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First published online 30 August 2006
doi: 10.1242/dev.02538
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1 The 21st Century Center of Excellence Program, Akita University School of
Medicine, Hondo 1-1-1, Akita, Akita 010-8543, Japan.
2 The J. David Gladstone Institutes, 1650 Owen Street, San Francisco, CA94158,
USA.
3 Department of Biological Sciences, Graduate School of Science, University of
Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
4 Department of Medicine and Human Genetics, David Geffen School of Medicine,
University of California at Los Angeles, 650 Charles E. Young Dr South, 47-123
CHS, Los Angeles, CA 90095, USA.
* Author for correspondence (e-mail: osada{at}med.akita-u.ac.jp)
Accepted 18 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Man1 (Lemd3), Inner nuclear membrane protein, Transforming growth factor ß signaling, Smad, Angiogenesis, Vascular remodeling, Xenopus, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
nuclear lamins and the LEM domain proteins interact with
barrier-to-autointegration factor, through which they are involved in
higher-order chromatin organization, nuclear assembly and gene regulation
(Gruenbaum et al., 2005).
However, the functions of a large number of putative integral NE proteins
remain to be elucidated (Schirmer et al.,
2003).
Human MAN1 was originally identified as one of three antigens recognized by
autoantibodies from an individual with a collagen vascular disease
(Lin et al., 2000). Man1 has a
N-terminal LEM domain, two putative transmembrane domains, a Man1-SRC1P
C-terminal domain of unknown function
(Mans et al., 2004) and an RNA
recognition motif. An RNA interference-mediated loss-of-function experiment
with the Man1 ortholog in C. elegans, Ce-MAN1, provides evidence that
Ce-MAN1, in combination with Ce-emerin, plays an essential role in chromosome
segregation and cell division (Liu et al.,
2003).
The linkage between the INM and growth factor-related signal transduction
was initially discovered by analyzing the roles of XMAN1 and SANE (Smad1
antagonistic effector), both of which are Xenopus orthologs of Man1,
in early embryogenesis (Osada et al.,
2003; Raju et al.,
2003). We identified XMAN1 as a novel factor that neuralizes the
ectoderm and dorsalizes the ventral mesoderm. In Xenopus, inhibition
of the bone morphogenetic protein (Bmp) pathway is a crucial step for neural
induction (Harland, 2000). We
have demonstrated that XMAN1 antagonizes the Bmp pathway by interacting with
the MH2 domain of the receptor-associated Smads (R-Smads) that mediate Bmp
signaling (Smad1, Smad5 and Smad8) through its C-terminal region. SANE was
isolated as a novel Smad1-interacting protein by the yeast two-hybrid system
and shows essentially the same activities as XMAN1.
Abnormal function of the NE is implicated in a wide range of human
diseases, collectively termed laminopathies
(Burke and Stewart, 2002).
Heterozygous loss-of-function mutations in human MAN1 (LEMD3
- Human Gene Nomenclature Database) cause osteopoikilosis, Buschke-Ollendorf
syndrome and melorheostosis (Hellemans et
al., 2004), all of which are characterized by hyperostotic bones.
The associated mutations involve the deletion of the C-terminal region of
MAN1, which is the interaction domain for R-Smads. Interestingly, expression
of wild-type human LEMD3 in mammalian cells suppresses both transforming
growth factor (TGF) ß1-dependent and BMP-dependent reporter activation,
whereas expression of a mutant MAN1 with either of the LEMD3
mutations is unable to suppress TGFß1 signaling. These observations
indicate that human MAN1 antagonizes both the BMP- and
TGFß/activin-signaling pathways, a conclusion that was also demonstrated
independently by two other groups using mammalian cells
(Lin et al., 2005;
Pan et al., 2005). Thus,
increased bone density and disseminated connective tissue nevi found in
individuals with the LEMD3 mutations and elevated elastin production
in fibroblasts from patients with Buschke-Ollendorf syndrome
(Giro et al., 1992) might be
explained by augmented sensitivity to the bone-forming activity of BMPs and
the extracellular matrix-producing activity of TGFß1.
In this study, to better understand the role of Man1 in cellular and
developmental processes, we analyzed the consequence of Man1 deficiency in
vivo. We used gene trapping to generate Man1-deficient mice, in which
the C-terminal region containing the Smad-interaction domain was deleted. We
found that the angiogenesis processes required to build a mature capillary
were severely perturbed in Man1-deficient embryos. These embryos had
an abnormal augmentation of Smad2/3 signaling, resulting in increased
extracellular matrix deposition, which is believed to inhibit endothelial cell
proliferation and migration (Pepper,
1997). These results provide the first evidence that Man1 plays a
crucial role in angiogenesis, acting as a `gatekeeper' at the INM to modulate
the activity of the Tgfß signaling pathway through the interaction with
receptor-associated Smads.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Nieuwkoop and Faber, 1967).
Whole-mount in situ hybridization of Xenopus embryos was performed.
RT-PCR and luciferase assays using ectodermal explants were carried out as
previously described (Osada et al.,
2003).
Generation of Man1-deficient mice
A mouse embryonic stem cell line (cell line XST167, strain 129/Ola)
containing an insertional mutation in Man1 was created in a
gene-trapping program by BayGenomics
(http://baygenomics.ucsf.edu/).
The gene-trapping vector, pGT1Mpfs, was designed to create an in-frame fusion
between the 5' exons of the trapped gene and a reporter gene,
ßgeo. The ES cells were injected into C57BL/6J blastocysts to
create chimeric mice, which bred to generate heterozygous
Man1-deficient (Man1+/
) mice. Mouse
embryonic fibroblasts (MEFs) were established from embryonic day (E) 16.5
wild-type and Man1+/ embryos using standard
procedures (Nagy et al.,
2003).
Genotypes of embryos older than E9.5 were determined by Southern blotting. Genomic DNA isolated from tails, yolk sacs and embryos were digested with PstI and hybridized with a Man1 probe located upstream of the insertion site. The probe was amplified from mouse genomic DNA with primers 5'-GCGCTGGGTTACTTTGTGTGCTG-3' and 5'-GCTTCCCGTTCACCACACTTCTG-3'. Signals were visualized with the Gene Image Random-Prime Labeling and Detection System (Amersham).
RT-PCR
Genotypes of embryos from E7.0-E9.0 and gene expression profiles of
wild-type and mutant embryos were analyzed by RT-PCR. Total RNA was isolated
from yolk sacs, ectoplacental cones and embryos with the High Pure RNA Tissue
Isolation kit (Roche). First-strand cDNA was synthesized with Superscript
Reverse Transcriptase (RT) III (Invitrogen). Primer sequences and PCR
conditions are presented in Table
1.
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) with the exception that hybridization was performed at
70°C. Digoxygenin (DIG)-labeled antisense RNA probes were synthesized with
the DIG RNA Labeling mix (Roche).
X-gal staining and immunohistochemistry
X-gal staining of heterozygous embryos was performed using standard
procedures (Nagy et al.,
2003). For whole-mount immunohistochemistry, E9.5 yolk sacs and
embryos were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline
followed by dehydration. After blocking, the specimens were incubated with an
anti-Pecam-1 antibody (MEC13.3; BD Pharmingen; 1:400) and then treated with a
horseradish peroxidase (HRP)-coupled anti-rat IgG antibody (Biosource, 1:500)
as a secondary antibody. The signals were detected with 0.3 mg/ml
3,3'-diaminobenzidine (DAB) containing 0.8 mg/ml nickel chloride.
For section immunohistochemistry, dissected embryos were fixed in 4% PFA,
dehydrated, embedded in paraffin and sectioned. Antibodies to smooth muscle
-actin (1A4; Dako; 1:500), phospho-histone H3 (Sigma; 1:2000),
activated caspase 3 (1:2000), or ssDNA (Dako; 1:400) were used as primary
antibodies. Vecta stain Elite peroxidase kit (Vector) and the ImmunoPure Metal
Enhanced DAB Substrate kit (Pierce) were used for visualization
For immunofluorescence assays, paraffin-embedded sections were treated with the following primary antibodies: anti-fibronectin (Sigma; 1:300), anti-phospho-Smad1/5/8 (Cell Signaling; 1:100) and anti-phospho-Smad2 (Cell Signaling; 1:500). After washing, the sections were incubated with an Alexa 546-conjugated anti-rabbit IgG antibody (Invitrogen; 1:2000). Nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI). Images were obtained using a confocal microscope (model LSM 510; Carl Zeiss).
Transmission electron microscopy
E9.5 embryos were fixed with 3% glutaraldehyde in 0.1 M sodium cacodylate
buffer at pH 7.4 and post-fixed with 1% osmium tetroxide in the same buffer.
After dehydration, they were substituted by propylene oxide and embedded in
epoxy resin. Ultrathin sections were doubly stained with uranyl acetate and
lead citrate, and examined with an H-7650 transmission electron microscope
(Hitachi).
Western blotting
E9.5 embryos were dissected out and yolk sacs were removed for genotyping.
Dissected embryos and MEFs were lysed in modified RIPA buffer (50 mM Tris-HCl
at pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA
supplemented with phosphatase and protease inhibitors). Lysates were cleared
by centrifugation and quantitated with the BCA Protein Assay Kit (PIERCE). The
lysates (20 µg) were separated on a 5-10% gradient gel (BioRad). The
following primary antibodies were used: anti-ß-galactosidase (Promega;
1:1000), anti-N-terminal human MAN1 (Santa Cruz Biotechnology; 1:200),
anti-fibronectin (Sigma; 1:1000) and anti-ß-tubulin (Sigma; 1:5000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lin et al., 2005;
Pan et al., 2005), it is not
clear whether this is also the case in vivo. To address this, we first
examined whether a Xenopus ortholog of MAN1, XMAN1, inhibits
activin/nodal signaling during Xenopus development. Chordin is
expressed in the Spemann organizer at the early gastrula stages and is a
downstream target for activin/nodal signaling
(Sasai et al., 1994). When
XMAN1 mRNA was injected into the future organizer region, the
expression of chordin was almost completely suppressed
(Fig. 1A). Conversely, delivery
of the antisense morpholino oligonucleotides (MO), which specifically inhibit
XMAN1 functions in vivo (Osada et al.,
2003), caused a large expansion of the expression domain for
chordin, whereas it was unchanged in embryos injected with
four-base-mismatched MO.
The above results were supported by animal cap (ectodermal explant) assays.
Xenopus nodal-related factor 1 (Xnr1) induced mesodermal markers
(Xbra, chordin and goosecoid) in animal caps as previously
described (Jones et al.,
1995). Wild-type XMAN1 and the C-terminal fragment of XMAN1
(XMAN1-CT) containing the Smad-interacting domain, suppressed the induction of
these markers by Xnr1, but a mutant lacking the CT (XMAN1-CT) did not
(Fig. 1B).
Xnr1 expression is autoregulated through an activin/nodal response element
composed of three Foxh1 (FAST-1)-binding sites present in the first intron
(Osada et al., 2000). We
performed a reporter assay with a luciferase construct (Int1) containing this
element to study whether XMAN1 inhibits Xnr1-dependent transcription. As shown
in Fig. 1C, the activation of
Xnr1-dependent Int1 was suppressed by XMAN1 and XMAN1-CT, but not by
XMAN1-CT. We also found that XMAN1 could directly bind to Smad2 and
Smad3 via its C-terminal region (Osada et
al., 2003) (data not shown) in the Xenopus embryo,
consistent with the works using cultured cells
(Hellemans et al., 2004;
Lin et al., 2005;
Pan et al., 2005). These data
indicate that XMAN1 negatively regulates activin/nodal signaling in addition
to Bmp signaling in vivo.
Generation of Man1-deficient mice
Although Man1 loss-of-function analyses have been performed in C.
elegans (Liu et al.,
2003), Xenopus (Osada
et al., 2003) and cultured mammalian cells
(Hellemans et al., 2004;
Pan et al., 2005), the
consequence of the complete loss of functional Man1 in whole organisms is
unknown, even in human disease states. To obtain insights into the requirement
for Man1 in mammalian development and cellular functions, we generated
Man1-deficient mice using an embryonic stem cell line in which
Man1 had been disrupted by gene trapping. A DNA sequence tag
generated by 5' rapid amplification of cDNA ends analysis revealed that
the insertional mutation in Man1 occurred in intron 4
(Fig. 2A). Embryos were
genotyped by RT-PCR at E7.0-E9.0 with Man1 and ßgeo
primer pairs (Fig. 2B) and by
Southern blots at E9.5 and later with a probe located upstream of the
insertion site (Fig. 2C). We
amplified a fusion transcript composed of the fourth exon of Man1, an
Engrailed-2 splice acceptor and full-length ßgeo from
Man1-deficient embryos (Fig.
2D). The deduced gene product of this transcript indicates that a
Man1-ßgeo fusion protein lacking the C-terminal Smad-interacting
domain was generated in Man1-deficient embryos. We refer to the
genetrap allele as Man1 hereafter.
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100 kDa
band, which was comparable with exogenously overexpressed human MAN1 (not
shown), in lysates prepared from wild-type and
Man1+/ embryos and MEFs, but not in those from
Man1/ embryos
(Fig. 2E). Conversely, a band
of 230 kDa corresponding to the predicted Man1-ßgeo fusion protein
was detected by an antibody to ß-galactosidase in the
Man1+/ and Man1/
specimens, but not in the wild-type ones. Thus, the Man1-ßgeo fusion
protein in Man1/ embryos is unlikely to
retain regulatory activity on Smad signaling.
We analyzed the temporal and spatial expression domains for Man1 during
early embryogenesis by whole-mount X-gal staining in
Man1+/ embryos. lacZ expression was
detected in the epiblast, the allantois and the ectoplacental cone at E7.5
(Fig. 2L), and in the heart,
the branchial arches, the foregut, the paraxial mesoderm, the sinus venosus
and the tail mesenchyme at E8.5 (Fig.
2N). No signals were detected in a wild-type littermate embryo
(Fig. 2K). As for the vascular
system, lacZ was detected in visceral endodermal, mesodermal and
endothelial cells in the yolk sac (Fig.
2Q), and endothelial and vascular smooth muscle cells (VSMCs) in
the dorsal aorta (Fig. 2R).
lacZ was also observed in a wide range of tissue/cell populations in
the adult (see Fig. S1 in the supplementary material). These
lacZ-positive tissues correspond well to the localization of
Man1 transcripts (Fig.
2F-J,M,O), suggesting that the ßgeo gene was under the
control of endogenous Man1 promoter. Man1 is also strongly
expressed in the head mesenchyme at E9.0
(Fig. 2P). An antisense probe
specifically hybridizes to the sequence covering the C-terminal region (P2)
gave no signals in Man1/ embryos
(Fig. 2J), supporting further
that the C-terminal transcript was not expressed in
Man1/ embryos.
Man1 deficiency causes embryonic lethality
Man1+/ mice are normal in terms of growth,
fertility and lifespan. Although humans with heterozygous LEMD3
mutations exhibit prominent bone and connective tissue phenotypes
(Hellemans et al., 2004), we
did not detect any abnormalities in bone density by magnetic resonance imaging
or connective tissue nevi by skin histology in
Man1+/ mice (data not shown). No homozygous mice
(Man1/) were recovered at weaning. To
elucidate further the apparent embryonic lethal phenotype, we analyzed the
offspring of heterozygote intercrosses at various stages of development.
Although Man1/ embryos at E7.5, E8.5, E9.5
and E10.5 were recovered at the predicted mendelian frequencies (see Table S1
in the supplementary material), the number of
Man1/ embryos at E11.5 was lower than
predicted. No homozygous mutants were recovered at E12.5 or later; thus,
Man1/ embryos died at about E10.5-E11.5.
Nodal expression is deregulated in Man1-deficient embryos
Members of the TGFß superfamily play a crucial role in mesoderm
formation and patterning of the embryo
(Chang et al., 2002;
Whitman, 1998). We first
examined the possibility that Man1/ embryos
die from impaired mesoderm formation by performing whole-mount in situ
hybridization. Because Man1/ embryos showed
a 0.5- to 1.0-day delay in development, we compared the mutant embryos with
stage-matched wild-type embryos. We found no obvious differences in the
expression domains for the mesodermal markers Nodal
(Fig. 3A; +/+,
n=6; /, n=4), chordin
(Fig. 3B; +/+,
n=7; /, n=4), Bmp4
(Fig. 3C; +/+,
n=5; /, n=5) and brachyury
(Fig. 3E; +/+,
n=2; /, n=3), between the wild-type
and mutant embryos, although we observed consistently stronger intensity of
expression of Nodal and chordin in the mutants. Nodal
normally shows an asymmetric expression in the left lateral plate mesoderm
(LPM) at E8.25 (Lowe et al.,
1996). However, in the mutant embryos, Nodal was
ectopically expressed in the right LPM
(Fig. 3A';
+/+, n=2; /, n=5),
suggesting that Nodal expression is deregulated in
Man1/ embryos.
Man1 deficiency did not grossly affect the anteroposterior
patterning of the central nervous system or axial midline, as assessed by
examining the expression of cerberus-related 1
(Fig. 3D; +/+,
n=2; /, n=2), Otx2 and
Krox20 (Fig. 3F;
+/+, n=3; /, n=5),
Emx2 (Fig. 3G;
+/+, n=4; /, n=3), and
sonic hedgehog (Fig.
3H; +/+, n=5; /,
n=5).
Angiogenesis defects in Man1 mutant embryos
At E10.0, Man1/ embryos were recognizable
by their smaller and pale yolk sacs (Fig.
4A,B). Histological analysis of wild-type and mutant yolk sacs
revealed that the yolk sac vasculature was formed in the mutants but appeared
highly dilated (Fig. 4C,D).
Some mutant embryos died in the middle of turning
(Fig. 4F), and others showed
clumps of red blood cells and an enlarged pericardium
(Fig. 4G), suggesting that
vascular permeability and intracardiac pressure may have been elevated in the
mutants.
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/ embryos were
confirmed by whole-mount immunostaining with an antibody to platelet
endothelial cell adhesion molecule 1 (Pecam1), which specifically detects
endothelial cells. In the yolk sac of the wild-type embryo at E10.0, major
blood vessels and a well-branched capillary network were present
(Fig. 5A). By contrast, the
yolk sac of the mutant embryo at E10.0 displayed a primary capillary plexus
but no branching from pre-existing vessels
(Fig. 5B). No significant
difference in endothelial cell proliferation was observed between wild-type
and mutant yolk sacs (data not shown). Moreover, in the wild-type embryo
proper, well-developed and defined capillary networks were apparent in the
head and intersomitic regions (Fig.
5C,E,G), whereas the fine capillary network in the cranial,
branchial, cardiac and intersomitic regions of mutant embryos was absent
(Fig. 5D,F,H). These data imply
that endothelial cell differentiation occurred normally in
Man1/ embryos, but that vascular remodeling
to build a mature vascular network was severely perturbed.
Interactions between endothelial cells and mural cells (pericytes and
VSMCs) are essential for vascular development
(Armulik et al., 2005;
Carmeliet, 2000). To determine
whether differentiation or recruitment of VSMCs is affected in mutant embryos,
we examined the expression of smooth muscle -actin, a marker for VSMC
differentiation, by immunohistochemistry. In E10.0 wild-type embryos, the
dorsal aorta was surrounded by VSMCs (Fig.
5I). However, in E10.0 mutant embryos, smooth muscle -actin
expression was barely detected around the dorsal aorta
(Fig. 5J), although its
expression was detected in other tissues, including the heart (data not
shown). Electron microscopic analysis supported the results of
immunohistochemistry (Fig.
5K-P); in wild-type embryos, endothelial cells in the dorsal aorta
were surrounded by mural cells and directly in contact with them
(Fig. 5K,M), whereas those in
mutant embryos were not supported by mural cells, resulting in a collapsed
dorsal aorta (Fig. 5L,N). Tight
junctions between endothelial cells were formed in both wild-type and
Man1/ embryos
(Fig. 5O,P). These results
indicate that mural cells were differentiated normally, but their recruitment
to the vascular wall was severely impaired in
Man1/ embryos.
The Smad2/3 pathway is abnormally activated in Man1/ embryos
Null mutants of central components of the Tgfß pathway demonstrate
early embryonic death with growth retardation at about E10.0, defects in
vascular development, and abnormal VMSC recruitment
(Chang et al., 2002;
Goumans et al., 2003). As
such, there is a remarkable correlation with the defects observed in
Man1/ embryos, consistent with the idea that
Man1 is required for manifestation of the Tgfß signal transduction
effect. Thus, we examined the activation status of the Smad1/5 and Smad2/3
pathways using a cellular resolution assay by immunohistochemistry with
antibodies to phospho-Smad1/5/8 and phospho-Smad2/3. Compared with wild-type
embryos, the intensity of nuclear phospho-Smad2 was significantly elevated in
mesenchymal, endothelial, and neural cells of
Man1/ embryos
(Fig. 6I-P, Fig. S3).
Quantitative analysis revealed that accumulation of phospho-Smad2 was
significantly increased in these cells in the mutants
(Fig. 6R). By contrast, the
difference in the intensity of phospho-Smad1 between wild-type and mutant
embryos was not significant in all cell types examined
(Fig. 6A-H,Q, see Fig. S2 in
the supplementary material).
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/ embryos, the expression level of
Id1, a specific downstream target for the Smad1/5 pathway in
endothelial cells (Goumans et al.,
2002; Ota et al.,
2002), was comparable with that in wild-type and heterozygous
embryos. By contrast, the expression levels of plasminogen activator inhibitor
1 (PAI-1; Serpine1 - Mouse Genome Informatics) and
fibronectin 1, both of which are specific downstream targets for the Smad2/3
pathway in endothelial cells, were significantly upregulated. The expression
of Smad7, which is a downstream target for both the Smad1/5 and Smad2/3
pathways, was unchanged in Man1/ embryos. We
obtained similar results in embryos from another cross (see Fig. S3 in the
supplementary material). Notably, the expression level of Tgfb1 was
also elevated in the mutants.
Tgfß1 strongly stimulates synthesis of extracellular matrix (ECM) and
deposition of ECM inhibits vascular remodeling
(Ignotz et al., 1987;
Pepper, 1997). In line with
the elevation of Tgfb1 expression and the augmentation of the Smad2/3
signaling, the expression of fibronectin 1 was increased in
Man1/ embryos
(Fig. 7A). This was further
supported by western blots, where fibronectin synthesis was highly elevated
(Fig. 7B), and by
immunofluorescence analyses, where fibronectin was strongly deposited in
Man1/ embryos and yolk sacs
(Fig. 7C-H). Occasionally,
fibronectin deposition was extremely prominent around endothelial cells
(Fig. 7D). These results
suggest that abnormal activation of Smad2/3 signaling leads to aberrant
deposition of ECM to inhibit migration of endothelial cells.
Man1 is required for cell survival and normal nuclear morphology
Because NE proteins are implicated in cell proliferation and apoptosis
(Cohen et al., 2001;
Gruenbaum et al., 2005), we
examined these processes in Man1/ embryos.
We first analyzed cell proliferation in
Man1/ embryos using immunohistochemistry
with an antibody against phospho-histone H3, a marker for cells in mitotic
prophase. As shown in Fig.
8A,B, the number of phospho-histone H3-positive nuclei was not
significantly different in wild-type and mutant embryos at the 17-somite
stage, indicating that cell proliferation was normal in the mutants.
Consistent with this, the expression of cyclin-dependent kinase inhibitor 1A
(Cdkn1a or p21), a cyclin-dependent kinase inhibitor and
mediator of the cellular growth arrest program of the Tgfß pathway, was
unchanged in Man1/ embryos
(Fig. 7A). By contrast,
immunohistochemistry with antibodies against activated caspase 3 and
single-strand DNA (ssDNA, a marker for DNA fragmentation during programmed
cell death) revealed massive apoptosis in
Man1/ embryos
(Fig. 8D,F), especially in
mesenchymal tissues where Man1 is strongly expressed
(Fig. 2P). Electron microscopic
analysis clarified that the mesenchymal cells in the mutants shrunk and their
nuclei were condensed and fragmented, which are the typical characteristics of
apoptotic cells (Fig. 8J).
Wild-type embryos had few apoptotic cells
(Fig. 7C,E). Notably, apoptotic
cells were seldom detected in other tissues even in
Man1/ embryos. These results suggest that
Man1 is essential for cell survival, but the degree of cell death is dependent
on the different sensitivity to the loss of Man1 between cells.
The INM proteins bind to nuclear lamins, which are essential for
maintaining nuclear shape (Gruenbaum et
al., 2005); emerin, an INM protein, is required for normal nuclear
shape (Lammerding et al.,
2005). We next investigated whether the loss of Man1 affects the
nuclear morphology by electron microscopy
(Fig. 8K-O). The outlines of
wild-type mesenchymal nuclei were tense and smooth, whereas the most of mutant
mesenchymal nuclei were irregularly shaped, although continuity of their NE
was apparently intact. At higher magnification, we found mesenchymal cells
with herniated nuclei (Fig.
8M,N) and those with an expanded perinuclear space
(Fig. 8O) at low frequency
(1%) in the Man1/ embryos.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), we have
proposed that XMAN1 is a novel type of negative regulator for Smad signaling.
A similar hypothesis has also been proposed by four other groups
(Hellemans et al., 2004;
Lin et al., 2005;
Pan et al., 2005;
Raju et al., 2003). We have
verified this hypothesis in vivo by analyzing Man1-deficient mice
lacking the Smad-interacting domain. We have found that
Man1/ embryos died during embryonic
development because of defects in vascular remodeling. We have demonstrated
that abnormal deposition of ECM caused by the augmented Smad2/3 pathway and
the disrupted intercellular communication between endothelial and mural cells
underlie the perturbed vascular remodeling in
Man1/ embryos. Our results have shed light
on the importance of Man1 in fine-tuning the activity of Tgfß signaling
during angiogenesis.
Man1 acts as a Smad regulator in angiogenesis
The Man1-ßgeo fusion generated in
Man1/ embryo contained the N-terminal LEM
domain and the first transmembrane domain, both of which are necessary and
sufficient for targeting Man1 to the INM in mammalian cells
(Wu et al., 2002), but lacked
the C-terminal Smad-interacting domain. Thus, it is likely that the phenotypes
observed in the mutants are caused by abnormal Smad regulation at the INM.
We suspect that deregulation of the Smad2/3 pathway is fundamental to the
defects in vascular remodeling in Man1/
embryos. We have demonstrated abnormal activation of the Smad2/3 pathway in
Man1/ embryos by immunofluorescence
microscopy (Fig. 6, see Fig. S2
in the supplementary material), where nuclear accumulation of phospho-Smad2 is
increased, and by RT-PCR (Fig.
7A), where the expression of its downstream targets is
upregulated. Importantly, the expression of Tgfb1 is elevated in
Man1/ embryos. The increased accumulation of
nuclear Smad2 in the mutants may lead to the activation of Tgfb1
promoter, resulting in Tgfß1 autoinduction
(Kim et al., 1989). Tgfß1
stimulates the synthesis of fibronectin
(Ignotz et al., 1987), which
inhibits proliferation and migration of endothelial cells, impairing vascular
remodeling (Pepper, 1997). We
have shown that the expression of fibronectin is upregulated in
Man1/ embryos at the mRNA and protein levels
(Fig. 7). In addition, the
expression level of Serpine1 is also elevated in mutant embryos.
Serpine1 is a potent inhibitor of endothelial cell migration in vitro
(Stefansson and Lawrence,
1996) and angiogenesis in vivo
(Stefansson et al., 2001).
Therefore, abnormal deposition of ECM caused by elevated Smad2/3 signaling
could underlie the defects in vascular remodeling in
Man1/ embryos.
Our quantitative analyses of phospho-Smads have revealed that Smad2/3
signaling is preferentially activated in endothelial cells of
Man1/ embryos
(Fig. 6R). Tgfß1 can
stimulate two Smad pathways in endothelial cells - the canonical Alk5-Smad2/3
pathway and the Alk1-Smad1/5 pathway
(Goumans et al., 2002;
Oh et al., 2000). It has been
proposed that Alk1-Smad1/5 signaling promotes endothelial cell proliferation
and migration and Alk5-Smad2/3 signaling inhibits them
(Goumans et al., 2002).
Tgfß and activin also inhibit proliferation and sheet formation of ES
cell-derived endothelial cells, probably by activating the Smad2/3 pathway
(Watabe et al., 2003).
Although an opposite model on the roles of the Alk1 and Alk5 pathways during
angiogenesis has been proposed (Lamouille
et al., 2002; Oh et al.,
2000; Seki et al.,
2003), the balance between the two pathways in endothelial cells
appears to be crucial in determining the activation state of the endothelium.
In Man1/ embryos at E9.5, the expression of
specific targets of the Smad2/3 pathway (Serpine1 and Fn1)
was significantly elevated (Fig.
7A), whereas that of Id1, a specific target for the
Smad1/5 pathway, was unchanged, suggesting that the balance between these two
pathways was disrupted in mutant endothelial cells.
It has been proposed that interactions between endothelial and mural cells
in the blood vessel wall play an important role in the regulation of vascular
remodeling (Armulik et al.,
2005). In Fig.
5I-N, we have clearly shown that the recruitment of mural cells to
the vascular wall was severely impaired in
Man1/ embryos. Because mural cells are
generally thought to be mesenchymal origin, we speculated that the mesenchymal
apoptosis observed in E9.5 Man1/ embryos
(Fig. 8D,E) led to the
decreased numbers of VSMCs. However, we detected the elevated expression of
trangelin (Tagln) and Acta2
(Fig. 7A) and accumulation of
SMA-positive cells around the heart in the mutants (data not shown),
suggesting that proliferation and differentiation of VSMCs was normal. The
activated Tgfß1-Smad2/3 signaling, which plays an important role in VSMC
development, may result in the elevation of these markers. Thus, VSMC
migration rather than proliferation and differentiation may be affected in the
mutants. The abnormal deposition of fibronectin around blood vessels observed
in Man1/ embryos would be also associated
with the inhibition of VMSC migration.
Man1 in cell survival and nuclear integrity
Man1 is highly expressed in the head mesenchyme
(Fig. 2P), where massive
apoptosis was observed in Man1/ embryos.
Loss of Ce-MAN1 in C. elegans and siRNA interference of human MAN1 in
cultured cells also induce apoptosis (Liu
et al., 2003; Pan et al.,
2005). Although these results suggest that the mesenchymal
apoptosis in Man1/ embryos is probably a
direct consequence of the loss of Man1, the elevated Tgfb1 expression
may be implicated in initiation or deterioration of the apoptotic processes.
We have clarified that most of the apoptotic cells in the mesenchyme are
caspase 3-dependent (Fig. 7D),
consistent with the observation that caspase 3 is activated during
Tgfß1-induced apoptosis in many types of cultured cells
(Schuster and Krieglstein,
2002). In addition, several lines of evidence have shown that
Smad3 is an important mediator of Tgfß1-induced apoptosis
(Kim et al., 2002;
Yamamura et al., 2000;
Yanagisawa et al., 1998).
Thus, the elevated Smad2/3 pathway in the mutants may also be involved in the
Tgfß1-induced caspase 3-dependent apoptosis.
Nuclear lamins and the INM proteins play a key role in maintaining nuclear
morphology (Burke and Stewart,
2002). We observed nuclear anomalies, especially in mesenchymal
cells of Man1/ mutants
(Fig. 8L-O), although we seldom
found the cells with discontinuous NE. It remains to be elucidated whether the
nuclear abnormalities in the mutants were caused by the abnormal regulation of
Smad signaling at the INM, or by the presence of structurally aberrant Man1
with the long C-terminal tail of ßgeo, and they are associated with the
increased mesenchymal apoptosis. Emerin-deficient MEFs show abnormal nuclear
shape and increased apoptosis, but impaired mechanotransduction rather than
nuclear fragility induces their apoptosis
(Lammerding et al., 2005).
Man1 in axis formation
Nodal expression in the node and left LPM during early
embryogenesis plays a crucial role in determining the left-right axis of the
embryo (Hamada et al., 2002;
Lowe et al., 1996). In
Man1/ embryos, initiation of Nodal
expression and gastrulation occurred normally. However, asymmetric
Nodal expression was impaired to become expressed bilaterally in
mutant embryos (Fig.
3A'). It would be intriguing to examine whether Man1 is
involved in establishing left-right asymmetry through the regulation of
Nodal expression.
In Man1/ embryos, the overall
anteroposterior patterning of the embryo proper and the initiation of the
expression of neural markers are normal
(Fig. 3), which is consistent
with previous data in XMAN1 morphants
(Osada et al., 2003). However,
in contrast to the XMAN1 morphants that show eyeless phenotypes with anterior
truncations, the anterior structures are relatively normal in Man1
mutants. Early embryonic lethality at about E10.5 in
Man1/ embryos prevented us from examining
the role of Man1 in neural development. Generation of conditional
Man1 mutant mice would be useful for analyzing the cell- and
tissue-specific roles of Man1 more precisely.
In cultured cells, depletion of human MAN1 leads to elevated responsiveness
to both TGFß and BMP (Hellemans et
al., 2004; Lin et al.,
2005; Pan et al.,
2005). Accordingly, the sensitivity to both activin/nodal and Bmp
stimulation was expected to be elevated in the setting of Man1
deficiency. The mechanism by which the Smad2/3 pathway was preferentially
augmented in Man1/ embryos is currently
unknown. Intracellular antagonism between Bmp4 and activin signaling through
competition for a limited pool of Smad4 has been proposed
(Candia et al., 1997). Thus, it
is possible that the Smad2/3 pathway activated by continuous Tgfß1
stimulation consumed more Smad4 than the Smad1/5 pathway, leading to the
dominance of the Smad2/3 pathway in Man1 mutants.
We did not detect overt morphological defects caused by the abnormal
activation of the Bmp-Smad1/5 pathway in
Man1/ embryos, aside from the fact that the
allantois was enlarged in the mutant embryos
(Fig. 3E). Bmp signaling plays
a crucial role in the development of the allantois
(Fujiwara et al., 2001). It
would be important to examine the development of tissues/cell population where
the involvement of the Bmp-Smad1/5 pathway is implicated, such as the
formation of the heart and primordial germ cells, and the dorsoventral
patterning of the neural tube (Hogan,
1996).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/19/3919/DC1
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ACKNOWLEDGMENTS |
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