1 Division of Biological Science, Graduate School of Science, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
2 CREST, Japan Science and Technology Corporation (JST), Division of Biological
Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya, 464-8602 Japan
* Author for correspondence (e-mail: i45240a{at}nucc.cc.nagoya-u.ac.jp)
Accepted 10 December 2002
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SUMMARY |
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Key words: Lung bud, Tracheo-esophageal fistula, Tbx4, Fgf10, Nkx2.1, In ovo electroporation, Chick
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INTRODUCTION |
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It is proposed that positional information in the gut tube is reflected by
the region-specific expression pattern of transcription factors in the
endoderm and visceral mesoderm as in the CNS and paraxial mesoderm. Several
transcription factors are expressed in the visceral mesoderm and/or the
endoderm of a specific region of the gut tube along the AP axis: Hox genes,
Nkx2.5 and Bapx1, etc. in the visceral mesoderm; Sox2,
CdxA, CdxC and Pdx1, etc. in the endoderm
(Grapin-Botton and Melton,
2000; Roberts,
2000
). The boundaries of the expression domains of these
transcription factors correspond to the boundaries of the presumptive
territories of different organ primordia. Actually, some of these genes
control the region-specific or, in some cases, the organ-specific signaling
systems for the specification of the presumptive organ territory and
organ-specific morphogenesis
(Grapin-Botton and Melton,
2000
; Roberts,
2000
). In addition, signaling crosstalk between the different
tissues or organ primordia is mediated by secreted proteins such as Fgfs, Bmps
and Wnts, etc. In this way, signaling systems that are spatiotemporally
coordinated by transcription factors and secreted proteins are thought to
control the region-specific organogenesis in the overall gut tube along the AP
axis.
The lung originates from the ventral wall of the foregut of the
esophagus-respiratory region, which lies between the pharynx and stomach. Lung
development begins with the establishment and swelling of the primordium in
the ventral wall of the foregut. Subsequently, the lung primordium forms a
pair of primary lung buds and then the lung bud endoderm (bronchus)
repetitively undergoes budding and branching. Because of the characteristic
budding and branching involved in lung morphogenesis, this process has been
studied extensively by experimental embryology in which dissected and
recombined lung primordia were cultured in vitro
(Alescio and Cassini, 1962;
Spooner and Wessels, 1970
).
These classical tissue recombination experiments revealed that: (1) the
presumptive field of the lung primordium is already established in the early
gut tube; (2) the tissue interaction between the mesoderm and the endoderm
plays an essential role in budding and branching morphogenesis; and (3) lung
morphogenesis consists of two independent sequential processes the
establishment of the lung primordium and primary lung bud formation in the
early stage, and the stereotypic budding and branching morphogenesis at the
following stage.
Recent molecular biological studies revealed that spatial-and
temporal-coordinated signaling systems, which are mediated by transcription
factors and secreted proteins, control the proper stereotypic morphological
pattering and differentiation in lung development
(Hogan, 1999;
Warburton et al., 2000
). The
elongation of the bronchial bud and bronchial branching, the later processes
of lung morphogenesis, are coordinated along the proximodistal (PD) axis. This
later phase of lung morphogenesis is controlled by the region-specific
signaling system along the PD axis and signaling crosstalk between the
endoderm and mesoderm, which are mediated by several transcription factors,
e.g. Foxf1 and Gli proteins, and secreted proteins, e.g. Fgfs, Bmp4 and Shh
(Bellusci et al., 1997a
;
Bellusci et al., 1997b
;
Colvin et al., 2001
;
Lebeche et al., 1999
;
Litingtung et al., 1998
;
Mahlapuu et al., 2001
;
Motoyama et al., 1998
;
Pepicelli et al., 1998
;
Weaver et al., 2000
;
Weaver et al., 1999
). To date,
the mechanisms of bronchial branching and cytodifferentiation, the later
processes of lung development, have been well studied. However, the mechanism
of the early processes of lung development, the positioning/establishment of
the lung primordium and primary lung bud formation, are less well
understood.
The positioning and establishment of the lung primordium are expected to
reflect the positional information of the gut tube along the AP and DV axes.
In mice, Nkx2.1, which encodes a homeobox transcription factor, is
specifically expressed in the ventral wall of the foregut endoderm: the
presumptive respiratory endoderm (Lazzaro
et al., 1991; Minoo et al.,
1999
). Nkx2.1 knockout mice exhibit immature bronchial
cytodifferentiation, abnormal branching, and the DV patterning defect of the
foregut (tracheo-esophageal fistula)
(Minoo et al., 1999
).
Therefore, Nkx2.1 plays a role in the regulation of the lung
morphogenesis specific pathway and the DV patterning of the foregut, which
reflects the AP and DV positional information in the gut endoderm. However,
the transcription factors that show lung-specific expression in the mesoderm
have not been identified. Furthermore, as Fgf10 and Bmp4 are
specifically expressed in the lung primordium mesoderm
(Bellusci et al., 1997b
;
Weaver et al., 1999
),
transcriptional regulation specific for the lung primordium mesoderm is
assumed. Moreover, homozygous Fgf10-null mice do not generate primary
lung buds from the ventral foregut (Min et
al., 1998
; Sekine et al.,
1999
), indicating that Fgf10 acts as an essential component of the
inductive signaling systems necessary for primary lung bud formation. From
this evidence it is clear that the signaling systems specific for the lung
primordium mesoderm, especially the mechanisms of transcriptional regulation
of Fgf10 expression in the lung primordium mesoderm, is very
important for understanding how the primary lung bud develops from a distinct
area of the gut tube.
T-box transcription factor family genes (Tbx) function as key regulatory
genes in many cases of vertebrate development
(Bruneau et al., 2001;
Chapman and Papaioannou, 1998
;
Rodriguez-Esteban et al.,
1999
; Takeuchi et al.,
1999
; Zhang et al.,
1998
). Among the T-box gene family, Tbx4 is specifically
expressed in the hindlimb, and misexpression of Tbx4 in the forelimb
induces a leg-like limb, indicating that this gene plays a crucial role in the
specification of hindlimb patterning
(Rodriguez-Esteban et al.,
1999
; Takeuchi et al.,
1999
). In addition, Tbx4, was briefly reported to be
expressed in the lung mesoderm
(Gibson-Brown et al., 1998
),
raising the possibility that Tbx4 is involved in lung
development.
In this study, we analyzed the expression profile of Tbx4 in chick embryos in detail and found that Tbx4 was specifically expressed in the visceral mesoderm of the lung primordium, and its expression overlapped with mesodermal Fgf10 and endodermal Nkx2.1 expression. There is a discrepancy between the Tbx4 and Fgf10/Nkx2.1 expression domains along the DV axis. Misexpression of Tbx4 induced ectopic Fgf10 and Nkx2.1 in the Tbx4-introduced mesoderm and only in the ventral region of the underlying endoderm, respectively, followed by ectopic endodermal bud formation. Our results suggest that: (1) Tbx4 defines the anterior and posterior boundaries of Fgf10 expression; and (2) Tbx4 is involved in the demarcation of the posterior border of the Nkx2.1 expression domain, but is independent from the system that determines the DV border of Nkx2.1 expression.
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MATERIALS AND METHODS |
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Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed essentially according to
Dietrich et al. (Dietrich et al.,
1997). Prior to pre-hybridization treatment, the foreguts were
dissected and isolated to allow penetration of the RNA probe. For two-color
whole-mount in situ hybridization, one probe was labeled with digoxigenin
(DIG) and the other was labeled with fluorescein (FITC). Alkaline phosphatase
(AP)-conjugated anti-DIG and anti-FITC antibodies (Roche) were colored with
Fast Red/Naphthol (Sigma) in NTMT (pH 8.0) (red), NBT (Roche, 3.5
µl/ml)/BCIP (Roche, 3.5 µl/ml) in NTMT (pH 9.5) (purple) and NBT (3.5
nl/ml)/BCIP (3.5 µl/ml) in NTMT (pH 9.5) (blue).
DNA constructs
Chicken Tbx4 cDNA was cloned into the internal ribosome entry site
(IRES)-carrying vector and then the IRES-Tbx4 fragment was isolated
and cloned into pCMV-Script (Stratagene). RCAS-Tbx4 and
RCAS-Tbx4-EGFP were kindly provided by Dr T. Ogura
(Takeuchi et al., 1999).
RCAS-Fgf10 was kindly provided by Dr S. Noji. ClaI fragments
of Tbx4, Tbx4-EGFP and Fgf10 were isolated from
RCAS-Tbx4, RCAS-Tbx4-EGFP and RCAS-Fgf10,
respectively, and inserted into the ClaI site of a modified pCAGGS
expression vector. The dominant-activator and dominant-repressor forms of Tbx4
(Tbx4-VP16, Tbx4-EnR) were generated by fusion of a truncated chick
Tbx4 fragment including the T-box (amino acid 1 to 273) with the
activator domain of VP16 and with the repressor domain of the En2 protein (eh1
domain) (Matsunaga et al.,
2000
). The Tbx4-VP16 and Tbx4-EnR fragment were
cloned into the ClaI site of a modified pCAGGS expression vector.
In ovo electroporation
In ovo electroporation was performed according to Momose et al.
(Momose et al., 1999). Eggs of
HH stage 7-12 embryos were opened, and the vitelline membranes were removed to
expose the embryo. A platinum electrode (0.5 mm diameter) was used as a
positive electrode. A sharpened tungsten needle or a platinum electrode (0.5
mm diameter) was used as a negative electrode. A positive electrode was placed
beneath the embryo at the prospective esophago-tracheobronchial region. A
positive electrode was placed into the coelomic cavity (when using a sharpened
tungsten needle) or on the embryo (when using a platinum electrode 0.5 mm
diameter). Plasmid solutions (8-15 µg/µl in T1/4E/0.1-1% Fast Green)
were injected into the coelomic cavity with a sharp glass pipette and an
electric pulse was immediately applied [5-9 V (HH7-10), 9-14 V (HH10-12);
pulse length 50-90 ms, once or twice times) with a CUY21 electroporator
(Tokiwa). Eggs were sealed and incubated in a humidified incubator until
analyzed further. Embryos were fixed with 4% paraformaldehyde at 4°C
overnight. The foreguts were isolated and GFP fluorescence was photographed,
and then the foreguts were processed for wholemount in situ hybridization.
Electroporation performed on total 1297 embryos, and 663 survival embryos
(51.1%) were recovered 1-3 days after electroporation. Among the recovered
embryos, 348 embryos (26.8%) exhibited efficient transfection. The efficiency
indicated in Results is always based on the number of the embryos exhibited
efficient transfection.
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RESULTS |
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Expression of Tbx4 in the developing respiratory system
First, we carefully analyzed the expression profiles of Tbx4
during chick lung development. In chick, the lung primordium appears between
50 and 60 hours after egg laying (stage 15/16) as a swelling of the ventral
wall and is recognized by the expression of Nkx2.1, the earliest
marker gene specific for the respiratory endoderm
(Lazzaro et al., 1991;
Minoo et al., 1999
). At stage
15 (25 somites), Tbx4 was expressed in the visceral mesoderm of the
distinct area of the esophagus-respiratory region covering the ventral
swelling (Fig. 1B).
Tbx4 was expressed in both the ventral and dorsal regions
(Fig. 1B) and the mesodermal
Tbx4 expression domain overlapped the Nkx2.1-expressing
endoderm except for the anterior end region of the Nkx2.1 domain
(Fig. 1D, compare B with C). We
could not detect Tbx4 expression at stage 14 (21 somites), whereas
Nkx2.1 was already expressed in the ventral endoderm of the foregut
in the esophagus-respiratory region (data not shown). On day 3 of incubation
(stage 18-20), when the lung buds were evident, Tbx4 was expressed in
the mesoderm of the lung buds, but not in the esophagus
(Fig. 1E,F). The anterior
boundary of the endodermal Nkx2.1 expression domain (purple staining
in Fig. 1E) was clearly more
anterior to the anterior boundary of the mesodermal Tbx4 expression
domain (mesodermal red staining in Fig.
1E). This expression profile of Tbx4 implies that the
Tbx4 expression domain at stage 15 contribute to the posterior
structure of the respiratory tract: the lung bud. To verify this possibility,
we carried out cell lineage analysis of the foregut visceral mesoderm at stage
15 and confirmed that the ventral Tbx4 expression domain contributes
to the lung bud (data not shown). On day 4 of incubation (stage 24/25), when
the separation of the trachea and the esophagus was progressing, Tbx4
was expressed in the mesoderm of the respiratory tract, posterior to the
bifurcation point of the main bronchus
(Fig. 1G,H), but not in the
esophagus (Fig. 1H).
We compared the Tbx4 expression domain with the expression domain
of Fgf10 that is expressed in the lung primordium mesoderm
(Bellusci et al., 1997b) and
required for primary lung bud formation in mice
(Min et al., 1998
;
Sekine et al., 1999
). At stage
15 (25 somites), Fgf10 was expressed in the visceral mesoderm of the
chick lung primordium, as in the mouse embryo
(Fig. 1I,J, purple staining).
Both the anterior and posterior boundaries of the Fgf10 domain
overlapped with those of Tbx4, as judged from the relationship to the
Nkx2.1 domain (blue staining in
Fig. 1I) and by the morphology
(compare Fig. 1I,J with 1B,D).
However, the Fgf10 domain was restricted to the ventral region of the
Tbx4 domain where a pair of endodermal buds would arise
(Fig. 1K, compare
Fig. 1I with 1B).
Misexpression of Tbx4 caused ectopic endodermal bud formation
To analyze the role of Tbx4 in lung development, we transiently
misexpressed Tbx4 in the visceral mesoderm of the presumptive
esophagus-respiratory region using in ovo electroporation
(Momose et al., 1999). When
the expression construct for the Tbx4-EGFP fusion protein
(Takeuchi et al., 1999
) or the
Tbx4 expression plasmids together with GFP plasmids were electroporated into
the prospective esophagus-respiratory region
(Matsushita, 1995
) at stage 7
to 12, evagination of the ectopic endodermal bud was observed [31/141 (22.0%)]
(Fig. 2A, yellow arrowhead). By
monitoring the expression of the foreign genes by GFP fluorescence, we found
that the ectopic buds always underlie the Tbx4-transfected mesoderm
(Fig. 2B,C). The ectopic buds
were induced in both the ventral (Fig.
2A, yellow arrowhead) and dorsal
(Fig. 2D, yellow arrowhead)
endoderm. In some cases, the ectopic endodermal bud was induced between the
primary bronchi, which resulted in trifurcation of the bronchi
(Fig. 2E,F). Misexpression of
Tbx4 caused endodermal budding, but this morphogenesis did not proceed to bud
elongation and branching. In addition, misexpression of Tbx4 caused a failure
of tracheo-esophageal septum formation, so-called tracheo-esophageal fistula
[13/141 (9.2%)] (Fig. 2G). By
contrast, misexpression of irrelevant genes, i.e. those encoding GFP or
alkaline phosphatase, did not cause ectopic endodermal bud formation and
tracheo-esophageal fistula [0/49 (0%)] (data not shown). Therefore, ectopic
endodermal bud induction by electroporation was a specific effect of Tbx4
misexpression.
|
Ectopic Tbx4 induced ectopic Fgf10 expression in the
mesoderm
As Tbx4 is a transcription factor, downstream signaling molecule(s) of Tbx4
in the visceral mesoderm are likely to mediate budding morphogenesis in the
underlying endoderm. The best candidate signaling molecule is Fgf10 because,
in normal development, Fgf10 is expressed in the visceral mesoderm of
the lung primordium, overlapping with Tbx4
(Fig. 1I,J compare with 1B,D).
In addition, Fgf10 is essential for budding morphogenesis of the
primary bud and bronchial branching
(Bellusci et al., 1997b;
Mailleux et al., 2001
;
Min et al., 1998
;
Sekine et al., 1999
). To
examine this possibility, we analyzed Fgf10 expression in the Tbx4
misexpressed embryos. In the Tbx4 misexpressed embryo, ectopic Fgf10
expression was observed in the visceral mesoderm at precisely the same
locations as exogenous Tbx4 expression
(Fig. 3A,B, blue arrowheads).
Tbx4 misexpression induced ectopic Fgf10 expression in the mesoderm
irrespective of the DV territory (Fig.
3C). We also examined another signaling molecule, Bmp4,
which is specifically expressed in the respiratory mesoderm of chicks
(Sakiyama et al., 2000
) and
plays role in the proximodistal patterning of mouse lung development
(Weaver et al., 1999
). Unlike
Fgf10, Bmp4 expression was not affected by Tbx4 misexpression
(Fig. 3D,E, red arrowheads).
These results indicate that Tbx4 promotes Fgf10 expression, but not
Bmp4 expression.
|
Ectopic Fgf10 induced ectopic endodermal buds
To examine whether Fgf10 has endodermal bud inducing activity, we
introduced Fgf10 expression plasmids together with GFP plasmids into the
visceral mesoderm of the prospective esophagus-respiratory region. As a
result, ectopic Fgf10 induced ectopic budding morphogenesis in the endoderm
underlying the Fgf10-transfected mesoderm [14/26 (53.8%)]
(Fig. 4A,B, yellow arrowheads).
Fgf10 also induced ectopic endodermal buds in both the dorsal
(Fig. 4A, yellow arrowheads)
and ventral (Fig. 4C, yellow
arrowhead) region. These results indicate that Fgf10 has endodermal bud
inducing activity. Fgf10 misexpression also caused ectopic Tbx4
expression in the esophagus mesoderm (Fig.
4E,F, red arrowheads). Ectopic Tbx4 expression in the
esophagus, however, was weaker than its endogenous expression in the lung bud
mesoderm (Fig. 4F). These
observations indicate the presence of a feedback loop between Tbx4
and Fgf10 in the regulation of lung development. Furthermore,
misexpression of Fgf10 also caused tracheo-esophageal fistula [15/26 (57.7%)]
(Fig. 4G).
|
Activity of Tbx4 was necessary for Fgf10 expression in
ovo
For further analysis on the regulatory role of Tbx4 on Fgf10
expression, we designed misexpression assays of a variant form of Tbx4.
Tbx4-VP16 and Tbx4-EnR were constructed as a constitutive active form and a
dominant-negative form of Tbx4, respectively. Electroporation was carried out
at stage 7-10 in order to express transgenes prior to endogeneous
Tbx4 and Fgf10 expression. Then, Fgf10 expression
was examined 24-30 hours after electroporation when development of the
manipulated embryos reached stage 17-19. Tbx4 misexpression in the presumptive
esophagus-respiratory region (Fig.
5A, bracket) resulted in a marked expansion of the Fgf10
expression domain throughout the Tbx4-transfected mesoderm (100%,
n=6) (Fig. 5B). In the
case of Tbx4-VP16 misexpression, the Fgf10 expression domain expanded
throughout the Tbx4-VP16-transfected mesoderm as in the case of Tbx4
misexpression (26%, n=19) (Fig.
5D, bracket), but the level of Fgf10 expression was lower
than normal in the lung bud on the contralateral side
(Fig. 5D, compare RL side with
LL side). Tbx4-EnR misexpression resulted in significant reduction or
elimination of Fgf10 expression in the Tbx4-EnR-transfected mesoderm
(62%, n=21) (Fig. 5F,
bracket). In severely affected cases, budding morphogenesis of the primary bud
was blocked (11/30 [36.7%]) (Fig.
5F,H). Surprisingly, Tbx4-EnR misexpression caused ectopic
Fgf10 expression in the distal esophagus mesoderm posterior adjacent
to the Tbx4-EnR-transfected mesoderm (Fig.
5F,G, red arrow). Corresponding to this ectopic Fgf10
induction, 2 days after electroporation of Tbx4-EnR, an ectopic endodermal bud
was observed in the distal esophagus (Fig.
5H, arrowheads) even though Tbx4-EnR was transfected into the
mesoderm of the lung field (Fig.
5H, green). In control experiments in which GFP was
electroporated, ectopic expression of Fgf10 was never observed (0%,
n=27), and downregulation of Fgf10 expression was rarely
observed (22%, n=27) (Fig.
5I,J).
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Tbx4 and Fgf10 induced ectopic Nkx2.1 expression in the
underlying endoderm
During gut organogenesis, the visceral mesoderm influences both endodermal
morphogenesis and endodermal differentiation
(Yasugi, 1993). It is likely
that the lung primordium mesoderm influences differentiation of the
respiratory endoderm. As described above, the Tbx4-expressing
mesoderm overlies the Nkx2.1-expressing endoderm
(Fig. 1D). To examine whether
Tbx4 influences differentiation of the underlying endoderm, we
analyzed the expression of Nkx2.1 in the Tbx4 misexpressed embryo.
When Tbx4 was misexpressed throughout the esophagus-respiratory region, the
expression domain of Nkx2.1 expanded posteriorly
(Fig. 6A,B, compare with 6G). The ectopic Nkx2.1 domain was observed in the distal esophagus
endoderm that underlies the Tbx4-transfected mesoderm
(Fig. 6A), but ectopic
Nkx2.1 expression was restricted to the ventral wall of the distal
esophageal endoderm of the Tbx4-transfected side
(Fig. 6C). The ectopic bud in
the ventral esophagus expressed Nkx2.1 at levels as high as in the
normal lung bud (Fig. 2A,
Fig. 6B, yellow arrowhead). By
contrast, the ectopic bud in the dorsal esophagus did not express
Nkx2.1 (Fig. 2D,
yellow arrowhead). Fgf10 misexpression also caused ectopic Nkx2.1
expression in the ventral endoderm, but not in the dorsal endoderm, as seen in
Tbx4 misexpression (Fig. 6E,F compare with
6H).
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DISCUSSION |
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The primordia of the visceral organs are established through a
regionalization process of the gut tube along the AP axis. During region
specific gut morphogenesis, a restriction of cell intermingling within a
single organ primordium occurs by approximately stage 15-16 in chicks
(Smith and Tabin, 2000). Thus,
it is likely that the compartment boundaries of the organ primordia and the
compartment-specific, i.e. organ-specific, identities are established by
approximately stage 15-16 in the developing chick gut tube. Tbx4
expression initiates in the mesoderm of the presumptive lung primordium area
around stage 14-15 (21-25 somites) (Fig.
1B,D). Thus, the expression of Tbx4 is coincident with
the establishment of the compartment of the lung primordium field, indicating
that Tbx4 is involved in the initial specification process of the
territory for the lung primordium mesoderm.
Once the lung primordium is established, the lung primordium mesoderm
acquires an inductive capability for the initial budding morphogenesis of
primary lung buds (Spooner and Wessels,
1970). Fgf10 is specifically expressed in the lung
primordium mesoderm (Bellusci et al.,
1997b
) and acts as an essential component of the inductive signal
for the initial budding morphogenesis of primary lung buds
(Min et al., 1998
;
Sekine et al., 1999
). In our
study, misexpression of Tbx4 in the esophagus mesoderm induced ectopic
Fgf10 expression in the mesoderm
(Fig. 3A-C) and subsequent
ectopic budding morphogenesis in the underlying esophagus endoderm
(Fig. 2A-F). The results of the
Tbx4 misexpression studies suggest that Tbx4 is able to trigger the specific
signaling pathway for endodermal budding morphogenesis in the ectopic area of
the esophagus. Furthermore, misexpression of Tbx4 in the esophagus mesoderm
induced the expression of Nkx2.1, a specific marker for the
respiratory endoderm, in the underlying esophagus endoderm
(Fig. 6A; discussed below).
Taken together, Tbx4 plays a role in the acquisition of the inductive
capability that is specific for the lung primordium mesoderm.
At later stage, Tbx4 was still expressed in the lung bud mesoderm
and the expression boundary of Tbx4 clearly demarcated the
respiratory tract (Tbx4-expressing) and the esophagus
(Tbx4-non-expressing) (Fig.
1H). When the border of the Tbx4 expression domain was
disturbed by misexpression of Tbx4, separation of the trachea and
esophagus failed (Fig. 2G).
Fgf10 misexpression also caused an identical phenotype
(Fig. 4G). Therefore, the
respiratory-specific expression of Tbx4 is crucial for
tracheo-esophageal septum formation, the structural separation of the
respiratory tract and the esophagus through Fgf10 activation.
However, Tbx4 or Fgf10 misexpression did not cause bud elongation following
budding morphogenesis (Fig.
2A,D, Fig. 4A,C).
One interpretation of this result is that another cascade(s) independent of
the Tbx4-Fgf10 system (e.g. the Bmp4 cascade) may be necessary for bud
elongation and bronchial branching. For bud elongation process, dynamic and
localized Fgf10 expression around the tip of the endodermal bud is
crucial (Bellusci et al.,
1997b; Weaver et al.,
2000
). In our Tbx4 misexpression study, ectopic Fgf10 was
expressed throughout the transfected mesoderm. If restriction of the Fgf10
source around the tip of the endodermal bud is crucial for normal development,
such a situation was difficult to reproduce by our gene transfer method.
Tbx4 defines the Fgf10 expression domain during early lung
development
During normal development, the anterior and posterior boundaries of
Tbx4 and Fgf10 expression in the lung primordium mesoderm
were overlapping (compare Fig. 1D with
1J). When the Tbx4 expression domain was expanded beyond the lung
primordium field by Tbx4 misexpression, the Fgf10 expression domain
expanded throughout the Tbx4-misexpressing mesoderm, including the outside of
the lung primordium field (Fig.
3A,B, Fig. 5A,B).
These observations suggest that the lung primordium-specific transcription
factor Tbx4 defines the Fgf10 expression domain, especially at both
the anterior and posterior boundaries, by activating Fgf10 expression
in a cell-autonomous manner. Because misexpression of Tbx4-VP16, a
dominant-activator form of Tbx4, caused ectopic activation of Fgf10
expression equal to that of full-length Tbx4
(Fig. 5D), Tbx4 acts as a
transcription activator for Fgf10 expression. Conversely,
interference with the transcription activation function of endogenous Tbx4 by
misexpression of Tbx4-EnR, a dominant-repressor form of Tbx4, resulted in
repression of the endogenous Fgf10 expression in the lung primordium
mesoderm (Fig. 5F). Therefore,
the transcription activation function of Tbx4 in the lung primordium mesoderm
is necessary for Fgf10 expression. We could not discriminate,
however, whether Tbx4 is necessary for initiation or maintenance of
Fgf10 expression under the strict criteria. The results of our
misexpression studies do not exclude the possibility that exogenous Tbx4
activates and Tbx4-EnR blocks the maintenance circuit for Fgf10
expression. An answer to this issue will be provided by the analysis of
molecular mechanism for Fgf10 transcription regulation.
Although Tbx4-VP16 construct exhibited stronger transcriptional stimulation than native Tbx4 in reporter transcription assay (data not shown), Tbx4-VP16 construct induced ectopic Fgf10 expression but to a lesser extent than native Tbx4 in the embryo (Fig. 5D). For constructing Tbx4-VP16, we have eliminated the region downstream of the T-box and replaced with VP-16 transcriptional activation domain. These results imply that Tbx4 may be not a simple transcriptional activator and behave as a repressor depend on the context of regulatory element of the target genes. It is possible that both activities are required for effective Fgf10 transcription through complex transcriptional network.
Interestingly, we found that misexpression of Tbx4-EnR induced ectopic Fgf10 expression in the distal esophagus mesoderm in the posterior region adjacent to the Tbx4-EnR-transfected mesoderm (Fig. 5F,G, red arrow). This result indicates that Fgf10 is capable of being expressed in the distal esophagus mesoderm if the transcription activation function of Tbx4 in the lung primordium mesoderm is disrupted. The simplest interpretation of this result is that Tbx4 normally activates the target genes that repress Fgf10 expression in a non-cell-autonomous manner as a form of lateral inhibition. If this lateral inhibition system malfunctions, the distal esophagus, the posterior neighbor to the lung field, may develop an additional Nkx2.1-expressing endodermal bud as observed during Tbx4-EnR misexpression (Fig. 5H). Taken together, Tbx4 regulates Fgf10 expression by binary pathways: a cell-autonomous activation pathway and a non-cell-autonomous repression pathway, and these dual regulation pathways of Tbx4 precisely define the Fgf10 expression domain within the lung primordium mesoderm.
During normal development, the Fgf10 expression domain was
restricted to the ventral half of the Tbx4 expression domain where
the primary bud would arise (compare Fig.
1I with 1B). This discrepancy between Tbx4 and
Fgf10 expression domains suggests two mechanisms for Fgf10
activation. First possibility is that the dosage of Tbx proteins is crucial
for Fgf10 activation. Tbx2, Tbx3 and Tbx5 have been
briefly reported to be expressed in the lung bud mesoderm of chick and mouse
(Chapman et al., 1996;
Gibson-Brown et al., 1998
). In
chicken foregut mesoderm, Tbx2, Tbx4 and Tbx5 showed
overlapping and slightly different expression pattern. Among them, only
Tbx4 exhibited common anterior and posterior boundaries with the
Fgf10 expression domain (J. S. and A. K., unpublished). It is
possible that the Fgf10 expression domains may be defined by
redundant/cooperative action of Tbx4 and other Tbx protein(s), which is
exclusively expressed in the ventral mesoderm. The second possibility is the
presence of the co-factor(s) that activate Fgf10 transcription
synergistically with Tbx4 on the ventral side and/or the presence of the
antagonist of Tbx4 that represses Fgf10 expression on the dorsal
side. Our results showed that Tbx4 misexpression alone induced the expression
of Fgf10. In this case, it is likely that the high dose of the
foreign Tbx4 exceeds the threshold for Fgf10 activation in the
absence of a co-factor and/or in the presence of an antagonistic factor.
During limb development, Tbx4 and Tbx5 are also expressed
in the hindlimb and forelimb mesenchyme, respectively, and this pattern is
conserved in the tetrapod and fish
(Gibson-Brown et al., 1996;
Gibson-Brown et al., 1998
;
Tamura et al., 1999
).
Mesenchymal Fgf10 expression is also crucial for growth of the limb
bud (Min et al., 1998
;
Sekine et al., 1999
).
Recently, it has been reported that Tbx5 is necessary for Fgf10
expression and fin bud formation in zebrafish
(Ng et al., 2002
). Thus,
Tbx-Fgf system may be extensively used in the bud formation during vertebrate
embryogenesis.
The Tbx4-Fgf10 system defines the Nkx2.1 expression domain
in the underlying endoderm
Previous studies did not mention whether the lung primordium mesoderm
induced the underlying endoderm to differentiate into the respiratory endoderm
(Spooner and Wessels, 1970).
We found that Tbx4 or Fgf10 misexpression induced ectopic Nkx2.1
expression in the underlying endoderm (Fig.
6A,E). Tbx4-Fgf10 system acts as a signaling component for the
inductive interactions specific to the lung primordium mesoderm. Thus, results
of Tbx4 or Fgf10 misexpression studies indicate that the lung primordium
mesoderm also influences respiratory endoderm differentiation. As the
expression of Nkx2.1 in the ventral endoderm precedes Tbx4
and Fgf10 expression (data not shown), the Tbx4-Fgf10 system does not
appear to be involved in the initiation of Nkx2.1 expression or the
initiation of respiratory endoderm differentiation. Once the expression of
Tbx4 and Fgf10 is initiated, the posterior boundaries of the
Tbx4, Fgf10 and Nkx2.1 expression domains are localized to
the same position in the respiratory-esophagus region of the foregut
(Fig. 1B-D,I,J). When the
posterior boundaries of the Tbx4 or Fgf10 expression domains
were shifted posteriorly by Tbx4 or Fgf10 misexpression, the Nkx2.1
expression domain expanded posteriorly to the distal esophagus endoderm
underlying the Tbx4- or Fgf10-miexpressing mesoderm
(Fig. 6A,E). This coincidence
of the posterior boundaries of Tbx4/Fgf10 and Nkx2.1 in the
normal and Tbx4 or Fgf10 misexpressed embryos suggests that the Tbx4-Fgf10
system defines the posterior boundary of the Nkx2.1 expression
domain, i.e. the posterior boundary of the respiratory endoderm, in the
maintenance pathway. However, misexpression of Tbx4 or Fgf10 induced ectopic
Nkx2.1 expression only in the ventral endoderm
(Fig. 6C,F). These results
suggest that another system(s) independent of Tbx4-Fgf10 regulates the ventral
restricted Nkx2.1 expression.
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ACKNOWLEDGMENTS |
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