A New Cre Driver Mouse Line, Tcf21/Pod1-Cre, Targets
Yoshiro Maezawa1, Matthew Binnie2, Chengjin Li1, Paul Thorner3, Chi-Chung Hui4, Benjamin Alman4,5,
Makoto Mark Taketo6, Susan E. Quaggin1,7,8*
1The Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada, 2Division of Respirology, St. Michael’s Hospital, University of Toronto,
Toronto, Ontario, Canada, 3Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada, 4Program in Developmental and Stem
Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada, 5Division of Orthopaedic Surgery, University of Toronto, Toronto, Ontario, Canada, 6Department of
Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 7Division of Nephrology, St. Michael’s Hospital, University of Toronto, Toronto, Ontario,
Canada, 8Department of Medicine, University Health Network, University of Toronto, Toronto, Ontario, Canada
Conditional gene targeting in mice has provided great insight into the role of gene function in kidney development and
disease. Although a number of Cre-driver mouse strains already exist for the kidney, development of additional strains with
unique expression patterns is needed. Here we report the generation and validation of a Tcf21/Pod1-Cre driver strain that
expresses Cre recombinase throughout the condensing and stromal mesenchyme of developing kidneys and in their
derivatives including epithelial components of the nephron and interstitial cells. To test the efficiency of this line, we crossed
it to mice transgenic for either loss or gain of function b-catenin conditional alleles. Mice with deletion of b-catenin from
Tcf21-expressing cells are born with hypoplastic kidneys, hydroureters and hydronephrosis. By contrast, Tcf21-Cre driven
gain of function for b-catenin in mice results in fused midline kidneys and hypoplastic kidneys. Finally, we report the first
renal mesenchymal deletion of Patched1 (Ptch1), the receptor for sonic hedgehog (Shh), which results in renal cysts
demonstrating a functional role of Shh signaling pathway in renal cystogensis. In summary, we report the generation and
validation of a new Cre driver strain that provides robust excision in metanephric mesenchyme.
Citation: Maezawa Y, Binnie M, Li C, Thorner P, Hui C-C, et al. (2012) A New Cre Driver Mouse Line, Tcf21/Pod1-Cre, Targets Metanephric Mesenchyme. PLoS
ONE 7(7): e40547. doi:10.1371/journal.pone.0040547
Editor: Christos Chatziantoniou, Institut National de la Sante ´ et de la Recherche Me ´dicale, France
Received May 3, 2012; Accepted June 8, 2012; Published July 6, 2012
Copyright: ? 2012 Maezawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by Canadian Institutes of Health Research grants MOP62931, Terry Fox grant 016002 to S.E.Q. who holds the Gabor-Zellerman
Chair in Renal Research, University of Toronto. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The introduction of gene targeting in mouse embryonic stem
cells in 1985 provided an efficient means to determine the function
of gene(s) in mammalian tissues in vivo. More recent advances
have permitted cell specific and temporal regulation of gene
deletion and overexpression, providing investigators more refined
tools to study the role of genes and molecular pathways in specific
organs and tissues. The Cre-loxP system is most broadly used,
largely due to the availability of a wide variety of floxed mouse
lines in public consortia, pharmaceutical companies and academic
In order to take full advantage of available floxed lines, renal
researchers must be able to choose from a wide variety of
complementary, robust and unique Cre-driver strains. Metanephric
mammalian kidneys derive from two cell lineages: metanephric
mesenchyme and ureteric bud that both arise from the intermediate
mesoderm . The metanephric mesenchyme is comprised of
subpopulations including the self-renewing cap mesenchyme and
stromal mesenchyme. The cap mesenchyme gives rise to all of the
epithelial cell derivatives of the nephron from the glomerulus to the
distal nephron, whereas the stromal mesenchyme is believed to give
the precise molecular steps have not been determined  . A
number of Cre driver strains exist for early kidney studies; e.g.
HoxB7  for the ureteric bud lineage, Six2 and Pax3 for the cap
mesenhcyme [4,6,7], Foxd1 for the stromal population  and Wnt4
for the renal vesicles . In addition, a number of Cre driver strains
exist for fully differentiated cells in the nephron (reviewed in ).
Tcf21/Pod1 is a basic helix loop helix transcription factor with a
unique expression pattern in both cap and stromal mesenchyme,
providing an opportunity to develop a Cre-driver strain capable of
Here we report the generation of a Cre-driver line using the
endogenous Tcf21 promoter and describe robust renal phenotypes
when this Cre driver strain is crossed to mice carrying a floxed b-
catenin allele causing loss of function, an allele resulting in b-catenin
gain of function and a floxed Ptch1 receptor allele. The Tcf21-Cre line
provides a robust and valuable alternative genetic tool for the
developmental renal biologist.
Materials and Methods
All mouse experiments were approved by the animal ethics
committee at the Toronto Center for Phenogenomics, and were
performed in accordance with ‘Canadian Council of Animal Care’
PLoS ONE | www.plosone.org1July 2012 | Volume 7 | Issue 7 | e40547
Construction of a Tcf21-Cre targeting vector
To make the Tcf21-Cre targeting vector, a 3.1 kb fragment
including intron 1, exon 2 and the 39 UTR of the Tcf21 gene
(accession number AF296764) was amplified from murine
genomic DNA by PCR and subcloned into the KpnI site of the
pKO (knockout) vector (a kind gift from Dr. J. Rossant, The
Hospital for Sick Children, Toronto, Canada). For the 59
homology arm, a 3.5 kb fragment was amplified from the 59
flanking region of exon 1, subcloned into pBluescript KS+, and
excised using SmaI and SalI. The Cre recombinase cassette and b-
actin polyA fragment were excised from the NLS-Cre plasmid
(kind gift from Dr. Brian Sauer, Oklahoma Medical Research
Foundation, Oklahoma City, OK) and Nephrin promoter NLS-
Cre plasmid  using EcoRI/SalI sites and EcoRI/XbaI sites,
respectively. The Tcf21-PKO vector was digested with XbaI and
PmlI, and the four fragments were subjected to ligation to
construct the final Tcf21-Cre targeting vector.
ES cell culture and generation of chimeras
The Tcf21-Cre vector was linearized by Pme1 and electroporated
into murine R1 ES cells derived from male blastocyst, hybrid of two
129 substrains (129X1/SvJ and 129S1/SV-+p+Tyr-cKitlSl-J/+) .
After selection with G418 and FIAU (5-Iodo-29-fluoroarauracil),
resistant clones were selected and subjected to Southern analysis
using a 39 Tcf21 genomic 500 bp probe outside the region of
homology that recognized a 5.3 kb and a 9.1 kb HindIII fragment
for the mutant and wild-type alleles, respectively. Embryo
manipulation and aggregation of the ES cell clones were carried
out. One ES cell line generated chimeras that gave germline
Breedings of mouse lines
The Tcf21-Cre heterozygous founder mice were crossed with a
Z/EG reporter murine line (kind gift from Dr. A Nagy, the Samuel
Lunenfeld Research Institute). The Z/EG reporter construct is
comprised of a systemic pCAGGS promoter which directs the
loxP-flanked b-Geo (lacZ/neomycin-resistance and STOP signal)
fusion gene followed by a green fluorescent protein (GFP) cassette
. Site-specific Cre recombinase expression leads to removal of
the STOP signal resulting in GFP expression. Tcf21-Cre mice were
then bred to b-catenin conditional loss of function (LOF) mice
(Ctnnb1fl/fl) , b-catenin conditional gain of function (GOF) mice
(Ctnnb1ex3/+) , and conditional loss of function Ptch1 mice
(Ptch1fl/fl)  to examine their phenotypes. Six2-Cre mouse line
was a kind gift from Dr. Andrew P. McMahon (Harvard
University, Cambridge), and crossed with Z/EG mouse line.
Tcf21-Cre mice offsprings were genotyped by 59mPod-1 primer
(59-CGGGACTGCCAGATCCCACC-39), AS-mPod-1 primer
primer (TTCTCCCACCGTCAGTACGTCA) that produce a
353 bp product for the wild type allele and a 719 bp product for
the mutant allele, respectively. Genotyping of the LOF and GOF
b-catenin mice and Ptch1 mice have been described previously
Murine embryos were dissected at specific time points described,
and macroscopic phenotypes of each organ were photographed
using a Leica dissecting microscope (Leica, MZ6). Whole embryos
or organs were fixed in 10% formalin in phosphate-buffered saline
(PBS) and subjected to histological analysis.
For immunohistochemistry, tissues were dissected and fixed in
10% formalin/PBS and embedded in paraffin. 5 mm-thick sections
were then rehydrated, boiled in citrate buffer for antigen retrieval,
and endogeneous peroxidases were quenched with 3% hydrogen
peroxide. For immunofluorescence, samples were fixed overnight
in 4% paraformaldehyde at 4uC, cryoprotected in 30% sucrose
overnight, embedded in Tissue-Tek OCT 4583 compound (Sakura
Finetek USA Inc., Torrance, CA, USA) and 10 mm sections were
cut by cryostat (Leica CM3050S). After blocking (10% goat serum,
3% albumin and 0.3% TritonX100 in PBS), samples were
incubated with primary antibodies at 4uC overnight, washed and
incubated with secondary antibodies for 1 h at room temperature.
Primary antibodies used for this study included anti-GFP (A11122,
Invitrogen, Camarillo, CA, USA), anti-alpha-smooth muscle actin
(a-Sma, ab5694, Abcam, Cambridge, MA, USA) and anti-Tamm
Horsfall protein (BT-590, Biomed Technologies, Inc. Stoughton,
MA, USA). Immunohistochemical staining was carried out using
Vectastain ABC kit (Vector Laboratories). Diaminobenzidine
(Vector Laboratories) was used for the color reaction.
Two lectins were utilized to stain specific renal segments.
Biotinylated Dolichos biflorus agglutinin (DBA, B-1035, Vector
Laboratories, Burlingame, CA, USA) was used as a specific marker
of the collecting duct, and visualized by FITC labeled avidin
(Vector Laboratories). FITC labeled Lotus tetragolonobus lectin
(LTL, FL-1321, Vector Laboratories) is a specific marker of the
proximal tubule and was visualized directly with a fluorescent
In situ hybridization
The samples were fixed overnight in DEPC-treated 4%
paraformaldehyde at 4uC, cryoprotected in 30% sucrose overnight
at 4uC, embedded in Tissue-Tek OCT 4583 compound and snap
frozen. 10 mm sections were cut on a cryostat and transferred to
Superfrost microscope slides (Fisher Scientific Co., Pittsburgh,
Pennsylvania, USA). Digoxigenin-labeled probes were prepared
according to the manufacturer’s instruction (Roche Molecular
Biochemicals, Mannheim, Germany). Probes used for in situ
analysis were Bmp4, Fgf8 (a kind gift from Dr. Janet Rossant, the
Hospital for Sick Children, Toronto), Wnt4 (a kind gift from Dr.
Andy McMahon at Harvard University), and Gli1 (a kind gift from
Dr. Alexandra Joyner, Sloan Kettering Institute, New York).
Details of the in situ analysis protocol may be obtained upon
Generation of a Tcf21-Cre founder mouse line
The targeting construct for Tcf21-Cre was designed to replace
the first exon of the Tcf21 gene with Cre recombinase and a
neomycin cassette (Fig. 1A). After electroporation, murine ES cell
clones were screened by Southern blot analysis using a HindIII
digest and 39 probe outside the region of homology. One correctly
targeted ES cell clone was used to generate chimeric mice that
gave germline transmission (Fig. 1B).
Tcf21-Cre driver mouse line excises genes in the
metanephric mesenchyme and its derivatives
Tcf21-Cre mice were bred with Z/EG reporter mice. In
embryonic day 10.5 metanephros, expression of the GFP reporter
(denoting successful Cre-mediated excision) was observed in the
condensing metanephric mesenchyme surrounding the invading
ureteric bud (Fig. 1C). At this stage, GFP staining appeared to be
thin and mosaic. At E11.5, GFP expression in metanephric
Generation of Tcf21/Pod1-Cre Mouse
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mesenchyme was evident (Fig. 1D). By E13.5, gene deletion
activity was shown in broad areas in the mesenchyme including
condensing mesenchyme, developing nephrons such as pretubular
aggregates and comma-shaped bodies, interstitial stromal cells,
and the mesenchyme surrounding the stalk of ureteric bud
(Fig. 1E). At E16.5, in addition to the GFP expression observed at
E13.5, GFP expression was also appreciated in S-shaped bodies,
immature podocytes in capillary loop glomeruli, and immature
tubular cells (Fig. 1F). We compared the domain of Tcf21-driven
Cre excision to that of the Six2-Cre mice. As seen in Fig. 1G, Six2-
Cre activity is restricted to cap mesenchyme and its derivatives. At
postnatal day 0, in Tcf21-Cre;Z/EG mice, GFP was observed in
podocytes, tubules and cells of Bowman’s capsule, though it
appeared mosaic in the tubules (Fig. 1H). Double immunostaining
for GFP and pancytokeratin showed no overlap, demonstrating
that Tcf21-Cre is not active in collecting duct cells that are derived
from the ureteric bud lineage (Fig. 1I). Taken together, Tcf21-Cre
has the capacity for broad gene deletion in the mesenchyme and
its derivatives, including both interstitial cells and epithelial
components of the nephron. Thus, Tcf21-Cre is a valuable resource
permitting the use of a single mouse Cre-driver strain to gain
insight into multiple metanephric populations.
Tcf21-Cre excises genes in mesenchymes in multiple
To explore the full potential of the Tcf21-Cre line, extrarenal
Cre activity was assessed. At E10.5, GFP expression was observed
in the 1stto 3rdbranchial arches (Fig. 2 AB), the heart, and in
metanephric mesenchyme surrounding the ureteric bud (Fig. 2C
and 1B). E11.5 embryos showed a similar pattern (data not
shown). At E13.5 and E16.5, GFP expression was prominent in
epicardium, lung mesenchyme, kidney mesenchyme, lamina
propria and smooth muscle layer of the gastrointestinal tract,
pancreas, adrenal gland, gonads, proximal part of aorta, facial and
sublingual muscles, and in the choroid plexus of the ventricles
Ctnnb1fl/fl;Tcf21-Cre mice develop hydroureter and/or
hypoplastic rudimentary kidneys
Wnt-b-catenin signaling plays a key role in the interaction
between the metanephric mesenchyme and the ureteric bud during
renal development [19,20]. To validate the Tcf21-Cre line, we bred
Tcf21-Cre mice with a b-catenin conditional loss of function (LOF)
mouse (Ctnnb1fl/fl;Tcf21-Cre, referred to as b-catenin LOF mutant,
hereafter). The b-catenin LOF mutants were born with the expected
Mendelian ratio, but died within a few hours after birth. The
deletion of b-catenin gene was confirmed by PCR, and was observed
in the kidney and extrarenal tissues where Tcf21 is expressed
including heart, lung, pancreas and gastrointestinal tract (Fig. 3A).
Examination at E18.5 and in P0 pups demonstrated hydroureter
with hypoplasia in 73.5% of the mutants, whereas 20.5% of mutants
showed hypoplastic rudimental kidneys (Fig. 3B–D). Most of the
mutant kidneys showed various degrees of hypoplasia. Kidneys with
hydroureter demonstrated dilated tubules (Fig. 3F) and expansion of
capillary loops, but some glomeruli appeared to be intact (Fig. 3I).
The rudimentary kidneys were cystic, had only a few immature
glomeruli, and showed random, disorganized nephrogenic aggre-
gates at the periphery (Fig. 3GJ). As expected from this phenotype,
Figure 1. Tcf21-Cre mouse delete genes in metanephric
mesenchyme and its derivatives. (A) Scheme of Tcf21-Cre targeted
allele: Exon1 of the Tcf21 gene was replaced by a Cre transgene and
neomycin cassette. (B) Genomic DNA from ES cell clones was isolated
and digested with HindIII. The 500 bps probe outside the region of 39
homology arm recognized a 5.3 kb and a 9.1 kb fragment for the
mutant and wild-type alleles, respectively. Tcf21-Cre (C–F and H, I) and
Six2-Cre (G) mice were bred to the Z/EG reporter mousline and Cre
dependent gene deletion was examined by immunostaining for GFP.
(C) At E10.5, thin and mosaic expression of GFP is present in
metanephric mesenchyme (black arrowhead). (D) E11.5 embryo shows
distinct expression of GFP in metanephric mesenchyme. Ureteric bud is
undergoing primary branching. (E) GFP expression is observed in
condensing mesenchyme (black arrowhead), pretubular aggregates
(white arrowhead), interstitial stromal cells (black arrow), mesenchyme
surrounding the ureteric bud (white arrow) at E13.5 embryo. (F) At
E16.5, GFP is expressed in developing nephrons and stromal cells.
Staining in S shaped body (white arrowhead) and presumptive
podocytes (black arrowhead) can be seen in this picture. (G) Six2-Cre
gene deletion is restricted to condensing mesenchyme (black
arrowhead), developing nephrons (large black arrowhead) and podo-
cytes (black arrow) at E16.5. (H) At postnatal day 0, gene deletion in
podocytes (black arrowhead) and Bowman’s capsule (white arrowhead)
is evident, but tubular staining was mosaic (white arrows). (I) P0 kidney
was stained with GFP (green) and pancytokeratin (red). There was no
overlap between the two stainings. Magnification: C–H 2006, I 406.
Generation of Tcf21/Pod1-Cre Mouse
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expression of nephrogenic induction markers, Wnt4 and Fgf8, were
markedly reduced and only detectable in the disorganized nephro-
genic area (Fig. 3K–N). Neither tubular structures nor interstitial
space were present.
In order to highlight the unique characteristics of Tcf21-Cre, we
compared the phenotype to that of previously published mice with
b-catenin deletion in the cap mesenchyme (Six2-Cre) or stromal cell
(FoxD1-Cre) populations. Six2-Cre;Ctnnb1fl/flmice were deficient
in nephrogenesis in the nephrogenic zone, whereas FoxD1-
Cre;Ctnnb1fl/flmice failed to develop the interstitial space [20,21].
Compared to these compartmentalized phenotypes, the Tcf21-Cre
phenotype extends from the nephrogenic zone, the interstitial
space to the ureter, and also provides a range of severity of
phenotypes among littermates due to a variable degree of
mosaicism (Table 1). Together, these results demonstrate that
the use of Tcf21-Cre allows the assessment of gene function in both
the stromal and cap mesenchymes.
In addition to the previously reported phenotypes, we observed
a high incidence of hydroureter in b-catenin LOF mutants, likely a
result of deletion in the periureteric bud mesenchyme that gives
rise to smooth muscle cells. In keeping with this possibility, we
examined the expression of a-Sma and Bmp4 , which is known to
play a key role in the development of the organized smooth muscle
layer in the ureter [22,23]. Although a-Sma positive cells were
present in the dilated ureter wall, the smooth muscle layer was
extremely thin (Fig. 3OP). Notably, Bmp4 could not be detected in
the mesenchyme surrounding the ureteric bud at E13.5 (Fig.
3QR), or in the dilated ureter at E18.5 (data not shown).
Ctnnb1ex3/+;Tcf21-Cre mice develop fusion kidney or
hypoplastic rudimentary kidney
Several recent papers have demonstrated that tight regulation of
b-catenin expression is required for tissue homeostasis [24,25].
Thus, as a second test to validate the Tcf21-Cre driver strain, we
bred it with the conditional gain of function b-catenin mice
(Ctnnb1ex3/+;Tcf21-Cre, referred to as b-catenin GOF mutant).
Exon 3 of the mutant allele is flanked by loxP sequences. Upon
Cre-mediated excision, the serine and threonine residues in exon 3
are removed, preventing phosphorylation and resulting in a
stabilized b-catenin molecule that translocates and accumulates in
the nucleus escaping ubiquitination and degradation .
b-catenin GOF mutants die during embryonic life between
E12.5 and P0 (data not shown). At E18.5, 66% of mutants
demonstrated fusion kidneys at midline and 33% had severely
hypoplastic/dysplastic kidneys with a few disorganized glomeruli
(Fig. 4A–E). These phenotypes were striking and differed from the
previously reported Six2-Cre;b-catenin gain of function phenotype,
which is renal agenesis at birth . Rudimentary kidneys of
Tcf21-Cre mutants and agenesis observed in Six2-Cre mutants may
both be due to failure of nephrogenesis in the cap mesenchyme
and its derivatives, but midline fusion appears to arise as a result of
the broader gene deletion of Tcf21-Cre.
Strikingly, 100% of the b-catenin GOF mutants also exhibited
embryonic tumor that engulfed the kidney, gut, heart, and lungs,
and appeared to arise from multiple mesenchymal tissues (Fig. S1).
Importantly, this phenotype did not obscure the renal phenotype
but as discussed below, provided new insight into possible genetic
interactions between the b-catenin and Shh pathways.
Ptch1fl/fl;Tcf21-Cre kidneys develop multiple renal cysts
As a final test of Tcf21-Cre line, we deleted Ptch1, the receptor for
Shh. The Shh signaling pathway is crucial for the development of
virtually all organs, and regulates cell fate determination,
proliferation and tissue patterning . After birth, Shh signaling
is associated with certain types of malignancy [27,28,29]. Shh null
mice show renal aplasia . Shh and Ptch1 deletion from ureteric
bud result in hydroureter and hypoplastic kidney, respectively
[31,32]. However, the deletion of Ptch1 in the metanephric
mesenchyme has not been previously reported.
Ptch1fl/fl;Tcf21-Cre mice (referred to as Ptch1 mutants) die at
varying time points between E12.5 to E18.5 due to an aggressive
embryonic tumor with striking similarities to the tumors observed
in the b-catenin GOF mutants described above (Fig. S1). Ptch1
mutant kidneys were of a similar size to those of Ptch1fl/fl
littermates (Fig. 5A). Renal invasion of the tumor appeared to be
minimal (Fig. 5C). Histological examination of E18.5 mutant
kidneys demonstrated dilation of the pelvic area and multiple cysts
Figure 2. Tcf21- Cre mouse deletes genes in the mesenchyme of
multiple organs. (A) Dissecting microscope photograph of E10.5
Tcf21-Cre;Z/EG embryo. (B) GFP expression of E10.5 Tcf21-Cre;Z/EG
embryo was visualized by fluorescent microscopy. (C–Q) Tcf21-Cre mice
were bred to a Z/EG reporter mouse line and Cre dependent gene
deletion was examined by immunostaining for GFP. (C) E10.5 embryo
shows GFP staining in heart and metanephric mesenchyme. (D) E13.5
and (E) E16.5 embryos show gene deletion in multiple organs. At E 16.5,
GFP expression was observed in (F) some part of the epicardium and
endocardium, (G) lung mesenchyme, (H) kidney mesenchyme, (I) proper
muscle and lamina propria of esophagus, (J) proper muscle and lamina
propria of intestine, (K) some part of pancreas, (L) adrenal gland, (M)
stroma of gonad, (N) proximal part of aorta, (O) facial muscle, (P) lingual
and sublingual muscle, (Q) choroid plexus of ventricles. Magnification: C
406, F, H, O, P 1006, I, L, N 2006, G, J, K, M, Q 4006. Many 406pictures
were taken for D and E, and stitched together to construct the image of
the whole embryo.
Generation of Tcf21/Pod1-Cre Mouse
PLoS ONE | www.plosone.org4July 2012 | Volume 7 | Issue 7 | e40547
(Fig. 5CD). In some cysts, glomeruli were observed, whereas other
cysts arose in the renal tubules (Fig. 5D).
Stainings with Lotus tetragolonobus lectin (LTL), Tamm
Horsfall protein (Thp), and Dolichos biflorus agglutinin (DBA)
which marks the proximal tubules, loop of Henle, and collecting
ducts respectively, showed that the cysts arose along the entire
nephron (Fig. 5E–J). Mutant collecting ducts were dilated, distorted
and winding (Fig. 5J). Additionally, immunostaining for Ki67
showed increased cell proliferation in the cystic wall, suggesting a
possible mechanism for cyst formation (Fig. 5L).
We examined the expression of Gli1, a well-established marker
of Shh activation. As Ptch1 suppresses the Shh pathway, genetic
deletion of the Ptch1 gene should result in upregulation of Gli1
expression. In keeping with this, in situ hybridization showed
Figure 3. Ctnnb1fl/fl;Tcf21-Cre mice shows hydroureter or rudimentary kidneys. (A) DNA extracted from whole organs was subjected to PCR
that detects wild type, floxed allele, and deleted allele of b-catenin. Deleted alleles were detected in kidney, heart, lung, pancreas and gastrointestinal
tract. (B, E, H) Control kidney, (C, F, I) Hydroureter and relatively normal kidney of Ctnnb1fl/fl;Tcf21-Cre mice, (D, G, J) Rudimentary kidney and
hypoplastic kidney with hydroureter of Ctnnb1fl/fl;Tcf21-Cre mouse. (F) Hydroureter kidneys had relatively normal appearance, but show dilation of
tubules, (I) dilation of glomerular capillaries. Some of the glomeruli appear to be normal. (G) Rudimentary kidney was cystic, (J) had random
nephrogenic area (black arrowhead) with a few disorganized glomeruli. (K) Fgf8 expression in control kidney. (L) Fgf8 is observed only in the
disorganized nephrogenesis area (black arrowhead). (M) Wnt4 expression in control kidney. (N) Wnt4 is observed only in the disorganized
nephrogenesis area (Black arrowhead). (O) Expression of a-Sma (green) in control mouse. Blue shows DAPI staining. (P) In hydroureter mutant, a-Sma
(green) is present in the ureter wall, but its layer is extremely thin. (Q) Bmp4 expression surrounding the ureteric bud in E13.5 control. (R) Expression
of Bmp4 surrounding the ureteric bud is lost in E13.5 Ctnnb1fl/fl;Tcf21-Cre. However, Bmp4 expression is restored in developing nephrons. Experiments
were performed on E18.5 embryos if not indicated. B–D are taken at the same magnification. Mice that lack at least one of the transgenes were used
as controls. Magnification: E–G, K–P 1006, H–J 2006, Q, R 4006.
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increased expression of Gli1 in the condensing mesenchyme and
interstitial cells at E16.5 (Fig. 5N). However, surprisingly, Gli1
expression was not seen in developing nephrons, such as
pretubular aggregates and S-shaped bodies.
Here we report the generation of a novel Cre transgenic mouse
line, Tcf21-Cre, that allows gene excision in the metanephric
mesenchyme and its derivatives, including interstitial cells and all
epithelial components of the nephron from podocytes to distal
tubules. It also results in gene deletion throughout the mesen-
chyme of other organs including lung, heart, gastrointestinal tract,
pancreas, gonad and adrenal gland. Gene deletion begins at E10.5
in the condensing metanephric mesenchyme, and is consistently
observed in its derivatives throughout renal development.
Currently, Six2-Cre and Pax3-Cre are most widely used to
investigate the role of genes in the developing kidney and
metanephric mesenchyme [4,6,7]. Is there any advantage or need
for Tcf21-Cre? In contrast to Tcf21, Six2-Cre expression is restricted
to the cap mesenchyme. While this is useful to determine gene
function in this specific compartment, Tcf21-Cre provides a
broader mesenchymal excision from both stromal and cap
compartments as well as mesenchymal cells that give rise to
periureteric bud smooth muscle cells. Similar to Six2-Cre, Tcf21-Cre
expression remains active in epithelial nephric derivatives but
unlike Six2, Tcf21 is also expressed in interstitial lineages. As a first
step in validation, we compared and contrasted phenotypes in
mice lacking b-catenin following Six2 versus Tcf21-Cre deletion. As
expected, we observed many similarities but also important
differences. Given the broader expression domain of Tcf21, we
observed hydroureters and a smooth muscle cell phenotype with
down-regulation of Bmp4. Similarly, gain of function studies of b-
catenin provided similarities but also some differences, with Tcf21-
Cre causing midline fusion of the kidneys in b-catenin GOF
How does Tcf21-Cre compare to the Pax3-Cre? Similar to Tcf21-
Cre, Pax3-Cre reportedly excises in both epithelial and interstitial
lineages that derive from cap and stromal metanephric mesen-
chyme. However, the extrarenal sites of Pax3-Cre activation are
largely different, and include derivatives of neural crest and
somites, such as dorsal root ganglia, skeletal muscle, adrenal
medulla, and some subsets of colon epithelium, which do not
overlap with the mesenchymal distribution of Tcf21-Cre expression
An additional benefit of Tcf21-Cre includes the method of
generation of the Cre line through homologous recombination in
ES cells. This allows faithful recapitulation of expression pattern
ensuring stable expression over subsequent breedings. Randomly
inserted or BAC transgenic lines may or may not provide this
asset. It is worthwhile to note that Tcf21 is not expressed in
intermediate mesoderm [33,34], which suggests that the GFP
expression of Tcf21-Cre; Z/EG mice doesn’t include an excision
prior to the development of metanephros. In addition, we noted
mosaicism of the Tcf21-Cre expression and excision. Although this
might be viewed as a negative (incomplete excision), we found it to
be an asset in our experiments. As demonstrated with our proof of
principle studies in this report, a wide spectrum of phenotypes can
be observed in each litter ranging from severe to mild, allowing a
more complete picture of gene function– i.e. permitting a dose
response experiment in a single litter. Finally, the extrarenal
expression of Tcf21 in other mesenchymal tissues allows insight
from other organs. Although extrarenal expression of a Cre driver
strain is often viewed as a weakness, it may provide additional
information for the investigator. In this study, although we
observed robust extrarenal phenotypes in all of the mutants, they
did not preclude analysis of phenotype in kidney. One of the
reasons for this is the relatively late onset of Tcf21 expression at
mid-gestation. In turn, the development of aggressive embryonic
sarcomas in both b-catenin GOF and Ptch1 LOF mutants points
Table 1. Comparison of beta-catenin loss of function mutants by various renal Cre lines.
Nephrogenic Zone Hypoplastic kidney and reduced nephron
number. Lack of nephrogenic zone. No
expression of inductive markers, such
as Wnt4, FGF8 .
Relatively normal nephron
Wide spectrum of hypoplastic kidneys, from
almost normal to rudimentary kidneys.
Reduced expression of inductive markers.
Interstitial spaceNoneFailure of medulla development Failure of medulla development (Various)
UreterNone None Hydroureter
Figure 4. Ctnnb1ex3/+; Tcf21-Cre mice show fusion kidney and
rudimentary kidney. (A) Control kidney (left) and fusion kidney of
Ctnnb1ex3/+;Tcf21-Cre mouse (right). (B) Severely hypoplastic/dysplastic
rudimentary kidney of Ctnnb1ex3/+;Tcf21-Cre mouse. (C) Normal histol-
ogy of control kidney. (D) Histology of the fusion kidney. (E) Histology
of the rudimentary kidney. Magnification: C 406, E 1006. Many 406
pictures were taken for D, and stitched together to construct the image
of the whole fusion kidney.
Generation of Tcf21/Pod1-Cre Mouse
PLoS ONE | www.plosone.org6 July 2012 | Volume 7 | Issue 7 | e40547
to a common genetic pathway – providing direction for future
Utility of Tcf21-Cre strain is further underscored by the
phenotypes observed in the Ptch1 mutants. Although a role for
Shh pathway in renal cystic diseases is supported by dysregulation
of Gli1 in nephronophthisis , there has not been any direct genetic
evidence [35,36]. Here, we show the development of a dramatic
cystic renal phenotype in mice lacking the Ptch1 receptor with data
to support a role for proliferation. Interestingly, we did not detect
Shh pathway activation in the developing nephrons of Ptch1fl/
fl;Tcf21-Cre mice. It remains to be determined whether the
nephrons found in the mutant kidney are derived from wild type
cells that escape Cre excision due to the inability of Ptch1 null cells
to form nephrons.
In summary, we report a new Cre driver strain that provides
robust excision in the metanephric mesenchyme. It provides
researchers with a useful tool to delete genes broadly from multiple
metanephric mesenchyme subpopulations, resulting in phenotypes
in the stromal cells, the cap mesenchyme and the periureteric bud
Ptch1fl/fl;Tcf21-Cre and Ctnnb1ex3/+;Tcf21-Cre
mice show strikingly similar sarcomas. Hematoxylin and
eosin staining of (A, D, G, J) Control, (B, E, H, K) Ptch1fl/fl;Tcf21-
Cre, (C, F, I, L) Ctnnb1ex3/+;Tcf21-Cre. (A–C) lungs, (D–F) liver, (G–I)
gastrointestinal tract, (J–L) pancreas. The sections were examined
by two experienced pathologists and diagnosed as sarcomas, which
invade multiple organs. Magnification: all 406.
We gratefully acknowledge the technical support of Ken Harpal for
histological analysis. We also thank Dr. Andrew P. McMahon at Harvard
Medical School for providing us with Six2-Cre mice.
Conceived and designed the experiments: SEQ. Performed the experi-
ments: YM MB CL. Analyzed the data: YM PT SEQ. Contributed
reagents/materials/analysis tools: CCH BA MMT. Wrote the paper: YM
CCH MMT SEQ.
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Generation of Tcf21/Pod1-Cre Mouse
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Generation of Tcf21/Pod1-Cre Mouse
PLoS ONE | www.plosone.org8 July 2012 | Volume 7 | Issue 7 | e40547