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Measuring Dorsoventral Pattern and Morphogen Signaling Profiles in the Growing Neural Tube

  • Marcin Zagorski
  • Anna Kicheva
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1863)

Abstract

Developmental processes are inherently dynamic and understanding them requires quantitative measurements of gene and protein expression levels in space and time. While live imaging is a powerful approach for obtaining such data, it is still a challenge to apply it over long periods of time to large tissues, such as the embryonic spinal cord in mouse and chick. Nevertheless, dynamics of gene expression and signaling activity patterns in this organ can be studied by collecting tissue sections at different developmental stages. In combination with immunohistochemistry, this allows for measuring the levels of multiple developmental regulators in a quantitative manner with high spatiotemporal resolution. The mean protein expression levels over time, as well as embryo-to-embryo variability can be analyzed. A key aspect of the approach is the ability to compare protein levels across different samples. This requires a number of considerations in sample preparation, imaging and data analysis. Here we present a protocol for obtaining time course data of dorsoventral expression patterns from mouse and chick neural tube in the first 3 days of neural tube development. The described workflow starts from embryo dissection and ends with a processed dataset. Software scripts for data analysis are included. The protocol is adaptable and instructions that allow the user to modify different steps are provided. Thus, the procedure can be altered for analysis of time-lapse images and applied to systems other than the neural tube.

Key words

Neural tube Spinal cord Tissue development Morphogen gradient Pattern formation Quantitative imaging 

Notes

Acknowledgments

We thank J. Briscoe and T. Bollenbach for comments on the manuscript. Funding: IST Austria and European Research Council under European Union’s Horizon 2020 research and innovation programme (680037) (MZ, AK).

Supplementary material

427781_1_En_4_MOESM1_ESM.ijm (1 kb)
Supplementary File 1 Fiji script “maximum_projections.ijm”. (IJM 1 KB)
427781_1_En_4_MOESM2_ESM.ijm (4 kb)
Supplementary File 2 Fiji script “profile_quantification.ijm”. (IJM 4 KB)
427781_1_En_4_MOESM3_ESM.m (22 kb)
Supplementary File 3 Matlab script “data_analysis.m”. (M 22 KB)
427781_1_En_4_MOESM4_ESM.zip (64.5 mb)
Supplementary File 4 Test dataset: folders “Images” and “Profiles_FI”. (ZIP 69256 KB)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Science and Technology Austria (IST Austria)KlosterneuburgAustria

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