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P Hardman

Publications and source records attributed to P Hardman.

13 recordsLinked to original sources
PubMed1997

A molecular and genetic analysis of renalglomerular capillary development.

The adult kidney is highly vascular and receives about 20% of the cardiac output, yet the mode of development of the glomerular capillaries is not fully understood. At the inception of nephrogenesis the condensed metanephric mesenchyme contains no patent capillaries. However, in this current study we detected vascular endothelial growth factor (VEGF) mRNA and protein in uninduced mouse E11 metanephric mesenchyme and in cell lines from this tissue. Moreover, transcripts for receptor tyrosine kinases which are markers of endothelial precursors (VEGFR-1/Flt-1, VEGFR-2/Flk-1 and Tie-1) were expressed by the E11 mesenchyme. In transgenic mice, Tie1/LacZ-expressing cells were identified in E11 renal mesenchyme when patent vessels were absent. Moreover, a similar pattern of transgene expression was detected within intermediate mesoderm condensing to form metanephric mesenchyme. When Tie-1/LacZ E11 metanephroi were transplanted into the nephrogenic cortex of wild-type mice, transgene-expressing capillary loops were detected in glomeruli developing in donor tissue. In contrast, glomerular Tie-1/LacZ-positive vessels never developed in rudiments in organ culture. We postulate that endothelial precursors are present at the inception of the mouse nephrogenesis, and these differentiate and undergo morphogenesis into glomerular capillaries when experimental conditions resemble those found in the metanephros in vivo.

Journal Article↗
PubMed1996

Deregulation of cell survival in cystic and dysplastic renal development.

Various aberrations of cell biology have been reported in polycystic kidney diseases and in cystic renal dysplasias. A common theme in these disorders is failure of maturation of renal cells which superficially resemble embryonic tissue. Apoptosis is a feature of normal murine nephrogenesis, where it has been implicated in morphogenesis, and fulminant apoptosis occurs in the small, cystic kidneys which develop in mice with null mutations of bcl-2. Therefore, we examined the location and extent of apoptosis in pre- and postnatal samples of human polycystic and dysplastic kidney diseases using propidium iodide staining, in situ end-labeling and electron microscopy. In dysplastic kidneys cell death was prominent in undifferentiated cells around dysplastic tubules and was occasionally found in cystic epithelia. The incidence of apoptosis was significantly greater than in normal controls of comparable age both pre- and postnatally. In the polycystic kidneys there was widespread apoptosis in the interstitium around undilated tubules distant from cysts, in undilated tubules between cysts and in cystic epithelia. The level of apoptosis compared to controls was significantly increased postnatally. A similar increase of cell death was also noted in the early and late stages of renal disease in the polycystic cpk/cpk mouse model. We speculate that deregulation of cell survival in these kidneys may reflect incomplete tissue maturation, and may contribute to the progressive destruction of functional kidney tissue in polycystic kidneys and the spontaneous involution reported in cystic dysplastic kidneys.

Animals↗
PubMed1995

Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros.

Several lines of evidence suggest that hepatocyte growth factor/scatter factor (HGF/SF), a soluble protein secreted by embryo fibroblasts and several fibroblast lines, may elicit morphogenesis in adjacent epithelial cells. We investigated the role of HGF/SF and its membrane receptor, the product of the c-met protooncogene, in the early development of the metanephric kidney. At the inception of the mouse metanephros at embryonic day 11, HGF/SF was expressed in the mesenchyme, while met was expressed in both the ureteric bud and the mesenchyme, as assessed by reverse transcription PCR, in situ hybridization, and immunohistochemistry. To further investigate the expression of met in renal mesenchyme, we isolated 13 conditionally immortal clonal cell lines from transgenic mice expressing a temperature-sensitive mutant of the SV-40 large T antigen. Five had the HGF/SF+/met+ phenotype and eight had the HGF/SF-/met+ phenotype. None had the HGF/SF+/met- nor the HGF/SF-/met- phenotypes. Thus the renal mesenchyme contains cells that express HGF/SF and met or met alone. When metanephric rudiments were grown in serum-free organ culture, anti-HGF/SF antibodies (a) inhibited the differentiation of metanephric mesenchymal cells into the epithelial precursors of the nephron; (b) increased cell death within the renal mesenchyme; and (c) perturbed branching morphogenesis of the ureteric bud. These data provide the first demonstration for coexpression of the HGF/SF and met genes in mesenchymal cells during embryonic development and also imply an autocrine and/or paracrine role for HGF/SF and met in the survival of the renal mesenchyme and in the mesenchymal-epithelial transition that occurs during nephrogenesis. They also confirm the postulated paracrine role of HGF/SF in the branching of the ureteric bud.

Animals↗
PubMed1995

The hepatocyte growth factor/scatter factor (HGF/SF) receptor, met, transduces a morphogenetic signal in renal glomerular fibromuscular mesangial cells.

Previous studies have demonstrated that hepatocyte growth factor/scatter factor (HGF/SF) is secreted by mesenchymal cells and that it elicits motility, morphogenesis and proliferation of epithelia expressing the met receptor. We now report that HGF/SF may act as an autocrine factor in fibromuscular renal mesangial cells. These cells mechanically support glomerular endothelia, control the rate of plasma ultrafiltration and are implicated in the pathogenesis of a variety of chronic renal diseases. We detected met protein in the vascular stalk of metanephric glomeruli and in the mature mesangium. Mesangial lines from a mouse transgenic for a temperature-sensitive simian virus 40 T antigen expressed met mRNA and protein, and recombinant HGF/SF phosphorylated the met receptor tyrosine kinase. Cells were immortal in the permissive condition and HGF/SF enhanced proliferation in a defined medium. In the absence of the immortalising protein, division ceased and recombinant HGF/SF caused multipolar cells to become bipolar. The factor diminished stress fibres, their focal contacts and immunostaining for extracellular fibronectin, hence suggesting reduced substratum adhesion and enhanced motility. Mesangial lines also expressed HGF/SF mRNA and secreted bioactive factor; immunocytochemistry showed both ligand and receptor in individual cells. HGF/SF blocking antibody aggregated the cells, suggesting that mesangial-derived factor affects basal cell conformation in an autocrine manner. We conclude that mesangial cells express both HGF/SF and met, and the factor induces morphogenesis of cultured mesangial cells. Therefore HGF/SF may have an autocrine role in mesangial biology but further studies are now required to investigate the potential importance of the factor in vivo.

Animals↗
PubMed1994

Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight.

Organ culture of embryonic mouse lung and pancreas rudiments has been used to investigate development and differentiation, and to assess the effects of microgravity on culture differentiation, during orbital spaceflight of the shuttle Endeavour (mission STS-54). Lung rudiments continue to grow and branch during spaceflight, an initial result that should allow future detailed study of lung morphogenesis in microgravity. Cultured embryonic pancreas undergoes characteristic exocrine acinar tissue and endocrine islet tissue differentiation during spaceflight, and in ground controls. The rudiments developing in the microgravity environment of spaceflight appear to grow larger than their ground counterparts, and they may have differentiated more rapidly than controls, as judged by exocrine zymogen granule presence.

Animals↗
PubMed1993

Growth and morphogenesis of embryonic mouse organs on non-coated and extracellular matrix-coated Biopore membrane.

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non-coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM-coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.

Animals↗
PubMed1993

Extracellular matrix and growth factors in branching morphogenesis.

The unifying hypothesis of the NSCORT in gravitational biology postulates that the ECM and growth factors are key interrelated components of a macromolecular regulatory system. The ECM is known to be important in growth and branching morphogenesis of embryonic organs. Growth factors have been detected in the developing embryo, and often the pattern of localization is associated with areas undergoing epithelial-mesenchymal interactions. Causal relationships between these components may be of fundamental importance in control of branching morphogenesis.

Animals↗
PubMed1992

Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy.

The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

Animals↗
PubMed1992

Collagen in organ development.

It is important to know whether microgravity will adversely affect developmental processes. Collagens are macromolecular structural components of the extracellular matrix (ECM) which may be altered by perturbations in gravity. Interstitial collagens have been shown to be necessary for normal growth and morphogenesis in some embryonic organs, and in the mouse salivary gland, the biosynthetic pattern of these molecules changes during development. Determination of the effects of microgravity on epithelial organ development must be preceded by crucial ground-based studies. These will define control of normal synthesis, secretion, and deposition of ECM macromolecules and the relationship of these processes to morphogenesis.

Animals↗
PubMed1992

Alterations in biosynthetic accumulation of collagen types I and III during growth and morphogenesis of embryonic mouse salivary glands.

We examined the biosynthetic patterns of interstitial collagens in mouse embryonic submandibular and sublingual glands cultured in vitro. Rudiments explanted on day 13 of gestation and cultured for 24, 48, and 72 h all synthesized collagen types I, III, and V. However, while the total incorporation of label into collagenous proteins did not change over the three-day culture period, the rate of accumulation of newly synthesized types I and III did change. At 24 h, the ratio of newly synthesized collagen types I:III was approximately 2, whereas at 72 h, the ratio was approximately 5. These data suggest that collagen types I and III may be important in initiation of branching in this organ, but that type I may become dominant in the later stages of development and in maintenance of the adult organ.

Animals↗
PubMedDate not supplied

Glial cell line-derived neurotrophic factor stimulates ureteric bud outgrowth and enhances survival of ureteric bud cells in vitro.

Development of epithelial organs requires co-ordinated interactions between epithelial and mesenchymal tissues. Studies using null mutant mice have indicated that the ret receptor and its ligand, glial cell line-derived neurotrophic factor (GDNF), are crucial for initiation of development of the metanephric kidney. However, the role of this signalling system in other branching organs has not been analysed. Here we describe expression studies of ret, GDNF, and a co-receptor for GDNF (GDNFR alpha) in the developing mouse metanephros, lung, and submandibular salivary gland. Also, we examined the role of this signalling system in the development of these organs in vitro. In situ hybridisation revealed differences in the spatial distribution of the three transcripts in the different organs. At the initiation of metanephric development, late on embryonic day 10 (E10), ret and GDNFR alpha were detected in the Wolffian duct (including the presumptive ureteric bud) whilst the presumptive metanephric, mesenchyme expressed GDNFR alpha and GDNF. Later in development, all three transcripts were restricted to the nephrogenic zone. In contrast, expression in the lung was not detectable by in situ hybridisation until after initiation of development, at E13.5. At this time ret was expressed throughout the epithelium; GDNF was detected throughout the mesenchyme, and GDNFR alpha was present in the proximal epithelium and mesenchyme only. Ret and GDNF were not detected in the epithelium or mesenchyme of the developing salivary gland, however, GDNFR alpha was expressed in the mesenchyme at E13.5 and E16.5. Functional studies demonstrated that in organ culture, GDNF significantly increased branching morphogenesis of the E11.5 metanephros, and induced the formation of ectopic ureteric buds from the base of the bud and from the Wolffian duct. The development of lung and salivary primordia were not affected under similar growth conditions. In a novel ureteric bud primary culture system, GDNF significantly increased cell numbers at 24 and 48 h. In cells cultured on laminin this increase was due to increased survival and proliferation, whereas in cells cultured on fibronectin, only survival was enhanced. Our data suggest that GDNF stimulates outgrowth of the ureteric bud, in part, by enhancing cell survival and possibly by increasing proliferation.

Aging↗