Pretreatment of chick embryos with estrogen in ovo prevents mullerian duct regression in organ culture

Diethylstilbestrol (DES) feminizes chick embryos in ovo, preventing regression of the Mullerian ducts (MDs). The feminized testis, however, despite its ovarian-like appearance, continues to produce Mullerian inhibiting substance (MIS). This study was designed to test whether exogenous DES preserved the MD against endogenous MIS by affecting the target organ (feminization). DES (0.1 mg) was injected into the air sacs of 5-day-old chick embryos. Testes were obtained from 15-day-old chick embryos and MDs from 8-day-old chick embryos and combined in organ culture for 72 h. MD regression was examined macroscopically and microscopically after 3 days in vitro in medium without DES (group A: n = 155) and with 10(-6) M DES (group B: n = 169). Four types of coculture were set up as follows: 1) Control testis with control MD; 2) DES-treated testis with DES-treated MD; 3) Control testis with DES-treated MD; 4) DES-treated testis with control MD. In medium without DES (group A), MD regression was inhibited significantly when the duct was pretreated with DES [2) and 3) vs. 1) and 4)]. The pretreated testis produced slightly less regression [4) vs. 1)], but this was not significant. Previous studies in ovo had suggested that estrogen prevented MD regression directly as well as causing feminization of the testis, because MIS secretion was not inhibited from the feminized gonad. These results show that pretreatment of the MD with DES blocks regression in vitro by a normal testis, confirming that the primary site of action of estrogen is on the duct itself.     

Focal Müllerian duct retention in male mice with constitutively activated β-catenin expression in the Müllerian duct mesenchyme

Müllerian-inhibiting substance (MIS), which is produced by fetal Sertoli cells shortly after commitment of the bipotential gonads to testicular differentiation, causes Müllerian duct (MD) regression. In the fetal female gonads, MIS is not expressed and the MDs will differentiate into the internal female reproductive tract. We have investigated whether dysregulated β-catenin activity affects MD regression by expressing a constitutively activated nuclear form of β-catenin in the MD mesenchyme. We show that constitutively activated (CA) β-catenin causes focal retention of MD tissue in the epididymides and vasa deferentia. In adult mutant mice, the retained MD tissues express α-smooth muscle actin and desmin, which are markers for uterine differentiation. MD retention inhibited the folding complexity of the developing epididymides and usually led to obstructive azoospermia by spermatoceles. The MDs of urogenital ridges from mutant female embryos showed less regression with added MIS in organ culture compared with control MDs when analyzed by whole mount in situ hybridization for Wnt7a as a marker for the MD epithelium. CA β-catenin did not appear to affect expression of either MIS in the embryonic testes or its type II receptor (AMHR2) in the MD mesenchyme nor did it inhibit pSmad1/5/8 nuclear accumulation, suggesting that dysregulated β-catenin must inhibit MD regression independently of MIS signaling. These studies suggest that dysregulated Wnt/β-catenin signaling in the MD mesenchyme might also be a contributing factor in persistent Müllerian duct syndrome, a form of male pseudohermaphroditism, and development of spermatoceles.     

Cyclic adenosine 3',5'-monophosphate modulation of mullerian duct regression

(Bu)2cAMP inhibits the action of testicular Mullerian inhibiting substance (MIS) in vitro, but it is unknown whether the intracellular nucleotide diminishes production of MIS by the testis or interferes with its action at the Mullerian duct. When added to the 14 1/2-day old rat embryo Mullerian duct in organ culture, (Bu)2cAMP (0.1 or 1.0 mM) inhibited regression caused by biologically active exogenous MIS fractions, as well as that produced by the fetal testis (1.0 mM). Dibutyryl cyclic guanosine 3',5'-monophosphate was ineffective against exogenous MIS fractions or the fetal testis. The phosphodiesterase inhibitor, methyl-isobutyl-xanthine, had the same inhibitory effect on the MIS-fraction (0.1 or 1.0 mM), as well as against MIS secreted from the fetal testis (1.0 mM). Theophylline at 1.0 mM inhibited the action of the MIS fraction. The presence of (Bu)2cAMP in the medium was required for at least the first 24 h of the 72-h incubation to significantly inhibit MIS action. These results indicate that intracellular cAMP inhibits the action of MIS at the Mullerian duct itself by a potentially reversible change in the cells. We speculate that cAMP may act by altering the state of differentiation in the cells, perhaps by mediating phosphorylation of intracellular (and extracellular) proteins.     

Autoradiographic studies of androgen-binding sites in the rat urogenital sinus and postnatal prostate

Binding sites of [3H]testosterone and [3H]dihydrotestosterone in the rat fetal urogenital sinus and postnatal prostate and vagina grown in vitro were examined by steroid autoradiography. Distinct nuclear incorporation of both androgens appeared between 14.5 and 16.5 days of gestation in rat fetuses. Nuclear labelling in the sinus was restricted to the mesenchyme surrounding the epithelium which showed no nuclear labelling. A similar distribution of labelled cells was observed in male and female sinuses up to 18.5 days of gestation. By 20.5 days of gestation, the labelling in the ventral mesenchyme of female urogenital sinuses became less intense but persisted in the mesenchyme of the dorsal sinus wall from which the vagina is formed. In the postnatal prostate, the epithelium showed nuclear [3H]testosterone labelling at 10 days coinciding with the onset of its functional differentiation. Epithelial labelling became more intensive at 4 weeks post partum while that of the mesenchyme declined. The results suggest two phases of androgen action: formation of the prostatic buds mediated by the androgen-activated mesenchyme of the fetal urogenital sinus and the differentiation of the postnatal prostatic epithelium directly stimulated by androgens.     

Change of mosaic pattern by androgens during prostatic bud formation in XTfm/X+ heterozygous female mice

The testicular feminization (Tfm) gene, which is characterized by a deficiency in androgen receptors, is located on the X-chromosome. Using steroid autoradiography, the mosaicism of the Tfm gene has been demonstrated in the androgen target tissues of XTfm/X+ heterozygous female mouse fetuses and the effects of androgens on the mosaic pattern analysed. In the mesenchyme of urogenital sinuses of wild-type female fetuses (X+/X+), more than 95% of the cells were androgen-receptor positive (labelled with [3H]testosterone) while in that of heterozygous fetuses (XTfm/X+), only half of the cells were receptor positive (Tfm gene inactive), and receptor-positive cells and -negative cells formed small irregular patches. When the heterozygous sinuses were cultured in vitro in the presence of androgens, the sinuses underwent male sexual development and formed epithelial buds (prostate gland rudiments) projecting into the surrounding mesenchyme. Autoradiographic analysis revealed that the mosaicism of the mesenchyme disappeared around the developing epithelial buds: almost all the mesenchymal cells in close vicinity to the buds were receptor positive while in the outer layers receptor-positive and -negative cells coexisted. The proportion of receptor-positive cells was greatly increased in the mesenchyme beneath the non-budding area of the sinus epithelium. This androgen-induced increase was observed before the onset of bud formation. The results obtained in the thymidine incorporation experiments suggest that the increase of receptor-positive cells beneath the sinus epithelium might be explained by the migratory behaviour of the androgen-incorporating cells rather than by their selective proliferation.     

Endometrial cancer is a receptor-mediated target for Mullerian Inhibiting Substance

Mullerian Inhibiting Substance (MIS), a 140-kDa homodimer glycoprotein member of the TGF-beta superfamily of biological-response modifiers, causes regression of the Mullerian ducts in developing male embryos. MIS also can induce growth arrest and apoptosis in ovarian and cervical cancer cell lines. The embryonic progenitor of the ovarian and cervical epithelium is the coelomic epithelium, the same tissue that regresses under the direction of MIS in the male. The endometrium and uterus also arise from the coelomic epithelium and the Mullerian ducts. Here, we show that both normal human endometrium and endometrial cancers express the receptor for MIS and that MIS can inhibit the proliferation of a number of human endometrial cancer cell lines that express the MIS type II receptor. In the representative endometrial cancer cell line AN3CA, MIS affects the expression of key cell-cycle regulatory proteins. This work broadens the scope of tumors that MIS can potentially control and, by elucidating the MIS signaling pathway, identifies other potential avenues for intervention.     

Mullerian duct regression and antiproliferative bioactivities of mullerian inhibiting substance reside in its carboxy-terminal domain.

A 25-kilodalton dimeric carboxy-terminal fragment of the recombinant human Mullerian inhibiting substance protein (rhMIS) was produced by proteolytic cleavage with plasmin and purified by size-exclusion chromatography. The identity of the isolated dimer as the carboxy-terminal fragment was confirmed by gel electrophoresis and Western analysis. As was true of every sample of the holo molecule, all preparations of the carboxy-terminal domain of rhMIS (n = 10), when added in the 0.5-5.0 micrograms/ml range, exhibited a dose-dependent partial to complete regression of the 14.5-day fetal rat Mullerian duct in an organ culture assay. The carboxy-terminal dimer also inhibited, in a dose-dependent manner, the growth of A431 cells in monolayer cultures. Daily addition of 5, 10, or 20 micrograms carboxy-terminus for 3 days resulted in 0%, 25%, and 100% inhibition of cell proliferation, respectively. Similar and higher doses of holo rhMIS had no or inconsistent antiproliferative activity (0-34% inhibition), even though the preparations caused Mullerian duct regression. All amino-terminal fragments prepared using this separation protocol were found to be inactive in these assays. These findings suggest that the bioactivity of rhMIS as a regressor of fetal Mullerian ducts and an inhibitor of A431 cell growth resides in its carboxy-terminal domain. These results indicate that the urogenital ridge tissue, but not A431 cells in culture, may be capable of cleaving intact MIS to a biologically active conformation.     

Steroid modulation of Mullerian duct regression in the chick embryo

The recent demonstrations that Mullerian inhibiting substance (MIS) is present in embryonic chick ovaries (P. K. Hutson, H. Ikawa, and P. K. Donahoe (1981). J. Pediatr. Surg. 16, 822-827), and that exogenous diethylstilbestrol does not significantly inhibit MIS secretion from feminized testes (Hutson et al. (1982) J. Pediatr. Surg. 17, 953-959), suggest that ovarian estrogens protect the female left Mullerian duct from MIS-induced regression. The possibility exists, however, that ovarian MIS may be inactive. This study was designed to see if interference with estrogen action in ovo would allow MIS to cause regression of the female left Mullerian duct. The "antiestrogens," tamoxifen and LY117018, had little effect on the female Mullerian ducts unless given in high doses or with added testosterone (greater than 0.1 mg). Two compounds known to inhibit estrogen synthesis, aminoglutethimide and 4-hydroxyandrostenedione, had no effect on their own, even in high doses (less than 1.0 mg/egg). However, when administered together (0.5 mg each) there was significant disappearance of the lower ends of both Mullerian ducts. Norethindrone, which has been described recently as an aromatase inhibitor (Y. Osawa, C. Yarborough, and V. Osawa (1982). Science (Washington, D. C.) 215, 1249-1251) caused partial regression of the upper end of the left Mullerian duct as well as complete loss of the lower ends of both ducts in the female. These results suggest that the steroid environment is a critical factor in the response of the Mullerian ducts to MIS, and that estrogen blockage may allow endogenous MIS from the ovary to induce partial regression of the Mullerian ducts in the female chick embryo.     

Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7).

The definitive mammalian kidney forms as the result of reciprocal interactions between the ureteric bud epithelium and metanephric mesenchyme. As osteogenic protein 1 (OP-1/bone morphogenetic protein 7), a member of the TGF-beta superfamily of proteins, is expressed predominantly in the kidney, we examined its involvement during metanephric induction and kidney differentiation. We found that OP-1 mRNA is expressed in the ureteric bud epithelium before mesenchymal condensation and is subsequently seen in the condensing mesenchyme and during glomerulogenesis. Mouse kidney metanephric rudiments cultured without ureteric bud epithelium failed to undergo mesenchymal condensation and further epithelialization, while exogenously added recombinant OP-1 was able to substitute for ureteric bud epithelium in restoring the induction of metanephric mesenchyme. This OP-1-induced nephrogenic mesenchyme differentiation follows a developmental pattern similar to that observed in the presence of the spinal cord, a metanephric inducer. Blocking OP-1 activity using either neutralizing antibodies or antisense oligonucleotides in mouse embryonic day 11.5 mesenchyme, cultured in the presence of metanephric inducers or in intact embryonic day 11.5 kidney rudiment, greatly reduced metanephric differentiation. These results demonstrate that OP-1 is required for metanephric mesenchyme differentiation and plays a functional role during kidney development.     

Mullerian Inhibiting Substance inhibits cervical cancer cell growth via a pathway involving p130 and p107

In addition to causing regression of the Mullerian duct in the male embryo, Mullerian Inhibiting Substance (MIS) inhibits the growth of epithelial ovarian cancer cells, which are known to be of Mullerian origin. Because the uterine cervix is derived from the same Mullerian duct precursor as the epithelium of the ovary, we tested the hypothesis that cervical cancer cells might also respond to MIS. A number of cervical cancer cell lines express the MIS type II receptor, and MIS inhibits the growth of both human papilloma virus-transformed and non-human papilloma virus-transformed cervical cell lines, with a more dramatic effect seen in the latter. As in the ovarian cancer cell line OVCAR8, suppression of growth of the C33A cervical cancer cell line by MIS is associated with induction of the p16 tumor suppressor protein. However, in contrast to OVCAR8 cells, induction of p130 and p107 appears to play an important role in the inhibition of growth of C33A cells by MIS. Finally, normal cervical tissue expresses the MIS type II receptor in vivo, supporting the idea that MIS could be a targeted therapy for cervical cancer.     

Specific estradiol binding in embryonic Mullerian ducts: a potential modulator of regression in the male and female chick

The total content of putative estradiol-specific cytosolic type I and nuclear type I and II estradiol-specific binders was measured in 8- and 9-day-old male and female chick embryo Mullerian ducts. Cytosolic and nuclear type I estradiol-specific binding levels were similar in males and females, and no significant differences were noted among right vs. left and 8-day-old vs. 9-day-old embryo Mullerian ducts. The levels of nuclear type I estradiol binder were consistently higher than the cytosolic type I binder, but this difference was not significant. Nuclear type II estradiol-specific binding, however, was significantly higher in the left Mullerian ducts of both male and female embryos. The significance of these findings in relation to the regression of Mullerian ducts in male and female chick embryos is discussed.     

Human recombinant mullerian inhibiting substance inhibition of rat oocyte meiosis is reversed by epidermal growth factor in vitro

Mullerian inhibiting substance (MIS), a glycoprotein responsible for the regression of Mullerian duct in the male mammalian embryo, was recently localized not only in the fetal and newborn testis, but also in the older ovary throughout reproductive life. Bovine MIS purified from newborn testicular tissue inhibited spontaneous oocyte meiosis in vitro in the rat. These studies show that partially purified recombinant MIS produced from a human MIS genomic construct caused inhibition of oocyte meiosis, but when purified to homogeneity, the effect was lost. Addition of low concentrations of the detergent Nonidet P-40, used to maintain stability in the purified bovine preparations, however, restored the MIS inhibitory effect to the human preparation, which could, in turn, be blocked by a polyclonal antibody raised to human recombinant MIS; Nonidet P-40 itself caused no inhibition. Since we have shown that epidermal growth factor (EGF) and MIS are antagonistic in a number of other systems, we tested the effect of EGF on the ability of MIS to inhibit oocyte meiosis. EGF added to the medium at a dose that caused no effect on oocytes (25 ng/ml) blocked the MIS inhibitory action on spontaneous rat oocyte meiosis, while EGF had no effect on a known oocyte meiosis inhibitor, 3-isobutyl-1-methylxanthine. These data indicate that human recombinant MIS can inhibit oocyte meiosis in the ovary and that its regulatory effect can be modulated by EGF, which appears to be an antagonist of MIS.     

Metabolism of testosterone by the epithelium and mesenchyme of the rat urogenital sinus

Testosterone metabolism was measured in separated epithelium and mesenchyme from the urogenital sinuses of 17- and 19-day-old male and female rat embryos and compared with testosterone metabolism in the intact sinus. Both the epithelium and the mesenchyme converted testosterone to 5 alpha-dihydrotestosterone. The epithelium produced much more androstanedione and androsterone but less 3 alpha, 17 beta-androstanediol than did the mesenchyme. The whole sinus synthesized all four metabolites, but in different proportions, producing relatively more androsterone than either of its two component tissues. These data suggest that androsterone is formed by the joint action of epithelium and mesenchyme. Metabolism of testosterone did not differ with sex or foetal age in either of the separated tissues or in the intact sinus, implying that the failure of urogenital mesenchyme from 19-day-old female foetuses to induce prostatic morphogenesis is not due to the loss of 5 alpha-reductase. It is suggested that this lack of inductive capacity may be attributable to a decline in androgen levels with age in female mesenchyme.     

Morphogenetic and proliferative effects of testosterone and insulin on the neonatal mouse seminal vesicle in vitro

Effects of testosterone (T) and insulin on epithelial branching morphogenesis were investigated in cultured seminal vesicles (SVs) of neonatal mice. SVs from 0-day-old male mice were cultured for 0.5-6 days in serum-free chemically defined medium in the presence of T (10(-7) M), insulin (10 micrograms/ml), or T plus insulin or in medium lacking both hormones. Without the addition of both hormones, SVs failed to grow, based on DNA and protein contents, and did not show any epithelial branching morphogenesis. T induced a 2.5-fold increase in protein content in SVs cultured for 6 days and elicited modest epithelial branching morphogenesis. Insulin increased the protein content of cultured SV rudiments as much as T, but failed to elicit epithelial branching morphogenesis. The combination of both hormones induced a 4.3-fold increase in protein content in cultured SVs and elicited more extensive epithelial branching morphogenesis than T alone. Epithelial and mesenchymal DNA contents in SVs declined slightly during the first 12 h of culture in all treatment groups. The epithelial DNA content in SVs grown with insulin alone remained constant thereafter, while that of SVs grown with T alone or in combination with insulin increased 1.2- and 2.6-fold, respectively. In the absence of both hormones, the mesenchymal DNA content remained constant in SVs grown for 6 days after the initial decline in DNA content. In contrast, mesenchymal DNA content was increased to the same degree (1.4-fold) by either T or insulin alone or in combination. The labeling index with [3H]thymidine of SV epithelium and mesenchyme grown under the hormonal conditions described above corroborated the results of epithelial and mesenchymal DNA contents. These data indicate that insulin by itself has no effect on epithelial proliferation and branching morphogenesis in the neonatal mouse SV, but, instead, amplifies the morphogenetic and proliferative effects of androgen on the developing mouse SV, thus eliciting extensive branching morphogenesis. Both insulin and T have a slight (nonsynergistic) effect on proliferation of SV mesenchyme. Analysis of androgen metabolism in developing mouse SVs indicated that dihydrotestosterone was the major product when T was used as substrate in SVs grown under all hormonal conditions. The rate of DHT production per 100 mg protein was not significantly different among the different treatment groups. Higher levels of 5 alpha-androstane-3 alpha,17 beta-diol were detected in SVs grown in the absence vs. the presence of T regardless of the presence or absence of insulin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Visualization of X-chromosome inactivation mosaicism of Tfm gene in XTfm/X+ heterozygous female mice

The testicular feminization (Tfm) locus, which produces a deficiency in androgen receptors, is located on the X-chromosome. Steroid autoradiographic techniques were used to demonstrate the mosaicism of the X-chromosome inactivation in two androgen target tissues of XTfm/X+ heterozygous female mice. In the mesenchyme of urogenital sinuses of wild-type female fetuses (X+/X+), more than 95% of the cells were androgen-receptor positive (labelled with [3H]testosterone) while in that of heterozygous fetuses (XTfm/X+), about half of the cells were receptor positive (Tfm gene inactive). Statistical analysis of coherent clone size was applied to the heterozygous mesenchyme of the urogenital sinus and the coherent clone size of receptor-positive cells was estimated to be two or three cells per clone. This small clone size suggests that considerable cell mixing occurred in the tissue during embryonic development. Androgen binding in the mammary gland rudiments was restricted to the mesenchymal cells only in close vicinity to the epithelial mammary bud. In the wild-type rudiments most of the mesenchymal cells beneath the epithelium were receptor positive, while in heterozygous rudiments, receptor-positive and -negative cells intermingled. This observation suggests that in the wild-type mammary gland rudiments the epithelial bud may induce the formation of androgen receptors in adjacent mesenchymal cells rather than attract pre-existing receptor-rich mesenchymal cells.     

Insensitivity of the chick embryo müllerian duct to human recombinant müllerian-inhibiting substance.

To determine whether mammalian Müllerian-inhibiting substance (MIS) is active in birds, Müllerian ducts from 7- to 8-day-old male or female chick embryos were cultured in the presence of human recombinant MIS at concentrations between 2.5 and 12.5 micrograms/ml. None of 20 ducts regressed at any concentration. In contrast, at concentrations of 2.5-5 micrograms/ml, all 12 Müllerian ducts from 13-day-old male mouse embryos and 13 out of 14 female ducts were inhibited to varying degrees. It is concluded that avian Müllerian ducts are unresponsive to mammalian MIS. There may be a difference in structure between the MIS of birds and mammals, or the signal-transduction system may be different.     

Relationships between plasma progesterone levels and testicular testosterone production in the rat fetus: effects of maternal ovariectomy

The present study was performed to examine whether circulating progesterone regulates testicular testosterone production in the fetal rat. Progesterone levels in fetal plasma were found to increase from day 14.5 to day 16.5; thereafter they reached a plateau between days 16.5 and 18.5 (80 nmol/l) and decreased threefold between days 18.5 and 21.5. The addition of progesterone, within the range of normal plasma concentrations, induced a dose-dependent increase in testosterone produced in vitro by the testes on days 16.5 and 18.5 but not on day 20.5. However, in 18.5-day-old fetuses, individual plasma progesterone levels were not correlated with testicular testosterone production in vivo and in vitro. Furthermore, maternal bilateral ovariectomy induced a significant fall in plasma progesterone in 18.5-day-old fetuses; this was not associated with a reduction in plasma testosterone nor in testicular testosterone content, although the amount of testosterone secreted by the testis incubated in vitro was slightly but significantly reduced. It is concluded that circulating progesterone does not regulate testicular testosterone production in vivo although the testis may use plasma progesterone as a substrate. On day 18.5 after maternal ovariectomy, the decrease in plasma progesterone levels was similar in fetuses and mothers, suggesting that most fetal progesterone originates from maternal plasma.     

Induction of androgen receptor formation by epithelium-mesenchyme interaction in embryonic mouse mammary gland.

The role of tissue interaction in the development of hormone responsiveness was studied in the embryonic mammary gland of the mouse, which becomes sensitive to testosterone on day 14. Previously, the mesenchyme had been identified as the sole target tissue for the hormone, although it was also demonstrated that its response to testosterone required the presence of mammary epithelium. Using autoradiography, we now show that [3H]testosterone or [3H]5 alpha-dihydrotestosterone is bound only by those mesenchymal cells closest to the epithelial mammary bud. When mammary epithelia were experimentally associated with mesenchyme of the mammary region and cultured together for 3 days in vitro, they also became surrounded by several layers of [3H]testosterone-binding mesenchymal cells. Correspondingly, this tissue association was accompanied by a substantial increase of androgen-binding sites in the explants. No hormone-building mesenchymal cells were seen in combinations with epidermis or pancreas epithelium; only salivary epithelium showed a weak positive effect. From these results we conclude that mammary epithelium induces the formation of androgen receptors in adjacent mesenchyme and thereby controls the development of androgen responsiveness in this tissue.     

Branching morphogenesis independent of mesenchymal–epithelial contact in the developing kidney

Whether mesenchymal-epithelial interactions leading to branching morphogenesis in developing epithelial tissues such as the kidney require direct cell-cell contact or are due to soluble mediators elaborated by the inducing tissue has been the subject of much debate. Here we demonstrate that ureteric bud (UB) epithelium, from which the kidney collecting system and upper urinary tract are derived, can undergo impressive three-dimensional branching morphogenesis when cultured in the appropriate extracellular matrix context in the absence of direct contact with mesenchymal tissue, indicating that the program for branching morphogenesis is inherent to the UB. Both a soluble factor in BSN cell-conditioned medium (BSN-CM) derived from an immortalized cell line thought to originate in the early metanephric mesenchyme and glial cell line-derived neurotrophic factor (GDNF) were required for early and later events in branching morphogenesis. In the absence of BSN-CM, the isolated UB did not survive; a similar result was obtained in the presence of neutralizing antibodies against glial cell line-derived neurotrophic factor. Preliminary analysis of key activity present in BSN-CM indicates that it is a heat-sensitive, heparin-binding factor with a probable molecular mass greater than 100 kDa. When the in vitro cultured UB was recombined with freshly isolated metanephric mesenchyme, nephric units were induced in the mesenchyme, and the UB branches underwent elongation. Our data suggest that, although UB branching morphogenesis per se does not require direct mesenchymal contact, such contact may play a key role in regulating branch elongation and establishing the pattern of branching. The results also suggest an approach to in vitro engineering of nephron.