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First published online 5 January 2006
doi: 10.1242/dev.02220
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1 Program in Developmental Biology, Hospital for Sick Children, University of
Toronto, Toronto, Canada.
2 Division of Nephrology, The Hospital for Sick Children, University of Toronto,
Toronto, Canada.
3 Cardiovascular Research Institute, University of California, San Francisco,
CA, USA.
4 Department of Medical and Molecular Genetics, University of Toronto, Toronto,
Canada.
5 Department of Paediatrics, University of Toronto, Toronto, Canada.
6 Department of Physiology, University of Toronto, Toronto, Canada.
7 Department of Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, Canada.
* Author for correspondence (e-mail: norman.rosenblum{at}sickkids.ca)
Accepted 24 November 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Kidney development, Sonic Hedgehog, GLI3
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Moreover, mice homozygous for a targeted mutation that
generates a 699 amino acid N-terminal GLI3 protein exhibit numerous
malformations, including renal aplasia/dysplasia as observed in PHS
(Bose et al., 2002). The
molecular mechanisms that control these deleterious GLI3-dependent effects are
unknown.
GLI3 is an intracellular transcriptional effector in the sonic hedgehog
(SHH) signaling pathway. Among the members of the hedgehog (HH) family of
secreted proteins, SHH controls cell fate, determination, proliferation and
tissue patterning during embryogenesis (reviewed by
Ingham and McMahon, 2001). In
Drosophila, Hh signaling is mediated, at the transcriptional level,
by a single zinc-finger protein, Cubitus interruptus (Ci)
(Methot and Basler, 2001). In
the absence of a Hh signal, Ci is processed by proteolysis into an
N-terminal fragment that includes the zinc finger region and acts to repress
gene transcription (Aza-Blanc et al.,
1997). In vertebrates, three Ci homologs, GLI1, GLI2 and
GLI3, mediate Hh signals by controlling gene expression. GLI1 and GLI2 exist
as full length proteins in cultured mammalian cells
(Dai et al., 1999) and act
primarily as transcriptional activators during murine embryogenesis
(Bai et al., 2002;
Park et al., 2000). Absence of
a SHH signal provokes processing of GLI3 into a shortened form that can act as
a transcriptional repressor in cultured cells and limb explant cultures
(Litingtung et al., 2002;
Wang et al., 2000). Genetic
analyses in mice demonstrate that the predominant activity of GLI3 is to
repress expression of Shh dependent genes
(Bai et al., 2002;
Park et al., 2000).
Integration of opposite GLI activities appears to be critical. Genetic
inactivation of Gli3 can rescue Shh mutant phenotypes in
embryonic tissues including the neural tube
(Litingtung and Chiang, 2000),
limb (Litingtung et al.,
2002), face and forebrain
(Rallu et al., 2002), and skin
(Mill et al., 2005) suggesting
a dynamic interplay between GLI signals during tissue formation. The molecular
mechanisms that control such interactions are largely undefined.
The mammalian kidney is a model with which to study embryonic
epithelial-mesenchymal interactions and growth factor signaling. In the
kidney, interactions between the ureteric bud, an epithelial structure, and
the metanephric mesenchyme, a mesenchymal tissue, instigate growth and
branching of the ureteric bud, a process known as renal branching
morphogenesis, and conversion of the metanephric mesenchyme to those
epithelial elements that exist proximal to the ureteric bud branches and their
daughter collecting ducts. Growth factors secreted by the ureteric bud and
metanephric mesenchymal cells act in a autocrine and paracrine manner to
control cellular events including cell proliferation and to control the
expression of genes such as Pax2, Sall1 and Mycn, each of
which performs crucial functions (reviewed by
Bouchard, 2004). The presence
of renal hypoplasia/dysplasia in mice deficient in Shh in the
ureteric bud lineage (Yu et al.,
2002) and in mice expressing a truncated form of GLI3
(Bose et al., 2002)
demonstrates a crucial role for SHH-GLI signaling during mammalian renal
development. However, the molecular mechanisms by which GLI3 controls renal
embryogenesis are unknown.
To identify mechanisms by which SHH-GLI signaling controls renal
organogenesis, we generated models of deficient SHH or SMO signaling in mutant
mice and cultured embryonic kidneys. Homozygous inactivation of Shh
or treatment of pregnant wild-type mice with cyclopamine, a steroidal alkaloid
that blocks SMO activity (Chen et al.,
2002), caused renal aplasia or dysplasia. Although these states
lowered the expression of both GLI1 and GLI2 protein, the relative expression
of a shortened form of GLI3 was increased relative to full-length GLI3
protein. Homozygous inactivation of Gli3 in a Shh-deficient
background normalized the renal phenotype and rescued kidney expression of
Pax2, Sall1, cyclin D1 and MYCN, as well as GLI1 and GLI2. We defined
mechanisms by which GLI3 controls expression or these SHH targets using
chromatin immunoprecipitation. In normal kidney tissue, GLI1 and/or GLI2 bound
a GLI-binding consensus region within each target gene 5' flanking
region. By contrast, in cyclopamine-treated malformed cultured kidneys, we
observed decreased associations with GLI1/GLI2 and de novo associations with
GLI3. Genetic inactivation of Gli3 alone blocked inhibition of GLI1
and GLI2 expression, and renal branching morphogenesis by cyclopamine. We
propose a model in which SHH-SMO signaling controls generation of a GLI3
repressor, which, in turn, controls Gli1 and Gli2 as well as
non-Gli target genes crucial to embryonic kidney development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
Gli2+/– (Mo et
al., 1997) and Gli3+/–
(Schimmang et al., 1992)
CD1/129 mice were housed in the Animal Facility of The Hospital for Sick
Children (Toronto, Canada) and genotyped by PCR as described previously
(Chiang et al., 1996;
Mo et al., 1997). Animal
experiments were approved by the ethics committee at The Hospital for Sick
Children.
Antibodies and specialized reagents
Antibodies and reagents were as follows: GLI1 (Abcam, Cambridge, MA);
cyclin D1 (BD PharMingen, San Diego, CA); GLI3, MYCN, MYC, cyclin D2 (Santa
Cruz Biotechnology, Santa Cruz CA); cyclopamine (Toronto Research Chemicals,
Toronto, ON); and SHH-N (B & D Systems, Minneapolis, MN). A partial mouse
Gli2 cDNA corresponding to amino acids 327-442 was cloned into the
pGEX1 vector (Amersham). Fusion protein was expressed in E. coli
(BL21) and purified on glutathione sepharose (Amersham) according to the
manufacturer's instructions. Antibodies were affinity purified using a column
of Affi-gel 10 beads (BioRad) conjugated with His-tagged GLI2 antigen.
Cyclopamine was dissolved in 100% ethanol for culture of kidney explants and
dissolved in 45% 2-hydroxypropyl-beta-cyclodextrin (HBC, Sigma-Aldrich, St
Louis, MO) in PBS for intraperitoneal injection. Shh-N was dissolved in 1% FBS
in PBS.
Immunohistochemistry and immunoblotting
Immunohistochemistry in paraffin wax-embedded sections (4 µm) of kidney
tissue was performed (Hu et al.,
2003) with the following antibodies: mouse anti-GLI1 (1:10),
rabbit anti-GLI2 (1:700) and rabbit anti-GLI3 (1:50). Biotinylated secondary
antibodies were used in a biotin-avidin complex assay (Vector laboratories,
Burlingame, CA). Immunoblotting of proteins transferred to nylon membrane was
performed using the following antibodies, anti-GLI1 (1:3300 dilution),
anti-GLI2 (1: 250 dilution), anti-GLI3 (1:250 dilution, Santa Cruz Biotech),
anti-MYC (1:250 dilution), anti-MYCN (1:250 dilution), anti-cyclin D1 (1:250
dilution), anti-cyclin D2 (1:250 dilution) and anti-ß-actin (1:3000
dilution, Sigma, St Louis, MO). Secondary anti-mouse IgG and anti-rabbit IgG
conjugated HRP (Amersham Pharmacia Biotech, Piscataway, NJ) were used at a
1:3000 and 1:5000 dilution, respectively. Chemiluminescence was performed
using commercially available reagents (ECL kit; Amersham Pharmacia Biotech,
Piscataway, NJ).
Embryonic kidney organ culture
Kidneys were surgically dissected from mouse embryos and cultured on 0.45
mm polyethylene terephthalate membranes (Corning) in multiwell plates in the
presence of Richter's modified Dulbecco's modified Eagle's medium-Ham's F-12
nutrient mixture (DMEM-F12) containing 50 µg/ml transferrin (Sigma)
(Pichel et al., 1996).
Ureteric bud-derived structures were identified in whole-mount kidney
specimens with fluorescein isothiocyanate (FITC)-conjugated DBA (20 µg/ml;
Vector Labs, Burlington, ON) (Piscione et
al., 1997). A ureteric bud branch point was defined as the
intersection between two connected branches.
Effect of cyclopamine on kidney development
Pregnant wild-type or Gli3+/– mice were injected
via the peritoneum at E 9.5 with cyclopamine (6 mg/kg body weight) once per
day for 4 consecutive days. Embryos were then isolated surgically and fixed in
4% paraformaldehyde-PBS. Alternatively, for treatment of cultured embryonic
kidney explants, cyclopamine was added to culture medium at a final
concentration of 10 µM. Shh-N was dissolved in 1% FBS and added to culture
medium at final concentration of 1.0 µg/ml for 4 days.
In situ mRNA hybridization
In situ hybridization was performed in paraffin wax-embedded tissue
sections as described (Mo et al.,
1997) using probes encoding Pax2 [kindly provided by Dr
Peter Gruss (Stoykova and Gruss,
1994)] and Sall1 [kindly provided by Dr Ryuichi
Nishinakamura (Nishinakamura et al.,
2001)]. For whole-mount in situ hybridization, embryos were fixed
in 4% paraformaldehyde at 4°C and processed according to published methods
(Conlon and Herrmann,
1993).
In situ cell proliferation assay
Cell proliferation in kidneys was assayed by incorporation of
5-bromo-2'-deoxyuridine (BrdU, Roche Molecular Biochemicals, Mannheim,
Germany). Pregnant female mice received an intraperitoneal injection of BrdU
(100 µg/g body weight) 4 hours prior to scarifying these mice.
Identification of BrdU-positive cells was performed by immunostaining as
described previously (Cano-Gauci et al.,
1999).
Chromatin immunoprecipitation
Chromatin immunoprecipitation was carried out using published methods
(Hu and Rosenblum, 2005). GLI
consensus binding sites were identified as TGGGTGGTC or GACCACCCA
(Lai et al., 2004).
Oligodeoxynucleotide primers were designed to encompass these binding sites as
follows. Mouse cyclin D1 promoter (GenBank Accession Number AF212040):
forward, 5'-AATTCTAAAGGTGGGGGAACA-3', reverse
5'-GAGACACGATAGGCTCCTTCC-3' (148 bp PCR product). Mouse
Mycn promoter (GenBank Accession Number X06993): forward
5'-TAATATCCCCCGAGCTTCAA-3', reverse
5'-AGCTTCGCAAGTACCGCTTC-3' (254 bp PCR product at annealing
temperature 56°C for 30 seconds). Mouse Sall1 promoter (GenBank
Accession Number AC147558): forward 5'-AGGCGGTGCCTAGGGTCT, reverse
5'-CTGAAGTTTTCGGGAGAAGC-3' (299 bp PCR product at annealing
temperature 54°C for 45 seconds). Mouse Pax2 promoter (GenBank
Accession Number MMU13975): forward 5'-GGGCTTTGCAGCTTTTAGAG-3',
reverse 5'-TTGGCAGAGAAGTAGCAATCC-3' (256 bp PCR product at
annealing temperature 54°C for 45 seconds), Mouse Gli1 promoter
(ENSEMBL gene ID ENSMUSG00000025407): forward
5'-AGCTTCAGTGCCACCCCAC-3', reverse
5'-TCGGTCCGAAGGAAGGATATA-3' (197 bp PCR product at annealing
temperature 55°C for 40 seconds). Mouse Gli2 promoter (ENSEMBL
gene ID ENSMUSG00000051835): forward 5'-GTGCTGAGGCTCCTGACAAT-3'
and reverse 5'-CAGCAGAGCAAGGTGAACAG-3' (124 bp PCR product at
annealing temperature 55°C for 40 seconds).
Reverse transcriptase-PCR
Total RNA from frozen tissue samples was extracted using the RNeasy Mini
kit (Qiagen, Mississauga, ON, Canada) according to the manufacture's protocol.
First-strand cDNA was generated from 2 µg total RNA using random hexamers
(SuperScript First Strand Synthesis System, Invitrogen). The following primers
were used. Gli1: forward 5'-ATCACCTGTTGGGGATGCTGGAT-3',
reverse 5'-GGCGTGAATAGGACTTCCGACAG-3' (317 bp product at annealing
temperature 56°C for 45 seconds). Gli2: forward
5'-GTTCCAAGGCCTACTCTCGCCTG-3', reverse
5'-CTTGAGCAGTGGAGCACGGACAT-3' (304 bp product at annealing
temperature 56°C for 45 seconds). Gli3: forward
5'-AGCAACCAGGAGCCTGAAGTCAT-3', reverse
5'-GTCTTGAGTAGGCTTTTGTGCAA-3' (270 bp product at annealing
temperature 56°C for 40 seconds). ß-actin: forward
5'-TGTTACCAACTGGGACGACA-3', reverse
5'-CTCTCAGCTGTGGTGGTGAA-3' (393 bp product). PCR was performed
using HotStarTag. The RT-PCR amplified products were separated by agarose gel
electrophoresis and stained with ethidium bromide.
Data analysis
Mean differences between groups were analyzed using Student's
t-test (two-tailed) or by ANOVA using Stat-View statistical analysis
program (version 4.01; Abacus Concepts, Berkeley, CA) for more than two
groups. Statistical significance was taken at a value of
P<0.05.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (Fig. 1E).
Using a complementary strategy, we used cyclopamine to block SMO function and
investigate the function of the SHH-SMO signaling during renal morphogenesis.
An intraperitoneal injection of 6 mg/kg cyclopamine daily for 4 consecutive
days starting at E9.5 exerted a major deleterious effect on kidney development
(Fig. 1H,I). Compared with
embryos treated with drug vehicle alone
(Fig. 1F,G), the kidneys in
cyclopamine treated embryos demonstrated primordial branching of the ureteric
bud and a paucity of metanephric-derived epithelial structures. To determine
SMO functions after the onset of mesenchymal-epithelial interactions, kidneys
were harvested at E11.5 and cultured in the presence of cyclopamine for 4
consecutive days. Qualitative analysis demonstrated marked inhibition of
ureteric bud branching identified by Dolichos Biflorus Agglutinin (compare
Fig. 1K with 1J). Quantitation
of this effect, performed by counting branch point number, showed that
cyclopamine inhibited formation of ureteric bud branch points by 40% (branch
point number – drug vehicle versus cyclopamine: 61.6±2.4 versus
37.6±1.6; P<0.0001. n=5 kidneys/group). Analysis
of histological sections generated from these kidneys revealed a paucity of
metanephric-derived epithelial elements (see Fig. S1 in the supplementary
material). Taken together, these results in two complementary models of
disrupted SHH-SMO signaling demonstrate a crucial role for SHH-SMO signaling
during induction of the metanephric blastema by the ureteric bud and during
branching morphogenesis and nephrogenesis.
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Gli3 inactivation rescues kidney dysplasia and SHH target gene expression
To investigate the impact of increased GLI3 repressor on kidney
development, we analyzed
Shh–/–;Gli3–/– mice at
different developmental stages. During murine kidney development,
Pax2, a member of the paired box family of transcription factors, is
expressed in the Wolffian Duct, ureteric bud and induced metanephric blastema
and is required for metanephric development
(Dressler et al., 1990;
Torres et al., 1995)
(Fig. 3A). Thus, we used
Pax2 mRNA expression as a marker of early inductive events
(Fig. 3A). Consistent with the
finding of renal aplasia in 50% Shh–/– mice,
Pax2 mRNA expression was barely detectable in 
50% of
Shh–/– mice
(Fig. 3B). In the remaining
Shh–/– embryos, Pax2 mRNA was
detected in the metanephros (Fig.
3C). Although loss of Gli3 function alone had no effect
on Pax2 mRNA expression (Fig.
3D), it restored Pax2 mRNA expression in all
Shh–/–;Gli3–/– mice
(Fig. 3E). Next, we
investigated the role of Gli3 at a later developmental stage when
branching morphogenesis and metanephric epithelialization are established. By
E13.5, Pax2 is normally expressed in both the induced metanephric
mesenchyme and ureteric bud branches (Fig.
3K). In addition, Sall1 expression is localized to the
metanephric mesenchyme (Fig.
3P). Both Pax2 and Sall1 are required for
metanephrogenesis at this stage
(Nishinakamura et al., 2001;
Rothenpieler and Dressler,
1993). Using Pax2 and Sall1 as markers of
ureteric bud and metanephric development, we observed rescue of kidney number,
histology and Pax2 and Sall1 expression in
Shh–/–;Gli3–/– mice
(Fig. 3J,O,T) compared with
Shh–/– mice
(Fig. 3G,L,Q). Interestingly,
deficiency of neither Gli2 nor Gli3 interfered with
expression of Pax2 (Fig.
3M,R) and Sall1 (Fig.
3N,S). Taken together, these data demonstrate that the deleterious
effect of SHH deficiency on kidney development is dependent on GLI3.
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). We determined the impact of altered SHH signaling on cell
proliferation by measuring incorporation of 5-bromodeoxyuridine (BrdU), a
surrogate marker of cell proliferation, in embryonic kidneys
(Fig. 4). Qualitative analysis
of kidney sections stained with anti-BrdU antibody
(Fig. 4A-D) revealed
incorporation of BrdU in epithelial and mesenchymal derivatives located on the
outer regions of kidneys isolated from wild-type,
Gli3–/– and
Shh–/–;Gli3–/– kidneys.
By contrast, BrdU incorporation was markedly diminished in
Shh–/– kidneys. Quantitation of the number of
BrdU-positive cells in ureteric bud branches, identified by Dolichos Biflorus
Agglutinin (Grisaru et al.,
2001), and in mesenchymal cells demonstrated an approximate
twofold and fourfold diminution of cell proliferation in the ureteric bud and
metanephric mesenchyme, respectively, in kidney tissue from
Shh–/– mice compared with wild type
(Fig. 4E). By contrast, in
kidneys isolated from
Shh–/–;Gli3–/– mice,
cell proliferation in both these tissue compartments was rescued to levels
observed in wild-type tissue. Interestingly, a higher rate of ureteric bud
cell proliferation was observed in Gli3 deficient mice compared with
wild type.
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;
Mill et al., 2005). Thus, we
determined the effect of SHH deficiency on the expression of cyclin D1, cyclin
D2, MYCN and MYC in the kidney of SHH-deficient mice using specific antibodies
and western analysis (Fig. 4F).
Although each of these gene products is expressed in the embryonic kidney in
wild-type mice, the expression of both cyclin D1 and MYCN was greatly
diminished in kidneys isolated from Shh–/–
mice but was rescued in the kidneys of
Shh–/–;Gli3–/– mice.
Together, these data demonstrate a crucial role for GLI3 in controlling the
response to SHH deficiency at the level of cell proliferation and target gene
expression.
Inhibition of SMO changes the association of GLI proteins with GLI-binding consensus regions within SHH target genes
We determined how differential control of GLI1, GLI2 and GLI3 by SHH-SMO
signaling controls the expression of SHH target genes in the kidney. First, we
identified putative GLI-binding consensus sequences
(Ikram et al., 2004;
Kinzler and Vogelstein, 1990;
Sasaki et al., 1997) in the
5' flanking regions of Pax2, Sall1, cyclin D1 and Mycn
(Fig. 5A). Next, we used
chromatin immunoprecipitation to analyze the association of GLI1, GLI2 or GLI3
with regions encompassing these sequences. Our results indicate that GLI1
and/or GLI2, but not GLI3, bind these promoter elements in wild-type kidneys
either cultured in vitro (Fig.
5B) or processed immediately after isolation (see Fig. S2 in the
supplementary material). Treatment of embryonic kidney explants with
cyclopamine induced changes in GLI protein binding with these target promoters
(Fig. 5B). Most remarkably, the
binding of GLI1 and GLI2 with Pax2 and Sall1 was barely
detectable. Strikingly, we observed de novo binding of GLI3 with each of these
promoters. These results demonstrate that decreased SMO signaling changes
induces the de novo association of GLI3 and decreases the association of GLI1
and GLI2 with target promoters.
GLI3 controls decreased GLI1 and GLI2 expression in SHH deficient mice
Our genetic analyses in Shh–/– mice
demonstrated that Shh deficiency decreases GLI1 and GLI2 as well as
expression of Shh target genes. By contrast, removal of both
Gli3 and Shh restored expression of Shh target
genes. To determine whether Gli3 plays a primary role in orchestrating these
events, we investigated whether Gli3 controls Gli1 and Gli2 in
Shh–/–;Gli3–/– mice.
Analysis of GLI protein expression in kidney tissue lysates demonstrated
rescue of GLI1 and GLI2 levels in
Shh–/–;Gli3–/– mice to
the levels observed in wild-type mice (Fig.
6A). To determine whether GLI3 controls GLI1 and GLI2 expression
at a transcriptional level, we examined mRNA in kidney tissue isolated from
wild-type and
Shh–/–;Gli3–/– mice
using reverse transcriptase PCR (Fig.
6B). Although Gli1 and Gli2 mRNA levels were
decreased in Shh–/– mice, the levels of these
mRNAs were rescued to wild-type levels in
Shh–/–;Gli3–/– mice.
These results provided a basis for determining how GLI3 controls Gli1
and Gli2 transcription. First, we identified GLI binding sites in the
5' flanking regions of Gli1 and Gli2
(Fig. 6C). Next, we examined
the association of GLI proteins with these sites by chromatin
immunoprecipitation. Analysis of the regions encoding GLI binding sequences in
wild-type kidneys either cultured in vitro
(Fig. 6D) or processed
immediately after isolation (see Fig. S3 in the supplementary material)
demonstrated that GLI2 bound both Gli1 and Gli2. A weaker
association was observed between GLI1 and Gli1 or Gli2 and
between GLI3 and Gli2. cyclopamine enhanced binding between GLI3 and
the Gli1 promoter and between GLI3 and the Gli2 promoter. By
contrast, the associations with GLI2 were decreased to almost undetectable
levels. Our concurrent finding that cyclopamine decreased mRNAs encoding
Gli1 and Gli2 and Pax2 showed that these effects in
vitro paralleled our findings in vivo (see Fig. S4 in the supplementary
material). Together, these results demonstrate that GLI3 binds to the
Gli1 and Gli2 promoters when SHH-SMO signaling is
inhibited.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Buttitta et al., 2003;
McDermott et al., 2005). Yet,
homozygous deficiency of Gli1, Gli2 and Gli3 does not
negatively impact murine kidney development
(Bai et al., 2002;
Kim et al., 2001;
Schimmang et al., 1992). By
contrast, mutations that generate expression of a shortened repressor-like
form of GLI3 in humans with Pallister-Hall syndrome
(Kang et al., 1997) and in
genetically engineered mice (Bose et al.,
2002) also disrupt kidney development. Our studies provide novel
insights into the potential actions of GLI3 repressor showing that when SMO is
inhibited, GLI3 controls Gli1 and Gli2 expression and
associates with the Gli1 and Gli2 promoters. These findings
suggest that in Pallister-Hall syndrome, GLI3 represses Gli1 and
Gli2 as well as Pax2, Sall1, cyclin D1 and Mycn via
interactions with their respective promoter elements. More generally, our
findings provide insight into the observation that genetic inactivation of
Gli3 can rescue Shh mutant phenotypes in nonrenal tissues
(Litingtung and Chiang, 2000;
Litingtung et al., 2002;
Mill et al., 2005;
Rallu et al., 2002). Our
results suggest that in these different developmental contexts GLI3 repressor
directly controls Gli transcription and that of
Gli-dependent target genes.
Kidney patterning genes are direct GLI transcriptional targets
Our results provide new insights into the target genes regulated by SHH and
SMO, demonstrating that Shh acts upstream of Pax2 and
Sall1. Both Pax2 and Sall1 perform crucial
functions during murine renal organogenesis
(Nishinakamura et al., 2001;
Rothenpieler and Dressler,
1993). Mutations in PAX2 are associated with renal
coloboma syndrome and renal hypoplasia
(Sanyanusin et al., 1995).
Homozygous inactivation of Pax2 in mice results in renal aplasia or
severe dysgenesis (Rothenpieler and
Dressler, 1993), indicating a requirement for Pax2 during
outgrowth of the ureteric bud and invasion of the metanephric blastema. The
observation that Pax2 controls expression of Gdnf
(Brophy et al., 2001), which
encodes a secreted growth factor essential for outgrowth and invasion by the
ureteric bud (Moore et al.,
1996), suggests that Shh acts upstream of the GDNF-RET
signaling pathway during renal organogenesis. SALL1 mutations are
found in individuals with Townes Brock syndrome and renal aplasia/dysplasia
(Kohlhase et al., 1998).
During murine kidney development, Sall1 expression is restricted to
the metanephric mesenchyme where it is essential as homozygous inactivation of
Sall1 abrogates outgrowth of the ureteric bud
(Nishinakamura et al., 2001).
Our results provide novel insight into Sall1 regulation showing that
it is controlled by Shh.
Our results using chromatin immunoprecipitation extend our genetic analyses and demonstrate that promoter elements in both Pax2 and Sall1 are bound by GLI1, GLI2 and GLI3 to variable degrees and in different combinations, depending, in part, on the state of SHH-SMO signaling.
GLI transcriptional control of target genes is dependent on context
Our results demonstrate that SHH controls cell proliferation and expression
of cyclin D1 and MYCN during kidney development as in non-renal embryonic
tissues (Long et al., 2001;
Mill et al., 2005).
Interestingly, while cyclin D2 is regulated by SHH in the developing hair
follicle (Mill et al., 2005),
cyclin D2 expression is independent of Shh expression in the
embryonic kidney. The mechanisms by which GLI proteins mediate the actions of
SHH to control cell proliferation appear to differ between skin and kidney. In
skin, both Shh and Gli2 control cell proliferation
(Mill et al., 2005). In
kidney, Gli2 deficiency does not affect cell proliferation (data not
shown). In both skin and kidney Gli3 deficiency rescues decreased
cell proliferation observed in Shh–/– mice.
However, the degree of rescue is much greater in kidney compared with skin,
suggesting that other factors modulate the actions of GLI proteins to control
cell proliferation. These factors may include GSK3ß and ß-catenin
(Mill et al., 2005). The
relevance of this mode of regulation to kidney development requires further
investigation.
A model of gene regulation in states of decreased SHH-SMO activity
The experiments reported here provide a basis for a model that predicts
that Gli3 acts downstream of SMO and upstream of Gli1 and
Gli2 in the developing kidney and that Shh programs kidney
morphogenesis by restricting the activity of GLI3. In our model, GLI3
repressor orchestrates the effects of SMO inhibition, thereby disrupting
kidney morphogenesis (Fig. 7C).
GLI3 repressor acts by inhibiting the transcription of several classes of
genes, the actions of which are crucial to kidney development
(Fig. 7C). The first is the Gli
family, specifically Gli1 and Gli2. The second class of
genes includes those crucial to renal patterning. Members of this class
include Pax2 and Sall1. The third class of genes is that
controlling cell proliferation and includes cyclin D1 and MYCN. Formation of
GLI3 repressor is critical to repression of these genes. Our findings provide
a basis for investigating molecular mechanisms that control the interplay of
GLI1, GLI2 and GLI3 in the regulation of these SHH-SMO target genes.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/3/569/DC1
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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