Intrinsic Differences Among Individual Retinal Progenitor Cells

Jeff Trimarchi (Postdoctoral Fellow)
Doug Kim (Postdoctoral Fellow)
Ashu Jadhav (Graduate Student)
Timothy Cherry (Graduate Student)

We are interested in how much of the heterogeneity in cell fates produced by retinal progenitor cells is due to intrinsic differences in the progenitor cells themselves. To this end, we have taken genomics approaches to determine the gene expression programs, and in particular, the differences, among retinal progenitor cells. Individual cells taken from the retina at different times are made into cDNA probes. The probes are hybridized to cDNA microarrays prepared from cDNA collections contributed by the Brain Molecular Anatomy Project (Bento Soares, University of Iowa). Each single cell probe is compared to many other single cell probes made from retinal tissue at the same and at different times in development (Figures 1 and 2). In addition to profiling progenitor cells, we are profiling single cells that are at various, early stages in the differentiation process. This allows us to identify how a cell changes its expression profile as it commits to a cell fate and initiates its differentiation.

Figure 1. Retinal progenitor cells are hypothesized to undergo changes in their competence to make different retinal cell fates. The changes in competence are likely produced by changes in gene expression within retinal progenitor cells. To determine these gene expression differences, single progenitor cells, as well as single cells just entering their differentiation program, are used to prepare cDNA probes. These probes are then applied to a cDNA microarray.

Figure 2. Expression levels of retinal progenitor genes. The signals on a cDNA microarray generated by a collection of single retinal cells are shown. Each colored bar represents the signal from the probe of an individual cell. Cyclin D1 is a known retinal progenitor gene expressed by the majority, or all, progenitor cells. FGF15 and sFRP2 can be seen to track with the expression of cyclinD1 in this graph. The location of cells expressing these genes was then investigated by in situ hybridization on retinal tissue sections. Progenitor cells are located in the areas where signal is seen for these two genes, consistent with the prediction of the data from the microarray.

To confirm the gene expression profiles of individual cells using an independent method, we are using in situ hybridization. Retinal cells are dissociated and plated on a slide, then hybridized with one or two probes that are detected fluorescently (Figures 3 and 4). This allows quantification of the % of cells that express a gene, or coexpress two genes, at different times in retinal development. Many pair wise analyses allow confirmation of the microarray data. In addition, the phase of the cell cycle in which a gene is expressed can be determined by this method. A retina is pulsed with 3H-thymidine for 1 hour and then either harvested immediately, or chased in cold thymidine for various time periods. By performing autoradiography in combination with in situ hybridization on dissociated cells, one can determine in which phase of the cell cycle a progenitor gene is expressed. One can also do this in conjunction with long chase periods to determine the order in which genes are expressed in postmitotic cells as they enter their differentiation programs.

Figure 3. FGF15 is expressed in cycling retinal progenitor cells. To further determine that FGF15 is expressed in cycling retinal cells, the retina was pulsed with 3H-thymidine for one hour and then the tissue was dissociated and placed on a slide. The cells were fixed and probed for FGF15 (red) and processed for autoradiography (black dots). It can be seen that approximately 50% of cells in S phase express FGF15 (ex. Red arrow), and that not all FGF15 cells are in S phase. Systematic analyses such as these allow alignment of gene expression patterns with the cell cycle.

Figure 4. Genes expressed in postmitotic cells. The suggestion by the microarray data and from the literature is that GAP43 would not be expressed in cycling retinal cells. This was confirmed using the method outlined in Figure 3. Red signal marks cells that express GAP43 RNA (red arrow) and the black dots mark cells in S phase or early G2 (Green arrow).