Zuch Daniel T, Bradham Cynthia A
Department of Biology, Boston University, Boston, MA, United States; Program in Molecular Biology, Cell Biology and Biochemistry, Boston University, Boston, MA, United States.
Department of Biology, Boston University, Boston, MA, United States; Program in Molecular Biology, Cell Biology and Biochemistry, Boston University, Boston, MA, United States; Program in Bioinformatics, Boston University, Boston, MA, United States.
Methods Cell Biol. 2019;151:433-442. doi: 10.1016/bs.mcb.2019.01.006. Epub 2019 Feb 18.
During sea urchin embryogenesis, primary mesenchyme cells (PMCs) follow a stereotypical migratory program, arrange into a primary pattern, then begin to secrete a bilaterally symmetric calcium carbonate skeleton. Recently identified genes are expressed in spatially-restricted domains within the PMC population (Sun & Ettensohn, 2014). To better understand the molecular mechanisms orchestrating PMC positioning, we are characterizing the expression profiles of PMC subset-specific genes. To deconvolve the spatiotemporal expression patterns within PMCs, we detect cell-specific mRNA expression with combined RNA fluorescence in situ hybridization and immunolabeling of PMCs. Subsequent confocal microscopy provides 3D position and expression information for individual PMCs. We extract PMC positions and relative gene expression levels, then model these results using open-source 3D modeling software. This versatile protocol can be extended to other models and systems.
在海胆胚胎发育过程中，初级间充质细胞（PMC）遵循一种刻板的迁移程序，排列成初级模式，然后开始分泌双侧对称的碳酸钙骨骼。最近鉴定出的基因在PMC群体内的空间受限区域表达（Sun & Ettensohn，2014）。为了更好地理解协调PMC定位的分子机制，我们正在表征PMC亚群特异性基因的表达谱。为了解析PMC内的时空表达模式，我们通过RNA荧光原位杂交和PMC免疫标记相结合的方法检测细胞特异性mRNA表达。随后的共聚焦显微镜提供了单个PMC的三维位置和表达信息。我们提取PMC位置和相对基因表达水平，然后使用开源三维建模软件对这些结果进行建模。这种通用方案可以扩展到其他模型和系统。
