Zinski Joseph, Tuazon Francesca, Huang Yan, Mullins Mary, Umulis David
Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN, USA.
Methods Mol Biol. 2019;1891:135-154. doi: 10.1007/978-1-4939-8904-1_10.
Spatiotemporal patterns of morphogen activity drive differential gene expression with a high degree of precision within a developing embryo and reproducibly between embryos. Understanding the formation and function of a morphogen gradient during development requires quantitative measurement of morphogen activity throughout an individual embryo and also between embryos within a population. Quantification of morphogen gradients in to presents unique challenges in imaging and image processing to minimize error and maximize the quality of the data so it may be used in computational models of development and in statistically testing hypotheses. Here we present methods for the preparation, immunostaining, imaging, and quantification of a bone morphogenetic protein (BMP) activity gradient in individual zebrafish embryos as well as methods for acquiring population-level statistics after embryo grouping and alignment. This quantitative approach can be extended to other morphogen systems, and the computational codes can be adapted to other imaging contexts in zebrafish and other organisms.
形态发生素活性的时空模式在发育中的胚胎内以高度的精确性驱动差异基因表达，并且在不同胚胎之间具有可重复性。了解发育过程中形态发生素梯度的形成和功能需要对单个胚胎以及群体内不同胚胎之间的形态发生素活性进行定量测量。对形态发生素梯度进行定量在成像和图像处理方面提出了独特的挑战，即要尽量减少误差并最大化数据质量，以便其可用于发育的计算模型和统计假设检验。在这里，我们展示了在单个斑马鱼胚胎中制备、免疫染色、成像和定量骨形态发生蛋白（BMP）活性梯度的方法，以及在胚胎分组和对齐后获取群体水平统计数据的方法。这种定量方法可以扩展到其他形态发生素系统，并且计算代码可以适用于斑马鱼和其他生物体的其他成像情况。
