Expression of bHLH Gene ash4 in the Developing Chick Retina

Abstract

Abstract: : Purpose: Neural development and differentiation involve gene regulation by transcription factors in the basic Helix–Loop–Helix family. To understand how retinal neurogenesis is governed by proneural bHLH genes, our laboratory has begun to investigate their involvement in the development of the embryonic chick retina. This study focuses on ash4. Methods: Chick ash4 was RT–PCR amplified according to published information. Antisense RNA probes were generated and used to examine the spatial and temporal pattern of ash4 expression. Results: In situ hybridization with digoxigenin–labeled probes detected ash4 expression in the developing chick retina. Unlike most other bHLH genes, the mRNA level of ash4 displayed a gradient across the thickness of the retina. The gradient was observed in both pseudostratified and stratified retinas. Spatially, the strongest in situ hybridization signals were found on the vitreal side or in the ganglion cell layer. The intensity of the signals gradually tapered off towards the outer retina and became undetectable at the outer portion of the inner nuclear layer. Nonetheless, cells expressing ath4 included Muller glia and those bipolar cells at the middle of the inner nuclear layer. Developing photoreceptor cells, either in a pseudostratified retina or in the outer nuclear layer of a stratified retina, lacked ash4 expression. Conclusions: The developing chick retina expressed ash4 in a remarkable gradient across the retinal thickness. This implies that the formation of, or retinal cell response to, a gradient of a developmental cue may involve ash4. Gain– and loss–of–function experiments are underway to examine whether retinal development will be altered when this ash4 expression gradient is perturbed.

Keywords: gene/expression • retinal development • transcription factors