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First published online 18 July 2007
doi: 10.1242/dev.006221
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1 Pulmonary Center, Boston University School of Medicine, Boston, MA 02118,
USA.
2 Biochemistry Department, Stanford University, Stanford, CA 94305, USA.
3 Baylor College of Medicine, Houston, TX 77030, USA.
* Author for correspondence (e-mail: wcardoso{at}bu.edu)
Accepted 17 June 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Retinoic acid, Fgf10, Fibroblast growth factor, Tgfß, Transforming growth factor, Lung development, Foregut development, Organogenesis, Mouse, Raldh2 (Aldh1a2)
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Respiratory progenitors (lung and trachea) can be identified in the foregut
by E9.0 as a group of endodermal cells posterior to the thyroid that expresses
the transcription factor Nkx2.1 (also known as Titf1 - Mouse Genome
Informatics) (Minoo et al.,
1999). Subsequently, Fgf10, a fibroblast growth factor that is
crucial for budding, is expressed locally in the mesoderm adjacent to these
Nkx2.1-expressing endodermal cells to trigger Fgf receptor 2b (Fgfr2b)
signaling, resulting in primary lung bud formation. Once primary lung buds
form, epithelial tubules undergo extensive branching morphogenesis, which
ultimately results in the formation of the bronchial tree and the future
alveolar region of the lung (Cardoso and
Lu, 2006; Shannon and Hyatt,
2004).
Lung morphogenesis depends on complex interactions between local signals
present in this prespecified foregut endoderm and signals from the adjacent
mesoderm (Cardoso and Lu,
2006; Shannon and Hyatt,
2004). The mechanisms that control gene expression and cellular
activities in the lung field of the foregut at the onset of lung development
are still poorly understood. Several studies have implicated retinoic acid
(RA) signaling as a key regulator of these functions during development of the
foregut and its derivatives. Genetic deletion of retinaldehyde dehydrogenase 2
(Raldh2; also known as Aldh1a2 - Mouse Genome Informatics),
an enzyme essential for RA synthesis in the mouse embryo, results in multiple
organ defects and death at around E10.5
(Niederreither et al., 1999).
RA signals through nuclear receptors Rars and Rxrs (each with isotypes
, ß and 
), which are found as heterodimers bound to
RA-responsive elements (RAREs) of target genes
(Chambon, 1996). These
receptors are expressed in the developing lung from its earliest stages
(Mollard et al., 2000b);
double-null mutant (Rara/Rarb or Rara/Rxrb) mice show
several features previously described in vitamin A-deficient animals
(Chambon, 1996;
Clagett-Dame and DeLuca, 2002;
Kastner et al., 1997;
Mendelsohn et al., 1994;
Wilson et al., 1953). Maternal
deficiency of vitamin A results in dramatic abnormalities in the respiratory
system of the embryo, which include tracheoesophageal fistula, lung hypoplasia
and lung agenesis (Dickman et al.,
1997).
In the developing lung, RA synthesis and utilization are most prominent
when primary buds are emerging from the primitive foregut
(Malpel et al., 2000).
Treatment of the whole E8.5 mouse embryo or isolated E8.5 foregut explants
with the pan-RA receptor (RAR) antagonist BMS493 completely abrogates
development of the lung and the neighboring stomach
(Desai et al., 2004;
Mollard et al., 2000a). We
found that this phenotype results from failure to induce Fgf10
expression in the foregut mesoderm at the prospective lung field. The
regulation of Fgf10 by RA occurs within a defined developmental
window and is not seen in other foregut derivatives (such as thyroid and
pancreas), where Fgf10 is also required for normal development
(Desai et al., 2004). These
observations have been confirmed in Raldh2-/- mice and
vitamin A-deficient rats (Desai et al.,
2004; Desai et al.,
2006; Wang et al.,
2006). Furthermore, we have shown that RA is not required for the
initiation of lung endodermal cell fate in the foregut
(Desai et al., 2006).
These studies raised the intriguing possibility that at the onset of lung development, RA-responsive genes are selectively activated or repressed in the prospective lung field of the foregut to allow bud formation. It was not known which genes present in the developing foregut could be involved in this process. Here we addressed this problem using oligonucleotide microarray and functional analyses in the models of RA deficiency that we had previously characterized, and at a stage in which lung development is crucially dependent on the RA status of the embryo. We provide evidence of a novel regulatory mechanism implicating RA, transforming growth factor ß (Tgfß) and Fgf10 interactions in primary lung bud induction. Our results suggest that at the onset of lung development, endogenous RA controls Tgfß signaling in the prospective lung field of the foregut to allow Fgf10 expression and induction of primary lung buds.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Raldh2-/- homozygous embryos were distinguished from their
heterozygous and wild-type (WT) littermates by their distinct phenotypic
features and by genotyping by PCR
(Niederreither et al., 1999).
Detection of RA signaling was achieved using the RARElacZ reporter
mouse line. These mice carry the bacterial lacZ gene under the
control of a heat shock protein promoter (Hsp68) and RAREs from the
Rarb promoter (Rossant et al.,
1991). X-Gal staining is used to visualize lacZ
expression (Malpel et al.,
2000). Fgf10lacZ reporter mice have been reported
previously (Kelly et al.,
2001). In this mutant, a myosin light chain-lacZ
(Mlc1v-nlacZ-24) transgene was integrated upstream of Fgf10,
which allows control of lacZ expression by Fgf10 regulatory
sequences; X-Gal staining reveals sites of Fgf10 expression in the
embryo, including the lung (Mailleux et
al., 2005). For all experiments, conclusions were based on the
evaluation of three independent specimens per culture condition.
Foregut explant cultures
The foregut culture system has been reported previously
(Desai et al., 2004;
Desai et al., 2006). Briefly,
timed-pregnant mice were sacrificed at E8.5 and foreguts were isolated from
the embryos (8- to 12-somite stage) in phosphate-buffered saline (PBS) using
tungsten needles. Extra-embryonic tissues and dorsal structures were removed.
Explants were cultured for 1-3 days on 6-well Transwell-Col dishes (Costar)
containing 1.5 ml of BGJb medium (Gibco-BRL), 0.3 mg of vitamin C (Sigma) and
10% fetal calf serum (FCS, Gibco-BRL) with or without the specific modulators
of RA or Tgfß signaling (see below). Cultures were shielded from light
and incubated at 37°C in 95% air and 5% CO2. Media were changed
daily. Under control conditions, lung buds, stomach and pancreas formed within
24 hours of culture. In some experiments, heparin beads soaked in 100 ng/ml
FGF10 (R&D systems) or PBS buffer were grafted onto the foregut after 24
hours of culture.
Modulation of RA and Tgfß signaling
BMS493 (Bristol Meyers Squibb), a pan-RAR antagonist, dissolved in BGJb
medium (10-6 M) was used to antagonize RAR-dependent signaling, as
previously reported in this and other systems
(Mollard et al., 2000b;
Wendling et al., 2000).
All-trans RA (Sigma) dissolved in BGJb medium (10-7 M) was used to
rescue RA signaling in Raldh2-/- foreguts in culture.
Foreguts were treated for 3 days with recombinant human TGFß1 (5-20
ng/ml, R&D Systems) or a pan-specific TGFß-blocking antibody (200
µg/ml, R&D Systems) dissolved in BGJb medium to activate or inhibit
Tgfß signaling, respectively.
Microarray analysis of RA-responsive genes in the developing foregut
E8.5 WT (control and BMS493-treated, n=3 each) and
Raldh2-/- (control and RA-treated, n=3 each)
foreguts were cultured for 24 hours. Total RNA was isolated using the RNeasy
Kit (Qiagen), and subjected to amplification, labeling, and fragmentation
according to Affymetrix's recommendations. cRNA was hybridized to Affymetrix's
Mouse Genome 430 2.0 array chips. Three array chips were used per experimental
condition. A single weighted mean expression level for each gene per condition
along with a detection P value was calculated using Affymetrix
Microarray Suite 5.0 software. Data from each array were scaled to the target
intensity of 500 to normalize the results for inter-array comparisons. Quality
control parameters of each chip met the acceptable criteria provided by
Affymetrix. Genes with detection P values greater than 0.05
(considered to be `absent') in all twelve chips were eliminated from the
analysis. Gene expression profiles were compared (WT control versus BMS493;
Raldh2-/- control versus RA). The difference in expression
of each gene was considered to be significant if the P value was
lower than 0.05 (Cyber t-test,
http://visitor.ics.uci.edu/genex/cybert).
To further increase the specificity of the analysis, only genes whose
expression levels were significantly changed in both comparisons were retained
on the final list of potential RA targets.
Western blotting
After 24 hours of culture, the heart of the foregut explant was separated
from the foregut region and discarded. The protein extract from individual
foregut explants was subjected to SDS-PAGE, blotted onto nitrocellulose,
washed in TBST [Tris-buffered saline (TBS) with 0.1% Tween 20], blocked with
5% milk in TBST, and incubated with polyclonal antibody (1:400) to
phosphorylated Smad2 (pSmad2, Cell Signaling) in TBST with 5% milk overnight.
The Immun-Star HRP Chemiluminescent Kit (Bio-Rad) was used for signal
development according to the manufacturer's instructions. Total Smad2 (tSmad2,
Cell Signaling) was used for normalization.
Whole-mount in situ hybridization
Whole-mount in situ hybridization (WMISH) of explants and embryos was
performed in a 96-well plate as previously described
(Lu et al., 2004;
Wertz and Herrmann, 2000).
Briefly, digoxigenin (DIG)-labeled riboprobes (Maxiscript kit, Ambion) were
generated and amplified from total embryonic cDNA (Col1a2, Ctgf, Tgfb2,
Tgfbr1, Tgfbr2) or plasmids carrying cDNA for the genes of interest
(Tgfb1, Nkx2.1, Sftpc, Fgf10, Tgfb3, Tgfbi). Specimens were
rehydrated, digested with proteinase K (Boehringer Mannheim), prehybridized (1
hour, 70°C) in buffer containing 50% formamide, 5xSSC, 1% SDS, 50
mg/ml yeast RNA and heparin followed by overnight hybridization with
DIG-labeled RNA probes, and another overnight incubation with anti-DIG
alkaline phosphatase conjugate (Boehringer Mannheim) at 4°C. Signal was
visualized with BM Purple substrate (Roche Diagnostics). Conclusions were
based on the evaluation of at least three independent specimens per probe per
condition.
Immunohistochemistry
Tissues were fixed in 4% paraformaldehyde in PBS overnight at 4°C,
washed twice in TBS with 0.1% Triton X-100 (Sigma), and blocked for 1 hour in
blocking buffer (1xTBS with 5% donkey sera, 0.1% Triton X-100). Samples
were incubated with primary antibody in blocking buffer overnight [1:150
dilution of rabbit anti-mouse pSmad2 antibody (Cell Signaling), 1 µg/ml
sheep anti-mouse Tgfbi antibody (R&D systems)] at 4°C, washed for 1
hour and incubated with secondary antibody overnight (1:750 donkey anti-rabbit
antibody conjugated to AF488 and donkey anti-sheep antibody conjugated to
AF598, both from Molecular Probes), washed five times for 1 hour each in
blocking buffer, then stored in SlowFade Gold Antifade buffer (Molecular
Probes) and photographed with a laser confocal microscope.
Mesenchymal lung cell culture and real-time PCR
Mouse neonatal lung mesenchymal (MLg) cells were cultured in DMEM, 10% FCS
with or without the specified modulator (all-trans RA or TGFß1) for 8-24
hours (n=3 100-mm dish plates per condition). Total RNA was isolated
(Trizol, Invitrogen), reverse transcribed (1 µg RNA) and amplified by
real-time PCR (SYBR Green qPCR Kit, Applied Biosystems). A dissociation curve
was used to determine the relative concentration of the single PCR product.
18S RNA was used for normalization.
Cell proliferation and cell death assay
Cell proliferation was assessed by expression of PCNA protein (PCNA
Staining Kit, Zymed). Apoptosis was evaluated by TUNEL (ApopTag Plus,
Chemicon). These assays were performed in paraffin sections (5 µm) of
foregut explants according to the manufacturers' recommendations. Sections
were counterstained with Methyl Green.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the
genetic model, Raldh2-/- foreguts were similarly cultured
in control medium (in which no lung forms) or in medium containing RA
(10-7 M), which allowed lung formation in these mutants
(Desai et al., 2006).
Microarray analysis was performed on RNA isolated from these samples (as
described in Materials and methods). Modulation of RA signaling was confirmed
by altered expression of known RA targets, such as Rarb, Hoxa1 and
Hoxb1 (Chambon, 1996;
Niederreither et al., 1999). A
comprehensive description of these transcriptional profiles will be reported
elsewhere. Analysis of these profiles using the EASE (Expression Analysis
Systematic Explorer,
http://david.abcc.ncifcrf.gov)
software revealed a striking over-representation of `Tgfß signaling
pathway-related genes' upregulated in the RA-deficient foreguts
(P=0.009). This list was expanded, as we searched for additional
Tgfß targets based on published reports
(Table 1, description
below).
|
Tgfß targets identify sites of Tgfß hyperactivation in the mesoderm of RA-deficient foreguts
The Tgfß targets upregulated in RA-deficient foreguts encode a diverse
group of molecules, and include several mediators of the fibrogenic activities
of Tgfß [connective tissue growth factor (Ctgf), cysteine rich
protein 61 (Cyr61), procollagen type I alpha 2 (Col1a2),
procollagen type III alpha 1 (Col3a1), procollagen C-endopeptidase
enhancer protein (Pcolce), tissue inhibitor of metalloproteinase 1
and 3 (Timp1 and Timp3, respectively)], secreted proteins
[biglycan (Bgn), transforming growth factor ß induced
(Tgfbi), secreted acidic cysteine rich glycoprotein (Sparc),
secreted phosphoprotein 1 (Spp1)], cell surface receptors [CD44
antigen (Cd44)], transcription factors [activating transcription
factor 3 (Atf3)], colony stimulating factor (Csf1), as well
as insulin-like growth factor binding protein 4 (Igfbp4) (see
Table 1 for references).
Previous studies and an initial assessment of the expression pattern of
these genes at the onset of lung development showed them to be predominantly
transcribed in mesodermal tissues (Chuva
de Sousa Lopes et al., 2004;
Ferguson et al., 2003;
Ponticos et al., 2004). The
presence of these targets in the mesoderm, where Raldh2 and
RARElacZ are also expressed
(Malpel et al., 2000),
suggests that RA and Tgfß pathways interact locally in the foregut. Thus,
we assessed expression of three representative Tgfß targets, Tgfbi,
Ctgf and Col1a2, in our system.
Tgfbi (also called ßig-h3) is a 68 kDa secreted cell-adhesion
molecule, known to bind collagen, fibronectin and sulfated glycosaminoglycans,
which has been shown to be induced by TGFß1 in various cells lines
(Billings et al., 2002;
Skonier et al., 1994). First,
we validated Tgfbi as a read out of Tgfß activation in lung
mesoderm-derived cells (MLg) by showing a dose-dependent induction of
Tgfbi at 8 hours with recombinant TGFß1 treatment
(Fig. 1B). Then, to investigate
the RA-Tgfbi relationship in the foregut, we compared sites of
Rar-dependent signaling and Tgfbi transcription by X-Gal staining of
RARElacZ reporter mice and WMISH of Tgfbi, respectively. In
wild-type (WT) control cultures, Tgfbi was localized to the foregut
mesoderm associated with the proximal (stalk) region of lung primordium, where
RA signaling was reported by lacZ expression
(Fig. 1C-E).
BMS493 disruption of RA signaling abolished RARElacZ expression and resulted in strong Tgfbi signals in the foregut mesoderm, particularly obvious where the lung bud and stomach failed to form (n=6) (Fig. 1G-I). Immunostaining of Tgfbi showed a marked increase of Tgfbi protein locally in the lung field; the broader domain of expression, compared with that of mRNA, suggested accumulation that is likely to be due to a longer half-life of Tgfbi protein. The stronger pSmad2 staining in BMS493-treated foreguts compared with controls, as revealed by confocal image analysis, further suggested an association between increased Tgfbi expression and hyperactivation of Tgfß signaling (Fig. 1F,J). The negative spatial correlation between Tgfbi and RA-dependent signaling was further supported by comparing the expression of Tgfbi and RARElacZ in E9.0-9.5 embryos. In WT embryos, Tgfbi transcripts were almost undetectable in the RARElacZ-expressing region that encompasses most of the foregut (Fig. 2A,B; see also Fig. 2C, asterisk on the right). This contrasted with the abundant Tgfbi signals observed in the corresponding region of Raldh2-/- mice in vivo (Fig. 2C, arrowhead on the left), or in Raldh2-/- foreguts cultured without RA supplementation (Fig. 2D). Although in Raldh2-/- explants Tgfbi signals were highly induced in both thyroid and lung fields, bud formation and Nkx2.1 expression were disrupted only in the lung field (Fig. 2D,E). Strikingly, Tgfbi signals were dramatically reduced in Raldh2-/- foregut explants in which exogenous RA rescued budding and expression of Nkx2.1 in the lung field (Fig. 2F,G). Independent evidence that activation of RA signaling (by treatment with RA at 10-7 M) inhibits Tgfbi expression in lung mesoderm-derived MLg cells (which also respond to TGFß1) is provided in Fig. 2H. Tgfbi levels are not affected by BMS493 treatment in these cells, as they lack endogenous RA signaling (data not shown).
|
|
).
Col1a2 is one of the subunits of type I collagen, which represents a major
structural component of most connective tissues
(Olsen et al., 2003).
Ctgf signals could not be identified in the WT control foreguts
(Fig. 3A), but were
dramatically induced in the foregut mesoderm associated with the presumptive
lung field of BMS493-treated WT and non-RA-supplemented
Raldh2-/- foreguts
(Fig. 3B,C). Rescue of the lung
bud in RA-treated Raldh2-/- cultures was associated with a
marked decline in Ctgf expression
(Fig. 3D). Analysis of
Col1a2 expression showed a similar behavior under the experimental
conditions described above (Fig.
3E-H).
|
). Foregut treatment with 5 ng/ml of recombinant TGFß1
resulted in only a small endodermal pouch where lungs were expected to develop
(n=3, data not shown). At 20 ng/ml, TGFß1 consistently blocked
induction of lung buds and stomach (n=15), reminiscent of the effect
seen in BMS493-treated cultures (Fig.
4A,B) (Desai et al.,
2004). Tgfß activation was confirmed by widespread induction
of Tgfbi mRNA and protein throughout the explant
(Fig. 4C,D). Unlike BMS493,
TGFß1 affected other foregut-derived structures. For example, albumin
gene expression, not altered by BMS493 treatment, was inhibited by TGFß1
(Desai et al., 2004) (data not
shown). The disruption of lung development was confirmed by the absence of the
endodermal markers Nkx2.1 and surfactant associated protein
C(Sftpc) transcripts in the presumptive lung field of
TGFß1-treated foreguts (Fig.
4I-L). The presence of abundant proliferating cell nuclear antigen
(PCNA) and control-like pattern of TUNEL staining in all layers suggested that
TGFß1 was not inducing toxic effects in these explants
(Fig. 4E-H and data not
shown). We asked whether the effects above could be secondary to TGFß1-mediated disruption of RA signaling. This was ruled out by experiments in which RARElacZ foreguts were shown to maintain strong lacZ signals after 3 hours and 72 hours of culture in TGFß1-containing medium (Fig. 5A,B).
TGFß1 disrupts Fgf10 expression in the foregut mesoderm
Based on the reported role of Fgf10 in organogenesis
(Min et al., 1998;
Sekine et al., 1999), we
proposed that the disruption of lung bud formation by TGFß1 was due to
loss of Fgf10 expression in the mesoderm. WMISH analysis confirmed
that this was the case, as Fgf10 expression was almost abolished
throughout the TGFß1-treated foreguts
(Fig. 5C,D). The effect did not
seem to result from loss of Fgf10-expressing mesodermal cells, but
rather from downregulation of Fgf10 expression in these cells.
Indeed, treating MLg cells with TGFß1 also resulted in marked
downregulation of Fgf10 mRNA (Fig.
5E), in agreement with observations in other systems
(Beer et al., 1997;
Lebeche et al., 1999;
Tomlinson et al., 2004).
Interestingly, engrafting a heparin bead soaked in FGF10 protein onto
TGFß1-treated foreguts rescued bud outgrowth and Nkx2.1
expression in the foregut endoderm, in spite of the widespread hyperactivation
of Tgfß signaling (Fig.
5F,G). This suggests that the effects of TGFß1 in the
prospective lung endoderm are likely to be secondary to the loss of
Fgf10 in the mesoderm, because when Fgf10 is replaced exogenously,
Fgfr2b signaling appears to stimulate lung gene expression and growth even in
the context of Tgfß pathway hyperactivation.
|
|
), in agreement with our findings
(Fig. 1F). Expression of
components of the Tgfß pathway has been extensively reported in the
developing foregut, although these studies have not focused specifically on
the lung domain (Millan et al.,
1991; Roelen et al.,
1994). To address this issue, we performed a comprehensive
expression pattern analysis of the Tgfß subfamily ligands
(Tgfb1-3) and receptors (Tgfbr1-2) both in vivo (E8.5-9.5
embryo and E12.5 lung) and in foregut cultures. At E8.5, both Tgfbr1
and Tgfbr2 were expressed diffusely in all layers of the foregut
(Fig. 6A,E). This diffuse
pattern was also seen after 24 hours in culture, but by 72 hours both
receptors were predominantly expressed in the foregut endoderm with lower
signals in the mesoderm (Fig.
6B,C,F,G,L,M). Strong Tgfbr1/2 signals were detected
later in the distal E12.5 lung in vivo
(Fig. 6D,H). Interestingly,
analysis of Fgf10 expression either by WMISH or by X-Gal staining of
an Fgf10lacZ reporter mouse
(Kelly et al., 2001;
Mailleux et al., 2005) showed
that sites where Fgf10 is induced in the lung field are also enriched
in Tgfbr1/2 transcripts (Fig.
6I-M). This suggests that Fgf10-expressing mesodermal
cells in the lung field are able to respond to Tgfß ligands (either
endogenous or exogenous) and activate Tgfß signaling. Together with the
observations from the previous section, the data further support the idea that
Tgfß signaling in the mesoderm influences Fgf10 expression.
Which ligands are expressed in the lung field?
Fig. 6N-V confirms previous
reports showing that the foregut mesoderm expresses Tgfb1-3 at E8.5
and at 24 hours in culture. Later, Tgfb2 became more restricted to
the epithelium (in the E12.5 lung, Fig.
6S); Tgfb3 expression could also be seen in the mesoderm
and mesothelial surfaces (see pleura in
Fig. 6V).
|
;
Cardoso and Lu, 2006;
Letterio et al., 1994).
To investigate the role of endogenous Tgfß signaling in lung
formation, first we asked whether we could reliably prevent activation of
signaling by all Tgfß subfamily ligands in our foregut explants. For
this, we cultured E8.5 foreguts in medium containing a pan-specific
TGFß-blocking antibody (referred to here as TAb) or control
isotype-matched immunoglobulins. Changes in expression of pSmad2 in
TAb-treated explants could not be detected, given that pSmad2 signals were
already relatively low in untreated WT foreguts. To circumvent this problem,
we used WMISH to assess expression of the Tgfß targets Tgfbi,
Col1a2 and Ctgf as `reporters' of Tgfß activity in these
cultures. Tgfbi and Col1a2 transcripts were significantly
reduced in WT foregut explants treated with TAb (200 µg/ml)
(Fig. 7A-D). Efficient
inhibition of Tgfß signaling was also suggested by the dramatic reduction
in the Ctgf levels observed in Raldh2-/- foreguts
cultured in TAb-containing medium (Fig.
7E,F, also compare with WT in
Fig. 3A). Analysis of
TAb-treated WT foreguts showed that blocking endogenous Tgfß signaling
does not interfere with primary lung bud formation. This was in full agreement
with results from Tgfb-knockout mouse models
(Bartram and Speer, 2004), and
further supported the idea that an excess, but not deficiency, of Tgfß
function is deleterious to early lung bud morphogenesis.
|
) (Fig.
8A). By contrast, bud formation and high levels of Nkx2.1
expression were consistently observed in BMS493 plus TAb-treated WT foreguts
(n=13/13) (Fig. 8B).
TAb also seemed to have rescued the formation of the stomach
(n=13/13), which is suppressed by BMS493
(Desai et al., 2004).
Remarkably, suppression of Tgfß signaling by TAb was also effective in
rescuing bud formation and Nkx2.1 expression in the lung field of
Raldh2-/- foreguts (n=11/13)
(Fig. 8C,D). Interestingly, in
none of the TAb-supplemented cultures (either from the BMS493 or the
Raldh2-/- model) did the rescue of lung buds occur
bilaterally. Whether the right or the left lung was rescued was not
determined. Presumably, it was the right lung, because other RA-deficient
models (Rara/Rarb-null mice, vitamin A-deficient rats, and
Raldh2-/- embryos rescued with low RA doses)
characteristically show left lung agenesis and right lung hypoplasia
(Mendelsohn et al., 1994;
Wang et al., 2006;
Wilson et al., 1953).
How could TAb prevent the total lung agenesis characteristic of the
RA-deficient foreguts? WMISH assessment of Fgf10 expression confirmed
that this gene is selectively downregulated in the lung field of foregut
cultures treated with BMS493 alone, as previously reported
(Desai et al., 2004). The
local disruption of Fgf10 by BMS493 contrasted with the strong
Fgf10 expression associated with the rescued lung bud in BMS493 plus
TAb-treated foreguts (Fig.
8E,F). Nevertheless, the distribution of Fgf10 mRNA in
the lung field in these cultures was overall more diffuse than in control
foreguts (compare with Fig.
5C). This is likely to have contributed to the prevention of full
rescue of the lung phenotype in BMS493 plus TAb-treated cultures. Endogenous
RA may be crucial in establishing local gradients of signaling molecules, such
as Fgf10, in the prospective lung field. Our data support a model in which
endogenous RA maintains low levels of Tgfß signaling in the lung field to
allow proper expression of Fgf10 and initiation of lung bud
morphogenesis (Fig. 9).
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Danielpour, 1996;
Falk et al., 1991;
Glick et al., 1989;
Imai et al., 1997;
Kojima and Rifkin, 1993). RA,
however, has also been reported to inhibit endogenous Tgfß signaling in
the mesenchyme of the developing inner ear
(Frenz and Liu, 2000) and
palate (Yu and Xing, 2006) in
mice. Disruption of Rxra in mice results in abnormal upregulation of
Tgfb2 in cardiac tissue (Kubalak
et al., 2002). A microarray analysis of vitamin A-deficient rat
embryos revealed upregulation of some of the genes (such as procollagens)
found in our study, suggestive of increased Tgfß-dependent activity
(Flentke et al., 2004). By
contrast, in the yolk sac, disruption of RA signaling inhibits
Tgfß-dependent expression of fibronectin and integrin and disrupts
visceral endoderm and vascular development
(Bohnsack et al., 2004). The
discordant data suggest that the way RA and Tgfß interact is strongly
influenced by the local milieu and thus depends on the particular system
studied.
|
|
), was not observed. Moreover, at the mRNA level, none
of the Tgfß ligands was upregulated by loss of RA signaling (data not
shown). We considered the possibility that a RA-regulated mechanism leading to
conversion of latent Tgfß ligand into an active form could be enhanced in
RA-deficient foreguts. This idea was attractive, as several genes associated
with this function were differentially upregulated by RA deficiency in both
models, such as annexin A2, cathepsin H, mannose-6-phosphate receptor, matrix
metallopeptidase 9 and S100 calcium binding protein A10
(Harpel et al., 1993;
Krishnan et al., 2004;
Ling et al., 2004;
Lyons et al., 1988;
Nunes et al., 1997;
Rifkin et al., 1997;
Taipale et al., 1994;
Yu and Stamenkovic, 2000;
Zhang et al., 2004) (F.C. and
W.V.C., unpublished). Using an immunofluorescence protocol to detect latent
and active TGFß1 (Ewan et al.,
2002), we could not identify significant differences in staining
pattern that could account for the major changes described here. Demonstration
of active Tgfß ligand is technically challenging, even when Tgfß
signaling is evident, in part owing to the short half-life of the active
Tgfß ligand (Araya et al.,
2006; Flanders et al.,
2001; Wakefield et al.,
1990). In our RA-deficient foreguts, it is possible that the
activation of Tgfß signaling would require only minimal increase in the
active Tgfß ligand, as at least one of the Tgfß receptors is also
being upregulated. Further studies will be required to clarify these
issues. |
ACKNOWLEDGMENTS |
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REFERENCES |
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