Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation.

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RNA in situ hybridization (ISH) has been widely used in cell and developmental biology research to study gene expression. Classical ISH protocols use colorimetric staining approaches, such as the assay with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP), which do not allow the implementation of multiple probe analyses and do not enable investigators to achieve cellular resolution. Here we describe a protocol to determine the presence of target cytoplasmic RNA via cytoplasmic fluorescence ISH (cFISH), an approach that renders possible the visualization of specific RNA strands from the whole tissue down to the cell. This fluorescence technique, adapted here for use in mouse embryos, enables researchers to implement multiple labeling by combining several RNA probes and/or antibodies in immuno-cFISH. Depending on the options chosen, the protocol can be completed within 2 or 3 d.

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The hypothesis of this research was that the maps based on remotely-sensed images would create zones of different vigor, yield, water status, winter hardiness and berry composition and the wines from the unique zones would show different chemical and sensorial profiles. A second hypothesis was that titer of grapevine leafroll-associated virus (GLRaV) could be correlated spatially to NDVI and other spectral indices. To determine zonation, unmanned aerial vehicles (UAVs) with multispectral and thermal sensors were flown over six Cabernet Franc vineyard blocks in Ontario, Canada. Zonation was based on NDVI values, and spatial correlations were examined between the NDVI and leaf water potential (Ψ), soil water content (SWC), stomatal conductance (gs), winter hardiness (LT50), vine size, yield, and berry composition. Additional NDVI data were acquired using GreenSeeker (proximal sensing), and both NDVI data sets produced maps of similar configuration. Several direct correlations were found between UAV-based NDVI and vine size, berry weight, yield, titratable acidity, SWC, leaf Ψ, gs, and NDVI from GreenSeeker. Inverse correlations included thermal data, Brix, color/ anthocyanins/ phenols, and LT50. The pattern of UAV-based NDVI and other variables corresponded to the PCA results. Thermal scan and GreenSeeker were useful tools for mapping variability in water status, yield components, and berry composition. In 2016, zoned maps were created based on UAV NDVI data, and grapes were harvested according to the separate zones. Additionally, spatial correlations between GLRaV titer and NDVI were observed. Use of UAVs may be able to delineate zones of differing vine size, yield components, and berry composition, as well as areas of different virus status and winter hardiness.

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