Abstract
Purpose: :
To better understand the mechanisms that control rod differentiation, we investigated the hypothesis that neural retina leucine zipper Nrl transcription factor can reprogram eye progenitor cells to different fates.
Methods: :
We used lipofection of stage17/18 Xenopus embryos (Ohnuma et al., (2002) Methods 28, 411–419) to ectopically express large Maf transcription factors in early dividing retinal progenitors. Xenopus LNrl or human Nrl were cloned under control of the CMV promoter into the pCS2 expression vector. A pCS2–GFP plasmid was included in all experiments in order to identify co–transfected cells and allow us to follow their fate in the developing retina. Thus, this method only detects cell autonomous outcomes. Only a few cells are transfected in each eye anlage so that the differentiated retina is largely intact. Retinas (stage 41) were cryosectioned, immmunostained with various antibodies and then analyzed for cell–types expressing GFP.
Results: :
Lipofection of eye anlagen (stage 17/18) with pCS2–GFP produced fluorescent cells in all layers and in a few lens cells by stage 41. Ectopic expression of hNrl or xLNrl in primordial retina caused a three–fold increase in the number of rhodopsin–positive cells and compensatory decrease in the number of cells expressing cone–specific markers. Ectopic expression of xLNrl also decreased the number of GFP–positive bipolar cells by 2.5 fold, while hNRL did not affect bipolar cell number. In addition, xLNrl expression caused a five–fold increase in GFP positive lens fiber cells compared to either control (GFP only) or hNrl transfections. Transfection of hNrl or xLNrl induced the expression of rhodopsin in other retinal cell layers in some but not all animals.
Conclusions: :
These results show that Nrl/LNrl can function cell–autonomously to change the fate of retinal progenitor cells, promoting the formation of rods and suppressing the formation of cones. We have also uncovered an evolutionary divergence in the functions of this large Maf subfamily. We have found that that the lower vertebrate ortholog has dual roles in retina and lens development while the mammalian ortholog is specialized for rod development.
Keywords: differentiation • photoreceptors • transcription factors