Müllerian inhibiting substance (MIS; also known as anti-Müllerian
hormone, AMH) is essential for normal male sexual differentiation.
In mammalian males, the fetal testes produce and secrete both MIS,
which causes Müllerian (paramesonephric) ducts to regress, and
testosterone, which promotes the differentiation of Wolffian
(mesonephric) ducts. Müllerian ducts, in the absence of MIS,
continue to develop and differentiate as the oviduct, uterus, cervix
and upper part of the vagina, whereas Wolffian ducts, which give rise
to the male internal reproductive tract structures, epididymides, vas
deferens and seminal vesicles, degenerate without testosterone
stimulation. Defects in either the gene for MIS or its receptor can
result in a form of male pseudohermaphroditism characterized by
retained Müllerian ducts (Behringer et al., 1994; Mishina et al.,
1996; Belville et al., 1999; Hoshiya et al., 2003).
The molecular mechanisms leading to Müllerian duct regression
have yet to be clarified. MIS functions, like other members of the
transforming growth factor ?(TGF?) superfamily, by binding to its
specific type II receptor (MISRII), which presumably must recruit
and phosphorylate a type I receptor to initiate a downstream
signaling cascade (for a review, see Teixeira et al., 2001; Josso and
di Clemente, 2003). When the Müllerian duct is first developing, the
coelomic epithelial cells are thought to invaginate and migrate in a
cranial-to-caudal manner to form the Müllerian duct (Gruenwald,
1941). The Müllerian duct is subsequently eliminated in a cranial-
to-caudal fashion as a result of MIS action (Picon, 1969; Tsuji et al.,
1992), which is attributed to the cranial-to-caudal expression of
MISRII (Allard et al., 2000). Expression of MISRII was found in the
mesenchyme but not in the Müllerian duct epithelial cells at the time
of regression in the male (Baarends et al., 1994; di Clemente et al.,
1994; Teixeira et al., 1996), thus MIS is believed to function via a
paracrine mechanism to cause apoptosis in the Müllerian duct (Tsuji
et al., 1992; Catlin et al., 1997; Roberts et al., 1999; Allard et al.,
2000). By contrast, MISRII transcripts are present in a polarized
pattern in the coelomic epithelium of female urogential ridges during
the corresponding period (Parr and McMahon, 1998; Clarke et al.,
2001). The cause of this sexually dimorphic pattern of MISRII
expression has heretofore been uncharacterized.
Whereas the type II receptor is unique for MIS signaling, several
type I receptors may mediate MIS signaling in different tissue
contexts. Dominant-negative (Clarke et al., 2001) and antisense
(Visser et al., 2001) Alk2 can reverse the function of MIS in p19
embryonic carcinoma cells and in the rat urogenital ridge in organ
culture, respectively. ALK6 can have MIS ligand-dependent
interaction with MISRII in Chinese hamster ovary (CHO) cells
(Gouédard et al., 2000); however, Müllerian ducts regress normally
in male Alk6 (Bmpr1b) knockout mice (Clarke et al., 2001).
Conditional inactivation of Alk3 (Bmpr1a) prevents Müllerian duct
regression in male mice (Jamin et al., 2002), creating a phenotype
identical to that seen by inactivating the MIS ligand or its type II
receptor, and thus providing strong evidence that ALK3 is an MIS
type I receptor in the mouse. When transgenic mice carrying the
conditional mutation of Alk3 were bred with transgenic mice
overexpressing human MIS, the female progeny had no uterus
(Jamin et al., 2003), suggesting possible redundancy among
different MIS type I receptors in the presence of high levels of MIS.
ALK2, ALK3 and ALK6 also mediate the signaling of bone
morphogenetic proteins (BMPs). These type I receptors
phosphorylate receptor-regulated SMADs (R-SMADs) 1, 5 and 8 at
the C-terminal SSXS motifs to transduce BMP signals. The
Müllerian inhibiting substance regulates its receptor/SMAD
signaling and causes mesenchymal transition of the coelomic
epithelial cells early in Müllerian duct regression
Yong Zhan, Akihiro Fujino, David T. MacLaughlin, Thomas F. Manganaro, Paul P. Szotek, Nelson A. Arango,
Jose Teixeira and Patricia K. Donahoe*
Examination of Müllerian inhibiting substance (MIS) signaling in the rat in vivo and in vitro revealed novel developmental stage-
and tissue-specific events that contributed to a window of MIS responsiveness in Müllerian duct regression. The MIS type II receptor
(MISRII)-expressing cells are initially present in the coelomic epithelium of both male and female urogenital ridges, and then
migrate into the mesenchyme surrounding the male Müllerian duct under the influence of MIS. Expression of the genes encoding
MIS type I receptors, Alk2 and Alk3, is also spatiotemporally controlled; Alk2 expression appears earlier and increases
predominantly in the coelomic epithelium, whereas Alk3 expression appears later and is restricted to the mesenchyme, suggesting
sequential roles in Müllerian duct regression. MIS induces expression of Alk2, Alk3 and Smad8, but downregulates Smad5 in the
urogenital ridge. Alk2-specific small interfering RNA (siRNA) blocks both the transition of MISRII expression from the coelomic
epithelium to the mesenchyme and Müllerian duct regression in organ culture. Müllerian duct regression can also be inhibited or
accelerated by siRNA targeting Smad8 and Smad5, respectively. Thus, the early action of MIS is to initiate an epithelial-to-
mesenchymal transition of MISRII-expressing cells and to specify the components of the receptor/SMAD signaling pathway by
differentially regulating their expression.
KEY WORDS: MIS, MIS type I/II receptor, SMAD, Epithelial-to-mesenchymal transition, RNA interference, Organ culture, Rat
Development 133, 2359-2369 (2006) doi:10.1242/dev.02383
Pediatric Surgical Research Laboratories, Department of Surgery, Massachusetts
General Hospital and Harvard Medical School, Boston, MA 02114, USA.
*Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 30 March 2006
phosphorylated SMADs translocate into the nucleus complexed
with SMAD4 and transcriptionally regulate specific sets of targeted
genes (for reviews, see Massagué, 2000; Attisano and Wrana, 2002).
MIS has been shown to activate SMAD1 (Gouédard et al., 2000;
Clarke et al., 2001; Visser et al., 2001) and SMAD5 (Visser et al.,
2001) in vitro, implying that R-SMADs 1, 5 and 8 may mediate
Müllerian duct regression (Kobayashi and Behringer, 2003).
The present study was undertaken to define when and where the
MIS type I receptors are employed and to determine which SMADs
transduce MIS signals in the urogenital ridge during Müllerian duct
regression. We adapted RNA interference (RNAi) (Calegari et al.,
2002; Sakai et al., 2003; Soutschek et al., 2004) to test functional
activity of the components of the MIS signaling pathway in a
urogenital ridge organ culture assay, which recapitulates the
morphological events occurring in vivo during Müllerian duct
regression (Donahoe et al., 1977). We show that ALK2-mediated MIS
signaling induces migration of MISRII-expressing cells from the
coelomic epithelium into the Müllerian duct mesenchyme, and thus is
responsible for the sexual dimorphism of MISRII expression. MIS
also orchestrates the spatiotemporal expression of its type I receptors
and R-SMADs, which is necessary for Müllerian duct regression.
MATERIALS AND METHODS
Animals, organ culture and recombinant human MIS
Urogenital ridges were dissected from the embryos of timed pregnant rats
(Harlan) and studied at developmental stages from E14 to E15 to determine
gene expression patterns and morphological changes in vivo. Male or female
urogenital ridges from timed pregnant rats at E14.5 were also dissected and
then cultured, either immediately or after special treatment, on MilliCell-
CM membranes (Millipore) over CMRL1066 medium (Life Technologies)
supplemented with 10% female (to avoid an effect of bovine MIS in male
serum) fetal bovine serum, penicillin/streptomycin and 10 nM testosterone.
Cultures were carried out with or without recombinant human MIS at a final
concentration of 6 ?g/ml (42.5 nM).
To obtain bioactive recombinant MIS, the human MIS cDNA was stably
transfected into CHO cells. MIS was purified from the serum containing
media by immunoaffinity chromatography as described previously in detail
(Ragin et al., 1992), using a monoclonal antibody developed in this
laboratory (Hudson et al., 1990).
In situ hybridization
Immediately after dissection at various times of gestation or after
organ culture, urogenital ridges were fixed overnight at 4°C in 4%
paraformaldehyde. Tissues were dehydrated, rehydrated, treated with
proteinase K, pre-hybridized and then hybridized with sense or antisense
riboprobes (1 ng/?l) overnight at 65-70°C. After hybridization, samples
were placed in 1% blocking solution (Roche) for 1.5 hours at room
temperature, then incubated with anti-digoxigenin-AP antibody (Roche) at
1:1000 overnight at 4°C. BM-Purple AP substrate (Roche) was used to
detect probe hybridization colorimetrically. Samples were subsequently
cryosectioned at 10 ?m.
Riboprobes were synthesized with digoxigenin-labeled nucleotide mix
(Roche). A full-length coding sequence of Wnt7a was subcloned from an
IMAGE consortium clone (GenBank Accession Number BC049093) into
pYX-ASC vector using EcoRI and NotI restriction sites. The Wnt7aplasmid
was digested with EcoRI and transcribed with T3 RNA polymerase to make
antisense probes. A full-length coding sequence of Alk3was subcloned from
an IMAGE consortium clone (GenBank Accession Number BI735174) into
the pCMV-sport6 vector. To make antisense probes, the Alk3 plasmid was
digested with SalI and transcribed with T7 RNA polymerase. The antisense
probes for Smad1 and Smad5 were made from linearized IMAGE
consortium clones (GenBank Accession Numbers BI695704 and BI695413)
and produced with T7 RNA polymerase. The probes for Smad8, Misr2 and
Alk2, all cloned in the laboratory, were made as described previously (Clarke
et al., 2001).
Histology, immunofluorescent staining and
For histology, urogenital ridges were fixed in Bouin’s fixative, dehydrated
and embedded in paraffin. Sections were cut at 8 ?m and stained with
Hematoxylin and Eosin (HE). For immunofluorescent staining, urogenital
ridges were fixed in 4% paraformaldehyde, embedded and cut at 7-10 ?m.
For vimentin staining, sections were blocked using 5% normal donkey
serum, then incubated with anti-vimentin antibody at a dilution of 1:100
(Santa Cruz Biotechnology) and FITC-conjugated secondary antibody. For
laminin staining, sections were blocked using 3% BSA, and then incubated
with anti-laminin ?1 antibody (1:50, Santa Cruz Biotechnology) and Alexa
fluor 568 secondary antibody (Invitrogen). For immunohistochemistry,
urogenital ridges were fixed overnight at 4°C in 4% paraformaldehyde,
embedded in paraffin wax and sectioned at 6 ?m. Deparaffinized and
hydrated sections were microwaved in 0.01 M sodium citrate to unmask
antigens by heating at 80-85°C for 10 minutes. Sections were blocked with
5% normal goat serum; incubated with rabbit anti-phosphoSMAD1/5/8
antibody (Cell Signaling) diluted at 1:100, with biotin-labeled goat anti-
rabbit antibody (Vector) and with ABC reagent (Vector); developed with
DAB reagent; and counterstained with 1% Methyl Green.
Whole-mount immunofluorescent microscopy
Urogenital ridges were fixed in 4% paraformaldehyde overnight at 4°C,
followed by washes with PBS, and permeabilized in 0.2% Triton X-100 in
PBS for 15 minutes at room temperature. Samples were quenched in 0.1%
sodium borohydride for 10 minutes at room temperature, blocked (1%
BSA/5% normal goat serum in PBS) for 3 hours at room temperature, and
incubated with rabbit anti-phosphoSMAD1/5/8 antibody (1:100, Cell
Signaling) in 1% BSA/PBST overnight at 4°C and FITC-conjugated goat
anti-rabbit IgG for 1 hour at room temperature.
siRNAs and RNAi in organ culture
After testing multiple small interfering RNAs (siRNAs), the optimal
targeting siRNA for each gene was selected as indicated in Table 1. The
siRNAs were chemically synthesized, purified and duplexed by Qiagen-
Xeragon, and resuspended to 20 ?M following the manufacture’s protocol.
siRNA concentrations between 50 and 400 nM were tested for optimal
silencing efficiency with less toxicity, and 200 nM was selected for further
studies. Urogenital ridges were transfected with siRNA duplex in serum-free
culture medium by using Oligofectamine reagent (Invitrogen). siRNAs and
Oligofectamine were diluted in separate tubes, combined and incubated for
20 minutes at room temperature. The siRNA:Oligofectamine mixture was
added to the medium and incubated with immersed urogenital ridges for 10-
12 hours. The urogenital ridges were subsequently placed on MilliCell-CM
membranes (Millipore) to continue culture at the air media interface over
Development 133 (12)
Table 1. The sequences for siRNA targeting
Accession Number NameSequence (5?-3?) (in coding sequence)
– Random sequence
CM-DiI labeling and tracking of cell migration
Urogenital ridges at E14.5 were incubated with Cell Tracker CM-DiI
(chloromethylbenzamido, Molecular Probes) at 1 ?M in CMRL 1066
medium for 10 minutes at 37°C. Then, the tissues were extensively washed,
directly fixed or cultured on MilliCell-CM membranes with or without MIS
followed by fixation in 4% paraformaldehyde overnight at 4°C. Tissues were
subsequently cryosectioned at 8 ?m and localization of DiI labeled cells was
examined by fluorescent microscopy.
Dynamic expression of MIS receptors and SMADs
early in Müllerian duct regression
In the rat embryo, the window of MIS responsiveness is from E14-
E15. MIS has to be present during this period in order to achieve
complete regression of the Müllerian ducts (Picon, 1969; Josso et
al., 1976; Donahoe et al., 1977; Tsuji et al., 1992). Regression
events, i.e. disruption of the basement membrane of the Müllerian
duct and apoptosis of the Müllerian duct epithelial cells, occur after
E15 in male rat urogenital ridges (Price et al., 1977; Trelstad et al.,
1982; Allard et al., 2000). These facts prompted us to examine the
expression profiles of the MIS receptors and SMADs in male rat
urogenital ridges at E14-E15 in order to understand how they
participate and cooperate in MIS signaling. At early E14 (E14.25),
MISRII mRNA was expressed strongly in the cranial urogenital
ridge (Fig. 1A), and then the expression was seen to extend
craniocaudally along the urogenital ridge (Fig. 1B,C). Unexpectedly,
MISRII expression was found predominantly in the coelomic
epithelium, but not in the mesenchyme between the Müllerian and
Wolffian ducts in male urogenital ridges (Fig. 1D). At this time, the
pattern of MISRII expression was seen as different from that
detected at E15.5 when MISRII expression appeared in a
circumferential pattern around the male Müllerian duct epithelium
(Clarke et al., 2001).
Previous studies have shown that MIS can activate or
phosphorylate R-SMADs1, 5 and 8 in cell culture (Gouédard et
al., 2000; Clarke et al., 2001; Visser et al., 2001). In this study,
we examined the expression of phosphorylated SMAD1/
SMAD5/SMAD8 (P-SMAD) in the urogenital ridge. Whole-
mount immunofluorescence analysis showed no obvious P-
SMAD expression in the urogenital ridge at early E14 (data not
shown). After E14.5, expression of P-SMAD could be detected
in male urogenital ridges. It appeared at low level at E14.5 and
increased craniocaudally thereafter (Fig. 1M-O). Presence of P-
SMAD in the male urogenital ridge after E14.5 implies that MIS
is eliciting a functional response and MIS signaling may
contribute to subsequent molecular events in the male urogenital
Sexually dimorphic pattern of Alk2 and Smad8 expression has
previously been found in rat urogenital ridges at E15.5 (Clarke et
al., 2001), and we examined their expression in male rat urogenital
ridges at earlier stages. Little Alk2 expression was detected in the
urogenital ridge before E14.5 (Fig. 1E). Thereafter, increased
expression of Alk2 was seen in the anterior male urogenital ridge
and extended craniocaudally (Fig. 1F,G). More Smad8 transcripts
were also detected after E14.5 in male rat urogenital ridges (Fig.
1J,K). Cryosections showed that Alk2 and Smad8 mRNA was
Early MIS signaling in Müllerian duct regression
Fig. 1. MIS signaling and dynamic expression of
Misr2, Alk2 and Smad8 in male rat urogenital ridges.
The mRNAs of Misr2 (A-D), Alk2 (E-H), and SMAD8 (I-L)
were detected by in situ hybridization (whole-mount and
cryosection) in the urogenital ridges of E14.25 (A), E14.5
(B,E,I), E14.75 (C,F,J) and E15 (D,G,K). (H,L) Cryosections
at the at the level of the broken lines in G and K,
respectively. (M-O) Expression of phosphorylated
SMADs1, 5, 8 (P-SMAD) was detected by whole-mount
immunofluorescence analysis in E14.5 (M), E14.75 (N)
and E15 (O) urogenital ridges of male rats. Arrows
indicate the first detected substantial expressions.
Arrowheads indicate expressions in the coelomic
epithelium (CE). Cranial is oriented towards the top and
Müllerian duct to the right of individual images. M,
Müllerian duct; T, testis; W, Wolffian duct. Scale bar: 500
?m for all the whole-mount samples (A-C,E-G,I-K,M-O);
50 ?m for all the sections (D,H,L).
mainly localized in the coelomic epithelium (Fig. 1H,L). At this
developmental stage, Alk3 expression was not detected in the
coelomic epithelium but in the mesenchyme (data not shown).
At E15.5, Alk2 expression was increased in the fetal gonad but
markedly reduced in the coelomic epithelium of the male urogenital
ridge, and it began to disappear craniocaudally (Fig. 2A, arrow).
Meanwhile, more Alk3 was detected in the mesenchyme, and its
expression was much higher at E15.5 (Fig. 2B) than at E14.5 in the
Müllerian duct mesenchyme in male urogenital ridges (data not
shown). Concomitantly, prominent expression of P-SMAD was
detected in the mesenchymal cells surrounding the Müllerian ducts
of male urogenital ridges (Fig. 2D,F), but absent in the same area in
the female (Fig. 2C,E), suggesting that functional MIS signaling
continues in the peri-Müllerian duct mesenchyme. Smad5 also has
a sexually dimorphic expression pattern at E15.5; its transcripts were
expressed in the coelomic epithelium of female urogenital ridges
(Fig. 2G), whereas male urogenital ridges expressed much less
Smad5 in the coelomic epithelium adjacent to the Müllerian duct
(Fig. 2H, arrow). Smad1expression was weak and indistinguishable
between male and female urogenital ridges from E14.5 to E15.5
(data not shown) (Clarke et al., 2001).
MIS signaling induces a shift of MISRII expression
from the coelomic epithelium to the mesenchyme
To confirm that the expression of P-SMAD in male urogenital ridges
at E14.5-E15.5 was the result of MIS action, we treated E14.5
female rat urogenital ridges in organ culture (Donahoe et al., 1977)
with MIS at concentrations known to cause Müllerian duct
regression (Ragin et al., 1992; Lorenzo et al., 2002). MIS treatment
induced P-SMAD expression in two hours in female urogenital
ridges (Fig. 3B). P-SMAD expression was first noted to increase
along the outer region of the urogenital ridge lateral to the Müllerian
duct (Fig. 3B,C, arrows), and later was also visualized medial to the
Müllerian duct following treatment with MIS for 30 hours (Fig. 3D,
arrowhead). This pattern is similar to that normally seen in male
urogenital ridges (Fig. 3F), but not in untreated female urogenital
ridges (Fig. 3E). These data suggest that the dynamic change
of P-SMAD in male urogenital ridges at the corresponding
developmental stage resulted from MIS activity.
We next investigated whether MIS directs Misr2expression from
the coelomic epithelium into the mesenchyme of the Müllerian duct.
Female urogenital ridges were treated with MIS in organ culture, and
the pattern of Misr2expression was compared with that observed in
untreated female counterparts. At E14.5, the coelomic epithelium
adjacent to the Müllerian duct appeared thicker than that in other
regions in both female (Fig. 4A, arrow) and male urogenital ridges
(data not shown). The coelomic epithelium was separated from
subjacent mesenchyme by a prominent basement membrane (Fig.
4A,G) (Ikawa et al., 1984), and was noted to have less vimentin
expression (Fig. 4B, arrow). Before treatment commenced, Misr2
transcripts were localized to the coelomic epithelium lateral to the
Müllerian duct (Fig. 4C). After treatment with MIS for 20 hours,
Misr2 mRNA was observed in the mesenchyme adjacent to the
Müllerian duct with reduced expression in the coelomic epithelium
(Fig. 4H), in contrast to the untreated conterpart (Fig. 4E), in which
Misr2 expression is indistinguishable from that at E14.5 (Fig. 4C).
Prolonged treatment with MIS for 40 hours caused expression of
Misr2to diminish markedly in the coelomic epithelium and increase
in the mesenchyme surrounding the Müllerian duct, notably,
between the Müllerian and Wolffian ducts (Fig. 4K, arrowhead). In
the untreated female urogenital ridges (without MIS for 40 hours),
expression of Misr2 remained lateral to the Müllerian duct,
predominantly in the coelomic epithelium (Fig. 4M). When MIS
was removed from organ culture before Misr2 expression appeared
around the Müllerian duct, the change of Misr2 expression did not
proceed (data not shown). These data indicate that constitutive MIS
signaling early in Müllerian duct regression contributes to the
distinct male pattern of Misr2 expression.
MIS induces migration of Misr2-expressing cells
To determine whether a mechanism of epithelial-to-mesenchymal
transition underlies the switch of Misr2 expression, we labeled the
coelomic epithelium of female urogenital ridges at E14.5 with CM-
DiI, which incorporates into cell membranes, with photostable
fluorescence and no apparent adverse effects (Austin, 1995; Karl and
Capel, 1998), and tracked the migration of fluorescent-labeled cells
Development 133 (12)
Fig. 2. Expression patterns of the MIS type I receptors (Alk2 and
Alk3), Smad5 and P-SMAD in E15.5 rat urogenital ridges.
(A,B) Whole-mount in situ hybridization shows decreased Alk2
expression in the cranial coelomic epithelium (A, arrow) and increased
Alk3 expression in the mesenchyme (B, black arrowhead) of male
urogenital ridges. White arrowhead indicates the coelomic epithelium.
(C-F) Sexually dimorphic expression of P-SMAD detected by
immunohistochemistry. (E,F) Higher magnifications of the boxed area
around the Müllerian ducts in C,D. Arrowheads in D,F indicate P-Smad
in mesenchyme surrounding Müllerian duct. (G,H) Smad5 expression
detected in the coelomic epithelium (indicated by arrows) adjacent to
the Müllerian duct of female (G) but not male (H) urogenital ridges.
Cranial is oriented towards the top and Müllerian duct to the right of
individual images (A,B). M, Müllerian duct; Ov, ovary; T, testis; W,
Wolffian duct. Scale bar: 500 ?m in A-D; 100 ?m in E,F; 200 ?m in
in the presence of MIS. After a short incubation with CM-DiI,
fluorescence could be detected in the coelomic epithelium (Fig. 4D,
arrow). The fluorescence-labeled coelomic epithelium adjacent to
the Müllerian duct comprises two to three layers of cells, thicker than
that in other regions. Deeper uptake of CM-DiI beyond the coelomic
epithelial cells appeared to be prohibited by the basement
membrane. In female urogenital ridges cultured at E14.5 for 20
hours, the basement membrane was continuous (Fig. 4G,
arrowheads) and CM-DiI remained in the coelomic epithelium (Fig.
4F). However, in the urogenital ridges treated with MIS for 20 hours,
CM-DiI fluorescence appeared beneath the disrupted basement
membrane, which was shown with loss of laminin staining (Fig. 4J,
arrowheads), and was detected in the area adjacent to the Müllerian
duct (Fig. 4I, arrowheads). The extension of CM-DiI was
colocalized with Misr2 expression (Fig. 4H). Longer treatment
resulted in localization of fluorescence around the Müllerian duct
(Fig. 4L, arrowhead). At this time, Misr2expression was also found
in the mesenchyme around the Müllerian duct (Fig. 4K). The Misr2-
expressing mesenchymal cells were stained for vimentin (data not
shown). In untreated urogenital ridges, CM-DiI labeled cells
remained in the thick surface epithelium (Fig. 4N). Our data suggest
that one of the earliest actions of MIS is to cause epithelial-to-
mesenchymal transition and drive the Misr2-expressing cells to
migrate from the coelomic epithelium into the mesenchyme
surrounding the Müllerian duct.
Exogenous MIS differentially regulates R-SMADs
1, 5 and 8 expression
To investigate whether the sexually dimorphic expression of Smad8
and Smad5 is dependent upon MIS, we treated E14.5 female
urogenital ridges in organ culture with MIS and examined their
expression. MIS treatment of female urogenital ridges resulted in
increased expression of Smad8 (Fig. 5B,E). Smad8 expression was
also induced by MIS added to female urogenital ridges after removal
of the gonad (data not shown), indicating that this effect is not a
result of other gonadal factors. In situ hybridization with probes
targeting different regions in the Smad8 transcript (data not shown)
confirmed that the regulated Smad8 was full length, not an isoform
encoding C-terminus deleted Smad8 (Nishita et al., 1999).
Treatment of E14.5 female urogenital ridges with MIS also resulted
in decreased Smad5expression in the coelomic epithelium adjacent
to the Müllerian duct (Fig. 5H,K), similar to that seen in male
urogenital ridges at the same developmental stage in vivo and in
vitro (Fig. 2H, Fig. 5I,L). MIS had no noticeable effect on Smad1
expression in urogenital ridges (data not shown).
MIS spatiotemporally regulates Alk2 and Alk3
To investigate whether MIS regulates Alk2 and Alk3 expression
during Müllerian duct regression, we treated E14.5 female
urogenital ridges in organ culture with MIS and examined
expression over time. Treatment of E14.5 female urogenital ridges
with MIS for 12 hours induced Alk2 expression (Fig. 5N,P) when
compared with untreated ridges (Fig. 5M,O). Moreover, increased
Alk2expression was detected in the coelomic epithelium as early as
4-6 hours after treatment, and decreased after treatment for 24 hours
(data not shown). Alk3 expression was increased only after culture
for more than 24 hours with MIS. It was upregulated predominantly
in the mesenchyme surrounding the Müllerian duct (Fig. 5R,T)
when compared with untreated ridges (Fig. 5Q,S). Upregulation of
both Alk2 and Alk3 both followed a cranial-to-caudal pattern.
Alk2 mediates the change of MISRII expression
and is required for Müllerian duct regression
The functional importance of the MIS type I receptors and R-
SMADs1, 5 and 8 in Müllerian duct regression was investigated by
RNAi in organ culture of male rat urogenital ridges. Multiple
siRNAs designed to target Alk2, Alk3, Smad1, Smad5 and Smad8
were first studied in cultured MIS-responsive and MISRII-
expressing R2C rat Leydig cells (data not shown) (Teixeira et al.,
1999). The siRNAs that showed significant silencing of mRNA
expression for each gene in cell culture were selected for subsequent
use in organ culture (Table 1). Transfection of fluorescein-labeled
siRNA into urogenital ridges could be visualized in the urogenital
ridge, where it was seen to penetrate the coelomic epithelium, but
not beyond (data not shown).
Male urogenital ridges were treated with control- or Alk2-siRNA,
and expression of Misr2 and P-SMAD was examined. P-SMAD
expression was markedly decreased in Alk2-siRNA treated male
urogenital ridges (compare Fig. 6B with Fig. 6A, arrows). In the
urogenital ridges treated with control-siRNA, Misr2 mRNA was
detected in the mesenchyme around the Müllerian duct (Fig. 6C,
arrowhead); however, in those treated with Alk2-siRNA, Misr2
expression was not evident in the area between the Müllerian and
Wolffian ducts (Fig. 6D, arrowhead).
The selective expression of Wnt7a, which drives the expression
of MISRII, in the Müllerian duct epithelium of urogenital ridges
(Parr and McMahon, 1998) makes it a particularly useful marker
Early MIS signaling in Müllerian duct regression
Fig. 3. MIS activates R-SMADs 1, 5 and 8 in urogenital ridges in
organ culture. (A-E) Female urogential ridges were harvested at E14.5,
directly fixed (A) or cultured for 2 hours (B), 6 hours (C), 30 hours (D-F),
in the presence (B-D) or absence (E) of MIS. (F) Male urogenital ridges
cultured at E14.5 for 30 hours. P-SMAD expression was detected by
whole-mount immunofluorescent analysis. Arrows and arrowheads
mark the expression of P-SMAD in the areas lateral and medial to the
Müllerian duct, respectively. Cranial is oriented towards the top and
Müllerian duct to the right of individual images. Ov, ovary; T, testis.
Scale bar: 500 ?m.
with which to study the Müllerian duct (data not shown), as it
faithfully reflects Mullerian duct formation and regression.
Detection of Wnt7a expression, which was able to locate remaining
Müllerian duct epithelium in urogenital ridges, allowed us to
monitor the effects of RNAi on Müllerian duct regression in organ
culture and to examine the contribution of Alk2 as an MIS type I
receptor in Müllerian duct regression.
Male urogenital ridges were treated with siRNAs and then
cultured for additional 2 days. In situ hybridization showed that the
Müllerian duct epithelium expressing Wnt7a was retained in the
urogenital ridges treated with Alk2-siRNA (Fig. 6F,H, arrows), but
not in control-siRNA treated urogenital ridges (Fig. 6E,G). Multiple
siRNAs targeting different regions of Alk2had similar effects (data
not shown). These results suggest that Alk2mediates essential MIS
signaling in the transition of Misr2-expressing cells from the
coelomic epithelium to the peri-Müllerian duct mesenchyme.
SMAD8 but not SMAD5 mediates MIS signaling in
Müllerian duct regression
The role of SMAD1, SMAD5 or SMAD8 in MIS signaling and
Müllerian duct regression was also investigated by RNAi. When
male ridges were treated with control-siRNA for 12 hours, the
entire Müllerian duct was still evident after culture for additional
36 hours (Fig. 7A). However, in Smad5-siRNA-treated urogenital
ridges, regression was accelerated, as discontinuous Wnt7a
expression was seen in the cranial area after culture for the same
period (Fig. 7B, arrowheads). Moreover, when RNAi effect was
examined in urogenital ridges after prolonged culture for additional
12 hours, Wnt7aexpression still remained in the posterior region of
control-siRNA urogenital ridges (Fig. 7C, arrow), but not in Smad5-
siRNA-treated ridges (Fig. 7D), indicating that SMAD5 deficiency
led to enhanced Müllerian duct regression. By contrast, treatment
with Smad8-siRNA delayed Müllerian duct regression in male
urogenital ridges, as Wnt7a expression was detected in the Smad8-
siRNA (Fig. 7F, arrow) but not control-siRNA treated urogenital
ridges (Fig. 7E). Moreover, the effect of Smad8-siRNA on
Müllerian duct regression was consistent with its specific gene
silencing in cell culture, demonstrated by both RT-PCR and western
(data not shown). Smad1-siRNA had no effect alone, and RNAi
with both Smad1-siRNA and Smad8-siRNA simultaneously did not
show a further inhibitory effect on Müllerian duct regression than
that caused by Smad8-siRNA alone (data not shown).
MIS-induced epithelial-to-mesenchymal transition
underlies the change of MISRII expression
During male sexual development, Müllerian ducts first form and
then are eliminated as a consequence of MIS signaling. In the rat,
MIS expression is first detected at E13 in fetal testes (Hirobe et al.,
1992). However, a functional signaling pathway is not initiated until
MISRII appears in the urogenital ridge. Our present work shows that
functional MIS signaling, as documented by activation of R-
SMADs1, 5 and 8, is not observed in the male urogenital ridge
immediately until after the expression of MISRII. Interestingly,
expression of MISRII and phosphorylated SMAD1/5/8 are localized
in the coelomic epithelium at this stage. The downstream signaling
events (e.g. upregulation of Alk2and Smad8, and downregulation of
Smad5) also appear initially in the coelomic epithelium. This
explains why RNAi with the lipid-based transfection technique,
which could only penetrate the surface coelomic epithelium, was
effective in knocking down the components of MIS signaling in the
Development 133 (12)
Fig. 4. MIS causes migration of Misr2-expressing coelomic
epithelial cells to the Müllerian duct mesenchyme. Hematoxylin
and Eosin (HE) staining (A), immunofluorescent analysis of vimentin
expression (B) of cranial transverse sections from E14.5 female
urogenital ridges. (C-N) Dynamic change of Misr2 expression, CM-DiI
localization and laminin-stained basement membrane in cultured
female urogenital ridges with or without MIS treatment. Female
urogenital ridges were harvested at E14.5, unlabeled or labeled with
CM-DiI (D,F,I,L,N), directly fixed (C,D), cultured with no treatment for
20 hours (E-G) or 40 hours (M,N), or treated with MIS for 20 hours
(H-J) or 40 hours (K,L). Arrows indicate the coelomic epithelium (CE).
White arrowheads (A,B,J) indicate the basement membrane that
separates the coelomic epithelium and subjacent mesenchyme.
Arrowheads in H-L indicate the extension of Misr2 expression (H,K),
CM-DiI detection in the mesenchyme (I,L) and disruption of the
basement membrane (J) under the influence of MIS. M, Müllerian
duct; W, Wolffian duct. Scale bar: 50 ?m for all the sections.
The epithelial cells of the Müllerian duct originate from the
coelomic epithelium (Gruenwald, 1941), where expression of Misr2
is also first detected (Fig. 1A-D). MIS signaling leads to the
appearance of Misr2 expression in the peri-Müllerian duct
mesenchyme. By tracking the migration of DiI-labeled cells, we
found that MIS induces the Misr2-expressing cells that originally
reside in the coelomic epithelium to migrate into the mesenchymal
compartment around the Müllerian duct, following disruption of
basement membrane. MIS causes epithelial cells to become
migratory, and thereby, initiates an epithelial-to-mesenchymal
transition (for a review, see Thiery, 2002), driving Misr2 expression
into the peri-Müllerian duct mesenchyme. Although we cannot
completely rule out that non-Misr2-expressing cells migrate in
response to an indirect effect of MIS and then begin to express Misr2
in the mesenchyme, our data strongly suggest that MIS directs
Misr2-expressing cells from the coelomic epithelium into
mesenchyme. The timeframe of the migration is in agreement with
the period required for apoptosis to be observed in the Müllerian
duct epithelium (Price et al., 1977; Roberts et al., 1999; Allard et al.,
2000), implying that the Misr2-expressing cells as MIS effectors
may have to arrive in the peri-Müllerian duct mesechyme and/or
become mesenchymal cells to exert significant paracrine effects on
Müllerian ducts. This early epithelio-mesenchymal transformation
is reminiscent of the subsequent transition of the epithelial duct cells
to mesenchyme later during the regression phase (Trelstad et al.,
1982; Austin, 1995; Allard et al., 2000), and illustrates that this
cellular process is a key mechanism in Müllerian duct regression.
Before the Müllerian ducts develop, the Wolffian ducts occupy
the lateral area of urogenital ridges beneath the coelomic
epithelium where the Müllerian ducts are later destined to emerge
(Gruenwald, 1941; Trelstad et al., 1982). The Müllerian duct
forms between the Wolffian duct and the coelomic epithelium,
and the Müllerian duct is initially separated from the coelomic
epithelium only by a shared basement membrane and no
intervening mesenchyme (Trelstad et al., 1982; Ikawa et al.,
1984). The coelomic epithelium adjacent to the Müllerian duct
expresses Misr2 and appears thicker than the epithelium covering
other regions of the urogenital ridge (Fig. 4A-G). After peri-
Müllerian duct mesenchyme forms under the influence of MIS,
the coelomic epithelium adjacent to the male Müllerian duct
becomes thinner and indistinguishable from that in lateral regions
(Trelstad et al., 1982). MIS induces the Misr2-expressing
epithelial cells to lose polarity and manifest a migratory
phenotype, and thus facilitates the formation and patterning of the
peri-Müllerian duct mesenchyme (Fig. 8). WNT signaling is
associated with the epithelial and mesenchymal patterning of the
female reproductive tract (Miller and Sassoon, 1998). ?-Catenin,
which transduces canonical WNT signaling, has been linked to
the regulation of epithelial cell migration and epithelial-to-
mesenchymal transition (Müller et al., 2002; Lu et al., 2003).
Misr2-directed ?-catenin knockout mice show defects in
Müllerian mesenchymal development (Arango et al., 2005). MIS
is able to activate the NF-?B pathway (Segev et al., 2001; Segev
et al., 2002), which is also a stimulatory signal leading to
epithelial-to-mesenchymal transition (Sosic et al., 2003; Huber et
al., 2004). Translocation of ?-catenin to the nucleus has also been
correlated with MIS signaling (Allard et al., 2000). Therefore,
MIS and WNT signaling pathways may function cooperatively in
mediating epithelial-to-mesenchymal transition early in Müllerian
Early MIS signaling in Müllerian duct regression
Fig. 5. MIS upregulates SMAD8,
downregulates SMAD5 and induces
Alk2 and Alk3 expression in
urogenital ridges. Male or female
urogenital ridges harvested at E14.5 were
untreated or treated with MIS in organ
culture for 20 hours (A-L), 12 hours (M-P)
or 30 hours (Q-T). Transcripts of SMAD8
(A-F), SMAD5 (G-L), ALK2 (M-P) and
ALK3 (Q-T) were detected by whole-
mount in situ hybridization (A-C,G-
I,M,N,Q,R), and their expressions are also
shown after cryosectioning (D-F,J-
L,O,P,S,T) at the broken lines. Arrows
indicate the presence of expression.
Cranial is oriented towards the top and
Müllerian duct towards the right of
individual whole-mount images. M,
Müllerian duct; Ov, ovary; T, testis; W,
Wolffian duct. Scale bar: 500 ?m for all
the whole-mount samples and 50 ?m for
all the cross-sections.
Alk2 and Alk3 may act as sequential MIS type I
receptors in Müllerian duct regression
Specificity and versatility in the signaling responses of TGF-?
family members are defined particularly by the type I receptors that
a ligand can activate. For example, TGF-? activates ALK5 in Mink
lung cells (Bassing et al., 1994), while ALK1 acts as a TGF-? type
I receptor in vascular smooth muscle differentiation (Oh et al.,
2000). Alk3 and ALK6 can serves as sequential type I receptors in
BMP signaling, and control the production and fate of dorsal
precursor cells from neural stem cells (Panchision et al., 2001). Alk2,
as a functionally essential MIS type I receptor in the rat urogenital
ridge (Visser et al., 2001), mediates the change of MISRII
expression, and thus the migration and transition of the coelomic
epithelial cells (Fig. 6). Alk2 has also been shown to regulate
epithelial-to-mesenchymal transition during cardiac valve formation
(Desgrosellier et al., 2005). Interestingly, constitutively active Alk3
can stimulate E-cadherin expression and antagonize the process of
epithelial-to-mesenchymal transition (Zeisberg et al., 2003).
Analysis of Alk2 expression in the male urogenital ridge of the
rat has previously shown that it is present in the Müllerian
mesenchyme at E15, but not at E16 (He et al., 1993) when
Müllerian duct regression is not yet complete. Alk3 is ubiquitously
expressed in embryonic organs including the urogenital system by
the time that MIS and MISRII are expressed (Dewulf et al., 1995).
However, we noted that ALK3 expression favors the mesenchyme
of the urogenital ridge instead of the coelomic epithelium where
functional MIS signaling initially occurs (Fig. 2B). Alk3expression
is increased after E15.5, coincident with the appearance of Misr2
in the peri-Müllerian duct mesenchyme in male rat urogenital
ridges (Fig. 2B), and this could also be recapitulated in female
urogenital ridges upon MIS treatment (Fig. 5Q-T). Moreover,
regulation of Alk3 expression appears to be a downstream event, as
diminution of Alk2-mediated signaling with Alk2-siRNA also
inhibits the upregulation of Alk3in male urogenital ridges (data not
shown). The spatiotemporal patterns of Alk2 and Alk3 expression
imply that they may act sequentially as type I receptors for MIS
signaling (Fig. 8); Alk2 functions early in MIS signaling and
mediates the migration and transition of coelomic epithelial cells,
Development 133 (12)
Fig. 6. Alk2 is essential for MIS signaling, transition of Misr2
expression and Müllerian duct regression in the rat. E14.5 male
urogenital ridges were treated with control-siRNA (A,C,E) or Alk2-siRNA
(B,D,F) for 10 hours. (A,B) Cultured for additional 10 hours followed by
whole-mount immunofluorescence analysis of activated R-SMAD1, 5, 8
(P-SMAD). (C,D) Cultured for an additional 20 hours followed by in situ
hybridization to detect Misr2. (E-H) Cultured for an additional 48 hours
followed by in situ hybridization to detect Wnt7a expression. White
arrows indicate the cranial regions with high (A) or low (B) P-SMAD
expression for comparison. The presence (C) or absence (D) of Misr2
expression can be noted in the regions between the Müllerian and
Wolffian ducts (arrowheads). Black arrows indicate the persistence of
Wnt7a expression in the remaining Müllerian duct epithelium (F,H). The
position of transverse sections (G,H) is marked by broken lines on E and
F, respectively. Cranial is oriented towards the top and Müllerian duct to
the right of individual images. M, Müllerian duct; T, testis; W, Wolffian
duct. Scale bar: 500 ?m for all the whole-mount samples; 50 ?m for all
Fig. 7. Smad5 RNAi enhances and Smad8 RNAi inhibits Müllerian
duct regression. E14.5 male rat urogenital ridges were treated with
control-siRNA (A,C,E), Smad5-siRNA (B,D) or Smad8-siRNA (F) for 12
hours, and subsequently cultured for indicated periods. Whole-mount
in situ hybridization was performed to detect Wnt7a expression.
Arrowheads or arrows indicate the disappearing and remaining Wnt7a
expression, respectively. Cranial is oriented towards the top and
Müllerian duct towards the right of individual images. Scale bar:
whereas ALK3may participate in later MIS signaling in Müllerian
duct regression, which has been well documented in the mouse
(Jamin et al., 2002). The indispensable role of ALK2 in Müllerian
duct regression was also documented in mouse urogenital ridges by
performing RNAi at E12.5 (data not shown). Moreover, we also
observed increased expression of Alk3 in the mouse at E14.5
(developmentally equivalent to ~E15.5 in the rat) in the Müllerian
duct mesenchyme (data not shown), which appears later than Alk2
(Visser et al., 2001). Although incubation with siRNAs for Alk3was
able to knockdown Alk3 expression in cultured cells (data not
shown), we could not achieve a commensurate decrease in ALK3
expression in the peri-Müllerian mesenchyme owing to limited
penetration of siRNAs (data not shown). This precluded further
pursuit of the ALK3 function in rat Müllerian duct regression using
our current RNAi techniques. However, given that the timing of
increased Alk3 expression seen in Müllerian duct mesenchyme
coincides with regression of Müllerian duct epithelium, which
occurs predominantly after E15.5 in male rat urogenital ridges, it
would be reasonable to speculate that in the rat (like the mouse)
Misr2-expressing mesenchymal cells favor Alk3 as the type I
receptor in late stage Müllerian duct regression.
Specificity of SMADs in MIS signaling during
Müllerian duct regression
Regression of the Müllerian duct requires the simultaneous action
of the MIS ligand, the type II receptor and the type I receptor(s). R-
SMADs 1, 5 and 8 are the intracellular effectors of Alk2/Alk3
signaling, and their functional redundancy has been suggested in
BMP signaling. It is also noteworthy that they can function
specifically in particular tissue and developmental contexts. SMAD5
mediates BMP2 signaling in developing cerebellum (Rios et al.,
2004) and knockout of SMAD5 reveals its importance in regulating
angiogenesis (Yang et al., 1999), whereas SMAD1 signaling
controls the growth of extra-embryonic structures at
postimplantation stages (Tremblay et al., 2001). MIS-induced
upregulation of Smad8and downregulation of Smad5correlate with
Müllerian duct regression, suggesting that SMAD5 and SMAD8 can
transduce specific signals in MIS pathways. In addition, targeting
Smad5expression with siRNA promoted Müllerian duct regression.
SMAD5 has been shown to mediate BMP7 signaling and to cause
reversal of TGF-?1-induced epithelial-to-mesenchymal transition
(Zeisberg et al., 2003). Downregulation of SMAD5 by MIS seems
to favor the transition of the coelomic epithelial cells to
Increased SMAD8, similar to upregulated ALK2, can act to
sustain and amplify the signaling cascade. Disruption of the feed-
forward circuit by RNAi-mediated gene silencing of either SMAD8
or ALK2 affected the subsequent downstream signaling events,
resulting in retained Müllerian ducts. However, our investigation did
not reveal a clear role for SMAD8 in MIS-induced earlier epithelial-
to-mesenchymal transition (data not shown). It is possible that this
process is independent of SMAD signaling. Prolonged induction of
Smad8 at E15~E16 over Alk2 expression was seen in the male peri-
Müllerian mesenchyme (data not shown), suggesting that SMAD8
may play a role in later ALK3-mediated molecular events during
Müllerian duct regression.
In conclusion, we identified the coelomic epithelium as the first
target for MIS and found that MIS exerts a profound influence on the
expression of its own signaling components early in Müllerian duct
regression. These events elicit epithelial-to-mesenchymal transition
and amplify the MIS signaling for subsequent regression of the
Müllerian duct. Knowledge of the downstream MIS signaling events
in the urogenital ridge will be important to the study of MIS at other
target sites such as the coelomic epithelium of the ovary where
oncogenic changes lead to ovarian cancer in mouse models (Orsulic
et al., 2002; Connolly et al., 2003; Dinulescu et al., 2005) and
presumably in humans.
We thank Drs Allan Goldstein, Liz Perkins and Drucilla Roberts for
suggestions and critical reading of the manuscript; Drs Trent Clarke,
Makiko Hoshiya and Yasunori Hoshiya for sharing techniques and
reagents, and members of the Donahoe laboratory for helpful discussions.
This work was supported by a grant form the NIH (NICHD-HD-32112 to
P.K.D. and D.T.M., and J.T.), a fellowship from the Ovarian Cancer
Research Training Program of the Department of Defense (Y.Z.), and a
National Research Service Award (NRSA) fellowship (to N.A.A.).
Recombinant human MIS used in this study was provided under the
auspices of NCI-CA-17393 (D.T.M. and P.K.D.).
Allard, S., Adin, P., Gouédard, L., di Clemente, N., Josso, N., Orgebin-Crist,
M. C., Picard, J. Y. and Xavier, F. (2000). Molecular mechanisms of hormone-
mediated Müllerian duct regression: involvement of ?-catenin. Development
Arango, N. A., Szotek, P. P., Manganaro, T. F., Oliva, E., Donahoe, P. K. and
Teixeira, J. (2005). Conditional deletion of ?-catenin in the mesenchyme of the
developing mouse uterus results in a switch to adipogenesis in the myometrium.
Dev. Biol. 288, 276-283.
Attisano, L. and Wrana, J. L. (2002). Signal transduction by the TGF-?
superfamily. Science 296, 1646-1647.
Austin, H. B. (1995). DiI analysis of cell migration during Müllerian duct
regression. Dev. Biol. 169, 29-36.
Baarends, W. M., van Helmond, M. J., Post, M., van der Schoot, P. J.,
Early MIS signaling in Müllerian duct regression
Fig. 8. A schematic model of MIS actions at the early stage of
Müllerian duct regression. Müllerian duct (M) formation and initial
MISRII expression (dark blue) in the coelomic epithelium (gray) are
similar in male and female urogenital ridges at E13 and early E14. After
~E14.5, MIS signaling (yellow) becomes functional in the male, driving
the MISRII-expressing cells into the area adjacent to the Müllerian duct
and eventually around the Müllerian duct at ~E15.5. This is an
epithelial-to-mesenchymal transition. Meanwhile, MIS also upregulates
ALK2 and SMAD8 and downregulates SMAD5. These combined
activities have roles in Müllerian duct regression, as noted by the
smaller Müllerian duct after E15.5, which disappears eventually. At this
time, ALK3 and SMAD8, which are highly expressed in the Müllerian
duct mesenchyme may mediate MIS signaling and Müllerian duct
regression. Expression of MISRII remains in the coelomic epithelium of
female urogenital ridges during this period. M, Müllerian duct; W,
Hoogerbrugge, J. W., de Winter, J. P., Uilenbroek, J. T., Karels, B.,
Wilming, L. G., Meijers, J. H. et al. (1994). A novel member of the
transmembrane serine/threonine kinase receptor family is specifically expressed
in the gonads and in mesenchymal cells adjacent to the Müllerian duct.
Development 120, 189-197.
Bassing, C. H., Yingling, J. M., Howe, D. J., Wang, T., He, W. W., Gustafson,
M. L., Shah, P., Donahoe, P. K. and Wang, X. F. (1994). A transforming
growth factor ? type I receptor that signals to activate gene expression. Science
Behringer, R. R., Finegold, M. J. and Cate, R. L. (1994). Müllerian-inhibiting
substance function during mammalian sexual development. Cell 79, 415-425.
Belville, C., Josso, N. and Picard, J. Y. (1999). Persistence of Müllerian derivatives
in males. Am. J. Med. Genet. 89, 218-223.
Calegari, F., Haubensak, W., Yang, D., Huttner, W. B. and Buchholz, F. (2002).
Tissue-specific RNA interference in postimplantation mouse embryos with
endoribonuclease-prepared short interfering RNA. Proc. Natl. Acad. Sci. USA 99,
Clarke, T. R., Hoshiya, Y., Yi, S. E., Liu, X., Lyons, K. M. and Donahoe, P. K.
(2001). Müllerian inhibiting substance signaling uses a bone morphogenetic
protein (BMP)-like pathway mediated by ALK2 and induces Smad6 expression.
Mol. Endocrinol. 15, 946-959.
Catlin, E. A., Tonnu, V. C., Ebb, R. G., Pacheco, B. A., Manganaro, T. F., Ezzell,
R. M., Donahoe, P. K. and Teixeira, J. (1997). Müllerian inhibiting substance
inhibits branching morphogenesis and induces apoptosis in fetal rat lung.
Endocrinology 138, 790-796.
Connolly, D. C., Bao, R., Nikitin, A. Y., Stephens, K. C., Poole, T. W., Hua, X.,
Harris, S. S., Vanderhyden, B. C. and Hamilton, T. C. (2003). Female mice
chimeric for expression of the simian virus 40 TAg under control of the MISIIR
promoter develop epithelial ovarian cancer. Cancer Res. 63, 1389-1397.
Desgrosellier, J. S., Mundell, N. A., McDonnell, M. A., Moses, H. L. and
Barnett, J. V. (2005). Activin receptor-like kinase 2 and Smad6 regulate
epithelial-mesenchymal transformation during cardiac valve formation. Dev. Biol.
Dewulf, N., Verschueren, K., Lonnoy, O., Moren, A., Grimsby, S., Vande
Spiegle, K., Miyazono, K., Huylebroeck, D. and Ten Dijke, P. (1995). Distinct
spatial and temporal expression patterns of two type I receptors for bone
morphogenetic proteins during mouse embryogenesis. Endocrinology 136,
di Clemente, N., Wilson, C., Faure, E., Boussin, L., Carmillo, P., Tizard, R.,
Picard, J. Y., Vigier, B., Josso, N. and Cate, R. (1994). Cloning, expression,
and alternative splicing of the receptor for anti-Müllerian hormone. Mol.
Endocrinol. 8, 1006-1020.
Dinulescu, D. M., Ince, T. A., Quade, B. J., Shafer, S. A., Crowley, D. and
Jacks, T. (2005). Role of K-ras and Pten in the development of mouse models of
endometriosis and endometrioid ovarian cancer. Nat. Med. 11, 63-70.
Donahoe, P. K., Ito, Y. and Hendren, W. H., 3rd (1977). A graded organ culture
assay for the detection of Müllerian inhibiting substance. J. Surg. Res. 23, 141-
Gouédard, L., Chen, Y. G., Thevenet, L., Racine, C., Borie, S., Lamarre, I.,
Josso, N., Massagué, J. and di Clemente, N. (2000). Engagement of bone
morphogenetic protein type IB receptor and Smad1 signaling by anti-Müllerian
hormone and its type II receptor. J. Biol. Chem. 275, 27973-27978.
Gruenwald, P. (1941). The relation of the growing Müllerian duct to the Wolffian
duct and its importance for the genesis of malformations. Anat. Rec. 81, 1-19.
He, W. W., Gustafson, M. L., Hirobe, S. and Donahoe, P. K. (1993).
Developmental expression of four novel serine/threonine kinase receptors
homologous to the activin/transforming growth factor-? type II receptor family.
Dev. Dyn. 196, 133-142.
Hirobe, S., He, W. W., Lee, M. M. and Donahoe, P. K. (1992). Müllerian
inhibiting substance messenger ribonucleic acid expression in granulosa and
Sertoli cells coincides with their mitotic activity. Endocrinology 131, 854-862.
Hoshiya, M., Christian, B. P., Cromie, W. J., Kim, H., Zhan, Y., MacLaughlin,
D. T. and Donahoe, P. K. (2003). Persistent Müllerian duct syndrome caused by
both a 27-bp deletion and a novel splice mutation in the MIS type II receptor
gene. Birth Defects Res. Part A Clin. Mol. Teratol. 67, 868-874.
Huber, M. A., Azoitei, N., Baumann, B., Grunert, S., Sommer, A.,
Pehamberger, H., Kraut, N., Beug, H. and Wirth, T. (2004). NF-?B is essential
for epithelial-mesenchymal transition and metastasis in a model of breast cancer
progression. J. Clin. Invest. 114, 569-581.
Hudson, P. L., Dougas, I., Donahoe, P. K., Cate, R. L., Epstein, J., Pepinsky, R.
B. and MacLaughlin, D. T. (1990). An immunoassay to detect human Müllerian
inhibiting substance in males and females during normal development. J. Clin.
Endocrinol. Metab. 70, 16-22.
Ikawa, H., Trelstad, R. L., Hutson, J. M., Manganaro, T. F. and Donahoe, P. K.
(1984). Changing patterns of fibronectin, laminin, type IV collagen, and a
basement membrane proteoglycan during rat Müllerian duct regression. Dev.
Biol. 102, 260-263.
Jamin, S. P., Arango, N. A., Mishina, Y., Hanks, M. C. and Behringer, R. R.
(2002). Requirement of Bmpr1a for Müllerian duct regression during male sexual
development. Nat. Genet. 32, 408-410.
Jamin, S. P., Arango, N. A., Mishina, Y., Hanks, M. C. and Behringer, R. R.
(2003). Genetic studies of the AMH/MIS signaling pathway for Müllerian duct
regression. Mol. Cell. Endocrinol. 211, 15-19.
Josso, N. and di Clemente, N. (2003). Transduction pathway of anti-Müllerian
hormone, a sex-specific member of the TGF-? family. Trends Endocrinol. Metab.
Josso, N., Picard, J. Y. and Trah, D. (1976). The antimüllerian hormone. Recent
Prog. Horm. Res. 33, 117-167.
Karl, J. and Capel, B. (1998). Sertoli cells of the mouse testis originate from the
coelomic epithelium. Dev. Biol. 203, 323-333.
Kobayashi, A. and Behringer, R. R. (2003). Developmental genetics of the
female reproductive tract in mammals. Nat. Rev. Genet. 4, 969-980.
Lorenzo, H. K., Teixeira, J., Pahlavan, N., Laurich, V. M., Donahoe, P. K. and
MacLaughlin, D. T. (2002). New approaches for high-yield purification of
Müllerian inhibiting substance improve its bioactivity. J. Chromatogr. B Analyt.
Technol. Biomed. Life Sci. 766, 89-98.
Lu, Z., Ghosh, S., Wang, Z. and Hunter, T. (2003). Downregulation of
caveolin-1 function by EGF leads to the loss of E-cadherin, increased
transcriptional activity of ?-catenin, and enhanced tumor cell invasion.
Cancer Cell 4, 499-515.
Massagué, J. (2000). How cells read TGF-? signals. Nat. Rev. Mol. Cell Biol. 1,
Miller, C. and Sassoon, D. A. (1998). Wnt-7a maintains appropriate uterine
patterning during the development of the mouse female reproductive tract.
Development 125, 3201-3211.
Mishina, Y., Rey, R., Finegold, M. J., Matzuk, M. M., Josso, N., Cate, R. L. and
Behringer, R. R. (1996). Genetic analysis of the Müllerian-inhibiting substance
signal transduction pathway in mammalian sexual differentiation. Genes Dev.
Müller, T., Bain, G., Wang, X. and Papkoff, J. (2002). Regulation of epithelial
cell migration and tumor formation by ?-catenin signaling. Exp. Cell Res. 280,
Nishita, M., Ueno, N. and Shibuya, H. (1999). Smad8B, a Smad8 splice variant
lacking the SSXS site that inhibits Smad8-mediated signalling. Genes Cells 4,
Oh, S. P., Seki, T., Goss, K. A., Imamura, T., Yi, Y., Donahoe, P. K., Li, L.,
Miyazono, K., ten Dijke, P., Kim, S. et al. (2000). Activin receptor-like kinase 1
modulates transforming growth factor-? 1 signaling in the regulation of
angiogenesis. Proc. Natl. Acad. Sci. USA 97, 2626-2631.
Orsulic, S., Li, Y., Soslow, R. A., Vitale-Cross, L. A., Gutkind, J. S. and Varmus,
H. E. (2002). Induction of ovarian cancer by defined multiple genetic changes in
a mouse model system. Cancer Cell 1, 53-62.
Panchision, D. M., Pickel, J. M., Studer, L., Lee, S. H., Turner, P. A., Hazel, T. G.
and McKay, R. D. (2001). Sequential actions of BMP receptors control neural
precursor cell production and fate. Genes Dev. 15, 2094-2110.
Parr, B. A. and McMahon, A. P. (1998). Sexually dimorphic development of the
mammalian reproductive tract requires Wnt-7a. Nature 395, 707-710.
Picon, R. (1969). Action of the fetal testis on the development in vitro of the
Müllerian ducts in the rat. Arch. Anat. Microsc. Morphol. Exp. 58, 1-19.
Price, J. M., Donahoe, P. K., Ito, Y. and Hendren, W. H., 3rd (1977).
Programmed cell death in the Müllerian duct induced by Müllerian inhibiting
substance. Am. J. Anat. 149, 353-375.
Ragin, R. C., Donahoe, P. K., Kenneally, M. K., Ahmad, M. F. and
MacLaughlin, D. T. (1992). Human Müllerian inhibiting substance: enhanced
purification imparts biochemical stability and restores antiproliferative effects.
Protein Expr. Purif. 3, 236-245.
Rios, I., Alvarez-Rodriguez, R., Marti, E. and Pons, S. (2004). Bmp2
antagonizes sonic hedgehog-mediated proliferation of cerebellar granule
neurones through Smad5 signalling. Development 131, 3159-3168.
Roberts, L. M., Hirokawa, Y., Nachtigal, M. W. and Ingraham, H. A. (1999).
Paracrine-mediated apoptosis in reproductive tract development. Dev. Biol. 208,
Sakai, T., Larsen, M. and Yamada, K. M. (2003). Fibronectin requirement in
branching morphogenesis. Nature 423, 876-881.
Segev, D. L., Hoshiya, Y., Stephen, A. E., Hoshiya, M., Tran, T. T.,
MacLaughlin, D. T., Donahoe, P. K. and Maheswaran, S. (2001). Müllerian
inhibiting substance regulates NF-?B signaling and growth of mammary
epithelial cells in vivo. J. Biol. Chem. 276, 26799-26806.
Segev, D. L., Hoshiya, Y., Hoshiya, M., Tran, T. T., Carey, J. L., Stephen, A. E.,
MacLaughlin, D. T., Donahoe, P. K. and Maheswaran, S. (2002). Müllerian-
inhibiting substance regulates NF-?B signaling in the prostate in vitro and in vivo.
Proc. Natl. Acad. Sci. USA 99, 239-244.
Sosic, D., Richardson, J. A., Yu, K., Ornitz, D. M. and Olson, E. N. (2003). Twist
regulates cytokine gene expression through a negative feedback loop that
represses NF-?B activity. Cell 112, 169-180.
Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R., Donoghue,
M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J. et al. (2004).
Therapeutic silencing of an endogenous gene by systemic administration of
modified siRNAs. Nature 432, 173-178.
Teixeira, J., He, W. W., Shah, P. C., Morikawa, N., Lee, M. M., Catlin, E. A.,
Development 133 (12)
Hudson, P. L., Wing, J., Maclaughlin, D. T. and Donahoe, P. K. (1996).
Developmental expression of a candidate Müllerian inhibiting substance type II
receptor. Endocrinology 137, 160-165.
Teixeira, J., Kehas, D. J., Antun, R. and Donahoe, P. K. (1999). Transcriptional
regulation of the rat Müllerian inhibiting substance type II receptor in rodent
Leydig cells. Proc. Natl. Acad. Sci. USA 96, 13831-13838.
Teixeira, J., Maheswaran, S. and Donahoe, P. K. (2001). Müllerian inhibiting
substance: an instructive developmental hormone with diagnostic and possible
therapeutic applications. Endocr. Rev. 22, 657-674.
Thiery, J. P. (2002). Epithelial-mesenchymal transitions in tumour progression. Nat.
Rev. Cancer 2, 442-454.
Trelstad, R. L., Hayashi, A., Hayashi, K. and Donahoe, P. K. (1982). The
epithelial-mesenchymal interface of the male rat Müllerian duct: loss of
basement membrane integrity and ductal regression. Dev. Biol. 92, 27-40.
Tremblay, K. D., Dunn, N. R. and Robertson, E. J. (2001). Mouse embryos
lacking Smad1 signals display defects in extra-embryonic tissues and germ cell
formation. Development 128, 3609-3621.
Tsuji, M., Shima, H., Yonemura, C. Y., Brody, J., Donahoe, P. K. and Cunha, G.
R. (1992). Effect of human recombinant Müllerian inhibiting substance on
isolated epithelial and mesenchymal cells during Müllerian duct regression in the
rat. Endocrinology 131, 1481-1488.
Visser, J. A., Olaso, R., Verhoef-Post, M., Kramer, P., Themmen, A. P. and
Ingraham, H. A. (2001). The serine/threonine transmembrane receptor ALK2
mediates Müllerian inhibiting substance signaling. Mol. Endocrinol. 15, 936-945.
Yang, X., Castilla, L. H., Xu, X., Li, C., Gotay, J., Weinstein, M., Liu, P. P. and
Deng, C. X. (1999). Angiogenesis defects and mesenchymal apoptosis in mice
lacking Smad5. Development 126, 1571-1580.
Zeisberg, M., Hanai, J., Sugimoto, H., Mammoto, T., Charytan, D., Strutz, F.
and Kalluri, R. (2003). BMP-7 counteracts TGF-?1-induced epithelial-to-
mesenchymal transition and reverses chronic renal injury. Nat. Med. 9, 964-968.
Early MIS signaling in Müllerian duct regression