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Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: relevance to optic nerve coloboma ¹

Department of Anatomy and Histology, Institute for Biomedical Research, University of Sydney, Sydney, NSW 2006, Australia

²Correspondence: Department of Anatomy and Histology (F13), Room S466, Anderson-Stuart Bldg., University of Sydney, Sydney, NSW 2006, Australia. E-mail: tailoi{at}anatomy.usyd.edu.au

SPECIFIC AIMS

The aims of the present study were to 1) to demonstrate the presence of astrocyte precursor cells (APCs) in intact human retina; 2) characterize the various stages of astrocyte differentiation, the time course of the appearance of these cells, and the topography of their spread in this extension of the central nervous system (CNS); and 3) determine whether the expression of Pax2 is specific to cells of the astrocytic lineage in the developing and adult human retina.

PRINCIPAL FINDINGS

1. Pax2 expression is specific to cells of the astrocytic lineage in the developing and adult human retina
Triple-label immunohistochemistry with whole-mount preparations and transverse sections of fetal and adult human retinas revealed that antibodies to Pax2 labeled only cells that were positive for vimentin, glial fibrillary acidic protein (GFAP), or both of these markers of the astrocytic lineage. Double labeling with antibodies to CD34 and antibodies to Pax2 showed that Pax2 is not expressed by endothelial cells.

2. Pax2⁺, vimentin⁺, and GFAP^- APCs are present in the intact human retina
Pax2⁺, vimentin⁺, and GFAP^- APCs were detected in the human optic nerve head (ONH) and retina at 8 wk of gestation (WG). They reached the edge of the retina by 28 WG and persisted at reduced densities throughout the retina at 32 WG, the oldest fetal age examined. However, such APCs were not evident in adult human retinas derived from individuals more than 65 years of age.

3. Characterization of four distinct stages of astrocyte differentiation in the human retina
Three populations of Pax2⁺ cells were identified in the developing retina: 1) APCs, which are characterized by the antigenic phenotype Pax2⁺, vimentin⁺, and GFAP^-; 2) immature perinatal astrocytes, characterized as Pax2⁺, vimentin⁺, and GFAP⁺; and 3) mature perinatal astrocytes, characterized as Pax2⁺, vimentin^-, and GFAP⁺. Thus, the transition from an APC to an immature perinatal astrocyte in vivo is characterized by the onset of expression of GFAP and the transition from immature to mature perinatal astrocytes is characterized by the loss of expression of vimentin. Consistent with these designations, most of the committed astrocytes in the retina at 12 and 32 WG were immature perinatal astrocytes and mature perinatal astrocytes, respectively. All three of these stages of astrocyte differentiation in the developing human retina thus retain Pax2 expression.

APCs exhibited a Pax2⁺ soma and vimentin⁺, GFAP^- bipolar processes. Immature perinatal astrocytes at the leading edge of GFAP immunoreactivity possessed bipolar GFAP⁺ processes, whereas mature perinatal astrocytes exhibited multiple GFAP⁺ processes that were closely associated with nerve fiber bundles or blood vessels. The presence of substantial numbers of APCs in the human retina at 32 WG and the persistence of expression of Pax2 throughout human retinal development prompted us to examine the astrocytic lineage in the adult retina. Triple-label (Pax2-vimentin-GFAP) immunohistochemistry applied to retinal whole mounts and transverse sections prepared from three aged human eyes revealed a fourth stage of astrocyte maturation, characterized as Pax2^-, vimentin^-, and GFAP⁺ and designated adult astrocyte. These cells possessed multiple GFAP⁺ processes and were closely associated with blood vessels and, to a lesser extent, with nerve fiber bundles.

4. APCs migrate into the retina from the ONH and precede perinatal astrocytes
Consistent with the previous demonstration of Pax2 gene expression in the region of the human optic disk and optic nerve, Pax2 immunoreactivity was detected in the optic nerve and at the ONH at 8 WG (Fig. 1G , H ). In the ONH at this time, 34% of the cells were APCs, with the remainder being perinatal astrocytes. APCs were consistently detected in a small region ahead of the perinatal astrocytes during development of the human retina (Fig. 1A -F ). The spread of APCs and perinatal astrocytes was centered on the ONH; these cells followed a curved pattern of migration in the temporal retina, mimicking the pattern of nerve fiber bundles and blood vessels. Throughout the observation period, neither APCs nor committed astrocytes were detected in the incipient foveal zone. APCs migrated superficially over regions of the retina containing immature perinatal astrocytes (Fig. 1A -D ). Perinatal astrocytes were abundant in the central region of the retina (Fig. 1A -C ), whereas only APCs were evident more peripherally (Fig. 1D , E ). At the edge of the retina, no Pax2⁺ cells were evident at 24 to 26 WG (Fig. 1F ).

View larger version (83K): [in this window] [in a new window]   Figure 1. A–F) Cryostat section of a retina at 24 to 26 WG labeled with anti-Pax2 (red) and anti-GFAP (green) antibodies. Posterior (A, B), equatorial (C, D), and peripheral (E, F) regions are shown. APCs (arrows) and perinatal astrocytes (arrowheads) are indicated. A, B) Pax2+, GFAP- APCs were observed in the superficial layer of the nerve fiber layer. E) Only Pax2+, GFAP- APCs were apparent peripherally. F) At the retinal edge, neither Pax2+, GFAP- APCs nor Pax2+, GFAP+ perinatal astrocytes were detected. G) Photographic montage of the ONH region of a retina at 8 WG labeled with anti-Pax2 (red) and anti-GFAP (green). H) A tracing of the montage in panel G showing the location of individual Pax2+, GFAP- APCs and Pax2+, GFAP+ perinatal astrocytes. Two clusters of APCs and perinatal astrocytes are present at the ventricular zone surrounding the ONH. I, J) Adjacent sections of a retina at 24 to 26 WG double-labeled with anti-Pax2 (red) and anti-GFAP (green) (I) or stained with toluidine blue (J), showing the ventricular zone at high magnification. I) APCs (arrow) and perinatal astrocytes (arrowhead) were observed in the ventricular region. Autofluorescent granules were apparent in the retinal pigment epithelium (RPE).

Topographical analysis of the distribution of cells of the astrocytic lineage in the aged adult human retina revealed two such populations of cells: Pax2⁺, vimentin^-, and GFAP⁺ mature perinatal astrocytes were restricted to the region surrounding the ONH whereas Pax2^-, vimentin^-, and GFAP⁺ adult astrocytes were present throughout the retina with the exception of the foveal region. Thus, in the aged human retina, with the exception of cells in a small region surrounding the ONH, cells of the astrocytic lineage no longer express Pax2.

5. APCs and perinatal astrocytes are present in the ventricular zone surrounding the ONH of the human fetal retina
Transverse sections revealed a cluster of Pax2⁺ somas present in a small region surrounding the ONH at the ventricular surface of the developing (8 to 32 WG) human retina (Fig. 1G-J ). This cluster of Pax2⁺ somas was located at the innermost margin of the ventricular zone of the retina at its junction with the numerous optic nerve axons that exit the retina to form the optic nerve (Fig. 1I , J ). These cells comprised Pax2⁺, vimentin⁺, and GFAP^- APCs and Pax2⁺, vimentin⁺, and GFAP⁺ immature perinatal astrocytes (Fig. 1I ). Such cells were no longer present in the ventricular zone of the adult human retina.

CONCLUSIONS AND SIGNIFICANCE

Unlike the oligodendrocyte lineage, the early stages of astrocyte differentiation have not been well characterized. Given that the retina forms as an extension of the midbrain during embryonic development, any understanding of the developmental biology of astrocytes gained by studies of the human retina is applicable to other regions of the human CNS. We have now provided evidence for the existence of APCs in intact human retina. Previous studies presenting in vitro evidence for the existence of APCs in rats did not exclude the possibility that other cell types, especially oligodendrocytes, might have been generated by these cells in a permissive environment. Indeed, no in vivo or in vitro studies have previously demonstrated the existence of an astrocyte-restricted differentiation pathway for APCs, leaving open the possibility that astrocytes are derived from glial precursor cells that have the ability to give rise to different cell types. However, since the retina appears to be permissive to oligodendrocyte differentiation and the normal human retina is devoid of oligodendrocytes, it is likely that APCs in the human retina do only give rise to astrocytes in vivo.

We have identified three stages of differentiation (APCs and immature and mature perinatal astrocytes) for cells of the astrocytic lineage during development of the human retina, with a fourth stage (adult astrocytes) apparent in the adult retina. Figure 2 A presents a schematic representation of the characteristics of the four stages of astrocyte differentiation based on the results of the present study as well as on data provided by previous in vivo and in vitro studies. APCs and perinatal astrocytes are proliferative and migratory cells. Various growth factors, including ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, bone morphogenetic protein, epidermal growth factor, and transforming growth factor ß, induce APCs to differentiate and express GFAP, whereas platelet-derived growth factor enhances astrocyte proliferation. The identification of four distinct stages of astrocyte differentiation in the fetal and adult human CNS raises the possibility that, like neonatal and adult oligodendrocyte precursor cells, adult human astrocytes differ substantially from perinatal astrocytes in their functional properties (such as response to growth factors, cell cycle time, and pattern of cell division) and in their potential for repair of tissue damage. The migratory and proliferative potential of adult astrocytes remains unknown.

View larger version (42K): [in this window] [in a new window]   Figure 2. A) Schematic representation of the differentiation pathway of cells of the astrocytic lineage based on the results of in vivo and in vitro studies. Four stages of astrocyte differentiation have been characterized. B, C) Schematic representation of the structure of the human ONH and surrounding retina at 25 WG in a normal fetus (B) and in a fetus with impaired Pax2 expression (C). This model of optic nerve coloboma formation is based on studies of Pax2 mutants (murine and human) in which colobomas occur. APCs are shown as red somas and perinatal astrocytes are shown as red somas surrounded by green processes. NFL, nerve fiber layer; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.

Our study is the first to show cells of the astrocytic lineage (APCs and perinatal astrocytes) at the ventricular surface of the developing human retina. These cells appear to correspond to the narrow cuff of Pax2⁺ cells at the ventricular zone previously shown to encircle the ONH during mouse embryonic development. The location and timing of differentiation of these cells suggest that they might be responsible for the formation of Kuhnt’s intermediary tissue and Jacoby’s tissue, layers of GFAP⁺ astrocytes that separate the retinal ganglion cell axons from the neural retina and retinal pigment epithelium. Although it is well established that retinal astrocytes are immigrants from the optic nerve, the presence of APCs and perinatal astrocytes in the ventricular zone raises the possibility that a subpopulation of retinal astrocytes may also be derived from the neuroepithelium of the retina.

Our observation that Pax2 expression is specific to cells of the astrocytic lineage in intact human fetal retina also suggests that congenital optic nerve colobomas might result from aberrant astrocytic differentiation at the ventricular zone during embryonic development. Colobomas are caused by imperfect formation or closure of the fetal cleft of the optic vesicle during embryogenesis. Clinical studies have described the presence of a variable amount of glial tissue within the enlarged optic disk of optic nerve colobomas, especially the deposition of glial material at the cup margin.

Some human optic nerve colobomas are associated with abnormalities in Pax2 gene expression. During normal development, Pax2 mRNA is abundant in the human optic nerve and ONH during the period of expected closure of the choroidal fissure. In mice with impaired expression of Pax2, the tight band of Pax2⁺ cells that normally demarcates the retina from the ganglion cell axons as they exit the retina becomes dispersed over a larger region and the surrounding retinal tissue is no longer clearly separated from the axons, resulting in the spread of the axons over a much wider area. Schematic representations of the effect of such impaired Pax2 expression on the structure of the retina and ONH and the consequent optic nerve coloboma formation are presented in Fig. 2B , C . Our observation that the Pax2⁺ cells of this cuff in the human retina comprise APCs and perinatal astrocytes thus indicates that Pax2⁺ cells of the astrocytic lineage play a critical role both in delineating the axons of the ONH from the surrounding retinal tissue during development and in funneling and restricting the pathway of axonal exit from the retina.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0868fje ; to cite this article, use FASEB J. (July 9, 2001) 10.1096/fj.00-0868fje

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