a-simple-and-effective-transplantation-device-for-zebrafish-embryos

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

Friedrich Miescher Laboratory of the Max Planck Society

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.

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

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.

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

Morphogens are signaling molecules that provide positional information to cells during development. They must move through embryonic tissues in order to coordinate patterning. The rate of a morphogen's movement through a tissue-its effective diffusivity-affects the morphogen's distribution and therefore influences patterning. Fluorescence recovery after photobleaching (FRAP) is a powerful method to measure the effective diffusion of molecules through cells and tissues, and has been successfully employed to examine morphogen mobility and gain important insights into embryogenesis. Here, we provide detailed protocols for FRAP assays in vitro and in living zebrafish embryos, and we explain how to analyze FRAP data using the open-source software PyFRAP to determine effective diffusion coefficients.

Article	Year
Dynamics of BMP signaling and distribution during zebrafish dorsal-ventral patterning. eLife\| PubMed ID: 28857744	2017
Scale-invariant patterning by size-dependent inhibition of Nodal signalling. Nature cell biology\| PubMed ID: 30061678	2018
FRAP Analysis of Extracellular Diffusion in Zebrafish Embryos. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30324594	2018

Gary H. Soh

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