Constance Cepko and her colleagues focus their studies on the retina, a tractable portion of the central nervous system. They study the mechanisms that determine cell fate during development and the death of photoreceptor cells, which are frequently the target of diseases that lead to blindness. They have been developing methods to map neuronal circuits as well as novel methods to manipulate biological activities specifically in cells that express green fluorescent protein (GFP).
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, an accessible protein of the central nervous system. 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) or other proteins expressed in a cell type-specific manner, as a scaffold for nanobody fusion proteins. This method allows the manipulation of gene expression, or other activities, in specific cell types. Finally, we are 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
Many 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. Our goal is to continue to discover the types of progenitor cells and the mechanisms that they use to generate the final complement of retinal cell types. We study the gene regulatory networks that operate within these cells, and their newly postmitotic progeny, by discovery of enhancers and their regulatory proteins.
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. The roles of extrinsic cues and transcription factors that set up areas of high acuity are being investigated.
In many forms of human blindness, rod photoreceptors express a mutant gene, whereas cone photoreceptors do not. Unfortunately, the cones still die, implying a non-autonomous 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 (Figure 1), which has been shown to occur in human diseases that lead to blindness. These vectors prolong vision in mouse models of human blindness and are now being tested on large animal models of these diseases.
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 (Figure 2). We are now creating vectors that are less toxic than the current tracers, and are examining the response of the immune system to the vectors, both to further our understanding of these interactions, as well as enhance the transmission of the vectors among neurons.
Cepko Research Abstract Slideshow 2
Figure 2a: Circuitry tracing using VSV. VSV can be engineered to discover the presynaptic circuitry partners for initially infected cells. VSV virions with two types of glycoproteins on the surface can be created in tissue culture. One type of glycoprotein, the rabies virus glycoprotein (RABV-G), is supplied by the tissue culture cells. Another type, a fusion of the extracellular domain of the avian EnvA glycoprotein and the cytoplasmic domain of RABV-G, is encoded by the VSV genome. The virus preparation is injected into the lateral geniculate nucleus (LGN), a retinorecipeint area. Virions can enter the terminals of retinal ganglion cells (RGCs) and travel to the cell body, where they can replicate, and produce GFP. The viral particles produced by the infected RGCs will have on their surface only the EnvA fusion protein, due to the fact that only this glycoprotein is encoded within the VSV genome. The viral particles will exit from the soma and dendrites and infect any presynaptic cells which express TVA, the receptor for EnvA. The Chat-Cre mouse was crossed to a floxed TVA strain, as well as a floxed tdtomato strain (Ai9 created by the Allen Brain Institute). The Chat gene is only expressed in cholinergic cells, which in the retina, are the starburst amacrine cells (SACs). By tracking which types of GFP+ RGCs transmit to the SACs, one can infer the type of RGCs that are postsynaptic to SACs.
See also Beir, K.T. et al. 2011 Developmental Biology 353:309–320.
Figure 2b: An image of an area of a flat mount of a retina infected as described in figure 2a.The GFP+ cell is an OFF-alpha retinal ganglion cell and the red cells are starburst amacrine cells labeled by tdtomato. Two starburst amacrine cells were infected from VSV transmitted by the OFF-alpha ganglion cell.
See also Beier, K. et al. 2013 Journal of Neuroscience 33:35-51.
Nanobody Fusion Proteins for Manipulation of Biological Activities
Many transgenic organisms express GFP, or other specific gene products, in specific cell types. We wished to develop a method that would restrict the manipulation of biological activities to cells that express a specific gene product. Initially, this method relied upon GFP-binding proteins (GBPs), nanobodies derived from camelid antibodies. Pairs of GBPs were made into fusion proteins, with each GBP fused to a different partner which would then, in the presence of GFP, constitute a biological activity, such as transcription activation (Figure 3). A set of fusions were made that 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 then developed single protein nanobody fusions that are conditionally stable, with stability dependent upon the presence of epitope. These fusions can be used to detect antigen, e.g. HIV capsid protein, perform recombination, e.g. Flp fusion, or carry out gene editing, e.g. Cas9 fusion, only in cells that express the cognate epitope. This method should allow the targeting of activities in non-model organisms, with the only requirement being the production of a nanobody (or other protein binder) that has specificity for the cell type of interest.
Cepko Research Abstract Slideshow 3
Figure 3a: Scheme for use of GFP as a scaffold to control biological activities. Pairs of fusion proteins that bind to GFP (in green and orange) can activate a reporter whose regulatory region (e.g., UAS) is recognized by a specific DNA binding domain (DBD). Reporter activation (up to 200-fold) is dependent upon GFP. The reporter plasmid can encode virtually any gene, such as Cre, channelrhodopsin, etc. The system also works with GFP derivatives, such as YFP. The modularity of the components allows for the use of different DBDs and has so far been developed for the tet, lexA, and Gal4 DBDs and several different activation domains (AD).
From Tang, J.D. et al. 2013 Cell 154:928–939. © 2013, with permission from Elsevier.
Figure 3b: The use of GFP as a scaffold to create biological activities only in GFP-expressing cells is illustrated. Delivery of GFP-binding proteins fused to transcription activation and DNA binding domains can lead to the expression of specific genes, here a red fluorescent protein, in a subset of bipolar neurons in the murine retina.
Image: Jonathan Tang. See also Tang, J.D. et al. 2013 Cell 154:928–939.
As of April 29, 2016