1 Laboratory of Comparative Carcinogenesis, National Cancer Institute,
NCI-Frederick, Frederick, MD 21702, USA
2 Cancer and Developmental Biology Laboratory, National Cancer Institute,
NCI-Frederick, Frederick, MD 21702, USA
3 Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology,
University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland
Author for correspondence (e-mail:
mlewandoski{at}mail.ncifcrf.gov)
Accepted 17 June 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cre recombinase, Fgf8, Kidney, Lim1, Nephron, Somitogenesis, T-Cre, Wnt4, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fgf8 is first expressed in the pre-gastrulation epiblast and then
in the primitive streak (Crossley and
Martin, 1995). Gastrulation fails in
Fgf8/ embryos
(Sun et al., 1999
). To bypass
this gastrulation requirement, mice carrying Fgf8 hypomorphic alleles
or tissue-specific gene inactivations have been studied. In the
midbrain-hindbrain boundary, Fgf8 inactivation results in aberrant
cell death of the prospective midbrain and cerebellum
(Chi et al., 2003
;
Meyers et al., 1998
), whereas
later cerebellar development requires both FGF8 and FGF17
(Xu et al., 2000
). In the
first branchial arch, Fgf8 inactivation causes abnormalities in
cardiovascular and smooth muscle development, mandibles, the submandibular
salivary gland and teeth (Abu-Issa et al.,
2002
; Macatee et al.,
2003
; Trumpp et al.,
1999
). In the limb bud, apical ectodermal ridge (AER)-specific
Fgf8 expression is required for limb patterning
(Lewandoski et al., 2000
;
Moon and Capecchi, 2000
). If
Fgf4 and Fgf8 are deleted in the AER, limb development fails
completely, indicating genetic redundancy
(Boulet et al., 2004
;
Sun et al., 2002
).
Additionally, from the analyses of expression patterns and experimental
manipulations in chick embryos, Fgf8 may act in other embryonic
locations. In presomitic mesoderm, a rostrocaudal gradient of Fgf8
expression (with higher caudal levels) may define a `differentiation front' in
which presomitic mesoderm becomes segmented as it leaves the Fgf8
expression domain during axis extension
(Dubrulle et al., 2001).
Myotome-specific Fgf8 expression
(Crossley and Martin, 1995
;
Stolte et al., 2002
) is
implicated in the development of two other somitic subcompartments, the
sclerotome (Huang et al.,
2003
) and the syndetome (Brent
et al., 2003
). Finally, it has been suggested that FGF8 activity
from the nephrogenic cord (NC) of the mesonephros initiates limb bud formation
(Crossley et al., 1996
;
Martin, 1998
). Because
gastrulation fails in Fgf8/ embryos,
loss-of-function tests of these models require the proper tissue-specific Cre
mouse lines for conditional Fgf8 deletion
(Lewandoski, 2001
).
Fgf8 expression has also been detected in primitive nephronic
structures, although there has been no exploration into its role
(Mahmood et al., 1995). In the
developing urinary tract, the mesodermally derived Wolffian duct (WD) induces
a wave of tubule formation in the adjacent NC to generate the mesonephros as
the WD extends caudally, and Fgf8 is expressed in these tubules
(Crossley et al., 1996
;
Fernandez-Teran et al., 1997
;
Vogel et al., 1996
).
Development of the metanephros begins as the WD reaches the cloaca. There, it
develops a diverticulum called the ureteric bud (UB), which grows into the
surrounding metanephric mesenchyme (MM) to produce the collecting duct network
and to induce mesenchymal-epithelial conversion of MM beneath the UB tips. The
conversion of MM is defined by a series of morphogenetic stages, involving
condensation and pretubular aggregation (renal vesicles), then tubular
aggregation (comma- and S-shaped bodies) and eventual formation of the various
epithelial segments of the nephron, including glomerular epithelium and
proximal/distal tubules. Genetic targeting studies have, thus far, implicated
only a few genes expressed by the MM that are critical to nephron
differentiation (reviewed by Perantoni,
2003
). Of these, arguably only Wnt4 is essential for the
normal conversion of MM to the epithelia of the nephron
(Stark et al., 1994
).
To examine the function of Fgf8 in mesodermal lineages, including
the metanephros, we have produced and characterized a transgenic mouse line in
which a Cre transgene is driven by the T (brachyury) promoter
(Clements et al., 1996). This
promoter is active in the primitive streak, allowing us to use T-Cre mice to
delete a conditional Fgf8 allele in the mesodermal lineage. We report
no Fgf8 requirement for somitic segmentation, differentiation of
somitic subcompartments, or limb bud initiation. However, we discover a major
role for Fgf8 in metanephric development. Like Wnt4, Fgf8 is
expressed in the condensing MM during normal development, and its conditional
loss causes severe renal hypoplasia. Fgf8 inhibits apoptosis in the
MM and acts in combination with Wnt4 to sustain Lim1
expression, which is required for normal nephrogenesis
(Kobayashi et al., 2005
).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In situ hybridization (ISH)
Embryos or dissected urogenital tracts were fixed and processed for
whole-mount ISH as described (Pizard et
al., 2004). For paraffin-wax embedding, mutant and control
metanephroi were fixed as described
(Karavanova et al., 1996
).
Thin section ISH was performed as described
(Wilkinson and Green, 1990
),
using 35S-labeled probes.
Immunohistochemistry (IHC)
For whole-mount IHC, isolated metanephroi were processed as described
(Yoshino et al., 2001). UBs
were visualized with a rabbit polyclonal pan-cytokeratin antibody (Santa Cruz)
at 1:50 and FITC-labeled anti-rabbit IgG antibody at 1:100. For thin sections,
deparaffinized tissues were probed with a rabbit polyclonal Cited1 antibody
(NeoMarkers) at 1:200, and visualized with a Vectastain ABC kit according to
the manufacturer's recommendations.
Proliferation/apoptosis studies
Proliferation was analyzed by examining histone H3 phosphorylation using a
rabbit polyclonal antibody against the Ser10 phosphopeptide of histone H3
(Upstate). Cell death was examined with confocal laser microscopy of kidneys
stained with LysoTracker Red (Invitrogen)
(Zucker et al., 1999).
RNA preparation and microarray analysis
Metanephroi were dissected from E12.5 or E14.5 embryos. For E12.5 control
and mutant embryos, MMs and UBs were isolated as described
(Karavanova et al., 1996).
Tissues were either snap frozen on dry ice, or incubated in Hams
F12:Dulbecco's MEM (50:50) with or without FGF8B (200 ng/ml) for 4 hours, and
then stored at 70°C. Total RNA was isolated from tissues using
Trizol Reagent (Invitrogen), and an RNeasy kit (Qiagen). Total RNA (1-2 µg)
was used in cDNA synthesis, and biotinylated cRNA was subsequently generated
using the manufacturer's protocols (Affymetrix). Microarray hybridizations to
GeneChip Mouse Expression Set 430A and measurement of hybridization
intensities were performed and analyzed according to the manufacturer's
methods (Affymetrix). Duplicate sets of GeneChips were used to allow
statistical evaluation (t-test and Change Call).
Tissue explant/recombination studies
For recombination studies, caudal portions of spinal cord (SC) from the
E11.5 embryos were stripped of somatic mesoderm and placed on type IV
collagen-coated filters in direct contact with isolated MMs. Filters were
floated on culture medium (50:50 DMEM:HamsF12) with 10% fetal bovine serum and
evaluated grossly for tubule formation at 3 and 6 days. Mutant and control SCs
were equally efficient at inducing tubule formation in control MMs, and
equally inefficient at inducing tubules in mutant MMs.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At E6.5, no ß-gal activity is evident in embryos heterozygous for both R26R and T-Cre (TCre; R26R) (data not shown). At E7.5, recombination is evident primarily in the primitive streak and migrating mesoderm (Fig. 1C). At E8.0, recombination is widespread in mesodermal lineages, including the allantois (see Fig. 1F), but is mostly absent in the node (Fig. 1D and data not shown). A dorsal view of an E8.5 embryo demonstrates extensive recombination in paraxial, intermediate and lateral mesoderm, but not in neural tissues (Fig. 1E). At E9.0, most mesodermal lineages are ß-gal+, but recombination is incomplete in the heart (Fig. 1F). At this stage, recombination is evident at midaxial levels in the notochord and neural tube floorplate, but is absent in gut endoderm (Fig. 1G). More caudally, recombination is extensive in neural tube and gut endoderm (Fig. 1H and data not shown), creating a gradient with higher levels in more posterior positions. Recombination is also mostly absent in surface ectoderm (Fig. 1G,H). By E10.5, Cre has activated R26R in the lens (Fig. 1I). A horizontal section through the E10.5 head reveals that lacZ has been extensively activated in head mesenchyme, but only marginally in neuronal epithelium (Fig. 1J). Also, recombination has occurred in posterior surface ectoderm, as shown in the hindlimb AER in Fig. 1K. Finally, the examination of sections through an E14.5 kidney demonstrates that all metanephric tissue is recombined (Fig. 1L). Thus, T-Cre recombination and Fgf8 mesodermal expression domains overlap sufficiently to use this Cre transgenic line to study the effects of Fgf8 inactivation in mesoderm.
|
Fgf8 is not required for somitogenesis, somite differentiation or limb bud induction
We examined mutants for aspects of embryogenesis proposed to be under
Fgf8 control, including somitogenesis
(Dubrulle et al., 2001),
somite differentiation (Brent et al.,
2003
; Brent and Tabin,
2004
; Huang et al.,
2003
) and early induction of the limb bud
(Crossley et al., 1996
).
Throughout development somites formed normally
(Fig. 2G, see also Fig. S1 in
the supplementary material) despite the lack of Fgf8 expression in
the presomitic mesoderm. Furthermore, no defects were observed in any aspect
of skeletal development, including the ribs (Fig. S1B,D,G in the supplementary
material), which were thought to require myotomal Fgf8 expression
(Huang et al., 2003
). To test
whether myotomal Fgf8 expression was required to maintain the tendon
progenitor population in the syndetome subcompartment of the somite, we
performed ISH for scleraxis, an early marker of this lineage
(Brent et al., 2003
). Scleraxis
expression was normal from E10.5 through E14.5 (Fig. S1A-D in the
supplementary material), indicating that Fgf8 is not essential for
this aspect of somite differentiation. Finally, we confirmed the observation
that NC-specific Fgf8 expression is not required for limb bud induction
(Boulet et al., 2004
), as limbs
formed at the normal position and time in mutants (Fig. S1E,F in the
supplementary material). However, most mutants lacked at least one hindlimb
digit (data not shown), presumably because of the Cre-mediated deletion of
Fgf8 in the hindlimb AER (Fig.
1K) (Lewandoski et al.,
2000
; Moon and Capecchi,
2000
).
Fgf8 is required for urogenital development
Mutant offspring appeared normal at birth, but died shortly thereafter and
upon dissection showed abnormally small kidneys. Other tissues in the
urogenital tract appeared to be grossly and histologically normal, including
the adrenals, ureters, bladder and ovaries
(Fig. 3A). Accessory tissues in
the male reproductive tract, however, were affected, but these alterations
will be described in detail elsewhere. Analysis of T-Cre;
Fgf8Flox2,3 mutants, therefore,
provides evidence that the primary requirement for FGF8 in post-primitive
streak mesoderm is in kidney development.
|
To ensure that Fgf8 expression has been eliminated in the metanephros by T-Cre activity at the time of tubule induction, E11.5 and E14.5 kidneys were hybridized using the exon2,3-specific probe (Fig. 2B). As expected, Fgf8 expression was absent in the mutant metanephros at E11.5 (Fig. 3C) and at E14.5, as branching progresses (Fig. 3F). However, using a longer probe that detects mutant transcripts, we observed focal Fgf8 expression in control and mutant metanephroi at E14.5 (Fig. 3G), indicating that the absence of Fgf8 exon2,3-specific transcripts is not due to the loss of Fgf8-expressing cells and that Fgf8 expression is not autoregulatory.
Fgf8 is required for nephron development
Histological examination reveals morphologically similar control and mutant
metanephroi at E12.5 (Fig.
4A,B). The UB is comparably branched (see Fig. S2A,B in the
supplementary material), and caps of condensed mesenchyme and renal vesicles
(arrowheads) are readily apparent at UB tips in both mutants and controls
(Fig. 4A,B). By E14.5, cap
condensation, tubulogenesis (comma- and S-shaped bodies) and glomerulogenesis
have occurred in control metanephroi, whereas mutant kidneys show only
condensation and renal vesicle formation (arrowheads,
Fig. 4C,D), and dramatically
reduced UB branching (Fig. S2C,D in the supplementary material). By E16.5,
evidence of cap formation is largely absent and few vesicles remain in the
mutant kidneys. Furthermore, vesicles do not progress to form comma- and
S-shaped bodies (Fig. 4E,F). At
E18.5, the hypocellular mutant rudiment is largely devoid of nephron
epithelia, and the remaining few UB radii fail to bifurcate in the cortical
nephrogenic region (Fig. 4G,H;
Fig. S2E,F in the supplementary material). Interstitial stromal cells populate
the increased expanse between UB radii, and hypercellular areas in the
nephrogenic zone are limited to regions directly around the UB radii.
Glomeruli are absent during any stage of development in mutants, indicating
that these kidneys are nonfunctional and that renal failure probably causes
neonatal death.
Mutants exhibit aberrant patterns of apoptosis
To understand the biological basis for the aberrant development and
decreased size of the kidney at E14.5 and E16.5, we compared cell
proliferation and death in mutant and control kidneys. No apparent differences
in proliferation were detected at E12.5, 14.5 and E16.5, as determined by
immunostaining for phosphorylated histone H3 (see Fig. S3 in the supplementary
material), which marks mitotic cells
(Gurley et al., 1978). Also,
the extent of cell death in E12.5 mutant metanephroi, as determined by
staining with the fluorophore Lysotracker Red
(Fig. 5) or TUNEL assay (not
shown), was similar to control tissues. Apoptotic cells were localized
primarily to the interstitial stroma and were not associated with mesenchyme
in the cortical nephrogenic zone or in condensates
(Fig. 5A,B). Cell death,
however, was significantly increased in E14.5
(Fig. 5C-E) and E16.5
(Fig. 5F-H) mutant kidneys,
where differential staining was most prominent in the cortical mesenchyme
(Fig. 5D,G arrows), and, at
E16.5, in primitive epithelia (Fig.
5H, arrowheads). Thus, MM-derived cells, especially those in the
nephrogenic zone where mesenchymal-epithelial differentiation occurs, depend
upon Fgf8 expression for survival.
|
Marker gene expression in T-Cre; Fgf8Flox/2,3 mice
To assess the molecular effects of Fgf8 loss during metanephric
development, we evaluated the expression of mesenchymal and epithelial markers
required for metanephric development. The Wilms tumor suppressor Wt1
is normally expressed at moderate levels in induced and condensed MM, and is
upregulated in podocytes (arrows in Fig.
7) (Armstrong et al.,
1993). In mutants, no strong focal expression is detected,
confirming the histological absence of podocytes/glomeruli. Nmyc
(Nmyc1 Mouse Genome Informatics) and Pax2 are both
highly expressed in condensed mesenchyme and pretubular aggregates of MM in
control and mutant E14.5 metanephroi, but are lost from mutant E16.5 MMs
(Fig. 7), consistent with the
depletion of nephrogenic precursors indicated in our Cited1 studies. By
contrast, UB-specific Pax2 expression persists in mutant and control
tissues. UB-limited Gata3 expression
(Lim et al., 2000
) occurs in
control and mutant E14.5 metanephroi, and demonstrates that epithelial
structures under the UB tips in mutants are not of UB origin but rather are
renal vesicles. Mutant UBs lose Gata3 expression by E16.5. The
Gdnf-receptor Ret, a critical factor in UB branching and a marker of
UB tips, is depleted in mutants at the E14.5 to E16.5 transition
(Fig. 7) but is present during
early branching. These markers document the precipitous decline of metanephric
differentiation between E14.5 and E16.5. Importantly, the decline of
Nmyc and Pax2 expression in condensed mesenchyme and
pretubular aggregates demonstrates that the precursors for nephron development
exist in E14.5 mutant renal anlage but are depleted at E16.5, which is
consistent with histological observations.
Identification of early responsive genes for Fgf8 signaling in the developing kidney
To assay for molecular events preceding the morphological changes observed
in E14.5 and E16 mutants, we performed microarray analysis on isolated MMs at
E12.5, when UB branching and cell death patterns in mutants appear normal.
Mutant and normal tissues were evaluated directly upon dissection or, in the
case of mutant tissues, some were incubated for 4 hours with FGF8b (200
ng/ml). This permitted identification of immediate-early response genes and
also helped to confirm the changes in gene expression profiles observed when
comparing genes downregulated in the absence of FGF8 with those induced with
FGF8 treatment. Following change call and t-test statistical
analyses, 31 genes were significantly both decreased in E12.5 mutant MMs and
increased with the brief FGF8 treatments (see Table S1 in the supplementary
material). Of particular interest were Egr1
(Rackley et al., 1995),
Nmyc (Bates et al.,
2000
), Lim1
(Kobayashi et al., 2005
), and
an inhibitor of receptor tyrosine kinases, sprouty 1
(Gross et al., 2003
), all of
which have been previously implicated in nephrogenesis through mouse genetic
analyses. Wnt4, which plays a central role in nephron development
(Stark et al., 1994
), was
upregulated in MM after a brief treatment with FGF8, and it was decreased more
than 2-fold in mutants when microarray results were confirmed using
semi-quantitative RT-PCR (Fig. S4 in the supplementary material).
|
In addition to Wnt4, microarray analysis revealed that expression
of the homeodomain gene Lim1 was less than 8% of normal levels in MMs
and could be induced 2-fold in FGF8-treated mutant MMs (Table S1 in the
supplementary material). Normally, Lim1 is expressed in the UB and in
pretubular aggregates formed from induced MM
(Fujii et al., 1994;
Karavanov et al., 1998
).
MM-specific inactivation of Lim1 results in a failure of renal
vesicles to form tubules (Kobayashi et
al., 2005
). In whole-mount ISH studies, Lim1 expression
was largely deficient in the MM from E12.5 mutant metanephroi, but was evident
in the UB (Fig. 8B,C), thus
Lim1 may function downstream of FGF8 in nephron development.
|
Wnt4null/null metanephroi were also
subjected to microarray analysis. A comparison with microarray results from
T-Cre; Fgf8Flox2,3 MM (Table S1
in the supplementary material) and intact metanephroi (not shown) revealed
some commonality in regulation at E12.5; for example, loss of either
Wnt4 or Fgf8 caused a decreased expression of Egr1,
Nmyc and Lim1 by more than 2-fold, which may underlie the common
defects shared by T-Cre;
Fgf8Flox
2,3 and
Wnt4null/null metanephroi. Also,
Wnt4null/null metanephroi showed a
significant reduction (>2-fold) in Fgf8 expression. To establish
the distribution of Fgf8 in the absence of Wnt4, E12.5 and
E14.5 Wnt4null/null metanephroi were
probed by ISH. Both whole-mount and thin-section ISH demonstrated focal
expression of Fgf8 in pretubular aggregates from control and mutant
tissues. However, numbers of positive foci were reduced in mutants
(Fig. 9C-F and data not shown),
suggesting that the decreased Fgf8 expression noted in microarray
studies was due to the reduced formation of Fgf8-expressing
pretubular aggregates. This finding is consistent with a role for
Fgf8 upstream of Wnt4 expression. A decrease in
Lim1 expression was also noted in microarray studies of
Wnt4null/null metanephroi (Table S1 in
the supplementary material). ISH analysis revealed a loss of Lim1
expression specifically in populations of MM underlying UB branch termini, but
not in the UB itself (Fig. 9H),
conforming to the Lim1 expression pattern observed in the T-Cre;
Fgf8Flox
2,3 mutant kidney.
Taken together, these data suggest an epistatic relationship in which FGF8
induces Wnt4, which plays a major role in nephron formation, possibly
through the induction of Lim1. Alternatively, these data are
consistent with a model in which FGF8 induces Wnt4, and then both
WNT4 and FGF8 act in parallel to activate Lim1 expression and induce
tubule formation.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Given the failure of primary mesoderm migration that occurs in
Fgf8/ embryos
(Sun et al., 1999), our
observation that mutants gastrulate normally demonstrates that the small
amount of FGF8 present in mutants is above a threshold requirement at this
stage. The observation that segmentation and somitic differentiation are
normal in our mutants can be reconciled with existing models by speculating
that Fgf8 function is redundant with other FGF genes, such as
Fgf3, Fgf4, Fgf5, Fgf17 or Fgf18, which are all expressed in
the primitive streak/tail bud (Hebert et
al., 1990
; Maruoka et al.,
1998
; Niswander and Martin,
1992
; Wilkinson et al.,
1988
; Xu et al.,
1999
). Furthermore, during somite differentiation, Fgf4
and Fgf8 are both expressed in the myotome
(Crossley and Martin, 1995
;
Kahane et al., 2001
;
Niswander and Martin, 1992
;
Stolte et al., 2002
), and may
together regulate scleraxis (Brent et al.,
2003
). Alternatively, as the role of Fgf8 in segmentation
and somitic development has been explored in the chick and zebrafish
(Dubrulle and Pourquie, 2004
),
a perhaps less likely explanation postulates that the Fgf8
requirement in these processes differs between these organisms and the
mouse.
Expression studies and in vitro manipulation suggest that FGFs play a role
in nephrogenesis, although, heretofore, murine gene targeting approaches have
provided a limited understanding of their contribution(s) to this process.
Fgf2 is expressed first in the UB during metanephric development and,
in explant cultures, induces condensation
(Barasch et al., 1997;
Perantoni et al., 1995
) and
some tubule formation in isolated MMs
(Karavanov et al., 1998
).
However, as Fgf2 null homozygotes have no obvious renal defects
(Dono et al., 1998
;
Ortega et al., 1998
), any in
vivo role of Fgf2 must be redundant with other FGF genes, possibly
Fgf9, which is similarly expressed and also inductive (A.O.P.,
unpublished). Other FGF genes are expressed by stromal cells in the
nephrogenic zone and primarily affect branching morphogenesis
(Ohuchi et al., 2000
;
Qiao et al., 2001
;
Qiao et al., 1999
).
|
We find Fgf8 expression first in pretubular aggregates at E11.5 in
the MM, below the UB at its first bifurcation, and expression is sustained in
these and primitive epithelial structures of the nephron. Despite this early
expression period, E12.5 mutant metanephroi show normal branching. The
presence of appropriate markers for nephron differentiation in condensations
and pretubular aggregates of E14.5 mutants, e.g. Nmyc and
Pax2, and the mutant Fgf8 transcript, indicate that
nephrogenic precursors persist at this stage, although histological data
demonstrate that this lineage arrests at the renal vesicle stage. This
suggests that early stages of metanephric morphogenesis (E12.5) are FGF8
independent, and this is supported by cell death studies, which show no
differences between E12.5 mutant and control metanephroi. By E14.5, aberrant
apoptotic figures pervade the cortical MM. In addition, there is a depletion
of condensed mesenchymal cap cells (and not stromal precursors) that overlay
the UB in the cortex and provide precursors for nephron epithelia
(Sariola, 2002). On this
basis, Fgf8 may play a role in sustaining nephrogenic mesenchymal
progenitors populating the cortical nephrogenic zone either directly
by promoting nephrogenic mesenchyme survival or indirectly through the UB or
stroma. By whichever mechanism, the gradual depletion of these progenitors
ultimately degrades nephron morphogenesis, which in turn perturbs UB
branching, leading to the late loss (E16.5) of Gata3 and
Ret, markers of UB morphogenesis. These observations are consistent
with previous studies suggesting that FGF8 is a survival factor during
development of the first branchial arch
(Trumpp et al., 1999
), the
brain (Chi et al., 2003
;
Storm et al., 2003
), and the
limb bud (Moon and Capecchi,
2000
; Sun et al.,
2002
).
|
|
|
Nevertheless, despite these phenotypic similarities, some nephron-like
structures occur in Wnt4 null homozygotes but not in T-Cre;
Fgf8Flox/2,3 metanephroi. Thus,
there is apparently an absolute in vivo requirement for Fgf8, but not
Wnt4, in nephron formation. We speculate that in Wnt4 null
homozygotes, other WNTs (reviewed by
Perantoni, 2003
;
Vainio, 2003
) may partially
compensate for the lack of WNT4, and, together with FGF8, induce nephron
formation. In T-Cre;
Fgf8Flox/
2,3 metanephroi, which
lack both Fgf8 and Wnt4 expression, these other WNTs cannot
support tubulogenesis.
In addition to early Wnt4 loss, Lim1 expression is
downregulated at E12.5 in both T-Cre;
Fgf8Flox/2,3 and Wnt4
null homozygous mutants, and is upregulated in
Fgf8Flox/
2,3 mutant MMs
following brief FGF8 treatment. Lim1 expression is maintained in some
mesodermal lineages, including the nephric duct, UB and MM, and MM-specific
inactivation of Lim1 arrests tubulogenesis at the level of renal
vesicle development (Kobayashi et al.,
2005
), as observed in T-Cre; Fgf8
Flox/
2,3 mutants. Therefore, FGF8, as well as WNT4, may
direct morphogenesis of the MM principally through Lim1
induction.
Microarray analysis of mutant E12.5 MMs revealed other candidate genes that
may also impact on tubular epithelia differentiation. For example,
Nmyc depletion causes a reduction of cell proliferation in the MM,
leading to fewer nephrons (Bates et al.,
2000), and inhibition of sprouty 1 gene expression reduces MM
condensation in explant culture to decrease nephron numbers
(Gross et al., 2003
). Thus,
several of the genes identified in microarray studies may also contribute in
part to the observed phenotype.
However, the loss of Wnt4 or Lim1 is apparently
sufficient to generate the observed renal phenotype in T-Cre;
Fgf8Flox/2,3 mutants.
Recombination studies support the concept that both FGF8 and a WNT are
necessary to induce tubule formation in MM, although the loss of either gene
may affect kidney development by either of two mechanisms. One possibility is
that differentiation cannot be sustained due to the loss (through aberrant
cell death) of nephrogenic progenitors in the cortical MM and pretubular
aggregates. Another possibility is that the pretubular aggregates have lost
the capacity to undergo tubular expansion. More likely, both mechanisms are in
play; the monitoring of individual populations within the metanephros will
delineate the exact role of each factor.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/17/3859/DC1
* These authors contributed equally to this work
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abu-Issa, R., Smyth, G., Smoak, I., Yamamura, K. and Meyers, E.
N. (2002). Fgf8 is required for pharyngeal arch and
cardiovascular development in the mouse. Development
129,4613
-4625.
Armstrong, J. F., Pritchard-Jones, K., Bickmore, W. A., Hastie, N. D. and Bard, J. B. (1993). The expression of the Wilms' tumour gene, WT1, in the developing mammalian embryo. Mech. Dev. 40,85 -97.[CrossRef][Medline]
Barasch, J., Qiao, J., McWilliams, G., Chen, D., Oliver, J. A. and Herzlinger, D. (1997). Ureteric bud cells secrete multiple factors, including bFGF, which rescue renal progenitors from apoptosis. Am. J. Physiol. 273,F757 -F767.[Medline]
Bates, C. M., Kharzai, S., Erwin, T., Rossant, J. and Parada, L. F. (2000). Role of N-myc in the developing mouse kidney. Dev. Biol. 222,317 -325.[CrossRef][Medline]
Boulet, A. M., Moon, A. M., Arenkiel, B. R. and Capecchi, M. R. (2004). The roles of Fgf4 and Fgf8 in limb bud initiation and outgrowth. Dev. Biol. 273,361 -372.[CrossRef][Medline]
Brenner, R. M., Slayden, O. D., Rodgers, W. H., Critchley, H.
O., Carroll, R., Nie, X. J. and Mah, K. (2003).
Immunocytochemical assessment of mitotic activity with an antibody to
phosphorylated histone H3 in the macaque and human endometrium.
Hum. Reprod. 18,1185
-1193.
Brent, A. E. and Tabin, C. J. (2004). FGF acts
directly on the somitic tendon progenitors through the Ets transcription
factors Pea3 and Erm to regulate scleraxis expression.
Development 131,3885
-3896.
Brent, A. E., Schweitzer, R. and Tabin, C. J. (2003). A somitic compartment of tendon progenitors. Cell 113,235 -248.[CrossRef][Medline]
Chi, C. L., Martinez, S., Wurst, W. and Martin, G. R.
(2003). The isthmic organizer signal FGF8 is required for cell
survival in the prospective midbrain and cerebellum.
Development 130,2633
-2644.
Chi, L., Zhang, S., Lin, Y., Prunskaite-Hyyrylainen, R.,
Vuolteenaho, R., Itaranta, P. and Vainio, S. (2004).
Sprouty proteins regulate ureteric branching by coordinating reciprocal
epithelial Wnt11, mesenchymal Gdnf and stromal Fgf7 signalling during kidney
development. Development
131,3345
-3356.
Clements, D., Taylor, H. C., Herrmann, B. G. and Stott, D. (1996). Distinct regulatory control of the Brachyury gene in axial and non-axial mesoderm suggests separation of mesoderm lineages early in mouse gastrulation. Mech. Dev. 56,139 -149.[CrossRef][Medline]
Colvin, J. S., Bohne, B. A., Harding, G. W., McEwen, D. G. and Ornitz, D. M. (1996). Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat. Genet. 12,390 -397.[CrossRef][Medline]
Crossley, P. H. and Martin, G. R. (1995). The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121,4439 -4351.
Crossley, P. H., Minowada, G., MacArthur, C. A. and Martin, G. R. (1996). Roles for FGF8 in the induction, initiation, and maintenance of chick limb development. Cell 84,127 -136.[CrossRef][Medline]
Dono, R., Texido, G., Dussel, R., Ehmke, H. and Zeller, R.
(1998). Impaired cerebral cortex development and blood pressure
regulation in FGF-2-deficient mice. EMBO J.
17,4213
-4225.
Dubrulle, J. and Pourquie, O. (2004). Coupling
segmentation to axis formation. Development
131,5783
-5793.
Dubrulle, J., McGrew, M. J. and Pourquie, O. (2001). FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106,219 -232.[CrossRef][Medline]
Eswarakumar, V. P., Monsonego-Ornan, E., Pines, M., Antonopoulou, I., Morriss-Kay, G. M. and Lonai, P. (2002). The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development 129,3783 -3793.[Medline]
Fernandez-Teran, M., Piedra, M. E., Simandl, B. K., Fallon, J. F. and Ros, M. A. (1997). Limb initiation and development is normal in the absence of the mesonephros. Dev. Biol. 189,246 -255.[CrossRef][Medline]
Fujii, T., Pichel, J. G., Taira, M., Toyama, R., Dawid, I. B. and Westphal, H. (1994). Expression patterns of the murine LIM class homeobox gene lim1 in the developing brain and excretory system. Dev. Dyn. 199,73 -83.[Medline]
Grieshammer, U., Cebrián, C., Ilagan, R., Meyers, E. N.,
Herzlinger, D. and Martin, G. R. (2005). FGF8 is required for
cell survival at distinct stages of nephrogenesis and for regulation of gene
expression in nascent nephrons. Development.
132,3847
-3857.
Gross, I., Morrison, D. J., Hyink, D. P., Georgas, K., English,
M. A., Mericskay, M., Hosono, S., Sassoon, D., Wilson, P. D., Little,
M. et al. (2003). The receptor tyrosine kinase regulator
Sprouty1 is a target of the tumor suppressor WT1 and important for kidney
development. J. Biol. Chem.
278,41420
-41430.
Gurley, L. R., D'Anna, J. A., Barham, S. S., Deaven, L. L. and Tobey, R. A. (1978). Histone phosphorylation and chromatin structure during mitosis in Chinese hamster cells. Eur. J. Biochem. 84,1 -15.[CrossRef][Medline]
Hatini, V., Huh, S. O., Herzlinger, D., Soares, V. C. and Lai, E. (1996). Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2. Genes Dev. 10,1467 -1478.[Abstract]
Hebert, J. M., Basilico, C., Goldfarb, M., Haub, O. and Martin, G. R. (1990). Isolation of cDNAs encoding four mouse FGF family members and characterization of their expression patterns during embryogenesis. Dev. Biol. 138,454 -463.[CrossRef][Medline]
Huang, R., Stolte, D., Kurz, H., Ehehalt, F., Cann, G. M., Stockdale, F. E., Patel, K. and Christ, B. (2003). Ventral axial organs regulate expression of myotomal Fgf-8 that influences rib development. Dev. Biol. 255, 30-47.[CrossRef][Medline]
Kahane, N., Cinnamon, Y., Bachelet, I. and Kalcheim, C. (2001). The third wave of myotome colonization by mitotically competent progenitors: regulating the balance between differentiation and proliferation during muscle development. Development 128,2187 -2198.[Medline]
Karavanova, I. D., Dove, L. F., Resau, J. H. and Perantoni, A.
O. (1996). Conditioned medium from a rat ureteric bud cell
line in combination with bFGF induces complete differentiation of isolated
metanephric mesenchyme. Development
122,4159
-4167.
Karavanov, A. A., Karavanova, I., Perantoni, A. and Dawid, I. B. (1998). Expression pattern of the rat Lim-1 homeobox gene suggests a dual role during kidney development. Int. J. Dev. Biol. 42,61 -66.[Medline]
Kispert, A., Vainio, S. and McMahon, A. P.
(1998). Wnt-4 is a mesenchymal signal for epithelial
transformation of metanephric mesenchyme in the developing kidney.
Development 125,4225
-4234.
Kobayashi, A., Kwan, K. M., Carroll, T. J., McMahon, A. P.,
Mendelsohn, C. L. and Behringer, R. R. (2005).
Distinct and sequential tissue-specific activities of the LIM-class homeobox
gene Lim1 for tubular morphogenesis during kidney development.
Development 132,2809
-2823.
Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A.
P. (1997). Evidence that FGF8 signalling from the
midbrain-hindbrain junction regulates growth and polarity in the developing
midbrain. Development
124,959
-969.
Lewandoski, M. (2001). Conditional control of gene expression in the mouse. Nat. Rev. Genet. 2, 743-755.[CrossRef][Medline]
Lewandoski, M., Sun, X. and Martin, G. R. (2000). Fgf8 signalling from the AER is essential for normal limb development. Nat. Genet. 26,460 -463.[CrossRef][Medline]
Lim, K. C., Lakshmanan, G., Crawford, S. E., Gu, Y., Grosveld, F. and Engel, J. D. (2000). Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system. Nat. Genet. 25,209 -212.[CrossRef][Medline]
Macatee, T. L., Hammond, B. P., Arenkiel, B. R., Francis, L.,
Frank, D. U. and Moon, A. M. (2003). Ablation of
specific expression domains reveals discrete functions of ectoderm- and
endoderm-derived FGF8 during cardiovascular and pharyngeal development.
Development 130,6361
-6374.
Mahmood, R., Bresnick, J., Hornbruch, A., Mahony, C., Morton, N., Colquhoun, K., Martin, P., Lumsden, A., Dickson, C. and Mason, I. (1995). A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth. Curr. Biol. 5, 797-806.[CrossRef][Medline]
Martin, G. R. (1998). The roles of FGFs in the
early development of vertebrate limbs. Genes Dev.
12,1571
-1586.
Maruoka, Y., Ohbayashi, N., Hoshikawa, M., Itoh, N., Hogan, B. L. and Furuta, Y. (1998). Comparison of the expression of three highly related genes, Fgf8, Fgf17 and Fgf18, in the mouse embryo. Mech. Dev. 74,175 -177.[CrossRef][Medline]
Meyers, E. N., Lewandoski, M. and Martin, G. R. (1998). An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat. Genet. 18,136 -141.[CrossRef][Medline]
Moon, A. M. and Capecchi, M. R. (2000). Fgf8 is required for outgrowth and patterning of the limbs. Nat. Genet. 26,455 -459.[CrossRef][Medline]
Niswander, L. and Martin, G. R. (1992). Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114,755 -768.[Abstract]
Ohuchi, H., Hori, Y., Yamasaki, M., Harada, H., Sekine, K., Kato, S. and Itoh, N. (2000). FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development. Biochem. Biophys. Res. Commun. 277,643 -649.[CrossRef][Medline]
Ornitz, D. M. and Itoh, N. (2001). Fibroblast growth factors. Genome Biol. 2,REVIEWS3005 .[Medline]
Ortega, S., Ittmann, M., Tsang, S. H., Ehrlich, M. and Basilico,
C. (1998). Neuronal defects and delayed wound healing in mice
lacking fibroblast growth factor 2. Proc. Natl. Acad. Sci.
USA 95,5672
-5677.
Perantoni, A. O. (2003). Renal development: perspectives on a Wnt-dependent process. Semin. Cell Dev. Biol. 14,201 -208.[CrossRef][Medline]
Perantoni, A. O., Dove, L. F. and Karavanova, I.
(1995). Basic fibroblast growth factor can mediate the early
inductive events in renal development. Proc. Natl. Acad. Sci.
USA 92,4696
-4700.
Pizard, A., Haramis, A., Carrasco, A. E., Franco, P., López, S. and Paganelli, A. (2004). Whole-Mount In Situ Hybridization and Detection of RNAs in Vertebrate Embryos and Isolated Organs. New York: John Wiley & Sons.
Plisov, S., Tsang, M., Shi, G., Boyle, S., Yoshino, K.,
Dunwoodie, S. L., Dawid, I. B., Shioda, T., Perantoni, A. O. and de
Caestecker, M. P. (2005). Cited1 is a bifunctional
transcriptional cofactor that regulates early nephronic patterning.
J. Am. Soc. Nephrol. 16,1632
-1644.
Qiao, J., Uzzo, R., Obara-Ishihara, T., Degenstein, L., Fuchs,
E. and Herzlinger, D. (1999). FGF-7 modulates ureteric
bud growth and nephron number in the developing kidney.
Development 126,547
-554.
Qiao, J., Bush, K. T., Steer, D. L., Stuart, R. O., Sakurai, H., Wachsman, W. and Nigam, S. K. (2001). Multiple fibroblast growth factors support growth of the ureteric bud but have different effects on branching morphogenesis. Mech. Dev. 109,123 -135.[CrossRef][Medline]
Rackley, R. R., Kessler, P. M., Campbell, C. and Williams, B. R. (1995). In situ expression of the early growth response gene-1 during murine nephrogenesis. J. Urol. 154,700 -705.[CrossRef][Medline]
Revest, J. M., Spencer-Dene, B., Kerr, K., De Moerlooze, L., Rosewell, I. and Dickson, C. (2001). Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4. Dev. Biol. 231,47 -62.[CrossRef][Medline]
Sariola, H. (2002). Nephron induction revisited: from caps to condensates. Curr. Opin. Nephrol. Hypertens. 11,17 -21.[CrossRef][Medline]
Shawlot, W. and Behringer, R. R. (1995). Requirement for Lim1 in head-organizer function. Nature 374,425 -430.[CrossRef][Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Stark, K., Vainio, S., Vassileva, G. and McMahon, A. P. (1994). Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature 372,679 -683.[CrossRef][Medline]
Stolte, D., Huang, R. and Christ, B. (2002). Spatial and temporal pattern of Fgf-8 expression during chicken development. Anat. Embryol. 205,1 -6.[CrossRef][Medline]
Storm, E. E., Rubenstein, J. L. and Martin, G. R.
(2003). Dosage of Fgf8 determines whether cell survival is
positively or negatively regulated in the developing forebrain.
Proc. Natl. Acad. Sci. USA
100,1757
-1762.
Sun, X., Meyers, E. N., Lewandoski, M. and Martin, G. R.
(1999). Targeted disruption of Fgf8 causes failure of cell
migration in the gastrulating mouse embryo. Genes Dev.
13,1834
-1846.
Sun, X., Mariani, F. V. and Martin, G. R. (2002). Functions of FGF signalling from the apical ectodermal ridge in limb development. Nature 418,501 -508.[CrossRef][Medline]
Trumpp, A., Depew, M. J., Rubenstein, J. L., Bishop, J. M. and
Martin, G. R. (1999). Cre-mediated gene inactivation
demonstrates that FGF8 is required for cell survival and patterning of the
first branchial arch. Genes Dev.
13,3136
-3148.
Vainio, S. J. (2003). Nephrogenesis regulated by Wnt signaling. J. Nephrol. 16,279 -285.[Medline]
Vogel, A., Rodriguez, C. and Izpisua-Belmonte, J. C.
(1996). Involvement of FGF-8 in initiation, outgrowth and
patterning of the vertebrate limb. Development
122,1737
-1750.
Weinstein, M., Xu, X., Ohyama, K. and Deng, C. X.
(1998). FGFR-3 and FGFR-4 function cooperatively to direct
alveogenesis in the murine lung. Development
125,3615
-3623.
Wilkinson, D. and Green, J. (1990). In Situ Hybridization and the Three-Dimensional Reconstruction Of Series Sections. London: Oxford University Press.
Wilkinson, D. G., Peters, G., Dickson, C. and McMahon, A. P. (1988). Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse. EMBO J. 7, 691-695.[Abstract]
Wilkinson, D. G., Bhatt, S. and Herrmann, B. G. (1990). Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343,657 -659.[CrossRef][Medline]
Xu, J., Lawshe, A., MacArthur, C. A. and Ornitz, D. M. (1999). Genomic structure, mapping, activity and expression of fibroblast growth factor 17. Mech. Dev. 83,165 -178.[CrossRef][Medline]
Xu, J., Liu, Z. and Ornitz, D. M. (2000).
Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and
differentiation of midline cerebellar structures.
Development 127,1833
-1843.
Yoshino, K., Rubin, J. S., Higinbotham, K. G., Uren, A., Anest, V., Plisov, S. Y. and Perantoni, A. O. (2001). Secreted Frizzled-related proteins can regulate metanephric development. Mech. Dev. 102,45 -55.[CrossRef][Medline]
Zucker, R. M., Hunter, E. S., 3rd and Rogers, J. M. (1999). Apoptosis and morphology in mouse embryos by confocal laser scanning microscopy. Methods 18,473 -480.[CrossRef][Medline]
Related articles in Development: