* Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago,
Illinois 60637; and Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
Tensin is a focal adhesion phosphoprotein
that binds to F-actin and contains a functional Src homology 2 domain. To explore the biological functions
of tensin, we cloned the mouse tensin gene, determined
its program of expression, and used gene targeting to
generate mice lacking tensin. Even though tensin is expressed in many different tissues during embryogenesis,
tensin null mice developed normally and appeared
healthy postnatally for at least several months. Over
time, /
mice became frail because of abnormalities in their kidneys, an organ that expresses high levels of
tensin. Mice with overt signs of weakness exhibited
signs of renal failure and possessed multiple large cysts
in the proximal kidney tubules, but even in tensin null
mice with normal blood analysis, cysts were prevalent.
Ultrastructurally, noncystic areas showed typical cell-
matrix junctions that readily labeled with antibodies
against other focal adhesion molecules. In abnormal regions, cell-matrix junctions were disrupted and tubule
cells lacked polarity. Taken together, our data imply
that, in the kidney, loss of tensin leads to a weakening,
rather than a severing, of focal adhesion. All other tissues appeared normal, suggesting that, in most cases, tensin's diverse functions are redundant and may be
compensated for by other focal adhesion proteins.
Focal adhesions are specialized cell-substratum junctions that are nearly ubiquitous among cells that attach to an extracellular matrix. At the core of the
focal adhesion is a cluster of activated integrin heterodimers, which are transmembrane signaling proteins that
attach cells to their ligands, i.e., extracellular matrix (ECM)1
(Hynes, 1992 Focal adhesions were first identified in tissue-culture
cells, as sites of contact between a cell and its underlying
substrate (Abercrombie et al., 1971 Of the myriad of focal adhesion proteins, tensin is particularly interesting because it has the ability to bind to actin microfilaments at multiple sites, enabling tensin both to
cap the growing (barbed) ends of actin filaments and to
cross-link actin filaments (Lo et al., 1994a To understand more about tensin's possible roles in tissues in vivo, we have isolated and characterized a mouse
tensin cDNA and gene, and we have analyzed the expression
of tensin during embryogenesis and postnatal development. To assess the extent to which tensin is functionally
important in these tissues and to elucidate the significance
of tensin's many associations, we have used gene targeting
in embryonic stem cells to generate tensin null mice. Analysis of the tensin null mice and the expression patterns of
tensin during embryonic and postnatal development reveals a number of surprising and intriguing results. Specifically, we find that (a) tensin is not required for mouse embryogenesis, or for the postnatal functioning of many
tissues that normally express tensin; and (b) tensin null
mice develop kidneys that harbor multiple tubular cysts
and progressive dilatation of the renal pelvis often without
leading to mortality.
Isolation and Characterization of Mouse cDNA and
Genomic Clones for Tensin
An 890-bp mouse tensin cDNA fragment was generated by PCR using
primers 5 Generation of Tensin The targeting vector was electroporated into embryonic stem (ES) cells
(R1 strain from Andreas Nagy and Janet Rossant, University of Toronto,
Canada), treated at 300 V and 500 µF in a GenePulser (Bio Rad Laboratories, Hercules, CA). Cells were then plated on eight 10-cm plates containing G418-resistant mouse embryonic fibroblast feeder cells. 24 h later,
selection was initiated using 250 µg/ml active G418 and 2 µM gancyclovir.
After 10 d of selection, double-resistant colonies were picked and transferred in duplicate onto fresh feeder cells for expansion of the ES colonies. Cells from one plate of each clone were trypsinized and stored in
10% DMSO at Cells from the other plate were incubated for 12 h at 55°C in the presence of DNA extraction buffer (20 mM NaCl, 1% SDS, 1 mM EDTA, 0.2 mg/ml proteinase K, 50 mM Tris-HCl, pH 8). These cell solutions were
then phenol-extracted and precipitated with 20 mM sodium acetate in
95% ethanol. DNAs were assayed by Southern blot analysis.
ES cells carrying the mutant allele were injected into the inner cell
mass of C57BL6 blastocysts. Chimeric mice with a high (>65%) contribution to coat color from 129SV were bred for germline transmission. For
genotyping, genomic DNAs isolated from ES cells or mouse tails were digested with EcoRI for 5 Immunoblot, Northern, and Southern Analyses and In
Situ Hybridizations
Immunoblot, Northern, and Southern analyses were performed as described (Guo et al., 1995 Generation of Tensin Antibodies
Glutathione-S-transferase (GST)-tensin fusion proteins engineered to
contain the L52 tensin fragment, encoding amino acid residues 338-748
(corresponding to the chick tensin coding sequence), were purified and injected to rabbits for antisera as described (Lo et al., 1994b Ultrastructural Analysis
For regular EM, tissues were fixed at room temperature for at least 1 h
with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.2 M sodium cacodylate buffer (pH 7.4). Samples were trimmed and washed three times
with the same buffer, and then postfixed with 1% aqueous osmium tetroxide for 1 h at room temperature. These samples were further washed with
sodium cacodylate buffer, followed by maleate buffer (pH 5.1), and
stained en bloc with 1% uranyl acetate for 1 h at room temperature. The
samples were then washed several times with maleate buffer, dehydrated
with cold ascending grades of ethanol and propylene oxide, embedded with LX-112 medium, and polymerized at 70°C for 48 h before sectioning.
Ultrathin sections (~80 nm) were cut with a regular diamond knife,
collected on 200-mesh, uncoated copper grids, and double stained with
50% saturated uranyl acetate and 0.2% lead citrate. The sections were
then examined with a JEOL-CX100 transmission electron microscope
(JEOL USA, Peabody, MA) operated at 60 kV.
For postembedding immunolabeling, samples were placed in warm fixative containing 2% fresh paraformaldehyde and 0.05% glutaraldehyde in
0.1 M PBS (pH 7.4) for 10 min. The samples were trimmed, washed several times with the same PBS at 4°C, dehydrated at Ultrathin sections (~90 nm) were cut with a diamond knife, collected
on Nickel grids coated with Formvar and thin carbon films, and labeled
with specific antibodies according to the tested dilution, followed by incubation with 10 nm colloidal gold-conjugated secondary antibodies. The
sections were briefly stained with uranyl acetate and lead citrate and examined with a JEOL-CX100 electron microscope operated at 60 kV (Yu
et al., 1996 Histological Analysis
Tissues were fixed in 4% formaldehyde, processed, and embedded in paraffin. Sections were cut at 5-µm thickness, mounted, and stained with hematoxylin and eosin. Sections were examined using an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY).
Tensin Is Expressed Broadly in Murine
Embryonic Development
To examine the expression of tensin during murine development, we first needed to isolate a mouse tensin cDNA
and to raise mouse tensin antibodies. We used PCR on a
pooled library of mouse lung cDNAs to isolate an 890-bp
tensin cDNA. Sequence analysis revealed a strong (~90%)
amino acid sequence identity between this fragment and
residues 181 to 477 of the previously reported chicken tensin
cDNA (Lo et al., 1994b We first performed Northern blot analysis, hybridizing
our mouse tensin cDNA (L52) to mouse RNAs isolated
from whole embryos taken at different ages. By gastrulation, tensin RNAs were abundantly expressed (Fig. 1). At
E7, a major RNA band of 10 kb and a minor band of 7 kb
hybridized with the probe under stringent conditions (Fig.
1). The same probe detected a single 6.7-kb EcoRI mouse genomic fragment on a Southern blot, and a 210-bp cDNA
probe complementary to a further upstream 5
Tensin RNAs remained prominent at all embryonic
ages examined. While expression of
Tensin Is Expressed in a Variety of Adult Tissues
To examine tensin expression in the adult mouse, we performed Northern blot analysis on mRNAs isolated from
various adult tissues. RNAs from the kidney and heart hybridized most strongly to the tensin cDNA probe (Fig. 3).
Tensin-hybridizing mRNAs were also readily detected in
lung, liver, and skeletal muscle, but only very weak signals
were detected in the mRNAs from brain, spleen, and testis
(Fig. 3). These expression patterns were very similar to
those seen by immunoblot analysis using an anti-tensin antiserum (not shown). Taken together, the expression of
tensin varied quite markedly among adult tissues.
Isolation of Mouse Tensin Gene and Generation of
Tensin Null Mice
The tissue-specific differences in tensin RNA levels suggested the possibility that the relative importance of tensin
might vary among different tissues. To explore tensin's
function in vivo, we focused on using homologous recombination and embryonic stem cell technology to ablate
tensin expression in mice. The 890-bp mouse tensin cDNA
was used to screen a 129SV mouse genomic library. We
identified one positive clone, mtensin
The mouse tensin targeting vector was constructed from
mtensin Of 121 G418-gancyclovir-resistant ES clones, one was
determined to have homologous recombination, and this
clone was eventually transmitted to germline (see Materials and Methods). Heterozygous mice were interbred to
produce litters that included homozygous offspring. Southern blot analysis of mouse DNAs isolated from the tails of
these offspring revealed the expected three genotypes (33+/+, 53+/ To assess whether the targeted homologous recombination event had resulted in the loss of tensin expression, we
used our monospecific mouse anti-tensin antisera (see
Materials and Methods) to conduct immunoblot analysis
on total proteins extracted from cultured fibroblasts that
we derived from the skin of our mice. As shown in Fig. 4
C, the anti-tensin antibody labeled a single band of ~200
kD in fibroblast protein extracts prepared from +/+ and
+/ Tensin Null Mice Display Striking Abnormalities in
Their Kidneys
Tensin Kidneys from mutant mice were pale and had a granular
appearance on their surface (Fig. 5 A). Often, the
Histologic examination of sections of kidneys from
tensin null animals revealed various degrees of cystic defects that were not seen in control sections (Fig. 5, C-H).
These defects ranged from extremely dilated cysts in the
cortex and medulla of severely affected kidneys (Fig. 5, D
and F) to small cysts in the cortex of the less affected organs
(Fig. 5 H). Most small cysts arose from proximal tubules,
as suggested by Periodic Acid-Schiff staining and as further judged by EM (see below). Upon closer inspection of
these cysts, it appeared that they derived from an expansion of the tubular lumen (Fig. 5 H). This was less obvious
in the larger cysts, where gross distortions often precluded
identification of the origin of cells that lined the cysts.
Higher magnification revealed additional abnormalities
in the kidneys from the tensin null mice (Fig. 5, I-N).
Some cysts contained atypical casts, i.e., amorphous materials, which seemed to be noncellular (Fig. 5 I, see also
double arrows in 5 F). Many glomeruli displayed enlarged
Bowman's spaces, i.e., the regions where small molecules
filtered from the blood will normally collect and pass into
the proximal tubules (Fig. 5 F, single arrow, and 5 J, double
arrows). In addition, the epithelial cells that surround each
glomerulus were often enlarged and cuboidal (Fig. 5 L, arrowheads), in contrast with the flattened, barely visible
simple squamous epithelium that surrounded control glomeruli (Fig. 5 K). In the most severely affected regions of
the The pale and granular surface of Despite the dramatic histological abnormalities in the
Tensin Is Expressed in the Epithelial Cells of
Kidney Tubules
Since kidney was one of the mouse organs displaying a
high level of tensin mRNA and protein, and since it was
the only tissue where major pathology was detected, we
were interested in where tensin was localized within the
kidney. To answer this question, we performed immunohistochemistry on tissue sections taken from a mouse kidney. Antibodies monospecific for tensin showed staining in many areas within the kidney, including the collecting
tubules within the medulla; however, staining was most
concentrated over the proximal and distal tubules in the
cortex region (Fig. 7).
Detection and Localization of Tensin at Cell-Matrix
Junctions of Kidney Tubules
The presence of tensin in the tubule epithelia was interesting, given that tubular cysts were an early sign of pathology in the tensin null mice. To further localize tensin within
the kidney tubule cells, regular and immunoelectron microscopy were performed on wild-type mouse kidneys.
Prominent antitensin labeling was detected at the basal
membrane of the tubular and endothelial cells (Fig. 6,
schematic; Fig. 8 A, proximal tubule cell; Fig. 8 B, antitensin labeling, restricted to the base of the tubule cell).
Periodically along the base of the basolateral labyrinth of
each kidney tubule cell were sites of markedly heavy antitensin labeling (Fig. 8 B, arrows; see also inset). In addition, numerous smaller clusters of antitensin labelings were
evident. The presence of small and large clusters of gold
particles at the base of the epithelium from antitensin labeling suggested that two kinds of focal adhesion complexes might exist in the kidney tubule cells. Support of
this hypothesis came from seeing similar labeling with an
antibody against p130cas, another focal adhesion protein
(data not shown).
A more diffuse antitensin labeling was seen at both edges
of the basal membrane (Fig. 8 B). This labeling was not
present in control tissue labeled with secondary antibodies
alone. Thus, despite the fact that the labeling seemed to be
extracellular rather than intracellular, we think it most
likely that this labeling reflects an intracellular residence
of tensin at these locations. Perhaps slight retraction of
cells during the fixation process left the leading edges of
the tubule and endothelial membranes within a region that
appeared by ultrastructure to be pure basal membrane.
Antitensin labeling was not detected elsewhere within the
tubule cells of the kidney, consistent with the notion that focal adhesion and cell-substratum contacts play an exclusive role at the base of the basolateral labyrinth, and not at
the lateral or apical regions of the tubule cells.
Focal Adhesions Are Present in Seemingly Normal
Tubules of Tensin Null Kidneys but Are Missing in the
Cystic Tubular Cells
In a normal kidney, the proximal tubules are lined by a
simple cuboidal epithelium. The apex of each epithelial
cell possesses abundant microvilli that form a brush border (see Fig. 6). The basal portion of the cell has abundant
membrane invaginations, referred to as the basal lateral
labyrinth. Numerous elongated mitochondria are concentrated within this region of each cell, and the locations of
mitochondria and increased cell membrane invaginations
are characteristic of cells engaged in active ion transport
(Junqueira et al., 1995
Most interestingly, in the more mildly affected regions,
immunogold labeling with antibodies against p130cas displayed both large (not shown) and small (shown) clusters
of gold particles at the base of the basal lateral labyrinth
(Fig. 9 C). This labeling was indistinguishable from that
which we had seen with antitensin (Fig. 8 B) and with antip130cas (not shown) in the proximal tubules of the wildtype kidney. In contrast with the normal-looking proximal tubules, the dilated, cystic proximal tubules of tensin null mice displayed an overall paucity of anti-p130cas labeling, even
when some cell-substratum adhesion still occurred (Fig. 9 D).
In regions where the tubules showed signs of cyst formation, labeling with antibodies to the sodium-potassium
ATPase was also significantly reduced (Fig. 9 F, compare
with largely unaffected region in Fig. 9 E). This finding
was interesting because this protein typically localizes in
the membrane, along the basolateral zones of the proximal
epithelial cells (Fig. 9 E). Thus, the reduction in epithelial
polarity was not restricted to the basal surface of the cells,
but rather extended to lateral and apical surfaces as well.
Given the localization of tensin in these cells, we presume that the abnormalities in lateral and apical markers are
secondary and arise as a consequence of a change at the
basal surface, initiated by the loss of tensin.
Although we detected mild antitensin labeling at the interface between podocyte foot processes and the basement
membrane of wild-type kidney glomeruli, no significant ultrastructural changes were detected in the tensin Tensin Although both male and female tensin Table I.
Summary of Litter Sizes in Different Matings
).
1 integrin, coupled with one of its many
partners, is thought to play a central role in focal adhesion
formation. Inside the cell, activated integrins anchor the
actin cytoskeleton to the plasma membrane (Burridge et al.,
1988
; Jockusch et al., 1995
). Focal adhesions are thought
to participate in many diverse biological processes including cell attachment, migration, polarization, growth, death,
differentiation, embryogenesis, and tissue development (Burridge et al., 1988
; Hynes, 1992
; Jockusch et al., 1995
;
Schwartz et al., 1995
).
). Terminating at these
sites are bundles of actin microfilaments, referred to as
stress fibers. At the interface between activated
1 integrins and stress fibers are a number of structural and signaling proteins, including talin, vinculin,
-actinin, paxillin, Src, protein kinase C, focal adhesion kinase, zyxin,
p130cas, and tensin (Burridge et al., 1988
; Jockusch et al.,
1995
). These proteins form a complex around the cytoplasmic domains of the integrin subunits, suggesting a dual
role for this complex in cytoskeletal architecture and in
signal transduction. In vitro binding assays have suggested
that talin and
-actinin may associate directly with
1 integrin (Horwitz et al., 1986
; Otey et al., 1990
). Kinetic studies have suggested that localization of tensin and focal adhesion kinase to sites of integrin clustering are also early
events in the formation of focal adhesions (Miyamoto et al.,
1995
).
; Chuang et al.,
1995
). In addition, tensin is phosphorylated on tyrosine,
threonine, and serine residues (Lo et al., 1994c
). The phosphorylation of tensin's tyrosine residues occurs upon integrin activation by ECM (Bockholt and Burridge, 1993
), as
well as by transformation of cells with oncogenes such as
v-src, BCR/ABL (Davis et al., 1991
; Salgia et al., 1995
).
Tensin also has an Src homology 2 (SH2) domain, which
makes it a candidate to bind to other proteins which themselves are tyrosine phosphorylated. One association mediated through tensin's SH2 domain occurs with PI3 kinase:
upon PDGF stimulation, ~1% of the total PI3 kinase activity is present in tensin immunoprecipitates (Auger et al., 1996
). Another interaction likely to be mediated by tensin's SH2 domain is the focal adhesion molecule p130cas:
in this case, tensin-conjugated beads associate specifically
with the p130cas present in lysates isolated from Rous sarcoma virus-transformed fibroblasts (Lo et al., 1994c
). The
diversity of interactions between tensin and other cellular
proteins, coupled with the activation of these associations
by growth factors or integrins, suggests that tensin is not
only a cytoskeletal-membrane linker protein, but also a signal transducer (Lo et al., 1994c
).
Materials and Methods
-TACATGCACTACAGCAA-3
and 5
-GTGGAGTTGCCCG
AGTC-3
and cDNAs from a mouse lung cDNA library (Clontech, Palo
Alto, CA). Primers were based on the published chicken cDNA sequence (Lo et al., 1994b
). The identity of the tensin cDNA fragment was confirmed by DNA sequencing, and the cDNA was then radiolabeled and
used to screen the same lung cDNA library, as well as a 129SV mouse genomic library (a gift from P. Soriano, University of Washington). Six cDNA
clones and one genomic clone were identified and subsequently purified.
Clone L52 contained a 1.2-kb cDNA insert, which was subsequently used
as a probe for Northern blot analysis and for in situ hybridization. The genomic clone was subjected to restriction map analysis and used for making
the targeting vector.
/
Mice
80°C.
probe or BamHI for 3
and Neo probes.
). In situ hybridization of paraffin sections was
performed using digoxigenin-labeled antisense riboprobes as described
(Chiang and Flanagan, 1996
).
). The p130cas
antibodies were raised against GST fusion proteins containing amino acid
residues 487-968 of rat p130cas (Sakai et al., 1994
). Antisera were purified
by passage through GST Sepharose columns (Sigma Chemical Co., St.
Louis, MO).
25°C with ascending
grades of ethanol, infiltrated with different concentrations of Lowicryl K4M
medium, embedded in gelatin capsules with fresh 100% Lowicryl K4M
medium, and then polymerized at
25°C with UV light for 5-7 d.
).
Results
). This fragment, encompassing a
5
segment of the tensin coding region, was subsequently
used as a probe to screen the same library for additional
mouse tensin clones. The cDNA insert of one clone (L52)
was then used to engineer a GST fusion protein, which we
subsequently used for antibody generation. Since most
available tensin antibodies were raised against chick tensin
(Glenney and Zokas, 1989
; Kanner et al., 1990
; Lo et al.,
1994b
), they either did not cross-react with mouse tensin,
or they recognized the mouse protein weakly. Our tensin
GST fusion protein reacted strongly with mouse tensin in tissues and on immunoblots (see below). We were now
equipped with the necessary molecular tools to examine
tensin expression during mouse development.
sequence
of mouse tensin RNA detected the same two bands (data
not shown). Thus, it is most likely that these size differences in tensin transcripts reflect either alternative usage
of polyadenylation signals or differential splicing. The 10kb RNA band corresponded most closely in size to that
expected based on knowledge of the chicken tensin
mRNA (Davis et al., 1991
; Lo et al., 1994b
; Chuang et al.,
1995
).
Fig. 1.
Tensin mRNA expression during embryonic development in the mouse. Northern blots of mRNAs isolated from mouse
embryos at 7, 11, 15, and 17 d of gestation were hybridized with
radiolabeled cDNA probes corresponding to tensin, p130cas, and
-actin (normalization control; the smaller sized band appearing
in E15 and E17 samples corresponds to the expected size of skeletal muscle actin mRNA). Bars represent the migration of RNA
molecular standards of 9.5 and 7.5 kb.
[View Larger Version of this Image (77K GIF file)]
-actin mRNA remained relatively constant in these embryo preparations
of different ages, expression of tensin mRNAs decreased
between E7 and E11 and then increased again between
E11 and E17 (Fig. 1). RNAs encoding another focal adhesion protein, p130cas, followed a profile more similar to
that of the
-actin control. We have not yet explored the
underlying reason for the unusually high level of tensin
mRNA expression at very early embryonic ages. However, in situ hybridization analyses revealed widespread
tensin mRNA expression in many embryonic tissues including nasal processes, walls of the midbrain, branchial
arches, centrum, heart, hepatic primordium, midgut, and
lung (Fig. 2).
Fig. 2.
Expression of
tensin mRNA in E10 mouse
embryos. Sagittal paraffin
sections (5 µm) of E10
mouse embryos were hybridized with a 1,200 nucleotide,
digegoxygenin-labeled antisense tensin cRNA probe
(A). No hybridization was
detected with the corresponding sense-strand probe
(B). MB, wall of the midbrain; NP, nasal process; BA,
branchial arch; H, heart; Lu,
lung; C, centrum; G, midgut; Li, liver. Bar, 420 µm.
[View Larger Version of this Image (130K GIF file)]
Fig. 3.
Expression of tensin mRNA in adult tissues. A Northern blot of mRNAs from the adult mouse tissues indicated was
hybridized sequentially with radiolabeled cDNA probes corresponding to tensin and -actin. Bars represent the migration of
RNA molecular standards of 9.5, 7.5, 2.4, and 1.4 kb.
[View Larger Version of this Image (68K GIF file)]
A, which contained a 13-kb fragment of the 5
end of the mouse tensin gene. A
partial restriction endonuclease map of this sequence is
shown in Fig. 4 A. Sequence analyses revealed that the
segment between the SpeI and the 3
EcoRI sites encompasses the portion of the tensin gene that encodes amino
acid residues 110-180 corresponding to the chicken tensin
cDNA (Lo et al., 1994b
).
Fig. 4.
Disruption of the tensin gene in embryonic stem cells
and in mice. (A) Targeting vector and homologous recombination at the tensin locus. Shown is a partial restriction map of a
portion of the mouse tensin gene. (Open boxes) 5 and 3
flanking
sequences used for targeting; (black boxes) 5
and 3
genomic
DNA probes used for Southern blot analysis; (PGK-Neo and
PGK-TK) neomycin resistance and Herpes thymidine kinase genes,
under the control of the phosphoglycerate kinase 1 promoter,
used for positive and negative selection, respectively. B, BamHI;
E, EcoRI; Sp, SpeI; S, SacI; X, XhoI. (B) Southern blot analyses
of DNA isolated from the tails of +/+, +/
, and
/
mice.
Mouse tail DNAs were digested with restriction endonucleases
EcoRI (left) or BamHI (middle and right) and resolved by electrophoresis through 0.8% agarose gels. After transferring to nitrocellulose paper, blots were hybridized with radiolabeled
cDNA probes corresponding to the 5
and 3
fragments indicated
in A, and to the PGK-Neo gene (5
EcoRI-BamHI fragment) as
indicated. (Asterisks) Bands expected for a successful homologous recombination event (2.8 kb, 5
probe; 0.4 kb, 3
probe); (arrows) bands expected for the wild-type genome (4 kb, 5
probe;
6.8 kb, 3
probe); (arrowhead) band for neomycin probe (3.8 kb).
(C) Immunoblot analysis of tensin protein in fibroblasts derived
from +/+, +/
, and
/
15-d mouse embryos. Fibroblasts were
cultured from the skin of 15-d mouse embryos. Whole-cell lysates
(30 µg) were subjected to electrophoresis through a 7% polyacrylamide SDS gel, and then transferred to nitrocellulose. After
staining with Ponceau S to confirm equal loading of proteins (not
shown), the blot was reacted with an anti-tensin antiserum. The
blot was developed with enhanced chemiluminescence (Amersham Corp., Arlington Heights, IL). A single 200-kD band, corresponding to the size of mouse tensin, was detected in protein
samples from wild-type and heterozygote fibroblasts, but not
from the tensin null fibroblasts.
[View Larger Versions of these Images (69 + 10K GIF file)]
A (Fig. 4 A). The ~4-kb SpeI-EcoRI fragment
of the mouse tensin gene was targeted for deletion. Since
tensin has two transcripts and the 210-bp cDNA fragment
targeted for deletion detects both RNA forms by Northern blot analysis (see above), we predicted that the deletion of this region of the genome should result in the disruption of both transcripts. Thus, we replaced this segment with a 1.8-kb fragment containing a pgk1 neomycin resistance gene for positive selection. The pgk1-neo gene was
flanked 3
with a 3-kb mouse tensin fragment and 5
with a
2.2-kb mouse tensin fragment. A pgk1-Herpes thymidine
kinase gene (Adra et al., 1987
) was used for negative selection as outlined in Fig. 4 A.
, 25
/
), as judged by diagnostic probes
for the correct 5
and 3
homologous recombination events
(Fig. 4 B). In addition, only a single genomic band of the
expected size was shown to hybridize to the neomycin
probe, suggesting that no additional recombination events
had occurred.
mice, but not from those of
/
mice. This result
confirmed the monospecificity of our mouse anti-tensin
antibody and also verified that the targeting event to disrupt the tensin gene was successful.
/
mice appeared normal at birth. As judged by
external appearance, body weight, and general behavior,
the developing postnatal tensin null mice did not seem to
differ from their wild-type or heterozygote littermates. At
2 mo old, animals were killed for further analysis. Gross
anatomical examination of the brain, liver, heart, spleen,
stomach, skin, lung, eyes, and intestine revealed no obvious abnormalities. In contrast, the kidneys of tensin null
animals exhibited significant and consistent abnormalities. The defects were bilateral and genotype related: they were
not found in any +/+ or +/
mice examined. We have not
yet examined whether the defects in the
/
mice vary
with mouse strain; as yet all our findings are confined to an
analysis of the 129sv/C57BL6 hybrid knockout animals.
/
kidneys were slightly larger than age-matched wild-type
kidneys. When hemisected, the +/+ kidneys always displayed a dark reddish brown cortex, i.e., the region of the
kidney that contains the proximal and distal tubules and
the glomeruli (Fig. 5 B). In +/+ kidneys, this region was
readily distinguishable from the lighter medulla, where the
collecting ducts and Henle's loops reside (for reference see diagram in Fig. 6). This distinction in coloring within the
wild-type kidneys was notably less prominent in the
/
kidneys (Fig. 5 B). In addition, the renal pelvis opening
was markedly enlarged in many of the kidneys from tensin
null mice. In severe cases, cortex and medulla regions
were often so compressed that the mutant kidneys were
nearly empty (Fig. 5 B).
Fig. 5.
Abnormalities in the overall appearance and histopathology of the kidneys of tensin /
mice. (A) Wild-type or tensin null mice were killed at various ages, and their kidneys were isolated and examined. Shown are representative kidneys from (left to right): 23 wk +/+, 6 wk
/
, 14 wk
/
, 28 wk
/
, and 36 wk
/
mice. Note the pale color of the tensin null kidneys at all ages. (B) Shown
are representative hemisected kidneys from (left to right): 23 wk +/+, 28 wk
/
, and 14 wk
/
mice. (C-F) Kidneys from 18-wk-old tensin null mice and their control wild-type littermates were placed in formaldehyde fixative, sectioned (5 µm), and stained with hematoxylin and eosin. Shown are representative sections from the kidneys of +/+ (C; box denotes region magnified in E) and
/
(D; box denotes region magnified in F) mice. Note the enlarged space, corresponding to the renal pelvis, in the knockout (asterisk in D). Note the
presence of numerous cysts in the
/
kidney sections, a typical feature of the knockout animals. (G-I) Kidneys from 4-wk-old tensin
null mice and their control littermates were fixed and stained as outlined above. Shown are representative sections of (G-H) the cortical
regions of +/+ (G) and
/
(H) kidneys; note again the prevalence of cysts (open circles) in the knockout kidney. G, glomeruli. (I) Section of
/
cortex to illustrate cysts containing amorphous materials (C), often referred to as casts (double arrow in F). (J) Section of
/
glomeruli, revealing large space in Bowman's space (double arrow), commonly seen when fluid backs up in the capsule as a result of a
malfunctioning of the kidney (see also arrow in F). (K) Glomeruli (G) from wild-type kidney; note the normal spacing surrounding
Bowman's space. (L) Glomeruli from tensin null kidney; note that these glomeruli were surrounded by atypical cuboidal epithelial cells
with a prominent cytoplasm (arrowheads), rather than the normal thin flattened epithelia, barely visible in the control glomeruli. (Open
circles) cysts. (M) Glomerulus from a tensin
/
kidney, surrounded by a highly irregular epithelium; also note what appears to be extracellular material, possibly collagen, similar to that seen in focal segment glomerular sclerosis in humans. (N) Region from a tensin null kidney, depicting marked signs of focal interstitial inflammatory infiltrates. Bar: (C and D) 740 µm; (E and F) 400 µm; (G and H) 100 µm;
(I and N) 50 µm; (K-M) 25 µm; (J) 17 µm.
[View Larger Versions of these Images (104 + 170K GIF file)]
Fig. 6.
Schematic illustration of a nephron unit of the kidney
and of the epithelium of a proximal tubule. The kidney comprises
more than a million nephrons, the functional unit of the kidney.
Blood enters the glomerulus at the start of each nephron. After
filtration to remove proteins and cells, the remaining fluid, composed largely of water and small molecules, such as salts, amino
acids, and glucose, passes through the Bowman's capsule surrounding the glomerulus and into the proximal tubule. Here most
of the nutrients are reabsorbed across the polarized epithelium of
the tubule (boxed area is enlarged to illustrate the epithelium at
right). The portion of fluid that is not reabsorbed continues to
pass through the kidney tubules, where it exits as urine. The epithelium of the proximal tubule differs from the distal tubule epithelium in that it contains a thick brush border for high efficiency
absorption of small molecules. The basal side of the epithelium
undulates to form what is called a basolateral labyrinth, which encases the numerous mitochondria necessary to provide the energy for transport. Separating the basal surface of the epithelium
from the endothelial walls of the capillaries is a basement membrane composed of ECM. Integrin-mediated attachment of the
basal surface to the underlying ECM is thought to be essential for
polarizing the epithelium and for enabling proper transport to
take place. The glomeruli and the proximal and distal tubules reside in the outermost region, i.e., cortex, of the kidney; the collecting ducts, located in the medulla of the kidney, pass urinary
fluid into the renal pelvis.
[View Larger Version of this Image (60K GIF file)]
/
kidneys, signs of focal segmental glomerular sclerosis were found. These highly abnormal areas of the
glomerulus are thickened and appear to contain extracellular depositions, which could be an increase in mesangial
matrix (Fig. 5 M, arrowhead). Finally, interspersed throughout the affected kidneys were focal interstitial inflammatory infiltrates (Fig. 5 N, arrowheads). Taken together,
these histological findings suggested strongly that large regions of the kidneys of tensin null mice were not functioning properly, and that fluid flow from the glomeruli to the
collecting ducts was aberrant.
/
kidneys was a
consistent feature of tensin null animals and was usually
evident by ~4-8 wk after birth. In addition, very small
cysts in the
/
kidneys could often be detected as early
as 1-2 wk postnatally, while the gross abnormalities in the
glomeruli were often not seen until the adult stage. In general, older animals had more severe kidney defects. However, this was not always the case, as some older mice, i.e.,
>6 mo old, displayed only mild cystic defects (but kidneys
were always pale). Conversely, some juvenile mice as young as 2 wk old sometimes possessed kidneys with an enlarged
renal pelvis space. Taken together, these data suggest that
the kidney abnormalities occurring in these animals were
not reflective of a specific developmental or temporally
defined defect. Rather, the deterioration appeared to be
slow, progressive, and triggered by some as yet unidentified stress, initiated by the absence of tensin. This said, we
cannot exclude the possibility that the mixed genetic background (129sv/C57BL6) played a role in the variable onset
time of kidney degeneration that was observed in the
tensin null progeny.
/
kidneys, the tensin null mice most often appeared
clinically normal. To date, only three of 44 mice (18 wk,
8 mo, and 9 mo of age) have become visibly ill in a fashion
that might be consistent with kidney failure. The 18-wk-old
female mouse had delivered once with two pups (one born
dead). In comparison with her littermates, her body weight
was less (19 g vs 21 g), her kidneys were double their normal size (0.25 g and 0.26 g vs 0.12 g and 0.12 g), and her
liver was smaller (0.5 g vs 1.0 g). Spleen, heart, and lung sizes were similar to those of her littermates. The 8-moold female (24 g) delivered once and similarly displayed
enlarged kidneys (1.0 g and 0.6 g) that were filled with
fluid. In this case, the heart appeared hardened, but it was
not analyzed further. Finally, the 9-mo-old male mouse was
thin (19 g), and both kidneys were enlarged, displaying
cysts on their surfaces and large renal pelvic cavities. This
mouse was the most affected, with many organs showing signs of deterioration. The chest of this animal was full of
fluid, and signs of scarring were seen on the heart. Since
we have not yet been able to generate sufficient numbers
of tensin null mice that are overtly ill, we are not able to say
unequivocally whether the ill mice in the colony suffer or
die from kidney failure. However, we have conducted blood
tests on 15 clinically normal, adult tensin null mice and 26 of
their control littermates. Glucose levels were largely normal
(161 ± 30 mg/dl
/
vs 164 ± 25 mg/dl control), as were creatinine levels (0.33 ± 0.08 mg/dl
/
vs 0.22 ± 0.04 mg/dl
control) and blood urea nitrogen levels (36 ± 10 mg/dl
/
vs 21 ± 4 mg/dl control). In contrast, the Bcl-2 null mice
displaying full-blown polycystic kidney disease have creatinine levels (0.77 ± 0.54 mg/dl
/
vs 0.19 ± 0.07 mg/dl control) and blood urea nitrogen levels (116 ± 87.6 mg/dl
/
vs 21 ± 5 mg/dl control) that are considerably abnormal (Veis et al., 1993
). These findings are consistent with
the notion that only a small fraction of the kidney needs to
be healthy to provide a seemingly normal urine output
(Junqueira et al., 1995
; Tisher and Brenner, 1994
).
Fig. 7.
Tensin protein is expressed in the epithelial tubules of
the mouse kidney. A kidney from a wild-type mouse was formaldehyde fixed, processed, embedded in paraffin, and sectioned
sagitally. Sections (5 µm) were then labeled with primary antibodies against mouse tensin, followed by enhancement with immunogold-conjugated goat anti-rabbit IgG secondary antibodies
(A). In the control section in B, the preimmune serum was used
in the labeling reaction. C, cortex, containing the glomeruli and
proximal/distal tubules (note strong labeling over the tubules);
M, medulla, containing the collecting ducts and Henle's loops
(note weaker labeling over the collecting tubules); P, renal pelvis.
Bar, 260 µm.
[View Larger Version of this Image (103K GIF file)]
Fig. 8.
Immunoelectron microscopy reveals tensin in two types of focal contacts along the basal surface of wild-type kidney tubules. Kidneys from wild-type mice were processed for regular or immunoelectron microscopy. Shown are ultrathin sections of epithelial cells
from the proximal tubules. (A) Polarized epithelial cells illustrating the presence of invaginations at the base of the epithelium, adjacent to the basal lamina and numerous microvilli (arrows) at the apical surface. Note the numerous mitochondria (m), reflective of transport activity, near the basal end of the cells. m, mitochondria; Nu, nucleus. (B) Epithelial cells labeled with antibodies against tensin. Note
two very large clusters of dense grains at the base of the epithelium (also at higher magnification in inset), next to the basal lamina (BL).
Note also more numerous smaller clusters of grains near the tips of the epithelial invaginations, where the epithelium made contact with
the basal lamina. Both types of labelings were seen throughout the tissue, wherever the tubule epithelium was in contact with the underlying basal lamina. Similar labelings were also seen with antibodies against p130cas, another focal adhesion protein (not shown). Note
we routinely observed gold particles in regions that appeared to be within the basal lamina; this (and other) labeling was specific for antibodies against focal adhesion proteins. We presume that the highly localized, but seemingly extracellular, labeling is an artifact, perhaps generated by a slight retraction of the cell's surface during fixation. EC, endothelial cell. Bar: (A) 3 µm; and (B) 0.5 µm.
[View Larger Versions of these Images (222 + 105K GIF file)]
). Throughout the histologically unaffected or mildly affected areas of the kidneys of tensin
null mice, the cuboidal epithelium of the proximal tubules
was ultrastructually normal (Fig. 9 A). This was in striking
contrast with the severely affected regions, where microvilli were largely deteriorated, mitochondria were less
organized and less abundant, and the basal membrane displayed fewer invaginations (Fig. 9 B). Many cells exhibited
irregularities in cell shape, and the epithelium often appeared to be less polarized. Despite these aberrations,
tight junctions could be detected between the defective
cells, suggesting that polarization was not completely lost.
Fig. 9.
Immunoelectron microscopy reveals the presence of focal adhesions in seemingly normal areas of the tensin null kidney tubules and the absence of focal adhesions in cystic tubules. Kidneys from tensin null mice were processed for regular or immunoelectron microscopy (see Materials and Methods). Shown are ultrathin sections of epithelial cells from the proximal tubules of mildly affected or
seemingly unaffected areas (A, C, and E) and of severely affected areas (B, D, and F). (A) Proximal tubule cells from a mildly affected region. Note that the epithelium is polarized, with numerous microvilli on the apical surface. In some regions such as this one, the only abnormality detected was fewer undulations of the basal membrane; however, such differences were not always seen. (B) Proximal tubule cells from a severely affected region. Note that only a few residual microvilli remain, and the epithelium shows few signs of polarization. (C-F). Tubular epithelial cells labeled with antibodies against p130 (C and D). Note that the mildly affected epithelial cells still
display labeling of focal adhesions (arrows in C). Occasional large focal contacts similar to those depicted in Fig. 8 were also seen. Note
the absence of significant labeling in the severely affected regions, suggestive of a loss of focal adhesions. (E and F). Tubular epithelial
cells labeled with antibodies against the sodium-potassium ATPase (E and F). Note that the mildly affected regions still showed labeling
of the basolateral surface, as is typical of wild-type controls (not shown). Note the absence of significant labeling in the severely affected
regions, suggestive of a loss of cell polarity. Nu, nucleus, M, mitochondria. Bar: (A) 2.3 µm; (B) 1.3 µm; (C) 0.6 µm; (D) 0.4 µm; (E and
F) 0.7 µm.
[View Larger Version of this Image (171K GIF file)]
/
glomeruli. Moreover, we did not detect fusions of foot processes nor separations of podocytes from their underlying
basement membranes (data not shown).
/
Female Mice Produce Few Live Pups
/
mice are fertile,
our homozygous mutant females mated to homozygous
mutant males have produced few live offspring. Thus far,
in 14 matings of homozygous females and males, only nine
have produced offspring, and the average litter size has
been only three pups (Table I). In contrast, crosses between homozygous males and either wild-type or heterozygous females have produced normal litters, ranging from
seven to nine pups. These data suggest a defect in the breeding potential of homozygous female mice. To further explore this issue, we killed one pregnant
/
female to examine her embryos at 15 d of gestation: three were alive, one
was dead, and one had been adsorbed. While additional
studies will be necessary to determine the mechanism underlying this deficiency, our findings suggest that normal
fertilization occurs, although a higher incidence of spontaneous abortion may be taking place in these mothers.
Inactivation of tensin gene expression by targeted ablation
has unveiled a specialized role for tensin in the kidney.
Given the widespread importance of focal adhesions in development and differentiation, and the fact that many focal adhesion molecules including 5 and
1 integrins and
focal adhesion kinase (Fassler and Meyer, 1995
; Furuta et al.,
1995
; Stephens et al., 1995
) lead to early embryonic lethality when targeted for removal, we were not expecting live
homozygous pups when we crossed heterozygous mice.
Moreover, since (a) tensin has an SH2 domain and is one of the few known targets for tyrosine phosphorylation in
the focal adhesion complex, (b) tensin associates with the
actin cytoskeleton in several different ways, and (c) we discovered that tensin is broadly expressed in embryonic development and in a variety of postnatal tissues, we expected
that the loss of tensin would be felt by many different cell
types and at many stages of development and differentiation.
Our results demonstrated that tensin is not necessary for mouse embryogenesis. While we cannot rule out the possibility that a related protein compensates for tensin in embryogenesis, a variety of different tensin cDNA probes and antibodies have failed thus far to suggest the existence of another tensin family member. This said, other actinbinding proteins have been identified at focal adhesions, and these may perform similar functions to tensin in tissues besides the kidney.
Given the results of our knockout, it was intriguing that
the highest levels of tensin we detected were in the kidney
and heart. The appearance and function of the tensin /
heart seemed normal, although additional studies will be
necessary to determine whether subtle defects might be
present. In contrast, the kidneys from our postnatal tensin
null mice displayed abnormalities that seemed to become
progressively more pronounced with age. Tensin null kidneys were uniformly pale, ranging from brown to light yellow, but never the reddish brown that is characteristic of normal kidneys. Since kidney color is reflective of the flow
of blood and/or its components, the paleness of the tensin
null kidneys immediately suggested that fluid flow through
the kidneys was compromised in our animals.
The rough and granular surface of the kidneys of our tensin null mice was likely due to the presence of numerous cysts under the surface. Most, but not all, of these mutant kidneys also possessed an enlarged renal pelvic cavity, and, in some cases, only a very thin parenchymal layer remained. Since we found compressed cortical and medullar regions in mildly affected kidneys, the hollow kidneys that we saw in severe instances most likely arise from enlargement of the renal pelvis, a feature that is often reflective of obstruction of fluid flow in the kidney.
A partial block in urine flow could certainly lead to an
enlargement of the renal pelvis and eventual parenchymal
loss, concomitant with the loss of color in the kidney. To
some extent, this condition resembles that seen in the human kidney disease known as hydronephrosis, where dilation
of the renal pelvis and calyces are associated with progressive atrophy of the kidney resulting from obstruction of
the urinary tract (Tisher and Brenner, 1994). Hydronephrosis can be congenital or acquired, unilateral or bilateral,
partial or complete; it tends to remain asymptomatic until
a very late stage in progression (Tisher and Brenner, 1994
).
Several observations suggest that dilation of the proximal tubules might initiate the kidney defects that we see in tensin mutant mice: (a) in mildly affected kidneys, dilated tubules were concentrated in the cortex of the kidneys; (b) tensin appeared to be most highly expressed in the cortical tubules; (c) glomerular abnormalities were not present, except in situations where tubular cysts were also prevalent; and (d) the majority of the dilated tubules displayed residual microvilli, exclusive to the proximal tubules of the kidney.
Based on our immunoelectron microscopy, we know
that tensin is localized at the cell-matrix junctions of the
tubular cells in kidneys. Our labeling patterns revealed the
existence of two kinds of in vivo focal adhesion complexes.
We have observed similar labeling patterns at the basement
membrane junction in the epidermis (unpublished results),
indicating that these hitherto unrecognized junctions are
likely to be broadly present in a variety of epithelia. The
relative importance of such junctions in kidney tubular cells vs the epidermis or other stratified epithelia may be rooted
in the fact that kidney epithelia do not possess hemidesmosomes, which are the major forms of cell-substratum
attachment in stratified epithelia (Dowling et al., 1996;
Georges-Labouesse et al., 1996
; Van der Neut et al., 1996).
We cannot yet fully explain how the loss of tensin might lead to an imbalance of fluid flow in the kidney. One possibility consistent with our knowledge of how tensin might function is that, without tensin, focal adhesions might be weakened in the kidney, leading to the loss of polarity seen in some tubular cells. The loss in polarity might compromise the transport of water and other small molecules across the proximal tubules, leading to cyst formation and a partial block in urine flow, resulting in an increased pelvic pressure.
A number of lines of evidence point to the importance
of cell-matrix attachment in epithelial polarity and directional transportation of molecules in the proximal tubules
of the kidney (Klein et al., 1988; Sorokin et al., 1990
; for
review see Eaton and Simons, 1995
). These tubules play a
critical role in reabsorption of small molecules that are
passed through the glomeruli. Proximal tubules are responsible for reabsorption of 100% of the glucose and
amino acids, 85% of the water that diffuses passively following an osmotic gradient, and 85% of the sodium chloride, which is transported by an active process involving
the Na+/K+ -ATPase in the basolateral cell membranes.
The proximal tubules also secrete into the lumen creatinine and foreign substances obtained from the interstitial
plasma (Tisher and Brenner, 1994
). Given these rigorous
absorption and secretion activities, proximal tubular cells
might be more susceptible to mildly defective focal adhesion complexes than to other regions of the kidney. Thus, even though tensin ablation does not prevent focal adhesion assembly per se, it may weaken it in a fashion that
could be responsive to fluid stresses. This notion is consistent with the reduction in focal adhesion immunolabeling
in the cystic tubular cells (Fig. 9).
It is well documented from studies on cultured kidney
epithelial cells that polarization is controlled not only by cell-
substratum but also by cell-cell interactions (Wang et al.,
1990a,b; for reviews see Drubin and Nelson, 1996
; Eaton
and Simons, 1995
). While integrin-mediated cell-substratum
interactions determine the orientation of the apicobasal
axis (Wang et al., 1990b
; Ojakian and Schwimmer, 1994
),
E-cadherin-mediated cell-cell contacts are required for
basolateral formation (Vega-Salas et al., 1987
; Gumbiner et al., 1988
). In the future, it will be important to explore whether E-cadherin adherens junctions are perturbed in
the cystic tubular cells of the tensin
/
kidneys. In addition, as we identify the specific domains of tensin that
when missing are responsible for the defects we see in the
kidneys, we should be able to assess whether the defects
stem from loss of structural (i.e., actin-binding) activities,
or alternatively from aberrancies in signal transduction. Finally, a key hypothesis as yet untested is the notion that
structural deficiencies exist in the focal adhesions of tensin
null proximal tubule cells, and that changes in focal adhesions precede the abnormalities seen in the tensin null kidneys. Sorting out the sequence of events between the loss
of tensin and the development of abnormal kidney histology will be essential to our understanding of tensin's role
in focal adhesion structure and function.
Received for publication 2 December 1996 and in revised form 17 January 1997.
We especially thank Dr. Vikas Sukhatme and Dr. Mark Haas for their valuable discussions regarding the kidney defects observed in our knockout animals. Blood analysis was performed by the Animal Resource Center at the University of Chicago.This work was supported by the Howard Hughes Medical Institutes (HHMI). S.H. Lo is a postdoctoral fellow of the HHMI; E. Fuchs is an investigator of the HHMI.
ECM, extracellular matrix; ES, embryonic stem; GST, glutathione-S-transferase; SH2, Src homology 2.