RESEARCH REPORT 1975
Development 137, 1975-1979 (2010) doi:10.1242/dev.051656
© 2010. Published by The Company of Biologists Ltd
The transcription factors Etv4 and Etv5 mediate formation of
the ureteric bud tip domain during kidney development
Satu Kuure*, Xuan Chi†, Benson Lu and Frank Costantini‡
SUMMARY
Signaling by the Ret receptor tyrosine kinase promotes cell movements in the Wolffian duct that give rise to the first ureteric bud
tip, initiating kidney development. Although the ETS transcription factors Etv4 and Etv5 are known to be required for mouse kidney
development and to act downstream of Ret, their specific functions are unclear. Here, we examine their role by analyzing the ability
of Etv4 Etv5 compound mutant cells to contribute to chimeric kidneys. Etv4–/–;Etv5+/– cells show a limited distribution in the caudal
Wolffian duct and ureteric bud, similar to Ret–/– cells, revealing a cell-autonomous role for Etv4 and Etv5 in the cell rearrangements
promoted by Ret. By contrast, Etv4–/–;Etv5–/– cells display more severe developmental limitations, suggesting a broad role for Etv4 and
Etv5 downstream of multiple signals, which are together important for Wolffian duct and ureteric bud morphogenesis.
INTRODUCTION
One of the key signals that promote metanephric kidney
development is glial cell-derived neurotrophic factor (Gdnf), which
is secreted by metanephric mesenchyme (MM) cells and signals
through the receptor tyrosine kinase (RTK) Ret and co-receptor
Gfr1 (Takahashi, 2001). Ret and Gfra1 are expressed in the
Wolffian duct (WD) and then in the tips of the ureteric bud (UB), an
outgrowth from the caudal WD that branches extensively to form the
renal collecting system, while inducing MM progenitor cells to
generate the nephron epithelia (Carroll and McMahon, 2003;
Costantini, 2006; Dressler, 2009). Mice lacking Ret, Gfr1 or Gdnf
fail to make normal kidneys owing to defects in UB outgrowth or
subsequent branching (Costantini and Shakya, 2006). Sprouty 1
(Spry1), a negative regulator of RTK signaling, is expressed in the
UB and upregulated by Ret signaling to generate a crucial negativefeedback loop (Basson et al., 2005; Basson et al., 2006).
One of the main challenges in understanding how Gdnf
promotes branching morphogenesis is to identify the specific
cellular behaviors that are altered by Ret signaling. A fruitful
approach has been to generate chimeras in which a subset of
WD/UB cells lack Ret and examine their ability to contribute to a
chimeric kidney (Chi et al., 2009b; Shakya et al., 2005). These
studies have shown that an early role of Ret is to promote
competitive cell rearrangements in the caudal WD, in which cells
with the strongest Ret signaling preferentially form a localized
epithelial domain, which emerges as the first UB tip. In
Ret–/–}wild-type chimeras (i.e. those generated by injecting Ret–/–
cells into wild-type blastocyts), wild-type cells converge to form
this ‘primary tip domain’, whereas Ret–/– cells are excluded. By
Department of Genetics and Development, Columbia University Medical Center,
New York, NY 10032, USA.
*Present address: Institute of Biotechnology, University of Helsinki, Viikki Biocenter,
Viikinkaari 9, FIN-00014 Helsinki, Finland
†
Present address: Division of Hematology, Center for Biologics Evaluation and
Research, Food and Drug Administration, Bethesda, MD 20892, USA
‡
Author for correspondence ([email protected])
Accepted 19 April 2010
contrast, in Spry1–/–}wild-type chimeras, the Spry1–/– cells (with
elevated Ret activity) preferentially form the primary tip domain,
while excluding the wild-type cells (Chi et al., 2009b).
How does Ret signaling exert these effects on WD and UB cells?
Several genes have been identified that are regulated by Ret
signaling and are likely to contribute to altered cellular phenotypes
and behaviors in response to Gdnf (Lu et al., 2009; Pepicelli et al.,
1997). Among them are two closely related ETS transcription
factors, Etv4 (Pea3) and Etv5 (Erm), which are expressed in the
WD/UB lineage in a temporal and spatial pattern similar to that of
Ret (as well as in the MM and nascent nephrons). Their expression
in UB tips can be upregulated by exogenous Gdnf or Fgf10 and is
greatly reduced in a hypomorphic Ret mutant (de Graaff et al.,
2001; Lu et al., 2009). However, Etv4/Etv5 expression remains
normal in Fgf10–/– or Fgfr2 mutant kidneys, despite reduced UB
branching (Lu et al., 2009; Ohuchi et al., 2000; Zhao et al., 2004).
Thus, whereas FGF signaling is a major effector of Etv4/Etv5
expression in several organs (Brent and Tabin, 2004; Firnberg and
Neubuser, 2002; Liu et al., 2003; Mao et al., 2009), Gdnf is the
main stimulus for their expression in the UB.
The roles of Etv4 and Etv5 in kidney development have been
examined in mouse using an Etv4 knockout allele (Livet et al.,
2002) and two Etv5 loss-of-function alleles, Etv5tm1Hass (Lu et al.,
2009) and Etv5tm1Kmm (Chen et al., 2005). Etv5tm1Hass (which lacks
the DNA-binding domain) is a stronger allele, with homozygotes
dying at E8.5 (Lu et al., 2009), whereas homozygotes for
Etv5tm1Kmm (exons 2-5 deleted) have normal kidneys (Chen et al.,
2005). Etv5tm1Hass, the allele used in this study, will be referred to
as ‘Etv5–’. Etv4–/– or Etv4+/–;Etv5+/– mice have low frequencies of
renal agenesis or hypoplasia, whereas nearly all Etv4–/–;Etv5+/–
compound mutants have renal agenesis or hypodysplasia, with
greatly reduced UB branching (Lu et al., 2009). Etv4 Etv5 double
homozygotes have been generated using the weaker Etv5tm1Kmm
allele (to allow development past E8.5) and lack kidneys entirely
(Lu et al., 2009). Thus, Etv4 and Etv5 are jointly required for
kidney development.
Although several downstream genes regulated by Etv4/Etv5 in
the UB have been identified (Lu et al., 2009) (see below), how the
activity of these transcription factors (and hence their target genes)
DEVELOPMENT
KEY WORDS: Kidney, Morphogenesis, Cell movement, Mouse, Wolffian duct
1976 RESEARCH REPORT
Development 137 (12)
alters the behavior of WD and UB cells, compromising UB
outgrowth and branching, remains to be elucidated. To address this
question, we have employed chimeric assays with Etv4/Etv5
mutant cells.
MATERIALS AND METHODS
Derivation and genotyping of ES cells
ES cell lines were derived from blastocysts from Hoxb7-myrVenus;Etv4–/–
or Hoxb7-myrVenus;Etv4–/–;Etv5+/– females crossed to Hoxb7myrVenus;Etv4+/–;Etv5+/– males, as described (Shakya et al., 2005). Etv4,
Etv5 and Hoxb7-myrVenus alleles were genotyped as described (Chi et al.,
2009a; Lu et al., 2009); wild-type Etv5 was amplified with primers 5⬘GACCCCAGGCTGTACTTTGA-3⬘ and 5⬘-CAGTCCAGGCGATGAAGTG-3⬘.
Generation of chimeric embryos and genotyping of Rethypomorphic hosts
ES cell injections into wild-type and Rettm2(RET)Vpa/tm2(RET)Vpa hosts and
genotyping of host embryos were performed as described (Chi et al.,
2009b; Hogan et al., 1994; Shakya et al., 2005). To generate CFP+ host
embryos, Hoxb7/Cre;R26RCFP/CFP males (Srinivas et al., 2001; Yu et al.,
2002) were mated with R26RCFP/CFP females.
Kidney culture and antibody staining
Urogenital regions were dissected, cultured and imaged as described
(Watanabe and Costantini, 2004). Antibodies against pan-cytokeratin
(Sigma), calbindin (Santa Cruz), GFP (Molecular Probes) and
phosphohistone H3 (Ser10) (6G3) (Cell Signaling) were used to stain
whole-mount specimens (Kuure et al., 2005) and cryosections (Chi et al.,
2009b).
Mitotic index
RESULTS AND DISCUSSION
To investigate the cellular defects that lead to failure of renal
development in Etv4/Etv5 mouse mutants, we generated both
Etv4–/–;Etv5+/– and Etv4–/–;Etv5–/– embryonic stem (ES) cells,
injected them into wild-type blastocysts, and analyzed their ability
to contribute to the WD and UB in the resulting chimeric kidneys.
We compared their behavior with Ret–/– ES cells (Chi et al., 2009b;
Shakya et al., 2005). All the mutant ES cell lines carried a
transgene expressing a fluorescent protein in the WD and UB,
either Hoxb7-GFP (Srinivas et al., 1999) or Hoxb7-myrVenus (Chi
et al., 2009a), whereas the wild-type host embryos expressed cyan
fluorescent protein (CFP) in the same cell lineage (see Materials
and methods).
Fig. 1 shows whole-mount images of chimeras made with
Etv4–/–;Etv5+/– cells, at stages from the initial WD swelling
(E10.5) through early UB branching (E12.5), as compared with
Ret–/– chimeras. As previously observed, the Ret–/– cells were
specifically excluded from a dorsal domain of the E10.5 WD,
termed the ‘primary tip domain’ because it gives rise to the first
UB tip (Chi et al., 2009b) (Fig. 1A). As the UB emerged, Ret–/–
cells were absent from the tip, but contributed to the trunk (Fig.
1B,C). In Etv4–/–;Etv5+/–}wild-type chimeras, the mutant cells
behaved very similarly to Ret–/– cells, as seen in whole-mount
specimens (Fig. 1D-F) or in cross-sections (Fig. 2). During the
first UB branching at E11.5, the Etv4–/–;Etv5+/– cells (Fig. 1J,M),
like Ret–/– cells (Fig. 1G), contributed preferentially to the
proximal side of the initial UB branches. During subsequent
Fig. 1. Etv4–/–;Etv5+/– cells, like Ret–/– cells, are excluded from the
ureteric bud tip domain in mutant}wild-type chimeric mouse
kidneys. Wild-type or mutant ES cells, carrying Hoxb7-GFP or Hoxb7myrVenus, were injected into host blastocysts carrying Hoxb7/Cre and
R26R-CFP. In the chimeric embryos, host-derived Wolffian duct (WD)
and ureteric bud (UB) cells express CFP (cyan), and ES-derived cells
express GFP or myrVenus (green). (A)As previously reported (Chi et al.,
2009b), Ret–/– ES cells contribute poorly to the primary UB tip domain
(arrow) and common nephric duct (CND; asterisk) at E10.5. (B,C)At
E11.0, the UB tip is composed mainly of wild-type cells.
(D-F)Etv4–/–;Etv5+/– cells are mostly excluded from the UB tip domain
(arrows) (n11/13), similar to Ret–/– cells (Chi et al., 2009b). However,
they contributed normally to the CND (asterisks) (n14/16). (G-I)During
initial UB branching, Ret–/– cells contribute mainly to the proximal side
of branches (G), and during subsequent branching (H,I) to some trunks,
but not tips (arrows) (Chi et al., 2009b; Shakya et al., 2005). (J-O)Two
Etv4–/–;Etv5+/–}wild-type chimeras, isolated at E11.5 (J,M) and cultured
for 2 days (K,L,N,O). Like Ret–/– cells, Etv4–/–;Etv5+/– mutant cells
contribute to UB trunks but not tips (arrows) (n17/17). (P-R)Control
ES cells (Etv4+/–) contribute throughout the trunks and tips (arrows).
branching (E12.5), both types of mutant cells contributed to
some of the trunks, but to few, if any, of the tips (Fig.
1H,I,K,L,N,O). By contrast, control (Etv4+/–) ES cells
contributed without restriction throughout the WD (not shown)
and UB epithelium (Fig. 1P-R). Thus, Etv4–/–;Etv5+/– mutant
cells displayed essentially the same cell-autonomous defect as
Ret–/– cells: an inability to contribute to the UB tip domain.
One significant difference was observed between Ret–/– and
Etv4–/–;Etv5+/– cells in the formation of the common nephric duct
(CND), the caudal-most segment of the WD that transiently
connects the WD and UB to the urogenital sinus. Between E11.5
and E12.5 the CND is remodeled, allowing the ureter to detach
DEVELOPMENT
Total and phosphohistone H3+ cells were counted in 20-25 serial sections
(per specimen) through the posterior ends of six Etv4+/– and five
Etv4–/–;Etv5+/– E10.5 WDs and the UB of four Etv4+/– and four
Etv4–/–;Etv5+/– E11.5 kidneys. P-value was calculated using Student’s t-test
(two-tailed, equal variance).
Fig. 2. Distribution of Etv4–/–;Etv5+/– cells in chimeric Wolffian
duct and ureteric bud epithelium. (A-F)Sections through
Etv4–/–;Etv5+/–}wild-type mouse chimeras at the 36-somite (A-C) and
39-somite (D-F) stages, stained with anti-GFP (green) to reveal mutant
cells, with anti-calbindin (red) to label all WD and UB cells, and with
Hoechst nuclear stain (blue; A,D). B and C are enlargements of the
WDs in A; E and F are enlargements of the emerging UBs in D. The
dorsal regions of the WD at the 36-somite stage (i.e. the primary UB tip
domains; dashed lines) are devoid of mutant cells, as are the UB tips at
the 39-somite stage (arrows). cl, cloaca; nt, neural tube.
from the WD and move to the bladder (Batourina et al., 2005).
Whereas Ret–/– cells are excluded from the CND (Chi et al.,
2009b), as confirmed here (Fig. 1A-C, asterisks), Etv4–/–;Etv5+/–
mutant cells contributed extensively to the CND (Fig. 1E,F,
asterisks). Thus, the roles of Ret in the formation of the UB tip
domain and the CND, two different WD derivatives with distinct
fates, appear to be mediated through different downstream
pathways: contribution to the UB tip, but not the CND, depends on
Etv4/Etv5. This is consistent with the finding that although the
ureter often fails to connect to the bladder (a process that is
dependent on the proper formation and remodeling of the CND) in
Ret–/– mice (Batourina et al., 2002), it apparently connects to the
bladder in Etv4–/–;Etv5+/– mice (Lu et al., 2009) (our unpublished
data).
Rettm2(RET)Vpa is a hypomorphic allele with reduced signaling,
causing renal hypoplasia with defective UB branching (de Graaff
et al., 2001). Unlike in wild-type hosts, Ret–/– cells contribute
extensively to the tips in Ret-hypomorphic host embryos (Chi et al.,
2009b). Together with the behavior of Spry1–/– cells in
Spry1–/–}wild-type chimeras, this has suggested that WD cells
compete with each other, based on the level of Ret signaling, to
contribute to the UB tip domain (Chi et al., 2009b). When
Etv4–/–;Etv5+/– ES cells were injected into Ret-hypomorphic hosts
they contributed extensively to the UB tips as well as the trunks
(Fig. 3). Their unrestricted contribution was similar to that of
control Etv4+/– cells in wild-type hosts (Fig. 1P-R). Thus,
Etv4–/–;Etv5+/– cells are not inherently unable to contribute to UB
tips: they fail to compete successfully with wild-type cells, but
RESEARCH REPORT 1977
Fig. 3. In Ret-hypomorphic host embryos, Etv4–/–;Etv5+/– ES cells
contribute strongly to ureteric bud tips. (A-D)Etv4–/–;Etv5+/– ES cells
were injected into wild-type (A,C) or Ret-hypomorphic (B,D) mouse
blastocysts. Kidneys were cultured from E12.5 for 24 (A,B) or 72 (C,D)
hours, then stained with anti-pan-cytokeratin (red), which stains all UB
cells, and with anti-GFP (green), which stains the Etv4–/–;Etv5+/– cells. In
wild-type hosts, the Etv4–/–;Etv5+/– ES cells contribute to UB trunks, but
not (or weakly) to the tips (n8/8 kidneys). However, in Rethypomorphic hosts, the Etv4–/–;Etv5+/– cells usually contribute strongly
to the entire UB epithelium, including the tips (n4/5 kidneys).
compete effectively with cells in which Ret signaling is reduced.
This supports the hypothesis that Ret signaling and transcriptional
regulation by Etv4/Etv5 control similar cellular behaviors.
Ret signaling has been implicated in both cell proliferation
(Pepicelli et al., 1997) and cell movement (Chi et al., 2009b; Tang
et al., 1998) in the WD/UB lineage. As such, the failure of
Etv4–/–;Etv5+/– cells to contribute to the UB tip domain in wild-type
hosts might reflect a competitive disadvantage in cell movement or
reduced proliferation in the primary UB tip domain. To examine
proliferation, we counted phosphohistone H3+ (pH3+) cells in
Etv4–/–;Etv5+/– mutant embryos and Etv4+/– controls, in the E10.5
caudal WD and E11.5 UB (see Fig. S1 in the supplementary
material). In the UB, pH3+ cells were 3.5-fold less abundant in the
mutant (0.6% versus 2.1%, P<0.005); thus, the severely reduced
growth of Etv4–/–;Etv5+/– UBs (Lu et al., 2009) can be attributed,
at least in part, to reduced cell proliferation. However, in the caudal
WD at E10.5, before UB outgrowth, mutants and controls had the
same percentage of pH3+ cells (2.6% versus 2.3%, P0.487).
Therefore, the failure of Etv4–/–;Etv5+/– cells to contribute to the
UB tip is not due to a proliferative defect in the WD. In
Ret–/–}wild-type chimeras, time-lapse imaging of organ cultures
showed that Ret–/– cells were excluded from the primary UB tip
domain by wild-type cells because the mutant cells failed to
participate in cell rearrangements that generate this localized
domain (Chi et al., 2009b). The nearly identical distributions of
Etv4–/–;Etv5+/– and Ret–/– cells in wild-type chimeras, and the lack
of a proliferative disadvantage, indicate that these cells share the
same deficiency in cell rearrangement.
To examine the effects of complete loss of Etv4 and Etv5 we
generated chimeras with Etv4–/–;Etv5–/– cells. Etv4–/–;Etv5–/–}wildtype chimeras developed past the stage when Etv4–/–;Etv5–/–
embryos die (E8.5), and remained grossly normal at E11.5-12.5.
The Etv4–/–;Etv5–/– cells were able to contribute (albeit usually very
weakly) to the WD (Fig. 4), but were very rarely found in the UB,
even in the trunks (Fig. 4C-G). They often contributed weakly to
DEVELOPMENT
Etv4 and Etv5 in ureteric bud morphogenesis
the rostral and mid-WD and less often to the caudal WD (e.g. Fig.
4G). This graded distribution suggests a possible defect in cell
movement or proliferation during WD elongation in cells lacking
both Etv4 and Etv5. By contrast, Etv4–/–;Etv5+/– or Ret–/– cells
extensively populated the entire WD and showed no proliferative
defect in the WD, but failed to contribute to the UB tip domain. In
those chimeras in which Etv4–/–;Etv5–/– cells were found in the
caudal WD, the mutant cells were able to contribute to the CND
(Fig. 4B,F,G), like Etv4–/–;Etv5+/– cells (Fig. 1E,F), further
supporting the conclusion that Etv4 and Etv5 are not required for
CND formation. The nature of the defect that restricts Ret–/–, but
not Etv4–/–;Etv5–/–, cells from contributing to the CND remains to
be identified.
Ureter and kidney development are affected very similarly in
Ret–/– and Etv4/Etv5 mutant embryos, and expression of Etv4 and
Etv5 requires normal Ret signaling. This has suggested that the
effects of Ret mutations on the developing WD/UB are caused, at
least in part, by loss of Etv4 and Etv5 expression (Lu et al., 2009).
However, the conclusions that could previously be drawn from a
comparison of Ret and Etv4/Etv5 mutant phenotypes were limited,
and the present study provides additional insight into the role of
Etv4 and Etv5. One issue is that these genes are expressed in the
MM as well as in the WD and UB (Lu et al., 2009), and signals
from the mesenchyme are crucial for UB morphogenesis (Airik and
Kispert, 2007; Dressler, 2006; Schedl, 2007). Therefore, the
branching defects in Etv4 Etv5 compound mutants could be due, at
least in part, to a failure of induction by the mutant MM. In the
chimera studies, however, Etv4–/–;Etv5+/– compound mutant WD
and UB cells showed cell-autonomous defects that were very
similar to those displayed by Ret–/– cells, which strongly supports
a direct role of Etv4/Etv5 in UB epithelial cells, downstream of Ret.
Furthermore, Ret and Etv4/Etv5 do not function in a linear
pathway, but in a more complex signaling network. For example,
several genes regulated by Ret (e.g. Wnt11, Crlf1, Dusp6) are
expressed normally in Etv4–/–;Etv5+/– mutant kidneys, suggesting
that their regulation uses different downstream pathways (Lu et al.,
2009). In addition, Etv4 and Etv5 function downstream of multiple
RTKs, including FGF receptors (Brent and Tabin, 2004; Firnberg
and Neubuser, 2002; Liu et al., 2003), that signal to promote UB
branching morphogenesis (Maeshima et al., 2007; Michos et al.,
2010; Ohuchi et al., 2000; Zhao et al., 2004), so not all the effects
of Etv4/Etv5 mutations can be attributed to a block in Ret signaling.
As such, it could not be predicted to what extent the behavior of
Etv4/Etv5 mutant cells would resemble that of Ret–/– cells in
chimeric embryos.
We observed that Etv4–/–;Etv5+/– cells behave similarly to Ret–/–
cells, revealing a role of Etv4 and Etv5 in the WD cell
rearrangements that are promoted by Ret signaling (Chi et al.,
2009b). Etv4 and Etv5 have been implicated in cell migration in
other contexts, such as in the radial migration of cortical neurons and
in invasive cancer cells (Firlej et al., 2008; Hasegawa et al., 2004).
Furthermore, several genes regulated by Etv4 or Etv5 (or other ETS
transcription factors) in other cell types, and implicated in cell
migration, are also strongly downregulated in the Etv4–/–;Etv5+/–
mutant UB (Lu et al., 2009). These include chemokine receptor
Cxcr4, Met [the RTK for hepatocyte growth factor (Hgf)], and the
matrix metalloprotease Mmp14 (Belien et al., 1999; Peschard and
Park, 2007; Schier, 2003). Future studies will seek to identify the full
set of genes regulated by Etv4 and Etv5 in the WD and UB.
In contrast to Etv4–/–;Etv5+/– cells, Etv4–/–;Etv5–/– cells were
more severely compromised than Ret–/– cells. WD/UB
morphogenesis is promoted not only by Gdnf, but also by the
Development 137 (12)
Fig. 4. Etv4–/–;Etv5–/– cells contribute weakly to the Wolffian duct
but not to the ureteric bud in chimeric mouse embryos.
(A-F)Etv4–/–;Etv5–/–}wild-type chimeras analyzed at E10.5-12.0. Rostral,
left; caudal, right. Mutant cells are green (Venus+); wild-type cells are
blue (CFP+). Six specimens are shown arranged from top to bottom
by developmental stage, illustrating the range of mutant cell
contributions. B has the most extensive contribution observed in the
WD (white arrowheads indicate some of the areas with Venus+ mutant
cells), but the forming UB tip domain is devoid of mutant cells (open
arrowhead). A,D,F show mutant cells at different positions along the
WD (white arrowheads) and in a mesonephric tubule (A, yellow
arrowhead), but not in the extreme caudal WD (A) or in UBs (D,F)
(open arrowheads). In C and E, a longer segment of caudal WD (as well
as the UB) is devoid of mutant cells. Asterisks in B and F indicate
mutant cells in the CND. The Etv4–/–;Etv5–/– cells contribute to the
mesonephric tubules, primitive nephrons that form near the rostral end
of the WD (A), as do Etv4–/–;Etv5+/– cells and Ret–/– cells (data not
shown). (G)The percentage of specimens showing strong, weak or no
contribution to different regions. N indicates the number of informative
specimens.
DEVELOPMENT
1978 RESEARCH REPORT
combined effects of many factors, including several that signal via
RTKs, such as FGFs, Hgf and epidermal growth factor (Egf)
(Ishibe et al., 2009; Michos et al., 2010; Tufro et al., 2007; Zhao et
al., 2004). The fact that the defect is more severe in Etv4–/–;Etv5–/–
than Ret–/– cells supports the conclusion that Etv4 and Etv5 not only
function downstream of Ret in kidney development, but also have
a broader role downstream of multiple RTKs (Lu et al., 2009;
Michos et al., 2010).
Acknowledgements
We thank Zaiqi Wu and Linda Williams for expert technical assistance. This
work was supported by NIH grants 1R01DK075578 and 1R01DK083289 (F.C.),
and fellowships from the National Kidney Foundation, Sigrid Juselius
Foundation and Finnish Culture Foundation (S.K.) and the American Heart
Association (X.C.). Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.051656/-/DC1
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DEVELOPMENT
Etv4 and Etv5 in ureteric bud morphogenesis