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First published online 15 March 2006
doi: 10.1242/dev.02313
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1 Department of Molecular Biology and Pharmacology, Washington University
Medical School, St Louis, MO 63110, USA.
2 Brigham and Women's Hospital, Division of Critical Care and Pulmonary
Medicine, Boston, MA 02115, USA.
* Author for correspondence (e-mail: dornitz{at}wustl.edu)
Accepted 30 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fibroblast growth factor 9 (FGF9), Sonic hedgehog (SHH), Lung development, Branching morphogenesis, Mesothelium, Epithelium, Mesenchyme, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Several studies have identified fibroblast growth factors (FGFs) that
signal across epithelial and mesenchymal boundaries to regulate both the
pseudoglandular and subsequent stages of lung development
(Shannon and Hyatt, 2004). In
vitro studies have shown that mesenchymally expressed FGF10 activates the `b'
splice form of FGF receptor 2 (FGFR2b) in lung epithelium to direct budding by
stimulating epithelial cell migration and proliferation
(Bellusci et al., 1997b;
Park et al., 1998;
Peters et al., 1992;
Weaver et al., 2000).
Consistent with this activity, loss of function of Fgf10 or
Fgfr2b results in the absence of primary branching in vivo, causing
the trachea to terminate as a blind sac
(Arman et al., 1999;
Celli et al., 1998;
De Moerlooze et al., 2000;
Min et al., 1998;
Peters et al., 1995;
Sekine et al., 1999).
Fgf7, also a ligand for FGFR2b, is expressed in developing lung
mesenchyme, but its function during normal lung development is not clear
(Bellusci et al., 1997b;
Cardoso et al., 1997;
Guo et al., 1996;
Park et al., 1998;
Post et al., 1996;
Simonet et al., 1995;
Tichelaar et al., 2000).
Spry2, an inducible inhibitor of FGF and EGF receptor tyrosine kinase
signaling, is expressed in the distal epithelial tips and is functionally
downstream of the mesenchymal FGF genes, acting to suppress mesenchymal FGF
signaling to epithelium (Hanafusa et al.,
2002; Mailleux et al.,
2001; Tefft et al.,
2002; Tefft et al.,
1999; Zhang et al.,
2001).
FGF9 fulfills the role of a reciprocal epithelial to mesenchymal signal in
the lung. At E10.5, Fgf9 is expressed in both the mesothelial lining
(future pleura) of the lung bud and in the epithelium of the developing
airways. As lung development progresses through the pseudoglandular stage,
Fgf9 expression persists in the mesothelium but can no longer be
detected in the lung epithelium (Colvin et
al., 1999). Fgf9–/– mice have
severe lung hypoplasia and die in the perinatal period. The most
characteristic feature of Fgf9–/– lungs is a
reduced ratio of mesenchyme to epithelium caused by a reduction in mesenchymal
(but not epithelial) proliferation at E10.5-E11.5. In addition, a secondary
consequence of Fgf9 gene inactivation is reduced branching of the
epithelial tubules after 
E12.5. Previous studies suggested that a
molecular etiology of reduced branching morphogenesis may be decreased
mesenchymal Fgf10 at actively branching regions of the lung
(Colvin et al., 2001).
Sonic hedgehog (SHH) and bone morphogenic protein 4 (BMP4) also modulate
lung mesenchymal and epithelial development. Bmp4 is expressed in the
distal epithelium, where it appears to have a primary role in promoting distal
epithelial differentiation and antagonizing FGF-mediated epithelial budding,
and has a suggested role in specifying smooth muscle precursors
(Bellusci et al., 1996;
Bitgood and McMahon, 1995;
Mailleux et al., 2005;
Weaver et al., 2000).
Additionally, Bmp4 is upregulated by FGF10 in vitro and in vivo
(Lebeche et al., 1999;
Mailleux et al., 2005;
Mailleux et al., 2001;
Weaver et al., 2000).
Shh is expressed in the distal epithelium of the lung throughout the
pseudoglandular stage and binds to its receptor, patched 1 (Ptch1),
in the adjacent sub-epithelial mesenchyme
(Bellusci et al., 1997a;
Bitgood and McMahon, 1995;
Miller et al., 2001;
Weaver et al., 2003). Other
members of this signaling pathway, such as Gli1 and Hip1,
colocalize with Ptch1 (Chuang et
al., 2003; Grindley et al.,
1997). Both Shh gain-of-function and loss-of-function
mutations, as well as combinatorial Gli loss-of-function mutations,
demonstrate that SHH signaling positively affects both mesenchymal and
epithelial growth, as well as lobe formation (reviewed by
van Tuyl and Post, 2000). SHH
is proposed to modulate the epithelial branching pattern by focally
suppressing Fgf10 in distal mesenchyme and upregulating Fgf7
(Lebeche et al., 1999;
Pepicelli et al., 1998).
Conversely, FGF10 does not affect Shh, whereas high levels of FGF7
suppresses both Shh expression and signaling
(Lebeche et al., 1999;
Park et al., 1998). Although
Shh appears unaffected at late stages of branching in
Fgf9–/– lungs
(Colvin et al., 2001), we
hypothesized that FGF9 may directly or indirectly regulate SHH signaling at
early stages of branching, which in turn may affect mesenchymal proliferation
and the level and pattern of Fgf10.
To investigate further the mechanisms by which FGF9 regulates lung development, we have developed two gain-of-function models: an in vivo method to transiently express ectopic FGF9 in lung epithelium, and in vitro organ culture that mimics FGF9 overexpression in the mesothelium. Combined with the Fgf9 loss-of-function mouse, these two systems facilitated an examination of the role of FGF9 on sub-mesothelial and sub-epithelial mesenchyme.
Here, we identify two distinct mesenchymal zones that are differentially regulated by FGF9 and SHH. We show that FGF9 stimulates proliferation of the sub-mesothelial population of mesenchymal cells, and is an upstream regulator of mesenchymal SHH signaling, which is required for cell proliferation and cell survival in the sub-epithelial mesenchymal compartment. Finally, FGF9 regulates expression of the mesenchymal to epithelial signals that may generate the crucial developmental switch from branching to dilation during lung development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of transgenic mice and doxycycline administration
The TRE-Fgf9-IRES-eGfp transgene was constructed by cloning the
Fgf9 cDNA (Santos-Ocampo et al.,
1996) upstream of the IRES-eGfp-SV40 polyA
cassette (pIRES2-eGfp vector, Clontech) in the pTRE2 vector
(Clontech). This TRE-Fgf9-IRES-eGfp transgene was released with
NotI and used to generate five founder lines of transgenic mice.
TRE-Fgf9-IRES-eGfp mice were mated to SPC-rtTA mice
(provided by Dr Jeff Whitsett) (Perl et
al., 2002; Tichelaar et al.,
2000). Two bi-transgenic
TRE-Fgf9-IRES-eGfp;SPC-rtTA lines exhibited a
doxycycline-inducible embryonic phenotype. One line was used for these
studies. Doxycycline-containing water (200 µg/ml) can activate a
TRE transgene within 16 hours
(Perl et al., 2002) and was
given to time-mated females 48 hours before embryo harvest. Mice were
genotyped with PCR primers specific for SPC-rtTA
(Tichelaar et al., 2000) and
eGfp (primers 5'-CGTAAACGGCCACAAGTTCAG-3' and
5'-ATGCCGTTCTTCTGCTTGTCG-3').
Whole mount in situ hybridization, lacZ staining and immunohistochemistry
Whole-mount in situ hybridization was performed as described previously
(Colvin et al., 2001).
Tie2LacZ+/– and Noggin-lacZ expression was
visualized by staining whole lobes with 1 mg/ml X-gal, 2 mM MgCl2,
35 mM Ke3F(CN)6, 35 mM Ke4F(CN)6
and 1x PBS at room temperature in the dark. Following adequate color
reaction, tissues were washed and dehydrated to methanol. Tissues were then
sectioned in paraffin wax at 5 µM (Tie2LacZ+/–)
or cryosectioned at 12 µM (Noggin-lacZ) and counterstained with
Eosin. Panel comparisons were paired with littermate controls, ensuring
similar developmental time points, tissue processing and staining incubation
periods (8-10 hours).
For whole-mount immunohistochemistry, dissected lung tissues were fixed overnight in 4% PFA, dehydrated to 100% methanol and stored at –20°C. All incubations and washes were performed at 4°C while shaking. Tissues were rehydrated, incubated with a blocking solution (2% skim milk, 1% sheep serum, 0.1% Triton X-100) for 2 hours, primary and secondary antibodies, in blocking solution, overnight at 4°C and then washed (2% skim milk, 0.1% Triton X-100) five times at hourly intervals. Visualization of secondary antibody conjugated to HRP was performed using the DAB kit (Vector Laboratories), according to manufacturer's instructions. PECAM1-stained tissues were sectioned in paraffin wax at 5 µM and counterstained with Hematoxylin. Rat anti-PECAM1 (BD) was incubated at 1:500, monoclonal anti-TTF1 (DakoCytomation) at 1:200, monoclonal anti-phosphohistone H3 (Sigma) at 1:500 and rabbit anti-active caspase 3 (R&D) at 1:600. Secondary HRP conjugated anti-mouse antibody (Chemicon) was incubated at 1:300, HRP anti-rat at 1:200 and HRP anti-rabbit at 1:400. Immunohistochemistry on paraffin embedded 5 µM sections was performed using a standard protocol with citrate buffer antigen retrieval. Rat anti-SMA (Sigma) was incubated at 1:500 overnight at 4°C, followed by a secondary HRP-conjugated anti-rat antibody (Chemicon) at 1:200. All expression studies are representative of at least three embryos.
Mesenchyme proliferation analysis
Mitotic cells were identified with anti-phospho histone H3 antibodies and
photographed at 90x magnification in whole-lung quadrants. Distal
mesenchyme was defined as the area between the outermost edge of the distal
epithelium and the mesothelium. Sub-mesothelial and sub-epithelial regions
were defined by dividing the mesenchyme at the relative halfway mark between
epithelium and mesothelium. Labeled cells present in the mesenchyme were
counted and averaged over the total or regional area, determined by tracing
and calculating the area using Canvas 9 software. Both 24 and 48 hour data
represent littermate biological samples. Each proliferation index was averaged
over each of the four quadrants per sample. Error bars represent one s.d., and
Student's t-test values indicate statistical significance for
comparable tissues.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Embryonic day 11.5 (E11.5) lung explants cultured alone or with a
BSA-soaked bead demonstrated mesenchymal and epithelial growth and a
reproducible pattern of epithelial branching
(Fig. 1A,D). To model
overexpression of Fgf9 from its normal mesothelial location during
early lung development, the central buds of the left lobe of E11.5 lung
explants were exposed to FGF9-saturated heparin beads or FGF9 in media. This
resulted in a significantly increased area of mesenchyme adjacent to the bead
after 24 hours (red line, Fig.
1B). Consistent with this, distal mesenchymal proliferation,
marked by staining with an antibody to phospho histone H3 (PH3), was increased
by 38±8% following 24 hours of incubation with FGF9 in media (see
below). After 48 hours in culture, the FGF9-exposed explants developed a
single extended epithelial tubule that often completely enveloped the adjacent
bead (Fig. 1E). Notably, this
luminal enlargement was only readily apparent after 48 hours in culture,
suggesting that a secondary signal is required to mediate this epithelial
phenotype.
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The mesenchymal receptor(s) that mediates the FGF9 response is not known.
Both Fgfr1 and Fgfr2 are expressed in the developing lung
mesenchyme (Arman et al., 1999;
Peters et al., 1992) and are
thus candidate receptors to transduce the FGF9 signal. To determine if one or
both of these receptors are necessary for lung mesenchymal development, we
used the Dermo1(Twist2)-Cre allele to target floxed
alleles of Fgfr1 and Fgfr2
(Trokovic et al., 2003;
Yu et al., 2003).
Dermo1-Cre activity in the developing lung was assessed by mating to
the Rosa26 reporter line (Soriano,
1999). This analysis showed that by E13.5, most lung mesenchymal
cells have been exposed to functional Cre activity
(Fig. 1G). Conditional knockout
mice lacking Fgfr1 and one copy of Fgfr2
(Fgfr1–/–;
Fgfr2+/–), or heterozygous for both receptors
(Fgfr1+/–; Fgfr2+/–), did
not exhibit a lung phenotype when compared with wild-type and
Dermo1-Cre controls (Fig.
1H; data not shown). By contrast, mice lacking both Fgfr1
and Fgfr2 (Fgfr1–/–;
Fgfr2–/–) showed lung hypoplasia at E12.5
through E18.5 (Fig. 1J,K; data
not shown). These double conditional knockout lungs failed to fill the
thoracic cavity and exhibited variable morphologies. Lungs lacking
Fgfr2 and one allele of Fgfr1
(Fgfr1+/–; Fgfr2–/–)
also exhibited a reduction in size, although less severe than double receptor
knockout lungs (Fig. 1I). These
data demonstrate that Fgfr1 and Fgfr2 are redundant but that
one wild-type allele of Fgfr2 is sufficient to transduce an FGF
signal to lung mesenchyme.
Induced expression of Fgf9 in lung epithelial cells results in lung hyperplasia in vivo
Lung organ culture experiments predict that in vivo overexpression of
Fgf9 will promote mesenchymal growth but will also alter lung
branching, possibly by disrupting the pattern of mesenchymal to epithelial
signals. To express Fgf9 in developing lung epithelium with temporal
and spatial specificity, we constructed transgenic mouse lines that contain
the Fgf9 cDNA under the control of seven tetracycline-inducible
regulatory elements (TREs). To monitor transgenic Fgf9 expression, an
IRES-eGfp (enhanced green fluorescent protein) gene was placed
3' to the Fgf9 cDNA, which allowed for co-expression of
eGfp with Fgf9. The TRE-Fgf9-IRES-eGfp mice were
mated to a transgenic line containing the 3.7 kb human surfactant C promoter
driving expression of the reverse tetracycline activator (SPC-rtTA).
The SPC-rtTA transgene has been used to induce genes efficiently in
developing lung epithelium, as early as E10, and in adult type II pneumocytes
(Perl et al., 2002;
Tichelaar et al., 2000). Two
out of five of the transgenic lines demonstrated robust and reproducible
doxycycline-inducible Fgf9 and eGfp expression in lung
epithelium. One of these lines was used for the following studies.
At all time points examined, following 48 hours of doxycycline administration, control embryos containing only the TRE-Fgf9-IRES-eGfp gene were phenotypically normal with no detectable eGFP activity or Fgf9 expression (Fig. 2). Transgenic pups that contained both the SPC-rtTA gene and the TRE-Fgf9-IRES-eGfp gene, but which were not exposed to doxycycline, were also phenotypically normal and showed no eGFP activity (data not shown), indicating tight regulation of transgene expression. By contrast, Fgf9 mRNA was detected throughout the lung epithelium of mice positive for both the TRE-Fgf9-IRES-eGfp and SPC-rtTA transgenes and exposed to doxycycline for 48 hours (Fgf9dox(48) mice) (Fig. 2J). Visual inspection of these lungs showed eGFP fluorescence under UV illumination (Fig. 2F). No additional sites of eGFP fluorescence were observed. Control lungs from the same litter demonstrated no fluorescence (Fig. 2E).
Fgf9 expression was induced for 48 hours prior to embryo harvest and lungs were examined at E11.5, E12.5 and E14.5 (Fig. 2). Similar to the in vitro response to FGF9, in vivo overexpression of Fgf9 during early lung morphogenesis induced a large mesenchymal expansion and a reduction in epithelial branching (Fig. 2A-D). Beginning at E12.5 (doxycycline at E10.5), the dramatic enlargement in lung size was characterized by an arrest in branching and luminal dilation, with morphology resembling a lung at the onset of doxycycline induction (Fig. 2C,D,G,H; data not shown). These observations suggest that the arrest in branching morphogenesis was initiated soon after doxycycline administration. Littermate control embryos treated with doxycycline and singly positive for SPC-rtTA, TRE-Fgf9-IRES-eGfp, or genotypically wild type, exhibited no difference in epithelial or mesenchymal morphology from non-doxycycline treated wild-type lungs (Fig. 2A,C,G).
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) showed an increased number of vessels in
Fgf9dox(48);Tie2-lacZ lungs
(Fig. 3C,D). The pattern of
Tie2-lacZ expression demonstrated the formation of a multilayered
capillary network in the presence of excess FGF9, in contrast to a single
layered vascular domain in control tissue. This pattern was confirmed by using
an anti PECAM1 antibody (Fig.
3E,F). E14.5 lungs were characterized by rapidly expanding
epithelial airways embedded in wide bands of mesenchyme
(Fig. 3H). At this stage, a
smooth muscle layer is formed in the sub-epithelial mesenchyme adjacent to
proximal airways (Fig. 3I). In
Fgf9dox(48) lungs, smooth muscle is notably absent from
the sub-epithelial compartment, although still present surrounding large blood
vessels (Fig. 3J). This is
consistent with in vitro data showing FGF9 inhibition of smooth muscle actin
(SMA) expression in mesenchyme-only explants in the presence of SHH
(Weaver et al., 2003), and
suggests that the FGF10-expressing lineage that gives rise to sub-epithelial
smooth muscle (Mailleux et al.,
2005) may be inhibited by epithelial expression of
Fgf9.
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;
Chuang et al., 2003;
Lebeche et al., 1999;
Litingtung et al., 1998;
Pepicelli et al., 1998;
Weaver et al., 2003). Because
Fgf9 also regulates mesenchymal proliferation and branching
morphogenesis, we hypothesized that early modulation of SHH signaling by FGF9
could account for some or all of these effects. Therefore, Shh and
Ptch1 expression were examined in
Fgf9–/– and Fgf9dox(48)
lungs during early stages of lung development. Ptch1 was of
particular interest, as it acts as transcriptional reporter of SHH signaling
activity (Bellusci et al.,
1997a; Grindley et al.,
1997; Ingham and McMahon,
2001; Litingtung et al.,
1998; Pepicelli et al.,
1998). At E11.5 and E12.5, the relative expression of epithelial
Shh was decreased in Fgf9–/– lungs,
but remained unchanged in the epithelium of the attached esophagus
(Fig. 4A,B,I,J). Importantly,
the expression of Ptch1 was significantly decreased in sub-epithelial
mesenchyme at E11.5 and E12.5, suggesting a decrease in SHH signaling in
Fgf9–/– lungs
(Fig. 4C,D,K,L). Gli1,
another transcriptional reporter of SHH signaling, was downregulated in distal
sub-epithelial mesenchyme in E12.5 Fgf9–/–
lungs (see Fig. S1 in the supplementary material). These data indicate that
Fgf9 is necessary to maintain normal levels of Shh
expression and SHH signaling to sub-epithelial mesenchyme during early lung
development. To determine whether FGF9 is sufficient to enhance SHH signaling, Ptch1 expression was examined in response to increased levels of mesothelial or epithelial FGF9. Consistent with the loss-of-function data, expansion of Ptch1 expression was observed in the mesenchyme of lung explant cultures exposed to FGF9 containing media (see below, Fig. 6B). Moreover, in E11.5 and E12.5 Fgf9dox(48) lungs, both Shh and Ptch1 were expressed at significantly higher levels throughout both proximal and distal regions (Fig. 4E-H,M-P). Taken together, these data indicate that FGF9 is both necessary and sufficient to induce Shh expression and SHH signaling.
FGF9 and SHH promote proliferation in distinct mesenchymal compartments of the developing lung
Lung histology at E12.5 revealed two morphologically distinct populations
of mesenchyme. The sub-epithelial mesenchymal cells appear condensed and
orient circumferentially around epithelial ducts in transverse sections, while
sub-mesothelial mesenchymal cells appear as a loose, non-oriented mesenchyme
(Fig. 5A). Noggin-lacZ
(Brunet et al., 1998) labels
sub-epithelial mesenchyme in both distal and proximal regions at E12.5 (see
Fig. S2). As both Fgf9 or Shh regulate mesenchyme
proliferation (Colvin et al.,
2001; Litingtung et al.,
1998) and FGF9 signaling maintains SHH signaling, we hypothesized
that FGF9 promoted sub-mesothelial proliferation directly and sub-epithelial
proliferation indirectly through SHH signaling.
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). By
contrast, a wide expanse of loose mesenchyme was evident prior to epithelial
expansion in E12.5 Fgf9dox(48) lungs
(Fig. 5C). Anti-phosphohistone
H3 labeling of mitotic cells in whole lung explants revealed a 38±8%
and 62±7% increase in the distal mesenchyme when treated with FGF9 for
24 and 48 hours, respectively (Fig.
5D,E,L,M). This increased proliferation primarily affected the
sub-mesothelial region (57±12% increase) versus the sub-epithelial
region (21±12% increase) at 24 hours. Lung explants treated with FGF9
appeared significantly more rounded and epithelial tubules were difficult to
visualize beneath the thickened mesenchyme
(Fig. 5D,E; data not shown).
Examination of these explants at high magnification showed increased labeling
of the sub-mesothelial region (Fig.
5D',E'). To determine whether FGF9-mediated mesenchymal proliferation required SHH signaling, the ratio of sub-epithelial to sub-mesothelial mesenchyme proliferation was assessed in response to FGF9 and cyclopamine. Treatment with cyclopamine inhibited Ptch1 expression (Fig. 6A,B) and resulted in a 31±4% (24 hours) and 66±19% (48 hours) decrease in distal mesenchyme proliferation (Fig. 5G,L,M). When explant cultures were incubated with both cyclopamine and FGF9, distal mesenchyme proliferation was at an intermediate level between the two conditions (Fig. 5F,L,M). However, when anti-phosphohistone H3-labeled cells were examined at high magnification, the sub-mesothelial region resembled explants treated only with FGF9 (39±10% increase), while proliferation was reduced in the sub-epithelial region (12±10% decrease) (Fig. 5E',F'). Furthermore, Ptch1 was absent in FGF9/cyclopamine co-incubated explants, similar to cyclopamine alone (data not shown). This suggests that mesothelial FGF9 does not require SHH signaling to stimulate sub-mesothelial mesenchymal proliferation, but that epithelial FGF9 may positively influence sub-epithelial mesenchymal proliferation by stimulating or maintaining SHH signaling (increased Ptch1 expression is present in FGF9-treated explants, Fig. 6B). These data indicate that FGF9 secretion from the mesothelium during the pseudoglandular stage acts to regulate lung volume by inducing the growth of sub-mesothelial mesenchyme, and indirectly regulates sub-epithelial mesenchyme through SHH signaling.
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). To determine
if SHH signaling is required for mesenchymal cell survival in whole lung
explants, an antibody to active caspase 3 was used to assess cell death in
cyclopamine treated lung explant cultures. Anti-caspase 3 staining was
increased when cultured with cyclopamine or with FGF9 and cyclopamine,
indicating that SHH signaling is necessary for cell survival as well as
proliferation, and that FGF9 does not completely rescue sub-epithelial
viability (Fig. 5J,K).
Models of lung development suggest that high levels of SHH signaling
decrease expression levels of Fgf10 but upregulate Fgf7
(Bellusci et al., 1997a;
Chuang et al., 2003;
Lebeche et al., 1999;
Pepicelli et al., 1998).
Consistent with this, at E12.5, the domain of Fgf10 expression was
expanded in Shh–/– lungs
(Pepicelli et al., 1998). We
hypothesized that loss of SHH signaling would affect epithelial branching
morphogenesis by enhancing budding through a de-repression of Fgf10,
and that FGF9 would result in a decrease in budding through increased
repression of Fgf10 (through enhanced SHH signaling) and resemble the
branching pattern seen in vivo. To test this hypothesis, we examined patterns
of epithelial branching in lung explants incubated with FGF9 and
cyclopamine.
Cyclopamine-treated E12.5 lung explants incubated for 48 hours exhibited a
complete absence of Ptch1 expression
(Fig. 6A,C), and a
statistically significant increase in the number of distal epithelial buds,
which also appeared elongated (Fig.
6D,E,G). This phenotype is similar to that observed in
Fgf10-treated lung epithelial cultures
(Bellusci et al., 1997b). By
contrast, FGF9-treated E12.5 lung explants showed increased Ptch1
expression (Fig. 6B) and were
characterized by increased distal mesenchyme, decreased distal buds and
dilated epithelial lumens (Fig.
6D-F).
FGF9 mediated epithelial expansion results from induction of mesenchymal FGF expression
FGF9 signals to mesenchymal splice forms of FGFRs and is, therefore,
unlikely to directly signal to lung epithelium. We hypothesized that FGF9
regulates the expression or activity of lung mesenchymal genes in addition to
SHH signaling, which in turn act as effectors of epithelial morphogenesis.
FGF7 and FGF10 signal to epithelial FGFRs to regulate epithelial development
and are likely candidates for this mesenchymal to epithelial signal.
In Fgf9–/– lungs, regional expression of
Fgf10 was decreased in sub-mesothelial mesenchyme at E13.5-E14.5,
resulting in a decrease in branching morphogenesis
(Colvin et al., 2001). To
further explore the epithelial-mesenchymal relationship between FGF9 and
FGF10, and to determine whether FGF10 could contribute to all or part of the
Fgf9dox(48) epithelial phenotype, Fgf10
expression was examined following induced expression of FGF9 from E12.5 to
E14.5. In control tissues, the intensity of Fgf10 expression varied
between distal and lateral locations around epithelial structures, with
highest expression between epithelial tubules
(Fig. 7A, arrowheads). By
contrast, these regional variations in Fgf10 expression were no
longer present in tissues overexpressing Fgf9, and Fgf10 was
expressed at high levels throughout the distal sub-mesothelial mesenchyme
(Fig. 7B). The location of
Fgf10 expression appeared distal to the domain of Ptch1,
consistent with the proposed ability of SHH to repress Fgf10.
Loss of focal Fgf10 expression in the distal mesenchyme does not
account for the observed epithelial dilation throughout both the proximal and
distal epithelium. We hypothesized that another mesenchymal factor must be
responsible for the luminal dilation. Fgf7 is diffusely expressed at
low levels throughout the mesenchyme during the pseudoglandular stage
(Fig. 7C) and has a stronger
proliferative effect on epithelium than FGF10
(Bellusci et al., 1997b;
Park et al., 1998).
Furthermore, Fgf7 overexpression in vitro and in lung epithelium in
vivo resulted in a luminal dilation
(Cardoso et al., 1997;
Park et al., 1998;
Simonet et al., 1995;
Tichelaar et al., 2000).
Examination of Fgf7 expression in Fgf9dox(48)
lungs demonstrated upregulation throughout the sub-epithelial mesenchyme
(Fig. 7C,D). These observations
suggest that both Fgf7 and Fgf10 are induced by FGF9 and
together may mediate the Fgf9dox(48) lung epithelial
phenotype.
To further test this hypothesis, the expression of Spry2 and
Bmp4 was examined following induction of Fgf9 in vivo or
exposure to FGF9 in vitro. Both Spry2 and Bmp4 are normally
expressed in the distal tip epithelium and are upregulated in response to
FGF10 signaling to FGFR2b in vitro
(Lebeche et al., 1999;
Mailleux et al., 2001;
Weaver et al., 2000).
Consistent with increased mesenchymal to epithelial FGF signaling, both
Spry2 and Bmp4 expression were significantly upregulated in
both proximal and distal epithelium in Fgf9dox(48) lungs
and in explants exposed to FGF7, FGF9 or FGF10 beads
(Fig. 7F,J; data not shown).
The extension of Spry2 and Bmp4 expression in both proximal
and distal locations is consistent with mesenchymal to epithelial FGF signals
in both proximal (FGF7) and distal (FGF7 and FGF10) locations. We conclude
from these data that FGF9 is sufficient to induce Spry2 and
Bmp4 through induction of Fgf7 and Fgf10.
To determine whether Fgf9 is necessary to maintain Spry2
and Bmp4 expression in vivo, Spry2 and Bmp4
expression patterns were examined in Fgf9–/–
lungs. Spry2 was decreased in lateral epithelial branches at E13.5
(Fig. 7G,H). At E12.5,
Bmp4 was expressed in locations comparable with controls
(Fig. 7K,L). By contrast, at
E14.5 Bmp4 was dramatically reduced in the distal epithelium
(Fig. 7M,N). These data are in
agreement with the observed reduced expression of Fgf10 in
Fgf9–/– lungs at mid to late stages of
branching morphogenesis (Colvin et al.,
2001) and suggest regulation of Spry2 and Bmp4
by FGF10 in vivo.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), is located in a position where it could fulfill a function
in sensing the size and shape of the thoracic cavity. Regulation of the
expansion of lung mesenchyme serves at least two roles: to allow the lung to
fill the thoracic space and to create a sufficient volume of mesenchyme
through which branching morphogenesis can proceed. This model suggests that
Fgf9 expression or activity could be spatially regulated (possibly by
mechanical forces) to allow the growing lung to contour the growing thoracic
cavity.
Using both in vitro organ culture and in vivo gain- and loss-of-function
models, we demonstrated that FGF9 is both necessary and sufficient for growth
of distal mesenchyme. Because SHH signaling appears to affect both mesenchymal
proliferation and epithelial development
(Litingtung et al., 1998;
Pepicelli et al., 1998), we
hypothesized that FGF9 could regulate mesenchymal proliferation both directly
and indirectly through upregulation of SHH signaling at early stages. We
showed that increased FGF9 signaling enhanced HH signaling and that loss of
Fgf9 reduced HH signaling in the sub-epithelial compartment. Because
both of these effects are modulated by epithelial Shh, we posit that
FGF9 should regulate a mesenchymal to epithelial signal that controls
Shh expression. Our preliminary data excludes BMP signaling, and
although retinoic acid (RA) has been shown to induce Shh expression
in culture, previous studies indicate that RA signaling is not active in the
epithelium at this stage (Bellusci et al.,
1997a; Malpel et al.,
2000). Additionally, FGF10 does not affect epithelial expression
of Shh (Lebeche et al.,
1999).
Given this relationship between FGF9 and SHH signaling, we asked whether HH signaling contributes to FGF9-induced mesenchymal proliferation. As Fgf9 is expressed in mesothelium throughout pseudoglandular development, and Shh is expressed exclusively in the epithelium, we propose a model (Fig. 8A) in which FGF9 primarily functions to stimulate the sub-mesothelial mesenchyme, whereas HH signaling functions to increase proliferation and maintain cell survival in the sub-epithelial compartment. In vitro organ cultures, in which lung explants were treated with FGF9 and cyclopamine, support a model in which SHH acts in an independent, spatially specific manner to regulate mesenchymal proliferation. Additionally, FGF9 may indirectly contribute to sub-epithelial mesenchymal proliferation through regulation of Shh.
|
), which
suggests that FGF9 does not induce proliferation in SHH treated
mesenchyme-only explants (Weaver et al.,
2003). By contrast, we show that FGF9 is both necessary and
sufficient to promote mesenchymal proliferation in vivo and in vitro. A
possible explanation for the lack of observed FGF9-induced proliferation in
the mesenchyme-only explant system may be due to the requirement of SHH for
both survival and proliferation, which we suggest will enrich for
sub-epithelial cells that are unable to proliferate in response to FGF9.
Interestingly, in the Weaver et al. study, some markers in the mesenchyme-only
explants demonstrated an unusual distribution, which supports a mixture of
distinct mesenchymal cells.
Additionally, the model presented by Weaver et al.
(Weaver et al., 2003)
suggested that an FGF9-promoted zone of undifferentiated cells is present in
the sub-mesothelial mesenchyme. We show that FGF9 is sufficient to expand
Tie2-lacZ and PECAM-positive cells into the sub-mesothelial region in
vivo, which probably represents increased vascular development. Thus, FGF9 may
signal to undifferentiated mesenchyme and/or vascular progenitor cells to
control vascular development. Interestingly, a recent study suggests that
early lung vascular development occurs through distal angiogenesis
(Parera et al., 2005) and
would suggest that FGF9 promotes blood vessel formation from pre-existing
vessels. Studies are under way to determine the relationship between FGF9 and
vascular development.
FGF9 regulates epithelial development through mesenchymal FGF expression
A key requirement for the formation of an epithelial branch is a focal
source of a ligand that is capable of stimulating epithelial migration. A
mesenchymal FGF10 to epithelial FGFR2b signal fulfills this role
(Arman et al., 1999;
Bellusci et al., 1997b;
Celli et al., 1998;
De Moerlooze et al., 2000;
Min et al., 1998;
Park et al., 1998;
Peters et al., 1992;
Sekine et al., 1999). Because
branching morphogenesis is severely decreased in lungs lacking FGF9 and absent
in lungs overexpressing FGF9, we sought to further explore the relationship
between FGF9 signaling and Fgf10 expression and signaling.
Overexpression of FGF9 caused dramatic epithelial expansion in vitro and in
vivo, and loss of FGF9 resulted in mesenchymal depletion, decreased
Fgf10 expression and decreased branching morphogenesis
(Fig. 8B,D). Because FGF9 is
unlikely to signal to epithelial splice forms of FGFR1 and FGFR2
(Ornitz et al., 1996), we
hypothesized that FGF9 overexpression induces a secondary mesenchymal
signal(s), such as Fgf10, which could mediate epithelial luminal
expansion. To characterize this mesenchymal to epithelial signal, we examined
the expression of Fgf10 and the distal epithelial markers,
Spry2 and Bmp4, both of which are positively regulated by
FGF10 in vitro (Lebeche et al.,
1999; Mailleux et al.,
2001; Weaver et al.,
2000). At E11.5, Fgf10 expression appeared normal in
Fgf9–/– lungs but was greatly reduced at later
stages (Colvin et al., 2001).
Consistent with FGF10 being a primary mesenchymal to epithelial signal,
expression of Spry2 and Bmp4 were downregulated over a
similar time course, and ectopic FGF9 enhanced expression of Fgf10,
Bmp4 and Spry2 both in vivo and in vitro. Interestingly, in FGF9
gain-of-function experiments, Bmp4 and Spry2 expression were
upregulated ectopically throughout the proximal and distal airway epithelium,
whereas Fgf10 expression was restricted to the distal mesenchyme
(Fig. 8A,D). This suggested
that the hypothesized FGF9-induced mesenchymal to epithelial signal may
originate from both proximal and distal mesenchyme. Consistent with this,
ectopic FGF9 upregulated Fgf7 throughout the sub-epithelial
mesenchyme, making FGF7 a likely candidate to mediate FGF9-induced epithelial
expansion and ectopic Spry2 and Bmp4 expression
(Fig. 8A,D). These data suggest
that during normal lung development, an epithelial to mesenchymal FGF signal,
such as FGF9, induces Fgf7 following branching morphogenesis. FGF7
may then facilitate the transition from the pseudoglandular to the canalicular
phase of lung development.
SHH is expressed at the highest levels in the distal tip of the epithelial
ducts and has been shown to suppress Fgf10 expression
(Lebeche et al., 1999;
Pepicelli et al., 1998).
Consistent with this, FGF10 expression
(Mailleux et al., 2005) and
branching (Fig. 6;
Fig. 8E) is increased in
cyclopamine treated lung explants. In Fgf9-overexpressing lungs, the
uniformity of sub-mesothelial Fgf10 in the distal mesenchyme and the
upregulation of SHH signaling represents an apparent paradox as enhanced SHH
signaling should repress Fgf10. Additionally, current models predict
an increase in branching with increased Fgf10 expression. Instead,
branching morphogenesis abruptly stops when Fgf9 is overexpressed.
This paradox can be resolved by a model in which increased HH signaling in the
immediate sub-epithelial mesenchyme (induced by FGF9) represses Fgf10
expression through a HH-dependent pathway, resulting in the termination of
branching morphogenesis. In the sub-mesothelial region, distal from the source
of SHH, FGF9 induces Fgf10 expression. Distal non-focal expression of
FGF10 may be insufficient to induce epithelial branching, but sufficient to
enhance tubule elongation (Fig.
8D).
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/8/1507/DC1
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REFERENCES |
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