1 Department of Cell Biology and Anatomy, Louisiana State University Health
Sciences Center, New Orleans, LA 70112, USA
2 Molecular and Human Genetics Center, Louisiana State University Health
Sciences Center, New Orleans, LA 70112, USA
* Author for correspondence (e-mail: pcserj{at}lsuhsc.edu)
Accepted 16 January 2004
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SUMMARY |
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Key words: bHLH, HAND1, Angiogenesis, Smooth muscle, Yolk sac
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Introduction |
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Previous studies have shown that the Hand1 gene is essential for
embryonic viability beyond 9.5 dpc
(Firulli et al., 1998;
Riley et al., 1998
).
Hand1-null mice exhibit numerous embryonic and extra-embryonic
defects. Based on the expression pattern of HAND1, many abnormalities in
Hand1-null mice are probably indirect and arise as a consequence of
defects in extra-embryonic tissues. A requirement for Hand1 during
trophectoderm development suggests that part of the embryonic defects could be
due to the failure to fully develop this lineage
(Riley et al., 1998
). However,
experiments designed to rescue the trophectoderm-induced defects did not
significantly rescue the extra-embryonic or the embryonic defects
(Riley et al., 2000
). Another
defect in extra-embryonic tissues that could account for the extensive
developmental abnormalities found in Hand1-null embryos is defects in
the yolk sac. A major defect in Hand1-null yolk sac is the lack of
fully developed vasculature (Firulli et
al., 1998
). However, the regulation of extra-embryonic membrane
development by HAND1 has not been investigated.
Embryonic vasculature development proceeds though a multi-step processes
(Conway et al., 2001;
Neufeld et al., 1999
;
Patan, 2000
). Embryonic blood
vessels initially form in the yolk sac early during development with
angioblasts first appearing around 7.0-7.5 dpc in mice. These migrate,
aggregate, proliferate and eventually differentiate to form a vascular plexus
through the process called vasculogenesis. Endothelial cells form from the
angioblasts within the mesoderm adjacent to the extra-embryonic endoderm in
part through signaling by vascular endothelial growth factor (VEGF). VEGF
binds two tyrosine kinase receptors implicated in the VEGF-directed
vasculogenesis and hematopoiesis. One receptor, FLK1 (KDR Mouse Genome
Informatics), is required for vasculogenesis and blood island formation
(Shalaby et al., 1995
).
Another VEGF receptor FLT1 regulates blood island formation but is not
required for endothelium differentiation
(Fong et al., 1995
).
Angiogenesis is the expansion and elongation of the primitive vascular
network through sprouting and remodeling from pre-existing vessels. Angiogenic
growth factors angiopoietin 1 and 2 (ANG1 and ANG2) play a key role in this
process. ANG1 directs phosphorylation of the endothelial tyrosine kinase
receptor TIE2 (TEK Mouse Genome Informatics) and acts as a
chemoattractant for endothelial cells
(Gale and Yancopoulos, 1999;
Suri et al., 1996
). ANG2
destabilizes the smooth muscle cells that form around the endothelial cells
and also induces endothelial sprouting by antagonizing ANG1
(Gale and Yancopoulos, 1999
;
Maisonpierre et al., 1997
).
Mice lacking Tie2 show extensive vascular remodeling defects, while
loss of Tie1, a gene closely related to Tie2, shows impaired
blood vessel integrity (Sato et al.,
1995
). Vasculature refinement is thought to occur through a
combination of activation and inhibition of the ANG/TIE signal transduction
pathways.
In addition to their role in vasculogenesis, VEGF and its receptors FLK1
and FLT1 are required for angiogenesis by promoting the migration,
proliferation and tube formation in endothelial cells
(Patan, 2000). Loss of a
single copy of Vegf results in embryonic lethality between 8.0 and
9.0 dpc resulting in vasculature defects, suggesting that the concentration of
VEGF is crucial for angiogenesis (Carmeliet
et al., 1996
; Ferrara et al.,
1996
). Endothelial cells respond to VEGF through the
VEGF165 specific receptors neuropilin 1 and 2 (NRP1 and NRP2)
(Gitay-Goren et al., 1992
).
Mice lacking neuropilins have angiogenic defects similar to mice lacking VEGF
(Takashima et al., 2002
),
suggesting that they are key receptors of VEGF mediated vascular
formation.
After endothelial tubes form, vessel maturation requires recruitment of
peri-endothelial cells, including vascular smooth muscle cells (SMCs) and
pericytes. Platelet-derived growth factor (PDGF) BB and PDGFRß are
recruitment factors for SMCs (Hellstrom et
al., 1999). Transforming growth factor-ß1 (TGFß1) also
regulates SMC differentiation and recruitment although its exact roles are
unknown (Orlandi et al.,
1994
). Mice lacking the TGFß1 receptors TGFßRII
(Hirschi et al., 1998
) and
endoglin (Li et al., 1999
),
and the TGFß signal transduction molecule Smad5
(Yang et al., 1999
) all show a
decrease in the number of SMCs surrounding endothelial tubes.
Several transcription factors have been identified as regulators of
vasculogenesis, angiogenesis and SMC recruitment
(Oettgen, 2001). We examined
the role of the bHLH transcription factor Hand1 during vascular
development in yolk sacs, the tissue where blood vessel formation originates
in the embryo. We have generated a Hand1 knockout (KO) mouse line and
examined the role of Hand1 in vasculature development during yolk sac
development. Our data shows that Hand1 is not required for
vasculogenesis, but is required for elaboration of the primitive endothelial
plexus to refine into a functional vascular system. One function of
Hand1 during yolk sac development is the recruitment of SMCs to the
endothelial network.
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Materials and methods |
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ß-Gal staining
Embryos were dissected and fixed briefly in 4% paraformaldehyde/PBS.
Embryos were rinsed three times with PBS and stained in 5 mM
K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM
MgCl2/PBS containing 1 mg/ml X-gal.
Whole-mount PECAM staining
Yolk sacs were fixed in 4% paraformaldehyde/PBS for 30 minutes, blocked in
immunoblock reagent (PBS, 5% goat serum, 1% DMSO) for 1 hour at room
temperature and incubated with 1:200 anti-PECAM antibody (PharMingen)
overnight at 4°C. Yolk sacs were washed five times with PBS and incubated
with 1:500 anti-rat AP-conjugated antibody (Zymed) overnight at 4°C. After
washing, a chromogen was generated using BCIP and NBT as substrates.
Section immunofluorescence and immunohistochemical analysis
Immunohistochemistry was performed on frozen sections. For detection of
ß-gal and PECAM, sections were incubated with rabbit anti-ß-gal (5
prime-3 prime, Boulder, CO) and rat anti-PECAM antibodies, washed, and
incubated with anti-rabbit rhodamine conjugated and anti-rat FITC conjugated
antibodies (Santa Cruz). To detect smooth muscle cells, rhodamine conjugated
anti-mouse smooth muscle -actin antibody (Sigma) was used.
RT-PCR analysis
Total RNA was extracted from extra-embryonic membranes using Trizole
reagent (Gibco). RNA samples were treated with RNAse free DNase I prior to
reverse transcription. RNA was primed with oligo(dT) and the first strand was
synthesized using SuperscriptII (Invitrogen). PCR amplification was with
MasterPCR mix (Qiagen) with gene specific primer pairs. All samples were
normalized to Gapd. Each PCR reaction was performed with dilution of
cDNA samples to maintain the products in a linear range. Each primer pair was
tested in three independent PCR amplifications for each sample. RT reactions
were performed three times with different membranes. Primer sequences are
available upon request.
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Results |
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Hand1ß-gal/ß-gal
embryos develop normally up to 7.5 dpc at which time they reabsorb when grown
in a 129sv background. When the mice were outcrossed to C57/Bl6 or
Swiss-Webster mice,
Hand1ß-gal/ß-gal
embryos were found in the predicted Mendelian frequency up to 7.75 dpc after
which the frequency of
Hand1ß-gal/ß-gal
embryos decreased with a corresponding increase in the number of absorption
sites. As reported previously (Firulli et
al., 1998; Riley et al.,
1998
),
Hand1ß-gal/ß-gal
embryos out-crossed appear grossly normal up to
8.0 dpc after which time
development is severely retarded and embryonic defects are more severe.
However, we found a large number of embryos survive up to 9.5 dpc although
they did not develop past the formation of nine somites.
Analysis of the expression of the inserted ß-gal gene in
Hand1ß-gal/+ embryos during
development revealed that the expression pattern recapitulates the expression
of the endogenous HAND1 gene (Cserjesi et
al., 1995) (Fig.
1B-E). HAND1 is first observed in the extra-embryonic mesoderm at
7.5 dpc. High expression levels of ß-gal expression are maintained in the
yolk sac and the amnion in the mesodermal layer throughout development. By
7.75 dpc ß-gal expression was seen throughout the lateral plate mesoderm
and in the forming heart (Fig.
1B). Expression of Hand1 is maintained in the SMCs of the
gut, a lateral plate derivative, throughout development and in adult mice.
ß-gal expression in the heart become progressively restricted and by 10.5 dpc ß-gal activity was seen predominantly in the left ventricle and outflow tract. Expression was also seen in neural crest derived tissues of the first and second branchial arches and in the forming sympathetic nervous system (Fig. 1C). Expression of ß-gal is high in components of the umbilicus including the blood vessels and smooth muscle of the umbilical gut (Fig. 1C).
To better visualize the expression of the ß-gal knock-in gene within developing embryos, we cleared later stages with benzyl benzoate and benzyl alcohol (Fig. 1D,E). By 12.5 dpc, expression of ß-gal is restricted to the superior-lateral region of the left ventricle (Fig. 1D). Expression continues in the branchial arch-derived tissues, most prominently in the tongue and mandible and thymus. Extensive expression was seen throughout the developing sympathetic nervous system, including the trunk and splanchnic ganglia. Expression was also observed in the developing adrenal gland in cells of the adrenal medulla, the exocrine component of the sympathetic nervous system. Extensive expression of ß-gal continues in the developing gut and by 14.5 dpc, expression of ß-gal was seen throughout the gut distal to the duodenum (Fig. 1E). Within the heart, expression was found only in the apex of the left ventricle. Expression continues in the tongue and mandible at the midline and in the sympatho/adrenal lineage. After 14.5 dpc, expression decreases in all tissues, except the gut and a subset of cells of the sympatho/adrenal lineages.
Vascular abnormalities in Hand1 mutant yolk sac
The yolk sac of
Hand1ß-gal/ß-gal
embryos shows multiple abnormalities soon after formation
(Firulli et al., 1998;
Riley et al., 1998
). Wild-type
yolk sac has an extensive and highly organized vasculature filled with blood
by 9.5 dpc (Fig. 2A,C) while
the vasculature within the
Hand1ß-gal/ß-gal
yolk sac was absent or poorly developed
(Fig. 2B,D,E). In
Hand1ß-gal/ß-gal
embryos, blood formation proceeds without an intact vasculature leading to
extensive leakage of hematopoietic cells
(Fig. 2B).
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Lack of blood vessel remodeling in Hand1 mutant yolk sac
During normal vessel development, formation begins with vasculogenesis, the
aggregation of endothelial cells. In the yolk sac, vasculogenesis occurs in
conjunction with hematopoiesis during the formation of blood islands. Blood
islands are composed of aggregates of endothelial and hematopoietic precursor
cells that form distinct lineages. We examined the organization of endothelial
cells in Hand1 mutant yolk sacs using the endothelial marker platelet
endothelial cell adhesion molecule, PECAM. Immunofluorescence analysis of
PECAM and ß-gal expression was used to localize endothelial cells and
HAND1 expressing cells in yolk sacs. Endothelial cells were located between
the extra-embryonic endoderm and mesoderm layers in both
Hand1ß-gal/+
(Fig. 3C) and
Hand1ß-gal/ß-gal
(Fig. 3D) embryos. HAND1 is
only expressed in extra-embryonic mesoderm and its expression pattern does not
overlap with PECAM (Fig. 3E-H). The presence of endothelial cells suggests the early steps of vasculogenesis,
the formation and clustering of endothelial cells is not dependent on
Hand1.
|
Hand1 regulates the expression of angiogenic genes
Numerous signaling molecules, receptors and signaling transduction pathways
regulate angiogenesis (Conway et al.,
2001; Patan, 2000
;
Sullivan and Bicknell, 2003
).
To determine which genes were misregulated in Hand1 mutant
extra-embryonic membranes, we analyzed their expression using
semi-quantitative RT-PCR (Fig.
4). All genes examined were expressed in Hand1 mutant
extra-embryonic membranes, indicating that Hand1 is not required for
their activation. However, several of the genes involved in vasculature
development were misexpressed in Hand1 mutant extra-embryonic
membranes, suggesting that Hand1 is required for their
regulation.
|
Angiogenesis is regulated through interactions between soluble angiogenic factors and their receptors. As VEGF and ANG1 can signal through a number of different receptors, we examined the expression of the VEGF receptors Flk1, Flt1/4 and Nrp1/2, and the ANG1 receptor gene Tie1/2, all of which are expressed in endothelial cells. Of these receptors, the expression of Flk1, Flt1, Nrp1 and Tie1 were upregulated in Hand1 mutant extra-embryonic membranes, while the expression of Flt4, Nrp2 and Tie2 was unchanged (Fig. 4).
The Eph/ephrin pathways play complex roles during vessel formation during
development (Adams, 2002). Both
ephrin B ligands and EphB receptors are expressed in yolk sac blood vessels
and disruption of this pathway leads to extra-embryonic vascular defects
morphologically similar to those of the Hand1-null mice
(Adams et al., 1999
). We
therefore examined if Eph/ephrin signaling in extra-embryonic membranes were
affected in
Hand1ß-gal/ß-gal
mice. Efnb2 was upregulated while Efnb1 and the
Ephb receptor expression were unaffected in Hand1 mutant
extra-embryonic membranes (Fig.
4).
Hedgehog (HH) signaling is also required during yolk sac angiogenesis,
although the action of the HH family member Indian hedgehog (IHH)
(Byrd et al., 2002) and the
loss of IHH results in severe vascular defects. In the extra-embryonic
membrane, IHH acts through binding to its receptor patched 1 (Ptch)
(Byrd et al., 2002
). In
Hand1 mutant extra-embryonic membranes, neither IHH nor
Ptch1 transcript levels were affected (Fig.
4).
Another signaling pathway that plays numerous roles during yolk sac
vasculature development is the Notch pathway
(Iso et al., 2003). Loss of
Notch1 arrests angiogenesis in the yolk sac and embryo, while the
loss of Notch4 has minor effects on vascular development. The
combined loss of both Notch1 and Notch4 leads to a more
severe vascular phenotype (Krebs et al.,
2000
). When we examined the effect of the loss of Hand1
on expression of Notch1/4 (Fig.
4) we found that both Notch1 and Notch 4 were
expressed at higher levels in Hand1 mutant membranes. To determine if
the enhanced expression of the Notch genes correlates with enhanced activity,
we examined the expression of a target of notch signaling, Hey1
(Maier and Gessler, 2000
). We
found that Hey1 was also upregulated in Hand1 mutant
extra-embryonic membranes (Fig.
4), suggesting that the enhanced Notch expression
corresponds to enhanced signaling.
Foxf1, a winged helix transcription factor, is involved in epithelial-mesenchymal interactions and loss of Foxf1 leads to vasculature defects. The extra-embryonic membranes of Foxf1-null embryos morphologically resemble those of the Hand1ß-gal/ß-gal mice. To determine if Hand1 regulates vascular development through the regulation of Foxf1, we examined the level of Foxf1 transcripts in Hand1ß-gal/ß-gal membranes. Foxf1 transcript levels were unaffected by the loss of the Hand1 gene (data not shown), suggesting that the genes regulate vascular development through different mechanisms.
Regulation of Hand2 expression by Hand1
The two HAND proteins share many structural features and function in a
similar manner to regulate limb polarity
(McFadden et al., 2002).
However, during heart development, the Hand genes have complementary
expression patterns (Cserjesi et al.,
1995
; Srivastava et al.,
1995
) suggesting that they may repress each other's expression.
Both Hand1 and Hand2 are expressed in the yolk sac mesoderm
and Hand2 mutant embryos show defects in yolk sac angiogenesis and in
neural crest-derived vascular smooth muscle development within the embryo. In
the yolk sac, Hand2 regulates the VEGF receptor Npr1,
suggesting a direct role in yolk sac vascular development
(Yamagishi et al., 2000
). We
examined the potential interaction between the two Hand genes during yolk sac
formation. Hand2 transcripts are upregulated in Hand1 mutant
embryos (Fig. 4). This is
consistent with the proposed role of HAND1 as a negative regulator of
Hand2 (Bounpheng et al.,
2000
; Cross et al.,
1995
). As the upregulation of Hand2 did not rescue the
Hand1 mutant phenotype, Hand1 and Hand2 appear to
regulate vascular development through different pathways.
To determine if Hand2 also regulates Hand1, we examined the expression of Hand1 in Hand2 mutant embryos (a gift from Dr Eric Olson, University of Texas Southwestern Medical Center, Dallas) by whole embryo ß-gal staining. When we examined the expression of the ß-gal knock-in gene in a Hand2-/- background (Fig. 5). We found that both the level and pattern of Hand1 expression were the same in wild type and Hand2 mutant backgrounds. RT-PCR was used to quantify the levels of Hand1 transcripts in Hand2 mutant and wild-type extra-embryonic membranes. Hand1 transcript levels were not affected by the genetic backgrounds (data not shown), indicating that Hand2 does not regulate Hand1 expression in extra-embryonic membranes.
|
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As VEGF and ANG1 are known to regulate smooth muscle recruitment as well as vasculature remodeling, and the levels of VEGF and ANG1 were upregulated in Hand1 mutant yolk sacs (Fig. 4), we examined the localization of both proteins in yolk sac using anti-VEGF and anti-ANG1 antibodies. The distribution of VEGF and ANG1 was unaffected in Hand1 mutant yolk sac (data not shown).
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Discussion |
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The extra-embryonic membrane is a region where Hand1 is normally expressed that is severely affected by loss of Hand1 function. Severe disruption of the vasculature occurs in the yolk sac of Hand1-null embryos. The data presented here suggest that Hand1 is required for maturation of the vasculature after extensive vasculogenesis has occurred and that Hand1 is required for both elaboration of the primitive vasculature and recruitment of SMCs. Interestingly, the lack of recruitment of SMC to the vasculature did not arrest SMC development.
Hand1 is expressed in the extra-embryonic mesoderm throughout development. The number of mesodermal cells in the Hand1-null membranes does not appear to be affected, suggesting that Hand1 is not required for their formation and survival. However, the yolk sac mesodermal layer is disorganized with extensive detachments of the mesoderm from the endoderm. Another major defect in Hand1-null yolk sacs is the loss of blood from the vasculature because of vascular defects. In the yolk sac, hematopoiesis accompanies angiogenesis in discrete clusters of cells that form the blood islands. This represents the beginnings of vascular development and hematopoiesis in mammals.
Regulation of angiogenesis by Hand1
Vascular development occurs in two stages: vasculogenesis, in which
angioblasts differentiate into endothelial cells to form the vascular
primordium; followed by angiogenesis, when the primitive vascular network is
extended by budding and branching. While endothelial cells differentiate in
Hand1 mutant yolk sacs, remodeling of the endothelial cells is
defective. The distribution of endothelial cells in a honeycomb-like plexus in
Hand1 mutant yolk sacs is similar to that seen in mice carrying
mutations for the angiogenic receptor tyrosine kinases Tie1/2
(Sato et al., 1995) and VEGF
receptor neuropilin 1/2 (Takashima et al.,
2002
). Thus, in yolk sacs lacking Hand1, vasculogenesis
occurs but angiogenesis is arrested at an early stage.
Angiogenesis is directed by a signaling system combining a number of
soluble growth factors that signal through a set of receptors. In
Hand1 mutant yolk sac, angiogenic genes are expressed showing that
Hand1 is not required for their activation. However, several
angiogenic genes are misexpressed in Hand1 mutant extra-embryonic
membranes. The angiogenic growth factor genes, Vegf and Ang1
are upregulated in Hand1 mutant membranes. Overexpression of VEGF
(Larcher et al., 1998) or ANG1
(Suri et al., 1998
) in
transgenic mice produces increased vascularization during embryogenesis and in
adults. It is unclear why increased expression of VEGF and ANG1 would lead to
a defect in vascular development. It is possible that the misregulation of
these factors in combination with other angiogenic genes leads to the vascular
phenotype. It has been reported that ANG1 regulates angiogenesis in a
paracrine manner (Suri et al.,
1996
) and that ANG1 acts as a chemoattractant; thus, the local
concentration of the signal is likely to influence angiogenesis.
The expression of the VEGF receptors Flk1, Flt1 and Nrp1,
and ANG1 receptor Tie1 is all upregulated in Hand1-null yolk
sac. As these genes are expressed in the endothelial lineage that does not
express Hand1, upregulation of these genes is not a cell autonomous
effect. Because expression of Flk1, Flt1, Nrp1 and Tie1 is
enhanced by VEGF (Barleon et al.,
1997; Kremer et al.,
1997
; McCarthy et al.,
1998
; Oh et al.,
2002
), the increased expression of these genes may be a result of
enhanced VEGF signaling in Hand1 mutant mice. The levels of the VEGF
receptor genes that are not regulated by VEGF signaling, Flt4 and
Nrp2 (Oh et al.,
2002
), were not affected in Hand1-null yolk sacs,
supporting an indirect role for HAND1 in regulating VEGF receptor genes.
VEGF is an upstream signal of the Notch pathway in arterial endothelial
differentiation (Lawson et al.,
2002). The enhanced activity of Notch in combination with other
signaling pathways may lead to the vascular phenotype we observe in
Hand1-null mice. The enhanced expression of the Notch1/4
genes in Hand1 mutant yolk sac, and the upregulation of the
downstream gene Hey1, suggests enhanced Notch function. Enhanced
Notch4 activity produces angiogenic defects
(Leong et al., 2002
;
Uyttendaele et al., 2001
)
suggesting that HAND1 functions in part by regulating the Notch pathway during
vascular development. Hand1 may regulate the Notch pathway through
enhanced expression of Vegf or by direct regulation of the Notch
genes.
Another signaling pathway that is enhanced in the absence of Hand1
is the Eph/ephrin family of receptors and ligand. In the absence of Hand1,
Efnb2 is up-regulated. Overexpression of ephrin B2 in mouse embryos
results in abnormal blood vessel formation
(Oike et al., 2002),
suggesting its overexpression in the yolk sac may contribute to the vascular
defects.
Regulation of Hand2 expression by Hand1
A wide and diverse array of transcription factors is required for
development of the vasculature (Oettgen,
2001). For example, the Ets family members ELF1, FLI1 and TEL; the
MADS box family member MEF2C; bHLH family members SCL/tal1 and EPAS and the
nuclear receptor COUP-TFII (NR2F2 Mouse Genome Informatics) are known
to regulate yolk sac vascular development. A number of these factors have been
shown to regulate angiogenesis through the direct regulation of angiogenic
genes. Loss of the Ets family transcription factor members
Elf1 (Dube et al.,
2001
), Fli1 (Hart et
al., 2000
) and Nerf2
(Dube et al., 1999
) leads to
reduced expression of the ANG1 receptor Tie2 while expression of
Ang1 is reduced in COUP-TFII knockout mice
(Pereira et al., 1999
). The
lack of total loss of angiogenic gene expression by the loss of an individual
transcription factor suggests that several different transcription factors
simultaneously regulate angiogenesis. In contrast to other transcriptional
regulators of angiogenic genes, where loss of function leads to decreased
expression, the loss of Hand1 results in enhanced expression of
angiogenic genes. If HAND1 regulates angiogenic genes directly, it appears to
suppress their expression. This is consistent with previous results that show
that HAND1 can act as a negative or positive regulator of transcription
(Bounpheng et al., 2000
;
Cross et al., 1995
).
HAND1 also suppresses expression of the bHLH factor Hand2. Hand2
is expressed in the yolk sac and is essential for vascular development, but
these defects in Hand2-null mice have not been examined in detail
(Yamagishi et al., 2000). As
the two Hand genes are expressed in a complementary manner during heart
development, this suggests that they may negatively regulate each other's
expression during heart development. Our results argue that although
Hand1 regulates Hand2 expression in the yolk sac,
Hand2 does not regulate Hand1. The inability of enhanced
Hand2 expression to compensate for the loss of Hand1
suggests the Hand genes have unique functions during extra-embryonic vascular
development. This is supported by the observation that the yolk sac
vasculature defects in Hand2-null mice are not identical to those
seen in the Hand1 mutant mice.
Regulation of smooth muscle cell development by Hand1
During vasculature maturation, endothelial cells are surrounded by
peri-endothelial cells and SMCs that give the vessels strength. In
Hand1 mutant yolk sacs, SMCs differentiate but do not surround the
endothelial tubes. The loss of SMCs in the peri-endothelial region may account
for the leakage of hematopoietic cells from the yolk sacs into the yolk
sac-amniotic space. Mice lacking Tie2 also exhibit hemorrhaging and
the absence of peri-endothelial cells observed in Hand1-null yolk
sacs (Sato et al., 1995).
However, in Hand1 null yolk sacs, Tie2 expression does not
appear to be affected, precluding Tie2 misregulation as the
Hand1 target causing the abnormal distribution of the SMCs.
A number of other transcription factors are involved in vascular SMC
development. Mice lacking Fli1
(Hart et al., 2000),
Mef2c (Lin et al.,
1998
), Smad5 (Yang et
al., 1999
), endoglin (Li et
al., 1999
) and Hand2
(Yamagishi et al., 2000
) show
reduced smooth muscle development around the vessel. It is not known whether
these genes are directly involved in SMC recruitment.
The abnormal distribution of SMCs in Hand1 yolk sac mesoderm suggests SMC migration is defective. TGFß1 and PDGFBB regulate vascular SMC migration through their receptors, TGFßRII, endoglin, PDGFRß and signal transduction factor SMAD5. We examined the expression of these genes in Hand1 mutant yolk sac and found the levels of these transcripts are unaffected. This suggests that regulation of SMC migration by HAND1 is downstream of, or independent from, these pathways. Vegf and Ang1 are also involved in smooth muscle recruitment, based on analysis of their loss of function, but it has not been determined whether enhanced expression of these two genes affects SMC recruitment. All signaling pathways that account for the SMC migration defect and whether Hand1 acts solely through these pathways are unknown.
Upregulation of angiogenic genes in the Hand1-null mice can account for defective vasculature remodeling and smooth muscle migration. In Hand1 mutant yolk sac, a number of angiogenic genes, especially the genes involving in signaling, are upregulated. It is unclear whether misregulation of these genes is the cause or the effect of the defective vasculature development.
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ACKNOWLEDGMENTS |
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