The brain is a complex tissue, comprising myriad cell types and more than 109 cells and 1015 connections in humans. Through cell division, commitment, and differentiation, cells are generated and become the final cell types observed in mature tissue. We are studying these processes in the retina of rodents and chicks, which exhibit patterns of development and final structures very similar to those of humans. We are also interested in the circuitry of retinal neurons and have developed viral vectors that transmit across synaptically connected cells, revealing both local and long distance connections. To aid in this endeavor, and to more broadly allow the manipulation of biological activities within specific cell types, we have also developed a novel method that utilizes green fluorescent protein (GFP) as a scaffold. This method allows us to manipulate gene expression, or other activities, specifically in GFP-positive cells. Finally, we are also committed to preserving vision in people who inherit disease genes leading to blindness. To this end, we have been studying why retinal photoreceptors die and then designing gene therapy approaches to combat these mechanisms.
Retinal Cell Fate Determination
More than 25 years ago, we developed a lineage marking technique that we used to discover that the mitotic progenitor cells that generate the various neuronal and glial cell types of the retina are multipotent. We have since been investigating the nature of these multipotent progenitor cells to learn whether there are distinct types that make particular types of daughter cells or whether the environment or stochastic mechanisms guide the formation of different cell types. We used microarrays to profile the transcriptomes of individual retinal progenitor cells and found a great deal of heterogeneity among them. To address whether the heterogeneity was correlated with the formation of different types of progeny, we modified our retroviral tracing method to target specific progenitor cells. We found that retinal progenitor cells that express particular genes make distinct daughter cell types and have specific proliferation patterns. These data suggest that the strategy used by the retina to make the >60 types of retinal neurons is very similar to that of the Drosophila ventral nerve cord. Our goal is to continue to outline the types of progenitor cells and the mechanisms they use to generate the final complement of retinal cell types.
Photoreceptors in Development and in Disease
Rod and cone photoreceptor cells detect light and thereby initiate vision, with cones being responsible for high acuity and color vision during the day. The mechanisms that direct the formation of photoreceptors, including the choice of rod versus cone, have been a focus of our studies. Using the method of electroporation for mapping the regulatory elements of genes, we defined regulatory regions for multiple early photoreceptor genes. One of these regions led us to a specific progenitor cell type, and the transcription factors that are necessary and sufficient, for cone photoreceptor determination. We are using these genes to further understand the mechanisms of photoreceptor determination and the patterning of cones across the retina in different species. We are also working with groups who wish to engraft photoreceptor cells into individuals who have lost them as a result of disease processes, using these genes to direct formation of cone photoreceptors from stem cells.
In many forms of human blindness, rod photoreceptors express a mutant gene, whereas cone photoreceptors do not. Unfortunately, the cones still die, implying a nonautonomous process in the death of cones. Humans rely most heavily on cones; therefore, we would like to understand why the cones die, so that a pharmaceutical or genetic intervention can be developed. To this end, we have used microarrays as well as other methods to examine several mouse models of human disease and found that the cones are starving subsequent to the death of rods. We are developing viral vectors to deliver genes that improve nutrition within cones, to promote their survival in a degenerating retina. In addition, we have created viral vectors that fight oxidation within cones (Figures 1a and 1b), which has been shown to occur in human diseases that lead to blindness.
Development of Transsynaptic Tracers
One goal of neuroscientists is to discover and understand the circuits that define the various functions carried out by the nervous system. To this end, we developed a new viral tracer, the vesicular stomatitis virus (VSV). We demonstrated that the VSV can travel between synaptically connected cells in many types of organisms, including mammals, birds, fish, and amphibians. We were also able to direct its transmission either anterograde or retrograde using different types of viral glycoproteins. Furthermore, we could alter the properties of the VSV so that it crossed one synapse and then stopped transmitting, or continued to transmit across several synapses. We used these vectors to begin to examine the direction-selective circuits in the mouse retina. Previously unknown synaptic partners were detected, and their connections were confirmed using electrophysiology, in collaborative studies (Figures 2a and 2b).
GFP as a Scaffold for Biological Activities
Many transgenic organisms express GFP in specific cell types. To expand the utility of these lines to include biological perturbations only in GFP-positive cells, we developed a method to use GFP as a scaffold. This method relies on GFP-binding proteins (GBPs), derived from camelid antibodies previously characterized by Ulrich Rothbauer and colleagues. We used pairs of GBPs as fusion partners for protein domains that, in the presence of GFP, would constitute a biological activity. For example, one GBP fusion that binds to a specific DNA sequence can be used in combination with another that activates transcription. GFP can bring these two fusions together to activate transcription from a specific promoter (Figures 3a and 3b). Another set of fusions reconstitutes Cre recombinase, allowing the manipulation of gene structure only in GFP-positive cells. These constructs are modular and are relatively straightforward to engineer to create many types of activities. We are currently using GBPs in combination with the transsynaptic tracing methods as a means to map circuitry using optogenetic, and other, methods.
These studies have been funded in part by grants from the National Eye Institute, the Harvard Neuro Discovery Center, Foundation Fighting Blindness, and the Thome Foundation.
As of April 04, 2013