3457
Development 127, 3457-3466 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
DEV4396
Abnormal gastrointestinal development in PDGF-A and PDGFR-α deficient
mice implicates a novel mesenchymal structure with putative instructive
properties in villus morphogenesis
Linda Karlsson1,*, Per Lindahl1, John K. Heath2 and Christer Betsholtz1
1Department of Medical
2School of Biosciences,
Biochemistry, Göteborg University, Medicinaregatan 9A, Box 440, SE 405 30 Göteborg, Sweden
University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
*Author for correspondence (e-mail: [email protected])
Accepted 1 June; published on WWW 20 July 2000
SUMMARY
Development of the gastrointestinal (GI) tract depends on
reciprocal epithelial-mesenchymal cell signaling. Here, we
demonstrate a role for platelet-derived growth factor-A
(PDGF-A) and its receptor, PDGFR-α, in this process. Mice
lacking PDGF-A or PDGFR-α were found to develop
an abnormal GI mucosal lining, including fewer and
misshapen villi and loss of pericryptal mesenchyme. Onset
of villus morphogenesis correlated with the formation of
clusters of PDGFR-α positive cells, ‘villus clusters’, which
remained located at the tip of the mesenchymal core of the
growing villus. Lack of PDGF-A or PDGFR-α resulted in
progressive depletion of PDGFR-α positive mesenchymal
cells, the formation of fewer villus clusters, and premature
expression of smooth muscle actin (SMA) in the villus
mesenchyme. We found that the villus clusters were
postmitotic, expressed BMP-2 and BMP-4, and that their
formation correlated with downregulated DNA synthesis in
adjacent intestinal epithelium. We propose a model in
which villus morphogenesis is initiated as a result of
aggregation of PDGFR-α positive cells into cell clusters
that subsequently function as mesenchymal centers of
signaling to the epithelium. The role of PDGF-A seems to
be to secure renewal of PDGFR-α positive cells when they
are consumed in the initial rounds of cluster formation.
INTRODUCTION
and the outer muscle layers. It is thought that while the
mesenchyme dictates the morphology and the cellular
organization of the digestive mucosa, the epithelial cells induce
the mesenchyme to develop the typical intestinal mesenchymal
structures (Kedinger et al., 1981; Duluc et al., 1994; reviewed
in Kedinger et al., 1998). Thus, development of the GI tract,
like other epithelial organ systems, depends on reciprocal
signaling between the epithelium and the mesenchyme
(reviewed in Birchmeier and Birchmeier, 1993).
The mechanisms involved in the inductive events of gut
development are poorly understood. However, expression
patterns and the results of gene targeting have implicated a
variety of signaling molecules and transcription factors in GI
development. Growth and differentiation factors implicated in
different aspects of GI development include members of the
fibroblast growth factor (FGF) family (Colvin et al., 1999; Jung
et al., 1999), epidermal growth factor (EGF) family (Luetteke
et al., 1994; Miettinen et al., 1995), transforming growth
factor-β (TGF-β) (Kaartinen et al., 1995), insulin-like growth
factors (IGF)-1 and -2 (Liu et al., 1993), hepatocyte growth
factor (HGF)/scatter factor (Sonnenberg et al., 1993; Schmidt
et al., 1995; Uehara et al., 1995; Kermorgant et al., 1997), sonic
and indian hedgehog (Bitgood and McMahon, 1995; Apelqvist
et al., 1997; Roberts et al., 1995), wnts and bone
The fully developed gastrointestinal (GI) tract displays a high
degree of organization at both structural and cellular levels.
Villus and crypt morphologies vary between different regions
of the intestine, but are in any selected region regularly spaced
and uniform in shape. Four component cell lineages are rapidly
renewed in the intestinal epithelium through a geographically
orchestrated sequence of proliferation, commitment and
migration-associated differentiation. The connective tissue
core of the villus is also highly organized, containing
subepithelial smooth muscle cells, blood vessels and nerve
fibers. Formation of the gastrointestinal (GI) tract starts during
development by the association of the visceral endoderm with
the splanchnic mesoderm. Intestinal organogenesis proceeds as
a proximal-to-distal wave of morphogenetic events (Kaufman
and Bard, 1999). First, the pseudostratified endoderm
differentiates into a single-layered epithelium. Secondly, the
epithelium folds to form intestinal villi and becomes
compartmentalized into differentiating villus and proliferating
intervillus epithelium. Basal crypts subsequently form by
reshaping of the intervillus epithelium (reviewed in Gordon
and Hermiston, 1994). In parallel, the GI tract mesenchyme
differentiates into mucosal and submucosal connective tissue,
Key words: Intestinal villus, Development, Platelet-derived growth
factor, Bone morphogenetic protein, Epithelial-mesenchymal
interaction
3458 L. Karlsson and others
morphogenetic proteins (BMPs) (Bitgood and McMahon,
1995; Simon and Gordon, 1995; Roberts et al., 1995).
Knowledge of the GI cellular functions controlled by these
factors is, however, limited (reviewed in Traber and Wu, 1995
and Montgomery et al., 1999).
In this paper we investigate the action of platelet derived
growth factor-A (PDGF-A) and the PDGF α-receptor
(PDGFR-α) in the development of the GI tract by analysis of
PDGF-A−/− and PDGFR-α−/− mice (Boström et al., 1996;
Soriano, 1997). The platelet-derived growth factor (PDGF)
family of ligands and receptors are known to have critical roles
in a number of developmental processes. The three PDGF
gene products, PDGF-A, PDGF-B (reviewed in Heldin, 1992)
and PDGF-C (Li et al., 2000), signal through two related
receptor tyrosine kinases, PDGFR-α and PDGFR-β. Gene
knockout studies have already established roles for PDGF-A/R-α signaling in the development of lung alveoli (Boström et
al., 1996; Lindahl et al., 1997a), central nervous system
oligodendrocytes (Calver et al., 1998; Fruttiger et al., 1999),
hair follicles (Karlsson et al., 1999) and neural crest-derived
mesenchyme (Soriano, 1997). PDGF-B and PDGFR-β
signaling has proven critical in the development of the
vascular wall (Levéen et al., 1994; Soriano, 1994; Lindahl et
al., 1997b, 1998; Hellström et al., 1999). Here we show that
PDGF-A and its receptor, PDGFR-α, are necessary for the
correct structuring of the mucosal lining of the GI tract. Our
data suggest that PDGF-A, acting by paracrine signaling
through PDGFR-α, controls the multiplication and
differentiation of a subset of the mucosal mesenchymal cells.
This activity of PDGF-A is required for coordinated GI villus
morphogenesis.
RESULTS
Abnormal intestinal villi in PDGF-A−/− mice
Intestinal villus formation starts around E14 and proceeds until
postnatal day 28 (P28) (Simon and Gordon, 1995). Dissection
of the PDGF-A−/− GI tract at different postnatal ages (P10 until
P42) revealed an abnormal morphology of the mucosal lining.
The abnormality was most prominent in the upper small
intestine, but extended throughout the GI tract. In the small
intestine, villi were lower in abundance, irregular in length,
thickness and spacing, and were sometimes branched (Figs 1A,
2A-F). The reduction in villus number in PDGF-A−/− mutants
was estimated to approximately 45% at P16-19 (Table 1). The
PDGF-A−/− large intestine displayed abnormally shaped folds
and a 30% reduction in the number of crypts at P16 (Table 1
and Figs 1B, 3A-F).
Histological analysis revealed tissue disorganization in the
PDGF-A−/− small (Fig. 2) and large intestines (Fig. 3). A
typical feature of the PDGF-A−/− villi in the small intestine was
the presence of epithelial pleats (Fig. 2B,F, arrows). These
pleats were not misplaced crypts as they lacked BrdU-labeled
cells (Fig. 2F). In the PDGF-A−/− colon, folds contained fewer
cuffs (Fig. 3). No individual epithelial subtypes were missing
in PDGF-A−/− intestines and cellular morphologies appeared
MATERIALS AND METHODS
PDGF-A+/− mice (Boström et al., 1996) and PDGFR-α+/− mice
(Soriano, 1997) were bred as 129/C57BL6 hybrids. Heterozygotes
were intercrossed to produce homozygous mutants. In C57BL6/129
hybrid genetic background, PDGF-A−/− mice display a range of
survival restriction points, ranging between approximately 10 days
post coitum (E10) and 6 weeks of postnatal age (P42). In this study,
PDGF-A−/− mice surviving the E10 restriction point were subject to
analysis of the GI tract.
Standard
histological
techniques
were
used.
For
immunohistochemistry (IHC), the following antibodies were used:
anti-α-smooth muscle actin (SMA) (clone 1A4, Dako); biotinconjugated anti-mouse IgM (Dako); anti-BrdU (#347580, Becton
Dickinson); biotin-conjugated rabbit anti-mouse Ig (Dako). Nonradioactive in situ hybridization was performed as described by
Boström et al. (1996) and combined with IHC as described by
Karlsson et al. (1999). PDGF-A and PDGFR-α probes were
generated as described previously (Boström et al., 1996). Bmp2 and
Bmp4 probes were transcribed from 1.2 and 1.0 kb mouse cDNA
fragments provided by Dr A. McMahon. For BrdU labeling, BrdU
(Sigma; 100 mg/g body mass) was injected intraperitoneally to
pregnant female mice or postnatal pups. Injected animals were killed
2 hours later, and tissues or embryos were fixed and processed for
IHC.
The number of villi, crypts and villus clusters were counted on
sections and related to the perimeter. The perimeter was measured
using Easy Image Measurement software (Bergstrom Instruments)
and expressed as arbitrary units. The results were statistically
analyzed using the Student’s two-tailed t-test.
Fig. 1. Abnormal intestinal mucosa in PDGF-A−/− mice. Dissection
views of PDGF-A+/− (left) and PDGF-A−/− (right) upper small
intestine (A) and colon (B) at postnatal day 25 revealed abnormal
mucosal lining. The upper small intestine of PDGF-A−/− mice
showed a disorganized mucosa with fewer, differently shaped villi
(arrows in A point to villi that are thicker and longer than normal).
The colon of PDGF-A mutants had abnormally shaped cuffs (arrow
in B).
PDGF-A in gut development 3459
largely normal; however, some cell types were abnormally
represented or distributed. In particular, the submucosal
mesenchyme contained a reduced amount of cells, especially
in older PDGF-A−/− pups (>P30) in which there was an almost
complete lack of pericryptal fibroblasts in the small intestine,
bringing the crypt epithelium in apposition to the smooth
muscle cell wall (Fig. 2C,D, arrows). Also in the colon folds,
the submucosal mesenchyme appeared more sparse (Fig. 3C,D,
arrows). In particular, α-smooth muscle actin (SMA) labeled
mesenchyme was thinner and showed abnormal distribution
in PDGF-A−/− cuffs (Fig. 3E,F). In addition, goblet cells
Fig. 2. Histological abnormalities of the upper small intestine in
PDGF-A−/− mice. (A-D) Hematoxylin/Eosin staining at P25 revealed
a lower abundance and disturbed morphology of villi in PDGF-A−/−
(B) compared to wild-type (A) intestine, including epithelial pleats
along villi (arrows in B,F). (C,D) The submucosal mesenchyme
(indicated by arrows) was reduced in thickness in PDGF-A−/− (D)
compared to wild-type (C) intestine. (E,F) Staining for goblet cells
(blue) using Alcian Blue/Fast Red at P42 revealed a lower abundance
of such cells in the PDGF-A−/− small intestine. (G,H) BrdU-labeling,
detected with FITC (yellow) and counterstained with propidium
iodide (red), at P10 showed that epithelial proliferation was
associated with crypts and not with villus pleats.
were reduced in number in the PDGF-A−/− small intestine
(Fig. 2E,F) and abnormally distributed in the PDGF-A−/− colon
cuffs (Fig. 3C,D).
PDGF-A and PDGFR-α expression in the developing
intestine
Before the onset of villus formation PDGF-A and PDGFR-α
were expressed in adjacent, alternating cell layers in the
developing intestine. PDGF-A was uniformly expressed in the
pseudostratified epithelium and in the developing intestinal
wall smooth muscle layers (Fig. 4A), while PDGFR-α was
expressed in the submucosal mesenchyme (strongest in cells
just beneath the epithelium) and in the mesenchymal cells
between the muscle layers (Fig. 4B).
At the onset of villus formation the pseudostratified
epithelium becomes compartmentalized into differentiating
villus and proliferating intervillus epithelium. This transition
was accompanied with changed expression patterns for
Fig. 3. Histological abnormalities in the colon of PDGF-A mutant
mice. (A,B) Hematoxylin/Eosin staining at P19 revealed an abnormal
morphology of colonic mucosal folds in PDGF-A−/− (B) compared to
wild-type (A) colon. (C,D) Alcian Blue/Fast Red staining of colon.
Note the reduction of mesenchymal cells in the submucosa of the
folds (arrows). Goblet cells (blue) were abnormally distributed in
PDGF-A−/− colon. (E,F) α-smooth muscle actin (SMA) staining
demonstrated reduced thickness of the SMA positive mesenchyme at
the core of the folds, and a smaller number of epithelial crypts along
each fold.
3460 L. Karlsson and others
Table 1. Reduction in villus number in PDGF-A−/− mutants compared to wild type
Wild type
PDGF-A−/−
P-value
Reduction in
PDGF-A−/−(%)
P16 small intestine
Number of villi/cross section
Number of villi/10,000 perimeter units
48 (±5.6)
130 (±37)
26.5 (±15)
106 (±30)
<1.3×10−6
P<0.008
45
20
P17-19 small intestine
Number of villi/cross section
Number of villi/10,000 perimeter units
52 (±9.9)
150 (±24)
27 (±7.9)
130 (±21)
<1.2×10−6
>0.1
47
P16 large intestine
Number of crypts/10,000 perimeter units
210 (±20)
150 (±28)
<2×10−8
30
Villi were counted on Haematoxylin/Eosin-stained 4 µm sections photographed with a Nikon SMZ-U dissection microscope. Only villi that were connected to
the submucosa were counted. The perimeter is reduced in PDGF-A−/− intestines (data not shown), resulting in only a minor reduction in number of villi/perimeter
at P16, and a non-significant reduction at P17-19. One individual from each age and genotype was quantified. Perimeter unit is an arbitrary number generated by
the software. Values are means (s.d. in parentheses).
PDGF-A and PDGFR-α. PDGF-A expression became
restricted to the intervillus epithelium (Fig. 4C), and later to
the crypt epithelium (Fig. 4E,G). Villus formation correlated
spatially and temporally with the formation of discrete
strongly PDGFR-α positive clusters of mesenchymal cells
(Figs 4D,F, 5). To our knowledge, this cell cluster
has not been described before, and it will hereafter
be referred to as the ‘villus cluster’. Villus clusters
were seen underneath the earliest protrusions of
epithelium (Fig. 4D arrowheads, Fig. 5C), and
remained associated with the tip of mesenchymal
core of the growing villus. During elongation of the
villus, PDGFR-α positive cells became distributed
along the villus epithelium (Fig. 4F). Accordingly,
full-length villi in postnatal gut (P10-P30) lacked
a villus cluster at the villus tip, while the
discontinuous lining of the epithelium by PDGFRα positive mesenchymal cells remained (Fig. 4F,
arrow). A similar lining of the epithelial basement
membrane by PDGFR-α positive cells was seen in
the intervillus region (Fig. 4D,H arrows).
Patterns of PDGF-A and PDGFR-α expression
similar in principal to the ones described for the upper
Fig. 4. Expression patterns of PDGF-A and PDGFR-α in
the gastrointestinal tract of wild-type mice at E15.5 until
P30, revealed by in situ hybridization. Before initiation of
villus formation, PDGF-A was expressed by the
pseudostratified epithelium (pep) and in the forming
muscle layers (ml) (A). Coincidentally with villus
formation, epithelial PDGF-A expression became
restricted to the intervillus epithelium (arrowheads in C)
and postnatally to the crypts (E, G arrows). PDGFR-α was
expressed in the submucosal mesenchyme (sm) and in the
mesenchymal cells between the muscle layers (B).
Particularly strong PDGFR-α expression was seen in a
single cell layer beneath the epithelium (B). After onset of
villus formation, strong PDGFR-α expression was seen in
clusters of mesenchymal cells (villus clusters) associated
with the growing villus (D). Cluster formation appeared to
precede or be associated with the earliest steps in villus
formation (arrowheads in D). Strong PDGFR-α expression
was also seen in a discontinuous layer of mesenchymal
cells beneath the intervillus epithelium (arrows in D). Fulllength villi lacked villus clusters (F and H and data not
shown).
small intestine were also seen in the developing ventricular
antrum and in the developing colon (data not shown). In the
colon, small clusters of PDGFR-α positive mesenchymal cells
were located to the developing cuff regions, i.e. sites analogous
to the villi of the small intestine.
PDGF-A in gut development 3461
Table 2. Reduction in villus cluster formation in PDGF-A−/− mutants compared to wild type
E15.5
Wild type
PDGF-A−/−
P-value
Reduction in
PDGF-A−/−
(%)
Number of villus clusters/10,000 perimeter units
39 (±10)
28 (±10)
<7×10−5
30
Approximately 30 cross-sections of small intestine from four E15.5 embryos of each genotype were quantified.
Values are means (s.d. in parentheses).
Abnormal pattern of PDGFR-α expression in PDGFA−/− intestine
PDGF-A−/− mice displayed altered expression of PDGFR-α
during development of the GI tract. Before villus formation,
the layer of PDGFR-α expressing cells underlying the early
epithelium was discontinuous and less prominent (Fig. 5A,B).
After onset of villus formation, the number of villus clusters
formed per perimeter unit was reduced by 30% in E15.5
PDGF-A−/− embryos (Table 2 and Fig. 5E,F). The PDGF-A−/−
intestine also displayed a reduction in the number of PDGFRα positive villus clusters associated with the formation of new
villi also at late embryonic and postnatal time-points (data not
shown). There was also a significant loss of PDGFR-α positive
cells subjacent to the intervillus epithelium at E15.5 (arrows in
Fig. 5C,D).
Reduced proliferation of PDGFR-α positive cells in
PDGF-A−/− intestine at E12.5
Proliferation studies using BrdU labeling showed
that the layer of strongly PDGFR-α positive cells
underlying the epithelium proliferated, whereas
the clusters were postmitotic (see below).
Quantification of the BrdU-labeling index in the
total submucosa at E12.5 and E17.5 revealed no
significant difference between PDGF-A mutant
and wild type. We next determined the BrdUlabeling index in the innermost mesenchymal cell
layer, where the strongly PDGFR-α positive cells
are found. At E12.5, i.e. at a time point before the
PDGFR-α positive cells are lost at this location in
PDGF-A−/− intestine, we found that the BrdUlabeling index was reduced by 30% in PDGF-A−/−
compared to PDGF-A+/+ intestine (Table 3). At
E17.5, when the strongly PDGFR-α positive cell
layer is depleted in the PDGF-A−/− intestine, BrdU
labeling was indistinguishable between PDGFA+/+ and PDGF-A−/− intestine. Determination of
total cell numbers in the submucosal compartment
of the intestine at E12.5 and E17.5 showed that
there were approximately 30% less cells in the
PDGF-A−/− submucosa (Table 3). TUNEL-labeling
failed to reveal any difference between wild-type
Fig. 5. Abnormal pattern of PDGFR-α expression in
PDGF-A−/− intestines. Before onset of villus formation,
PDGF-A−/− mutants displayed a reduced PDGFR-α
positive layer of cells beneath the pseudostratified
epithelium (A,B). After villus formation, PDGF-A−/−
mutants showed a lower abundance of villus clusters
(C-F) and loss of PDGFR-α positive cells situated
beneath the intervillus epithelium (arrows in C,D).
Abbreviations as in Fig. 4.
and PDGF-A−/− tissue, arguing against increased apoptosis in
mutant submucosa (data not shown). Our data are compatible
with the idea that PDGF-A promotes proliferation (but has no
influence on survival) in a layer of strongly PDGFR-α
positive cells located immediately beneath the intestinal
epithelium.
Differentiation of villus smooth muscle cells
As the development of smooth muscle cells (SMC) in lung
alveoli and in small blood vessels depends on PDGFs, we
investigated the expression of the SMC marker α-smooth
muscle actin (SMA) during embryonic and postnatal GI
development. SMA staining at different postnatal ages
demonstrated the presence of SMC in wild type as well as in
PDGF-A−/− villi in the small intestine (not shown), hence
PDGF-A is not required for villus SMC formation per se.
Differentiation of villus SMC normally occurs postnatally.
3462 L. Karlsson and others
Table 3. BrdU-labeling index in PDGF-A−/− cells at different locations in the intestine
Wild type
PDGF-A−/−
Significance of
size difference
E12.5
Number of cells in the submucosa
BrdU-labeling index in the innermost cell layer
BrdU-labeling index in the total submucosa
537 (±132)
0.554 (±0.12)
0.467 (±0.068)
358 (±111)
0.367 (±0.19)
0.367 (±0.12)
P<0.003
P<0.03
P=0.28
E17.5
Number of cells in the submucosa
BrdU-labeling index in the innermost cell layer
BrdU-labeling index in the total submucosa
86.6 (±26)
0.38 (±0.087)
0.25 (±0.045)
66.1 (±17)
0.31 (±0.09)
0.25 (±0.06)
P<0.005
P=0.60
P<0.10
The number of BrdU-labeled and propidium iodide-labeled nuclei were counted in cross-sections of embryonic upper small intestine. Note that there is a
significant difference in BrdU-labeling index of the cells just beneath the epithelium (i.e. where the strongly α-receptor positive cells are located) but not in the
total submucosa.Values are means (s.d. in parentheses).
SMA staining was evident in the embryonic mesenchymal villus
core in the PDGF-A−/− intestine at E18.5 when no or little SMA
expression was seen in the wild-type mesenchymal villus core
(Fig. 6A,B). This may suggest that premature differentiation of
villus SMC occurs in the absence of PDGF-A.
The expression patterns for PDGFR-α and SMA in the
mesenchymal villus core seemed spatially similar, indicating
that the PDGFR-α positive cells might constitute villus SMC
or their progenitors. However, double-labeling of P10
intestine showed that SMA and PDGFR-α are not expressed
by the same cells (Fig. 6C). It also showed that the PDGFRα positive cells are found closely associated with the
epithelium, while the SMA-expressing cells are found more
centrally in the villus mesenchyme, possibly in association
with its blood vessels.
Abnormal intestinal development in PDGFR-α−/−
mice
PDGFR-α−/− embryos usually die before E15, but in a
C57Bl6/129sv hybrid genetic background, an appreciable
number of mutants survive until E16.5 or later (Soriano, 1997).
This permits analysis of the initial steps in GI villus formation.
The intestines of two E16.5 PDGFR-α−/− embryos were
compared with wild-type littermates. The mutants showed
fewer and broader villi (Fig. 7A-D) and less mesenchymal cells
in the submucosal compartment (Fig. 7C,D arrows). Thus,
PDGFR-α−/− embryos display a villus phenotype similar to
PDGF-A−/− embryos. Like the PDGF-A−/− embryos, PDGFRα−/− villi showed premature expression of SMA in the villus
mesenchyme (Fig. 7E,F arrow). Although PDGFR-α could not
be used to stain the villus clusters in PDGFR-α−/− embryos,
mesenchymal condensations associated with villus formation
suggested normal induction of villus clusters, similar to the
situation in PDGF-A−/− mice (Fig. 7C,D arrowheads).
Fig. 6. Alpha-smooth muscle actin (SMA) expression during
gastrointestinal development. (A,B) SMA expression at E18.5 in
wild-type (A) and PDGF-A−/− upper small intestine (B) revealed
premature expression of SMA in the PDGF-A−/− intestine (arrow in
B). (C) Combined PDGFR-α in situ hybridisation (blue) and SMA
antibody staining (brown) at P10 showed that the two markers are
expressed by different cells and that these cells have a slightly
different position (C and data not shown). The PDGFR-α positive
cells were located just beneath the villus epithelium and the SMA
expressing cells were located at the centre of the villus core.
PDGF-A in gut development 3463
Proliferative arrest and BMP expression in the
PDGFR-α positive villus cluster of mesenchymal
cells
Cell proliferation in embryonic intestines was examined using
BrdU-labeling. Before the onset of villus formation, a BrdU
pulse uniformly labeled cell nuclei in the epithelium and in the
mesenchyme (data not shown), in agreement with earlier
studies (Calvert and Pothier, 1990). Following villus
formation, epithelial proliferation became progressively
restricted to the intervillus region, and later to the crypts (Fig.
8A,B and data not shown). In the mesenchyme, BrdU labeling
was evident in the layer of PDGFR-α positive cells subjacent
to the PDGF-A positive intervillus epithelium. The villus
clusters, however, were invariably unlabeled by BrdU, arguing
that these structures consisted of non-cycling or slowly cycling
cells (Fig. 8B, cluster indicated by black hatched line). Since
the zone of epithelial mitotic arrest correlates spatially and
temporally with the presence of villus clusters (arrows and
arrowheads in Fig. 8B), we hypothesize that the clusters may
produce inhibitor(s) of epithelial cell proliferation. BMP-2 and
BMP-4 have been reported to be expressed in
intestinal mesenchyme, and are candidate inhibitors
of epithelial cell growth in the intestine and
elsewhere (Bitgood and McMahon, 1995; Roberts et
al., 1995; Bellusci et al., 1996; Kaestner et al., 1997;
Jung et al., 1998; Roberts et al., 1998; Pabst et al.,
1999). Indeed, non-radioactive in situ hybridization
localized the expression of both BMP-2 and BMP-4
mRNA to the villus cluster (Fig. 8C,D). PDGF-A−/−
intestine displayed the formation of fewer villus
clusters and, consequently, fewer sites of BMP
expression (data not shown).
condensation of the mesenchyme. We show that the generation
of the villus epithelial folds coincides with the formation of
mesenchymal clusters that express PDGFR-α, BMP-2 and
BMP-4. These clusters remain beneath the villus tip during its
invagination into the gut lumen. The growing villus tip has
previously been reported to be associated with condensed
mesenchymal cells and extracellular matrix with specific
ultrastructural features (Colony and Conforti, 1993).
The villus cluster was not found in full-length villi by
PDGFR-α labeling and histological analysis of serial sections.
We could not detect signs of cell death in the villus
mesenchyme by TUNEL labeling or standard histology, hence
the loss of the villus cluster does not appear to result from cell
death. The PDGFR-α positive cells distributed along the villus
basement membrane may instead have detached from the
cluster during villus elongation. Some PDGFR-α positive
cells remain at this location in adult intestine. It is not known
whether these cells represent quiescent mesenchymal stem
or progenitor cells, or whether they exert a differentiated
function.
DISCUSSION
Development of the GI tract requires reciprocal
epithelial-mesenchymal signaling. Several growth
and differentiation factors have been implicated in
these processes but the mechanisms by which GI
epithelial-mesenchymal interactions proceed in vivo
are still poorly understood. Here we show that
PDGF-A is necessary for the normal development of
the mucosal lining of the GI tract. In the absence of
PDGF-A the structure of the GI tract is disorganized,
presumably as a result of loss of a PDGF-A target
cell population in the mesenchyme of the developing
submucosa. The analysis also reveals some new
aspects of villus formation. Our data suggest
that PDGF-A/PDGFR-α signaling promotes
proliferation and prevents premature differentiation
of intestinal mesenchymal progenitor cells. We
propose that these mesenchymal cells are recruited
to a transient structure that we have tentatively
named the ‘villus cluster’. The villus cluster appears
to play a critical role during GI villus morphogenesis
(see below).
The villus cluster
At E14, GI villus formation starts in the anterior
portion of the small intestine and is first seen as a
Fig. 7. PDGFR-α−/− mice display a similar phenotype to PDGF-A−/− mice, but
earlier during development. (A-D) Hematoxylin/Eosin staining at E16.5 revealed
fewer and thicker villi in PDGFR-α−/− embryos (B, D) and reduced cellularity in
the sub-villus mesenchymal compartment (indicated by hatched white lines and
arrows in C,D). Villus clusters in C and D are indicated with a black hatched line
and arrowhead. (E,F) Staining for SMA at E16 was increased in the mesenchymal
villus core of in PDGFR-α−/− (F) compared with wild-type (E) intestine.
3464 L. Karlsson and others
Fig. 8. The villus cluster expresses BMP-2 and BMP-4 and is
postmitotic. Upper small intestine of E15.5 wild-type mouse
embryos was double-stained for BrdU (immunohistochemistry;
brown) and PDGFR-α (in situ hybridization; blue) (A), or single
stained for BrdU (B), Bmp-2 (in situ hybridization; blue) (C) or
Bmp-4 (in situ hybridization; blue) (D). Note that the villus clusters
express PDGFR-α (A) and BMPs (C,D), but lack BrdU staining (B).
Arrows in B point at intervillus epithelium in which BrdU labeling is
abundant. Arrowheads in (B) point at the villus tip epithelium in
which BrdU labeling is sparse. White dashed lines outline the
epithelium. Black dashed lines in B outline villus clusters.
Villus morphogenesis in wild-type and PDGF-A−/−
mice
Based on our analyses of GI villus morphogenesis
in normal and PDGF-A−/− or PDGFR-α−/− mice, we
hypothesize the following sequence of events in
normal villus morphogenesis (Fig. 9A): (1) PDGF-
A produced by the pseudostratified epithelium drives the
proliferation of PDGFR-α positive cells subjacent to the
epithelium. (2) PDGFR-α positive cells aggregate, exit the
cell cycle and initiate expression of BMP-2 and -4. (3) The
villus clusters trigger epithelial folding and mitogenic arrest
of the epithelium at the tip of the fold, perhaps through
signals mediated by BMP-2 and -4. PDGF-A expression
becomes restricted to the proliferating epithelium at the
bottom of the folds (the intervillus epithelium), at which site
PDGF-A promotes proliferation and prevents premature
differentiation of non-clustered PDGFR-α positive cells. This
is necessary to secure mesenchymal cell renewal. (4) During
elongation of the villus, PDGFR-α positive cells (possibly
detached from the cluster) distribute along the basement
membrane, at which location they remain. As the gut
diameter and distance between villi increases, new villi form
between already existing ones through the same sequence of
events. In the PDGF-A−/− intestine (Fig. 9B), PDGFR-α
positive cells are recruited to villus clusters, but become
depleted during the initial rounds of cluster formation as they
fail to undergo PDGF-A dependent renewal. As a result, new
rounds of villus formation do not occur properly.
The proposed model would explain the lower abundance of
villi in PDGF-A deficient intestine, but does not offer an
immediate explanation for the abnormal villus shapes. It is
possible that the premature SMA expression seen in PDGFA−/− and PDGFR-α−/− villi, possibly reflecting premature
mesenchymal differentiation, leads to the formation of
abnormal villus shapes. Alternatively, as the spatial
arrangement and the size of the villi in the normal gut are
well ordered (O’Connor, 1966) and probably a result of
periodic patterning, low villus abundance per se may have
secondary influences on villus shapes. Finally, although less
likely, systemic effects on GI development may result from
other defects in the PDGF-A−/− mice, such as the lung
emphysema.
Fig. 9. Model of villus formation in (A) normal and (B)
PDGF-A−/− intestine. (A) Normal villus formation. Stage
1: PDGF-A secreted from epithelium (I) stimulates
proliferation of PDGFR-α positive mesenchymal cells.
Stage 2: an inducing signal (II) promotes clustering of
PDGFR-α positive mesenchymal cells. A cluster-derived
signal (III, BMP-2 and BMP-4?) triggers mitogenic arrest
in adjacent epithelium. Stage 3: PDGF-A secreted from
proliferating intervillus epithelium (I) promotes renewal of
PDGFR-α positive mesenchymal cells. PDGFR-α positive
cells detach from the villus cluster (IV) during villus
elongation. Stage 4: in full-length villi all cells have
detached from the cluster. In the expanding intervillus
region, new cluster induction and initiation of villus
formation occurs (II, III). (B) Villus formation in PDGFA−/− mice. Stage 1: lack of PDGF-A secreted from
epithelium leads to fewer PDGFR-α positive
mesenchymal cells. At stage 3, no renewal of PDGFR-α
positive mesenchymal cells beneath the intervillus epithelium takes place. There is premature expression of SMA. Stage 4: the full-length villus
is deformed. In the expanding intervillus region, new cluster induction and initiation of villus formation is deficient, due to lack of PDGFR-α
positive cells. See text for explanations.
PDGF-A in gut development 3465
Role of BMP-2 and BMP-4 in villus formation
The expression of BMPs by the villus cluster may suggest a
role in periodic patterning of villi, similar to what has been
suggested for the periodic patterning of feather anlagen (Jung
et al., 1998). It is possible that BMP-2 and -4 expressed by the
cluster induce differentiation and mitogenic arrest in nearby
epithelium, hence limiting the epithelial proliferation (and
epithelial stem cells) to the intervillus and prospective crypt
regions. This idea lends support from the knockout of two
transcription factors, in which downregulated BMP-2 and -4
expression has been correlated with increased epithelial
proliferation in the intestine. First, a null mutation in the
winged helix transcription factor Fkh-6/FoxL1 led to abnormal
villus formation and to the distribution of proliferating
epithelial cells outside the crypts (Kaestner et al., 1997). Fkh6/ FoxL1−/− intestines showed perturbed mesenchymal
aggregation and reduced expression of BMP-2 and -4 in
RNAse protection assays. It is an interesting possibility that
Fkh-6/FoxL1 is essential for the formation of villus clusters.
Second, targeted disruption of the homeobox transcription
factor Nkx2-3, which is normally expressed by intestinal
mesenchyme, led to lowered expression of BMP-2 and -4,
increased epithelial proliferation and perturbed villus
morphology (Pabst et al., 1999).
positive dermal mesenchymal progenitors (Karlsson et al.,
1999) and male spermatogenic arrest due to deficient postnatal
formation of Leydig cells (Gnessi et al., 2000). We present here
the first evidence for a role of PDGF-A in the development of
an appropriately sized mucosal lining of the GI-tract. Our study
also reveals key events in the morphogenesis of GI villi and
highlights villus formation as a useful model system for the
study of epithelial-mesenchymal interaction. Together with the
previously established roles for PDGF-A in the formation of
lung alveoli and PDGF-B in kidney glomeruli (Boström et al,
1996; Levéen et al, 1994; Soriano, 1994), the present study
implicates a function for PDGFs in promoting the generation of
cells involved in the morphogenesis of folded epithelial sheaths.
Similarities between the villus cluster and the
dermal papilla
Skin appendages such as hairs and feathers involve the
formation of mesenchymal clusters with critical
morphogenetic properties (Kollar, 1970; Jahoda et al., 1984;
Hardy, 1992). It has occurred to us that similarities exist
between the villus clusters studied here and the dermal papillae
of hair follicles. In both cases, the mesenchymal clusters
constitute structures around which the epithelium folds. In the
hair follicle this results in the embedding of the dermal papilla
within an epithelial pocket located at the base of the follicle.
In the gut, the epithelium folds around the mesenchymal cluster
to form the prospective villus tip. Both mesenchymal clusters
consist of postmitotic PDGFR-α and BMP-4 positive cells (this
paper and Karlsson et al., 1999). However, in the hair follicle
BMP-2 is not expressed by the dermal papillae, but is instead
expressed by the adjacent follicular germinative epithelium
(Karlsson et al., 1999). Nevertheless, the morphological and
genetic similarities between villus clusters and dermal papillae
suggest that similar signaling events and associated
morphogenetic processes may be operating in the generation
and periodic patterning of both ectodermal and endodermal
epithelial derivatives.
REFERENCES
Multiple roles of PDGF-A in development
PDGF-A null mice have previously been shown to develop a
number of phenotypes that reflect impaired proliferation and
spreading of progenitor cells. These phenotypes include lung
emphysema due to failure of proliferation and distal spreading
of PDGFR-α positive alveolar smooth muscle progenitors along
the peripheral branches of the respiratory tract (Boström et al.,
1996; Lindahl et al., 1997), hypomyelination of the central
nervous system due to failure of proliferation and distant
spreading of PDGFR-α positive oligodendrocyte progenitors
(Calver et al., 1998; Fruttiger et al., 1999), skin and hair
abnormalities due to failure of proliferation of PDGFR-α
We thank Sara Beckman for excellent handling of mutant mouse
breeding programs, Brigid Hogan and William Richardson for DNA
probes, Philippe Soriano for PDGFR-α knockout mice, and members
of the Betsholtz and Heath laboratories for valuable discussions. P.L.
is supported by a fellowship from the Swedish Society for Medical
Research. The study was supported by grants from the Swedish
Cancer Foundation, the Cancer Research Campaign UK, the Swedish
Medical Research Council, The Göran Gustafsson Foundation and
The Inga-Britt and Arne Lundberg Foundation.
Apelqvist, A., Ahlgren, U. and Edlund, H. (1997). Sonic hedgehog directs
specialised mesoderm differentiation in the intestine and pancreas
[published erratum appears in Curr. Biol., 1997 Dec 1;7(12):R809]. Curr.
Biol. 7, 801-804.
Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. M.
(1996). Evidence from normal expression and targeted misexpression that
bone morphogenetic protein-4 (Bmp-4) plays a role in mouse embryonic
lung morphogenesis. Development 122, 1693-1702.
Birchmeier, C. and Birchmeier, W. (1993). Molecular aspects of
mesenchymal-epithelial interactions. Annu. Rev. Cell Biol. 9, 511-540.
Bitgood, M. J. and McMahon, A. P. (1995). Hedgehog and Bmp genes are
coexpressed at many diverse sites of cell-cell interaction in the mouse
embryo. Dev. Biol. 172, 126-138.
Boström, H., Willetts, K., Pekny, M., Levéen, P., Lindahl, P., Hedstrand,
H., Pekna, M., Hellström, M., Gebre-Medhin, S., Schalling, M., Nilsson,
M., Kurland, S., Törnell, J., Heath, J. K. and Betsholtz, C. (1996).
PDGF-A signaling is a critical event in lung alveolar myofibroblast
development and alveogenesis. Cell 85, 863-873.
Calver, A. R., Hall, A. C., Yu, W.-P., Walsh, F. S., Heath, J. K., Betsholtz,
C. and Richardson, W. D. (1998). Oligodendrocyte population dynamics
and the role of PDGF in vivo. Neuron 20, 869-882.
Calvert, R. and Pothier, P. (1990). Migration of fetal intestinal intervillous
cells in neonatal mice. Anat. Rec. 227, 199-206.
Colony, P. C. and Conforti, J. C. (1993). Morphogenesis in the fetal rat
proximal colon: effects of cytochalasin D. Anat. Rec. 235, 241-252.
Colvin, J. S., Feldman, B., Nadeau, J. H., Goldfarb, M. and Ornitz, D. M.
(1999). Genomic organization and embryonic expression of the mouse
fibroblast growth factor 9 gene. Dev. Dyn. 216, 72-88.
Duluc, I., Freund, J.N., Leberquier, C. and Kedinger, M. (1994). Fetal
endoderm primarily holds the temporal and positional information required
for mammalian intestinal development. J. Cell Biol. 126, 211-221.
Fruttiger, M., Karlsson, L., Hall, A. C., Abramsson, A., Calver, A. R.,
Boström, H., Willetts, K., Berthold, C.-H., Heath, J. K., Betsholtz, C.
and Richardson, W. D. (1999). Defective oligodendrocyte development
and severe hypomyelination in PDGF-A knockout mice. Development 126,
457-467.
Gnessi, L., Basciani, S., Mariani, S., Arizzi, M., Spera, G., Wang, C.,
Bondjers, C., Karlsson, L. and Betsholtz, C. (2000). Leydig cell loss and
spermatogenic arrest in platelet-derived growth factor (PDGF)-A-deficient
mice. J. Cell Biol. 149, 1019-1026.
Gordon, J.I. and Hermiston, M. L. (1994). Differentiation and self-renewal
in the mouse gastrointestinal epithelium. Curr. Opin. Cell Biol. 6, 795-803.
3466 L. Karlsson and others
Hardy, M. H. (1992. The secret life of the hair follicle. Trends Genet. 8, 5561.
Heldin, C.-H. (1992). Structural and functional studies of platelet-derived
growth factor. EMBO J. 11, 4251-4259.
Hellström, M., Kalén, M., Lindahl, P., Abramsson, A. and Betsholtz, C.
(1999). Role of PDGF-B and PDGFR-β in recruitment of vascular smooth
muscle cells and pericytes during embryonic blood vessel formation in the
mouse. Development 126, 3047-3055.
Jahoda, C. A. B., Horne, K. A. and Oliver, R. F. (1984). Induction of hair
growth by implantation of cultured dermal papilla cells. Nature 311, 560562.
Jung, H.-S., Francis-West, P. H., Widelitz, R. B., Jiang, T.-X., TingBerreth, S., Tickle, C., Wolpert, L. and Chuong, C.-M. (1998). Local
inhibitory action of BMPs and their relationship with activators in feather
formation: Implications for periodic patterning. Dev. Biol., 196, 11-23.
Jung, J., Zheng, M., Goldfarb, M. and Zaret, K. S. (1999). Initiation of
mammalian liver development from endoderm by fibroblast growth factors.
Science 284, 1998-2003.
Kaartinen, V., Voncken, J. W., Shuler, C., Warburton, D., Bu, D.,
Heisterkamp, N. and Groffen, J. (1995). Abnormal lung development and
cleft palate in mice lacking TGF-beta3 indicates defects of epithelialmesenchymal interaction. Nature Genetics 11, 415-421.
Kaestner, K. H., Silberg, D. G., Traber, P. G. and Schutz, G. (1997). The
mesenchymal winged helix transcription factor Fkh6 is required for the
control of gastrointestinal proliferation and differentiation. Genes Dev. 11,
1583-1595.
Karlsson, L., Bondjers, C. and Betsholtz, C. (1999). Roles for PDGF-A and
sonic hedgehog in development of mesenchymal components of the hair
follicle. Development 126, 2611-2621.
Kaufman, M. H. and Bard, J. B. L. (1999). The gut and its associated tissues.
In The Anatomical Basis of Mouse Development, pp. 129-152. Academic
Press, London.
Kedinger, M., Lefebvre, O., Duluc, I., Freund, J. N. and Simon-Assmann,
P. (1998). Cellular and molecular partners involved in gut morphogenesis
and differentiation. Phil. Trans. R. Soc. Lond. B 353, 847-856.
Kedinger, M., Simon, P. M., Grenier, J. F. and Haffen, K. (1981). Role of
epithelial-mesenchymal interactions in the ontogenesis of intestinal brushborder enzymes. Dev. Biol. 86, 339-347.
Kermorgant, S., Walker, F., Hormi, K., Dessirier, V., Lewin, M. J. and
Lehy, T. (1997). Developmental expression and functionality of hepatocyte
growth factor and c-Met in human fetal digestive tissues. Gastroenterology
112, 1635-1647.
Kollar, E. J. (1970). The induction of hair follicles by embryonic dermal
papillae. J. Invest. Dermatol. 55, 374-378.
Levéen, P., Pekny, M., Gebre-Medhin, S., Swolin, B., Larsson, E. and
Betsholtz, C. (1994). Mice deficient for PDGF B show renal,
cardiovascular, and hematological abnormalities. Genes Dev. 8, 1875-1887.
Li, X., Pontén, A., Aase, K., Karlsson, L., Abramsson, A., Uutela, M.,
Bäckström, G., Hellström, M., Boström, H., Li, H., Soriano, P.,
Betsholtz, C., Heldin, C.-H., Alitalo, K., Östman, A. and Eriksson, U.
(2000). PDGF-C is a new protease-activated ligand for the PDGF alpha
receptor. Nature Cell Biol. 2, 302-309.
Lindahl, P., Karlsson, L., Hellström, M., Gebre-Medhin, S., Willetts, K.,
Heath, J. K. and Betsholtz, C. (1997a). Alveogenesis failure in PDGF-A
deficient mice is coupled to lack of distal spreading of alveolar smooth
muscle cell progenitors during lung development. Development 124, 39433953.
Lindahl, P., Johansson, B. R., Levéen, P. and Betsholtz, C. (1997b). Pericyte
loss and microaneurysm formation in PDGF-B-deficient mice. Science 277,
242-245.
Lindahl, P., Hellström, M., Kalén, M., Karlsson, L., Pekny, M., Pekna, M.,
Soriano, P. and Betsholtz, C. (1998). Paracrine PDGF-B/PDGF-Rβ
signaling controls mesangial cell growth in the development of kidney
glomeruli. Development 125, 3313-3322.
Liu, J.-P., Baker, J., Perkins, A. S., Robertson, E. J. and Efstratiadis, A.
(1993). Mice carrying null mutations of the genes encoding insulin-like
growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75, 59-72.
Luetteke, N.C., Phillips, H. K., Qiu, T. H., Copeland, N. G., Earp, H. S.,
Jenkins, N. A. and Lee, D. C. (1994). The mouse waved-2 phenotype
results from a point mutation in the EGF receptor tyrosine kinase. Genes
Dev. 8, 399-413.
Miettinen, P.J., Berger, J. E., Meneses, J., Phung, Y., Pedersen, R. A.,
Werb, Z. and Derynck, R. (1995). Epithelial immaturity and multiorgan
failure in mice lacking epidermal growth factor receptor. Nature 376, 337341.
Montgomery, R. K., Mulberg, A. E. and Grand, R. J. (1999). Development
of the human gastrointestinal tract: twenty years of progress.
Gastroenterology 116, 702-731.
O’Connor, T. M. (1966). Cell dynamics in the intestine of the mouse from
late fetal life to maturity. Am. J. Anat. 118, 525-536.
Pabst, O., Zweigerdt, R. and Arnold, H. H. (1999). Targeted disruption of
the homeobox transcription factor Nkx2-3 in mice results in postnatal
lethality and abnormal development of small intestine and spleen.
Development 126, 2215-2225.
Roberts, D. J., Johnson, R. L., Burke, A. C., Nelson, C. E., Morgan, B. A.
and Tabin, C. (1995). Sonic hedgehog is an endodermal signal inducing
Bmp-4 and Hox genes during induction and regionalization of the chick
hindgut. Development 121, 3163-3174.
Roberts, D. J., Smith, D. M., Goff, D. J. and Tabin, C. J. (1998). Epithelialmesenchymal signaling during the regionalization of the chick gut.
Development 125, 2791-2801.
Schmidt, C., Bladt, F., Goedecke, S., Brinkmann, V., Zschiesche, W.,
Sharpe, M., Gherardi, E. and Birchmeier, C. (1995). Scatter
factor/hepatocyte growth factor is essential for liver development. Nature
373, 699-702.
Simon, T. C. and Gordon, J. I. (1995). Intestinal epithelial cell
differentiation: new insights from mice, flies and nematodes. Curr. Opin.
Genet. Dev. 5, 577-586.
Sonnenberg, E., Meyer, D., Weidner, K. M. and Birchmeier, C. (1993).
Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine
kinase, can mediate a signal exchange between mesenchyme and epithelia
during mouse development. J. Cell Biol. 123, 223-235.
Soriano, P. (1994). Abnormal kidney development and hematological
disorders in PDGF b-receptor mutant mice. Genes Dev. 8, 1888-1896.
Soriano, P. (1997). The PDGF alpha receptor is required for neural crest cell
survival and for normal patterning of the somites. Development 124, 26912700.
Traber, P. G. and Wu, G. D. (1995). Intestinal Development and
Differentiation. In Gastrointestinal Cancers: Biology, Diagnosis and
Therapy (ed. A. K. Rustgi), pp. 21-43. Lippincott-Raven Publishers,
Philadelphia.
Uehara, Y., Minowa, O., Mori, C., Shiota, K., Kuno, J., Noda, T. and
Kitamura, N. (1995). Placental defect and embryonic lethality in mice
lacking hepatocyte growth factor/scatter factor. Nature 373, 702-705.