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 Müller 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).

Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4.

Five newly isolated mycobacteriophages--Angelica, CrimD, Adephagia, Anaya, and Pixie--have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them--with the exception of TM4--form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species.