Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney

Signaling by the ureteric bud epithelium is essential for survival, proliferation and differentiation of the metanephric mesenchyme during kidney development. Most studies that have addressed ureteric signaling have focused on the proximal, branching, ureteric epithelium. We demonstrate that sonic hedgehog is expressed in the ureteric epithelium of the distal, non-branching medullary collecting ducts and continues into the epithelium of the ureter -- the urinary outflow tract that connects the kidney with the bladder. Upregulation of patched 1, the sonic hedgehog receptor and a downstream target gene of the signaling pathway in the mesenchyme surrounding the distal collecting ducts and the ureter suggests that sonic hedgehog acts as a paracrine signal. In vivo and in vitro analyses demonstrate that sonic hedgehog promotes mesenchymal cell proliferation, regulates the timing of differentiation of smooth muscle progenitor cells, and sets the pattern of mesenchymal differentiation through its dose-dependent inhibition of smooth muscle formation. In addition, we also show that bone morphogenetic protein 4 is a downstream target gene of sonic hedgehog signaling in kidney stroma and ureteral mesenchyme, but does not mediate the effects of sonic hedgehog in the control of mesenchymal proliferation.     

GDNF and its receptors in the regulation of the ureteric branching.

Recent transgenic and organ culture experiments have inevitably shown that glial cell line-derived neurotrophic factor (GDNF) is a mesenchyme-derived signal for ureteric budding and branching. The signalling receptor complex for GDNF includes a dimer of Ret receptor tyrosine kinase and two molecules of GDNF family receptor alpha1. Alpha-receptors are not only needed for the ligand binding and Ret activation, but they might mediate signals without Ret. While GDNF is clearly required for ureteric branching, tissue recombination studies have shown that it is not sufficient for the completion of ureteric morphogenesis, and other signalling molecules are needed. Different experimental models have resulted in somewhat contradictory results on their molecular identity, but transforming growth factor-beta1, -beta2, fibroblast growth factor-7 and hepatocyte growth factor form, obviously among others, a redundant set of growth factors in ureteric differentiation. Three other members of the GDNF family, neurturin, artemin and persephin, are also expressed in the developing kidney, and at least neurturin and persephin promote ureteric branching in vitro, but their true in vivo roles are still unclear.     

Identification of pleiotrophin as a mesenchymal factor involved in ureteric bud branching morphogenesis

Branching morphogenesis is central to epithelial organogenesis. In the developing kidney, the epithelial ureteric bud invades the metanephric mesenchyme, which directs the ureteric bud to undergo repeated branching. A soluble factor(s) in the conditioned medium of a metanephric mesenchyme cell line is essential for multiple branching morphogenesis of the isolated ureteric bud. The identity of this factor had proved elusive, but it appeared distinct from factors such as HGF and EGF receptor ligands that have been previously implicated in branching morphogenesis of mature epithelial cell lines. Using sequential column chromatography, we have now purified to apparent homogeneity an 18 kDa protein, pleiotrophin, from the conditioned medium of a metanephric mesenchyme cell line that induces isolated ureteric bud branching morphogenesis in the presence of glial cell-derived neurotrophic factor. Pleiotrophin alone was also found to induce the formation of branching tubules in an immortalized ureteric bud cell line cultured three-dimensionally in an extracellular matrix gel. Consistent with an important role in ureteric bud morphogenesis during kidney development, pleiotrophin was found to localize to the basement membrane of the developing ureteric bud in the embryonic kidney. We suggest that pleiotrophin could act as a key mesenchymally derived factor regulating branching morphogenesis of the ureteric bud and perhaps other embryonic epithelial structures.     

Light and Electron Microscopic Investigation of Equine Synovial Membrane

Light and electron microscopic examination was made on equine synovial membrane from 23 healthy joints, nine joints with synovitis caused by intraarticular fracture and 10 joints with synovitis caused by osteochondrosis dissecans. Histologically as well as ultrastructurally the equine synovial membrane from healthy joints was of principally the same character as described in other species. Three types of synovial membrane — areolar, fibrous and adipose — and two types of lining cell were distinguished histologically. Ultrastructurally three types of lining cells were distinguished: A and Β type and an intermediate cell type. In healthy joints they were loosely arranged, parallel to the joint surface in an intercellular matrix, which was in direct continuity with the joint space. In joints with intraarticular fracture there was mild inflammation of the synovial membrane. There was elongation and hyperplasia of the lining cells with a relative increase in type A cells. The cell surface of lining cells was increased through filopodia. There was also an increase in cytoplasmic organelles i.e. hyperplasia of rough endoplasmic reticulum and Golgi complexes in Β type cells and an increase in lysosomes, and increased numbers of vesicles of varying types in A cells. In joints with osteochondrosis dissecans the lining cell hyperplasia and the inflammation in the synovial membrane were more prominent. Ultrastructurally the same alterations as in the previous group were seen including a relative increase in the number of A cells but degenerative changes were common in the lining cells. These changes were dilatation and vesiculation of rough endoplasmic reticulum, mitochondrial condensation, dilatation of the nuclear envelope and loss of plasma membranes, leading to disintegration of cells.     

Distribution of immunoreactive fibronectin and collagen (type I, III, IV) in mouse joints. Fibronectin, an essential component of the synovial cavity border

Hyaluronic acid traditionally has been held to play a major role in the control of transsynovial exchanges and in the biomechanical properties of synovial fluid and cartilage surfaces. As with previous ultrastructural observations, immunohistochemical data show that a more complex differentiation must be envisaged for interstitial tissues bordering the synovial cavity. In particular, the elective concentration of fibronectin in the lining layer of the synovial membrane and its presence as a fine layer at the articular cartilage surfaces indicate that this glycoprotein, along with hyaluronic acid, may play an important role in joint physiology. Only immunoreactivity for type III procollagen is diffusely distributed in the entire synovial membrane and, in addition, is found associated with fibronectin immunoreactivity at the articular cartilage surfaces.     

Self-differentiation of human fetal lung organ culture: the role of prostaglandins PGE2 and PGF2 alpha

Addition of PGE2, but not PGF2 alpha, to fetal lung organ cultures accelerates the process of self-differentiation with increased dilatation of terminal airsacs and differentiation of the epithelial lining. Indomethacin reduces the endogenous production by organ cultures of PGE2, PGF2 alpha, 13,14-dihydro-15-keto-PGE2, and 13,14-dihydro-15-keto-PGF2 alpha and retards the process of self-differentiation. Prolonged exposure of cultures to indomethacin results in cell necrosis. Indomethacin inhibition of self-differentiation can be reversed and accelerated by the addition of PGE2. Addition of PGF2 alpha in the presence of indomethacin prevents indomethacin-associated cell necrosis but does not accelerate dilatation or differentiation beyond that of cultures in sera-free media without additions. We propose that the endogenous production of PGE2 is a key process in the mechanism of self-differentiation of human fetal lung in organ culture.     

A comparative study of the urinary bladder and the intramural portion of the ureter.

The bladder belonging to eleven mammalian species were investigated, and as a result the following observations were made: (1) a submucous ureter was found in the case of most of the species examined; (2) histological investigation revealed three types of intramural ureters, and (3) downward extension of ureteric musculature, separate from bladder musculature, to the seminal colliculus in the male, or to the lower end of the urethra in the female, was found. A suggestion is presented to explain the manner by which the ureteric openings change their relations to those of the mesonephric ducts development. It seems that 'after absorption of the common segment of the Wolffian duct, breaking down of the ventral wall of ureter' is the most likely process.     

Ultrasonography compared with intravenous urography in the investigation of adults with haematuria.

OBJECTIVE--To compare ultrasonography with intravenous urography in the investigation of adults with haematuria. DESIGN--Prospective study entailing the examination of all patients with both investigations concurrently. The investigations were performed independently on routine lists by different duty radiologists. Each was aware of the details of the request form but not of the findings of the other investigation. SETTING--Radiology department of a teaching hospital. PATIENTS--155 Consecutive adult patients (aged 18-93) referred from general practitioners and hospital outpatient clinics with a history of haematuria. FOLLOW UP--When results of both examinations proved normal no clinical or radiological follow up was sought. All abnormal findings of either investigation were correlated with results of subsequent imaging studies or operative findings. RESULTS--81 Patients (52%) had normal findings on urography and ultrasonography. Overall, the findings of ultrasonography concurred with those of urography in 144 cases (93%). Among the discrepant findings of the two investigations ultrasonography missed two ureteric calculi; one was in a non-dilated ureter, and in the other case ultrasonography detected the secondary ureteric dilatation. Ultrasound examination alone detected four bladder tumours not visible on urography with sizes ranging from 5 to 21 mm, representing one fifth of the 20 cystoscopically proved bladder tumours detected in the series. Ultrasonography detected all the 22 neoplastic lesions discovered in the study (20 bladder, two renal). Ultrasonography clarified the nature of renal masses evident in three urograms (simple cysts). CONCLUSIONS--Ultrasonography is a safe and accurate method of investigating the urinary tract in adults with haematuria. When combined with a single plain abdominal radiograph it proved to be superior to urography as the primary imaging study in this series. Ultrasonography should certainly be preferred to urography if cystoscopy is not planned. No urothelial tumours of the upper urinary tract were found in the series, reflecting their rarity. For those patients in whom ultrasonography and plain radiography have shown no abnormality and in whom cystoscopic appearances are normal urography would be advisable to exclude urothelial tumours of the upper urinary tract.     

An ultrastructural study of the cercarial excretory system in Bucephaloides gracilescens and Prosorhynchus squamatus

The ultrastructure of the flame cells, capillaries, collecting tubes, excretory bladder, excretory atrium, caudal vesicle, lateral caudal ducts and excretory pores of cercariae of Bucephaloides gracilescens (Rudolphi, 1819) Hopkins, 1954 and Prosorhynchus squamatus Odhner, 1905 (Digenea: Bucephalidae) is described. Both species are essentially similar except for some details. The terminal parts of the protonephridia have all the structural features that are typical of trematodes. The collecting tubes in the cercarial body are composed of cells that are wrapped around the lumen. The main collecting tubes are joined to the excretory bladder syncytium by septate junctions. Features of P. squamatus excretory bladder epithelium indicate that it is involved in secretory activity, but this is not the case in B. gracilescens. In both species the luminal surface of the excretory bladder epithelium is increased by lamellae, and the basal plasma membrane forms invaginations. In the bladder syncytium of P. squamatus both apical lamellae and basal invaginations are more developed and mitochondria are also more numerous. The excretory atrium is lined by a syncytium with nucleated cytons located in the surrounding parenchyma. The atrium lining is not continuous with the body tegument and possesses specific secretory inclusions and a thick glycocalyx. Septate junctions connect the atrium syncytium to the excretory bladder epithelium at its anterior end and to the syncytial excretory epithelium lining the caudal vesicle and the lateral caudal ducts at its posterior. In the excretory pores the caudal duct syncytium is joined to the tegument by septate desmosomes.     

Organotypic growth and differentiation of human mammary gland in sponge-gel matrix supported histoculture

Summary Blocks of breast tissue obtained during radical mastectomies from 23 patients with mammary gland carcinomas were used for cultivation in native-state, gel-supported histocultures. We show that the human mammary gland can be successfully maintained in this system so that normal epithelial breast structures proliferate and undergo differentiation for several weeks and a well-developed system of ducts and lobules is formed. Using antibodies to individual keratins 17 and 8 we have shown for the first time that ducts and alveoles developing in vitro undergo differentiation into the lining epithelium and myoepithelium in the same way as mammary gland epithelium in vivo. Growth of epithelial structures in vitro is also accompanied by the development of continuous basal membrane.     

The importance of Rho-associated kinase-induced Ca2+ sensitization as a component of electromechanical and pharmacomechanical coupling in rat ureteric smooth muscle

Ureteric peristalsis, which occurs via alternating contraction and relaxation of ureteric smooth muscle, ensures the unidirectional flow of urine from the kidney to the bladder. Understanding of the molecular mechanisms underlying ureteric excitation–contraction coupling, however, is limited. To address these knowledge deficits, and in particular to test the hypothesis that Ca²⁺ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway plays an important role in ureteric smooth muscle contraction, we carried out a thorough characterization of the electrical activity, Ca²⁺ signaling, MYPT1 (myosin targeting subunit of myosin light chain phosphatase, MLCP) and myosin regulatory light chain (LC₂₀) phosphorylation, and force responses to membrane depolarization induced by KCl (electromechanical coupling) and carbachol (CCh) (pharmacomechanical coupling). The effects of ROK inhibition on these parameters were investigated. We conclude that the tonic, but not the phasic component of KCl- or CCh-induced ureteric smooth muscle contraction is highly dependent on ROK-catalyzed phosphorylation of MYPT1 at T855, leading to inhibition of MLCP and increased LC₂₀ phosphorylation.     

Hoxa11 and Hoxd11 regulate branching morphogenesis of the ureteric bud in the developing kidney

Hoxa11 and Hoxd11 are functionally redundant during kidney development. Mice with homozygous null mutation of either gene have normal kidneys, but double mutants have rudimentary, or in extreme cases, absent kidneys. We have examined the mechanism for renal growth failure in this mouse model and find defects in ureteric bud branching morphogenesis. The ureteric buds are either unbranched or have an atypical pattern characterized by lack of terminal branches in the midventral renal cortex. The mutant embryos show that Hoxa11 and Hoxd11 control development of a dorsoventral renal axis. By immunohistochemical analysis, Hoxa11 expression is restricted to the early metanephric mesenchyme, which induces ureteric bud formation and branching. It is not found in the ureteric bud. This suggests that the branching defect had been caused by failure of mesenchyme to epithelium signaling. In situ hybridizations with Wnt7b, a marker of the metanephric kidney, show that the branching defect was not simply the result of homeotic transformation of metanephros to mesonephros. Absent Bf2 and Gdnf expression in the midventral mesenchyme, findings that could by themselves account for branching defects, shows that Hoxa11 and Hoxd11 are necessary for normal gene expression in the ventral mesenchyme. Attenuation of normal gene expression along with the absence of a detectable proliferative or apoptotic change in the mutants show that one function of Hoxa11 and Hoxd11 in the developing renal mesenchyme is to regulate differentiation necessary for mesenchymal-epithelial reciprocal inductive interactions.     

B-cells of the synovial membrane. II. Differentiation during development of the synovial cavity in the mouse

Summary Study of pre- and postnatal development of the metatarsophalangeal joint of the mouse shows that the synovial cavity (SC) forms before any differentiation of the synovial mesenchyme. The primitive cleft results from degradation of a thin vascular mesenchymal layer in direct contact with the chondrogenic layers. Differentiation of the synovial membrane coincides with clarification of the SC (3rd to 6th day of postnatal life). When dilatation of the SC occurs (6th to 8th day), the two intimal cells types (A- and B-cells) are well identified. The B-cells already show typical features at day 6; their content of typical dense secretory vesicles is comparable to that of the adult B-cells at day 13. The specific secretory function of B-cells could be correlated with the particular structure of the intimal interstitial tissue and could account for the origin of some protein(s) of the synovial fluid.ERA 178 (Neuroendocrinologie Comparée) du CNRS et INSERM     

Muscle cell differentiation in the ascidian heart.

The simple tubular heart of tunicates consists of a single layer of striated muscle cells which display distinct electrical properties at the luminal and extraluminal surfaces. We have investigated heart morphogenesis and cytodifferentiation in the ascidian, Botryllus schlosseri. Myocardium is formed by invagination from the wall of the heart primordium. Cell polarity is clearly apparent in the undifferentiated cells of the heart primordium and is maintained throughout the whole course of cardiac muscle differentiation. Myocardium cells are initially cubic in appearance, then undergo a progressive flattening with the formation of characteristic protrusions at the luminal surface. The first sign of muscle cell differentiation is the formation of close associations between sarcoplasmic reticulum cisterns and the plasma membrane at the luminal and junctional surface. Myofibrillogenesis also occurs near the luminal surface, whereas the cell portion facing the pericardial cavity maintains an undifferentiated structure. The findings support the hypothesis that membrane changes precede and influence myofibril formation in developing muscle cells.     

Ultrastructure of cell surface coat (glycocalyx) in rat urinary bladder epithelium

Earlier statements to the contrary, the present study demonstrates the presence of a cell surface coat (glycocalyx) on the luminal plasma membrane of the superficial transitional epithelial cells lining the urinary bladder of male Buffalo rats. This coat was demonstrated with ruthenium red, an electron dense stain, which revealed a surface layer, 60-80 A thick, separated from the outer leaflet of the plasma membrane by an electron lucent layer, approximately 30 A thick. The structure of the glycocalyx was not affected by 12 weeks of treatment with dibutylnitrosamine, a known bladder carcinogen.     

Resistance of the alimentary canal of Daphnia magna Straus to the effect of enteropathogenic NAG-vibrios

The interrelations between 30 Daphnias and enteropathogenic NAG-vibrios were experimentally investigated. Using histological and immunoserological techniques, NAG-vibrio administration into the medium inhabited by Crustaceous was shown to cause no pathological changes in the animal alimentary canal. Daphnias used these organisms as food. The intestinal epithelium is well protected from mechanical injury by peritrophic membrane, chitinous lining and peritrophic cavity where digestion takes place.     

ELECTRON MICROSCOPY OF THE HUMAN SYNOVIAL MEMBRANE

The structure of the lining cells at the surface of the synovial membrane facing the joint cavity has been studied by electron microscopy. The long cytoplasmic processes of these cells appear to be oriented toward the surface of the membrane, where they overlap and intertwine. The matrix of the lining cells contains dense material but no fibers with the periodicity of collagen. The lining cells are divided into two cell types or states of activity on the basis of their cytoplasmic contents. Type A is more numerous and contains a prominent Golgi apparatus, numerous vacuoles (0.4 to 1.5 microns in diameter) containing varying amounts of a dense granular material, many filopodia, mitochondria, intracellular fibrils, and micropinocytotic-like vesicles. Type B contains large amounts of ergastoplasm with fewer large vacuoles, micropinocytotic-like vesicles, and mitochondria. The probable functions of these cells are discussed in the light of current knowledge of the metabolism and function of the synovial membrane.     

Stromal progenitors are important for patterning epithelial and mesenchymal cell types in the embryonic kidney

Growth and expansion of the embryonic kidney is driven in large part by continuous branching morphogenesis and nephron induction that occurs in a restricted domain beneath the renal capsule called the nephrogenic zone. Here, new ureteric bud branches and nephron aggregates form surrounded by a layer of cortical stromal cell progenitors. The boundaries and inductive activities of the nephrogenic zone are maintained as the kidney grows. As new ureteric bud branches and nephrogenic aggregates form, older generations of ureteric bud branches, renal vesicles and stromal progenitors are displaced from the nephrogenic zone and undergo further differentiation surrounded by medullary stroma, a different population of stromal cells. Recent studies suggest that cortical and medullary stromal progenitors may be an important source of signals that maintain outer and inner zones of differentiation in the embryonic kidney, and regulate distinct events important for differentiation of nephrons and the collecting duct system.