Tbx2 Controls Lung Growth by Direct Repression of the Cell Cycle Inhibitor Genes Cdkn1a and Cdkn1b
et al. (2013) Tbx2 Controls Lung Growth by Direct Repression of the Cell Cycle Inhibitor
Genes Cdkn1a and Cdkn1b. PLoS Genet 9(1): e1003189. doi:10.1371/journal.pgen.1003189
Tbx2 Controls Lung Growth by Direct Repression of the Cell Cycle Inhibitor Genes Cdkn1a and Cdkn1b
Timo H.-W. Lu dtke 0
Henner F. Farin 0
Carsten Rudat 0
Karin Schuster-Gossler 0
Marianne Petry 0
Phil Barnett 0
Vincent M. Christoffels 0
Andreas Kispert 0
Virginia E. Papaioannou, Columbia University Medical Center, United States of America
0 1 Institute for Molecular Biology, Medizinische Hochschule Hannover, Hannover, Germany, 2 Department of Anatomy, Embryology and Physiology, Heart Failure Research Center, Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands
Vertebrate organ development relies on the precise spatiotemporal orchestration of proliferation rates and differentiation patterns in adjacent tissue compartments. The underlying integration of patterning and cell cycle control during organogenesis is insufficiently understood. Here, we have investigated the function of the patterning T-box transcription factor gene Tbx2 in lung development. We show that lungs of Tbx2-deficient mice are markedly hypoplastic and exhibit reduced branching morphogenesis. Mesenchymal proliferation was severely decreased, while mesenchymal differentiation into fibrocytes was prematurely induced. In the epithelial compartment, proliferation was reduced and differentiation of alveolar epithelial cells type 1 was compromised. Prior to the observed cellular changes, canonical Wnt signaling was downregulated, and Cdkn1a (p21) and Cdkn1b (p27) (two members of the Cip/Kip family of cell cycle inhibitors) were strongly induced in the Tbx2-deficient lung mesenchyme. Deletion of both Cdkn1a and Cdkn1b rescued, to a large degree, the growth deficits of Tbx2-deficient lungs. Prolongation of Tbx2 expression into adulthood led to hyperproliferation and maintenance of mesenchymal progenitor cells, with branching morphogenesis remaining unaffected. Expression of Cdkn1a and Cdkn1b was ablated from the lung mesenchyme in this gain-of-function setting. We further show by ChIP experiments that Tbx2 directly binds to Cdkn1a and Cdkn1b loci in vivo, defining these two genes as direct targets of Tbx2 repressive activity in the lung mesenchyme. We conclude that Tbx2-mediated regulation of Cdkn1a and Cdkn1b represents a crucial node in the network integrating patterning information and cell cycle regulation that underlies growth, differentiation, and branching morphogenesis of this organ.
Funding: Work in the laboratory of VMC was supported by the European Communitys Seventh Framework Programme contract (CardioGeNet 223463). Work
in the laboratory of AK was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence
REBIRTH (From Regenerative Biology to Reconstructive Therapy) and for the publication charges in the program Open Access Publishing at Medizinische
Hochschule Hannover. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The development of organs and organisms depends on the
precise control of the progression through and the exit from the
cell cycle to achieve appropriate patterns of proliferation and
differentiation in time and space. Progression through the cell
cycle is regulated predominantly by a series of serine/threonine
kinases, the cyclin-dependent kinases (CDKs) that link proliferative
signals with mechanical aspects of cell duplication. CDK function
is controlled by a variety of mechanisms, including a group of
molecules that inhibits CDK activity by complex formation. These
CDK inhibitors (CKIs) have been categorized into two families,
the Cip/Kip (Cdkn1) family with three members in mammals
(Cdkn1a, Cdkn1b, Cdkn1c (also known as p21, p27 and p57)), that
inhibit all kinases involved in G1/S transition, and the Ink4
(Cdkn2) family with four mammalian members (Cdkn2a, Cdkn2b,
Cdkn2c, Cdkn2d (also known as p16/p19ARF, p15, p18, p19))
that specifically inhibit Cdk4 and Cdk6. Biochemical and cell
culture experiments have identified CKIs as primary effectors of
signaling pathways that control cell cycle exit, an event critical for
differentiation. Expression or stability of CKIs is reduced in
tumors, and deletion of six of the seven family members leads to
organ hyperplasia and increased tumor susceptibility. In contrast
to the obvious relevance of CKIs in tissue homeostasis, their role in
development of tissues and organs, and the transcriptional
mechanisms that mediate their precise temporal and spatial
expression in the embryo have been much less well defined. This
may relate to functional redundancy between family members as
well as to the complexity of their regulatory modules (for reviews
on CKIs see ).
T-box (Tbx) genes encode an evolutionary conserved family of
transcription factors that regulate patterning and differentiation
processes during vertebrate development . Tbx2 and Tbx3 are
two closely related members of the Tbx2-subfamily that are
required in the development of numerous organs during
mammalian embryogenesis including the heart, the palate, the
limbs, and the liver . In these contexts, these two
transcriptional repressors mainly seem to regulate cell fate
decisions and differentiation. However, in vitro studies indicated a
role for Tbx2 and Tbx3 in the progression of the cell cycle [11
During organ formation, proliferation rates and
differentiation patterns vary widely between different stages and
tissue compartments. It is poorly understood how cell
cycle progression is locally controlled and integrated with
patterning processes in these developmental programs.
Here, we used the mouse lung as a model to study how
growth and differentiation are controlled on a
transcriptional level. Combining genetic loss- and gain-of-function
approaches, we show that the T-box transcription factor
gene Tbx2 is required and sufficient to direct appropriate
lung growth by maintaining proliferation and inhibiting
differentiation in the mesenchymal compartment of the
lung. We found that expression of the cell cycle inhibitor
genes Cdkn1a (p21) and Cdkn1b (p27) inversely correlates
with expression of Tbx2 and that deletion of both genes
rescues, to a large degree, the growth deficits of
Tbx2mutant lungs. We further show by biochemical assays that
Tbx2 directly binds to Cdkn1a and Cdkn1b loci in vivo,
defining these two genes as direct targets of Tbx2
repressive activity in the lung mesenchyme. We conclude
that Tbx2-mediated regulation of Cdkn1a and Cdkn1b
represents a crucial module for the tissue-specific control
of cell cycle progression that underlies growth,
differentiation, and branching morphogenesis of this organ.
13]. Expression of Tbx2 and Tbx3 is upregulated in a number of
tumors including those of the breast, pancreas, liver and bladder,
and in melanomas, and both genes can function as immortalizing
agents to bypass senescence, i.e. escape irreversible growth arrest
(for reviews see [14,15]). In cell culture assays, this phenomenon is
mediated by transcriptional repression of Cdkn1a and Cdkn2a
[12,13,16,17]. Although often speculated (e.g. ), the relevance
for this molecular function in a developmental context has
remained unclear. Intriguingly, mice analyzed in our lab that
were mutant for Tbx2, showed severely hypoplastic lungs, pointing
to a possible role of this T-box factor in the regulation of
proliferation and/or differentiation during development of this
The architecture of the mammalian lung arises from a complex
developmental program in which the tight orchestration of
proliferation and differentiation processes assures the formation
of an appropriately sized organ with a correct distribution of
differentiated cell types for air-conduction and gas-exchange. In
the mouse, the conducting airways develop from two primary buds
that emerge from the ventral wall of the foregut endoderm at
embryonic day (E) 9.5 by iterative rounds of stereotyped
outgrowth and branching until E16.5. The gas-exchange units,
the alveoli, only arise subsequently to this pseudoglandular stage
from the terminal buds until late in postnatal life. Normal
morphogenesis and patterning of the bronchial tree critically
depends on the underlying mesenchyme that is derived from the
splanchnic mesoderm. This mesenchyme is a source of signals that
mediate proliferation of epithelial precursors, and direct their
correct spatial differentiation. It also gives rise to a number of
different cell types, including parabronchial and vascular smooth
muscle cells, lipocytes, fibrocytes and endothelial cells. In turn,
epithelial signals from the endoderm but also from the
mesothelium maintain proliferation of mesenchymal precursors, closing a
reciprocal signaling loop that directs outgrowth of the distal
epithelial buds (for a recent review see ).
Previous work reported the expression of Tbx2 and Tbx3 in the
mesenchymal compartment of the developing lung, but a
functional significance has not been assigned to this expression
[19,20]. Here, we show by loss- and gain-of-function experiments
in the mouse that Tbx2 is required and sufficient to maintain
proliferation and inhibit differentiation in the mesenchymal
compartment of the developing lung. Expression, organ culture
and biochemical assays identify the cell cycle inhibitors encoded by
the Cdkn1a and Cdkn1b genes as direct targets of Tbx2 repressive
activity in this developmental program in vivo. Lung growth was
substantially rescued by genetically limiting Cdkn1a and Cdkn1b
expression in Tbx2-deficient mice, indicating that suppression of
these genes is a critical function of Tbx2 in the control of organ
growth during development.
Tbx2-deficient mice exhibit hypoplastic lungs
Mice homozygous for a null allele of Tbx2 (Tbx2cre) that is
maintained on an NMRI outbred background survive
embryogenesis but die shortly after birth due to a cleft palate [9,21].
Morphological and histological examination of mutant embryos at
E18.5 revealed hypoplastic lungs that frequently manifested with
alveolar haemorrhages. Air was present in the bronchial network
but the lung was poorly inflated. Lobulation was normal but all
four right lung lobes and the left lung lobe were reduced in size;
the tissue appeared thickened. The weight of the mutant lung was
reduced to approx. 50% of that of the littermate control whereas
the liver and the spleen were unaffected excluding a general
growth retardation problem (Figure 1A1C). At E16.5, the mutant
lung was visibly smaller and haemorrhagic. Its weight was reduced
to 33% of the wildtype level (Figure 1D1F). No obvious difference
in morphology, histology and weight of the lung between wildtype
and Tbx2-deficient embryos was observed at E14.5 (Figure 1G1I).
To evaluate whether the decreased size of Tbx2-deficient lungs
after E14.5 relates to a reduction in branching morphogenesis, we
explanted E11.5 lung rudiments and analyzed their
(2-dimensional) outgrowth after 6 days of culture. Whole-mount in situ
hybridization analysis for expression of the epithelial tip marker
gene Id2 showed an almost 3-fold reduction of branching
endpoints in the Tbx2-mutant lung explants suggesting that
epithelial branching morphogenesis is indeed severely hampered
by loss of Tbx2 (Figure 1J1L). However, reduction of branching
morphogenesis was restricted to the late phase of lung outgrowth
as revealed by non-significant changes of the number of branching
endpoints in Tbx2-deficient cultures at 2 and 4 days (Figure S1).
We conclude that Tbx2 is required to maintain normal branching
morphogenesis and growth of the developing lung after E14.5.
Decrease in proliferation of mesenchymal progenitor
Lung growth during the pseudoglandular stage is driven by
branching morphogenesis of the distal lung buds. This, in turn,
relies on rapid proliferation of the precursor cells in the bud
epithelium and its underlying mesenchyme. Reduced size of
Tbx2deficient lungs could therefore relate to increased apoptosis and/or
to decreased proliferation of distal epithelial and mesenchymal
tissue compartments as shown for other models of lung hypoplasia
. Terminal deoxynucleotidyl transferase-mediated nick-end
labeling (TUNEL) staining revealed that apoptosis was absent both
in wildtype and mutant lungs at E14.5 and E16.5 but was
increased in Tbx2-deficient lungs at E18.5 indicating a late
contribution to the hypoplasia of this organ (Figure 2A and 2A9,
2D and 2D9, 2G and 2G9).
Analysis of 5-bromo-29-deoxyuridine (BrdU) incorporation
showed that the epithelial and mesenchymal tissue compartments
of the lung were highly proliferative irrespective of the genotype at
Figure 1. Tbx2-deficient lungs become hypoplastic at the late pseudoglandular stage. (A,A9,D,D9,G,G9) Ventral views of whole isolated
lungs, (B,B9,E,E9,H,H9) histological analysis by haematoxylin and eosin staining of frontal sections of wildtype and Tbx22/2 embryos, and (C,F,I)
statistical analysis of relative lung per body weight; liver was analyzed as a control organ. Reduction of the lung weight to about half of the wildtype
value (100%) at E18.5, and a third at E16.5 was statistically highly significant (**) whereas liver weights and lung weights at E14.5 were without
significant change in Tbx2-deficient embryos. Stages and genotypes are as indicated. (JL) Analysis of branching morphogenesis of E12.0 lung
rudiments cultured for 6 days by in situ hybridization of the epithelial tip marker Id2 (JK9), and subsequent statistical analysis reveal a highly
significant reduction of branching end-points in Tbx2-deficient cultures (L). Scale bars represent 1 mm in A,A9,B,B9,D,D9,E,E9, 500 mm in G,G9,H,H9,J,J9
and 100 mm in K,K9. For statistics see Table S1A.
Figure 2. Decreased proliferation and increased apoptosis contribute to hypoplasia of Tbx2-deficient lungs. (A,A9,D,D9,G,G9) Analysis of
apoptosis by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL), and (B,B9,E,E9,H,H9) of proliferation by anti-BrdU
immunohistochemistry. (C,F,I) Statistical analysis of the BrdU labeling index for the lung mesenchyme, the proximal and distal epithelium, and the
diaphragm at different developmental stages. Genotypes and stages are as indicated. Scale bars represent 100 mm in A,A9,D,D9,G,G9, and 50 mm in
B,B9,E,E9,H,H9. For statistics see Table S1B.
E14.5 (Figure 2B2C). However, at E16.5 the BrdU labeling index
showed a highly significant reduction from 29.6+/21.5% in the
wildtype to 16.0+/23.4% in the mutant mesenchyme, and a
significant reduction from 19.7+/21.6% in the wildtype to 14.2+/
24.7% in the mutant distal lung epithelium (marked by expression
of SRY-box containing protein (Sox)9 [23,24]) while the proximal
lung epithelium (marked by expression of Sox2 [25,26]) or a
control tissue (the diaphragm) were unaffected (Figure 2E2F,
Figure S2). At E18.5, proliferation as indicated by the BrdU
labeling index was dramatically decreased in the whole lung to
levels similar in wildtype and mutant embryos (Figure 2H2I).
Thus, Tbx2 is required to maintain normal proliferation of the
mesenchyme and distal epithelium of the lung in a narrow
Premature differentiation of the Tbx2-deficient lung
As proliferation and differentiation are often inversely
correlated, we next investigated the occurrence of changes in the
differentiation patterns of both mesenchyme and epithelium in
Tbx2-deficient lungs at E14.5, E16.5 and E18.5 to cover the
period before, around and after the histological and cellular
defects were apparent in the mutant (Figure 3). At all analyzed
stages a normal distribution of networks of endomucin
(Emcn)positive endothelial cells  was present throughout the mutant
lung. In the mutant mesenchyme, transgelin (Tagln)-positive
smooth muscle cells were restricted to the proximal airways as in
the wildtype. It has recently been shown that expression of S100
calcium binding protein A4 (S100a4) marks fibroblasts that are
highly proliferative and express low levels of extracellular matrix
proteins indicating the precursor character of these cells .
We found that in E14.5 wildtype lungs all mesenchymal cells that
were positive for S100a4 also incorporated BrdU confirming the
proliferative character of this cell type (Figure S3). In the
Tbx2deficient lung, expression of S100a4 was completely abolished.
Fibronectin (Fn) and periostin (Postn), extracellular matrix
proteins that are secreted by mature fibrocytes at low levels in
proximal airways in the wildtype , were expressed
throughout the mesenchyme starting from E14.5 (Fn) and
E16.5 (Postn) in the mutant lung (Figure 3). This suggests that
Tbx2 is required to maintain the precursor state of a
subpopulation of future fibrocytes in the lung mesenchyme.
We next investigated whether these mesenchymal changes are
accompanied by alterations in proximal-distal patterning of the
respiratory tree and cell differentiation in the epithelium during
development in Tbx2-deficient lungs (Figure S4). In the wildtype
lung, Sox2 was expressed in the trachea and proximal airways
and was excluded from distal endoderm at all analyzed stages.
Sox9 was expressed in the distal tip endoderm and excluded
proximally at E14.5 and E16.5. At E18.5, Sox9 was
downregulated distally and reactivated in the mesenchyme of the proximal
airways possibly indicating onset of cartilage formation in this
Figure 3. Precocious differentiation in the Tbx2-deficient lung mesenchyme. Immunohistochemistry for the endothelial marker Emcn and
immunofluorescence analysis for smooth muscle cells (Tagln), immature fibroblasts (S100a4) and fibrocyte-secreted extracellular matrix proteins (Fn1,
Postn) on frontal sections of wildtype (wt) and Tbx2-deficient (Tbx22/2) lungs at E14.5, E16.5 and E18.5 as indicated. S100a4 is lost in Tbx2-deficient
lungs whereas deposition of Fn1 and Postn is greatly increased. Scale bars represent 100 mm.
region. Expression of keratin 14 (Krt14, also known as
cytokeratin 14) in basal cells in the trachea, of tubulin, beta 4A
class IVA (Tubb4a) in ciliated cells, and of secretoglobin, family
1A, member 1 (Scgb1a1, also known as CC10 and uteroglobin) in
secretory or Clara cells was activated at E16.5 and maintained at
E18.5. Krt14 was also found in myofibroblasts surrounding the
proximal airways at E16.5. Surfactant associated protein C
(Sftpc1, also known as SP-C) was expressed in alveolar epithelial
cells type II (AEC2) from E16.5. Expression of podoplanin (Pdpn)
and aquaporin 5 (Aqp5) was activated in AEC1 at E18.5. All of
these markers (described in ) were appropriately activated
and maintained in Tbx2-deficient lungs with the exception of
Pdpn and Aqp5 that showed reduced expression levels at E18.5.
We conclude that mesenchymal loss of Tbx2 does not affect
proximal-distal patterning of the lung epithelium. Reduced or
delayed differentiation of AEC1 from AEC2 may relate to the
loss of appropriate signaling from the prematurely differentiated
Coexpression of T-box genes in the developing lung
Phenotypic changes of Tbx2-deficient lungs were confined to
the late phase of branching morphogenesis suggesting a narrow
temporal window of expression and/or activity of this gene.
Alternatively, Tbx2 may act redundantly with other T-box
genes during early lung development. In fact, previous work
reported expression of Tbx2 as well as of Tbx3, Tbx4 and Tbx5
in the pulmonary mesenchyme [20,33]. To assess the
comparative temporal expression patterns of these genes
during lung development, we performed in situ hybridization
analysis on sagittal sections of the lung (Figure 4). We observed
coexpression of Tbx2 and Tbx3 in the mesenchymal
compartment from E10.5 to E14.5. Expression of Tbx3 declined
sharply after this stage, whereas Tbx2 was maintained at high
levels at subsequent embryonic stages. Coexpression of Tbx4
and Tbx5 was found between E10.5 to E16.5 in the lung
mesenchyme. Hence, late onset of phenotypic changes in
Tbx2deficient lungs may relate to functional redundancy with the
closely related Tbx3 gene during the initial phase of branching
morphogenesis. This notion is supported by the finding that
mice homozygous for a null allele of Tbx3 exhibit lungs
morphologically and histologically indistinguishable from the
wildtype at E14.5, shortly before these mice die ( and data
not shown). Since mice with more than two mutant alleles of
Tbx2 and Tbx3 die around E9.5 due to cardiac defects ,
analysis of the functional redundancy of the two genes in early
lung development was not possible with the mouse lines
available to us.
Figure 4. Expression analysis of T-box genes suggests a non-redundant role of Tbx2 in late lung development. Analysis of Tbx gene
expression during lung development by RNA in situ hybridization on serial sagittal sections of wildtype embryos. The closely related genes Tbx2 and
Tbx3 are co-expressed until E14.5. Thereafter, only Tbx2 expression is maintained in the lung mesenchyme. Developmental stages and probes are as
indicated in the figure. e, epithelium; m, mesenchyme. Scale bars represent 50 mm at E10.5 and E12.5, 100 mm at E14.5 through E18.5.
De-repression of cell cycle inhibitors in Tbx2-deficient
An antisense oligonucleotide approach with cultured lung
rudiments and more recently conditional gene targeting
demonstrated a requirement for mesenchymal Tbx4 and Tbx5 in the
regulation of pulmonary branching morphogenesis [19,33]. Tbx4
and Tbx5 genetically interact with Fgf10 during lung growth and
branching, and may direct transcriptional activation of Fgf10 that
encodes a potent growth factor in the lung but also in other
developmental contexts [33,34]. Given the molecular nature of
Tbx2 and Tbx3 as transcriptional repressors, Tbx2 and Tbx3 may
compete with Tbx4 and Tbx5 for binding to conserved
DNAbinding sites in the promoter of Fgf10, similar to the antagonistic
control of Nppa expression in the heart by Tbx5 and Tbx2/Tbx3
To test this hypothesis and determine the molecular changes
underlying the lung phenotype, we analyzed components as
well as targets of bone morphogenetic protein (Bmp)-,
fibroblast growth factor (Fgf), sonic hedgehog (Shh) and
wingless-related MMTV integration site (Wnt) pathways that
collectively confer outgrowth and branching morphogenesis of
the respiratory tree . To accurately identify expression
changes we used quantitative RT-PCR of whole lung extracts
at different developmental stages. We started our analysis with
lungs at E16.5, when morphological, histological and
proliferation defects were fully apparent (Figure 5A, grey bars). At
this stage, we observed a significant downregulation of
components of the Bmp pathway such as Bmp4 and Bmp
receptor (Bmpr)2 as well as the Bmp target gene homeobox, msh-like
(Msx)1 . Bmp2 and Bmpr1a expression, however, was not
significantly altered. Expression of Shh was markedly reduced
but not accompanied by decreased intracellular signaling as
revealed by almost normal expression of the target gene patched
(Ptch)1 . Wnt ligands Wnt2 and Wnt5a were strongly
reduced in their expression as was the target of the canonical
(Ctnnb1-dependent) sub-branch of Wnt signaling, Axin2 .
Unexpectedly, no changes in Fgf pathway components were
found. Fgf10 expression was at wildtype level as was the
receptor Fgfr2 and the known Fgf target ets variant gene 4 (Etv4,
also known as Pea3) . At E14.5, i.e. prior to the observed
phenotypic changes, components and targets of Shh-, Fgf- and
Bmp-activity were unchanged in their expression. The
canonical Wnt target gene Axin2 and the non-canonical ligand Wnt5a
were strongly and Wnt2 expression was slightly reduced in
mutant lungs (Figure 5A, black bars). In situ hybridization
analysis showed that downregulation of Wnt2, Wnt5a and Axin2
was confined to the mesenchymal compartment of E14.5
Tbx22/2 lungs (Figure 5B). These data suggest, that Tbx2 does
branching morphogenesis of the respiratory tree (A), of cell cycle
regulators that influence proliferation of the pulmonary tissue and of
Tbx3 to check for compensatory regulation (C) on mRNA harvested from
wildtype and Tbx2-deficient lungs at E14.5 and E16.5. (B,D) Analysis of
expression of Wnt2, Wnt5a, Axin2 by in situ hybridization (B), and of
Cdkn1a/Cdkn1a and Cdkn1/Cdkn1b by in situ hybridization and
immunofluorescence (D) on frontal sections of lungs at E14.5 in
wildtype (wt) and Tbx22/2 lungs. Scale bars in B and D represent
100 mm. For statistics see Table S1C.
not counteract the transcriptional activation of Fgf10
transcription and Fgf signaling by Tbx4/Tbx5 but targets
canonical Wnt signaling in the lung mesenchyme, what, in
turn, may secondarily affect Bmp signaling.
Next, we analyzed expression of cell cycle regulators potentially
involved in proliferation control of lung mesenchyme (Figure 5C,
grey bars). Among the tested cell cycle activators cyclin-dependent
kinase (Cdk)1 and cyclin D (Ccnd)1 showed significant reduction
whereas Ccnd2 and Ccnd3 expression was unchanged at E16.5. As
Ccnd1 has been described as target of canonical Wnt signaling ,
its reduced expression may relate to the observed downregulation
of this pathway. The cell cycle inhibitors Cdkn1a, Cdkn1c, Cdkn2a
and Cdkn2d were unchanged whereas Cdkn1b was upregulated
more than 7 times in the mutant at this stage. At E14.5, all cell
cycle regulators were unaffected except Cdkn1b and Cdkn1a that
were upregulated 4 and 3.5 times, respectively, in the
Tbx2deficient lung (Figure 5C, black bars). Expression of Tbx3 was
unaltered at both analyzed stages excluding a compensatory
upregulation of this gene in the Tbx2-mutant background
(Figure 5C). In situ hybridization and immunofluorescence analyses
confirmed strong upregulation of Cdkn1a/Cdkn1b mRNA and
Cdkn1a/Cdkn1b protein both in the mesenchymal and in the
distal epithelial compartment of E14.5 Tbx22/2 lungs (Figure 5D).
These results argue that reduced proliferation in the mesenchyme
and distal epithelium (that are probably secondary to altered
mesenchymal signals) of E16.5 Tbx2-deficient lungs may be caused
by de-repression of cell cycle inhibitors Cdkn1a and Cdkn1b.
Decreased (canonical) Wnt signaling in Tbx2-deficient lungs may
reflect an independent branch of Tbx2 activity, or may merely
present a secondary consequence of de-repression of cell cycle
Repression of Cdkn1a and Cdkn1b by Tbx2 is direct and
contributes to lung growth
To unravel the contribution of increased expression of Cdkn1a
and Cdkn1b to the growth deficit of Tbx2-deficient lungs, we
ablated the two genes in the mutant background. Compound
Tbx2;Cdkn1a and Tbx2;Cdkn1b mutants, respectively, exhibited
lungs that were morphologically indistinguishable from the
Tbx2single mutant organ. In contrast, triple Tbx2;Cdkn1a;Cdkn1b
mutants exhibited visibly larger lungs at E18.5 (Figure 6A). To
quantify the observed changes, we determined the relative lung
weight (lung weight to body weight ratios, normalized to that of
Tbx2+/2 control embryos) of the different compound mutants.
Statistical analysis did not detect significant weight changes
between Tbx22/2;Cdkn1a2/2 and Tbx22/2;Cdkn1b2/2 lungs,
whereas the increase in weight in Tbx22/2;Cdkn1a2/2;Cdkn1b2/2
lungs was highly significant (Figure 6B). Although Tbx22/2;
Cdkn1a2/2;Cdkn1b2/2 lungs reached 80% of the control weight,
the difference remained significant indicating an incomplete
rescue. This suggests that the combined de-repression of Cdkn1a
and Cdkn1b accounts predominantly but not completely for
hypoplasia of Tbx2-deficient lungs.
harboring the site AGGTGTGTG amplified with the primer pair #1, and
an intronic element in the Cdkn1b locus harboring an inverse TBE site
CACACCT amplified with the primer pair #2. For statistics see Table
Since the individual deletion of Cdkn1a and Cdkn1b in the
Tbx2mutant background did not lead to even a partial rescue of growth,
we tested for the presence of a compensatory mechanism by
analyzing expression of Cdkn1a and Cdkn1b, respectively, by
quantitative RT-PCR analysis on mRNA of E16.5 (compound)
mutant lungs (Figure 6C). Cdkn1a expression was increased 2-fold
in Cdkn1b2/2 lungs and 9-fold in Tbx22/;2Cdkn1b2/2 lungs
whereas Cdkn1b was increased 2-fold in Cdkn1a2/2 lungs and
3fold in Tbx22/2;Cdkn1a2/2 lungs at this stage. Thus, either gene
shows a compensatory upregulation upon loss of the other gene.
Binding sites for TBX2 within the Cdkn1a promoter have
recently been described in cell culture experiments  whereas
Cdkn1b has not been recognized as a direct target of Tbx2
repressive activity before. In silico analysis of the mouse Cdkn1a and
Cdkn1b genes identified a consensus DNA-binding site for T-box
proteins (T-box binding element (TBE): AGGTGTGA)  in the
Cdkn1a promoter and two putative TBEs in the Cdkn1b locus. The
first element (AGGTGTGTG) was detected 3 kbp upstream of the
start codon, the second element with the reverse complementary
sequence CACACCT was localized within an intron of that gene
(Figure S5). ChIP experiments with E15.5 lung tissue revealed in
vivo binding of Tbx2 to the known TBE in the Cdkn1a locus and to
the 59 located but not the intronic TBE in the Cdkn1b gene
(Figure 6D) compatible with the notion that Cdkn1a and Cdkn1b
represent direct targets of Tbx2 repressive activity in the lung
It has previously been shown that Cdkn1a expression is elevated
on inactivation of endogenous Tbx2 in the murine B16 melanoma
and the human MCF-7 breast cancer cell line . Using the
previously published conditions , we downregulated Tbx2 in
both cell lines using a Tbx2-specific siRNA approach.
Immunofluorescence analysis showed that in the non-silencing control
nuclear Tbx2 protein was present in all cells whereas Cdkn1b was
not detected. In contrast, in cells treated with the Tbx2-specific
siRNA Tbx2 expression was extinguished in almost all cells
examined whereas Cdkn1b expression was strongly upregulated in
the cytoplasm (in MCF-7 cells) and in the nucleus (in B16
melanoma cells) (Figure S6). This further supports that Cdkn1b
similar to Cdkn1a is a true target of Tbx2.
Maintenance of Tbx2 expression prevents terminal
differentiation of lung fibrocytes
To further evaluate the mechanistic role of Tbx2 in the lung
mesenchyme, we additionally employed an in vivo gain-of-function
approach. For this, we crossed the Tbx2cre line and an
HprtTBX2allele, that was generated by integration of a bicistronic
transgenecassette containing the human TBX2 ORF followed by IRES-GFP
in the ubiquitously expressed X-chromosomal Hypoxanthine guanine
phosphoribosyl transferase (Hprt) locus [10,42] to maintain TBX2
expression in its endogenous domains including the lung
mesenchyme. Male (Tbx2cre/+;HprtTBX2/y) embryos were not
recovered after E12.5 most likely due to cardiac defects. In
contrast, female (Tbx2cre/+;HprtTBX2/+) embryos, which exhibit a
mosaic expression due to random X-chromosome inactivation,
survived embryogenesis and puberty. Lungs of E18.5 Tbx2cre/+;
HprtTBX2/+ embryos were slightly bigger than those of control
littermates and showed a looser tissue organization (Figure 7A).
Apoptosis was not detected in either genotype, but Tbx2cre/+;
HprtTBX2/+ lungs exhibited a strong increase of proliferation in the
mesenchyme as shown by the BrdU assay (Figure 7B). Notably,
Western blot analysis of lungs of E18.5 Tbx2cre/+;HprtTBX2/+
embryos showed that transgenic TBX2 expression did not reach
unphysiologically high levels (Figure S7).
Branching morphogenesis is downregulated after E16.5
concomitant with the shut-down of signaling pathways involved in
epithelial-mesenchymal tissue interactions at the distal lung buds.
Therefore, increased proliferation in Tbx2cre/+;HprtTBX2/+ lungs
may relate to continued branching by maintained activity of these
signaling pathways. Morphological inspection did not detect
changes of branching between E12.0 wildtype and Tbx2cre/+;
HprtTBX2/+ lung rudiments cultured for 6 days (Figure S8).
Furthermore, RT-PCR analysis found unchanged expression of
targets of Shh (Ptch1), Fgf (Etv4), Bmp (Msx1) and canonical Wnt
(Axin2) pathways in Tbx2cre/+;HprtTBX2/+ lungs at E18.5 showing
that Tbx2 is not sufficient to induce these pathways, thus,
branching morphogenesis (Figure 7C). However, when testing cell
cycle regulators in this assay, we detected a selective
downregulation of Cdkn1a and Cdkn1b in Tbx2cre/+;HprtTBX2/+ lungs showing
that Tbx2 is not only required but also sufficient to repress
expression of Cdkn1a and Cdkn1b (Figure 7D).
To evaluate long-term consequences of prolonged TBX2
expression in the lung mesenchyme, we analyzed Tbx2cre/+;
HprtTBX2/+ mice at postnatal day (P) 40, a stage when they were
present in the expected numbers. Although Tbx2cre/+;HprtTBX2/+
mice were visibly smaller than their littermate controls at this
stage, the relative lung mass was increased by a factor of 1.27
(Figure 8A and 8B). Immunofluorescence for GFP and TBX2
expression on lung sections confirmed the widespread expression
of the transgene in the mesenchymal compartment of P40
Tbx2cre/+;HprtTBX2/+ mice. Histological analysis by haematoxylin
and eosin staining uncovered clusters of tissue thickenings, and
alveolar air spaces were surrounded by multiple cell layers in these
transgenic lungs. Histological staining for keratin and collagen
(Massons trichrome) did not detect changes in the transgenic lung,
excluding the possibility that tissue thickening is caused by
excessive deposition of extracellular matrix (Figure 8C). Analysis
of BrdU incorporation showed that the lung tissue was highly
proliferative in the transgenic animals at P40 (Tbx2cre/+;HprtTBX2/+:
31.0%65.2, control: 2.1%60.8) (Figure 8D). Apoptosis as
detected by TUNEL staining was similarly absent from control
and transgenic lungs (Figure 8E).
Analysis of cell differentiation by immunofluorescence of
marker proteins showed normal presence of lung epithelial cell
types, of endothelial cells, and of mesenchymal smooth muscle
cells around the proximal airways of Tbx2cre/+;HprtTBX2/+ lungs
(Figure S9). Fn1 and Postn deposition in the extracellular matrix
was augmented, and S100a4-positive cells were increased in
number. Expression of the cell cycle inhibitors Cdkn1a and
Cdkn1b was dramatically downregulated in the mesenchymal
compartment (Figure 8F). Together these findings indicate that
prolonged expression of TBX2 maintains mesenchymal
proliferation at a high level. While a part of these mesenchymal cells
differentiate into ECM-producing cell-types, a substantial fraction
retains a S100a4-positive precursor character.
To determine the contribution of reduced expression of Cdkn1a
and Cdkn1b to the observed histological, immunohistochemical
and molecular changes in Tbx2cre/+;HprtTBX2/+ mice, we
additionally analyzed Cdkn1a2/2;Cdkn1b2/2 mice at P40 using a similar
panel of assays. Double mutant lungs, normalized against the
increased body weight, were significantly larger than lungs of their
littermates (Figure S10A, S10B). Histological analysis did not find
changes in the tissue organization (Figure S10C) but Cdkn1a2/2;
Cdkn1b2/2 lungs exhibited a 5-fold increase of proliferation as
shown by the BrdU assay compared to the wildtype. Apoptosis was
unaffected (Figure S10D, S10E). Fn1 and Postn deposition in the
extracellular matrix was normal and immature fibroblasts
(S100a4) were absent as in the wildtype. Immunofluorescence
analysis of Cdkn1a and Cdkn1b confirmed that both proteins were
completely absent in the Cdkn1a2/2;Cdkn1b2/2 lung (Figure
S10F). Hence, Cdkn1a2/2;Cdkn1b2/2 mice do not feature the
histological and cellular changes seen in Tbx2cre/+;HprtTBX2/+ mice,
but exhibit increased lung mass due to increased proliferation. We
conclude that downregulation of Cdkn1a and Cdkn1b mediates
the pro-proliferative effects of Tbx2 overexpression to a large
degree but may not account for changes in tissue architecture and
Branching morphogenesis and growth of the lung requires the
coordination of cellular behaviors of its epithelial and
mesenchymal tissue compartments. Here, we have identified Tbx2 as a
crucial mesenchymal factor that maintains the mesenchymal
signaling center for epithelial branching morphogenesis. We
suggest that Tbx2 promotes mesenchymal proliferation and
inhibits terminal differentiation partly via direct transcriptional
repression of cell cycle inhibitor genes. Irrespective of its precise
mode, Tbx2 additionally maintains canonical Wnt signaling in the
mesenchyme, which, in turn, may account for maintenance of
epithelial growth and branching at the distal tips of the lung buds
Tbx2 directly represses cell cycle regulators in the lung
Cdkn1a, Cdkn1b together with Cdkn1c constitute the Cip/Kip
family of CKIs that inhibit cell cycle progression by binding to and
inhibition of a broad range of cyclin-CDK complexes via a shared
N-terminal cyclin-CDK binding domain. Cdkn1 activity correlates
with cell cycle exit and differentiation, and is, thus, under tight
control of anti-mitogenic signals. In tissue homeostasis, expression
and activity of CKIs is regulated by a large number of molecular
mechanisms including protein binding and posttranslational
modification that affect cyclin/CDK binding as well as stability
and degradation of CKIs (for a review see ). Gene targeting
experiments have unambiguously shown that all three members
are important players in tissue homeostasis and cancer (for a
review see ) whereas Cdkn1c is the only CKI to be uniquely
required for embryonic development [44,45]. However, additional
congenital defects have been described in mice lacking more than
one member of this gene family pointing to redundant functions in
some but not all developmental processes (see e.g. [46,47].
To exert a precise timing of cell cycle exit and differentiation in
development, expression of Cdkn1 genes must be tightly controlled
on the transcriptional level. In fact, Cdkn1a and Cdkn1c have
specific patterns of expression in development that correlate with
terminal differentiation of multiple cell lineages including skeletal
muscle, cartilage, skin, and nasal epithelium. In contrast, Cdkn1b
expression appears more widespread (for a review see . Cell
culture experiments identified Cdkn1a as a transcriptional target of
p53 [48,49] whereas the transcriptional regulation of Cdkn1c is
mediated by factors that play critical roles during embryogenesis
such as Notch/Hes1, MyoD and p73 . To our knowledge,
the in vivo relevance of these regulatory modules has remained
unclear. Interestingly, previous efforts were largely directed
towards the identification of transcriptional activators of Cdkn1
genes, and the possibility that these genes are subject to negative
regulation in vivo, i.e. that activation of expression in a certain cell
type results from attenuation or abolition of a prior transcriptional
repression, was neglected.
Here, we have shown that Cdkn1a and Cdkn1b are derepressed in
the pulmonary mesenchyme in Tbx2-deficient mice prior to other
molecular changes, that Cdkn1a and Cdkn1b are repressed upon
ectopic expression of TBX2 in mature lung mesenchyme, and that
deletion of Cdkn1a and Cdkn1b largely rescued the growth defects of
Tbx2-deficient lungs. Furthermore, we identified by ChIP analysis
Tbx2 binding to Cdkn1a and Cdkn1b loci in the developing lung.
Together, our genetic and biochemical analyses provide evidence
that Cdkn1a and Cdkn1b are subject to direct repression by Tbx2
and are crucial downstream mediators of this gene in the
mesenchymal compartment of the developing lung. In turn, it is
the first clear evidence, that Tbx2 directly regulates cell cycle
control genes in a developmental context in vivo. Intriguingly,
ChIP-seq analysis of genomic binding of Tbx3 in cardiomyocytes
in vivo, identified a large number of loci with binding peaks
containing a variant TBE . Tbx3 and Tbx2 are closely related
family members that recognize the same DNA binding site.
Reinspection of this data set identified binding peaks of Tbx3 in both
the Cdkn1a and Cdkn1b loci. In fact, the DNA-element used in our
ChIP analysis precisely mapped to a major Tbx3 peak in the
promoter of the Cdkn1a locus which contained additional less
conserved TBEs, whereas the DNA element used for our Cdkn1b
ChIP located closely to a minor peak (Figure S11). This together
with enhanced expression of Cdkn1a and Cdkn1b in melanoma
and breast cancer cell lines depleted of endogenous Tbx2
[12,13,17] (and this study), indicates that Tbx3 and the closely
related Tbx2 protein occupy DNA sites in the Cdkn1a and Cdkn1b
loci in other cell types and may regulate these genes in other
developmental contexts as well.
It should be noted that changes of Tbx2 did not only (inversely)
affect proliferation in the lung mesenchyme but directly correlated
with the precursor state of at least one mesenchymal
subpopulation, S100a4-positive fibroblasts. Although Cdkn1-mediated
cell cycle arrest has been associated with cellular differentiation in
different developmental contexts [44,54,55], we did not observe
differentiation defects in lungs double mutant for Cdkn1a and
Cdkn1b. This may indicate that in this developmental context
negative control of cell differentiation by Tbx2 is not mediated by
repression of Cdkn1 and Cdkn1b. Changes of Tbx2 expression did
not affect differentiation of other mesenchyme-derived cell types
including smooth muscle cells. This may indicate that Tbx2 does
not control differentiation of these cell types, or it may simply
reflect the fact that these cell types differentiate prior to E14.5
when Tbx3 expression is downregulated and Tbx2 is uniquely
required. In the future, it will be interesting to study the relation
between mesenchymal proliferation and differentiation in mice
deficient for both Tbx2 and Tbx3, which are likely to act
redundantly throughout the pseudoglandular stage until E14.5.
Tbx2 maintains canonical Wnt signaling in the lung
Combined deletion of Cdkn1a and Cdkn1b function in
Tbx2deficient embryos restored lung growth largely but not completely
suggesting that additional factors or pathways may act
downstream of Tbx2 to mediate mesenchymal proliferation. Our
RTPCR analysis of signaling pathways relevant for branching
morphogenesis did not detect changes of Fgf and Shh signaling
but uncovered reduced activity of canonical Wnt and Bmp
signaling. Notably, we detected decreased expression of Wnt
components and signaling as early as E14.5 in the pulmonary
mesenchyme, whereas Bmp4 expression and Bmp signaling was
unchanged at that stage suggesting a secondary mode of change of
the latter. As Bmp4 was shown to act as an autocrine signal for
distal endoderm proliferation , reduced expression may
contribute to the reduced proliferation in the distal endoderm at
E16.5 in Tbx2-deficient lungs.
A number of studies have implicated different Wnt genes in lung
development. Mice deficient for the non-canonical Wnt ligand
gene Wnt5a, which is expressed in the distal lung mesenchyme,
exhibit increased cell proliferation in both epithelium and
mesenchyme with a resulting expansion of the distal lung and
increased lung size . Wnt2 is a canonical Wnt ligand robustly
expressed in the mesenchyme of the developing lung. Wnt2, in
cooperation with Wnt2b, is essential for specification of the
respiratory lineage in the anterior foregut endoderm . Later,
Wnt2 acts upstream of Fgf10 and the critical transcription factor
myocardin to regulate early airway smooth muscle cell
differentiation in the multipotent lung mesenchyme . Finally, Wnt7b,
a canonical ligand expressed in the pulmonary epithelium
stimulates embryonic lung growth by increasing proliferation in
both tissue compartments of the developing lung without affecting
the differentiation patterns . Furthermore, tissue-specific
deletion of the unique signaling mediator of the canonical
pathway, Ctnnb1, in the epithelium led to defects in
proximaldistal differentiation of airway epithelium  whereas
mesenchymal deletion of Ctnnb1 resulted in hypoplasia due to reduced
epithelial and mesenchymal proliferation .
Maintained differentiation of airway smooth muscle cells but
decreased proliferation in the epithelial and mesenchymal
compartments during the late phase of branching morphogenesis
is compatible with the idea that loss of Tbx2 affects the canonical
Wnt pathway in the mesenchyme triggered by the epithelial
Wnt7b signal. The growth-promoting effect of this pathway may
at least partly be mediated by activation of the pro-proliferative
gene Ccnd1 that was previously recognized as a target of Wnt
signaling . This is compatible with the finding that
proliferation defects observed in Tbx2-mutant lungs at E16.5 coincide
with a strong decline of expression of this gene at this stage.
However, the significance of downregulation of Wnt2 and Wnt5a
in the Tbx2-deficient lung remains unclear. We assume that it
provides only a minor contribution to the observed changes.
At present, we cannot distinguish whether changes of Wnt
signaling activity are secondary to cell cycle exit and/or
upregulation of Cdkn1a and Cdkn1b or represent an independent
branch of Tbx2 transcriptional activity in the lung mesenchyme.
Unfortunately, the recovery of mice triple mutant for Tbx2, Cdkn1a
and Cdkn1b for analysis of signaling pathways at E14.5 is extremely
inefficient. The finding that constitutive expression of Tbx2 in the
lung mesenchyme of adult mice did not increase canonical Wnt
signaling, suggests that Tbx2 is not sufficient to activate this
pathway. However, Tbx2 may be required for repression of an
inhibitor of Wnt signaling to maintain this pathway during
branching morphogenesis. The relevance of the control of
canonical Wnt signaling by Tbx2 in the lung mesenchyme will
be addressed in future experiments.
Materials and Methods
Mice and genonotyping
Mice carrying a null allele of Cdkn1a (Cdkn1atm1Tyj, synonym
Cdkn1a2) , a null allele of Cdkn1b (Cdkn1btm1Mlf, synonym:
Cdkn1b2)  or a null allele of Tbx2 (Tbx2tm1.1(cre)Vmc, synonyms:
Tbx22, Tbx2cre) , and mice with integration of the human
TBX2 gene in the Hprt locus (Hprttm2(CAG-TBX2,-EGFP)Akis, synonym:
HprtTBX2)  were maintained on an outbred (NMRI)
background. For timed pregnancies, vaginal plugs were checked in the
morning after mating; noon was taken as embryonic day (E) 0.5.
Pregnant females were sacrificed by cervical dislocation; embryos
were harvested in phosphate-buffered saline, decapitated, fixed in
4% paraformaldehyde overnight, and stored in 100% methanol at
220uC before further use. Genomic DNA prepared from yolk sacs
or tail biopsies was used for genotyping by polymerase chain
reaction (PCR). For primers and conditions see Table S2.
Histological analysis and immunofluorescence
Embryos were embedded in paraffin and sectioned to 5 mm.
For histological analyses, sections were stained with
haematoxylin and eosin (HE), Massons trichrome (Massons) and
picrosirius red (Sirius red) following standard protocols. For
the detection of antigens, antigen retrieval was performed
using citrate-based antigen unmasking solution (H-3300,
Vector Laboratories Inc). Sections were pressure-cooked for
5 min and signal amplification was performed with the
Tyramide Signal Amplification (TSA) system (NEL702001KT,
Perkin Elmer LAS) or the DAB substrate kit (SK-4100, Vector
Laboratories Inc). The following primary antibodies were used:
rabbit anti-mouse E-cadherin (gift from Rolf Kemler, MPI for
Immunobiology and Epigenetics, Freiburg/Germany) ,
rabbit polyclonal antibody against GFP (1:200, sc-8334, Santa
Cruz), mouse monoclonal antibody against GFP (1:200,
11814460001, Roche), monoclonal antibody against alpha
smooth muscle actin, Cy3-conjugate (1:200, C 6198, Sigma),
monoclonal antibody against alpha smooth muscle actin,
FITC-conjugate (1:200, F3777, Sigma), rabbit polyclonal
against SM22a (transgelin, 1:200, ab14106, Abcam), rat
monoclonal antibody against endomucin (1:2, gift from
Dietmar Vestweber, MPI for Molecular Medicine, Mu nster/
Germany) , rabbit polyclonal antibodies against Tbx2
(1:100, ab33298, Abcam), Cdkn1a (1:200, sc-397, SantaCruz),
Cdkn1b (1:200, 554069, BD Biosciences), uteroglobin (1:200,
ab40873, Abcam), cytokeratin14 (1:200, ab7800, Abcam),
Tubb4a (1:100, ab11315, Abcam), prosurfactant protein C
(1:200, ab40879, Abcam), Sox2 (1:100, ab97959, Abcam),
Sox9 (1:200, ab5535, Millipore), aquaporin5 (1:100, ab92320,
Abcam), hamster monoclonal against podoplanin (1:50,
ab11936, Abcam) and mouse monoclonal against BrdU
(1:100, 1170376, Roche). For immunofluorescent stainings on
adult sections or double immunofluorescent stainings with two
primary mouse antibodies the Biotinylated Mouse on Mouse
(M.O.M.) Anti-Mouse Ig Reagent (Vector laboratories) was
For analysis of branching morphogenesis E11.5 or E12.0
lung rudiments were dissected and kept on Transwell
permeable 0.4-mm pore size, PET 6-well plates (Corning)
supplied with DMEM supplemented with 10% fetal calf serum
(Biowest), 2 mM Glutamax, 100 units/ml Penicillin, 100 mg/
ml Streptomycin (Gibco). Lungs were cultivated at 37uC and
5% CO2 for 2 to 6 days and the number of branching
endpoints was counted.
Cell culture and siRNA
Human MCF-7 breast adenocarcinoma cell line was cultured in
RPMI 1640 with Glutamax (Gibco) supplemented with 10% FBS,
MEM non-essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), 10 mg/ml human insulin (Roche) and 100 units/ml
Penicillin, 100 mg/ml Streptomycin (Gibco). Mouse B16
melanoma cells were cultured in RPMI 1640 with glutamax,
supplemented with 10% FBS and 100 units/ml Penicillin, 100 mg/ml
Downregulation of TBX2 or Tbx2 was achieved by siRNA
exactly as recently described .
In situ hybridization analysis
Whole-mount in situ hybridization was performed following
a standard procedure with digoxigenin-labeled antisense
riboprobes . Stained specimens were transferred in 80%
glycerol prior to documentation. In situ hybridization on 10 mm
paraffin sections was done essentially as described . For
each marker at least three independent specimens were
Proliferation and apoptosis assays
Cell proliferation in embryonic and adult lungs was investigated
by detection of incorporated 5-bromo-29-deoxyuridine (BrdU)
similar to published protocols . At least nine sections from
three individual embryos per genotype and stage were used for
quantification. Statistical analysis was performed using the
twotailed Students t-test. Data were expressed as mean 6 standard
deviation. Differences were considered significant when the
Pvalue was below 0.05.
For detection of apoptotic cells in 5 mm paraffin sections of
embryos, the terminal deoxynucleotidyl transferase-mediated
nickend labeling (TUNEL) assay was performed as recommended by
the manufacturer (Serologicals Corp.) of the ApopTag kit used.
Semi-quantitative reverse transcription PCR
Total RNA was extracted from dissected lungs with RNAPure
reagent (Peqlab). RNA (500 ng) was reverse transcribed with
RevertAid H Minus reverse transcriptase (Fermentas). For
semiquantitative PCR, the number of cycles was adjusted to the
mid-logarithmic phase. Quantification was performed with
Quantity One software (Bio-Rad). Assays were performed at least
twice in duplicate, and statistical analysis was done as previously
described . For primers and PCR conditions see Table S3.
Chromatin immunoprecipitation (ChIP) assays
2ChIP was performed essentially as previously described .
Dissected E15.5 lung tissue was treated with 4% paraformaldehyde
overnight. The DNA-containing supernatants were incubated
overnight with anti-Tbx2 antibodies and collected on protein G
beads. Cross-linked products were reversed by cooking for 15 min,
treated with Proteinase K and RNAse H at 56uC for 30 min and the
immunoprecipitated DNA was purified. Primers for PCR
amplification were 59-CCGAGAGGTGTGAGCCGC-39 (Cdkn1a-f1)
and 59- GTCATCCACCTGCCGCGG-39 (Cdkn1a-r1); 59-GGC
TTAGATTCCCAGAGGG-39 (Cdkn1af2) and 59-TTCTGGG
GACACCCACTGG-39 (Cdkn1a-r2) for the Cdkn1a promoter and
59- CAAGTTCAGTAAACTAAGTAGG-39 (Cdkn1b-f1) and
59GCACATATGTGGACAAACTCG-39 (Cdkn1b-r1) for the
59-Tsite in the Cdkn1b promoter. For the intron located T-site
59ATATACCTTCTACAGACATAGC-39 (Cdkn1b-f2) and
59GCTTTTGACTAGAGTCTTATGG-39 (Cdkn1b-r2) primers
were used. Primers for the negative control region were
59CTCTGAAACTCGAACAGGCC-39 (ncr-f1) and 59-
Sections were photographed using a Leica DM5000 microscope
with a Leica DFC300FX digital camera. Whole mount specimens
were photographed on a Leica M420 microscope with a Fujix
digital camera HC-300Z. Images were processed in Adobe
Figure S1 Tbx2 is required for lung epithelial branching. (A)
Morphology of lung explants from E12.0 wildtype and Tbx22/
2 embryos at the start and after 6 days of culture. Boxes show
regions that were magnified to see branching endpoints in the
lower panel. (B) Quantitative and statistical analysis of
branching morphogenesis of E12.0 lung rudiments cultured
for 0, 2, 4 and 6 days by counting of peripheral branching
endpoints. Branching endpoints were not significantly (ns)
reduced after four days of culture (p = 0.08). After 6 days of
culture branching endpoints were highly significantly reduced
from 116+/29 in wildtype to 70+/22 in Tbx2-deficient
cultures (p = 161024). Scale bars represent 500 mm. For
statistics see Table S1G.
Figure S2 Determination of proliferation in the epithelium of
Tbx2-deficient lungs. (Co-) immunofluorescence analysis of BrdU
and the distal epithelial marker Sox2 (A,C) and the proximal
marker Sox9 (B,D) in sections of E14.5 and E16.5 wildtype and
Tbx2-deficient (Tbx22/2) lungs. Reduced proliferation was found
in the distal but not in the proximal region of the lung epithelium.
Scale bars represent 50 mm.
Figure S3 S100a4-positive fibroblasts are highly proliferative.
BrdU incorporation assay analyzed by immunofluorescence on
E14.5 lung sections co-stained for S100a4 shows that all
S100a4positive fibroblasts proliferate. Arrowheads mark S100a4-positive
cells. Scale bars represent 50 mm.
Figure S4 Minor changes of epithelial differentiation in
Tbx2deficient lungs. Immunohistochemistry on frontal section of
wildtype (wt) and Tbx2-deficient (Tbx22/2) lungs for
regionalization of proximal (Sox2) and distal airways (Sox9), for
differentiation of proximal airway epithelium into tracheal basal cells
(Krt14), ciliated cells (Tubb4a) and Clara cells (Scgb1a1), and for
differentiation of alveolar epithelial cells type 1 (Pdpn, Aqp5) and
type 2 (Sftpc1). Genotypes, probes and stages are as indicated. All
markers are appropriately activated in the Tbx2-deficient lung
epithelium. Pdpn and Aqp5, however, are not maintained at the
appropriate levels. Scale bars represent 100 mm.
Figure S5 Cdkn1a and Cdkn1b loci harbor binding sites for
Tbox proteins. Schemes depicting the genomic organization of the
Cdkn1a and the Cdkn1b locus. Exon coding sequences are
indicated as black boxes, white boxes refer to untranslated
sequences within exons. Arrows mark positions of primers used to
amplify DNA fragments for ChIP analysis. Nucleotide sequences
refer to conserved binding sites for T-box proteins. Black
numbers refer to exons, red numbers indicate the size of the
genomic fragments in bp.
Figure S6 Endogenous Tbx2/TBX2 represses endogenous
Cdkn1b in B16 melanoma and MCF-7 breast cancer cell lines.
Immunofluorescent stainings of TBX2/Tbx2 and CDKN1B/
Cdkn1b protein in human MCF-7 (human) and mouse B16 cell
lines transfected with siRNA specific for TBX2/Tbx2 or a
nonsilencing control siRNA. Knock-down of TBX2/Tbx2 results in
upregulation of CDKN1B/Cdkn1b expression in both cell lines
three days after treatment. Scale bars represent 50 mm.
Figure S7 Quantification of Tbx2/TBX2 protein in
TBX2overexpressing lungs by Western blot analysis. 4 lungs each of E18.5
control and Tbx2cre/+;HprtTBX2/+ embryos were pooled and lysed in
1 ml of Nonidet-P40 buffer. After sonification, 2 ml of the lysate
(1:500) and 0.2 ml (1:5000), respectively, were loaded on the gel and
analyzed with an anti-Tbx2 antibody after blotting. Levels of Tbx2/
TBX2 protein were comparable between samples indicating that
TBX2 misexpression occurs within physiological range.
Figure S8 Branching morphogenesis is not affected in explant
cultures of Tbx2cre/+;HprtTBX2/+ lungs. (A) Morphology of lung explants
from E12.0 wildtype and Tbx2cre/+;HprtTBX2/+ embryos at the start and
after 6 days of culture. Boxes show regions that were magnified to see
branching endpoints in the lower panel. (B) Quantitative and statistical
analysis of branching morphogenesis of E12.0 lung rudiments cultured
for 0, 2, 4 and 6 days by counting of peripheral branching endpoints
does not detect differences in branching morphogenesis between
wildtype and Tbx2cre/+;HprtTBX2/+embryos. Scale bars represent
500 mm. For statistics see Table S1H.
Figure S9 Normal epithelial differentiation in P40 Tbx2cre/+;
HprtTBX2/+ mice. Immunofluorescence (Tagln) and
immunohistochemistry (Emcn, Sox2, Sox9, Tubb4a, Scgb1a1, Aqp5,
Pdpn, Sftpc1) on frontal section of wildtype (wt) and
TBX2overexpressing (Tbx2cre/+;HprtTBX2/+) lungs for endothelial cells
(Emcn), for smooth muscle cells surrounding the proximal
airways (Tagln), for regionalization of proximal airways (Sox2)
and distal airways (Sox9), for differentiation of proximal
airway epithelium into ciliated cells (Tubb4a) and Clara cells
(Scgb1a1), and for differentiation of alveolar epithelial cells
type 1 (Aqp5, Pdpn) and type 2 (Sftpc1). Genotypes, probes
and stages are as indicated. All markers are appropriately
expressed in Tbx2cre/+;HprtTBX2/+ mice. Scale bars represent
Figure S10 Loss of Cdkn1a and Cdkn1b increases lung
growth. Analysis of wildtype, Cdkn1a2/2;Cdkn1b2/2 and
Cdkn1a2/2;Cdkn1b+/2 mice at P40. (A) Morphology of P40 mice
and lungs. (B) Statistical analysis of relative body and lung weight.
Relative body weight is increased by 20% both in Cdkn1a2/2;
Cdkn1b2/2 and Cdkn1a2/2;Cdkn1b+/2 mice; the lung weight
(relative to the body weight) is increased in Cdkn1a2/2;
Cdkn1b2/2 1.35-fold (n = 2). (C) Histological analysis by
haematoxylin and eosin (HE), Sirius red and Massons staining on
sections of P40 lungs. (D) BrdU incorporation assay of frontal
sections of the lung. Cdkn1a/Cdkn1b double-mutant mice show
increased BrdU incorporation. (E) Analysis of apoptosis by
TUNEL staining. (F) Immunofluorescence analysis of Fn1, Postn,
S100a4, Cdkn1a and Cdkn1b expression on lung sections at P40.
Genotypes are as indicated. Scale bars in A represent 1 cm and
2.5 mm, respectively. Scale bars in C,D,E,F represent 50 mm. For
statistics see Table S1I.
Figure S11 ChIP-Seq analysis of Tbx3-binding to Cdkn1a and
Cdkn1b loci in atrial cardiomyocytes as identified in .
Graphical representation of binding peaks (in red) of the
transcriptional repressor Tbx3 to the genomic region of Cdkn1a
(A) and Cdkn1b (B). Boxes show genomic regions that are
subsequently represented in a magnified fashion. Scale bars are
indicated. Arrows refer to primers used to amplify genomic
fragments that harbor TBEs (see Figure 6).
We thank R. Kemler and D. Vestweber for antibodies, K.
ReimersFadhlaoui and C. Mayer for cell lines, U. Kossatz-Bohlert and M. Mai for
antibodies and discussions, and J. Norden and M.-O. Trowe for critical
reading of the manuscript.
Performed the experiments: TH-WL CR MP. Conceived and designed the
experiments: TH-WL HFF VMC AK. Contributed reagents/materials/
analysis tools: HFF KS-G PB VMC. Analyzed the data: TH-WL CR AK.
Wrote the paper: TH-WL VMC AK.
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