Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels ¹. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest ². As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens ³. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources ^4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure ¹². Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.     

Freshwater salinization syndrome: from emerging global problem to managing risks

Freshwater salinization is an emerging global problem impacting safe drinking water, ecosystem health and biodiversity, infrastructure corrosion, and food production. Freshwater salinization originates from diverse anthropogenic and geologic sources including road salts, human-accelerated weathering, sewage, urban construction, fertilizer, mine drainage, resource extraction, water softeners, saltwater intrusion, and evaporative concentration of ions due to hydrologic alterations and climate change. The complex interrelationships between salt ions and chemical, biological, and geologic parameters and consequences on the natural, social, and built environment are called Freshwater Salinization Syndrome (FSS). Here, we provide a comprehensive overview of salinization issues (past, present, and future), and we investigate drivers and solutions. We analyze the expanding global magnitude and scope of FSS including its discovery in humid regions, connections to human-accelerated weathering and mobilization of ‘chemical cocktails.’ We also present data illustrating: (1) increasing trends in salt ion concentrations in some of the world’s major freshwaters, including critical drinking water supplies; (2) decreasing trends in nutrient concentrations in rivers due to regulations but increasing trends in salinization, which have been due to lack of adequate management and regulations; (3) regional trends in atmospheric deposition of salt ions and storage of salt ions in soils and groundwater, and (4) applications of specific conductance as a proxy for tracking sources and concentrations of groups of elements in freshwaters. We prioritize FSS research needs related to better understanding: (1) effects of saltwater intrusion on ecosystem processes, (2) potential health risks from groundwater contamination of home wells, (3) potential risks to clean and safe drinking water sources, (4) economic and safety impacts of infrastructure corrosion, (5) alteration of biodiversity and ecosystem functions, and (6) application of high-frequency sensors in state-of-the art monitoring and management. We evaluate management solutions using a watershed approach spanning air, land, and water to explore variations in sources, fate and transport of different salt ions (e.g. monitoring of atmospheric deposition of ions, stormwater management, groundwater remediation, and managing road runoff). We also identify tradeoffs in management approaches such as unanticipated retention and release of chemical cocktails from urban stormwater management best management practices (BMPs) and unintended consequences of alternative deicers on water quality. Overall, we show that FSS has direct and indirect effects on mobilization of diverse chemical cocktails of ions, metals, nutrients, organics, and radionuclides in freshwaters with mounting impacts. Our comprehensive review suggests what could happen if FSS were not managed into the future and evaluates strategies for reducing increasing risks to clean and safe drinking water, human health, costly infrastructure, biodiversity, and critical ecosystem services.

     

Improved environmental DNA sampling scheme for Alabama sturgeon provides new insight into a species once presumed extinct

Detection of rare species can be challenging and time-consuming using conventional methods, but environmental DNA (eDNA) is becoming a commonly used tool for detection in conservation and management of species. This study demonstrates the utility of the precipitation method (precipitated and preserved in 3 M sodium acetate and 95% ethanol) for collection of eDNA to detect the seasonal distribution of the critically endangered Alabama sturgeon (Scaphirhynchus suttkusi). Surface and benthic water samples were collected across a wider geographic area than previously published for Alabama sturgeon eDNA. Surface and benthic samples both yielded detections and resulted in a similar proportion of positive detections to previous work. However, by sampling a greater portion of the distribution of the Alabama sturgeon, further insight was provided on potential sturgeon movement. The results of the precipitation method show that Alabama sturgeon detections increase during spawning months, and that the fish may be overwintering in the Tombigbee River. High detections from winter benthic samples suggest that habitat choice may play a role in detectability and highlight the need to consider natural history when designing environmental DNA studies. When designing environmental DNA collection for rare species, sampling design should factor in species ecology, habitat use, site characteristics, and specific questions driving the research.     

Functional gap junctions accumulate at the immunological synapse and contribute to T cell activation

Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca(2+) responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell-T cell signaling machinery that regulates T cell activation.     

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al³⁺ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell.     

N-Glycosylation Determines the Abundance of the Transient Receptor Potential Channel TRPP2

Glycosylation plays a critical role in the biogenesis and function of membrane proteins. Transient receptor potential channel TRPP2 is a nonselective cation channel that is mutated in autosomal dominant polycystic kidney disease. TRPP2 has been shown to be heavily N-glycosylated, but the glycosylation sites and the biological role of N-linked glycosylation have not been investigated. Here we show, using a combination of mass spectrometry and biochemical approaches, that native TRPP2 is glycosylated at five asparagines in the first extracellular loop. Glycosylation is required for the efficient biogenesis of TRPP2 because mutations of the glycosylated asparagines result in strongly decreased protein expression of the ion channel. Wild-type and N-glycosylation-deficient TRPP2 is degraded in lysosomes, as shown by increased TRPP2 protein levels upon chemical inhibition of lysosomal degradation. In addition, using pharmacological and genetic approaches, we demonstrate that glucosidase II (GII) mediates glycan trimming of TRPP2. The non-catalytic β subunit of glucosidase II (GIIβ) is encoded by PRKCSH, one of the genes causing autosomal dominant polycystic liver disease (ADPLD). The impaired GIIβ-dependent glucose trimming of TRPP2 glycosylation in ADPLD may explain the decreased TRPP2 protein expression in Prkcsh^−/− mice and the genetic interaction observed between TRPP2 and PRKCSH in ADPLD. These results highlight the biological importance of N-linked glycosylation and GII-mediated glycan trimming in the control of biogenesis and stability of TRPP2.     

Evidence for selection on a CONSTANS-like gene between two red oak species

Background and Aims

Hybridizing species such as oaks may provide a model to study the role of selection in speciation with gene flow. Discrete species'' identities and different adaptations are maintained among closely related oak species despite recurrent gene flow. This is probably due to ecologically mediated selection at a few key genes or genomic regions. Neutrality tests can be applied to identify so-called outlier loci, which demonstrate locus-specific signatures of divergent selection and are candidate genes for further study.

Methods

Thirty-six genic microsatellite markers, some with putative functions in flowering time and drought tolerance, and eight non-genic microsatellite markers were screened in two population pairs (n = 160) of the interfertile species Quercus rubra and Q. ellipsoidalis, which are characterized by contrasting adaptations to drought. Putative outliers were then tested in additional population pairs from two different geographic regions (n = 159) to support further their potential role in adaptive divergence.

Key Results

A marker located in the coding sequence of a putative CONSTANS-like (COL) gene was repeatedly identified as under strong divergent selection across all three geographically disjunct population pairs. COL genes are involved in the photoperiodic control of growth and development and are implicated in the regulation of flowering time.

Conclusions

The location of the polymorphism in the Quercus COL gene and given the potential role of COL genes in adaptive divergence and reproductive isolation makes this a promising candidate speciation gene. Further investigation of the phenological characteristics of both species and flowering time pathway genes is suggested in order to elucidate the importance of phenology genes for the maintenance of species integrity. Next-generation sequencing in multiple population pairs in combination with high-density genetic linkage maps could reveal the genome-wide distribution of outlier genes and their potential role in reproductive isolation between these species.     

Isolation of cellular membranes from lignin‐producing tissues of Norway spruce and analysis of redox enzymes

There are no earlier reports with successful isolation of plasma membranes from lignin‐forming tissues of conifers. A method to isolate cellular membranes from extracellular lignin‐producing tissue‐cultured cells and developing xylem of Norway spruce was optimized. Modifications to the homogenization buffer were needed to obtain membranes from these phenolics‐rich tissues. Membranes were separated by aqueous polymer two‐phase partitioning. Chlorophyll a determination, marker enzyme assays and western blot analyses using antibodies for each membrane type showed that mitochondrial, chloroplastic and to a certain extent also ER and Golgi membranes were efficiently diminished from the upper phase, but tonoplast and plasma membranes distributed evenly between the upper and lower phases. Redox enzymes present in the partially purified membrane fractions were assayed in order to reveal the origin of H₂O₂ needed for lignification. The membranes of spruce contained enzymes able to generate superoxide in the presence of NAD(P)H. Besides members of the flavodoxin and flavodoxin‐like family proteins, cytochrome b5, cytochrome P450 and several stress responsive proteins were identified by nitroblue tetrazolium staining of isoelectric focusing gels and by mass spectrometry. Naphthoquinones juglone and menadione increased superoxide production in activity‐stained gels. Some juglone‐activated enzymes were preferentially using NADH. With NADH, menadione activated only some of the enzymes that juglone did, whereas with NADPH the activation patterns were identical. Duroquinone, a benzoquinone, did not affect superoxide production. Superoxide dismutase, ascorbate peroxidase, catalase and an acidic class III peroxidase isoenzyme were detected in partially purified spruce membranes. The possible locations and functions of these enzymes are discussed.     

Detection of the edible ectomycorrhizal fungus Lyophyllum shimeji colonising seedlings of cultivated conifer species in New Zealand

Lyophyllum shimeji is an edible ectomycorrhizal fungus that is widely distributed in East Asia and also present in the northern regions of Europe. In Japan, L. shimeji is a culinary delicacy, considered amongst all edible mushrooms to have the best taste and to be second only to Tricholoma matsutake in price. Traditionally, fruiting bodies of L. shimeji have been collected from the wild but fruiting of L. shimeji is now relatively uncommon and cannot keep up with increasing consumer demand. As a result, methods for its cultivation are being developed for commercial production in Japan and other countries. In this work, techniques were developed to cultivate L. shimeji on coniferous seedlings using a pure culture inoculum. They resulted in successful mycorrhization of Pinus pinaster and Picea abies in only 8 to 10 months. As ectomycorrhizae of L. shimeji are difficult to identify morphologically, mycorrhization was confirmed using an L. shimeji-specific PCR diagnostic, which was designed following a phylogenetic analysis of the Lyophyllum section Difformia using DNA sequences of the internal transcribed spacer (ITS), intergenic spacer (IGS) and elongation factor 1-α (EF1-α) gene. L. shimeji is a member of the Lyophyllum decastes complex in section Difformia, which also includes Lyophyllum fumosum and L. decastes. This analysis confirmed the separation of L. shimeji from closely related Lyophyllum spp. and enabled its unambiguous detection using an IGS-based PCR diagnostic. This is the first report of successful mycorrhization of L. shimeji on P. pinaster and P. abies and provides an opportunity for its commercial cultivation on conifers in New Zealand.