Cellular localization of immunoreactive epidermal growth factor during Wolffian duct differentiation of the fetal mouse

Previous studies from this laboratory indicated a role for epidermal growth factor (EGF) in androgen-dependent male reproductive tract differentiation of the fetal mouse. Expression of an EGF-like protein during Wolffian duct differentiation was indicated from the determinations by radioimmunoassay (RIA) and radioreceptor assay. To further characterize the protein and to assess its role in male sexual differentiation, expression of the protein has been analyzed by Western blot assay and its tissue-specific cellular expression has been determined by immunocytochemical assay in the present study. Western blot analysis of the 18-day fetal male reproductive tract detected an immunoreactive band of the predicted 6-kDa size. Immunocytochemical analysis also detected EGF-specific immunostaining in the Wolffian duct derivatives. At day 18 of gestation, the staining was localized predominantly in the epithelial nuclei of the Wolffian duct derivatives whereas at days 14 and 16 of gestation, the staining was equally distributed in the mesenchymal and epithelial sites of the Wolffian duct derivative. The intensity of the staining increased with progression of differentiation during the 14th–18th days of gestation. Prenatal exposure to the antiandrogen flutamide significantly reduced the immunostaining of the duct. Thus, a role for EGF in Wolffian duct differentiation is indicated.     

Paradigm shift from classic anatomic theories to contemporary cell biological views of CAKUT

Ectopic budding of the ureter from the Wolffian duct is the first ontogenic misstep that leads to many-if not all-congenital anomalies of the kidney and urinary tract (CAKUT). The ectopia results in hypoplastic kidney, ectopia of ureterovesical orifice, urinary outflow obstruction and/or reflux. Studies in several mutant mouse models have verified that ectopic ureteric budding indeed precedes formation of CAKUT. Often, the genes involved in navigating ureteric budding to the correct site also regulate later ontogenic events of the kidney and urinary tract. The wide spectrum of CAKUT, for example, multicystic dysplastic kidney, megaureter and atretic ureter, portray the additional important functions of these same genes that are activated at multiple sites and stages during the normal morphogenesis of the kidney and urinary tract     

How do you get six meters of epididymis inside a human scrotum?

It is very clear that the epididymis plays a crucial role in the maturation of spermatozoa, and without a fully developed and functional epididymis, male infertility will result. We are especially interested in understanding the mechanisms that regulate the development of this important organ because disruptions to epididymal function will also arise as a consequence of abnormal development. Very little is known either of the process of epididymal development or the nature and causes of congenital defects that lead to male infertility. A major event during Wolffian/epididymal duct embryonic development is elongation and coiling and this short review outlines potential mechanisms by which these events occur. It is hypothesized that elongation is the result of cell proliferation coupled with directed cell rearrangements, the later regulated by the planar cell polarity signaling pathway. Coiling proceeds in a proximal to distal manner, with three-dimensional coiling beginning approximately embryonic day 16.5 to 18.5 in the mouse. The exact mechanisms of coiling are not known but we hypothesize that it involves an interaction between the Wolffian duct epithelium and the surrounding mesenchyme cells, such that the extracellular matrix is remodeled to allow coiling and growth of the duct. Cell proliferation in the Wolffian duct appears to be dependent on the presence of androgens and mesenchymal factors during embryonic development, but lumicrine factors play an additional role during postnatal development.     

To bud or not to bud: the RET perspective in CAKUT

Congenital anomalies of the kidneys or lower urinary tract (CAKUT) encompass a spectrum of anomalies that result from aberrations in spatio-temporal regulation of genetic, epigenetic, environmental, and molecular signals at key stages of urinary tract development. The Rearranged in Transfection (RET) tyrosine kinase signaling system is a major pathway required for normal development of the kidneys, ureters, peripheral and enteric nervous systems. In the kidneys, RET is activated by interaction with the ligand glial cell line-derived neurotrophic factor (GDNF) and coreceptor GFRα1. This activated complex regulates a number of downstream signaling cascades (PLCγ, MAPK, and PI3K) that control proliferation, migration, renewal, and apoptosis. Disruption of these events is thought to underlie diseases arising from aberrant RET signaling. RET mutations are found in 5–30 % of CAKUT patients and a number of Ret mouse mutants show a spectrum of kidney and lower urinary tract defects reminiscent of CAKUT in humans. The remarkable similarities between mouse and human kidney development and in defects due to RET mutations has led to using RET signaling as a paradigm for determining the fundamental principles in patterning of the upper and lower urinary tract and for understanding CAKUT pathogenesis. In this review, we provide an overview of studies in vivo that delineate expression and the functional importance of RET signaling complex during different stages of development of the upper and lower urinary tracts. We discuss how RET signaling balances activating and inhibitory signals emanating from its docking tyrosines and its interaction with upstream and downstream regulators to precisely modulate different aspects of Wolffian duct patterning and branching morphogenesis. We outline the diversity of cellular mechanisms regulated by RET, disruption of which causes malformations ranging from renal agenesis to multicystic dysplastic kidneys in the upper tract and vesicoureteral reflux or ureteropelvic junction obstruction in the lower tract.     

Fibroblast growth factor receptor signaling in kidney and lower urinary tract development

Fibroblast growth factor receptors (FGFRs) and FGF ligands are highly expressed in the developing kidney and lower urinary tract. Several classic studies showed many effects of exogenous FGF ligands on embryonic renal tissues in vitro and in vivo. Another older landmark publication showed that mice with a dominant negative Fgfr fragment had severe renal dysplasia. Together, these studies revealed the importance of FGFR signaling in kidney and lower urinary tract development. With the advent of modern gene targeting techniques, including conditional knockout approaches, several publications have revealed critical roles for FGFR signaling in many lineages of the kidney and lower urinary tract at different stages of development. FGFR signaling has been shown to be critical for early metanephric mesenchymal patterning, Wolffian duct patterning including induction of the ureteric bud, ureteric bud branching morphogenesis, nephron progenitor survival and nephrogenesis, and bladder mesenchyme patterning. FGFRs pattern these tissues by interacting with many other growth factor signaling pathways. Moreover, the many genetic Fgfr and Fgf animal models have structural defects mimicking numerous congenital anomalies of the kidney and urinary tract seen in humans. Finally, many studies have shown how FGFR signaling is critical for kidney and lower urinary tract patterning in humans.     

Localization of protein gene product 9.5 immunoreactivity in derivatives of the human Wolffian duct and in prostate cancer

BACKGROUND: Protein gene product 9.5 (PGP 9.5) has been considered to be a neuronal marker, but it is also present in extraneuronal tissues, e.g., the human mammary gland and rat epididymis. Its presence and distribution in the developing and adult male human genital tract have been unknown. METHODS: Immunohistochemical reactions were performed on human embryonic and postnatal specimens of the male genital tract, using commercial monoclonal and polyclonal antibodies. RESULTS: PGP 9.5 immunoreactivity was found in the Wolffian duct of human embryos (55-85 mm crown-rump length). Strong reactivity was observed in mesonephric tubular cells and at the apical rim of Wolffian duct cells. Owing to their PGP 9.5 immunoreactivity, these cells could also be identified on the surface of the embryonic verumontanum, extending from the orifices of the Wolffian duct to a small stretch of the urogenital sinus. There they contrasted sharply against non-Wolffian cells. In the adult human genital tract, PGP 9.5 immunoreactive material was present in the supranuclear portion of some epithelial cells of the epididymal efferent ductules, in isolated cells of the ejaculatory ducts, and in prostate cancer specimens. In the ejaculatory ducts, the PGP 9.5-immunoreactive cells were free of immunoreactivity for semenogelin, the major secretory product of the ejaculatory-vesicular-ampullary complex, and they also lacked chromogranin A-immunoreactivity. In prostate cancer specimens, PGP 9.5 immunoreactivity was never observed in secretory cells (immunoreactive for prostate-specific antigen), but was restricted to neuroendocrine cells, where it occurred either alone or coexpressed with chromogranin A-immunoreactivity. CONCLUSIONS: PGP 9.5-immunoreactivity is prenatally distributed in the Wolffian duct and its derivations; postnatally, it is restricted to a few cells derived from the initial and terminal segment of the Wolffian duct, and to neuroendocrine cells in prostate cancer specimens.     

Giant cyst of the seminal vesicle associated with ipsilateral renal agenesis

Cyst of the seminal vesicles represent a rare but illustrative type of embryologic malformation whose etiology is associated with an abnormal development of the mesonephric or Wolffian duct. Frequently these malformations are associated with an abnormal development of the ipsilateral upper urinary tract. The initial evaluation of the majority of cases is performed with abdominal or transrectal ultrasound. Considering the possible need of other diagnostic procedures to confirm the diagnosis, ultrasonography is safe in the majority of cases. The treatment of these urologic malformations should be restricted to symptomatic cases and usually consists of vesiculectomy, with of without, removal of the displastic or histoplastic kidney. We present a case of a right mesonephric duct malformation with a giant seminal vesicle associated with ipsilateral kidney agenesis and severe oligozoospermia, that presented with sporadic episodes of hemospermia and urinary complaints.     

Ureter malformations and renal dysplasia--an embryological concept (author's transl)

A study was made of 32 cases of single ectopic ureters (without duplication) to assess the abnormalities observed in the corresponding renal parenchyma and to correlate renal status with location of the ectopic uretic opening. Specimens were obtained from 26 nephrectomies and renal dysplasia was found in 22 cases. There was a close relationship between renal anomalies and location of the ureteric opening. The more remote the ectopic orifice from its normal position, the more severe were the associated renal lesions. Dysplasia was always present when the ectopic ureter opened outside the urinary tract (vagina, Gartner's duct, vestibule, seminal tract). Am embryological concept is proposed to explain the association of renal dysplasia with abnormal location of the ureter opening. It is based on the abnormal--premature or late--appearance of the ureteral bud on the Wolffian duct resulting in the abnormal positioning of the bud on the Wolffian duct with the ureter growing into nephrogenic tissue lacking the potential for normal renal development and leading to renal dysplasia.     

Molecular biology of ureteral bud and trigonal development

Advances in molecular biology have provided valuable insight into the development of the urinary tract, particularly ureteral bud formation. Reciprocal inductive signals between the ureteral bud and growing kidney are crucial for normal development. The Wolffian duct serves as the site of origin of the ureteral bud and forms distal excretory ducts that are incorporated into the developing bladder to become the trigone. Vesicoureteral reflux and renal dysplasia can result from abnormal position of the ureteral orifice on the trigone. The presumed origin of trigone formation is based largely on evaluation of human and animal models performed nearly a century ago. The trigone is thought to develop from the mesodermal germ cell layer; however, several recent studies have shown that endoderm may be the tissue of origin. This review highlights important discoveries in the field of molecular biology as it relates to the development of normal and abnormal ureteral bud formation. It also describes the anatomic relationship between the developing bud and trigone as it pertains to clinically relevant urinary tract anomalies, including recent discoveries that attempt to prove the origin of the trigone.     

Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract

Ksp-cadherin is a unique, tissue-specific member of the cadherin family of cell adhesion molecules that is expressed in tubular epithelial cells in the kidney and developing genitourinary (GU) tract. It has recently been shown that a 1341-bp fragment of the 5' flanking region containing the Ksp-cadherin gene promoter can recapitulate the complete expression pattern of the gene in the developing kidney and GU tract. Similar to the endogenous Ksp-cadherin gene, transgenes containing 1341 bp of the 5' flanking region are expressed in developing nephrons, ureteric bud, mesonephric tubules, Wolffian duct, and Müllerian duct. In adult mice, the expression is restricted to renal tubules. In the current study, Ksp1.3/Cre transgenic mice carrying 1329 bp of the Ksp-cadherin 5' flanking region linked to the Cre recombinase gene were produced. Adult transgenic mice expressed Cre recombinase in renal tubules, especially collecting ducts and thick ascending limbs of Henle's loops. Transgenic embryos expressed Cre recombinase in the branching ureteric bud, developing renal tubules, and sex ducts. Ksp1.3/Cre transgenic mice were crossed with mice carrying a lacZ reporter gene that is activated by Cre/lox-mediated genetic recombination. Bitransgenic progeny expressed lacZ exclusively in renal tubules, mesonephric tubules, ureteric bud, developing ureter, and Wolffian duct. These results demonstrate that Ksp1.3/Cre transgenic mice express Cre recombinase exclusively in the kidney and developing GU tract and can mediate epithelial-specific Cre/lox recombination in these tissues. Ksp1.3/Cre transgenic mice should be useful for cell lineage studies and kidney-specific gene targeting.     

Congenital anomalies of the kidney and urinary tract--role of the loss of function mutation in the pluripotent angiotensin type 2 receptor gene

PURPOSE: Recent studies of the human genome and genetic engineering experiments in mice revealed that congenital anomalies of the kidney and urinary tract commonly seen in newborns in various anatomical forms are polygenic disease, that is a disease caused by simultaneous defects in multiple genes. We discuss some possible genetic explanations of the classic theories of the formation of congenital kidney and urinary tract anomalies. MATERIALS AND METHODS: We reviewed classic and current theories regarding urinary tract development. Included in our review are recent results from our laboratory evaluating the genetic role of normal and abnormal urinary tract development. RESULTS: We observed a genetic abnormality that may explain many classic anatomical theories of congenital kidney and urinary tract anomalies. One of the genes involved in urinary tract ontogenesis is the angiotensin type 2 receptor gene, which is the "other" angiotensin receptor. While the type 1 receptor mediates essentially all known actions of angiotensin, including its hypertensive effect, relatively little is known about the angiotensin type 2 receptor. Careful dissection studies in mutant mouse embryos selectively lacking the angiotensin type 2 receptor gene revealed that this gene is pleiotropic, that is its defect causes not only ectopic ureteral budding from the wolffian duct, but also disturbance in other subsequent ontogenic events that are critical for the normal growth of the kidney and urinary tract. CONCLUSIONS: Many congenital anomalies of the kidney and urinary tract appear to share a common genetic cause. While these anomalies are caused by various genetic hits, abnormalities in the angiotensin type 2 receptor gene are often involved in this anomalous development. This review article offers a better understanding of the genetics involved in urinary tract development and ties some of the newly emerging genetic theories with classic anatomical theories.     

Male adnexal tumour of probable Wolffian duct origin

Male adnexal tumours of Wolffian duct origin are very rare. They have variable malignant potential and surgical excision is recommended. We report the case of a 56-year-old male who presented with a swelling in the perineum thought to be a haematoma as the result of a complication of a laparoscopic inguinal hernia repair. It recurred after incision and two attempts at drainage and was then completely excised. Histology revealed the lesion to be an adnexal tumour of probable Wolffian duct origin. To our knowledge there have only been two previous cases of male adnexal tumour of Wolffian duct origin reported in the literature.     

Glycogen synthase kinase-3 inactivation and stabilization of beta-catenin induce nephron differentiation in isolated mouse and rat kidney mesenchymes

Wnt proteins are required for induction of nephrons in mouse metanephric kidneys, but the downstream pathways that mediate tubule induction and epithelial differentiation have remained obscure. The intracellular mechanisms by which Wnt signaling mediates nephron induction in embryonic kidney mesenchymes were studied. First is shown that transient exposure of isolated kidney mesenchymes to structurally different glycogen synthase kinase-3 (GSK3) inhibitors lithium or 6-bromoindirubin-3'-oxime results in abundant epithelial differentiation and full segregation of nephrons. Shown further by mice with genetically disrupted ureteric bud or Wolffian duct development is that this nephrogenic competence arises independent of the influence of Wolffian duct-derived epithelia. Analysis of the intracellular signaling cascades downstream of GSK3 inhibition revealed stabilization of beta-catenin and upregulation of Lef1 and Tcf1, both events that are associated with the active canonical Wnt signaling. Last, genetic evidence that metanephric mesenchyme-specific stabilization of beta-catenin is sufficient to induce nephron differentiation in isolated kidney mesenchymes, similar to that induced by GSK3 inhibitors, is provided. These data show that activation of canonical Wnt pathway is sufficient to induce nephrogenesis and suggest that this pathway mediates the nephron induction in murine kidney mesenchymes.     

A minimal Ksp-cadherin promoter linked to a green fluorescent protein reporter gene exhibits tissue-specific expression in the developing kidney and genitourinary tract

Ksp-cadherin is a unique, tissue-specific member of the cadherin family of cell adhesion molecules that is expressed exclusively in tubular epithelial cells in the kidney and developing genitourinary (GU) tract. Transgenic mice carrying 3425 bp of the Ksp-cadherin 5' flanking region linked to a lacZ reporter gene express beta-galactosidase exclusively in the kidney, although the expression pattern is incomplete (Am J Physiol 277: F599-F610, 1999). To further define the region that mediates tissue-specific expression, transgenic mice carrying 1341 bp or 324 bp of the 5' flanking region linked to a green fluorescent protein (GFP) reporter gene were produced. Transgenic mice carrying 1341 bp of the 5' flanking region expressed GFP in all embryonic tissues that endogenously express Ksp-cadherin, including the ureteric bud, Wolffian duct, Müllerian duct, and developing tubules in the mesonephros and metanephros. In the adult kidney, GFP was highly expressed in thick ascending limbs of Henle's loops and collecting ducts and weakly expressed in proximal tubules and Bowman's capsules. Transgenic mice carrying 324 bp of the 5' flanking region exhibited expression exclusively in tubular epithelial cells in the developing kidney and GU tract. Immunoblot analysis showed that the expression of GFP was restricted to the kidney in adult mice. Taken together, these results demonstrate that 324 bp of the Ksp-cadherin 5' flanking region is sufficient to direct epithelial-specific expression in the developing kidney and GU tract. Transgenic mice that express GFP in the mesonephros, metanephros, ureteric bud, and sex ducts may be useful for cell lineage studies.     

How they begin and how they end: classic and new theories for the development and deterioration of congenital anomalies of the kidney and urinary tract, CAKUT.

CAKUT are problems that often require surgical intervention or, in the worst case, lead to renal failure and the need for dialysis and/or renal transplantation. It is believed that these anomalies share a common genetic cause and to date there has been no good animal model with which to study these abnormalities. Although the abnormal interaction between the ureteral bud and metanephric blastema leads to renal hypodysplasia, vesicoureteral reflux, and ectopic ureters to name a few, the genetic and biochemical modulation of urinary tract development is not understood. Studies using the mouse strain mutant for angiotensin type 2 (AT2) receptors have given new insight into this mystery. The animals show defective apoptosis of undifferentiated mesenchymal cells in the area surrounding the developing kidney and urinary tract. This abnormal apoptosis may well interfere with the normal interaction between the ureteral bud and metanephric blastema resulting in CAKUT. This abnormal interaction would theoretically lead to preexisting intrinsic abnormalities of the kidney, which are programmed and take effect early in embryonic development. In the worst cases, the renal abnormalities would lead to progressive deterioration of renal function. Undoubtedly, there are more genes and biochemical modulators involved in this process other than the RAS and AT2 receptors. Our current animal model gives new and unique possibilities with which to study development of the kidney and urinary tract and ultimately seek ways of preventing an often debilitating disease process.