Abstract
Abstract v
During embryonic development neural tissues are generated by multipo-tent progenitor cells; this requires an high coordination of proliferation, cell cycle exit and dierentiation.
Xenopus retina is an ideal system for studying these processes, given its simple anatomical structure. Vertebrate retina is composed by six neural cell types (ganglion cells, cones, horizontal cells, amacrine cells, rods and bipolar cells) and one type of glial cell (Muller glia); each retinal progenitor is able to generate all retinal cell types in an evolutionary ordered timing schedule. Retinal cell fate determination requires the activation of cell fate genes speci c for each cell type, such as Xotx5b (photoreceptors), Xotx2 and Xvsx1 (bipolar cells). The timing of expression of these genes parallels the timing of generation of corresponding cell types (Decembrini et al., 2006). As recently shown, these homeobox genes are regulated at the translational level, that is mRNAs are expressed during all retinogenesis but proteins are translated only in post-mitotic cells fated to dierentiate in photoreceptors or bipolar cells. This translational control depends on unknown factors able to bind cis-acting signals in the 3'UTR of mRNAs (Decembrini et al., 2006). It is unknown if other genes required for retinal cell fate determination are regulated at the translational level.
It is known from literature that microRNAs and RNA binding proteins (RBPs) are able to regulate translation after binding to the 3'UTR of target mRNAs. In this study we analysed some candidate RBPs, in order to in-vestigate their possible role during retinogenesis, and to understand if they could regulate retinal homeobox genes translation.
First, we analysed the expression pattern of selected RBPs at dierent developmental stages by whole mount in situ hybridization and in situ hybri-dization on cryosections. In this way we attempted to identify RBPs expres-sed in proliferating progenitor cells. After these experiments, we performed a functional analysis on the most interesting genes, XLin-28 and Nrp-1. We used gain of function experiments in order to understand if these RBPs might be involved in retinal dierentiation. We overexpressed candidate genes by lipofection of retinal progenitors at stage 17, and we analysed lipofected re-tinas at stage 42 (mature embryonic retina). Then we identi ed retinal cell types using both morphological criteria and speci c cell markers. These
ex-Abstract vi
periments show that XLin-28A overexpression increases amacrine cell type and decreases late neurons (photoreceptors and bipolar cells). Nrp-1 overex-pression instead increases glial cell number and decreases photoreceptors cell number. These phenotypes could be caused by an eect on cell prolifera-tion. To address this issue, we performed birthdating experiments by BrdU labelling of dividing cells in lipofected embryos at dierent developmental stages. These experiments show that XLin-28A doesn't aect the overall cell proliferation, but it changes the timing of generation of amacrine cells thus resulting in an heterochronic phenotype. Instead Nrp-1 overexpression induces early cell cycle exit and early dierentiation of retinal progenitors.
These results show that these two RBPs have dierent roles during re-tinal development. Nrp-1 probably doesn't control the timing of generation of retinal cells. XLin-28A changes this timing and could regulate the trans-lation of retinal cell fate genes. Future experiments are required to identify mRNAs regulated by XLin-28A and to elucidate the molecular mechanisms underlying its activity.
