Final Activity Report Summary - RETINA BHLH (Study of the role of the basic helix-loop-helix transcription factors Nhlh1 and Nhlh2 in the transcriptional network regulating retinogenesis)
During retinal development six classes of neurons and one type of glia are generated from a common pool of proliferating retinal progenitor cells (RPCs). The cellular and molecular mechanisms that specify the fate of each RPC are still vague. It has already been established that these events are controlled by a hierarchy of genes encoding extrinsic and intrinsic factors. Among the extrinsic factors are mitogens and morphogens. Among the intrinsic factors are components of the cell cycle machinery and transcription factors (TFs). Members of the basic helix-loop-helix (bHLH) family of TFs have been shown to promote neuronal versus glial fates and to regulate retinal subtype specification. Nhlh1 and Nhlh2 constitute a subfamily of neuronal-specific bHLH TFs which are expressed in post-mitotic neurons in the developing nervous system, including the retina. However, limited data is available on their function in the retina. The aim of this study was to investigate the role of Nhlh1 and Nhlh2 in the developing retina by characterising their expression and the retinal phenotype associated with their deletion (Nhlh1-/-, Nhlh2-/-) in mice.
Nhlh1 and Nhlh2 showed overlapping, yet distinct, patterns of expression in the developing retina. Both genes were expressed by embryonic day (E)12.5 in the neo-formed retinal ganglion cell (RGC) layer (GCL) as well as in the neuroblast layer (NBL). Nevertheless, the times of expression and patterns were different, with Nhlh1 being down-regulated by E18.5 while Nhlh2 was expressed throughout embyonic retinal development. Similarly, both genes were up-regulated postnatally, with peak expression around postnatal day P6 for Nhlh1 and P4 for Nhlh2 in the GCL and inner nuclear layer (INL), yet with different spatial distribution. Low expression of Nhlh1 and Nhlh2 persisted in the adult retina. Expression studies suggested distinct functions for Nhlh1 and Nhlh2 in the development of RGCs and amacrine cells and in the maintenance of differentiated retinal cell populations.
Deletion of Nhlh1 or Nhlh2 did not affect the overall retinal morphology. All cell layers were present in Nhlh1-/- and Nhlh2-/- retinas and their thickness was comparable to the wild type (wt). No obvious abnormalities in the total number or location of amacrine or RGCs were observed in the mature retina. Structural abnormalities were observed in a proportion of Nhlh1-/- retinas, at times exhibiting the presence of extra-retinal tissue. These data were suggestive of over-production of RPCs during retinogenesis and mirrored the small eye phenotype obtained in Nhlh1 misexpression experiments in the chicken. Nhlh1-/- neural stem cells (NSCs) that were isolated and cultured in vitro proliferated at higher rate than their wt counterparts, suggesting a potential role for Nhlh1 in regulating cell proliferation or differentiation. Immunohistochemical analysis revealed an increase in the proportion of S-opsin, but not of M-opsin immunoreactive cones, in the Nhlh2-/- compared to wt retina. These data were consistent with an indirect involvement of Nhlh2 in the specification of cone photoreceptor subpopulations or in the expression of cell subtype specific markers in mature cones and suggested that Nhlh2 might contribute to specification of retinal cell subtypes.
Overall, the phenotype associated with the deletion of Nhlh1 or Nhlh2 in the mouse retina was very mild. This suggested that Nhlh1 and Nhlh2 were dispensable for normal completion of retinal development. The high homology between these two TF raised the possibility of a compensatory effect. Investigation of the Nhlh1-/- and Nhlh2-/- double knockout mice might contribute a better insight into the function of these genes in retinal development in general and in RGC, amacrine and cone-photoreceptor cells in particular. In vitro studies overexpressing Nhlh1 and Nhlh2 in the retinoblastoma cell line Weri-Rb-1 using the constructs which were generated by this project provided a gain-of-function approach to study the molecular function of these genes and their reciprocal interactions.
Nhlh1 and Nhlh2 showed overlapping, yet distinct, patterns of expression in the developing retina. Both genes were expressed by embryonic day (E)12.5 in the neo-formed retinal ganglion cell (RGC) layer (GCL) as well as in the neuroblast layer (NBL). Nevertheless, the times of expression and patterns were different, with Nhlh1 being down-regulated by E18.5 while Nhlh2 was expressed throughout embyonic retinal development. Similarly, both genes were up-regulated postnatally, with peak expression around postnatal day P6 for Nhlh1 and P4 for Nhlh2 in the GCL and inner nuclear layer (INL), yet with different spatial distribution. Low expression of Nhlh1 and Nhlh2 persisted in the adult retina. Expression studies suggested distinct functions for Nhlh1 and Nhlh2 in the development of RGCs and amacrine cells and in the maintenance of differentiated retinal cell populations.
Deletion of Nhlh1 or Nhlh2 did not affect the overall retinal morphology. All cell layers were present in Nhlh1-/- and Nhlh2-/- retinas and their thickness was comparable to the wild type (wt). No obvious abnormalities in the total number or location of amacrine or RGCs were observed in the mature retina. Structural abnormalities were observed in a proportion of Nhlh1-/- retinas, at times exhibiting the presence of extra-retinal tissue. These data were suggestive of over-production of RPCs during retinogenesis and mirrored the small eye phenotype obtained in Nhlh1 misexpression experiments in the chicken. Nhlh1-/- neural stem cells (NSCs) that were isolated and cultured in vitro proliferated at higher rate than their wt counterparts, suggesting a potential role for Nhlh1 in regulating cell proliferation or differentiation. Immunohistochemical analysis revealed an increase in the proportion of S-opsin, but not of M-opsin immunoreactive cones, in the Nhlh2-/- compared to wt retina. These data were consistent with an indirect involvement of Nhlh2 in the specification of cone photoreceptor subpopulations or in the expression of cell subtype specific markers in mature cones and suggested that Nhlh2 might contribute to specification of retinal cell subtypes.
Overall, the phenotype associated with the deletion of Nhlh1 or Nhlh2 in the mouse retina was very mild. This suggested that Nhlh1 and Nhlh2 were dispensable for normal completion of retinal development. The high homology between these two TF raised the possibility of a compensatory effect. Investigation of the Nhlh1-/- and Nhlh2-/- double knockout mice might contribute a better insight into the function of these genes in retinal development in general and in RGC, amacrine and cone-photoreceptor cells in particular. In vitro studies overexpressing Nhlh1 and Nhlh2 in the retinoblastoma cell line Weri-Rb-1 using the constructs which were generated by this project provided a gain-of-function approach to study the molecular function of these genes and their reciprocal interactions.