Download Mesenchymal expression of Tbx4 gene is not altered

Pediatr Surg Int (2010) 26:407–411
DOI 10.1007/s00383-010-2580-y
ORIGINAL ARTICLE
Mesenchymal expression of Tbx4 gene is not altered
in Adriamycin mouse model
Piotr Hajduk • Paula Murphy • Prem Puri
Accepted: 2 February 2010 / Published online: 25 February 2010
Ó Springer-Verlag 2010
Abstract
Purpose The Adriamycin mouse model (AMM) is a
reproducible teratogenic model of esophageal atresia/tracheo-esophageal fistula (EA/TEF). Tbx4 is a member of
the T-box family of transcription factor genes, which is
reported to play a key role in separation of the respiratory
tract and the esophagus. Up-regulation of Tbx4 is reported
to cause TEF in the chick. Optical projection tomography
(OPT) is a technique that allows three-dimensional (3D)
imaging of gene expression in small tissue specimens in an
anatomical context. The aim of this study was to investigate the temporo-spatial expression of Tbx4 during the
critical period of separation of the trachea and esophagus in
normal and Adriamycin treated embryos using OPT.
Materials and methods Time-mated CBA/Ca mice
received intraperitoneal injections of Adriamycin (6 mg/kg)
or saline on days 7 and 8 of gestation. Embryos were
harvested on days 9–12, stained following whole mount in
situ hybridization with labeled RNA probes to detect Tbx4
transcripts (n = 5 for each treatment/day of gestation).
Immunolocalization with the endoderm marker Hnf3b was
used to visualize morphology. Embryos were scanned by
OPT to obtain 3D representations of gene expression
domains. Animal licence no. B100/4106.
Results OPT elegantly revealed Tbx4 gene expression
in both controls and in the disorganized pulmonary
P. Hajduk (&) P. Puri
Children’s Research Centre,
Our Lady’s Children’s Hospital,
Crumlin, Dublin 12, Ireland
e-mail: [email protected]
P. Hajduk P. Murphy
Department of Zoology, Trinity College,
University of Dublin, Dublin, Ireland
mesenchyme in the treated embryos. Although characteristic morphological abnormalities were observed in Adriamycin treated embryos, there was no significant difference
in Tbx4 transcript distribution around lung primordia in
comparison with control embryos.
Conclusion Although previously reported morphological
abnormalities of notochord and esophagus were observed
in AMM, Tbx4 gene expression was unaltered, suggesting
that esophageal anomalies can occur in the presence of
normal Tbx4 gene expression in this model.
Keywords Foregut Lung Notochord Tbx4 Optical projection tomography
Introduction
Esophageal atresia/tracheo-esophageal fistula (EA/TEF)
are common malformations of the foregut in humans which
require emergency surgery in the newborn period. EA/TEF
can occur as part of a heterogenous group of non-randomly
associated anomalies known by the acronym of VACTERL
[1–3]. This association includes vertebral defects, anal
atresia, cardiovascular anomalies, tracheo-oesophageal
fistula, renal anomalies and limb defects. The developmental link between the systems affected in these cases has
been intriguing.
Adriamycin, a chemotherapeutic agent, was originally
found to cause birth defects in rats that resemble the
VACTERL association [4]. Detailed studies of the development of EA/TEF in the Adriamycin rat model (ARM)
have provided an understanding of the morphological
stages involved in their development. However, the mouse
is the developmental biologist’s mammalian model of
choice, providing much greater availability of molecular
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probes, antibodies and transferable knowledge from mutant
mouse models. Previous work has established that the
Adriamycin mouse model (AMM) also demonstrates
VACTERL association similar to ARM and these mice
develop EA/TEF [5, 6].
A number of genes have been implicated in the normal
development of trachea and esophagus. Tbx4 is a member
of the T-box transcription factor gene family. It is
expressed in the visceral mesoderm around the lung primordia [7, 8]. Ectopic Tbx4 expression in the mesoderm of
manipulated chick embryos induced ectopic lung bud formation in the foregut, activating the expression of Fgf10,
which is essential for lung budding morphogenesis [8].
Furthermore ectopic expression of Tbx4 caused the failure
of trachea-esophageal septum formation resulting in TEF.
HNF3b is a forkhead domain transcription factor
expressed throughout the definitive endoderm of the
respiratory and gastrointestinal tract and notochord [9, 10].
HNF3b antibody has been used to label the developing
endoderm and the floorplate [11]. Optical projection
tomography (OPT) is a new, rapid and non-invasive technique for 3D imaging of small biological tissue specimens
that allow visualization of the tissue distribution of RNA,
protein or histological stains in developing organs while
also recording morphology [11].
The aim of this study was to investigate the temporospatial expression of the Tbx4 gene during the critical
period of separation of the trachea and esophagus in normal
and Adriamycin treated embryos using OPT.
Materials and methods
Animals
Male and female CBA/Ca mice (Harlan UK, Bicester,
England) were accurately time-mated over a 4-h period
starting at 8 a.m. Identification of a vaginal plug at the end
of the mating period was taken to be the start of gestation.
Pregnant mice received two intraperitoneal injections on
embryonic days E7 and E8. The Adriamycin treated mice
received a dose of 6 mg/kg of Adriamycin (Doxorubicin,
EBEWE Pharma Ges.m.b.H Nfg.KG, A-4866 Unterach,
Austria). The control mice received an equivalent volume
of 0.9% sodium chloride. The Adriamycin was diluted with
0.9% sodium chloride and stored appropriately. Dams were
humanely killed by swift cervical dislocation between E9
and E12. The embryos were dissected in RNase-free
phosphate buffered saline (PBS) and then fixed in 4%
paraformaldehyde in PBS overnight. E12 embryos were
dissected to include only the trunk region from the first
branchial arch to the base of the liver to ensure penetration
of RNA probes and antibodies.
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Pediatr Surg Int (2010) 26:407–411
Whole mount in situ hybridization
and immunolocalization
The plasmid including a subclone of the mouse Tbx4 gene
was generously gifted by Dr Saverio. The region used
as a probe extends from nucleotide 526 to 1821 on
NM_172798.1 (GenBank Sequence). The restriction
enzymes NotI and EcoRI were used to linearize the plasmid
DNA and in vitro transcription using RNA polymerases
and digoxigenin–dUTP was performed to generate antisense and sense Tbx4 RNA probes.
Whole embryos stocked in 100% methanol were subsequently rehydrated and underwent in situ hybridization
as previously described [12]. The sense probe was used as a
control. Immunolocalization with HNF3b primary antibody (07-633 Upstate Cell Signaling Solution, Lake Placid,
NY), diluted 1/500, and Cy3 conjugated goat anti-rabbit
IgG (111-165-144 Jackson Immno Research) at 1/200
dilution as secondary antibody was performed. Embryos
were double labeled for Tbx4 expression by in situ
hybridization and HNF3b immunolocalization by simultaneous incubation with antibodies to digoxigenin and
HNF3b.
Optical projection tomography
Stained embryos were embedded in 1% low melting point
agarose, attached to a metal mount, dehydrated in MeOH
overnight and cleared in benzyl benzoate/benzyl alcohol
(1:2) (BABB) for at least 5 h [11, 12]. Projection images of
the specimens were captured in a prototype OPT scanner
constructed at the MRC Human Genetics Unit, Edinburgh,
and installed in the Zoology Department, Trinity College,
Dublin [11, 12]. The raw data (400 projected images) from
each of the scans were loaded onto a Linux workstation,
reconstructed and analyzed using custom made software
(MA3DView and MAPaint), provided by the Edinburgh
Mouse Atlas Project.
All experiments were carried out in compliance with
current European Union regulations for animal investigation (ED86/609/EC), with prior ethical approval under
licence number B100/4106 from the Department of Health,
Ireland.
Results
The Tbx4 antisense probe was first tested on normal
embryos and found to give a clear and specific pattern of
expression in the mesenchyme around the site of the lung
bud, trachea, in a specific patch in the forelimb and
extensively in the hindlimb (Fig. 1). Figure 1 illustrates the
3D nature of the data generated through in situ
Pediatr Surg Int (2010) 26:407–411
409
Fig. 1 The expression of the
Tbx4 gene at E11.
a Photomicrograph of a lateral
view of a whole mount Tbx4 in
situ hybridized specimen. b The
3D nature of the data analyzed
in this study is shown in a still
shot taken from a 3D
reconstruction of the externally
visible pattern of Tbx4
expression following the
merging of two OPT scans
where the tissue (pseudocolored
in green) and the gene
expression (pseudocolored in
red) were captured separately.
c The pattern of Tbx4
expression around the trachea
and lung buds is shown in more
detail in this surface rendered
image. fg foregut, fl forelimb, hl
hindlimb, tr trachea, lb lung bud
hybridization and OPT scanning to visualize features of the
Tbx4 expression domain in detail.
Analysis of 3D computer reconstructed specimens of
control and treated embryos across stages E9–E12 following OPT allowed the Tbx4 gene expression patterns to
be viewed in 2D virtual sections throughout the developing
tissue (Fig. 2). The intensity of staining and the spatial
territory within which the Tbx4 gene was expressed with
respect to the forming foregut was compared visually
between control and Adriamycin treated groups at each
time point of collection.
In E9 Adriamycin treated and control embryos Tbx4
was expressed in the mesenchyme around the site of the
lung buds (Fig. 2a, b, i, j). Any apparent difference in Tbx4
expression pattern viewed on coronal sections (Fig. 2b, j)
are due to relatively more advanced lung development in
control embryos.
At E10 Tbx4 expression was clearly detected in the
mesenchyme around the trachea (Fig. 2c) and in the mesenchyme around the lung bud (Fig. 2d). At this stage, Tbx4
expression persisted in the lung bud region mesenchyme in
treated embryos (Fig. 2k, l), even in cases where lung bud
formation is lacking (not shown).
At E11, virtual sections through the embryos showed
Tbx4 expression in the mesenchyme around the trachea in
both control and treated embryos (Fig. 2e, m), and around
the lung bud (Fig. 2f, n) without obvious changes in the
spatial distribution or level of gene expression.
Fig. 2 Control (a–h) and Adriamycin treated (i–p) mouse embryos at
embryonic days E9–E12 following whole mount in situ hybridization
show mesenchymal Tbx4 gene expression represented by virtual
sections taken through the 3D data in different saggital and coronal
planes indicated by orange lines. Red arrows indicate Tbx4 expression around site of the lung buds (a–p). White arrow indicates Tbx4
gene expression around tracheal mesenchyme (c, e, m, o). e–f, m–n
virtual sections taken through the 3D data following the merging of
two OPT scans where the tissue (pseudocolored in green) and the
Tbx4 gene expression (pseudocolored in red) were captured separately. tr trachea
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At E12, embryos were enlarged considerably and the
complexity of tissue increased. To ensure penetration of
the RNA probe, the embryos were dissected to include only
the trunk region from the first branchial arch to the base
of the liver. Following OPT scanning and computer
reconstruction Tbx4 gene expression was detected in the
mesenchyme around the lung bud in control embryos
(Fig. 2g, h) and treated embryos (Fig. 2o, p) without any
difference in spatio-temporal expression between control
and Adriamycin treated embryos.
Although the Adriamycin treated embryos showed a
variety of the expected foregut abnormalities including
complete laryngotracheo-esophageal cleft (in 6 of 22
embryos), OA (in 6 of 22 embryos) and TOF (in 5 of 22
embryos), there were no differences in Tbx4 gene expression pattern in the Adriamycin treated groups, irrespective
of the severity of the morphological abnormalities.
Discussion
The development of the embryo is a complex process in
which tissues and organs undergo an intricate sequence of
movements relative to each other, controlled by interaction
through diffusible signaling molecules [13]. In normal
development, the respiratory and digestive tubes are
derived by division of the foregut primordium that is surrounded by splanchnic mesoderm [14]. In mice, the bronchi are the first respiratory structures to be generated as
bulges on the ventrolateral wall of the foregut at E9.5. At
E10, the laryngo-tracheal groove gives rise to the trachea,
which connects to the left and right main bronchi. The lung
buds are located on the ventral aspect of the gastric dilatation of the foregut. The tracheal and bronchial stalks
extend to form the main bronchi, a process completed by
approximately E10.5. Stereotypic branching and budding
occurs as the bronchial and bronchiolar tubules form
between approximately E11 and E16 [15]. The embryonic
development of the foregut into a respiratory and a digestive part is a complicated process, of which many aspects
have not yet been clarified, particularly the factors that
trigger the normal progression of events described above.
An understanding of normal development of the foregut is
essential to define the mechanism underlying the origin of
OA/TOF in mammalian embryos.
A number of mouse mutants replicate aspects of OA/
TOF shown in the Adriamycin model indicating that
complex signaling events involving the mutant gene
products are involved [16, 17]. The morphological similarity of foregut abnormalities seen in a number of mutants
with different genetic lesions produced by Adriamycin
treatment in chick, rat and mice indicate that common
processes are being disturbed [4–6, 18].
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Tbx4 is a member of the T-box transcription factor gene
family which controls multiple processes during respiratory
tract development such as initial endodermal bud formation, respiratory endoderm formation and separation of the
respiratory tract and the esophagus. It is expressed in the
visceral mesoderm at the position where lung primordial
form overlapping with Fgf10 expression [7, 8]. Ectopic
Tbx4 expression induced ectopic lung bud formation in the
foregut, by activating the expression of Fgf10 in chick [8].
It has also been reported that when the border of the Tbx4
expression domain was disturbed by misexpression of
Tbx4, there was failure in tracheo-esophageal separation.
Fgf10 misexpression also led to failure in tracheooesophageal formation [8]. Inactivation of Tbx4 function is
reported to repress Fgf10 expression and lead to failure of
lung bud formation. This suggests that a Tbx4–Fgf10
system, in mouse and chick, acts as a signaling component
for the inductive interactions specific to the lung primordium mesoderm and that altered boundaries of expression
can affect tracheo-esophageal separation.
In a previous study, we showed a delay in Fgf10 gene
expression in Adriamycin treated embryos and a lack of
Fgf10 gene expression in cases associated with tracheal
atresia [19] in contrast to the lack of effect on Tbx4
expression shown here. This differential effect indicates the
level at which Adriamycin impacts this particular signaling
system.
In this study, we used OPT to visualize the distribution
of the transcripts of Tbx4 gene across early stages of
development, when malformations occur in the AMM.
OPT is a relatively new technique for 3D imaging of small
whole embryos, revealing morphological details of the
developing embryos as well as recording gene expression
patterns in mouse and chick embryos [11, 12, 20, 21]. It
takes 400 projected, optical density images from one
complete revolution of the embryo and then uses computer
software to reconstruct the original 3D object digitally.
This information can be viewed as a whole 3D model
which can be rotated to view it from any angle or as virtual
sections cut in any plane. The technique is non-invasive
(does not require sectioning) (Fig. 1), avoiding the problem
of section loss and distortion, allowing large numbers of
specimen be screened to show colored or fluorescent
stained tissue for gene expression patterns and distribution
of other molecules within the anatomical context of the
developing embryos.
Here we show successful staining and visualization of
lung buds and tracheal mesenchyme by combining in situ
hybridization to detect Tbx4 expression with OPT scaning
and computer reconstruction across the E9–E12 stages of
embryonic development. In the present study, Tbx4
expression was detected adjacent to the foregut at an
equivalent antero/posterior position and at approximately
Pediatr Surg Int (2010) 26:407–411
the same level in control and Adriamycin treated embryos.
This indicates that the morphological abnormalities previously reported in AMM [5, 6] are not accompanied by
obvious changes in the spatial distribution or level of gene
expression of Tbx4 suggesting that Tbx4 regulation is not
altered in this model. This implies that the morphological
abnormalities in AMM can occur in the presence of Tbx4
transcripts.
References
1. Quan L, Smith DW (1973) The VATER association: vertebral
defects, anal atresia, T-E fistula with esophageal atresia, radial
and renal dysplasia: a spectrum of associated defects. J Pediatr
82:104–107
2. Temtamy SA, Miller JD (1974) Extending the scope of the
VATER association: definition of the VATER syndrome.
J Pediatr 85:345–349
3. Khoury MJ, Cordero JF, Greenberg F, James LM, Erickson JD
(1983) A population study of the VACTERL association: evidence for its etiologic heterogeneity. Pediatrics 71:815–820
4. Diez-Pardo JA, Qi B, Navarro C, Tovar JA (1996) A new rodent
experimental model of esophageal atresia and tracheoesophageal
fistula: preliminary report. J Pediatr Surg 31:498–502
5. Ioannides AS, Chaudhry B, Henderson DJ, Spitz L, Copp AJ
(2002) Dorsoventral patterning in oesophageal atresia with
tracheo-oesophageal fistula: evidence from a new mouse model.
J Pediatr Surg 37:185–191
6. Dawrant M, Giles S, Bannigan J, Puri P (2007) Adriamycin
mouse model: a variable but reproducible model of tracheooesophageal malformations. Pediatr Surg Int 23:469–472
7. Chapman DL, Garvey N, Hancock S, Alexiou M, Agulnik SI,
Gibson-Brown JJ, Cebra-Thomas J, Bollag RJ, Silver LM,
Papaioannou VE (1996) Expression of the T-box family genes,
Tbx1–Tbx5, during early mouse development. Dev Dyn
206:379–390
8. Sakiyama J, Yamagishi A, Kuroiwa A (2003) Tbx4-Fgf10 system
controls lung bud formation during chicken embryonic development. Development 130:1225–1234
411
9. Ang SL, Wierda A, Wong D, Stevens KA, Cascio S, Rossant J,
Zaret KS (1993) The formation and maintenance of the definitive
endoderm lineage in the mouse: involvement of HNF3/forkhead
proteins. Development 119:1301–1315
10. Ang SL, Rossant J (1994) HNF-3 beta is essential for node and
notochord formation in mouse development. Cell 78:561–574
11. Sharpe J, Ahlgren U, Perry P, Hill B, Ross A, Hecksher-Sorensen
J, Baldock R, Davidson D (2002) Optical projection tomography
as a tool for 3D microscopy and gene expression studies. Science
296:541–545
12. Summerhurst K, Stark M, Sharpe J, Davidson D, Murphy P
(2008) 3D representation of Wnt and Frizzled gene expression
patterns in the mouse embryo at embryonic day 11.5 (Ts19).
Gene Expr Patterns 8(5):331–348
13. Costa RH, Kalinichenko VV, Lim L (2001) Transcription factors
in mouse lung development and function. Am J Physiol Lung
Cell Mol Physiol 280:L823–L838
14. Merei JM, Hutson JM (2002) Embryogenesis of tracheo-esophageal anomalies: a review. Pediatr Surg Int 18:319–326
15. Maeda Y, Dave V, Whitsett JA (2007) Transcriptional control of
lung morphogenesis. Physiol Rev 87:219–244
16. Litingtung Y, Lei L, Westphal H, Chiang C (1998) Sonic
hedgehog is essential to foregut development. Nat Genet 20:58–
6139
17. Que J, Choi M, Ziel JW, Klingensmith J, Hogan BL (2006)
Morphogenesis of the trachea and esophagus: current players and
new roles for noggin and Bmps. Differentiation 74(7):422–437
18. Naito Y, Kimura T, Aramaki M, Izumi K, Okada Y, Suzuki H,
Takahashi T, Kosaki K (2009) Caudal regression and tracheoesophageal malformation induced by adriamycin: a novel chick
model of VATER association. Pediatr Res 65:607–612
19. Hajduk P, Murphy P, Puri P (2009) Fgf10 gene expression is
delayed in the embryonic lung mesenchyme in the adriamycin
mouse model. Pediatr Surg Int 26:23–27
20. Lioubinski O, Alonso MT, Alvarez Y, Vendrell V, Garrosa M,
Murphy P, Schimmang T (2006) FGF signalling controls
expression of vomeronasal receptors during embryogenesis.
Mech Dev 123(1):17–23
21. Sato H, Murphy P, Giles S, Bannigan J, Takayasu H, Puri P
(2008) Visualizing expression patterns of Shh and Foxf1 genes in
the foregut and lung buds by optical projection tomography.
Pediatr Surg Int 24:3–11
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