The vertebrate retina is a multilayered tissue acting at the interface of input light and visual perception. In the mouse, the formation of this complex structure takes place between embryonic day E11.5 and post-natal day P8 requiring a combination of intrinsic and extrinsic factors (1
). Six neuronal cell types [retinal ganglion cell (RGC), amacrine, bipolar, horizontal, rod and cone] and a single glial cell type (Müller) are formed when actively dividing neuroblasts leave the cell cycle and commit to cell fates in a temporally ordered sequence.
Differentiation occurs along all three axes. Along the anterior–posterior axis, uncommitted cells differentiate into distinct retinal cell types and migrate to their final positions within either the ganglion, inner nuclear or outer nuclear layer. Along the dorsal–ventral axis, RGCs and other cell types acquire a dorsal–ventral pattern that can be observed by retinotectal projection maps or by molecular markers that distinguish dorsal axons from ventral axons (2
). Along the nasal–temporal axis, differentiation proceeds as a bidirectional propagation wave from the center of the developing retina to the peripheral regions (4
Environmental cues are particularly crucial in retinal development because naïve neuroblasts are not intrinsically programmed for any particular lineage (1
). In the zebrafish retina, hedgehog signaling factors have been implicated as essential components of the propagation event (4
), and sonic hedgehog appears to negatively regulate RGC formation behind the wave front (6
). Other secreted factors have been shown to play key roles in retinal development. Fibroblast growth factors (FGFs) emanating from the surface ectoderm appear to control the decision to become neural retina or pigmented epithelium (7
), and FGFs play roles in other aspects of retinal patterning and differentiation (9
The Notch–Delta pathway negatively regulates the first cell-fate commitment made in the retina, which is to become a RGC (10
). Recently, the proneural gene math5
, which encodes a bHLH transcription factor and is repressed by Notch signaling, was shown to be required for RGC formation (13
). Brn-3b, a Class IV POU domain protein, acts downstream of Math5 as an essential transcription factor in RGC differentiation, axon outgrowth and cell survival (14
). Brn-3b may also play a role in RGC specification and may be a direct target of Math5 (15
). Pax6 is required upstream of several bHLH factors to maintain the multipotent state of retinal progenitor cells (18
), and a number of bHLH factors and other transcription factors have been implicated in the formation of RGCs, bipolar cells, amacrine cells and photoreceptor cells (1
Progress in defining the genetic regulatory events that lead to the formation of the vertebrate retina has been substantial, particularly over the past decade. However, many of the regulatory pathways and genes found to be critical for retinal development are known to occur in the development of other vertebrate tissues and organs. What is unclear is how these pathways ultimately lead to the unique repertoire of expressed genes that define the functioning retina. As a starting point, it would be valuable to define the complete set of expressed genes for the developing retina. This would enable comparisons to be made with other neuronal tissues, and perhaps more important, would provide the basis for establishing a well-defined platform upon which gene expression profiling experiments could be based and new genes could be discovered.
At E14.5, most cells in the developing retina are either uncommitted neuroblasts or newly differentiated retinal neurons (20
). It is clear that in this heterogeneous population of cells, the complete expressed gene set will provide information not only on the events occurring at E14.5 but on past and future events as well. Here we report the sequence analysis of an E14.5 retinal cDNA library and the establishment of an EST database. The database currently contains 15 268 ESTs, of which 9035 have been annotated and represent 5288 genes. From the current database, we estimated that there could be as many as 27 000 genes expressed in the E14.5 retina. A pilot microarray gene expression profiling analysis using 864 clones identified GAP-43
, encoding a protein required for proper RGC axon growth and pathfinding, as a potential target of Brn-3b. The retinal EST database can therefore be used successfully to reveal novel gene regulatory linkages involved in retinal development. We expect that the embryonic retinal EST database will be an accessible, valuable resource for the biomedical research community.