Program in Developmental Biology and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA
* Author for correspondence (e-mail: psoriano{at}fhcrc.org)
SUMMARY
Recent advances in genetic manipulation have greatly expanded our understanding of cellular responses to platelet-derived growth factors (PDGFs) during animal development. In addition to driving mesenchymal proliferation, PDGFs have been shown to direct the migration, differentiation and function of a variety of specialized mesenchymal and migratory cell types, both during development and in the adult animal. Furthermore, the availability of genomic sequence data has facilitated the identification of novel PDGF and PDGF receptor (PDGFR) family members in C. elegans, Drosophila, Xenopus, zebrafish and mouse. Early data from these different systems suggest that some functions of PDGFs have been evolutionarily conserved.
Introduction
Platelet-derived growth factor (PDGF) was first identified in a search for
serum factors that stimulate the proliferation of arterial smooth muscle cells
(Ross et al., 1974). Since
then, mammalian PDGFs have been extensively characterized in culture-based
assays, where they have been shown to drive cellular responses including
proliferation, survival, migration, and the deposition of extracellular matrix
(ECM) and tissue remodeling factors. Knockout studies have demonstrated that
many of these cellular responses to PDGFs are essential during mouse
development. The genes that encode two ligands, Pdgfa and
Pdgfb, and both receptors, PDGF receptor alpha and PDGF receptor beta
(Pdgfra, Pdgfrb), have been knocked out in the mouse. These studies
have demonstrated that PDGFB and PDGFRß are essential for the development
of support cells in the vasculature, whereas PDGFA and PDGFR
are more
broadly required during embryogenesis, with essential roles in numerous
contexts, including central nervous system, neural crest and organ development
(Levéen et al., 1994
;
Soriano, 1994
;
Boström et al., 1996
;
Soriano, 1997
;
Fruttiger et al., 1999
;
Karlsson et al., 1999
;
Gnessi et al., 2000
;
Karlsson et al., 2000
).
Because of the severe and pleiotropic phenotypes of Pdgfa and
Pdgfra knockout mouse embryos, many primary functions of PDGFs
remained elusive until being addressed in experiments using conditional gene
ablation and gain-of-function transgenics. Pdgfr signaling mutants
have also been generated in which specific tyrosine residues in the receptor
cytoplasmic domains have been mutated to phenylalanines. These mutations
disrupt the interactions of PDGFRs with individual cytoplasmic signaling
proteins and, in some cases, abrogate a subset of receptor functions
(Heuchel et al., 1999
;
Tallquist et al., 2000
;
Klinghoffer et al., 2002
) (M.
Tallquist and P.S., unpublished). Together, such in vivo studies have
demonstrated that the PDGFs perform distinct cellular roles at successive
stages of mouse embryogenesis. In many contexts, PDGFs are mitogenic during
early developmental stages, driving the proliferation of undifferentiated
mesenchyme and some progenitor populations (reviewed by
Betsholtz et al., 2001
). During
later maturation stages, PDGF signaling has been implicated in tissue
remodeling and cellular differentiation, and in inductive events involved in
patterning and morphogenesis. In mouse and Drosophila, PDGFs also
direct cell migration, both at short and long distances from signal
sources.
This review discusses the known roles of PDGFs in development, with emphasis on cellular responses to PDGFs and how they contribute to neural/oligodendrocyte development, vascular and hematopoietic development, neural crest cell development, organogenesis, somitogenesis and skeletal patterning. Although most published studies of PDGF functions in vivo have been performed in mouse, early studies of PDGF- and PDGFR-related proteins in other model organisms suggest that some known PDGF roles (e.g. in glial/neural development) are conserved from fly to man, whereas others (e.g. in the neural crest) are specific to, but conserved among, vertebrates (Table 1).
|
In both mouse and human, the PDGF signaling network consists of four
ligands, PDGFA-D, and two receptors, PDGFR and PDGFRß
(Fig. 1). All PDGFs function as
secreted, disulfide-linked homodimers, but only PDGFA and B can form
functional heterodimers. PDGFRs also function as homo- and heterodimers, and
in vitro assays have demonstrated that the ligands differ in their affinities
for the
,
ß and ßß receptors, as indicated
in Fig. 2
(Heldin and Westermark, 1999
;
Li et al., 2000
;
Bergsten et al., 2001
;
LaRochelle et al., 2001
). All
known PDGFs have characteristic `PDGF domains', which include eight conserved
cysteines that are involved in inter- and intramolecular bonds. However,
differences in structure and proteolytic processing segregate the ligands into
two subfamilies. PDGFA and B comprise one subfamily: the amino terminal
prodomains of these proteins are cleaved intracellularly so that they are
secreted in their active forms (Heldin and
Westermark, 1999
). In addition, PDGFB and one splice form of PDGFA
have negatively charged motifs near their carboxy termini that are cleaved
extracellularly. These `retention motifs' may interact with ECM components
and/or retain these ligands near their producing cells until being cleaved
(Heldin and Westermark, 1999
).
Unlike PDGFA and PDGFB, the C and D ligands are activated post-secretion by
cleavage of their N-terminal CUB domains, which are repeat regions first
identified in complement subcomponents C1r/C1s, sea urchin
uEGF and human BMP-1
(Bork, 1991
;
Li et al., 2000
;
Bergsten et al., 2001
). The
extracellular proteases that activate PDGFC and D in vivo have not been
identified, although plasmin can cleave and activate them in cell culture
(Li et al., 2000
;
Bergsten et al., 2001
).
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|
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PDGF roles in the development of neural and vascular support cells
Oligodendrocyte and neural development in the CNS
In the mammalian central nervous system (CNS), oligodendrocytes deposit an
insulating layer of myelin around neuronal projections; these myelin sheaths
are essential in facilitating neurotransmission. Oligodendrocytes
differentiate postnatally from PDGFR-expressing progenitor (O2A) cells,
which arise in the mouse around embryonic day 12.5 (E12.5) in the
periventricular zone of the neuroepithelium. From E12.5-E15.5, O2A cells
proliferate and migrate to fill the neural tube/spinal cord
(Calver et al., 1998
). During
this time, Pdgfa is expressed by neurons and astrocytes throughout
the spinal cord, and PDGFC is expressed in the floor plate and ventral horn
regions (Fig. 4A)
(Fruttiger et al., 1999
;
Aase et al., 2002
). Although
the role of PDGFC in the embryonic spinal cord is not yet known, the expansion
of the O2A population (E12.5-E15.5) is significantly impaired in spinal cords
of Pdgfa null and Pdgfra signaling mutant mouse embryos;
consequently, mutant pups exhibit CNS hypomyelination and tremor phenotypes
(Fruttiger et al., 1999
;
Klinghoffer et al., 2002
).
|
In Pdgfa-null mice, graded dysmyelination has been observed in the
brain and optic nerve: the most severely hypomyelinated regions are those
farthest from the origins of oligodendrocyte progenitors
(Fruttiger et al., 1999).
Similarly, in Pdgfra signaling mutants, O2A cells fail to reach the
dorsal region of the spinal cord, and the initial migration of these cells
away from the ventricular zone is delayed
(Klinghoffer et al., 2002
).
These observations suggest that the migration of oligodendrocytes or their
progenitors is impaired in the absence of normal PDGF signaling. PDGF is
capable of driving chemotactic migrations of oligodendrocytes and neural stem
cells in primary cultures
(Forsberg-Nilsson et al.,
1998
; Simpson and Armstrong,
1999
), but further studies are needed to determine whether this is
a primary physiological function of PDGF signaling, or whether the in vivo
observations reflect proliferation defects prior to migration.
Mutant analysis has not revealed roles for PDGF in mouse neurogenesis,
although PDGFR is expressed in some neuronal populations in the
developing CNS, and PDGFB and PDGFRß are expressed in postnatal neurons
(Vignais et al., 1995
;
Nait-Oumesmar et al., 1997
;
Fruttiger et al., 1999
;
Enge et al., 2003
). However,
preliminary evidence suggests that PDGF-related signals are involved in neural
and glial development in C. elegans and Drosophila. C.
elegans VER proteins are expressed by specific neurons and sheath cells
(analogous to glial cells), but the functions of VER signaling are not yet
known (Table 1)
(Popovici et al., 2002
). In
Drosophila, Pvr is expressed by the ventral midline glia, and
Pvf2 and Pvf3 are expressed in the ventral nerve cord
(Cho et al., 2002
).
Overexpression of Pvf1 in postmitotic neurons induces neuronal
pathfinding and synaptogenic defects
(Kraut et al., 2001
).
Functional studies in these organisms may help elucidate conserved roles of
PDGFs in neural development.
PDGF roles in vascular mural cell development
During angiogenesis, primitive vascular networks are remodeled through
endothelial sprouting, branching and pruning, and contractile mesenchymal
cells, known as mural cells, are recruited to coat nascent vessels. The two
major classes of mural cells are vascular smooth muscle cells (VSMCs) and
pericytes; these cells provide survival and antiproliferative factors that
stabilize nascent vascular endothelial sprouts
(Lindahl et al., 1997a;
Benjamin et al., 1998
;
Hellström et al., 2001
).
The development of vascular mural cells requires PDGFB/PDGFRß signaling
(Levéen et al., 1994
;
Soriano, 1994
;
Lindahl et al., 1997a
;
Hellström et al., 1999
).
In Pdgfb and Pdgfrb knockout mice, which die perinatally
with extensive hemorrhaging, numerous vessels lack or are incompletely covered
by mural cells (Levéen et al.,
1994
; Soriano,
1994
). Consequently, endothelial sprouts hyperproliferate and give
rise to dilated, ectopic capillaries that are unstable, hyperpermeable and
vulnerable to degeneration or regression
(Lindahl et al., 1997a
;
Hellström et al., 2001
;
Enge et al., 2002
).
In the mouse embryo, PDGFB secreted by vascular endothelial cells is a
chemotactic, and perhaps survival, signal for Pdgfrb-expressing VSMC
and pericyte progenitors as they leave the arterial walls and primitive plexa
to migrate along newly formed endothelial sprouts
(Fig. 4B)
(Lindahl et al., 1997a;
Hellström et al., 1999
).
There is also evidence that PDGFB signaling drives the proliferation of VSMC
and pericyte progenitors. In wild-type mice, Pdgfb is expressed at
sites of pericyte proliferation, and there is a twofold decrease in VSMC
proliferation around developing arteries in Pdgfb-null mice
(Hellström et al., 1999
).
Furthermore, there are reduced numbers of spinal cord pericytes in embryos
homozygous for Pdgfrb signaling mutant alleles, which could be
attributed to deficient pericyte proliferation or survival (M. Tallquist and
P.S., unpublished). In vitro, PDGFs (AB, BB, CC) can directly induce
angiogenic sprouting and branching of vascular endothelium
(Cao et al., 2002
), but this
role has not been demonstrated in vivo.
The analysis of chimeric mice that comprise both wild-type and
Pdgfrb-null cells has demonstrated that in a competitive in vivo
context, there is a selection for Pdgfrb-positive cells in all VSMCs
and pericytes examined (Crosby et al.,
1998; Lindahl et al.,
1998
). Although this suggests a requirement for PDGFRß in
these cells, observations in knockout embryos indicate that some VSMC and
pericyte lineages are not affected, or are only mildly affected, by the
disruption of PDGFB/PDGFRß signaling
(Lindahl et al., 1997a
;
Fruttiger et al., 1999
;
Hellström et al., 1999
).
For example, in Pdgfb-null animals, pericytes are present, albeit in
low numbers, in skeletal muscle, skin and adrenal gland; Itoh cells
(pericyte-like cells in the liver) and VSMCs around developing arteries appear
to develop normally in the absence of PDGFB/PDGFRß signaling
(Soriano, 1994
;
Lindahl et al., 1997a
;
Hellström et al., 1999
).
These results may reflect tissue-specific functions of other factors in mural
cell development. Although their functions in the vasculature are not known,
PDGFC and D are expressed in VSMCs and in connective tissue surrounding
arteries, respectively, and both are expressed by endothelial cells
(Uutela et al., 2001
).
PDGF/VEGF signaling in embryonic hematopoeitic cell migration
In Drosophila, PVR signaling is essential for the embryonic
migration of hemocytes, the precursors of the fly blood cell lineage
(Cho et al., 2002). These
cells arise as bilaterally symmetric clusters of mesoderm in the head region
of stage 8 Drosophila embryos, and subsequently undergo stereotyped
anterior, ventral and posterior migrations
(Fig. 5A). The posterior
migration requires PVR signaling: a mutation in Pvr blocks hemocyte
migration into the tail and results in their clustering in the head region
(Cho et al., 2002
). All three
Pvfs are expressed along the hemocyte migration route and must
simultaneously be knocked down to mimic the receptor mutant phenotype
(Cho et al., 2002
). However,
results from ectopic expression experiments indicate that the ligands differ
in their capacity to influence hemocyte behavior in vivo. For example,
hemocyte migration is re-directed to sites of high ectopic Pvf2, but
not Pvf1, expression (Cho et al.,
2002
). Similarly, overexpression of Pvf2, but not of
Pvf1, drives hemocyte hyperproliferation in Drosophila
larvae (Munier et al., 2002
).
PVF2 (and/or PVF3) may therefore be the relevant ligand(s) for hemocyte
development in vivo.
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PDGF functions in neural crest: proliferation, migration and tissue remodeling
In the mouse, PDGFR is required cell autonomously for the
development of a subset of non-neuronal neural crest cell derivatives in the
cardiac and cranial regions
(Morrison-Graham et al., 1992
;
Soriano, 1997
;
Tallquist and Soriano, 2003
).
Deletion of Pdgfra in the murine neural crest cell lineage leads to
defects in palatal closure and fusion, nasal and cardiac septation, and the
development of several bone and cartilage structures
(Morrison-Graham et al., 1992
;
Soriano, 1997
;
Tallquist and Soriano, 2003
).
Furthermore, thymus size is often reduced in the absence of PDGFR
signaling (Morrison-Graham et al.,
1992
; Tallquist and Soriano,
2003
). Homozygous Patch mutant embryos (which carry a
large genomic deletion that encompasses the Pdgfra gene) and
NCC-Pdgfra embryos (which have Pdgfra conditionally deleted
in neural crest cells) exhibit several cardiac septation and remodeling
defects similar to those observed in neural crest ablation studies
(Kirby et al., 1983
;
Kirby and Waldo, 1990
;
Morrison-Graham et al., 1992
;
Tallquist and Soriano, 2003
).
This implies that these Pdgfra mutants are deficient for functional
cardiac neural crest cells, though it is unclear whether the deficiency is in
the number of cells that reach target tissues, or in neural crest cell
differentiation or function at target sites. Pdgfa and Pdgfc
are highly expressed in neural crest target tissues in the mouse, including
the epithelial lining of the branchial arches and branchial pouches
(Orr-Urtreger and Lonai, 1992
;
Ding et al., 2000
;
Aase et al., 2002
). These
ligands may act as long-range migration cues or post-migratory signals for
neural crest cells in the cranial region. In Xenopus and zebrafish
embryos, Pdgfa and Pdgfra are similarly expressed in the
pharyngeal region and neural crest, respectively, indicating that PDGF roles
in neural crest are likely to be conserved among vertebrates
(Table 1)
(Ho et al., 1994
;
Liu et al., 2002a
;
Liu et al., 2002b
).
PDGFR signaling has been implicated in the migration and survival of
cranial neural crest cells. In explant experiments, PDGFA enhances neural
crest cell motility (without affecting proliferation) and stimulates cultured
neural crest cells to secrete matrix metalloproteinase 2 (MMP2) and its
activator, MT-MMP (Robbins et al.,
1999
; Li et al.,
2001
). MMP2 influences neural crest cell migration and may play a
role in tissue remodeling (Robbins et al.,
1999
). In Pdgfra-null embryos, increased apoptosis and
differences in ECM deposition have been observed along the neural crest
migratory pathway (Morrison-Graham et al.,
1992
; Soriano,
1997
). However, NCC-Pdgfra mutant embryos do not exhibit
defects in neural crest survival, migration or proliferation, and phenotypes
observed in these embryos suggest that PDGFR
is required for
postmigratory neural crest functions
(Tallquist and Soriano,
2003
). The discrepancy between the Pdgfra-null and
conditional-null data could reflect cell non-autonomous requirements for PDGF
signaling in neural crest cell migration and/or survival, or could be due to
differences in the precise location or stage at which the neural crest defects
were analyzed in the different studies.
PDGF roles in organogenesis
PDGFs play distinct roles at successive stages of mammalian organogenesis
(Table 2,
Fig. 6). During early stages,
PDGFs drive mesenchymal proliferation. For example, PDGFR signaling is
essential for interstitial cell proliferation in the early embryonic testis
and kidney, and for mesenchymal proliferation in early intestine, skin and
lung development (Karlsson et al.,
1999
; Karlsson et al.,
2000
; Li et al.,
2000
; Sun et al.,
2000
; Li and Hoyle,
2001
; Brennan et al.,
2003
). In each of these contexts, PDGFR
is broadly
expressed in the mesenchyme, and the mitogenic PDGF function is elicited by
paracrine signals from local epithelium. There is no evidence that PDGFs act
as long-range proliferative signals during development, and although ligands
and receptors are coexpressed in some cell types, the role(s) and regulation
of autocrine signaling in organogenesis are not yet understood.
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PDGF functions in the developing kidney
PDGFB/PDGFRß signaling is required for the development of capillary
tufts in glomeruli, which are filtration units in the kidney. In
Pdgfb and Pdgfrb knockout mice, these tufts either do not
form or consist of enlarged, unbranched capillary loops that lack mesangial
cells (pericyte-like cells surrounding glomerular capillaries)
(Levéen et al., 1994;
Soriano, 1994
). There is
evidence that Pdgfrb-positive mesenchymal progenitors give rise to
both endothelial and mesangial cells of glomerular capillary tufts, which are
thought to form by de novo vasculogenesis and angiogenesis.
Pdgfrb-expressing cells originate in the metanephric mesenchyme and
migrate, first to the nephron cleft (before glomerulogenesis) and then into
the glomerular space where vasculogenesis and angiogenesis take place (see
Fig. 6A)
(Hyink et al., 1996
;
Woolf and Loughna, 1998
;
Ricono et al., 2003
). In
Pdgfb- and Pdgfrb-null embryos, there is a reduction, and in
some cases a loss, of the Pdgfrb-positive mesenchymal population at
the nephron cleft. This suggests that although these cells are capable of
reaching the cleft, their migration or premigratory proliferation is impaired
(Levéen et al., 1994
;
Lindahl et al., 1998
).
PDGFB/PDGFRß signaling is not required for proliferation or survival of
the putative progenitor cells at the nephron cleft, but is absolutely required
for their migration into the glomerular space
(Fig. 6A) (Lindahl et al., 1998
;
Arar et al., 2000
;
Ricono et al., 2003
). Given
the angiogenic potential of PDGFs demonstrated in vitro
(Cao et al., 2002
), PDGFs may
also induce branching of the glomerular capillary endothelium.
PDGF functions in mouse and Drosophila gonad
development
PDGF signaling is required for the development of the interstitium and
vasculature in the mouse testis (Fig.
6A,B). Perhaps due to these roles, Pdgfra-null embryos
have enlarged testis cords, a phenotype also seen in wild-type embryonic
testes treated with PDGFR inhibitors
(Uzumcu et al., 2002;
Brennan et al., 2003
). Early
in embryonic testis development, Pdgfa, Pdgfb and Pdgfra are
expressed in scattered cells within the XY gonad, and Pdgfc and
Pdgfra are expressed at the coelomic surface and the
mesonephros/gonad boundary (Brennan et
al., 2003
). At this time, PDGFR
signaling is required for
the proliferation and/or differentiation of interstitial cells, including
fetal Leydig cells (Fig. 6B)
(Brennan et al., 2003
), which
are testosterone-producing interstitial cells that differentiate shortly after
testis cord formation. Culture-based studies indicate that PDGF signaling may
also be involved in the development or function of perimyoid cells (PMCs)
later in embryonic testis development. PMCs are smooth muscle-like
interstitial cells that associate tightly with testis cords and, together with
Sertoli cells, secrete components of the basement membrane that surrounds
testis cords (Hadley et al.,
1985
; Skinner et al.,
1985
). At the time of PMC differentiation, Pdgfa
expression becomes restricted to testis cords and Pdgfrs are
expressed by all interstitial cells
(Uzumcu et al., 2002
;
Brennan et al., 2003
). PDGF
stimulation of cultured PMCs induces the expression of smooth muscle-specific
genes, enhanced ECM production, cellular contraction and stress fiber
formation (Gnessi et al.,
1993
; Chiarenza et al.,
2000
). These in vitro findings may reflect in vivo roles of PDGFs
in PMC differentiation or function. In the postnatal testis, PDGFs continue to
be essential for interstitial development: PDGFA (which does not appear to be
essential in the embryonic testis) is required postnatally for adult Leydig
cell development, interstitial cell proliferation and completion of
spermatogenesis (Gnessi et al.,
2000
). The molecular mechanisms underlying these roles await
further investigation.
PDGF signaling is also essential for the development of the testis
vasculature in the mouse (Brennan et al.,
2003). The embryonic testis becomes vascularized through the
angiogenic branching of the coelomic vessel, which forms at the testis surface
and extends branches between the testis cords
(Brennan et al., 2002
).
Endothelial cells migrate from the mesonephros into the testis and contribute
to the coelomic vessel branches. This migration is thought to be driven by an
attractive signal from Sertoli cells
(Merchant-Larios and Moreno-Mendoza,
1998
; Nishino et al.,
2001
). PDGFR
signaling is required for both coelomic vessel
branching and endothelial migration (Fig.
6A) (Brennan et al.,
2003
). PDGF signals may directly induce angiogenic branching of
the coelomic vessel. However, co-culture experiments have demonstrated that
endothelial migration requires PDGFR
signaling specifically in the
testis (Brennan et al., 2003
),
suggesting that PDGFs are not themselves long-range chemoattractants for
endothelial cells, but are required upstream of the activation or secretion of
a different signaling protein.
In contrast to this indirect role of PDGF in cell migration during
mammalian testis development, PVF1 directly induces migration during the
development of the Drosophila egg chamber. In this context, PVF1 is
secreted by the oocyte and acts as a long range chemoattractant for border
cells, a cluster of somatic cells that express PVR
(Fig. 5B)
(Duchek et al., 2001;
McDonald et al., 2003
). In
response to a graded PVF1 signal, one border cell of the cluster sends out a
long cellular extension (LCE) towards the oocyte. The LCE adheres to nurse
cells along the migration route and guides the migration of the border cell
cluster from the anterior cortex of the egg chamber to its final localization
adjacent to the oocyte (Fulga and
Rørth, 2002
). PVR signaling is partially redundant with
EGFR (epidermal growth factor receptor) signaling in directing border cell
migration (Duchek et al.,
2001
). However, ligand specificity has been demonstrated in
misexpression experiments in which border cell migration is redirected to
sites of high PVF1, but not PVF2 or Gurken (an EGFR ligand), expression
(McDonald et al., 2003
). PVF1
is also required for the normal distribution of Drosophila E-Cadherin
in migrating border cells (McDonald et
al., 2003
). These cellular responses to a PVF1 gradient in the
developing egg chamber may represent common mechanisms by which PDGFs induce
and guide cell migration.
Lung and intestine development: signaling centers at sites of
epithelial folding?
Epithelia undergo patterned folding or invagination during the maturation
stages of mammalian lung, intestine and skin development. In each of these
organs, PDGF signaling is required for the development of mesenchymal cells
that express Pdgfra and localize at sites of future folding
(Fig. 6C). The roles of these
cells, and specifically of PDGF signaling to these cells, are unknown, but
phenotypic data suggest that they may play an active role in inducing or
regulating morphogenesis or differentiation.
In the mammalian lung, PDGFA/PDGFR signaling is required for
alveolar septation, a postnatal process whereby the air sac epithelium
invaginates to form septa and is lined by specialized matrix and mesenchymal
cells (Boström et al.,
1996
; Klinghoffer et al.,
2002
). This process depends on a Pdgfra-positive
mesenchymal population, the embryonic development of which requires PDGFA.
Pdgfra-positive cells originate subjacent to distal branches of
embryonic lung epithelium; these cells associate tightly with growing tubules
and may modulate embryonic lung growth and/or branching
(Sun et al., 2000
). Late in
gestation, Pdgfra-positive cells undergo PDGFA-dependent migrations,
dispersing as single cells to line prospective terminal sacs throughout the
lung (Lindahl et al., 1997b
).
These cells localize near sites of future septation at the onset of
alveogenesis (Boström et al.,
1996
; Lindahl et al.,
1997b
). Pdgfra-positive mesenchymal cells in the lung may
be precursors to alveolar myofibroblasts
(Lindahl et al., 1997b
), or
might provide signals that induce septum formation, matrix deposition or
differentiation (e.g. of alveolar smooth muscle cells) during alveogenesis.
Further studies are needed to distinguish between these models, and to
determine whether postnatal PDGFR
-mediated signals are required for
alveogenesis.
In the developing skin and intestine, postmitotic Pdgfra-positive
mesenchymal cells similarly migrate to form clusters that underlie sites of
future epithelial folding (Karlsson et
al., 1999; Karlsson et al.,
2000
). In the intestine, these cells are called villus cluster
cells and pericryptal fibroblasts. Although their functions in morphogenesis
are not known, phenotypic data suggest that these clusters provide instructive
or permissive signals for villus outgrowth
(Karlsson et al., 2000
). For
example, Pdgfa-null animals have fewer villus clusters, possibly due
to an earlier mitogenic role of PDGFA, and fewer villi extend into the
intestinal lumen (Karlsson et al.,
2000
). Furthermore, in Pdgfa- and Pdgfra-null
mice, villi are often pleated with aberrant distributions of differentiated
cell types. These phenotypes could be secondary to the loss of earlier roles
of PDGFs in villus cluster cells. However, mesenchymal cells from the clusters
migrate into growing villi during villus outgrowth and maintain
Pdgfra expression throughout intestinal development
(Karlsson et al., 2000
).
Pdgfa is not expressed in the villus epithelium
(Karlsson et al., 2000
), but
conditional inactivation of Pdgfra and/or Pdgfc in the
intestine may reveal PDGF roles in villus maturation.
PDGF signaling in somite and skeletal patterning
Phenotypic analysis of Pdgfra-null mouse embryos suggests that, as
in organogenesis, PDGFs play successive roles in somite and skeletal
development. PDGF signals have been implicated in driving somitic or
presomitic mesoderm proliferation: Pdgfra is expressed throughout
epithelial somites, and the somites of Pdgfra-null mouse embryos are
smaller than those of wild-type littermates
(Soriano, 1997). Later in
development, Pdgfra expression becomes restricted, with high
expression in sclerotome and lower expression in dermatome. Pdgfa and
Pdgfc are both expressed in the myotome
(Orr-Urtreger and Lonai, 1992
;
Ding et al., 2000
;
Aase et al., 2002
). In
Pdgfra-null embryos, myotome compartments in rostral somites are
abnormally shaped and are frequently fused with the myotome of adjacent
somites. These abnormalities are paralleled by later skeletal phenotypes,
which are, in many cases, characterized by the aberrant growth or fusion of
skeletal elements (Soriano,
1997
). Together, expression and phenotypic data suggest that
PDGFR
signaling is involved in feedback signaling between the myotome
and the sclerotome, although the specific cellular responses to PDGF signaling
during somitogenesis are unknown
(Soriano, 1997
;
Tallquist et al., 2000
).
Experimental data suggest that PDGFs induce chondrocyte differentiation
during the outgrowth of limbs and of somitederived skeletal elements (such as
the ribs). In somite micromass cultures, PDGF-AA and -BB are potent effectors
of chondrogenesis, driving chondrocyte maturation without inducing
proliferation (Tallquist et al.,
2000). Similarly, studies in the chick have demonstrated that
PDGF-AA induces cartilage formation in vivo and in limb bud micromass cultures
(Ataliotis, 2000
).
PDGFs and human disease
Pdgf and Pdgfr mutant mice exhibit several phenotypes
that are reminiscent of human diseases. These mice can serve as experimental
models in which to study the etiology, progression and treatment of these
diseases, and the contributions of different genes and/or pathways to disease
development. For example, Pdgfra mutant mice model neural crest
deficiency disorders, such as cardiac and nasal septation defects, cleft face
and cleft palate (Grüneberg and
Truslove, 1960;
Morrison-Graham et al., 1992
;
Soriano, 1997
;
Tallquist and Soriano, 2003
).
In addition, knockout and transgenic studies have highlighted two distinct
modes by which aberrant PDGF function can lead to the lung disease emphysema.
Pdgfa-null mice and Pdgfra signaling mutants that survive
past birth exhibit early postnatal emphysema as a result of failed alveolar
septation (Boström et al.,
1996
; Lindahl et al.,
1997b
; Sun et al.,
2000
; Klinghoffer et al.,
2002
). By contrast, transgenic overexpression of Pdgfb in
the lung gives rise to emphysema in adult mice; these animals have thickened
septa, as well as enlarged saccules, severe inflammation and fibrosis
(Hoyle et al., 1999
).
The relevance of these and other mouse mutant phenotypes to human disease
and development has been highlighted by findings of abnormal PDGF/PDGFR
expression or activity in human patients. A common phenotype in
Pdgfra-null mouse embryos is a failure in neural tube closure;
genetic studies have demonstrated that Pdgfra and Pax1
interact genetically in the development of spina bifida occulta
(Grüneberg and Truslove,
1960; Helwig et al.,
1995
; Soriano,
1997
; Payne et al.,
1997
; Joosten et al.,
1998
). In humans, there are common Pdgfra promoter
polymorphisms that underlie variation in transcriptional activity, and
specific haplotype combinations correlate with a predisposition to neural tube
defects (Joosten et al.,
2001
). Promoter haplotypes with higher Pdgfra
transcriptional activity are over-represented in patients with sporadic spina
bifida; conversely, homozygosity for a common promoter haplotype with low
transcriptional activity was not observed in any cases of sporadic spina
bifida, suggesting that this variant protects or selects against the
development of this condition (Joosten et
al., 2001
).
Although Pdgfb and Pdgfrb knockout mice die perinatally,
PDGFB roles in adult vascular, kidney and retina pathogenesis have been
elucidated through studies using conditional knockout mice, transgenic mice
and mice harboring mutations that alter PDGF activity without causing early
lethality. In the vasculature, Pdgfrb activation caused by loss of
the low density lipoprotein receptor-related protein (LRP1) in VSMCs
leads to aneurysms and the development of atherosclerotic lesions. These
vascular defects are associated with the disruption of the elastic lamina
around vessels, and with hyperproliferation of VSMCs
(Boucher et al., 2003). In
addition, mice that lack Pdgfb or Pdgfrb, as well as some
Pdgfrb signaling mutants, exhibit cardiac hypertrophy
(Levéen et al., 1994
;
Soriano, 1994
;
Klinghoffer et al., 2001
).
Whether this reflects a primary defect in the cardiomyocytes or a
physiological response to other vascular defects remains unclear. The form of
cardiac hypertrophy in Pdgfrb signaling mutants resembles that
commonly associated with hypertension
(Klinghoffer et al., 2001
).
However, hyperproliferation of cardiac fibroblasts was observed in transgenic
mice overexpressing Pdgfc, which also have cardiac hypertrophy
(Li et al., 2000
). The disease
states in the Pdgfb and Pdgfrb mutant mice may be
etiologically distinct from the hypertrophy in the Pdgfc
overexpression model.
The kidney and the retina are both particularly sensitive to mutations in
the Pdgfrb signaling domain: some signaling mutant mice (which
survive to adulthood) exhibit the hallmarks of glomerulosclerosis and
proliferative retinopathy (Klinghoffer et
al., 2001) (M. Tallquist and P.S., unpublished). In humans, PDGFs
have been shown to be upregulated in glomerulosclerosis, as well as in
diabetic and allograft-related nephropathies
(Langham et al.,
2003
; Eitner et al.,
2003
), and PDGF inhibitors can inhibit pathogenic mesenchymal
proliferation in the kidney (Savikko et
al., 2003
). PDGFA and PDGFB are also upregulated in human
patients with diabetic proliferative retinopathy
(Freyberger et al., 2000
), and
PDGF inhibition using a dominant-negative PDGFR
can inhibit the
progression of proliferative vitreoretinopathy in an ex vivo model
(Ikuno and Kazlauskas, 2002
).
In the mouse retina, proliferative disease can result from either excessive
PDGF in astrocytes or deficient PDGF in the vascular endothelium, both of
which result in a shortage of pericytes
(Fruttiger et al., 1996
;
Andrews et al., 1999
;
Klinghoffer et al., 2001
;
Enge et al., 2002
;
Forsberg-Nilsson et al.,
2003
). These responses to the up- and downregulation of PDGF
indicate that PDGF-directed retinopathy therapies will probably need to be
targeted in a cell type-(or PDGFR-) specific manner.
PDGFs have also been implicated in the etiology of human cancer. Many years
ago, the transforming gene in Simian Sarcoma Virus, sis, was found to
encode PDGFB (Doolittle et al.,
1983; Waterfield et al.,
1983
). Since then, PDGF hyperactivity has been observed in
invasive gastric carcinomas and gliomas, and in several other types of human
cancer (Nakamura et al., 1997
;
Hermanson et al., 1992
;
Hermanson et al., 1996
). In
human gastric cancers, PDGF has been found to be an effective prognostic
marker: high levels of PDGFA correlate with high grade carcinomas and reduced
patient survival (Katano et al.,
1998
). A recent study also identified Pdgfra-activating
mutations in a subset of human gastrointestinal stromal tumors, for which
Pdgfra may prove to be a useful molecular marker and therapeutic
target (Heinrich et al.,
2003
). Further studies of PDGF function in both normal and
diseased gastrointestinal tracts may shed light on how gastric tumors
originate and progress.
Both PDGFs and PDGFRs are upregulated in human gliomas and astrocytomas,
and Pdgfra expression levels are higher in more advanced forms of
gliomas than in less malignant glial tumors
(Hermanson et al., 1992;
Hermanson et al., 1996
).
Transgenic overexpression of PDGFB in neural progenitors or glial cells
induces the formation of oligodendrogliomas and oligoastrocytomas in the mouse
(Dai et al., 2001
). In these
mouse models, it was recently shown that Ink4a-Arf, a tumor
suppressor gene that is commonly mutated in high-grade human gliomas,
cooperates with PDGF in the development and malignant progression of gliomas
(Dai et al., 2001
). This is
one example of a disease model, generated by genetic manipulation of PDGF in
the mouse, which could prove useful in elucidating both the cellular roles of
PDGFs in tumorigenesis, and the way(s) in which PDGFs or PDGFRs interact with
other oncogenes and tumor suppressor genes in the progression of human
cancer.
Closing remarks
Over the next few years, our knowledge of PDGF-dependent developmental processes should be vastly expanded through functional studies of recently identified PDGF family members in various organisms, and through experiments in which multiple ligands or receptors are simultaneously disrupted. Because requirements for PDGF signaling in early development can obscure later roles, it will also be necessary to use conditional mutagenesis to elucidate the serial roles of PDGFs within specific developmental contexts. Although expression data suggest that some PDGF roles are conserved among different organisms, this needs to be investigated through functional studies. The different model systems each have their strengths that will be invaluable in elucidating how the PDGFs and receptors are regulated, the cellular mechanisms by which they exert their actions, and how they interact with other signaling systems during development. In addition to elucidating normal developmental functions of PDGFs, genetic manipulations of PDGF function have identified disease states that are induced and/or influenced by aberrant PDGF activity. These model systems are invaluable tools for investigating disease etiology, therapeutic approaches and the interactions of PDGFs with other genes that contribute to multigenic diseases. To address these problems effectively, we need to understand the specific cellular functions that are driven by PDGF signaling in normal development and physiology.
As the roles of recently identified factors become known, we will be able
to address the question of how receptor mutant phenotypes relate to those of
the ligands. In both flies and mammals, questions remain as to the genetic
interactions between PDGF ligands and their receptors. In the mouse, the
Pdgfrb and Pdgfb knockout phenotypes appear virtually the
same (Levéen et al.,
1994; Soriano,
1994
), suggesting that PDGFD may have non-essential or subtle
roles during embryogenesis. However, the Pdgfa- and
Pdgfra-null phenotypes differ dramatically in their severity:
Pdgfra-null embryos do not survive past E15, with most dying by
E11.5, whereas many Pdgfa-null mice survive past birth (M.
Hellström, C. Betsholtz and P.S., unpublished)
(Boström et al., 1996
;
Tallquist and Soriano, 2003
).
This discrepancy is likely to be due to PDGFR
-mediated functions in
response to PDGFB and PDGFC. Preliminary evidence indeed suggests that
Pdgfc-null embryos exhibit cleft palate and spina bifida, and die
perinatally. In addition, Pdgfa/Pdgfc double-null embryos
recapitulate phenotypes associated with Pdgfra deficiency (H. Ding
and A. Nagy, personal communication).
The early lethality of Pdgfra-knockout mice indicates that the
PDGFs play essential roles in early embryonic development that are not yet
fully understood. Studies in Xenopus have implicated PDGF signaling
in gastrulation, neural tube closure, and mesoderm adhesion or migration
during early embryonic development
(Ataliotis et al., 1995;
Symes and Mercola, 1996
).
Only a subset of the phenotypes observed in Xenopus experiments are
recapitulated in Pdgfra/Pdgfrb double-null mouse embryos, which
exhibit failed anterior neural fold closure but no gastrulation defects (P.S.,
unpublished results). This may reflect the functional divergence of PDGFs
between amniotic and anamniotic species. Recent studies have demonstrated that
PDGFR
and PDGFRß are essential for extraembryonic development in
the mouse. PDGFR
is expressed in the parietal endoderm (E8-E10) and in
cells lining the chorioallantoic plate (E9-E13). In wild-type embryos,
PDGFR
-expressing cells migrate from the periphery to populate the
chorioallantoic plate. This migration fails to occur in Pdgfra-null
embryos, and the chorioallantoic plate vasculature does not develop normally
(Hamilton et al., 2003
),
perhaps contributing to their early lethality. PDGFB/PDGFRß signaling is
required in extraembryonic tissues at later stages (E13-E17) for the
maturation of the vasculature in the labyrinthine layer of the placenta, where
fetal/maternal gas and nutrient exchange takes place. The labyrinthine layers
of Pdgfb- and Pdgfrb-null embryos have deficient numbers of
pericytes and trophoblasts, abnormally large vessels and a reduction in
vascular surface area (Ohlsson et al.,
1999
). It is not known how these extraembryonic vascular defects
impact embryonic development.
Another unresolved question is how PDGFs and PDGFRs are regulated at the
levels of transcription, splicing, and ligand maturation or cleavage.
Establishing what factors contribute to these different aspects of PDGF
regulation will help associate PDGF signaling with other genetic pathways. For
example, mouse PAX1 has been shown to regulate Pdgfra transcription
and to interact genetically with Pdgfra in mouse neural tube
development (Helwig et al.,
1995; Joosten et al.,
1998
). Alternative splicing of Pdgfa is conserved among
vertebrates, and in vitro studies suggest that this modulates the range of
PDGFA activity by dictating the usage of the retention motif
(Mercola et al., 1988
;
Heldin, 1998
;
Heldin and Westermark, 1999
;
Horiuchi et al., 2001
).
However, the in vivo function(s) and developmental utilization of PDGFA
variants and PDGF retention motifs have not yet been determined, nor is it
known which proteases cleave the retention motifs of PDGFA and B, or the CUB
domains of PDGFC and D, in vivo. These proteases define the diffusibility and
sites of action of PDGFs, and so studies of their localization and regulation
will greatly further our understanding of PDGF functional regulation in vivo.
It is not clear whether the activity of invertebrate ligands is similarly
regulated by cleavage of retention motifs or CUB-like domains. However,
Drosophila Pvf1 is subject to alternative splicing, and the two known
variants differ at their amino termini
(Cho et al., 2002
).
Splice-form-specific analyses of PDGFs, PVFs and PVR are needed to clarify how
alternative splicing modulates PDGF signaling in vivo.
To understand why cellular responses to PDGFs differ at distinct times or
locations in development, we need to determine what other signals influence
PDGF-mediated responses, and to identify transcriptional targets of PDGF
signaling in different cellular contexts. A cell's response to PDGF is
probably influenced by its expression of PDGF receptors and ligands as well as
by its differentiation state and local signaling environment. Different PDGF
ligands may drive distinct responses in some developmental contexts. However,
the response of a given cell to the same ligand may change over time because
of crosstalk with other signaling pathways. For example, a recent study
demonstrated that antagonistic crosstalk occurs between LRP1 and PDGFRß
signaling in the vasculature (Boucher et
al., 2003). In addition, PDGF signals might drive different
transcriptional responses in naive versus committed or differentiated cells
due to developmental changes in chromatin structure or the activity of
transcriptional regulatory factors. The identification of transcriptional
targets of distinct PDGFs in different contexts should shed light on the
mechanisms by which cellular responses to PDGFs are specified.
ACKNOWLEDGMENTS
We thank Christer Betsholtz, Rich Klinghoffer, Susan Parkhurst, Pernille Rørth, Michelle Tallquist and members of the Soriano lab for critical comments on the manuscript. We also thank Hao Ding, Andras Nagy and Pernille Rørth for allowing us to discuss their results prior to publication. R.H. is supported in part by a National Science Foundation Predoctoral Fellowship, and by a Public Health Service, National Research Service Award from the National Institute of General Medical Sciences. Work in the authors' laboratory is supported by grants from the National Institute of Child Health and Human Development.
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