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First published online 20 August 2008
doi: 10.1242/dev.023697
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Centro Andaluz de Biología del Desarrollo, CSIC/UPO, Carretera de Utrera Km1, 41013 Sevilla, Spain.
Author for correspondence (e-mail:
jlgomska{at}upo.es)
Accepted 28 July 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Iroquois, Kidney, Xenopus, Regulation, Patterning
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Dressler, 2006;
Ryffel, 2003). Moreover, these
similarities at the molecular level correlate with physiological homologies.
Thus, the tubules of all nephrons have similar subdivisions along the
anteroposterior axis with analogous distribution of transporters of small
molecules and ions along this axis
(Reggiani et al., 2007;
Wingert et al., 2007;
Zhou and Vize, 2004). This
fact, and the accessibility of Xenopus to genetic manipulation, makes
this animal an excellent model system with which to study kidney development.
In Xenopus, the specification of the pronephric anlage occurs in the
late gastrula/early neurula (stage 12). However, the first sign of pronephric
morphogenesis, the thickening of the lateral mesoderm, is detected 10 hours
later, in the late neurula (stage 20/21). At the tailbud stage (stage 25-30),
differentiation of the three basic segments occurs: the corpuscle, the tubules
and the duct. This is followed by the final maturation of the organ, which is
associated with the physiological specialization of the pronephric tubules
along the proximal-distal axis, as observed by the differential activation of
several genes encoding different transport proteins
(Carroll et al., 1999;
Reggiani et al., 2007;
Ryffel, 2003;
Zhou and Vize, 2004).
The Iroquois (Irx) genes encode homeoproteins conserved during evolution
with multiple functions during animal development
(Gómez-Skarmeta and Modolell,
2002). Their role during patterning of the vertebrate nervous
system has been studied in detail
(Gómez-Skarmeta and Modolell,
2002). By contrast, their participation in the development of
other organs is less well understood. Recently, it has been reported that
Xenopus Irx1, Irx2 and Irx3 are expressed from the tailbud
stage in the intermediate tubule segment of the pronephros, immediately prior
to regionalization of the proximal-distal axis
(Reggiani et al., 2007). This
study further showed that Irx3, but not Irx1 or
Irx2, is required for development of this region
(Reggiani et al., 2007).
We report that Xenopus Irx1, Irx2 and Irx3 are also
expressed in the pronephric territory during earlier mid-neurula stages.
Morpholino loss-of-function analyses, together with misexpression of inducible
forms of wild-type and dominant-negative Irx proteins, reveal a two-step
requirement for Irx1 and Irx3 during kidney development.
Initially, Irx1 and Irx3 maintain the identity of the
pronephric territory and define its size. This seems to occur, at least in
part, through positive regulation of Bmp signalling. Later, both Irx genes are
required for the formation of the intermediate tubule segment of the
pronephros, as reported only for Irx3
(Reggiani et al., 2007). In
addition, we show that both genes are regulated by retinoic acid, which is
known to be necessary to activate early kidney genes and to participate in the
segmentation of the pronephros in the proximal-distal axis
(Cartry et al., 2006;
Wingert et al., 2007).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To generate MT-Irx4 and MT-Irx5, a
fragment from the 5' region of each cDNA, including unique sites within
the ORF (KpnI in Irx4 and ClaI in Irx5),
were PCR amplified. These sites allowed us to fuse the PCR fragments to the
rest of each cDNA. 5' primers contained a XhoI (Irx4)
or EcoRI (Irx5) site, allowing us to clone these PCRs
fragments in frame within the pCS2-MT plasmid
(Turner and Weintraub, 1994)
at XhoI (MT-Irx4) or between EcoRI and
XhoI (MT-Irx5). Primers used were: Irx4,
5'-ccctcgagATGTCATATCCTCAGTTTGGC-3' and
5'-GCTCCCATCCATGGTACCATACC-3'; Irx5,
5'-gggaattcaCATGTCCTATCCGCAGGGC-3' and
5'-ATCCCCTGCATCTCCATC-3'. Bold nucleotides indicate restriction
sites used for cloning procedures; capitals indicate sequences present in the
cDNAs.
Irx-MT constructs
To generate Irx-MT constructs, a fragment from the 3' region
of each cDNA, including unique sites within the ORF (SacI in
Irx1, PstI in Irx2, SacII in Irx3, KpnI in
Irx4 and XbaI in Irx5 cDNAs), were PCR amplified.
For Irx1, Irx2, Irx3 and Irx5, the fusion to the rest of
each cDNA in pCS2-MT was carried out as follows: EcoRI/SacI,
PstI, SacII or XbaI fragments that contain the
5' cDNA regions of Irx1, Irx2, Irx3 or Irx5,
respectively, were cloned into pBluescript. These fragments were excised with
HindIII, SacI, PstI, SacII or
XbaI and ligated with the PCR fragment in pCS2-MT between the
HindIII and ClaI (Irx1) or BamHI
(Irx2, Irx3 and Irx5) sites. The full Irx ORF in frame with
the Myc tag was then transferred into the EcoRI site of pCS2+. For
Irx4, a ClaI/KpnI fragment containing the 5' cDNA was
ligated with the corresponding 3' PCR fragment, expanding the 3'
cDNA into the ClaI site of pCS2-MT. Primers used were: for
Irx1, 5'-GCAACAAGCCCAGATGG-3' and
5'-ccaatcgatGGCAGAGGGAAGTGCTG-3'; for Irx2,
5'-GCCGACCATCTTTGCG-3' and
5'-ggggatccTGGGTATGGTTGTACTCC-3'; for Irx3,
5'-CACAGCCCCATGTTCTGG-3' and
5'-ggggatccGGATGAGGATAAAGCGGA-3'; for Irx4,
5'-CCATGGTACCTACCCTCG-3' and
5'-ccaatcgatAGCAAGATGTTCTGTTCCT-3'; for Irx5,
5'-CTTCTCCATCTAGATCTCC-3' and
5'-ggggatccAATGCTAGACATACCTTTCTTC-3'. Bold nucleotides
indicate restriction sites used for cloning procedures; capitals indicate
sequences present in the cDNAs.
MT-Irx-GR constructs
We first generated Irx-MT-GR derivatives by cloning the
GR domain within the XhoI and XbaI fragment located 3'
of the Irx-MT in the pCS2 Irx-MT vectors. The
hormone-inducible GR domain was obtained by PCR using the oligonucleotides
5'-cccctcgagATCCCCTCTGAAAATCC-3' and
5'-ctctagaCACTTTTGATGAAACAGAAG-3' from a MyoD-GR plasmid
kindly donated by H. Sive. To make the chimeric mRNAs of these constructs
insensitive to the MOs, we introduced a MT 5' by fusing these constructs
with their corresponding MT-Irx as follows: for Irx1, a
5' EcoRI-SacI fragment from MT-Irx1 was
ligated to a 3' SacI-NotI fragment from
MT-Irx1-GR in pCS2-MT; for Irx2, a 5'
EcoRI-ApaI fragment from MT-Irx2 was ligated to a
3' ApaI-NotI fragment from MT-Irx2-GR in
pCS2-MT; for Irx3, a 5' EcoRI-SacII fragment
from MT-Irx3 was ligated to a 3' SacII-NotI
fragment from MT-Irx3-GR in pCS2-MT.
In situ hybridization, X-Gal and antibody staining
Antisense RNA probes were prepared from cDNAs using digoxigenin or
fluorescein labels (Roche). Xenopus specimens were prepared,
hybridized and stained as described
(Harland, 1991). X-Gal
staining was performed accordingly to Coffman et al.
(Coffman et al., 1993). Double
fluorescent in situ hybridization was performed as previously described
(Zhou and Vize, 2004).
Antibody staining was performed as previously described
(Gómez-Skarmeta et al.,
2001). Monoclonal antibodies 3G8 and 4A6 were kindly provided by
E. Jones. The monoclonal antibody 12/101, generated by J. P. Brockes, was
obtained from the Developmental Studies Hybridoma Bank (NICHD and The
University of Iowa, Department of Biological Science, Iowa City, IA
52242).
In vitro RNA synthesis, microinjection of mRNA and morpholinos, and grafts
DNAs were linearized and transcribed as described
(Harland and Weintraub, 1985)
with GTP cap analogue (New England Biolabs). SP6, T3 or T7 RNA polymerases
were used. After DNAse treatment, RNA was extracted with phenol-chloroform,
column purified and precipitated with ethanol. mRNAs for injection were
resuspended in water. Synthetic mRNAs or MOs were injected into V2.2
blastomeres with 2-4 nl solutions. The following morpholinos were used in this
study: MOIrx1, 5'-CATGTCTCTCCGGCAGGGAAATCGC-3';
MOIrx2, 5'-AGGTAACCCTGAGGATAGGACATGG-3'; MOIrx3,
5'-CTGTGGGAAGGACATGGTGCAGCCG-3'; MOIrx3.2,
5'-AGCTGTGGGAAGGACATGGTGCAGC-3'; MOIrx4,
5'-GTAGCCAAACTGAGGATATGACATT-3'; and MOIrx5,
5'-CAAGTAGCCCTGCGGATAGGACATG-3'. MOIrx1 and
MOIrx5 are 100% homologous to the Irx1 and Irx5
alleles used in this study. The second Irx5 and Irx1 alleles
contain 1 and 2 sequence mismatches, respectively, with their corresponding
MOs. The other Irx MOs have 100% homology with all their
corresponding Irx alleles. In the MO- or mRNA-injected embryos, in
images taken at the same magnification, we used the histogram function of
Photoshop to compare the size of the area expressing different markers in the
injected versus the uninjected sides of the same embryo. Grafts were performed
as previously described
(Gómez-Skarmeta et al.,
1999).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Irx1, Irx2 and Irx3 were initially
expressed at mid neurula stages in the pronephric anlage
(Fig. 1A,E,I). Double staining
with the early pronephric markers Lim1 and Pax8
(Carroll and Vize, 1999;
Heller and Brandli, 1999)
confirmed that Irx1 and Irx3 were expressed in the
pronephric field (insets in Fig.
1A,I; Fig. 1M,Q).
Moreover, although the Irx3 pronephric domain was broad and
encompasses most, if not all, of the Lim1 territory, Irx1
was confined to the dorsal area of the Lim1 field. At late neurula
stage, Irx1 was still confined to the dorsal pronephric domain
(Fig. 1B,N). By contrast,
Irx3 became now restricted to the ventral pronephric territory
(Fig. 1J,R). During tailbud
stages, Irx1 expression became localized in the intermediate tubule,
as judged by double staining with Pax8
(Fig. 1C, inset) or with the
proximal tubule marker Sglt1k
(Fig. 1O)
(Reggiani et al., 2007;
Zhou and Vize, 2004). At this
stage, Irx3 also became confined to the intermediate tubule, but its
expression extended into the distal area of the proximal tubule and into the
distal tubule, as determined by double staining with Pax8
(Fig. 1K, inset) or with the
intermediate tubule marker Nkcc2
(Fig. 1S)
(Reggiani et al., 2007;
Zhou and Vize, 2004). The
expression patterns of Irx1 and Irx3 were maintained at
tadpole stages (Fig. 1D,L,P,T).
In addition, late Irx1 expression was also observed in migrating
ventral mesoderm (Fig. 1D, blue
arrowhead). We conclude that Irx genes have dynamic patterns of expression
during pronephros development and that their expression in the pronephric
territory starts much earlier than recently reported
(Reggiani et al., 2007).
Loss of Irx1 and Irx3 function impairs pronephros development
To examine the requirement for Irx genes during Xenopus pronephros
development, we interfered with the activity of each Irx mRNA by injecting
specific translation-blocking morpholinos (MOs). As Xenopus laevis is
pseudotretaploid, we identified all ESTs available in the database for each
Irx gene and found one allele for Irx4 and two alleles for Irx1,
Irx2, Irx3 and Irx5. We designed specific Irx MOs that block
translation from both Irx alleles when present in the genome. The specificity
of these MOs is shown in Fig. S2 in the supplementary material. Embryos
injected with any of these Irx MOs showed different degrees of neural defects
(E.R.-S., P.A. and J.L.G.-S., unpublished) indicating that they are effective
in blocking the activity of their respective Irx genes. To reduce the MO
effects on off-target tissues, in all experiments, we targeted the pronephric
anlage by injecting the V2.2 blastomere of 8-16-cell stage embryos. We then
evaluated the effect these injections on the early renal markers Lim1
and Pax8. We considered that an embryo had an altered pronephros when
the area expressing the corresponding marker on the injected side varied by
more than 20% relative to the uninjected control side. Differences greater
that 20% between the two pronephros of a single embryo were very rarely
observed in non-injected embryos or in embryos injected with a control MO
(<2%, n=83). Injection of 8 ng of MOs against Irx1 or
Irx3 (Fig. 2), but not
Irx2, Irx4, Irx5 or a control MO (Fig. S3 in the supplementary
material; not shown), caused renal defects. Thus, at mid-late neurula stage,
the territory expressing Lim1 or Pax8 was reduced in most
Irx1 or Irx3 morphant embryos
(Fig. 2A,B,I,J, and not shown).
The average of pronephros size reduction was around 40-50%, and was observed
in 57% (n=159) and 83% or (n=74) of the MOIrx1- and
MOIrx3-injected embryos, respectively. As muscles are a source of
signals that influence kidney development
(Seufert et al., 1999), we
determined whether, in the Irx morphant embryos, muscle development
was altered. By staining with the muscle-specific antibody 12/101, we found
that this was not the case (Fig.
2A,B,I,J). We also monitored the effect of injecting
MOIrx1 or MOIrx3 on the expression of genes expressed at
tadpole stages, during the maturation of the pronephros. All three genes
examined, Sglt1k, Nkcc2 and Nbcc1 [which are expressed in
proximal, intermediate and distal tubule, respectively
(Reggiani et al., 2007;
Zhou and Vize, 2004)], were
downregulated in MOIrx1 (38-47%, n=34-39) or MOIrx3
(51-96%, n=24-41) morphants (Fig.
2C-H,K-P). The reduction of Irx function did not significant
altered the rate of cell proliferation or cell death in the kidney territory
(not shown).
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), and 12/101 (Fig.
3G-J). Interestingly, in these Irx-depleted embryos, the number of
ventral muscle fibres was reduced (Fig.
3G,H, blue arrowheads). This suggests that the expression of
Irx1 in ventral migrating muscle cells may be required for proper
development of these muscles. Sections through these injected embryos
suggested that cells that lose their kidney fate are likely to end up as
fibroblasts, as an increased number of cells with fibroblast shape are
detected in the Irx depleted side
(Fig. 3J).
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). All three MT-Irx-GR proteins behaved similarly in
overexpression studies (see below and not shown). Consistent with a
requirement for Irx genes during pronephric development, overexpression of
MT-Irx1-GR or MT-Irx3-GR mRNAs, upon addition of
dexamethasone (Dex) at stage 14, triggered a ventral expansion of
Lim1 and Pax8 (Fig.
4A-H; 60% of the embryos showing enlarged pronephros,
n=132). In most embryos, the pronephros at the injected side was
about 50% larger than the pronephros at the control non-injected side. This
expansion enlarged the differentiated kidney tissue, as determined by staining
with the tubules antibody (3G8) (Fig.
4Q-T). We next determined the ability of these MT-Irx-GR
constructs to rescue the defects observed in Irx morphant embryos. Although
interference with Irx1 and Irx3 function with a mix of MOs
caused downregulation of Lim1 and Pax8
(Fig. 4I-L;
Fig. 3C-F), co-injection of
Irx MOs with MT-Irx1-GR or MT-Irx3-GR mRNAs rescued
the expression of these genes, but only upon hormone addition at early neurula
stage (Fig. 4M-P and not shown;
15% reduced and 55% enlarged pronephros, n=100). These results
indicate that the pronephric expression of Irx genes is required to maintain
the transcription of the key renal genes Lim1 and Pax8, and
to define the size of the pronephric anlage.
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). By contrast, during kidney development, overexpression of
HD-GR-E1A mRNA (Fig.
5A-H) mimicked the ventral expansion of Lim1 (64% of the
embryos with enlarged pronephros, n=48) caused by wild-type Irx mRNAs
(Fig. 4). Conversely, the
overexpression of HD-GR (Fig.
5I-P) or HD-GR-EnR (inset in
Fig. 5I, M, and not shown)
mRNAs promoted downregulation of Lim1 (88% of the embryos showing
reduced pronephros, n=54). Therefore, during pronephros development,
HD-GR and HD-GR-EnR proteins behave as dominant-negative molecules that
interfere with Irx function. Similar results were found when Pax8
expression was examined (not shown). In addition, by providing Dex at
different stages of development, we also found that the requirement for Irx
function during pronephros development occurred around mid neurula (stages
15-17). Overexpression of Irx proteins at later stages (20-22) had little
effect on Lim1 and Pax8
(Fig. 5). These data suggest
that Irx proteins act as transcriptional activators to maintain the kidney
anlage properly and to define the size of this territory, and confirm that
they are initially required before pronephros morphogenesis takes place.
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). A recent report showed that blocking this pathway during
Xenopus neurula stages impaired pronephros formation
(Bracken et al., 2008). These
results resemble those observed by reducing Irx function. As Irx genes
modulate Bmp signalling during neural development
(Gómez-Skarmeta et al.,
2001), a similar situation may occur during kidney development. To
evaluate this, we increased Bmp signalling at neurula stages, by
overexpressing an inducible Smad1 construct (Smad1GR) in the absence of
Irx1 and Irx3 activity. Induction of Smad1GR at stage 14
caused an expansion of kidney territory (51%, n=35), as determined by
Pax8 expression (Fig.
6A,B). In the absence of Dex, embryos co-injected with
Smad1GR mRNA and Irx1 and Irx3 MOs showed
downregulation of Pax8 (Fig.
6C,D; 41%, n=51). This phenotype is partially rescued by
Dex treatment at stage 14 (Fig.
6E,F; 21% with Pax8 downregulated, n=48). Thus,
part of Irx function seems to be to positively modulate the Bmp pathway.
Bmp7 is expressed and required for kidney development
(Dudley et al., 1995;
Luo et al., 1995;
Wang et al., 1997). We
examined whether its expression depended on Irx activity. Indeed, as for other
kidney markers, in double Irx1 and Irx3 morphant embryos
Bmp7 expression was downregulated
(Fig. 6G,H).
Irx1 and Irx3 genes are required for proximal-distal patterning of the pronephros
Irx3, but not Irx1, has been shown to be required for
formation of the intermediate tubule segment of the pronephros
(Reggiani et al., 2007). We
examined whether Irx1 was also required in this late process.
Injection of high doses of Irx MOs downregulated all proximal-distal
genes, probably because of the early requirement of Irx genes for maintaining
the kidney anlage, thus preventing examination of later Irx functions. To try
to overcome this problem, we partially reduced Irx function by injecting
Irx1 and Irx3 MOs at lower doses. We complement these
experiments injecting a second Irx3 MO (MOIrx3.2) that, in a
previous report, was unable to reveal an early Irx3 requirement
(Reggiani et al., 2007).
Blastomere injection of 4 ng of Irx1 MO or either one of the two
Irx3 MOs caused little effect on Lim1 and Pax8
expression in late neurula or tailbud stages (not shown). By contrast, at
tadpole stage, embryos injected with Irx1 or with any of the
Irx3 MOs showed downregulation of the proximal domain of
Nkcc2 (40-50%, n=19-26)
(Fig. 7C,D,G,H,K,L). This was
also accompanied by a reduction in the distal expression of Sglt1k in
the MOIrx3, but not in the MOIrx1, injected embryos
(Fig. 7A,B,E,F,I,J). These
results are consistent with the expression domains of Irx1 and
Irx3, and suggest that both genes are required for proximal-distal
patterning, as was already reported for Irx3
(Reggiani et al., 2007). We
also tried to address why, in the previous report
(Reggiani et al., 2007), an
early requirement for Irx3 was not detected. For that, we injected
one of the Irx3 MO they used (MOIrx3.2) at higher (8 ng)
doses. At this concentration, most embryos injected with our Irx3 MO
were healthy and showed the strong reduction of all segment markers shown in
Fig. 2. By contrast, the
majority of the embryos injected with 8 ng of MOIrx3.2 were
malformed. These malformed embryos also lost most markers
(Fig. 7M-P). This might explain
the discrepancy if those embryos with stronger phenotypes and malformations
were not taken into account in the previous report
(Reggiani et al., 2007).
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;
Wingert et al., 2007;
Zhou and Vize, 2004). By
contrast, the same construct induced at stage 27 downregulated Nkcc2
(59%, n=17), whereas Sglt1k and Gata3 were not
affected (Fig. 8E-H). These
results are consistent with an early and a late requirement for Irx function.
A further confirmation of this dual function was obtained by examining these
proximal-distal markers in embryos injected with Dex-inducible Irx1 or Irx3
proteins. Incubation of injected embryos with Dex from stage 13 caused ectopic
patches of Sglt1K (38-47%, n=15-24)
(Fig. 8I,J,Q,R) and an enlarged
Nkcc2 domain (52-60%, n=17-24)
(Fig. 8K,L,S,T). No clear
effect was found on Gata3 expression
(Fig. 8I,J,Q,R). Addition of
hormone at stage 27, expanded the Nkcc2 expression domain (50-60%,
n=14-22) (Fig.
8O,P,W,X), but did not affect Sglt1K expression
(Fig. 8M,N,U,V).
Retinoic acid regulates pronephric expression of Irx1 and Irx3
Retinoic acid (RA) is a requisite for the activation of early kidney genes
and for the late segmentation of the pronephros
(Cartry et al., 2006;
Wingert et al., 2007).
Therefore, RA may regulate Irx genes during kidney development. To test this,
we examined Irx expression in embryos injected with 100 pg of mRNAs encoding
either a dominant-negative (RAR-DN) or a constitutively active (RAR-Vp16) RA
receptor (Blumberg et al.,
1997). Embryos with reduced or increased RA signalling showed down
or upregulation, respectively, of the expression of Irx genes in the kidney
(Fig. 9A-H). Thus, RA
positively regulates Irx genes in the pronephros. We then determined when RA
signalling is required for kidney Irx expression. To this end, we incubated
Xenopus embryos at different developmental stages (12.5, 15 or 25)
for 1 hour with a control solution (DMSO), with 4-diethylaminobenzaldehyde
(DEAB 30 µM), the chemical inhibitor of the RA producing enzyme Raldh2 or
with RA (10 µM). Reducing or increasing RA signalling, down or upregulated,
respectively, the expression of both genes, but only when the drug treatments
were done at late gastrula stage (12.5)
(Fig. 9I-Y).
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Irx genes are required to maintain the kidney anlage before pronephros morphogenesis
Irx1 and Irx3 show dynamic expression patterns during
pronephros development. We find that Irx genes are initially expressed in the
pronephric territory at neurula stage. This occurs after the initial
specification of this territory by Ors, Lim1 and Pax8 genes
at gastrula stage, but before morphological or molecular signs of kidney
morphogenesis at late neurula-early tailbud sages. Irx1 is initially
activated in the dorsal pronephric territory. By contrast, Irx3 is
initially expressed in most of the kidney anlage, but it becomes confined to
the ventral pronephros territory. This dorsal-ventral subdivision of the
prospective kidney may reflect the initial subdivision of the pronephric
territory by Notch signalling into a dorsal region that will generate glomus
and proximal tubule, and a ventral domain that will give rise to distal tubule
and duct (McLaughlin et al.,
2000; Taelman et al.,
2006). However, we have not detected an alteration of
Irx1 or Irx3 expression by manipulating Notch signalling
(not shown), which suggests that Notch does not regulate Irx genes.
Interestingly, the expression of Irx genes slightly precedes the onset of
expression of Dl1 and Notch1, which is compatible with Irx
genes participating in the regulation of Notch signalling in the pronephros.
Indeed, Irx genes play a pivotal role in the regulation of Notch signalling
during Xenopus neural crest formation
(Glavic et al., 2004) and
during Drosophila eye development
(Domínguez and de Celis,
1998). The relationship between Irx genes and Notch signalling is
currently under investigation.
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; Heller and Brandli,
1999; Tena et al.,
2007). However, depletion of Irx1 or Irx3
impairs the expression of all kidney genes examined at tailbud and tadpole
stages. This Irx function seems to be autonomous, as downregulation of kidney
genes occurs without affecting neural or other mesodermal tissues. Consistent
with a requirement for Irx genes before pronephros morphogenesis,
time-controlled loss or gain of Irx function during neurula, but not during
tailbud stages, reduces or expands the pronephric field, respectively.
Interestingly, gain of Irx function expands but does not promote ectopic
expression of Xlim1 and Pax8, as found when Osr genes or
Pax8 and Xlim1 are overexpressed
(Carroll and Vize, 1999;
Tena et al., 2007). This
suggests that Irx genes alone are unable to trigger the kidney program. Our
results indicate that Irx genes are expressed and required before appearance
of any sign of pronephros morphogenesis. This early Irx gene requirement for
kidney development is likely to be conserved in other vertebrates as these
genes are also expressed in the early kidney anlage of other vertebrates
(Houweling et al., 2001;
Lecaudey et al., 2005).
During neural development, Irx proteins act as repressors and downregulate
Bmp signalling to allow neural plate formation
(Gómez-Skarmeta et al.,
2001; Itoh et al.,
2002). In this work we show that Irx proteins act as activators
during kidney formation. Thus, one possible mechanism of action of Irx
proteins could be to upregulate Bmp signalling, which is known to participate
at many steps during vertebrate kidney formation
(Cain et al., 2008). Consistent
with this idea, the reduction of Bmp signalling during Xenopus
neurula stages causes defects similar to those produced by Irx gene impairment
(Bracken et al., 2008).
Furthermore, we show that increasing Bmp signalling partially rescue the
kidney defects observed in Irx morphant and that Bmp7 expression is
downregulated in Irx-deficient embryos. Further experiments are required to
determine more precisely the interaction between the Bmp pathway and Irx
genes.
Irx genes are required at later stages for proximal-distal pronephric patterning
Recently, it has been reported that, within the pronephros field, Irx genes
are initially expressed at tailbud stages and that only Irx3 is
required for proximal-distal pronephric patterning
(Reggiani et al., 2007). As
indicated above, we detect an earlier expression (at neurula stage) of
Irx1, Irx2 and Irx3 in that territory, and a requirement for
both Irx1 and Irx3 for the proper development of the kidney
territory before pronephric morphogenesis. As it was possible that the late
Irx3 described function might be an indirect effect of the earlier
Irx requirement, we have re-examined the participation of Irx genes in
proximal-distal patterning of the pronephros. Although MOs are very useful
reagents to reduce gene activity, their injections into blastomeres may impair
gene function from early stages and make it difficult to recognize a late
requirement. To try to uncouple early and late Irx requirements, we injected
low doses of Irx MOs. In these hypomorphic conditions, embryos did not show
early kidney phenotypes but the late pronephric segmentation was affected.
This suggests that both genes are required for this late process but does not
exclude that this could be an indirect consequence of an early requirement for
Irx genes. To determine Irx protein function unambiguously during
proximal-distal pronephric patterning, we have used conditional loss- and
gain-of-function of Irx genes. Overexpression of an inducible
dominant-negative construct demonstrates that early impairment of Irx gene
activity downregulates all proximal-distal markers examined. By contrast, late
impairment of Irx activity prevents only intermediate tubule formation. We
confirmed this dual Irx protein function by overexpressing Irx1 or
Irx3 at neurula or tailbud stages. Early Irx activation
expands or promotes ectopic expression of different segment markers, whereas
late overexpression expands only the intermediate tubule marker
Nkcc2. Thus, our study reveals an earlier Irx gene requirement for
most of the phronephric field and also, in agreement with Reggiani et al.
(Reggiani et al., 2007), a
late requirement, although in contrast to this report, our data support the
necessity for both Irx1 and Irx3 for proximal-distal
patterning.
Irx genes are regulated by RA signalling
RA is required for the activation of the early kidney genes Lim1
and Pax8 (Cartry et al.,
2006), and for the late regionalization of the pronephros
(Wingert et al., 2007). We
observe that the expansion of the kidney field associated with overexpression
of Irx genes are similar to that found upon increasing retinoic acid activity
(Cartry et al., 2006). This
suggests a possible link between Irx genes and RA signalling. Indeed, we find
that both Irx1 and Irx3 are activated by RA signalling. This
is consistent with the fact that the pronephric expression of the gene
encoding the RA producing enzyme Raldh2 and the RA receptor
RAR
precedes that of the Irx genes. Thus, RA probably lies
upstream of Irx genes during pronephros development. We also find that RA is
necessary for Irx gene expression at late gastrula/early neurula stages, but
not later. This developmental period is when RA is required for the activation
of the early kidney genes Lim1 and Pax8
(Cartry et al., 2006), and it
is well before Irx genes are initially expressed. Thus, the RA effect on Irx
gene expression is likely to be indirect, probably through Lim1 and
Pax8. In addition, as we do not detect alteration of the Irx
expression patterns when RA signalling is modulated just before
proximal-distal patterning, RA influence on proximal-distal patterning
(Wingert et al., 2007) is
likely to occur as an indirect consequence of its effect on early kidney genes
such as Lim1 and Pax8
(Cartry et al., 2006).
In a functional survey of the enhancer activity of highly conserved
non-coding elements present in the IrxB complex
(de la Calle-Mustienes et al.,
2005) we identified two ultraconserved non-coding regions (UCRs)
that activate expression in the pronephros, as determined in Xenopus
transgenic experiments (de la
Calle-Mustienes et al., 2005). Each UCR is located in each Irx
gene cluster in relatively close proximity to Irx1 and Irx3.
These regulatory regions are likely to contribute to the regulation of Irx
genes during kidney development by early kidney specification genes. The
detailed analyses of these regions should help unravel the molecular
mechanisms that control Irx gene expression during pronephros formation and to
define the genetic cascade that operates during this process.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/19/3197/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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