Modulation of retinal cell populations and eye size in retinoic acid receptor knockout mice.

PURPOSE: The retinoic acid receptors are expressed from early stages of development in the diverse tissues that make up the vertebrate eye. Their loss has subtle effects on eye development. We adapted sensitive quantitative trait locus (QTL) mapping methods to assess consequences of inactivating alleles of the alpha and beta receptors, Rara and Rarb, on eye and retinal development. Rara is of particular interest because this gene is a candidate for Nnc1, a QTL that controls retinal ganglion cell proliferation. METHODS: We studied lines of mice in which expression of the a1 isoform of Rara or all isoforms of Rarb had been disrupted by gene targeting. We measured eye weight, lens weight, retinal area, and retinal ganglion cell number in each of six genotypes (Rara and Rarb -/-, +/-, +/+; 10-25 cases/genotype). RESULTS: Loss of either protein is associated with a small but significant loss of eye weight and retinal area. However, only the Rarb knockout has a significant effect on the ganglion cell population and the loss of both wildtype alleles leads to an 8,000 cell deficit. Surprisingly, loss of the Rara a1 isoform that is expressed in this cell population from early stages has no effect on number. Null alleles of both genes have little if any effect on lens growth. CONCLUSIONS: Despite its expression in embryonic retina, Rara is unlikely to be the Nnc1 QTL. In contrast, Rarb, a gene that maps to Chr 14 and which is not an Nnc1 candidate gene, has a significant effect on cell number and is therefore a QTL controlling this key population. This raises the intriguing possibility that normal allelic variants of Rarb modulate the ganglion cell population in other vertebrates, including humans.

Cell production and cell death in the generation of variation in neuron number.

Retinal ganglion cell numbers in adult mice vary from 40,000 to 80, 000. Much of this variation and the prominent bimodality of strain averages are generated by allelic variants at the neuron number control 1 (Nnc1) locus on chromosome 11. The Nnc1 locus may modulate either ganglion cell production or the severity of ganglion cell death. Here we have determined what the relative contributions of these two processes are to variation in adult cell number by estimating total ganglion cell production in 10 strains of mice (A/J, BALB/cJ, BXD32, C57BL/6J, CAST/Ei, CARL/ChGo, CE/J, C3H/HeSnJ, DBA/2J, and LP/J). These strains have adult populations that range from 45,000 to 76,000 (data available at http://qtl.ml.org). We estimated cell production by counting ganglion cell axons after ganglion cell neurogenesis but before the onset of significant cell death. Total cell production ranges from 131,000 to 224,000, and most of the variation in adult ganglion cell number is explained by this significant variation in cell production. In contrast, the percentage of cell death is relatively uniform in most strains (approximately 69% cell loss). The exceptions are BXD32, a strain that has an extremely high adult cell population, and Mus caroli (CARL/ChGo), a wild southeast Asian species that is distantly related to laboratory strains. In BXD32 and M. caroli, approximately 62% of the population dies. Our analysis indicates that substitutions of single alleles at the Nnc1 locus are responsible for production differences of approximately 8000 ganglion cells.

Genetic dissection of retinal development.

Retinal development depends on complex interactions between products of thousands of genes and numerous cellular and environmental factors. We are using novel quantitative genetic methods to map and characterize genes that are responsible for the pervasive quantitative differences in the architecture of the eye and the retina. These genes, known as quantitative trait loci (QTLs), may also determine susceptibility to common eye diseases. To map QTLs that generate variation among normal individuals we have analyzed several traits in a wide variety of mice, including standard inbred strains, recombinant inbred strains, wild mice, F1 hybrids and intercross progeny. Here we review this approach and give three specific examples of how genes with well-defined functions in retinal development are being mapped and characterized.

Natural variation in neuron number in mice is linked to a major quantitative trait locus on Chr 11.

Common genetic polymorphisms-as opposed to rare mutations-generate almost all heritable differences in the size and structure of the CNS. Surprisingly, these normal variants have not previously been mapped or cloned in any vertebrate species. In a recent paper (), we suggested that much of the variation in retinal ganglion cell number in mice, and the striking bimodality of strain averages, are caused by one or two quantitative trait loci (QTLs). To test this idea, and to map genes linked to this variable and highly heritable quantitative trait, we have counted ganglion cells in 38 recombinant inbred strains (BXD and BXH) derived from parental strains that have high and low cell numbers. A genome-wide search using simple and composite interval-mapping techniques revealed a major QTL on chromosome (Chr) 11 in a 3 cM interval between Hoxb and Krt1 (LOD = 6.8; genome-wide p = 0.001) and possible subsidiary QTLs on Chr 2 and Chr 8. The Chr 11 locus, neuron number control 1 (Nnc1), accounts for one third of the genetic variance among BXH strains and more than half of that among BXD strains, but Nnc1 has no known effects on brain weight, eye weight, or total retinal cell number. Three strong candidate genes have been mapped previously to the same region as Nnc1. These genes-Rara, Thra, and Erbb2- encode receptors for retinoic acid, thyroxine, and neuregulin, respectively. Each receptor is expressed in the retina during development, and their ligands affect the proliferation or survival of retinal cells.

Genetic and environmental control of variation in retinal ganglion cell number in mice.

How much of the remarkable variation in neuron number within a species is generated by genetic differences, and how much is generated by environmental factors? We address this problem for a single population of neurons in the mouse CNS. Retinal ganglion cells of inbred and outbred strains, wild species and subspecies, and F1 hybrids were studied using an unbiased electron microscopic method with known technical reliability. Ganglion cell numbers among diverse types of mice are highly variable, ranging from 32,000 to 87,000. The distribution of all cases (n = 252) is close to normal, with a mean of 58,500 and an SD of 7800. Genetic factors are most important in controlling this variation; 76% of the variance is heritable and up to 90% is attributable to genetic factors in a broad sense. Strain averages have an unanticipated bimodal distribution, with distinct peaks at 55,500 and 63,500 cells. Three pairs of closely related strains have ganglion cell populations that differ by > 20% (10,000 cells). These findings indicate that different alleles at one or two genes have major effects on normal variation in ganglion cell number. Nongenetic factors are still appreciable and account for a coefficient of variation that averages approximately 3.6% within inbred strains and isogenic F1 hybrids. Age- and sex-related differences in neuron number are negligible. Variation within isogenic strains appears to be generated mainly by developmental noise.