Müllerian Inhibiting Substance Signaling Uses a Bone Morphogenetic Protein (BMP)-Like Pathway Mediated by ALK2 and Induces Smad6 Expression
Trent R. Clarke,
Yasunori Hoshiya,
Soyun E. Yi,
Xiaohong Liu,
Karen M. Lyons and
Patricia K. Donahoe
Pediatric Surgical Research Laboratories (T.R.C., Y.H., X.L.,
P.K.D.) Department of Surgery Massachusetts General Hospital
and Harvard Medical School Boston, Massachusetts 02114
Department of Orthopaedic Surgery (S.E.Y., K.M.L.) Department
of Molecular, Cellular and Developmental Biology Department of
Biological Chemistry University of California, Los Angeles,
California 90095
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ABSTRACT
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Signal reception of Müllerian inhibiting
substance (MIS) in the mesenchyme around the embryonic Müllerian
duct in the male is essential for regression of the duct. Deficiency of
MIS or of the MIS type II receptor, MISRII, results in abnormal
reproductive development in the male due to the maintenance of the
duct. MIS is a member of the transforming growth factor-ß (TGFß)
superfamily of secreted protein hormones that signal through receptor
complexes of type I and type II serine/threonine kinase receptors. To
investigate candidate MIS type I receptors, we examined reporter
construct activation by MIS. The bone morphogenetic protein
(BMP)-responsive Tlx2 and Xvent2 promoter-driven reporter constructs
were stimulated by MIS but the TGFß/activin-induced p3TP-lux or
CAGA-luc reporter constructs were not. The induction of Tlx2-luc was
dependent upon the kinase activity of MISRII and was blocked by a
dominant negative truncated ALK2 (tALK2) receptor but not by truncated
forms of the other BMP type I receptors ALK1, ALK3, or ALK6. MIS
induced activation of a Gal4DBD-Smad1 but not a Gal4DBD-Smad2 fusion
protein. This activation could also be blocked by tALK2. The
BMP-induced inhibitory Smad, Smad6, was up-regulated by MIS
endogenously in Leydig cell-derived lines and is expressed in male but
not female Müllerian duct mesenchyme. ALK6 has been shown to
function as an MIS type I receptor. Investigation of the pattern of
ALK2, MISRII, and ALK6 in the developing urogenital system demonstrated
overlapping expression of ALK2 and MISRII in the mesenchyme surrounding
the duct while ALK6 was observed only in the epithelium. Examination of
ALK6 -/- male animals revealed no defect in duct regression. The
reporter construct analysis, pattern of expression of the receptors,
and analysis of ALK6-deficient animals suggest that ALK2 is the MIS
type I receptor involved in Müllerian duct regression.
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INTRODUCTION
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Müllerian inhibiting substance (MIS) is a developmentally
regulated protein hormone that belongs to the transforming growth
factor-ß (TGFß) superfamily of extracellular ligands (1, 2). MIS,
also known as anti-Müllerian hormone, is essential for the proper
primary organization of internal reproductive structures of male
mammals (3, 4, 5). The developing mammalian reproductive system is
initially identical in males and females with both sexes having two
sets of ducts in the mesonephros, the Müllerian and Wolffian
ducts, and bipotential gonads. In males, differentiation of the gonad
into a testis is initiated by SRY and results in the differentiation of
Sertoli and Leydig cells, which produce the hormones MIS and
testosterone, respectively. Expression of MIS is necessary for
regression of the Müllerian duct, and synthesis of testosterone
is essential for the maintenance of the Wolffian duct, which develops
into the vas deferens and epididymis (3). In females, the absence of
MIS allows the maintenance of the Müllerian duct, which develops
into the fallopian tubes, uterus, and upper vagina. Lack of
testosterone support in females results in the degeneration of the
Wolffian duct. MIS is also important for male fecundity and length of
female reproductive life span in mice (6, 7). Most MIS-deficient males
are sterile due to an anatomical block to sperm release but they still
produce functional sperm (6). MIS null females exhibit normal fertility
but with abnormal follicle recruitment. This results in higher numbers
of primary follicles, less primordial follicles, and a greater number
of total follicles undergoing atresia during the lifespan of the animal
(7).
MIS is a divergent member of the TGFß superfamily that does not fall
neatly into the subfamilies of activins, TGFßs, or bone morphogenetic
proteins (BMPs)/growth and differentiation factors (GDFs). Many reports
have characterized the signaling mechanism of TGFß superfamily
members and have shown that a general paradigm is conserved (reviewed
in Ref. 8). Ligand binds to or induces the formation of a receptor
complex consisting of type I and type II receptors. The type II
receptors are constitutively active serine/threonine kinases which,
when complexed with ligand and type I receptor, phosphorylate a motif
of the type I receptors called the GS box (for its Gly-Ser repeats)
(8). This activates type I receptor serine/threonine kinase activity
and allows the type I receptors to be recognized by pathway-regulated
Smad proteins (R-Smads) (9). The type I receptors phosphorylate the
R-Smads, which in turn dissociate from the receptor complex and
associate with the coactivator Smad, Smad4. This heteromeric Smad
complex translocates to the nucleus and modulates gene transcription by
affecting transcription factor complexes at regulated promoters (9).
Gene regulation seems to involve many mechanisms, the simplest of which
is the direct binding of the Smads to DNA and functioning as
transcriptional activators (10). Other described mechanisms include
binding to transcription factor complexes through protein-protein
interactions, recruitment of transcriptional coactivators or
corepressors, blocking or supplanting other transcription factors that
have overlapping binding sites with the Smads, and increasing the
degradation of transcriptional repressors or other signaling molecules
(10).
Five type II receptors have been identified in mammals. TßRII
is used for TGFß signaling, ActRIIA and ActRIIB for activin
signaling, BMPRII as well as ActRIIA and ActRIIB for BMP/GDF signaling,
and MISRII for MIS signaling (8). Seven type I receptors have been
identified in mammals. Activin receptor like kinase 1 (ALK1) and ALK5
(TßRI) are activated by TGFß, while ALK4 (ActRIB) is used by
activin. ALK7 is an orphan type I receptor whose physiological ligand
has not yet been determined. ALK1, ALK2 (ActRIA), ALK3 (BMPRIA), and
ALK6 (BMPRIB) are activated by various BMP ligands (8); however, the
relative abilities of different BMP/GDF ligands to activate specific
ALKs 1, 2, 3, and 6 are not completely understood. The identity of the
MIS type I receptor and the Smad-mediated downstream pathway activated
by MIS is the topic of this study.
Smad proteins are a family of intracellular signal mediators that fall
into three broad categories consisting of R-Smads, coactivator Smads,
and inhibitory Smads (I-Smads). R-Smads are described above, and the
only identified mammalian coactivator Smad, Smad4, complexes with
activated R-Smads. This causes translocation into the nucleus to
participate in gene regulation. Inhibitory Smads (I-Smads) inhibit the
signaling pathways by binding and sequestering activated type I
receptors or activated R-Smads, thus preventing signal transduction
(8). The I-Smads are often up-regulated by the specific pathways that
they inhibit, thus providing a negative feedback mechanism ensuring
that the magnitude and duration of the intracellular response
correlates with the concentration and continuing presence of the
extracellular ligand (11, 12, 13). This effect is due to direct activation
of the promoters of the I-Smads by activated R-Smad/Smad4 heteromeric
complexes (14, 15).
Smad2 and Smad3 are R-Smads used by both the TGFß and activin
pathways, whereas the other R-Smads, Smad1, Smad5, and Smad8, are
activated by BMP/GDF pathways (8). No differential activation between
the BMP-specific Smads has been demonstrated downstream of different
BMP ligands or type I receptors (ALKs 1, 2, 3, and 6), although they
may play different roles during development due to varied patterns of
expression in different tissues or, perhaps, to intrinsic functional
differences (16). Smad5 expression has been shown near the newly formed
Müllerian duct in day 12 mouse embryos (17). The I-Smad, Smad7,
is induced by TGFß and activin signaling (11), while the expression
of the other I-Smad, Smad6, has been demonstrated in response to BMP
signaling (18).
Previously, an orphan type II receptor that had an expression pattern
consistent with it being the MIS type II receptor was identified
(19, 20, 21). Its identity as the MIS type II receptor was unequivocally
verified by generating homozygous knockout mice that exhibited
phenotypes identical to homozygous MIS ligand knockout mice (22).
Previous biochemical analyses identified ALK6 as an MIS type I receptor
(23). Here we show that ALK2 can function as an MIS type I receptor,
that its pattern of expression in the Müllerian duct is
consistent with this role in vivo, and that male mice in
which ALK6 has been homozygously inactivated exhibit normal
Müllerian duct regression. Based on these data, we propose that
ALK2 is likely to be a physiologically relevant type I receptor for
Müllerian duct regression.
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RESULTS
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Smad6 Induction by MIS in Leydig Cell Tumor Cell Lines
Since TGFß and activin signaling has been shown to induce the
expression of Smad7, and BMP signaling has been shown to induce the
expression of Smad6, we investigated which I-Smad (if any) is induced
by MIS, which could provide insight into its signaling pathway and the
identity of the MIS type I receptor. Leydig cells have been shown to
respond to MIS (24, 25, 26, 27, 28, 29), so we examined the induction of Smad6 and
Smad7 in the MA-10 and R2C Leydig cell-derived immortalized cell lines
(28). Treatment of the mouse MA-10 Leydig cell tumor cell line with 100
nM MIS increased expression of Smad6 and, to a much lesser
extent, Smad7 (Fig. 1A
). Robust induction
of Smad6 over a low basal level was observed by 2 h (5-fold by
densitometry scans), reached a maximum at 6 h (8-fold), and
decreased by 10 h (6-fold), whereas a slight induction of Smad7
was observed by 2 h and remained constant for the next 8 h.
Examination of earlier timepoints revealed that the first detectable
increase in Smad6 expression by Northern analysis was at 90 min after
MIS treatment (data not shown). The Smad6 and Smad7 probes used in Fig. 1
were of similar specific activity, suggesting that the induction of
Smad7 in MA-10 cells never reaches even the basal level of observed
Smad6 expression. To determine whether this induction could be repeated
in a different species, the rat R2C Leydig tumor cell line was
investigated. Figure 1B
demonstrates that MIS treatment induces Smad6
and slightly increases Smad7 expression in R2C cells but,
interestingly, with a different time course than in MA-10 cells. Smad6
increased over a basal level by 6 h, was maximal at 12 h, and
was beginning to decrease by 24 h. Smad7 expression peaked
slightly over basal at 12 h. It is unclear whether the different
time courses for Smad induction and magnitude of the Smad6 induction in
the two Leydig tumor cell lines is due to the species difference,
expression levels of the receptors, or some unknown factor. To examine
whether the induction of the Smad mRNAs by MIS was a direct effect or
required the induction of another protein, cycloheximide (CHX)
treatment was used to block new protein synthesis. Smad6 and Smad7
expression was similarly induced in the presence and absence of 10
µg/ml of CHX, demonstrating that the increase in Smad6 and Smad7 mRNA
was directly regulated downstream of MIS receptor activation (Fig. 1C
).
Smad6 was induced 7-fold in the presence of CHX and 5-fold in the
absence of CHX after 2.5 h of MIS treatment. Smad7 exhibited an
increased level of mRNA due to the treatment of CHX alone, which was
additive with the MIS induction. This increase with CHX treatment is
often seen with highly labile mRNAs. This may indicate that the rate of
degradation of Smad7 message is slowed by CHX blocking production of
short-lived proteins important for destabilization of the mRNA (11).
This would result in a greater steady-state level of the message in the
presence of protein synthesis inhibitors.

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Figure 1. Smad6 Induction by MIS in Leydig Cells
Northern blots of polyA+ mRNA generated from serum-starved (1/20 normal
serum) MA-10 (A and C) or R2C cells (B) treated with or without MIS
were hybridized with Smad6 and Smad7 probes. The lower
panels show the same blots probed for ß-actin mRNA. A, Blot
of 5 µg of mRNA from serum starved (15 h) MA-10 cells treated with or
without 100 nM MIS for 010 h demonstrating that Smad6
and, to a lesser extent, Smad7 mRNAs are induced by MIS. B, Blot of 3.8
µg of polyA+ mRNA from serum-starved (15 h) R2C cells treated with
100 nM MIS for 024 h showing that Smad6 is induced in
this Leydig cell line as well. C, Blot of 5 µg of polyA+ mRNA from
serum-starved (18.5 h) MA-10 cells pretreated for 30 min with or
without 10 µg/mg CHX and then incubated with or without 188
nM MIS for 2.5 or 5 h. Smad6 and Smad7 induction by
MIS does not require protein synthesis, demonstrating that their
induction is a direct effect of MIS signaling. These experiments were
performed on serum-starved cells but identical results were obtained in
the presence of serum (data not shown).
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Smad6 Expression in Male Müllerian Duct Mesenchyme
To investigate whether the induction of Smad6 by MIS observed in
the Leydig tumor cell lines also occurred in the Müllerian duct
in vivo, whole-mount in situ hybridization was
performed. Smad6 expression is observed in the mesenchyme surrounding
the regressing Müllerian duct in male rat embryos at 14.5 days
post coitum (E14.5) (Fig. 2
, A and B) and
with greater intensity at 15.5 days post coitum (E15.5) (Fig. 2
, E and
F). Smad6 expression was not observed in the mesenchyme surrounding the
Müllerian duct in female rat embryos at E14.5 or E15.5 (Fig. 2
, C, D, G, and H). The localization of this expression is similar to that
of MISRII (see Fig. 6
, IL); however, MISRII is expressed in both male
and female embryos. This pattern is consistent with Smad6 being induced
by MIS in vivo in male embryos. The greater induction of
Smad6 by MIS compared with that of Smad7 in Leydig cell-derived lines
and the male-specific expression of Smad6 at the site of MIS action
suggests that MIS signaling is more similar to the BMP pathway than the
TGFß/activin pathway.

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Figure 2. Smad6 Induction in the Male Müllerian Duct
Mesenchyme
In situ hybridization of Smad6 RNA was performed on
E14.5 (AD) and E15.5 (EH) male (A, B, E, and F) and female (C, D,
G, and H) embryos. The ventral body wall and viscera were removed from
the embryos. They are oriented ventral side up, anterior to the
upper left. The dotted line in the
whole-mount pictures (A, C, E, and G) represents the approximate plane
from which the sections (B, D, F, and H) were taken. The sections were
redetected with BM-Purple for 24 h at 25 C. The black
arrowheads point to the Müllerian duct in the pictures of
the embryos. Note the equivalent expression of Smad6 in both male (E)
and female (G) kidney at E15.5, presumably due to induction by another
BMP-like signaling pathway, demonstrating the integrity of the RNA in
the female embryo. These embryos are representative of five different
embryos examined at each time point for both sexes. W, Wolffian duct;
M, Müllerian duct; K, kidney.
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Figure 6. ALK2, ALK6, and MISRII Expression in the
Müllerian Duct Mesenchyme
In situ hybridization of ALK2 (AD), ALK6 (EH), and
MISRII (IL) mRNA was performed on E15.5 male (A, B, E, F, I, and J)
and female (C, D, G, H, K, and L) embryos. The ventral body wall and
viscera were removed from the embryos. They are oriented ventral side
toward the camera, anterior to the top. The dotted line
in the whole-mount pictures (A, C, E, G, I, and K) represents the
approximate plane from which the sections (B, D, F, H, J, and L) were
taken. B and D, The ALK2 sections were redetected with BM-Purple for
20 h at 25 C. F and H, The ALK6 sections were redetected for
50 h. J and L, The MISRII sections did not require redetection.
The black arrowheads point to the Müllerian duct
in panels A, E, I, and K. The gray arrowheads in panels
A, E, I, and K point to gonadal expression of the receptors. The
white arrowheads in panels C and G point to nonspecific
background staining between the ovary and mesonephros. W, Wolffian
duct; M, Müllerian duct. Note the expression of ALK2 in the
mesenchyme around the Müllerian duct and the expression of ALK6
in the epithelium of the Müllerian and Wolffian ducts. Expression
of the MIS type II receptor overlaps the expression of ALK2 and Smad6
in the Müllerian mesenchyme. These embryos are representative of
three different embryos examined for both sexes with each probe. ALK6
is not required for Müllerian duct regression in males. The
internal reproductive structures of an adult wild type (M) and BMPRIB
(ALK6) homozygous null male mouse (N). Note the absence of any retained
Müllerian-derived structures such as are observed in MIS and MIS
type II receptor homozygous knockout mice. T, Testis; e, epididymis; v,
vas deferens; p, prostate; sv, seminal vesicle.
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MIS Activation of BMP-Responsive Promoters
To investigate further whether MIS signaling shares similarities
with BMP signaling, transient transfection assays with promoters
regulated by the TGFß, activin, or BMP pathways were analyzed for
their regulation by MIS. The TGFß- and activin-responsive reporter
constructs 3TP-lux and CAGA-luc (30, 31), and the BMP-inducible
reporter constructs Tlx2-luc and Xvent2-luc (32, 33) did not show
regulation by MIS in MA-10, COS7, or Mv1Lu cells (data not shown).
While the 3TP-lux and CAGA-luc reporters utilize artificial promoters
that are regulated by TGFß and activin in a variety of cell lines
including Mv1Lu cells, the Tlx2 and Xvent2 constructs employ endogenous
promoters. Both Tlx2 and Xvent2 are expressed in very restricted
domains exclusively in the early embryo (33, 34); hence, it is likely
that several tissue-specific transcription factors are required for
these promoters in addition to BMP-regulated transcription factors
and/or Smads. Since previous analyses of the Tlx2 and Xvent2 reporter
constructs were performed in the embryonic carcinoma-derived P19 cell
line, the regulation of the reporters by MIS in these cells was
investigated. The activity of the Tlx2 reporter construct is not
affected by MIS in P19 cells unless an expression construct for MISRII
is also transfected, indicating that these cells do not express
endogenous MISRII (Fig. 3A
). When MISRII
is transfected, the Tlx2 reporter construct exhibited 8- to 12-fold
induction by MIS (Fig. 3A
). The Xvent2 reporter construct also
demonstrated an MISRII-dependent regulation by MIS in P19 cells,
although the fold induction was less, approximately 3- to 5-fold (Fig. 3A
). To examine specificity of the MISRII-mediated MIS induction of the
reporters, other type II receptors were examined for their ability to
transduce MIS-mediated reporter activation. When TßRII and BMPRII
were expressed, treatment with MIS was unable to activate Tlx2 reporter
expression (Fig. 3B
). To demonstrate that the activation of the Tlx2
promoter was dependent upon the kinase activity of MISRII, a conserved
lysine (K228) in the ATP binding region of the kinase domain was
mutated to an arginine (K228R). This should make a form of MISRII
which, based on analogy with other kinase domains, should be unable to
bind ATP and thus have no kinase activity. Overexpression of
MISRII(K228R) does not allow P19 cells to respond to MIS, demonstrating
that the Tlx2 promoter induction observed with MIS is dependent upon
MISRII serine/threonine kinase activity (Fig. 3C
). MISRII-transfected
P19 cells were treated with different MIS concentrations to examine the
sensitivity of the Tlx2 promoter to its activation. The Tlx2 reporter
construct was maximally activated at concentrations as low as 10
nM MIS and still exhibited 80% activation at 1
nM MIS (Fig. 3D
). No activation of the 3TP-lux and CAGA-luc
reporters by MIS was observed in MISRII-transfected P19 cells (data not
shown). The induction of the Tlx2-luc and Xvent2-luc reporter
constructs by MIS and the lack of MIS-mediated activation of the
3TP-lux and CAGA-luc reporters further confirms that MIS signaling is
more similar to BMP signaling than to either TGFß or activin
signaling.

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Figure 3. BMP-Responsive Promoter Induction by MIS
P19 cells were transfected with 400 ng of the Tlx2-luc or Xvent2-luc
firefly luciferase reporter constructs, various expression constructs,
and 100 ng of the thymidine kinase promoter-driven Renilla luciferase.
Twenty four hours after transfection the cells were treated with or
without MIS; 24 h later the lysates were assayed for firefly and
Renilla luciferase. The results are presented as relative luciferase
units (R.L.U.) of the mean ± SD of triplicate samples
for each representative experiment. A, MISRII mediated activation of
Tlx2-luc and Xvent2-luc reporter constructs. Three hundred nanograms of
an MISRII-expressing or empty expression vector and the Tlx2-luc or
Xvent2-luc reporter constructs were transfected into P19 cells. The
Tlx2 and Xvent2 reporter constructs are induced by 100 nM
MIS in P19 cells only when transfected with the MIS type II receptor.
B, Type II receptor specificity of MIS-mediated reporter activation.
P19 cells were transfected with Tlx2-luc and 300 ng of type II receptor
expression construct. Tlx2-luc is activated by 100 nM MIS
only in MISRII-expressing P19 cells, not in cells transfected with
TßRII or BMPRII expression constructs. C, Tlx2 reporter activation by
MIS depends on MISRII kinase activity. P19 cells were transfected with
Tlx2-luc and 300 ng of expression constructs for MISRII or the ATP
binding site mutant receptor MISRII(K228R); 100 nM MIS
induction of Tlx2-luc was only mediated by MISRII with an intact kinase
domain. D, Response of the Tlx2 promoter to exposure to different
concentrations of MIS. P19 cells were transfected with 300 ng of the
MISRII expression vector and treated with the indicated concentrations
of MIS. Tlx2 promoter activation was still maximal with 10
nM MIS. E, Effect of dominant negative truncated receptors
on Tlx2 promoter induction by MIS. Five hundred nanograms of expression
constructs for forms of ALK1, ALK2, ALK3, and ALK6, which terminate
2024 amino acids beyond the transmembrane domain, and 50 ng of the
MISRII expression construct were transfected into P19 cells along with
Tlx2-luc and treated with 100 nM MIS. Only truncated ALK2
was able to block reporter activation.
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Dominant Negative ALK2 Blocks Tlx2 Promoter Activation by MIS
Because the Tlx2 promoter reporter construct responds to low
concentrations of MIS with robust activation, it was employed to
investigate which type I receptor(s) MIS uses for signal transduction.
Because ALK1, ALK2, ALK3, and ALK6 are activated by BMP signaling,
these type I receptors were investigated as candidate MIS type I
receptor(s). The fact that MISRII-transfected P19 cells can support MIS
signaling suggested that these cells must endogenously express at least
one MIS type I receptor(s). To examine which type I receptor(s) MIS is
using, we constructed truncated forms of ALK1 (tALK1), ALK2 (tALK2),
ALK3 (tALK3), and ALK6 (tALK6), which terminate 2024 amino acids
after the transmembrane domain, and replaced the GS box and kinase
domain with a hemagglutinin (HA) epitope tag. The truncated receptors
should act as dominant negative forms because they are able to bind and
sequester ligand but, lacking the kinase domain, cannot initiate
downstream signaling. The constructs were transfected into COS7 cells,
and lysates were prepared and analyzed by Western blotting with
HA
antibody to verify that the receptors were truncated and expressed
(data not shown). When expressed along with MISRII in P19 cells, tALK2
completely blocked MIS-mediated Tlx2 promoter activation. However,
tALK1, tALK3, and tALK6 had little effect (Fig. 3E
). This indicates
that while ALK1, ALK3, and ALK6 are dispensable for MIS signaling in
P19 cells, ALK2 is essential for MIS-mediated Tlx2 promoter activation.
Taken together, these experiments suggest that MIS signaling is
dependent upon the kinase activity of MISRII and ALK2.
MIS Activates Smad1
Knowing that BMP signaling activates Smad1 (35), we investigated
the ability of MIS to activate Smad1. It has previously been shown that
a Gal4-Smad1 fusion protein with the DNA binding domain of the Gal4
transcription factor (Gal4DBD) fused to the N-terminus of Smad1 can be
used to measure BMP signaling (35). When the Gal4-Smad1 fusion protein
is phosphorylated and translocated to the nucleus, it activates a
reporter construct of multiple Gal4 binding sites driving the
expression of a luciferase gene. When cotransfected with MISRII in P19
cells, this Gal4 reporter system gives a small, reproducible response
to MIS (Fig. 4A
). Cotransfection of a
constitutively active form of ALK2, ALK2(Q207D), with Gal4-Smad1
produces a robust activation of the Gal4 binding site reporter
construct (Fig. 4A
). In the absence of the Gal4-Smad1 fusion protein,
ALK2(Q207D) cannot induce the reporter construct, showing the
specificity of the assay (Fig. 4A
). The Gal4-Smad1-mediated induction
of the promoter by MIS was abrogated when truncated ALK2 was included
in the transfection (Fig. 4B
). Similar Gal4-Smad2 and Gal4-Smad3 fusion
proteins have been used to assay TGFß signaling (36, 37). When the
Gal4-Smad2 fusion protein was used, no induction of the reporter by MIS
was observed (data not shown). These experiments suggest that MIS can
induce the activation of Smad1 via the function of MISRII and ALK2.

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Figure 4. Smad1 Activation by MIS
P19 cells were transfected with 400 ng of the pG5E1b-luc firefly
luciferase reporter construct, the expression of which is driven by
five Gal4 binding sites, various expression constructs, and 100 ng of
the thymidine kinase promoter-driven Renilla luciferase. Twenty four
hours after transfection the cells were treated with or without 100
nM MIS; 24 h later the lysates were assayed for
firefly and Renilla luciferase. The results are presented as relative
luciferase units (R.L.U.) of the mean ± SD of
triplicate samples for each representative experiment. A, Activation of
a Gal4 responsive promoter by MIS-mediated activation of a
Gal4DBD-Smad1 fusion protein. P19 cells were transfected with
pG5E1b-luc, 200 ng of an expression construct for a fusion protein
consisting of the DNA binding domain of Gal4 fused to the N terminus of
Smad1, and 300 ng of expression constructs for MISRII or ALK2Q207D, the
constitutively active form of ALK2. MIS treatment of the cells
expressing MISRII increased reporter activity but less than the
activation seen with the constitutively active form of ALK2. B,
Truncated ALK2 blocks the MIS-induced activation of pG5E1b-luc by
Gal4-Smad1. P19 cells were transfected with pG5E1b, 50 ng of the MISRII
and Gal4-Smad1 expression constructs, and 400 ng of the truncated ALK2
expression construct or parental vector. MIS treatment was not able to
induce activation of pG5E1b by Gal4-Smad1 when truncated ALK2 was
overexpressed.
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Expression of BMP-Regulated R-Smads during Müllerian Duct
Regression
Although we had observed Smad1 activation in tissue culture,
it did not necessarily imply a role for Smad1 in MIS signaling, since
ALK2 has been shown to activate Smad1, Smad5, as well as Smad8 in
overexpression systems (38, 39, 40). To identify which of the BMP-regulated
Smads are possible physiological mediators of MIS signaling, we
investigated the pattern of expression of these Smads in the urogenital
ridge at the time of Müllerian duct regression (E15.5). Smad1
shows robust expression in the mesenchyme of male and female
mesonephros in the regions surrounding the Müllerian duct and
adjacent to the Wolffian duct (Fig. 5
, A,
D, and G). Smad5 is expressed at substantially lower levels than that
observed with Smad1 in both male and female mesenchyme (Fig. 5
, B, E,
and H). Smad8 exhibited low levels of expression throughout the
mesenchyme of the mesonephros in the female, but in the male,
interestingly, the expression is much greater in the mesenchyme
surrounding the Müllerian duct (Fig. 5
, C, F, I, and J). These
results give the impression that Smad1 and Smad8 are most likely to be
the mediators of MIS signaling.

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Figure 5. BMP-Regulated R-Smad Expression in the
Müllerian Duct Mesenchyme at the Time of Duct Regression
In situ hybridization of Smad1 (A, D, and G), Smad5 (B,
E, and H), and Smad8 (C, F, I, and J) mRNA was performed on E15.5 male
(AC and GI) and female (DF and J) embryos. The ventral body wall
and viscera were removed from the embryos. The embryos in the
whole-mount images (AF) are oriented ventral side toward the camera,
anterior to the top. The dotted line in whole-mount
pictures A, B, C, and F represents the approximate plane from which the
sections G, H, I, and J were taken, respectively. The sections were
redetected with BM-Purple for 42 h at 25 C. The black
arrowheads point to the Müllerian duct in panels AF.
The gray arrowheads in panels AF point to gonadal
expression of the Smads. W, Wolffian duct; M, Müllerian duct.
Note the higher expression of Smad1 compared with Smad5 and the greater
expression of Smad8 in the mesenchyme around the male compared with the
female Müllerian duct. Expression of the MIS type II receptor
overlaps the expression of all three Smads in the Müllerian
mesenchyme (see Fig. 6 , IL). These embryos are representative of at
least six different embryos examined for both sexes with each probe.
|
|
Colocalization of ALK2 and MISRII in the Developing Urogenital
System
Because both ALK2 (this study) and ALK6 (23) have been
implicated in MIS signal transduction, we investigated the expression
of ALK2 and ALK6 in the Müllerian duct. ALK2 is observed in male
and, to a lesser extent, in female rat embryos at E15.5 in the
mesenchyme of the Müllerian duct (Fig. 6
, AD). In contrast, ALK6 is observed
in male and female embryos at E15.5 in the epithelium of the
Müllerian and Wolffian ducts, but not in the mesenchyme
surrounding the ducts (Fig. 6
, EH). When compared with the expression
of MISRII in male and female embryos at E15.5 (Fig. 6
, IL), ALK2
expression overlaps that of MISRII, while ALK6 is expressed in the
adjacent epithelial layer. It has been demonstrated previously that the
mesenchyme around the Müllerian duct, but not the epithelium of
the duct, is the site of MIS action (41, 42, 43). The pattern of expression
exhibited by ALK2 suggests that it is more likely the physiologically
relevant type I receptor for Müllerian duct regression.
ALK6 Null Male Mice Do Not Have Retained Müllerian
Ducts
Recently, we generated ALK6 knockout mice (44). Upon
inspection of male mice homozygous for the inactive allele of ALK6, we
discovered that they do not exhibit any evidence of retained
Müllerian duct structures (Fig. 5
, compare panel N to panel M).
They do exhibit a defect in seminal fluid production that is reflected
in the deflated and discolored appearance of the seminal vesicles (Fig. 5N
). This defect is currently being investigated. Female ALK6-deficient
mice have oviducts and uteri (data not shown), indicating that
Müllerian duct structures in these mice form and are capable of
differentiating into adult structures. This argues against a defect in
Müllerian duct formation as is observed in Wnt4-deficient animals
(45) in which both females and males lack Müllerian ducts. These
observations suggest that ALK6 is not essential for Müllerian
duct formation or regression. The expression data for MISRII, ALK2, and
ALK6 combined with the absence of a Müllerian duct defect in
ALK6-deficient male mice suggest that ALK2, not ALK6, is involved in
regression of the Müllerian duct, but does not rule out a role
for ALK6 as an essential MIS type I receptor in other MIS
functions.
 |
DISCUSSION
|
---|
In this report, we have shown that MIS can activate reporter
constructs driven by the BMP-responsive promoters, Tlx2 and Xvent2, in
P19 cells. This induction was dependent upon the presence of functional
MISRII, because expression of a form of the receptor that has an
altered ATP binding pocket, MISRII(K228R), did not respond to MIS
stimulation. Previous studies analyzing BMP function in Xenopus
laevis have used forms of receptors lacking the intracellular
domain to act as dominant negative receptors (46). When truncated forms
of ALK1, ALK2, ALK3, and ALK6 were expressed, only tALK2 blocked the
activation of the Tlx2 reporter by MIS. Thus, MIS signaling in P19
cells is dependent on ALK2 while ALK1, ALK3, and ALK6 are dispensable.
A previous report by Macias-Silva et al. (39) has
demonstrated the presence of ALK2, ALK3, and ALK6 in P19 cells by
immunoprecipitating the receptors cross-linked to radiolabeled BMP2 or
BMP7 using receptor-specific antibodies. This would suggest that in
these cells MIS signaling requires ALK2 even though ALK3 and ALK6 are
present. The previous report by Gouédard et al. (23)
indicates that ALK6 can mediate MIS function. Our results suggest
that ALK2 can also mediate MIS function. Taken together, these studies
may indicate that the signaling pathway used by MIS may depend on the
cell type or tissue. To determine which receptor is most likely the MIS
type I receptor mediating Müllerian duct regression, we analyzed
the expression of ALK2 and ALK6. ALK2 was found in the mesenchyme
surrounding the Müllerian duct. In contrast, ALK6 localized to
the epithelium of both the Müllerian and Wolffian ducts. These
results are consistent with our previous examination of ALK2 expression
(47) and the previously reported expression pattern of ALK6 (48). The
expression of ALK2 overlaps the expression of MISRII in the mesenchyme
adjacent to the Müllerian duct. The mesenchyme of the duct has
previously been identified as the site of MIS action (41, 42, 43). The
epithelium is not directly responsive to MIS, but undergoes apoptosis
during duct regression as a secondary effect to signal reception in the
mesenchyme (41, 42, 43).
Knockout mice homozygously deficient in ALK2 and ALK6 have been
generated. Male ALK6 knockout mice survive to adulthood and do not
exhibit any defects in Müllerian duct regression, demonstrating
that ALK6 is not essential for this process (Fig. 5
, panel M
vs. panel N). ALK2 knockout mice die before embryonic day
9.5 (49, 50), making it impossible to investigate the effect of an ALK2
null genotype on Müllerian duct regression. The production of
mice with tissue-specific inactivation of ALK2 may be the most
definitive way to determine whether ALK2 function is indispensable for
duct regression. Despite the fact that ALK6 is not required for
regression of the duct, ALK6 may be essential for other functions of
MIS. MIS has been shown to decrease steroid synthesis in Leydig cells
(24, 25, 26, 27, 28, 29) and to play a role in follicle recruitment (7). The existence
of viable animals with homozygous deficiency of ALK6 will allow us in
future studies to determine whether ALK6 plays an indispensable role in
either of these functions of MIS.
Interestingly, the E15.5 male Müllerian duct mesenchyme exhibits
a greater level of expression of ALK2 and Smad8 than is observed in the
female. In the rat, MIS expression initiates before E13.5, so the E15.5
male rat embryos have already had considerable exposure to MIS. Whether
this increased expression in the male is a consequence of ongoing MIS
signaling, the presence of testosterone, or another male-specific
signal is the topic of ongoing investigations. Sexual differentiation
is a prototypical example of a "genetic switch" in which the
outcome is normally one of two finite end states. It is tempting to
speculate that up-regulation of ALK2 and Smad8 in response to MIS
generates a feed-forward mechanism sensitizing the tissue such that
once the regression of the Müllerian duct has begun it is more
likely to proceed to completion. On the other hand, if the greater
expression of ALK2 and Smad8 in the male is due to testosterone or
another testicular signal, it would suggest a new dimension to
interactions between testicular hormones.
Many BMP ligands have been demonstrated to activate Smad1. MIS was
shown to induce a Gal4 reporter construct by activating a Gal4DBD-Smad1
fusion protein. This activation was blocked with truncated ALK2,
suggesting that the induction of the reporter was dependent upon ALK2
as well as MISRII. The activation of Smad1 by MIS is consistent with
the report by Gouédard et al. (23) in which they
demonstrated phosphorylation of Smad1 by MIS signaling with a
phospho-specific Smad1 antibody. We also observed activation of the
Gal4DBD-Smad1 fusion protein by constitutively active ALK2, which is
consistent with the reports by Macias-Silva et al. (39) and
Chen and Massague (40) in which they demonstrate that ALK2 can
phosphorylate Smad1. ALK2 is used by BMPs also, most notably BMP7 (39).
MIS shares with some BMP/GDFs the roles of developmental tissue
remodeling through apoptosis and epithelial-mesenchymal transformation
(42, 43, 51, 52, 53, 54, 55, 56, 57). Other BMP/GDF ligands, including BMP8A, BMP8B, GDF9,
and BMP15, have also been shown to have important roles in reproductive
function. Deficiency of BMP8B or BMP8A causes defects in initiation or
maintenance of spermatogenesis, respectively (58, 59), GDF9 null
females have defective follicle maturation resulting in sterility (60),
and homozygous deficiency of BMP15 results in infertility (61, 62).
Whether ALK2 is used in these other BMP signaling pathways impacting
reproduction is an interesting question that will need to be addressed
with conditional inactivation of ALK2 in specific tissues using Cre-lox
technology in mice.
Smad 5 has been demonstrated previously in the early Müllerian
duct (17). In this study, in situ hybridization of embryos
at a later developmental stage revealed expression of Smad1 and Smad8
in the mesenchyme around the Müllerian duct whereas Smad5 was
barely detectable. The intriguing sexual dimorphism exhibited by Smad8
will be investigated in future studies to determine whether this is a
direct effect of MIS signaling. Since each of these R-Smads have been
shown to be activated by ALK2 (38, 39, 40), it will be interesting in the
future to investigate whether they have nonredundant functions in
Müllerian duct regression or whether they can functionally
substitute for each other.
MIS induces Smad6 transcription strongly and, to a much lesser extent,
Smad7 transcription. BMPs have previously been shown to induce
expression of Smad6, and TGFß and activin have been shown to induce
expression of Smad7 (11, 18). The induction of Smad6 by MIS did not
require protein synthesis, indicating that Smad6 induction occurs
immediately downstream of MIS function. Both MIS and BMPs induce Smad6
with a similar time course in which maximal expression was 46 h after
exposure (18). Smad7 induction by TGFß (11), on the other hand,
appears within 30 min. Despite the much longer time course observed for
Smad6 induction by MIS, it appears to be an immediate early effect as
well since it does not require protein synthesis. These observations
are consistent with the study by Ishida et al. (15), which
identified a BMP-responsive Smad binding site in the Smad6 promoter.
Future studies will determine whether MIS induction of Smad6 functions
through this same region. The expression of Smad6 in the mesenchyme of
the male Müllerian duct and its absence in the mesenchyme of the
female duct suggests that Smad6 is induced by MIS in vivo.
The induction of negative regulators by signaling pathways seems to be
a common paradigm. Several examples include the induction of patched in
Leydig cells by desert hedgehog secreted from Sertoli cells (63), the
induction of Argos by EGF signaling (64), the induction of Sprouty by
FGF signaling (65), and the induction of naked cuticle by Wnt signaling
(66). The possibility of the induction of the ALK2 type I receptor and
the R-Smad, Smad8, by MIS signaling and the increased expression of the
intracellular antagonist Smad6 by MIS suggest that even a seemingly
straightforward biological pathway has robust mechanisms of ensuring
appropriate response in the target tissues. In conclusion, the MIS
signaling pathway appears to have many similarities with previously
described BMP signaling pathways. MIS signaling activates Smad1,
induces the expression of Smad6, and relies upon the kinase activity of
MISRII and ALK2.
 |
MATERIALS AND METHODS
|
---|
Reagents
R2C cells, P19 cells, and COS7 cells were purchased from the
American Type Culture Collection (Manassas, VA). MA-10
cells were a gift from Dr. Mario Ascoli (Department of Pharmacology,
The University of Iowa College of Medicine, Iowa City, IA). Chemicals,
unless specified otherwise, were from Sigma (St. Louis,
MO). Human MIS was prepared from stably transfected CHO cells as
described previously (67).
Plasmids and Constructs
The MISRII expression construct was made by mutating the stop
codon of the rat MISRII cDNA from pBS7 (21) to a BamHI
site using the Quick Change mutagenesis system
(Stratagene, La Jolla, CA) following manufacturers
instructions using the primers
5'-GCAAGGCTCTGGCTCTAAGTCCGGATCCTGTAAGTGCCACTG-3' and
5'-CAGTGGCACTTACAGGATCCGGACTTAGAGCCAGAGCCTTGC-3' and
subcloning into EcoRI-BamHI-digested pFLAG-CMV5a
(Sigma). The MISRII(K228R) expression construct was
created by changing nt 683 of the coding region from A to G, which
changes lysine 228 to an arginine, by PCR-mediated mutagenesis using
the primer 5'-TGGGGGGAAGGCCCTGATGGCTACC-3' and the M13 -40
vector sequencing primer (5'-GTTTTCCCAGTCACGACGTTGTA-3') to amplify the
N-terminal part of the coding sequence and using the primers
5'-GGTAGCCATCAGGGCCTTCCCCCCA-3' and
5'-ACATCCGCTCTCTGGAGAGC-3' (RIIMB) to amplify the C-terminal part of
the cDNA. The PCR products were mixed and reamplified using the M13
-40 and RIIMB primers to give a cDNA fragment spanning the mutation.
Phosphorylated PCR product was cloned into phosphatase-treated
SmaI-digested pBluescript. The region between
BstXI and SfiI was verified by sequencing and
subcloned into the MISRII cDNA in pUC19. This altered cDNA was inserted
into the pFLAG-CMV5a expression construct as described above for
MISRII.
The cDNAs for the ALK1 (R3) and ALK2 (R1) type I receptors were
isolated from a rat E14.5 urogenital ridge cDNA library as described
previously (47). The ALK3 and ALK6 type I receptor cDNAs were a kind
gift from Dr. C. H. Heldin (Ludwig Institute for Cancer Research,
Uppsala, Sweden).
An expression construct (pCMVHA) that contains the HA epitope tag
(YPYDVPDYA) followed by a stop codon was created by annealing the
oligonucleotides 5'-TCGACTATCCATATGATGTACCAGATTATGCATGAGCGGCCGCG-3'
and
5'-GATCCGCGGCCGCTCATGCATAATCTGGTACATCATATGGATAG-3'
and then cloning the annealed oligonucleotides into the
SalI-BamHI sites of pCMV5 (68).
A SalI site was introduced into the coding sequence of
the ALK1, ALK2, ALK3, and ALK6 cDNA constructs at codons 20 to 24 amino
acids C-terminal to the transmembrane domain by using the Quick Change
mutagenesis system using the
primers 5'-GGCGAGTCCAGTGTCGACCTGAAGGCATCG-3' and
5'-CGATGCCTTCAGGTCGACACTGGACTCGCC-3' for ALK1,
5'-CTATCGAAGGGGTCGACACCACCAAC-3' and
5'-GTTGGTGGTGTCGACCCCTTCGATAG-3' for ALK2,
5'-GATTTGGAACAGGTCGACGCATTTATTCCAGTTG-3' and
5'-CAACTGGAATAAATGCGTCGACCTGTTCCAAATC-3' for ALK3, and
5'-CTGGAGCAGGTCGACACATACATTCCTCCTG-3' and
5'-CAGGAGGAATGTATGTGTCGACCTGCTCCAG-3' for ALK6.
The mutagenized cDNA fragments coding for the extracellular and
transmembrane domains were subcloned into pCMVHA using
EcoRI (for ALK1, ALK2, and ALK6) or HindIII
(ALK3) sites in the polylinkers of the cDNA constructs and
SalI. This allowed the open reading frame of the receptors
to be in frame with the HA epitope tag and to terminate at the stop
codon after the tag. The production of tagged, truncated receptor by
the expression constructs was verified by Western blotting lysates from
transfected COS7 cells following standard procedures (57).
Northern Blot Analysis
MA-10 cells were maintained in Waymouths MB 752/1 medium plus
L-glutamine (Life Technologies, Inc.,
Gaithersburg, MD) with 50 µg/ml gentamycin sulfate, 20 mM
HEPES, pH 7.4, and 15% horse serum (Life Technologies, Inc.). R2C cells were grown in Hams F10 plus
L-glutamine (Mediatech, Inc., Herndon, VA) with 15% horse
serum, 2.5% female FBS (Biologos, Inc., Montgomery, IL), 100 U/ml
penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). mRNA was prepared from MA-10 or R2C cell cultures in T75
flasks (Corning, Inc., Corning, NY) by using Quickprep
(Amersham Pharmacia Biotech, Piscataway, NJ)
following the manufacturers protocol. Poly(A)+
mRNA from each sample was electrophoresed on 1.2% agarose-formaldehyde
gels. The gels were blotted to nylon membrane (Hybond-N; Amersham Pharmacia Biotech), and the RNA was UV cross-linked. A 1.5-kb
BamHIHindIII fragment of a rat Smad6 cDNA clone
isolated from a rat E14.5 urogenital ridge cDNA library, a 1.1-kb
EcoRINotI fragment of a mouse Smad7 partial
cDNA clone obtained from the IMAGE consortium (GenBank accession no.
AA022262), or a 0.4-kb BamHIHindIII fragment of
human ß-actin (nt 82463 of the coding region) was used to prepare
32P-labeled probes by the random hexamer method.
The blots were prehybridized, hybridized with the probes, washed, and
exposed to film as described previously (69).
Transient Transfections and Luciferase Assays
P19 cells were cultured in
MEM plus L-glutamine
and nucleosides (Mediatech, Inc., Herndon, VA) with 5% female
FBS (Biologos, Inc., Montgomery, IL) and 100 U/ml penicillin and 100
µg/ml streptomycin (Life Technologies, Inc.). For
transient transfections, 2 x 105 P19 cells
in 9-cm2 wells were transfected using 3 µl
FuGENE6 (Roche Molecular Biochemicals, Indianapolis, IN)
with 400 ng of luciferase reporter constructs, various expression
constructs, and 100 ng of thymidine kinase minimal promoter-driven
Renilla luciferase vector as a control for transfection efficiency. The
total amount of expression construct was kept constant with parental
expression vector. After 24 h of incubation, the transfected cells
were treated with or without MIS. After 48 h, the cells were
washed two times with 2 ml of HBBS, 250 µl of passive lysis buffer
was added per well, the cells were agitated for 15 min. and 20 µl
were analyzed for firefly and Renilla luciferase activity by detection
of chemiluminescence with luciferin substrate (Dual Luciferase kit,
Promega Corp., Inc., Madison, WI) using a AutoLumat LB953
luminometer (Perkin-Elmer Corp., Norwalk, CT) and
expressed as mean ± SD from triplicate transfections
after normalization to Renilla luciferase activity. Each separate
experiment was done at least three times.
In Situ Hybridization And Tissue Sections
Embryos were collected from timed pregnant rats (Charles River Laboratories, Inc., Wilmington, MA), after which the
ventral body wall and viscera were removed and the embryos were fixed
in 4% paraformaldehyde in PBS overnight. Embryos were dehydrated,
treated with proteinase K, prehybridized, and hybridized as previously
described (70). Probes were labeled with digoxigenin and the
digoxigenin probes were identified with Fab fragments conjugated to
alkaline phosphatase. BM-Purple precipitation (Roche Molecular Biochemicals) was used to detect the signal. The Smad6
probe was made by linearizing a mouse Smad6 partial cDNA clone obtained
from the IMAGE consortium (GenBank accession no. W41111) and producing
the antisense RNA with T3 RNA polymerase (
600 bp). The MISRII probe
was made by digesting pBS7 (21) with SalI and synthesizing
with T3 RNA polymerase (2 kb). The ALK2 probe was created by digesting
pBS-ALK2 with HindIII and making antisense RNA with T3 RNA
polymerase (1.5 kb). The ALK6 probe was made by digesting pBS-ALK6 with
BamHI and synthesizing probe with T7 RNA polymerase (1 kb).
The Smad1 probe was made from a linearized IMAGE consortium clone
(GenBank accession no. AA177814) and producing the antisense RNA with
T3 RNA polymerase (
700 bp). The
500 bp antisense Smad5 probe was
produced with T3 RNA polymerase from linearized IMAGE consortium clone
(GenBank accession no. AI573395). The plasmid for the Smad8 probe was
created by subcloning an RT-PCR product from rat testis RNA. The Smad8
probe was created by linearizing this plasmid and making antisense RNA
with T3 RNA polymerase (
1.3 kb). To assay background signal, control
hybridization was performed with sense probe, which did not show any
signal.
After whole-mount in situ hybridization, embryos were
equilibrated overnight in 30% sucrose in PBS, embedded in tissue
freezing medium (Triangle Biomedical Sciences, Durham, NC), and
cryosectioned with a thickness of 14 µm. As indicated in the figure
legends, some sections were redetected with BM-Purple after sectioning
to enhance signal intensity.
Analysis of Transgenic Animals
The generation of BMPRIB-/- mice has been previously reported
(44). The animals were genotyped as described (44), wild-type or
BMPRIB-/- adult male littermates were euthanized, and the internal
reproductive system was dissected.
Experimental Animals
All animals were housed in an AAALAC-certified facility
and treated according to the guidelines for animal husbandry at the
Massachusetts General Hospital (Protocol no. 1999N001138) and at the
University of California, Los Angeles (Protocol no. 95018-13) and are
in accordance with the NIH Guide for the Care and Use of Laboratory
Animals.
Note Added in Proof
Visser et al. (71) in the preceding article (pp.
936945) present data complementary to that presented here which also
supports ALK2 mediating MIS signaling in the Müllerian duct.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Drs. J. Wrana and L. Attisano for the
TßRII, BMPRII, and ALK2(Q207D) expression constructs, for the
Tlx2-luc reporter construct, and for Mv1Lu cells; J. Massague for the
Gal4DBD-Smad1 and Gal4DBD-Smad2 expression constructs and the Gal4-luc
and 3Tp-lux reporter construct; J.-M. Gauthier for the CAGA-luc
reporter construct; K. W. Y. Cho for the Xvent2-luc reporter
construct; C. H. Heldin (Ludwig Institute for Cancer Research,
Uppsala, Sweden) for the ALK3 and ALK6 cDNAs; G. Gil for the M13 human
ß-actin construct; D. Russell for the pCMV5 expression vector; and to
Mario Ascoli for MA-10 cells. We thank Francis H. ONeill for
technical assistance. We thank Drs. J. Lorenzen, A. Trbovich, and J.
Teixeira for critical evaluation of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Patricia K. Donahoe, Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Warren 11, Boston, Massachusetts 02114. E-mail:
donahoe.patricia{at}mgh.harvard.edu
This work was supported by grants from the NIH to P.K.D.
(NICHD-HD-32112, NCI-CA-17393, and NICHD-HD-28138) and to K.M.L.
(AR-44528) and from the American Cancer Society to T.R.C. (postdoctoral
fellowship).
Received for publication February 9, 2001.
Revision received March 20, 2001.
Accepted for publication March 22, 2001.
 |
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