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Molecular and Cellular Biology, July 2000, p. 5208-5215, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Fatal Bilateral Chylothorax in Mice Lacking the
Integrin 
9
1
X. Z.
Huang,1,2
J. F.
Wu,1,2
R.
Ferrando,1,2
J. H.
Lee,1,2
Y. L.
Wang,1,2
R. V.
Farese Jr.,2,3 and
D.
Sheppard1,2,*
Lung Biology Center, Center for Occupational
and Environmental Health, Cardiovascular Research
Institute,1 and Department of
Medicine,2 University of California, San
Francisco, and Gladstone Institute of Cardiovascular
Disease,3 San Francisco, California
Received 29 February 2000/Accepted 28 March 2000
 |
ABSTRACT |
Members of the integrin family of adhesion receptors mediate both
cell-cell and cell-matrix interactions and have been shown to play
vital roles in embryonic development, wound healing, metastasis, and
other biological processes. The integrin 
9
1 is a receptor for the
extracellular matrix proteins osteopontin and tenacsin C and the cell
surface immunoglobulin vascular cell adhesion molecule-1. This receptor
is widely expressed in smooth muscle, hepatocytes, and some epithelia.
To examine the in vivo function of 91, we have generated mice
lacking expression of the 9 subunit. Mice homozygous for a null
mutation in the 9 subunit gene appear normal at birth but develop
respiratory failure and die between 6 and 12 days of age. The
respiratory failure is caused by an accumulation of large volumes of
pleural fluid which is rich in triglyceride, cholesterol, and
lymphocytes. 9
/ mice also develop edema
and lymphocytic infiltration in the chest wall that appears to
originate around lymphatics. 9 protein is transiently expressed in
the developing thoracic duct at embryonic day 14, but expression is
rapidly lost during later stages of development. Our results suggest
that the 9 integrin is required for the normal development of the
lymphatic system, including the thoracic duct, and that 9 deficiency
could be one cause of congenital chylothorax.
| |
INTRODUCTION |
Integrins are heterodimeric
receptors for extracellular matrix and cell surface counterreceptors,
which play important roles in embryonic development, inflammation,
wound healing, and tumorigenesis (8, 9, 15). The integrin
1 subunit pairs with at least 12 subunits, forming the largest
subfamily of integrins. Ablation of the 1 gene produces early
embryonic lethality (3, 17), and most null mutations
described for individual 1-associated subunits cause severe but
individually distinct developmental phenotypes (10).
91 is a member of the 1 family that recognizes tenascin C
(23, 26), osteopontin (16, 24), and vasular cell
adhesion molecule-1 (VCAM-1) (20) as ligands. Immunostaining
of mouse tissue has shown that this integrin is expressed in skeletal
and cardiac muscle, visceral smooth muscle, hepatocytes, airway
epithelium, squamous epithelium, and choroid plexus epithelium
(13, 21). Wound healing experiments performed on mouse
corneas (18, 19) suggest that 9 may play a role in
corneal epithelial migration and differentiation. In mouse embryos,
9 expression was not detected prior to embryonic day 12.5 (E12.5)
(21), suggesting that this integrin is unlikely to play a
general role in the earliest stages of tissue morphogenesis.
In vitro experiments demonstrate that 91 mediates cell adhesion
as well as cell migration on all three known ligands (16, 20,
26). In addition, 9-transfected SW480 cells use 91 to
proliferate on tenascin-C (25), and these effects are
associated with phosphorylation of the integrin signaling intermediates
focal adhesion kinase and paxillin and with activation of the
mitogen-activated protein kinase isoform, erk2. However, the in vivo
function(s) of 91 is unknown.
In this study, we have generated mice homozygous for a null mutation of
the 9 subunit gene to directly examine the role(s) of 9 integrins
in vivo. 9/ mice were born at the
expected Mendelian frequency but developed bilateral chylothorax within
6 to 12 days after birth and died of respiratory failure. The presence
of edema and extravascular lymphocytes surrounding the thoracic duct
and other lymphatic vessels suggested a defect in lymphatic
development. These results suggest that 91 plays a critical role
in development of the thoracic duct and other lymphatic vessels and
that mutations in the 9 subunit gene could be one cause of
congenital chylothorax.
| |
MATERIALS AND METHODS |
Constructing the mouse 9 targeting vector.
A 240-bp
fragment of murine 9 cDNA was amplified from RNA obtained from
murine liver using the degenerate integrin subunit PCR primers A14F
and A2AR that we have previously described (2, 14). This
fragment was cloned into pBluescript and completely sequenced from both
strands. The resultant sequence was used to design the murine
9-specific PCR primers, m9-1F (5'-TCCTCCTTGTGTGCAGTCGACC-3') and m9-3R (5'-TCTTGAATTCTCATCTCTGATCTCAGAA-3').
These primers were used to identify an approximately 80-kb
genomic P1 clone (clone 2532; Genome System, Inc., St. Louis, Mo.)
containing the mouse 9 gene. A 7.4-kb BglII fragment
containing two exons from this clone was used for making a replacement
targeting vector. The vector contained a neomycin resistance gene
inserted into the first of the two exons in the clone and a thymidine
kinase gene at the 5' end.
Detection of recombinant clones by Southern blotting.
As
described previously (7), RF8 embryonic stem (ES) cells were
grown in ES complete medium. The targeting vector was linearized at a
unique SacII site and electroporated into RF8 ES cells.
Selection medium containing G418 plus
1-(2'-deoxy-2'-fluoro--D-arabinofuranosyl)-5-iodouracil (FIAU) was used to obtain resistant clones. Individual colonies were
screened by Southern blotting. Genomic DNA digested with SacI was blotted with two different probes, one specific for
mouse 9 and the other for the neo gene. Only clones with
single integration were used for blastocyst injection.
Generation of germ line chimeras.
Chimeras were generated as
described by Bradley (1). Targeted ES cells were injected
into C57BL/6 blastocysts, and injected embryos were transferred into
the uteri of pseudopregnant recipients. Male chimeras were mated with
C57BL/6 females. Agouti offspring were tested for the targeted 9
gene by PCR and Southern blotting.
Reverse transcription-PCR (RT-PCR).
Total RNA was extracted
from freshly isolated mouse tissue using TRIzol solution (Gibco/BRL)
according to the company's recommendations. Single-stranded cDNA was
transcribed from RNA using the superscript cDNA synthesis system and
random hexamers (Gibco/BRL). PCR was performed with the 9-specific
primers m9-1F and m9-3R to show that the transcription of 9
was disrupted in 9/ mice.
Western blot analysis.
Freshly isolated mouse liver was
minced and then ultrasonicated in lysis buffer (150 mM NaCl, 50 mM
Tris, 1% Triton X-100, 0.1% sodium dodecyl sulfate). The homogenates
were centrifuged, and the supernatant was saved. Eighty micrograms of
total protein from both 9+/+ and
9/ lysate was separated on a 7.5%
polyacrylamide gel and transferred onto Immobilon membrane (Millipore,
Bedford, Mass.) using a Hoefer transfer apparatus. The membrane was
blocked with 5% milk overnight at 4°C (or 2 h at room
temperature) and blotted with rabbit anti-9 antiserum 1057, raised
against a portion of the cytoplasmic domain of human 9
(13). The membrane was exposed to film after a brief incubation in Luminol (Amersham, Arlington Heights, Ill.).
Histology and immunohistochemistry.
For histology, mouse
tissue was fixed in 10% formalin for 48 h and embedded in
paraffin. Sections (5 µm thick) were stained with hematoxylin and
eosin (H&E). For immunohistochemistry, freshly isolated organs were
embedded in OCT and quick frozen in liquid nitrogen. Unfixed sections
were air dried for 30 min and washed twice with phosphate-buffered
saline (PBS). Sections were blocked for endogenous peroxidase and
biotin activities with Peroxoblock solution (Zymed Labs) and an
avidin-biotin blocking kit (Vector) at room temperature. After rinsing,
sections were preblocked with 3% normal goat serum in PBS for 15 min
and then incubated overnight at 4°C (or 1 h at room temperature)
in primary antibody. After being rinsed in PBS, sections were incubated
in biotin-labeled secondary antibody for 1 h, which was followed
by incubation in ABC avidin-peroxidase reagent (Vector Labs) for
another hour at room temperature. Chromagen was developed using the DAB
Plus kit (Zymed Labs). Finally, sections were dehydrated, cleared, and coverslipped using Permount.
| |
RESULTS |
Generation of 9-deficient mice.
The targeting strategy to
inactivate the 9 subunit gene is outlined in Fig.
1A. The linearized construct was
electroporated into RF8 embryonic stem cells and the resultant targeted
ES cell clones were used to generate 9-deficient mice. Offspring
were tested for the targeted 9 gene by Southern blotting (Fig. 1B) and PCR.
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FIG. 1.
Disruption of the mouse 9 gene by homologous
recombination. (A) Structures of mouse 9 wild-type allele, targeting
vector, and targeted allele. Two exons are shown as solid boxes. The
expected fragment size after SacI digestion is 3.4 kb for
the wild-type allele and 4.9 kb for the targeted allele. (B) Southern
blot analysis of genomic DNA from mouse tail digested with
SacI and hybridized with the external specific probe of
mouse 9 indicated in panel A. (C) RT-PCR analysis of mRNA from
9+/+ and 9/
mice. Total RNA was extracted from mouse liver and transcribed to
complementary DNA (cDNA). A 95-bp fragment was amplified from
9+/+ mouse but not from
9/ mouse using primers specific for
wild-type 9 cDNA. (D) Western blotting of cell lysate of mouse liver
with a polyclonal antiserum against 9. A band of the appropriate
size to be 9 is demonstrated in 9+/+ but
not in 9/ mice. The positions of
molecular mass markers (in kilodaltons) are shown to the right.
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9 mRNA and protein expression were also examined. RT-PCR analysis of
RNA derived from mouse liver showed that an expected
95-bp fragment was
amplified from the cDNA of
9+/+ mice, but no
amplification product was detectable from the cDNA
of
9/ mice (Fig.
1C). To confirm that
9/ mice did not produce
9 protein, we
performed Western blot analysis
using a polyclonal antiserum against
the cytoplasmic domain of
human
9. The
9 antibody detected a band
of the appropriate molecular
mass to be
9 in
9+/+ mouse liver homogenates; however, no
band was detected in
9/ mouse liver (Fig.
1D).
9-deficient mice are born alive but develop bilateral
chylothorax.
Cross-breeding of heterozygous
9+/ mice gave rise to viable homozygous
offspring in the expected Mendelian distribution
(9+/+, 25.5%;
9+/, 51.8%;
9/, 22.7%), indicating that 9
integrins are not essential for intrauterine development. For the first
4 to 6 days after birth, 9/ mice appeared
normal. However, by 6 to 8 days of age the
9/ mice appeared smaller than control
littermates and began to show signs of respiratory distress. The body
weights of 6- to 8-day-old 9/ mice were
less than those of wild-type littermates
(9/, 4.98 ± 0.43 g;
9+/+, 6.12 ± 0.17 g).
Between 6 and 12 days of age, 100% of
9/
mice showed signs of respiratory distress and were physically inactive.
9/ mice died within 2 days of the onset
of respiratory distress.
To determine the cause of death, the whole
mouse body was embedded
and frozen in OCT, and cross sections were
made. Sections through
the thorax of
9/
mice revealed a marked increase in the space between the outer
surface
of the lung and the chest wall, and this space was filled
with opaque
milky fluid (Fig.
2A). This pleural fluid
was separated
into three layers (Fig.
2B) by centrifugation. Cell
differential
counts showed more than 90% of the cells in this fluid
were lymphocytes.
Analysis of pleural fluid demonstrated a very high
concentration
of both triglycerides and cholesterol, confirming that
these mice
had developed chylothorax (Table
1).
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FIG. 2.
Appearance of pleural effusion in
9/ mice. (A) Cross section through mouse
thorax shows fluid filling an expanded pleural space (demarcated by
arrowheads) in an 9/ mouse compared to
the absence of any identifiable space in an
9+/+ littermate. Arrowheads indicate
locations of visceral pleura and the external surface of the ribs. (B)
Fluid collected from the pleural space of
9/ mice is milky prior to centrifugation
(left) and can be separated into three layers by centrifugation
(right): top, white lipid layer; middle, transparent aqueous layer;
bottom, cell pellet, typical of chylous effusion.
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To date, from more than 100
9/ mice
analyzed not a single mouse has survived from either a 129/C57BL/6
mixed or pure 129 inbred
genetic background, suggesting that a
strain-specific modifier
is not the cause of the defect in
9/ mice. By draining 50 to 200 µl of
chyle daily with a syringe,
we were able to postpone respiratory
failure and extend the life
of
9/ mice up
to 21 days, confirming that respiratory failure from
chylothorax
contributed to their death. However, treated mice
failed to gain weight
and eventually died, presumably as a consequence
of
malnutrition.
Because we have previously reported that
9 is expressed in skeletal
muscle, visceral smooth muscle, hepatocyte, squamous
epithelium, and
airway epithelium (
13,
21), we analyzed the
effects of
9
deficiency on all of the organs in which we have
detected
9
expression. However no gross or microscopic abnormalities
were found in
these
tissues.
Lymphatic vessels in 9/ mice.
A
common cause of chylothorax is leakage of chyle from the thoracic duct.
We therefore examined the thoracic duct, grossly and microscopically,
in wild-type and 9/ mice. The thoracic
duct was easily visualized in 6- to 10-day-old wild type and
9/ mice on the basis of its white color,
and no gross defects were apparent in 9/
mice in either the integrity or diameter of this structure (Fig. 3A and B).
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FIG. 3.
Anatomy and histology of thoracic duct in
9/ mouse and a wild-type littermate at 8 days of age. Presented are photographs of the opened thorax of
9+/+ (A) and 9/
(B) mice, showing the presence of a visible thoracic duct in both
groups (the white tubular structure along the spine denoted by
arrowheads). (C and D) H&E-stained sections of the region including the
thoracic duct (T) and the adjacent aorta (A) in
9+/+ (C) and 9/
mice (D), demonstrating edema and extravascular lymphocytes surrounding
the thoracic duct in 9/ but not in
9+/+ mice.
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Microscopically (Fig.
3C and D), we were able to identify the thoracic
duct in H&E-stained sections from both wild-type and
9/ mice as a thin-walled structure
immediately to the right of the
posterior portion of the aorta. This
structure was composed of
a tubular endothelial layer with no luminal
red blood cells surrounded
by a thin layer of smooth muscle. The
microscopic structure of
the thoracic duct was not consistently
different between
9/ and
9+/+ mice. However, in several
9/ mice obvious edema and extravascular
lymphocytes were present
in the tissue surrounding the thoracic duct,
findings that were
not observed in sections from wild-type
mice.
9/ mice also develop edema and
inflammatory cell accumulation in the chest wall.
To determine
whether the apparent extravasation of fluid from the thoracic duct in
9/ mice reflects a more generalized
defect in lymphatic development, we also examined the morphology of the
lymphatics in the chest wall. Sections from the chest wall of
9/ but not wild-type mice demonstrated
numerous areas of tissue edema and lymphocyte accumulation, and
extravascular lymphocytes were most prominent in close proximity to
lymphatic vessels (Fig. 4A and B). Such
accumulations of lymphocytes could be seen throughout the skeletal
muscle of the chest wall (Fig. 4C and D) and even within the dermis
(Fig. 4F).
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FIG. 4.
Lymphocyte infiltration in 9 null mice. Presented are
H&E-stained cross sections from the chest wall of
9/ mice, showing mononuclear cell
infiltration around lymphatic vessels adjacent to the parietal pleura
(A and B), within skeletal muscle and interstitial tissue (C and D),
and within the dermis (F) in the 9/ mice.
No such findings were ever observed in these locations in
9+/+ mice. One such example is shown for the
dermis (E).
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Transient 9 expression in the developing thoracic duct.
Because no previous reports have described expression of 9 in the
thoracic duct, the phenotype of 9/ mice
was unexpected. Our initial efforts to identify expression of this
integrin subunit in the thoracic duct of adult and newborn animals were
also unsuccessful. We therefore undertook a comprehensive evaluation of
thoracic expression of 9 during mouse embryonic development. We
performed immunohistochemistry with affinity-purified rabbit polyclonal
anti-9 antiserum on E12, E13, E14, E15, E17, and E19 embryos and on
mice at 1, 6, 8, or 60 days after birth. 9 was clearly expressed
from day E12 onward in the smooth muscle cells of the aorta,
immediately adjacent to the developing thoracic duct, raising the
possibility that normal thoracic duct development might be dependent on
an inductive signal from aortic smooth muscle cells. At only a single
time point, E14, did the cluster of cells adjacent to the aorta that
ultimately form the thoracic duct clearly demonstrate 9
immunoreactivity (Fig. 5A and B). This
staining, as well as the immunoreactivity in the adjacent aorta, was
specific, since no such staining was seen in corresponding tissue from
any 9/ E14 embryo (data not shown). 9
expression in the developing thoracic duct was transient, since
immunoreactivity was not seen in this region at any other time during
embryonic or postnatal development (Fig. 5A).
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FIG. 5.
Transient 9 expression in the
primordial thoracic duct and adjacent aorta of E14 mouse embryo. Frozen
sections fixed with acetone were stained with affinity-purified
anti-9 polyclonal antiserum (brown staining). (A) Section of an
9+/+ E19 embryo (original magnification,
×200), demonstrating staining of the aorta but not of the adjacent
thoracic duct (Td). (B) Section of an 9+/+
E14 embryo at the same magnification, demonstrating staining of both
the aorta and the adjacent tissue that will ultimately form the
thoracic duct. Airway smooth muscle (ASM) also demonstrates 9
immunoreactivity. (C) Enlargement of the same section shown in panel
B.
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By comparing the pattern of
9 expression in
9/ and wild-type mice, we were able to
confirm the specificity of our immunodetection
methods and our
previously reported cell and tissue distribution
of
9. By both
immunohistochemistry and Western blotting,
9 immunoreactivity
was
never seen in
9/ mice, and was found in a
pattern similar to those described in
our previous reports in wild-type
mice (
13,
21).
| |
DISCUSSION |
In this report, we describe a novel and completely unexpected
phenotype in mice expressing a null mutation of the integrin 9
subunit, congenital chylothorax and defective development of the
thoracic duct and other lymphatics. Our finding that 9 is transiently expressed during the early stages of thoracic duct development suggests that an 9-containing integrin, presumably 91, plays some temporally restricted critical role in formation of a functionally intact thoracic duct. Alternatively, given the expression of 9 in the adjacent aortic smooth muscle before and during the period of thoracic duct development, it is possible that an
9-containing integrin is required for the expression of an inductive
signal arising from aortic smooth muscle. Interestingly, despite
considerable indirect evidence of abnormal leakage of chylous fluid and
lymphocytes from the thoracic duct and other lymphatics in these
animals, we could not identify obvious structural abnormalities in
these vessels, suggesting that the developmental defect is subtle.
These results, while surprising, are consistent with a pattern that has
emerged from other reports of the phenotypes of mice expressing null
mutations of individual integrin subunits. Despite in vitro evidence
demonstrating considerable functional overlap among integrin
heterodimers, in virtually every case, inactivation of individual
integrins in mice has produced unique phenotypes (10). These
findings suggest that the large numbers of integrins seen in mammals
has evolved, in part, to support an array of unique functions. The
molecular mechanisms responsible for this specificity are only
beginning to emerge.
As noted in the introduction, three proteins, tenascin C, osteopontin,
and VCAM-1, have been described as ligands for 91. The effects of
null mutations in each of these 91 ligands have been reported.
Mice expressing a null mutation of the tenascin C gene have been
described by two groups (4, 5) and have either a minimal
phenotype or subtle changes in behavior, but do not develop
chylothorax. Mice expressing a null mutation of the osteopontin gene
are similarly viable and fertile, with no thoracic or lymphatic
pathology (11). Inactivation of the VCAM-1 gene does produce
a dramatic phenotype with usually lethal defects in the development of
both the placenta and the heart (6). The allantois of VCAM-1
knockout mice fails to fuse to the chorion, resulting in a defective
vasculature. A strikingly similar phenotype is seen in mice expressing
a null mutation in the integrin 4 subunit gene (22).
These data suggest that interactions between VCAM-1 and the integrin
41 play a critical role in vascular development. However, a small
number of VCAM-deficient embryos survive, and they can become viable
and fertile adults without chylothorax or other described lymphatic
abnormalities (6). Taken together, these results suggest
that the abnormality in lymphatic development we describe is not
explained by an interaction of 91 with any single ligand thus far reported.
A somewhat surprising finding from the current study was the absence of
any phenotype in nonlymphatic tissues in which 9 expression is
temporally and/or spatially regulated during development, including
squamous and airway epithelium, airway and gut smooth muscle, choroid
plexus, and liver (13, 21). However, this result is also
consistent with results seen with many other integrin knockouts and is
probably explained, at least in part, by the functional redundancy
among integrins alluded to above. Because of the early mortality of
9/ mice, it was not possible to examine
the potential functional consequences of loss of this integrin on organ
physiology or in disease models requiring adult animals.
The results of the current study provide little insight into the
mechanism(s) by which an 9-containing integrin(s) contributes to
lymphatic development. Our inability to identify any discrete structural abnormality compounds this problem, as does the scarcity of
information from previous studies on the general mechanisms underlying
lymphatic development. Nonetheless, our results suggest a potential
genetic cause of chylothorax. Congenital chylothorax is a rare disorder
in humans and has thus far been largely unexplained (12).
The results of the present study raise the possibility that some of
these cases might be due to inactivating or null mutations in the
integrin 9 subunit.
| |
ACKNOWLEDGMENTS |
This work was supported in part by National Institutes of Health
grants HL/AI33259, HL47412, HL53949, and HL56385 (D.S.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Lung Biology
Center, UCSF Box 0854, San Francisco, CA 94143-0854. Phone: (415)
206-5901. Fax: (415) 206-4123. E-mail:
deans{at}itsa.ucsf.edu.
| |
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Molecular and Cellular Biology, July 2000, p. 5208-5215, Vol. 20, No. 14
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
