1 Department of Cell and Developmental Biology, Cornell University, Weill
Medical School, New York, NY, 10021, USA
2 Dipartimento di Biologia Animale, Universita' di Modena e Reggio Emilia, Via
Università 4, 41100, Modena, Italy
3 Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX 77030, USA
4 The Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, University of
Toronto, Toronto, Ontario M5G 1X5, Canada
5 Università Vita-Salute San Raffaele, 20132 Milan, Italy
6 Department of Pathology, Stanford University School of Medicine, Stanford, CA
94305, USA
* Author for correspondence (e-mail: lis2008{at}med.cornell.edu)
Accepted 29 April 2005
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SUMMARY |
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Key words: Spleen ontogeny, Organogenesis, Pbx1, Hox11 (Tlx1), Nkx2.5, Nkx3.2 (Bapx1), Pod1 (capsulin, Tcf21), Wt1, Transcriptional regulation, Pbx1 targets, Proliferation, Mouse
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Introduction |
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In vertebrates, the mesodermally derived spleen normally displays
left-handed asymmetry (Boorman and
Shimeld, 2002) and has been considered to be a landmark organ for
detecting laterality defects (Aylsworth,
2001
). But, interestingly, mice with left-right (LR) asymmetry
defects, such as the Inv/Inv
(Yokoyama et al., 1993
) and
ActRIIB (Oh et al., 2002) mutant mice exhibit either normally
developed spleens or, infrequently, splenic hypoplasia. Furthermore, asplenic
mouse models, such as the Dh spontaneous mutant
(Green, 1967
) and the
Nkx3.2 (Bapx1 - Mouse Genome Informatics) mutant mouse
(Lettice et al., 1999
;
Tribioli et al., 1999), appear to exhibit only regional perturbations of LR
asymmetry in the primordial splenopancreatic mesoderm
(Hecksher-Sorensen et al.,
2004
). Other asplenic mouse models, such as those mutant for
Hox11 (Tlx1 - Mouse Genome Informatics)
(Roberts et al., 1994
;
Dear et al., 1995
), display
asplenia as the sole organ abnormality. Likewise, in humans, asplenia may
present as the sole organ anomaly, without perturbations of LR asymmetry
(Rose et al., 1975
;
Waldman et al., 1977
).
Overall, these findings underscore the notion that, both in mice and humans,
mechanisms other than the regulation of LR asymmetry must be responsible for
the control of splenic cell fate specification and morphogenesis.
Recent advances in mouse genetics have led to the discovery of novel genes
required for early spleen ontogeny. These include Hox11
(Roberts et al., 1994;
Dear et al., 1995
;
Kanzler and Dear, 2001
),
Nkx3.2 (Lettice et al.,
1999
; Tribioli et al., 1999), Pod1
(Quaggin et al., 1999
;
Lu et al., 2000
) and
Wt1 (Herzer et al.,
1999
); however, the hierarchical relationships among these genes
remain unknown. This limited collection of genes also includes Pbx1
(Nourse et al., 1990
;
Kamps et al., 1990
), which
encodes a TALE class (Burglin,
1997
) homeodomain protein, the absence of which results in
embryonic asplenia with 100% penetrance
(Selleri et al., 2001
).
Although the role of Pbx1 in spleen development is undefined, its reported
biochemical in vitro interaction with homeodomain protein Hox11 through the
hexapeptide motif (Shen et al.,
1996
) raises the possibility that these two homeoproteins may
cooperate in spleen ontogeny, as Hox11 is also required for spleen formation
(Roberts et al., 1994
;
Dear et al., 1995
).
Heterodimers of Pbx and other TALE proteins of the Meinox family, such as
Meis (Bischof et al., 1998;
Chang et al., 1997
) and
Pknox/Prep1 (Berthelsen et al.,
1998
; Knoepfler et al.,
1997
; Fognani et al.,
2002
), form stable nuclear complexes, and biochemical analyses
suggest that these complexes regulate several genes
(Swift et al., 1998
). Indeed,
we have found that loss of Pbx1 causes multiple organogenesis defects in the
mouse and lethality in utero at E15.5
(Selleri et al., 2001
). These
defects include abnormalities in patterning and development of the skeleton
(Selleri et al., 2001
), in
pancreas morphogenesis and function (Kim
and Selleri et al., 2002
), in adrenal/urogenital development
(Schnabel et al., 2003a
;
Schnabel et al., 2003b
), and
in caudal pharyngeal pouch-derived organ formation and patterning
(Manley and Selleri et al.,
2004
), as well as impaired hematopoiesis
(DiMartino and Selleri et al.,
2001
). These findings underscore the notion that Pbx1 serves as a
key developmental regulator, although the crucial genetic and transcriptional
pathways underlying its specific developmental roles have not been
established.
In this report, we investigated spleen ontogeny by analyzing asplenic mouse models lacking Pbx1, Hox11, Nkx3.2 or Pod1 (capsulin). Our studies define a genetic hierarchy in which Pbx1 serves a central and crucial role as a common co-regulator in spleen ontogeny.
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Materials and methods |
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Histological analysis, immunohistochemistry and in situ hybridization
Embryos from E10.5 to E15.5 were harvested and fixed overnight at 4°C
in phosphate-buffered saline (PBS) containing 4% (w/v) paraformaldehyde (PFA).
For histological analysis and immunohistochemistry with a mouse anti-Pbx1b
primary antibody (Jacobs et al.,
1999), protocols were followed as described
(Selleri et al., 2001
).
Single-stranded sense and antisense riboprobes for in situ hybridization on
frozen sections were specific for Hox11
(Dear et al., 1995
),
Nkx3.2 (Tribioli et al.,
1997
), Nkx2.5 (Lyons
et al., 1995
), Pbx1 (3' UTR), Pod1
(Quaggin et al., 1998
) and
Wt1 (Herzer et al.,
1999
).
Assessment of ß-galactosidase activity
Embryos heterozygous for Hox11lacZ were collected at
E11.5 and E12.5 and stained for ß-galactosidase as described previously
(Dear et al., 1995).
Staining of germinal centers
Mice 6-8 weeks old were immunized by intravenous (i.v.) injection of
5x107 sheep red blood cells (SRBC) in PBS. Mice were
sacrificed and the spleens processed for immunohistochemistry with
biotinylated lectin peanut agglutinin (PNA; Vector Laboratories, Burlingame,
CA) as described (Inada et al.,
1998).
BrdU analysis
Pregnant Pbx1+/- and Hox11+/-
female mice, carrying embryos at E12.5 and E13.5, respectively, were injected
intraperitoneally with 5-bromo-2-deoxy-uridine (BrdU) (50 µg/g of body
weight) and BrdU incorporation was assayed as previously described
(Selleri et al., 2001). The
number of BrdU-positive cells (dark brown nuclei) within the developing spleen
were counted in six to eight sagittal sections (10 µm thickness) for each
genotype. Quantitative analysis of BrdU immunoperoxidase-stained sections was
made on a Nikon microscope equipped with a video camera. TUNEL assays were
performed as described by Gavrieli et al.
(Gavrieli et al., 1992
).
Cell culture and immunostaining
Embryonic spleens at day E16-17 were dissected and trypsinized (0.25% final
concentration) for 10-15 minutes at 37°C. The cell suspension was washed
twice and cultured in Dulbecco's Modified Eagle Medium (D-MEM), supplemented
with 10% fetal calf serum (Celbio), 2 mM L-glutamine (Invitrogen), 100 U/ml
penicillin and 100 mg/ml streptomycin (Invitrogen) in humidified 5%
CO2, and used as a primary cell culture for chromatin
immunoprecipitation (ChIP) assays. Primary spleen cultures were
immunophenotyped by using an -smooth muscle actin Ab (ASMA; Santa Cruz
Biotech), which stains mesoderm-derived cells. To generate immortalized spleen
embryonic cell lines from Pbx2-/- embryos, the NIH 3T3
protocol (Todaro et al., 1963) was used.
Western blot analysis
Western blot analysis was performed as described previously
(Berthelsen et al., 1996;
Jacobs et al., 1999
). The
following antibodies were used: anti-Hox11 (1:1,000) (Santa Cruz Biotech, CA),
anti-Pbx1b (1:1,500) (Jacobs et al.,
1999
) and anti-Prep 1 (1:1,500; Upstate Biotechnology).
In vitro transcription assays
NIH 3T3 cells were cultured in D-MEM supplemented with 5% fetal calf serum
and 5% delipidated fetal calf serum. Transient transfections were performed
using Lipofectamine 2000 (Invitrogen) according to the manufacturer's
instructions. For Hox11 promoter analysis, the following constructs
were used: a luciferase reporter vector (pGL2); a pGL2 construct carrying an
EcoRI-SalI 0.9 kb fragment, corresponding to the
Hox11 promoter region (pGL2-540)
(Arai et al., 1997); pcDNA3
constructs containing the cDNA of Pbx1a or Prep1
(Berthelsen et al., 1998
) and a
pBlueScript-Hox11 construct containing the full-length cDNA of Hox11 (obtained
from Dr N. Hoehler) (Koehler et al.,
2000
). Cells were lysed 40-45 hours after transfection and assayed
for luciferase activity (Benasciutti et
al., 2004
). Values were normalized for ß-gal activity. Data
represent means of triplicate values from a representative experiment. All
transfections were independently performed three times.
Electrophoretic mobility shift assays
Electrophoretic mobility shift assays (EMSA) were performed as described
(Berthelsen et al., 1996;
Jacobs et al., 1999
) using
nuclear extracts from spleen cells. The following oligonucleotides, spanning
Pbx1-binding sites within the Hox11 promoter (AB 000681)
(Arai et al., 1997
), were
employed in EMSA reactions: 5'-CCAAAGGCTTGTGACTGCTTTTCAGG-3' PX1
and 5'-CCAAAGGCTTGTCGACGCTTTTCAGG-3' PX1 mutated.
The following antibodies were used: rabbit polyclonal antibodies specific for Pbx1 (P-20) and Hox11 (C-18; Santa Cruz Biotech, CA).
Chromatin immunoprecipitation assay
Formaldehyde crosslinking and chromatin immunoprecipitation were performed
as described (Frank et al.,
2001). Samples were immunoprecipitated overnight at 4°C with
the following antibodies: mouse monoclonal specific for Pbx1b
(Jacobs et al., 1999
), rabbit
polyclonal specific for Hox11 (Santa Cruz Biotech, CA), mouse monoclonal
anti-Green Fluorescent Protein (anti-GFP; Santa Cruz Biotech, CA) and normal
rabbit serum (Covenge Research). Immune complexes were recovered by adding 30
µl of blocked protein G beads and incubated for 2 hours at 4°C. Beads
were washed and eluted, and crosslinks were reversed as described (Aparicio et
al., 1999). The eluted DNA was resuspended in 30-60 µl water. A region
within the Hox11 promoter containing the Pbx1-binding sites (-359
s/-115 as and -258 s/-54 as) and a control region within the same promoter
(-1170 s/-891 as) were amplified by PCR using specific primer pairs. One
primer pair that amplifies a region of the Bmp4 promoter was also
used as additional negative control: 5'-ACGCACTTCCCTGATTCTCGTC-3'
(-359 s) and 5'-AGCAGTCACAAGCCTTTGGATTAC-3' (-115 as) product size
244 bp; 5'-TCTCACAAACCCAGAGCCATTC-3' (-258 s) and
5'-TAGCAGCCACTCCAACTCAGTCTC-3' (-54 as) product size 204 bp;
5'-TGAGAACAACTACCTGCTTCGTGC-3' (-1170 s) and
5'-TGGAGACTTGACTTGCCCAACC-3' (-891 as) product size 279 bp; and
5'-AATGAACAAACACCACTCTCCCTC (Bmp4 s) and
5'-AACACCAGACCGAAAAGATGACTG (Bmp4 as) product size 350 bp.
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Results |
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Pbx1 is required for onset of Hox11 (Tlx1) and Nkx2.5 gene expression in the splenic anlage
Visualization of gene transcripts known to be present in the condensing
splenic mesenchyme within the Dm was conducted to trace spleen ontogeny at
early embryonic stages (E11-12.5) and to establish hierarchical requirements
for specific transcription factors in splenic cell fate specification and
morphogenesis. Using in situ hybridization, we assessed expression of
Hox11 (Koehler et al.,
2000; Kanzler and Dear
2001
) (Fig. 2A-D)
and Nkx2.5 (Patterson et al.,
2000
; Hecksher-Sorensen et
al., 2004
), which are first observed in Dm mesenchyme between
E10.5 and E11 and are regarded as the earliest known markers for splenic cell
fate.
|
|
Expression of Wt1 was severely down regulated in the condensing mesenchyme of Pbx1-/- Dm, when compared with wild-type littermates. However, its expression was well maintained in the outer mesothelial lining of the Pbx1-/- splenic primordium, both at E11-11.5 and E12-12.5 (Fig. 2E-H). As Hox11 is absent in Pbx1-/- embryos, and Wt1 is regulated by Hox11 (Koehler, 2000) (data not shown), these findings suggest a genetic hierarchy whereby Pbx1 regulates Hox11, which in turn regulates Wt1 in condensing splenic mesenchyme. Nevertheless, while Wt1 expression in Dm splenic mesenchyme is hierarchically dependent upon Pbx1, probably through Hox11, its expression is independent of Pbx1 in the developing splenic capsule.
Pbx1 and Pod1 independently regulate Nkx2.5 in the splenic anlage
Expression of Pod1 (Robb et
al., 1998; Lu et al.,
2000
) (Fig. 2I-L) and Nkx3.2 (Fig. 2M-P)
overlaps with Hox11 and Nkx2.5 in the condensing splenic
mesenchyme. In addition, gene targeting studies have shown that Pod1
(Quaggin et al., 1999
;
Lu et al., 2000
) and
Nkx3.2 (Lettice et al.,
1999
; Tribioli et al., 1999) are required for spleen development.
Pod1 (Fig. 2I-L) and
Nkx3.2 (Fig. 2M-P)
expression is maintained in Pbx1-/- embryos, and
Pbx1 expression is unperturbed in the condensing mesenchyme of both
Pod1-/- (Fig.
3C,D) and Nkx3.2-/-
(Fig. 3E,F) embryos at
E12-12.5. Thus, Pbx1 expression in Dm condensing mesenchyme is not
dependent on the presence of either Pod1 or Nkx3.2. We also confirm that
Nkx3.2 is expressed in the condensing mesenchyme of
Pod1-/- embryos at E12-12.5 (see Fig. S1A,B in the
supplementary material), as already reported by others
(Lu et al., 2000
).
Additionally, Pod1-/- expression is unperturbed in
Nkx3.2-/- embryos at E12-12.5 (see Fig. S1C,D in the
supplementary material). Thus, the Pod1 and Nkx3.2 pathways
appear to be separate in early spleen development. Furthermore, our present
studies (Fig. 2A-D) and work by
Lettice et al. (Lettice et al.,
1999
) reveal that Pbx1 and Nkx3.2 independently
regulate Hox11 in a hierarchical fashion. Therefore, Hox11
expression is dependent on both Pbx1 (Fig.
2A-D) and Nkx3.2 (Lettice et
al., 1999
). Moreover, no expression of Nkx2.5 was
observed in the condensing splenic mesenchyme of Pod1-/-
or Pbx1-/- embryos
(Fig. 3I,J; K,L). Conversely,
we demonstrated that Nkx2.5 is still expressed in the condensing
splenic mesenchyme of Nkx3.2-/- embryos (see Fig. S1E,F in
the supplementary material) at E12-12.5. Thus, Nkx2.5 expression is
dependent on both Pbx1 (Fig.
3K,L) and Pod1 (Fig.
3I,J), although it is not dependent on Nkx3.2 (see Fig. S1E,F in
the supplementary material). Taken together, these results suggest that
Pbx1 impinges on the control of the separate Pod1 and
Nkx3.2 pathways, both essential for spleen development, by
functioning upstream of Hox11 and Nkx2.5, respectively.
|
Given the requirement for both Hox11
(Roberts et al., 1994;
Dear et al., 1995
) and
Pbx1 in spleen development and their cellular colocalization in the
splenic anlage, their potential genetic interaction during spleen ontogeny was
assessed. Pbx1+/- and Hox11+/- mice
were intercrossed and offspring were examined at 6 to 8 weeks of age
(Table 1). A high percentage
(80%) of Pbx1+/-;Hox11+/- double
heterozygous mice displayed hypoplastic and malformed spleens, compared with
wild-type or single heterozygous littermates
(Fig. 4E,F;
Table 1), of which a very low
percentage exhibited splenic morphological abnormalities such as minor
indentations (Table 1). The
spectrum of malformations of double heterozygous spleens
(Fig. 4F) comprised sickle
shapes, presence of indentations, tubercles and nodules, as well as fusions of
two spleens (polysplenia). Thus, Pbx1 and Hox11 genetically
interact in spleen development. Despite the observed morphological
abnormalities, Pbx1+/-;Hox11+/- double
heterozygous mice exhibited normal splenic architecture
(Fig. 4G,H), germinal center
(GC) (Dent et al., 1997
)
formation (Fig. 4I,J) and
primary immune function (not shown).
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|
Given the in vivo genetic interaction of Pbx1 and Hox11
in spleen development and their cellular colocalization, BrdU in vivo labeling
was also performed in E13.5 Hox11-/- embryos. The splenic
anlage of Hox11-/- embryos
(Fig. 5C,D) was remarkably
similar to that of Pbx1-/- embryos
(Fig. 5A,B), with a reduction
in the percentage of S-phase cells of 50% in both mutants. Interestingly,
the splenic primordium of E13.5 Hox11-/- embryos, unlike
that of Pbx1-/- embryos, also exhibited a modest increase
in apoptosis, mostly localized to the mesothelium surrounding the mesenchyme
of the splenic primordium (not shown). In sum, loss of either Pbx1 or
Hox11 presents a comparable phenotype, affecting the proliferation of
mesenchymal splenic progenitor cells and preventing normal expansion of the
splenic anlage.
Hox11 is a direct in vivo target of Pbx1 in spleen ontogeny
To investigate the possibility that Pbx1 may directly regulate
Hox11 expression, sequences of the Hox11 promoter that are
conserved between the mouse and human genes were examined for Pbx-binding
sites. A potential Pbx-binding site (PX1) was identified within the 540 bp
Hox11 region that displays promoter activity
(Arai et al., 1997), as
indicated in Fig. 6A. To
determine if the PX1 element could support the formation of a Pbx1 DNA-binding
complex, EMSA assays were performed using nuclear extracts from primary
embryonic spleen cells. A slow-migrating band containing Pbx1, as demonstrated
by its specific competition with an anti-Pbx1 antibody, was observed to form
on an oligonucleotide containing the PX1 site
(Fig. 6B: left panel, lanes 1
and 2).
|
Possible in vivo Hox11 promoter-specific binding by Pbx1 and Hox11
was examined by chromatin immunoprecipitation (ChIP) performed on primary cell
cultures established from murine embryonic spleens at E16. These primary
murine spleen cultures, which uniformly expressed the mesodermal marker
alpha-smooth muscle actin (green fluorescence;
Fig. 6C: top panel), exhibited
high levels of Pbx1b, Hox11 and the Meinox co-factor protein Prep1, as
detected by western blot analysis (Fig.
6C: bottom panel). The binding of Pbx1 and Hox11 was examined at
the region of the Hox11 promoter
(Arai et al., 1997;
Fig. 6A) that contains the PX1
site, where assembly of the Pbx1-Hox11 heterodimer had been detected by EMSA.
Two different pairs of primers within this region (spanning regions -258 to
-54, depicted in red, and -359 to -115, depicted in teal) amplified sequences
within the Hox11 promoter that had been immunoprecipitated by the
anti-Pbx1b antibody (Fig. 6D:
left and middle panels). As a control for Pbx1b antibody specificity, two
different sets of primers (one on the Hox11 promoter: -1170 to -891;
depicted in blue; and one on the Bmp4 promoter) did not amplify their
respective intervening sequences after immunoprecipitation
(Fig. 6D: right panel).
The potential binding of Hox11 at its own promoter in vivo in embryonic murine spleen cells was also examined by ChIP assay. Similar to Pbx1, Hox11 was present at the region of Hox11 promoter activity (Fig. 6E), as detected by both primer pairs that revealed Pbx1 on the Hox11 promoter (Fig. 6E: left panel and data not shown). Overall, these results indicate recruitment of both Pbx1 and Hox11 on the Hox11 promoter in vivo in spleen embryonic cells. Thus, Hox11 is a direct target of Pbx1. The simultaneous binding of Hox11 to its own promoter suggests that it may contribute to an auto-regulatory circuit in spleen development.
Hox11 autoregulates its own promoter with Pbx1
The functional consequences of potential interactions of Pbx1 and Hox11 for
Hox11 expression were tested in transient transcription assays using
NIH 3T3 fibroblasts. For these studies, we employed a luciferase reporter
construct containing the promoter regulatory region of Hox11 (p540)
(Arai et al., 1997), which
spans the PX1 site. When the p540 reporter gene was co-transfected with the
Pbx1 construct no activation above background was observed
(Fig. 6F). Co-expression of
Pbx1 and a representative Meinox family protein, Prep1 (highly expressed in
spleen mesenchyme; Fig. 6C: bottom panel), resulted in transcriptional activation of two- to threefold
above background levels (Fig.
6F). Significantly, co-transfection of Hox11 with Pbx1 and Prep1
resulted in an eight- to ninefold increase in transcription above the baseline
(Fig. 6F). These results
demonstrate that synergistic activation of the Hox11 promoter is
achieved by the association of the three homeodomain proteins, consistent with
a Pbx1-dependent autoregulatory role for Hox11 to enhance and/or maintain its
own expression during spleen development.
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Discussion |
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Loss of Pbx1 does not affect LR decisions or development of the splanchnic mesoderm
Regional perturbations of LR asymmetry have been associated with spleen
agenesis in Dh spontaneous mutants and Nkx3.2-/-
embryos (Hecksher-Sorensen et al.,
2004). However, our studies demonstrate that asplenia in
Pbx1-/- embryos is not the result of perturbations of LR
asymmetry, providing further evidence that asplenia is not always associated
with LR asymmetry defects. This notion is best supported by the presence of
spleen agenesis as the sole organ abnormality, without perturbations of LR
asymmetry, in mice that lack Pod1
(Quaggin et al., 1999
) or
Hox11 (Roberts et al.,
1994
; Dear et al.,
1995
) (N. Dear, personal communication). Similarly,
Pbx1-/- embryos do not exhibit anomalies of LR asymmetry
in the heart (C. P. Chang, unpublished), lungs (B. Hogan, personal
communication), liver or stomach (data not shown). Although the
Pbx1-/- pancreas displays an anteroposterior (AP)
patterning defect, it does not present LR asymmetry abnormalities
(Kim and Selleri et al.,
2002
).
Furthermore, at E10-10.5, the architecture of the splanchnic mesoderm,
which normally exhibits an epithelial-like cellular organization, remains well
preserved in Pbx1-/- embryos. This contrasts with
Dh mutant embryos, in which the splanchnic mesoderm is replaced by
unorganized mesenchyme, and Nkx3.2-/- embryos, in which it
is in part defective (Green,
1967; Hecksher-Sorensen et
al., 2004
). Thus, loss of Pbx1 does not result in
perturbations of very early developmental choices, such as LR position and
differentiation of the splanchnic mesoderm.
Reiterative requirement for Pbx1 in early splenic morphogenesis and later anlage expansion
In vertebrates Pbx1 is a key developmental regulator required for
the ontogeny of most organ systems
(Selleri et al., 2001). In
this study, we demonstrate that Pbx1 is reiteratively required in
spleen development. First, it affects, at least in part, the fate of
condensing mesenchymal cells during early organogenesis, at E11-11.5, as
demonstrated by the absence of Nkx2.5 and Hox11 in
Pbx1-/- Dm. Therefore, Pbx1 genetically regulates
both Hox11 and Nkx2.5 in early spleen morphogenesis.
Nkx2.5 is regarded as one of the earliest known markers for spleen
progenitor cells (Patterson et al.,
2000
), and its expression overlaps with Hox11, although
its requirement in spleen genesis has not yet been shown since Nkx2.5
loss causes lethality in utero at E9-10, before development of the splenic
anlage (Lyons et al., 1995
).
Second, at later stages of organogenesis, by E13.5, Pbx1 is required
again for splenic progenitor cell proliferation, as detected by BrdU
incorporation experiments, underscoring the essential role of Pbx1 in
organ expansion. Thus, Pbx1 exhibits temporally distinct roles in
spleen ontogeny, reminiscent of its dual contributions to skeletal development
(Selleri et al., 2001
).
Regulation of another marker of early splenic anlage, Wt1, is also dependent on Pbx1, possibly through regulation of Hox11, in the splenic condensing mesenchyme. It is of interest that regulation of Wt1 expression is independent of Pbx1 in the outer mesothelial lining of the splenic anlage, which normally does not express Hox11, and will give rise to the splenic capsule. Taken together, these results support a scenario where Pbx1 regulates Hox11, which in turn regulates Wt1 in the splenic mesenchyme, whereas in the mesothelial lining of the developing spleen regulation of Wt1 is uncoupled from Pbx1. Thus, Pbx1 can be considered as the uppermost known genetic regulator within the Hox11-Wt1 pathway in the non-mesothelial splenic mesenchyme (Fig. 7A).
|
Although previous work has demonstrated asplenia in Hox11 mutants
(Roberts et al., 1994;
Dear et al., 1995
), until now
the cellular basis of this defect was mostly unknown. Indeed, apoptosis was
not detected in Hox11-/- splenic primordium in a previous
study (Roberts et al., 1995
),
while it was documented in another report that used a different
Hox11-deficient model (Dear et
al., 1995
). In the present study, we observed a modest increase of
apoptosis in Hox11-/- splenic primordium. The apoptotic
cells were mostly localized to the outer mesothelial lining of the splenic
anlage, which gives rise to the spleen capsule and does not normally express
Hox11. Thus, it appears that such a subtle increase of apoptosis
cannot be responsible for the complete lack of spleen development in
Hox11-deficient embryos. Conversely, our finding that, by E13.5,
Hox11-/- spleen progenitor cells exhibit a marked defect
in cellular proliferation comparable with that in Pbx1-/-
embryos is consistent with the hypoplasia of Hox11-/-
splenic anlage (Roberts et al.,
1994
; Dear et al.,
1995
) and the demonstrated involvement of Hox11 in
cellular proliferation and cell cycle control
(Kawabe et al., 1997
;
Hough et al., 1998
;
Owens et al., 2003
). Finally,
the finding of a common cellular defect (i.e. impaired progenitor cell
proliferation) in spleen development of Pbx1-/- and
Hox11-/- embryos further corroborates the observation that
Pbx1 genetically regulates Hox11.
In sum, the requirement for Pbx1 in spleen ontogeny appears to be reiterative. This reiterative role can account for the complete absence of the spleen, which would not otherwise be explained either by a partial impairment of splenic cell fate specification or by a 50% decrease in progenitor cellular proliferation in the splenic anlage, but probably results from the summation of these defects.
Genetic interaction of Pbx1 and Hox11 in spleen ontogeny
The finding of a high percentage (80%) of
Pbx1+/-;Hox11+/- double heterozygous
mice displaying severely hypoplastic and malformed spleens
(Fig. 4E,F;
Table 1), compared with single
heterozygotes, demonstrates that Pbx1 and Hox11 genetically
interact in vivo in spleen development. The wide spectrum of malformations of
Pbx1+/-;Hox11+/- double heterozygous
spleens, which includes fusions of two spleens, mimics polysplenia, a human
congenital condition. In polysplenia two or more splenic masses, hypoplastic
and irregularly shaped (splenules), are present lateral to the stomach
(Lodewyk et al., 1972). Unlike
human asplenia, which involves life-threatening infections in children
(Waldman et al., 1977
),
polysplenia is associated with normal splenic function
(Lodewyk et al., 1972
).
Despite their morphological abnormalities,
Pbx1+/-;Hox11+/- double heterozygous mice
exhibit normal splenic architecture, germinal center formation and primary
immune function (not shown), thus closely modeling the human polysplenic
condition.
Hox11 is a direct in vivo target of Pbx1 and autoregulates its own promoter with Pbx1 in spleen ontogeny
Despite the growing understanding of Hox and TALE homeoprotein functions in
development (Krumlauf, 1994;
Mann and Affolter, 1998
;
Popperl et al., 2000
;
Selleri et al., 2001
;
Waskiewicz et al., 2002
;
Hisa et al., 2004
;
Selleri et al., 2004
) and
their functional interactions (Popperl et
al., 1995
; Maconochie et al.,
1997
; Jacobs et al.,
1999
; Ferretti et al.,
2000
; Manzanares et al.,
2001
; Samad et al.,
2004
), to date, only a few direct target genes have been reported
(Rauskolb et al., 1993; Graba et al.,
1997
; Bromleigh and Freedman,
2000
; Theokli et al.,
2003
). In this study, we provide the first in vivo evidence that
Pbx1 directly regulates Hox11 in embryonic spleen cells.
Interestingly, at E9.5, Pbx1 is already expressed in the mid-gut
mesenchyme (Schnabel et al.,
2001
), from which the spleen is derived, well before the onset of
Hox11 expression (Kanzler and
Dear, 2001
). And indeed, Pbx1 controls the onset of
Hox11 expression at E11 within the Dm, as demonstrated by our in situ
hybridization experiments. Although Pbx1 expression starts to
decrease in the splenic anlage after E13.5 (data not shown), Hox11
persists in the spleen until birth
(Kanzler and Dear, 2001
),
suggesting that Pbx1 is required for the onset of Hox11 expression
and for its continued expression in early spleen development, until E13.5,
although it is not necessary for Hox11 maintenance in later phases of
organogenesis.
Hox11 is one of the earliest known markers for spleen cell
progenitors (Dear et al.,
1995). A useful tool to monitor Hox11 transcription in
the developing spleen is provided by Hox11lacZ mice
(Dear et al., 1995
), in which
lacZ expression is dependent on Hox11 regulatory sequences
and faithfully recapitulates Hox11 expression. Analysis of
Hox11lacZ/lacZ embryos previously demonstrated that
lacZ expression is normally initiated in the absence of
Hox11 (Dear et al.,
1995
), suggesting that the Hox11 protein is not required for
initiation of its own transcription in the splenic mesenchyme. These findings
indicate that other factors might be necessary for the onset of Hox11
transcription. Here, we identify Pbx1 as one such factor that activates
Hox11 transcriptional onset and early expression in the splenic
anlage until E 13.5.
Pbx1 and Hox11 bind to a potential Pbx-binding site (PX1) within the
Hox11 promoter, as shown by EMSA assays conducted on embryonic spleen
primary cells. Additionally, Pbx1 and Hox11 bind the Hox11 promoter
in vivo in embryonic spleen cells, as revealed by ChIP assays. Regulatory
interactions of Hox genes, such as the induction of Hoxb1 segmental
expression by Hoxb1 and Hoxa1 through auto- and cross-regulatory loops, have
been documented in developmental processes
(Popperl et al., 1995;
Studer et al., 1996
;
Studer et al., 1998
). Here, we
reveal an autoregulatory loop for an orphan Hox gene, Hox11, which is
non-clustered but bears a hexapeptide motif
(Shen et al., 1996
). Taken
together, our findings establish that Hox11 is a direct target of
Pbx1 and that, simultaneously, it regulates its own promoter. Significantly,
co-transfection of Hox11 with Pbx1 and Prep1 resulted in a striking increase
in transcription above baseline, demonstrating that synergistic activation of
the Hox11 promoter is achieved by the association of the three
homeoproteins, consistent with a Pbx1-dependent autoregulatory role for Hox11
to enhance and/or maintain, at least in part, its own expression during spleen
development. Interestingly, additional potential Pbx-Meinox-binding sites were
identified within the Hox11 promoter downstream of the PX1 site (not
shown), suggesting that multiple binding sites within close proximity might be
used, simultaneously or at different times, for a complex, multi-faceted
transcriptional regulation of Hox11 in spleen development.
Establishment of a Pbx1-dependent genetic and transcriptional network that regulates spleen ontogeny
In addition to establishing that Pbx1 is the most upstream known
direct regulator of Hox11 in spleen ontogeny
(Fig. 7A), our studies
demonstrate an even broader role for Pbx1 in spleen development.
Pbx1 regulates key genes downstream of Nkx3.2 and
Pod1, which we show to control spleen development through separate
genetic pathways (Fig. 7A).
Both Nkx3.2 (Lettice et al.,
1999; Tribioli et al., 1999) and Pod1
(Quaggin et al., 1999
;
Lu et al., 2000
) are essential
for spleen development, and their expression in condensing splenic mesenchyme
overlaps with Hox11 and Nkx2.5. The specific mechanisms and
cellular behaviors by which the Nkx3.2 transcription factor regulates spleen
development are as yet unknown, while Pod1 has been proposed to control
splenic cell survival (Lu et al.,
2000
). Interestingly, Lu et al. have reported that the spleen
primordium of Pod1-/- embryos does not further expand
after E12.5 and starts to undergo apoptotic cell death. As a result, after
E12.5, expression of all splenic markers, including Nkx3.2,
disappears from the degenerating splenic primordium of
Pod1-/- embryos (Lu et
al., 2000
). Our studies demonstrate that Pbx1 expression
is not dependent on the presence of either Nkx3.2 or Pod1 in the splenic
mesenchyme (Fig. 7A). Likewise,
the requirement for both of these transcription factors in splenic development
is Pbx1 independent (Fig.
7A). In addition, our findings that Nkx3.2 is expressed
in the condensing mesenchyme of Pod1-/- embryos at
E12-12.5, and that Pod1 expression is also unperturbed in
Nkx3.2-/- embryos, indicate that the Pod1 and Nkx3.2
transcription factors use separate pathways to regulate early spleen
development.
Furthermore, our studies reveal that Pbx1 and Nkx3.2
independently regulate Hox11 in a hierarchical fashion
(Fig. 2M-P;
Fig. 3E,F)
(Lettice et al., 1999). And,
in a similar scenario, Pbx1 and Pod1, but not
Nkx3.2 (see Fig. S1E,F), independently control Nkx2.5 gene
expression in a hierarchical fashion (Fig.
7A). Thus, Pbx1 impinges on the separate Nkx3.2 and
Pod1 pathways by genetically regulating key players in both of these
pathways, i.e. Hox11 and Nkx2.5
(Fig. 7A). As a result,
Pbx1 emerges as a central hierarchical co-regulator in spleen
ontogeny (Fig. 7A). It will be
of interest to determine the roles of additional transcription factors
required for spleen development, such as Sox11
(Sock et al., 2004
) and Nkx2.3
(Pabst et al., 1999
;
Wang et al., 2000
;
Tarlinton et al., 2003
),
within the genetic pathways established by our study.
In conclusion, we demonstrate here the essential role of the Pbx1-Hox11 transcriptional pathway in spleen ontogeny. We provide evidence that Pbx1 is reiteratively required during spleen development, as it is implicated, at least in part, in splenic cell fate specification and morphogenesis, and then is essential again, later in organogenesis, for anlage expansion through control of progenitor cell proliferation. Finally, we demonstrate that spleen ontogeny is dependent on the orchestration of a complex network of transcription factors, among which Pbx1 emerges as a central, master co-regulator. Overall, our study takes a significant first step towards understanding the genetic and transcriptional control of spleen development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/13/3113/DC1
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ACKNOWLEDGMENTS |
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