1 Department of Pathology, University of Colorado Health Sciences Center, The
Children's Hospital, 1056 East 19th Avenue, Denver, CO 80218, USA
2 Division of Developmental Biology, Cincinnati Children's Hospital Medical
Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
3 Division of Pathology, Cincinnati Children's Hospital Medical Center, 3333
Burnet Avenue, Cincinnati, OH 45229-3039, USA
* Author for correspondence (e-mail: gail.deutsch{at}chmcc.org)
Accepted 2 November 2004
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SUMMARY |
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Key words: Lung, Liver, Endoderm, Heart, NKX2.1, FGF signaling, Mouse
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Introduction |
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Lung specification, along with its neighboring foregut derivatives
(including pancreas, liver, thyroid, esophagus, stomach), occurs within the
domain of FOXA2 (formally designated HNF3ß) gene expression; members in a
family of developmentally regulated transcription factors that play a
significant role in regional differentiation of the gut endoderm
(Sasaki and Hogan, 1993;
Monaghan et al., 1993
;
Ang et al., 1993
). In vitro,
FOXA2 (HNF3ß) activates transcription of the homeodomain protein NKX2.1
(Ikeda et al., 1996
) and is
thought to cooperate with NKX2.1 to define cell lineage within the pulmonary
epithelium (Guazzi et al.,
1990
; Kimura et al.,
1996
; Minoo et al.,
1999
). NKX2.1 [also named thyroid transcription factor 1 (TTF1)
and thyroid enhancer binding protein (T/ebp)] is the earliest known marker of
the respiratory epithelium, with reported onset of expression in the
developing lung, thyroid and ventral forebrain at 9-9.5 days mouse gestation
(Lazzaro et al., 1991
;
Minoo et al., 1999
). Early in
morphogenesis, NKX2.1 is expressed in all pulmonary epithelial cells, but
becomes progressively restricted to the distal alveolar type II cells and
proximal Clara cells with development
(Lazzaro et al., 1991
;
Ikeda et al., 1995
). Targeted
disruption of the gene has demonstrated that although NKX2.1 is essential for
lung branching and terminal cell differentiation (including expression of
surfactant proteins SP-A and SP-C, and Clara cell secretory protein CC10), it
is not required for the initial specification of the lung or thyroid
(Kimura et al., 1996
;
Minoo et al., 1999
;
Kimura et al., 1999
). The
early onset and essential requirement for NKX2.1 in lung development implies
that this transcription factor is activated by the primary signals that induce
pulmonary specification from the endoderm.
Experimental systems using embryo tissue isolation and culture have proven
valuable in defining inductive interactions crucial for endoderm patterning as
well as liver and pancreas specification
(Gualdi et al., 1996;
Jung et al., 1999
;
Wells and Melton, 2000
;
Deutsch et al., 2001
;
Rossi et al., 2001
;
Kumar et al., 2003
;
Bort et al., 2004
). Chick
embryo transplant studies and mouse embryo explants have established that in
liver development, cardiac mesoderm is an absolute requirement for activation
of liver-specific genes from the ventral foregut endoderm and for subsequent
morphogenesis of the hepatic bud
(LeDouarin, 1975
;
Houssaint, 1980
;
Fukuda-Taira, 1981
;
Gualdi et al., 1996
). FGF1 and
FGF2, diffusable signals secreted from cardiac mesoderm at the time of liver
specification in the mouse embryo, can efficiently induce hepatic
differentiation within isolated ventral endoderm explants
(Jung et al., 1999
). Within
the same assay, the absence of cardiac mesoderm and FGF signaling leads to
expression of genes specific to the pancreas lineage
(Deutsch et al., 2001
),
confirming the multipotent capacity of the ventral foregut endoderm and the
ability to study cell specification in vitro.
In the developing lung bud, FGF receptor type 2 isoform IIIb and its high
affinity mesenchymal ligands (notably FGF10, FGF7 and FGF1) are essential for
patterning and morphogenesis of the respiratory tract, after specification has
occurred (Peters et al., 1994;
Bellusci et al., 1997
;
Celli et al., 1998
;
De Moerlooze et al., 2000
).
Disruption of FGFRIIIb signaling in transgenic mice completely blocks airway
branching and distal lung differentiation; the persistence of NKX2.1
expression indicates that specification of the lung remains unperturbed
(Peters et al., 1994
;
Celli et al., 1998
). Mice
deficient in FGF10, which is expressed in the early pulmonary mesenchyme at
E9.5 (Bellusci et al., 1997
),
have a similar phenotype with normal trachea but no mainstem bronchi
(Min et al., 1998
;
Sekine et al., 1999
). Although
these models underscore the integral requirement of FGF signaling in early
branching morphogenesis, they fail to define which signals are important in
initiating a lung pathway from multipotential endoderm.
In the present study we assess the tissue interactions necessary for the initial specification of a pulmonary cell fate, using embryonic tissue cultures. The results indicate that FGF signaling from the cardiac mesoderm is a crucial early determinant of lung specification, which functions in an instructive rather than permissive manner. In an in vitro system, induction of a liver versus lung cell fate is controlled by FGFs acting at different concentration thresholds and upon selective activation of FGFR1 and FGFR4 expressed in the ventral foregut endoderm. Together, the findings imply that temporally specific FGF signaling from the developing cardiac domain plays a role in patterning pluripotent endoderm during embryogenesis.
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Materials and methods |
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Explant cultures with growth factors, FGF receptor inhibition assays, analyses for cell proliferation and apoptosis
Immediately upon placing explants in culture, different concentrations of
recombinant FGFs were added to culture medium containing heparin sulfate
proteoglycan (50 ng/ml) and 0.1% bovine serum albumin. The following
concentrations of FGFs were used: human FGF1 and human FGF2 (Boehringer
Mannheim) at 5, 15, 30, 50, 125, 250, 500 and 1000 ng/ml; and mouse FGF8B
(R&D Systems) at 5, 50 and 500 ng/ml. Control cultures without FGFs were
always grown in parallel. Growth and changes in morphology were monitored
daily.
For FGF receptor inhibition assays antiserum to FGFR1 or FGFR4 (Santa Cruz Biotechnology; final concentration 1 µg/ml) or recombinant human soluble FGFR1 (IIIC)/Fc and FGFR4/Fc complexes (obtained from Axxora, LLC, San Diego, CA; final concentration 500 ng/ml) were added to the culture medium containing heparin sulfate (100 ng/ml) prior to tissue isolation. The same concentration of rabbit IgG was used as a control.
SU5402 (Calbiochem, La Jolla, CA; final concentration 25 or 50 µM) was added to the culture medium immediately after tissue isolation. Control explants were cultured with an equal volume of the vehicle DMSO.
For statistical analysis of FGF-treated and inhibition assays the Fisher's exact test or chi-square test were used to compare control with experimental groups; the Student's t-test (two-tailed) was also used in the inhibition assays. P values of less than 0.05 were considered to indicate significant differences.
BrdU incorporation was detected using the BrdU labeling and Detection Kit II (Roche). At the end of a 24-hour culture period, BrdU was added at a concentration of 10 µM for 1 hour. BrdU immunostaining was carried out using the manufacturer's protocol. Apoptotic cells were detected using the In Situ Cell Death Detection Kit (Roche).
Whole-mount and section immunofluorescence
Embryos and endoderm cultures were fixed overnight at 4°C in 4%
paraformaldehyde buffered to pH 7.4 with PBS, washed three times with PBS then
sequentially dehydrated and stored in 100% methanol at -20°C until used
for whole-mount or sectioned immunostaining. Embryo and explant sections were
generated by embedding the rehydrated tissue in tissue freezing medium, and
serially sectioned in a Leitz cryostat at 8 µm at -20°C. The following
antibodies and dilutions were used for both whole-mount double immunostaining
and embryo/tissue section double immunofluorescence: mouse anti-TTF1 (1:150,
Neomarkers, Fremont, CA); sheep anti-albumin (1:300, The Binding Site,
Birmingham, UK); sheep anti-HNF3ß (FOXA2) 1:100, Upstate Biotechnology,
Lake Placid, NY); muscle-specific mouse actin (1:500, Neomarkers); goat
anti-SP-C and anti-CC10; rabbit anti-FGFR1 and FGFR4 (all 1:100, Santa Cruz
Biotechnology). The rabbit PDX1 antibody was generously provided by C. Wright
(dilution 1:1500). In all experiments, normal serum was used as a control (the
latter giving no signals; data not shown). All antibody incubations took place
overnight at room temperature. Immunofluorescence of embryo and explant
sections was detected with fluorescein or Texas Red anti-sheep, anti-mouse,
anti-rabbit or anti-goat IgG and mounted with DAPI media (all from Vector).
All results were photographed with a SPOT RT digital camera (Diagnostic
Instruments) mounted on a Nikon E400 microscope.
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Results |
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To better resolve the expression pattern seen within sectioned embryos, single cell cytospin slides were generated from dissected ventral foregut endoderm at 8, 10 and 14 somites and analyzed by double immunofluorescence for NKX2.1 and albumin (Fig. 1K-S). Quantification of the material confirmed the early onset of albumin versus NKX2.1 protein expression in the ventral foregut, as well as the existence of co-expressing cells that diminished with advancing somite age (Fig. 1S). The co-expression of NKX2.1 and albumin cells within the foregut endoderm in vivo led us to hypothesize that the cardiac signals that induce liver specification may impact lung specification as well.
Lung specification is induced by adjacent cardiac mesoderm
To examine the potential relationship between lung and liver specification,
we used an embryo explant assay. Hepatic specification can be reconstituted in
vitro by culturing ventral endoderm from the two- to five-somite stages in
close proximity to cardiac mesoderm
(Gualdi et al., 1996). Cardiac
mesoderm cultivated in DMEM + 10% calf serum progresses to the beating stage
within 1 day of culture and is thus readily distinguishable from co-cultured
endoderm fragments; we also stained explants for expression of muscle specific
actin (MSA) which delineates the cardiac domain
(Fig. 2B). Ventral endoderm
from the two- to five-somite embryo, cultured in the absence of cardiac
mesoderm for 48 hours and assayed by RT-PCR or immunostaining, survived and
expressed PDX1 but failed to express NKX2.1 (n=24) or albumin
(n=17) (summarized in Table
1; Fig. 2D, lane 4;
Fig. 2F-H; see
Fig. 5A). This finding was
anticipated by prior studies (Deutsch et
al., 2001
; Rossi et al.,
2001
), which demonstrated that ventral endoderm, in the absence of
signals required for hepatic specification, activates pancreas-specific genes.
Ventral endoderm co-cultured with beating cardiac mesoderm predictably
expressed albumin protein and mRNA in 53 of 56 explants (95%), but also
expressed NKX2.1 in 45 of 82 explants (55%)
(Fig. 2C,D, lanes 2-3;
Fig. 2I-N;
Table 1). Examination for the
activation of NKX2.1 by immunostaining (19 positive of 32 explants, 59%)
yielded similar findings to analysis by RT-PCR (28 positive of 52 explants,
54%). Double-labeling assays corroborated that the cells positive for NKX2.1
overlapped with FOXA2 signal (Fig.
2I,J), a marker of definitive endoderm
(Sasaki and Hogan, 1993
;
Ang et al., 1993
;
Monaghan et al., 1993
). The
specificity of cardiac mesoderm as an inducer of NKX2.1 was tested by
co-culturing ventral endoderm with other ectodermal/mesodermal tissues,
including isolated neural tube, optic lobes and caudal segments, comprising
allantois and extremity of neural plate
(Fig. 1A). These explants
survived and grew but failed to express NKX2.1 by immunostaining after 2 days
in culture (data not shown). Furthermore, isolated mesodermal and lateral
ectodermal (non-NKX2.1 expressing in vivo) tissues cultured under identical
conditions never expressed NKX2.1.
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Activation of a lung pathway is related to quantity of and proximity to cardiac mesoderm
In contrast to liver specification, cardiac mesoderm did not consistently
induce lung markers in cultured ventral endoderm in vitro, doing so in
50% of explants tested (Table
1). The in vivo expression of NKX2.1, which was limited to
endoderm neighboring the developing heart, implied that lung specification
might require close proximity and/or high levels of signaling from the cardiac
domain. Our data support this, showing that explants in which NKX2.1, as well
as SP-C and CC10, were activated had more cardiac mesoderm than did explants
in which only albumin was detected (Fig.
2I-N; data not shown). Furthermore, in explants in which NKX2.1
was expressed, the signal was invariably confined to endoderm that was
directly adjacent to the beating cardiac domain,
(Fig. 2J), whereas albumin
expression often extended well beyond the beating areas
(Fig. 2C,K,N)
(Gualdi et al., 1996
;
Deutsch et al., 2001
). These
results suggested that induction of a hepatic versus pulmonary cell fate
within the ventral endoderm rests on the extent of signal emanating from the
cardiac mesoderm.
Dosage effect of FGF signaling on endoderm cell differentiation
As indicated in the Introduction, prior work has demonstrated that FGF1 or
FGF2 can substitute for the cardiac mesoderm as an inducer of liver-specific
genes within the ventral endoderm (Jung et
al., 1999). Both FGF1 and FGF2 bind with high affinity to FGF
receptor 1 (FGFR1) and FGFR4 (Ornitz et
al., 1996
), which are expressed in the E8.5 ventral endoderm at
the time of hepatic specification (see Fig.
7A-C) (Stark et al.,
1991
; Sugi et al.,
1995
) (data not shown). To investigate whether the concentration
of FGF ligand determines a liver versus lung fate, we treated ventral endoderm
explants at a range of FGF concentrations. Ventral foregut endoderm was
obtained from two- to five-somite embryos and cultured with either recombinant
FGF1 or FGF2 (5-1000 ng/ml tested). Over 48 hours of monitoring, we did not
observe a significant difference in the morphological appearance or growth of
explants treated with low versus high dose of FGF1 or FGF2; analyses for cell
proliferation (BrdU incorporation) and apoptosis (TUNEL staining) confirmed
this impression (data not shown). After one or two days of culture, the
expression of NKX2.1 and albumin were assayed for by RT-PCR or double
immunofluorescence. Using both methods we observed a dose-dependent expression
of NKX2.1 and albumin in response to exogenous FGF2 and FGF1, which was
statistically significant (P<0.0001, Fisher's exact test). A low
concentration of FGF2 (2-5 ng/ml), which effectively suppresses the expression
of PDX1 (Fig. 3I)
(Deutsch et al., 2001
),
consistently induced albumin expression (10 out of 11 explants) but not
expression of NKX2.1 (none of nine explants)
(Fig. 3A,B,I,J). By contrast,
10 to 500 ng/ml of FGF2 induced NKX2.1 expression (26 out of 31 explants) but
typically not expression of albumin (four out of 26 explants)
(Fig. 3C-F,I,J). Neither NKX2.1
(two out of nine explants) nor albumin (none of six explants) was reliably
activated in the presence of 1000 ng/ml of FGF2, suggesting an upper limit of
dose responsiveness (Fig.
3G-J).
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In the presence of all doses of FGFs tested, none of the 29 control
cultured ectodermal/mesodermal tissues, which express FGFR1 in vivo
(Yamaguchi et al., 1992),
activated NKX2.1 or albumin (Fig.
3I; Table 1).
FGF signaling is sufficient to activate downstream targets of NKX2.1
Concentrations of FGF2 (500 ng/ml) that reliably activated NKX2.1 from the
ventral endoderm in vitro also elicited expression of SP-C and CC10
(Fig. 5B-F), as well as of
thyroglobulin, expression of which is restricted to the thyroid lineage (data
not shown). From this we conclude that FGF signaling is sufficient to induce
not only NKX2.1 but also the lung and thyroid genes that it regulates within
the foregut.
FGF signaling is instructive in lung specification
Although dorsal endoderm of the mid- and hindgut gives rise to the small
and large intestine, studies have demonstrated that the 8.5-13.5 day
dorsoposterior endoderm is competent to adopt a liver fate when isolated from
adjacent mesoderm that inhibits albumin expression in co-culture assays
(Gualdi et al., 1996;
Bossard and Zaret, 2000
).
We used midgut endoderm to establish whether FGF signaling is instructive,
rather than permissive, in activating a lung pathway in endoderm fated to
become the intestinal tract. Dorsal endoderm from the prospective midgut
region (Fig. 1A) was dissected
from two- to five-somite embryos, and treated with FGFs at a range of
concentrations. In the absence of FGF, dorsal endoderm isolated from the
prospective midgut region and cultured for 2 days did not activate NKX2.1
expression by RT-PCR analysis (none of four explants) or immunostaining (none
of seven explants), although it did activate albumin
(Fig. 6A, lane 1)
(Gualdi et al., 1996;
Bossard and Zaret, 2000
).
However, dorsal midgut endoderm treated with exogenous FGF2 exhibited a
concentration-dependent induction of NKX2.1 in a manner analogous to the
ventral endoderm. Specifically, NKX2.1 was efficiently induced at high (50 and
500 ng/ml) (six out of six explants) rather than low concentrations of FGF2
(1-5 ng/ml) (none of three explants) and inefficiently by FGF8B at identical
concentrations (two out of six explants)
(Fig. 6A, lanes 2-4; 6B-E).
Furthermore, exposure of dorsal midgut endoderm to high concentrations of FGF2
suppressed activation of albumin in all five isolates tested, similar to what
was observed with ventral foregut endoderm.
|
To test the necessity of FGF signaling and possible receptor specificity for NKX2.1 activation by the cardiac mesoderm, we used several approaches. To perturb signaling through FGFR1 or FGFR4, explants were treated with an antibody that binds to the extracellular, ligand-binding domain of the receptor or exposed to a soluble recombinant FGFR/IgG-Fc chimeric protein. Both approaches yielded a comparable effect on the expression of albumin and NKX2.1 (see below). Ventral endoderm was dissected from four- to five-somite mouse embryos along with its associated cardiac mesoderm and cultured in the presence of IgG to FGFR1 or FGFR4 (1 µg/ml) or the soluble FR-IgG complex (500 ng/nl), and heparin sulfate at 100 ng/ml. As a control, we used rabbit IgG with heparin sulfate. We modified our dissection technique to ensure that all explants contained an increased proportion of cardiac mesoderm to ventral endoderm. Over 48 hours, we did not see a significant difference between the treatment groups in the growth or viability of cultures. However, compared with controls treated with rabbit IgG, there was a 3- to 4-hour delay in the onset of beating within explants exposed to FGFR-IgG and soluble FR-IgG; by 18 hours this disparity in activity of the cardiac domain between groups was no longer evident. After 2 days, the presence and amount of NKX2.1 and albumin signal were assessed by sectioned double immunostaining and RT-PCR. Compared with the consistent induction of albumin and NKX2.1 within the control group (10/10 and 9/10 positive explants, respectively) (Fig. 7H,Q, lane 1), FGFR1-IgG and soluble FR1-IgG effectively diminished cardiac induction of albumin (two out of 10 and two out of seven positive explants, respectively) and NKX2.1 (one out of 11 and two out of seven positive explants, respectively), with only few labeled cells by immunostaining when present (Fig. 7K,L,P,Q, lane 3) (P<0.0003, Fisher's exact test). Explants treated with a single dose of SU5402, a FGFR1 tyrosine kinase inhibitor, yielded comparable findings with only two out of nine explants expressing albumin and NKX2.1 (Fig. 7Q, lane 4). Notably, interfering with signaling through FGFR1 did not result in an appreciable defect of cardiac or endoderm viability, confirmed by morphology and gene expression (Fig. 7Q, lanes 1-4).
In agreement with prior work (Jung et
al., 1999), FGFR4-IgG and soluble FR4-IgG were less effective then
FR1-IgG in diminishing albumin expression (eight out of 10 and four out of
seven explants positive, respectively), and we expected analogous results for
NKX2.1. Surprisingly, compared with the controls, FGFR4-IgG and soluble
FR4-IgG efficiently inhibited the expression of NKX2.1, with only two out of
10 and one out of seven explants demonstrating limited expression in
cryosections and by RT-PCR (Fig.
7O-Q, lane 2); this was significantly different from that observed
with albumin (P<0.0019, chi-square test).
These data support the hypothesis that the cardiac mesoderm is the endogenous activator of NKX2.1 expression in the endoderm and that this induction is mediated, at least in part, by FGFs. Furthermore, the ability to effectively suppress NKX2.1 expression by selective inhibition of both FGFR1 and FGFR4 implies that the FGF receptor composition on the surface of the endoderm cell is as important in lung versus liver specification as is the concentration of mesoderm-derived ligand.
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Discussion |
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Supportive evidence that cell fate within the foregut endoderm is driven by
a threshold of FGF signaling comes from a detailed knowledge of FGF ligand
expression within the developing heart. Jung et al.
(Jung et al., 1999) determined
that FGF2 mRNA is present, but barely detectable in the cardiac mesoderm of
the five-somite stage embryo; by the seven- to eight-somite stages FGF1 first
appears while FGF2 levels are now robust
(Fig. 8). Additionally, a low
level of FGF8 persists in an area of the cardiac mesoderm bordering ventral
endoderm (Jung et al., 1999
).
Thus, the intensity of total FGF expressed in the heart in vivo positively
correlates with the successive induction of albumin and NKX2.1 in the adjacent
endoderm. The onset and expansion of FGF2 and FGF1 in the developing heart
also parallels the temporal and spatial expression of FGFR4 in the endoderm,
upon which NKX2.1 is functionally dependent in our in vitro assay
(Fig. 7). Our data suggests
that expression of FGFR4 within endoderm is induced by FGF from cardiac
mesoderm. Ventral endoderm explants co-cultured with cardiac mesoderm or with
exogenous FGF1 or FGF2 show elevated levels of FGFR4 expression by
immunostaining and RT-PCR (Fig.
7D-G; data not shown) implying that FGFR4 is regulated by FGF
signaling. Induction of NKX2.1 in the dorsal endoderm by FGF2 may also be
mediated through FGFR4. Further studies will be required to assess the precise
relationship between cardiac signaling and the FGFR4 responsive endoderm in
promoting a pulmonary cell fate. However, the data indicate that the total
level of FGF signaling, both at the ligand and receptor level, is necessary
for lung specification (Fig.
8).
Despite the described influence of FGF1 and FGF2, as well as FGFR4 in
vitro, mice lacking these genes are viable and have no major abnormalities in
organ development (Ortega et al.,
1998; Dono et al.,
1998
; Weinstein et al.,
1998
; Miller et al.,
2000
; Yu et al.,
2000
). However, the presence and requirement for FGFR1 during
embryogenesis (Deng et al.,
1994
; Yamaguchi et al.,
1994
) as well as FGF4, FGF8 and FGF17 expressed in the cardiac
mesoderm at the time of liver and lung specification
(Crossley and Martin, 1995
;
Zhu et al., 1996
;
Maruoka et al., 1998
) indicate
that related molecules can probably function in a redundant capacity.
We acknowledge that the threshold effect of exogenous FGF on cell differentiation might be related to mesoderm cells (FOXA2 negative), which are frequently present within our endoderm isolates. Such cells could be a source of FGF, or another as yet unidentified inducer, and thus induction of albumin versus NKX2.1 may be an indirect phenomenon (see Fig. 8). Although the poor viability of individual endoderm cells in culture precludes systematic analysis at the single cell level, we have observed isolated and small clusters of endoderm cells expressing albumin in the presence of low FGF and NKX2.1 in the presence of high FGF (data not shown), supporting primary induction.
Interestingly, FGF1 and FGF2 lack a definitive signal sequence and are
inefficiently secreted into the extracellular environment by nonclassical
pathways (for a review, see Powers et.
al., 2000), implying that FGFs may pattern the ventral foregut
through short-range signaling.
The dorsoposterior endoderm of the midgut is competent to activate NKX2.1
by FGF when isolated from adjacent mesoderm and ectoderm
(Fig. 6). Prior studies
(Gualdi et al., 1996;
Bossard and Zaret, 2000
) have
demonstrated that dorsal endoderm is also able to activate liver-specific
genes when cultured alone. This competency, which is lost at E13.5 prior to
intestinal differentiation, is theorized to be endowed by the transient
occupancy of FOXA2 and GATA4 factors bound to the albumin enhancer
(Bossard and Zaret, 2000
). The
authors surmise that active repression of the gut mesoderm on a foregut fate
involves a secreted factor, as fragments of dorsal mesoderm and ectoderm, in
tissue recombination assays, inhibits liver genes in ventral endoderm
co-cultured with cardiac mesoderm (Gualdi
et al., 1996
). Elucidating the nature of repressive mesodermal
interactions in patterning the endoderm is of considerable interest,
especially as the posterior mesoderm expresses high levels of FGFs
(Crossley and Martin, 1995
;
Maruoka et al., 1998
;
Wells and Melton, 2000
;
Karabagli et al., 2002
).
Low levels of FGF2 expressed by the notochord
(Zuniga Mejia Borja et al.,
1996; Hebrok et al.,
1998
) might explain the paradoxical finding that rare dorsal
endoderm cells in the foregut, at least transiently, express albumin in vivo
(Fig. 1B,E). This expression
could be lost with the midline fusion of the paired dorsal aortas, which
separate notochord from the foregut slightly later in development
(Hebrok et al., 1998
).
We have found that the ability of the endoderm to be programmed by FGF
signaling is temporally restricted, for a 6 hour delay in exposure of explant
cultures to FGF2 results in activation of PDX1 at low doses, and albumin at
high doses, with minimal expression of NKX2.1 (data not shown). This latter
data supports prior in vitro studies, which indicate that in the absence of
cardiac mesoderm or FGF, the default fate of the early somite ventral foregut
endoderm is to activate a pancreas gene program
(Gamer and Wright, 1995;
Deutsch et al., 2001
). This
finding brings into question whether endoderm cells retain plasticity in fate
after specification has occurred.
It is intriguing that early genes specific to the liver and lung
transiently co-express within the ventral endoderm soon after hepatic and
pulmonary initiation in the mouse embryo. It unclear at this time what refines
the expression pattern of these genes by E9.5
(Fig. 1G-J,O-S), but multiple
factors, including continued induction, lack of induction and/or suppression,
particularly from adjacent germ layers, may play a role
(Fig. 8). Prior work, using an
identical in vitro system, has implicated BMP signaling from the septum
transversum, cardiac/FGF-induced sonic hedgehog and Hex-dependent cell
proliferation as contributing to hepatic versus pancreatic induction
(Rossi et al., 2001;
Deutsch et al., 2001
;
Bort et al., 2004
). Within the
developing respiratory tract of the chick embryo the TBX4-FGF10 system
regulates early lung bud formation and defines the posterior boundary of
NKX2.1 expression (Sakiyama et al.,
2003
). We are currently investigating the possibility that after
NKX2.1 is activated combinatorial interactions with pathways implicated in
lung development establish a pulmonary fate in the endoderm.
NKX2.1 is an early marker, as well as transcriptional regulator, of the
thyroid as well as the lung (Lazzaro et
al., 1991; Kimura et al.,
1996
; Kimura et al.,
1999
), and data drawn from the isolated expression of NKX2.1
cannot distinguish between the two. In preliminary studies, we have found that
ventral endoderm explants require cardiac mesoderm or FGF to activate the
thyroid specific marker thyroglobulin (G.H.D., unpublished). How these
requirements modify specification of one organ versus the other remains to be
determined.
The model described here for patterning of the ventral endoderm
(Fig. 8) is reminiscent of the
developmental processes involved in generating the limb bud, which is the
source of soluble FGFs that confer proximodistal identity to the skeletal
elements (reviewed by Martin,
1998). Reexamination of the vertebrate limb model
(Sun et al., 2002
;
Dudley et al., 2002
) supports
early specification of the proximal and distal limb domains, of which the
respective precursor populations expand and differentiate with subsequent
development. Likewise in the ventral endoderm, cell fate choice is not related
to the degree of cell proliferation or cell death
(Deutsch et al., 2001
) (data
not shown); however, these processes might be involved in acquisition of a
definitive fate after specification occurs. For example, the dose of FGF8
directly regulates cell survival in the developing forebrain
(Storm et al., 2003
). Defining
lineage relationships and the factors that restrict cell fates in the
embryonic foregut endoderm may facilitate the identification and manipulation
of progenitor cells to treat congenital and adult diseases.
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ACKNOWLEDGMENTS |
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