Maintaining cell fate relies on robust mechanisms that prevent the differentiation of specified cells into other cell types. This is especially critical during embryogenesis, when extensive cell proliferation, patterning, and migration events take place. Here we show that vertebrate primordial germ cells (PGCs) are protected from reprogramming into other cell types by the RNA-binding protein Dead end (Dnd). PGCs knocked down for Dnd lose their characteristic morphology and adopt various somatic cell fates. Concomitantly, they gain a gene expression profile reflecting differentiation into cells of different germ layers, in a process that we could direct by expression of specific cell-fate determinants. Importantly, we visualized these events within live zebrafish embryos, which provide temporal information regarding cell reprogramming. Our results shed light on the mechanisms controlling germ cell fate maintenance and are relevant for the formation of teratoma, a tumor class composed of cells from more than one germ layer.

BackgroundWhole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting the expression of protein molecules.ResultsThe protocol we have developed and present here is based on the RNAscope technology that is currently employed on formalin-fixed, paraffin-embedded and frozen tissue sections for research and clinical applications. By using zebrafish embryos as an example, we provide a robust and rapid method that allows the simultaneous visualization of multiple transcripts, demonstrated here for three different RNA molecules. The optimized procedure allows the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios. Employing this method thus allows the determination of the spatial expression pattern and subcellular localization of multiple RNA molecules relative to each other at high resolution, in the three-dimensional context of the developing embryo or tissue under investigation. Lastly, we show that this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity, allowing protein detection using antibodies.ConclusionsBy fine-tuning the RNAscope technology, we have successfully redesigned the protocol to be compatible with whole-mount embryo samples. Using this robust method for zebrafish and extending it to other organisms would have a strong impact on research in developmental, molecular and cell biology. Of similar significance would be the adaptation of the method to whole-mount clinical samples. Such a protocol would contribute to biomedical research and clinical diagnostics by providing information regarding the three-dimensional expression pattern of clinical markers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0055-7) contains supplementary material, which is available to authorized users.

BackgroundWhole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting the expression of protein molecules.ResultsThe protocol we have developed and present here is based on the RNAscope technology that is currently employed on formalin-fixed, paraffin-embedded and frozen tissue sections for research and clinical applications. By using zebrafish embryos as an example, we provide a robust and rapid method that allows the simultaneous visualization of multiple transcripts, demonstrated here for three different RNA molecules. The optimized procedure allows the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios. Employing this method thus allows the determination of the spatial expression pattern and subcellular localization of multiple RNA molecules relative to each other at high resolution, in the three-dimensional context of the developing embryo or tissue under investigation. Lastly, we show that this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity, allowing protein detection using antibodies.ConclusionsBy fine-tuning the RNAscope technology, we have successfully redesigned the protocol to be compatible with whole-mount embryo samples. Using this robust method for zebrafish and extending it to other organisms would have a strong impact on research in developmental, molecular and cell biology. Of similar significance would be the adaptation of the method to whole-mount clinical samples. Such a protocol would contribute to biomedical research and clinical diagnostics by providing information regarding the three-dimensional expression pattern of clinical markers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0055-7) contains supplementary material, which is available to authorized users.

The control over the acquisition of cell motility is central for a variety of biological processes in development, homeostasis, and disease. An attractive in vivo model for investigating the regulation of migration initiation is that of primordial germ cells (PGCs) in zebrafish embryos. In this study, we show that, following PGC specification, the cells can polarize but do not migrate before the time chemokine-encoded directional cues are established. We found that the regulator of G-protein signaling 14a protein, whose RNA is a newly identified germ plasm component, regulates the temporal relations between the appearance of the guidance molecules and the acquisition of cellular motility by regulating E-cadherin levels.rgs protein | cell adhesion

Live imaging of mRNA in cells and organisms is important for understanding the dynamic aspects underlying its function.