Differentiation and migration of astrocyte precursor

cells and astrocytes in human fetal retina: relevance

to optic nerve coloboma1
YI CHU, SUZANNE HUGHES, AND TAILOI CHAN-LING2
Department of Anatomy and Histology, Institute for Biomedical Research, University of Sydney,
Sydney, NSW 2006, Australia
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
0892-6638/01/0015-2013 FASEB

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
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
2
Correspondence: Department of Anatomy and Histology
(F13), Room S466, Anderson-Stuart Bldg., University of Sydney,
Sydney, NSW 2006, Australia. E-mail: tailoi@anatomy.usyd.edu.au
2013

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. 1AF). 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

Figure 1. AF) 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).
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Vol. 15

September 2001

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.

regions of the retina containing immature perinatal

astrocytes (Fig. 1AD). Perinatal astrocytes were
abundant in the central region of the retina (Fig.
1AC), 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).
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.

The FASEB Journal

CHU ET AL.

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. 1GJ). 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 2A 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 , induce APCs to differentiate and express GFAP, whereas platelet-derived
growth factor enhances astrocyte proliferation. The
identification of four distinct stages of astrocyte differ-

ASTROCYTE PRECURSOR CELLS IN HUMAN RETINA

entiation 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.
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 Kuhnts intermediary tissue and Jacobys 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.

2015