Patterning of the circumferential marginal zone of progenitors in the chicken retina

Abstract

A circumferential marginal zone (CMZ) of retinal progenitors has been identified in most vertebrate classes, with the exception of mammals. Little is known about the formation of the CMZ during late stages of embryonic retinal histogenesis. Thus, the purpose of this study was to characterize the formation and patterning of the CMZ in the embryonic chicken retina. We identified progenitors by assaying for the expression of proliferating cell nuclear antigen (PCNA), N-cadherin and the nestin-related filament transitin, and newly generated cells by using BrdU-birthdating. We found that there is a gradual spatial restriction of progenitors into a discreet CMZ during late stages of embryonic development between E16 and hatching, at about E21. In addition, we found that retinal neurons remain immature for prolonged periods of time in far peripheral regions of the retina. Early markers of neuronal differentiation (such as HuC/D, calretinin and visinin) are expressed by neurons that are found directly adjacent to the CMZ. By contrast, genes (protein kinase C, calbindin, red/green opsin) that are expressed with a delay (7–10 days) after terminal mitosis in the central retina are not expressed until as many as 30 days after terminal mitosis in the far peripheral retina. We conclude that the neurons that are generated by late-stage CMZ progenitors differentiate much more slowly than neurons generated during early stages of retinal development. We propose that the microenvironment within the far peripheral retina at late stages of development permits the maintenance of a zone of progenitors and slows the differentiation of neurons.

Introduction

Post-embryonic retinal growth results from on-going neurogenesis and the addition of new neurons to the peripheral edge of the retina. A zone of proliferating retinal stem cells exists at the far peripheral edge of the retinas of fish, frogs and birds (reviewed by Fischer, 2005, Hitchcock et al., 2004, Otteson and Hitchcock, 2003, Reh and Levine, 1998). This zone of stem cells has been termed the ciliary or circumferential marginal zone (CMZ). In the normal mammalian retina, there is no evidence for the persistence of neural progenitors that are organized into a CMZ (Close et al., 2005, Kubota et al., 2002, Moshiri and Reh, 2004). The CMZ is a relatively narrow band (< 100 μm in diameter) of cells that line the periphery of the neural retina. It is located at the transition between the multilayered retina and the non-pigmented epithelium (NPE) of the ciliary body, a pseudostratified columnar monolayer of cells that lines the vitread surface of the ciliary body. The far periphery of the retina narrows and tapers down to the CMZ, which further tapers and is continuous with the NPE of the ciliary body. In the frog eye, Perron et al. (Perron et al., 1998) have demonstrated that there is a gradient of maturity that extends through the CMZ, with retinal stem cells residing in the most anterior region of the CMZ, restricted-fate progenitors residing in middle, and postmitotic differentiating neurons residing in the posterior region of the CMZ.

In comparison to the CMZ of frogs and fish, the avian CMZ adds relatively few retinal cells to the peripheral edge of the retina, but appears to persist into adulthood (Fischer and Reh, 2000, Kubota et al., 2002, Reh and Fischer, 2001). Although the CMZ of the chick can be discreetly identified with progenitor-specific markers such as transitin (Fischer and Omar, 2005), Notch1/cHairy (Fischer, 2005), and the combination of Pax6/Chx10 (Fischer and Reh, 2000), progenitor-like cells may extend into the NPE of the ciliary body and retain the ability to proliferate and generate new neurons (Fischer and Reh, 2003). Under normal conditions, the progenitors in the CMZ are relatively quiescent, but can be stimulated to proliferate by intraocular injections of insulin, insulin-like growth factor-I, epidermal growth factor or sonic hedgehog, and by increasing rates of ocular growth via form-deprivation (Fischer et al., 2002b, Fischer and Reh, 2000, Fischer and Reh, 2003, Kubota et al., 2002, Moshiri et al., 2005). Conversely, the proliferation of progenitors can be suppressed by intraocular injections of glucagon or glucagon-like peptide 1 (Fischer et al., 2005). The quiescent state of the postnatal CMZ may, in part, result from glucagon-mediated inhibitory input from a unique type of retinal neuron that has been termed “bullwhip cell” (Fischer et al., 2005, Fischer et al., 2006). Furthermore, under normal conditions, the progenitors in the postnatal avian CMZ do not generate photoreceptors (Fischer and Reh, 2000), or ganglion cells unless stimulated by the combination of insulin and fibroblast growth factor-2 (Fischer et al., 2002a).

Although the CMZ of the chick retina has been well described in the postnatal eye, nothing is known about the formation and patterning of the CMZ in the embryonic eye. Accordingly, the purpose of this study was to characterize the development of the CMZ in the embryonic chicken eye. We find that the CMZ is formed late during embryonic development and that there is a gradual restriction of progenitor cells to the CMZ during this time. In addition, we find that the differentiation of retinal neurons near the postnatal CMZ is much slower compared to that of neurons found in central regions of the retina. We propose that the microenvironment that permits the persistence of retinal progenitors in the CMZ also slows the differentiation of neurons in the periphery of the retina.