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Lung mesenchyme cell biology

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Lung mesenchyme cell biology

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LUNG MESENCHYME CELL BIOLOGY

by
Wenming Zhang

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CRANIOFACIAL BIOLOGY)

August 2014
Copyright 2014 Wenming Zhang
2

ACKNOWLEDGEMENTS
Over the past four and half years I have received tremendous support and kind
encouragement from a great number of outstanding individuals, without whom I
would not have been able to complete my years of study.
I would like to express my deep appreciation and gratitude to my mentor and
advisor, Dr. Wei Shi, for his fundamental role in my doctoral work. For years,
he has been offering me every bit of his patient guidance, assistance, expertise,
and encouragement.
Thanks to the research environment sustained by Dr. Shi, I have crossed paths
with many excellent postdocs and graduate students who have influenced and
enhanced my research. Hui Chen, Yongfeng Luo, Wei Xu, Ying Huang, Yang
Tang, Gianluca Turcatel, my colleagues and friends. They not only provided me
with valuable academic advices, helped me with trouble shooting when
problems arise towards experiments, strengthened my lab work skills, but also
kept my company and helped me solved issues that came up in my life. They
feel like family to me while I am away from my own for years. The memory we
shared together shall be missed.
Dr. Esteban Fernandez makes the empowering training on Confocal imaging
available and even open up several supportive workshops towards imaging
3

software, without which I would not be able to acquire the images of my
projects or put all my figures together.
I feel fortunate to have Dr. Songtao Shi on my committee who has been
generously sharing his knowledge and resources on mesenchymal stem cell
studies. My brief participation in his lab was beneficial and pleasant. His lab
staff, Chider Chen, as my peer and my friend, spared huge amount of time
helping me with flow cytometry analysis conducted at USC CCMB center.
Things could have been much more difficult without him around and I owe him
a huge thanks.
As the graduate program director, Dr. Michael Paine deserves thanks for many
things. Upon my admission to USC, I was an international student who had
never been to the United States. He took time to sit with me and acquaint me
with the program and the school. Along the years he always provided practical
advice and constructive instructions in the time of needs.
I am honored to have Dr. David Warburton, Dr. Tracy Grikscheit, Dr. Michael
Paine, and Dr. Songtao Shi as members of my graduation committee. They all
have very demanding schedules and yet would consider taking the
responsibilities and attending to the research work of a graduate student. Thank
you all very much.
Parents and family members should not be forgotten when I am listing names
here. My parents and grandparents nurtured my interests in biological science
4

since my childhood, enlightened me to pursue the career that I pursue, and they
are always there welcoming when I seek spiritual comfort. All the time, my
uncle and aunt offered thoughtful suggestions that helped to shape my
perspectives of my work and life. I am blessed to have all these caring and
knowledgeable people around me within my family.
I would love to express my appreciation towards Shui Yu and Yunsi Shang, my
inspiring friends, whose contagious enthusiasm and consistent dedication
towards the goals of their life has affected me profoundly. I would hate to miss
the opportunity to mention this in the permanent record.
5

TABLE OF CONTENTS

CHAPTER 1 Development and characterization of an in vivo approach to specifically
target lung mesenchymal cells ...................................................................................................... 6
LIST OF ANTIBODIES............................................................................................................... 7
LIST OF FIGURES ..................................................................................................................... 8
LIST OF ABBREVIATIONS ................................................................................................... 10
ABSTRACT ............................................................................................................................... 11
INTRODUCTION ...................................................................................................................... 12
METHOD ................................................................................................................................... 16
RESULT .................................................................................................................................... 20
DISCUSSION ............................................................................................................................. 64
CONCLUSION .......................................................................................................................... 69

CHAPTER 2 Lung Mesenchymal Stem Cell ............................................................................. 70
LIST OF FIGURES ................................................................................................................... 71
ABSTRACT ............................................................................................................................... 72
INTRODUCTION ...................................................................................................................... 74
METHOD ................................................................................................................................... 76
RESULT .................................................................................................................................... 82
DISCUSSION ............................................................................................................................. 99

REFERENCES .......................................................................................................................... 102

6

CHAPTER 1

DEVELOPMENT AND CHARACTERIZATION OF AN IN
VIVO APPROACH TO SPECIFICALLY TARGET LUNG
MESENCHYMAL CELLS

7

LIST OF ANTIBODIES
GFP: Rabbit anti-GFP, Santa Cruz Biotechnology, Santa Cruz, CA;
Mouse anti-EGFP, Clonetech, Mountain View, CA;
Goat anti-GFP, Abcam, Cambridge, MA;
SMA: Anti- -smooth muscle actin 1:2000, Sigma, Saint Louis, MO;
LacZ: Rabbit β-Galactosidase, 1:1000, MP Biomedicals, Solon, OH;
Mouse Anti-β-Galactosidase, 1:200, Promega, Madison, WI;
Cytokeratin: Mouse Anti-Cytokeratin, Sigma, Saint Louis, MO;
PECAM1: Rabbit anti-platelet endothelial cell adhesion molecule-1, LSBio, Seattle, WA;
NG2: Rabbit anti-NG2, chondroitin sulfate proteoglycan, Millipore, Billerica, MA;
Hamster anti- , DSHB at the University of Iowa;
ADRP: Mouse anti-adipophilin, BioGenex, Fremont, CA;
CGRP: Mouse anti-calcitonin gene-related peptide, Sigma, Saint Louis, MO.

8

LIST OF FIGURES
Figure 1 Analysis of the 5.5kb genomic DNA sequences of the potential mouse Tbx4
lung enhancer
Figure 2 Generation of an embryonic/fetal lung-specific Tbx4-rtTA transgenic mouse line
Figure 3 Expression of rtTA-IRES-LacZ transgene in E13.5 lung was verified by X-gal
staining
Figure 4 Schematic diagram for generating Tbx4-rtTA/TetO-Cre/mT-mG reporter mice
and The Dox induction strategy
Figure 5 Cre-mediated mGFP expression in E13.5 mouse embryos
Figure 6 Cre-mediated mGFP expression in E13.5 mouse lung
Figure 7 Sagittal frozen section of E10.5 embryo with Dox induction from E6.5 to E10.5
Figure 8 Fluorescence microscopic examination of tissue frozen sections of E18.5 triple
transgenic mice
Figure 9 X-gal staining of the E13.5 triple transgenic mouse lung frozen section
Figure 10 Dynamic expression profile of Tbx4-rtTA-mediated Tet-On targeting system
Figure 11 Efficiencies of cell targeting by the Tbx4-rtTA mediated Tet-On inducible
system at different prenatal ages
Figure 12 Efficiencies of cell targeting by the Tbx4-rtTA mediated Tet-On inducible
system at different postnatal stages
Figure 13 The E18.5 lung of the triple transgenic reporter (Tbx4-rtTA/TetO-Cre/mT-mG)
mouse and Tbx4-rtTA/mT-mG mouse with Dox induction from E6.5 – E10.5
Figure 14 Cre-mediated mGFP expression (green) was detected in the mesenchyme of
the triple transgenic mouse (Tbx4-rtTA/TetO-Cre/mT-mG) lung at E14.5, but not in the
other organs
9

Figure 15 Tbx4 lung enhancer-mediated Tet-On induction activity was only detected in
cytokeratin-negative cells
Figure 16 Not all GFP-positive cells were lacZ-positive
Figure 17 Differential targeting of lung smooth muscle cells by the Tbx4 lung enhancer at
different gestational ages.
Figure 18 Differential targeting of lung vascular endothelial cells by the Tbx4 lung
enhancer at different gestational ages
Figure 19 Lipofibroblasts were marked by the Tbx4 lung enhancer driven Tet-On system
Figure 20 Myofibroblasts were marked by the Tbx4 lung enhancer driven Tet-On system
Figure 21 NG2-positive cells were targeted by the Tbx4-lung enhancer driven Tet-On
system
Figure 22 Lung neuroendocrine cells were not targeted by the Tbx4 lung enhancer
Figure 23 Cells with Tbx4 lung enhancer activity in adult lungs
Figure 24 Evaluation of adult transgenic reporter mouse lungs in the absence of Dox
induction
Figure 25 Cre-mediated mGFP expression (green) was detected in the lung of the triple
transgenic mice (Tbx4-rtTA/TetO-Cre/mT-mG) in both line 8 and line 6
Figure 26 In line 8 Cre-mediated mGFP expression was detected in the mesenchyme of
the triple transgenic mouse (Tbx4-rtTA/TetO-Cre/mT-mG) lung of E13.5
Figure 27 In line 8 mGFP expression was detected in the mesenchyme of the triple
transgenic mouse (Tbx4-rtTA/TetO-Cre/mT-mG) lung of E18.5
Figure 28 In line 8, no mGFP expression was detected in the 8-week lung of the triple
transgenic mouse
10

LIST OF ABBREVIATIONS

GFP: Green Fluorescent Protein;
RFP: Red Fluorescent Protein;
Dox: Doxycycline;
rtTA: reverse tetracycline transactivator;
mT-mG: loxP-membrane Tomato-STOP-loxP-membrane GFP;
PBS: Phosphate Buffered Saline;
PBST: Phosphate Buffered Saline with Tween-20;
E: embryonic day;
P: postnatal day;
Dox: doxycycline;
-smooth muscle actin;
PECAM-1: platelet endothelial cell adhesion molecule;
ADRP: Adipose differentiation-related protein or adipophilin;
CGRP: calcitonin gene-related peptide

11

ABSTRACT

Background
Reciprocal interactions between lung mesenchymal and epithelial cells play essential
roles in lung organogenesis and homeostasis. Altered lung mesenchymal cell number and
function are related to many chronic lung diseases, such as pulmonary fibrosis and
emphysema. Although the molecular markers and related animal models that target lung
epithelial cells are relatively well studied, molecular markers of lung mesenchymal cells
and the genetic tools to target and/or manipulate gene expression in a lung mesenchyme-
specific manner are not available. The ability to manipulate gene expression in specific
cell type in the lung mesenchyme is important for understanding the molecular
mechanisms of the lung mesenchymal biology and the related pulmonary diseases. Lack
of knowledge of gene regulatory elements that contribute to lung mesenchymal cell
specificity is a major barrier for developing such genetic tools.

Result
We have characterized a mouse Tbx4 gene enhancer that contains conserved DNA
sequences across many vertebrate species with lung or lung-like gas exchange organ.
Two transgenic mouse lines were then generated to express rtTA/LacZ under the control
of this Tbx4 lung enhancer. By combining the Tbx4-rtTA driven Tet-On inducible Cre
recombinase expression mouse lines with a mT-mG dual fluorescence reporter mouse
line, the spatial-temporal patterns of Tbx4 lung enhancer-targeted lung mesenchymal
cells were defined. The Tbx4 lung enhancer was active only in lung mesenchymal cells,
but not in other tissues, from prenatal developmental stage to adult. Pulmonary
12

endothelial cells and vascular smooth muscle cells were only targeted by the Tbx4-rtTA
driver line prior to E11.5 and E15.5, respectively, while other subtypes of lung
mesenchymal cells including airway smooth muscle cells, fibroblasts, pericytes could be
targeted during the entire developmental stage.

Conclusion
Activation of the Tbx4 lung enhancer is only detected in mesenchymal cells of
developing lung. With our newly created Tbx4 lung enhancer-driven Tet-On inducible
system, lung mesenchymal cells can be specifically and differentially targeted in vivo at
the first time by controlling the doxycycline induction time window. This novel system
provides a unique tool to study lung mesenchymal cell lineages and gene functions in
lung mesenchymal development, injury repair, and regeneration in mice.

13

INTRODUCTION

Lung is originally developed from ventral foregut endoderm and surrounding splanchnic
mesoderm (Cardoso and Whitsett, 2008; Warburton et al., 2010a). Mammalian lung is
comprised of epithelial, endothelial and interstitial cell populations. The honeycomb-like
structure of functional mammalian lung enables efficient ventilation and gas exchange
between air and the circulation blood. The complex development process of mammalian
lung includes lung airway branching morphogenesis and alveolarization, together with
angiogenesis and vasculogenesis. (Shi et al., 2007).
Reciprocal interactions between lung mesenchymal and epithelial cells play essential
roles in lung organogenesis and homeostasis. In fetal mice, lung epithelial cells are
initially specified by Nkx2.1 expression around embryonic day (E) 9.5, followed by lung
bud growth, airway branching morphogenesis, and terminal saccular formation (Morrisey
and Hogan, 2010). During this developmental process, a wide variety of lung-specific
epithelial cells are differentiated from their epithelial progenitor cells. The molecular
markers and related animal models to target these epithelial cells are relatively well
studied. However, developmental lung mesenchymal progenitor cells and their
differentiation are poorly understood. As defects in lung mesenchymal compartment will
have significant impacts on both fetal lung formation and postnatal lung injury repair,
giving rise to lung diseases, many unsolved issues of lung mesenchymal biology remain
critical questions in the field of lung research, such as whether mesenchymal cells in
developing lung are different from those in other organs and whether lung smooth muscle
cells in airways and vasculature are derived from the same lung mesenchymal progenitors.
Furthermore, no animal model is available to specifically target lung mesenchymal cells
14

in order to manipulate gene expression in these cells (Rawlins and Perl, 2012). Therefore,
novel molecular approaches and genetic tools to specifically target lung mesenchyme
from the beginning of lung formation are urgently needed.
T-box gene family, a group of transcription factors, are involved in early embryonic cell
fate decisions, regulation of the development of extraembryonic structures, embryonic
patterning, and many aspects of organogenesis. Many T-box genes, such as
eomesodermin (TBR2), spadetail, and tbxo play important roles in mesoderm formation
and patterning during vertebrate gastrulation (Naiche et al., 2005). Tbx4 is known to play
important roles during embryonic development through modulating gene expression.
(Naiche et al., 2005). Endogenous Tbx4 gene expression is detected in many mesoderm-
derived tissues including lung mesenchyme (Chapman et al., 1996), but is not specific for
lung (Naiche et al., 2011). However, Menke et al recently reported that Tbx4 expression
in different tissues is controlled by a dispersed group of enhancers at different loci within
the Tbx4 genomic structure. One of these is located in the third intron and is conserved
among several mammalian species (Menke et al., 2008). A 5.5kb DNA segment from this
region is able to drive transgenic reporter expression in the developing lung and trachea
at E12.5. However, detailed characteristics of this lung enhancer, including the spatial-
temporal pattern of the enhancer activity at different developmental or post-
developmental stages, are not known. By taking advantage of this potential lung-specific
enhancer of the mouse Tbx4 gene expression, we have generated new Tbx4 lung enhancer
driven-reverse tetracycline transactivator (Tbx4-rtTA) transgenic mouse line. By
combining Cre/loxP recombination system, we then developed the lung mesenchyme-
specific Tet-On inducible transgenic mouse models by crossing Tbx4-rtTA mice with
15

TetO-Cre mice. With the loxP-mTomato-STOP-loxP-mGFP (mT-mG) dual-fluorescence
reporter mice from Jackson Labs, we were thus able to identify and define the spatial-
temporal pattern of Tbx4-rtTA-targeted lung mesenchymal progenitors, and the derived
cells during different stages of lung development and adulthood. Hence, our new lung
mesenchymal-specific Tet-On inducible genetic system serves as a valuable tool for the
study of lung mesenchymal cells under both physiological and pathophysiological
conditions.

16

METHOD
DNA vector construction
rtTA2
s
-M2 DNA fragment was amplified from pTet-On Advanced vector (Clontech)
using primers 5’-CGGCCCCGAATTCACCATGTCTAGA-3’ and 5’-
ACGCGTCGACACTTAGTTACCCGGGGAGCATG-3’, and subcloned to EcoR1/SalI
digested pBluescript KS II to generate pBS-rtTA. A 5.5kb DNA fragment of mouse Tbx4
lung enhancer and a 0.9kb DNA fragment of HSP68 minimal promoter were obtained by
NotI and SmaI/SfoI digestion of pDBM3 (Menke et al., 2008), respectively, and
subcloned into pBS-rtTA to generate pBS-Tbx4-rtTA. Finally, IRES-T-LacZ DNA
fragment was obtained by digesting pNTR-lacZ-PGKNeolox plasmid provided by Dr.
Vesa Kaartinen at University of Michigan, and inserted to pBS-Tbx4-rtTA to produce an
intact transgenic vector pBS-Tbx4-rtTA-LacZ. The prokaryotic part of the vector was
then removed by NotI digestion and the 12kb transgenic DNA fragment was isolated for
C57BL/6 pronuclear injection.

Mouse strains, breeding, and genotyping
Tbx4-rtTA mouse lines were generated at the UCLA Transgenic Core facility. The
founders of transgenic mouse lines (6 and 8) were identified by genomic DNA PCR
using primers 5’-GGA AGG CGA GTC ATG GCA AGA-3’ and 5’-AGG TCA AAG
TCG TCA AGG GCA T-3’. The mice of transgenic line 6 were further verified by DNA
Southern blot for genomic DNA digested with XhoI and SalI. DIG-labeled
nonradioactive detecting system (Roche, Indianapolis, IN) was used. Both line 6 and line
17

8 of the Tbx4 transgenic mice were studied while line 6 was the major subject of the
study.
TetO-Cre mouse line was originally provided by Dr. Jeffrey Whitsett at Cincinnati
Children’s Hospital (Perl et al., 2002; Sun et al., 2008). The mT-mG double fluorescent
Cre/loxP reporter mice were obtained from Jackson Laboratory (Muzumdar et al., 2007).
All mice were bred in C57BL/6 strain background. Fetal lung mesenchymal-specific Cre
recombinase expression was induced through doxycycline (Dox) oral administration at
different gestational stages by feeding the pregnant mice with both Dox diet (625 mg/kg;

TestDiet, Richmond, IN) and Dox water (0.5 mg/ml; Sigma,

St. Louis, MO). In postnatal
pups, Cre induction was initiated by single intraperitoneal injection of Dox (100 mg/kg
body weight) followed by Dox oral administration with both Dox diet (625 mg/kg) and
Dox water (0.5 mg/ml) for the entire induction period. All reported mouse studies were
approved by the Institutional Animal Care

and Use Committee (IACUC) at The Saban
Research Institute of Children’s Hospital of Los Angeles.

Fluorescence protein and immunofluorescence detection
Tissue specimens were isolated from mice at multiple time points. Mouse embryos, lung
tissues, and other organs such as kidneys, spleens, and hearts were dissected. For
membrane-tdTomato and membrane-GFP detection, mouse embryos and/or tissues were
directly imaged under a Leica MZFLIII fluorescence dissecting microscope in PBS. The
embryos or tissues were then fixed with 4% Paraformaldehyde in phosphate buffer
solution (Wako Pure Chemical Industries, Ltd) at 4 °C, washed with PBS, and processed
either for frozen section or regular histological section. For frozen section, fixed embryos
18

and/or tissues were cryoprotected in sucrose gradient (15%, 30% w/v in PBS) and
embedded in O.C.T. (Tissue-Tek). Cryostat sections were cut with the thickness of 5 to
8μm. For the regular histological sections, fixed embryos and/or tissues were paraffin-
embedded and cut at 5μm.
The Tomato or GFP fluorescence on frozen tissues was sufficient to visualize without
antibody staining. The embryo or tissue frozen sections were washed in PBS,
counterstained with DAPI in PBS, and mounted with either VECTASHIELD medium
(Vector Laboratories, Burlingame, CA), or Mowiol mounting medium prepared in the lab
(12.5% of Mowiol 4-88, 12.5% of glycerin, 0.625% of N-propyl Gallat, stored at -20 °C).
Immunofluorescence staining was performed for the paraffin-tissue sections. The sections
were deparaffinized at 55°C for 20 minutes, and rehydrated by immersion through the
following series of solutions: Xylene (10 minutes; repeat once in fresh xylene for 5
minutes), 100% Ethanol (5 minutes; repeat three times), 95% Ethanol (5 minutes; repeat
three times), 70% Ethanol(5 minutes, twice), 1xPBST (2 minutes, twice). Antigen
retrieval was conducted with boiling Tris-EDTA solution (1X, with 0.5% Tween-20) for
30 minutes. After cooling down, the slides were wash with PBST (1X, 2 minutes; twice),
and blocked with 10% normal donkey serum containing 1% BSA and 0.5% Triton X-100
at room temperature for one hour. The primary antibodies were diluted in the same
blocking solution, applied and incubated overnight at 4 °C. After being washed in PBS,
the slides were incubated with secondary antibodies at room temperature for 1 hour.
These secondary antibodies include Alexa 488 (1:500; green fluorescence), Alexa 647
(1:400; red fluorescence), Alexa 594 (1:500; red fluorescence). The cell nuclei of tissue
specimens were then counterstained with DAPI (1:1000 in PBS), and the slides were
19

mounted with VECTASHIELD medium or Mowiol mounting medium as described
above.
The related primary antibodies were: rabbit anti-GFP (1: 200, Santa Cruz Biotechnology,
Santa Cruz, CA), goat anti-GFP (1:500, Abcam, Cambridge, MA), mouse anti-EGFP
(1:1000, Clonetech, Mountain View, CA), mouse anti-cytokeratin (1:200, Sigma, Saint
Louis, MO), mouse anti- -smooth muscle actin (1:2000, Sigma, Saint Louis, MO), rabbit
anti-platelet endothelial cell adhesion molecule-1 (PECAM-1, LSBio, Seattle, WA),
rabbit β-Galactosidase (1:1000, MP Biomedicals, Solon, OH), mouse Anti-β-
Galactosidase (1:200, Promega, Madison, WI), mouse anti-adipophilin (ADRP,
BioGenex, Fremont, CA), rabbit anti-NG2 (chondroitin sulfate proteoglycan, Millipore,
Billerica, MA), hamster anti-T1 (DSHB at the University of Iowa), and mouse anti-
calcitonin gene-related peptide (CGRP, Sigma, Saint Louis, MO). Fluorescent signals
were detected using a Zeiss LSM710 confocal microscope at the Imaging Core Facility of
the Saban Research Institute of Children’s Hospital of Los Angeles. All experiments
were repeated at least three times, and data

represent consistent results.

X-gal staining
Lung specimen were briefly fixed at 4 °C with 4% paraformaldehyde in phosphate
buffered saline containing 2mM MgCl2, and embedded in O.C.T. Lung frozen sections
were incubated with X-gal reaction buffer (35mM potassium ferrocyanide, 35mM
potassium ferriccyanide, 2mM MgCl2, 0.02% NP-40, 0.01% sodium deoxycholate)
following the procedures described in previous publication (Chen et al., 2008).

20

RESULT
The lung enhancer of the mouse Tbx4 gene contains genomic DNA sequence
elements that are highly conserved across species that have lung or lung-like gas
exchange organ.
In order to understand the role of the 5.5kb DNA fragment in mouse Tbx4 intron 3 that
has the potential to drive gene expression in mouse lung (Menke et al., 2008), the
evolutionary conservation of this region was analyzed across 60 different vertebrate
species using the BLAT program available through UCSC Genome Bioinformatics
(Kent, 2002). Interestingly, several fragments of the DNA sequences exhibited high
similarity in 40 placental mammals (Fig.1A). In particular, a DNA fragment of about 500
bp in the middle of the region of interest appeared highly conserved in most vertebrate
species that have a lung-like respiratory organ. For instance, coelacanth, an ancient kind
of fish close to lungfishes and tetrapods, not only has a fat-filled single-lobed lung (Brito
et al., 2010), but also a sequence homologous to this mouse Tbx4 region. In contrast, no
such DNA sequence similarity was found in other fishes that do not develop lung-like
structures (Fig.1B). This suggests that this Tbx4 DNA segment may be an early
evolutionary adaptation that is important for successful morphogenesis of gas exchange
organs for air breathing.

Generation of the Tbx4-lung enhancer transgenic mouse lines
Tbx4-rtTA transgenic mice were generated using a DNA vector as illustrated in Figure
2A, in which simultaneous expression of a reverse tetracycline transactivator (rtTA) and
a lacZ (rtTA-IRES-LacZ) was controlled by the 5.5kb mouse Tbx4 lung enhancer. Four
21

founder lines of mice, line 5, 6, 7, and 8, were positive for Tbx4-rtTA transgene
integration as detected by genomic DNA PCR, while the transgene in mouse line 6 and 7
were easily detected by Southern blot (Fig.2B-C). These 4 lines with positive Tbx4-rtTA
transgene were then crossed to TetO-Cre/mT-mG reporter line. The efficacy and lung
specificity of transgene expression were compared based on Cre-mediated mGFP
expression pattern. The transgenic mice from line 6 and line 7 have stronger mGFP
expression in lung, while the mice of line 5 and 8 have relatively weak mGFP expression
under fluorescence dissection microscopy. Mice of all transgenic mouse lines appeared to
be healthy and fertile. Therefore, Tbx4-rtTA transgenic mice of line 6 were used for
studies on cell type specificity and lineage tracing, while Tbx4-rtTA transgenic mice of
line 8 were characterized separately.

Generation of the Tbx4-lung enhancer driven Tet-On system that specifically targets
lung mesenchyme
Active expression of the transgene rtTA-IRES-LacZ driven by the Tbx4 lung enhancer
was easily verified by detecting nuclear LacZ activity with X-gal staining (Fig.3). In
order to further trace and characterize the cell types with Tbx4-driven rtTA expression,
mice with Tbx4-rtTA mediated Tet-On controlled Cre expression in combination with
fluorescence protein reporter expression were generated using the breeding strategy
depicted in Figure 4 (Upper panel). The Tbx4-rtTA mice were first crossed to TetO-Cre
mice to make a Tet-On inducible Cre expression system (Kistner et al., 1996), in which
the Cre DNA recombinase expression was only induced in rtTA expressing cells
whenever doxycycline (Dox) is added. By further crossing to a mT-mG (loxP-mTomato-
22

STOP-loxP-mGFP) fluorescence protein reporter mouse line (Muzumdar et al., 2007),
cells with Cre DNA recombinase expression can be easily detected by mGFP expression
as result of Cre-mediated floxed-mTomato deletion, while Cre-negative cells still express
mTomato. As shown in Figure 5, Dox induction (E6.5 to E13.5) resulted in mGFP
expression only in embryonic lung of the triple transgenic mice (Tbx4-rtTA/TetO-
Cre/mT-mG) by gross view. In contrast, control mice without Tbx4-rtTA transgene
(TetO-Cre/mT-mG) did not express mGFP, confirming the specificity of the Tbx4-rtTA
transgene-mediated Cre reporter system. Figure 6 gives a closer look to the E13.5 lung of
the triple transgenic mice (Tbx4-rtTA/TetO-Cre/mT-mG). Moreover, sagittal sections of
the E10.5 triple transgenic embryo showed that Cre-mediated mGFP expression was
restricted to the lung buds and not detected in other organs. Within the lung bud, mGFP
expression was restricted to mesenchymal cells surrounding the epithelial cells, including
the outer layer of mesothelial progenitor cells of the visceral pleural membrane (Fig.7).
This lung/tracheal mesenchyme-specific pattern of Cre induction by the Tbx4-rtTA
transgene persisted to the end of gestation, since no mGFP was detected in organs other
than lung and trachea of the triple transgenic mice with continuous Dox induction from
E6.5 to E18.5 (Fig.8). Likewise, the section of the E14.5 embryo of the triple transgenic
mouse with Dox induction from E6.5 to E14.5 shows mGFP expression restricted to lung
mesenchyme (Fig.14). The mesenchyme-specific Tbx4-rtTA expression was also verified
by X-gal staining on frozen lung tissue section from E13.5 triple transgenic mice, of
which the Dox was administered from E6.5 to E13.5 (Fig. 9).

23

Dynamic pattern of Tbx4 lung enhancer-driven lung mesenchymal cell targeting
during and after development
As mentioned above, mouse lung development starts around E9.5. Based on the
morphological features, lung development can be arbitrarily divided into pseudoglandular
(E10.5-E16.5), canacular (E16.5-E17.5), saccular (E17.5-P5), and alveolar (P5-P30)
stages. Lung mesenchymal progenitor cells proliferate and differentiate into different
types of cells in concert with lung epithelial growth. Therefore, Tbx4-rtTA targeted lung
mesenchymal cells may undergo developmental changes at different lung developmental
stages as well as in post-development of the adult lung. Thus, the Tbx4-rtTA mediated
Tet-On Cre inducible triple transgenic reporter mice were examined when Dox induction
was initiated at different gestation or postnatal ages. The Dox induction strategy was
designed as shown in Figure 4 lower panel.
As shown in Figure 10, Dox administration before lung formation (E8.5) was not able to
induce Cre expression in any tissues including lung, illustrated by negative mGFP
expression in E13.5 embryos, while Dox administration from E8.5 to E9.5 started to
induce Cre expression in some lung mesenchymal cells. During prenatal and early
postnatal lung development, the Tbx4-rtTA driven Tet-On system was very efficient in
inducing Cre expression in lung mesenchyme (Fig.11). For example, about 91% of
cytokeratin-negative cells in peripheral lung saccular structure at E18.5 had inducible Cre
expression when Dox was given to the triple transgenic mice (Tbx4-rtTA/TetO-Cre/mT-
mG) from E6.5 to E18.5 (Fig.15). Moreover, as shown in Figure 13, Cre expression in
the E18.5 lung of the triple transgenic reporter (Tbx4-rtTA/TetO-Cre/mT-mG) mouse
24

with Dox induction from E6.5 – E10.5 was sufficient to label the entire lung mesenchyme
with mGFP.
Although Cre induction in adult lung was still detected in many lung mesenchymal cells
including airway smooth muscle cells, the efficiency of Cre induction was reduced
compared to that seen during lung development, as shown by reduction in the proportion
of mGFP-positive cells (Fig.12).

Differential targeting of smooth muscle cells by the Tbx4 lung enhancer in
developing lung
In the triple transgenic (Tbx4-rtTA/TetO-Cre/mT-mG) mouse system described above,
mGFP expression persists within cells once Cre-mediated floxed-mTomato deletion has
occurred. Thus, mGFP-positive cells do not necessarily represent an active status of the
Tbx4 lung enhancer-driven rtTA/LacZ expression. Since the TetO-Cre transgene is
ubiquitously expressed, the Tet-On induction is dependent upon Tbx4-rtTA/LacZ
transgenic expression. Therefore, by comparing the expression pattern of mGFP and
LacZ in mouse lungs with different Dox induction time windows, cells that previously
expressed Cre in early lung development could be distinguished from the cells that were
still actively expressing Cre. Hence the activity of Tbx4 lung enhancer-mediated targeting
to a variety of differentiated lung mesenchymal cells could be analyzed.
With continuous induction of Dox during the entire period of fetal lung development
(E6.5 to E18.5), no mGFP-positive epithelial cells were detected as demonstrated by GFP
and cytokeratin co-immunostaining (Fig.15).
25

Furthermore, not all mGFP-positive cells were LacZ positive, while LacZ-positive cells
were all mGFP-positive (Fig.16), suggesting that some of the targeted lung mesenchymal
cells lost the Tbx4 lung enhancer activity during lung development.
In order to understand whether loss of the Tbx4 lung enhancer activity is related to
changes in differentiation status of lung mesenchymal cells, we first looked at lung
smooth muscle cells by examining co-expression of SMA and mGFP or SMA and LacZ.
As shown in Figure 17, Dox induction from E6.5 to E18.5 resulted in mGFP expression
in both airway and vascular smooth muscle cells, suggesting that the smooth muscle cells
of these structures were originally targeted by Tbx4 lung enhancer during fetal lung
development. However, LacZ expression was only detected in smooth muscle cells of the
E18.5 airways, but not in the vascular smooth muscle cells, indicating that the Tbx4 lung
enhancer was not active in differentiated vascular smooth muscle cells at late gestation
(E18.5). We then further determined the developmental time windows during which the
Tbx4-lung enhancer was turned off in vascular smooth muscle cells. In E15.5 lung with
Dox induction from E11.5, co-expression of mGFP and LacZ was seen in smooth muscle
cells of both airway and vasculature, however, the intensity of lacZ staining in vascular
smooth muscle cells was significantly reduced compared to that seen in airway smooth
muscle cells. In contrast, in E18.5 lung with Dox induction from E15.5, co-expression of
mGFP and LacZ was detected in airway smooth muscle cells only, with no expression of
mGFP and LacZ in the vascular smooth muscle cells, suggesting that the Tbx4-lung
enhancer was not active in differentiated vasculature smooth muscle cells after E15.5.
Therefore, the Tbx4 lung enhancer is able to target smooth muscle cells of both airway
26

and vascular lineages prior to E15.5, but mainly airway smooth muscle cells at later times
in gestation.

Differential targeting of pulmonary endothelial cells by the Tbx4 lung enhancer in
developing lung
Endothelial cells in the pulmonary vasculature were also mGFP-positive in the triple
transgenic lung specimens with Dox induction from E6.5 to E18.5 (Fig.18A). However,
these mGFP-positive endothelial cells were LacZ-negative at E18.5, suggesting that the
Tbx4 lung enhancer might target endothelial progenitor cells during early lung
morphogenesis. We then looked at lung specimens at different developmental stages in
combination with different Dox induction time windows.
In E15.5 triple transgenic lung with Dox induction from E6.5 to 10.5, endothelial cells,
detected by PECAM-1 immunostaining, were positive for mGFP, but not for LacZ
(Fig.18B). Moreover, in E15.5 triple transgenic lung with Dox induction from E11.5 to
15.5, all PECAM-1-positive cells were negative for both mGFP and LacZ (Fig.18C).
These data indicate that pulmonary endothelial cells may be derived from lung
mesenchymal progenitors at an early gestation stage prior to E11.5, and that the Tbx4
lung enhancer-mediated Tet-On system is able to target these progenitor cells if Dox
induction is given before E11.5.

Other lung cell lineages targeted by the Tbx4 lung enhancer during lung
development
27

In addition to smooth muscle cells and endothelial cells, we also looked at other
differentiated lung mesenchymal cells targeted by the Tbx4 lung enhancer. Lung
myofibroblasts and lipofibroblasts in the alveolar septal structures are important for lung
alveolar formation and maintenance, which can be identified by SMA and ADRP
immunostaining, respectively (Bostrom et al., 1996; Rehan et al., 2006). In the primary
alveolar sacs of E18.5 triple transgenic mice with Dox induction from either E6.5 to
E18.5, or E15.5 to E18.5, both myofibroblasts and lipofibroblasts were all GFP-positive
(Fig.19, Fig.20), suggesting that fetal Tbx4-rtTA expressing cells can be the progenitors
for both of these specialized subpopulations of lung fibroblasts throughout the entire
prenatal stage.
Moreover, Tbx4 lung enhancer-targeting was also detected in potential lung pericytes by
co-immunostaining of GFP and NG2, one of the markers for pericytes (Fig.21). In E15.5
lungs of the triple transgenic mice with Dox induction either from E6.5 to 10.5 or 11.5 to
E15.5, NG2-positive staining was detected in both vascular smooth muscle cells and
dispersed individual mesenchymal cells, which were all GFP-positive (Fig.21B-C). This
suggests that the NG2-positive cells are derived from the Tbx4 lung enhancer targeted
cells.
In contrast, pulmonary neuroendocrine cells, as identified by CGRP expression, were not
targeted by the Tbx4-lung enhancer driver, as determined by negative GFP staining in the
CGRP-positive cells of the triple transgenic mouse lung (Fig.22).
Cells with Tbx4 lung enhancer activity were also studied in adult lungs of triple
transgenic (Tbx4-rtTA/TetO-Cre/mT-mG) mice with Dox administration from 6th week
to 8th week. The co-staining results showed cells with GFP expression were not positive
28

for T1α and PECAM-1, while most GFP-positive cells were positive for NG2, and a few
GFP-positive cells were positive for SMA (Fig.23). Besides, adult transgenic reporter
mouse lungs were evaluated in the absence of Dox induction. As Figure 24 shows, mGFP
expression in lung mesenchyme cells without the induction of Dox is rare.

Evaluation of the Cre-mediated mGFP expression in the triple transgenic mice
(Tbx4-rtTA/TetO-Cre/mT-mG) in line 8
Both transgenic mouse line 6 and line 8 were identified by genomic DNA PCR, while
Southern blot for line 8 yield negative results. The E13.5 embryos of the triple transgenic
mice of line 8 with Dox induction from E6.5 to E13.5 were examined. Figure 25 showed
a side by side comparison of the E13.5 embryos of line 8 and line 6. Cre-mediated mGFP
expression (green) was detected in both triple transgenic mouse (Tbx4-rtTA/TetO-
Cre/mT-mG) lungs of line 6 and 8. However, at the gross-view, mGFP expression in the
lung of line 8 mouse was considerably lower than that of line 6 under the same imaging
conditions.
The E13.5 lung frozen section of line 8 triple transgenic (Tbx4-rtTA/TetO-Cre/mT-mG)
mouse with Dox induction E6.5 - E13.5 (Fig.26) was further examined under microscope.
Only a small portion of lung mesenchymal cells were targeted by the Tbx4 lung enhancer
transgene in line 8.
Even in the E18.5 lung frozen section of line 8 triple transgenic (Tbx4-rtTA/TetO-
Cre/mT-mG) mouse with Dox induction E6.5 - E18.5 (Fig.27), there were only a small
number of cells, which were labeled with mGFP and mainly localized in visceral pleural
29

membrane, while all mesenchyme cells labeled with mGFP in the E18.5 lung of line 6
with same treatment.
No mGFP expression was identified in the 8-week lung of the triple transgenic mice of
line 8 with Dox induction from week 6 to week 8, suggesting that the transgenic Tbx4
lung enhancer of line 8 may not be active in adulthood (Fig. 28).
30

Figure 1 Analysis of the 5.5kb genomic DNA sequences of the potential mouse Tbx4
lung enhancer. A. Placental mammal basewise conservation scores determined by
phyloP program. Positive scores are assigned to the sites with conserved DNA sequences
among the studied vertebrate species, which include mouse, rat, kangaroo rat, naked mole
rat, guinea pig, squirrel, rabbit, pika, human, chimp, gorilla, orangutan, gibbon, rhesus,
baboon, marmoset, squirrel monkey, tarsier, mouse lemur, bushbaby, treeshrew, pig,
alpaca, dolphin, sheep, cow, cat, dog, panda, horse, microbat, megabat, hedgehog, shrew,
elephant, rock hyrax, tenrecs, manatee, armadillo, sloth, opossum, Tasmanian devil,
wallaby, platypus, turkey, chicken, zebra finch, budgerigar, lizard, painted turtle, xenopus
31

tropicalis, coelacanth, tetraodon, fugu, nile tilapia, stickleback, medaka, Atlantic cod,
zebrafish, lamprey. B. The related sequence alignments of the above vertebrates to mouse
Tbx4 genomic DNA sequence by Multiz program. Most sequence alignments were
omitted due to limited space. Identical nucleotide sequences are indicated by the vertical
black bars.

32

Figure 2 Generation of an embryonic/fetal lung-specific Tbx4-rtTA transgenic
mouse line. A. Schematic diagram of transgenic DNA construct. The position of the
DNA probe for Southern blot is indicated. B. Screen of transgenic founder lines by
genomic DNA PCR. 2C. Verification of transgenic lines by Southern blot. Genomic
DNA was digested by XhoI and SalI prior to separation by gel electrophoresis.
B.
C.
33

Figure 3 Expression of rtTA-IRES-LacZ transgene in E13.5 lung was verified by X-
gal staining (blue).

50 μm
34

Figure 4 Upper: Schematic diagram for generating Tbx4-rtTA/TetO-Cre/mT-mG
reporter mice. Lower: The Dox induction strategy is designed to track the mouse lung
developmental stages. E stands for embryonic day; P stands for postnatal day; WK stands
for week. Each induction period is highlighted in yellow, and the time points at which the
samples are harvested are marked with red short lines.
35

Dox E6.5 - E13.5
Figure 5 Cre-mediated mGFP expression (green) was detected in the lung of the triple
transgenic mice (Tbx4-rtTA/TetO-Cre/mT-mG), but not in the double transgenic control
mice (TetO-Cre/mT-mG), in which floxed-mTomato expression (red) was not affected.
Dox induction was started from E6.5, and E13.5 mouse embryos were isolated and
visualized under Leica fluorescence dissecting microscope.

36

Dox E6.5 - E13.5
Figure 6 Dox induction of the triple transgenic mice (Tbx4-rtTA/TetO-Cre/mT-mG)
was from E6.5 to E13.5. mGFP expression is only in the embryonic lung mesenchyme of
the triple transgenic mice by gross view. Mouse lung were dissected and visualized under
the Leica fluorescence dissecting microscope .

E13.5
37

Figure 7 Sagittal frozen section of E10.5 embryo of the triple transgenic mice
(Tbx4-rtTA/TetO-Cre/mT-mG) with Dox induction from E6.5 to E10.5. Dox
induction from E6.5 – E10.5 was sufficient to label the entire lung mesenchyme with
mGFP. Cre-mediated mGFP expression was restricted to the lung buds and not detected
in other organs. Within the lung bud, mGFP expression was restricted to mesenchymal
cells surrounding the epithelial cells, including the outer layer of mesothelial progenitor
cells of the visceral pleural membrane. Right panel shows the lung bud structure under a
higher magnification. Both images were acquired with Zeiss LSM710 confocal
microscope.

38

Figure 8 Fluorescence microscopic examination of tissue frozen sections from E18.5
triple transgenic mice (Tbx4-rtTA/TetO-Cre/mT-mG), in which Dox induction was
initiated from E6.5. mGFP was detected only in lung and tracheal mesenchyme, but not
in other tissues. Dox, doxycycline; E, embryonic day.

39

Figure 9 X-gal staining of the E13.5 triple transgenic mouse lung frozen section. Dox
induction last from E6.5 to E13.5. The sample was harvested at E13.5 and fixed to X-gal
staining standards. The mesenchyme-specific Tbx4-rtTA expression was verified by X-
gal staining on frozen lung tissue section from E13.5 triple transgenic mice. Green
fluorescence: GFP. Magnification: Upper panel, 40x; Lower panel: 63x.
GFP/DAPI GFP/Xgal
GFP/DAPI GFP/Xgal
40

Figure 10 Dynamic expression profile of Tbx4-rtTA-mediated Tet-On targeting
system shown by the triple transgenic reporter (Tbx4-rtTA/TetO-Cre/mT-mG)
No GFP fluorescence was observed on the sagittal frozen sections of E13.5 lungs of the
triple transgenic reporter (Tbx4-rtTA/TetO-Cre/mT-mG) mice with Dox induction from

41

E0.5 – E6.5, or E6.5- E8.5. Partial mesenchyme was labeled with GFP fluorescence in
E13.5 lungs of the triple transgenic reporter (Tbx4-rtTA/TetO-Cre/mT-mG) mice with
Dox induction from E8.5-E9.5. Tbx4-rtTA-mediated Tet-On system targeted lung
mesenchyme was initiated immediately after lung morphogenesis at E9.5. Dox induction
was given at different time windows of early gestation, and the sagittal frozen sections of
E13.5 embryos were used to detect mGFP (green) and mTomato (red) expression. Nuclei
were counterstained with DAPI (blue). Lung tissue (L) was marked on the left side of the
dotted line, and vertebrae (V) were located on the right side.

42

Figure 11 Efficiencies of cell targeting by the Tbx4-rtTA mediated Tet-On inducible
system at different prenatal ages. The time windows of Dox induction are indicated
above each panel, and the ages of the examined lung specimens are specified inside each
panel. DAPI, 4',6-diamidino-2-phenylindole; Dox, doxycycline; E, embryonic day

Dox (E15.5-E18.5) Dox (E6.5-E18.5)
E18.5

43

Figure 12 Efficiencies of cell targeting by the Tbx4-rtTA mediated Tet-On inducible
system at different postnatal stages. The time windows of Dox induction are indicated
above each panel, and the ages of the examined lung specimens are specified inside each
panel. DAPI, 4',6-diamidino-2-phenylindole; Dox, doxycycline; E, embryonic day

Dox (E18.5-P10)
P10
44

Tbx4-rtTA/TetO-Cre/mT-mG Tbx4-rtTA/mT-mG

Figure 13 The E18.5 lung of the triple transgenic reporter (Tbx4-rtTA/TetO-
Cre/mT-mG) mouse and Tbx4-rtTA/mT-mG mouse with Dox induction from E6.5
– E10.5. Cre expression in the E18.5 lung of the triple transgenic reporter (Tbx4-
rtTA/TetO-Cre/mT-mG) mouse with Dox induction from E6.5 – E10.5 was sufficient to
label the entire lung mesenchyme with mGFP.

45

Figure 14 Cre-mediated mGFP expression (green) was detected in the mesenchyme
of the triple transgenic mouse (Tbx4-rtTA/TetO-Cre/mT-mG) lung at E14.5, but not
in the other organs. Dox induction was initiated at E6.5, and at E14.5 mouse embryos
were harvested and processed for sagittal frozen section. The specimen was visualized
under the Zeiss LSM710 confocal microscope with tiling function. Magnification, 6x; E,
embryonic day.
46

Dox (E15.5-E18.5) Dox(E6.5-E18.5)

Figure 15 Tbx4 lung enhancer-mediated Tet-On induction activity was only
detected in cytokeratin (Cyt)-negative cells, shown by GFP and Cyt co-
immunostaining. The lung specimens were taken from the E15.5 and E18.5 triple
transgenic fetuses with Dox induction time windows indicated above the panels. Red:
Cytokeratin; Green: GFP.

GFP/CYTO
47

Dox E6.5- E18.5

Figure 16 Not all GFP-positive cells were lacZ-positive, shown by GFP and LacZ co-
immunostaining. The lung specimens were taken from the E18.5 triple transgenic fetuses
with Dox induction fron E6.5 to E18.5. Red: LacZ; Green: GFP; Blue: Dapi.

GFP/LacZ
48

A
B
49

Figure 17 Differential targeting of lung smooth muscle cells by the Tbx4 lung
enhancer at different gestational ages. Co-immunostaining of GFP, LacZ and SMA for
the lungs of E18.5 (A), E15.5 (B), and E18.5 (C) triple transgenic fetuses with different
Dox inductions indicated above the panels. Smooth muscle cells were originally targeted
by Tbx4 lung enhancer during fetal lung development. LacZ expression was only
detected in smooth muscle cells of the E18.5 airways, but not in the vascular smooth
muscle cells indicating Tbx4 lung enhancer was not active in differentiated vascular
smooth muscle cells at E18.5. Tbx4-lung enhancer was not active in differentiated
vasculature smooth muscle cells after E15.5. Tbx4 lung enhancer is able to target smooth
muscle cells of both airway and vascular lineages prior to E15.5, but mainly airway
smooth muscle cells at later times in gestation. a: airway; v: vasculature. Dox,
doxycycline; SMA, α-smooth muscle actin.
C
50

51

Figure 18 Differential targeting of lung vascular endothelial cells by the Tbx4 lung
enhancer at different gestational ages. A. In E18.5 lung of the triple transgenic mice
with Dox induction from E6.5, PECAM-1-positive endothelial cells were GFP positive,
but LacZ negative. B-C. In the E15.5 lung of the triple transgenic mice with different
Dox induction time windows as indicated above the panel, vascular endothelial cells were
GFP-positive if Dox induction was given prior to E10.5 (B), but LacZ expression was
already negative. Consistently, Dox induction from E11.5 to E15.5 was not able to target
vascular endothelial cells, shown by double negative staining for GFP and LacZ (C). V:
vasculature. Dox, doxycycline; E, embryonic day; PECAM-1, platelet endothelial cell
adhesion molecule.

52

Figure 19 Lipofibroblasts (ADRP positive) were marked by the Tbx4 lung enhancer
driven Tet-On system. E18.5 lung tissue sections of triple transgenic mice (Tbx4-
rtTA/TetO-Cre/mT-mG) with different Dox induction during early or late gestation as
indicated were co-stained with mGFP/ADRP (A). Cell nuclei, counter-stained with DAPI
(blue), were not included in the merged panels. ADRP, adipophilin; DAPI, 4',6-
diamidino-2-phenylindole; Dox, doxycycline; E, embryonic day.

53

Figure 20 Myofibroblasts (SMA positive) were marked by the Tbx4 lung enhancer
driven Tet-On system. E18.5 lung tissue sections of triple transgenic mice (Tbx4-
rtTA/TetO-Cre/mT-mG) with different Dox induction during early or late gestation as
indicated were co-stained with mGFP/SMA (B). Cell nuclei, counter-stained with DAPI
(blue), were not included in the merged panels. DAPI, 4',6-diamidino-2-phenylindole;
Dox, doxycycline; E, embryonic day; SMA, α-smooth muscle actin.

54

55

Figure 21 NG2-positive cells were targeted by the Tbx4-lung enhancer driven Tet-
On system. Lung tissue sections of the triple transgenic mice (Tbx4-rtTA/TetO-Cre/mT-
mG) with different Dox induction as indicated were co-immunostained by GFP, NG2,
and SMA or PECAM-1. A. In the E18.5 lung of the triple transgenic mice with Dox
induction from E6.5 to E18.5, NG2-positive cells, including SMA-positive vasculature
smooth muscle cells and SMA-negative pericytes, were all marked by GFP expression,
suggesting that these cells were targeted by the Tet-On system in fetuses. B-C. With Dox
induction at either mid-gestation (E11.5 to E15.5) or late gestation (E15.5 to E18.5), most
NG-2 positive cells of the triple transgenic mouse lungs at E15.5 (B) or E18.5 (C) were
positive for GFP. These NG2-positive cells were adjacent to PECAM-1 positive
endothelial cells. Dox, doxycycline; E, embryonic day; PECAM-1, platelet endothelial
cell adhesion molecule; SMA, α-smooth muscle actin.

56

Figure 22 Lung neuroendocrine cells were not targeted by the Tbx4 lung enhancer.
Co-immunostaining of GFP (green) and CGRP (red) for E18.5 lung tissue section of the
triple transgenic mouse (Tbx4-rtTA/TetO-Cre/mT-mG) with Dox induction from E6.5
to E18.5. Pulmonary neuroendocrine cells, as identified by CGRP expression, were not
targeted by the Tbx4-lung enhancer driver, as determined by negative GFP staining in the
CGRP-positive cells of the triple transgenic mouse lung. Blue: DAPI nuclear
counterstaining.

57

Figure 23 Cells with Tbx4 lung enhancer activity in adult lungs. Adult mice with
triple transgenic genotypes (Tbx4-rtTA/TetO-Cre/mT-mG) were induced by Dox
administration for 2 weeks. The lung tissue sections were co-immunostained with GFP
and one of the cell markers as indicated. Cells with GFP expression (green) were not
positive for T1α and PECAM-1 (red). However, most GFP-positive cells were positive
for NG2, and a few GFP-positive cells were positive for SMA, shown by overlapped co-
staining (yellow color). DAPI was used for nuclear counterstaining (blue).

58

Figure 24 Evaluation of adult transgenic reporter mouse lungs in the absence of
Dox induction. Mouse genotypes are indicated above the panel. mGFP expression in
lung mesenchyme cells without the induction of Dox is rare. Expression of endogenous
mTomato (red) and mGFP (green) was examined for the lung frozen sections under
fluorescence microscope. DAPI was used for nuclear counterstaining (blue)

59

Line 8 Line 6

Figure 25 Cre-mediated mGFP expression (green) was detected in the lung of the
triple transgenic mice (Tbx4-rtTA/TetO-Cre/mT-mG) in both line 8 and line 6,
while The GFP signal from the lung of line 8 mouse was significantly weaker than of line
6 under the same imaging conditions. Dox induction was started from E6.5, and E13.5;
mouse embryos were isolated and visualized under the Leica fluorescence dissecting
microscope.

E13.5 E13.5
60

E13.5
E13.5
61

Figure 26 In line 8, Cre-mediated mGFP expression (green) was detected in the
mesenchyme of the triple transgenic mouse (Tbx4-rtTA/TetO-Cre/mT-mG) lung of E13.5.
Only a small portion of lung mesenchymal cells were targeted by the Tbx4 lung enhancer
transgene in line 8. Induction was from E6.5 - E13.5 and the mouse was harvested on
E13.5. Upper panel: 10x; Lower Panel: 20x.
62

Line 8 Line 6

Figure 27 In line 8, mGFP expression (green) was detected in the mesenchyme of the
triple transgenic mouse (Tbx4-rtTA/TetO-Cre/mT-mG) lung of E18.5. There were only a
small number of cells, which were labeled with mGFP and mainly localized in visceral
pleural membrane, while all mesenchyme cells labeled with mGFP in the E18.5 lung of
line 6 with same treatment. Induction was from E6.5 - E18.5 and the mice were harvested
on E18.5. Magnification: 20x.

E18.5 E18.5
63

Dox 6wk - 8wk

Figure 28 In line 8, no mGFP expression (green) was detected in the 8-week lung of the
triple transgenic mouse (Tbx4-rtTA/TetO-Cre/mT-mG). The transgenic Tbx4 lung
enhancer of line 8 may not be active in adulthood. Induction was from postnatal 6th week
to 8th week and the sample was harvested at week 8.

8 week
64

DISCUSSION
Mesenchymal cells in a variety of tissues are mainly derived from mesoderm during
organogenesis. They play important roles in guiding organogenesis, generating the tissue-
specific mesenchymal progenitor/stem cells needed for homeostasis in developed organs,
and can differentiate into fibroblasts, smooth muscle cells, chondrocytes, and other types
of mesenchymal cells that support specific tissue functions. However, genetic markers
specifically for mesenchymal cells in individual visceral tissue, including lung
mesenchyme, have not been well defined. For example, in Dermo1-Cre knockin mice,
Dermo1 promoter drives Cre expression in many mesoderm-derived tissues including
lung mesenchyme, diaphragm, and ventral body walls (Chen et al., 2008; Sun et al.,
2007; Yu et al., 2003). Similarly, although endogenous Tbx4 expression was detected in
the mesenchyme of lung and trachea around E9.25, a few hours after the specification of
the lung and trachea primordia by Nxk2.1 and Tbx5 (Arora et al., 2012), expression of
both Tbx4 and Tbx5 genes is not restricted to lung tissue even though they are important
in lung organogenesis (Naiche et al., 2011). Interestingly, a dispersed group of Tbx4 gene
enhancers are found to be responsible for the distinct tissue locations of this gene (Menke
et al., 2008). Among these, a 5.5kb fragment of genomic sequences in Tbx4 intron 3 is
related to its expression in early embryonic lung. Our DNA sequence analysis suggests
that several DNA fragments in this region, particularly a ~500bp fragment in the middle
region, are highly conserved in vertebrate species that develop lungs or lung-like gas
exchange structures. This suggests that this DNA regulatory element may be important
for lung structure formation. However, further experiments to examine individual
fragments of mouse Tbx4 genomic region and compare their gene regulatory activities
65

among different specimens will be needed to fully understand the role of mouse Tbx4
lung enhancer in lung morphogenesis. Furthermore, whether these conserved fragments
of DNA sequences in each species are involved in regulating Tbx4 gene expression also
needs to be experimentally analyzed.
We have used this lung-specific DNA enhancer of Tbx4 gene to generate a Tet-On
inducible transgenic system in mice. In combination with Cre-mediated reporter mice, we
have clearly demonstrated that developing lung mesenchymal cells can be specifically
marked from the beginning of lung formation, which makes these cells distinguishable
from the mesenchymal cells arising from other organs. We have shown for the first time
that lung mesenchymal cells can be targeted in an organ-specific manner by using the
Tbx4 lung enhancer, rather than the entire endogenous Tbx4 promoter as in the Tbx4-Cre
knock-in model (Naiche et al., 2011). This provides a powerful genetic tool with which to
study the functions of genes in lung mesenchymal development, and to isolate and trace
lung mesenchymal cell lineages during prenatal development through postnatal
development to adulthood. More importantly, it provides the potential to generate unique
mouse models mimicking lung interstitial diseases without adversely affecting other
organs and systems. The molecular mechanisms by which the Tbx4 lung enhancer is
activated specifically in lung mesenchymal cells are not yet completely clear, and also
will need further investigation. It is possible that specific lung mesenchymal cells may
express a unique array of transcription factors that interact and activate the Tbx4 lung
enhancer. In contrast, mesenchymal cells in other tissues may lack some of these specific
transcriptional activators, resulting in suppression of this particular Tbx4 gene regulatory
element, but not endogenous Tbx4 gene expression that can be activated by multiple
66

regulatory elements. Furthermore, our experiments also show that the Tbx4 lung enhancer
is not persistently active in all lung mesenchymal cells. Its activity is turned off in some
committed and/or differentiated mesenchymal cell lineages at various developmental
stages, particularly in pulmonary endothelial cells and vascular smooth muscle cells.
Whether active Tbx4 lung enhancer activity is related to lung mesenchymal progenitor
cells, or is associated with cell differentiation status remains to be determined.
Another feature of our Tbx4-rtTA-mediated Tet-On Cre inducible system is the
combination of the Cre-mediated reporter and LacZ reporter systems. Cre-mediated
mGFP expression is the marker for cells that had and/or have induced Cre expression,
while LacZ expression indicates active Tbx4 lung enhancer activity at the time of
examination. Therefore, using this system, we have been able to determine the dynamic
changes and fates of lung mesenchymal cells with Tbx4 lung enhancer activity. In early
lung buds, the majority of these mesenchymal progenitors can be marked using this Tbx4
lung enhancer, and these cells are able to differentiate to endothelial cells, and smooth
muscle cells of both airway and vasculature. However, the multi-potent differentiation
potential of these mesenchymal progenitor cells is reduced during the course of lung
development, as the majority of endothelial cells and vascular smooth muscle cells are
neither labeled by Cre-mediated mGFP expression if Dox induction is given after E11.5
and E15.5, respectively, nor detected for LacZ expression. However, lung airway smooth
muscle cells and subtypes of fibroblasts (lipofibroblast and myofibroblast) are positive
for induced mGFP expression even if Dox is given at late gestation. Therefore,
differential targeting of mouse lung mesenchymal progenitors and related cell lineages
can be achieved by controlling the time windows of Dox induction.
67

Interestingly, a proportion of mesenchymal cells in postnatal developing and even adult
lung are still active for the Tbx4-lung enhancer. These cells are located adjacent to
alveolar epithelial cells and alveolar endothelial cells of adult lung alveolar structure, and
are positive for NG2 or SMA staining (Figure 23), suggesting that these may be pericytes
and myofibroblasts. Therefore, our Tbx4-lung enhancer driven targeting driver line has
the potential to be used in the study of adult lung diseases, such as asthma, emphysema,
and interstitial pulmonary fibrosis. It could be used to create new disease models, to
determine the response of targeted cell lineages to specific injuries, and/or to test
intervention approaches to prevent or attenuate pathological processes. Moreover,
understanding developmental lung mesenchymal specificity is also important when
considering the future design of cell-based therapies. For example, the role of bone
marrow-derived mesenchymal stem cells in lung injury repair and regeneration is
controversial, although their effects on modulating immune and inflammatory responses
are well recognized (Bernardo and Fibbe, 2012). This raises the question whether
mesenchymal stem cells derived from lung may function better in repairing lung
structures than those originating from other organs such as bone marrow (Bianco et al.,
2013). Our newly generated Tet-On system will make it possible to compare
mesenchymal stem cells of lung with those of non-lung origins in mice by marking
developing lung mesenchymal cells with prenatal Dox induction. Comparison of these
two groups of cells and characterization of their molecular signatures under both
physiological and pathological conditions will greatly advance our knowledge about lung
biology and pulmonary diseases.
68

By comparing line 6 and line 8 of the Tbx4-rtTA mice, we found that Tbx4 lung
enhancer-driven rtTA expression in line 8 is extremely low, resulting in Cre-mediated
mGFP expression only in a small portion of lung mesenchymal cells. This could be
caused by an integration site of transgene in the genome, and/or low copy number of the
inserted transgene. Indeed, our genomic PCR and southern blot data have shown that the
copy number of the transgene is lower in line 8 than that in line 6. Further prenatal and
postnatal characterization could be done with the triple transgenic mouse of line 8 to
explore its utility as a different tool to study lung mesenchyme biology. For example, it
will be easy to achieve single cell labeling in line 8 for cell lineage tracing by controlling
the concentration of inducing agent doxycycline.

69

CONCLUSION
Although mesenchymal cells in developing lung display complicated heterogeneous
phenotypes, they are probably derived from splanchnic mesoderm surrounding lung
progenitors of foregut endoderm. We have found an evolutionally conserved Tbx4 lung
enhancer that is active specifically in lung mesenchymal progenitors and subsets of their
derived cells. Therefore, with our newly created Tbx4 lung enhancer-driven Tet-On
inducible system, lung mesenchymal cells can be specifically and differentially targeted
at the first time by controlling the doxycycline induction time window. This novel system
provides a unique tool to study lung mesenchymal cell lineages and gene functions in
lung mesenchymal development, injury repair, and regeneration in mice.

70

CHAPTER 2

LUNG MESENCHYMAL STEM CELL

71

LIST OF FIGURES
Figure 1 Isolation and characterization of lung mesenchymal stem cells
Figure 2 Verification of isolated lung resident MSC by immunostaining
Figure 3 Verification of isolated lung resident MSC by flow cytometry analysis
Figure 4 Isolated lung MSCs were able to differentiate to adipocytes
Figure 5 Proliferation of the isolated lung MSCs was detected by BrdU incorporation to
nuclear DNA
Figure 6 Flow cytometry analysis of the mGFP-labeled and mRFP-labeled lung
mesenchymal stem cell
Figure 7 Smad3-/- mice has reduced number of lung MSCs than Wildtype mice
Figure 8 MSCs from Smad3-/- lung has reduced proliferation rate
Figure 9 MSCs from Smad3-/- lung has reduced passage doubling rate
Figure 10 Adipogenesis of the lung MSCs from wildtype mouse and Smad3 -/- mouse
Figure 11 Lung MSCs isolated from Smad3 knockout lungs exhibited reduced expression
of CD105

72

ABSTRACT
Background
Although bone marrow mesenchymal stem cells (BMMSCs) have been extensively
studied, tissue specific MSCs, including lung resident MSCs, are poorly defined and
characterized. Studies have shown that stem cell-mediated injury repair and regeneration
is critical to maintain normal lung structure and function. The increasing recognition of
the properties of mesenchymal stem cells has led to various pathogenesis-oriented studies.
The roles of lung resident MSCs in lung diseases have never been studied.
Emphysema is the third leading cause of death in U.S, the pathogenesis of emphysema
has not been fully elucidated. No effective therapy is available to treat this disease.
Recent studies in our lab have demonstrated that Smad3 knockout mice can
spontaneously develop emphysema, which offers a great tool for us to investigate the
pathogenic mechanisms and test novel therapeutic approaches for emphysema. Therefore,
we have used this emphysema-mouse model to study the role of lung resident
mesenchymal progenitor cells in emphysema pathogenesis.

Result
We have successfully isolated potential lung mesenchymal stem cells from mouse lung
tissue using modified method of bone marrow MSC isolation. These cells are plastic-
adherent, capable of self-renew and differentiation, and expressing certain mesenchymal
stem cell markers, same features as described for BMMSC. Furthermore, with the
aforementioned Tbx4-rtTA/TetO-Cre/mT-mG transgenic mice of line 6 in last chapter,
we found that the isolated lung resident MSCs consist of at least two subpopulations with
73

different developmental origins and repair/regeneration capacities. Lung resident MSCs
isolated from our newly developed transgenic mice, in which MSCs with embryonic lung
mesenchyme origin are marked with membrane GFP protein, while MSCs derived from
other organs are labeled with membrane Tomato protein.
The deficiency of lung resident MSCs and their pathogenic roles in a Smad3 knockout
emphysema mouse model were studied. Impaired lung MSC-mediated
repair/regeneration may contribute to emphysema pathogenesis. The quantitative and
qualitative alterations of lung MSCs subsets in Smad3 knockout mice need further
investigation. To determine the therapeutic efficacy of lung resident mesenchymal stem
cells, exogenous MSCs administration into emphysema mice should be considered.

74

INTRODUCTION
Although MSCs were first isolated from bone marrow (Friedenstein, 1970) ，it is now
generally accepted that most organs carry their own population of MSCs including lung
(Da Silva Meirelles L et al., 2008). Identification of lung resident MSCs have been
reported by several different approaches. Based on the ability to efflux the vital dye
Hoechst 33342, Summer et al reported a rare population of mesenchymal progenitors
resides within the CD45-CD31- Hoechst low fraction of the adult murine lung (Summer
R, Fitzsimmons K. et al., 2007). Lung mesenchymal stem cells were also isolated from
bronchoalveolar lavage fluid in adult patient allograft tissue and tracheal aspirates of
ventilated neonates, respectively, by their plastic adherence and colony forming ability
(Popova AP, 2010; Lama VN, 2007). All the lung MSCs obtained by different
approaches have self-renewal and multi-potent differentiation potential.
It is widely accepted that MSCs have the capacity to self-renew and differentiate into
different cell lineages, including mesodermal, endodermal, and ectodermal cells
(Pittenger MF et al., 1999; Qunzhou Zhang et al., 2009). Stem cell-mediated injury repair
and regeneration is critical to maintaining normal lung structure and function. Although
bone marrow mesenchymal stem cells (MSCs) have been extensively studied, lung tissue
resident MSCs and their roles in lung diseases, such as emphysema, are poorly defined.
Emphysema, the major component of chronic obstructive pulmonary disease (COPD), is
one of the most common lung diseases, and the third leading cause of death in the United
State. With the characteristic of alveolar destruction, emphysema results from a complex
interaction between various genetic and environmental factors including smoking. The
75

pathogenic mechanisms of emphysema are not completely understood, and currently no
effective approach to prevent or treat emphysema has ever been found. Decreased TGF-
beta-Smad3 activity was detected in lung tissue specimens from COPD patients
(Zandvoort et al., 2006). Reduced Smad3 and increased Smad7 (TGF-beta-pathway
inhibitor) expression was also detected in cultured COPD fibroblasts, which was
exacerbated by addition of cigarette smoke extract (Zandvoort et al., 2008). All these
observations suggest that reduced TGF-beta-Smad3 mediated signaling is associated with
emphysema pathogenesis of some COPD patients. Furthermore, Smad3 knockout in mice
results in spontaneous development of emphysema in young adult, as reported by our lab
(Hui Chen, 2005). Therefore, Smad3 knockout mice offer a great tool for us to
investigate the pathogenic mechanisms and test novel therapeutic approaches for
emphysema. Herein, we hypothesize that Lung resident MSCs consist of at least two
subpopulations with different developmental origins and repair/regeneration capacities;
impaired lung MSC-mediated repair/regeneration may contribute to emphysema
pathogenesis.

76

METHOD
Mouse breeding
All mice were bred in C57BL/6 strain background. Wildtype and the Tbx4-rtTA/TetO-
cre/mT-mG transgenic mice have been described in previous chapter. Fetal lung
mesenchymal cells were permanently labeled with Cre-mediated mGFP expression by
feeding the pregnant mice with both Dox diet (625 mg/kg; TestDiet, Richmond, IN) and
Dox water (0.5 mg/ml; Sigma, St. Louis, MO) from embryonic day 6.5 to 18.5. Smad3
heterozygous knockout mice were originally provided by Dr. Xiao-Fan Wang at Duke
University, and maintained in our lab. Mice were euthanized in a CO
2
chamber, and lung
specimens were isolated at different ages. All reported mouse studies were approved by
the Institutional Animal Care and Use Committee (IACUC) at The Saban Research
Institute of Children’s Hospital of Los Angeles.

Lung mesenchymal stem cell isolation
Potential lung mesenchymal stem cells are isolated and cultured following a modified
method used for bone marrow MSCs (Sun et al., 2009). Briefly, the de-blooded lung was
first dissected into individual lobes. One lobe was fixed in 4% Paraformaldehyde in PBS
(Wako Pure Chemical Industries, Ltd), while the rest lung tissues are minced into 1mm
3

pieces inside the hood and digested with 6ml of enzyme cocktail containing 2mg/ml
Dispase II and 1mg/ml Collagenase I in PBS. Tissue digestion tubes were placed in a
shaking water bath at 37° C ，with intermittent mixing by pipetting every 10 minutes in
the cell culture hood. After 45-60 min digestion, the cell suspension was filtered through
a 70 m cell strainer (BD) ，and collected in a 50ml tube. The strainer was then flushed
77

with 10ml MSC culture medium (Dex-, see below). The cells were pelleted by
centrifugation at 4 °C, 1600 rpm for 6 minutes. The resuspended cells were plated in
plastic cell culture dishes with the density from 1x10
4
to 5x10
4
per 100mm dish.

Cell culture
MSC culture medium was Alpha MEM (Life Technologies/Gibco) supplemented with 20%
of fetal bovine serum (Equitech-Bio Inc, TX), 100 U/ml penicillin/streptomycin (Life
Technologies/Biosource,), 2 mM L-Glutamine (Life Technologies/Biosource), 55uM
beta-mercaptoethanol (Life Technologies/Gibco), but not dexamethasone (Dex-). Culture
medium of the primary MSC culture was changed twice in the first week. On Day 8, half
of the medium was replaced with the above medium containing 10
-8
M dexamethasone
(Sigma) (Dex+). On Day 10, the entire medium is replaced with Dex+ medium. After at
least 2-week selection, cells remained growing on the dish were colonized, These
adherent cells were then trypsinized and passed to fresh dishes. The newly seeded cells
were regarded as passage 1 of potential lung MSCs, and were maintained in the Dex+
medium and passed to fresh dishes whenever they reach 80% confluence. These lung
MSCs within 10 passages were used for the experiments described below.

Colony forming unit-fibroblastic (CFU-F) assay
To quantify the lung resident MSC pool, we took advantage of colony forming unit assay.
The freshly isolated cells were seeded on to T25 cell culture flasks at serials dilutions
(1,000~100,000 cells per flask). After the 2-week colonization in MSC culture medium
without dexamethasone, culture medium is removed and adherent cells are washed with
78

PBS, fixed and stained with 1% Toluidine Blue in 2% paraformaldehyde PBS overnight.
The staining solution was then removed on the second day and the flask was washed with
deionized H
2
O to rinse off excessive staining. The flasks were left at room temperature to
air dry. The numbers from flasks of each group with moderate colony density were
counted.

BrdU labeling
To determine the potential lung mesenchymal stem cell proliferation rate, P1 of the lung
MSCs were seeded at a density of 5,000 cells per well on 4-well chamber slides, BrdU
label (1:2000 in cell culture medium, Calbiochem) was added. After 24 hours of BrdU
labeling, cells were fixed, and the percentage of cells that were positively stained with the
BrdU staining kit (Invitrogen) was counted and analyzed.

Immunofluorescent microscopy
Lung mesenchymal stem cells (passage 1), isolated from wildtype mice, were seeded at a
density of 1,000 cells per well on 8-well chamber slides and cultured overnight. The 50%
confluent cells were rinsed in PBS and fixed with 4% paraformaldehyde. These cells
were analyzed by immunostaining for specific markers of MSCs (CD105, CD73, CD90,
Sca-1) and markers for hematopoietic stem cells (HSCs: CD11b, CD34, CD45) as well as
marker for endothelial cells (PECAM1). Briefly, slides were incubated with normal
serum, which was from the same species of secondary antibody. After blocking, the
slides were incubated with the specific primary antibodies overnight at 4° C, followed by
the incubation with Alexa Fluor® 488-conjugated or Alexa Fluor® 594-conjugated
79

secondary antibody (1:200, Invitrogen) for 30min at room temperature in dark. Slides
were counterstained for nuclei with DAPI and mounted with VECTASHIELD®
Mounting Media (Vector Laboratories).

Flow cytometry analysis and sorting
The isolated lung MSCs were also evaluated by flow cytometry (FCM) for specific
markers of MSCs (CD105, CD73, CD90, Sca-1) and other markers for hematopoietic
stem cells (HSCs) (CD11b, CD34, CD45). Lung MSCs within passage 3 were trypsinized
from 100 mm dishes, pelleted by centrifugation at 4° C, 1600 rpm for 6 minutes, washed
with PBS, and finally resuspended in FACS blocking buffer (HBSS without phenol
red/Calcium/Magnesium, supplemented with 5% FBS, 1% BSA, 5% normal serum, 1%
penicillin/streptomycin). The total cell number was determined per standard protocol and
0.2-0.3x10
5
cells in 100μL were aliquot into each FACS tube. Each sample was
incubated with 4μL APC-conjugated primary antibodies (Biolegend) or APC-conjugated
isotype-matched control immunoglobulin IgG2a, IgG2b, IgG1 (Biolegend) on ice for 45
minutes, washed and suspended in 100μl FACS wash buffer (HBSS without phenol
red/Calcium/Magnesium and supplemented with 5% FBS). When in need, 100μL of 4%
paraformaldehyde can be added to each sample for the preservation of the stained
samples at 4° C in dark for a week. The FACS analysis was performed on FACS
Calibur

(BD Bioscience) at the Center for Craniofacial Molecular Biology of University of
Southern California with the help from Dr. Chider Chen.
The lung MSCs isolated from aforementioned Tbx4-rtTA/TetO-cre/mTmG transgenic
mice were directly analyzed with flow cytometry sorting. The endogenous GFP or RFP
80

fluorescence of the isolated cells could be detected by FACS sorting without antibody
enhancement. Lung MSCs were harvested and single cell suspension was prepared in
FACS sorting buffer (1mM EDTA, 25mM HEPE pH 7.0, 1% FBS in 1 x PBS without
Calcium/Magnesium). The FACS sorting was processed on FACSDIVA at the Flow
cytometry core of Children’s Hospital of Los Angeles with the help from Ann George.
Sorted GFP and RFP lung MSCs subpopulations were separately collected in Dex+
medium and plated for further analysis.

In vitro adipogenic assay
Adipocyte differentiation was induced in expanded lung mesenchymal cell cultures
(passage 3) for nine days by treating cells with the Dex+ medium containing 0.5 mM l-
fiiethyl-3-isobutylxanthine, 10μg/ml insulin, 0.5μM hydrocortisone (Sigma), 0.1 mM L-
ascorbic acid phosphate (Wako), and 60μM indomethacin (Sigma) (Mark F. Pittenger,
1999), which was changed every three day.
The intracellular lipid droplets were detected with freshly prepared 0.3% Ohil red O
staining working solution, which was diluted and filtered Oil red isopropanol saturated
solution in ddH
2
O. Briefly, cell culture medium was removed, and the adherent cells
were washed with PBS twice and fixed with 4% paraformaldehyde at room temperature
for 10 minutes, washed with PBS twice again and treated with 60% isopropanol for 1
minute, then incubated with the Oil red working solution for about 20 minutes at room
temperature. The samples were washed with PBS twice and examined under transmitted
light microscope.

81

Doxycycline induced Cre-recombinase expression in lung MSC culture
Lung mesenchymal cells were isolated from the triple transgenic Tbx4-rtTA/TetO-
Cre/mT-mG mice which have not been treated with doxycycline in their prenatal and
postnatal life. In vitro doxycycline induction was initiated with change of medium to the
Dex+ MSC culture medium supplemented with 1μm/ml Doxycycline, which was
reported to be an effective dose for rtTA-M2-mediated TetO promoter activation
(Urlinger et al., 2000). After 48 hours of induction, these cells were examined under
Leica fluorescence microscope for GFP or Tomato expression. The doxycycline solution
was prepared by dissolving 100mg doxycycline in 1ml ddH
2
O and filtered through a
0.02μm syringe filter.

82

RESULT
Isolation and verification of lung resident mesenchymal stem cells from wild type
adult mice
The potential lung mesenchymal stem cells were isolated from wildtype mice initially to
establish and optimize the isolation procedures. Lung MSCs were also isolated from
Tbx4-rtTA/TetO-Cre/mT-mG transgenic mice and Smad3 knockout mice. After two
weeks of colonization and selection in mesenchymal stem cell culture medium, the
plastic-adherent cells were considered as passage 0 of lung MSCs. The cells were
maintained in the MSC medium containing dexamethasone. The medium is replaced
twice a week. When adherent cells grow to about 80% confluence, they will be
trypsinized and passed to fresh dishes at the density of 20,000 ~ 50,000 cells/10cm
2
dish.
To measure colony forming units of lung resident mesenchymal stem cells,
1,000~100,000 fresh lung cells from the wildtype mouse were plated onto the T25 flask,
after 14 days of cell culture in Dex- medium, the total colony number was collected.
75,000 cells from fresh adult lung were plated onto T25 Flask, and 121 colonies were
formed after 14 days of cell culture (Fig.1)
The potential lung MSCs were verified by positive MSC marker staining: CD73, CD90,
CD105; and negative HSC marker staining: CD45, CD34, CD14.as shown in Figure 2.
Flow cytometry analysis was also employed to examine expression of MSC-specific
markers in these potential lung MSCs isolated from wildtype mice (Fig.3). 80.8% of the
cell population expressed positive for CD105, 83.1% for Sca-1, and 66.7% for CD90,
while only 0.44% of the population was positive for CD34, 0.81% for CD45, and 0.77%
for CD11b.
83

Adipogenic differentiation was performed in expanded lung mesenchymal cell cultures
for nine days by treatment with the differentiation medium specified in the method
section above (Mark F. Pittenger, 1999). The differentiated adipocytes were visualized
with Oil red staining. As shown in Figure 4, the isolated lung mesenchymal stem cells
were able to differentiate into adipocytes when cultured with the adipogenic medium.
The lipid droplets were stained red with Oil red, while no Oil Red staining was observed
in the control group in which the isolated lung MSCs were cultured with regular MSC
Dex+ medium.
Furthermore, lung MSCs of wild type mice were plated on 4-well chamber slides,
followed by 24-hour incubation with bromodeoxyuridine (BrdU) at 37 °C (Akiyama et
al., 2012). 76% of the adherent cells were labeled by BrdU incorporation into nuclear
DNA as a result of cell proliferation related DNA synthesis, detected by anti-BrdU
immunostaining (Fig.5).

Identification of lung resident MSC subpopulations with different developmental
origins
The triple transgenic (Tbx4-rtTA/TetO-Cre/mT-mG) reporter mice with Dox induction
from E6.5 to E18.5 were generated as described previously in Chapter 1. Mouse lung
resident MSCs were then isolated from a 3-month old mouse. Surprisingly, isolated lung
resident MSCs were composed of two different types of cells, (1) mGFP+ cells and (2)
mTomato+ cells. From the data shown in Chapter 1, Dox induction from E6.5 to E18.5
resulted in Cre-mediated mGFP expression in almost all mesenchymal cells in
developing lung. All descent cells from these mesenchymal cells in developing lung
84

should still be mGFP-positive. Therefore, mTomato positive MSCs were derived from
some cells that was not targeted by the Tbx4-rtTA during development, most probably
mesenchymal progenitor cells of other organs (Fig. 1B). The established mesenchymal
stem cell culture at passage two was then sorted by flow cytometry. About 59% of the
total population was mGFP-positive, while 41% of the sorted cells were mTomato-
positive. These sorted two subpopulations were collected, and cultured in Dex+ MSC
medium.
The expanded cultures were used for their cell surface marker analyses using
immunostaining and subsequent FACS. As shown in Figure 6, both mGFP and mRFP
subpopulations were verified by positive MSC markers staining (Sca-1, CD105, CD90),
and were negative for other cell markers, including CD34, CD11b, CD19, CD14, CD31,
CD326, and CD45. However, the expression level of CD105 is noticeably lower in
mTomato population than that in mGFP population (27.9% Versus 82.2%).

TGF- -Smad3 pathway plays important role in regulating lung resident MSCs in
vivo
As mentioned in the introduction above, spontaneous emphysema-like pathology occurs
in Smad3 knockout mice at the age of 1 month. One potential mechanism is disruption of
cell homeostasis, caused by increased cell damage and/or reduced cell repair and
regeneration. We then determine the possibility of changes in lung resident MSCs in
Smad3 knockout mice. Fresh lungs were isolated from Smad3 knockout mice and their
wild type littermate controls at the age of 4 weeks. The lung tissues were enzymatically
digested per the methods discussed above. The freshly isolated cells were seeded on to
85

T25 cell culture flask at densities of 1,000 cells ~ 100,000 cells per flask. After a 2-week
colonization in MSC culture medium without dexamethasone, adherent cells are fixed
and stained as described in the methods (Fig. 7). The numbers of MSC colonies were
counted under microscopy. Compared to the wild type controls, the colonies of lung
MSCs in Smad3 knockout lungs was decreased by 40% ~ 60%.
Proliferations of these lung MSCs between Smad3 knockout and wild type control were
also assessed based on the rate of BrdU incorporation of the passage 1 (Fig. 8). After 24-
hour incubation with BrdU, about 50% of lung MSCs from wildtype mice were BrdU
positive, while only about 30% of lung MSCs from Smad3 knockout mice were BrdU
positive. Lung MSCs from Smad3 knockout mice also had slower passage double rate.
As shown in Figure 9, the same number (1,000

or 5,000

) of established lung
mesenchymal stem cells from wildtype mice or Smad3 KO mice were plated on T25
flasks. After 7 days, wild type lung MSC culture reached 100% confluence, while Smad3
knockout lung MSC culture were about 50 ~ 80% confluence. These suggest that TGF-b-
Smad3 signaling is required for normal lung MSC proliferation and maintenance of the
appropriate MSC pool size.
In addition, induction of adipocyte differentiation from these lung MSCs did not have
significant difference (Figure 10), suggesting that disrupted TGF-b-Smad3 signaling may
not have impact on the cell differentiation potency.
86

A

B

Figure 1 Isolation and characterization of lung mesenchymal stem cells. A. Colony
Forming Unit Assay: 75,000cells from fresh adult lung were plated onto T25 Flask. 121
colonies were formed after 14 days of cell culture in Dex+- MSC medium. B. mGFP
Lung resident MSCs were isolated from a 2-month-old triple transgenic reporter mouse
(Tbx4-rtTA/TetO-Cre/mT-mG) with Dox induction from E6.5 to E18.5. Therefore, the
fetal lung mesenchymal cells were labeled by mGFP expression while the rest cells
remained mTomato expression. The established lung MSC culture had two populations,
fetal lung origin (green) and other tissue origin (red).
87

Figure 2 Verification of isolated lung resident MSC. By immunostaining, these cells
were positive for MSC markers including CD73, CD90, and CD105; and negative for
HSC markers including CD45, CD34, and CD14.

88

Figure 3 Verification of isolated lung resident MSC. Cell surface markers of the
isolated lung MSC were also determined by flow cytometry analysis.
CD105 SCA-1 CD90

80.8% 83.1% 66.7%

CD34 CD45 CD11b

0.44%
0.81% 0.77%
89

Figure 4 Isolated lung MSCs were able to differentiate to adipocytes. MSCs were
cultured in adipogenesis medium for 9-day induction, and the adipocytes were detected
by oil red staining.
AD medium
Dex(+)
90

Figure 5 Proliferation of the isolated lung MSCs was detected by 24 hour BrdU
incorporation to nuclear DNA.

91

92

Figure 6 Flow cytometry analysis of the mGFP-labeled and mRFP-labeled lung
mesenchymal stem cell. The positive percentage of each sample is marked as shown.
APC-conjugated antibodies were used to analyze the mGFP and mRFP cells.
93

WT Smad3 KO

Figure 7 Smad3-/- mice has reduced number of lung MSCs than Wildtype mice.
Colony forming unit assay was employed to compare the MSC pool between Smad3-/-
mice and Wildtype mice. Approximately 1000 fresh isolated lung cells were seeded on to
T25 flask. The staining was performed after 13 days of colonization.

94

Figure 8 MSCs from Smad3-/- lung has reduced proliferation rate. BrdU labeling
was used to contrast the MSC proliferation rate of Smad3-/- mice and of Wildtype mice.
After 24-hour incubation with BrdU, about 50% of lung MSCs from wildtype mice were
BrdU positive, while only about 30% of lung MSCs from Smad3 knockout mice were
BrdU positive.
WT
KO
95

Figure 9 MSCs from Smad3-/- lung has reduced passage doubling rate The same
number (1,000

and 5,000

) of established lung mesenchymal stem cells from wildtype
mice or Smad3 KO mice were plated on T25 flasks. After 7 days, wild type lung MSC
1000

cells/flask 5000

cells/flask
WT KO
WT KO
96

culture reached 100% confluence, while Smad3 knockout lung MSC culture were about
50 ~ 80% confluence.

97

Figure 10 Adipogenesis of the lung MSCs from wildtype mouse and Smad3 -/-
mouse. Induction of adipocyte differentiation from these lung MSCs did not have
significant difference, suggesting that disrupted TGF-b-Smad3 signaling may not have
impact on the cell differentiation potency.

WT
KO
98

Figure 11 Lung MSCs isolated from Smad3 knockout lungs exhibited reduced
expression of CD105

WT
Smad3 KO
99

DISCUSSION
In this project, we have established a method to isolate lung resident MSCs by adopting
the methods used for bone marrow mesenchymal stem cell isolation. According to the
International Society for Cellular Therapy (ISCT) (M Dominici et al., 2006), MSCs are
defined based on the following criteria: (1) MSC must be plastic-adherent when
maintained in standard culture conditions using tissue culture flasks; (2) 95% of the
MSC population must express CD105, CD73 and CD90, lack expression of CD45, CD34,
CD14 or CD11b, CD79  or CD19 and HLA class II, as measured by flow cytometry; (3),
the cells must be able to differentiate to osteoblasts, adipocytes and chondroblasts under
standard in vitro differentiating conditions.
We also applied our novel Tbx4-rtTA/TetO-Cre/mT-mG transgenic mouse model to
study the origins of these lung resident MSCs, and identified two subsets of lung resident
mesenchymal stem cells distinguished by their different fluorescence protein expression
(mGFP versus mTomato). Based on the characterization of this triple transgenic line as
described in Chapter 1, mGFP cells are derived from embryonic/fetal lung mesenchymal
cells, and mRFP cells are derived from cells other than these embryonic lung
mesenchymal cells. Further studies are needed to determine the source of these
mTomato-positive MSCs.
TGF-  signaling plays an important role in regulating fetal lung morphogenesis, postnatal
lung growth, injury repair, and remodeling. Blockade of TGF-  activation by knocking
out fibrillin 1, latent TGF-  binding protein 4, and 6-intergin in mice causes alveolar
simplification and emphysema-like pathology, suggesting an important regulatory
function for TGF-  signaling in lung cell homeostasis (Warburton et al., 2010b). In our
100

Smad3 KO emphysema mouse model, we have observed retardation of lung
alveolarization in early postnatal development and subsequently centrilobular
emphysema starting from postnatal day 28, while disruption of TGF-beta signaling in
lung epithelial cells alone did not cause subsequent emphysema. The mechanisms of
emphysema pathogenesis in this mouse model have not been defined. Recently autocrine
production of TGF- 1 was reported to promote myofibroblastic differentiation of
neonatal LMSCs (Popova AP and MB., 2010). TGF-  signaling pathway may plays an
essential role in mesenchymal stem cells and the related mesenchymal cell homeostasis.
We determined the quantitative and qualitative change of MSCs in the emphysema lung
of Smad3 KO mice. The difference in the number, proliferation, and differentiation
ability of lung MSCs between wild type and Smad3 knockout lung was investigated. My
preliminary data suggested that Smad3 knockout lung had deficiency of lung MSCs,
further effort needs to be spare to understand whether this deficiency contributes to
reduced tissue injury repair and emphysema in vivo. For example, the comparison of
MSC-treated and untreated Smad3 knockout mouse lung will indicate whether exogenous
wild-type MSCs are able to correct abnormal lung homeostasis and prevent or postpone
emphysema onset, and whether administration of normal MSCs will relief or slow down
alveolar destruction and emphysema progression. We expect that replenishment of lung
MSC subsets may prevent or delay alveolar enlargement and improve the lung function
of adult Smad3 KO mice.
The mGFP and mRFP subpopulations of lung MSCs isolated from the Tbx4-rtTA/TetO-
Cre/mT-mG mice showed different expression level of CD105. Lung MSCs isolated
from Smad3 knockout also expressed lower level of CD105 compared to the expression
101

level of wildtype lung MSCs (22.77% versus 55.69% in Figure 11). Whether the
deficiency of lung MSCs of Smad3 knockout mice is a result from the insufficiency of
mGFP lung MSC population could also be studied.

102

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Abstract (if available)

Abstract Chapter 1 ❧ Background: Reciprocal interactions between lung mesenchymal and epithelial cells play essential roles in lung organogenesis and homeostasis. Altered lung mesenchymal cell number and function are related to many chronic lung diseases, such as pulmonary fibrosis and emphysema. Although the molecular markers and related animal models that target lung epithelial cells are relatively well studied, molecular markers of lung mesenchymal cells and the genetic tools to target and/or manipulate gene expression in a lung mesenchyme‐specific manner are not available. The ability to manipulate gene expression in specific cell type in the lung mesenchyme is important for understanding the molecular mechanisms of the lung mesenchymal biology and the related pulmonary diseases. Lack of knowledge of gene regulatory elements that contribute to lung mesenchymal cell specificity is a major barrier for developing such genetic tools. ❧ Result: We have characterized a mouse Tbx4 gene enhancer that contains conserved DNA sequences across many vertebrate species with lung or lung‐like gas exchange organ. Two transgenic mouse lines were then generated to express rtTA/LacZ under the control of this Tbx4 lung enhancer. By combining the Tbx4-rtTA driven Tet‐On inducible Cre recombinase expression mouse lines with a mT‐mG dual fluorescence reporter mouse line, the spatial‐temporal patterns of Tbx4 lung enhancer‐targeted lung mesenchymal cells were defined. The Tbx4 lung enhancer was active only in lung mesenchymal cells, but not in other tissues, from prenatal developmental stage to adult. Pulmonary endothelial cells and vascular smooth muscle cells were only targeted by the Tbx4-rtTA driver line prior to E11.5 and E15.5, respectively, while other subtypes of lung mesenchymal cells including airway smooth muscle cells, fibroblasts, pericytes could be targeted during the entire developmental stage. ❧ Conclusion: Activation of the Tbx4 lung enhancer is only detected in mesenchymal cells of developing lung. With our newly created Tbx4 lung enhancer‐driven Tet‐On inducible system, lung mesenchymal cells can be specifically and differentially targeted in vivo at the first time by controlling the doxycycline induction time window. This novel system provides a unique tool to study lung mesenchymal cell lineages and gene functions in lung mesenchymal development, injury repair, and regeneration in mice. ❧ Chapter 2 ❧ Background: Although bone marrow mesenchymal stem cells (BMMSCs) have been extensively studied, tissue specific MSCs, including lung resident MSCs, are poorly defined and characterized. Studies have shown that stem cell‐mediated injury repair and regeneration is critical to maintain normal lung structure and function. The increasing recognition of the properties of mesenchymal stem cells has led to various pathogenesis‐oriented studies. The roles of lung resident MSCs in lung diseases have never been studied. Emphysema is the third leading cause of death in the U.S.

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Asset Metadata

Creator Zhang, Wenming (author)

Core Title Lung mesenchyme cell biology

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Publication Date 08/21/2014

Defense Date 05/15/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag lung mesenchyme,mesenchymal stem cell,OAI-PMH Harvest,Smad3,Tbx4 lung enhancer,Tet‐On system

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Shi, Wei (committee chair), Grikscheit, Tracy (committee member), Paine, Michael L. (committee member), Shi, Songtao (committee member), Warburton, David (committee member)

Creator Email wenmingz@outlook.com,wenmingz@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-462894

Unique identifier UC11287589

Identifier etd-ZhangWenmi-2837.pdf (filename),usctheses-c3-462894 (legacy record id)

Legacy Identifier etd-ZhangWenmi-2837.pdf

Dmrecord 462894

Document Type Dissertation

Format application/pdf (imt)

Rights Zhang, Wenming

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA