Abstract vi During embryonic development neural tissues are generated from multipotent progenitor cells that require a high coordination of proliferation, cell cycle exit and activation of factors involved in cell different

Abstract

Abstract vi During embryonic development neural tissues are generated from multipotent progenitor cells that require a high coordination of proliferation, cell cycle exit and activation of factors involved in cell differentiation.

Xenopus retina is an ideal model for studying cell diversity as it is composed by six major types of neurons (cones, rods, horizontal cells, amacrine cells, bipolar cells, ganglion cells) and a type of glial cell (Müller glia) that are easily recognizable by their morphology and localization in the retinal layers. The arise from a single layer of multipotent progenitor cells, in an evolutionary ordered timing schedule.

In this context, cell fate is determined by the activation of specific genes, including homeobox genes Xotx5 (photoreceptors), Xotx2 and Xvsx1 (bipolar cells). The activation of these genes, as expected, parallels the generation timing of corresponding retinal cell types. Recently it was shown that the activation of these genes is regulated at the translational level: the three genes are transcribed in all early progenitor cells but are translated only in the cells fated to differentiate in the corresponding retinal cell type, photoreceptors or bipolar cells (Decembrini et al., 2006). Such translational control seems to be due to factors in cis recognising signals on the 3’UTR of mRNAs.

The main factors, known in literature, able to regulate the translation by

3'UTR recognition, are mRNA binding proteins (RBP) and microRNA

(miRNA). In my thesis I studied the possible regulative role of four

miRNA, previously identified in Cell Biology laboratory of the

Department of Biology, on the mRNAs of the homeobox genes Xotx2 and

Xvsx1.

Abstract vii Firstly, to understand if these miRNAs were actually involved in the repression of translation of Xotx2 and Xvsx1, I conducted loss of function experiments, by lipofection in Xenopus embryos optical vesicles at the embryonic stage 17-18. In these experiments I co-lipofected pCS2-GFP vector (to identify transfected cells), with antisense chemically modified oligoribonucleotides (Decoy), each of which is able to inhibit a particular miRNA. Then, I determined the percentage of Xotx2 or Xvsx1 positive cells on the total lipofected cells using antibodies against the two proteins by immunostaining experiments.

Secondly, I conducted a complementary gain of function experiment, co- transfecting pCS2-GFP vector (to identify lipofected cells ) and a mixture of the four miRNAs (or an unrelated miRNA, as control) in the optical vesicles and then determining the percentage of Xotx2 or Xvsx1 positive cells on the total lipofected cells. Some of these embryos were treated with a molecule that can lengthen the cell cycle of retinal cells (cyclopamine) to check the possible effects of the cell cycle length on regulation of the two transcripts.

Finally, I conducted a similar gain of function experiment and treatment with cyclopamine, co-transfecting the mixture of miRNAs, the pCS2-RFP vector, as internal standard, and a Sensor-GFP (a vector containing the 3'UTR of one of the two genes, cloned downstream of the fluorescent protein GFP). The use of these Sensors allows to verify potential interactions between miRNA and 3'UTR of the two transcripts, by measuring the expression ratio between sensor and internal standard.

The results show that the inhibition of the four miRNAs, in general, leads

to a strong increase in the percentage of Xotx2 positive cells. In contrast

the inhibition of only two of these four miRNAs leads to a weaker increase

of Xvsx1 positive cells. Furthermore, if we consider data deriving from

gain of function experiments, we can assume that repression by miRNAs,

Abstract viii in early retinal progenitor cells during retinal development, is stronger for Xotx2 than Xvsx1, confirming the results obtained by Decembrini et al., (2006). Finally, the effects of cyclopamine seem to confirm the involvement of the cell cycle for the differentiation of retinal progenitor cells in different cell types in mature retina.

In conclusion, we presume that the differentiation of retinal cells, particularly bipolar cells, is subjected to a clear translational control by miRNAs and that these are largely influenced by the cell cycle progression.

Nevertheless some of the results suggest possible involvement of other

miRNAs, still unidentified, or other mechanisms of control to allow

differentiation of the proper proportion of bipolar cells. So we can assume

that early progenitor cells rapidly divide themself under the influence of

proliferative stimuli, as the signal factor sonic hedgehog, maintaining high

levels of certain miRNAs that inhibit Xotx2 translation, to prevent their

differentiation in late bipolar cells .