The nervous system is an extraordinary array of different cell types that are generated at predictable times in developing brain structures. Several studies on how this variety is achieved, have revealed that for any specific cell type a combination of both intrinsic and extrinsic signals is required.
The vertebrate retina is a very useful model to investigate the generation of such cell diversity. We demonstrated that retinal progenitors exit from the cell cycle in distinct waves, thus giving rise to the cell types they have competence to generate in that specific retinogenetic time. This means that, each retinal cell type has a retinogenetic timing to withdraw from the cell cycle.
In the vertebrate retina, homeobox genes play crucial role in establishing different cell identities. Basing our studies on this knowledge, in this thesis work we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. We found that the three Xenopus homeobox genes Xotx5b, Xvsx1 and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b) and bipolar cells (Xvsx1, Xotx2). Furthermore, by in vivo lipofection of “sensors” in which GFP translation is under control of the 3’ untranslated region (UTR), we found that the 3’UTRs of Xotx5b, Xvsx1 and Xotx2 are sufficient to drive a spatio-temporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequence, respectively. This is the first evidence that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities. Finally our observations suggest that this clock measures cell cycle length and that translational inhibitors are part of the clock machinery, while its molecular nature is completely unknown.
We then focused our attention to find out how cell cycle progression can remove the translational inhibition over developmental time. Using an in silico approach, we found that the 3’UTR of Xotx5b, Xvsx1, and Xotx2 contain widley dispersed candidate microRNAs (miRNAs) domains for 42 distinct miRNAs. To assay the role of miRNAs during retinal development, we performed a high throughput analysis by microarrays followed by whole mount in situ hybridization.
We found that several miRNAs are expressed in the frog developing retina and that their expression pattern changes during retinogenesis.
Subsequently, we performed loss of function experiments on selected miRNAs (222, 155, 129). The inhibition of each miRNA caused an increase in the proportion of bipolar cells. This could be due to an increase in the translation of the corresponding target mRNAs.
To investigate the role of mature miRNAs in regulating cell proliferation, survival and differentiation of retinal cells, we downregulated dicer activity in Xenopus embryos by antisense morpholino (Mo) oligonucleotides. The RNaseIII enzyme Dicer has been implicated in the maturation of most miRNAs. This means that blocking Dicer function removes mature miRNAs from a cell. The eyes of Mo-injected embryos (morphants) at the stage of swimming tadpole (st. 42) showed several defects such as: a reduced eye size, an increased retinal cell death, an incomplete retinal lamination and an increase of retinal cycling cells. Whereas Xotx5b and Xotx2 mRNAs were expressed from early developmental stages in morphants, the onset of both Xotx5b and Xotx2 protein detection was delayed in morphants compared to controls. Thus, dicer inactivation affects the timing of retinal differentiation by delaying progenitor cell divisions and the translation of key genes required for the generation of late retinal cell types.
Our data suggest a role for miRNAs in the regulation of the homeobox genes translation, but do not rule out the possibility that specific RNA-binding proteins (RBPs) might also be involved. Our idea is that miRNAs could co-operate with RBPs, using a combinatorial and co-operative code, to regulate gene expression.