Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation(1-16). The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Muller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates (12,14-18). While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells (7,19-23). For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues (8,19-22,24-33). Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level (5,8,21,24,27-30,33-39). Xenopus laevis, a classic model system for the study of early neural development (19,27,29,31-32,40-42), serves as a particularly suitable system for retinal primary cell culture (10,38,43-45). Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction (25,38,43). In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products (10,24,44-45). However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1)

The efficiency of indicator groups for the conservation of amphibians in the Brazilian Atlantic Forest

The adequate selection of indicator groups of biodiversity is an important aspect of the systematic conservation planning. However, these assessments differ in the spatial scale, in the methods used and in the groups considered to accomplish this task, which generally produces contradictory results. The quantification of the spatial congruence between species richness and complementarity among different taxonomic groups is a fundamental step to identify potential indicator groups. Using a constructive approach, the main purposes of this study were to evaluate the performance and efficiency of eight potential indicator groups representing amphibian diversity in the Brazilian Atlantic Forest. Data on the geographic range of amphibian species that occur in the Brazilian Atlantic Forest was overlapped to the full geographic extent of the biome, which was divided into a regular equal-area grid. Optimization routines based on the concept of complementarily were applied to verify the performance of each indicator group selected in relation to the representativeness of the amphibians in the Brazilian Atlantic Forest as a whole, which were solved by the algorithm"simulated annealing", through the use of the software MARXAN. Some indicator groups were substantially more effective than others in regards to the representation of the taxonomic groups assessed, which was confirmed by the high significance of data (F = 312.76; p < 0.01). Leiuperidae was considered as the best indicator group among the families analyzed, as it showed a good performance, representing 71% of amphibian species in the Brazilian Atlantic Forest (i.e. 290 species), which may be associated with the diffuse geographic distribution of its species. This study promotes understanding of how the diversity standards of amphibians can be informative for systematic conservation planning on a regional scale

Dismissive and deceptive car dealerships create barriers to electric vehicle adoption at the point of sale

This study investigates the role of car dealerships in the electrification of passenger transport, namely their sales advice about the purchase and use of electric vehicles (EVs). Because most consumers do not have pre-existing knowledge of EVs, and current market conditions favour petrol and diesel vehicles, car dealership experiences may strongly influence EV purchasing decisions. Here we show that car dealerships pose a significant barrier at the point of sale due to a perceived lack of business case viability in relation to petrol and diesel vehicles. In 126 shopping experiences at 82 car dealerships across Denmark, Finland, Iceland, Norway, and Sweden, we find dealers were dismissive of EVs, misinformed shoppers on vehicle specifications, omitted EVs from the sales conversation and strongly oriented customers towards petrol and diesel vehicle options. Dealer’s technological orientation, willingness to sell, and displayed knowledge of EVs were the main contributors to likely purchase intentions. These findings combined with expert interviews suggest that government and industry signalling affect sales strategies and purchasing trends. Policy and business strategies that address barriers at the point of sale are needed to accelerate EV adoption

High Levels of Diversity Uncovered in a Widespread Nominal Taxon: Continental Phylogeography of the Neotropical Tree Frog

Species distributed across vast continental areas and across major biomes provide unique model systems for studies of biotic diversification, yet also constitute daunting financial, logistic and political challenges for data collection across such regions. The tree frog Dendropsophus minutus (Anura: Hylidae) is a nominal species, continentally distributed in South America, that may represent a complex of multiple species, each with a more limited distribution. To understand the spatial pattern of molecular diversity throughout the range of this species complex, we obtained DNA sequence data from two mitochondrial genes, cytochrome oxidase I (COI) and the 16S rhibosomal gene (16S) for 407 samples of D. minutus and closely related species distributed across eleven countries, effectively comprising the entire range of the group. We performed phylogenetic and spatially explicit phylogeographic analyses to assess the genetic structure of lineages and infer ancestral areas. We found 43 statistically supported, deep mitochondrial lineages, several of which may represent currently unrecognized distinct species. One major clade, containing 25 divergent lineages, includes samples from the type locality of D. minutus. We defined that clade as the D. minutus complex. The remaining lineages together with the D. minutus complex constitute the D. minutus species group. Historical analyses support an Amazonian origin for the D. minutus species group with a subsequent dispersal to eastern Brazil where the D. minutus complex originated. According to our dataset, a total of eight mtDNA lineages have ranges >100,000 km2. One of them occupies an area of almost one million km2 encompassing multiple biomes. Our results, at a spatial scale and resolution unprecedented for a Neotropical vertebrate, confirm that widespread amphibian species occur in lowland South America, yet at the same time a large proportion of cryptic diversity still remains to be discovered