|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 12 September 2007
doi: 10.1242/dev.009308
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115,
USA.
2 Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
3 Department of Craniofacial Development, Dental Institute, Kings College,
London SE1 9RT, UK.
4 Department of Molecular and Cellular Biology, Harvard University, Cambridge,
MA 02138, USA.
5 Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115,
USA.
* Author for correspondence (e-mail: ramesh_shivdasani{at}dfci.harvard.edu)
Accepted 5 August 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Barx1, Mesenchyme-epithelium interactions, Stomach development, Spleen development, Wnt signaling, Organogenesis, Wt1
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Hecksher-Sorensen et al.,
2004; Thiel and Downey,
1921). Stomach rotation and leftward movement of the dorsal
pancreas subsequently juxtapose the dorsal and ventral pancreatic buds, which
fuse and come to lie near the duodenum, whereas the spleen remains associated
with the lateral stomach wall, near its site of origin. Patterning of the
rostral gut and its neighboring organs is poorly understood and little is
known about the role of mesothelium in their development.
Stomach mesenchymal expression of the homeobox gene Barx1 seems to
be required to suppress regional Wnt activity in prospective gastric endoderm
and thus allows stomach-specific epithelial differentiation
(Kim et al., 2005). During the
period of gastric morphogenesis and gut endoderm specification, Barx1
is expressed selectively in stomach mesenchyme
(Kim et al., 2005;
Tissier-Seta et al., 1995).
Small-interfering (si) RNA-induced loss of Barx1 in recombinant
cultures of embryonic day (E) 12 mouse fetal tissues profoundly affects
differentiation of overlying stomach endoderm: intestinal marker genes are
robustly activated at the expense of stomach epithelial transcripts
(Kim et al., 2005).
Barx1-null E12 embryos have normal intestines and a small, aberrantly
shaped stomach with atypical endodermal lining; Cdx2, a specific marker of
intestinal epithelium (Silberg et al.,
2000), is expressed ectopically in the distal stomach. Levels of
the secreted frizzled-related proteins Sfrp1 and Sfrp2, soluble antagonists of
Wnt signaling (Finch et al.,
1997; Rattner et al.,
1997), are reduced in the absence of Barx1, and forced Sfrp
expression in Barx1-deficient stomach mesoderm restored gastric markers in
co-cultured endoderm (Kim et al.,
2005).
On the inbred 129/Sv genetic background, Barx1-/- mouse embryos die at E13 of unknown causes and so we could not study their subsequent development. Moreover, recombinant fetal tissue cultures convey information about molecular markers but not about histomorphology. Breeding the Barx1 mutation into a mixed genetic background with contribution from the C57BL/6 strain circumvented embryonic lethality and allowed us to elucidate an unprecedented and completely penetrant patterning defect of the stomach. In addition to this homeotic aberration, Barx1 loss causes a unique defect in development of the spleen, which is consistently mislocalized and severely hypoplastic. As Barx1 is never present in the spleen primordium but highly expressed in surrounding mesogastrium, its effects on spleen development, like those on stomach epithelial specification, must also occur across tissue planes. We confirmed the role of Barx1 in suppressing stomach endodermal Wnt activity, but our studies suggest that its role in spleen development is exerted through a different mechanism. In particular, absence of Barx1 specifically reduces mesothelial expression of Wt1, a transcription factor known to be required for spleen morphogenesis. These findings help define the basis for the diverse functions of a homeodomain transcription factor in the development of abdominal organs.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
Catnb+/lox(ex3) mice carry an allele with loxP sites
flanking exon 3 of the ß-catenin (Catnb; Ctnnb1 - Mouse
Genome Informatics) gene (Harada et al.,
1999) and were generously provided by Mark Taketo (Kyoto
University, Japan). Axin2lacZ mice have lacZ cDNA
embedded in the Axin2 locus (Yu
et al., 2005) and were kindly provided by Walter Birchmeier
(Max-Delbrück Center, Berlin, Germany). Animals were handled according to
protocols approved by an institutional committee. The morning following
vaginal plugging was regarded as day 0.5 of gestation.
Histology and immunohistochemistry
After overnight fixation in Bouin's solution or 4% paraformaldehyde, whole
embryos or isolated organs were dehydrated, embedded in paraffin and sections
of 5-6 µm were prepared. Hematoxylin and Eosin (H&E), PAS and Alcian
Blue staining were performed using routine methods. For antigen retrieval
prior to immunostaining, specimens were heated in 10 mM Na citrate buffer (pH
6.0) in a decloaking chamber (Biocare Medical, Concord, CA), then cooled for
60 minutes at room temperature. To eliminate endogenous peroxidases, tissues
were treated in methanol containing 0.5% H2O2 for 30
minutes. After blocking with normal goat serum, samples were incubated for 24
hours at 4°C with one of the following monoclonal antibodies (Ab): Cdx2
(1:20; Biogenex, San Ramon, CA), activated ß-catenin (1:500; Upstate
Millipore, Charlottesville, VA), Ter119, B220 (Ly76 and Ptprc, respectively -
Mouse Genome Informatics) (1:100; B-D Pharmingen, Franklin Lakes, NJ),
H+/K+-ATPase (2B6, 1:1000; MBL, Nagoya, Japan), smooth
muscle actin (1A4, 1:3000; Biogenex) and Muc5ac (45M1, 1:500; Novocastra,
Newcastle, UK), or rabbit antisera against gastrin (1:1000; Novocastra), Pdx1
(1:6000; gift of Christopher Wright, Vanderbilt University, TN), insulin
(1:1000; Santa Cruz Biotech, Santa Cruz, CA), Barx1 [1:9000
(Kim et al., 2005)], Wt1
(1:3000; Santa Cruz) or Sox2 (1:1000; Chemicon, Temecula, CA). Samples were
washed, incubated with biotinylated goat anti-mouse, anti-rabbit or anti-rat
IgG and treated with avidin-biotin-peroxidase complex (Vector Laboratories,
Burlingame, CA). Color reactions were developed with diaminobenzidine
hydrochloride solution (Sigma, St Louis, MO).
ß-galactosidase staining
Pregnant dams were sacrificed at various stages and embryos exposed to a
ß-galactosidase (ß-gal) staining protocol that yielded no background
in non-transgenic animals (Kim et al.,
2005). Briefly, mouse embryos or organs were isolated in
Ca2+- and Mg2+-free Hanks' Balanced Salt Solution
(Invitrogen, Carlsbad, CA), fixed for 15 minutes with 4% paraformaldehyde in
PBS, washed three times in PBS, and incubated in staining solution [PBS (pH
7.2), 1 mg/ml 5-bromo-4-chloro-3-indoyl-ß-D-galactoside, 5 mM
K3Fe(CN)6, 5 mM
K4Fe(CN)6·3H2O, 1 mM MgCl2,
0.01% sodium deoxycholate, 0.02 % NP40] for 9-10 hours at 37°C.
In situ hybridization
Sections (6 µm) were cut and mounted on SuperFrost Plus slides (Fisher
Scientific, Kalamazoo, MI), deparaffinized, rehydrated, washed in PBS and
treated with 1 µg/ml proteinase K (Roche, Indianapolis, IN) for 10 minutes.
After acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine (pH
8.0), slides were washed in 2xSSC and air-dried. Hybridization was
performed overnight at 60°C with digoxigenin-labeled antisense riboprobes
in 50% formamide, 5xSSC, 2xDenhardt's solution, 0.02% bovine serum
albumin, 0.1% Tween-20, 0.25% sodium dodecyl sulfate, 5 mM EDTA (pH 8.0) and
50 µg/ml yeast tRNA. Slides were subsequently washed in 2x or
0.2x SSC between 60 and 65°C and again in PBS, followed by
incubation for 90 minutes with 20% sheep serum. The hybridized probe was
detected by incubating tissue sections overnight at 4°C with alkaline
phosphatase-conjugated sheep anti-digoxigenin Ab diluted 1:2000 in PBS
supplemented with 5% sheep serum and 5% fetal bovine serum. Color reactions
were developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate
(Roche); slides were monitored until color development was observed and the
reaction was terminated with distilled water. In situ hybridization with
radioactively labeled Barx1 probe was performed as described
previously (Kim et al.,
2005).
Transmission electron microscopy
Embryonic stomachs were fixed overnight at 4°C in a solution containing
2.5% paraformaldehyde, 5% glutaraldehyde, 0.06% picric acid, 0.1 M Na
cacodylate, and 0.06% CaCl2, post-fixed in OsO4, and
embedded in Epon 812. Thin (0.1 µm) sections were stained with uranyl
acetate and lead citrate and examined in a JEOL 1200 electron microscope at an
accelerating voltage of 80 kV.
Reverse-transcription (RT)-PCR
Total RNA was extracted using Trizol (Invitrogen), treated with RNase-free
DNase (Ambion, Austin, TX) and reverse transcribed using oligo-(dT) primers.
Wt1 mRNA levels were assessed by conventional and SYBR Green
real-time quantitative RT-PCR (Applied Biosystems, Foster City, CA) using a
common forward primer (5'-GCCTTCACCTTGCACTTCTC-3') and the reverse
primers 5'-CATTCAAGCTGGGAGGTCAT-3' and
5'-GACCGTGCTGTATCCTTGGT-3' for conventional and real-time PCR,
respectively.
Flow cytometry
Neonatal spleen cells were dislodged with forceps and a single-cell
suspension prepared by filtering through a 30-µm strainer. Cells were
incubated on ice for 1 hour with 1 µg/ml Ter119, B220, Gr1 (Ly6g - Mouse
Genome Informatics), Cd4, Cd8a or Mac1 (Itgam - Mouse Genome Informatics)
primary Ab (B-D Pharmingen), followed by washing in PBS and further incubation
on ice for 30 minutes with fluorophore-conjugated secondary Ab. Flow cytometry
was performed on a Becton Dickinson FACScan and the data were analyzed using
FlowJo software (Tree Star, Ashland, OR).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
)] to
introduce the C57BL/6 genetic background permitted
Barx1-/- mice to survive to birth, although they succumb
soon thereafter to respiratory distress that is likely to result from cleft
palate; this defect probably reflects significant Barx1 expression in
developing branchial arches (see Fig. S1A in the supplementary material)
(Tissier-Seta et al., 1995).
Barx1-/- pups appeared in the expected proportion in over
25 litters and our findings did not vary with C57BL/6 contributions between
50% and 98%. Barx1-/- stomach was found to be greatly
reduced in size (Fig. 1A) and
escaped leftward rotation, thus presenting as a midline structure (data not
shown). The villiform lining of neonatal Barx1-/- stomach
(Fig. 1B) contrasts with the
flat mucosa in control mice (Fig.
1D) and carried two distinct surfaces: the distal 1/4 to 1/3 is
lined by villi of the intestinal variety, whereas the proximal stomach has a
highly atypical mucosa with gastric features. Periodic acid Schiff (PAS),
which stains gastric but not intestinal epithelium uniformly, highlighted this
difference (Fig. 1C).
|
). In
mice, Muc5ac, H+/K+-ATPase and pepsinogen or intrinsic
factor (IF; also known as Gif - Mouse Genome Informatics) are specific
molecular markers of these respective lineages: foveolar (pit), parietal
(oxyntic) and chief (zymogenic) cells. The distal (antral-pyloric) stomach has
a similar lining, with modified cell ratios and folds that impart a scalloped
appearance (Fig. 1D,G). Neutral
mucins in gastric pit cells stain with PAS, whereas intestinal goblet cells
are fewer in number and produce acidic mucins with affinity for both PAS and
Alcian Blue; intestinal goblet cells also express trefoil factor 3 (Tff3)
abundantly (Chinery et al.,
1992). Two homeodomain transcription factors permit further
distinction between gut segments: Pdx1 expression is scattered in distal
stomach and uniform in duodenal epithelium
(Offield et al., 1996),
whereas Cdx2 is exquisitely specific to the intestine
(Silberg et al., 2000). We
used these features to characterize Barx1-/- stomach.
The mucosal lining of Barx1-/- mid-stomach was found to
be thickly folded and the glandular morphology highly disorganized, with
branching structures and epithelial nests deep in the mesenchyme
(Fig. 1H,I). Although this
mucosa was folded, it lacked authentic villi and showed characteristic stomach
features: apical PAS staining of pit cells, scattered Pdx1 expression and
complete absence of Cdx2 or Alcian Blue staining
(Fig. 1J-M). By contrast, the
intestinal features of the caudal 
1/3 of Barx1-/-
stomach mucosa were signified by the classic villus morphology, uniform
expression of Cdx2 and Pdx1, and presence of goblet cells that stain with
Alcian Blue, PAS and Tff3 antibody (Fig.
1J-M,O). A sharp boundary (dotted lines in
Fig. 1C,J-O) always separated
the intestinal (distal) and gastric (proximal) types of epithelia; cells
expressing gastric intrinsic factor (Fig.
1N) and Cdx2 (Fig.
1M) or Tff3 (Fig.
1O), for example, were never mixed. By contrast, although
molecular markers of stomach glands were well represented in the proximal
mucosa, there was a loss of the usual boundary between corpus and antral
epithelia in the Barx1-/- mutant. In particular,
H+/K+-ATPase- and Pdx1-expressing cells, which normally
show little overlap in distribution, mixed freely in the reduced glandular
zone in the Barx1-/- stomach and Pdx1, normally an antral
marker, was expressed in cells abutting the squamous mucosa
(Fig. 2A,B).
Proximal Barx1-/- foregut showed additional anomalies
and even more extensive mixing of cell types. First, radial asymmetry was
evident, with a squamous mucosa on one surface and a cuboidal epithelium on
the other (Fig. 2C). Second,
the differentiated squamous epithelium was interspersed with strongly
PAS+ cells of the foveolar type
(Fig. 2D). These cells, which
typify stomach glands and are never seen in normal forestomach or esophagus,
appeared throughout the tubular portion of the mutant foregut; we also
observed ectopic expression of Muc5ac, the mucin responsible for PAS staining
in gastric glands (Fig. 2E).
Third, many cells lining the Barx1-/- proximal foregut
extended numerous apical cilia, a feature restricted to a few scattered cells
in control littermates. Ultrastructural analysis highlighted this finding
(Fig. 2F), which is
characteristic of prospective squamous epithelia and is likely to indicate
incomplete differentiation of Barx1-/- proximal foregut.
Finally, the esophagus was significantly truncated and we never identified a
passage lined by a contiguous squamous epithelium; probably as a result of
this, the stomach lay mainly in the thorax instead of the abdomen. Thus,
judging by its epithelium, the structure that overtly resembles an esophagus
appeared to be a highly dysmorphic fundus with a mixed squamo-glandular
lining. Sox2, a molecular marker of foregut squamous epithelium
(Que et al., 2007), was
expressed in proximal but not distal Barx1-/- foregut
(Fig. 2G,H). Cells with
smooth-muscle morphology and expression of smooth muscle actin appeared in the
correct distribution in the peripheral sub-epithelium, although the muscle
layer was discontinuous and less well differentiated than in controls
(Fig. 2I,J).
|
;
Tissier-Seta et al., 1995),
the mucosal anomalies in Barx1-/- mice must reflect
mesenchymal influence over the differentiation of the overlying endoderm.
Structural defects such as reduced stomach size, fusiform shape, fundic
dysmorphogenesis and pyloric sphincter agenesis, are likely to represent
functions intrinsic to the mesenchyme.
Genetic evidence that Barx1 inhibits stomach endodermal Wnt signaling
We previously proposed that Wnt antagonists are prominent targets of Barx1
regulation in gastric mesenchyme (Kim et
al., 2005). The prospective stomach shows a wave of Wnt activity
after E9, and we proposed that the usual decline in this activity results from
Barx1-regulated production of secreted frizzled-related proteins (Sfrps).
Recombinant fetal cell culture results supported this idea, but death of
Barx1-/- embryos precluded direct genetic confirmation.
Having overcome fetal lethality, we crossed 129/Sv-C57BL/6 hybrid
Barx1+/- and TOPGAL transgenic (Tg) mice, which carry
lacZ cDNA linked to multimerized Wnt-response elements and report
faithfully on Wnt signaling (DasGupta and
Fuchs, 1999). If the model is correct, proximal stomach endoderm
in Barx1-/-;TOPGALTg embryos should, unlike
control TOPGALTg embryos, continue to express ß-gal late in
gestation. Indeed, between E16.5 (Fig.
3B) and birth, Barx1-/- embryos carrying one
copy of the Wnt-reporter transgene showed prominent ß-gal activity
throughout the proximal foregut, a region we characterized as an atypical
gastric fundus with mixed squamous-glandular epithelium
(Fig. 2). Residual ß-gal
activity in control transgenic stomachs was minimal by E16.5
(Fig. 3A,C) and undetectable in
E18.5 stomach (data not shown) and at any stage in the developing esophagus.
By contrast, the signal in Barx1-/-;TOPGALTg
fundic stomach appeared sooner and stronger than in any other site of
embryonic Wnt activity; this signal localized to the endoderm
(Fig. 3D). We confirmed
lacZ expression by RNA in situ hybridization in E16.5 foregut, where
signal was readily detected in mutant (Fig.
3F) but not control TOPGALTg
(Fig. 3E) samples.
To monitor Wnt signaling independent of the TOPGAL reporter, we examined
ß-catenin localization. In E18.5 Barx1-/- foregut,
innumerable cells showed unambiguous localization in the nucleus
(Fig. 3G,H), whereas the signal
in littermate control foregut always appeared at cell-cell junctions
(Fig. 3I). We also mated
Barx1+/- mice with another Wnt-reporter strain,
Axin2lacZ. Insertion of lacZ cDNA into the mouse
Axin2 locus, a ubiquitous target of canonical Wnt signaling
(Jho et al., 2002), accurately
marks sites of Wnt activity (Yu et al.,
2005). Again, we readily detected prominent ß-gal activity in
the atypical fundus in E18.5 Barx1-/-;Axin2lacZ
embryos (Fig. 3K), but only
weak residual signal in the stomach and none in the esophagus of
Barx1+/-;Axin2lacZ littermates
(Fig. 3J). Together, these data
powerfully validate the idea that Barx1 functions in part to attenuate Wnt
signaling in developing stomach endoderm.
|
). We
crossed mice carrying a floxed, activating ß-catenin allele
(Harada et al., 1999) with
those expressing Cre recombinase under control of the endogenous sonic
hedgehog (Shh) gene (Harfe et
al., 2004). As Shh is highly expressed in stomach
endoderm by E8.5 (Echelard et al.,
1993), the progeny from this mating should express unrestrained
Wnt activity in epithelial progenitors. Crosses between
ShhCre/+ and ROSA26R mice
(Soriano, 1999) indicated
efficient Cre-mediated recombination in E10.5 embryos (data not shown) and we
detected significant nuclear ß-catenin in E18.5
ShhCre/+;Catnb+/lox(ex3) foregut mucosa
(Fig. 4A,B). This experimental
model mimics local Wnt activation, although differences in timing and dose
might not replicate the Barx1-/- stomach exactly.
Nevertheless, stomach size was reduced in
ShhCre/+;Catnb+/lox(ex3) embryos and its
glandular epithelium resembled that of the Barx1-/-
gastric corpus (Fig. 1B,H,I):
it was thickly folded, branched and contained many epithelial nests deep in
the mesenchyme (Fig. 4C,E). As
in Barx1-/- fundus
(Fig. 2C,D), the lining of the
distal esophagus was radially asymmetric, with well-formed squamous mucosa on
one side and cuboidal epithelium containing PAS-staining cells on the other
(Fig. 4F,G). Although
intestinal villi were absent from
ShhCre/+;Catnb+/lox(ex3) stomach, the abnormal
gastric epithelium contained many cells expressing Cdx2
(Fig. 4H,I), an intestinal
epithelial marker (Silberg et al.,
2000) that is never found in normal stomach epithelium
(Fig. 4J and data not shown).
Ectopic ß-catenin activation in the developing mouse stomach therefore
mimics the heterotopia seen in Barx1-null embryos, albeit with some
differences, and confirms that attenuation of endogenous Wnt activity is
required for normal stomach epithelial differentiation.
Unexpected and unusual requirement for Barx1 in spleen development
The position, size, morphology and histology of lower abdominal organs are
preserved in Barx1-/- embryos and neonates (data not
shown). By contrast, the spleen never appeared in the usual position, apposed
to the greater curvature of the stomach, as shown in
Fig. 5A for a control neonate;
instead, it was markedly hypoplastic and embedded within the dorsal pancreas
(Fig. 5B). Associated with this
fully penetrant anomaly was failure of the dorsal and ventral pancreatic buds
to fuse (Fig. 5B), a defect we
attribute to the absence of stomach rotation. Barx1-/-
spleen harbored typical blood cells, including those with the size and
features of megakaryocytes (Fig.
5C and data not shown), and insulin
(Fig. 5D) and Pdx1 (red box in
Fig. 7D) immunostaining
confirmed that they reside in the immediate vicinity of the pancreas. Flow
cytometric and immunohistochemical analyses revealed normal proportions of all
blood lineages (Fig. 5E and
data not shown). Thus, Barx1 loss mispositions the spleen and causes marked
hypoplasia without compromising blood or lymphocyte colonization per se.
As in control littermates (Fig.
6A), the pre-splenic mesenchyme appeared in E9.5
Barx1-/- mouse embryos as a cell aggregate within the
dorsal mesogastrium, next to the dorsal pancreatic anlage
(Fig. 6B). Focal ß-gal
activity in E10.5 and E11.5 Barx1-/- embryos that also
carry the Tlx1lacZ knock-in reporter gene
(Kanzler and Dear, 2001)
confirmed activation of a genetic program for spleen specification (data not
shown; Tlx1 is also known as Hox11). Whereas mesothelial invagination normally
separates the spleen and dorsal pancreas as they enlarge in the ensuing 2 days
(Fig. 6C),
Barx1-/- spleen showed little growth and remained attached
to the pancreatic primordium (Fig.
6D). To understand the basis for the unexpected role of Barx1 in
spleen development, we re-examined its expression domain. At E9.5 and E10.5,
Barx1 expression is reported in a columnar cell layer termed the splanchnic
mesodermal plate, which is likely to correspond to the future spleen capsule
(Hecksher-Sorensen et al.,
2004). We observed that the level of Barx1 mRNA in this
structure, which is contiguous with the mesogastrium, was comparable to that
in stomach mesenchyme, but Barx1 mRNA was excluded from wild-type
spleen anlage at all stages, including and beyond E9.5
(Fig. 6E-G and see Fig. S1A in
the supplementary material). A specific antiserum helped verify prominent
mesothelial expression of Barx1 protein
(Fig. 6J,K). Both mRNA and
protein staining indicated that mesothelial Barx1 expression is limited to the
region surrounding the stomach, spleen and caudal surface of the liver, and
does not extend into the mesenteric lining of intestinal loops
(Fig. 6H,I and see Fig. S1 in
the supplementary material). These data implicate mesothelial Barx1 expression
in expansion and morphogenesis of adjacent spleen mesenchyme and segregation
of the spleen from the dorsal pancreas.
|
|
|
|
|
;
Roberts et al., 1994),
Bapx1 (Nkx3-2 - Mouse Genome Informatics)
(Lettice et al., 1999;
Tribioli and Lufkin, 1999),
Pbx1 (Brendolan et al.,
2005), Wt1 (Herzer et
al., 1999) or Pod1 (Tcf21 - Mouse Genome
Informatics) (Lu et al., 2000)
genes show splenic atrophy or asplenia, usually in conjunction with other
defects. Mutant embryos typically initiate but fail to sustain spleen
development as cells die, fail to proliferate, or change potential. Bapx1,
Pbx1 and Tlx1 are early splenic markers and another homeobox
gene, Nkx2-5, is also suspected to regulate spleen development
(Brendolan et al., 2007). These
factors therefore represent good candidates for dependence on Barx1 and their
deficiencies might in part mediate aberrant spleen development in its absence.
To address this possibility, we assessed expression in
Barx1-/- mice of transcription factor genes implicated in
spleen development. Expression of Tlx1, Nkx2-5, Pbx1, Bapx1 and
Pod1 mRNAs appeared identical in Barx1-/- and
control embryos (Fig. 7D) and
ß-gal staining in the splenic anlage of
Barx1-/-;Tlx1lacZ embryos was indistinguishable
from that in Barx1+/-;Tlx1lacZ littermates
(data not shown). Each of the transcripts we tested was expressed in the spleen primordium in wild-type mice, and incidentally also in stomach mesenchyme, but was absent from the mesothelial envelope (Fig. 7D). Only Wt1 showed a distinctive pattern, with prominent mesothelial expression, similar or lower levels in splenic mesenchyme, and absence from stomach tissue (Fig. 8A). Thus, among the genes previously implicated in spleen development, Wt1 is the best candidate for cell-autonomous regulation by mesothelial Barx1. Indeed, Wt1 mRNA is appreciably reduced in Barx1-/- mesothelium (Fig. 8B), whereas Barx1 mRNA expression is preserved in the embryonic stomach and mesothelium of Wt1 mutants (B.-M.K., J. Alberta, D. Housman and R.A.S., unpublished). Conventional and quantitative RT-PCR confirmed reduced Wt1 mRNA levels in isolated Barx1-/- spleen (Fig. 8C; residual expression is likely to derive from spleen mesenchyme), and Wt1 immunostaining in Barx1-/- and control embryos matched results from RNA in situ hybridization. We detected Wt1 in both wild-type and Barx1-/- spleen anlagen; signals were prominent in wild-type mesothelium and substantially reduced in Barx1-null spleen, particularly in the mesothelium (Fig. 8D). By contrast, Wt1 signals were preserved in embryonic kidney (Fig. 8E), the site of highest native expression.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mice lacking Barx1 present a severe, invariant and completely penetrant form of visceral homeosis, with posteriorization of the proximal foregut. The esophagus is considerably shortened or, in the absence of markers that can distinguish mouse esophagus from squamous forestomach, might be missing entirely. Instead of the usual domed morphology, the fundic stomach is tubular, and cuboidal cells expressing neutral mucins and Muc5ac, which are usually confined to the glandular stomach, interrupt its squamous lining. Zones similarly blur in the body of the stomach, where cells normally restricted to the antrum/pylorus mix freely with corpus gland cells. By contrast, the next epithelial boundary is strictly preserved; intestinal villi occupy the entire distal stomach, but stomach and intestinal cells do not overlap in morphology or expression of regional markers. Homeosis in the Barx1-/- gut thus harbors unique features, with blurring of rostral organ and epithelial boundaries and anterior shifting of intestinal mucosa.
These gastrointestinal abnormalities extend away from the Barx1
expression domain both rostrally (esophagus) and caudally (pyloric sphincter),
a phenomenon that is reminiscent of homeotic transformations in the limbs and
axial skeleton (Capecchi, 1996;
Izpisua-Belmonte and Duboule,
1992). However, the major anomalies occur precisely in the domain
of fetal Barx1 expression, in the gastric fundus and body, and suggest a dual
role for Barx1 in stomach mesenchyme. One group of functions, likely to be
intrinsic to the mesenchyme, drives sub-epithelial differentiation and
generates the correct organ size and shape. Mesenchymal mass is reduced in
Barx1-/- mice but its viability seems intact and smooth
muscle appears in the right location; we have not addressed the mechanisms
behind the role of Barx1 in stomach morphogenesis. A second group of
non-cell-autonomous functions helps specify the overlying endoderm, as we
previously inferred in part from findings in recombinant embryonic cell
cultures (Kim et al., 2005).
Our characterization of Barx1-/- stomach reinforces this
function, extends our understanding and establishes the role of Barx1 in
suppressing endodermal Wnt activity. We demonstrate that its absence permits
persistent Wnt signaling in stomach endoderm, which is likely to disrupt
mucosal specification and differentiation as a direct consequence. However,
the scope of stomach and spleen defects in Barx1-/-
embryos, coupled with the lack of canonical Wnt signaling in normal spleen
primordium, implies that Wnt inhibition represents only a facet of Barx1
mechanisms, albeit one that is vital in stomach differentiation. Furthermore,
we cannot rule out the possibility that Barx1 regulation of spleen
morphogenesis also involves Wnt signaling through non-canonical pathways.
Unexpectedly, Barx1-/- mice have a misplaced and
severely hypoplastic spleen of a form not observed in other animal models.
Some reptiles (Falkmer, 1985)
and mice lacking the pancreas-determining factor Ptf1a
(Krapp et al., 1998) show
isolated endocrine pancreatic progenitors scattered within the spleen. By
contrast, Barx1-/- mice reveal a novel phenotype in which
a discrete spleen is embedded within intact pancreatic parenchyma. Molecular
understanding of spleen development is incomplete, but the organ is known to
originate as a mesenchymal condensation within dorsal mesogastrium, in close
apposition to the dorsal pancreas
(Brendolan et al., 2007;
Hecksher-Sorensen et al.,
2004; Thiel and Downey,
1921). Mice with defects in late pancreas development, in which
the mesenchyme is unaffected, usually have an intact spleen, whereas loss of
pancreas mesenchyme, as observed, for example, in transgenic mice with ectopic
Shh expression, is strongly correlated with asplenia
(Ahlgren et al., 1996;
Apelqvist et al., 1997;
Harrison et al., 1999); these
observations signify a role for dorsal pancreatic mesenchyme in some aspects
of spleen development. However, Barx1 mRNA and protein are
conspicuously absent from spleen and pancreas anlagen, but appear at high
levels in the epitheliod lining of these organ primordia. Barx1 is thus unique
among regulators of spleen development in exerting a pivotal influence
exclusively from the mesothelium and its expression pattern suggests that it
moderates spleen development indirectly, much as mesenchymal Barx1 helps
specify adjacent stomach endoderm. The splanchnic mesodermal plate is a known
source of developmental signals, including fibroblast growth factors 9 and 10
(Hecksher-Sorensen et al.,
2004). A key role for the prospective capsule in spleen
development is independently revealed in dominant hemimelia (Dh)
mutant mice, which lack this layer and are asplenic
(Green, 1967;
Hecksher-Sorensen et al.,
2004); our findings suggest that some Dh effects might be
mediated through Barx1.
All transcription factor genes expressed only in spleen primordium and
previously implicated in its maturation are expressed normally in
Barx1-/- spleen. These findings are consistent with the
preservation of hematopoietic potential and indicate that Barx1 is dispensable
for their expression. Features of the mutant phenotype point instead to
functions not previously explored in spleen development. First, normal
mesothelium seems to exert a Barx1-dependent trophic effect that enlarges the
organ and imparts its characteristic shape. Alternatively, the mutant
mesothelium might limit expansive and morphogenetic capacities inherent to the
spleen anlage, and we cannot exclude the possibility that the spleen defects
in Barx1-/- mice follow mainly from stomach malrotation
and attendant disturbance in configuration of the omental bursa. A second
function, separation of the spleen from the dorsal pancreas, is arguably
better attributed to cell-autonomous properties of the mesothelium, and it is
here that Wt1 loss might be especially pertinent. Unlike other genes
implicated in the specification, survival or expansion of the spleen
primordium, Wt1 alone is expressed in the mesothelium (in addition to
spleen mesenchyme); this overlap with the Barx1 expression domain
adds plausibility and significance to the result that mesothelial Wt1
expression depends on Barx1. In both Barx1-/- and
Wt1-/- embryos, the spleen is initially specified in the
correct location and ultimately much reduced in size but not absent, and
Tlx1 expression is not perturbed. Wt1-/- spleen
primordium is also reported to have a shorter connection to the prospective
pancreas (Herzer et al.,
1999), although perhaps not as short as we observe in
Barx1-/- mice. Taken together, these observations raise
the possibility that Barx1 control over spleen development might be exercised
in part through Wt1 gene regulation in the dorsal mesothelium. It is
interesting that Wt1 mRNA is reduced in Tlx1-/-
splenic mesenchyme, but not in Tlx1-/- or
Pbx1-/- mesothelium
(Brendolan et al., 2005;
Koehler et al., 2000).
Mice with targeted disruption of another homeobox gene, Bapx1,
reveal markedly different consequences of failure of the spleen and dorsal
pancreas to separate (Asayesh et al.,
2006). Bapx1-/- pancreatic endoderm undergoes
metaplastic conversion to intestinal cyst-like structures, a defect attributed
to persistent contact with spleen mesenchyme past E13.5, the stage by which
the two organs have normally separated. The authors argued that other mouse
models of asplenia avoid the same outcome because they do not expose the
pancreatic epithelial primordium directly to spleen mesenchyme
(Asayesh et al., 2006). As such
contact is evident in Barx1-/- embryos, we suggest that
either the metaplastic defect in Bapx1-/- pancreas is
unique to that genotype, or the Barx1-/- spleen lacks the
putative required factors.
Abdominal Barx1 expression is restricted to the stomach wall and mesothelium and we identify significant and distinct developmental functions in each of these locations. Our results also make a persuasive argument for Barx1-mediated inhibition of Wnt signaling in stomach endoderm and against a role for canonical Wnt signaling in spleen development. They hence demonstrate that positional and morphogenetic functions conferred by this homeobox gene occur through distinct mechanisms, even over the short distance that separates the stomach wall from its mesothelium. The pathways we have elucidated thus far - inhibition of canonical Wnt signaling in endoderm and regulation of Wt1 gene expression in mesothelial cells - represent early steps in appreciating the basis for homeobox gene functions in the gastrointestinal tract. Barx1 is likely to regulate additional events that contribute not only to foregut patterning and spleen expansion, but also to control of stomach size and shape and pyloric sphincter formation. Characterization of other such pathways will add to the growing understanding of abdominal organogenesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/20/3603/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ahlgren, U., Jonsson, J. and Edlund, H. (1996). The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development 122,1409 -1416.[Abstract]
Apelqvist, A., Ahlgren, U. and Edlund, H. (1997). Sonic hedgehog directs specialized mesoderm differentiation in the intestine and pancreas. Curr. Biol. 7,801 -804.[CrossRef][Medline]
Asayesh, A., Sharpe, J., Watson, R. P., Hecksher-Sorensen, J.,
Hastie, N. D., Hill, R. E. and Ahlgren, U. (2006). Spleen
versus pancreas: strict control of organ interrelationship revealed by
analyses of Bapx1-/- mice. Genes Dev.
20,2208
-2213.
Brendolan, A., Ferretti, E., Salsi, V., Moses, K., Quaggin, S.,
Blasi, F., Cleary, M. L. and Selleri, L. (2005). A
Pbx1-dependent genetic and transcriptional network regulates spleen ontogeny.
Development 132,3113
-3126.
Brendolan, A., Rosado, M. M., Carsetti, R., Selleri, L. and Dear, T. N. (2007). Development and function of the mammalian spleen. BioEssays 29,166 -177.[CrossRef][Medline]
Capecchi, M. R. (1996). Function of homeobox genes in skeletal development. Ann. N. Y. Acad. Sci. 785, 34-37.[Medline]
Chinery, R., Poulsom, R., Rogers, L. A., Jeffery, R. E., Longcroft, J. M., Hanby, A. M. and Wright, N. A. (1992). Localization of intestinal trefoil-factor mRNA in rat stomach and intestine by hybridization in situ. Biochem. J. 285, 5-8.[Medline]
Clevers, H. (2006). Wnt/beta-catenin signaling in development and disease. Cell 127,469 -480.[CrossRef][Medline]
DasGupta, R. and Fuchs, E. (1999). Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126,4557 -4568.[Abstract]
Dear, T. N., Colledge, W. H., Carlton, M. B., Lavenir, I., Larson, T., Smith, A. J., Warren, A. J., Evans, M. J., Sofroniew, M. V. and Rabbitts, T. H. (1995). The Hox11 gene is essential for cell survival during spleen development. Development 121,2909 -2915.[Abstract]
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75,1417 -1430.[CrossRef][Medline]
Falkmer, S. (1985). Comparative morphology of pancreatic islets in animals. In The Diabetic Pancreas (ed. B. W. Volk and K. F. Wellmann), pp. 17-52. New York: Plenum Press.
Finch, P. W., He, X., Kelley, M. J., Uren, A., Schaudies, R. P.,
Popescu, N. C., Rudikoff, S., Aaronson, S. A., Varmus, H. E. and Rubin, J.
S. (1997). Purification and molecular cloning of a secreted,
Frizzled-related antagonist of Wnt action. Proc. Natl. Acad. Sci.
USA 94,6770
-6775.
Green, M. C. (1967). A defect of the splanchnic mesoderm caused by the mutant gene dominant hemimelia in the mouse. Dev. Biol. 15,62 -89.[CrossRef][Medline]
Harada, N., Tamai, Y., Ishikawa, T., Sauer, B., Takaku, K., Oshima, M. and Taketo, M. M. (1999). Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. EMBO J. 18,5931 -5942.[CrossRef][Medline]
Harfe, B. D., Scherz, P. J., Nissim, S., Tian, H., McMahon, A. P. and Tabin, C. J. (2004). Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118,517 -528.[CrossRef][Medline]
Harrison, K. A., Thaler, J., Pfaff, S. L., Gu, H. and Kehrl, J. H. (1999). Pancreas dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9-deficient mice. Nat. Genet. 23, 71-75.[Medline]
Hecksher-Sorensen, J., Watson, R. P., Lettice, L. A., Serup, P.,
Eley, L., De Angelis, C., Ahlgren, U. and Hill, R. E. (2004).
The splanchnic mesodermal plate directs spleen and pancreatic laterality, and
is regulated by Bapx1/Nkx3.2. Development
131,4665
-4675.
Herzer, U., Crocoll, A., Barton, D., Howells, N. and Englert, C. (1999). The Wilms tumor suppressor gene wt1 is required for development of the spleen. Curr. Biol. 9, 837-840.[CrossRef][Medline]
Izpisua-Belmonte, J. C. and Duboule, D. (1992). Homeobox genes and pattern formation in the vertebrate limb. Dev. Biol. 152,26 -36.[CrossRef][Medline]
Jho, E. H., Zhang, T., Domon, C., Joo, C. K., Freund, J. N. and
Costantini, F. (2002). Wnt/beta-catenin/Tcf signaling induces
the transcription of Axin2, a negative regulator of the signaling pathway.
Mol. Cell. Biol. 22,1172
-1183.
Kanzler, B. and Dear, T. N. (2001). Hox11 acts cell autonomously in spleen development and its absence results in altered cell fate of mesenchymal spleen precursors. Dev. Biol. 234,231 -243.[CrossRef][Medline]
Karam, S. M. and Leblond, C. P. (1992). Identifying and counting epithelial cell types in the "corpus" of the mouse stomach. Anat. Rec. 232,231 -246.[CrossRef][Medline]
Kim, B. M., Buchner, G., Miletich, I., Sharpe, P. T. and Shivdasani, R. A. (2005). The stomach mesenchymal transcription factor Barx1 specifies gastric epithelial identity through inhibition of transient Wnt signaling. Dev. Cell 8, 611-622.[CrossRef][Medline]
Koehler, K., Franz, T. and Dear, T. N. (2000). Hox11 is required to maintain normal Wt1 mRNA levels in the developing spleen. Dev. Dyn. 218,201 -216.[CrossRef][Medline]
Krapp, A., Knofler, M., Ledermann, B., Burki, K., Berney, C.,
Zoerkler, N., Hagenbuchle, O. and Wellauer, P. K. (1998). The
bHLH protein PTF1-p48 is essential for the formation of the exocrine and the
correct spatial organization of the endocrine pancreas. Genes
Dev. 12,3752
-3763.
Lettice, L. A., Purdie, L. A., Carlson, G. J., Kilanowski, F.,
Dorin, J. and Hill, R. E. (1999). The mouse bagpipe gene
controls development of axial skeleton, skull, and spleen. Proc.
Natl. Acad. Sci. USA 96,9695
-9700.
Lu, J., Chang, P., Richardson, J. A., Gan, L., Weiler, H. and
Olson, E. N. (2000). The basic helix-loop-helix transcription
factor capsulin controls spleen organogenesis. Proc. Natl. Acad.
Sci. USA 97,9525
-9530.
Offield, M. F., Jetton, T. L., Labosky, P. A., Ray, M., Stein, R. W., Magnuson, M. A., Hogan, B. L. and Wright, C. V. (1996). PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122,983 -995.[Abstract]
Que, J., Okubo, T., Goldenring, J. R., Nam, K. T., Kurotani, R.,
Morrisey, E. E., Taranova, O., Pevny, L. H. and Hogan, B. L.
(2007). Multiple dose-dependent roles for Sox2 in the patterning
and differentiation of anterior foregut endoderm.
Development 134,2521
-2531.
Rattner, A., Hsieh, J. C., Smallwood, P. M., Gilbert, D. J.,
Copeland, N. G., Jenkins, N. A. and Nathans, J. (1997). A
family of secreted proteins contains homology to the cysteine-rich
ligand-binding domain of frizzled receptors. Proc. Natl. Acad. Sci.
USA 94,2859
-2863.
Roberts, C. W., Shutter, J. R. and Korsmeyer, S. J. (1994). Hox11 controls the genesis of the spleen. Nature 368,747 -749.[CrossRef][Medline]
Silberg, D. G., Swain, G. P., Suh, E. R. and Traber, P. G. (2000). Cdx1 and cdx2 expression during intestinal development. Gastroenterology 119,961 -971.[CrossRef][Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Thiel, G. A. and Downey, H. (1921). The development of the mammalian spleen with special reference to its hematopoietic activity. Am. J. Anat. 28,279 -339.[CrossRef]
Tissier-Seta, J. P., Mucchielli, M. L., Mark, M., Mattei, M. G., Goridis, C. and Brunet, J. F. (1995). Barx1, a new mouse homeodomain transcription factor expressed in cranio-facial ectomesenchyme and the stomach. Mech. Dev. 51, 3-15.[CrossRef][Medline]
Tribioli, C. and Lufkin, T. (1999). The murine Bapx1 homeobox gene plays a critical role in embryonic development of the axial skeleton and spleen. Development 126,5699 -5711.[Abstract]
Yu, H. M., Jerchow, B., Sheu, T. J., Liu, B., Costantini, F.,
Puzas, J. E., Birchmeier, W. and Hsu, W. (2005). The role of
Axin2 in calvarial morphogenesis and craniosynostosis.
Development 132,1995
-2005.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Grigoryan, P. Wend, A. Klaus, and W. Birchmeier Deciphering the function of canonical Wnt signals in development and disease: conditional loss- and gain-of-function mutations of {beta}-catenin in mice Genes & Dev., September 1, 2008; 22(17): 2308 - 2341. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
