1 UMR144-CNRS/Institut Curie, 75248 Paris cedex 05, France
2 URA 2578 CNRS, Department of Developmental Biology, Institut Pasteur, 25 rue
du Dr Roux, 75015 Paris, France
3 Department for Molecular Biomedical Research, Flanders Interuniversity
Institute for Biotechnology (VIB)-Ghent University, Technologiepark 927,
B-9052 Ghent, Zwijnaarde, Belgium
Author for correspondence (e-mail:
sbellusci{at}chla.usc.edu)
Accepted 27 February 2005
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SUMMARY |
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Key words: Fgf10, Bmp4, Smooth muscle cells, Lung, Progenitors, Differentiation, Epithelial-mesenchymal interaction, Mouse
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Introduction |
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Given the significance of the distal lung tips for the establishment of
lung epithelial lineages (reviewed by
Warburton et al., 2000), we
considered the possibility that the distal tips have a similar significance
for the lung mesenchymal lineages. We focused in particular on a putative role
for fibroblast growth factor 10 (FGF10) in this process, as it is specifically
expressed in the distal lung mesenchyme
(Bellusci et al., 1997b
).
Homozygous null mutants for Fgf10 display complete lung agenesis
(Min et al., 1998
;
Sekine et al., 1999
), and can
therefore not be used to study the role of Fgf10 in the establishment
of lung mesenchymal lineages. Instead, we took advantage of the recently
reported Mlcv1v-nLacZ-24 transgenic mouse strain
(Kelly et al., 2001
). In this
strain the transgene was inserted 120 kb upstream of the Fgf10 gene,
and due to positional effects, LacZ expression appeared to be a true
reporter for Fgf10 expression in the developing heart
(Kelly et al., 2001
). Given
that tissue-specific enhancer sequences drive tissue-specific gene expression,
it remains to be determined whether this mouse strain is also a useful
reporter for Fgf10 expression in other organs, in this case the
lung.
Here, we first demonstrate that LacZ expression in Mlc1v-nLacZ-24 mice faithfully mimicked Fgf10 expression in the developing lung, and excluded a contribution of the Mlc1v promoter sequences to LacZ expression. Analyzing the expression profile of Mlc1v-nLacZ-24 (hereafter named Fgf10LacZ for simplicity), we show that Fgf10-positive cells in the distal mesenchyme were progenitors for the parabronchial SMCs. Next, we demonstrate that the integration of the transgenic cassette resulted in decreased expression from the Fgf10 gene. We obtained Fgf10 hypomorphic mutants, by generating heterozygous Fgf10 embryos that were hemizygous for transgenic insertion: Fgf10+/;Mlc1v-nLacZ-24+/ (hereafter called Fgf10LacZ/). In Fgf10LacZ/ lungs, part of the ß-galactosidase/Fgf10-positive cells failed to leave the distal tip mesenchyme to become SMCs. Therefore, we postulate that Fgf10-positive cells in the distal lung mesenchyme are progenitors for parabronchial SMCs and, moreover, that Fgf10 is required for their entry into the smooth muscle cell lineage.
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Materials and methods |
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Mutant embryos
The Mlc1v-nLacZ-24 line is bred in a mixed background and has been
previously described (Kelly et al.,
2001). The transgene containing an nLacZ reporter gene is
integrated upstream of the Fgf10 gene.
Fgf10LacZ/ mouse embryos were generated by crossing
Fgf10+/ on a C57Bl/6 background
(Sekine et al., 1999
) with
Mlc1v-nLacZ-24+/ mice
(Kelly et al., 2001
). Wild-type
littermates were used as control embryos at different developmental stages.
The Fgf10 and Mlc1v-nLacZ-24+
alleles were genotyped as described previously
(Mailleux et al., 2002
;
Kelly et al., 2001
). The number
of Fgf10LacZ/ embryos used in this study (52 in
total) at the different stages was as follows: embryonic day (E) 12.5
(n=7); E13.5 (n=3); E14.5 (n=6); E16.5
(n=2); E17.5 (n=10); E18.5 (n=7); P0
(n=17). For simplicity, the
Mlc1v-nLacZ-24+/ is called
Fgf10LacZ/+; the Mlc1v-nLacZ-24+/+ is
called Fgf10LacZ/LacZ; the
Fgf10+/+;Mlc1v-nLacZ-24+/ is called
Fgf10LacZ/+ while the
Fgf10+/;Mlc1v-nLacZ-24+/
is called Fgf10LacZ/. The SpC-Bmp4
(n=3 at E16.5) and SpC-Shh (n=4 at E16.5)
transgenic embryos have been generated previously
(Bellusci et al., 1996
;
Bellusci et al., 1997a
).
Whole-lung culture, cyclopamine treatment and distal mesenchyme grafting
Embryonic lungs were removed at E11.5 and placed on Nuclepore Filters (8
µm pore diameter) in 30 µl of a 1:1 mixture of Matrigel and culture
medium (DMEM/F12 medium containing penicillin/streptomycin and 0.1%
heat-inactivated fetal bovine serum). After 30 minutes at 37°C to allow
the Matrigel to polymerize, these filters were laid on the surface of 500
µl culture medium containing cyclopamine (TRC Biomedical Research
Chemicals, Canada) in Nunclon dishes (technique adapted from
Lebeche et al., 1999).
Concentrations of 1, 2, 5, 10 and 15 µmol/l cyclopamine were tested as
previously described (Yao et al.,
2002
). At 10 and 15 µmol/l, cyclopamine was toxic. Explants
were cultured for 1 day in the presence of cyclopamine and then fixed in 4%
PFA (15 minutes at 4°C) before X-gal staining. For distal mesenchyme
grafting, the distal part (mesenchyme and mesothelium) of the accessory or
median lobes of wild-type and Fgf10LacZ/+ lungs at E12.5
were dissected with tungsten needles in culture medium.
Fgf10LacZ/+ and wild-type lungs were reciprocally grafted
in 1:1 Matrigel:culture medium on Nuclepore Filters using tungsten needles.
After 30 minutes polymerization of the Matrigel at 37°C, 500 µl culture
medium was added under the filter. Grafts were cultured for 44 hours
(37°C, 6% CO2), fixed in 4% PFA and X-gal stained as described
above.
Distal mesenchyme labeling and video-cinematography
The distal part of wild-type accessory lobe mesenchyme was dissected and
incubated with the Cell Tracker Green CMFDA (Molecular Probe; 20 µmol/l in
DMEM/F12 medium) for 20 minutes in the dark at room temperature. The labeling
mixture was prepared following the manufacturer's instructions. The distal
parts were then grafted back at the tip of the accessory lobe they were
derived from. Time-lapse video-cinematography was started 18 hours
post-grafting using a LEICA inverted microscope equipped with a temperature
and CO2 controlled chamber and a Princeton Micromax CDD camera as
described previously (Murase and Horwitz,
2002). The experiments have been carried out over a 24-48 hour
period. With this in-vitro system, the position of the most external edges of
the lung mesenchyme did not significantly change and was used as the reference
point in order to follow the putative migration of the mesenchymal cells
within this time frame.
Semi-quantitative RT-PCR
Total RNA was extracted from a pool of three lungs at E14.5 per genotype
using the RNeasy Total RNA kit (Qiagen). The RNA was reverse-transcribed using
hexamers with TaqMan Transcript Reagent kit (Applied Biosystem) according to
the manufacturer's conditions. One-fiftieth of the cDNA prepared from 1 µg
RNA was subjected to PCR using murine gene-specific primers for Fgf10
(5'-TGTTTTTTTGTCCTCTCCTGGGAG-3' and
5'-GGATACTGACACATTGTGCCTCAG-3'), Bmp4
(5'-GAACAGGGCTTCCACCGTA-3' and
5'-TGAGGTGTCCAGGAACCAT-3'), Spry2
(5'-CTCCACTCAGCACAAACAT-3' and
5'-TTGTCCTTGTATGCTCCGA-3') and tubulin
(5'-TGGCCAGATCTTCAGACCAG-3' and
5'-GTAAGTTCAGGCACAGTGAG-3') as internal control. No amplicons were
detected in water and minus RT control. For semiquantitative PCR, target
sequences were amplified within the linear amplification range between 25 and
34 cycles at 55°C in order to yield visible products. PCR products were
separated by electrophoresis on a 1.5% agarose gel and stained with ethidium
bromide. Photographs were taken with a Vilbert-Loumart apparatus and the
intensity of the bands was determined by densitometry with NIH Image software
(http://rsb.info.nih.gov/nih-image/)
using Gel Plotting Macros
(ftp://rsbweb.hih.gov/pub/nih-image/macros/).
Expression of Fgf10, Bmp4 and Spry2 were determined relative
to tubulin expression.
Cultures of isolated lung mesenchyme explants
Explants consisted of the total mesenchyme distal to the primary bronchi of
wild-type and Flk1LacZ/+
(Shalaby et al., 1995) E11.5
lungs. They were cultured in MatrigelTM
(Bellusci et al., 1997b
) for 48
hours in the presence of 0, 50, 100 and 150 ng/ml BMP4 (R&D). BMP4 (100
ng/ml) was the optimal dose to induce
-SMA expression in the
mesenchyme, and this dose was used throughout the study (n=4). The
explants were then fixed in PFA 4%, dehydrated, embedded in paraffin and
sectioned at 7 µm. Flk1LacZ/+ explants were stained
with X-gal before embedding.
Preparation of mesenchymal cell cultures and FGF treatment
Whole lungs were dissected at E13.5 and subjected to trypsin digestion to
give rise to single cells. Mesenchymal cells were separated from epithelial
cells by differential adhesion as described previously
(Lebeche et al., 1999;
Yang et al., 1999
).
Mesenchymal cells were cultured for 4 hours (undifferentiated cells) or 48
hours (differentiated cells) in DMEM/F12 medium containing
penicillin/streptomycin and 0.5% heat-inactivated fetal bovine serum for
starvation. They were subsequently cultured during 20 minutes with 100 ng/ml
of recombinant FGF1, FGF7 and FGF10, respectively (R&D Systems). Protein
extracts and immunoblot analysis were performed as described previously
(Yang et al., 1999
).
Antibodies
A Cy3-conjugated mouse monoclonal antibody against -SMA, (Sigma,
C-6198), pan-Cytokeratine (Dako), ß-galactosidase (US Biological,
G1041-42) were used at a dilution of 1/200 for immunohistochemistry. For
immunoblot analysis, rabbit polyclonal antibodies against phospho-AKT (Ser473,
#9271), AKT (#9272), phospho-p44/42 MAP kinase (#9101) and p44/42 MAP kinase
(#9102) were obtained from Cell Signalling Technology, and used according to
the manufacturer's instructions.
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Results |
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We next compared the expression pattern of the transgene in lungs of
Mlc1v-nLacZ-24 hemizygous embryos to Fgf10 expression in
wild type lungs, at the level of either LacZ mRNA expression or
ß-galactosidase activity. At E10.5, the distal tip of the right lobe
(black arrow), but not the left lobe, expressed Fgf10 mRNA
(Fig. 1A). One day later, the
right lobe had subdivided into four lobes. Each of these lobes expressed
Fgf10 mRNA at the distal tips
(Fig. 1C). At this time, the
left lobe had also started to express Fgf10 mRNA (dashed boxed area
in Fig. 1C). We have previously
shown that the distal Fgf10 expression is exclusively mesenchymal
(Bellusci et al., 1997b). We
detected ß-galactosidase activity in a very similar pattern at E10.5
(Fig. 1B) and E11.5
(Fig. 1D). The absence of
ß-galactosidase activity in the left lobe at E11.5 (boxed area in
Fig. 1D) may reflect the
delayed onset of expression of Fgf10 in the left lobe, as already
observed at E10.5. Analysis of LacZ expression as mRNA
(Fig. 1G,H) or as
ß-galactosidase activity (Fig.
1I,J) at E12.5 revealed that its expression was still highly
similar to the expression pattern of Fgf10 mRNA at the mesenchymal
edges of each lobe adjacent to the distal ends of the bronchi
(Fig. 1E,F)
(Mailleux et al., 2001
).
However, at this stage (Fig.
1I,J) and later at E14.5, ß-galactosidase activity was
additionally observed at the level of the secondary bronchi (arrow in
Fig. 1L) by contrast to
Fgf10 mRNA expression (Fig.
1K). This expression corresponds to the mesenchyme adjacent to the
bronchial epithelium (Fig.
2E,F). Importantly, LacZ expression was essentially not
found in the mesenchyme of the primary bronchi except for the very distal part
(black arrowhead in Fig. 1I and
white arrowhead in Fig.
1L).
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To further validate the use of the Mlc1v-nLacZ-24 transgene as a
reporter for Fgf10 expression, we analyzed the control of
LacZ expression by Fgf10 regulatory elements in a functional
assay. We made use of the knowledge that sonic hedgehog (SHH) downregulates
Fgf10 expression in the lung
(Bellusci et al., 1997b;
Pepicelli et al., 1998
;
Lebeche et al., 1999
), and that
cyclopamine is a specific inhibitor of SHH function
(Yao et al., 2002
). Lungs
derived from E11.5 hemizygous Mlc1v-nLacZ-24 embryos, and cultured
for 28 hours in the presence of 5 µmol/l cyclopamine displayed dilated
epithelium (arrows in Fig.
1S,O). A similar phenotype was previously observed upon addition
of recombinant FGF10 on lung grown in vitro
(Bellusci et al., 1997b
),
suggesting that cyclopamine treatment triggers Fgf10 upregulation. An
increase in ß-galactosidase activity was observed compared with the
untreated lung (compare Fig.
1P-U). The effect of cyclopamine upon ß-galactosidase
activity was particularly apparent in the left lobe (arrows in
Fig. 1P,T). These observations
support our expression data and indicate that LacZ expression in the
Mlc1v-nLacZ-24 strain can be used as a reporter for Fgf10
expression. In accordance with this conclusion, we will hereafter refer to the
mice heterozygous for the Mlc1v-nLacZ-24 cassette as
Fgf10LacZ/+ mice.
Fgf10-positive cells in the distal mesenchyme give rise to parabronchial smooth muscle cells
The ß-galactosidase expression domain expanded progressively from the
peripheral distal mesenchyme toward the distal epithelium between E11.5 and
12.5 (Fig. 2A-D), whereas the
Fgf10 and LacZ mRNAs remained expressed only in the
peripheral mesenchyme at this time (Fig.
1D and double pointed arrows in
Fig. 1F,H). This supports the
presumption that cells from the peripheral distal mesenchyme or their daughter
cells can relocate to more proximal areas. Vibratome sections of the accessory
lobe at E12.5 illustrate that the ß-galactosidase-positive cells were
exclusively located in the mesenchyme throughout the distal tip mesenchyme and
as one layer adjacent to the proximal epithelium
(Fig. 2E). Transversal sections
in the most proximal part of E12.5LacZ/+ accessory lobe
show that ß-galactosidase expression entirely surrounded the epithelium
of the secondary bronchi (Fig.
2E'). In addition, at this stage not all the mesenchymal cells
directly adjacent to the epithelium were positive for ß-galactosidase
(Fig. 2E, red arrows), in
harmony with the patchy ß-galactosidase expression pattern in whole-mount
staining of the accessory lobe (arrow in
Fig. 2D). By E13.5, the
ß-galactosidase-positive cells had formed a continuous layer adjacent to
the epithelium (Fig. 2F).
The location of these cells suggested that they could be parabronchial
SMCs. Indeed, they co-expressed the smooth muscle cell marker -SMA
around the bronchial epithelium (Fig.
2G). Fig. 2H,I show
additionally that
-SMA was not expressed in the distal tip mesenchyme,
but was restricted to the layer of mesenchymal cells directly in contact with
the proximal bronchial epithelium.
In conclusion, the dynamics of the ß-galactosidase expression pattern strongly suggests that the mesenchymal Fgf10/ß-galactosidase-positive cells in the distal tip represent smooth muscle cell progenitors.
Fgf10-positive mesenchymal cells passively relocate around the bronchial epithelium
In order to assess whether these proximal ß-galactosidase-positive
cells are indeed derived from the distal mesenchyme, we carried out reciprocal
heterotypic grafts of pulmonary distal mesenchyme on lungs of wild-type and
Fgf10LacZ/+ embryos. The grafted lungs were cultured for 2
days and compared to unmanipulated Fgf10LacZ/+ cultured
lungs. In the unmanipulated lungs, the ß-galactosidase-positive cells
were located in the distal tip as well as in a single layer of cells around
the more proximal epithelium (Fig.
3A-D).
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To assess how distal mesenchymal cells populate the more proximal areas, the distal part of a wild-type lung (mesenchyme and mesothelium) was dissected out from the accessory lobe, re-implanted after staining with the fluorescent marker CMFDA and followed up for 25 hours by video-cinematography. Our results show no relative movement of the labeled cells with respect to the epithelium and no cells leaving the labeled explant (Fig. 3M,N). This suggests that mesenchymal cells do not actively migrate along the epithelium but passively relocate during epithelial outgrowth into the distal mesenchyme.
Reduction of Fgf10 expression in the lung leads to decreased smooth muscle actin expression around the bronchi
We noted that Fgf10LacZ/+ mice, similar to
Fgf10 heterozygous mice, have smaller eyelids and a moderate nervous
behavior compared with wild-type mice. We therefore tested whether the
insertion of the Mlvc1v-nLacZ cassette 120 kb upstream of the
transcriptional start site of the Fgf10 gene reduces Fgf10
expression. Indeed, whole-mount in-situ experiments showed decreased
Fgf10 expression in Fgf10LacZ/LacZ embryos when
compared with wild-type littermates (Fig.
4A,B). This indicates that the insertion of the transgenic
cassette creates a hypomorphic Fgf10 allele. It furthermore suggests
that an allelic series can be generated by crossing hemizygous or homozygous
Fgf10LacZ mice with heterozygous Fgf10 null
mice.
|
At E12.5, Fgf10LacZ/ embryos have a slightly
smaller lung with decreased branching morphogenesis compared with wild-type or
Fgf10LacZ/+ lungs (Fig.
4D,E). In addition, -SMA expression is decreased
specifically around the epithelium of the secondary bronchi (n=2;
Fig. 4F,G). The deposition of
laminin, an extracellular matrix protein synthesized by the lung epithelium
and involved in the differentiation of the SMC
(Zhang et al., 1999
), was not
perturbed around the bronchial epithelium of
Fgf10LacZ/ lungs (data not shown).
A similar decrease in -SMA expression was observed at E13.5
(n=2, data not shown), E14.5 (n=2, data not shown) and E17.5
(n=3, Fig. 4I,K)
compared with control lungs (Fig.
4H,J), as well as at E18.5 (n=3, data not shown) and
after birth (n=2, data not shown). Elastin deposition by SMCs around
the bronchi was also reduced in Fgf10LacZ/ lungs at
birth (data not shown). In addition, a specific decrease in gelatinase
activity at birth in Fgf10LacZ/ lung mesenchyme,
especially in parabronchial SMCs, was revealed by in-situ zymography (data not
shown). Therefore, decreased Fgf10 expression in the distal
mesenchyme clearly leads to a defect in the proper establishment of the
parabronchial SMC population from early embryonic stages.
Interestingly, less ß-galactosidase activity was observed around the epithelium of the secondary bronchi, while more activity was present in the distal mesenchyme of Fgf10LacZ/ lungs compared with Fgf10LacZ/+ lungs (compare Fig. 4L,M). By TUNEL or PCNA analyses (data not shown), we did not see any significant changes in apoptosis or in proliferation of the parabronchial SMCs between E14.5 and 17.5.
These results suggest that FGF10 is involved directly or indirectly in the entry of the Fgf10/ß-galactosidase-positive SMC progenitors into the SMC lineage rather than in their survival or proliferation.
FGF10 regulates SMC formation in a non-autocrine fashion, possibly via upregulation of epithelial BMP4
To test whether FGF10 acts in an autocrine fashion on the mesenchyme, or in
a paracrine fashion via the epithelium, primary cultures of mesenchymal cells
derived from the entire E13.5 wild-type lung were established. These were
either cultured for 4 hours, sufficient for the cells to start to
differentiate, or for 48 hours to lead to fully differentiated SMCs, before
addition of recombinant FGF1, FGF7 or FGF10. Unlike FGF1, FGF10 and FGF7 did
not upregulate ERK phosphorylation (Fig.
5A) nor AKT phosphorylation (data not shown) in undifferentiated
or differentiated cultures. These results suggest that FGF10 does not act on
mesenchymal cells in an autocrine fashion but via an epithelial intermediate.
Previous reports have shown that Bmp4 is a downstream epithelial
target of FGF10 in the lung (Lebeche et
al., 1999; Weaver et al.,
2000
; Mailleux et al.,
2001
). In harmony with these results, the expression of
Bmp4 was decreased in Fgf10LacZ/ lungs
(Fig. 5C,E). The expression of
Sprouty2, another FGF10 downstream target
(Mailleux et al., 2001
) was
also decreased (Fig. 5D,E),
while the expression of Shh, an upstream negative regulator of
Fgf10 expression (Bellusci et al.,
1997b
), was unchanged (Fig.
5B).
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Discussion |
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Fgf10 expression in the distal lung mesenchyme identifies progenitors for parabronchial SMCs
Our chimaeric lung cultures demonstrated that the distal mesenchyme gives
rise to the parabronchial SMC population in more proximal areas. To date, this
is the first evidence for the location of progenitors for parabronchial SMCs,
as well as for their identification by the expression of Fgf10.
Our data indicate that ß-galactosidase is essentially not present in
the mesenchyme around the primary bronchi of the
Fgf10LacZ/+ lungs, while -SMA is detected around
the primary bronchi at E12.5. This difference suggests that the smooth muscle
cells around the epithelium of the primary bronchi and the smooth muscle cells
around the epithelium of the secondary bronchi may originate from different
pools of progenitors. This is supported by the presence of
-SMA in the
secondary bronchi at E11 (Tollet et al.,
2001
), even before Fgf10/ß-galactosidase-positive
cells are relocalized around the secondary bronchi (this report). However,
from E13.5 onward, the smooth muscle cells around the secondary bronchi
originate mostly from the Fgf10-expression domain, as indicated by
the homogenous expression of ß-galactosidase in the mesenchyme adjacent
to the bronchial epithelium.
The distal location of this new subset of parabronchial SMC progenitors in
the lung is reminiscent of the distal location of the progenitors for the
various epithelial cell lineages of the lung (reviewed by
Warburton et al., 2000). It is
now of interest to determine whether the other mesenchymal lineages, including
other types of SMCs (e.g. the vascular SMCs), lipocytes and endothelial cells,
also originate in this domain. Interestingly, this domain of progenitor cells
is also the area where budding morphogenesis is initiated
(Bellusci et al., 1997b
) and
where many signaling molecules are expressed. This suggests that the distal
lung buds act as signaling centers that synchronize morphogenetic events with
differentiation.
FGF10 regulates the establishment of the parabronchial smooth muscle cell lineage via a non-autocrine effect on the lung bud epithelium
A difference in parabronchial SMC formation was observed in
Fgf10LacZ/ lungs at all the stages investigated.
The decreased ß-galactosidase expression around the bronchial epithelium
and the increased expression in the distal mesenchyme in
Fgf10LacZ/ lungs strongly suggest that FGF10
regulates the entry of the SMC progenitors into the SMC program, rather than
controlling the progenitor population. This is a new role for Fgf10,
as to date its major role has seemed to be the regulation of budding and
branching morphogenesis in a variety of organs
(Min et al., 1998;
Sekine et al., 1999
) including
the lung (Bellusci et al.,
1997b
). Interestingly, our in-vitro data indicate that the
relocalization of the Fgf10-positive cells in the distal mesenchyme
along the epithelium is essentially due to the growth of the distal epithelium
into the distal mesenchyme. However, a comparison of
Fig. 3D with
Fig. 2E demonstrates that the
domain of ß-galactosidase-positive cells does not expand as proximally in
vitro as it does in vivo, and that the branching pattern is disturbed.
Therefore, we cannot exclude the fact that active migration of the mesenchyme
does occur in vivo. It is therefore possible that the epithelium exerts a
chemoattractive activity on cells derived from the distal mesenchyme, similar
to the way the mesenchyme acts as a chemoattractant for the distal lung
epithelium (Park et al.,
1998
).
Our cultures of primary lung mesenchyme in the presence of various
recombinant FGFs demonstrated that FGF10, in contrast to FGF1, was not able to
phosphorylate ERK and AKT. This indicates that FGF10 does not act in an
autocrine fashion on the mesenchyme to fulfill its function in parabronchial
SMC formation. At present, only FGF9 secreted by the mesothelium is implied in
vivo in the biogenesis of parabronchial SMCs. It is proposed to signal through
the mesenchymal receptor FGFR2c, and to maintain the mesenchymal progenitors
in a proliferative and undifferentiated state
(Weaver et al., 2003). By
contrast, we propose that FGF10 uses an epithelial intermediate, BMP4.
Epithelial BMP4 is a candidate mediator of SMC formation
Our results indicate a decrease in Bmp4 expression in
Fgf10LacZ/ embryos. This reduction in Bmp4
expression seems to primarily occur in the epithelium. These results are
consistent with previous reports showing that FGF10 upregulates epithelial
Bmp4 transcription (Lebeche et
al., 1999; Weaver et al.,
2000
). Overexpression of Bmp4 in the distal lung
epithelium using the surfactant protein C promoter led to ectopic expression
of
-SMA in the distal mesenchyme. While addition of recombinant SHH
induces
-SMA expression on isolated lung mesenchymal explants
(Weaver et al., 2003
), we show
that overexpression of Shh in the distal lung epithelium in vivo does
not modify
-SMA expression. This may be explained by the lack of
upregulation of Bmp4 in the epithelium or the mesenchyme upon
overexpression of Shh in vivo
(Bellusci et al., 1997a
), by
contrast to the induction of Bmp4 expression by SHH in vitro
(Weaver et al., 2003
).
Consistent with a major role for Bmp4 in SMC differentiation,
recombinant BMP4 induced -SMA expression in lung mesenchyme explants in
vitro after 48 hours of culture. These results strongly suggest that BMP4
induces SMC formation by acting directly on the mesenchyme. We therefore
propose that FGF10 expressed by the distal mesenchyme may contribute to
parabronchial SMC formation via the upregulation of BMP4 synthesis by the
epithelium. The failure to induce
-SMA expression in all cells can be
explained by the presence of other cell types in the mesenchymal explants,
e.g. the endothelial cells. In addition, these explants also contain a layer
of mesothelium, producing FGF9, which has been shown to prevent the
differentiation of the smooth muscle cells
(Weaver et al., 2003
).
Independent reports support the proposed role of BMP4 in smooth muscle cell
differentiation. In the kidney, periureteral mesenchymal cells differentiate
into smooth muscle cells at a site where Bmp4 is highly expressed. In
addition, E15.5 Bmp4+/ ureters have fewer
-SMA-positive cells (Miyazaki et
al., 2003
). Furthermore, in human lung fibroblast cultures,
exogenous BMP4 inhibits proliferation and promotes smooth muscle cell
differentiation, as indicated by increased expression of
-SMA and
smooth muscle myosin (Jeffery et al.,
2004
).
The expression of the BMP antagonist noggin in parabronchial SMC
(Weaver et al., 2003) suggests
that regulation of BMP signaling may be important to finely tune SMC
differentiation.
In conclusion, we demonstrate that the mesenchyme of the distal lung tip contains a set of progenitors for parabronchial SMCs that can be identified by Fgf10, and that normal transcription levels of at least one Fgf10 allele are required for their entry into the parabronchial SMC lineage.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Developmental Biology Program, Saban Research Institute of
Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA
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