Developmental regulation of GDNF response and receptor expression in the enteric nervous system.

The development of the enteric nervous system is dependent upon the actions of glial cell line-derived neurotrophic factor (GDNF) on neural crest-derived precursor cells in the embryonic gut. GDNF treatment of cultured enteric precursor cells leads to an increase in the number of neurons that develop and/or survive. Here we demonstrate that, although GDNF promoted an increase in neuron number at all embryonic ages examined, there was a developmental shift from a mitogenic to a trophic response by the developing enteric neurons. The timing of this shift corresponded to developmental changes in gut expression of GFR alpha-1, a co-receptor in the GDNF-Ret signaling complex. GFR alpha-1 was broadly expressed in the gut at early developmental stages, at which times soluble GFR alpha-1 was released into the medium by cultured gut cells. At later times, GFR alpha-1 became restricted to neural crest-derived cells. GFR alpha-1 could participate in GDNF signaling when expressed in cis on the surface of enteric precursor cells, or as a soluble protein. The GDNF-mediated response was greater when cell surface, compared with soluble, GFR alpha-1 was present, with the maximal response seen the presence of both cis and trans forms of GFR alpha-1. In addition to contributing to GDNF signaling, cell-surface GFR alpha-1 modulated the specificity of interactions between GDNF and soluble GFR alphas. These experiments demonstrate that complex, developmentally regulated, signaling interactions contribute to the GDNF-dependent development of enteric neurons.

Expression of anti-Müllerian hormone during normal and pathological gonadal development: association with differentiation of Sertoli and granulosa cells.

The ontogeny of expression of anti-Müllerian hormone (AMH) was examined by immunohistochemistry in 135 human gonadal tissue specimens of various developmental age, ranging from 6 weeks of fetal development to 38 yr of postnatal age. The series included specimens from normal testes and ovaries and from individuals either with pathological conditions affecting gonadal development or with idiopathic infertility manifested as azoospermia or severe oligozoospermia. AMH expression was found only in Sertoli and granulosa cells. A 6-week-old fetal testis at the indifferent gonad stage did not yet express AMH. The protein was first visible at 8.5 weeks of development, when sex cords have not yet been formed. Afterward, a majority of testicular specimens, including those from pathological conditions, strongly expressed AMH through fetal development and childhood until puberty. Markedly prolonged expression of AMH was observed in a 20-yr-old 46,XY female with androgen insensitivity syndrome, who retained prepubertal testicular morphology. In normal testes, the switch-off of AMH expression was usually associated with the appearance of primary spermatocytes, suggesting that their presence had an inhibitory effect on AMH. However, in adolescent boys lacking germ cells because of cancer treatment and in a majority of infertile adult men with idiopathic germ cell aplasia, AMH expression was also down-regulated despite the complete lack of spermatogenesis. The decrease in AMH expression thus reflects the terminal differentiation of Sertoli cells and is probably only partially dependent upon a regulatory factor associated with the onset of meiosis. In fetal ovaries, AMH was first detected at 36 weeks gestation in granulosa cells of preantral follicles. Thus, the onset of ovarian expression is at the end of fetal life and not in infancy as previously reported.

Perturbation of RET signaling in the embryonic kidney.

We have used a RET-Ig fusion protein to disrupt signaling in the rat embryonic kidney development pathway. Treatment of embryonic kidney organ cultures with RET-Ig results in a decrease in branching of the ureteric bud and a down regulation in expression of the Wnt-11, Wnt-4, and ld genes. These data suggest that Wnt-11, Wnt-4, and ld function downstream of RET signaling in normal development. Expression of BMP-7, shh, and ptc were uneffected by RET-Ig treatment, implying that these genes are regulated independently of ret. We have also performed immunohistochemistry with a GFR alpha-1 specific polyclonal antisera to localize GFR alpha-1 protein expression in the developing kidney.

Genomic structure and chromosomal localization of the human GDNFR-alpha gene.

GDNFR-alpha is a glycosyl-phosphotidylinositol-linked receptor for glial cell line-derived neurotrophic factor (GDNF). GDNF binds to GDNFR-alpha and this complex, in turn, is believed to interact with the RET receptor tyrosine kinase to effect downstream signalling. GDNFR-alpha belongs to a novel gene family without strong homology to known genes. Thus, little information has been available to help predict genomic structure or location of this gene. In this study, the genomic organization of human GDNFR-alpha was delineated through a combination of PAC clone characterization, long distance PCR and sequence analyses. Exon-intron boundaries were defined by comparing the size and sequence of the genomic PCR products to those predicted by the cDNA sequence. The human GDNFR-alpha gene comprises 9 exons. GDNFR-alpha PAC clones were used for FISH analysis to map this gene to 10q26.

Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury.

We report the identification of rat and human cDNAs for a type 1 membrane protein that contains a novel six-cysteine immunoglobulin-like domain and a mucin domain; it is named kidney injury molecule-1 (KIM-1). Structurally, KIM-1 is a member of the immunoglobulin gene superfamily most reminiscent of mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Human KIM-1 exhibits homology to a monkey gene, hepatitis A virus cell receptor 1 (HAVcr-1), which was identified recently as a receptor for the hepatitis A virus. KIM-1 mRNA and protein are expressed at a low level in normal kidney but are increased dramatically in postischemic kidney. In situ hybridization and immunohistochemistry revealed that KIM-1 is expressed in proliferating bromodeoxyuridine-positive and dedifferentiated vimentin-positive epithelial cells in regenerating proximal tubules. Structure and expression data suggest that KIM-1 is an epithelial cell adhesion molecule up-regulated in the cells, which are dedifferentiated and undergoing replication. KIM-1 may play an important role in the restoration of the morphological integrity and function to postischemic kidney.

Glial cell line-derived neurotrophic factor-dependent RET activation can be mediated by two different cell-surface accessory proteins.

Glial cell line-derived neurotrophic factor (GDNF)-dependent activation of the tyrosine kinase receptor RET is necessary for kidney and enteric neuron development, and mutations in RET are associated with human diseases. Activation of RET by GDNF has been shown to require an accessory component, GDNFR-alpha (RETL1). We report the isolation and characterization of rat and human cDNAs for a novel cell-surface associated accessory protein, RETL2, that shares 49% identity with RETL1. Both RETL1 and RETL2 can mediate GDNF dependent phosphorylation of RET, but they exhibit different patterns of expression in fetal and adult tissues. The most striking differences in expression observed were in the adult central and peripheral nervous systems. In addition, the mechanisms by which the two accessory proteins facilitate the activation of RET by GDNF are quite distinct. In vitro binding experiments with soluble forms of RET, RETL1 and RETL2 demonstrate that while RETL1 binds GDNF tightly to form a membrane-associated complex which can then interact with RET, RETL2 only forms a high affinity complex with GDNF in the presence of RET. This strong RET dependence of the binding of RETL2 to GDNF was confirmed by FACS analysis on RETL1 and RETL2 expressing cells. Together with the recent discovery of a GDNF related protein, neurturin, these data raise the possibility that RETL1 and RETL2 have distinctive roles during development and in the nervous system of the adult. RETL1 and RETL2 represent new candidate susceptibility genes and/or modifier loci for RET-associated diseases.

Genetic analysis of the Müllerian-inhibiting substance signal transduction pathway in mammalian sexual differentiation.

Müllerian-inhibiting substance (MIS) is a member of the transforming growth factor-beta (TGF-beta) gene family. MIS expression in males causes the regression of the Müllerian ducts, an essential process in male sexual differentiation. Recently, an MIS type II receptor gene has been isolated that is expressed during embryogenesis in mesenchymal cells adjacent to the Müllerian duct epithelium and in Sertoli and granulosa cells of the fetal and adult, male and female gonads, respectively. MIS receptor mutant males develop as internal pseudohermaphrodites, possessing a complete male reproductive tract and also a uterus and oviducts, a phenocopy of MIS ligand-deficient male mice. They express both MIS mRNA and protein, showing that ligand was present, but target organs were hormone-insensitive. All produce sperm, but the majority were infertile because the presence of their female reproductive organs blocks sperm transfer into females. Focal seminiferous tubule atrophy accompanied by Leydig cell hyperplasia was observed and began as early as 2 months of age. The phenotype of MIS ligand/MIS receptor double mutant males was indistinguishable from those of each single mutant. MIS receptor/alpha-inhibin double mutant males developed testicular stromal tumors and large fluid-filled uteri that were identical in phenotype to MIS ligand/alpha-inhibin double mutant males. These studies provide in vivo evidence that MIS is the only ligand of the MIS type II receptor, in contrast to the complexity of other TGF-beta gene family signaling pathways.

In vivo administration of CD4-specific monoclonal antibody: effect on provirus load in rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques.

Since monoclonal antibodies (MAb) specific for CD4 are potent inhibitors of HIV and SIV replication in vitro, we explored their potential usefulness in vivo as an AIDS therapy. The anti-CD4 MAb 5A8 binds to domain 2 of the CD4 molecule and inhibits virus replication and virus-induced cell fusion at a postvirus binding step. Administration of this MAb to normal rhesus monkeys coats all circulating and lymph node CD4 cells and induces neither CD4 cell clearance nor measurable immunosuppression. In the present study, monkeys chronically infected with the simian immunodeficiency virus of macaques (SIVmac) had stable levels of SIVmac provirus in PBMC prior to treatment as measured by a quantitative polymerase chain reaction technique. Six infected monkeys treated with anti-CD4 MAb demonstrated a significant decrease in SIVmac provirus level after 9 days. Of these monkeys, 3 had > 800 CD4 cells/microliter and developed strong antimouse Ig responses that prevented further treatment. The remaining 3 monkeys had < 800 CD4 cell/microliter and failed to develop antimouse Ig antibody responses. When treatment was continued for 12-21 days in these monkeys, a sustained or further decrease in SIVmac provirus load occurred over the extended treatment period. Four monkeys that received a control MAb of irrelevant specificity for 9-22 days showed either no significant change or a transient increase in SIVmac provirus. Thus, the passive administration of anti-CD4 MAb may exert a specific antiviral effect in controlling immunodeficiency virus infection in vivo.

Müllerian-inhibiting substance function during mammalian sexual development.

To investigate the role of Müllerian-inhibiting substance (MIS) in mammalian sexual development, we generated MIS-deficient mice. Although MIS-deficient males had testes that were fully descended and produced functional sperm, they also developed female reproductive organs, which interfered with sperm transfer into females, rendering most of these males infertile. Their testes had Leydig cell hyperplasia and, in one instance, neoplasia. The actions of the two primary hormones of male sexual differentiation were genetically eliminated using the testicular feminization (Tfm) mutation in combination with the MIS mutant allele. XY Tfm/MIS double mutants developed as females, with a uterus, coiled oviducts, and no male reproductive organs except undescended dysfunctional testes. These results suggest that eliminating the presumptive female reproductive tract in male fetuses facilitates fertility and that in testes MIS is a negative regulator of Leydig cell proliferation. Eliminating the presumptive male reproductive tract is necessary for proper oviductal morphogenesis during female mouse development.

Directed expression of an oncogene to Sertoli cells in transgenic mice using mullerian inhibiting substance regulatory sequences.

Mullerian inhibiting substance (MIS) is a glycoprotein hormone expressed by Sertoli cells that induces the regression of Mullerian ducts during development of the male reproductive tract. Transgenic mice carrying a fusion gene composed of human MIS transcriptional regulatory sequences linked to the SV40 T-antigen gene specifically develop testicular tumors composed of a cell type histologically resembling the Sertoli cell. The lack of pathology at other sites suggests tissue-restricted expression of the transgene. A cell line derived from one of the testicular tumors has been established that continues to express markers associated with Sertoli cells, such as transferrin, sulfated glycoprotein-2, and inhibin-beta B. The cell line does not express detectable levels of inhibin-alpha, MIS, or FSH receptor. However, the cells have retained forskolin responsiveness. As adult Sertoli cells cannot be propagated in vitro, the availability of an immortal cell line displaying features characteristic of normal Sertoli cells should aid in subsequent analyses of the biology of this cell type.

A quantitative and interspecific test for biological activity of anti-müllerian hormone: the fetal ovary aromatase assay.

Anti-Müllerian hormone (AMH), also known as Müllerian-inhibiting substance or factor, has previously been shown to sex-reverse the steroidogenic pattern of fetal mammalian ovaries through repression of aromatase biosynthesis. Study of the ontogeny of the response of cyclic AMP-stimulated aromatase activity of rat fetal ovaries to AMH has allowed us to develop a quantitative bioassay for the hormone. Linear responses as a function of the logarithm of AMH concentration were observed over ranges of 0.2-7.5 micrograms/ml for the bovine protein and 0.15-2 micrograms/ml for the human protein, with a maximal decrease in aromatase activity of 90% for both proteins. Under the same in vitro conditions, AMH treatment did not affect cyclic AMP-stimulated fetal rat testicular aromatase activity. Partially purified chick AMH also decreased rat ovarian aromatase activity, allowing us to use this test to study AMH ontogeny in chick gonads. Analysis of the species specificity of AMH repression of ovarian aromatase activity indicated that turtle and rat fetal ovaries responded to AMH of other vertebrate classes, whereas aromatase activity of chick embryo ovaries could be repressed only by the homospecific hormone.

Imaging of DNA sequences with chemiluminescence.

We have coupled a chemiluminescent detection method that uses an alkaline phosphatase label to the genomic DNA sequencing protocol of Church and Gilbert [Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991-1995]. Images of sequence ladders are obtained on x-ray film with exposure times of less than 30 min, as compared to 40 h required for a similar exposure with a 32P-labeled oligomer. Chemically cleaved DNA from a sequencing gel is transferred to a nylon membrane, and specific sequence ladders are selected by hybridization to DNA oligonucleotides labeled with alkaline phosphatase or with biotin, leading directly or indirectly to deposition of enzyme. If a biotinylated probe is used, an incubation with avidin-alkaline phosphatase conjugate follows. The membrane is soaked in the chemiluminescent substrate (AMPPD) and is exposed to film. Dephosphorylation of AMPPD leads in a two-step pathway to a highly localized emission of visible light. The demonstrated shorter exposure times may improve the efficiency of a serial reprobing strategy such as the multiplex sequencing approach of Church and Kieffer-Higgins [Church, G. M. & Kieffer-Higgins, S. (1988) Science 240, 185-188].

An immunoassay to detect human müllerian inhibiting substance in males and females during normal development.

An enzyme-linked immunosorbent assay has been developed to measure human Müllerian inhibiting substance (MIS) in biological fluids. The enzyme-linked immunosorbent assay is specific for MIS, with a sensitivity in human serum to 0.5 ng/ml and does not recognize transforming growth factor-beta 1 or -beta 2, LH, or FSH. It similarly fails to recognize other proteins secreted from the cell type into which the MIS gene was cloned. MIS was detected in the serum of normal newborns, infants, children, and adults. In males the serum level of MIS is 10-70 ng/mL at birth. The level increases slightly after birth, and then decreases to a basal level of 2-5 ng/mL after the first 10 yr of life. Newborn male urine contains minimal amounts of MIS (0.5 ng/mL). In females MIS is barely detectable in serum at birth, but rises to the basal level equal to that seen in males after 10 yr of age. Similar basal levels of MIS were found in adult ovarian follicular fluid. MIS levels were high in the serum of a female patient with a sex cord tumor (3200 ng/mL), but fell to 100 ng/mL after multiple excisional operations. In addition, a serum MIS level of 20 ng/mL was detected in a patient with an ovarian granulosa cell tumor. A sensitive assay for MIS could be useful in the diagnosis of patients with congenital abnormalities of sexual development and patients with Sertoli cell and/or other MIS-producing neoplasms. Other applications may also be recognized as the biology of MIS in both males and females is further elucidated.

Minimal antiproliferative effect of recombinant müllerian inhibiting substance on gynecological tumor cell lines and tumor explants.

Múllerian Inhibiting Substance (MIS) is a testicular hormone that promotes involution of the Múllerian duct during embryogenesis. The Múllerian duct gives rise to adult female reproductive ducts including the fallopian tubes, uterus, and upper vagina. Thus, testicular MIS ensures the regression of female sex organ primordia. Partially purified bovine MIS was reported to inhibit proliferation of tumor cells derived from human gynecological cancers. These observations suggest that MIS might be an effective anticancer agent for some human tumors. Recombinant human MIS (rHu-MIS) has recently become available. To assess the antiproliferative activity of rHu-MIS, we examined its effects on 11 ovarian, six endometrial, and two nongynecological human tumor cell lines. rHu-MIS had no effect on proliferation of these cell lines in five independent assays. Forty-three primary human tumor explants were also examined in human tumor colony forming assays, gel-supported primary culture assays, and subrenal capsule assays. rHu-MIS significantly inhibited the growth of five of these tumors including four ovarian and one small cell lung cancer explant. The four ovarian cancer responses include three of 13 (23%) explants tested in human tumor colony-forming assays and one of eight (12.5%) explants tested in gel-supported primary culture assays. We conclude that rHu-MIS may have antiproliferative activity against some human ovarian cancers.

Comparison of the methylation patterns of the two rat insulin genes.

We have investigated whether DNA methylation is involved in regulating the expression of the rat insulin genes. We studied cytosine methylation in and near the two insulin genes to examine the correlation of site-specific methylation with expression in different tissues and to compare the demethylation patterns of the two genes. For both genes, we found certain sites undermethylated only in insulin-producing tissues. However, the overall patterns of the two genes in expressing tissues are quite different. The insulin I gene is significantly less methylated than the insulin II gene in a tumor that makes equal amounts of the two insulin RNAs. In another tumor and cell line that make 5-fold higher levels of insulin I RNA than insulin II RNA, the changes in the methylation levels of the two genes do not correlate with the increased expression of insulin I. In nonexpressing tissues, the insulin I gene is completely methylated, while the insulin II gene is demethylated at a number of sites. Thus, the general level of methylation of the insulin genes does not correlate with their differential expression. Furthermore, the methylation at specific sites does not correlate either; some CG dinucleotides that appear to be important for one gene are not present in the other. Our results suggest that there is no specific control by methylation of the expression of the insulin genes in the rat.