The retina of fish and amphibian contains genuine neural stem cells located at the most peripheral edge of the ciliary marginal zone (CMZ). However, their cell-of-origin as well as the mechanisms that sustain their maintenance during development are presently unknown. We identified Hes4 (previously named XHairy2), a gene encoding a bHLH-O transcriptional repressor, as a stem cell-specific marker of the Xenopus CMZ that is positively regulated by the canonical Wnt pathway and negatively by Hedgehog signaling. We found that during retinogenesis, Hes4 labels a small territory, located first at the pigmented epithelium (RPE)/neural retina (NR) border and later in the retinal margin, that likely gives rise to adult retinal stem cells. We next addressed whether Hes4 might impart this cell subpopulation with retinal stem cell features: inhibited RPE or NR differentiation programs, continuous proliferation, and slow cell cycle speed. We could indeed show that Hes4 overexpression cell autonomously prevents retinal precursor cells from commitment toward retinal fates and maintains them in a proliferative state. Besides, our data highlight for the first time that Hes4 may also constitute a crucial regulator of cell cycle kinetics. Hes4 gain of function indeed significantly slows down cell division, mainly through the lengthening of G1 phase. As a whole, we propose that Hes4 maintains particular stemness features in a cellular cohort dedicated to constitute the adult retinal stem cell pool, by keeping it in an undifferentiated and slowly proliferative state along embryonic retinogenesis. Stem Cells 2012;30:2784–2795

Neural stem cell research suffers from a lack of molecular markers to specifically assess stem or progenitor cell properties. The organization of the Xenopus ciliary marginal zone (CMZ) in the retina allows the spatial distinction of these two cell types: stem cells are confined to the most peripheral region, while progenitors are more central. Despite this clear advantage, very few genes specifically expressed in retinal stem cells have been discovered so far in this model. To gain insight into the molecular signature of these cells, we performed a large-scale expression screen in the Xenopus CMZ, establishing it as a model system for stem cell gene profiling. Eighteen genes expressed specifically in the CMZ stem cell compartment were retrieved and are discussed here. These encode various types of proteins, including factors associated with proliferation, mitotic spindle organization, DNA/RNA processing, and cell adhesion. In addition, the publication of this work in a special issue on Xenopus prompted us to give a more general illustration of the value of large-scale screens in this model species. Thus, beyond neural stem cell specific genes, we give a broader highlight of our screen outcome, describing in particular other retinal cell markers that we found. Finally, we present how these can all be easily retrieved through a novel module we developed in the web-based annotation tool XenMARK, and illustrate the potential of this powerful searchable database in the context of the retina.

In contrast with the wealth of data involving bHLH and homeodomain transcription factors in retinal cell type determination, the molecular bases underlying neurotransmitter subtype specification is far less understood. Using both gain and loss of function analyses in Xenopus, we investigated the putative implication of the bHLH factor Ascl1 in this process. We found that in addition to its previously characterized proneural function, Ascl1 also contributes to the specification of the GABAergic phenotype. We showed that it is necessary for retinal GABAergic cell genesis and sufficient in overexpression experiments to bias a subset of retinal precursor cells towards a GABAergic fate. We also analysed the relationships between Ascl1 and a set of other bHLH factors using an in vivo ectopic neurogenic assay. We demonstrated that Ascl1 has unique features as a GABAergic inducer and is epistatic over factors endowed with glutamatergic potentialities such as Neurog2, NeuroD1 or Atoh7. This functional specificity is conferred by the basic DNA binding domain of Ascl1 and involves a specific genetic network, distinct from that underlying its previously demonstrated effects on catecholaminergic differentiation. Our data show that GABAergic inducing activity of Ascl1 requires the direct transcriptional regulation of Ptf1a, providing therefore a new piece of the network governing neurotransmitter subtype specification during retinogenesis.