1 Comparative and Developmental Genetics Section, MRC Human Genetics Unit,
Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
2 Department of Developmental Biology, Hagedorn Research Institute, Niels
Steensens Vej 6, 2820 Gentofte, Denmark
3 The Institute of Human Genetics, The International Centre for Life, Central
Parkway, Newcastle upon Tyne NE1 3BZ, UK
Author for correspondence (e-mail:
bob.hill{at}hgu.mrc.ac.uk)
Accepted 19 July 2004
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SUMMARY |
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Key words: LR asymmetry, Bapx1, Pancreas, Spleen, SMP, Fgf regulation, Mouse
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Introduction |
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The normal disposition of organs is called situs solitus. Disruptions of
organ situs are common and heterotaxia conditions cover a broad range of
gastrointestinal and cardiac defects
(Burn, 1991;
Aylsworth, 2001
). In situs
inversus complete mirror-image reversal of situs occurs. More commonly
individuals have partial situs malformations owing to randomisation of
patterning information and only a few organs are affected (situs ambiguus or
heterotaxy). The mesenchymally derived spleen is normally situated on the left
side of the abdominal cavity and as such is a readily identifiable landmark
for detecting extensive laterality defects
(Aylsworth, 2001
). Conditions
such as asplenia, and polysplenia syndromes are often associated with situs
ambiguous, indicating an underlying, fundamental defect of asymmetric
organogenesis (Bowers et al.,
1996
). Loss of splenic tissue (asplenia or rudimentary spleen)
relates to bilateral right sidedness called right isomerism, whereas the
contrasting phenotype of additional splenic tissue (polysplenia, either as
additional splenules or multilobulated single spleen) relates to bilateral
left sidedness (left isomerism). Hence, spleen abnormalities are frequently
associated with situs malformations of other organ systems dependent on LR
signalling (Ivermark's syndrome) (Ivemark,
1955
).
In this report, we have examined two asplenic mouse mutations, dominant
hemimelia (Dh) and the Bapx1-targeted disruption, to
investigate the relationship of spleen development and LR asymmetry.
Dh is an established, spontaneously derived mouse mutation that
disrupts visceral and limb development
(Green, 1967). The
Bapx1 gene (also referred to as Nkx3.2) is a member of the
NK family of homeobox-containing genes
(Tribioli et al., 1997
) first
described in Drosophila and is most closely related to the
Drosophila bagpipe (bap) gene
(Azpiazu and Frasch, 1993
).
Targeted mutations of Bapx1 results in loss of the spleen and
vertebral defects (Lettice et al.,
1999
; Tribioli and Lufkin,
1999
; Akazawa et al.,
2000
). Examination of visceral development in Dh and
Bapx1 mutants led to the identification of a mechanism whereby the
primordial spleno-pancreatic mesoderm that surrounds the gut endoderm controls
localised asymmetric growth.
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Materials and methods |
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Probes and in situ hybridisation
DIG-in situ hybridisation was performed essentially as described by
Wilkinson (Wilkinson, 1992).
Prior to staining, the embryos were washed twice in 0.1 M Tris (pH 8.2) for 30
minutes and the signal was visualized in 2 ml 0.1 M Tris (pH 8.2) containing
one Fast Red tablet (Roche). The probes for Bapx1, Nkx2.5,
Hox11 (Tlx1 Mouse Genome Informatics), Wt1,
capsulin (Tcf2l Mouse Genome Informatics), Barx1, Pitx2,
Fgf9, Fgf10 and Fgfr3 have been described previously
(Mundlos et al., 1993
;
Peters et al., 1993
;
Roberts et al., 1994
;
Tissier-Seta et al., 1995
;
Semina et al., 1996
;
Bellusci et al., 1997
;
Lu et al., 1998
;
Colvin et al., 1999
;
Lettice et al., 2001
).
BrdU labelling
E10.5 embryos were labelled with BrdU by intraperitoneal injection of 200
µl BrdU (10 mg/ml) into pregnant females. After a 30 minute labelling
period, the embryos were dissected out and fixed in 4% PFA. Embryos were then
embedded in paraffin wax and sectioned. After rehydration, sections were
trypsinised for 10 minutes at 37°C, washed in PBS and then incubated for
10 minutes in 1 N HCl. Incorporation of BrdU was detected using anti-BrdU
monoclonal antibodies and Alexa Fluor® 488 goat anti-mouse secondary
antibody (1:200 dilution; Mol. Probes). Finally, sections were counterstained
with Propidium Iodide (1:5000).
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Results |
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We have identified the initial lateral position of the pre-spleen rudiment at a stage before the spleen is associated with the stomach. At E10.5, cross-sections taken through the embryo in the region of the developing forelimb reveal the spatial relationship between the stomach (Fig. 1A,B) and dorsal pancreas (Fig. 1A,C) during the early stages of leftward growth. At this stage, Hox11 expression is detected in the dorsal mesenchyme positioned on the left-lateral side, but lying posterior to the stomach primordium (Fig. 1D). The pancreas-specific anti-PDX1 antibody shows that this mesenchyme also supports the dorsal pancreatic bud (Fig. 1D) lying ventral to the splenic rudiment.
|
A distinct columnar epithelium surrounds the spleno-pancreatic mesenchyme
An outer layer of epithelium surrounding the splenopancreatic mesenchyme
was noted in the E10.5 embryos (broken lines in
Fig. 1D,E). Earlier stages were
examined to establish when this tissue originates. The splanchnic mesoderm is
the region of the lateral plate mesoderm (LPM) that surrounds the gut. In the
region of the dorsal pancreatic bud, we found that fluorescently tagged
phalloidin detects the accumulation of f-actin at the boundary between the
endoderm and splanchnic mesoderm and at the outer margin of the splanchnic
mesoderm, thus outlining a thickened epithelial structure
(Fig. 2A,B). The splanchnic
mesoderm surrounds the foregut and appears as a bilateral, highly organised
layer of elongated cells 50-60 µm thick that is easily distinguishable
from the unorganised mesenchymal cells that compose a large proportion of the
embryo. The position of the dorsal pancreatic bud was detected with the PDX1
antibody (Fig. 2A,C,D,O,P). The
pancreatic bud is situated at the embryonic midline
(Fig. 2A) and extends dorsally
flanked tightly on both sides by the splanchnic mesoderm.
|
By E10 the spleno-pancreatic region in wild-type embryos acquires a characteristic triangular shape in cross-section (Fig. 2C). The SMP is prominent and located on the left side (white arrowheads in Fig. 2C). The mesenchyme on the right side of the gut tube remains positioned near the embryonic midline and the thick epithelial plate-like structure is lost. TUNEL staining for apoptotic nuclei reveals negligible cell death in this region between the stages E9.5 and E10.5 (data not shown). This argues against apoptosis as the driving force behind the observed loss of the right-sided SMP. Cells of the right SMP persist after E9.5, most probably contributing to the underlying mesenchyme and the thin layer of cuboidal cells (indistinguishable from the mesothelial layer that lines the mesenchyme in the remainder of the coelomic cavity and shown by yellow arrowheads in Fig. 2C) that is found in place of the right SMP from E10.0 onwards. Analysis of nuclear morphology in the spleno-pancreatic region between E9.5 and E10.5 reveals that the elongated nuclear structure that characterises the left SMP is progressively lost from the right-hand side during this period (Fig. 2F-N). By E10.0, nuclei positioned ventrally along the right margin resemble those present in the underlying mesenchyme or in adjacent mesothelia (Fig. 2I,K). At this stage, nuclei positioned dorsally along the right margin retain their elongated morphology (Fig. 2I,J). By E10.5, nuclei along the length of the right-hand margin are overtly indistinguishable from those in the underlying mesenchyme or in adjacent mesothelia (Fig. 2L-N), suggesting that the observed loss of the right-hand SMP is actually the result of a spatiotemporal change in cell morphology.
Dorsal pancreatic bud growth accompanies the asymmetric expansion of the mesenchyme such that it becomes displaced to the left of the embryonic midline. The SMP, which by bright-field microscopy appears as a translucent cell layer (indicated by broken lines in Fig. 6A), entirely encompasses the left, lateral spleno-pancreatic mesenchyme extending from the posterior half of the stomach to the pancreatic buds. During the subsequent day of development, the shape and position of the region changes dramatically and by E11.5 the thick epithelial structure is no longer detected (data not shown).
|
The SMP is under control of the LR signalling cascade
To determine whether SMP asymmetry is under control of the LR signalling
pathway, we analysed expression of Pitx2, a gene known to be
expressed in the left LPM during early embryogenesis
(Logan et al., 1998;
Piedra et al., 1998
;
Shiratori et al., 2001
).
Pitx2 has a role in lung isomerism, heart development and rotation of
the duodenum (Gage et al.,
1999
; Kitamura et al.,
1999
; Lin et al.,
1999
; Lu et al.,
1999
; Liu et al.,
2001
). Expression of the Pitx2 gene persists later than
many other genes in the LR signalling cascade, with expression still
detectable at E9.5. At this stage, before asymmetric organogenesis is
apparent, Pitx2 is expressed predominantly in the left-sided SMP in
the region of the dorsal pancreatic bud
(Fig. 3A). Thus, Pitx2
expression is an indicator of left/right differences and shows that the left
SMP expresses LR-specific genes. The homeobox-containing Barx1
(Tissier-Seta et al., 1995
),
which has not previously been associated with LR asymmetry, also shows
left-specific mesenchymal expression, substantiating the phenotypic
differences between left and right mesenchyme at these early stages
(Fig. 3B).
|
To further investigate the link between SMP and the genetic LR asymmetry
program, the spleno-pancreatic region in mice carrying the inversion of body
turning (inv) mutation was analysed
(Fig. 3F,G). Homozygous
inv mutant embryos all show situs inversus
(Yokoyama et al., 1993;
Morgan et al., 1998
;
Watanabe et al., 2003
) with
both the spleen and pancreas developing on the right hand side. At E10.5 in
the inv/inv embryos, the SMP is positioned on the opposite,
right-hand side (Fig. 3G). The
underlying spleno-pancreatic mesenchyme has grown to the right and the region
is the mirror image of that in the wild type. These findings show that the
maintenance of the SMP, and subsequent growth of the spleno-pancreatic
mesenchyme, is a downstream consequence of the LR signalling pathway.
Leftward pancreatic growth is dependent on the SMP
The Dh mutant embryo enables the examination of left lateral
asymmetry in the absence of the SMP. In wild-type embryos at E10 the SMP and
associated mesenchyme grows laterally (Fig.
4A). By contrast, in the Dh/Dh embryos the SMP remains
undetectable and specific leftward growth of the mesenchyme is impaired
(Fig. 4C). The mesenchyme is
symmetrical and surrounds the dorsal pancreatic bud that remains situated
along the embryonic midline. This arrangement persists at later stages and at
E10.5 the pancreas is still observed at the midline (data not shown).
|
Thus the Dh and Fgf10/ mouse mutations provide two contrasting developmental conditions in which to examine leftward asymmetry. In the absence of the SMP, no leftward pancreatic growth was observed, whereas in the absence of the pancreatic bud, the SMP and underlying mesenchyme expand laterally. This suggests that asymmetry of the spleno-pancreatic mesenchyme and SMP is autonomous. The pancreatic endoderm in wild-type embryos is embedded in the left-sided mesenchyme and develops in close association with the SMP, but has no role, either physically or inductively, in the asymmetric growth of these structures.
Cells in the SMP proliferate at a high rate
As endodermal development is not required for asymmetric growth in the
spleno-pancreatic region, we hypothesised that regional variations in cellular
proliferation might be a key factor in the morphogenetic changes that
characterise the development of the SMP and the underlying tissue. At E9.5,
prior to overt LR asymmetry in this region, BrdU incorporation can be detected
at high levels in both the left and right sides of the SMP
(Fig. 5A). This pattern is
reiterated at E9.75, during the initial stages of asymmetric morphogenesis.
However, by this stage, regional differences in the BrdU-uptake pattern exist.
Comparable levels of BrdU incorporation are observed in the left and
right-sided SMP anterior to the dorsal pancreatic bud, with positive nuclei
distributed evenly along the dorsoventral axis
(Fig. 5B). However, in the
region of the dorsal pancreatic bud, BrdU is detected in clusters of nuclei
located around the apex of the outgrowing SMP
(Fig. 5C).
|
The Bapx1 gene regulates functions of the SMP
Similar to the Dh mutant, the
Bapx1/ embryos are asplenic. Mice homozygous
for the Bapx1 mutation were examined at the early stages of
spleno-pancreatic outgrowth, E10 and E10.5
(Fig. 6B,E, respectively). In
contrast to Dh embryos, the Bapx1 mutants retain the left
SMP throughout these stages. The spleno-pancreatic mesenchyme of the
Bapx1 mutant expands laterally but lags behind that of wild type and
the characteristic triangular shape of the wild-type SMP is compromised. Most
significantly, the dorsal pancreatic bud of the Bapx1 mutant does not
grow laterally but remains positioned along the embryonic midline.
To investigate the effect that the early malposition of the pancreas has on
later development, we examined both the Dh and Bapx1 mutants
at a later stage in organogenesis. Wholemount, three-dimensional analysis of
organ position by optical projection tomography (OPT)
(Sharpe et al., 2002) revealed
a significant change in the orientation
(Fig. 7A-C). At E13.5 in both
Dh and Bapx1 mutants, the pancreas is growing along a
different embryonic axis. In wild-type embryos, the dorsal pancreas grows
laterally in a plane that is nearly perpendicular to the stomach
(Fig. 7A). In both mutants, the
dorsal pancreas grows along embryonic axes that are close to parallel with the
stomach. In the Bapx1 embryos, the pancreas grows along the lateral
wall of the stomach (Fig. 7C),
whereas in the Dh embryo the pancreas is oriented ventrally to the
stomach (Fig. 7B).
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Discussion |
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Therefore, while determination of LR asymmetry is a general developmental
mechanism, it appears that LR information is interpreted differently by each
organ primordia. We suggest that Bapx1 and Dh mutants are
good models for heterotaxy syndromes that include asplenia (double right
isomerism). Neither mouse mutant shows cardiac, lung or liver malformations
(Green, 1967;
Herbrand et al., 2002
) (data
not shown) and, therefore, they provide insights into laterality disorders in
which only a restricted number of organ systems are affected. We propose that
the developmental mechanism that drives asymmetric organ morphogenesis in
spleen and pancreas differs from that responsible for lobation of the lung and
morphogenesis of the heart tube and that it is dependent on a
mesodermal-derived structure, the SMP.
Mechanism for spleno-pancreatic LR asymmetry
The SMP is central to our model for left lateral specific morphogenesis of
the spleen and the pancreas (Fig.
9). The process of LR asymmetry is divided into four steps
(Hamada et al., 2002). The
first three steps are responsible for transferring an initial breaking of
symmetry from near the node (step 1), to the LPM (step 2) with the subsequent
asymmetric expression of TGFß-related molecules (step 3). Less clearly
understood is the fourth step, which is the relay of information from the LMP
to the organ primordia. The SMP appears to be an important element in this
final step and in the spleno-pancreatic region may be the primary target for
the LR positional information.
|
The Dh mutation underscores the distinctive structural nature of
the SMP. The mutation operates early in embryogenesis to disrupt the bilateral
organised, columnar structure. We suggest that the highly organised structure
of the SMP plays a significant role in morphogenesis. In several cases,
disruption of the structural integrity of embryonic epithelia has resulted in
profound perturbations in subsequent morphogenesis. A similar highly organised
epithelia, which is derived from the lateral plate mesoderm, is key to the
process of gut looping in zebrafish
(Horne-Badovinac et al.,
2003). Genetic disruption of the cellular organisation results in
the lack of morphogenesis. Thus, the structural context of the SMP may be
crucial to lateral outgrowth and we speculate that the structure may provide a
rigid tissue layer for guidance. Accordingly, the SMP is characterised by the
accumulation of f-actin at the apical surfaces as detected by phalloidin
staining. Localisation of actin filaments to the apical surface of columnar
epithelial cells has been described in a number of organisms (reviewed by
Jacinto and Baum, 2003
) and
networks of actin filaments have recently been shown to play a crucial role
during the morphogenesis of the pharyngeal pouches by directing expansion
along specific axes (Quinlan et al.,
2004
).
A second characteristic of the SMP important for our model of lateral outgrowth is the high rate of cellular proliferation in this tissue layer. Outgrowth of the spleno-pancreatic mesenchyme and SMP is autonomous and entails no discernible input from the pancreatic endoderm. Proliferation within this cellular layer appears to be the motive force for driving lateral growth. We suggest that within a highly organised cellular framework proliferation within the SMP drives rapid, disproportionate expansion of this tissue layer behind which the mesenchyme, perhaps passively, populates.
Since the description of asymmetric Bapx1 expression during early
mouse and chick embryogenesis, it has been postulated that Bapx1 may
have a role to play in the establishment of LR asymmetry
(Rodriguez Esteban et al.,
1999; Schneider et al.,
1999
). The exact nature of involvement of Bapx1 in this
process has, however, remained undetermined. Based on the reported expression
patterns, it is tempting to speculate that Bapx1 could be playing a
role in the early establishment of positional information. Paradoxically, the
laterality of the expression is not conserved in evolution
(Schneider et al., 1999
). Our
observations suggest that Bapx1 in mouse loses side-specific
expression at a stage (E9.5) before any phenotype is observed in the
Bapx1/ spleno-pancreatic region. Expression
of the Bapx1 gene persists after that of Pitx2, a late stage
gene in the LR cascade. The defects observed in the
Bapx1/ embryos reported here relate
specifically to the process of morphogenesis and are in agreement with a role
for Bapx1 in the translation of positional information into
asymmetric morphogenesis. Thus, we suggest that Bapx1 has a key role
in the process of linking organ morphogenesis to events that define LR
positional information.
Two phenotypic consequences result from the lack of Bapx1
expression. The first is the lateral growth of the spleno-pancreatic
mesenchyme is reduced. The second is the downregulation of Fgf10
expression. Bapx1 is expressed in the ventral region of the SMP
overlapping the Fgf10 domain, consistent with Bapx1 as a
regulator of Fgf10 expression. FGF10 itself is responsible for early
pancreatic growth (Bhushan et al.,
2001), and in its absence the pancreas does not progress beyond
the early bud stage. Fgf10 is expressed in the SMP and neighbouring
mesenchyme at a source distal to the pancreatic bud. FGF10 is a known
chemotactic factor in lung development
(Weaver et al., 2000
). We
suggest that, in addition to its role in maintaining proliferation in the
pancreatic endoderm, a role for FGF10 is to promote leftward pancreatic growth
toward the source of high FGF10 production, thus resulting in the initial
pancreatic asymmetry.
Organ morphogenesis in other vertebrates
Recent analysis in the developing zebrafish has addressed the question of
gut asymmetry (Horne-Badovinac et al.,
2003). The digestive organs originate from a solid rod of
endodermal cells and the first leftward bend arises from a morphogenetic
process known as gut looping. The tissue layer that appears responsible for
the initial mechanism in gut looping is the LPM. The relationship of the LPM
in zebrafish and the SMP in mouse is not clear. The SMP is derived from the
mesoderm of the lateral plate and both the LPM described in fish and SMP
described here, are similar epithelial layers composed of columnar cells.
However the mechanism of LR organogenesis in which these embryonic tissues
participate is different. In the zebrafish, the lateral plate mesoderm (LPM)
flanks the gut tube on the left and right sides, and by coordinated tissue
migration drives the initial asymmetry of the gut tube by a pushing mechanism
(Horne-Badovinac et al.,
2003
). In the mouse embryo, the mechanism relies on the gut
mesoderm, in which leftward expansion of the mesenchyme and lateral
morphogenesis of the pancreas appears to follow growth of the SMP.
Is it possible that organ morphogenesis shows appreciable species
differences? Many of the genes that specify organ identity and cellular
differentiation are highly conserved. However species have different
requirements in the gut depending on food source and diet, and in vertebrates
separated by such an evolutionary distance as mammals and fish some
differences may be expected to occur. An important example is that the
zebrafish has no stomach (Smith,
1982). In addition, some endodermal components of the gut are
generated differently. It is clear, for example, that fish and mammals
generate the pancreatic rudiment in a different manner. In fish, the pancreas
does not bud from the endodermal gut tube, but instead is derived from
endoderm that is peripheral to the intestine
(Wallace and Pack, 2003
). The
mechanisms for organ morphogenesis must therefore respond to these different
requirements and may underlie species differences.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Developmental Biology, Hagedorn Research
Institute, Niels Steensens Vej 6, 2820 Gentofte, Denmark
Present address: Umeå Centre for Molecular Medicine, Umeå
University, 901 87, Umeå, Sweden
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