Sherry Thurig

Retinal ganglion cell-derived sonic hedgehog locally controls proliferation and the timing of RGC development in the embryonic mouse retina

The timing of cell cycle exit and temporal changes in the developmental competence of precursor cells are key components for the establishment of the normal complement of cell types in the mammalian retina. The identity of cell extrinsic cues that control these processes is largely unknown. We showed previously in mouse retina that sonic hedgehog (Shh) signalling from retinal ganglion cells (RGCs) to retinal precursor cells (RPC) is required for the establishment of normal retinal organization. Here, we show that conditional ablation of Shh expression in the peripheral mouse results in a depletion of the RPC pool, owing to precocious cell-cycle exit and neuronal differentiation. These changes were correlated with the downregulation of cyclin D1 and Hes1 gene expression. Shh inactivation also results in an increase in RGC number owing to a bias of RPC towards RGC production. In contrast to zebrafish, where Shh signalling drives cell cycle exit and RGC development, our findings indicate that in the mouse retina Shh signalling is required to maintain RPC proliferation and to control the timing of RGC development.

Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity

Sonic hedgehog (Shh) is an indispensable, extrinsic cue that regulates progenitor and stem cell behavior in the developing and adult mammalian central nervous system. Here, we investigate the link between the Shh signaling pathway and Hes1, a classical Notch target. We show that Shh-driven stabilization of Hes1 is independent of Notch signaling and requires the Shh effector Gli2. We identify Gli2 as a primary mediator of this response by showing that Gli2 is required for Hh (Hedgehog)-dependent up-regulation of Hes1. We also show using chromatin immunoprecipitation that Gli2 binds to the Hes1 promoter, which suggests that Hes1 is a Hh-dependent direct target of Gli2 signaling. Finally, we show that Shh stimulation of progenitor proliferation and cell diversification requires Gli2 and Hes1 activity. This paper is the first demonstration of the mechanistic and functional link between Shh, Gli, and Hes1 in the regulation of progenitor cell behavior.

Control of glial precursor cell development in the mouse optic nerve by sonic hedgehog from retinal ganglion cells

The development of glial precursor cells in the mammalian optic nerve depends on retinal ganglion cell (RGC) axons, but the signals that mediate this neuron-to-glia interaction have not been fully characterized. Sonic hedgehog (Shh) is expressed by RGCs, and we showed previously that it is required for the specification of astrocyte lineage cells at the optic disc. To study the role of RGC-derived Shh on astrocyte development at later developmental stages, we generated mice with a conditional ablation of Shh in the peripheral retina and analyzed gene expression and glial cell development in the optic nerve. Astrocyte development was initiated in the optic nerves of these mutant mice; however, the expression of Hedgehog (Hh) target genes, Gli1 and Ptch1 and cell cycle genes, Ccnd1 and Cdc25b in the optic nerves were downregulated. Astrocyte proliferation was markedly reduced. Oligodendrocyte precursor cells were fewer in the optic nerves of mutant mice, possibly as a consequence of reduced secretion of growth factors by astrocytes. At a later developmental stage, optic nerve axons displayed signs of Wallerian degeneration, including reduction of astrocyte processes, degenerating glial cells and formation of distended axons. We also demonstrate that the Hh pathway can be activated in optic nerve-derived astrocytes in vitro, but fails to induce cell cycle gene expression and proliferation. RGC-derived Shh signalling isthus necessary in vivo for maintenance of astrocyte proliferation, affecting both axo-glial and normal glial cell development in the optic nerve.

Direct and indirect effects of hedgehog pathway activation in the mammalian retina

The morphogen Sonic hedgehog (Shh) is expressed by the projection neurons of the retina, retinal ganglion cells (RGCs) and promotes retinal precursor cell (RPC) proliferation. To distinguish between direct and indirect effects of Hedgehog (Hh) pathway activation in the perinatal mouse retina, we followed the fate of cells that expressed a constitutively active allele of Smoothened (SMO-M2), the signal transduction component of the Hh pathway. SMO-M2 expression promoted a cell-autonomous increase in CyclinD1 expression and RPC proliferation and promoted the development of cells with an inner nuclear layer identity. SMO-M2 expression also inhibited rhodopsin expression in uninfected cells, thus highlighting an unexpected non-cell autonomous effect of Hh pathway activation on photoreceptor development.

Suppressor of Fused Is Required to Maintain the Multipotency of Neural Progenitor Cells in the Retina

The morphogen sonic hedgehog (Shh) plays a crucial role in development of the CNS, including the neural retina. Suppressor of fused (Sufu) has been recently identified as a critical regulator of Hh signaling in mammals. However, the precise roles that Sufu plays in the regulation of proliferation and cell-fate decisions in neural progenitors is unknown. Here, we have addressed these questions by conditionally deleting Sufu in mouse multipotent retinal progenitor cells (RPCs). Sufu deletion in RPCs results in transient increases in Hh activity and proliferation followed by developmentally premature cell-cycle exit. Importantly, we demonstrate a novel role for Sufu in the maintenance of multipotency in RPCs. Sufu-null RPCs downregulate transcription factors required to specify or maintain RPC identity (Rax, Vsx2) and multipotency (Pax6) but continue to express the neural progenitor marker Sox2. These cells fail to express retinal lineage-specific transcription factors, such as Math5, and adopt an amacrine or horizontal cell fate at the expense of all other classes of retinal neurons. Genetic elimination of Gli2 in Sufu-null RPCs attenuates Hh pathway activity and restores multipotency in neural progenitors. These data provide novel evidence that Sufu-mediated antagonism of Hh/Gli2 signaling is required to maintain RPC multipotency and identity.

Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line.

We report successful retinal cone enrichment and transplantation using a novel cone-GFP reporter mouse line. Using the putative cone photoreceptor-enriched transcript Coiled-Coil Domain Containing 136 (Ccdc136) GFP-trapped allele, we monitored developmental reporter expression, facilitated the enrichment of cones, and evaluated transplanted GFP-labeled cones in wildtype and retinal degeneration mutant retinas. GFP reporter and endogenous Ccdc136 transcripts exhibit overlapping temporal and spatial expression patterns, both initiated in cone precursors of the embryonic retina and persisting to the adult stage in S and S/M opsin+ cones as well as rod bipolar cells. The trapped allele does not affect cone function or survival in the adult mutant retina. When comparing the integration of GFP+ embryonic cones and postnatal Nrl−/− ‘cods’ into retinas of adult wildtype and blind mice, both cell types integrated and exhibited a degree of morphological maturation that was dependent on donor age. These results demonstrate the amenability of the adult retina to cone transplantation using a novel transgenic resource that can advance therapeutic cone transplantation in models of age-related macular degeneration.

Erratum: Corrigendum: Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line

Scientific Reports 6: Article number: 22867 ; published online: 11 March 2016; updated: 22 April 2016 The original version of this Article contained a typographical error in the spelling of the author Yves De Repentigny, which was incorrectly given as Yves De Repentingy. This has now been corrected in the PDF and HTML versions of the Article.