Müllerian Inhibiting Substance Signaling Uses a Bone Morphogenetic Protein (BMP)-Like Pathway Mediated by ALK2 and Induces Smad6 Expression

Trent R. Clarke, Yasunori Hoshiya, Soyun E. Yi, Xiaohong Liu, Karen M. Lyons and Patricia K. Donahoe

Pediatric Surgical Research Laboratories (T.R.C., Y.H., X.L., P.K.D.) Department of Surgery Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts 02114
Department of Orthopaedic Surgery (S.E.Y., K.M.L.) Department of Molecular, Cellular and Developmental Biology Department of Biological Chemistry University of California, Los Angeles, California 90095


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Signal reception of Müllerian inhibiting substance (MIS) in the mesenchyme around the embryonic Müllerian duct in the male is essential for regression of the duct. Deficiency of MIS or of the MIS type II receptor, MISRII, results in abnormal reproductive development in the male due to the maintenance of the duct. MIS is a member of the transforming growth factor-ß (TGFß) superfamily of secreted protein hormones that signal through receptor complexes of type I and type II serine/threonine kinase receptors. To investigate candidate MIS type I receptors, we examined reporter construct activation by MIS. The bone morphogenetic protein (BMP)-responsive Tlx2 and Xvent2 promoter-driven reporter constructs were stimulated by MIS but the TGFß/activin-induced p3TP-lux or CAGA-luc reporter constructs were not. The induction of Tlx2-luc was dependent upon the kinase activity of MISRII and was blocked by a dominant negative truncated ALK2 (tALK2) receptor but not by truncated forms of the other BMP type I receptors ALK1, ALK3, or ALK6. MIS induced activation of a Gal4DBD-Smad1 but not a Gal4DBD-Smad2 fusion protein. This activation could also be blocked by tALK2. The BMP-induced inhibitory Smad, Smad6, was up-regulated by MIS endogenously in Leydig cell-derived lines and is expressed in male but not female Müllerian duct mesenchyme. ALK6 has been shown to function as an MIS type I receptor. Investigation of the pattern of ALK2, MISRII, and ALK6 in the developing urogenital system demonstrated overlapping expression of ALK2 and MISRII in the mesenchyme surrounding the duct while ALK6 was observed only in the epithelium. Examination of ALK6 -/- male animals revealed no defect in duct regression. The reporter construct analysis, pattern of expression of the receptors, and analysis of ALK6-deficient animals suggest that ALK2 is the MIS type I receptor involved in Müllerian duct regression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Müllerian inhibiting substance (MIS) is a developmentally regulated protein hormone that belongs to the transforming growth factor-ß (TGFß) superfamily of extracellular ligands (1, 2). MIS, also known as anti-Müllerian hormone, is essential for the proper primary organization of internal reproductive structures of male mammals (3, 4, 5). The developing mammalian reproductive system is initially identical in males and females with both sexes having two sets of ducts in the mesonephros, the Müllerian and Wolffian ducts, and bipotential gonads. In males, differentiation of the gonad into a testis is initiated by SRY and results in the differentiation of Sertoli and Leydig cells, which produce the hormones MIS and testosterone, respectively. Expression of MIS is necessary for regression of the Müllerian duct, and synthesis of testosterone is essential for the maintenance of the Wolffian duct, which develops into the vas deferens and epididymis (3). In females, the absence of MIS allows the maintenance of the Müllerian duct, which develops into the fallopian tubes, uterus, and upper vagina. Lack of testosterone support in females results in the degeneration of the Wolffian duct. MIS is also important for male fecundity and length of female reproductive life span in mice (6, 7). Most MIS-deficient males are sterile due to an anatomical block to sperm release but they still produce functional sperm (6). MIS null females exhibit normal fertility but with abnormal follicle recruitment. This results in higher numbers of primary follicles, less primordial follicles, and a greater number of total follicles undergoing atresia during the lifespan of the animal (7).

MIS is a divergent member of the TGFß superfamily that does not fall neatly into the subfamilies of activins, TGFßs, or bone morphogenetic proteins (BMPs)/growth and differentiation factors (GDFs). Many reports have characterized the signaling mechanism of TGFß superfamily members and have shown that a general paradigm is conserved (reviewed in Ref. 8). Ligand binds to or induces the formation of a receptor complex consisting of type I and type II receptors. The type II receptors are constitutively active serine/threonine kinases which, when complexed with ligand and type I receptor, phosphorylate a motif of the type I receptors called the GS box (for its Gly-Ser repeats) (8). This activates type I receptor serine/threonine kinase activity and allows the type I receptors to be recognized by pathway-regulated Smad proteins (R-Smads) (9). The type I receptors phosphorylate the R-Smads, which in turn dissociate from the receptor complex and associate with the coactivator Smad, Smad4. This heteromeric Smad complex translocates to the nucleus and modulates gene transcription by affecting transcription factor complexes at regulated promoters (9). Gene regulation seems to involve many mechanisms, the simplest of which is the direct binding of the Smads to DNA and functioning as transcriptional activators (10). Other described mechanisms include binding to transcription factor complexes through protein-protein interactions, recruitment of transcriptional coactivators or corepressors, blocking or supplanting other transcription factors that have overlapping binding sites with the Smads, and increasing the degradation of transcriptional repressors or other signaling molecules (10).

Five type II receptors have been identified in mammals. TßRII is used for TGFß signaling, ActRIIA and ActRIIB for activin signaling, BMPRII as well as ActRIIA and ActRIIB for BMP/GDF signaling, and MISRII for MIS signaling (8). Seven type I receptors have been identified in mammals. Activin receptor like kinase 1 (ALK1) and ALK5 (TßRI) are activated by TGFß, while ALK4 (ActRIB) is used by activin. ALK7 is an orphan type I receptor whose physiological ligand has not yet been determined. ALK1, ALK2 (ActRIA), ALK3 (BMPRIA), and ALK6 (BMPRIB) are activated by various BMP ligands (8); however, the relative abilities of different BMP/GDF ligands to activate specific ALKs 1, 2, 3, and 6 are not completely understood. The identity of the MIS type I receptor and the Smad-mediated downstream pathway activated by MIS is the topic of this study.

Smad proteins are a family of intracellular signal mediators that fall into three broad categories consisting of R-Smads, coactivator Smads, and inhibitory Smads (I-Smads). R-Smads are described above, and the only identified mammalian coactivator Smad, Smad4, complexes with activated R-Smads. This causes translocation into the nucleus to participate in gene regulation. Inhibitory Smads (I-Smads) inhibit the signaling pathways by binding and sequestering activated type I receptors or activated R-Smads, thus preventing signal transduction (8). The I-Smads are often up-regulated by the specific pathways that they inhibit, thus providing a negative feedback mechanism ensuring that the magnitude and duration of the intracellular response correlates with the concentration and continuing presence of the extracellular ligand (11, 12, 13). This effect is due to direct activation of the promoters of the I-Smads by activated R-Smad/Smad4 heteromeric complexes (14, 15).

Smad2 and Smad3 are R-Smads used by both the TGFß and activin pathways, whereas the other R-Smads, Smad1, Smad5, and Smad8, are activated by BMP/GDF pathways (8). No differential activation between the BMP-specific Smads has been demonstrated downstream of different BMP ligands or type I receptors (ALKs 1, 2, 3, and 6), although they may play different roles during development due to varied patterns of expression in different tissues or, perhaps, to intrinsic functional differences (16). Smad5 expression has been shown near the newly formed Müllerian duct in day 12 mouse embryos (17). The I-Smad, Smad7, is induced by TGFß and activin signaling (11), while the expression of the other I-Smad, Smad6, has been demonstrated in response to BMP signaling (18).

Previously, an orphan type II receptor that had an expression pattern consistent with it being the MIS type II receptor was identified (19, 20, 21). Its identity as the MIS type II receptor was unequivocally verified by generating homozygous knockout mice that exhibited phenotypes identical to homozygous MIS ligand knockout mice (22).

Previous biochemical analyses identified ALK6 as an MIS type I receptor (23). Here we show that ALK2 can function as an MIS type I receptor, that its pattern of expression in the Müllerian duct is consistent with this role in vivo, and that male mice in which ALK6 has been homozygously inactivated exhibit normal Müllerian duct regression. Based on these data, we propose that ALK2 is likely to be a physiologically relevant type I receptor for Müllerian duct regression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Smad6 Induction by MIS in Leydig Cell Tumor Cell Lines
Since TGFß and activin signaling has been shown to induce the expression of Smad7, and BMP signaling has been shown to induce the expression of Smad6, we investigated which I-Smad (if any) is induced by MIS, which could provide insight into its signaling pathway and the identity of the MIS type I receptor. Leydig cells have been shown to respond to MIS (24, 25, 26, 27, 28, 29), so we examined the induction of Smad6 and Smad7 in the MA-10 and R2C Leydig cell-derived immortalized cell lines (28). Treatment of the mouse MA-10 Leydig cell tumor cell line with 100 nM MIS increased expression of Smad6 and, to a much lesser extent, Smad7 (Fig. 1AGo). Robust induction of Smad6 over a low basal level was observed by 2 h (5-fold by densitometry scans), reached a maximum at 6 h (8-fold), and decreased by 10 h (6-fold), whereas a slight induction of Smad7 was observed by 2 h and remained constant for the next 8 h. Examination of earlier timepoints revealed that the first detectable increase in Smad6 expression by Northern analysis was at 90 min after MIS treatment (data not shown). The Smad6 and Smad7 probes used in Fig. 1Go were of similar specific activity, suggesting that the induction of Smad7 in MA-10 cells never reaches even the basal level of observed Smad6 expression. To determine whether this induction could be repeated in a different species, the rat R2C Leydig tumor cell line was investigated. Figure 1BGo demonstrates that MIS treatment induces Smad6 and slightly increases Smad7 expression in R2C cells but, interestingly, with a different time course than in MA-10 cells. Smad6 increased over a basal level by 6 h, was maximal at 12 h, and was beginning to decrease by 24 h. Smad7 expression peaked slightly over basal at 12 h. It is unclear whether the different time courses for Smad induction and magnitude of the Smad6 induction in the two Leydig tumor cell lines is due to the species difference, expression levels of the receptors, or some unknown factor. To examine whether the induction of the Smad mRNAs by MIS was a direct effect or required the induction of another protein, cycloheximide (CHX) treatment was used to block new protein synthesis. Smad6 and Smad7 expression was similarly induced in the presence and absence of 10 µg/ml of CHX, demonstrating that the increase in Smad6 and Smad7 mRNA was directly regulated downstream of MIS receptor activation (Fig. 1CGo). Smad6 was induced 7-fold in the presence of CHX and 5-fold in the absence of CHX after 2.5 h of MIS treatment. Smad7 exhibited an increased level of mRNA due to the treatment of CHX alone, which was additive with the MIS induction. This increase with CHX treatment is often seen with highly labile mRNAs. This may indicate that the rate of degradation of Smad7 message is slowed by CHX blocking production of short-lived proteins important for destabilization of the mRNA (11). This would result in a greater steady-state level of the message in the presence of protein synthesis inhibitors.



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Figure 1. Smad6 Induction by MIS in Leydig Cells

Northern blots of polyA+ mRNA generated from serum-starved (1/20 normal serum) MA-10 (A and C) or R2C cells (B) treated with or without MIS were hybridized with Smad6 and Smad7 probes. The lower panels show the same blots probed for ß-actin mRNA. A, Blot of 5 µg of mRNA from serum starved (15 h) MA-10 cells treated with or without 100 nM MIS for 0–10 h demonstrating that Smad6 and, to a lesser extent, Smad7 mRNAs are induced by MIS. B, Blot of 3.8 µg of polyA+ mRNA from serum-starved (15 h) R2C cells treated with 100 nM MIS for 0–24 h showing that Smad6 is induced in this Leydig cell line as well. C, Blot of 5 µg of polyA+ mRNA from serum-starved (18.5 h) MA-10 cells pretreated for 30 min with or without 10 µg/mg CHX and then incubated with or without 188 nM MIS for 2.5 or 5 h. Smad6 and Smad7 induction by MIS does not require protein synthesis, demonstrating that their induction is a direct effect of MIS signaling. These experiments were performed on serum-starved cells but identical results were obtained in the presence of serum (data not shown).

 
Smad6 Expression in Male Müllerian Duct Mesenchyme
To investigate whether the induction of Smad6 by MIS observed in the Leydig tumor cell lines also occurred in the Müllerian duct in vivo, whole-mount in situ hybridization was performed. Smad6 expression is observed in the mesenchyme surrounding the regressing Müllerian duct in male rat embryos at 14.5 days post coitum (E14.5) (Fig. 2Go, A and B) and with greater intensity at 15.5 days post coitum (E15.5) (Fig. 2Go, E and F). Smad6 expression was not observed in the mesenchyme surrounding the Müllerian duct in female rat embryos at E14.5 or E15.5 (Fig. 2Go, C, D, G, and H). The localization of this expression is similar to that of MISRII (see Fig. 6Go, I–L); however, MISRII is expressed in both male and female embryos. This pattern is consistent with Smad6 being induced by MIS in vivo in male embryos. The greater induction of Smad6 by MIS compared with that of Smad7 in Leydig cell-derived lines and the male-specific expression of Smad6 at the site of MIS action suggests that MIS signaling is more similar to the BMP pathway than the TGFß/activin pathway.



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Figure 2. Smad6 Induction in the Male Müllerian Duct Mesenchyme

In situ hybridization of Smad6 RNA was performed on E14.5 (A–D) and E15.5 (E–H) male (A, B, E, and F) and female (C, D, G, and H) embryos. The ventral body wall and viscera were removed from the embryos. They are oriented ventral side up, anterior to the upper left. The dotted line in the whole-mount pictures (A, C, E, and G) represents the approximate plane from which the sections (B, D, F, and H) were taken. The sections were redetected with BM-Purple for 24 h at 25 C. The black arrowheads point to the Müllerian duct in the pictures of the embryos. Note the equivalent expression of Smad6 in both male (E) and female (G) kidney at E15.5, presumably due to induction by another BMP-like signaling pathway, demonstrating the integrity of the RNA in the female embryo. These embryos are representative of five different embryos examined at each time point for both sexes. W, Wolffian duct; M, Müllerian duct; K, kidney.

 


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Figure 6. ALK2, ALK6, and MISRII Expression in the Müllerian Duct Mesenchyme

In situ hybridization of ALK2 (A–D), ALK6 (E–H), and MISRII (I–L) mRNA was performed on E15.5 male (A, B, E, F, I, and J) and female (C, D, G, H, K, and L) embryos. The ventral body wall and viscera were removed from the embryos. They are oriented ventral side toward the camera, anterior to the top. The dotted line in the whole-mount pictures (A, C, E, G, I, and K) represents the approximate plane from which the sections (B, D, F, H, J, and L) were taken. B and D, The ALK2 sections were redetected with BM-Purple for 20 h at 25 C. F and H, The ALK6 sections were redetected for 50 h. J and L, The MISRII sections did not require redetection. The black arrowheads point to the Müllerian duct in panels A, E, I, and K. The gray arrowheads in panels A, E, I, and K point to gonadal expression of the receptors. The white arrowheads in panels C and G point to nonspecific background staining between the ovary and mesonephros. W, Wolffian duct; M, Müllerian duct. Note the expression of ALK2 in the mesenchyme around the Müllerian duct and the expression of ALK6 in the epithelium of the Müllerian and Wolffian ducts. Expression of the MIS type II receptor overlaps the expression of ALK2 and Smad6 in the Müllerian mesenchyme. These embryos are representative of three different embryos examined for both sexes with each probe. ALK6 is not required for Müllerian duct regression in males. The internal reproductive structures of an adult wild type (M) and BMPRIB (ALK6) homozygous null male mouse (N). Note the absence of any retained Müllerian-derived structures such as are observed in MIS and MIS type II receptor homozygous knockout mice. T, Testis; e, epididymis; v, vas deferens; p, prostate; sv, seminal vesicle.

 
MIS Activation of BMP-Responsive Promoters
To investigate further whether MIS signaling shares similarities with BMP signaling, transient transfection assays with promoters regulated by the TGFß, activin, or BMP pathways were analyzed for their regulation by MIS. The TGFß- and activin-responsive reporter constructs 3TP-lux and CAGA-luc (30, 31), and the BMP-inducible reporter constructs Tlx2-luc and Xvent2-luc (32, 33) did not show regulation by MIS in MA-10, COS7, or Mv1Lu cells (data not shown). While the 3TP-lux and CAGA-luc reporters utilize artificial promoters that are regulated by TGFß and activin in a variety of cell lines including Mv1Lu cells, the Tlx2 and Xvent2 constructs employ endogenous promoters. Both Tlx2 and Xvent2 are expressed in very restricted domains exclusively in the early embryo (33, 34); hence, it is likely that several tissue-specific transcription factors are required for these promoters in addition to BMP-regulated transcription factors and/or Smads. Since previous analyses of the Tlx2 and Xvent2 reporter constructs were performed in the embryonic carcinoma-derived P19 cell line, the regulation of the reporters by MIS in these cells was investigated. The activity of the Tlx2 reporter construct is not affected by MIS in P19 cells unless an expression construct for MISRII is also transfected, indicating that these cells do not express endogenous MISRII (Fig. 3AGo). When MISRII is transfected, the Tlx2 reporter construct exhibited 8- to 12-fold induction by MIS (Fig. 3AGo). The Xvent2 reporter construct also demonstrated an MISRII-dependent regulation by MIS in P19 cells, although the fold induction was less, approximately 3- to 5-fold (Fig. 3AGo). To examine specificity of the MISRII-mediated MIS induction of the reporters, other type II receptors were examined for their ability to transduce MIS-mediated reporter activation. When TßRII and BMPRII were expressed, treatment with MIS was unable to activate Tlx2 reporter expression (Fig. 3BGo). To demonstrate that the activation of the Tlx2 promoter was dependent upon the kinase activity of MISRII, a conserved lysine (K228) in the ATP binding region of the kinase domain was mutated to an arginine (K228R). This should make a form of MISRII which, based on analogy with other kinase domains, should be unable to bind ATP and thus have no kinase activity. Overexpression of MISRII(K228R) does not allow P19 cells to respond to MIS, demonstrating that the Tlx2 promoter induction observed with MIS is dependent upon MISRII serine/threonine kinase activity (Fig. 3CGo). MISRII-transfected P19 cells were treated with different MIS concentrations to examine the sensitivity of the Tlx2 promoter to its activation. The Tlx2 reporter construct was maximally activated at concentrations as low as 10 nM MIS and still exhibited 80% activation at 1 nM MIS (Fig. 3DGo). No activation of the 3TP-lux and CAGA-luc reporters by MIS was observed in MISRII-transfected P19 cells (data not shown). The induction of the Tlx2-luc and Xvent2-luc reporter constructs by MIS and the lack of MIS-mediated activation of the 3TP-lux and CAGA-luc reporters further confirms that MIS signaling is more similar to BMP signaling than to either TGFß or activin signaling.



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Figure 3. BMP-Responsive Promoter Induction by MIS

P19 cells were transfected with 400 ng of the Tlx2-luc or Xvent2-luc firefly luciferase reporter constructs, various expression constructs, and 100 ng of the thymidine kinase promoter-driven Renilla luciferase. Twenty four hours after transfection the cells were treated with or without MIS; 24 h later the lysates were assayed for firefly and Renilla luciferase. The results are presented as relative luciferase units (R.L.U.) of the mean ± SD of triplicate samples for each representative experiment. A, MISRII mediated activation of Tlx2-luc and Xvent2-luc reporter constructs. Three hundred nanograms of an MISRII-expressing or empty expression vector and the Tlx2-luc or Xvent2-luc reporter constructs were transfected into P19 cells. The Tlx2 and Xvent2 reporter constructs are induced by 100 nM MIS in P19 cells only when transfected with the MIS type II receptor. B, Type II receptor specificity of MIS-mediated reporter activation. P19 cells were transfected with Tlx2-luc and 300 ng of type II receptor expression construct. Tlx2-luc is activated by 100 nM MIS only in MISRII-expressing P19 cells, not in cells transfected with TßRII or BMPRII expression constructs. C, Tlx2 reporter activation by MIS depends on MISRII kinase activity. P19 cells were transfected with Tlx2-luc and 300 ng of expression constructs for MISRII or the ATP binding site mutant receptor MISRII(K228R); 100 nM MIS induction of Tlx2-luc was only mediated by MISRII with an intact kinase domain. D, Response of the Tlx2 promoter to exposure to different concentrations of MIS. P19 cells were transfected with 300 ng of the MISRII expression vector and treated with the indicated concentrations of MIS. Tlx2 promoter activation was still maximal with 10 nM MIS. E, Effect of dominant negative truncated receptors on Tlx2 promoter induction by MIS. Five hundred nanograms of expression constructs for forms of ALK1, ALK2, ALK3, and ALK6, which terminate 20–24 amino acids beyond the transmembrane domain, and 50 ng of the MISRII expression construct were transfected into P19 cells along with Tlx2-luc and treated with 100 nM MIS. Only truncated ALK2 was able to block reporter activation.

 
Dominant Negative ALK2 Blocks Tlx2 Promoter Activation by MIS
Because the Tlx2 promoter reporter construct responds to low concentrations of MIS with robust activation, it was employed to investigate which type I receptor(s) MIS uses for signal transduction. Because ALK1, ALK2, ALK3, and ALK6 are activated by BMP signaling, these type I receptors were investigated as candidate MIS type I receptor(s). The fact that MISRII-transfected P19 cells can support MIS signaling suggested that these cells must endogenously express at least one MIS type I receptor(s). To examine which type I receptor(s) MIS is using, we constructed truncated forms of ALK1 (tALK1), ALK2 (tALK2), ALK3 (tALK3), and ALK6 (tALK6), which terminate 20–24 amino acids after the transmembrane domain, and replaced the GS box and kinase domain with a hemagglutinin (HA) epitope tag. The truncated receptors should act as dominant negative forms because they are able to bind and sequester ligand but, lacking the kinase domain, cannot initiate downstream signaling. The constructs were transfected into COS7 cells, and lysates were prepared and analyzed by Western blotting with {alpha}HA antibody to verify that the receptors were truncated and expressed (data not shown). When expressed along with MISRII in P19 cells, tALK2 completely blocked MIS-mediated Tlx2 promoter activation. However, tALK1, tALK3, and tALK6 had little effect (Fig. 3EGo). This indicates that while ALK1, ALK3, and ALK6 are dispensable for MIS signaling in P19 cells, ALK2 is essential for MIS-mediated Tlx2 promoter activation. Taken together, these experiments suggest that MIS signaling is dependent upon the kinase activity of MISRII and ALK2.

MIS Activates Smad1
Knowing that BMP signaling activates Smad1 (35), we investigated the ability of MIS to activate Smad1. It has previously been shown that a Gal4-Smad1 fusion protein with the DNA binding domain of the Gal4 transcription factor (Gal4DBD) fused to the N-terminus of Smad1 can be used to measure BMP signaling (35). When the Gal4-Smad1 fusion protein is phosphorylated and translocated to the nucleus, it activates a reporter construct of multiple Gal4 binding sites driving the expression of a luciferase gene. When cotransfected with MISRII in P19 cells, this Gal4 reporter system gives a small, reproducible response to MIS (Fig. 4AGo). Cotransfection of a constitutively active form of ALK2, ALK2(Q207D), with Gal4-Smad1 produces a robust activation of the Gal4 binding site reporter construct (Fig. 4AGo). In the absence of the Gal4-Smad1 fusion protein, ALK2(Q207D) cannot induce the reporter construct, showing the specificity of the assay (Fig. 4AGo). The Gal4-Smad1-mediated induction of the promoter by MIS was abrogated when truncated ALK2 was included in the transfection (Fig. 4BGo). Similar Gal4-Smad2 and Gal4-Smad3 fusion proteins have been used to assay TGFß signaling (36, 37). When the Gal4-Smad2 fusion protein was used, no induction of the reporter by MIS was observed (data not shown). These experiments suggest that MIS can induce the activation of Smad1 via the function of MISRII and ALK2.



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Figure 4. Smad1 Activation by MIS

P19 cells were transfected with 400 ng of the pG5E1b-luc firefly luciferase reporter construct, the expression of which is driven by five Gal4 binding sites, various expression constructs, and 100 ng of the thymidine kinase promoter-driven Renilla luciferase. Twenty four hours after transfection the cells were treated with or without 100 nM MIS; 24 h later the lysates were assayed for firefly and Renilla luciferase. The results are presented as relative luciferase units (R.L.U.) of the mean ± SD of triplicate samples for each representative experiment. A, Activation of a Gal4 responsive promoter by MIS-mediated activation of a Gal4DBD-Smad1 fusion protein. P19 cells were transfected with pG5E1b-luc, 200 ng of an expression construct for a fusion protein consisting of the DNA binding domain of Gal4 fused to the N terminus of Smad1, and 300 ng of expression constructs for MISRII or ALK2Q207D, the constitutively active form of ALK2. MIS treatment of the cells expressing MISRII increased reporter activity but less than the activation seen with the constitutively active form of ALK2. B, Truncated ALK2 blocks the MIS-induced activation of pG5E1b-luc by Gal4-Smad1. P19 cells were transfected with pG5E1b, 50 ng of the MISRII and Gal4-Smad1 expression constructs, and 400 ng of the truncated ALK2 expression construct or parental vector. MIS treatment was not able to induce activation of pG5E1b by Gal4-Smad1 when truncated ALK2 was overexpressed.

 
Expression of BMP-Regulated R-Smads during Müllerian Duct Regression
Although we had observed Smad1 activation in tissue culture, it did not necessarily imply a role for Smad1 in MIS signaling, since ALK2 has been shown to activate Smad1, Smad5, as well as Smad8 in overexpression systems (38, 39, 40). To identify which of the BMP-regulated Smads are possible physiological mediators of MIS signaling, we investigated the pattern of expression of these Smads in the urogenital ridge at the time of Müllerian duct regression (E15.5). Smad1 shows robust expression in the mesenchyme of male and female mesonephros in the regions surrounding the Müllerian duct and adjacent to the Wolffian duct (Fig. 5Go, A, D, and G). Smad5 is expressed at substantially lower levels than that observed with Smad1 in both male and female mesenchyme (Fig. 5Go, B, E, and H). Smad8 exhibited low levels of expression throughout the mesenchyme of the mesonephros in the female, but in the male, interestingly, the expression is much greater in the mesenchyme surrounding the Müllerian duct (Fig. 5Go, C, F, I, and J). These results give the impression that Smad1 and Smad8 are most likely to be the mediators of MIS signaling.



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Figure 5. BMP-Regulated R-Smad Expression in the Müllerian Duct Mesenchyme at the Time of Duct Regression

In situ hybridization of Smad1 (A, D, and G), Smad5 (B, E, and H), and Smad8 (C, F, I, and J) mRNA was performed on E15.5 male (A–C and G–I) and female (D–F and J) embryos. The ventral body wall and viscera were removed from the embryos. The embryos in the whole-mount images (A–F) are oriented ventral side toward the camera, anterior to the top. The dotted line in whole-mount pictures A, B, C, and F represents the approximate plane from which the sections G, H, I, and J were taken, respectively. The sections were redetected with BM-Purple for 42 h at 25 C. The black arrowheads point to the Müllerian duct in panels A–F. The gray arrowheads in panels A–F point to gonadal expression of the Smads. W, Wolffian duct; M, Müllerian duct. Note the higher expression of Smad1 compared with Smad5 and the greater expression of Smad8 in the mesenchyme around the male compared with the female Müllerian duct. Expression of the MIS type II receptor overlaps the expression of all three Smads in the Müllerian mesenchyme (see Fig. 6Go, I–L). These embryos are representative of at least six different embryos examined for both sexes with each probe.

 
Colocalization of ALK2 and MISRII in the Developing Urogenital System
Because both ALK2 (this study) and ALK6 (23) have been implicated in MIS signal transduction, we investigated the expression of ALK2 and ALK6 in the Müllerian duct. ALK2 is observed in male and, to a lesser extent, in female rat embryos at E15.5 in the mesenchyme of the Müllerian duct (Fig. 6Go, A–D). In contrast, ALK6 is observed in male and female embryos at E15.5 in the epithelium of the Müllerian and Wolffian ducts, but not in the mesenchyme surrounding the ducts (Fig. 6Go, E–H). When compared with the expression of MISRII in male and female embryos at E15.5 (Fig. 6Go, I–L), ALK2 expression overlaps that of MISRII, while ALK6 is expressed in the adjacent epithelial layer. It has been demonstrated previously that the mesenchyme around the Müllerian duct, but not the epithelium of the duct, is the site of MIS action (41, 42, 43). The pattern of expression exhibited by ALK2 suggests that it is more likely the physiologically relevant type I receptor for Müllerian duct regression.

ALK6 Null Male Mice Do Not Have Retained Müllerian Ducts
Recently, we generated ALK6 knockout mice (44). Upon inspection of male mice homozygous for the inactive allele of ALK6, we discovered that they do not exhibit any evidence of retained Müllerian duct structures (Fig. 5Go, compare panel N to panel M). They do exhibit a defect in seminal fluid production that is reflected in the deflated and discolored appearance of the seminal vesicles (Fig. 5NGo). This defect is currently being investigated. Female ALK6-deficient mice have oviducts and uteri (data not shown), indicating that Müllerian duct structures in these mice form and are capable of differentiating into adult structures. This argues against a defect in Müllerian duct formation as is observed in Wnt4-deficient animals (45) in which both females and males lack Müllerian ducts. These observations suggest that ALK6 is not essential for Müllerian duct formation or regression. The expression data for MISRII, ALK2, and ALK6 combined with the absence of a Müllerian duct defect in ALK6-deficient male mice suggest that ALK2, not ALK6, is involved in regression of the Müllerian duct, but does not rule out a role for ALK6 as an essential MIS type I receptor in other MIS functions.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this report, we have shown that MIS can activate reporter constructs driven by the BMP-responsive promoters, Tlx2 and Xvent2, in P19 cells. This induction was dependent upon the presence of functional MISRII, because expression of a form of the receptor that has an altered ATP binding pocket, MISRII(K228R), did not respond to MIS stimulation. Previous studies analyzing BMP function in Xenopus laevis have used forms of receptors lacking the intracellular domain to act as dominant negative receptors (46). When truncated forms of ALK1, ALK2, ALK3, and ALK6 were expressed, only tALK2 blocked the activation of the Tlx2 reporter by MIS. Thus, MIS signaling in P19 cells is dependent on ALK2 while ALK1, ALK3, and ALK6 are dispensable. A previous report by Macias-Silva et al. (39) has demonstrated the presence of ALK2, ALK3, and ALK6 in P19 cells by immunoprecipitating the receptors cross-linked to radiolabeled BMP2 or BMP7 using receptor-specific antibodies. This would suggest that in these cells MIS signaling requires ALK2 even though ALK3 and ALK6 are present. The previous report by Gouédard et al. (23) indicates that ALK6 can mediate MIS function. Our results suggest that ALK2 can also mediate MIS function. Taken together, these studies may indicate that the signaling pathway used by MIS may depend on the cell type or tissue. To determine which receptor is most likely the MIS type I receptor mediating Müllerian duct regression, we analyzed the expression of ALK2 and ALK6. ALK2 was found in the mesenchyme surrounding the Müllerian duct. In contrast, ALK6 localized to the epithelium of both the Müllerian and Wolffian ducts. These results are consistent with our previous examination of ALK2 expression (47) and the previously reported expression pattern of ALK6 (48). The expression of ALK2 overlaps the expression of MISRII in the mesenchyme adjacent to the Müllerian duct. The mesenchyme of the duct has previously been identified as the site of MIS action (41, 42, 43). The epithelium is not directly responsive to MIS, but undergoes apoptosis during duct regression as a secondary effect to signal reception in the mesenchyme (41, 42, 43).

Knockout mice homozygously deficient in ALK2 and ALK6 have been generated. Male ALK6 knockout mice survive to adulthood and do not exhibit any defects in Müllerian duct regression, demonstrating that ALK6 is not essential for this process (Fig. 5Go, panel M vs. panel N). ALK2 knockout mice die before embryonic day 9.5 (49, 50), making it impossible to investigate the effect of an ALK2 null genotype on Müllerian duct regression. The production of mice with tissue-specific inactivation of ALK2 may be the most definitive way to determine whether ALK2 function is indispensable for duct regression. Despite the fact that ALK6 is not required for regression of the duct, ALK6 may be essential for other functions of MIS. MIS has been shown to decrease steroid synthesis in Leydig cells (24, 25, 26, 27, 28, 29) and to play a role in follicle recruitment (7). The existence of viable animals with homozygous deficiency of ALK6 will allow us in future studies to determine whether ALK6 plays an indispensable role in either of these functions of MIS.

Interestingly, the E15.5 male Müllerian duct mesenchyme exhibits a greater level of expression of ALK2 and Smad8 than is observed in the female. In the rat, MIS expression initiates before E13.5, so the E15.5 male rat embryos have already had considerable exposure to MIS. Whether this increased expression in the male is a consequence of ongoing MIS signaling, the presence of testosterone, or another male-specific signal is the topic of ongoing investigations. Sexual differentiation is a prototypical example of a "genetic switch" in which the outcome is normally one of two finite end states. It is tempting to speculate that up-regulation of ALK2 and Smad8 in response to MIS generates a feed-forward mechanism sensitizing the tissue such that once the regression of the Müllerian duct has begun it is more likely to proceed to completion. On the other hand, if the greater expression of ALK2 and Smad8 in the male is due to testosterone or another testicular signal, it would suggest a new dimension to interactions between testicular hormones.

Many BMP ligands have been demonstrated to activate Smad1. MIS was shown to induce a Gal4 reporter construct by activating a Gal4DBD-Smad1 fusion protein. This activation was blocked with truncated ALK2, suggesting that the induction of the reporter was dependent upon ALK2 as well as MISRII. The activation of Smad1 by MIS is consistent with the report by Gouédard et al. (23) in which they demonstrated phosphorylation of Smad1 by MIS signaling with a phospho-specific Smad1 antibody. We also observed activation of the Gal4DBD-Smad1 fusion protein by constitutively active ALK2, which is consistent with the reports by Macias-Silva et al. (39) and Chen and Massague (40) in which they demonstrate that ALK2 can phosphorylate Smad1. ALK2 is used by BMPs also, most notably BMP7 (39). MIS shares with some BMP/GDFs the roles of developmental tissue remodeling through apoptosis and epithelial-mesenchymal transformation (42, 43, 51, 52, 53, 54, 55, 56, 57). Other BMP/GDF ligands, including BMP8A, BMP8B, GDF9, and BMP15, have also been shown to have important roles in reproductive function. Deficiency of BMP8B or BMP8A causes defects in initiation or maintenance of spermatogenesis, respectively (58, 59), GDF9 null females have defective follicle maturation resulting in sterility (60), and homozygous deficiency of BMP15 results in infertility (61, 62). Whether ALK2 is used in these other BMP signaling pathways impacting reproduction is an interesting question that will need to be addressed with conditional inactivation of ALK2 in specific tissues using Cre-lox technology in mice.

Smad 5 has been demonstrated previously in the early Müllerian duct (17). In this study, in situ hybridization of embryos at a later developmental stage revealed expression of Smad1 and Smad8 in the mesenchyme around the Müllerian duct whereas Smad5 was barely detectable. The intriguing sexual dimorphism exhibited by Smad8 will be investigated in future studies to determine whether this is a direct effect of MIS signaling. Since each of these R-Smads have been shown to be activated by ALK2 (38, 39, 40), it will be interesting in the future to investigate whether they have nonredundant functions in Müllerian duct regression or whether they can functionally substitute for each other.

MIS induces Smad6 transcription strongly and, to a much lesser extent, Smad7 transcription. BMPs have previously been shown to induce expression of Smad6, and TGFß and activin have been shown to induce expression of Smad7 (11, 18). The induction of Smad6 by MIS did not require protein synthesis, indicating that Smad6 induction occurs immediately downstream of MIS function. Both MIS and BMPs induce Smad6 with a similar time course in which maximal expression was 4–6 h after exposure (18). Smad7 induction by TGFß (11), on the other hand, appears within 30 min. Despite the much longer time course observed for Smad6 induction by MIS, it appears to be an immediate early effect as well since it does not require protein synthesis. These observations are consistent with the study by Ishida et al. (15), which identified a BMP-responsive Smad binding site in the Smad6 promoter. Future studies will determine whether MIS induction of Smad6 functions through this same region. The expression of Smad6 in the mesenchyme of the male Müllerian duct and its absence in the mesenchyme of the female duct suggests that Smad6 is induced by MIS in vivo. The induction of negative regulators by signaling pathways seems to be a common paradigm. Several examples include the induction of patched in Leydig cells by desert hedgehog secreted from Sertoli cells (63), the induction of Argos by EGF signaling (64), the induction of Sprouty by FGF signaling (65), and the induction of naked cuticle by Wnt signaling (66). The possibility of the induction of the ALK2 type I receptor and the R-Smad, Smad8, by MIS signaling and the increased expression of the intracellular antagonist Smad6 by MIS suggest that even a seemingly straightforward biological pathway has robust mechanisms of ensuring appropriate response in the target tissues. In conclusion, the MIS signaling pathway appears to have many similarities with previously described BMP signaling pathways. MIS signaling activates Smad1, induces the expression of Smad6, and relies upon the kinase activity of MISRII and ALK2.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
R2C cells, P19 cells, and COS7 cells were purchased from the American Type Culture Collection (Manassas, VA). MA-10 cells were a gift from Dr. Mario Ascoli (Department of Pharmacology, The University of Iowa College of Medicine, Iowa City, IA). Chemicals, unless specified otherwise, were from Sigma (St. Louis, MO). Human MIS was prepared from stably transfected CHO cells as described previously (67).

Plasmids and Constructs
The MISRII expression construct was made by mutating the stop codon of the rat MISRII cDNA from pBS7 (21) to a BamHI site using the Quick Change mutagenesis system (Stratagene, La Jolla, CA) following manufacturer’s instructions using the primers 5'-GCAAGGCTCTGGCTCTAAGTCCGGATCCTGTAAGTGCCACTG-3' and 5'-CAGTGGCACTTACAGGATCCGGACTTAGAGCCAGAGCCTTGC-3' and subcloning into EcoRI-BamHI-digested pFLAG-CMV5a (Sigma). The MISRII(K228R) expression construct was created by changing nt 683 of the coding region from A to G, which changes lysine 228 to an arginine, by PCR-mediated mutagenesis using the primer 5'-TGGGGGGAAGGCCCTGATGGCTACC-3' and the M13 -40 vector sequencing primer (5'-GTTTTCCCAGTCACGACGTTGTA-3') to amplify the N-terminal part of the coding sequence and using the primers 5'-GGTAGCCATCAGGGCCTTCCCCCCA-3' and 5'-ACATCCGCTCTCTGGAGAGC-3' (RIIMB) to amplify the C-terminal part of the cDNA. The PCR products were mixed and reamplified using the M13 -40 and RIIMB primers to give a cDNA fragment spanning the mutation. Phosphorylated PCR product was cloned into phosphatase-treated SmaI-digested pBluescript. The region between BstXI and SfiI was verified by sequencing and subcloned into the MISRII cDNA in pUC19. This altered cDNA was inserted into the pFLAG-CMV5a expression construct as described above for MISRII.

The cDNAs for the ALK1 (R3) and ALK2 (R1) type I receptors were isolated from a rat E14.5 urogenital ridge cDNA library as described previously (47). The ALK3 and ALK6 type I receptor cDNAs were a kind gift from Dr. C. H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden).

An expression construct (pCMVHA) that contains the HA epitope tag (YPYDVPDYA) followed by a stop codon was created by annealing the oligonucleotides 5'-TCGACTATCCATATGATGTACCAGATTATGCATGAGCGGCCGCG-3' and 5'-GATCCGCGGCCGCTCATGCATAATCTGGTACATCATATGGATAG-3' and then cloning the annealed oligonucleotides into the SalI-BamHI sites of pCMV5 (68).

A SalI site was introduced into the coding sequence of the ALK1, ALK2, ALK3, and ALK6 cDNA constructs at codons 20 to 24 amino acids C-terminal to the transmembrane domain by using the Quick Change mutagenesis system using the primers 5'-GGCGAGTCCAGTGTCGACCTGAAGGCATCG-3' and 5'-CGATGCCTTCAGGTCGACACTGGACTCGCC-3' for ALK1, 5'-CTATCGAAGGGGTCGACACCACCAAC-3' and 5'-GTTGGTGGTGTCGACCCCTTCGATAG-3' for ALK2, 5'-GATTTGGAACAGGTCGACGCATTTATTCCAGTTG-3' and 5'-CAACTGGAATAAATGCGTCGACCTGTTCCAAATC-3' for ALK3, and 5'-CTGGAGCAGGTCGACACATACATTCCTCCTG-3' and 5'-CAGGAGGAATGTATGTGTCGACCTGCTCCAG-3' for ALK6.

The mutagenized cDNA fragments coding for the extracellular and transmembrane domains were subcloned into pCMVHA using EcoRI (for ALK1, ALK2, and ALK6) or HindIII (ALK3) sites in the polylinkers of the cDNA constructs and SalI. This allowed the open reading frame of the receptors to be in frame with the HA epitope tag and to terminate at the stop codon after the tag. The production of tagged, truncated receptor by the expression constructs was verified by Western blotting lysates from transfected COS7 cells following standard procedures (57).

Northern Blot Analysis
MA-10 cells were maintained in Waymouth’s MB 752/1 medium plus L-glutamine (Life Technologies, Inc., Gaithersburg, MD) with 50 µg/ml gentamycin sulfate, 20 mM HEPES, pH 7.4, and 15% horse serum (Life Technologies, Inc.). R2C cells were grown in Ham’s F10 plus L-glutamine (Mediatech, Inc., Herndon, VA) with 15% horse serum, 2.5% female FBS (Biologos, Inc., Montgomery, IL), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). mRNA was prepared from MA-10 or R2C cell cultures in T75 flasks (Corning, Inc., Corning, NY) by using Quickprep (Amersham Pharmacia Biotech, Piscataway, NJ) following the manufacturer’s protocol. Poly(A)+ mRNA from each sample was electrophoresed on 1.2% agarose-formaldehyde gels. The gels were blotted to nylon membrane (Hybond-N; Amersham Pharmacia Biotech), and the RNA was UV cross-linked. A 1.5-kb BamHI–HindIII fragment of a rat Smad6 cDNA clone isolated from a rat E14.5 urogenital ridge cDNA library, a 1.1-kb EcoRI–NotI fragment of a mouse Smad7 partial cDNA clone obtained from the IMAGE consortium (GenBank accession no. AA022262), or a 0.4-kb BamHI–HindIII fragment of human ß-actin (nt 82–463 of the coding region) was used to prepare 32P-labeled probes by the random hexamer method. The blots were prehybridized, hybridized with the probes, washed, and exposed to film as described previously (69).

Transient Transfections and Luciferase Assays
P19 cells were cultured in {alpha}MEM plus L-glutamine and nucleosides (Mediatech, Inc., Herndon, VA) with 5% female FBS (Biologos, Inc., Montgomery, IL) and 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Inc.). For transient transfections, 2 x 105 P19 cells in 9-cm2 wells were transfected using 3 µl FuGENE6 (Roche Molecular Biochemicals, Indianapolis, IN) with 400 ng of luciferase reporter constructs, various expression constructs, and 100 ng of thymidine kinase minimal promoter-driven Renilla luciferase vector as a control for transfection efficiency. The total amount of expression construct was kept constant with parental expression vector. After 24 h of incubation, the transfected cells were treated with or without MIS. After 48 h, the cells were washed two times with 2 ml of HBBS, 250 µl of passive lysis buffer was added per well, the cells were agitated for 15 min. and 20 µl were analyzed for firefly and Renilla luciferase activity by detection of chemiluminescence with luciferin substrate (Dual Luciferase kit, Promega Corp., Inc., Madison, WI) using a AutoLumat LB953 luminometer (Perkin-Elmer Corp., Norwalk, CT) and expressed as mean ± SD from triplicate transfections after normalization to Renilla luciferase activity. Each separate experiment was done at least three times.

In Situ Hybridization And Tissue Sections
Embryos were collected from timed pregnant rats (Charles River Laboratories, Inc., Wilmington, MA), after which the ventral body wall and viscera were removed and the embryos were fixed in 4% paraformaldehyde in PBS overnight. Embryos were dehydrated, treated with proteinase K, prehybridized, and hybridized as previously described (70). Probes were labeled with digoxigenin and the digoxigenin probes were identified with Fab fragments conjugated to alkaline phosphatase. BM-Purple precipitation (Roche Molecular Biochemicals) was used to detect the signal. The Smad6 probe was made by linearizing a mouse Smad6 partial cDNA clone obtained from the IMAGE consortium (GenBank accession no. W41111) and producing the antisense RNA with T3 RNA polymerase (~600 bp). The MISRII probe was made by digesting pBS7 (21) with SalI and synthesizing with T3 RNA polymerase (2 kb). The ALK2 probe was created by digesting pBS-ALK2 with HindIII and making antisense RNA with T3 RNA polymerase (1.5 kb). The ALK6 probe was made by digesting pBS-ALK6 with BamHI and synthesizing probe with T7 RNA polymerase (1 kb). The Smad1 probe was made from a linearized IMAGE consortium clone (GenBank accession no. AA177814) and producing the antisense RNA with T3 RNA polymerase (~700 bp). The ~500 bp antisense Smad5 probe was produced with T3 RNA polymerase from linearized IMAGE consortium clone (GenBank accession no. AI573395). The plasmid for the Smad8 probe was created by subcloning an RT-PCR product from rat testis RNA. The Smad8 probe was created by linearizing this plasmid and making antisense RNA with T3 RNA polymerase (~1.3 kb). To assay background signal, control hybridization was performed with sense probe, which did not show any signal.

After whole-mount in situ hybridization, embryos were equilibrated overnight in 30% sucrose in PBS, embedded in tissue freezing medium (Triangle Biomedical Sciences, Durham, NC), and cryosectioned with a thickness of 14 µm. As indicated in the figure legends, some sections were redetected with BM-Purple after sectioning to enhance signal intensity.

Analysis of Transgenic Animals
The generation of BMPRIB-/- mice has been previously reported (44). The animals were genotyped as described (44), wild-type or BMPRIB-/- adult male littermates were euthanized, and the internal reproductive system was dissected.

Experimental Animals
All animals were housed in an AAALAC-certified facility and treated according to the guidelines for animal husbandry at the Massachusetts General Hospital (Protocol no. 1999N001138) and at the University of California, Los Angeles (Protocol no. 95–018-13) and are in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Note Added in Proof
Visser et al. (71) in the preceding article (pp. 936–945) present data complementary to that presented here which also supports ALK2 mediating MIS signaling in the Müllerian duct.


    ACKNOWLEDGMENTS
 
We are grateful to Drs. J. Wrana and L. Attisano for the TßRII, BMPRII, and ALK2(Q207D) expression constructs, for the Tlx2-luc reporter construct, and for Mv1Lu cells; J. Massague for the Gal4DBD-Smad1 and Gal4DBD-Smad2 expression constructs and the Gal4-luc and 3Tp-lux reporter construct; J.-M. Gauthier for the CAGA-luc reporter construct; K. W. Y. Cho for the Xvent2-luc reporter construct; C. H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden) for the ALK3 and ALK6 cDNAs; G. Gil for the M13 human ß-actin construct; D. Russell for the pCMV5 expression vector; and to Mario Ascoli for MA-10 cells. We thank Francis H. O’Neill for technical assistance. We thank Drs. J. Lorenzen, A. Trbovich, and J. Teixeira for critical evaluation of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Patricia K. Donahoe, Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Warren 11, Boston, Massachusetts 02114. E-mail: donahoe.patricia{at}mgh.harvard.edu

This work was supported by grants from the NIH to P.K.D. (NICHD-HD-32112, NCI-CA-17393, and NICHD-HD-28138) and to K.M.L. (AR-44528) and from the American Cancer Society to T.R.C. (postdoctoral fellowship).

Received for publication February 9, 2001. Revision received March 20, 2001. Accepted for publication March 22, 2001.


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