| Popis: |
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 (SunEttensohn, 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. |
