analyzing-vivo-cell-migration-using-cell-transplantations-time-lapse

Analyzing <em>In Vivo</em> Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

deep-spatially-controlled-volume-ablations-using-two-photon

Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly ...

IBENS, Institut de Biologie de l'École Normale Supérieure

CNRS UMR7645, INSERM U1182, Institut Polytechnique de Paris

Germ-layer formation during gastrulation is both a fundamental step of development and a paradigm for tissue formation and remodeling. However, the cellular and molecular basis of germ-layer segregation is poorly understood, mostly because of the lack of direct in vivo observations. We used mosaic zebrafish embryos to investigate the formation of the endoderm. High-resolution live imaging and functional analyses revealed that endodermal cells reach their characteristic innermost position through an active, oriented, and actin-based migration dependent on Rac1, which contrasts with the previously proposed differential adhesion cell sorting. Rather than being attracted to their destination, the yolk syncytial layer, cells appear to migrate away from their neighbors. This migration depends on N-cadherin that, when imposed in ectodermal cells, is sufficient to trigger their internalization without affecting their fate. Overall, these results lead to a model of germ-layer formation in which, upon N-cadherin expression, endodermal cells actively migrate away from their epiblastic neighbors to reach their internal position, revealing cell-contact avoidance as an unexplored mechanism driving germ-layer formation.

Endodermal germ-layer formation through active actin-driven migration triggered by N-cadherin.

Being optically clear, the zebrafish embryo is a nice model system to analyze cell migration in vivo. This chapter describes a combination of injection and cell transplant procedures that allows creation of mosaic embryos, containing a few cells labeled differently from their neighbors. Rapid 5D confocal imaging of these embryos permits to simultaneously track and quantify the movement of large cell groups, as well as analyze the cellular or subcellular dynamics of transplanted cells during their migration. In addition, expression of a candidate gene can be modified in transplanted cells. Comparing behavior of these cells to control or neighboring cells allows determination of the role of the candidate gene in cell migration. We describe the procedure, focusing on one specific cell population during gastrulation, but it can easily be adapted to other cell populations and other migration events during early embryogenesis.

Nicolas B. David

CNRS UMR8197 – INSERM U1024

IBENS, Institut de Biologie de l'École Normale Supérieure

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

Nicolas B. David

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}CNRS UMR8197 – INSERM U1024

IBENS, Institut de Biologie de l'École Normale Supérieure

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

CNRS UMR8197 – INSERM U1024