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Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis

Nature Protocols volume 4, pages 1397–1412 (2009)Cite this article

Abstract

This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multi-photon microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and programming that is appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without experimental data to carry out the analysis. The protocol has offered new insights into the genetic control of cell migration during Drosophila gastrulation. With simple modifications, this systematic analysis could be applied to any developing system to define cell positions in accordance with the body plan, to decompose complex 3D movements and to quantify the collective nature of cell migration.

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Figure 1: Experimental work flow describing the five main parts of the protocol.
Figure 2: Optical properties of the mesoderm in early Drosophila and zebrafish embryos.
Figure 3: Typical image processing results with Imaris.
Figure 4: Cylinder fit on the spatial distribution of ectoderm cell positions obtained with EctodermCylinderFit.m script (Step 25).
Figure 5: Spatial and temporal registration (Steps 26 and 27).
Figure 6: Decomposition of mesoderm cell movements into their cylindrical components using MovementDecomp.m (Step 28A).
Figure 7: Analysis of mesoderm cell spreading using SpreadingAnalysis.m (Step 28B).

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Acknowledgements

We thank M. Liebling for advice on Imaris and Matlab, and the Caltech Biological Imaging Center for sharing equipment. This work was supported by grants to AS from National Institutes of Health (R01 GM078542), the Searle Scholars Program and the March of Dimes (Basil O'Conner Starter Scholar Award, 5-FY06-145), grants to SF from the Caltech Beckman Institute and National Institutes of Health (Center for Excellence in Genomic Science grant P50HG004071), and fellowship to W.S. from the Caltech Biology Division.

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Author notes

  1. Willy Supatto & Scott E Fraser

    Present address: Present address: Institut Jacques Monod, CNRS UMR 7592, Université Paris 7, Paris, France.,

Authors and Affiliations

  1. Division of Biology and Beckman Institute, California Institute of Technology, Pasadena, California, USA

    Willy Supatto, Amy McMahon, Scott E Fraser & Angelike Stathopoulos

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  1. Willy Supatto
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Contributions

W.S. designed the experimental workflow and the computational analysis. W.S. and A.M. acquired and processed the data. W.S., A.M., S.E.F. and A.S. wrote the paper.

Note: Supplementary information is available via the HTML version of this article.

Corresponding authors

Correspondence to Willy Supatto or Angelike Stathopoulos.

Supplementary information

Supplementary Data 1. Matlab scripts.

See Table 2 for details. (ZIP 15 kb)

Supplementary Data 2. Sample datasets.

Mesoderm, ectoderm, and midline tracking sample datasets are included. (ZIP 801 kb)

Supplementary Movie 1.

3D image of an entire klarsicht;nls-GFP embryo at the onset of mesoderm cell spreading. This movie shows the good signal obtained from the mesoderm furrow. The nls-GFP signal is diffuse during cell division (see the anterior pole). This dataset has been acquired in slightly different conditions compared to the rest of this protocol to image the embryo with a larger field of view using a 20x 0.95NA objective from Olympus. The nls-GFP Drosophila embryo (Bloomington Stock Center, stock number 5623) has been imaged on a TriMScope 2PEF setup provided LaVision BioTech company. (MOV 2754 kb)

Supplementary Movie 2.

3D schematic animation of quantitative imaging of Drosophila embryos. This movie successively shows (i) the Drosophila embryo shape and body plan with the position of the midline; (ii) the position of the image acquisition field of view on the ventral side of the embryo; (iii) the position and shape of the ectoderm and mesoderm layers at the onset of mesoderm spreading, with a representation of the ventral furrow; (iv) a 3D view of the cylindrical coordinate system adapted to the shape of the embryo in the imaging area; (v) a 3D representation of morphogenetic movements showing the ectoderm convergence-extension during germband extension (GBE), the mesoderm furrow collapse and the mesoderm spreading. (MOV 6389 kb)

Supplementary Movie 3.

Example of angular drift during image acquisition. View of the ectoderm layer from the ventral side in H2A-GFP expressing embryos. Arrow indicates the position of midline cells and highlights the angular drift. It corresponds to the drift corrected in Fig. 5. The specific shape of the nuclei from midline cells (elongated along the anterior-posterior axis) is clear. Anterior-posterior axis is vertical, posterior is up. (MOV 3432 kb)

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Supatto, W., McMahon, A., Fraser, S. et al. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis. Nat Protoc 4, 1397–1412 (2009). https://doi.org/10.1038/nprot.2009.130

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