Salybekov, A.A., Salybekova, A.K., Pola, R., and Asahara, T. (2018). Sonic Hedgehog Signaling Pathway in Endothelial Progenitor Cell Biology for Vascular Medicine. Int. J. Mol. Sci., 19.","DOI":"10.20944\/preprints201808.0110.v2"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"252","DOI":"10.3109\/13816810.2015.1071409","article-title":"Molecular biology and genetics of embryonic eyelid development","volume":"37","author":"Rubinstein","year":"2016","journal-title":"Ophthalmic Genet."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/S0047-6374(00)00168-8","article-title":"Temporal changes in collagen composition and metabolism during rodent palatogenesis","volume":"119","author":"Mansell","year":"2000","journal-title":"Mech. Ageing Dev."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1201","DOI":"10.1089\/ten.tea.2010.0590","article-title":"Crosslinking and mechanical properties significantly influence cell attachment, proliferation, and migration within collagen glycosaminoglycan scaffolds","volume":"17","author":"Haugh","year":"2011","journal-title":"Tissue Eng. Part A"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1016\/j.bihy.2009.07.009","article-title":"A possible explanation of what\u2019s happening at the moment of palatal shelf elevation","volume":"2","author":"Li","year":"2009","journal-title":"Biosci. Hypotheses"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"965","DOI":"10.1021\/acs.chemrestox.6b00350","article-title":"Computational Model of Secondary Palate Fusion and Disruption","volume":"30","author":"Hutson","year":"2017","journal-title":"Chem. Res. Toxicol."},{"key":"ref_37","first-page":"594","article-title":"Effect of Smo SiRNA-mediated Hedgehog Signaling Pathway Inhibition on Palatal Fusion","volume":"29","author":"Zhou","year":"2016","journal-title":"Biomed. Environ. Sci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.ydbio.2004.09.010","article-title":"The cellular and molecular etiology of the cleft secondary palate in Fgf10 mutant mice","volume":"277","author":"Alappat","year":"2005","journal-title":"Dev. Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/10\/4815\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:34:29Z","timestamp":1760031269000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/10\/4815"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,17]]},"references-count":38,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,5]]}},"alternative-id":["ijms26104815"],"URL":"https:\/\/doi.org\/10.3390\/ijms26104815","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2025,5,17]]}}}