Distinct Effects of Hedgehog Signaling on Neuronal Fate Specification and Cell Cycle Progression in the Embryonic Mouse Retina

Jules Stein Eye Institute and Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 06/2009; 29(21):6932-44. DOI: 10.1523/JNEUROSCI.0289-09.2009
Source: PubMed


Cell-extrinsic signals can profoundly influence the production of various neurons from common progenitors. Yet mechanisms by which extrinsic signals coordinate progenitor cell proliferation, cell cycle exit, and cell fate choices are not well understood. Here, we address whether Hedgehog (Hh) signals independently regulate progenitor proliferation and neuronal fate decisions in the embryonic mouse retina. Conditional ablation of the essential Hh signaling component Smoothened (Smo) in proliferating progenitors, rather than in nascent postmitotic neurons, leads to a dramatic increase of retinal ganglion cells (RGCs) and a mild increase of cone photoreceptor precursors without significantly affecting other early-born neuronal cell types. In addition, Smo-deficient progenitors exhibit aberrant expression of cell cycle regulators and delayed G(1)/S transition, especially during the late embryonic stages, resulting in a reduced progenitor pool by birth. Deficiency in Smo function also causes reduced expression of the basic helix-loop-helix transcription repressor Hes1 and preferential elevation of the proneural gene Math5. In Smo and Math5 double knock-out mutants, the enhanced RGC production observed in Smo-deficient retinas is abolished, whereas defects in the G(1)/S transition persist, suggesting that Math5 mediates the Hh effect on neuronal fate specification but not on cell proliferation. These findings demonstrate that Hh signals regulate progenitor pool expansion primarily by promoting cell cycle progression and influence cell cycle exit and neuronal fates by controlling specific proneural genes. Together, these distinct cellular effects of Hh signaling in neural progenitor cells coordinate a balanced production of diverse neuronal cell types.

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    • "HH is an established mitogen in the developing CNS, including the retina [51,52,81]. The deletion of Smoothened, an essential mediator of this signal-transduction pathway, from RPCs resulted in progenitor-pool depletion due to cell-cycle aberrations which included reduced expression of Ccnd1 and elevated expression of P27Kip1 [51,52]. "
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    ABSTRACT: The coupling between cell-cycle exit and onset of differentiation is a common feature throughout the developing nervous system, but the mechanisms that link these processes are mostly unknown. Although the transcription factor Pax6 has been implicated in both proliferation and differentiation of multiple regions within the central nervous system (CNS), its contribution to the transition between these successive states remains elusive. To gain insight into the role of Pax6 during the transition from proliferating progenitors to differentiating precursors, we investigated cell-cycle and transcriptomic changes occurring in Pax6 (-) retinal progenitor cells (RPCs). Our analyses revealed a unique cell-cycle phenotype of the Pax6-deficient RPCs, which included a reduced number of cells in the S phase, an increased number of cells exiting the cell cycle, and delayed differentiation kinetics of Pax6 (-) precursors. These alterations were accompanied by coexpression of factors that promote (Ccnd1, Ccnd2, Ccnd3) and inhibit (P27 (kip1) and P27 (kip2) ) the cell cycle. Further characterization of the changes in transcription profile of the Pax6-deficient RPCs revealed abrogated expression of multiple factors which are known to be involved in regulating proliferation of RPCs, including the transcription factors Vsx2, Nr2e1, Plagl1 and Hedgehog signaling. These findings provide novel insight into the molecular mechanism mediating the pleiotropic activity of Pax6 in RPCs. The results further suggest that rather than conveying a linear effect on RPCs, such as promoting their proliferation and inhibiting their differentiation, Pax6 regulates multiple transcriptional networks that function simultaneously, thereby conferring the capacity to proliferate, assume multiple cell fates and execute the differentiation program into retinal lineages.
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    • "FGF9 belongs to a different subfamily of FGF factors compared to bFGF (FGF2) and can inhibit the canonical Wnt pathway via upregulation of Dkk-1, a canonical Wnt antagonist, and regulate the transcription of Hedgehog targets patched homolog 1 (Ptch1) and glioma-associated zinc finger 1 (Gli1) independently of the Hedgehog ligand [77]. Both effects may promote NR differentiation [16,78]. Additional investigations are necessary to clearly delineate the role of FGF9 in NR differentiation. "
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    ABSTRACT: To examine the potential of NIH-maintained human embryonic stem cell (hESC) lines TE03 and UC06 to differentiate into retinal progenitor cells (hESC-RPCs) using the noggin/Dkk-1/IGF-1/FGF9 protocol. An additional goal is to examine the in vivo dynamics of maturation and retinal integration of subretinal and epiretinal (vitreous space) hESC-RPC grafts without immunosuppression. hESCs were neuralized in vitro with noggin for 2 weeks and expanded to derive neuroepithelial cells (hESC-neural precursors, NPs). Wnt (Integration 1 and wingless) blocking morphogens Dickkopf-1 (Dkk-1) and Insulin-like growth factor 1 (IGF-1) were used to direct NPs to a rostral neural fate, and fibroblast growth factor 9 (FGF9)/fibroblast growth factor-basic (bFGF) were added to bias the differentiation of developing anterior neuroectoderm cells to neural retina (NR) rather than retinal pigment epithelium (RPE). Cells were dissociated and grafted into the subretinal and epiretinal space of young adult (4-6-week-old) mice (C57BL/6J x129/Sv mixed background). Remaining cells were replated for (i) immunocytochemical analysis and (ii) used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Mice were sacrificed 3 weeks or 3 months after grafting, and the grafts were examined by histology and immunohistochemistry for survival of hESC-RPCs, presence of mature neuronal and retinal markers, and the dynamics of in vivo maturation and integration into the host retina. At the time of grafting, hESC-RPCs exhibited immature neural/neuronal immunophenotypes represented by nestin and neuronal class III β-tubulin, with about half of the cells positive for cell proliferation marker Kiel University -raised antibody number 67 (Ki67), and no recoverin-positive (recoverin [+]) cells. The grafted cells expressed eye field markers paired box 6 (PAX6), retina and anterior neural fold homeobox (RAX), sine oculis homeobox homolog 6 (SIX6), LIM homeobox 2 (LHX2), early NR markers (Ceh-10 homeodomain containing homolog [CHX10], achaete-scute complex homolog 1 [MASH1], mouse atonal homolog 5 [MATH5], neurogenic differentiation 1 [NEUROD1]), and some retinal cell fate markers (brain-specific homeobox/POU domain transcription factor 3B [BRN3B], prospero homeobox 1 [PROX1], and recoverin). The cells in the subretinal grafts matured to predominantly recoverin [+] phenotype by 3 months and survived in a xenogenic environment without immunosuppression as long as the blood-retinal barrier was not breached by the transplantation procedure. The epiretinal grafts survived but did not express markers of mature retinal cells. Retinal integration into the retinal ganglion cell (RGC) layer and the inner nuclear layer (INL) was efficient from the epiretinal but not subretinal grafts. The subretinal grafts showed limited ability to structurally integrate into the host retina and only in cases when NR was damaged during grafting. Only limited synaptogenesis and no tumorigenicity was observed in grafts. Our studies show that (i) immunosuppression is not mandatory to xenogenic graft survival in the retina, (ii) the subretinal but not the epiretinal niche can promote maturation of hESC-RPCs to photoreceptors, and (iii) the hESC-RPCs from epiretinal but not subretinal grafts can efficiently integrate into the RGC layer and INL. The latter could be of value for long-lasting neuroprotection of retina in some degenerative conditions and glaucoma. Overall, our results provide new insights into the technical aspects associated with cell-based therapy in the retina.
    Full-text · Article · Apr 2012 · Molecular vision
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    • "FGFs function as key initiators of RGC differentiation (Martinez-Morales et al., 2005), while Shh is critical for the propagation of the wave of RGC differentiation from the central retina out towards the periphery (Neumann and Nuesslein-Volhard, 2000; Esteve and Bovolenta, 2006). In contrast, RGC-derived Shh in the embryonic mouse retina maintains precursor proliferation and inhibits cell-cycle exit and neuronal differentiation (Wang et al., 2005; Sakagami et al., 2009). "
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