My lab studies a variety of processes associated with the zebrafish eye, including development of the retina and lens, retinal cell death (neuronal degeneration), and the role of adult stem cells and glial cells in regeneration of retinal neurons. We use genetic, cell biological, biochemical, and molecular approaches to examine the status of the retina and lens.
We use zebrafish because of the ease in isolating large numbers (over 100) from a single pair mating. Because the embryos develop external to the female, they are easy to examine and manipulate. We have generated a large number of transgenic zebrafish lines in the last 5 years. The ease in generating transgenic lines allows us to examine the roles of genes and their proteins in a variety of cellular processes and identify genetic interactions. The zebrafish retina is also structurally and functionally similar to the human eye, which makes the findings in zebrafish applicable to human vision and neurological disease.
The work in my lab can be divided into four main areas: 1) retinal development, 2) lens development and degeneration, 3) mechanisms of retinal regeneration, 4) development of genetic tools. Most students work in one of these areas, although some cross over between different research areas depending upon their interests.
Our overall goal is to understand the mechanisms that underlie a variety of human eye diseases, such as macular degeneration, retinitis pigmentosa, and diabetic retinopathy in the retina and cataracts in the lens. Understanding these mechanisms will provide clues as to potential therapies. One of the most promising therapies is the regeneration of tissue, particularly neurons. Zebrafish possess the capacity to regenerate retinal neurons and we are studying the signals that induce regeneration and the mechanisms employed by the adult stem cells to replace the correct neuronal types. Because this regeneration uses existing stem cells to regenerate a variety of different neurons, it might be possible to extend this work to restore neurons to specific brain areas, such as in Alzheimer’s disease or Parkinson’s disease, or to repair spinal cord injuries.
The retina develops from an undifferentiated neuroepithelial cell sheet that proceeds through coordinated waves of mitotic activity followed by cell differentiation to produce the mature laminated retina (Figure 1A). We are studying how the proteins that determine the neuroepithelial cell polarity control the development of the eyes, the patterning of the different layers in the retina, and the differentiation of the various retinal cell types. We are pursuing this work using a combination of classical and molecular genetic techniques.
For example, we cloned the zebrafish pard3 gene (Wei et al., 2004), which is alternatively spliced to encode two different proteins that localize to the apical side of the tight junction. Molecular genetic techniques that blocked the translation of the Pard3 protein produced two phenotypes. The first phenotype is cyclopia, which is the fusion of the eyes. We found that the absence of Pard3 expression in the developing brain ventral diencephalon resulted in cell death and the inability of the developing eye fields to separate. Later, the retina exhibits a lack of lamination (Figure 1B), even though all the different neuronal classes are present in the disorganized retina. Another protein that regulates cell polarity is Scribble, which is localized to the basolateral membrane of neuroepithelial cells. Preliminary experiments suggest that Scribble, unlike Pard3, is required for cell cycle exit and differentiation. We are studying if Scribble regulates cell cycle exit by either sequestering specific molecules to the basal region of the cells or by signaling the cell to accept terminal differentiation signals.
Figure 1. A wild-type retina (A) and a retina that develops in the absence of the Pard3 protein (B). The wild-type retina is laminated and contains the outer nuclear layer (ONL), which is where the rod and cone cell nuclei are located, the inner nuclear layer (INL), where the stem cell population is located, and the ganglion cell layer (GCL). These three nuclear layers are separated by two synaptic layers, the outer plexiform layer (IPL) and inner plexiform layer (IPL). The retina that develops in the absence of the Pard3 protein is not laminated and lacks any neuronal organization.
As an alternative approach to study retinal development, we identified several different zebrafish mutants that exhibit defects during retinal formation (Vihtelic and Hyde, 2002). These ENU-induced mutations are being characterized to determine if the mutation affects neuroepithelial cell proliferation, cell migration, neuronal differentiation, or apoptosis. We are using several approaches to characterize these processes, including immunohistochemistry and transgenic lines that express fluorescent markers in specific cell types.
We are using molecular genetic approaches to identify genes critical for development of the vertebrate lens. The ocular lens is an ideal model tissue because of its relatively simple structural organization, accessibility during the relevant developmental stages and rich historical role in embryological research. Studies of the lens have resulted in important contributions to understanding the roles of various transcription factors, cytoskeleton remodeling, apoptosis, and signal transduction. In addition, the lens is necessary for proper formation of the retina and other eye tissues.
Several different mutant lines exhibiting defects in lens development or maintenance were generated in a chemical mutagenesis screen for eye morphological mutants (Vihtelic and Hyde, 2002). These included the arrested lens (arl), disrupted lens (dsl), and lens opaque (lop) lines (Vihtelic et al., 2001; Vihtelic et al., 2005). The detailed phenotypic and genetic characterization of the arl mutant resulted in the identification of laminin a1 (lama1) as the mutant gene (Semina et al., 2006). These studies demonstrated the lama1 gene is necessary for both lens epithelial and fiber cell differentiation. In addition, focal adhesion kinase activation was affected in the mutant lens, which suggests the interrupted signaling pathways may be responsible for cytoskeletal rearrangments necessary for lens cell morphogenesis. Mutation mapping and further cellular and biochemical characterizations are being pursued for the other lens mutants.
Figure 2. Lens Mutant. Zebrafish mutational analysis identifies human lens disease genes. A: Wild-type lens displays a single layer of epithelial cells (arrowheads) that overlie the lens cortical fibers. B: A zebrafish lens mutant characterized by aberrant lens cell growth (arrows), which may model human anterior subcapsular cataracts
Zebrafish orthologs of lens development genes characterized in other species are also being identified and studied. For example, we characterized the zebrafish genes encoding the Pitx3 and Foxe3 transcription factors and demonstrated their genetic interactions during lens development (Shi et al., 2005; Shi et al., 2006). We determined Pitx3 is required for Foxe3 expression and Foxe3 regulates cell proliferation and differentiation in the developing lens (Figure 3). In addition, EST analysis of adult zebrafish eye tissues resulted in the identification of lengsin, a gene whose protein product is expressed exclusively in the lens differentiating secondary fibers (Vihtelic et al., 2005). Immunolocalizations, tissue in situ RNA hybridizations, antisense-mediated reduction in protein expression by morpholino injections, and transgenic techniques are being used to characterize the functions of additional zebrafish lens development genes.
Figure 3. Characterization of zebrafish lens gene orthologs. A: Wild-type lens actin (green) is localized along the cortical fiber cell membranes. B and C: Foxe3 anti-sense knockdown by morpholino injection. Reduction in Foxe3 during lens development resulted in fiber cell dysmorphogenesis (B, arrow) and a persistent connection to the cornea (C, double arrows). Nuclei in Panel B were stained with propidium iodide)
We developed two different paradigms to cause retinal damage and induce the regeneration of the lost neurons. Treating albino zebrafish with intense light causes apoptosis (cell death) of the rod and cone cells (Vihtelic and Hyde, 2000). Injection of a low concentration of ouabain causes cell death of the inner retinal neurons, with very little damage to the photoreceptors (Fimbel et al., in press). Both of these models that affect different classes of retinal neurons induce the proliferation of neuronal progenitor cells in the inner nuclear layer. These progenitor cells migrate to the correct retinal layers and differentiate into the missing neurons.
We recently found that the source of the retinal progenitor cells during regeneration are the Müller glial cells (Vihtelic et al., 2006; Fimbel et al., in press; Kassen et al., in press). Using a transgenic zebrafish line that expresses Enhanced Green Fluorescent Protein (EGFP) from the glial fibrillary acidic protein (gfap) promoter in the Müller glia, we demonstrated that the EGFP-expressing Müller glia reenetered the cell cycle shortly after the retinal damage. Subsequently, a subset of these Müller glia cease expressing EGFP from the gfap promoter. However, these Müller glial cells, that appear to dedifferentiate, continue to proliferate and produce the majority of the neuronal progenitor cells that regenerate the lost neurons.
To study the signaling mechanisms that induce the regeneration response, we carefully identified different time points that correspond to critical retinal changes (Figure 4). At the beginning of the light treatment (Figure 3A), the rod outer segments are full (green) and there is an absence of proliferating cells (red). At 16 hours into the light treatment (Figure 4B), increased proliferation of the rod precursor cells in the outer nuclear layer is apparent (red cells). At this same time, apoptosis is at the maximal level. By 31 hours (Figure 4C), there is a noticeable decrease in rod outer segment integrity, which is consistent with photoreceptor cell death. The inner nuclear layer cells also begin to proliferate at this time (Figure 4C, arrow). By 51 hours (Figure 4D), the inner nuclear layer cells exhibit a fusiform morphology and migrate to the outer nuclear layer. By 68 hours (Figure 4E), cell proliferation is maximal in both the inner and outer nuclear layers. By 96 hours into the light treatment (Figure 4F), differentiation of regenerated rods and cones has begun.
Figure 4. Timecourse of light-induced photoreceptor cell death and regeneration. Images correspond to albino zebrafish after 7 days of constant darkness (A), followed by constant light for 16 hours (B), 31 hours (C), 51 hours (D), 68 hours (E), and 96 hours (F). The retinas are labeled with antisera that detects rhodopsin in the rod outer segments (green), which corresponds to the presence of rod photoreceptors, and PCNA (red), which is expressed in proliferating cells. The retinal layers are: ROS, rod outer segments; CC, cone cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; and GCL, ganglion cell layer. Arrowheads point to proliferating rod precursor cells and the arrow shows the relative location of inner nuclear layer stem cells.
To identify the molecules involved in mediating the regeneration response, we isolated RNA from regenerating retinas at each of the above time points for a gene microarray analysis. We identified several genes that are upregulated at specific times during regeneration (Figure 5). For example, one of the upregulated genes is olig2, which begins to increase in expression at 51 hours into the light treatment. Using a transgenic zebrafish line that expresses EGFP from the olig2 promoter, we determined that transcription from the olig2 promoter increases in the neuronal progenitor cells beginning at 51 hours. This represents the earliest marker for these progenitor cells after they dedifferentiate from being Müller glial cells.
Figure 5. Microarray expression profiles during retinal regeneration. A. A heat map shows the change in expression of several different transcription factors through the timecourse. In this heatmap, red represents increasing gene expression and green decreasing expression. B. Different expression profiles are present within the microarray data. The top panel shows genes that increase in the first 16 hours. The middle panels shows genes that do not begin to increase in expression until between 31 and 51 hours. The lower panel shows genes that begin increasing in expression between 51 and 68 hours into the light treatment.
We are developing two different approaches to control gene expression in the zebrafish lens and retina. First, we cloned several different ubiquitous and cell-type-specific promoters to express desired transgenes in the eye. We tested these promoters by expressing a reporter, such as Enhanced Green Fluorescent Protein (EGFP). For example, the rod opsin promoter specifically directs EGFP expression to rod photoreceptors (Figure 6), while a glial fibrillary acidic protein (gfap) gene promoter drives EGFP in the retina specifically to the Müller glia (Figure 7). Similarly, we cloned several promoters that are expressed in the different lens cell types. We intend to use these zebrafish promoters to create transgenes that incorporate genetic regulatory systems, such as the Gal4-UAS or the yeast two-hybrid, to regulate the induction of transgenes to either mark or kill specific cell types.
Figure 6. Transgenic zebrafish developed at Notre Dame. A transgenic zebrafish line that expresses Green Fluorescent Protein (GFP) from the rhodopsin promoter in only the rod photoreceptor cells (A). The GFP is expressed from the ef1 ubiquitous promoter and is found throughout the zebrafish embryo (B, top). A control zebrafish embryo shows autofluorescence in the residual yolk sac (B, bottom).
Morpholinos are modified oligonucleotides that can block translation or splicing of a RNA in a gene-specific manner. These morpholinos are routinely used to mimic a mutant phenotype by knocking down the expression of specific proteins during development. We developed an electroporation technique to introduce morpholinos into the adult retina to knock down the expression of specific proteins during retinal regeneration. We are currently using this technique to examine the functions of genes identified from the microarray analysis of the regenerating light-damaged retina.
Figure 7. Transgenic expression of enhanced green fluorescent protein in Müller glia in the developing zebrafish retina. Confocal microscopic image of retinal cryosections of a 7 day post fertilization Tg(gfap:EGFP)nt embryo counter stained with the nuclear dye TO-PRO-3.
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