PyFDAP: automated analysis of fluorescence decay after photoconversion (FDAP) experiments.

UNLABELLED: We developed the graphical user interface PyFDAP for the fitting of linear and non-linear decay functions to data from fluorescence decay after photoconversion (FDAP) experiments. PyFDAP structures and analyses large FDAP datasets and features multiple fitting and plotting options. AVAILABILITY AND IMPLEMENTATION: PyFDAP was written in Python and runs on Ubuntu Linux, Mac OS X and Microsoft Windows operating systems. The software, a user guide and a test FDAP dataset are freely available for download from http://people.tuebingen.mpg.de/mueller-lab.

Quantitative diffusion measurements using the open-source software PyFRAP.

Fluorescence Recovery After Photobleaching (FRAP) and inverse FRAP (iFRAP) assays can be used to assess the mobility of fluorescent molecules. These assays measure diffusion by monitoring the return of fluorescence in bleached regions (FRAP), or the dissipation of fluorescence from photoconverted regions (iFRAP). However, current FRAP/iFRAP analysis methods suffer from simplified assumptions about sample geometry, bleaching/photoconversion inhomogeneities, and the underlying reaction-diffusion kinetics. To address these shortcomings, we developed the software PyFRAP, which fits numerical simulations of three-dimensional models to FRAP/iFRAP data and accounts for bleaching/photoconversion inhomogeneities. Using PyFRAP we determined the diffusivities of fluorescent molecules spanning two orders of magnitude in molecular weight. We measured the tortuous effects that cell-like obstacles exert on effective diffusivity and show that reaction kinetics can be accounted for by model selection. These applications demonstrate the utility of PyFRAP, which can be widely adapted as a new extensible standard for FRAP analysis.

Scale-invariant patterning by size-dependent inhibition of Nodal signalling.

Individuals can vary substantially in size, but the proportions of their body plans are often maintained. We generated smaller zebrafish by removing 30% of their cells at the blastula stages and found that these embryos developed into normally patterned individuals. Strikingly, the proportions of all germ layers adjusted to the new embryo size within 2 hours after cell removal. As Nodal-Lefty signalling controls germ-layer patterning, we performed a computational screen for scale-invariant models of this activator-inhibitor system. This analysis predicted that the concentration of the highly diffusive inhibitor Lefty increases in smaller embryos, leading to a decreased Nodal activity range and contracted germ-layer dimensions. In vivo studies confirmed that Lefty concentration increased in smaller embryos, and embryos with reduced Lefty levels or with diffusion-hindered Lefty failed to scale their tissue proportions. These results reveal that size-dependent inhibition of Nodal signalling allows scale-invariant patterning.

Dynamics of BMP signaling and distribution during zebrafish dorsal-ventral patterning.

During vertebrate embryogenesis, dorsal-ventral patterning is controlled by the BMP/Chordin activator/inhibitor system. BMP induces ventral fates, whereas Chordin inhibits BMP signaling on the dorsal side. Several theories can explain how the distributions of BMP and Chordin are regulated to achieve patterning, but the assumptions regarding activator/inhibitor diffusion and stability differ between models. Notably, 'shuttling' models in which the BMP distribution is modulated by a Chordin-mediated increase in BMP diffusivity have gained recent prominence. Here, we directly test five major models by measuring the biophysical properties of fluorescently tagged BMP2b and Chordin in zebrafish embryos. We found that BMP2b and Chordin diffuse and rapidly form extracellular protein gradients, Chordin does not modulate the diffusivity or distribution of BMP2b, and Chordin is not required to establish peak levels of BMP signaling. Our findings challenge current self-regulating reaction-diffusion and shuttling models and provide support for a graded source-sink mechanism underlying zebrafish dorsal-ventral patterning.

Measuring protein stability in living zebrafish embryos using fluorescence decay after photoconversion (FDAP).

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.