Quantitative imaging of morphogen gradients in drosophila imaginal discs - Kicheva, Anna; Holtzer, Laurent; Wartlick, Ortrud; Schmidt, Thomas; González Gaitán, Marcos (Cold Spring Harbor Protocols, 2013)
Cells at different positions in a developing tissue receive different concentrations of signaling molecules, called morphogens, and this influences their cell fate. Morphogen concentration gradients have been proposed to control patterning as well as growth in many developing tissues. Some outstanding questions about tissue patterning by morphogen gradients are the following: What are the mechanisms that regulate gradient formation and shape? Is the positional information encoded in the gradient sufficiently precise to determine the positions of target gene domain boundaries? What are the temporal dynamics of gradients and how do they relate to patterning and growth? These questions are inherently quantitative in nature and addressing them requires measuring morphogen concentrations in cells, levels of downstream signaling activity, and kinetics of morphogen transport. Here we first present methods for quantifying morphogen gradient shape in which the measurements can be calibrated to reflect actual morphogen concentrations. We then discuss using fluorescence recovery after photobleaching to study the kinetics of morphogen transport at the tissue level. Finally, we present particle tracking as a method to study morphogen intracellular trafficking.
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Keywords: article