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First published online 18 April 2007
doi: 10.1242/dev.004234
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1 Department of Physiology and Biophysics, Weill Medical College of Cornell
University, New York, NY 10021, USA.
2 Department of Genetics and Development, Columbia University Medical Center New
York, NY 10032, USA.
3 Department of Internal Medicine and Division of Basic Science, University of
Texas Southwestern Medical Center, Dallas, TX 75390, USA.
4 Ronald O. Perelman Department of Dermatology, Kaplan Comprehensive Cancer
Center, New York University School of Medicine, New York, NY 10016, USA.
* Author for correspondence (e-mail: daherzli{at}med.cornell.edu)
Accepted 8 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cell lineage, Tailbud, Urogenital system
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The junction between the kidneys and the conduit system is a common site
for congenital defects in humans, including obstructions that compromise renal
function (Becker and Baum,
2006
). This border lies between the intra-renal collecting tubule
system and ureter, both of which derive from an epithelial primordium called
the ureteric bud (UB). Cells at the proximal UB tip are induced to form the
intra-renal collecting system via their interactions with the mesenchymal
progenitors of the nephrons (Fig.
1), which secrete factors such as glial cell-derived neurotrophic
factor (GDNF) (for a review, see Costantini
and Shakya, 2006). The distal or tubular domain of the UB
differentiates into the ureter and is surrounded by a mesenchymal cell
population that can be distinguished from nephron progenitors by mRNA
expression patterns. Several genes, including the transcription factor
Tbx18, which are selectively expressed by this mesenchyme, are
required for ureter morphogenesis (Airik et
al., 2006; Miyazaki et al.,
2000; Oshima et al.,
2001). Thus, the different mesenchymal populations surrounding the
proximal and distal UB are essential for collecting system and ureter
morphogenesis, respectively.
The developmental process that establishes these distinct mesenchymal tissues has yet to be determined and can occur by at least two different mechanisms (Fig. 1C,D). The distal and proximal domains of the UB may be inherently different and direct a homogeneous population of intermediate mesoderm to either maintain a nephrogenic fate or differentiate into the unique population of mesenchyme surrounding the distal UB (Fig. 1C). Alternatively, the entire UB epithelium may be a homogeneous population of cells competent to differentiate into either collecting tubule or the ureter epithelium (Fig. 1D). In this model, distinct mesenchymal populations surrounding the proximal and distal UB are specified at a stage in development prior to their association with the UB; cell migration or tissue movements are required to establish the distinct mesenchymal populations around the UB. Discrimination between these two models is dependent on determining the origin of the mesenchymal cell populations surrounding the UB.
In this report we use fate mapping techniques to show that the tailbud, a population of undifferentiated cells that is located at the most caudal aspect of the embryo after the completion of gastrulation, contributes cells to the developing urinary tract. Specifically, our data show that tailbud-derived mesenchyme associates with the distal UB but not its proximal domains, which form the intra-renal collecting system. We found that tailbud-derived mesenchyme, including those derivatives that invest the distal UB or nascent ureter, expresses abundant levels of Bmp4 mRNA and our functional experiments show that the BMP4 secreted by this mesenchyme is required for ureter differentiation. Furthermore, our data demonstrate that ectopic BMP4 signaling can induce ureter differentiation in domains of the UB normally fated to form the intra-renal collecting system. Thus, cells located at the proximal UB tips are multipotent and competent to differentiate into either ureter or collecting tubule epithelia.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). All mice were housed in the Weill Medical College of
Cornell University Animal Facility and treated according to the Research
Animal Resource Center guidelines. Timed pregnant Swiss Webster female mice
were purchased from Taconic Farms (Germantown, NY). Mice heterozygous for a
Bmp4lacZ reporter allele
(Lawson et al., 1999), and
mice homozygous for the Bmp4 conditional allele created by the
introduction of Cre recombinase (loxP) sites into the BMP4 locus
(Kulessa and Hogan, 2002),
were provided by Brigid Hogan (Duke University, Durham, NC). The lacZ
transgene was detected by using the following primer pair: forward,
5'-ACCCAACTTAATCGCCTTGC-3'; reverse,
5'-AACAAACGGCGGCGGATTGACC-3'. The following primers were used to
genotype animals carrying the loxP sites in the Bmp4 allele:
#79, 5'-AGACTCTTTAGTGAGCATTTTCAAC-3'; #80,
5'-AGCCCAATTTCCACAACTTC-3'; #24,
5'-AGGTGAGCAGAGCTAAGATG-3'. The Bmp4flox/+
versus Bmp4flox/flox genotype was detected using primer
pair #79 and #80. The #79 and #24 primer pair was used to detect the
occurrence of a recombination event. Bmp4flox/flox females
were mated with Cre/Esr1 (estrogen receptor 1) heterozygous male mice
(strain name: B6.Cg-Tg(Cre/Esr1)5Amc/J; The Jackson Laboratory, Bar Harbor,
ME) (Hayashi and McMahon,
2002). To check for the presence of the Cre recombinase
transgene the following primer pair was used: forward,
5'-GTGAAACAGCATTGCTGTCACTT-3'; reverse,
5'-GCGGTCTGGCAGTAAAAACTATC-3'. Bmp4flox/+ male
offspring carrying the Cre transgene were selected and mated with
Bmp4flox/flox females to generate
Bmp4flox/flox;Cre/Esr1 embryos. Timed pregnant dams and
their subsequent litters were normal and did not appear to be affected by the
presence of the Cre transgene. Bmp4flox/flox
embryos carrying the Cre transgene died in utero approximately 24
hours after timed pregnant dams were injected with 5 mg tamoxifen (Sigma, St
Louis, MO) (Hayashi and McMahon,
2002).
Lineage analyses
Chick embryos at given stages were injected in ovo with 
1 nl of either
CMDiI (Molecular Probes) or 0.5x107-108
ml-1 virions of concentrated SNTZ retrovirus
(Hyer and Mikawa, 1997;
Obara-Ishihara et al., 1999).
Retroviral stocks were routinely assayed for replication competence as
described (Obara-Ishihara et al.,
1999). Eggs were resealed and incubated for given times before
fixation with 4% paraformaldehyde. Transverse vibratome sections or whole
mounts of DiI-injected embryos were examined using epifluorescence optics.
SNTZ-injected embryos were processed for ß-galactosidase activity
(Lobe et al., 1999), examined
as whole mounts, and then embedded in paraffin. Serial paraffin sections were
prepared and then stained with Hematoxylin and Eosin. Unless otherwise stated,
all fate mapping data are representative of a minimum of four embryos analyzed
for each experimental condition described.
Metanephric explant cultures
Kidney rudiments were isolated from wild-type mouse embryos at the
gestational stages noted; the day of vaginal plug was designated as E0.5.
Rudiments were cultured in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum and 1% penicillin/streptomyocin on polycarbonate
membrane filters (0.4 µm pore size, Transwell®; Corning, Wilkes Barre,
PA). Recombinant BMP4, BMP7, Noggin and Gremlin were purchased from R&D
Systems (Minneapolis, MN) and added to the culture medium daily. Timed
pregnant Bmp4flox/flox females that were mated with
Bmp4flox/+;Cre-Esr1 males were injected with tamoxifen at
E12 and sacrificed 6 hours later. Kidney rudiments were isolated from embryos
and cultured with 50 pM 4-hydroxytamoxifen (Sigma) to ensure complete
Bmp4 excision. The heads of isolated embryos were used for
genotyping.
In situ hybridization detection of mRNA expression
Embryos were fixed with 4% paraformaldehyde and processed for wholemount in
situ hybridization according to standard protocols
(Henrique et al., 1995).
Briefly, samples were treated with 10 µg/ml of proteinase K for 30 minutes
at 25°C. Hybridization with digoxigenin-labeled antisense RNA probes was
performed at 68°C. Reactions were developed with NBT/BCIP (Roche,
Indianapolis, IN). Bmp4 plasmids were kindly provided by Brigid
Hogan's laboratory. Detection of Bmp4 and Pax2 mRNAs in the
same sample was performed according to the double in situ hybridization
techniques of Hurtado and Mikawa (Hurtado
and Mikawa, 2006).
Immunofluorescent protein detection
Frozen sections, prepared according to standard protocol
(Stern, 1993), or whole
explants were blocked in 1% normal donkey serum (NDS)/PBS for 1 hour and
primary antibodies diluted to appropriate concentrations in the same solution.
Primary antibodies were incubated with samples for up to 3 hours at 37°C
and secondary antibodies at described dilutions were applied after samples
were thoroughly washed. Samples were examined with a Hamamatsu C4742-95
digital camera using a Metamorph Image capturing system (Universal Imaging of
Molecular Devices, Downingtown, PA). Primary and secondary antibodies and
their dilutions were as follows: uroplakin (rabbit anti-serum against total
bovine uroplakins, a gift from the Tung-Tien Sun laboratory, New York
University, 1:3000 dilution; secondary is donkey anti-rabbit, Molecular
Probes, Carlsbad, CA, 1:200 dilution); smooth muscle actin (SMA)
(Cy3-conjugated mouse monoclonal, Sigma, 1:400 dilution); E-cadherin (R&D
Systems, 1:200 dilution; secondary is donkey anti-goat, Molecular Probes,
1:200); DAPI (Sigma, 1:1000 dilution).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Replication-defective viral stocks were injected into the intermediate
mesoderm of Hamburger-Hamilton (HH) st10 embryos at the axial level of somites
6-10 (Fig. 2A-C); we have
previously shown that this domain of intermediate mesoderm gives rise to the
nephric duct. After 24 hours of incubation, the only lineage-tagged,
ß-galactosdiase-positive cells detected in the caudal aspect of the
embryo are localized to the nephric duct (data not shown)
(Obara-Ishihara et al., 1999).
After 6 days of incubation, lineage-tagged cells were detected within the
nephric duct, short tubules extending into the mesonephros, and the UB
(Fig. 2B). Epithelial cells of
the cloaca, which gives rise to the terminal portion of the conduit system,
were unlabeled (Fig. 2B). After
14 days of incubation, lineage-tagged cells were localized to the nephric
duct, ureter and intra-renal collecting tubules. These data demonstrate that
the intrarenal collecting tubule system and the ureters of the avian embryo,
as in the mouse, derive from the UB. By contrast, the epithelia of the cloaca
derive from another tissue present in the early chick embryo
(Fig. 2C).
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). Therefore, we transferred lacZ into this tissue to
test whether it gives rise to both mesenchymal nephron progenitors surrounding
the proximal UB as well as the mesenchyme surrounding the distal UB.
Intermediate mesoderm from the axial levels of somites 25-27 gave rise to
mesonephric nephrons (n=6; data not shown). Metanephric nephron
progenitors were efficiently labeled when SNTZ was injected into the
unsegmented mesoderm caudal to somite 28, approximating the position of the
intermediate mesoderm (n=3). Targeted lacZ transfer into
this site resulted in the presence of large populations of lineagetagged,
ß-galactosidase-positive nephron epithelia at later stages of development
(Fig. 2D). However,
lineage-tagged cells were not observed in tissues surrounding the ureter or
cloaca in any of the embryos analyzed. These data suggest that the proximal
and distal UB are associated with mesenchyme derived from different embryonic
sources.
To further test this hypothesis, we used two independent fate mapping
techniques to determine the origin of the mesenchymal cells surrounding the
distal UB or nascent ureter. DiI, a carbocyanine dye, was injected into HH
st14-16 embryos in ovo (Fig.
3A-F). Dye injections were targeted to focal domains of mesoderm
from the axial level of somite 23 through to the axial level of somite 27, and
unsegmented mesoderm rostral to the posterior neuropore. In addition, DiI was
injected into the medial aspect of the embryo caudal to the closed neuorpore,
which can be defined as the tailbud (Catala
et al., 1995; Krenn et al.,
1990; Wilson and Beddington,
1996). Embryos were analyzed 2-4 days post-injection and only the
six embryos that were injected with DiI into the tailbud exhibited large
populations of tagged cells immediately adjacent to the cloaca and the distal
domain of the UB or the nascent ureter
(Fig. 3E,F). Most importantly,
DiItagged cells derived from the tailbud were not present around the proximal
domain of the UB (Fig. 3D).
Instead, the proximal UB tips or nascent collecting tubules were surrounded by
unlabeled Pax2-expressing nephrogenic mesenchyme, which derives from
the intermediate mesoderm (Fig.
3D) (Dressler et al.,
1990). We also transferred lacZ into tailbud mesenchyme
to mark cells derived from this compartment in a heritable fashion as DiI can
be diluted by successive rounds of cell division
(Fig. 3G-I). These fate mapping
experiments, which were then analyzed at later stages of development, confirm
and extend DiI fate mapping results. Cells deriving from the tailbud
differentiate into sacral connective tissues, including muscle, and are
present in the connective tissue surrounding the cloaca and the distal-most
domain of the ureter (Fig.
3G-I). Tailbud-derived cells were not observed in the metanephros
of any of the seven embryos analyzed (data not shown). Collectively, these
data raise the possibility that signals secreted by tailbud-derived mesenchyme
surrounding the distal UB may play a role in controlling ureter
morphogenesis.
Bmp4 is expressed in the tailbud and the tailbudderived mesenchyme surrounding the cloaca and distal UB
We used a candidate gene approach to test whether tailbud mesoderm
expresses genes required for ureter morphogenesis. In both the developing
chick and mouse embryo, several genes, including Fgf8, Tbx6, Wnt5a
and Bmp4, are abundantly expressed in the tailbud
(Gofflot et al., 1997;
Knezevic et al., 1998). The
spatial and temporal expression patterns of one of these genes, Bmp4,
parallels the movement of tailbud-derived mesenchyme to a position surrounding
the epithelial tissues that give rise to the conduit system. As can be seen in
Fig. 4, Bmp4 is
abundantly expressed in the avian tailbud at HH st14
(Fig. 4A). Approximately 2 days
later, abundant Bmp4 expression is detected around the caudal-most
aspect of the developing urinary tract, including the cloaca and distal UB. By
contrast, the proximal UB is surrounded by Pax2-expressing nephron
progenitors (Fig. 4B-D).
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) and later, from E11.5-E13.5 by mesenchyme surrounding the
distal UB and urogenital sinus (Fig.
4E,F). Thus, the spatial and temporal expression patterns of
Bmp4 mRNA are consistent with the movement of tailbud-derived cells
to the developing conduit system and a possible role in ureter morphogenesis.
Moreover, by E15.5, overt ureter differentiation, as determined by the
upregulated expression of uroplakins in the UB epithelium and presence of an
organized, SMA-positive connective tissue coat, occurs only in the domain of
the UB invested with Bmp4-expressing mesenchyme
(Fig. 5A-C). Uroplakins are not
expressed by collecting tubule epithelia and although SMA-positive cells are
interspersed between collecting tubules in the medullary interstitium at later
stages of development, these SMA-positive cells are not organized into a
thick, circumferentially oriented connective tissue investment. Finally, we
show that ureter differentiation occurs in metanephric kidney explants
(Fig. 5D-F). Neither uroplakins
(Fig. 5D) nor SMA-positive
mesenchyme (data not shown) can be detected in freshly isolated E12.5
metanephric kidney rudiments, whereas these markers of ureter differentiation
are abundantly expressed after 4 days of culture
(Fig. 5E,F). Importantly, the
domain of the UB that displays the ureteral phenotype in vitro parallels the
spatially restricted expression of Bmp4 in adjacent mesenchyme
(Fig. 5G). Thus, this explant
system successfully recapitulates ureter differentiation.
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).
The Cre/ESR1 fusion protein can enter the nucleus only when bound to an
estrogen receptor 1 ligand. Therefore, Cremediated excision of the floxed
Bmp4 alleles is inducible by maternal administration of the synthetic
estrogen, tamoxifen. Timed pregnant mice were injected with tamoxifen at E12
and embryos harvested 6 hours post-injection because
Bmp4flox/flox embryos carrying the Cre
recombinase transgene begin to die in utero 24 hours after tamoxifeninduced
Bmp4 excision. Metanephric kidney rudiments were isolated and
cultured in vitro in the presence of tamoxifen to ensure complete gene
excision and Bmp4 expression levels were assessed after 4 days of
growth by in situ hybridization. Abundant Bmp4 mRNA expression was
seen in rudiments from Bmp4flox/flox embryos lacking the
Cre transgene (Fig.
6B), whereas Bmp4 mRNA expression was not detected in
rudiments isolated from Bmp4flox/flox, Cre/ESR1 embryos
(Fig. 6C). After 4 days of
culture, uroplakin and SMA expression were detected in the distal-most domain
of Bmp4flox/flox rudiments lacking the Cre
transgene (Fig. 6D,F,H,J),
whereas the expression of these markers indicative of ureter differentiation
was dramatically reduced when Bmp4 alleles were excised at E12
(n=4; Fig. 6E,G,I,K).
Excision of Bmp4 alleles at developmental stages later than E12.5 did
not perturb ureter differentiation, as determined by upregulated uroplakin
expression and the formation of a SMA-positive connective tissue coat
(n=4; data not shown). Collectively, these data demonstrate that BMP4
signaling prior to E12.5 is required for ureter morphogenesis.
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Recombinant BMP4 markedly alters the growth of metanephric kidney rudiments
(Bush et al., 2004;
Cain et al., 2005;
Raatikainen-Ahokas et al.,
2000). E12.5 rudiments cultured with increasing concentrations of
recombinant BMP4 exhibit a dose-dependent decrease in UB tip and nephron
number as compared with control cultures
(Fig. 7C-F). Most importantly,
in this report we show that exogenous BMP4 converted the proximal UB tips into
tubular structures that exhibit a ureteral phenotype, in a dose-dependent
manner (Fig. 7C-F). The
proximal UB tips exhibit upregulated uroplakin expression
(Fig. 7C,E) and become invested
with a condensed, SMA-positive smooth muscle coat
(Fig. 7D,F). Developmental
plasticity persists in the UB, as similar results were obtained when E14.5
rudiments were cultured in vitro with high concentrations of recombinant BMP4
(Fig. 7G-I). As expected, in
both control and BMP4-treated cultures, the distal domain of the UB
differentiated into the ureter (Fig.
7G,H). Strikingly, the proximal UB tips also acquired a ureteral
phenotype when older rudiments were cultured with recombinant BMP4, whereas
large portions of the UB network between the definitive ureter and the UB tips
did not develop ureteral characteristics
(Fig. 7I,J). These data
demonstrate that the proximal UB tips, normally fated to differentiate into
the collecting system, acquire the ureteral phenotype in the presence of
ectopic BMP4 signaling.
By contrast, high concentrations of BMP7, a TGFß family member
expressed by the UB and by nephrogenic mesenchyme around its proximal tips
(Dudley and Robertson, 1997),
were unable to induce ureter morphogenesis in the proximal domains of the UB
normally destined to form the collecting system in vitro (data not shown).
Together, these data demonstrate that ectoptic signaling by BMP4, a paracrine
factor normally present in mesenchyme surrounding the distal UB, is capable of
inducing ureter morphogenesis in domains of the UB fated to differentiate into
the intra-renal collecting system. Thus, spatially restricted Bmp4
expression plays a role in determining the location at which the UB is
subdivided into the ureter and the collecting system.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mesenchymal-to-epithelial signaling mediated by Bmp4 patterns the UB
BMP signaling is likely to play a role in multiple processes required for
ureter morphogenesis. Our results, combined with previous studies, implicate
BMP signaling in the control of ureteral smooth muscle formation
(Airik et al., 2006;
Miyazaki et al., 2003).
Moreover, targeted deletion of Shh, which is expressed in the
nascent ureter epithelium, results in a deficit of Bmp4-expressing
peri-ureteral mesenchyme and an abnormally thin ureteral smooth muscle coat
(Yu et al., 2002).
Collectively, these data indicate that formation of the ureteral smooth muscle
coat is dependent on signals from the nascent ureter epithelium, similar to
what has been shown by Baskin in the developing bladder, and signaling within
the mesenchymal population surrounding the nascent ureter
(Baskin et al., 1996).
In this study we show that Bmp4 is also required for the
upregulated expression of uroplakins in the differentiating ureter. Uroplakins
are a family of transmembrane proteins selectively expressed by the ureter
epithelium and required for its water-impermeable properties
(Hu et al., 2001). BMP4
secreted by periureteral mesenchyme may signal directly to the underlying
epithelium, as UBs isolated from the surrounding mesenchyme cease branching
when exposed to recombinant BMP4 in vitro
(Bush et al., 2004). However,
targeted deletion of SMAD4 in the UB epithelium does not grossly affect ureter
morphogenesis in vivo (Oxburgh et al.,
2004). Because SMAD4 is an obligatory component of SMAD-dependent
BMP signaling, if BMP4 directly signals to the UB epithelium, it is likely to
do so via SMAD-independent mechanisms such as activation of LIM1 kinase
(Lee-Hoeflich et al., 2004).
Alternatively or additionally, Bmp4 may upregulate the expression of
unknown mesenchymal signaling factors that then directly induce ureter
epithelial differentiation. Future experiments examining ureter
differentiation in animal models lacking BMP receptors within the UB will be
required to further dissect the mechanisms mediating BMP-dependent ureter
epithelial differentiation.
We have also shown that ectopic BMP4 signaling induces ureter
differentiation. Raatikainen-Ahokas et al. demonstrated that the large
portions of the UB network acquire a dense connective tissue coat
characteristic of the ureter when metanephric kidney explants are cultured
with recombinant BMP4 (Raatikainen-Ahokas
et al., 2000). Our experiments confirm and extend these
observations. We show that ectopic BMP4 signaling at the early stages of renal
development converts the entire UB network into tubules exhibiting a ureteral
phenotype as determined by the upregulated expression of uroplakins and the
acquisition of a thick connective tissue coat. This phenotypic conversion may
be due to BMP-dependent migration or overproliferation of connective tissue
and uroplakin-expressing cells from the nascent ureter to more proximal
domains of the UB network. However, experiments analyzing UB differentiation
in E14.5 kidney rudiments suggest that ectopic BMP4 signaling actually changes
the fate of the UB from a collecting tubule to ureteral phenotype. High
concentrations of recombinant BMP4 induce the most proximal UB tips to acquire
a ureteral phenotype, whereas large portions of the UB network located between
these abnormal UB tips and the definitive ureter do not develop ureteral
characteristics. This result strongly suggests that ectopic BMP4 signaling
changes the fate of the proximal UB from a collecting tubule to ureteral
phenotype, because it is extremely unlikely that large populations of both
uroplakin- and SMA-positive cells move from the distal-most domain of the UB
to its most proximal tips without populating the intervening UB segments.
These results are consistent with earlier studies demonstrating that the
differentiated phenotype of epithelia derived from the UB is controlled, in
part, by factors secreted by surrounding mesenchyme
(Lipschutz et al., 1996).
Modulation of the timing and level of BMP signaling may be essential for
the proper patterning of these tissues. Our results suggest that portions of
the UB network become refractory to BMP-induced ureter differentiation or
committed to a collecting tubule phenotype by E14.5. This early specification
of the UB appears to allow BMP4 signaling to mediate additional developmental
processes at later stages. For example, BMP4 expressed in the medullary
interstitium and glomerulus may pattern the renal vasculature, as signaling by
this TGFß family member has been implicated in blood vessel formation,
and antagonists of BMP signaling compromise the integrity of the glomerular
capillary tuft (Miyazaki et al.,
2006; Nimmagadda et al.,
2005).
Sensitivity to both specific ligands and the total level of BMP signaling
in the target tissue may also allow the BMPs to promote the formation of
different cell types even in the absence of temporal restriction.
Dose-response experiments included in this study demonstrate that high
concentrations of recombinant BMP4 induce ureter morphogenesis in domains of
the UB normally fated to differentiate into the renal collecting system
whereas BMP7 does not. BMP7 is expressed by the UB and by nephrogenic
mesenchyme surrounding its branching tips in vivo and is essential for
maintaining the viability of nephron progenitors within the developing kidney
(Dudley et al., 1995;
Dudley and Robertson, 1997;
Oxburgh et al., 2005). Since
the receptor specificity of BMP4 and BMP7 differ, it is possible that the
receptors mediating BMP4-dependent ureter morphogenesis do not respond to BMP7
signaling. However, despite the distinct receptor specificity and functions of
BMP4 and BMP7 in the developing kidney, Bmp4 can substitute for
Bmp7 in supporting nephron progenitor viability in vivo
(Oxburgh et al., 2005). We
hypothesize that the levels of ectopic Bmp4 mRNA expressed around the
UB tips when the gene is knocked into the Bmp7 locus are not
sufficient to induce ectopic ureter morphogenesis in vivo
(Oxburgh et al., 2005). This
hypothesis is consistent with data demonstrating that different levels of BMP
signaling promote diverse responses in many embryonic cell types
(James and Schultheiss, 2005;
Oxburgh et al., 2005;
Wilson et al., 1997).
Generation of restricted BMP expression
Collectively, our data indicate that spatially restricted BMP4 signaling is
essential for normal urinary tract patterning. Restricted BMP4 expression in
the developing urinary tract may be imposed by several different mechanisms.
Fate mapping experiments combined with mRNA expression analyses presented in
this study show that the mesenchyme surrounding the distal UB derives from the
tailbud and expresses Bmp4 prior to associating with the developing
urinary tract. By contrast, the mesenchyme surrounding the domain of the UB
fated to form the renal collecting system does not express Bmp4 and
derives from the intermediate mesoderm. Therefore, one of the mechanisms that
confines BMP4 signaling to the domain of the UB fated to become the ureter is
the association of tailbud-derived mesenchyme with the most caudal segments of
the developing urinary tract, including the distal UB.
Tailbud-derived mesenchyme also associates with the cloaca. The cloaca
derives from endoderm and its ventral wall forms the bladder, the structure
into which the ureter drains (Carlson,
1996). The bladder epithelium has different in vitro growth
characteristics from the ureter epithelium, which derives from mesoderm
(Liang et al., 2005). Despite
these differences, the ureter and bladder epithelium share many similar
features, including morphology and gene expression patterns
(Liang et al., 2005). We
suggest that the association of a common population of tailbud-derived,
BMP4-expressing mesenchyme with both the distal UB and cloaca accounts for
these similarities.
Our fate mapping results address the origin of the mesenchyme surrounding
the distal UB in the developing chick embryo and several lines of evidence
suggest that tailbud-derived mesenchyme plays a role in conduit system
formation in a variety of species. Sustained BMP signaling within the tailbud
is essential for the formation of the terminal portions of the zebrafish
urinary tract (Pyati et al.,
2006). Similarly, targeted deletion of the vitamin A degradation
enzyme cyp26, which is localized to the murine tailbud, results in both
massive tailbud apoptosis and gross ureter and bladder defects
(Sakai et al., 2001).
Mesenchyme from the tailbud remains poorly understood. As this tissue does
not arise from primary gastrulation, it is possible that it has a distinct
gene expression profile and developmental potential compared with other
embryonic mesenchyme (Catala et al.,
1995; Gofflot et al.,
1997; Knezevic et al.,
1998; Wilson and Beddington,
1996). Our data raise the possibility that this mesenchyme is
required for the formation of multiple structures within the murine urogenital
system. In support of this hypothesis, a recent study indicates that
mesenchyme surrounding the cloaca, which we show derives from the tailbud,
plays a fundamental role in the differentiation of the bladder, urethra and
genitalia (Haraguchi et al.,
2007). Thus, tailbud mesenchyme may be required for the formation
of multiple structures, both in the urogenital system and throughout the
lumbar and sacral regions of the embryo. We suggest that the fate and function
of tailbud mesenchyme merits further investigation.
Formation of the junction between the kidney and ureter is a complex, multi-stage process
Our data shows that a sharp boundary between the ureter and the branched
collection system can be detected in the developing murine urinary tract by
E15.5. This initial boundary undergoes further differentiation to create the
renal pelvis, which does not fully mature until birth and shares properties
with both the renal collecting system and ureter
(Miyazaki et al., 1998). The
mechanisms regulating renal pelvis formation are currently unclear, but it is
likely that a failure to properly form an initial boundary at this site will
ultimately result in general defects at the urteral-pelvic junction.
In conclusion, our results indicate that formation of the ureteralpelvic
junction, the border between the kidney and conduit system in the mature
animal, is a complex, multi-stage process that requires the integration of
cells from at least three different embryonic lineages into a single,
composite structure. These aspects of ureteralpelvic junction formation are
reminiscent of cardiac outflow tract morphogenesis, which requires the
integration of cells derived from the neural crest, lateral and cardiac
mesoderm into a single, functional structure
(Carlson, 1996). Quite
possibly, as a result of the complex tissue interactions involved, the outflow
tract is a major site of congenital malformations in humans. Our experiments
analyzing ureter morphogenesis suggest that the high incidence of congenital
defects localized to the ureteral-pelvic junction may also be related to the
complexities of integrating cells from a variety of lineages into a single
composite site.
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ACKNOWLEDGMENTS |
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REFERENCES |
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