Search

Epidermal growth factor (EGF) is known to promote proliferation of both retinal progenitors and Muller glia in vitro, but several questions remain concerning an in vivo role for this factor. In this study, we investigated whether the EGF receptor (EGFR) is necessary for the maintenance of normal levels of progenitor and Muller glial proliferation in vivo. Here, we show that (1) mice with homozygous deletion of the Egfr gene have reduced proliferation in late stages of retinal histogenesis, (2) EGF is mitogenic for Müller glia in vivo during the first two postnatal weeks in the rodent retina, (3) the effectiveness of EGF as a Müller glial mitogen declines in parallel with the decline in EGFR expression as the retina matures, and (4) following damage to the retina from continuous light exposure, EGFR expression is up-regulated in Müller glia to levels close to those in the neonatal retina, resulting in a renewed mitotic response to EGF. Together with previous results from other studies, these data indicate that the downregulation of a growth factor receptor is one mechanism by which glial cells maintain mitotic quiescence in the mature nervous system.

The optic vesicle gives rise to several very different epithelial tissues, including the neural retina, the pigmented epithelium, the iris, the ciliary epithelium of the ciliary body and the optic stalk. Retinal regeneration can arise from several different cellular sources; in some species, the process involves interconversion, or transdifferentiation, among cells of the different tissue types. Therefore, prior to a discussion of retinal regeneration, we will briefly discuss current knowledge about the influence of signaling molecules in cell fate determination in ocular tissues. Next, we will detail the evidence for neurogenesis in the mature retina. Lastly, we will describe various types of regenerative phenomena that occur in the retina, from complete regeneration of functional retina in fish and amphibians, to the more limited neuronal production that occurs in avian and mammalian retinas.

The cyclin-dependent kinase inhibitor protein, p27(Kip1), is necessary for the timing of cell cycle withdrawal that precedes terminal differentiation in oligodendrocytes of the optic nerve. Although p27(Kip1) is widely expressed in the developing central nervous system, it is not known whether this protein has a similar role in neuronal differentiation. To address this issue, we have examined the expression and function of p27(Kip1) in the developing retina, a well-characterized part of the central nervous system. p27(Kip1) is expressed in a pattern coincident with the onset of differentiation of most retinal cell types. In vitro analyses show that p27(Kip1) accumulation in retinal cells correlates with cell cycle withdrawal and differentiation, and when overexpressed, p27(Kip1) inhibits proliferation of the progenitor cells. Furthermore, the histogenesis of photoreceptors and Müller glia is extended in the retina of p27(Kip1)-deficient mice. Finally, we examined the adult retinal dysplasia in p27(Kip1)-deficient mice with cell-type-specific markers. Contrary to previous suggestions that the dysplasia is caused by excess production of photoreceptors, we suggest that the dysplasia is due to the displacement of reactive Müller glia into the layer of photoreceptor outer segments. These results demonstrate that p27(Kip1) is part of the molecular mechanism that controls the decision of multipotent central nervous system progenitors to withdraw from the cell cycle. Second, postmitotic Müller glia have a novel and intrinsic requirement for p27(Kip1) in maintaining their differentiated state.

The number of proliferating cells in the rodent retina declines dramatically after birth. To determine if extrinsic factors in the retinal micro-environment are responsible for this decline in proliferation, we established cultures of retinal progenitors or Muller glia, and added dissociated retinal neurons from older retinas. The older cells inhibited proliferation of progenitor cells and Muller glia. When these experiments were performed in the presence of TGF(beta)RII-Fc fusion protein, an inhibitor of TGF(beta) signaling, proliferation was restored. This suggests a retina-derived TGF(beta) signal is responsible for the developmental decline in retinal proliferation. TGFbeta receptors I and II are expressed in the retina and are located in nestin-positive progenitors early in development and glast-positive Muller glia later in development. RT-PCR and immunofluorescence data show TGF(beta)2 is the most highly expressed TGF(beta)ligand in the postnatal retina, and it is expressed by inner retinal neurons. Addition of either TGF(beta)1 or TGF(beta)2 to postnatal day 4 retinas significantly inhibited progenitor proliferation, while treatment of explanted postnatal day 6 retinas with TGF(beta) signaling inhibitors resulted in increased proliferation. Last, we tested the effects of TGF(beta) in vivo by injections of TGF(beta) signaling inhibitors: when TGF(beta) signaling is inhibited at postnatal day 5.5, proliferation is increased in the central retina; and when co-injected with EGF at postnatal day 10, TGF(beta)inhibitors stimulate Muller glial proliferation. In sum, these results show that retinal neurons produce a cytostatic TGF(beta) signal that maintains mitotic quiescence in the postnatal rat retina.