1 GSF, Institute of Experimental Genetics, Ingolstaedter Landstr. 1, D-85764
Neuherberg, Germany
2 GSF, Institute of Pathology, Ingolstaedter Landstr. 1, D-85764 Neuherberg,
Germany
Author for correspondence (e-mail:
hrabe{at}gsf.de)
Accepted 26 September 2002
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SUMMARY |
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Key words: Notch signalling, Gastrulation, Node formation, Notochord, Floorplate, Midline, Left-right asymmetry, Mouse
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INTRODUCTION |
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Several genes playing key roles in the LR pathway are conserved in
vertebrates. Among these genes are the TGF-ß superfamily members
Nodal and Leftb (Lefty2), which are asymmetrically
expressed during a narrow window of development in the left lateral plate
mesoderm (LPM) before morphological differences between the left and right
body halves are evident (Bisgrove et al.,
1999). In addition, both Nodal and Leftb
loss-of-function mutants die early in development and interfere with
gastrulation (Conlon et al.,
1994
; Meno et al.,
1999
). Embryos that are compound heterozygotes for a
Nodal hypomorphic and a Nodal null allele display LR
abnormalities, including randomised LR cardiac asymmetry
(Lowe et al., 2001
). Also
asymmetrically expressed in the LPM is the Drosophila bicoid-related
homeobox gene Pitx2, which is expressed predominantly in left halves
of developing organs, such as the heart and gut
(Ryan et al., 1998
).
Pitx2 null mutants show multiple abnormalities including right
isomerism of the lung and atria (Gage et
al., 1999
; Lin et al.,
1999
; Lu et al.,
1999
). The first visible morphological indication for a LR
sidedness in vertebrates is bending of the bilaterally organised, linear heart
tube to the right body side (Beddington and
Robertson, 1999
). In mammals, a twisting of the embryo along its
rostrocaudal axis follows this process
(Faisst et al., 2002
).
Subsequently, LR asymmetric morphogenesis of the visceral organs such as the
stomach and the spleen occurs.
Manipulating experiments in gastrulating embryos, for example by
extirpation of organiser cells in Xenopus or the ablation of the node
in mouse, identified the organiser or the node, respectively, as an early
inducer of laterality (Danos and Yost,
1995; Davidson et al.,
1999
). Experiments in Xenopus embryos and studies on
zebrafish mutants revealed that besides the organiser, intact axial midline
tissues such as the notochord and floorplate are required for correct LR
development (Bisgrove et al.,
2000
; Danos and Yost,
1995
; Danos and Yost,
1996
; Lohr et al.,
1998
). The existence of mouse mutants with abnormal midline tissue
and laterality defects strongly suggests that these processes may be a common
feature of vertebrates.
The breaking of the initial bilateral symmetry of vertebrates seems to be
caused by an asymmetrical signal associated with the organiser
(Fujinaga, 1997). In mammals,
for example, it has been suggested that the directional rotation of cilia in
the ventral node may generate a laminar leftward flow transporting a morphogen
(Nonaka et al., 1998
;
Okada et al., 1999
).
Interestingly, mouse mutants with immotile or absent cilia display laterality
defects reminiscent to human primary ciliary dyskinesia
(Ibanez-Tallon et al., 2002
).
Recently, it was demonstrated that an artificially generated rightward flow is
sufficient to reverse the situs of mouse embryos
(Nonaka et al., 2002
). In
Xenopus, chicken and zebrafish, such a mechanism has not yet been
identified, but the existence of cilia in the organiser suggests that they may
be required for LR asymmetry in all vertebrates
(Essner et al., 2002
).
However, it is not clear which kind of essential molecules are transported by
the nodal flow and which molecular factor triggers the expression of
Nodal, the earliest gene expressed asymmetrically in all vertebrates.
Sonic hedgehog (Shh) and Fgf8 are asymmetrically expressed
in and around the chicken organiser (Hensen's node), whereas the same genes in
mouse embryos have a symmetrical expression pattern
(Boettger et al., 1999
;
Levin et al., 1995
;
Meyers and Martin, 1999
).
However, both Shh and Fgf8 mouse mutants display numerous
LR-asymmetry abnormalities (Meyers and
Martin, 1999
; Tsukui et al.,
1999
). The identification of novel, so far unknown mutants with
impaired laterality is essential for the further understanding of LR-axis
formation in vertebrates.
Predominantly bilateral symmetric, LR differences are also present in
invertebrates. In C. elegans, for example, stereotyped cleavages of
early AB blastomere descendants lead to an invariant handedness of the
intestine (Hutter and Schnabel,
1995), which is dependent on the LIN-12/Notch-like signalling
pathway (Hermann et al.,
2000
).
In vertebrates, the evolutionarily conserved Notch-signalling pathway had
not been implicated in LR development to date. Notch signalling is thought to
act predominantly in a ligand/receptor-like manner and mediates various
cell-fate decisions, which are important for the morphogenesis and development
of numerous organs and tissues in many vertebrates and invertebrates
(Artavanis-Tsakonas et al.,
1995; Artavanis-Tsakonas et
al., 1999
; Lewis,
1998
). During neurogenesis, Notch signalling regulates the
proliferation of various neural stem cells, either keeping them in an
undifferentiated state or promoting glial differentiation
(De Bellard et al., 2002
;
de la Pompa et al., 1997
;
Frisen and Lendahl, 2001
).
Other important processes with a crucial involvement of Notch signalling are,
for example, somitogenesis, pancreas development and inner ear sensory
development (Apelqvist et al.,
1999
; Beckers et al.,
1999
; Hrabé de Angelis
et al., 1997
; Jiang et al.,
2000
; Kiernan et al.,
2001
; Kusumi et al.,
1998
; Morrison et al.,
1999
).
We describe an as yet unknown requirement of Notch signalling for the
normal development of LR asymmetry in vertebrate embryos. We observe
randomisation of the direction of heart looping and embryonic turning in
embryos homozygous for a loss-of-function allele of the Dll1 gene
(Hrabé de Angelis et al.,
1997). In addition, expression studies and scanning electron
microscopy analysis of late gastrulating embryos show that Dll1 function is
also required for the development of proper embryonic midline structures and
normal node morphology. The requirement of Dll1 function for node development
represents the earliest function of Notch signalling during mammalian
development described until now.
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MATERIALS AND METHODS |
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Whole mount lacZ staining and RNA in situ hybridisation
lacZ staining was carried out as described by Wurst and Gossler
(Wurst and Gossler, 2002). Antisense riboprobes were generated using the
DIG-RNA labelling system (Roche Molecular Biochemicals, Mannheim, Germany)
according to the manufacturer's instructions. In situ hybridisation was
performed using the InsituPro robot from ABIMED (Langenfeld, Germany)
following a protocol previously described (Spörle and Schughart, 1998).
Embryos were stained with BM Purple AP substrate (Roche Molecular
Biochemicals) and postfixed with 4% PFA in PBS. The following probes were
used: Hand1 (eHAND)
(Srivastava et al., 1995);
Nodal (Zhou et al.,
1993
); Lefty1 (Ebaf) and Lefty2
(Leftb) (Meno et al.,
1997
); Pitx2
(Campione et al., 1999
);
T (brachyury) (Herrmann,
1991
); Hnf3b (Ang et
al., 1993
); Dll1
(Bettenhausen et al., 1995
);
Dll3 (Dunwoodie et al.,
1997
); Jag1
(Mitsiadis et al., 1997
);
Notch1 (Del Amo et al.,
1992
); Notch2
(Larsson et al., 1994
); and
Lfng (Cohen et al.,
1997
).
Histological analysis
Stained embryos were dehydrated through an ethanol series, embedded in
Spurr's resin (Spurr, 1969),
sectioned at 7.5 µm and counterstained with safranin. Alternatively,
stained embryos were cryoprotected in 30% sucrose/PBS at 4°C overnight,
subsequently embedded in Cryoblock (Medite Medizintechnik GmbH, Burgdorf,
Germany) and cryosectioned (35 µm) at -25°C. Processed sections were
mounted under coverslips in KAISER'S glycerol gelatine (Merck, Darmstadt,
Germany). For quantitative analysis of cell numbers, embryos were fixed in 3%
glutaraldehyde, 4% sucrose, 0.1 M Na-cacodylate/HCl pH 7.6 and 2 mM
CaCl2; washed several times in the same solution without
glutaraldehyde; fixed for 2 hours with 2% OsO4; dehydrated in
ethanol; and embedded in EPON®. Embryos were cut at 1 µm and
counterstained with Toluidine Blue. Cell numbers were collected from five
sections (every 20 µm of the first segment posterior to the forelimb bud)
of five individuals each per age and genotype. Statistical significance was
proven by the repeated measures analysis of variance using the SAS 6.12
software.
Scanning electron microscopy
Dehydration of embryo samples (developmental stage E7.5 to E10.5) was
performed in a graded series of ethanol. The embryos were critical-point dried
from CO2 by a routine procedure and sputtercoated (K575 EMITECH
LTD, Ashford, UK) with 1-3 nm platinum. Coated specimens were examined in a
field emission scanning microscope (Jeol JSM-6300F, Tokyo, Japan) with
accelerating voltage of 2-10 kV in secondary electron mode.
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RESULTS |
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Whereas 100% of the wild-type embryos showed looping of the tubular heart to the right, close to 50% of homozygous Dll1lacZ mutant embryos at E8.5 and E9.5 had an abnormal heart looping, either completely or incompletely to the left (Table 1). As a consequence of the anti-clockwise rotation of wild-type embryos, the developing tail curves to the right side in the vast majority (97.6%) of E9.5 embryos (Fig. 1A, left). By contrast, axial rotation at E8.5 was clockwise in 50% and the positioning of the tail at E9.5 was either to the left in 48.5% or abnormal in Dll1-deficient embryos (Table 1; Fig. 1A, right). Apparently, the direction of heart looping and embryonic turning are not linked in mutant embryos, as all combinations between normal and abnormal tail placement and heart orientation were observed in homozygous Dll1lacZ mutant embryos at E9.5. These data show that, in Dll1-deficient embryos, the asymmetric development of the heart and the direction of embryonic turning are randomised.
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|
To investigate the morphology of Dll1 mutant hearts in more detail, we used scanning electron microscopy. Heart morphology at E10.5 was either as in wild-type embryos, with complete looping of the heart to the right in 39.4% of the mutants (Fig. 1C), or abnormal with incomplete looping to the right or left (in 18.2% of the mutants, Fig. 1D) or with complete looping to the left (in 42.2% of the mutants, Fig. 1E), resulting in mirror-imaged morphology when compared with wild-type hearts. Despite the reversed orientation all morphologically distinct subunits, such as the bulbus cordis, the future left and right ventricles, were present, including the beginning bifurcation into branchial arteries (Fig. 1E).
To confirm the anatomical reversion towards mirror-imaged hearts at the
molecular level, we analysed the expression of the bHLH transcription factor
Hand1 (also called eHAND)
(Srivastava et al., 1995).
Hand1 expression starts during preimplantation and is subsequently
restricted to the developing heart and to neural-crest derivatives
(Firulli et al., 1998
;
Riley et al., 1998
). In looped
wild-type hearts at E10.5, Hand1 is specifically expressed in a large
domain in the proampulla (future left or systemic ventricle) and in a
restricted region of the metampulla (future right or pulmonary ventricle,
Fig. 1B, left)
(Thomas et al., 1998
).
According to the mirror-imaged morphology in mutant hearts, we found that
Hand1 is expressed in a large domain reminiscent to the expression in
the proampulla of normal heart primordia on the right side and in a restricted
domain on the left side comparable with the expression in the right pulmonary
ventricle of wild-type embryos (Fig.
1B, right). Taken together, these data suggest that during
embryonic development, Dll1 function may be required for directional looping
of the heart primordial, but not for the differentiation of particular heart
subunits. The loss-of-function mutants display a randomisation characteristic
of a situs ambiguous phenotype.
To investigate whether this phenotype is specific for the Dll1
gene, we analysed LR sidedness in a second mutant allele of a putative Notch
ligand, the Jag1 gene (Kiernan et
al., 2001). Among 33 homozygous headturner (Htu) mutants
at E10.5, only one showed a complete reversed looping of the heart together
with a right-sided tail, whereas all others remained unchanged compared with
the wild type (data not shown). These data show that the situs ambiguous
phenotype is more pronounced in Dll1 mutant embryos.
Randomised expression of LR-specific genes
To investigate in more detail the determination of LR asymmetry in
Dll1 mutant embryos, we analysed the expression pattern of specific
molecular markers. The TGFß family members Nodal, Ebaf (also
called Lefty1) and Leftb (also called Lefty2),
together with the homeobox gene Pitx2, are either required for proper
LR development or are specific markers of LR determination
(Hamada et al., 2002). These
genes have side-specific expression patterns prior to heart looping and
embryonic turning (Capdevila et al.,
2000
; Hamada et al.,
2002
). To test whether Dll1 is required for the
asymmetric expression of these genes (Nodal, Ebaf, Leftb and
Pitx2) we performed whole-mount RNA in situ-hybridisation in
Dll1 mutant embryos.
In wild-type (Dll1+/+) and heterozygous mutant embryos
(Dll1+/lacZ) at the early somite stage (E8.5, 0
to 6 somite pairs) Nodal expression is confined to the left lateral
plate mesoderm (LPM) and to small domains to the left and right of the node,
which is stronger to the left and weaker to the right with an increasing
number of somites (Fig. 2A,B).
Later, embryos with more than six pairs of somites show no detectable
expression of Nodal adjacent to the node
(Table 2). In homozygous mutant
littermates at somite stages 0 to 6, expression of Nodal in the LPM
was either left-sided (4/21) or right-sided (4/21), bilateral (3/21) or not
detectable (10/21) by in situ hybridisation. Expression around the node was
either normal (4/21 with a left bias as in the wild type), changed (three out
of 21 with a right bias or equal to both sides of the node) or absent (14/21)
(Table 2;
Fig. 2C-E). Similarly,
expression of Leftb was altered. In wild-type and heterozygous
embryos with up to seven somites, Leftb expression was detected
exclusively in the left LPM (Fig.
2F). By contrast, expression of Leftb in homozygous
mutant embryos was randomised (2=0.5; 3 df; P=0.08),
with expression either in the left LPM (five out of 16), the right LPM (three
out of 16), bilateral expression (four out of 16), or was not detected in the
LPM (four out of 16) (Table 2;
Fig. 2G,H).
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|
At E8.5 Pitx2 is expressed in the head mesenchyme and in the left
LPM. The left-sided expression of Pitx2 in the LPM is thought to be
induced by Nodal (Shiratori et al.,
2001). Similar to the expression of Nodal and Leftb,
Pitx2 expression was altered in homozygous mutant embryos with six to 10
somites but did not follow the pattern found for Nodal
(
2=44,7; df=3; P
0). Pitx2 was expressed
either in the left LPM (five out of 25), in the right LPM (one out of 25), in
the left and right LPM (15/25), or was absent from the LPM (four out of 25).
In heterozygous and wild-type embryos, Pitx2 expression was detected
only in the left LPM as expected (Fig.
2I-L; Table 2).
It was suggested that expression of Ebaf (Lefty1) in the
left half of the floorplate may be required for midline structures to prevent,
for example, expression of left-sided genes on the right side of the embryo
(Meno et al., 1997;
Meno et al., 1998
). Because we
observed the expression of marker genes for left LPM also on the right, we
analysed expression of Ebaf in mutant embryos. Although Ebaf
was expressed in the left half of the floorplate of wild-type and heterozygous
embryos at the two to six somite stage, Ebaf expression was not
detected by in situ hybridisation in homozygous mutant embryos (0/11)
(Fig. 2M,N; Table 2).
The loss of the unilateral expression domains of Nodal, Leftb, Ebaf and Pitx2 is in accordance with the observed situs ambiguous phenotype in homozygous Dll1lacZ knockout mutants. The expression analysis demonstrates that Dll1 function is required for the consistent asymmetrical expression of these marker genes.
Altered development of midline structures
It has been suggested that the lack of intact midline structures such as
floorplate and notochord may cause defects in LR asymmetry
(Dufort et al., 1998;
Izraeli et al., 1999
;
King et al., 1998
;
Melloy et al., 1998
). Although
it is not clear how embryonic midline structures are functionally involved in
the determination and/or maintenance of LR sidedness, it has been argued that
axial structures might function as physical barrier for unilateral signals. To
investigate whether the defects in LR asymmetry are accompanied by defects in
the embryonic midline of Dll1 mutants, we analysed the morphology of
the floorplate and notochord, and examined the expression of essential midline
markers.
In wild-type embryos, the axial marker gene T (brachyury) is
expressed from early gastrulation onwards in the primitive streak and in the
developing notochord (Wilkinson et al.,
1990) (Fig. 2O). By
contrast, in homozygous Dll1lacZ mutant embryos T
expression was present in the primitive streak, but strongly reduced and often
absent along the notochord at E8.5 (Fig.
2P). Therefore, we examined the morphology of the neural tube and
notochord in Dll1 mutant embryos in serial cross-sections. Although
hyperplasic to a certain extent, the overall dorsoventral patterning of the
neural tube was unaltered (M. H. de A. and K. Wünsch, unpublished). We
found that the mutant floorplate was larger in histological preparations from
E8.5 to E10.5 when compared with wild-type littermates
(Fig. 3A-F). This finding was
further supported by the extended expression domain of the floorplate marker
gene Hnf3b in mutant embryos at E8.5
(Fig. 3A,B). In addition to the
floorplate defect, we observed regional abnormalities in the morphology of the
notochord (Fig. 3B). In some
regions along the AP axis, which coincide with absent T expression,
presumptive notochord cells appeared rather as a sheet tightly associated with
the dorsal primitive gut endoderm (Fig.
3B) when compared with the rod-like shape in the wild-type embryo
(Fig. 3A). To further analyse
these midline defects, we quantitated cell numbers in the floorplate and
notochord in serial cross-sections of embryos at E9.5 and E10.5. This analysis
revealed that the mutant floorplate contained significantly more cells,
whereas the number of cells in the notochord was reduced
(Fig. 3G,H). These data
indicate that in Dll1 mutant embryos, abnormal LR patterning is
associated with defects in the development of axial structures such as
floorplate and notochord.
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Expression of Notch-pathway genes during node formation
The major source of progenitor cells for midline structures in vertebrates
is the node (Kinder et al.,
2001; Tam and Behringer,
1997
). To investigate whether Notch signalling is required for
node formation in the node itself, or whether its function may be required in
a domain closely associated with the node, we analysed expression of the
Dll1 gene and other molecular factors of the Notch-signalling pathway
during node formation. Interestingly, Dll1 is expressed in a distinct
domain adjacent to the node of wild-type embryos and not in the node itself
(Bettenhausen et al., 1995
).
X-gal staining of Dll1lacZ heterozygous embryos at late
gastrulation [E7.5, Theiler stage (TS) 11] reveals strong expression of
ß-galactosidase to the posterior mesoderm but excluding the node
(Fig. 4A), whereas in
homozygous embryos weak staining was found also in the node and in more
anterior regions (Fig. 4B). To
further analyse the involvement of Notch-signalling pathway genes in the
formation and/or maintenance of the node, we analysed the expression patterns
of Dll3, Jag1, Lfng, Notch1 and Notch2 during late
gastrulation (E7.5, TS 11).
|
Like Dll1, the expression of the Notch ligand Dll3 is
restricted to the posterior mesoderm
(Dunwoodie et al., 1997). No
expression in the node was found (Fig.
4C). The Notch ligand Jag1 was expressed in the mesoderm
in an anterior domain complementary to the Dll1 expression domain
(Fig. 4D). At late
gastrulation, the expression pattern of Notch1 is similar to
Dll1 and Dll3. Transcripts are present in and adjacent to
the primitive streak, in posterior ectoderm and in the mesoderm
(Del Amo et al., 1992
;
Williams et al., 1995
). No
expression was detected in the node (Fig.
4E). Notch2 is expressed in a distinct pattern with sharp
boundaries (Williams et al.,
1995
) and was found in the node, notochord and in single stripes
lateral to each side of the node (Fig.
4G). In the fly, fringe is thought to participate in the
formation of cellular boundaries by modifying the ability of Notch to bind its
ligand Delta (Cohen et al.,
1997
; Johnston et al.,
1997
). At mid gastrulation (TS 10), the mouse gene Lunatic
fringe (Lfng) was expressed in a similar pattern like Dll1,
Dll3 and Notch1 (data not shown). At late gastrulation (TS 11),
Lfng expression was restricted to a distinct domain lateral to the
node (Fig. 4I).
Among the offspring of heterozygous intercrosses, all embryos tested (18
for each gene in three independent experiments) showed no obvious differences
in the expression of Dll3 and Jag1 when compared with
wild-type embryos (data not shown). By contrast, 25% of the embryos
revealed significant alterations in the expression of Lfng, Notch1
and Notch2. In particular, Lfng and Notch1 were
ectopically expressed in the node and the expression in the surrounding
tissues was not restricted to the characteristic domains
(Fig. 4F,K). In addition,
Notch2 expression in the node was strongly reduced or patchy and
expression adjacent to the node was diffuse
(Fig. 4H).
Taken together, these Notch-signalling pathway genes show a distinct expression pattern surrounding and/or including the node. This pattern might be of functional relevance with respect to the morphology of the node and for the maintenance of node integrity.
Structural abnormalities of the node in homozygous
Dll1lacZ mutants
To investigate whether the disrupted expression pattern of Notch-pathway
genes in and adjacent to the node might cause abnormal node morphology in
Dll1 mutant embryos, we analysed node structures with scanning
electron microscopy. In wild-type embryos at E7.5, the node has formed as a
distinct structure at the apex of the embryonic cone at the anterior end of
the primitive streak (Sulik et al.,
1994). The mesendodermal node cells are characterised by their
small surface area in comparison to the surrounding endodermal cells and a
single, central cilium on each cell (Fig.
5A). Approximately 25% of the offspring from
Dll1lacZ heterozygous intercrosses displayed morphological
changes in the node. These are evident as rupturing of the surface, bulging of
cells and loss of monociliated cells (Fig.
5B). Later, at the late headfold stage (TS 11), prior to heart
looping and embryonic turning, all wild-type embryos analysed had a
symmetrical, club-shaped node with evenly distributed ciliated cells
(Fig. 5C). By contrast,
homozygous mutants at the late headfold stage often displayed irregularities
in the node: cells with abnormal morphology disturbed the node symmetry and
the regular distribution of cilia was altered
(Fig. 5D). At E8.5 (TS 12), the
wild-type node consists of microvilli-lined, cone-shaped cells, each with a
single, motile cilium located on their ventral surface
(Sulik et al., 1994
)
(Fig. 5E). By comparison, the
mutant node contained enlarged cells of an unusual character with a smooth
surface that disrupted the regular array of ciliated cells
(Fig. 5F). Occasionally, we
observed characteristics of cell death in the mutant node and in cells along
the future gut endoderm (data not shown). Thus, although the node is formed in
homozygous Dll1lacZ mutants, its structural integrity is
not maintained. The requirement of Dll1 for the formation and/or
maintenance of a regular node is the earliest function associated with this
gene during embryonic development described so far.
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DISCUSSION |
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A complex regulatory network of genes required for the initiation,
formation and maintenance of LR asymmetry of vertebrates has been discovered
(Bisgrove and Yost, 2001;
Capdevila et al., 2000
;
Hamada et al., 2002
;
Wood, 1997
). The TGFß
family genes Nodal and Leftb, which are the earliest
asymmetrically expressed genes in mouse described so far, play pivotal roles
in this process. Together with the transcription factor Pitx2, they
are expressed in the left lateral plate mesoderm before morphological
differences between the left and right halves of the embryo are evident. The
Dll1lacZ allele interferes with the expression pattern of
these LR marker genes, such that their unilateral expression domain is altered
and accordingly randomised in homozygous mutants. Interestingly, our data show
with statistical significance, that the expression found for Pitx2 in
homozygous mutants does not follow Nodal expression, although
Pitx2 is a known target of Nodal
(Shiratori et al., 2001
). The
bilateral expression of both genes might be explained by an impaired midline
structure. This has been shown for several mutants in mouse and zebrafish
(Bisgrove and Yost, 2001
).
However, this does not explain why the percentage of mutants with bilateral
expression of Pitx2 was higher than with bilateral expression of
Nodal. These findings suggest, that there might be an additional
mechanism triggering Pitx2 expression. It has to be kept in mind that
we investigated expression of Pitx2 later in development (at the 6-10
somite stage) than expression of Nodal (at the 0-6 somite stage).
Nevertheless, the expression data reflect the morphological situation and
randomisation of LR sidedness, suggesting that Dll1 function may act before
expression of Nodal, Leftb and Pitx2.
Though laterally reversed in nearly 50% of mutant embryos, all
morphologically distinct segments of the embryonic heart are present. This is
consistent with the expression pattern of the Hand1 gene, which is
thought to be involved in the development of specific segments during
cardiogenesis (Thomas et al.,
1998). As in wild-type embryos, Hand1 expression in
Dll1 mutants is segment specific and independent from laterality.
Taken together, these results show that Dll1 is required for proper
heart looping and embryonic turning, but is not necessary for chamber
specification of the developing heart.
The Dll1 mutant is the first mouse mutant described so far with a
randomised expression pattern of the LR marker genes Nodal, Leftb and
Pitx2, together with a loss of Ebaf (Lefty1)
expression. Other mouse mutants with laterality defects also display a
strongly reduced or loss of Ebaf expression, but show invariable
bilateral expression of the LR marker genes, together with thoracic left
isomerism (Bisgrove and Yost,
2001), whereas the expression patterns of Nodal and
Pitx2 found in Dll1 mutant embryos are in accordance with a
situs ambiguous phenotype. Owing to the early lethality of Dll1
mutant embryos, it is not possible to investigate the situs of abdominal
organs. No differences in the arrangement of kidney primordia both in
wild-type and mutant embryos were found. In Ebaf mutants with
thoracic left isomerism, the LR-marker genes are also bilaterally expressed
(Meno et al., 1998
). It was
suggested that the asymmetrical, floorplate-specific expression of
Ebaf might be required in the midline to function as a molecular
barrier that prevents the expression of Nodal and Leftb in
the right side of the embryo. However, the absence of Ebaf is most
probably not the primary reason for the randomisation of heart looping and
turning in Dll1 mutants, because in Ebaf mutants these
processes are not affected (Meno et al.,
1998
).
Homozygous Dll1lacZ mutants show structural
abnormalities in midline tissues, such as an enlargement of the floorplate in
combination with a decrease in the number of notochord cells. Defects in axial
midline tissues are also reported from mouse mutants such as no turning,
Shh-/- and Sil-/-. In addition, these
mutants display a combination of randomised heart looping or embryonic turning
with a loss in Ebaf expression
(Izraeli et al., 1999;
Melloy et al., 1998
;
Meyers and Martin, 1999
). The
severe midline defects in Dll1 mutant embryos are consistent with the
observations that midline tissues may function as a physical barrier, which
might be a prerequisite for normal development and/or maintenance of
laterality in vertebrates (Klessinger and
Christ, 1996
; Levin et al.,
1996
; Lohr et al.,
1997
). The change in the number of floorplate and notochord cells
in Dll1 mutant embryos suggests that Notch signalling is involved in
the specification of midline cells. Interestingly, mutations in the zebrafish
homologs deltaA and deltaD cause deficiencies of cells in
the midline (Appel et al.,
1999
; Latimer et al.,
2002
). In particular, deltaA mutants have fewer cells in
the floorplate and an increase of cells in the notochord
(Appel et al., 1999
),
suggesting that Notch signalling is also required in other vertebrates for the
specification of midline cells.
Our analysis describes the earliest function associated with the Delta1 gene in vertebrates so far. The requirement for normal LR development in vertebrates is a novel function of Notch signalling that was not described before. However, there is no evidence to date, that Dll1 or any other molecular factor of the Notch-signalling pathway could be directly involved in the determination or maintenance of the LR axis in vertebrates. In particular, there is no description of an asymmetric expression pattern of any Notch-signalling pathway component either during early or late gastrulation in the node, in tissues adjacent to the node or in the paraxial mesoderm. All expression patterns of Notch-signalling pathway genes analysed in this study from the onset of expression at around midstreak stage (TS10, E6.5-7) until early organogenesis were symmetrical to the embryonic midline. The observed defect in the midline structure of Dll1 mutant embryos cannot fully explain the primary cause of the LR abnormalities. This led us to the hypothesis that Notch signalling might be involved in proper node development and gastrulation.
Based on known cellular movements in the node and fate maps of the node and
primitive streak (Kinder et al.,
2001; Sulik et al.,
1994
; Tam and Beddington,
1987
), it is likely that the midline defects of Dll1
mutant embryos may be caused by earlier defects in the differentiation of node
cells and node morphology. In fact, we observed severe morphological and
cellular defects in the node of Dll1 mutant embryos. It was suggested
that the shape of the node and the equal distribution of motile cilia on its
ventral surface are prerequisites to generate a nodal flow, which might
transport a not yet identified morphogen that triggers the
onset of asymmetric gene expression
(Nonaka et al., 1998
;
Okada et al., 1999
). Taken
together, the defects in LR-axis formation in Dll1 mutant embryos may
originate from a combination of altered node morphology and distorted
midline.
However, the question remains as to how Notch signalling participates in
proper development of the node. It is generally known that the
Notch-signalling pathway is involved in boundary formation (for a review, see
Irvine and Rauskolb, 2001). We
find that the early, distinct expression pattern of Notch-pathway genes at
E7.5 surrounding and/or within the node (summarised in
Fig. 6A) is in some way
reminiscent of the expression of these genes in the wing imaginal disc of
Drosophila, where Serrate/Jagged and Delta have
opposing expression domains and activation of Notch, modulated by
fringe, at the wing margin is required for dorsoventral lineage
restriction in the wing imaginal disc
(Doherty et al., 1996
;
Micchelli and Blair, 1999
;
Rauskolb et al., 1999
). When
Notch signalling is disrupted, cells can intermix and violate the compartment
border (Micchelli and Blair,
1999
; Rauskolb et al.,
1999
). It is tempting to speculate that a somewhat similar
mechanism may exist during early mouse embryogenesis and that this mechanism,
by restricting the allocation of cells to the node, might be required for its
proper differentiation. The loss of distinct expression boundaries of at least
some Notch-pathway components in homozygous Dll1lacZ
mutant embryos (summarised in Fig.
6B) would lead to a softening of the compartment boundary and thus
could lead to the observed defects in specification of node cells. The
appearance of large, non-ciliated cells in the ventral node of Dll1
mutant embryos could be deemed to be a result of an improper cell sorting
mechanism, which might be mediated by cell adhesion forces. This idea is also
supported by loosening of the tightly packed cells in the ventral node of
Dll1 mutant embryos. Currently, we do not have any evidence for an
involvement of Delta and Notch molecules in cell adhesion during node
formation, but it is well known that specific adhesive forces are required for
proper gastrulation (Ip and Gridley,
2002
). A relationship between cell adhesion molecules and LR
development was revealed by experiments in chicken embryos, where blocking of
N-cadherin function resulted in randomisation of heart looping and altered
expression of Pitx2
(Garcia-Castro et al.,
2000
).
|
Interestingly, embryos homozygous for a targeted mutation of
RBP-J (Rbpsuh Mouse Genome Informatics) a key
downstream component of the Notch-receptor signalling-pathway, shows severe
developmental abnormalities, including defective somitogenesis, neural tube
defects and an incomplete rotation of the body axis
(Oka et al., 1995
). Although
nothing is known about node defects and LR-asymmetry defects in
Notch1 and Notch2 mutants, the analysis of compound mutants,
homozygous for both Notch1 and Notch2 mutant alleles could
help to clarify the role of Notch signalling in node formation and in
LR-development, respectively.
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ACKNOWLEDGMENTS |
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Footnotes |
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