Name: live-cell imaging and quantitative analysis of embryonic epithelial cells in xenopus laevis
Uploaded: 2010-05-23T17:00:00
Description: Watch this Scientific Journal Video about Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis at JoVE.com

This work was supported by a National Institutes of Health (R01-HD044750), a CAREER grant from the National Science Foundation, and support from the American Heart Association (Beginning Grant-in-Aid). The authors thank Davidson Lab members: H.Y. Kim, M. von Dassow and J. Zhou (for help with daily lab responsibilities), and Lin Zhang for her assistance in molecular biology and synthesizing mRNA. We acknowledge experimental efforts from all the frog labs (especially Ray Keller's and Doug DeSimone's) who have contributed to some part of this protocol, by developing and testing various methods over the years. We thank Richard Harland and John Wallingford for their kind gifts of mem-gfp and moesin-gfp plasmids respectively.

Before you begin
<ol class="lalpha">
<li>	Synthesize capped mRNA from linearized DNA template encoding the fluorescently tagged protein and purify mRNA by standard methods (AmpliCap Transcription kit; Epicentre Biotechnologies, Madison WI). mRNA should be aliquoted for single time use in 0.2 ml centrifuge tubes and stored at -80&deg;C. </li>
<li>	Standard methods to obtain eggs, in vitro fertilization, and removal of the egg coat have been described previously2. A procedure to obtain eggs from a ...

<ol>
	<li>Ninomiya, H., Winklbauer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+coating+controls+mesenchymal+shape+change+through+tissue-positioning+effects+and+reduction+of+surface-minimizing+tension.">Epithelial coating controls mesenchymal shape change through tissue-positioning effects and reduction of surface-minimizing tension.</a> Nat Cell Biol. 10 (1), 61-61 (2008).</li><li>Kay, B. K., Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xenopus laevis: Practical Uses in Cell and Molecular Biology. , (1991).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+eggs+from+Xenopus+laevis+females.">Obtaining eggs from Xenopus laevis females.</a> J Vis Exp. , (2008).</li><li>Cohen, S., Au, S., Pante, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> J Vis Exp. , (2009).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Tables of Xenopus laevis (Daudin). , (1967).</li><li>Davidson, L. A., Ezin, A. M., Keller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+wound+healing+by+apical+contraction+and+ingression+in+Xenopus+laevis.">Embryonic wound healing by apical contraction and ingression in Xenopus laevis.</a> Cell Motil Cytoskeleton. 53 (3), 163-163 (2002).</li><li>Joshi, S. D., Dassow, M. v. o. n., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+control+of+excitable+embryonic+tissues:+three+stimuli+induce+rapid+epithelial+contraction.">Experimental control of excitable embryonic tissues: three stimuli induce rapid epithelial contraction.</a> Exp Cell Res. 316 (1), 103-103 (2010).</li></ol>

We have presented experimental protocols used regularly in our lab to image dynamically changing cytoskeleton in the apical cell cortex and oscillating cell membranes of epithelial cells in Xenopus embryos. One should use these protocols as a starting point for live-imaging of epithelial cell shapes; optimization of this protocol is essential and some of the settings will change depending on the type of microscope system one has and the type of proteins one is interested in imaging. Together, the protocol steps ...

live-cell imaging and quantitative analysis of embryonic epithelial cells in xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Watch this Scientific Journal Video about Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis at JoVE.com

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Research

JoVE Journal

Biology

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Facial Transplants in Xenopus laevis Embryos

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...