Although much is known about the transcriptional regulation that coordinates retinal cell fate determination, very little is known about the developmental processes that establish the characteristic laminar architecture of the retina, in particular, the specification of neuronal positioning. The LIM class homeodomain transcription factor Lim1 (Lhx1) is expressed in postmitotic, differentiating, and mature retinal horizontal cells. We show that conditional ablation of Lim1 results in the ectopic localization of horizontal cells to inner aspects of the inner nuclear layer, among the retinal amacrine cells. The ectopic cells maintain a molecular phenotype consistent with horizontal cell identity; however, these neurons adopt a unique morphology more reminiscent of amacrine cells, including a dendritic arbor positioned within the inner plexiform layer. All other retinal cell populations appear unaltered. Our data suggest a model whereby Lim1 lies downstream of horizontal cell fate determination factors and functions cell autonomously to instruct differentiating horizontal cells to the appropriate laminar position in the developing retina. This study is the first to describe a cell type-specific genetic program that is essential for targeting a discrete retinal neuron population to the proper lamina.
Horizontal cells are inhibitory interneurons with laterally oriented dendrites that overlap one another, contacting the pedicles of cone photoreceptors. Because of their regular spacing, the network of horizontal cells provides a uniform coverage of the retinal surface. The developmental processes establishing these network properties are undefined, but cell-intrinsic instructions and interactions with other cells have each been suggested to play a role. Here, we show that the intercellular spacing of horizontal cells is essentially independent of genetic background and is predicted by local density, suggesting that horizontal cell positioning is modulated by proximity to other horizontal cells. Dendritic field area compensates for this variation in intercellular spacing, maintaining constant dendritic coverage between strains. Functional dendritic overlap is achieved anatomically at the level of the pedicles, where horizontal cells interact with one another to establish their connectivity: the number of dendritic terminals contacting a pedicle changes, reciprocally, between neighboring horizontal cells during development based on their relative proximity to each pedicle. Cellular morphology is also shown to be regulated by the afferents themselves: afferent elimination before innervation does not alter dendritic field size nor stratification but compromises dendritic branching and prevents terminal formation. Afferent and homotypic interactions therefore generate the morphology, spacing, and connectivity of horizontal cells underlying their functional coverage of the retina.
The Nobel Prize in Chemistry was awarded in 2000 for the discovery of conductive organic polymers, which have subsequently been adapted for applications in ultrasensitive biological detection. Here, we report the first use of this new class of fluorescent probes in a diverse range of cytometric and imaging applications. We demonstrate that these ''Brilliant Violet'' reporters are dramatically brighter than other UV-violet excitable dyes, and are of similar utility to phycoerythrin (PE) and allophycocyanin (APC). They are thus ideally suited for cytometric assays requiring high sensitivity, such as MHC-multimer staining or detection of intracellular antigens. Furthermore, these reporters are sensitive and spectrally distinct options for fluorescence imaging, twophoton microscopy and imaging cytometry. These ultra-bright materials provide the first new high-sensitivity fluorescence probes in over 25 years and will have a dramatic impact on the design and implementation of multicolor panels for high-sensitivity immunofluorescence assays. Published 2012 Wiley Periodicals, Inc. limited by the variety of fluorochromes currently available (1). Existing fluorescent probes fall into three classes: large protein-based molecules (e.g., phycobiliproteins), inorganic fluorescent nanocrystals (e.g., quantum dots), and small organic dyes (e.g., fluorescein). All have been incorporated into standard immunofluorescence staining technology, but many exhibit undesirable qualities in terms of brightness, stability, or applicability to different techniques (like intracellular staining) (2,3). In this contribution, we examine a fourth class of fluorescent materials that offer distinct advantages in performance and versatility.In principle, the sensitivity of fluorescent probes in cell staining is limited by three factors. First, and most importantly, is the intrinsic brightness of the probe, quantified generally by its absorbance cross-section and quantum efficiency. Second is the degree to which intrinsic background (autofluorescence) of the sample overlaps with the dye's emission. And third, for multiparameter applications, is how broad the excitation and emission spectra are, leading to ''spillover'' or overlap of multiple probe emissions. This requires mathematical correction (known as compensation). For imaging applications, a fourth factor becomes important: resistance to photobleaching. The properties of phycoerythrin (PE) and (to a lesser extent) allophycocyanin (APC), two phycobiliproteins in use for the last 25 years, make them the brightest probes currently used in most immunofluorescence experiments. Both
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