both systems divide a complex sensory input into parallel streams (41). These data suggest a homology in the development of these two sensory systems, based on a partial overlap of transcriptional regulators.
RPF-1, the fourth POU domain sequence implicated in ganglion cell development, was identified in human and mouse genomic DNA and subsequently found in the human retina where it is expressed in subsets of ganglion and amacrine cells (H.Z. and J.N., unpublished data). As described above for the pattern of immunostaining for the Brn3 factors, immunostaining for RPF-1 shows a characteristic heterogeneity of nuclear staining intensity. In the cat retina, the highest levels of RPF-1 are found in medium (β) and small (γ) ganglion cells; the large (α) ganglion cells contain little or no RPF-1. In the macaque, many ganglion cells contain low levels of RPF-1 and a minority of cells in the ganglion cell layer contain high levels of RPF-1. In contrast to the eccentricity-dependent decrease in overall cell density in the ganglion cell layer, the density of cells that contain high levels of RPF-1 changes little with retinal eccentricity.
The most direct evidence that any of the POU domain transcription factors play a role in ganglion cell development comes from recent experiments in which the Brn3b gene has been inactivated by homologous recombination in embryonic stem cells (M.X., J.N., L. Gan, and W. Klein, unpublished data). Mice that are homozygous for the mutant allele are viable but show specific defects in retinal structure. While Brn3b knockout retinae resemble those of the wild type in overall structure, they have 70% fewer ganglion cells. Other neurons within the retina and brain appear to be minimally or not at all affected.
An intriguing aspect of the Brn3 and RPF-1 immunolabeling patterns is the characteristic heterogeneity in nuclear labeling intensity. This heterogeneity in levels of transcription factors suggests that stable differentiated states may be determined not only by the presence or absence of different transcription factors but by the maintenance of these factors at particular intermediate levels. A graded mechanism of this general type has been shown to mediate anterior–posterior fate determination in the Drosophila embryo, in which case concentration gradients of a small set of maternally derived regulatory proteins determine the level of expression of a larger set of target genes at different positions in the embryo (19).
The authors thank Dr. Stewart Hendry for helpful comments on the manuscript. This work was supported by the National Eye Institute (National Institutes of Health) and the Howard Hughes Medical Institute.
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