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.

Wnt/Hedgehog morphogens in retinal stem cell niche, (ii) analysed Wnt signalling activity (target gene expression, transgenic reporter line) following Hedgehog pathway pharmacological interference, or vice versa, and (iii) investigated retinal cell proliferation and determination phenotypes following simultaneous inhibition or activation of the two pathways. Altogether, our data suggest that Wnt and Hedgehog morphogens form opposite gradients within retinal stem cell niche and that these signalling pathways antagonize with each other to control retinal stem cell proliferation and multipotence. Interestingly, our retinal gradient model is reminiscent to the neural tube patterning model.

homeostasis is achieved, although the molecular mechanisms controlling this process are poorly understood. Previously we demonstrated that the transcription factor, Hoxa3, significantly promotes angiogenesis during tissue repair and regeneration (Mace et al., 2005 J Cell Sci). Subsequent analysis of GFP bone marrow chimeras during wound repair in mice with directed Hoxa3 expression revealed that Hoxa3 promotes the recruitment of HSCs and endothelial progenitor cells. We would like to better understand the role of Hoxa3 in the regulation of HSC migration and differentiation in response to injury. In this work, for the first time we report an increased myeloid (Gr-1+Cd11b+) differentiation potential for HSC as a result of Hoxa3 overexpression. Hoxa3 also enhances the migration of HSCs towards chemoattractant. Gr-1+Cd11b+ cells have an important role in tumor angiogenesisand we are investigating the involvement of these cells in neovascularisation during wound healing. This can be one mechanism by which Hoxa3 promotes angiogenesis via myeloid differentiation of HSCs. These data will provide avenues for furthering our understanding of how manipulation of adult stem cells ex vivo could be used as a potential therapy in patients with severe injuries or impaired wound healing. The adult subventricular zone (SVZ) is the major neurogenic region of the adult mammalian brain and a potential source of regenerative stem cells. SVZ stem cells respond to defined growth factor treatment both in vitro and in vivo. Moreover, the adult SVZ shows a remarkable capability for self-repair through activation of a quiescent pool of progenitor cells. We are addressing the role of the Notch pathway in the activation of the adult neural stem cells during growth factor treatment and SVZ regeneration. Taking advantage of a transgenic reporter mouse where GFP is expressed under the control of the Hes5 promoter (Basak and Taylor 2007. Eur J Neurosci 25:1006-22), we show that canonical Notch signalling is active in a distinct subpopulation of SVZ astrocytes. Notch signalling cells divide slowly or are quiescent in the SVZ but respond selectively to specific growth factor treatment in vivo by increasing their mitotic activity. Moreover, Hes5-GFP positive cells participate in regeneration of the neurogenic niche after ablation of fast dividing progenitors with the anti-mitotic Ara-C in vivo. Finally, using conditional gene inactivation in Nestin::creER T2 mice we show that Notch signalling cells not only contribute regeneration but that Notch1 signalling is required for effective regeneration of the adult SVZ. Our data suggest that Notch-dependent stem cells are activated by both growth factor treatment and lesion of the neurogenic niche. Elucidating the mechanisms by which Notch-dependent stem cells respond to growth factors and are activated during regeneration could be important for brain repair.

Recruitment of bone marrow derived cells (BMDC) to thewound site plays an essential role in wound healing as these can contribute directly to vasculogenesis.This process is dysregulated in diabetic wounds. It has been shown that injury-induced