Imaging and Quantification of P-Smad1/5 in Zebrafish Blastula and Gastrula Embryos.

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.