Atrophy of Müller Glia and Photoreceptor Cells in Chick Retina Misexpressing cNSCL2

The vertebrate retina contains six major types of cells: photoreceptors, horizontal cells, bipolar cells, amacrine cells, ganglion cells, and Muller glia. These cells are stereotypically organized into three nuclear layers separated by two synaptic layers. During retinal neurogenesis, precursors of each cell type migrate to their prospective location and undergo a unique differentiation program, enabling them to assume a distinct morphology and to perform a discrete function. The molecular mechanism underlying retinal development is not fully understood. The Drosophila proneural genes, achaete-scute complex and atonal, encode the basic helix-loop-helix (bHLH) family of transcription factors and play important roles in neurogenesis in the fly. In vertebrates, a number of homologues have been identified and shown to play important roles in nervous system development.1 In the developing retina, mash-1/cash-1, ngn1, math-5/cath5, and math3/cath3 are expressed in progenitor cells.2–9 math3/cath3 is also expressed in bipolar cells.10,11 Various spatial patterns of neuroD expression have been reported.7,8,11–15 Significant progress has been made in understanding how these proneural genes function during retinal neurogenesis. Mice without mash-1 do not exhibit any obvious abnormalities in eye development during embryogenesis and at birth, the time when the mutant mice die,16,17 but explant cultures of mash-1−/− retinas showed a delay in differentiation of rod photoreceptors, horizontal cells, and bipolar cells; a decrease in bipolar cell number, and an increase in glia.17 Retinal explant cultures were also used to examine retinal phenotype of neuroD null mutation, and it is reported that mouse neuroD plays multiple roles during retinal development.15 Studies from our laboratory indicated that chick neuroD is involved in specifying a photoreceptor cell fate.14,18,19 Misexpression of neuroD in the developing chick retina results in an overproduction of photoreceptor cells specifically, and ectopic neuroD expression in cultured RPE cells promotes de novo generation of photoreceptor cells selectively.14,18,19 NSCL is a two-member subfamily of bHLH genes homologous to Drosophila proneural gene atonal. Mammalian NSCL1 and NSCL2 were originally cloned by low-stringency hybridization.20,21 Northern blot analysis and in situ hybridization revealed that expression of mammalian NSCL1 and NSCL2 is specific to neural tissues.20–22 DNA microarray analysis has identified NSCL2 as a target of p53.23 Targeted deletion of NSCL2 has been reported to result in a disruption of the hypothalamic–pituitary axis and triggers adult-onset obesity in mice.24 The expression and function of mammalian NSCL1 and NSCL2 in retinal development remain to be studied. cNSCL1 is the chick homologue of mammalian NSCL1 and, similar to its mammalian counterpart, appears specific to neural tissues.25 In the developing retina, cNSCL1 is expressed transiently in the developing ganglion cells and later in Muller glia.26 Misexpression of cNSCL1 decreases cell proliferation activity in the retina and in the external granular layer of the developing cerebellum,25,26 suggesting that one function of cNSCL1 may be to prevent postmitotic cells from reentering the cell cycle. To learn whether and how NSCL2 is involved in retinal development, we cloned its chick homologue, cNSCL2, examined its expression pattern in the retina, and studied the effect of its misexpression on retinal development. We found that cNSCL2 was expressed in amacrine and bipolar cells. Misexpression of cNSCL2 in the developing retina resulted in retinal degeneration with profound atrophy of photoreceptors and glia.