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First published online 30 May 2007
doi: 10.1242/dev.005033
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8ß1-mediated stimulation of Gdnf expression
1 Department of Physiology, 1550 Fourth Street, University of California, San
Francisco, San Francisco, CA 94143, USA.
2 Department of Anatomy and 1550 Fourth Street, University of California, San
Francisco, San Francisco, CA 94143, USA.
3 Howard Hughes Medical Institute, 1550 Fourth Street, University of California,
San Francisco, San Francisco, CA 94143, USA.
* Author for correspondence (e-mail: lfr{at}cgl.ucsf.edu)
Accepted 2 May 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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8ß1 integrin,
invasion by the ureteric bud into the metanephric mesenchyme is inhibited,
resulting in renal agenesis. Here we present genetic evidence that the
extracellular matrix protein nephronectin is an essential ligand that engages
8ß1 integrin during early kidney development. We show that embryos
lacking a functional nephronectin gene frequently display kidney agenesis or
hypoplasia, which can be traced to a delay in the invasion of the metanephric
mesenchyme by the ureteric bud at an early stage of kidney development.
Significantly, we detected no defects in extracellular matrix organization in
the nascent kidneys of the nephronectin mutants. Instead, we found that
Gdnf expression was dramatically reduced in both nephronectin- and
8 integrin-null mutants specifically in the metanephric mesenchyme at
the time of ureteric bud invasion. We show that this reduction is sufficient
to explain the agenesis and hypoplasia observed in both mutants.
Interestingly, the reduction in Gdnf expression is transient, and its
resumption presumably enables the nephronectin-deficient ureteric buds to
invade the metanephric mesenchyme and begin branching. Our results thus place
nephronectin and 8ß1 integrin in a pathway that regulates
Gdnf expression and is essential for kidney development.
Key words: Kidney, Integrin, Extracellular matrix, GDNF, Mouse, Ureteric bud
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E11, a signal(s) from the MM elicits the formation of
the ureteric bud (UB) from the ND. Once formed, the UB extends toward and then
invades the MM, and by E11.5 the UB begins branching, thus giving rise to
a tubular network that will eventually mature into the collecting ducts of the
adult kidney. As the UB invades and branches, it expresses genes that induce
the MM to condense and differentiate into various cell types that comprise the
nephron. In the absence of invasion by the UB into the MM, the metanephric
kidney does not develop (reviewed in
Saxen, 1987
).
Glial cell line-derived neurotrophic factor (GDNF), a member of the
transforming growth factor-ß (TGFß) superfamily, is a key signal in
the initiation of UB formation and subsequent branching
(Costantini and Shakya, 2006).
GDNF is expressed in the MM and signals to the UB through a receptor complex
consisting of the receptor tyrosine kinase RET and a coreceptor, GDNF family
receptor alpha1 (GFR1). GDNF has been shown to induce ectopic UB
formation in culture, and mice lacking either the Gdnf, Ret or
Gfra1 genes display renal agenesis at high penetrance
(Cacalano et al., 1998;
Moore et al., 1996;
Pichel et al., 1996;
Sainio et al., 1997;
Sanchez et al., 1996;
Schuchardt et al., 1994).
Gdnf expression in the MM is regulated by a highly conserved network
of transcription factors, and can be activated or inhibited by extracellular
signaling molecules such as WNT11, GDF11 and SLIT2 at discrete stages of
development (Brodbeck and Englert,
2004; Esquela and Lee,
2003; Grieshammer et al.,
2004; Majumdar et al.,
2003). It is also known that GDNF signaling is dependent on
heparan sulphate glycosaminoglycans, which interact with or are constituents
of the extracellular matrix (ECM) (Barnett
et al., 2002).
Integrins are cell adhesion receptors that serve as a link between the ECM
and the cytoskeleton. Integrins are heterodimers, consisting of two
single-pass transmembrane proteins designated as the and ß
subunits. Integrins are thought to occupy either an inactive or active state
depending on cues received from the extracelluar environment in the form of an
appropriate ECM ligand or growth factor signal. They communicate information
from the ECM to the cell interior through recruitment of cytoskeletal proteins
and kinases. In addition, they are able to communicate information from the
cell interior to the ECM, resulting in its proper deposition and remodeling
(Delon and Brown, 2007;
ffrench-Constant and Colognato,
2004; Giancotti and Ruoslahti,
1999; Hynes,
2002).
Previously, we showed that the integrin subunit 8 (Itga8)
is expressed throughout the nephric cord, including the MM
(Muller et al., 1997).
Furthermore, we demonstrated that loss of Itga8 function invariably
results in a delay of invasion of the MM by the UB, which in turn results in a
high frequency of kidney agenesis. The molecular mechanism by which
Itga8 function in the MM influences the UB has yet to be determined.
Several ECM constituents are known ligands for 8ß1 integrin,
including fibronectin, osteopontin, tenascin C and vitronectin
(Denda et al., 1998a;
Denda et al., 1998b;
Varnum-Finney et al., 1995).
However, data from expression and loss-of-function analyses indicated that
these ligands are unlikely to be mediators of 8ß1 integrin
function in the developing kidney. Using an expression cloning strategy we
identified a gene that encodes a novel ECM molecule, nephronectin
(Npnt), that is expressed by the UB and the epithelia of several
developing organs (Brandenberger et al.,
2001). We demonstrated that 8ß1 integrin recognizes
nephronectin in binding assays and associates with nephronectin in vivo. In
addition, we found that the localization of nephronectin in the kidney is
consistent with it mediating 8ß1 integrin function during
development.
Here we report that mice lacking nephronectin frequently display kidney
agenesis. We show that the phenotype arises during the early stages of
metanephric development, when the UB is beginning to invade the MM, similar to
the phenotype of mice lacking Itga8 (and therefore 8ß1
integrin function). Thus, nephronectin is an ECM protein expressed by the UB
that is required for 8ß1 integrin function during early stages of
UB invasion and branching. Significantly, we demonstrate that Gdnf
expression is reduced in both Npnt and Itga8 mutants at the
time when the invasion of the MM by the UB is delayed. Finally, we present
genetic data indicating that Gdnf dosage as well as signaling from
the receptor tyrosine kinase, RET, impact the penetrance of the Itga8
mutant phenotype. Taken together, our results suggest that the observed
reduction of Gdnf expression in the MM is sufficient to explain the
phenotypes observed in mice lacking either nephronectin or the 8
integrin subunit.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Clones harboring BACs that were correctly modified were
identified by Southern blot hybridization, which revealed the presence of both
the 5' and 3' arms. One clone, designated 273P10 NN-1, was
electroporated into E14 embryonic stem (ES) cells.
Because of the large size of the targeting construct (300 kb), we used
TaqMan (Applied Biosystems) real-time quantitative PCR to identify ES cells in
which there was a reduction in copy number of a region that was targeted for
insertion (Valenzuela et al.,
2003). This involved the use of specific PCRs that amplify the
wild-type allele, but fail to amplify the mutated allele because of the
increase in sequence length. Candidate ES cell clones that were identified by
sequential PCR screens were assessed for reduced wild-type copy number by
quantitative Southern hybridization using densitometry. Two clones isolated
from the screen were injected into C57BL/6 blastocysts (Transgenic/Targeted
Mutagenesis Core, University of California, San Francisco, CA), and both were
incorporated into the germ line.
We generated a null allele for nephronectin by crossing animals
heterozygous for the Npntfloxneo allele with animals
expressing CRE recombinase under the ß-actin promoter
(Lewandoski et al., 1997).
This cross produced mice carrying the
Npnt
ex1 allele in which the
first exon of Npnt was excised without deletion of the
neomycin-resistance gene expression cassette
(Fig. 1D,E). All the analysis
described here was performed on mice carrying this allele on a mixed genetic
background (129Sv/J; C57BL/6; FVB/N).
In situ hybridization
To stage embryos, noon of the day on which a vaginal plug was detected was
considered E0.5. Embryos were collected at various stages and the region
containing the hindlimb buds was fixed in 4.0% paraformaldehyde
(PFA)/phosphate-buffered saline (PBS) overnight at 4°C and cryosectioned
at 14 µm. Analysis of gene expression using in situ hybridization with RNA
probes was performed according to standard protocols. Data using the following
probes are presented: Gdnf
(Srinivas et al., 1999),
Eya1 (Xu et al.,
1999), Six2 (Xu et
al., 2003), Pax2
(Dressler et al., 1990).
Histology and immunofluorescence
Embryos at various stages and kidneys from newborn animals were fixed in
4.0% PFA/PBS overnight at 4°C. Tissues were cryosectioned and stained with
hematoxylin and eosin according to standard protocols. Sections were stained
with the following antibodies: anti-nephronectin (1:100)
(Brandenberger et al., 2001),
anti-EHS laminin (1:500) (Sigma L9393), anti-fibronectin (1:300) (Sigma
F-6140), anti-calbindin D28K (1:600) (Swant CB-38a), anti-collagen type IV
(1:500) (LB-1403; Cosmo Bio., Tokyo, Japan), and anti-pax2 (1:100) (PRB-276P;
Covance, Princeton, NJ). Confocal imaging was performed on a Zeiss LSM 5
Pascal.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ex1, which lacks the first
exon of nephronectin (see Materials and methods;
Fig. 1). Animals homozygous for
Npntex1 express neither
nephronectin protein nor Npnt RNA (see
Fig. 1G,H), demonstrating that
Npntex1 is a null allele.
Npntex1 homozygotes, hereafter
referred to as Npnt mutants, were born at the expected Mendelian
frequency (52 out of 219 mice; 24%). At birth they appeared to be of normal
size without any obvious external defects. However, upon dissection, we found
that Npnt mutant animals (n=52) frequently lacked one (23%)
or both (46%) kidneys (Fig.
2A-C). The remaining Npnt mutants had two kidneys (31%),
on average resulting in 58% agenesis (see legend to
Fig. 2F). By contrast, 2 of 109
Npntex1 heterozygotes displayed
bilateral kidney agenesis and none displayed unilateral agenesis (2%
agenesis). No agenesis was observed in their wild-type littermates. In most
cases the Npnt mutant kidneys were smaller than those of their
wild-type littermates (Fig.
2A,B). Histological analysis revealed variability in development,
with most Npnt mutant kidneys containing essentially normal
nephrogenic regions (Fig.
2D,E), and a few displaying cystic and dysplastic elements (not
shown). Significantly, Npnt mutants lacking kidneys did not show
evidence of partial ureter development or survival of MM, suggesting that
development is perturbed at an early stage, when the UB has yet to invade the
MM.
Renal agenesis in nephronectin-null embryos results from a developmental delay
To determine the developmental origin of the kidney agenesis observed at
birth, we examined Npnt mutants and their wild-type littermates at
early stages of metanephric development. At E11.0, the UB had formed and
appeared similar in Npnt mutants (n=2) and their wild-type
littermates (Fig. 3A,B).
However, by E11.5, we found a significant difference: whereas in all wild-type
embryos the UB had invaded the MM and branched, none of the mutant embryos
examined, in wholemount or serial section (n=12), showed any evidence
of UB invasion of the MM or branching, on either side of the embryo
(Fig. 3C,D). By E12.5, the UB
had invaded the MM and undergone some branching in all Npnt mutants
examined (n=4). However, the extent of branching was variable,
ranging from a single branching event to two rounds of branching, and
therefore fewer than the three rounds in their wild-type littermates
(Fig. 3E-G).
To determine whether the UB branching observed was sufficient to promote
nephrogenesis, we analyzed kidney development at E13.5. In two of four
Npnt mutants examined, we found that kidneys were developing on both
sides in one embryo and on one side in the other. In all cases, the mutant
kidneys were markedly smaller than normal. In the other two of four
Npnt mutants, no kidney development was detected on either side and
the MM was beginning to atrophy (five out of eight kidneys expected did not
form), whereas kidneys were developing on both sides in four of four wild-type
littermates (no agenesis) (Fig.
3H-K', and data not shown). It seems likely that the three
kidneys that were observed at E13.5 developed in embryos like those in which
the UB had undergone several rounds of branching by E12.5 (see
Fig. 3F). By contrast, the
failure of kidney development at E13.5 may have occurred in embryos like those
in which only a single branching event had occurred by E12.5 (see
Fig. 3G). From these data it
appears that in Npnt mutants, UB formation is initially normal, but
then the UB fails to invade the MM at E11.5. Subsequently, invasion/branching
does occur, but in many cases it appears that the extent of branching is too
little to sustain kidney development, resulting in the kidney agenesis
observed at birth. Significantly, the lack of invasion of the MM by the UB at
E11.5 in the Npnt mutants appeared similar to that observed in
embryos lacking 8ß1 integrin function
(Muller et al., 1997).
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). At E13.5 we
found that the localization of this protein was similar in both a
Npnt mutant and a wild-type littermate
(Fig. 4K,L). From these
results, we conclude that the basement membrane is comparatively normal in
Npnt mutants.
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8ß1 integrin may facilitate
key signaling events within the MM that promote kidney development. To
investigate this possibility, we analyzed the expression, by in situ
hybridization and antibody staining, of various genes known to have roles in
kidney development. Because a lack of invasion of the MM by the UB was
invariably observed in both Npnt- and Itga8-null mutants at
E11.5, we performed our analysis at this stage. We examined the expression of
key genes expressed in the ND and UB, including Pax2, Ret, Gfra1 and
Emx2 and found that they all were expressed at comparable levels in
both Npnt mutants and wild-type controls at E11.5
(Fig. 5A-H, and data not
shown). The expression of genes that mark the MM was also assessed, including
Gdnf, Eya1, Six2 and Pax2. Interestingly, we did not detect
Gdnf RNA in the Npnt mutant MM. Importantly, however, the
level of Gdnf expression in the adjacent limb bud was comparable in
the mutant and control embryos (Fig.
5A,B). Similar results were obtained in seven separate experiments
with seven separate mutant embryos. The expression domains and levels of
Eya1 and Six2 RNA and PAX2 protein, which are expressed in
the MM and are known to have roles in controlling Gdnf expression
(Brodbeck et al., 2004;
Brophy et al., 2001;
Xu et al., 1999), were similar
in Npnt mutants and their wild-type littermates
(Fig. 5C-H). Taken together,
these results demonstrate that, at E11.5, the MM is present in Npnt
mutants and displays normal expression of several genes known to be upstream
of Gdnf, and provide evidence that nephronectin has a role in
promoting Gdnf expression in the MM at this stage.
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Gdnf expression is reduced in Itga8-null embryos at E11.5
In view of the apparent similarity between the kidney phenotypes of
Npnt- and Itga8-null mice, we were interested to examine
Gdnf expression in Itga8-null homozygotes, hereafter
referred to as Itga8 mutants. We compared the expression at E11.5 of
Gdnf and several other genes expressed in the MM, including the
signaling molecule genes Bmp4 and Gdf11 and the
transcription factor genes Eya1, Pax2, Six2, Wt1 and Sall1,
in Itga8 mutants and their control littermates. Consistent with what
we observed in Npnt mutants at E11.5, we detected no change in
expression of any of these genes in Itga8 mutants at E11.5 except
Gdnf (Fig. 6A-D and
data not shown). Again, Gdnf RNA was not detected in the MM, but
Gdnf expression appeared comparable in the adjacent limb buds of
Itga8 mutant and control embryos
(Fig. 6A,B). Similar results
were obtained in five separate experiments with five separate mutant embryos.
However, using a radiolabeled Gdnf probe we were able to detect a
signal at low level (data not shown). In addition, in situ hybridization
assays revealed no obvious difference between the Itga8 mutants and
controls at E10.5 and E13.5 (Fig.
6E-L), indicating that, as in Npnt mutants, 8
integrin is transiently required for normal Gdnf expression in the MM
at E11.5.
|
). To
assess kidney development we scored kidney agenesis as described in the legend
to Fig. 1F. We found that
animals heterozygous for either Itga8- or Gdnf-null alleles
displayed 0% and 9% agenesis, respectively. These data are consistent with
previously published frequencies of kidney agenesis in these mutant
heterozygotes (Moore et al.,
1996; Muller et al.,
1997). However, in animals heterozygous for both the
Itga8- and Gdnf-null alleles
(Itga8+/-;GdnflacZ/+), we observed 53%
agenesis (Table 1).
Furthermore, the frequency of kidney agenesis in animals homozygous for the
Itga8-null allele was increased from 59% to 96% by heterozygosity for
the Gdnf-null allele (Table
1). The one kidney that was found in the 11
Itga8-/-;GdnflacZ/+ animals examined
was reduced in size and histological analysis revealed it to be highly
dysplastic and most likely non-functional (data not shown). These results
support the hypothesis that the reduction in Gdnf expression that
results from a loss of 8ß1 integrin function is responsible for
the kidney agenesis in Npnt- and Itga8-null mutants.
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; Basson et al.,
2006). Spry1-null mutants display ectopic ureteric
budding from the ND, resulting in multiple ureters and multiplex kidneys as a
consequence of excess GDNF signaling. We reasoned that if the kidney agenesis
phenotype in the Itga8 mutants was due primarily to reduced
Gdnf expression, it should be possible to overcome this deficiency by
reducing Spry1 gene dosage and thereby increasing sensitivity of the
UB to the small amount of GDNF produced in the Itga8 MM at E11.5.
Indeed, we observed that heterozygosity for a Spry1-null allele in
Itga8-null animals resulted in a significant rescue of kidney
development: 25% agenesis in
Itga8-/-;Spry1+/- animals
(n=20) versus 71% agenesis in their
Itga8-/-;Spry1+/+ littermates
(n=14). In general,
Itga8-/-;Spry1+/- kidneys appeared to
be of normal size. Furthermore, no kidney agenesis was observed in animals
homozygous for both the Itga8- and Spry1-null alleles
(n=8) (Fig. 7A). These
data provide strong support for the hypothesis that the kidney agenesis that
results from the absence of the 8 integrin subunit is because of a
reduction in Gdnf expression.
Histological analysis revealed that the rescue of the Itga8-null
phenotype by reduction of Spry1 gene dosage takes place early in
kidney development. Like Npnt mutants, all Itga8-null
embryos displayed a lack of invasion of the MM by the UB at E11.5; however, in
all Itga8-null embryos that were heterozygous for a
Spry1-null allele (n=5) the UB on one or both sides was
found to have invaded the MM at this stage (9/10 UBs invaded)
(Fig. 7B,C). This finding
provided us with an opportunity to address an important question: is the
reduction in Gdnf expression that we observed at E11.5 in
Itga8 mutants secondary to the lack of invasion of the MM by the UB
at E11.5? The latter is a viable possibility because the UB is known to
promote Gdnf expression in the MM at later stages by producing
signaling molecules such as WNT11
(Majumdar et al., 2003).
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8 integrin subunit in the
rescued embryos. However, the signal was stronger than that found in
Itga8-null embryonic kidneys (Fig.
7B), most likely because the presence of the UB in the rescued
mutants produces signals that enhance Gdnf expression
(Majumdar et al., 2003). |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, loss-of-function studies have revealed that few
ECM proteins or their receptors have specific roles in the early events of
metanephric development (Lelongt and
Ronco, 2003). One exception is 8ß1 integrin, which we
have previously shown to be crucial for initial UB invasion into the MM and
its subsequent branching, as well as for epithelialization of the MM
(Muller et al., 1997). Our
finding that Npnt-null embryos phenocopy the early phenotype of
Itga8-null embryos (Muller et
al., 1997) strongly suggests that nephronectin is the ligand
mediating 8ß1 integrin function during the early events of kidney
development. In addition, we have found that the expression of Gdnf,
a key factor in kidney development, is severely reduced in Npnt as
well as in Itga8 mutant MM at E11.5, the time at which we invariably
observed a lack of invasion of the MM by the UB in both mutants. These data
suggested that 8ß1 integrin might be involved in a pathway
upstream of Gdnf expression. Our genetic analysis provides strong
support for this hypothesis. It therefore appears that we have uncovered a
novel pathway in which an integrin, through recognition of its ECM ligand, is
directly involved in the regulation of an essential signaling molecule in the
developing kidney.
Nephronectin is a crucial factor in kidney development
Assessment of kidney development at birth in Npnt mutants revealed
a high frequency of agenesis, with 46% of the mutants displaying bilateral
agenesis and 23% unilateral agenesis. Only 31% of mutant animals had two
kidneys, most of which were smaller than normal but otherwise appeared
unaffected. Surprisingly, although nephronectin is expressed in several other
organ systems during development, preliminary analysis has shown that all
organs except the kidney appear grossly normal in Npnt mutants at
birth. Consistent with this finding, Npnt mutants that survive beyond
birth are healthy and fertile, and have an apparently normal life span. This
suggests that in tissues other than the developing kidney, the presence of
other ECM proteins compensates for the absence of nephronectin. Among the
other ECM proteins that could replace nephronectin is Mam domain and EGf
domain-containing protein (MAEG) (Buchner
et al., 2000), which shares 41% overall amino acid identity with
nephronectin and has been shown to be a ligand for 8ß1 integrin
(Osada et al., 2005). However,
at present, little is known about MAEG function and expression, and it remains
to be determined whether this ECM protein has roles in organogenesis.
Nephronectin is an essential ligand for 8ß1 integrin during the initial events of kidney development
Our analysis has revealed that although a UB forms in Npnt
mutants, it consistently fails to invade the MM at E11.5. Significantly, this
phenotype very closely resembles the early phenotype of Itga8 mutants
(Muller et al., 1997)
(Fig. 6D). Since its
identification, nephronectin has been a candidate ligand for 8ß1
integrin in the developing kidney and this similarity in phenotype strongly
points to nephronectin as an essential ligand for 8ß1 integrin
during the crucial early process of UB invasion.
Although the Npnt and Itga8 mutant phenotypes appear very
similar at E11.5, there are some important differences at later stages. One is
that Npnt mutants display kidney agenesis at a lower frequency than
Itga8 mutants, 58% versus 83% agenesis, respectively
(Muller et al., 1997)
(Fig. 2F). A possible
explanation for this is that there may be functional redundancy with another
ligand(s) expressed by the UB, which can be recognized by 8ß1
integrin and can mediate responsiveness of the MM. If so, the expression of
this ligand might be responsible for enabling the UB in Npnt mutants
to undergo the delayed invasion and branching that we observed at E12.5, which
in some cases must be sufficient for kidney formation. By contrast,
Itga8 mutants should be unable to respond to any ligand, and
therefore display complete agenesis. The finding that kidneys occasionally
form in Itga8 mutants raises the possibility that another integrin
may compensate for the absence of 8ß1 integrin.
Differences between the Npnt- and Itga8-null mutants might also reflect differences in the genetic backgrounds of the mice. Although the background of the Itga8 mutants was largely C57BL/6, with some minor contribution remaining from 129Sv/J, the Npnt allele has been maintained on a mixed background with contributions from C57BL/6, 129Sv/J and FvB/N. In support of this explanation, we have observed that Itga8 mutant survival increases dramatically on an outbred background (J.M.L. and L.F.R., unpublished). Once the Npnt-null allele has been bred onto a pure background the penetrance of the homozygous phenotype may more closely resemble that in Itga8 mutants.
A role for nephronectin and 8ß1 integrin in regulating Gdnf expression in the developing kidney
Here we have presented data that support a role for 8ß1
integrin and its ligand nephronectin in a pathway that regulates the
expression of Gdnf, an essential growth factor in kidney development.
Using in situ hybridization, we have shown that Gdnf expression is
severely reduced in Npnt- and Itga8-null embryos at a time
when we invariably found that the UB has not invaded the MM. We have
demonstrated that Itga8;Gdnf-compound-null heterozygotes
display kidney agenesis at a fivefold higher frequency than is observed in
Gdnf-null heterozygotes, and that reducing the level of Gdnf
gene dosage increases the penetrance of the Itga8-null phenotype.
Furthermore, we found that by reducing the dosage of a gene that encodes an
attenuator of GDNF signaling, Sprouty1, and thus enhancing the sensitivity of
Itga8-null mutants to GDNF, we decreased the penetrance of the
Itga8-null phenotype. Taken together, these data provide genetic
evidence that 8ß1 integrin and GDNF function in a common pathway
and suggest that 8ß1 integrin and its ECM ligand play a role in
regulating the expression of Gdnf.
Of special interest, our results show that the severe reduction in
Gdnf expression in Npnt and Itga8 mutants is
transient: in Npnt and Itga8 mutants at E10.5, Gdnf
RNA levels appeared normal, at E11.5 Gdnf RNA was barely detectable,
and at E13.5 Gdnf RNA was readily detected in those mutants in which
sufficient UB branching had occurred such that kidney development proceeded.
This transient effect may be indicative of multiple factors working at
different times during kidney development to produce the normal pattern of
Gdnf expression. For example, WNT11, which has been shown to maintain
Gdnf expression in the MM, seems to be required only after UB
invasion (Majumdar et al.,
2003). According to this hypothesis, lack of either nephronectin
or 8ß1 integrin results in a severe decrease in Gdnf
expression, which causes a delay in UB invasion that is subsequently overcome
by the presence of other factors, possibly WNT11, or perhaps members of the
fibroblast growth factor (FGF) or TGF-ß families, which may have
facilitating roles in regulating Gdnf expression.
An alternative explanation for the reduction in Gdnf expression is
that it is a secondary effect of the absence of the UB from the MM in
Npnt and Itga8 mutants. We have addressed this possibility
by assaying for Gdnf expression in
Itga8-/-;Spry1+/- embryos, in which
8ß1 integrin function is lacking but the UB has invaded the MM at
E11.5. We found that in these
Itga8-/-;Spry1+/- embryos the level of
Gdnf expression at E11.5 was substantially reduced compared with that
in their Itga8+/-;Spry1+/-
littermates. This result demonstrates that loss of 8ß1 integrin
causes a substantial decrease in Gdnf expression in the MM even in
the presence of a UB and, therefore, strongly supports our hypothesis that the
recognition of nephronectin by 8ß1 integrin in the developing
kidney is necessary for robust Gdnf expression.
A possible mechanistic explanation of 8ß1 integrin-mediated effects on Gdnf expression in the developing kidney
How might an integrin and its ECM ligand regulate Gdnf expression?
Integrins are classically known as adhesion receptors, which have been shown
to play roles in organizing the cytoskeleton and activating intercellular
signaling pathways (ffrench-Constant and
Colognato, 2004; Humphries et
al., 2004; Hynes,
2002). There is an extensive literature demonstrating that in cell
culture, integrin-mediated cell adhesion together with growth factor signaling
can promote mitogenesis, cell viability and gene expression
(ffrench-Constant and Colognato,
2004; Giancotti and Ruoslahti,
1999). In mammary gland cultures, ß1 integrins have been
shown to synergize with prolactin signaling to activate Stat5 and thus to play
a role in maintaining the differentiated state of the glandular epithelium and
its expression of ß-casein (Akhtar and
Streuli, 2006; Faraldo et al.,
1998; Naylor et al.,
2005).
With respect to 8ß1 integrin, it has been shown that its
recognition of fibronectin activates both the MAPK and PI3K pathways in cell
culture systems (Farias et al.,
2005). These data raise the possibility that in the kidney,
8ß1 integrin activates the MAPK cascade in the MM. In support of
this, we have observed reduced levels of phospho-ERK in the MM of
Itga8 mutants (J.M.L. and L.F.R., unpublished). Therefore, it is
conceivable that signaling by 8ß1 integrin synergizes with a
growth factor signal in the MM to activate the MAPK cascade that then impinges
on the transcriptional network involved in regulating Gdnf
expression.
The signaling properties of integrins have been appreciated for some time, but there is as yet very little in vivo evidence demonstrating roles for integrin signaling in regulating gene expression. The data presented here suggest that this is a key function of integrin in the developing kidney. Further studies will be needed to identify the specific signaling pathway(s) and target genes involved.
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Akhtar, N. and Streuli, C. H. (2006). Rac1
links integrin-mediated adhesion to the control of lactational differentiation
in mammary epithelia. J. Cell Biol.
173,781
-793.
Barnett, M. W., Fisher, C. E., Perona-Wright, G. and Davies, J.
A. (2002). Signalling by glial cell line-derived neurotrophic
factor (GDNF) requires heparan sulphate glycosaminoglycan. J. Cell
Sci. 115,4495
-4503.
Basson, M. A., Akbulut, S., Watson-Johnson, J., Simon, R., Carroll, T. J., Shakya, R., Gross, I., Martin, G. R., Lufkin, T., McMahon, A. P. et al. (2005). Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction. Dev. Cell 8, 229-239.[CrossRef][Medline]
Basson, M. A., Watson-Johnson, J., Shakya, R., Akbulut, S., Hyink, D., Costantini, F. D., Wilson, P. D., Mason, I. J. and Licht, J. D. (2006). Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1. Dev. Biol. 299,466 -477.[CrossRef][Medline]
Brandenberger, R., Schmidt, A., Linton, J., Wang, D., Backus,
C., Denda, S., Muller, U. and Reichardt, L. F. (2001).
Identification and characterization of a novel extracellular matrix protein
nephronectin that is associated with integrin alpha8beta1 in the embryonic
kidney. J. Cell Biol.
154,447
-458.
Brodbeck, S. and Englert, C. (2004). Genetic determination of nephrogenesis: the Pax/Eya/Six gene network. Pediatr. Nephrol. 19,249 -255.[CrossRef][Medline]
Brodbeck, S., Besenbeck, B. and Englert, C. (2004). The transcription factor Six2 activates expression of the Gdnf gene as well as its own promoter. Mech. Dev. 121,1211 -1222.[CrossRef][Medline]
Brophy, P. D., Ostrom, L., Lang, K. M. and Dressler, G. R.
(2001). Regulation of ureteric bud outgrowth by Pax2-dependent
activation of the glial derived neurotrophic factor gene.
Development 128,4747
-4756.
Buchner, G., Orfanelli, U., Quaderi, N., Bassi, M. T., Andolfi, G., Ballabio, A. and Franco, B. (2000). Identification of a new EGF-repeat-containing gene from human Xp22: a candidate for developmental disorders. Genomics 65,16 -23.[CrossRef][Medline]
Cacalano, G., Farinas, I., Wang, L. C., Hagler, K., Forgie, A., Moore, M., Armanini, M., Phillips, H., Ryan, A. M., Reichardt, L. F. et al. (1998). GFRalpha1 is an essential receptor component for GDNF in the developing nervous system and kidney. Neuron 21,53 -62.[CrossRef][Medline]
Costantini, F. and Shakya, R. (2006). GDNF/Ret signaling and the development of the kidney. BioEssays 28,117 -127.[CrossRef][Medline]
Delon, I. and Brown, N. H. (2007). Integrins and the actin cytoskeleton. Curr. Opin. Cell Biol. 19, 43-50.[CrossRef][Medline]
Denda, S., Muller, U., Crossin, K. L., Erickson, H. P. and Reichardt, L. F. (1998a). Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C. Biochemistry 37,5464 -5474.[CrossRef][Medline]
Denda, S., Reichardt, L. F. and Muller, U.
(1998b). Identification of osteopontin as a novel ligand for the
integrin alpha8 beta1 and potential roles for this integrin-ligand interaction
in kidney morphogenesis. Mol. Biol. Cell
9,1425
-1435.
Dressler, G. R., Deutsch, U., Chowdhury, K., Nornes, H. O. and
Gruss, P. (1990). Pax2, a new murine paired-box-containing
gene and its expression in the developing excretory system.
Development 109,787
-795.
Esquela, A. F. and Lee, S. J. (2003). Regulation of metanephric kidney development by growth/differentiation factor 11. Dev. Biol. 257,356 -370.[CrossRef][Medline]
Faraldo, M. M., Deugnier, M. A., Lukashev, M., Thiery, J. P. and Glukhova, M. A. (1998). Perturbation of beta1-integrin function alters the development of murine mammary gland. EMBO J. 17,2139 -2147.[CrossRef][Medline]
Farias, E., Lu, M., Li, X. and Schnapp, L. M. (2005). Integrin alpha8beta1-fibronectin interactions promote cell survival via PI3 kinase pathway. Biochem. Biophys. Res. Commun. 329,305 -311.[CrossRef][Medline]
ffrench-Constant, C. and Colognato, H. (2004). Integrins: versatile integrators of extracellular signals. Trends Cell Biol. 14,678 -686.[CrossRef][Medline]
Giancotti, F. G. and Ruoslahti, E. (1999).
Integrin signaling. Science
285,1028
-1032.
Grieshammer, U., Le, M., Plump, A. S., Wang, F., Tessier-Lavigne, M. and Martin, G. R. (2004). SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site. Dev. Cell 6,709 -717.[CrossRef][Medline]
Grobstein, C. (1955). Inductive interaction in the development of the mouse metanephros. J. Exp. Zool. 130,319 -340.[CrossRef]
Humphries, M. J., Travis, M. A., Clark, K. and Mould, A. P. (2004). Mechanisms of integration of cells and extracellular matrices by integrins. Biochem. Soc. Trans. 32,822 -825.[CrossRef][Medline]
Hynes, R. O. (1986). Fibronectins. Sci. Am. 254,42 -51.[Medline]
Hynes, R. O. (2002). Integrins: bidirectional, allosteric signaling machines. Cell 110,673 -687.[CrossRef][Medline]
Lelongt, B. and Ronco, P. (2003). Role of extracellular matrix in kidney development and repair. Pediatr. Nephrol. 18,731 -742.[CrossRef][Medline]
Lewandoski, M., Meyers, E. N. and Martin, G. R. (1997). Analysis of Fgf8 gene function in vertebrate development. Cold Spring Harb. Symp. Quant. Biol. 62,159 -168.[Medline]
Majumdar, A., Vainio, S., Kispert, A., McMahon, J. and McMahon,
A. P. (2003). Wnt11 and Ret/Gdnf pathways cooperate in
regulating ureteric branching during metanephric kidney development.
Development 130,3175
-3185.
Moore, M. W., Klein, R. D., Farinas, I., Sauer, H., Armanini, M., Phillips, H., Reichardt, L. F., Ryan, A. M., Carver-Moore, K. and Rosenthal, A. (1996). Renal and neuronal abnormalities in mice lacking GDNF. Nature 382, 76-79.[CrossRef][Medline]
Muller, U., Wang, D., Denda, S., Meneses, J. J., Pedersen, R. A. and Reichardt, L. F. (1997). Integrin alpha8beta1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis. Cell 88,603 -613.[CrossRef][Medline]
Naylor, M. J., Li, N., Cheung, J., Lowe, E. T., Lambert, E.,
Marlow, R., Wang, P., Schatzmann, F., Wintermantel, T., Schuetz, G. et al.
(2005). Ablation of beta1 integrin in mammary epithelium reveals
a key role for integrin in glandular morphogenesis and differentiation.
J. Cell Biol. 171,717
-728.
Osada, A., Kiyozumi, D., Tsutsui, K., Ono, Y., Weber, C. N., Sugimoto, N., Imai, T., Okada, A. and Sekiguchi, K. (2005). Expression of MAEG, a novel basement membrane protein, in mouse hair follicle morphogenesis. Exp. Cell Res. 303,148 -159.[CrossRef][Medline]
Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A. C., Drago, J., Grinberg, A., Lee, E. J., Huang, S. P., Saarma, M., Hoffer, B. J. et al. (1996). Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 382, 73-76.[CrossRef][Medline]
Sainio, K., Suvanto, P., Davies, J., Wartiovaara, J., Wartiovaara, K., Saarma, M., Arumae, U., Meng, X., Lindahl, M., Pachnis, V. et al. (1997). Glial-cell-line-derived neurotrophic factor is required for bud initiation from ureteric epithelium. Development 124,4077 -4087.[Abstract]
Sanchez, M. P., Silos-Santiago, I., Frisen, J., He, B., Lira, S. A. and Barbacid, M. (1996). Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382, 70-73.[CrossRef][Medline]
Saxen, L. (1987). Organogenesis of the Kidney. Cambridge: Cambridge University Press.
Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis, V. (1994). Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367,380 -383.[CrossRef][Medline]
Srinivas, S., Wu, Z., Chen, C. M., D'Agati, V. and Costantini, F. (1999). Dominant effects of RET receptor misexpression and ligand-independent RET signaling on ureteric bud development. Development 126,1375 -1386.[Abstract]
Valenzuela, D. M., Murphy, A. J., Frendewey, D., Gale, N. W., Economides, A. N., Auerbach, W., Poueymirou, W. T., Adams, N. C., Rojas, J., Yasenchak, J. et al. (2003). High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotechnol. 21,652 -659.[CrossRef][Medline]
Varnum-Finney, B., Venstrom, K., Muller, U., Kypta, R., Backus, C., Chiquet, M. and Reichardt, L. F. (1995). The integrin receptor alpha 8 beta 1 mediates interactions of embryonic chick motor and sensory neurons with tenascin-C. Neuron 14,1213 -1222.[CrossRef][Medline]
Xu, P. X., Adams, J., Peters, H., Brown, M. C., Heaney, S. and Maas, R. (1999). Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nat. Genet. 23,113 -117.[CrossRef][Medline]
Xu, P. X., Zheng, W., Huang, L., Maire, P., Laclef, C. and
Silvius, D. (2003). Six1 is required for the early
organogenesis of mammalian kidney. Development
130,3085
-3094.
Zhang, Y., Buchholz, F., Muyrers, J. P. and Stewart, A. F. (1998). A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20,123 -128.[CrossRef][Medline]
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