Vsx2/Chx10 ensures the correct timing and magnitude of Hedgehog signaling in the mouse retina

Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.     

Insulin-like growth factor-1 regulation of retinal progenitor cell proliferation and differentiation

Strategies to improve retinal progenitor cell (RPC) capacity to yield proliferative and multipotent pools of cells that can efficiently differentiate into retinal neurons, including photoreceptors, could be vital for cell therapy in retinal degenerative diseases. In this study, we found that insulin-like growth factor-1 (IGF-1) plays a role in the regulation of proliferation and differentiation of RPCs. Our results show that IGF-1 promotes RPC proliferation via IGF-1 receptors (IGF-1Rs), stimulating increased phosphorylation in the PI3K/Akt and MAPK/Erk pathways. An inhibitor experiment revealed that IGF-1-induced RPC proliferation was inhibited when the PI3K/Akt and MAPK/Erk pathways were blocked. Furthermore, under the condition of differentiation, IGF-1-pretreated RPCs prefer to differentiate into retinal neurons, including photoreceptors, in vitro, which is crucial for visual formation and visual restoration. These results demonstrate that IGF-1 accelerates the proliferation of RPCs and IGF-1 pretreated RPCs may have shown an increased potential for retinal neuron differentiation, providing a novel strategy for regulating the proliferation and differentiation of retinal progenitors in vitro and shedding light upon the application of RPCs in retinal cell therapy.     

Step-wise specification of retinal stem cells during normal embryogenesis

The specification of embryonic cells to produce the retina begins at early embryonic stages as a multi-step process that gradually restricts fate potentials. First, a subset of embryonic cells becomes competent to form retina by their lack of expression of endo-mesoderm-specifying genes. From these cells, a more restricted subset is biased to form retina by virtue of their close proximity to sources of bone morphogenetic protein antagonists during neural induction. During gastrulation, the definitive RSCs (retinal stem cells) are specified as the eye field by interactions with underlying mesoderm and the expression of a network of retina-specifying genes. As the eye field is transformed into the optic vesicle and optic cup, a heterogeneous population of RPCs (retinal progenitor cells) forms to give rise to the different domains of the retina: the optic stalk, retinal pigmented epithelium and neural retina. Further diversity of RPCs appears to occur under the influences of cell-cell interactions, cytokines and combinations of regulatory genes, leading to the differentiation of a multitude of different retinal cell types. This review examines what is known about each sequential step in retinal specification during normal vertebrate development, and how that knowledge will be important to understand how RSCs might be manipulated for regenerative therapies to treat retinal diseases.     

Ganglion cells are required for normal progenitor- cell proliferation but not cell-fate determination or patterning in the developing mouse retina

The vertebrate retina develops from an amorphous sheet of dividing retinal progenitor cells (RPCs) through a sequential process that culminates in an exquisitely patterned neural tissue. A current model for retinal development posits that sequential cell-type differentiation is the result of changes in the intrinsic competence state of multipotent RPCs as they advance in time and that the intrinsic changes are influenced by continuous changes in the extracellular environment. Although several studies support the proposition that newly differentiated cells alter the extrinsic state of the developing retina, it is still far from clear what role they play in modifying the extracellular environment and in influencing the properties of RPCs. Here, we specifically ablate retinal ganglion cells (RGCs) as they differentiate, and we determine the impact of RGC absence on retinal development. We find that RGCs are not essential for changing the competence of RPCs, but they are necessary for maintaining sufficient numbers of RPCs by regulating cell proliferation via growth factors. Intrinsic rather than extrinsic factors are likely to play the critical roles in determining retinal cell fate.     

Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs’ ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2‐Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4‐Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC‐pre‐treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF‐pre‐treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.     

Beta-catenin is essential for lamination but not neurogenesis in mouse retinal development

During vertebrate retinal development, the seven retinal cell types differentiate sequentially from a single population of retinal progenitor cells (RPCs) and organize themselves into a distinct laminar structure. The purpose of this study was to determine whether beta-catenin, which functions both as a nuclear effector for the canonical Wnt signaling pathway and as a regulator of cell adhesion, is required for retinal neurogenesis or lamination. We used the Cre-loxP system to either eliminate beta-catenin or to express a constitutively active form during retinal neurogenesis. Eliminating beta-catenin did not affect cell differentiation, but did result in the loss of the radial arrangement of RPCs and caused abnormal migration of differentiated neurons. As a result, the laminar structure was massively disrupted in beta-catenin-null retinas, although all retinal cell types still formed. In contrast to other neural tissues, eliminating beta-catenin did not significantly reduce the proliferation rate of RPCs; likewise, activating beta-catenin ectopically in RPCs did not result in overproliferation, but loss of neural retinal identity. These results indicate that beta-catenin is essential during retinal neurogenesis as a regulator of cell adhesion but not as a nuclear effector of the canonical Wnt signaling pathway. The results further imply that retinal lamination and retinal cell differentiation are genetically separable processes.     

The Retinal Homeobox (Rx) gene is necessary for retinal regeneration

The Retinal Homeobox (Rx) gene is essential for vertebrate eye development. Rx function is required for the specification and maintenance of retinal progenitor cells (RPCs). Loss of Rx function leads to a lack of eye development in a variety of species. Here we show that Rx function is also necessary during retinal regeneration. We performed a thorough characterization of retinal regeneration after partial retinal resection in pre-metamorphic Xenopus laevis. We show that after injury the wound is repopulated with retinal progenitor cells (RPCs) that express Rx and other RPC marker genes. We used an shRNA-based approach to specifically silence Rx expression in vivo in tadpoles. We found that loss of Rx function results in impaired retinal regeneration, including defects in the cells that repopulate the wound and the RPE at the wound site. We show that the regeneration defects can be rescued by provision of exogenous Rx. These results demonstrate for the first time that Rx, in addition to being essential during retinal development, also functions during retinal regeneration.     

Reconstruction of rat retinal progenitor cell lineages in vitro reveals a surprising degree of stochasticity in cell fate decisions

In vivo cell lineage-tracing studies in the vertebrate retina have revealed that the sizes and cellular compositions of retinal clones are highly variable. It has been challenging to ascertain whether this variability reflects distinct but reproducible lineages among many different retinal progenitor cells (RPCs) or is the product of stochastic fate decisions operating within a population of more equivalent RPCs. To begin to distinguish these possibilities, we developed a method for long-term videomicroscopy to follow the lineages of rat perinatal RPCs cultured at clonal density. In such cultures, cell-cell interactions between two different clones are eliminated and the extracellular environment is kept constant, allowing us to study the cell-intrinsic potential of a given RPC. Quantitative analysis of the reconstructed lineages showed that the mode of division of RPCs is strikingly consistent with a simple stochastic pattern of behavior in which the decision to multiply or differentiate is set by fixed probabilities. The variability seen in the composition and order of cell type genesis within clones is well described by assuming that each of the four different retinal cell types generated at this stage is chosen stochastically by differentiating neurons, with relative probabilities of each type set by their abundance in the mature retina. Although a few of the many possible combinations of cell types within clones occur at frequencies that are incompatible with a fully stochastic model, our results support the notion that stochasticity has a major role during retinal development and therefore possibly in other parts of the central nervous system.     

Toll-like receptor 4 restricts retinal progenitor cell proliferation

Retinal neurogenesis ceases by the early postnatal period, although retinal progenitor cells (RPCs) persist throughout life. In this study, we show that in the mammalian eye, the function of Toll-like receptor 4 (TLR4) extends beyond regulation of the innate immune response; it restricts RPC proliferation. In TLR4-deficient mice, enhanced proliferation of cells reminiscent of RPCs is evident during the early postnatal period. In vitro experiments demonstrate that TLR4 acts as an intrinsic regulator of RPC fate decision. Increased TLR4 expression in the eye correlates with the postnatal cessation of cell proliferation. However, deficient TLR4 expression is not sufficient to extend the proliferative period but rather contributes to resumption of proliferation in combination with growth factors. Proliferation in vivo is inhibited by both MyD88-dependent and -independent pathways, similar to the mechanisms activated by TLR4 in immune cells. Thus, our study attributes a novel role to TLR4 as a negative regulator of RPC proliferation.