Role of the extracellular matrix during Wolffian/epididymal duct morphogenesis

细胞外基质在沃尔夫/附睾管形态发生过程中的作用

• 批准号: 10172943
• 负责人: Barry T. Hinton
• 金额: $ 32.84万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10172943
• 关键词: Area; Atomic Force Microscopy; Attention; Behavior; Biomechanics; Cardiomyopathies; Cells; Clinical; Computer software; Congenital Abnormality; Deposition; Development; Duct (organ) structure; Elasticity; Embryo; Embryonic Development; Environment; Epididymis; Epithelial; Epithelial Cells; Event; Extracellular Matrix; Failure; Fetal Development; Genes; Goals; Growth; In Vitro; Lead; Liquid substance; Liver Fibrosis; Male Infertility; Mesenchymal; Mesenchyme; Microscopy; Modulus; Molecular; Morphogenesis; Movement; Mus; Nature; Neural Tube Development; Organ Culture Techniques; Outcome; Outcome Study; Pathologic; Play; Process; Property; Protein Tyrosine Kinase; Radial; Regulation; Reproductive Biology; Role; Sperm Maturation; Structure of mesonephric duct; Testing; Tissues; Tubular formation; cardiogenesis; cell motility; fetal; gastrulation; hearing impairment; human disease; intercalation; interest; male; male fertility; organ growth; real-time images; reproductive tract; second harmonic; tumor progression; two-photon

PROJECT SUMMARY The epididymis provides a unique luminal fluid microenvironment that allows for sperm maturation and survival, and disruptions to this function lead to male infertility. Further, disruptions to epididymal function may also arise as a consequence of abnormal fetal development, although very little is known either of the process of Wolffian duct/epididymal development or of the nature and causes of congenital defects that lead to male infertility. The central hypothesis of this application is that elongation and coiling of the Wolffian duct is crucial for the function of the resulting epididymis, and that failure to elongate and coil leads to male infertility. The overall goal is two-fold, to examine: (a) the mechanisms by which cells move to elongate the duct and (b) to dissect the underlying cellular and molecular mechanisms that regulate those cell movements. Although we have evidence that epithelial cells move by intercalating, we will test the hypothesis that intercalation-type movements of the mesenchyme cells that surround the duct and the en masse movement of mesenchymal cells in the interstitium contribute to ductal elongation and coiling. A combination of genetically modified mice, advanced microscopy including confocal, second harmonic two-photon, and atomic force microscopy, in vitro organ culture and contemporary software analyses will be used to test the hypotheses outlined in the following three specific aims: (1) To test the hypothesis that the stiffness (modulus) of the ECM undergoes dynamic changes during Wolffian duct morphogenesis thereby providing a biomechanical environment that promotes epithelial and mesenchymal cell intercalation and en masse movement of mesenchymal cells within the interstitium. (2) To test the hypothesis that radial intercalation of mesenchymal cells and the en mass movements of mesenchymal cells within the interstitium are major drivers of Wolffian duct elongation and coiling. (3) To test the hypothesis that Ptk7 and Rac1 regulate radial intercalation and en masse movement of mesenchymal cells via regulating ECM biomechanical properties through changes in its deposition and assembly during Wolffian duct development, which in turn are important for male fertility. Therefore, this application focuses on the role of the ECM during Wolffian duct morphogenesis paying special attention to the importance of its assembly, distribution and stiffness, and how this is coordinated to regulate mediolateral and radial intercalation of epithelial and mesenchymal cells respectively, and the en masse movement of mesenchymal cells within the interstitium. Coordination of these events is critical for Wolffian/epididymal duct development, and therefore, male fertility. The anticipated outcomes of this study will not only have a major impact on an area of reproductive biology that has been poorly understood, but will also contribute to our understanding of the fundamental process of tubular morphogenesis. Specifically they will provide an understanding as to how the regulation of growth of the epididymis during development is important clinically.

项目总结 附睾提供了独特的管腔液体微环境，允许精子成熟和存活， 而这一功能的中断会导致男性不育。此外，还可能出现附睾功能中断。 作为胎儿发育异常的结果，尽管对沃尔夫的发育过程知之甚少 导管/附睾管发育或导致男性不育的先天性缺陷的性质和原因。这个 这一应用的中心假设是，沃尔夫管的延长和卷曲对 附睾的功能，不能伸长和卷曲导致男性不育。 总体目标是双重的，以检查：(A)细胞移动以拉长导管的机制和(B) 来剖析调节这些细胞运动的潜在的细胞和分子机制。虽然我们 有证据表明上皮细胞通过插层运动，我们将检验插层类型的假设 胆管周围间充质细胞的运动和间充质的集体运动 间质中的细胞有助于导管的延长和卷曲。转基因小鼠的组合， 高级显微镜，包括共焦显微镜、二次谐波双光子显微镜和原子力显微镜 器官培养和当代软件分析将被用来检验以下概述的假设 三个具体目标：(1)检验ECM的刚度(模数)经历动态变化的假设 在Wolffian管形态发生过程中的变化，从而提供了促进 上皮细胞和间充质细胞的嵌插和间充质细胞的集体运动 间质。(2)验证间充质细胞的放射状嵌入和En团块的假说 间充质细胞在间质内的运动是Wolffian管延长和 盘绕。(3)验证Ptk7和rac1对大鼠径向插层和集体运动的调控作用 间充质细胞通过改变其沉积和表达来调节ECM的生物力学特性 在沃尔夫管发育过程中组装，这反过来对男性生育能力很重要。因此，这 应用的重点是细胞外基质在Wolffian管形态发生中的作用，特别是对 它的组装、分布和硬度的重要性，以及如何协调这一点来调节内侧和外侧 上皮细胞和间充质细胞的放射状镶嵌和集落运动。 间质内的间质细胞。这些事件的协调对Wolffian/附睾管至关重要 发育，因此，男性生育能力。这项研究的预期结果不仅将具有重大的 对生殖生物学领域的影响一直知之甚少，但也将有助于我们的 了解肾小管形态发生的基本过程。具体地说，它们将提供 了解附睾体发育过程中的生长调控在临床上具有重要意义。