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.

Cell migration is essential for morphogenesis, for organ formation and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model to study this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility and with the ability of the cells to reach their target.

Cellular plasticity is now recognized as a fundamental feature of tissue biology. The steady-state differentiation of stem and progenitor cells into mature cells is, in itself, the index form of cellular plasticity in adult organisms. Following injury, when it is critical to quickly regenerate and restore tissue integrity and function, other types of cellular plasticity may be crucial for organismal survival. In these contexts, alterations in the epigenetic landscape of tissues are likely to occur in order to allow normally restricted cell fate transitions. Epigenetic mechanisms, particularly DNA methylation and histone modifications, have been shown to play an important role in regulating such plasticity. Relevant mechanisms have been well studied in the context of the direct reprograming of somatic cells into induced pluripotent stem cells. Indeed, epigenetic regulation of cell fate is part and parcel of normal embryonic development and is a central regulator of cellular diversity. This is normally thought to involve the establishment of divergent chromatin patterns that culminate in cells with distinct and what were previously thought to be irreversible fates. This brief review aims to put some of these new observations in the larger context of regeneration after injury.

Background: Anopheles stephensi is a key urban malaria vector in the Indian subcontinent and Middle East includ­ing south and southeast of Iran. Wide application of insecticides resulted in resistance of this species to various insec­ticides in these regions. This study was conducted to reveal the role of metabolic mechanisms in the development of resistance in An. stephensi to DDT and cyfluthrin. Methods: Field mosquito specimens were collected from Chabahar Seaport, southeast corner of Iran, in 2015. Insec­ticide susceptibility and enzyme assays were conducted as recommended by WHO. Results: Mean enzyme ratios were 3.95 and 3.04 for α- esterases and 2.40 and 1.97 for β- esterases in the DDT and cyfluthrin- resistant populations correspondingly compared with the susceptible strain. The GSTs enzyme mean ac­tivity ratios were 5.07 and 2.55 in the DDT and cyfluthrin- resistant populations compared with the susceptible beech strain. The cytochrome p450s enzyme ratios were 1.11 and 1.28 in the DDT and cyfluthrin- resistant populations respectively compared with the susceptible beech strain. Conclusion: Metabolic mechanisms play a crucial role in the development of DDT and cyfluthrin resistance in An. stephensi, therefore, further evaluation of the mechanisms involved as well as implementation of proper insecticide resistance management strategies are recommended.