Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: Dev Biol. 2013 Dec 19;386(1):227–236. doi: 10.1016/j.ydbio.2013.12.015

Induction of WNT inhibitory factor 1 expression by Müllerian inhibiting substance/AntiMullerian hormone in the Müllerian duct mesenchyme is linked to Müllerian duct regression

Joo Hyun Park 1,4,5, Yoshihiro Tanaka 1,4, Nelson A Arango 2,4,6, Lihua Zhang 1, L Andrew Benedict 2,4, Mi In Roh 3,4, Patricia K Donahoe 2,4, Jose M Teixeira 1,4,7
PMCID: PMC4047716  NIHMSID: NIHMS551275  PMID: 24362065

Abstract

A key event during mammalian sexual development is regression of the Müllerian ducts (MDs) in the bipotential urogenital ridges (UGRs) of fetal males, which is caused by the expression of Müllerian inhibiting substance (MIS) in the Sertoli cells of the differentiating testes. The paracrine signaling mechanisms involved in MD regression are not completely understood, particularly since the receptor for MIS, MISR2, is expressed in the mesenchyme surrounding the MD, but regression occurs in both the epithelium and mesenchyme. Microarray analysis comparing MIS signaling competent and Misr2 knockout embryonic UGRs was performed to identify secreted factors that might be important for MIS-mediated regression of the MD. A seven-fold increase in the expression of Wif1, an inhibitor of WNT/β-catenin signaling, was observed in the Misr2-expressing UGRs. Whole mount in situ hybridization of Wif1 revealed a spatial and temporal pattern of expression consistent with Misr2 during the window of MD regression in the mesenchyme surrounding the MD epithelium that was absent in both female UGRs and UGRs knocked out for Misr2. Knockdown of Wif1 expression in male UGRs by Wif1-specific siRNAs beginning on embryonic day 13.5 resulted in MD retention in an organ culture assay, and exposure of female UGRs to added recombinant human MIS induced Wif1 expression in the MD mesenchyme. Knockdown of Wif1 led to increased expression of β-catenin and its downstream targets TCF1/LEF1 in the MD mesenchyme and to decreased apoptosis, resulting in partial to complete retention of the MD. These results strongly suggest that WIF1 secretion by the MD mesenchyme plays a role in MD regression in fetal males.

Keywords: antiMüllerian hormone, WNT signaling, reproductive tract development

INTRODUCTION

Sexual differentiation of the bipotential embryonic urogenital ridge (UGR) into the testes and internal male reproductive tract is regulated by the Sex Determining Region Y (SRY) transcription factor expressed in XY gonads, which leads to the production of three hormones. The fetal testes produce and secrete both Müllerian inhibiting substance (MIS; also known as anti-Müllerian hormone, AMH), which causes Müllerian ducts (MDs) to regress, and testosterone, which promotes the differentiation of the Wolffian ducts into the internal male reproductive tract tissues, i.e., the epididymides, vasa deferentia, and seminal vesicles. In the absence of MIS, as is the case with females, MDs do not regress but continue to develop and differentiate into the oviducts, uterus, and upper vagina (reviewed in (Teixeira 2001)). Defects in either the gene for MIS or its receptors can result in a form of male pseudohermaphroditism characterized by retained MD-derived tissues (Behringer 1994; Mishina 1996; Jamin 2002). In humans, approximately 85% of patients with persistent Müllerian duct syndrome (PMDS) are thought to have underlying mutations in MIS or its receptors (Belville 2004; Josso 2005). The third hormone, insulin-like 3 (INSL3), is produced by the pre-and postnatal testes and is necessary for testicular descent (Nef and Parada 1999; Zimmermann 1999).

As with other members of the transforming growth factor β (TGFβ) super family, MIS binds to its specific type II receptor (MISR2), which activates its latent kinase, causing recruitment and phosphorylation of a type I receptor, either ACVR1 (ALK2) or BMPR1A (ALK3), to initiate a downstream signaling cascade that results in apoptosis and regression of the MDs (Teixeira 2001; Josso and Clemente 2003). The MDs first form from coelomic epithelial cells that invaginate, proliferate, and migrate in a cranial-to-caudal direction to merge with the urogenital sinus epithelium (Guioli 2007; Orvis and Behringer 2007; Fujino 2009). The MDs are similarly eliminated in a cranial-to-caudal manner as a result of MIS action (Picon 1969; Tsuji 1992), which is attributable to the cranial-to-caudal expression of Misr2 (Allard 2000; Arango 2008). We have recently shown that Misr2-expressing cells are initially present in the coelomic epithelium and subjacent mesenchyme of both male and female UGRs, but that under the influence of MIS, the Misr2-expressing cells proliferate and migrate into the mesenchyme surrounding the male MD (Hayashi 1982; Trelstad 1982; Zhan 2006). We and others have also shown that expression of the MIS type I receptors, is also spatiotemporally controlled, and that the SMAD1, 5 and 8 subfamily of intracellular signal transducers are involved in MD regression (Gouedard 2000; Clarke 2001; Visser 2001; Zhan 2006; Orvis 2008). One of the important unanswered questions in MD regression is how MISR2, which is expressed in the mesenchyme surrounding the MD but not in the MD epithelial cells at the time of regression (Baarends 1994; di Clemente 1994; Teixeira 1996), induces apoptosis in the neighboring epithelial cells (Tsuji 1992; Teixeira 1996; Catlin 1997; Allard 2000).

WNT/β-catenin signaling is known to be crucial for normal uterine development from the MDs in females (Kobayashi and Behringer 2003). For example, Wnt5a knockout mice display anomalous development of the uterine horns, cervix and vagina, and the uteri from Wnt7a knockout mice have defective myometria, endometrial glands, and oviductal structures (Miller and Sassoon 1998; Parr and McMahon 1998; Mericskay 2004). The MD mesenchyme-specific expression of Wnt5A and MD epithelium-specific expression of Wnt7A adds further complexity to the respective roles in uterine development (Mericskay 2004). The different phenotypes observed by the knockout of either Wnt7a or Wnt5a suggests that these ligands have different functional roles, which might be attributable to the separate signaling pathways used by the respective ligands, i.e., the canonical β-catenin pathway for WNT7A (Mikels and Nusse 2006b) and the Ca2+ or planar cell polarity pathway for WNT5A (Loscertales 2008; Romereim and Dudley 2011). However, WNT5A can signal through the canonical β-catenin pathway, depending on specific WNT receptor expression (He 1997; Mikels and Nusse 2006a; van Amerongen 2012).

WNT signaling is not only important for uterine development in females, it is also a key factor in MD regression in male fetuses. Male Wnt7a knockout mice have retained MDs (Miller and Sassoon 1998; Parr and McMahon 1998), so far the only Wnt family gene knockout reported to develop this phenotype. However, Misr2 mRNA expression is also lost in Wnt7a knockout mice, thereby abrogating MIS signaling and precluding any inference on its role in the downstream activity of β-catenin. Nuclear accumulation of β-catenin has been reported in the MD mesenchyme cells during MD regression (Allard 2000), and male mice with conditional knockout of β-catenin from the MD mesenchyme were shown to have retained MDs (Kobayashi 2011), indicating that nuclear β-catenin activity in the MD mesenchyme is necessary for MD regression. Taken together, these studies support a dual role for WNT/β-catenin signaling in MD biology, one for regression in males and another for differentiation in females. We have shown that either conditional knockout of β-catenin or constitutive activation of β-catenin in the MD mesenchyme leads to myometrial pathologies in female mice (Arango 2005; Tanwar 2009) and that constitutive activation of β-catenin in the MD mesenchyme predisposes male mice to focal MD retention (Tanwar 2010). Thus, it appears that the contradictory finding that MD retention in males with either β- catenin knockout (Kobayashi 2011) or constitutive activation of β-catenin (Tanwar 2010) using the same Misr2-driven Cre would suggest that MD regression is exquisitely sensitive to WNT/β-catenin signaling in the MD mesenchyme or that highly localized microenvironmental factors that modulate WNT/β- catenin signaling might need to act in concert with β-catenin to complete MD regression in males. In contrast to the critical roles played by WNT/β-catenin signaling in the MD mesenchyme, its expression in the MD epithelium does not appear to be necessary for MD regression (Kobayashi 2011).

We have identified WNT inhibitory factor 1 (WIF1) (Hsieh 1999) as a candidate intercellular factor involved in the MIS/MISR2-mediated regulation of MD regression using microarray analyses of Misr2 homozygous knockout UGRs compared with heterozygous controls. Here we show that MIS induces expression of Wif1 mRNA during normal MD regression in males and that knockdown of Wif1 by siRNA leads to partial retention of the MD epithelium in UGR assays. These results suggest that the dual roles played by β-catenin in MD retention and regression can be controlled by the local expression of WNT inhibitors such as WIF1.

METHODS

Microarray analysis of Misr2 knockout and control mice

Mice used in this study were housed under standard animal housing conditions and maintained on a C57BL/6;129/SvEv mixed genetic background. The Institutional Animal Care and Use Committee at Massachusetts General Hospital approved the protocols for animal experimentations performed in this study. YFP mice (Gt(ROSA)26Sortm1(EYFP)Cos, obtained from Dr. Frank Costantini (Srinivas 2001)) were mated with Misr2-LacZ mice (Amhr2tm2(LacZ)Bhr, obtained from Dr. Richard Behringer (Arango 2008)) to obtain homozygous YFP/YFP;Misr2-LacZ/Misr2-LacZ females, which were then mated with male Misr2-Cre/+ (Amhr2tm3(cre)Bhr, obtained from Dr. Richard Behringer (Jamin 2002)) mice to produce Misr2-Cre/Misr2-LacZ;YFP/+ embryos that are knocked out for Misr2 and cannot transduce MIS signaling but express the YFP reporter or Misr2-LacZ/+;YFP/+ embryos that can transduce MIS signaling and do not express the YFP reporter. Genotyping was performed when necessary with DNA collected from tail biopsies using standard PCR protocols. Timed pregnant matings were performed and the presence of a vaginal plug in the morning was considered embryonic day 0.5 (E0.5) at 12 p.m. UGRs of the male embryos were sorted manually by YFP fluorescence (YFP+ were Misr2 knockouts, YFP− were Misr2 heterozygotes) using a fluorescence dissecting microscope, and the mesonephroi were dissected away from the gonads with a scalpel as cleanly as possible, pooled, frozen and stored at −80 C. Female UGRs were discarded. Total RNA was prepared (~5 ug) from approximately 200 mesonephroi for each genotype, the mRNA converted to cRNA, and compared by microarray analyses using the Affymetrix oligonucleotide chips (Mouse430_2) at the Harvard/Partners Center for Personalized Genetic Medicine. Data from the microarray analysis has been deposited in the GEO database under the accession number GSE38009.

Animals, organ culture and recombinant human MIS

UGRs were dissected from wildtype embryos of timed pregnant mice and studied at developmental stages E12.5, E13.5 and E14.5, to determine normal gene expression patterns and morphological changes in vivo. UGRs collected from timed pregnant mice at E13.5 were also dissected and then cultured on MilliCell-CM membrane (Millipore, Bellerica, MA) over 2% agarose gel droplets immersed in CMRL 1066 medium (Life Technologies, Grand Island, NY) supplemented with 10% female fetal bovine serum (Aires Scientific/Bio-logos, Richardson, Texas) to avoid bovine MIS in male serum, 100 U/ml penicillin, 100 ug/ml streptomycin, and 10 nM testosterone, which stimulates Wolffian duct development, for comparison with MDs. Cultures were carried out with or without recombinant human MIS (rhMIS) at a final concentration of 6 ug/ml (42.5 nM). MIS was purified from serum-containing media of Chinese hamster ovary clonal cells expressing native MIS by immunoaffinity chromatography as described previously (Ragin 1992).

RNA probes

Digoxigenin-labeled antisense and sense riboprobes were generated by in vitro transcription using digoxigenin-labeled nucleotide mix (Roche, Indianapolis, IN) according to the manufacturer’s instructions. A PCR fragment of Wnt7a coding sequence (NM_009527; nucleotides 335–970) was subcloned into pCRII-TOPO (Life Technologies), digested with EcoRV, and transcribed with Sp6 RNA polymerase to make antisense probes. A PCR fragment of coding sequence of Wif1 (NM_011915; nucleotides 516–1131) was cloned from a mouse testis cDNA library into pCR2.1-TOPO (Life Technologies), digested with SpeI, and transcribed with T7 RNA polymerase to make antisense probes. The Mis plasmid (Swain 1998) was digested with SmaI and transcribed with T7 RNA polymerase for the antisense probe. The probe for Misr2 was made as previously described (Clarke 2001). All templates were purified using phenol-chloroform-isoamyl alcohol and ether for RNase-free conditions.

In situ hybridization

Immediately following dissection at various gestational ages or after organ culture in RNase-free conditions, UGRs were fixed for 2 h at 4 C in 4% paraformaldehyde (PFA). The UGRs were dehydrated and stored in 100% methanol until further use. Tissues were rehydrated, treated with hydrogen peroxide, then with 10 g/ml proteinase K, pre-hybridized, and hybridized with digoxigenin-labeled sense or antisense riboprobes overnight at 70 C. A minimum of 3 UGRs was used for each experimental condition. After high-stringency washing, the samples were placed in 10% Sheep serum/TBS-T for 1.5 h at room temperature for blocking, then incubated with anti-digoxigenin-AP antibody (Roche) at 1:2000 overnight at 4 C with gentle rocking. The signals were visualized using the BM-Purple alkaline phosphatase substrate (Roche). For frozen sections, samples were embedded in O.C.T. compound and subsequently cryosectioned at 16 um. Photos were taken with an Nikon SMZ1500 dissecting microscope with a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI)

RNAi in organ culture

A cocktail of siRNA duplex oligoribonucleotides of Wif1 (MSS280614, MSS280615, MSS280616) and negative control duplexes were purchased from Life Technologies. RNAi in organ culture was performed as described previously (Zhan 2006). siRNA concentrations between 50 and 400 nM were tested with murine embryonal fibroblasts using the Alexa Fluor-555 siRNA control (Life Technologies) for optimal silencing efficiency and minimal toxicity; as a result, 200 nM was selected for further studies. UGRs from E13.25 were transfected with siRNA duplex in serum-free culture medium by using the Oligofectamine RNAiMAX reagent (Life Technologies). siRNAs and Oligofectamine were diluted in separate tubes in OptiMEM (Life Technologies), combined and incubated for 20 min at room temperature. The siRNA:Oligofectamine mixture was added to the medium and incubated with immersed UGRs for 12 h. The UGRs were subsequently placed on MilliCell-CM membranes to continue culture for an additional 60 h at the air-media interface over complete medium. The tissues were then fixed in 4% PFA for either whole mount in situ hybridization or immunofluorescence staining.

Immunofluorescence staining following Wif1 siRNA treatment of UGRs

For immunofluorescence staining, UGRs were incubated in 15% sucrose solution for 2 h followed by embedding in 7.5% gelatin/15% sucrose. The embedded tissues were frozen at −60 C in isopentane and sectioned at 9 um. For β-catenin, TCF1 and LEF1 immunostaining, serial sections were washed in PBS three times consecutively. Sections were then washed in PBST and blocked with 10% donkey serum, 1% bovine serum albumin, and 0.1% triton-X in PBS for β-catenin or 5% goat serum and 0.1% triton-X for TCF1/LEF1 in TBST, 1 h at room temperature. Tissue sections were then incubated in a humidified chamber overnight at 4 C with anti-β-catenin primary antibody (1:250; BD Transduction Laboratories, San Jose, CA), anti-TCF1 and anti-LEF1 primary antibodies (1:500; Cell signaling Technologies, Danvers, MA). Tissue sections were subsequently washed with PBS-T, incubated for 1 h at room temperature with AlexaFluor secondary antibodies (1:250; Life Technologies), then counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Images were photographed using a microscope (Nikon Eclipse TE 2000-S) equipped with a Spot digital camera.

TUNEL assay

After transfecting E13.5 male UGRs with Wif1 siRNA and RNAi for 12 h, they were incubated for an additional 48 h in complete medium. The UGRs were fixed in 4% PFA at 4 C for 2 h, washed in PBS and kept at 4 C for immediate assay or stored in 70% ethanol at 4 C for later analysis. The UGRs were post fixed in cold ethanol:acetic acid at a 2:1 ratio for 5 min at −20 C for permeabilization. Terminal deoxynucleotidyl Transferase enzyme and anti-digoxigenin conjugates were applied according to the ApopTag Plus Fluorescein In Situ kit (Millipore) manufacturer’s protocol. Counterstaining was performed with DAPI, and the UGRs were examined with a dissecting microscope. Discrete TUNEL+ cells lining the Müllerian duct were manually counted in photos of longitudinal planes obtained after identifying the section where the Müllerian duct was present along with sections approximately 100 um above and below. As a result, 3 sections per ridge for 5 control ridges and 6 Wif1 siRNA treated ridges were counted.

Statistical Analysis

Results are expressed as mean values ± standard error of mean (SEM) from a minimum of three individual experiments. Statistical analyses were perfomed by ANOVA or unpaired t-test as appropriate with Prism 5 (GraphPad, La Jolla, CA). P-values ≤ 0.05 were considered statistically significant.

RESULTS

MIS induces Wif1 expression in the MD mesenchyme

The MIS ligand will not be able to bind to MD mesenchyme cells lacking the MISR2 receptor and initiate the signaling cascade required for MD regression. To determine which genes are induced by MIS signaling during MD regression, a mating scheme was developed for efficient sorting of heterozygous Misr2 UGRs from homozygous Misr2 knockout UGRs of E14.5 fetuses without resorting to PCR genotyping (Fig. 1A). The male gonads were dissected away from the UGRs so that only the duct-containing mesonephroi were collected and pooled for total RNA isolation enriched for agonadal mRNAs. The results of microarray analyses of the RNAs from Misr2-expressing UGRs compared to the Misr2 knockout UGRs are shown in Tables 1 and 2. The loss of Misr2 expression in the knockout RNA (not shown) was used to validate the ability of the experiment to differentiate between Misr2 knockout and heterozygous UGRs.

Figure 1. Wif1 expression is diminished with loss of MIS signaling in UGRs.

Figure 1

Open in a new tab

(A) Schematic showing the breeding and scoring used to distinguish UGRs from Misr2 knockout mice and heterozygous mice. Misr2 knockout UGRs have both the Misr2-Cre and Misr2-LacZ alleles. The Cre recombinase from the Misr2-Cre allele drives YFP expression, which is detected by fluorescence. The Misr2-Cre allele is not in the heterozygotes and YFP expression is not detected. Whole mount in situ hybridization for Wif1 mRNA in male UGRs from embryonic day 13.5 Misr2 knockout mice (B) displays downregulation of Wif1 as indicated by BM purple compared to Misr2 heterozygous control (C) and wildtype (D) UGRs, where there is a clear expression along the MD as well as in the gonads. (E) No Wif1 mRNA expression is detected with a sense RNA probe. Frozen sections of the whole mount in situ hybridized UGRs reveal noticeably higher levels of Wif1 mRNA detection in the mesenchyme surrounding the MD epithelium of the heterozygous and the wildtype controls (G and H, respectively) than in the Misr2 knockout (F) or in the sense control (I). Arrowheads indicate MDs in whole mounts. MD epithelium is outlined with a dashed line in panels with sections. T= testis, M= Müllerian duct, W= Wolffian duct. Bar=100μm

Table 1.

Genes upregulated with MIS signaling.

Gene Name Accession Fold
Sp6 NM_031183.2 7.715
Wif1 NM_011915.2 7.36
Msx2 NM_013601.2 6.688
Dlx5 NM_010056.2 4.069
Gata5 NM_008093.2 3.939
Ednrb NM_007904.4 3.633
Ablim3 NM_198649.3 3.503
Ncstn NM_021607.3 3.451
Ddx17 NM_199080.2 3.33
Syt1 NM_009306.3 3.273
H2-T23 NM_010398.1 3.219
Mbp NM_010777.3 3.023
Mael NM_175296.4 2.977
Stk31 NM_029916.2 2.951
Dppa4 NM_028610.2 2.937
Rpl39l NM_026594.2 2.914
Pou5f1 NM_013633.3 2.893
Zmat2 NM_025594.3 2.867
Nfatc1 NM_016791 2.76
Helb NM_080446.2 2.751
Dazl NM_010021.5 2.7
Gfra2 NM_008115.2 2.658
Epha4 NM_007936.3 2.634
Ptprd NM_011211.3 2.576
Open in a new tab

Table 2.

Genes downregulated with MIS signaling.

Gene Name Accession Fold
Fbxo10 NM_001024142.1 −2.744
Phip NM_001081216.1 −2.764
Cnn3 NM_028044.2 −2.944
Spry4 NM_011898.2 −3.112
Hspa14 NM_015765.2 −3.21
Bcas2 NM_026602.3 −3.405
Kif1c NM_153103.2 −4.155
Tfap2c NM_009335.2 −4.37
Kif5b NM_008448.3 −4.406
Mat2a NM_145569.4 −6.663
Fank1 NM_025850.2 −8.488
Open in a new tab

Wif1 mRNA expression was induced more than 7-fold in MIS-responsive agonadal UGRs (Table I). WIF1 belongs to the secreted Frizzled-related protein class of WNT signaling inhibitors that binds to WNTs, which in turn inhibits their binding to their receptors (Hsieh 1999). Its expression and activity has been reported in a variety of different developmental pathways that, when disrupted, can lead to developmental anomalies and cancer (Kawano and Kypta 2003). Because of the importance of WNT signaling for MD differentiation and regression, we performed whole-mount in situ hybridization to verify the induction of Wif1 mRNA by MIS. UGRs were collected from E13.5 embryos of the Misr2 knockout and heterozygous embryos. E13.5 was chosen because MD regression is well underway by E14.5, when it is more difficult to detect the MDs in males. Expression of Wif1 mRNA was clearly visible along the entire length of the MD and in the testis cords of both Misr2 wild type and heterozygous UGRs (Fig. 1C & D). In Misr2 knockout UGRs, only trace expression of Wif1 was observed (Fig. 1B) when stained for an equivalent amount of time as Misr2-expressing UGRs but which was still higher than that observed with a negative control sense probe (Fig. 1E). Analyses of the whole-mount UGRs in frozen sections were done to determine the spatial relationship of Wif1 mRNA expression with respect to the MD epithelium. In the Misr2 wild type and heterozygous UGRs, expression was localized to the mesenchyme surrounding the MD epithelium (Fig. 1G & H) where Misr2 is expressed and to the MD epithelial cells as well. However, the sections from the Misr2 knockout UGR showed what little Wif1 mRNA was observed localized to the MD epithelial cells (Fig. 1F).

The expression pattern of Wif1 mRNA over time was studied by whole mount in situ hybridization in male UGRs from E12.5, E13.5 and E14.5 embryos. At E12.5 Wif1 mRNA expression is essentially negative (Fig. 2A) and frozen section analysis shows that the light staining in the duct could be an artifact of substrate pooling in the MD lumen (Fig. 2D). However at E13.5 male UGRs are strongly positive for Wif1 in the mesenchyme surrounding the MD epithelium, particularly at the cranial end (Fig. 2B & E). In contrast, female UGRs at this age do not show Wif1 expression by whole mount in situ hybridization above background (Fig. 2H & I). As MD regression progresses caudally at E14.5, Wif1 expression was observed at the middle to caudal end of the MD where regression has not yet occurred (Fig. 2C & G). At the cranial end where MD regression has occurred, Wif1 expression is not observed (Fig. 2C & F). In addition to MIS, the embryonic male gonads produce testosterone, which is necessary for Wolffian duct development and has been shown to induce Wif1 expression (Keil 2012), and other secreted factors that could account for induced Wif1 expression in MD mesenchyme in male UGRs compared with females. To ensure that Wif1 expression is induced by MIS alone, without the need for any other factor produced by the fetal male gonads, E13.5 female UGRs were exposed to recombinant human MIS (6 μg/ml) for 48 h and then analyzed by whole mount in situ hybridization for Wif1 mRNA. Partial MD regression was observed in female UGRs (Fig. 2J), which like male UGRs express the Misr2 in the MD mesenchyme (Baarends 1994; di Clemente 1994; Teixeira 1996), precluding continued expression of Wif1 as was observed in male UGRs with MD regression (Fig. 2C). Also as with male UGRs (Fig. 2 G), analysis of frozen sections where MD regression was not complete showed expression of Wif1 in female UGRs exposed to MIS (Fig. 2K). These results clearly demonstrate the transitory nature of Wif1 expression in a cranial to caudal manner within a narrow window of time in the MD mesenchyme during male UGR development that is induced by MIS.

Figure 2. MIS induces Wif1 expression.

Figure 2

Open in a new tab

Time course analysis shows expression of Wif1 mRNA by in situ hybridization in male UGRs at E12.5 (A), E13.5 (B,) and E14.5 (C). Corresponding frozen sections (D, E) show strongest Wif1 mRNA expression is detected in the MD mesenchyme at E13.5. At E14.5, Wif1 mRNA is not detectable in rostral sections where the MDs have regressed (C, F) but more caudal expression is observed where the MD has not yet regressed (C, G). Wif1 mRNA expression is low or not detectable in female UGR at E13.5 (H, I). In contrast, Wif1 mRNA is detected in the surrounding mesenchyme of the MDs (arrowhead) in E13.5 female UGRs exposed to rhMIS for 60 h in organ culture (J, K). M= Müllerian duct, W= Wolffian duct. MD epithelium is outlined with a dashed line in panels with sections.

Wif1 knockdown inhibits complete MD regression in vitro

To determine whether MD regression in vitro is dependent on MIS-induced expression of Wif1, knockdown of Wif1 was performed with a cocktail of Wif1-specific siRNA in UGRs from E13.25 male embryos in organ culture. When the UGRs were treated with negative control siRNA, complete or near complete regression of the MDs was observed in the UGRs by whole mount in situ hybridization for Wnt7a mRNA (Fig. 3A), which is specific to the MD epithelium (Miller and Sassoon 1998; Parr and McMahon 1998). Occasional MD epithelium was detected, usually in the caudal end, which regresses last (Picon 1969; Tsuji 1992). The whole mount in situ hybridizations clearly demonstrate regression of the MD as reflected by the absence or reduced level of staining for Wnt7a (Fig. 3C & D). In contrast, when the UGRs were exposed to a cocktail of Wif1-specific siRNAs, significant retention of the MDs was observed (Fig. 3B). In some UGRs, Wnt7a expression was observed along the whole length of the MD (Fig. 3E), but in others caudal expression was most prominent (Fig. 3F). After arbitrarily dividing the MD into three vertical segments, a quantitative comparison of the rate of MD retention between the control and Wif1 siRNA-treated UGRs revealed a statistically significant difference (Fig. 3G) in each segment, confirming that knockdown of Wif1 expression negatively impacts MD regression.

Figure 3. Wif1 mRNA knockdown inhibits MD regression.

Figure 3

Open in a new tab

E13.5 male UGRs were treated with either control scrambled siRNA duplexes or with a cocktail of Wif1 siRNA duplexes for 12 h and assayed for MD regression after 60 h by whole mount in situ for Wnt7a, a MD epithelium-specific mRNA. (A) The absence of Wnt7a in most of the MD epithelium indicates nearly complete regression of the MDs was observed in most UGRs with control siRNA. (B) Complete MD regression was not observed in UGRs transfected with Wif1 siRNA. Higher magnification of representative UGRs boxed in A and B are shown in C–F. Black arrowheads indicate the Wolffian duct and red arrowheads indicate Wnt7a-postiive MDs. UGRs from three separate sets of experiments were scored for MD retention for semi-quantitative comparison with the UGRs divided into rostral, middle and caudal segments (G). Open circles (○) are male control siRNA-treated UGRs, and closed circles (●) are Wif1 siRNA-treated UGRs showing significantly fewer MD regression in all three segments analyzed. Neither Mis in the testes nor Misr2 mRNAs in the testes and remaining MD appeared to be affected by transfection with Wif1 siRNA (J, K) compared to controls (H, I). Arrowhead indicates remaining MD with Misr2 mRNA. T = testis.

To rule out the possibility that MD retention might be due to an indirect effect of Wif1 siRNA knockdown on the expression of either Mis or Misr2, the experiment was repeated and the URGs were examined by whole mount in situ hybridization with probes specific for Mis and Misr2. Neither Mis (Fig. 3H & J) nor Misr2 (Fig. 3I & K) mRNA expression in the gonads appeared to be affected in the Wif1 siRNA-treated UGRs compared to controls. Misr2 expression in the MD mesenchyme, while limited to the caudal regions in the control-treated UGRs undergoing regression, was also observed in the retained MD regions of the Wif1 siRNA-treated UGRs. In the face of MD regression in controls, changes in Misr2 expression are more difficult to interpret in the Wif1 knockdown UGRs because its expression is observed as an artifact of MD retention.

We next examined WNT/β-catenin signaling in control and Wif1 knockdown male UGRs before MD regression is normally completed. Although membranous β-catenin was observed in the control UGRs, increased β-catenin signal was observed in both the mesenchymal cells surrounding the MD and the MD epithelial cells of Wif1 siRNA-treated UGRs (Fig. 4A and B). Confirmation of increased nuclear β-catenin activity with Wif1 siRNA was observed by strongly increased expression of LEF1 (Fig. 4C and D) and a more subtle increase in TCF expression (Fig. 4E and F). However, the induction of both these β-catenin-regulated genes appeared limited to the MD mesenchyme cells, suggesting that the increased β-catenin expression resulting from Wif1 knockdown was not affecting nuclear β-catenin activity in the MD epithelial cells. These results would also suggest that during normal MD regression, MIS-induced WIF1 suppresses WNT/β-catenin signaling in the MD mesenchyme by an autocrine mechanism.

Figure 4. Downstream activities of β-catenin are induced by Wif1 knockdown.

Figure 4

Open in a new tab

Expression of β-catenin (A, B) and its regulated genes, TCF1 (C, D) and LEF1 (E, F), were analyzed by immunofluorescence in frozen sections in either control siRNA-treated or Wif1 siRNA-treated E13.5 male UGRs at 60 h post transfection. The degree of apoptosis in the MD was analyzed by determining the number of TUNEL-positive cells located mostly at the rostral tip (arrowhead) of the UGRs treated with either control siRNA (G) or with Wif1 siRNA (I). White boxed areas in G and I are shown at higher magnification in H and J. M= MD, W= Wolffian duct, T= testis. MD epithelium is outlined with a dashed line. Nuclei are stained with DAPI. Bar=100μm.

Because MIS-induced apoptosis in the Müllerian duct epithelium is a key phenotypical event in male MD regression, the impact of knocking down Wif1 mRNA on apoptosis was investigated. TUNEL staining for UGRs after Wif1 siRNA (Fig. 4G and H) versus negative control siRNA treatment (Fig. 4I and J) revealed a significantly increased distribution of apoptotic cells along the cranial portion of the ridges in the control siRNA treated UGRs (mean 211 ± 9 SEM vs. 32 ± 3 SEM; p=0.0009) at 60 h post transfection, confirming that induced WNT signaling negatively affects MD regression.

DISCUSSION

The development of the male or female sex phenotype from the bipotential embryonic gonads has fascinated reproductive biologists for nearly a century. Aside from its inherent allure, it is also important to understand sex differentiation because genitourinary disorders are the most common in human development and of significant clinical importance to human health and quality of life. These include congenital uterine anomalies, cryptorchidism, intersex anomalies, hypospadias, and exstrophies, which are currently treated by surgical intervention, and in some cases combined with hormonal therapy, with varying degrees of success. Thus, the basic developmental mechanisms underlying gonadal and reproductive tract development have to be further investigated in order to understand more fully the underlying etiologies of these genitourinary anomalies with the goal of developing better and more rational methods to treat these patients. For example, the basic mechanisms and the complex coordination of regression, retention and selective differentiation of the Müllerian and Wolffian ducts are not completely understood. Our results reported here showing that inhibition of WNT signaling in male UGRs after MIS binds to its receptor, MISR2, in the MD mesenchyme provide an important contribution to the study of the key mechanisms governing the regression of MD epithelial cells.

We show that inducing WNT signaling by blocking MIS-mediated induction of Wif1 with subsequent accumulation of β-catenin can result in partial MD retention and support our previous findings that male mice with constitutively activated β-catenin in the MD mesenchyme have a focal MD retention phenotype (Tanwar 2010). Conversely, another report using the same Misr2-Cre allele indicated that β- catenin in the MD mesenchyme is required for regression (Kobayashi 2011), supporting a previous study that suggested accumulation of β-catenin was required for MD regression (Allard 2000). Taken together, these studies suggest an intricately regulated balance of WNT/β-catenin signaling may be crucial for normal regression of MD in males and that too much or too little β-catenin activity might in fact interfere with MD regression (Tanwar 2010). A similar phenomenon has been observed during lung development, where altering the dosage of β-catenin activity in vivo by knockout or constitutively activated expression can have profound effects on epithelial cell differentiation (Mucenski 2003; Okubo and Hogan 2004; Mucenski 2005). Another possibility is that inhibition of Wif1 expression might be affecting WNT signaling pathways that do not involve canonical β-catenin activity.

A key finding from our studies shown in Fig. 4, suggests that decreased Wif1 expression (i.e., more WNT signaling) appears to increase cellular β-catenin levels in both the MD epithelial cells and in the surrounding mesenchymal cells but induced nuclear expression of the proteins encoded by the WNT target genes, Lef1 and Tcf1, was only observed in the mesenchymal cells. Although Wif1 expression appears to be limited to the MD mesenchyme, it is a secreted factor that binds to WNT proteins and inhibits their binding to cell surface receptors, and subsequent intracellular paracrine WNT signaling in both the mesenchyme and adjacent epithelium. These results support the observation that LEF1 expression is observed in the mesenchyme of β-catenin intact UGRs but not in β-catenin knockout UGRs (Kobayashi 2011). We speculate that the effects of WIF1 on canonical β-catenin activity appear to be autocrine. These results do not preclude a paracrine function for WIF1 in the epithelium but do suggest that if there is one, it probably does not require β-catenin-mediated transcriptional activation.

In addition to Wif1, our analyses comparing Misr2 heterozygous with Misr2 knockout UGRs showed that the expression of many other genes was changed by loss of MIS signaling (GSE38009). We chose Wif1 for further analyses because of our interest in β-catenin signaling during Müllerian duct differentiation. Msx2 and Dlx5 are two other genes highly induced by MIS signaling in the UGRs that we expect to study further. The induction of Msx2, a homeobox gene that encodes the muscle segment homeobox homolog 2 protein, in the male MDs during regression has been previously shown in chicken embryos (Ha 2008). Although loss of Msx2 has pleiotropic effects in mice, it does not appear to affect fertility (Satokata 2000), which would be expected in males with retained MDs (Mishina 1996). However, its loss does appear to exacerbate the deleterious effect of diethylstilbestrol on Müllerian duct development (Yin 2006) and conditional postnatal knockout of both Msx1 and Msx2 leads to defective uterine implantation (Daikoku 2011). Dlx5, another homeobox gene that encodes Distal-less homeobox 5 protein, has been implicated in craniofacial, limb, and bone development (Kraus and Lufkin 2006), but there are no reports linking Dlx5 to MD development or regression.

We examined whether evidence of MD retention could be observed in both neonatal and 4 week old male Wif1−/− mice (Kansara 2009) but did not observe any MD-derived tissues in these mice (data not shown). The Wif1−/− mice have yet to provide an overt phenotype other than an increased susceptibility to develop osteosarcomas, which is very surprising given the number of diverse processes that WIF1 has been implicated in modulating, including lung development (Xu 2011), hematopoiesis (Schaniel 2011), and carcinogenesis (Ramachandran 2012). A recent report showed that Wif1 expression in the mesenchyme of the urogenital sinus is regulated by androgens and that it enhances prostatic development (Keil 2012). However, they also reported no obvious prostate phenotype was observed in the Wif1−/− mice as well. The disparity between the results from the knockdown organ culture experiments shown in Fig. 3 and the absent Wif−/− knockout mouse phenotype is reminiscent of an earlier report showing that, although knockdown of matrix metalloproteinase 2 (MMP2) inhibited MD regression in organ culture, the Mmp2−/− male mice did not have retained MD tissues (Roberts 2002). Those authors speculated that functional redundancy of the MMP family could be mitigating the loss of MMP2 on UGR development in vivo, which could also be an issue in the Wif1−/− mice with the several other WNT inhibitors available to replace some WIF1 function. Resolution of these discrepancies with the numerous critical activities attributed WIF1 and the lack of a Wif1−/− phenotype is eagerly awaited.

In summary, we have reported gene expression analyses of murine UGRs during MD regression with and without MIS signaling in order to understand the mechanisms driving a fundamental aspect of sexual differentiation. We chose Wif1 for follow up studies and showed for the first time that MIS induces the expression of the Wif1 gene, which encodes an inhibitor of WNT signaling. Confirmatory studies showed that knockdown of Wif1 mRNA expression interferes with MD regression. Like the role of β-catenin itself during MD regression, we hypothesize that an exquisitely tuned amount of Wif1 expression is required for normal reproductive tract development.

HIGHLIGHTS.

  1. Müllerian inhibiting substance causes Müllerian duct regression in urogenital ridges.

  2. Gene expression analyses showed Wif1 induction during Müllerian duct regression.

  3. Whole mount in situ confirmed Müllerian inhibiting substance induces Wif1 expression.

  4. Knockdown of Wif1 inhibits apoptosis and Müllerian duct regression.

  5. Canonical β-catenin activity was induced in urogenital ridges by Wif1 knockdown.

Acknowledgments

We would like to thank Dr. Igor Dawid and Martha Rebbert at NICHD for generously sharing their Wif1−/− mice with us. We are indebted to Dr. Richard Behringer at MD Anderson for providing us with the Misr2-Cre and Misr2-LacZ mice. These studies were supported by NIH grants to JMT (OD012206) and to PKD (CA017393).

Footnotes

Conflict of Interest: Authors have no financial/conflicting interests to disclose.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Allard S, Adin P, Gouedard L, di Clemente N, Josso N, Orgebin-Crist MC, Picard JY, Xavier F. Molecular mechanisms of hormone-mediated Mullerian duct regression: involvement of beta-catenin. Development. 2000;127(15):3349–3360. doi: 10.1242/dev.127.15.3349. [DOI] [PubMed] [Google Scholar]
  2. Arango NA, Kobayashi A, Wang Y, Jamin SP, Lee HH, Orvis GD, Behringer RR. A mesenchymal perspective of Mullerian duct differentiation and regression in Amhr2-lacZ mice. Mol Reprod Dev. 2008;75(7):1154–1162. doi: 10.1002/mrd.20858. [DOI] [PubMed] [Google Scholar]
  3. Arango NA, Szotek PP, Manganaro TF, Oliva E, Donahoe PK, Teixeira J. Conditional deletion of beta-catenin in the mesenchyme of the developing mouse uterus results in a switch to adipogenesis in the myometrium. Dev Biol. 2005;288(1):276–283. doi: 10.1016/j.ydbio.2005.09.045. [DOI] [PubMed] [Google Scholar]
  4. Baarends WM, van Helmond MJ, Post M, van der Schoot PJ, Hoogerbrugge JW, de Winter JP, Uilenbroek JT, Karels B, Wilming LG, Meijers JH, et al. A novel member of the transmembrane serine/threonine kinase receptor family is specifically expressed in the gonads and in mesenchymal cells adjacent to the mullerian duct. Development. 1994;120(1):189–197. doi: 10.1242/dev.120.1.189. [DOI] [PubMed] [Google Scholar]
  5. Behringer RR, Finegold MJ, Cate RL. Mullerian-inhibiting substance function during mammalian sexual development. Cell. 1994;79(3):415–425. doi: 10.1016/0092-8674(94)90251-8. [DOI] [PubMed] [Google Scholar]
  6. Belville C, Van Vlijmen H, Ehrenfels C, Pepinsky B, Rezaie AR, Picard JY, Josso N, di Clemente N, Cate RL. Mutations of the anti-mullerian hormone gene in patients with persistent mullerian duct syndrome: biosynthesis, secretion, and processing of the abnormal proteins and analysis using a three-dimensional model. Mol Endocrinol. 2004;18 (3):708–721. doi: 10.1210/me.2003-0358. [DOI] [PubMed] [Google Scholar]
  7. Catlin EA, Tonnu VC, Ebb RG, Pacheco BA, Manganaro TF, Ezzell RM, Donahoe PK, Teixeira J. Mullerian inhibiting substance inhibits branching morphogenesis and induces apoptosis in fetal rat lung. Endocrinology. 1997;138(2):790–796. doi: 10.1210/endo.138.2.4906. [DOI] [PubMed] [Google Scholar]
  8. Clarke TR, Hoshiya Y, Yi SE, Liu X, Lyons KM, Donahoe PK. Mullerian inhibiting substance signaling uses a bone morphogenetic protein (BMP)-like pathway mediated by ALK2 and induces SMAD6 expression. Mol Endocrinol. 2001;15(6):946–959. doi: 10.1210/mend.15.6.0664. [DOI] [PubMed] [Google Scholar]
  9. Daikoku T, Cha J, Sun X, Tranguch S, Xie H, Fujita T, Hirota Y, Lydon J, DeMayo F, Maxson R, Dey SK. Conditional deletion of Msx homeobox genes in the uterus inhibits blastocyst implantation by altering uterine receptivity. Dev Cell. 2011;21(6):1014–1025. doi: 10.1016/j.devcel.2011.09.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. di Clemente N, Wilson C, Faure E, Boussin L, Carmillo P, Tizard R, Picard JY, Vigier B, Josso N, Cate R. Cloning, expression, and alternative splicing of the receptor for anti-Mullerian hormone. Mol Endocrinol. 1994;8(8):1006–1020. doi: 10.1210/mend.8.8.7997230. [DOI] [PubMed] [Google Scholar]
  11. Fujino A, Arango NA, Zhan Y, Manganaro TF, Li X, MacLaughlin DT, Donahoe PK. Cell migration and activated PI3K/AKT-directed elongation in the developing rat Mullerian duct. Dev Biol. 2009;325(2):351–362. doi: 10.1016/j.ydbio.2008.10.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gouedard L, Chen YG, Thevenet L, Racine C, Borie S, Lamarre I, Josso N, Massague J, di Clemente N. Engagement of bone morphogenetic protein type IB receptor and Smad1 signaling by anti-Mullerian hormone and its type II receptor. J Biol Chem. 2000;275(36):27973–27978. doi: 10.1074/jbc.M002704200. [DOI] [PubMed] [Google Scholar]
  13. Guioli S, Sekido R, Lovell-Badge R. The origin of the Mullerian duct in chick and mouse. Dev Biol. 2007;302(2):389–398. doi: 10.1016/j.ydbio.2006.09.046. [DOI] [PubMed] [Google Scholar]
  14. Ha Y, Tsukada A, Saito N, Zadworny D, Shimada K. Identification of differentially expressed genes involved in the regression and development of the chicken Mullerian duct. Int J Dev Biol. 2008;52(8):1135–1141. doi: 10.1387/ijdb.072441yh. [DOI] [PubMed] [Google Scholar]
  15. Hayashi A, Donahoe PK, Budzik GP, Trelstad RL. Periductal and matrix glycosaminoglycans in rat Mullerian duct development and regression. Dev Biol. 1982;92(1):16–26. doi: 10.1016/0012-1606(82)90146-4. [DOI] [PubMed] [Google Scholar]
  16. He X, Saint-Jeannet JP, Wang Y, Nathans J, Dawid I, Varmus H. A member of the Frizzled protein family mediating axis induction by Wnt-5A. Science. 1997;275(5306):1652–1654. doi: 10.1126/science.275.5306.1652. [DOI] [PubMed] [Google Scholar]
  17. Hsieh JC, Kodjabachian L, Rebbert ML, Rattner A, Smallwood PM, Samos CH, Nusse R, Dawid IB, Nathans J. A new secreted protein that binds to Wnt proteins and inhibits their activities. Nature. 1999;398(6726):431–436. doi: 10.1038/18899. [DOI] [PubMed] [Google Scholar]
  18. Jamin SP, Arango NA, Mishina Y, Hanks MC, Behringer RR. Requirement of Bmpr1a for Mullerian duct regression during male sexual development. Nat Genet. 2002;32 (3):408–410. doi: 10.1038/ng1003. [DOI] [PubMed] [Google Scholar]
  19. Josso N, Belville C, di Clemente N, Picard JY. AMH and AMH receptor defects in persistent Mullerian duct syndrome. Hum Reprod Update. 2005;11(4):351–356. doi: 10.1093/humupd/dmi014. [DOI] [PubMed] [Google Scholar]
  20. Josso N, Clemente N. Transduction pathway of anti-Mullerian hormone, a sex-specific member of the TGF-beta family. Trends Endocrinol Metab. 2003;14(2):91–97. doi: 10.1016/s1043-2760(03)00005-5. [DOI] [PubMed] [Google Scholar]
  21. Kansara M, Tsang M, Kodjabachian L, Sims NA, Trivett MK, Ehrich M, Dobrovic A, Slavin J, Choong PF, Simmons PJ, Dawid IB, Thomas DM. Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcoma, and targeted disruption accelerates osteosarcomagenesis in mice. J Clin Invest. 2009;119(4):837–851. doi: 10.1172/JCI37175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kawano Y, Kypta R. Secreted antagonists of the Wnt signalling pathway. J Cell Sci. 2003;116(Pt 13):2627–2634. doi: 10.1242/jcs.00623. [DOI] [PubMed] [Google Scholar]
  23. Keil KP, Mehta V, Branam AM, Abler LL, Buresh-Stiemke RA, Joshi PS, Schmitz CT, Marker PC, Vezina CM. Wnt Inhibitory Factor 1 (Wif1) Is Regulated by Androgens and Enhances Androgen-Dependent Prostate Development. Endocrinology. 2012 doi: 10.1210/en.2012-1564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kobayashi A, Behringer RR. Developmental genetics of the female reproductive tract in mammals. Nat Rev Genet. 2003;4(12):969–980. doi: 10.1038/nrg1225. [DOI] [PubMed] [Google Scholar]
  25. Kobayashi A, Stewart CA, Wang Y, Fujioka K, Thomas NC, Jamin SP, Behringer RR. beta-Catenin is essential for Mullerian duct regression during male sexual differentiation. Development. 2011;138(10):1967–1975. doi: 10.1242/dev.056143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kraus P, Lufkin T. Dlx homeobox gene control of mammalian limb and craniofacial development. Am J Med Genet A. 2006;140(13):1366–1374. doi: 10.1002/ajmg.a.31252. [DOI] [PubMed] [Google Scholar]
  27. Loscertales M, Mikels AJ, Hu JK, Donahoe PK, Roberts DJ. Chick pulmonary Wnt5a directs airway and vascular tubulogenesis. Development. 2008;135(7):1365–1376. doi: 10.1242/dev.010504. [DOI] [PubMed] [Google Scholar]
  28. Mericskay M, Kitajewski J, Sassoon D. Wnt5a is required for proper epithelial-mesenchymal interactions in the uterus. Development. 2004;131(9):2061–2072. doi: 10.1242/dev.01090. [DOI] [PubMed] [Google Scholar]
  29. Mikels AJ, Nusse R. Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context. PLoS Biol. 2006a;4(4):e115. doi: 10.1371/journal.pbio.0040115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mikels AJ, Nusse R. Wnts as ligands: processing, secretion and reception. Oncogene. 2006b;25(57):7461–7468. doi: 10.1038/sj.onc.1210053. [DOI] [PubMed] [Google Scholar]
  31. Miller C, Sassoon DA. Wnt-7a maintains appropriate uterine patterning during the development of the mouse female reproductive tract. Development. 1998;125(16):3201–3211. doi: 10.1242/dev.125.16.3201. [DOI] [PubMed] [Google Scholar]
  32. Mishina Y, Rey R, Finegold MJ, Matzuk MM, Josso N, Cate RL, Behringer RR. Genetic analysis of the Mullerian-inhibiting substance signal transduction pathway in mammalian sexual differentiation. Genes Dev. 1996;10(20):2577–2587. doi: 10.1101/gad.10.20.2577. [DOI] [PubMed] [Google Scholar]
  33. Mucenski ML, Nation JM, Thitoff AR, Besnard V, Xu Y, Wert SE, Harada N, Taketo MM, Stahlman MT, Whitsett JA. Beta-catenin regulates differentiation of respiratory epithelial cells in vivo. Am J Physiol Lung Cell Mol Physiol. 2005;289(6):L971–979. doi: 10.1152/ajplung.00172.2005. [DOI] [PubMed] [Google Scholar]
  34. Mucenski ML, Wert SE, Nation JM, Loudy DE, Huelsken J, Birchmeier W, Morrisey EE, Whitsett JA. beta-Catenin is required for specification of proximal/distal cell fate during lung morphogenesis. J Biol Chem. 2003;278(41):40231–40238. doi: 10.1074/jbc.M305892200. [DOI] [PubMed] [Google Scholar]
  35. Nef S, Parada LF. Cryptorchidism in mice mutant for Insl3. Nat Genet. 1999;22(3):295–299. doi: 10.1038/10364. [DOI] [PubMed] [Google Scholar]
  36. Okubo T, Hogan BL. Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm. J Biol. 2004;3(3):11. doi: 10.1186/jbiol3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Orvis GD, Behringer RR. Cellular mechanisms of Mullerian duct formation in the mouse. Dev Biol. 2007;306(2):493–504. doi: 10.1016/j.ydbio.2007.03.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Orvis GD, Jamin SP, Kwan KM, Mishina Y, Kaartinen VM, Huang S, Roberts AB, Umans L, Huylebroeck D, Zwijsen A, Wang D, Martin JF, Behringer RR. Functional redundancy of TGF-beta family type I receptors and receptor-Smads in mediating anti-Mullerian hormone-induced Mullerian duct regression in the mouse. Biol Reprod. 2008;78(6):994–1001. doi: 10.1095/biolreprod.107.066605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Parr BA, McMahon AP. Sexually dimorphic development of the mammalian reproductive tract requires Wnt-7a. Nature. 1998;395(6703):707–710. doi: 10.1038/27221. [DOI] [PubMed] [Google Scholar]
  40. Picon R. Action of the fetal testis on the development in vitro of the Mullerian ducts in the rat. Arch Anat Microsc Morphol Exp. 1969;58(1):1–19. [PubMed] [Google Scholar]
  41. Ragin RC, Donahoe PK, Kenneally MK, Ahmad MF, MacLaughlin DT. Human mullerian inhibiting substance: enhanced purification imparts biochemical stability and restores antiproliferative effects. Protein Expr Purif. 1992;3(3):236–245. doi: 10.1016/1046-5928(92)90020-w. [DOI] [PubMed] [Google Scholar]
  42. Ramachandran I, Thavathiru E, Ramalingam S, Natarajan G, Mills WK, Benbrook DM, Zuna R, Lightfoot S, Reis A, Anant S, Queimado L. Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth, invasion and angiogenesis in vivo. Oncogene. 2012;31(22):2725–2737. doi: 10.1038/onc.2011.455. [DOI] [PubMed] [Google Scholar]
  43. Roberts LM, Visser JA, Ingraham HA. Involvement of a matrix metalloproteinase in MIS-induced cell death during urogenital development. Development. 2002;129(6):1487–1496. doi: 10.1242/dev.129.6.1487. [DOI] [PubMed] [Google Scholar]
  44. Romereim SM, Dudley AT. Cell polarity: The missing link in skeletal morphogenesis? Organogenesis. 2011;7(3):217–228. doi: 10.4161/org.7.3.18583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Satokata I, Ma L, Ohshima H, Bei M, Woo I, Nishizawa K, Maeda T, Takano Y, Uchiyama M, Heaney S, Peters H, Tang Z, Maxson R, Maas R. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet. 2000;24(4):391–395. doi: 10.1038/74231. [DOI] [PubMed] [Google Scholar]
  46. Schaniel C, Sirabella D, Qiu J, Niu X, Lemischka IR, Moore KA. Wnt-inhibitory factor 1 dysregulation of the bone marrow niche exhausts hematopoietic stem cells. Blood. 2011;118(9):2420–2429. doi: 10.1182/blood-2010-09-305664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol. 2001;1:4. doi: 10.1186/1471-213X-1-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Swain A, Narvaez V, Burgoyne P, Camerino G, Lovell-Badge R. Dax1 antagonizes Sry action in mammalian sex determination. Nature. 1998;391(6669):761–767. doi: 10.1038/35799. [DOI] [PubMed] [Google Scholar]
  49. Tanwar PS, Lee HJ, Zhang L, Zukerberg LR, Taketo MM, Rueda BR, Teixeira JM. Constitutive activation of Beta-catenin in uterine stroma and smooth muscle leads to the development of mesenchymal tumors in mice. Biol Reprod. 2009;81(3):545–552. doi: 10.1095/biolreprod.108.075648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tanwar PS, Zhang L, Tanaka Y, Taketo MM, Donahoe PK, Teixeira JM. Focal Mullerian duct retention in male mice with constitutively activated beta-catenin expression in the Mullerian duct mesenchyme. Proc Natl Acad Sci U S A. 2010;107(37):16142–16147. doi: 10.1073/pnas.1011606107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Teixeira J, He WW, Shah PC, Morikawa N, Lee MM, Catlin EA, Hudson PL, Wing J, Maclaughlin DT, Donahoe PK. Developmental expression of a candidate mullerian inhibiting substance type II receptor. Endocrinology. 1996;137(1):160–165. doi: 10.1210/endo.137.1.8536608. [DOI] [PubMed] [Google Scholar]
  52. Teixeira J, Maheswaran S, Donahoe PK. Mullerian inhibiting substance: an instructive developmental hormone with diagnostic and possible therapeutic applications. Endocr Rev. 2001;22(5):657–674. doi: 10.1210/edrv.22.5.0445. [DOI] [PubMed] [Google Scholar]
  53. Trelstad RL, Hayashi A, Hayashi K, Donahoe PK. The epithelial-mesenchymal interface of the male rate Mullerian duct: loss of basement membrane integrity and ductal regression. Dev Biol. 1982;92(1):27–40. doi: 10.1016/0012-1606(82)90147-6. [DOI] [PubMed] [Google Scholar]
  54. Tsuji M, Shima H, Yonemura CY, Brody J, Donahoe PK, Cunha GR. Effect of human recombinant mullerian inhibiting substance on isolated epithelial and mesenchymal cells during mullerian duct regression in the rat. Endocrinology. 1992;131(3):1481–1488. doi: 10.1210/endo.131.3.1505479. [DOI] [PubMed] [Google Scholar]
  55. van Amerongen R, Fuerer C, Mizutani M, Nusse R. Wnt5a can both activate and repress Wnt/beta-catenin signaling during mouse embryonic development. Dev Biol. 2012;369(1):101–114. doi: 10.1016/j.ydbio.2012.06.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Visser JA, Olaso R, Verhoef-Post M, Kramer P, Themmen AP, Ingraham HA. The serine/threonine transmembrane receptor ALK2 mediates Mullerian inhibiting substance signaling. Mol Endocrinol. 2001;15(6):936–945. doi: 10.1210/mend.15.6.0645. [DOI] [PubMed] [Google Scholar]
  57. Xu B, Chen C, Chen H, Zheng SG, Bringas P, Jr, Xu M, Zhou X, Chen D, Umans L, Zwijsen A, Shi W. Smad1 and its target gene Wif1 coordinate BMP and Wnt signaling activities to regulate fetal lung development. Development. 2011;138(5):925–935. doi: 10.1242/dev.062687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yin Y, Lin C, Ma L. MSX2 promotes vaginal epithelial differentiation and wolffian duct regression and dampens the vaginal response to diethylstilbestrol. Mol Endocrinol. 2006;20(7):1535–1546. doi: 10.1210/me.2005-0451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zhan Y, Fujino A, MacLaughlin DT, Manganaro TF, Szotek PP, Arango NA, Teixeira J, Donahoe PK. Mullerian inhibiting substance regulates its receptor/SMAD signaling and causes mesenchymal transition of the coelomic epithelial cells early in Mullerian duct regression. Development. 2006;133(12):2359–2369. doi: 10.1242/dev.02383. [DOI] [PubMed] [Google Scholar]
  60. Zimmermann S, Steding G, Emmen JM, Brinkmann AO, Nayernia K, Holstein AF, Engel W, Adham IM. Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. Mol Endocrinol. 1999;13(5):681–691. doi: 10.1210/mend.13.5.0272. [DOI] [PubMed] [Google Scholar]

RESOURCES