|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 3 July 2006
doi: 10.1242/dev.02458
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Laboratoire d'Embryologie Moléculaire, Université Libre de
Bruxelles, Institut de Biologie et de Médecine Moléculaires
(IBMM), rue des Profs. Jeener et Brachet 12, B-6041 Gosselies, Belgium.
2 DFG-Center of Molecular Physiology of the Brain, Department of Developmental
Biochemistry, University of Göttingen, Justus-von-Liebig Weg 11, 37077
Göttingen, Germany.
* Author for correspondence (e-mail: ebellefr{at}ulb.ac.be)
Accepted 25 May 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: XHRT1, Notch, Pronephros
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Vize et al., 2003;
Ryffel, 2003;
Jones, 2005).
All three components of the pronephros develop within the intermediate
mesoderm right posterior to the head. In Xenopus, at late neurula
(around stage 21), cells in the lateral layer of the intermediate mesoderm
below somites 3-7 start to condense. Cells from the dorsoanterior region of
the pronephric field will form the tubules, while those from the
ventroposterior region migrate posteriorly out of the original kidney
primordium to give rise to the majority of the duct. Concomitantly, cells in
the adjacent medial layer undergo morphogenesis to form the glomus. The
molecular mechanisms that control the early specification of the pronephros
have been well studied in frog and chicken
(Brennan et al., 1998;
Brennan et al., 1999;
Seufert et al., 1999;
Obara-Ishihara et al., 1999;
Carroll and Vize, 1999;
Mauch et al., 2000;
Chan et al., 2000;
James and Schulteiss, 2005).
By contrast, much less is known about the gene products that pattern the
pronephric anlagen. The Wilms' tumor xWT1 gene encoding a zinc finger
transcription factor, which is expressed around the dorsal and anterior border
of the future pronephros, is thought to have a role in the specification of
the glomus by suppressing tubule and duct gene expression
(Carroll and Vize, 1996;
Wallingford et al., 1998).
Evi1 is another gene encoding a zinc finger transcription factor that
may play a role in the partitioning of the pronephros; it is selectively
expressed in the ventroposterior part of the pronephros anlagen, giving rise
to the distal tubule and duct compartments
(Van Campenhout et al., 2006).
Notch signaling has also been shown to play an important role in the
partitioning of the pronephros, inhibiting duct and distal tubule
differentiation in the dorsoanterior region of the anlagen, where cells are
normally fated to form proximal tubules and to increase the expression of the
xWT1 gene (McLaughlin et al., 2000;
Van Campenhout et al., 2006).
Studies in mice have demonstrated that Notch signaling is similarly required
during metanephros development for glomerular podocyte and proximal tubule
fates (McCright et al., 2001;
Wang et al., 2003a;
Cheng et al., 2003;
Cheng and Kopan, 2005).
However, the stages of nephron morphogenesis that are dependent upon the
activation of Notch remain unidentified.
In Xenopus, the XHRT1 gene (also named
Hey1/HERP2/Hesr-1/CHF2), encoding a
downstream basic helix-loop-helix Orange (bHLH-O) mediator of Notch signaling,
has been shown to be expressed in numerous tissues during development,
including the pronephros, and to be responsive to Notch signaling
(Rones et al., 2002;
Pichon et al., 2002). XHRT1 is
a member of the HRT subfamily of bHLH-O proteins that forms heterodimers with
hairy proteins through the bHLH-O and downstream sequences, and represses
transcription in a groucho-independent manner
(Iso et al., 2003;
Taelman et al., 2004;
Pichon et al., 2004). In the
embryonic mouse metanephros, several intracellular Notch effectors have been
found to be expressed in a segment-specific manner in early nephrons, but
nothing is known as yet about their role in patterning cell fate decisions
(Leimeister et al., 2003;
Piscione et al., 2004;
Chen and Al-Awqati, 2005).
Here, we show that XHRT1, when compared with the other bHLH-O factors expressed in the developing kidney, plays a predominant role in the pronephros as a Notch effector, being required for glomus formation and for proximodistal patterning of the pronephric primordium. We show that this is due not only to its earlier temporal expression pattern, but also to intrinsic properties of the protein that the HES proteins lack.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Wettstein et al.,
1997; Taelman et al.,
2004). The following plasmids were constructed: pCS2+MT
XHes2-hGR, encoding an inducible, Myc-tagged XHes2 protein; pCS2+MT
XHRT1-XHes2-hGR, encoding an inducible, truncated form of XHRT1 (amino
acids 1 to 160) to which has been attached the carboxy terminus of XHes2
(amino acids 129 to 191); and pCS2+MT Hes2-XHRT1-hGR, encoding an
inducible, truncated form of XHes2 (amino acids 1 to 127) with the carboxy
terminus of XHRT1 (amino acids 161 to 294). In addition,
pCS2+XHRT1a-mut-MT-hGR, derived from pCS2+XHRT1a-MT-hGR,
incorporates several mismatches (small letters) in the MO target sequence:
5'-ATGAAaaGaGGcCAtGAtTA (predicted start codon is underlined).
pCS2+XHRT1-eGFP encodes the full-length XHRT1 protein fused with eGFP
at its carboxy terminus. pCS2+xWT1-eGFP encodes a similar fusion with the
N-terminal part of xWT1 (amino acids 1-91).
Morpholino oligonucleotides
Antisense morpholinos for xWT1, XHRT1, esr9, esr10 and Xhairy2b (GeneTools)
consist of the following sequences (sequence complementary to the predicted
start codon is underlined):
XHRT1 MO, 5'-TAGTCGTGTCCCCGCTTCATGGCTG-3';
xWT1a MO, 5'-CATATCCCGGACATCAGACCCCATC-3';
xWT1b MO, 5'-CATATCCCGCACATCAGATCCCATC-3';
esr9 MO, 5'-CTGTCTGGTAATGGGATGTGATGGA-3';
esr10 MO, 5'-CTGTTTAGTAAGTGGATATGATGGA-3';
Xhairy2a MO, 5'-ATGGTATCTGCGGGCATGTTCAGTT-3';
Xhairy2b MO, 5'-GGCATGTTCAGATGTTGTATCCGGA-3'.
Individual MOs, or a mixture of both MOs for xWT1, were injected at 15 ng/blastomere.
Embryo and injections
Xenopus eggs were obtained from hormone-induced (chorionic
gonadotropin, Sigma) adult female frogs and fertilized using standard methods.
Capped mRNAs were transcribed using the mMessage mMachine Kit (Ambion). For
targeting the pronephros, synthetic RNA (500 pg) was injected into one
blastomere in the lateral marginal zone of 8-cell stage embryos.
nuc-lacZ mRNA (100-250 pg/blastomere) was used as a lineage tracer.
Induction of hGR constructs in embryos was performed by addition of
dexamethasone (Dex; 10 µM; Sigma). Injected embryos were fixed in MEMFA,
stained for ß-galactosidase activity with
5-bromo-4-chloro-3-indolyl-ß-galactopyranoside (X-Gal, Bioline) or
6-chloro-3-indolyl-ß-D-galactoside (Red-Gal, Research Organics) and
stored in ethanol at -20°C. Only embryos that were phenotypically normal
and show lacZ staining in the pronephic region were scored.
In situ hybridization
Whole-mount in situ hybridization was carried out as previously described
(Harland et al., 1991). X-Serrate-2 was identified through a search
of the EST database (NIBB #XL054p21). The X-Serrate-2 plasmid
linearized with EcoR1 was transcribed with T7 polymerase. Plasmids
used for generating the other in situ hybridization probes are: XHRT1
(Pichon et al., 2002),
XHairy2b, Xhairy1 (Tsuji et al.,
2003; Taelman et al.,
2004), Evi1, xSat1, xPDZK1
(Van Campenhout et al., 2006),
xCLC-K (Vize, 2003),
ESR-4, ESR-5 (Jen et al.,
1999), ESR-6e
(Deblandre et al., 1999),
esr9, esr10 (Li et al.,
2003), XHes2 (M.S., unpublished), Hes6
(Koyano-Nakagawa et al.,
2000), XSMP-30 (Sato
et al., 2000), xWT1
(Carroll and Vize, 1996),
Pax8 (Heller and Brandli,
1999), X-Delta-1, N-tubulin
(Chitnis et al., 1995),
Ep. keratin (Jonas et al.,
1985), X-Serrate-1
(Kiyota et al., 2001) and
Nephrin (Gerth et al.,
2005). For sections, embryos were gelatine embedded and vibratome
sectioned.
Western blots
Western blot analysis was performed as described
(Taelman et al., 2004) using
the 9E10 anti-Myc and anti-ß-tubulin monoclonal antibodies (Sigma).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), Xhairy1
(Taelman et al., 2004),
Xhairy2b (Tsuji et al.,
2003), ESR-1
(Wettstein et al., 1997),
ESR-4, ESR-5 (Jen et al.,
1999), ESR-6e
(Deblandre et al., 1999),
esr9, esr10 (Li et al.,
2003), XHes2 (M.S., unpublished) and Hes6
(Koyano-Nakagawa et al.,
2000), and compared their expression with that of the ligands
X-Delta-1 (Chitnis et al.,
1995), X-Serrate-1
(Kiyota et al., 2001) and
X-Serrate-2, identified by EST mining. ESR-1, ESR-4, ESR-5,
ESR-6e, XHes2 and Hes6 mRNA were not detected in the pronephric
anlagen between stages 20 and 36. XHRT1, Xhairy1, Xhairy2b, esr9 and
esr10 are all expressed from the early tailbud stage in the
dorsoanterior region of the developing pronephros, XHRT1 expression
being detectable slightly earlier than the others.
During early tailbud stages, XHRT1, esr9 and esr10
expression appears localized to the most dorsoanterior portion of the
pronephric anlagen, whereas Xhairy1 and Xhairy2b are more
broadly expressed within the pronephric mesoderm
(Fig. 1A,E,G,I,K,M). In
transverse sections of stage 20-23 embryos, XHRT1, esr9 and
esr10 transcripts are predominantly found, similarly to xWT1
transcripts (Carroll and Vize,
1996), around the dorsoanterior border of the pronephros anlagen
(Fig. 1B,D,F,H,J). By contrast,
Xhairy1 and Xhairy2b expression is detected both around and
inside the developing pronephros, strong Xhairy2b staining being also
observed in the sensorial layer of the ectoderm covering the pronephros
anlagen (Fig. 1L,N).
X-Delta-1 is the only Notch ligand to be expressed in the developing
pronephros at early tailbud stage. Its expression is detected in the lateral
mesodermal layer in cells surrounded by the XHRT1-positive cells
(Fig. 1C).
During late tailbud to early tadpole stages, XHRT1 expression demarcates the most dorsoanterior portion of the pronephros, whereas esr9, esr10, Xhairy1 and Xhairy2b occupy more ventral regions. Within this dorsoanterior portion of the pronephros, high levels of XHRT1 expression progressively become restricted to the tip of the forming tubules, while expression of the other bHLH repressors remains broader (Fig. 2A,C,E,G,I,K,R). Sectioning of those embryos revealed that during this period, expression of XHRT1, esr9 and esr10 disappear in the medial layer and that they are now actively transcribed in the dorsoanterior portion of the pronephros anlagen itself. Xhairy1 and Xhairy2b, which were initially transcribed in both layers, are now predominantly expressed in the lateral layer too (Fig. 2B,D,F,H,J,L). During this period, all Notch ligand genes are expressed in the dorsoanterior portion of the pronephros anlagen in a region slightly ventral to XHRT1 expression. Whereas X-Delta-1 expression is restricted to a band just below XHRT1, Serrate2 expression is broader (Fig. 2M-Q). X-Serrate-1 is expressed similarly to X-Serrate2 at that stage (data not shown). Although pronephric expression of XHRT1, esr9, esr10 and Xhairy1 is no longer detectable at late tadpole stages (stage 35), Xhairy2b expression is maintained in the proximal and distal tubules. X-Serrate-1 is the only Notch ligand to remain expressed in the dorsoanterior portion of the pronephros at that time, and its expression appears to be similar to that of Xhairy2b (Fig. 2S,T). The spatially and temporally distinct expression patterns of those bHLH-O repressor-encoding genes suggest that they may have non-identical functions during pronephros development.
|
). We investigated whether Notch signaling also affects the
expression of the other bHLH-O genes. To study the consequence of activation
of Notch signaling in the developing pronephros without affecting earlier
developmental steps, we used an hormone-inducible form of the transcription
factor Su(H) that mediates Notch signaling
(Wettstein et al., 1997).
Injected embryos were induced with dexamethasone at stage 18 and assayed for
expression of the different bHLH-O genes between stages 25-30. We observed
that, as in the case of XHRT1, activation of Notch signaling using an
inducible Notch ICD-ankyrin fusion of Su(H) increased the pronephric
expression of esr9 (n=24), esr10 (n=35),
Xhairy1 (n=26) and Xhairy2b (n=25) in all
injected embryos (Fig. 3A,
parts a-j). In many embryos, expression of those bHLH-O genes expands within
the posterior part of the intermediate mesoderm, the strongest staining being
detected in the case of esr9, esr10 and XHRT1 in the lateral
part of the intermediate mesoderm, while Xhairy1 and Xhairy2b expression is
found in both layers (arrowheads). Xhairy2b, which is expressed in
the ectoderm overlying the pronephros anlagen, was also strongly activated in
the ectoderm (Fig. 3B, parts
a-e). As reported in the case of XHRT1, suppression of Notch
signaling using a dominant-negative form of Su(H) decreased the expression in
the pronephros of esr9, esr10, Xhairy1 and Xhairy2b
(n=8 for esr9, 13 for esr10, 29 for
Xhairy1 and 31 for Xhairy2b;
Fig. 3A, parts k-t). Thus,
esr9, esr10, Xhairy1 and Xhairy2b may function together with
XHRT1 in the developing pronephros as downstream mediators of Notch
signaling. As our results indicate that XHRT1 is expressed in a dynamic manner in the pronephros, we wanted to know whether this reflects a difference in the temporal responsiveness of the medial and lateral layers to activation of Notch signaling. Therefore, we analysed XHRT1 expression in earlier embryos (stage 23). As observed in stage 25-30 embryos, activation of Notch induced XHRT1 expression in both layers (Fig. 3B, part f). By contrast, xWT1 is only activated in the medial layer at all stages analysed (Fig. 3B, part g). Thus, the successive expression of XHRT1 in the medial and lateral mesodermal layers is not a consequence of a difference in the temporal competence of the two layers to respond to Notch activation.
XHRT1 early expression in the developing glomus is affected by translational inhibition of xWT1
The xWT1 gene is thought to play an important role in the
development of the pronephros by repressing lateral-specific gene expression
in the portion of the pronephric mesoderm fated to form the glomus
(Wallingford et al., 1998;
Van Campenhout et al., 2006).
To determine whether xWT1, which is activated at about the same time
as XHRT1 in the pronephros, is required for the expression of bHLH-O
repressors in the forming glomus, we generated antisense MOs that block the
translation of both xWT1 pseudoalleles. Injection of those MOs
specifically blocked the translation in vitro and in vivo of its target mRNA
(Fig. 4A; data not shown).
Embryos injected with those xWT1 MOs were analysed by in situ hybridization
with XHRT1 and other pronephric markers. As expected, knockdown of
xWT1 abolished the expression of nephrin, a marker of glomerular
podocytes, which is directly activated in mice by WT1
(Wagner et al., 2004;
Guo et al., 2004;
Gerth et al., 2005) (100%
inhibited, n=199; Fig.
4B,C). Expression of the XSMP-30 proximal tubule (82%
unaffected, n=72) and the Pax8 (83% unaffected,
n=24) and Evi1 (90% unaffected, n=20) early
pronephric markers was not affected (Fig.
4D-K). Interestingly, knockdown of xWT1 decreased the early
glomus-specific expression of XHRT1 (48% inhibited, n=125)
but did not perturb its late expression in the pronephros anlagen (80%
unaffected, n=35) (Fig.
4L-O). Together, these experiments are consistent with the idea
that xWT1, which promotes glomus formation, may play a role in XHRT1
early expression. They also suggest that there may be factors other than xWT1
that repress the expression of lateral-specific genes in the developing
glomus.
|
|
).
Embryos were injected with the different hGR constructs mixed with
ß-galactosidase mRNA as a lineage tracer. In the injected embryos, we
examined how the expression of those bHLH-O genes affects pronephric gene
markers. We first examined the ability of XHRT1, which is the earliest and
strongest bHLH-O gene expressed in the pronephric primordium, to inhibit
distal tubule and duct cell fates. Addition of dexamethasone at the end of
neurulation (stage 18) resulted in a hormone-dependent inhibition at early
tadpole stage of the formation of the distal tubule and duct as revealed by
the Evi1 marker (94% inhibited, n=32)
(Van Campenhout et al., 2006).
This phenotype is similar to that previously described upon activation of
Notch signaling using a hGR/Su(H)/Ank construct (McLaughlin et al., 2000)
(Fig. 5A-F). Similar results
were observed using the xClC-K marker (data not shown). The same
experiments were repeated for esr9, esr10 and Xhairy2b. However, unlike XHRT1,
although effective in inhibiting primary neurogenesis, esr9, esr10 and
Xhairy2b did not inhibit Evi1 expression (unaffected: 72%,
n=21; 76%, n=33; 88%, n=40; for esr9, esr10 and
Xhairy2b, respectively; Fig.
5G-O). XHes2, another bHLH-O repressor, which is not found in the
pronephros (M.S., unpublished), was also inefficient at repressing
Evi1 (80% unaffected, n=30)
(Fig. 5P-R). It has been
suggested that XHRT1 may participate in regulating aspects of gene expression
that are linked to cell-cycle control and apoptosis
(Wang et al., 2003b;
Huang et al., 2004). The
inhibition of distal tubule and duct formation could thus be explained by a
decrease of proliferation or the apoptotic elimination of pronephric cells.
However, immunostaining using an antibody recognizing phosphorylated histone
H3, and TUNEL analysis of injected embryos, revealed no change in the pattern
of mitotic and apoptotic cells (data not shown). We therefore conclude that
activation of XHRT1, but not that of esr9, esr10 or
Xhairy2b, may play a crucial role during pronephros formation in the
inhibition of distal tubule and duct cell fates.
|
To further determine the importance of XHRT1 as a mediator of Notch signaling in the pronephros, we co-injected embryos with mRNA encoding Su(H)DBM together with XHRT1-MT-hGR mRNA. Injected embryos were induced at stage 22 and assayed for the expression of Evi1 and XSMP-30. We observed that early inhibition of Notch signaling in untreated embryos reduces the expression of the proximal tubule marker XSMP-30 and, as previously reported, elevates the expression of the distal tubule and duct marker Evi1 (Fig. 7A-D,I). By contrast, in dexamethasone-treated embryos, we observed a reduction of both XSMP-30 and Evi1 expression (Fig. 7E-H,I). Thus, XHRT1 could reverse the effect of Su(H)DBM on Evi1 but is not sufficient to restore the expression of XSMP-30. Together, these results indicate that XHRT1 functions as an important downstream effector of Notch signaling. Compared with the other bHLH-O repressors, it appears to play a specific role in early pronephros development, contributing to the inhibition of distal tubule and duct cell fates in cells that form the glomus and the proximal tubules.
XHRT1 depletion reduces the expression of proximal tubule and glomus markers
To determine whether XHRT1 is required for glomus and proximal tubule
development, we generated a MO that targets a 100% conserved 25 bp stretch,
including the AUG initiation codon, in the two XHRT1 pseudoalleles.
In vivo and in vitro, the XHRT1-MO specifically and efficiently blocks the
translation of the corresponding mRNA (Fig.
8A). XHRT1-MO-injected embryos displayed a decrease in the
expression of all proximal tubule-specific markers tested [including
XSMP-30 (72%, n=59), xPDZK1 (88%, n=17)
and xSat1 (68%, n=15)] and downregulated the expression of
xWT1 (77%, n=40) and nephrin (88% inhibited,
n=17; Fig. 8B-I; data
not shown). Transverse sections revealed that the glomus was present and that
in many injected embryos, the proximal tubules were reduced in size
(Fig. 8J,K). These effects are
not due to a change in the pattern of mitotic and apoptotic cells, as revealed
by analysis of the injected embryos by phosphorylated histone H3
immunostaining and TUNEL (data not shown). Expression of the distal tubule and
duct markers Evi1 and xClC-K appears unaffected
(Evi1 64%, n=37; xClC-K 72%, n=11;
Fig. 8L,M; data not shown).
Expression of other markers, such as Ep. keratin, and
N-tubulin, was also unaltered (Ep. Keratin, none inhibited,
n=27; N-tubulin, none inhibited, n=25;
Fig. 8N,O). Injection of an MO
designated against esr9, esr10 or xHairy2b that efficiently
inhibits their target mRNA did not affect the expression of any of the
pronephric genes tested (see Fig. S1 and S2 in the supplementary material),
which further supports the idea that XHRT1 has a specific function in the
partitioning of the pronephros anlagen.
|
The specific activity of XHRT1 is conferred by its C-terminal region
Our results support the idea that, compared with the other bHLH-O
repressors tested, XHRT1 has a specific function in the developing pronephros.
To identify the region(s) that are required for its activity, we performed
domain-swapping experiments between XHRT1 and one of the bHLH-O repressors
inactive in the pronephros. We chose the novel bHLH-O gene XHes2
because both genes are expressed in the retina where XHes2 but not XHRT1
promotes Müller glial development
(Satow et al., 2001) (M.S.,
unpublished), which may provide another assay to identify the regions required
for their distinct regulatory functions. As we and others have previously
shown that the bHLH and the Orange domains of XHRT1 are required for efficient
DNA-binding and homo- and heterodimerization
(Taelman et al., 2004), we
decided to keep the bHLH-O regions of both proteins intact and swap their
C-terminal sequences (Fig. 9A).
Both chimeric molecules were linked to glucocorticoid receptor ligand-binding
domain sequences to generate inducible constructs. Injected embryos were
induced at stage 18 and assayed for the expression of Evi1. All
constructs were effectively translated when expressed in embryos
(Fig. 9B). As shown in
Fig. 9C, only the hybrid
containing the XHRT1 C-terminal domain fused to the XHes2 bHLH-O domain
reduced xEvi1 expression to a similar extent as the wild-type XHRT1
protein. Thus, the specific properties of XHRT1 in the pronephros appear to be
linked to its C-terminal region.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
; Chen and Al-Awqati,
2005). We found that in Xenopus, XHRT1 is expressed early
in the developing pronephros region in the pronephric mesoderm that will give
rise to the glomus. X-Delta-1 is the only Notch ligand to be
expressed at those early stages in the pronephric region. It is expressed in
cells of the lateral layer in close contact with XHRT1-expressing
cells, suggesting that it may trigger XHRT1 activation in the
surrounding cells. xWT1 is another potential regulator of
XHRT1, as both genes are activated at about the same time in the
forming glomus and its translational inhibition decreased XHRT1
expression. Interestingly, we observed that the expression of lateral specific
genes is not expanded in the xWT1 morphants, suggesting that xWT1 is not the
only factor that controls mediolateral patterning of the pronephros. Later on,
expression of XHRT1 in the medial layer is downregulated and strong
staining is detected transiently in the dorsoanterior proximal-tubule forming
part of the pronephros anlagen itself. Thus, both in mouse and in Xenopus,
HRT1 is early and selectively expressed in proximal compartments of the
developing nephron, suggesting an early role in glomus and proximal tubule
formation. At early tadpole stage, we observed that XHRT1 expression
is restricted to the tip of the forming tubules, suggesting another later role
in the subcompartmentalization of the tubules.
|
;
Chen and Al-Awqati, 2005).
Similarly in Xenopus, Xhairy1 and Xhairy2b are initially
expressed in both pronephric mesodermal layers, suggesting that they may have
early a broader role than XHRT1. In contrast to the other bHLH-O
genes, Xhairy2b expression is maintained in late tadpoles. Its
expression at that stage resembles that of the Notch ligand
X-Serrate-1. As recent investigations have demonstrated that the
Notch ligands not only deliver their signal by binding to Notch receptors, but
also by playing a functional role in the cells in which they are expressed
(Ascano et al., 2003;
LaVoie and Selkoe, 2003;
Ikeuchi and Sisodia, 2003), it
is tempting to speculate that X-Serrate-1 may play a role in Xhairy2b
activation.
|
;
Chen and Al-Awqati, 2005).
Similarly, in Xenopus, esr9 and esr10 expression are
detected in the pronephros anlagen of late tailbud embryos in a region
slightly more ventral to XHRT1 expression. At that stage, expression
of esr9 and esr10 resembles that of the Notch ligands
X-Serrate-1 and X-Serrate-2, suggesting that they may be
involved in their regulation. Further experiments are needed to analyse the
contribution of the distinct Notch ligands to the spatiotemporal regulation of
bHLH-O genes in the pronephros.
Role of Notch signaling and downstream bHLH-O targets in the specification of glomus and proximal tubules within the pronephros anlagen
In the Xenopus pronephros and the mouse metanephroi, Notch
activation has been shown to be essential for proximal tubule and glomus
formation (McLaughlin et al., 2000;
McCright et al., 2001;
Wang et al., 2003a;
Cheng et al., 2003;
Cheng and Kopan, 2005;
Van Campenhout et al., 2006).
Here, we show that activation of Notch in the pronephric primordium favors
first glomus and later proximal tubule fates, which correlates with the
temporal expression pattern of XHRT1, esr9 and esr10. To the best of our
knowledge, this is the first time that the precise temporal dependence on
Notch activation of those two processes has been investigated. We observed
that overexpression and the morpholino knockdown of XHRT1, but not that of
esr9, esr10 or Xhairy2b, phenocopy the defects observed upon activation and
inhibition of Notch signaling. These results indicate that XHRT1 may act to
repress distal tubule and duct cell fates in the portion of pronephric
mesoderm fated to form the glomus and the proximal tubules. They also suggest
that XHRT1 has a role distinct to that of the other bHLH-O repressors in the
earliest stage of pronephros development. However, we cannot exclude that
these finding may arise from a reduced efficiency of the corresponding MOs.
Kidneys from mice where the Hes1 or Hes5 genes were deleted
show no defects, whereas compound homozygotes for both Hes1 and
Hes5 die before kidney development
(Chen and Al-Awqati, 2005).
Further evaluation of the targeted disruption of these genes in conditional
knockout mice is required to determine their contribution in nephron
patterning.
In contrast to Notch activation using an inducible form of an activated
Su(H) construct, XHRT1 overexpression does not increase
proximal tubule/glomus formation. We also observed that XHRT1 does not restore
the expression of proximal tubule markers in embryos where Notch has been
inhibited by injection of a Su(H)DBM construct, suggesting that it
mediates only part of the effects executed by Notch. xWT1 is another
transcriptional repressor that has also been suggested to have a role in the
repression of tubule and duct specific genes in the forming glomus
(Wallingford et al., 1998;
Van Campenhout et al., 2006).
Further investigations are required to elucidate the hierarchical relationship
that links XHRT1 and xWT1, and to identify other factors that may contribute
to glomus/proximal tubule cell fate decisions.
In HRT1 single mutant or in HRT1/HRT2 double
mutant mice, no kidney defects have been reported
(Fisher et al., 2004;
Kokubo et al., 2005). The
difference in phenotype between Xenopus and mouse may be due to
differential evolution or expression of the HRT genes in both species.
Differential evolution of the HRT2 gene has been recently reported in
fish (Winkler et al., 2003).
In the mouse, HRT1 and HRT3, but not HRT2, have
overlapping expression in the developing nephrons
(Leimeister et al., 2003). The
identification of the functional role of HRT1 in mouse nephrogenesis
will require the analysis of the phenotype through nephrogenesis of
HRT1/HRT3 double knockout mice.
XHRT1 specific function in the pronephros is dependent on its C-terminal sequences
Swapping experiments between XHRT1 and the related bHLH-O XHes2 that is
inactive in the pronephros have shown that the specific properties of XHRT1
are dependent on their divergent C-terminal sequences. Whereas the XHes2
protein has a C-terminal domain of 62 amino acids terminated by a WRPW motif,
the XHRT1 C-terminal domain is much longer (133 amino acids) and does not
contain the WRPW motif; this is replaced by a related sequence (YRPW) near its
C terminus. In E(spl) in Drosophila, this region has also been shown
to be important, as mutants that lack the sequences C terminal to the Orange
domain act as dominant-negative variants
(Giebel and Campos-Ortega,
1997). In zebrafish Her4, the Orange domain-WRPW interval is also
essential for its ability to block neurogenesis
(Takke et al., 1999). At
present, the functional role of the bHLH-O C-terminal sequences is unclear. In
XHRT1, those sequences are involved, together with the bHLH and Orange
domains, in dimerization and selection of the bHLH partners, and they possess
intrinsic repression activity (Taelman et
al., 2004). In HES1, the C-terminal domain allows interaction with
the Runt-related protein CbFa1 (McLarren
et al., 2000). Further studies are needed to clarify the role of
the C-terminal sequences in XHRT1-specific function.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/15/2961/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ascano, J. M., Beverly, L. J. and Capobianco, A. J.
(2003). The C-terminal PDZligand of Jagged1 is esential for
cellular transformation. J. Biol. Chem.
278,8771
-8779.
Brändli, A. W. (1999). Towards a molecular anatomy of the Xenopus pronephric kidney. Int. J. Dev. Biol. 43,381 -395.[Medline]
Brennan, H. C., Nijjar, S. and Jones, E. A. (1998). The specification of the pronephric tubules and duct in Xenopus laevis. Mech. Dev. 75,127 -137.[CrossRef][Medline]
Brennan, H. C., Nijjar, S. and Jones, E. A. (1999). The specification and growth factor inducibility of the pronephric glomus in Xenopus laevis. Development 126,5847 -5856.[Abstract]
Carroll, T. and Vize, P. D. (1996). Wilm's tumor suppressor gene is involved in the development of disparate kidney forms: evidence from expression in the Xenopus pronephros. Dev. Dyn. 206,131 -138.[CrossRef][Medline]
Carroll, T. and Vize, P. D. (1999). Synergism between Pax8 and lim-1 in embryonic kidney development. Dev. Biol. 214,46 -59.[CrossRef][Medline]
Chan, T.-C., Takahashi, S. and Asashima, M. (2000). A role for Xlim1 in pronephros development in Xenopus laevis. Dev. Biol. 228,256 -269.[CrossRef][Medline]
Chen, L. and Al-Awqati, Q. (2005). Segmental expression of Notch and Hairy genes in nephrogenesis. Am. J. Physiol. 288,939 -952.
Cheng, H. T. and Kopan, R. (2005). The role of Notch signaling in specification of podocyte and proximal tubules within the developing mouse kidney. Kidney Int. 68,1951 -1952.[CrossRef][Medline]
Cheng, H. T., Miner, J. H., Lin, M., Tansey, M. G., Roth, K. and
Kopan, R. (2003). 
-Secretase activity is dispensable
for mesenchyme-to-epithelium transition but required for podocyte and proximal
tubule formation in developing mouse kidney.
Development 130,5031
-5042.
Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. and Kintner, C. (1995). Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. Nature 375,761 -766.[CrossRef][Medline]
Deblandre, G. A., Wettstein, D. A., Koyano-Nakagawa, N. and Kintner, C. (1999). A two step mechanism generates the spacing pattern of the ciliated cells in the skin of Xenopus embryos. Development 126,4715 -4728.[Abstract]
Fisher, A., Schumacher, N., Maier, M., Sentner, M. and Gessler,
M. (2004). The Notch target genes Hey1 and Hey2 are required
for embryonic vascular development. Genes Dev.
18,901
-911.
Gerth, V. E., Zhou, X. and Vize, P. D. (2005). Nephrin expression and threedimensional morphogenesis of the Xenopus pronephric glomus. Dev. Dyn. 233,1131 -1139.[CrossRef][Medline]
Giebel, B. and Campos-Ortega, J. A. (1997).
Functional dissection of the Drosophila Enhancer of split protein, a
suppressor of neurogenesis. Proc. Natl. Acad. Sci. USA
94,6250
-6254.
Guo, G., Morrison, D. J., Licht, J. D. and Quaggin, S. E.
(2004). WT1 activates a glomerular-specific enhancer identified
from the human nephrin gene. J. Am. Soc. Nephrol.
15,2851
-2856.
Harland, R. M. (1991). In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36,685 -695.[Medline]
Heller, N. and Brandli, A. W. (1999). Xenopus Pax-2/5/8 orthologues: novel insights into Pax gene evolution and identification of Pax-8 as the earliest marker for otic and pronephric cell lineages. Dev. Genet. 24,208 -219.[CrossRef][Medline]
Huang, Q., Raya, A., DeJesus, P., Chao, S.-H., Quon, K. C.,
Caldwell, J. S., Chanda, S. K., Izpisua-Belmonte, J. C. and Schultz, P.
(2004). Identification of p53 regulators by genome-wide
functional analysis. Proc. Natl. Acad. Sci. USA
101,3456
-3461.
Ikeuchi, T. and Sisodia, S. (2003). The Notch
ligands, Delta1 and Jagged2, are substrates for presenilin-dependent
"-secretase" cleavage. J. Biol.
Chem. 278,7751
-7754.
Iso, T., Kedes, L. and Hamamori, Y. (2003). HES and HERP families: multiple effectors of the Notch signaling pathway. J. Cell. Physiol. 194,237 -255.[CrossRef][Medline]
James, R. G. and Schultheiss, T. M. (2005). BMP signaling promotes intermediate mesoderm gene expresssion in a dose-dependent, cellautonomous and translation-dependent manner. Dev. Biol. 288,113 -125.[CrossRef][Medline]
Jen, W. C., Gawantka, V., Pollet, N., Niehrs, C. and Kintner, C. (1999). Periodic repression of Notch pathway governs the segmentation of Xenopus embryos. Genes Dev. 13,486 -499.
Jonas, E., Sargent, T. D. and Dawid, I. B.
(1985). Epidermal keratin gene expressed in embryos of Xenopus
laevis. Proc. Natl. Acad. Sci. USA
82,5413
-5417.
Jones, E. A. (2005). Xenopus: a prince among
models for pronephric kidney development. J. Am. Soc.
Nephrol. 16,313
-321.
Kiyota, T., Jono, H., Kuriyama, S., Hasegawa, K., Miyatani, S. and Kinoshita, T. (2001). X-Serrate-1 is involved in primary neurogenesis in Xenopus laevis in a complementary manner with X-Delta-1. Dev. Genes Evol. 211,367 -376.[CrossRef][Medline]
Kokubo, H., Miyagawa-Tomita, S., Nakazawa, M., Saga, Y. and Johnson, R. L. (2005). Mouse hers1 and hesr2 genes are redundantly required to mediate Notch signaling in the developing cardiovascular system. Dev. Biol. 278,301 -309.[CrossRef][Medline]
Koyano-Nakagawa, N., Kim, J., Anderson, D. and Kintner, C. (2000). Hes6 acts in a positive feedback loop with the neurogenins to promote neuronal differentiation. Development 127,4203 -4216.[Abstract]
LaVoie, M. J. and Selkoe, D. J. (2003). The
Notch ligands, Jagged and Delta, are sequentially processed by
-secretase and presenilin/-secretase and release signaling
fragments. J. Biol. Chem.
278,34427
-34437.
Leimeister, C., Schumacher, N. and Gessler, M. (2003). Expression of Notch pathway genes in the embryonic mouse metanephros suggests a role in proximal tubule development. Gene Expr. Patterns 3,595 -598.[CrossRef][Medline]
Li, Y., Fenger, U., Niehrs, C. and Pollet, N. (2003). Cyclic expression of esr9 gene in Xenopus presomitic mesoderm. Differentiation 71, 83-89.[CrossRef][Medline]
Mauch, T. J., Yang, G., Wright, M., Smith, D. and Schoenwolf, G. C. (2000). Signals from the trunk paraxial mesoderm induce pronephros formation in chick intermediate mesoderm. Dev. Biol. 220,62 -75.[CrossRef][Medline]
McCright, B., Gao, X., Shen, L. Y., Lozier, J., Lan, Y., Maguire, M., Herzlinger, D., Weinmaster, G., Jiang, R. L. and Gridley, T. (2001). Defects in development of the kidney, heart and eye vasculature in mice homozygous for a hypomorphic Notch2 mutation. Development 128,491 -502.[Abstract]
McLarren, K. W., Lo, R., Grbavec, D., Thirunavukkarasu, K.,
Karsenty, G. and Stifani, S. (2000). The mammalian basic
helix-loop-helix protein HES-1 binds to and modulates the transactivating
function of the runt-related factor Cbfa1. J. Biol.
Chem. 275,530
-538.
McLaughin, K. A., Rones, M. S. and Mercola, M. (2000). Notch regulates cell fate in the developing pronephros. Dev. Biol. 227,567 -580.[CrossRef][Medline]
Nieuwkoop, P. D. and Faber, J. (1967). Normal Table of Xenopus laevis (Daudin). Amsterdam: North Holland.
Obara-Ishihara, T., Kuhlman, T., Niswander, L. and Herzlinger, D. (1999). The surface ectoderm is essential for nephric duct formation in intermediate mesoderm. Development 126,1103 -1108.[Abstract]
Pichon, B., Taelman, V., Kricha, S., Christophe, D. and Bellefroid, E. J. (2002). XHRT-1, a hairy and Enhancer of split related gene with expression in floor plate and hypochord during early Xenopus embryogenesis. Dev. Genes Evol. 212,491 -495.[CrossRef][Medline]
Pichon, B., Taelman, V., Bellefroid, E. J. and Christophe, D. (2004). Transcriptional repression by the bHLH-Orange factor XHRT1 does not involve the C-terminal YRPW motif. Biochim. Biophys. Acta 1680,46 -52.[Medline]
Piscione, T. D., Wu, M. Y. J. and Quaggin, S. E. (2004). Expression of Hairy/Enhancer of Split genes, Hes1 and Hes5, during murine nephron morphogenesis. Gene Expr. Patterns 4,707 -711.[CrossRef][Medline]
Rones, M. S., Woda, J., Mercola, M. and McLaughlin, K. A. (2002). Isolation and characterization of Xenopus Hey-1: a downstream mediator of Notch signaling. Dev. Dyn. 225,554 -560.[CrossRef][Medline]
Ryffel, G. U. (2003). What can a frog tell us about kidney development. Nephron Exp. Nephrol. 94, 35-43.[CrossRef]
Sato, A., Asashima, M., Yokota, T. and Nishinakamura, R. (2000). Cloning and expression pattern of a Xenopus pronephros-specific gene, XSMP-30. Mech. Dev. 92,273 -275.[CrossRef][Medline]
Satow, T., Bae, S.-K., Inoue, T., Miyoshi, G., Tomita, K.,
Bessho, Y., Hashimoto, N. and Kageyama, R. (2001). The basic
helix-loop-helix gene hesr2 promotes gliogenesis in mouse retina.
J. Neurosci. 21,1265
-1273.
Seufert, D. W., Brennan, H. C., DeGuire, J., Jones, E. A. and Vize, P. D. (1999). Developmental basis of pronephric defects in xenopus body plan phenotypes. Dev. Biol. 215,233 -242.[CrossRef][Medline]
Taelman, V., Van Wayenbergh, R., Sölter, M., Pichon, B., Pieler, T., Christophe, D. and Bellefroid, E. J. (2004). Sequences downstream of the bHLH domain of the Xenopus hairy-related transcription factor-1 act as an extended dimerization domain that contributes to the selection of the partners. Dev. Biol. 276, 47-63.[CrossRef][Medline]
Takke, C., Dornseifer, P., Weizcäcker, E. V. and Campos-Ortega, J. A. (1999). her4, a zebrafish homologue of the Drosophila neurogenic gene E(spl), is a target of Notch signaling. Development 126,1811 -1821.[Abstract]
Tsuji, S., Cho, K. W. and Hashimoto, C. (2003). Expression of a basic helix-loop-helix transcription factor Xhairy2b during Xenopus laevis development. Dev. Genes Evol. 213,407 -411.[CrossRef][Medline]
Van Campenhout, C., Nichane, M., Antoniou, A., Pendeville, H., Bronchain, O. J., Marine, J. C., Mazabraud, A., Voz, M. L. and Bellefroid, E. J. (2006). Evi1 is expressed specifically in the distal tubule and duct of the Xenopus pronephros and plays a role in its formation. Dev. Biol. 294,203 -219.[CrossRef][Medline]
Vize, P. D. (2003). The chloride conductance channel ClC-K is a specific marker for the Xenopus pronephric distal tubule and duct. Gene Expr. Patterns 3, 347-350.[CrossRef][Medline]
Vize, P. D., Woolf, A. S. and Bard, J. B. L. (2003). The Kidney. From Normal Development to Congenital Disease. Amsterdam: Academic Press.
Wagner, N., Wagner, K. D., Xing, Y., Scholz, H. and Schedl,
A. (2004). The major podocyte protein nephrin is
transcriptionally activated by the Wilms' tumor suppressor WT1. J.
Am. Soc. Nephrol. 15,3044
-3051.
Wallingford, J. B., Carroll, T. J. and Vize, P. D. (1998). Precocious expression of the Wilm's tumor gene xWT1 inhibits embryonic kidney development in Xenopus laevis. Dev. Biol. 202,103 -112.[CrossRef][Medline]
Wang, P., Pereira, F. A., Beasley, D. and Zheng, H.
(2003a). Preselinins are required for the formation of comma- and
S-shaped bodies during nephrogenesis. Development
130,5019
-5029.
Wang, W., Prince, C. Z., Hu, X. and Pollman, M. J. (2003b). HRT1 modulates vascular smooth cell proliferation and apoptosis. Biochem. Biophys. Res. Commun. 308,596 -601.[CrossRef][Medline]
Wettstein, D. A., Turner, D. L. and Kintner, C. (1997). The Xenopus homolog od Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis. Development 124,693 -702.[Abstract]
Winkler, C., Elmasri, H., Klamt, B., Nicolas, J. and Gessler, M. (2003). Characterization of hey bHLH genes in teleost fish. Dev. Genes Evol. 213,541 -553.[CrossRef][Medline]
Related articles in Development:
