Understanding how Connexin43 regulates joint formation in the regenerating zebrafish fin

了解 Connexin43 如何调节再生斑马鱼鳍的关节形成

• 批准号: 10291593
• 负责人: Mary Kathryn Iovine
• 金额: $ 48.3万
• 依托单位: LEHIGH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10291593
• 关键词: Address; Articular Range of Motion; Automobile Driving; Bacterial Artificial Chromosomes; Binding; Biological Models; Cell Fate Control; Cell Nucleus; Cells; Connexin 43; Connexins; Data; Degenerative polyarthritis; Development; Disease; Enhancers; Event; Felis catus; Gap Junctions; Gene Expression; Genetic Transcription; Goals; Injury; Joints; Knowledge; Lateral; Length; Location; Mechanics; Mediating; Membrane; Membrane Potentials; Mesenchyme; Molecular; Molecular Target; Natural regeneration; Nature; Nuclear; Osteoblasts; Outcome; Pathway interactions; Pattern; Play; Quality of life; Regulation; Regulatory Element; Regulatory Pathway; Reporter; Research; Rheumatoid Arthritis; Role; Signal Transduction; Skeletal Development; Skeleton; Source; Specific qualifier value; Synovial joint; Testing; Time; Zebrafish; arthropathies; bioelectricity; design; flexibility; insight; intercellular communication; joint formation; joint function; novel; novel therapeutics; precursor cell; premature; regenerative; skeletal; spatiotemporal; transcription factor

Summary Synovial joints are critical for skeletal form and function, but are susceptible to debilitating diseases such as osteoarthritis and injury. While much is known about the composition and mechanics of functioning joints, relatively little is known about how joint-forming cells (JFCs) are specified in a particular location, thereby permitting the development of joints that provide flexibility. Addressing this gap in knowledge could facilitate the development of novel therapies for joint disease by targeting molecular pathways influencing cell fate decisions. The zebrafish regenerating fin is an important model system for addressing fundamental questions of skeletal development, including the specification and commitment of JFCs. The fin skeleton is made of bony fin rays, and each fin ray is made of bony segments separated by joints. Thus, the fin is a rich source of joints, which are produced regularly during typical outgrowth and during regeneration. The osteoblasts and JFCs that build the fin skeleton are derived from a common skeletal precursor cell (SPC) located in the lateral fin ray mesenchyme. Recent studies from the Iovine lab provide strong evidence that Connexin43 (Cx43), via gap junctional intercellular communication (GJIC), influences JFC specification by suppressing expression of evx1. Evx1 is a transcription factor required for joint formation. The overall objectives for this proposal are to reveal the nature of Cx43-GJIC, and to reveal the subsequent mechanism of evx1 suppression. The central hypothesis of this proposal is that Cx43-GJIC influences joint formation by transducing changes in membrane potential to the SPCs; changes in membrane potential in turn trigger changes in gene expression that influence evx1 expression, JFC specification, and joint formation. This hypothesis was formulated on the basis of prior and preliminary data. For example, Cx43-GJIC promotes simplet (smp) expression in SPCs. Smp is required to bring -catenin to the nucleus. In concert with Lef1 or TCF7 transcription factors, -catenin (directly or indirectly) suppresses evx1 transcription. The Aims of this proposal are designed to test the central hypothesis. Aim 1 is to test if changes in membrane resting potential are sufficient to influence gene expression in SPCs. This will be accomplished by driving membrane hyperpolarization or depolarization in Cx43-expressing cells, and evaluating smp and evx1 expression in SPCs. Aim 2 is to identify cis-regulatory elements (CREs) that influence evx1 transcription. Putative CREs will be deleted from a BAC reporter for evx1, and impacts on reporter expression will be assessed to reveal likely enhancers or silencers acting on evx1. Completion of these Aims will significantly advance our understanding of signals mediated by GJIC influencing cell specification, and will further provide novel insights into the spatiotemporal regulation of JFC specification.

总结 滑膜关节对于骨骼的形态和功能至关重要，但易患使人衰弱的疾病，如 骨关节炎和损伤。虽然我们对功能关节的组成和力学有很多了解， 关于如何在特定位置指定关节形成细胞（JFC）， 允许形成提供柔韧性的关节。解决这一知识差距可以促进 通过靶向影响细胞命运的分子途径开发关节疾病的新疗法 决策斑马鱼再生鳍是解决基本问题的重要模型系统 包括JFC的规格和承诺。鳍的骨架是由多骨 鳍条，每个鳍条由关节分开的骨段组成。因此，鳍是关节的丰富来源， 其在典型的生长和再生过程中有规律地产生。成骨细胞和JFC， 建立鳍骨架来自一个共同的骨骼前体细胞（SPC）位于侧鳍射线 间充质Iovine实验室最近的研究提供了强有力的证据，表明连接蛋白43（Cx43），通过间隙 细胞间连接通讯（GJIC）通过抑制evx 1的表达影响JFC的特化。 Evx 1是关节形成所需的转录因子。该提案的总体目标是揭示 Cx43-GJIC的性质，并揭示evx 1抑制的后续机制。中央 这一建议的假设是Cx43-GJIC通过转导 膜电位的变化反过来触发基因的变化， 表达，影响evx 1表达，JFC规范和联合形成。了该假设 根据先前和初步数据制定。例如，Cx43-GJIC促进simlet（smp） 在SPC中的表达。需要Smp将β-连环蛋白带到细胞核。与Lef 1或TCF 7配合使用 在转录因子中，β-连环蛋白（直接或间接）抑制evx 1转录。本提案的目的 是为了检验中心假设目的1是测试膜静息电位的变化是否 足以影响SPC中的基因表达。这将通过驱动膜 在表达Cx43的细胞中的超极化或去极化，以及在表达Cx43的细胞中评估smp和evx 1表达。 SPC。目的2是确定影响evx 1转录的顺式调控元件（克雷斯）。假定的克雷斯将 从evx 1的BAC报告基因中删除，并评估对报告基因表达的影响，以揭示可能的 作用于EVX 1的增强剂或沉默剂。这些目标的实现将极大地促进我们的理解 的信号介导的GJIC影响细胞规格，并将进一步提供新的见解， JFC规范时空调节。

项目成果

Cx43 suppresses evx1 expression to regulate joint initiation in the regenerating fin.

• DOI: 10.1002/dvdy.24531
• 发表时间: 2017-09
• 期刊: Developmental dynamics : an official publication of the American Association of Anatomists
• 作者: Dardis G;Tryon R;Ton Q;Johnson SL;Iovine MK
• 通讯作者: Iovine MK

Cohesin: an emerging master regulator at the heart of cardiac development.

• DOI: 10.1091/mbc.e22-12-0557
• 发表时间: 2023-05-01
• 期刊: MOLECULAR BIOLOGY OF THE CELL
• 影响因子: 3.3
• 作者: Mfarej, Michael G.;Hyland, Caitlin A.;Sanchez, Annie C.;Falk, Matthias M.;Lovine, M. Kathryn;V. Skibbens, Robert
• 通讯作者: V. Skibbens, Robert

Impaired Cx43 gap junction endocytosis causes morphological and functional defects in zebrafish.

• DOI: 10.1091/mbc.e20-12-0797
• 发表时间: 2021-10-01
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Hyland C;Mfarej M;Hiotis G;Lancaster S;Novak N;Iovine MK;Falk MM
• 通讯作者: Falk MM