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First published online 21 June 2006
doi: 10.1242/dev.02442
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1 Molecular and Vascular Medicine Unit, Beth Israel Deaconess Medical Center,
330 Brookline Ave, RW-663, Boston, MA 02215, USA.
2 Program in Biological and Biomedical Sciences, Harvard Medical School, Boston,
MA, USA.
3 Center for Oral Biology and Department of Biomedical Genetics, University of
Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Author for correspondence (e-mail:
tschulth{at}bidmc.harvard.edu)
Accepted 15 May 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Kidney, Metanephros, Mesonephros, Chick embryo, Mouse, Osr1, Odd1
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In amniotes,
the IM gives rise to three types of kidney tissue, called (from anterior to
posterior) the pronephros (a transient embryonic structure), the mesonephros
(the functional embryonic kidney, which also contributes to the male
reproductive system) and the metanephros (the definitive adult kidney). While
all kidney types are comprised of the same fundamental functional unit (the
nephron), the size and organization of these kidney types is very different:
the mouse metanephros can contain up to 11,000 nephrons
(Yuan et al., 2002), while the
mesonephros has merely twelve (Sainio,
2003).
Kidney formation can be conceptualized as consisting of two stages:
establishment of nephrogenic mesenchyme within the IM, and differentiation of
that mesenchyme into functional nephrons. Through numerous studies over many
years, much has been learned concerning the second stage: differentiation of
nephrogenic mesenchyme. In the mouse metanephros, which is the best studied
example, kidney tissue differentiates as the result of interaction between the
metanephric mesenchyme, which is derived from the most posterior region of the
IM, and the nephric duct, another IM derivative that migrates into the
metanephric zone from a more anterior region of the embryo
(Sariola and Sainio, 1997;
Schultheiss et al., 2003;
Vainio and Muller, 1997).
Reciprocal signaling between the metanephric mesenchyme and a derivative of
the nephric duct known as the ureteric bud results in branching of the
ureteric bud and condensation of metanephric mesenchyme at its tips. The
condensed mesenchyme is thought to form a precursor cell population, which
both maintains itself at the tips of the ureteric bud (via proliferation
and/or addition from the surrounding non-condensed mesenchyme) and gives off
cells that differentiate into pretubular aggregates and renal vesicles, the
precursors of the kidney tubules (Cho and
Dressler, 2003).
The mechanisms that regulate the earlier phase of kidney development -
formation of nephrogenic mesenchyme - are less understood. Multiple genes have
been identified that are expressed specifically in the undifferentiated
metanephric mesenchyme; many of these are required for proper differentiation
of the metanephric kidney, including Pax2
(Torres et al., 1995),
Wt1 (Kreidberg et al.,
1993), Eya1 (Xu et
al., 1999), Six1 (Xu
et al., 2003), the Hox11 paralogous group
(Wellik et al., 2002) and
N-myc (Mycn - Mouse Genome Informatics)
(Bates et al., 2000). However,
with the exception of Eya1, all of the above genes are dispensable
for the initial formation of the metanephric mesenchyme, and are instead
required for its subsequent differentiation and/or survival.
The current study characterizes the role during kidney formation of odd-skipped related 1 (Odd1)(Osr1 - Mouse Genome Informatics), a zinc finger-containing transcription factor related to the Drosophila pair rule gene odd skipped (Odd). Odd1 is expressed before all previously described kidney regulatory genes, and its expression is confined to undifferentiated kidney precursor tissue. Loss-of-function studies in the mouse revealed a requirement for Odd1 in the establishment of the metanephric mesenchyme and in the activation of a set of the earliest known genes expressed during metanephric kidney formation. Gain-of-function studies in the chick embryo revealed that Odd1 can activate early markers of kidney precursor cells but inhibits the production of differentiated kidney tubules. On the basis of these studies, we propose that Odd1 plays an important role in the establishment and maintenance of the nephrogenic mesenchyme, the precursor population from which the kidney is derived.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
Pax2 (Burrill et al.,
1997; Herbrand et al.,
1998), Lim1 (Tsuchida
et al., 1994) and mouse Pax2
(Dressler et al., 1990),
Lim1 (Fujii et al.,
1994), Eya1 (Xu et
al., 1999), Six2 (Xu
et al., 1999), Gdnf
(Pichel et al., 1996),
Sall1 (Nishinakamura et al.,
2001) and Wt1
(Armstrong et al., 1993) using
standard methods described previously
(Schultheiss et al., 1995).
Whole-mount in situ hybridization was performed as described previously
(Schultheiss et al., 1995).
For in situ hybridization on sections, paraformaldehyde-fixed cryostat
sections were post-fixed, treated with 10 µg/ml proteinase K (Roche), and
hybridized overnight with labeled RNA probe (1 µg/ml) at 70°C. The
sections were then washed in 2x SSC (70°C), incubated with RNAse (1
µg/ml, 2x SSC, 37°C) and washed in 0.2x SSC (65°C).
After blocking in PBT (PBS with 2 mg/ml BSA, 0.1% Triton X-100), the sections
were incubated in alkaline phosphatase antibody solution (Roche, 1:2000, in
PBT + 10% sheep serum) overnight. Lastly, sections were washed and the color
was visualized using NBT and BCIP.
Immunohistochemistry and lacZ histochemistry
Immunofluorescence microscopy on paraformaldehyde-fixed cryostat sections
was performed as previously described
(James and Schultheiss, 2003).
The following primary antibodies were used: polyclonal guinea pig anti-Odd1
[1:750 (James and Schultheiss,
2005)], rabbit anti-Pax2 (BabCo, 1:250), mouse monoclonal and
rabbit polyclonal anti-Gfp (1:100, Molecular Probes) and mouse monoclonal
anti-Wt1 (1:100, Dako). Apoptotic cells were detected by TUNEL assay (Sigma)
as per the manufacturer's instructions. Staining for lacZ expression
was adapted from Lobe et al. (Lobe et al.,
1999).
Odd1 knockout mice
Generation of mice carrying a targeted in-frame fusion of a lacZ
gene into the first coding exon of the Odd1 gene has been described
(Wang et al., 2005). Embryos
were harvested at the indicated times and processed for in situ hybridization,
immunofluorescence or lacZ histochemistry as described above.
Plasmid and retroviral expression vectors
For electroporation studies, full-length chicken Odd1
(James and Schultheiss, 2005)
was cloned into the pMES expression vector
(Swartz et al., 2001), which
drives expression from a CMV/chicken ß-actin promoter/enhancer, and which
also expresses Gfp from an IRES element. For retroviral expression
studies, full-length Odd1 was cloned into the ClaI site of
the RCAS expression vector
(Fekete and Cepko, 1993). The
resulting RCAS-Odd1 vector was transfected into chick embryo
fibroblasts, and retroviral particles were harvested from culture supernatant,
concentrated and titered using previously described methods
(Fekete and Cepko, 1993).
RCAS-Gfp retroviral particles for control experiments were produced
in a similar manner (RCAS-Gfp plasmid was a kind gift from Cliff
Tabin).
Electroporation and infection of chicken embryos
Electroporation and culture of chicken embryos was performed as previously
described (James and Schultheiss,
2003; Wilm et al.,
2004). Briefly, embryos were collected at Stage 5 or younger
(Hamburger and Hamilton, 1951)
and attached to a doughnut-shaped paper ring (P5, Fisher). The embryo was
suspended in Tyrode's saline, ventral side down, above a 1 mm gauge platinum
wire (positive electrode). Through a small hole in the vitelline membrane, 1
µl of DNA solution (0.6 µg/µl) was injected into the space between
the embryo and the membrane. A 20 µm gauge tungsten wire (negative
electrode) was lowered above the embryo until it was submerged in Tyrode's and
the embryos were pulsed three times for 25 ms at 12 V using an electro-square
porator BTX-830 (BTX). Subsequently, embryos were incubated endoderm-side up
on albumin-agar culture dishes [50% Albumin, 1.5% Glucose, 0.9% NaCl
(Sundin and Eichele, 1992)] at
38°C for 24-48 hours.
Methods for infecting unincubated chicken eggs with retroviral vectors were
adapted from previous work (Andacht et al.,
2004; Sang, 2004).
Unincubated fertilized white-leghorn chicken eggs, obtained from Hy-Line
International (Elizabethtown, PA), were placed on their sides at room
temperature for 2 hours before microinjection. A small hole (approximately 5
mm in diameter) was ground into the shell using a blunt needle (cat. # 08-965A
Fisher), while leaving the underlying membrane intact. A drop of Tyrode's
saline was placed on top of the shell membrane, which was subsequently removed
using a microscalpel. In order to prevent air from entering the egg, Tyrode's
was continuously added to the hole; any eggs that developed air bubbles were
discarded. After locating the embryo, a pulled glass capillary (100-200 µm
in diameter), Picospritzer II injector and Leitz micromanipulator were used to
inject 1-2 µl of retrovirus (107-108 infectious
particles/ml) through the center of the embryo into the subgerminal space. The
hole was then sealed with hot glue from a Superbonder glue gun (FPC). The glue
was allowed to harden for 5 minutes, and the eggs were incubated glue-side
down at 38°C until they were harvested for analysis.
Quantification of mesonephros, tubule and duct size
For each embryo, two images were collected using a Zeiss Axiophot
microscope with a SPOT camera: (1) 10x DIC image of the entire section;
and (2) a 20x fluorescent image of the urogenital ridge stained with
anti-Pax2 antibody. For standardization, the ImageJ software package was used
to calculate the two-dimensional area of the notochord from the first image.
The freehand drawing tool was used to trace a line that encircled the
notochord borders, and the measure command was executed to calculate its area
in pixels. To calculate the total area of all tubular tissue from the second
image, we calculated the area of the individual Pax2-positive epithelial
condensates (groups of cells containing polarized nuclei) and lumenized
tubules and summed them. Duct area was determined in the same manner. Finally,
to control for variability of embryo size, the size of the notochord was used
to standardize the tubular tissue and duct area measurements. The graph in
Fig. 7 shows the mean of all
embryos analyzed and the error bar represents one standard error. Differences
between the mean values were tested for statistical significance at the 0.05
level by the two-sample Student's t-test.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) just lateral to Henson's node
(Fig. 1A). Shortly thereafter,
its expression pattern broadens to include the intermediate mesoderm and the
medial lateral plate (Fig.
1B,E). By contrast, other markers of kidney differentiation, such
as Pax2, are initially expressed later (HH9,
Fig. 1C), and in a more
spatially restricted pattern (Fig.
1D) (see James and
Schultheiss, 2003; Mauch et
al., 2000; Obara-Ishihara et
al., 1999).
Cellular morphogenesis begins shortly after kidney-specific gene expression
is initiated. Analysis of sections during the early stages of kidney
morphogenesis revealed that Odd1 is expressed only in the mesenchymal
components. This is true at several different stages during the development of
the urogenital region: initially at the onset of nephric duct differentiation
(Fig. 1E), and later during
mesonephric tubule differentiation (Fig.
1F,G). Pax2 and Odd1 have complementary
expression patterns in the mesonephros: while Pax2 is detectable in
the differentiated epithelium of the nephric duct and tubules
(Mauch et al., 2000),
Odd1 is limited to small regions of mesenchyme adjacent to the
tubules (Fig. 1G).
That Odd1 is present in kidney mesenchyme, but absent in differentiated epithelium at several stages of development, suggested that Odd1 is expressed in kidney precursors and subsequently downregulated as they undergo epithelial morphogenesis. To investigate this idea further, Odd1 expression during kidney tissue differentiation was examined at single cell resolution. Immediately before nephric duct formation, Pax2 expression is initiated in the medial part of the urogenital region (Fig. 1J). Subsequently, the nephric duct rudiment is formed, as evidenced by a subset of the Pax2-expressing cells that migrate toward the ectodermal layer (Fig. 1M). Odd1 protein is detectable only in Pax2-expressing cells that have not migrated dorsally (compare Fig. 1I,J with 1L,M), indicating that Odd1 is downregulated in cells that have begun to differentiate. Similarly, in the mesonephros Odd1 expression is detectable in Pax2-expressing nephrogenic mesenchyme, but is absent in cells that have organized into tubules (Fig. 1O,P).
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). At
embryonic day (E) 9.5, lacZ is localized to the intermediate and
lateral plate mesoderm (Fig.
2A), similar to the localization of the Odd1 transcript
described in chicken embryos (Fig.
1). LacZ expression is much higher in undifferentiated
mesonephric mesenchyme than in the tubules of the mesonephric kidney
(Fig. 2B). Starting at E10.5,
the mouse metanephric kidney develops via reciprocal interaction between the
ureter and metanephric mesenchyme (reviewed by
Bard, 2003;
Saxen, 1987). Initially, the
ureteric bud forms as a thickening of the nephric duct, which bulges into the
metanephric mesenchyme primordia (Fig.
2C, left). At this stage, Odd1 is expressed in the
metanephric mesenchyme and is absent in the ureteric bud
(Fig. 2D). Shortly thereafter,
signals from the mesenchyme induce branching within the ureter
(Fig. 2C middle, and
lacZ localization Fig.
2E,F). Lastly, tubular condensates begin to epithelialize adjacent
to the branching ureteric bud trunks (yellow circles,
Fig. 2C, right). At all stages
of ureteric bud branching, lacZ is detectable only in the condensing
mesenchyme that surrounds the branching ureteric bud and not in the tubular
tissue that differentiates from this mesenchyme
(Fig. 2E-I). As in the
mesonephros, Odd1 expression is downregulated in cells that have
formed pre-tubular aggregates (arrow, Fig.
2H) and comma/S-shaped bodies (arrow,
Fig. 2I). In summary, in both
mouse and chick embryos, and during nephric duct, mesonephros and metanephros
formation, Odd1 is expressed in kidney precursor cells and
downregulated upon the initiation of epithelial differentiation.
Odd1 is required for metanephric mesenchyme formation
In order to determine whether Odd1 is required for development of
the kidney, mice were examined that carry a targeted disruption of the
Odd1 locus (Wang et al.,
2005). The majority of mice that are homozygous for loss of
Odd1 function die at E11.5 due to cardiac abnormalities
(Wang et al., 2005). At E11.5
the ureteric bud has already invaded the condensed metanephric mesenchyme in
wild-type and Odd1 heterozygous embryos, but the ureteric bud and
metanephric mesenchyme condensation are completely absent in the homozygous
mutant embryos (Fig. 3A,B).
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); Eya1
(Fig. 3E)
(Kalatzis et al., 1998;
Xu et al., 1999); Gdnf
(Moore et al., 1996;
Pichel et al., 1996;
Sanchez et al., 1996); Pax2
(Fig. 3H)
(Dressler et al., 1990;
Torres et al., 1995); and
Sall1 (Nishinakamura et al.,
2001). Embryos mutant for Odd1 completely lack expression of Six2,
Eya1, Gdnf, Pax2 and Sall1 in the metanephric region
(Fig. 3 and data not shown).
Previous reports have shown that Eya1 mutants have similar defects in
metanephric mesenchyme formation
(Sajithlal et al., 2005). The
data reported here implicate Odd1 as epistatic to Eya1 and as the earliest
expressed factor known to be required specifically for metanephric mesenchyme
formation.
Nephric duct and mesonephros defects in Odd1 mutant embryos
The ureteric bud forms as an outcropping of the nephric duct
(Saxen, 1987). As ureteric bud
defects were observed in Odd1 mutant embryos
(Fig. 3B), the earlier
development of the nephric duct was analyzed. At E9.5, Lim1, which is
required for formation of the nephric duct
(Kobayashi et al., 2005), is a
specific marker for the nephric duct primordia
(Fig. 4A,B). In Odd1
mutant embryos (Fig. 4A,B),
Lim1 is expressed, but at significantly lower levels than in
heterozygous control embryos (Fig.
4C,D). Analysis of serial sections of Odd1 mutants
demonstrated that fewer cells express Lim1 at E9.5 relative to the
number seen in control embryos (Fig.
4B,D), and whole-mount in situ hybridization for Lim1
revealed significant gaps in the Odd1 mutant nephric ducts
(Fig. 4C,D). Decreased diameter
of the nephric duct as assessed by Lim1 and Pax2 expression
was also seen at E8.5 (data not shown), indicating that defects in the nephric
duct are present from close to the initiation of nephric duct formation.
Visualization of embryos stained for Pax2 at E10.5 also showed that
the nephric duct does not migrate toward the cloaca and metanephric mesenchyme
in Odd1 mutants (Fig.
4F) as it normally does in wild-type embryos
(Fig. 4E, arrow points to
cloaca).
A small number of mesonephric tubules differentiate in Odd1
mutants (Fig. 4F, arrowhead).
Normally, two types of mesonephric tubule form in mouse embryos: anterior
tubules, which are fused to the nephric duct, and posterior tubules, which
remain separate from the duct (Sainio et
al., 1997). Only anterior mesonephric tubules form in
Odd1 mutant mice (Fig.
4F), supporting the suggestion that anterior and posterior
mesonephric tubules form via different mechanisms
(Kobayashi et al., 2005;
Sainio et al., 1997). In order
to determine whether the mesonephric tubules in Odd1 null embryos
exhibit normal patterning, immunofluorescence was used to localize the
expression of Wt1 and Pax2. Normally, the duct and the distal tubule express
only Pax2 (Fig. 4G), while the
proximal tubule coexpresses Pax2 and Wt1 (arrow
Fig. 4G). Odd1 mutant
embryos exhibited normal regionalization of Pax2 and Wt1 expression within the
duct and tubule (Fig. 4H),
although the number and size of tubules was significantly smaller than in
control embryos.
Gene expression defects precede apoptosis in Odd1 mutants
In order to determine whether apoptosis was contributing to cell loss in
the developing meso- and metanephros, TUNEL assays were performed on sectioned
mouse embryos. At E8.5, the nephric duct of Odd1 mutant embryos, as
assessed by Lim1 and Pax2 expression, is already thinner than in control
embryos, while the levels of apoptosis in the mesonephric region of mutant
embryos are similar to those observed in controls (data not shown). At E9.5,
there is some normal apoptosis at the border of the proximal tubule and the
mesonephric mesenchyme (Fig.
5A, arrowhead). Odd1 mutants exhibit significant
inappropriate apoptosis in the mesenchyme surrounding the mesonephric tubules
(compare Fig. 5A,B), indicating
that increased programmed cell death is a probable contributor to the observed
mesonephric size decreases in Odd1 mutant embryos.
|
;
Dressler et al., 1990),
Pax8 (Bouchard et al.,
2002), Eya1
(Sajithlal et al., 2005) and
Wt1 (Armstrong et al.,
1993) is initiated by E9.5 (see also
Fig. 5C,E). The levels of
apoptosis in metanephric mesenchyme precursor cells at E9.5 are similarly low
in Odd1 mutant and control embryos (compare
Fig. 5C,D). Therefore,
apoptosis begins in Odd1 mutant mice after metanephric mesenchyme
gene expression is normally initiated (see absence of mesenchymal Pax2 in
Fig. 5D and Eya1 in
Fig. 5F). This implies that
cell death cannot explain the initial metanephric mesenchyme gene expression
defects observed in Odd1 mutant embryos, and that Odd1 is
required autonomously for normal gene expression in the metanephric
mesenchyme. Apoptosis does increase in Odd1 mutant mesenchyme by
E10.5 (Fig. 5G,H), and this may
contribute to the metanephric size decreases observed in Odd1
mutants. Even after the levels of apoptosis increase in the metanephric
mesenchyme at E10.5, there are still many mesenchymal cells in the
meso/metanephric region of Odd1 mutant embryos that express
nephrogenic genes, as evidenced by expression of Wt1
(Fig. 5I,J) and the detection
of many lacZ-positive cells exhibiting intact Odd1 promoter
activity (Fig. 5K,L).
Odd1 promotes kidney precursor gene expression
As described above, Odd1 is expressed in non-epithelial kidney
precursor cells and is required for expression of many metanephric
mesenchyme-specific factors. To test if Odd1 could function to
promote expression of kidney-specific genes, an Odd1-IRES-Gfp plasmid
was electroporated into HH4 (gastrula) chick embryos, specifically targeting
kidney and somite precursors. Morphologically, ectopic Odd1 often
disrupted somite formation (8/16 embryos; see
Fig. 6B,C,E). Ectopic
Odd1 promoted expression of the kidney transcription factors Pax2
(11/14 embryos; Fig. 6A,B) and
Lim1 (4/5 embryos; Fig.
6D,E) in somitic cells within 24 hours of electroporation.
Upregulation of Pax2 and Lim1 did not occur in all cells that were
electroporated. In particular, Gfp-labeled cells present in the lateral plate
did not express Pax2 (arrows in Fig.
6B,C). This is consistent with the absence of kidney gene
expression in the medial part of the lateral plate where Odd1 is
normally expressed (Fig.
1E,I,L), and indicates that the ability of Odd1 to
promote kidney gene expression is context-dependent.
Odd1 represses kidney tubule differentiation
We have found that Odd1 is normally downregulated as intermediate
mesoderm derivatives differentiate into epithelial structures (Figs
1,
2). In order to determine
whether downregulation of Odd1 is required for normal kidney tubule
differentiation, chicken mesonephroi were infected with RCAS retrovirus
expressing Odd1, which allows for the stable ectopic expression of
Odd1. Unincubated (pre-gastrula) chicken embryos were infected with
the RCAS-Odd1 retrovirus and, after incubation for 4-5 days, adjacent
sections were stained for Odd1 by in situ hybridization or for Pax2
by immunofluorescence. For analysis, `epithelialized tubules' were defined as
Pax2-positive cells arranged around a distinct lumen, while the broader term
`tubular tissue' was defined as Pax2-positive cells either in epithelialized
tubules or in cellular aggregates with polarized nuclei.
RCAS-Gfp-infected embryos served as controls.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Xu et al., 2003),
Pax2 (Torres et al.,
1995), Gdnf (Moore et
al., 1996; Pichel et al.,
1996; Sanchez et al.,
1996), Sall1
(Nishinakamura et al., 2001),
Wt1 (Kreidberg et al.,
1993) and Wnt4
(Kispert et al., 1998;
Stark et al., 1994), all of
which show some evidence of metanephric mesenchyme formation and expression of
at least a subset of metanephric genes. Additionally, we have shown here that
Odd1 is epistatic to Eya1, which has also been shown to be
crucial for the formation of the metanephric mesenchyme
(Sajithlal et al., 2005;
Xu et al., 1999). These data
implicate Odd1 as one of the earliest regulators of formation of the
metanephric mesenchyme. These findings are consistent with the report that
rare Odd1 null embryos surviving beyond E11.5 have no morphologically
detectable metanephric kidneys (Wang et
al., 2005).
|
). However, the
nephric duct is not required for initial expression of genes in the
metanephric mesenchyme, as seen for example in the Gata3 knockout
mouse, where Pax2-expressing metanephric mesenchyme cells are found
in the metanephric zone despite failure of the nephric duct to migrate into
this region of the embryo (Grote et al.,
2006; Sainio,
2003). Thus the effects of Odd1 on nephric duct
development cannot explain the effects of Odd1 loss on early
metanephric gene expression.
Another possibility is that, in the absence of Odd1, the tissue
that would give rise to the metanephric mesenchyme undergoes apoptosis before
the onset of metanephric mesenchyme gene expression, and that such apoptosis
eliminates all metanephric mesenchyme precursor cells. Several lines of
evidence point away from this explanation. While there is apoptosis in the
metanephric region in Odd1 null mice, the apoptosis begins after
E9.5, which is after the time that the early metanephric mesenchyme markers
Pax2 and Eya1 begin to be expressed
(Fig. 5). Also, abundant cells
with Odd1 promoter activity are found in Odd1 null mice at
E10.5, after the initiation of apoptosis
(Fig. 5L), indicating that many
cells that normally express Odd1 survive in the absence of
Odd1. Many Wt1-expressing cells are present in the metanephric region
of Odd1 null mice (Fig.
5J), further indicating that cells with nephrogenic potential
exist in the absence of Odd1, even if such cells do not proceed to
express other kidney markers. Metanephric mesenchyme apoptosis also occurs in
mouse lines mutant for Pax2, Wt1, Six1 and Eya1
(Kreidberg et al., 1993;
Torres et al., 1995;
Xu et al., 1999;
Xu et al., 2003). The
apoptosis seen in the Odd1 null metanephros after E9.5 could be due
to a combination of the absence of Odd1 as well as of these other
metanephric mesenchyme genes. Taken together, these data support an essential
role for Odd1 in the initial formation of metanephric mesenchyme.
|
;
Nishinakamura et al., 2001;
Xu et al., 1999;
Xu et al., 2003). However,
Odd1 mutant nephric duct defects are less severe than those of double
mutants for Pax2 and Pax8, which have no duct at all
(Bouchard et al., 2002).
Pax2 and Pax8 are expressed in the nephric duct itself,
suggesting a cell-autonomous requirement for Pax2/8 activity in the
nephric duct. By contrast, Odd1 is expressed in nephric duct
precursors and in nephrogenic mesenchyme, but not in the nephric duct itself
(Fig. 1). Thus, the nephric
duct defects seen in Odd1 mutant mice can be attributed either to
defects in duct precursor cells, or to defects in interactions between the
duct and Odd1-expressing mesenchyme, which may be necessary for
nephric duct maintenance (Obara-Ishihara
et al., 1999). Wang et al.
(Wang et al., 2005) have
reported that the nephric duct defects are consistently more severe on the
left side than on the right, suggesting an interaction of Odd1
activity with laterality regulation pathways.
While the number of mesonephric tubules that form in Odd1 mutants
is greatly reduced compared with wild type, it is nevertheless significant
that a few anterior tubules form in Odd1 mutant mice. It has been
reported that two types of mesonephric tubules develop in mice: anterior
tubules that fuse with the nephric duct and posterior tubules that remain
unfused with the duct (Sainio et al.,
1997). In Odd1 mutants, as well as in Wt1
mutants (Sainio et al., 1997),
only the anterior, fused tubules differentiate. It has been suggested that the
anterior, fused tubules differentiate simultaneously with the nephric duct
from common precursor cells or by direct extension from the developing duct
(Hiruma and Nakamura, 2003;
Sainio et al., 1997). Our data
indicate that Odd1 function is not required for the formation of this
anterior group of pro/mesonephric tubules.
By contrast, posterior mesonephric tubules develop from a band of
Pax2-expressing nephrogenic mesenchyme adjacent to the nephric duct.
In Odd1 mutants, there is no evidence for mesenchymal Pax2 expression
or formation of this nephrogenic mesenchyme
(Fig. 4), which would explain
the severe defects in posterior mesonephric tubule formation. Mutants for
Eya1, Six1 and Sall1, which are required for metanephros
development, have normal mesonephric tubule formation
(Nishinakamura et al., 2001;
Sajithlal et al., 2005;
Xu et al., 1999;
Xu et al., 2003). This
suggests that Odd1 is required for common processes that underlie
both mesonephric and metanephric tubule formation, while genes such as
Eya1, Six1 and Sall1 are required specifically for formation
of the metanephros.
The Odd1 mutant mesonephric tubule phenotype is less severe than
that seen in Pax2 mutants, which have no mesonephric tubules
(Brophy et al., 2001;
Torres et al., 1995). However,
Pax2, unlike Odd1, is expressed in tubules themselves and in
the nephric duct (Dressler et al.,
1990). Thus, the lack of all pro/mesonephric tubules in
Pax2 mutants may reflect an autonomous requirement for Pax2
at a later stage in tubule differentiation or in duct formation.
Odd1 may promote a nephric precursor state
Odd1 can activate the transcription of Pax2 and
Lim1 when transiently expressed ectopically by electroporation in the
paraxial mesoderm (Fig. 6).
However, when Odd1 is stably expressed ectopically via a retroviral
vector, and embryos are examined after 4 to 5 days, relatively little ectopic
expression of Pax2 or Lim1 is seen. Rather, the predominant
effect of long-term ectopic expression is the inhibition of tubule formation
in the mesonephric region (Fig.
7). Odd1-expressing mesonephric cells express
Pax2 but do not adopt an epithelial morphology. Indeed,
Odd1-expressing cells are selectively excluded from epithelial
tubules (Fig. 7). One way to
reconcile the results of short-term versus longer-term ectopic expression of
Odd1 is to postulate that Odd1 promotes the establishment of
a Pax2-positive nephric precursor fate. Odd1 may be able to
both produce such a state and maintain it, thereby blocking subsequent tubule
differentiation. The low degree of ectopic Pax2 expression in the
retrovirus-infected embryos compared with the electroporated embryos could be
due to levels of expression (the RCAS retroviral vector integrates only one
copy of the gene per cell, while electroporation can introduce many gene
copies per cell) or to the absence of factors needed to maintain kidney gene
expression in regions outside the intermediate mesoderm.
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ACKNOWLEDGMENTS |
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Footnotes |
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