Hedgehog signaling has been linked to cell proliferation in a variety of systems; however, its effects on the cell cycle have not been closely studied. In the vertebrate retina, Hedgehog's effects are controversial, with some reports emphasizing increased proliferation and others pointing to a role in cell cycle exit. Here we demonstrate a novel role for Hedgehog signaling in speeding up the cell cycle in the developing retina by reducing the length of G1 and G2 phases. These fast cycling cells tend to exit the cell cycle early. Conversely, retinal progenitors with blocked Hedgehog signaling cycle more slowly, with longer G1 and G2 phases, and remain in the cell cycle longer. Hedgehog may modulate cell cycle kinetics through activation of the key cell cycle activators cyclin D1, cyclin A2, cyclin B1, and cdc25C. These findings support a role for Hedgehog in regulating the conversion from slow cycling stem cells to fast cycling transient amplifying progenitors that are closer to cell cycle exit.[Keywords: Hedgehog; retinal stem cells; Xenopus; zebrafish; cell cycle kinetics; cell cycle exit; cyclin; Cdc25C] Supplemental material is available at http://www.genesdev.org.

Graphical Abstract Highlights d YAP is required for Xenopus M€ uller glia proliferation in response to injury d YAP is required for mouse M€ uller glia exit from quiescence upon degeneration d YAP5SA reprograms mouse M€ uller glia into highly proliferative cells d YAP functionally interacts with EGFR signaling in M€ uller cells (M.P.) In BriefWhile fish and amphibian M€ uller cells behave as retinal stem cells upon injury, their regenerative potential is limited in mammals. Hamon et al. show that YAP is required for their cell-cycle re-entry in Xenopus and is sufficient in mouse to awake them from quiescence and trigger their proliferative response. SUMMARYContrasting with fish or amphibian, retinal regeneration from M€ uller glia is largely limited in mammals. In our quest toward the identification of molecular cues that may boost their stemness potential, we investigated the involvement of the Hippo pathway effector YAP (Yes-associated protein), which is upregulated in M€ uller cells following retinal injury. Conditional Yap deletion in mouse M€ uller cells prevents cell-cycle gene upregulation that normally accompanies reactive gliosis upon photoreceptor cell death. We further show that, in Xenopus, a species endowed with efficient regenerative capacity, YAP is required for their injury-dependent proliferative response. In the mouse retina, where M€ uller cells do not spontaneously proliferate, YAP overactivation is sufficient to induce their reprogramming into highly proliferative cells. Overall, we unravel a pivotal role for YAP in tuning M€ uller cell proliferative response to injury and highlight a YAP-EGFR (epidermal growth factor receptor) axis by which M€ uller cells exit their quiescence state, a critical step toward regeneration.

SUMMARYContinuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/b-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. and a transgenic male. The latter was selected beforehand as having a single transgene insertion site (as inferred by mendelian ratios in its progeny) in order to ensure homogeneous levels of GFP expression in the offspring. Construction of the LEF1-VP16 and LEF1-EnR transgenesis vectors has been described previously (Denayer et al., 2008) and transgenic X. tropicalis lines (LEF1-VP16Tg and LEF1-EnRTg) were generated as described (Sekkali et al., 2008). These constructs are fused with the dexamethasone-responsive hormone-binding domain of the human glucocorticoid receptor (GR). Expression constructs and morpholinospCS2- TCF3-VP16GR and pCS2-dnTCF3-GR (de Croze et al., 2011), pCS2-Ihh-CD2 [previously called Bhh (Locker et al., 2006)], pCS2-Smo-M2 , pCS2-cyclinA2 and pCS2-cdk2 (Decembrini et al., 2006) and pCS2-GFP (a gift from David Turner, University of Michigan, Ann Arbor, USA) were described previously. pCS2-Shh-CD2 and pCS2-Dhh-CD2 (previously called Chh) were generated by subcloning the N-terminal coding regions (devoid of the C-terminal cleavage product) of Shh and Dhh cDNAs (Ekker et al., 1995) Microinjection and in vivo DNA lipofectionCapped mRNAs encoding TCF3-VP16GR and GFP were transcribed from pCS2 plasmids after NotI digestion using the mMessage mMachine SP6 Kit (Ambion). Then, 400 pg of each mRNA was injected into two blastomeres of four-cell stage embryos. Gli3, Sfrp-1 or standard control morpholino oligonucleotides (Gene Tools) were injected into one blastomere at the one-cell stage (30 ng). Their efficacy was tested by analysing in vivo GFP fluorescence following co-injection of a chimeric GFP construct fused downstream of the morpholino-complementary sequence (supplementary material Fig. S1).Lipofection experiments were performed by cotransfecting the indicated pCS2 constructs together with pCS2-GFP at stage 18 into the presumptive region of the retina, as previously...

Background: In recent years, considerable knowledge has been gained on the molecular mechanisms underlying retinal cell fate specification. However, hitherto studies focused primarily on the six major retinal cell classes (five types of neurons of one type of glial cell), and paid little attention to the specification of different neuronal subtypes within the same cell class. In particular, the molecular machinery governing the specification of the two most abundant neurotransmitter phenotypes in the retina, GABAergic and glutamatergic, is largely unknown. In the spinal cord and cerebellum, the transcription factor Ptf1a is essential for GABAergic neuron production. In the mouse retina, Ptf1a has been shown to be involved in horizontal and most amacrine neurons differentiation.

The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability.DOI: http://dx.doi.org/10.7554/eLife.08488.001

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The Hedgehog (Hh) pathway regulates proliferation in a variety of tissues, however its specific effects on the cell cycle are unclear. During retinal proliferation in particular, the role of Hh has been controversial, with studies variably suggesting a stimulatory or an inhibitory effect on proliferation. Our recent data provide an underlying mechanism, which reconciles these different views. We showed that Hh signaling in the retina accelerates the G 1 and G 2 phases of the cell cycle and then pushes these rapidly dividing cells out of the cell cycle prematurely. From this and other evidence, we propose that Hh converts quiescent retinal stem cells into fast-cycling transient amplifying progenitors that are closer to cell cycle exit and differentiation. This is, we suggest, likely to be a general role of Hh in the nervous system and other tissues. This function of Hh in cell cycle kinetics and cell cycle exit may have implications for tumorigenesis and brain evolution.