Effects of loss of Nhlh1 and Nhlh2 basic helix–loop–helix (bHLH) transcription factors on retinal development

Abstract

Abstract: : Purpose: Among the transcription factor families that play crucial roles in retinogenesis, basic helix–loop–helix (bHLH) factors have emerged as important regulators of neuronal identity in the retina. Nhlh1 and Nhlh2 constitute a subfamily of neuronal–specific bHLH transcription factors. They are expressed in postmitotic and differentiated neurons suggesting a function as late acting differentiation genes and in the maintenance of the differentiated state. Gain–of–function studies in the chicken suggest a role for Nhlh1 and Nhlh2 in retinogenesis. The purpose of this study is to gain insight into the function of Nhlh1 and Nhlh2 in the retina by investigating phenotypes associated with loss–of–function in mice carrying a homozygous deletion of these genes (knockout). Methods: Knockout mice for Nhlh1 and Nhlh2 gene were generated at the National Cancer Institute, NIH, Bethesda, MD. Phenotypes associated with loss of these genes have been published, but no data is available on their retinal phenotype. Expression studies in the developing retina were performed by in situ hybridization and real time RT–PCR on embryonic (E9.5 to E18.5) and post–natal (P0 to P21) wild type mice. Histological assessment of the phenotype associated with gene loss was performed by H+E staining, immunohistochemistry and immunofluorescence. Results: Both Nhlh1 and Nhlh2 were expressed in the developing eye by E9.5. Expression of Nhlh1 and Nhlh2 was maintained throughout development and in the adult. As in other areas of the developing nervous system, localization of Nhlh1 and Nhlh2 partially overlapped but the degree and time of expression were different. The Nhlh1 knockout (Nhlh1^–/–), the Nhlh2 knockout (Nhlh2^–/–) mice, and the double knockout neonates (Nhlh1^–/–Nhlh2^–/–) showed retinal dysplasia. The severity of the phenotype was variable, including the presence of ectopic tissue in extreme cases. Conclusions: Loss of Nhlh1 and Nhlh2 is associated with an abnormal retinal phenotype. Confirmation of our preliminary observations and identification of specific retinal cell populations affected by their deletion would further reinforce the notion of a role for Nhlh1 and Nhlh2 in retinogenesis. Expression of both genes in the adult eye also suggests a function in the maintenance of differentiated retinal cell populations.

Keywords: retinal development • transcription factors • gene/expression