1 The Wolfson Institute for Biomedical Research, University College London,
Gower Street, London WC1E 6BT, UK
2 MRC Laboratory for Molecular Cell Biology, University College London, Gower
Street, London WC1E 6BT, UK
3 Department of Biology, University College London, Gower Street, London WC1E
6BT, UK
Author for correspondence (e-mail:
m.fruttiger{at}ucl.ac.uk)
Accepted 2 February 2005
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SUMMARY |
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Key words: Astrocytes, Retina, Blood vessels, PDGF-A, Oxygen, Transgenic mice
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Introduction |
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Retinal astrocytes arise from a population of precursor cells in the optic
nerve head that express Pax2 and the platelet-derived growth factor receptor
alpha (PDGFR) (Mudhar et al.,
1993
; Otteson et al.,
1998
; Chu et al.,
2001
; Dakubo et al.,
2003
). As these astrocyte precursors invade the retina they
proliferate rapidly and start to express low levels of GFAP. As they mature,
retinal astrocytes become quiescent and display strong GFAP immunoreactivity
(Chu et al., 2001
;
Gariano, 2003
). PDGFR
continues to be expressed at all stages of maturation and is important for
retinal astrocyte proliferation and migration, being activated mainly by PDGFA
homodimers from retinal neurons (Mudhar et
al., 1993
; Fruttiger et al.,
1996
). In transgenic mice that overexpress PDGFA under the control
of the neuron-specific enolase gene promoter (NSE-PDGFA mice), the
number of retinal astrocytes is greatly increased, causing a proportional
overgrowth of the retinal vasculature
(Fruttiger et al., 1996
). This
indicates that PDGFA is normally a limiting factor for astrocyte proliferation
and subsequent angiogenesis. We speculated that if this limitation were
overcome by creating an autocrine mitogenic loop in astrocytes, then the
astrocytes might proliferate indefinitely and trigger uncontrolled
angiogenesis.
We tested this by engineering transgenic mice that express a PDGFA
transgene under control of the GFAP gene promoter
(GFAP-PDGFA mice) (Fruttiger et
al., 2000), so that astrocytes provide their own PDGFA. Despite
dramatic early hyperproliferation of astrocytes and blood vessels, cell
proliferation still slowed down and stopped within a week after birth
just as in wild type mice although final cell numbers were much higher
in the transgenics. We found no evidence that expression of the PDGFA
transgene or its receptor PDGFR
is extinguished in the transgenics, so
there must be other factors that limit retinal astrocyte proliferation even
when PDGFA is in excess.
In the present study, we tried to identify factors other than PDGF that limit retinal astrocyte proliferation. We noticed that, in neonatal mice, most astrocyte proliferation and VEGF expression occurs in the peripheral retina, ahead of the advancing vasculature. This suggested that blood vessels or their contents might negatively regulate both astrocyte proliferation and VEGF expression. We tested this by raising newborn mice in a high-oxygen atmosphere, which blocks development of retinal blood vessels and consequently leads to tissue hypoxia. This was accompanied by enhanced astrocyte proliferation and VEGF production, suggesting that blood vessels normally limit their own formation through a feedback signal(s) that serves to cut off the VEGF supply. The negative feedback could be mediated by a molecule(s) released from endothelial cells or the blood. We found that astrocyte proliferation and differentiation could be controlled in vitro by manipulating oxygen tension, raising the possibility that oxygen itself might be an active moiety.
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Materials and methods |
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Retinal whole-mount preparations
Eyes were fixed briefly in 2% (w/v) paraformaldehyde (PFA) in
phosphate-buffered salt solution (PBS) and then dissected in 2 xPBS.
Retinae were flattened after making radial incisions and then stored in
methanol at -20°C. After recovery from methanol, retinae were fixed for 5
minutes in 4% PFA in PBS and washed in PBS before further use.
Hypoxia staining with EF5
Mouse pups were injected with 50 µl of EF5 solution (10 mM in PBS) and
sacrificed 2 hours later. Retinae were prepared as described above but
methanol fixation was replaced with fixation in 4% (w/v) PFA in PBS for 10
minutes at room temperature.
Immunohistochemistry on retinal whole-mount preparations
Retinal wholemounts were incubated in PBS containing 5% fetal bovine serum
(FBS) and 0.5% Triton X-100 for one hour at room temperature. Incubations with
antibodies (diluted in PBS containing 1% FBS and 0.1% Triton X-100) were
carried out overnight at 4°C (primary antibodies) and for 3 hours at room
temperature (secondary antibodies). Antibodies used were mouse anti-GFAP
(clone G-A-5, Sigma), rabbit anti-GFAP (gift from Martin Raff), rabbit
anti-mouse collagen type IV (Biogenesis, Poole, UK), rabbit anti-Pax2 (Covance
Research Product, Princeton, USA), Cy3-conjugated ELK3.51 (monoclonal antibody
against EF5 adducts, provided by C. J. Koch, University of Pennsylvania,
Philadelphia, USA), Alexa Fluor 488 and 594 anti-rabbit IgG, and Alexa Fluor
594 anti-mouse IgG (Molecular Probes, Eugene, USA).
In situ hybridization of retinal wholemounts
Retinal wholemounts were prepared as described above, then digested
slightly for 5 minutes in proteinase K (80 µg/ml in PBS containing 1.3%
SDS) followed by fixation for 5 minutes in 4% PFA and 0.2% glutaraldehyde in
PBS. After a brief wash in PBS, retinae were pre-incubated in hybridization
buffer (Jensen and Wallace,
1997) for 10 minutes at 65°C and then incubated with RNA
probes diluted in hybridization buffer at 65°C overnight. Probe labeling
with digoxigenin-UTP and visualization of RNA hybrids with alkaline
phosphatase-conjugated anti-digoxigenin antibodies was carried out according
to the manufacturer's instructions using NBT/BCIP as a colour reagent (Roche
Diagnostics GmbH, Mannheim, Germany). For combined in situ hybridization and
immunohistochemistry, antibody labeling was performed after the in situ
hybridization protocol was completed. When double labeling for blood vessels
and BrdU incorporation, we first incubated retinae with an anti-collagen type
IV antibody, fixed with PFA (4% in PBS for 10 minutes), treated with 6 M HCl
containing 1% Triton X-100 for 45 minutes, and then stained with a mouse
anti-BrdU antibody (hybridoma supernatant BU209)
(Magaud et al., 1989
).
In situ hybridization on tissue sections
Whole eyes were fixed in 4% PFA in PBS overnight at 4°C, cryoprotected
in 20% (w/v) sucrose in PBS, embedded in OCT compound (Raymond and Lamb,
Sussex, UK), frozen and stored at -70°C. Cryosections (15 µm) were
collected on Vectabond (Vector Laboratories, Burlingham, USA)-coated slides
and air-dried for two hours. Digoxigenin-labeled probes diluted in
hybridization buffer were applied directly to sections and hybridized
overnight at 65°C. Subsequent visualization of RNA hybrids was carried out
as described above.
In situ hybridization on cultured cells
Cultured cells were fixed for 30 minutes in 4% PFA in 5% acetic acid,
dehydrated through an ascending series of alcohols and incubated in xylene for
10 minutes. After rehydrating in a descending alcohol series, cells were
digested for 10 minutes at 37°C in 0.1 M HCl containing 0.1% pepsin,
washed in PBS and post fixed in 1% PFA in PBS for 10 minutes. Cells were
dehydrated again in an ascending series of alcohols, air-dried and then
processed according to the same protocol described above for tissue
sections.
Cell culture
Retinae were dissected in PBS and then incubated for 30 minutes at 37°C
in Dulbecco's minimum essential medium (DMEM) containing 1% (w/v) collagenase
D (from Clostridium histolyticum) and 0.5 mg/ml papain. DNase (1
mg/ml) and 10% FBS were added before cells were dissociated by gentle
trituration through a fire-polished Pasteur pipette. Cells were washed and
re-dissociated in DMEM containing 10% FBS and then plated on poly D-lysine
coated coverslips in 24-well plates at a density of 1.5 x106
cells/well and incubated at 37°C, 5% CO2. For hypoxic culture,
an oxygen controller (PRO-OX 110; Biospherix, New York, USA) regulated
N2 influx into the tissue culture incubator to achieve 1.5%
O2.
Proliferation assays
Proliferation of cultured cells was assessed by adding BrdU to culture
medium (final concentration 10 µM) two hours before cells were fixed with
2% PFA in PBS. Retinal astrocytes were then stained using rabbit polyclonal
antibodies against GFAP or Pax2. Cells were further fixed using 4% PFA in PBS
for 10 minutes and then exposed to 6 M HCl containing 1% Triton X-100 for 45
minutes. The pH was neutralized with 0.1 M
Na2B4O7 (pH 8.5) and cells were incubated
with mouse anti-BrdU antibody (Magaud et
al., 1989), which was subsequently visualized with Alexa Fluor 488
anti-mouse IgG. In order to measure proliferation in vivo, mouse pups were
injected subcutaneously with BrdU (50 µg per gram body weight) two hours
before animals were sacrificed. Retinae where dissected, dissociated, cultured
overnight, fixed with 2% PFA in PBS and then stained as described above. For
each data point at least three coverslips were prepared and counted (
200
astrocytes/coverslip). BrdU-labeling index was calculated as the proportion of
GFAP- or Pax2-positive cells that were also BrdU positive.
Hyperoxia exposure in vivo
P0 mice with their mother were exposed to increased oxygen levels in a
modified, airtight cage. Oxygen concentrations were measured with a sensor
placed inside the cage and regulated by an oxygen controller (PRO-OX 110). A
small fan was installed inside the cage to mix inflowing pure oxygen with cage
air. Cage air was removed and replaced with normal room air at a constant rate
(approximately 6 cage volumes per hour) to prevent build-up of carbon dioxide
and humidity. Pups were removed from the hyperoxic chamber for injection with
BrdU and then returned for two hours before sacrificing.
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Results |
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Retinal astrocyte differentiation correlates with the presence of blood vessels
We assessed BrdU incorporation in retinal whole-mount preparations to gain
a better understanding of the spatial distribution of proliferating retinal
astrocytes in vivo. Retinal astrocytes were identified by in situ
hybridization for PDGFRa and blood vessels by immunolabeling for
collagen type IV. In P5 wild-type mice, retinal astrocytes in outer regions of
the retina, not yet occupied by the expanding vascular network, incorporated
BrdU readily (Fig. 2A,B),
whereas more central astrocytes, associated with blood vessels, did not
incorporate BrdU (Fig. 2A,C).
High magnification micrographs of the peripheral avascular area clearly reveal
that BrdU labeling is limited to retinal astrocytes, identified by
PDGFRa (Fig. 2E),
GFAP (Fig. 2F) and
VEGF (Fig. 2G) transcripts. We counted BrdU-positive cells in selected areas of five
different retinae and found that astrocytes in avascular regions undergo
proliferation, whereas they are virtually quiescent in vascularized regions
(Fig. 2D). Near the leading
edge of the vessel network (arrowheads in
Fig. 2A), cell proliferation
was pronounced but we were unable to distinguish between retinal astrocytes
and vascular cells due to the high cell density in this area. From the
distribution of PDGFRa-positive cells it appears that the retinal
astrocyte network is reorganized by the spreading vasculature as it advances
(Fig. 2A). A further effect on
retinal astrocytes, apparently caused by the presence of blood vessels, was a
marked downregulation of VEGF mRNA
(Fig. 2H). This is most likely
due to differences in tissue oxygenation in vascular and avascular areas of
the retina, as it is well known that VEGF transcripts are
specifically induced by hypoxia and suppressed by normoxia/hyperoxia.
Expression levels of PDGFRa in astrocytes are unaffected by the
presence or absence of blood vessels (Fig.
2A). However, we found an uneven distribution of GFAP
mRNA (Fig. 2I), which is low in
the avascular region but sharply increases in areas covered by vessels. GFAP
is upregulated during astrocyte differentiation in the retina
(Chu et al., 2001), and it
therefore appears that the proximity of blood vessels causes retinal
astrocytes to stop dividing and differentiate. This could be mediated by local
interactions between astrocytes and vascular cells, or by a diffusible agent
carried in the blood.
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An interesting exception to this rule is the small area in the immediate
vicinity of the optic nerve head. In this localized region astrocytes
downregulated VEGF, upregulated GFAP (arrows in
Fig. 3G,H) and did not
incorporate BrdU (Fig. 3F), in
stark contrast to astrocytes in the remainder of the retina. Thus, astrocytes
in this region behave as if they are experiencing normoxia, despite the
absence of retinal vessels. A likely explanation is that the optic nerve head
is oxygenated directly by the major hyaloid artery that passes along the optic
nerve, through the central retina to the lens
(Claxton and Fruttiger, 2003).
Note that this hyaloid artery is not visible in our whole-mount
preparations.
Retinal oxygen tension measured with the hypoxia marker EF5
In order to visualize oxygenation in whole-mount retinae we used the EF5
hypoxia marker system (Koch,
2002). Mouse pups were injected with the EF5 reagent, which is
reduced and covalently bound to cellular macromolecules under hypoxic
conditions. The drug-macromolecule adducts are then detected with specific
antibodies. In P6 mice the peripheral, not yet vascularized retina shows a
clear increase in EF5 staining when compared with the central, vascularized
retina (Fig. 4A,B), indicating
a low oxygen concentration in the avascular retina. In mice raised for 6 days
in 80% oxygen, the retina remains avascular and displays strong EF5 staining
throughout (Fig. 4C,D) with the
exception of a small circular area in the center (arrow in
Fig. 4D). Thus EF5 staining
confirms the existence of retinal hypoxia in a pattern that correlates closely
with VEGF mRNA distribution (compare
Fig. 3H).
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Discussion |
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In the present study we also present evidence suggesting that tissue
oxygenation might be the critical factor that drives differentiation of
retinal astrocytes. In vitro, low oxygen kept retinal astrocytes in a
proliferating, immature state, whereas increased oxygen inhibited their
proliferation and induced a more mature, GFAP-positive phenotype. From our
experiments it is not possible to determine whether oxygen levels affect
astrocytes directly or indirectly; for example, by stimulating the release of
differentiation factors from other retinal cells. It has been shown that
leukemia inhibitory factor (LIF) secreted from endothelial cells can induce
cultured, GFAP-negative astrocyte precursors from optic nerve to differentiate
into GFAP-positive astrocytes (Mi et al.,
2001). An analogous paracrine interaction between endothelial
cells and astrocytes in the retina is possible but, at least in our in vitro
experiment, seems unlikely because our retinal cultures contained very few, if
any, endothelial cells (as assessed by in situ hybridization with probes
against VEGFR1/2; data not shown). Moreover, we have failed to see any effects
of anti-LIF antibodies on retinal astrocytes in our culture system (data not
shown). However, we cannot exclude the possibility that other secreted factors
might mediate the effects of oxygen in our retinal cultures.
Manipulation of tissue oxygen levels in living mice is not as straightforward as in cell culture. It is well established that exposing newborn mouse pups to an 80% oxygen-containing atmosphere prevents retinal vessel development. The conventional explanation is that high atmospheric oxygen leads to high tissue oxygen levels, resulting in the downregulation of VEGF and consequent angiogenesis failure. This is a tenable explanation in situations where blood vessels have already become established; in mouse retinae one week or more after birth, for example. In such cases, the high atmospheric oxygen can be directly transported into the retina via the blood. However, the situation described in the present paper is very different; in newborn mice retinal vessels have not yet developed so the majority of the retina is never exposed to oxygenated blood and consequently remains hypoxic, despite higher than normal atmospheric oxygen.
This explains why VEGF is not downregulated across the retinae of newborn mice exposed to hyperoxia, but presents an apparent paradox as to why the high VEGF levels do not trigger angiogenesis as normal. The answer probably lies at the very centre of the retina around the hyaloid artery. Exposure to hyperoxia does cause local downregulation of VEGF mRNA around the hyaloid artery, because this central region becomes normoxic or hyperoxic, whereas the remainder of the (as yet avascular) retina remains hypoxic, as we confirmed by EF5 labeling. This presumably forms a hyperoxic barrier around the hyaloid vessel that prevents radial sprouting (angiogenesis).
It could be argued that the changes in astrocyte proliferation and GFAP
expression that we observe in normoxic and hyperoxic retinae result from
tissue oxygenation per se, rather than from cell-cell interactions between
vascular cells and astrocytes (Fig.
6). A link between oxygen and astrocyte differentiation was also
suggested by a previous study (Zhang et
al., 1999), but those authors proposed that oxygen was acting
indirectly via other retinal cells. As discussed above, our results do not
exclude such indirect mechanisms. It is also possible that multiple factors,
including oxygen, might mediate the differentiating effects of retinal blood
vessels.
Oxygen levels in most mammalian tissues range between 1% and 5% (8-38 mm of
Hg partial pressure) (Silver and
Erecinska, 1998). Therefore, the 20% O2 that cells
experience under standard cell culture conditions is highly non-physiological.
In general, culturing primary cells under hypoxic conditions might more
closely mimic their natural environment and allow them to behave more nearly
as they do in vivo. Under conditions of `physiological hypoxia' a multitude of
genes are differentially regulated
(Maltepe and Simon, 1998
), so
it is quite plausible that oxygen levels might affect more integrative aspects
of cell behaviour, such as proliferation and differentiation. There are some
concrete examples of this. Cultured CNS precursor cells and T lymphocytes both
display enhanced proliferation and survival under hypoxia
(Studer et al., 2000
;
Hale et al., 2002
), and
isolated neural crest stem cells show a wider differentiation potential
(Morrison et al., 2000
).
Cultured human cytotrophoblasts (specialized placental cells) proliferate in
low oxygen but differentiate in high oxygen, mimicking their response as they
migrate towards blood vessels in vivo
(Genbacev et al., 1997
;
Adelman et al., 2000
). Early
embryonic development is dominated by physiological hypoxia, whereas late
embryonic development is characterized by a mosaic of normoxic and hypoxic
regions giving rise to oxygen gradients
(Maltepe and Simon, 1998
).
Therefore, many cell types develop and differentiate in an environment of
changing oxygen concentrations and it seems likely that more examples will be
found of cells maturing in response to rising oxygen levels.
How do our studies of retinal astrocytes apply to other regions of the CNS?
Astrocytes in the gray matter of the brain or spinal cord are usually
GFAP-negative, apart from those that specifically contact blood vessels, which
express GFAP strongly (Bignami and Dahl,
1974). It is possible that astrocytes in contact with blood
vessels are induced to express GFAP by differentiation factors secreted by
vascular cells or, alternatively, because they experience higher oxygen
concentrations than astrocytes further away from vessels. Sequence analysis of
the GFAP promoter region reveals several hypoxia response elements
(not shown), but to date it has not been established how or whether these
affect GFAP transcription in vivo.
In future it will be interesting to explore the developmental relationships between astrocytes, blood vessels and oxygen in the brain and spinal cord. It is also an intriguing possibility that reciprocal feedback between vessel-inducing cells and their associated vessels might govern vascular development in other parts of the embryo outside the CNS.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adelman, D. M., Gertsenstein, M., Nagy, A., Simon, M. C. and
Maltepe, E. (2000). Placental cell fates are regulated in
vivo by HIF-mediated hypoxia responses. Genes Dev.
14,3191
-3203.
Alon, T., Hemo, I., Itin, A., Pe'er, J., Stone, J. and Keshet, E. (1995). Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat. Med. 1,1024 -1028.[CrossRef][Medline]
Bignami, A. and Dahl, D. (1974). Astrocyte-specific protein and radial glia in the cerebral cortex of newborn rat. Nature 252,55 -56.[Medline]
Brenner, M., Kisseberth, W. C., Su, Y., Besnard, F. and Messing, A. (1994). GFAP promoter directs astrocyte-specific expression in transgenic mice. J. Neurosci. 14,1030 -1037.[Abstract]
Chu, Y., Hughes, S. and Chan-Ling, T. (2001).
Differentiation and migration of astrocyte precursor cells and astrocytes in
human fetal retina: relevance to optic nerve coloboma. FASEB
J. 15,2013
-2015.
Claxton, S. and Fruttiger, M. (2003). Role of arteries in oxygen induced vaso-obliteration. Exp. Eye Res. 77,305 -311.[CrossRef][Medline]
Dakubo, G. D., Wang, Y. P., Mazerolle, C., Campsall, K.,
McMahon, A. P. and Wallace, V. A. (2003). Retinal ganglion
cell-derived sonic hedgehog signaling is required for optic disc and stalk
neuroepithelial cell development. Development
130,2967
-2980.
Dorrell, M. I., Aguilar, E. and Friedlander, M.
(2002). Retinal vascular development is mediated by endothelial
filopodia, a preexisting astrocytic template and specific R-cadherin adhesion.
Invest. Ophthalmol. Vis. Sci.
43,3500
-3510.
Fruttiger, M. (2002). Development of the mouse
retinal vasculature: angiogenesis versus vasculogenesis. Invest.
Ophthalmol. Vis. Sci. 43,522
-527.
Fruttiger, M., Calver, A. R., Kruger, W. H., Mudhar, H. S., Michalovich, D., Takakura, N., Nishikawa, S. and Richardson, W. D. (1996). PDGF mediates a neuron-astrocyte interaction in the developing retina. Neuron 17,1117 -1131.[CrossRef][Medline]
Fruttiger, M., Calver, A. R. and Richardson, W. D. (2000). Platelet-derived growth factor is constitutively secreted from neuronal cell bodies but not from axons. Curr. Biol. 10,1283 -1286.[CrossRef][Medline]
Gariano, R. F. (2003). Cellular mechanisms in retinal vascular development. Prog. Retin. Eye Res. 22,295 -306.[CrossRef][Medline]
Genbacev, O., Zhou, Y., Ludlow, J. W. and Fisher, S. J.
(1997). Regulation of human placental development by oxygen
tension. Science 277,1669
-1672.
Gerhardt, H., Golding, M., Fruttiger, M., Ruhrberg, C.,
Lundkvist, A., Abramsson, A., Jeltsch, M., Mitchell, C., Alitalo, K., Shima,
D. et al. (2003). VEGF guides angiogenic sprouting utilizing
endothelial tip cell filopodia. J. Cell Biol.
161,1163
-1177.
Hale, L. P., Braun, R. D., Gwinn, W. M., Greer, P. K. and
Dewhirst, M. W. (2002). Hypoxia in the thymus: role of oxygen
tension in thymocyte survival. Am. J. Physiol. Heart Circ.
Physiol. 282,H1467
-H1477.
Jensen, A. M. and Wallace, V. A. (1997).
Expression of Sonic hedgehog and its putative role as a precursor cell mitogen
in the developing mouse retina. Development
124,363
-371.
Jiang, B., Bezhadian, M. A. and Caldwell, R. B. (1995). Astrocytes modulate retinal vasculogenesis: effects on endothelial cell differentiation. Glia 15, 1-10.[Medline]
Koch, C. J. (2002). Measurement of absolute oxygen levels in cells and tissues using oxygen sensors and 2-nitroimidazole EF5. Meth. Enzymol. 352,3 -31.[Medline]
Ling, T. L., Mitrofanis, J. and Stone, J. (1989). Origin of retinal astrocytes in the rat: evidence of migration from the optic nerve. J. Comp. Neurol. 286,345 -352.[CrossRef][Medline]
Magaud, J. P., Sargent, I., Clarke, P. J., Ffrench, M., Rimokh, R. and Mason, D. Y. (1989). Double immunocytochemical labeling of cell and tissue samples with monoclonal anti-bromodeoxyuridine. J. Histochem. Cytochem. 37,1517 -1527.[Abstract]
Maltepe, E. and Simon, M. C. (1998). Oxygen, genes, and development: an analysis of the role of hypoxic gene regulation during murine vascular development. J. Mol. Med 76,391 -401.[CrossRef][Medline]
Mi, H., Haeberle, H. and Barres, B. A. (2001).
Induction of astrocyte differentiation by endothelial cells. J.
Neurosci. 21,1538
-1547.
Morrison, S. J., Csete, M., Groves, A. K., Melega, W., Wold, B.
and Anderson, D. J. (2000). Culture in reduced levels of
oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural
crest stem cells. J. Neurosci.
20,7370
-7376.
Mudhar, H. S., Pollock, R. A., Wang, C., Stiles, C. D. and
Richardson, W. D. (1993). PDGF and its receptors in the
developing rodent retina and optic nerve. Development
118,539
-552.
Otteson, D. C., Shelden, E., Jones, J. M., Kameoka, J. and Hitchcock, P. F. (1998). Pax2 expression and retinal morphogenesis in the normal and Krd mouse. Dev. Biol. 193,209 -224.[CrossRef][Medline]
Pierce, E. A., Foley, E. D. and Smith, L. E. (1996). Regulation of vascular endothelial growth factor by oxygen in a model of retinopathy of prematurity. Arch. Ophthalmol. 114,1219 -1228.[Abstract]
Pollock, R. A. and Richardson, W. D. (1992). The alternative-splice isoforms of the PDGF A-chain differ in their ability to associate with the extracellular matrix and to bind heparin in vitro. Growth Factors 7,267 -277.[Medline]
Provis, J. M., Leech, J., Diaz, C. M., Penfold, P. L., Stone, J. and Keshet, E. (1997). Development of the human retinal vasculature: cellular relations and VEGF expression. Exp. Eye Res. 65,555 -568.[CrossRef][Medline]
Sandercoe, T. M., Madigan, M. C., Billson, F. A., Penfold, P. L. and Provis, J. M. (1999). Astrocyte proliferation during development of the human retinal vasculature. Exp. Eye Res. 69,511 -523.[CrossRef][Medline]
Silver, I. and Erecinska, M. (1998). Oxygen and ion concentrations in normoxic and hypoxic brain cells. Adv. Exp. Med. Biol. 454,7 -16.[Medline]
Stone, J. and Dreher, Z. (1987). Relationship between astrocytes, ganglion cells and vasculature of the retina. J. Comp. Neurol. 255,35 -49.[CrossRef][Medline]
Stone, J., Itin, A., Alon, T., Pe'er, J., Gnessin, H., Chan-Ling, T. and Keshet, E. (1995). Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia. J. Neurosci. 15,4738 -4747.[Abstract]
Studer, L., Csete, M., Lee, S. H., Kabbani, N., Walikonis, J.,
Wold, B. and McKay, R. (2000). Enhanced proliferation,
survival, and dopaminergic differentiation of CNS precursors in lowered
oxygen. J. Neurosci. 20,7377
-7383.
van Heyningen, P., Calver, A. R. and Richardson, W. D. (2001). Control of progenitor cell number by mitogen supply and demand. Curr. Biol. 11,232 -241.[CrossRef][Medline]
Watanabe, T. and Raff, M. C. (1988). Retinal astrocytes are immigrants from the optic nerve. Nature 332,834 -837.[CrossRef][Medline]
Zhang, Y. and Stone, J. (1997). Role of astrocytes in the control of developing retinal vessels. Invest. Ophthalmol. Vis. Sci. 38,1653 -1666.[Abstract]
Zhang, Y., Porat, R. M., Alon, T., Keshet, E. and Stone, J. (1999). Tissue oxygen levels control astrocyte movement and differentiation in developing retina. Brain Res. Dev. Brain Res. 118,135 -145.[Medline]
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