ABSTRACT
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.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Retina/metabolism , Transcription, Genetic , Xenopus Proteins/metabolism , Xenopus laevis/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/metabolism , Receptors, GABA/genetics , Receptors, GABA/metabolism , Retina/cytology , Signal Transduction , Xenopus Proteins/genetics , Xenopus laevis/embryologyABSTRACT
Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/ß-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.
Subject(s)
Cell Proliferation , Hedgehog Proteins/physiology , Retina/embryology , Retina/growth & development , Wnt Signaling Pathway/physiology , Animals , Animals, Genetically Modified , Cell Proliferation/drug effects , Drug Antagonism , Embryo, Nonmammalian , Enzyme Inhibitors/pharmacology , Hedgehog Proteins/antagonists & inhibitors , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Indoles/pharmacology , Models, Biological , Organogenesis/drug effects , Organogenesis/genetics , Organogenesis/physiology , Oximes/pharmacology , Receptor Cross-Talk/drug effects , Receptor Cross-Talk/physiology , Retina/drug effects , Retina/metabolism , Teratogens/pharmacology , Veratrum Alkaloids/pharmacology , Wnt Signaling Pathway/drug effects , Xenopus laevis/embryologyABSTRACT
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.
