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.

概括 滑膜关节对于骨骼形式和功能至关重要，但易受使人衰弱的疾病（例如 骨关节炎和损伤。虽然对功能关节的组成和力学知之甚少，但 关于如何在特定位置指定联合形成单元（JFC）的知之甚少 允许开发提供灵活性的关节。解决这一知识的差距可以促进 通过靶向分子途径来开发新的关节疾病疗法 决定。斑马鱼再生鳍是解决基本问题的重要模型系统 骨骼发育，包括JFC的规范和承诺。鳍骨架是由奖励制成的 鳍射线和每个鳍片均由由关节隔开的骨段制成。那是鳍是一个丰富的关节来源， 这些是在典型的产物和再生过程中定期生产的。成骨细胞和JFC 构建鳍骨架源自位于侧鳍射线中的常见骨骼前体细胞（SPC） 间充质。 Iovine Lab的最新研究提供了有力的证据，表明Connexin43（CX43）通过GAP 连接间交流（GJIC）通过抑制EVX1的表达来影响JFC规范。 EVX1是关节形成所需的转录因子。该提议的总体目标是揭示 CX43-GJIC的性质，并揭示了EVX1抑制的后续机制。中央 该提议的假设是CX43-GJIC通过转导变化来影响关节形成 SPC的膜潜力；膜电位的变化反过来触发基因的变化 影响EVX1表达，JFC规范和关节形成的表达。这个假设是 例如，CX43-GJIC促进Simplet（SMP） 在SPC中的表达。需要SMP将-catenin带到核。与LEF1或TCF7协同 转录因子-catenin（直接或间接）抑制EVX1转录。该提议的目的 AIM 1是测试是否设计了膜静止电位的变化来检验中心假设。 足以影响SPC中的基因表达。这将通过驾驶膜来完成 表达CX43的细胞中的超极化或沉积，并评估SMP和EVX1的表达 SPC。目标2是确定影响EVX1转录的顺式调节元件（CRE）。假定的cr板 将从BAC记者中删除EVX1，并将评估对记者表达的影响，以揭示可能 作用于EVX1的增强剂或消音器。这些目标的完成将大大提高我们的理解 GJIC介导的信号影响细胞规范，并将进一步提供新的见解 JFC规范的时空调节。

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

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

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