Roles of homeobox and bHLH genes in specification of a retinal cell type

Previous analysis of mutant mice has revealed that the bHLH genes Mash1 and Math3, and the homeobox gene Chx10 are essential for generation of bipolar cells, the interneurons present in the inner nuclear layer of the retina. Thus, a combination of the bHLH and homeobox genes should be important for bipolar cell genesis, but the exact functions of each gene remain largely u nknown. We have found that in Mash1-Math3 double-mutant retina, which exhib its a complete loss of bipolar cells, Chx10 expression did not disappear bu t remained in Muller glial cells, suggesting that Chx10 expression per se i s compatible with gliogenesis. In agreement with this, misexpression of Chx 10 alone with retrovirus in the retinal explant cultures induced generation of the inner nuclear layer cells, including Muller glia, but few of them w ere mature bipolar cells. Misexpression of Mash1 or Math3 alone did not pro mote bipolar cell genesis either, but inhibited Muller gliogenesis. In cont rast, misexpression of Mash1 or Math3 together with Chx10 increased the pop ulation of mature bipolar cells and decreased that of Muller glia. Thus, th e homeobox gene provides the inner nuclear layer-specific identity while th e bHLH genes regulate the neuronal versus glial fate determination, and the se two classes of genes together specify the bipolar cell fate. Moreover, M ash1 and Math3 promoted the bipolar cell fate, but not the other inner nucl ear layer-specific neuronal subtypes in the presence of Chx10, raising the possibility that the bHLH genes may be involved in neuronal subtype specifi cation, in addition to simply making the neuronal versus glial fate choice.