Lee Jung-Mi, Jung Hunmin, de Paula Machado Pasqua Bruno, Park Yungki, Tang Qinghuang, Jeon Shin, Lee Soo-Kyung, Lee Jae W, Kwon Hyuk-Jae Edward
Department of Oral Biology, School of Dental Medicine, University at Buffalo, The State University of New York, Buffalo, NY 14214, U.S.A.
Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, Institute for Myelin and Glia Exploration, University at Buffalo, The State University of New York, Buffalo, NY 14203, U.S.A.
bioRxiv. 2024 Nov 28:2024.07.16.603832. doi: 10.1101/2024.07.16.603832.
MLL4, also known as KMT2D, is a histone methyltransferase that acts as an important epigenetic regulator in various organogenesis programs. Mutations in the gene are the major cause of Kabuki syndrome, a human developmental disorder that involves craniofacial birth defects, including anomalies in the palate. This study aimed to investigate the role of MLL4 and the underlying mechanisms in the development and growth of the palate. We generated a novel conditional knockout (cKO) mouse model with tissue-specific deletion of in the palatal mesenchyme. Using micro-computed tomography (CT), histological analysis, cell mechanism assays, and gene expression profiling, we examined palate development and growth in the -cKO mice. Gross craniofacial examination at adult stages revealed mild midfacial hypoplasia and midline defects of the palate in -cKO mice, including a widened midpalatal suture and disrupted midline rugae pattern. Micro-CT-based time-course skeletal analysis during postnatal palatogenesis through adulthood demonstrated a transverse growth deficit in overall palate width in -cKO mice. Whole-mount and histological staining at perinatal stages identified that the midline defects in the -cKO mice emerged as early as one day prior to birth, presenting as a widened midpalatal suture, accompanied by increased cell apoptosis in the suture mesenchyme. Genome-wide mRNA expression analysis of the midpalatal suture tissue revealed that MLL4 is essential for the timely expression of major cartilage development genes, such as and , at birth.Immunofluorescence staining for osteochondral differentiation markers demonstrated a marked decrease in the chondrogenic marker COL2A1, while the expression of the osteogenic marker RUNX2 remained unchanged, in the -cKO midpalatal suture. Additionally, SOX9, a master regulator of chondrogenesis, exhibited a significant decrease in protein expression. Indeed, time-course histological analysis during postnatal palate growth revealed retardation in the development of the suture cartilage in -cKO mice. Taken together, our results demonstrate that MLL4 is essential for orchestrating key cellular and molecular events that ensure proper midpalatal suture development and palate growth.
MLL4，也称为KMT2D，是一种组蛋白甲基转移酶，在各种器官发生程序中作为重要的表观遗传调节因子发挥作用。该基因的突变是歌舞伎综合征的主要原因，歌舞伎综合征是一种人类发育障碍，涉及颅面出生缺陷，包括腭裂异常。本研究旨在探讨MLL4在腭部发育和生长中的作用及其潜在机制。我们构建了一种新型条件性敲除（cKO）小鼠模型，该模型在腭间充质中组织特异性缺失MLL4。使用微型计算机断层扫描（CT）、组织学分析、细胞机制分析和基因表达谱分析，我们检测了MLL4基因敲除小鼠的腭部发育和生长情况。成年期的大体颅面检查显示，MLL4基因敲除小鼠存在轻度面中部发育不全和腭部中线缺陷，包括腭中缝增宽和中线嵴纹模式破坏。基于微型CT的从出生后腭部发育到成年期的时间进程骨骼分析表明，MLL4基因敲除小鼠的腭部整体宽度横向生长不足。围产期的整体和组织学染色表明，MLL4基因敲除小鼠的中线缺陷早在出生前一天就出现了，表现为腭中缝增宽，同时缝间质中的细胞凋亡增加。腭中缝组织的全基因组mRNA表达分析表明，MLL4对于出生时主要软骨发育基因（如[此处原文缺失相关基因名称]和[此处原文缺失相关基因名称]）的及时表达至关重要。对骨软骨分化标志物的免疫荧光染色显示，在MLL4基因敲除小鼠的腭中缝中，软骨生成标志物COL2A1显著减少，而成骨标志物RUNX2的表达保持不变。此外，软骨生成的主要调节因子SOX9的蛋白表达也显著降低。事实上，出生后腭部生长过程中的时间进程组织学分析显示，MLL4基因敲除小鼠的缝软骨发育迟缓。综上所述，我们的结果表明，MLL4对于协调关键的细胞和分子事件至关重要，这些事件确保了腭中缝的正常发育和腭部生长。
