Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme

Nephrogenesis in the mouse kidney begins at embryonic day 11 and ends approximately 10 days postpartum. During this period, new nephrons are continually being generated from a stem-cell population-the nephrogenic mesenchyme-in response to signals emanating from the tips of the branching ureter. Relatively little is known about the mechanism by which the nephrogenic mesenchyme cell population is maintained at the tips of the ureter in the presence of signals promoting tubulogenesis. Previous studies have shown that a loss of Bmp7 function leads to kidney defects that are a likely result of progressive loss of nephrogenic mesenchyme by apoptosis. The studies presented here demonstrate that BMP7 signaling can prevent apoptosis in explants of metanephric mesenchyme. The surviving mesenchyme cell population, however, is not competent to respond to signals promoting tubulogenesis. In conjunction with FGF2, BMP7 promotes growth and maintains competence of the mesenchyme in vitro. In addition, FGF2 and BMP7 signaling, both independently and in combination, inhibit tubulogenesis. Interestingly, FGF2 and BMP7 also promote expansion of the stromal progenitor cell population in whole kidney culture. Because BMP7 is not produced by stromal progenitor cells, these data suggest a novel interaction between the nephrogenic mesenchyme and stromal progenitor cell populations. A model for the regulation of nephrogenesis by FGF and BMP signaling is presented.     

Progranulin (acrogranin/PC cell-derived growth factor/granulin-epithelin precursor) is expressed in the placenta, epidermis, microvasculature, and brain during murine development.

The growth factor progranulin (acrogranin/PC-derived growth factor/granulin-epithelin precursor) promotes onset of blastocyst cavitation and is required for neonatal hypothalamic sexual differentiation. Little is known, however, of the range of developmental processes in which it is involved. We used in situ hybridization to investigate progranulin expression in murine embryos. Progranulin mRNA is expressed in maternal and embryonic components during early establishment of pregnancy. Abundant expression is observed in the early decidualizing uterine stroma and glands. In the embryo, the trophoblast giant cells at the interface of placental exchange sites (both choriovitelline and chorioallantoic placenta) show strong expression. The gastrulating epiblast and mesenchyme (intraembryonic and extraembryonic mesenchyme) all revealed activity. The allantois and yolk sac mesenchyme (site of early hemopoiesis) were positive, as were later phases of active vessel formation (pia mater of brain, epicardium of the heart). In the urogenital system, it was expressed in Sertoli cells and in kidney tubules. It was highly expressed in proliferating epidermal cells. During epidermal appendage formation, the early epithelial bud was positive, but the forming duct and differentiating adjacent mesenchyme was negative. It is widely distributed during central nervous system development and the peripheral nervous system (dorsal root ganglia and sympathetic ganglia). Based on the pattern of progranulin gene expression, we propose proliferative and developmental roles for progranulin in establishing pregnancy, during gastrulation, and during embryonic development of the epidermis, nervous system, blood vessel, formation, and spermatogenesis.     

Presence of the long chain form of polysialic acid of the neural cell adhesion molecule in Wilms'' tumor. Identification of a cell adhesion molecule as an oncodevelopmental antigen and implications for tumor histogenesis.

The long chain form of polysialic acid characteristic of the low adhesive embryonic form of the neural cell adhesion molecule NCAM is temporally and spatially expressed in developing kidney but undetectable in normal adult kidney. Therefore, this molecule represents a developmentally regulated antigen in kidney contrasted with neural tissue, where it is also detectable in the adult brain. This investigation of 25 Wilms' tumors comprising all different histologic types demonstrates expression of this molecule under conditions of malignant growth. Immunostaining was observed in Wilms' tumors with both a monoclonal anti-polysialic acid antibody and a polyclonal anti-NCAM polypeptide antiserum. Intense cell surface staining sensitive to endosialidases specifically hydrolyzing alpha 2,8 linked (poly)sialic acid was detectable in blastemal regions, and weaker, variable labeling was seen over tubules and glomeruloid bodies. The stroma was not stained. This is evidence indicating that Wilms' tumor originates from the embryonic equivalent of induced metanephrogenic mesenchyme. It seems unlikely however, that the stroma is derived from the blastema. The same high molecular mass broad band typical of the embryonic form of NCAM was revealed by immunoblot analysis of homogenates from Wilms' tumor as well as from embryonic kidney and brain. In situ hybridization demonstrated the presence of mRNA for NCAM in all but stromal elements of Wilm's tumors. Thus, polysialic acid is present on NCAM and represents a new oncodevelopmental antigen in human kidney. Polysialic acid was greatly reduced or absent by immunohistochemistry and immunoblotting in necrotic tumor areas.     

Disruption of anterior segment development by TGF-beta1 overexpression in the eyes of transgenic mice.

Previous experiments showed that transgenic mice expressing a secreted self-activating transforming growth factor (TGF) -beta1 did not show a phenotype in the lens and cornea until postnatal day 21, when anterior subcapsular cataracts, sporadic thickening of the corneal stroma, and thinning of the corneal epithelium were noted (Srinivasan et al., 1998). To examine the effects of higher concentrations of TGF-beta1 on the lens and cornea, we constructed transgenic mice harboring the strong, lens-specific chicken betaB1-crystallin promoter driving an activated porcine TGF-beta1 gene. In contrast to the earlier study, the transgenic mice had microphthalmic eyes with closed eyelids. Already at embryonic day (E) 13.5, the future cornea of the transgenic mice was threefold thicker than that of wild-type littermates due to increased proliferation of corneal stromal mesenchyme cells. Staining of fibronectin and thrombospondin-1 was increased in periocular mesenchyme. At E17.5, the thickened transgenic corneal stroma was vascularized and densely populated by abundant star-shaped, neural cell adhesion molecule-positive cells of mesenchymal appearance surrounded by irregular swirls of collagen and extracellular matrix. The corneal endothelium, anterior chamber, and stroma of iris/ciliary body did not develop, and the transgenic cornea was opaque. Fibronectin, perlecan, and thrombospondin-1 were elevated, whereas type VI collagen decreased in the transgenic corneal stroma. Stromal mesenchyme cells expressed alpha-smooth muscle actin as did lens epithelial cells and cells of the retinal pigmented epithelium. By E17.5, lens fiber cells underwent apoptotic cell death that was followed by apoptosis of the entire anterior lens epithelium between E18.5 and birth. Posteriorly, the vitreous humor was essentially absent; however, the retina appeared relatively normal. Thus, excess TGF-beta1, a mitogen for embryonic corneal mesenchyme, severely disrupts corneal and lens differentiation. Our findings profoundly contrast with the mild eye phenotype observed with presumably lower levels of ectopic TGF-beta and illustrate the complexity of TGF-beta utilization and the importance of dose when assessing the effects of this growth factor.     

Clear cell sarcoma of the kidney demonstrates an embryonic signature indicative of a primitive nephrogenic origin

Clear cell sarcoma of the kidney (CCSK) is a tumor affecting children with a median age of 3 years at diagnosis. The cell of origin of CCSK is unknown and data on the molecular changes giving rise to CCSK is scarce. This has hindered the identification of positive diagnostic markers and development of molecularly targeted treatment protocols for CCSK. We have characterized a panel of CCSK to gain information regarding its molecular profile and possible origin. High‐resolution genomic analysis with single nucleotide polymorphism array of 37 tumors did not reveal any clues to the mechanisms behind tumor development as remarkably few genetic imbalances were found. Gene expression analysis revealed a highly characteristic gene signature, enriched for pathways involved in embryonic development, including kidney formation. The presence of markers for two different developmental lineages in the embryonic kidney was therefore investigated in the tumor cells. FOXD1 which identifies cells giving rise to stromal elements, and CITED1, a marker for cells primed for nephrogenic epithelial differentiation, were both highly expressed in CCSK. In addition, the early embryonic marker OSR1 was expressed at higher levels in CCSK than in Wilms tumor, normal fetal kidney or adult kidney. As this marker discriminates the intermediate mesoderm from other mesodermal structures, our study could suggest that CCSK arises from a mesodermal cell type that retains the capacity to initiate differentiation towards both nephrons and stroma, but remains locked in a primitive state. © 2014 Wiley Periodicals, Inc.     

Essential roles of Sall family genes in kidney development

We isolated a mouse Sall1, a mammalian homologue of the Drosophila region-specific homeotic gene spalt (sal), and found that mice deficient in Sall1 die in the perinatal period from kidney agenesis. Sall1 is expressed in the metanephric mesenchyme surrounding the ureteric bud, and the homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth. Therefore Sall1 is essential for ureteric bud invasion, the initial key step for metanephros development. We also set up an in vitro culture system, using NIH3T3 cells stably expressing Wnt4 as a feeder layer, to identify kidney progenitors in the metanephric mesenchyme. In this culture condition, a single renal progenitor in the mesenchyme forms colonies consisting of several types of epithelial cells that exist in glomeruli and renal tubules. We found that only cells strongly expressing Sall1 (Sall1-GFP(high) cells) form colonies and that they reconstitute a three-dimensional kidney structure in an organ culture setting. Thus our colony-forming assay, which identifies multipotent progenitors in the embryonic mouse kidney, can be used for examining mechanisms of renal progenitor differentiation.     

Expression of tenascin, type IV collagen and laminin during human intrahepatic bile duct development and in intrahepatic cholangiocarcinoma

Expression of tenascin, type IV collagen and laminin during human intrahepatic bile duct development and in cholangiocarcinoma was examined by immunohistochemistry. In the developing hilar bile ducts, tenascin was expressed in the mesenchyme around the epithelial cells migrating from the ductal plate into the mesenchyme at 10–14 weeks of gestation. Tenascin was also expressed in the mesenchyme around newly formed hilar bile ducts at 15-20 weeks of gestation, but its expression disappeared after 21 weeks of gestation. Type IV collagen and laminin were expressed around the ductal plate, around epithelial cells migrating from the ductal plate into the mesenchyme, and around newly formed hilar bile ducts, and their expression was present throughout fetal life. By contrast, in the development of peripheral bile ducts, tenascin expression was not found. Type IV collagen and laminin were identified around the ductal plate, migrating epithelial cells and peripheral bile ducts. In cholangiocarcinoma, tenascin and type IV collagen were expressed in the stroma, but laminin was not identified. These findings suggest that tenascin may play a role in hilar bile duct development and that type IV collagen and laminin may play a role in both hilar and peripheral bile duct development. Expression of tenascin and type IV collagen in the stroma of cholangiocarcinoma may be the result of malignant transformation of intrahepatic biliary epithelium; tenascin in peritumoral stroma may stimulate carcinoma cell proliferation and growth in cholangiocarcinoma.     

Immunohistochemical localization of glutathione-S-transferase and glutathione peroxidase in adult Syrian hamster tissues and during kidney development.

Tissues from adult Syrian hamsters were studied with immunoperoxidase techniques using polyclonal antibodies to glutathione-S-transferase (rat liver and human placental enzymes) and human erythrocyte glutathione peroxidase. Most tissues immunostained similarly with these antibodies. Most notable was the cytoplasmic staining of mesenchyme tissues, especially smooth muscle, by all three antibodies. Epithelial cells stained distinctively, but usually less intensely than mesenchyme. Epithelial cells from all levels of the gastrointestinal tract, respiratory epithelium, transitional epithelium, and epidermis all showed strong staining with these antibodies. Other epithelial cell types were usually positive but showed less dramatic staining. Most epithelial tissues showed both nuclear and cytoplasmic staining; some also showed cell-surface (eg, cilia) staining. The role of these enzymes in cell differentiation of a stable organ was studied by immunostaining the kidney during its development. Early stroma (13- and 15-day fetuses) of the kidney (metanephric mesenchyme) showed strong cell-surface staining for glutathione transferases and moderate staining for glutathione peroxidase; renal tubules (which are epithelial cells) at this stage were negative for these markers. As renal tubules differentiated, first cytoplasm and then nuclei stained moderately, suggesting that glutathione-S-transferases and glutathione peroxidase are markers of both mesenchymal cells, including embryonic mesenchyme, and terminal differentiation of at least some epithelial cells.     

Mixed epithelial and stromal tumor of the kidney in a 12-year-old girl

Mixed epithelial and stromal tumor of the kidney (MESTK) is a rare kidney neoplasm that almost exclusively occurs in perimenopausal women, and long-term estrogen replacement is relevant to its pathogenesis. Herein is described an atypical case of MESTK uncovered in a 12-year-old premenarcheal girl without a history of prior estrogen use. On surgical specimen it was found that the well-circumscribed tumor measuring 14 cm arose from the lower pole of the right kidney, showing solid and fibrous-cystic areas. Microscopically, it was composed both of epithelial structures similar to renal tubules and stroma comprising non-specific spindle cells. Some intratumoral tubules showed affinities to distal-nephron-specific lectins, and those immunoreactive for proximal-tubule-specific CD15 were also present. In addition, primitive ductal structures were reactive both for CD15 and lectins, but immature epithelial elements typical of nephroblastoma were absent. Spindle cells were positive for actin, desmin and vimentin, and expressed progesterone and estrogen receptors. The tumor was comparable with MESTK, although some epithelia were associated with the immunophenotype of proximal tubules. The patient was free of disease postoperatively for 40 months. In the present case, remnants of the primitive periductal mesenchyme might be promoted to neoplastic cells by a sex-steroid surge during puberty.     

Developmental origins and functions of stromal cells in the normal and diseased mammalian kidney

The kidney is a model developmental paradigm of vertebrate organogenesis. As in many other organs, kidney development involves reciprocal inductive tissue interactions between multiple cell lineages. The most well defined of these interactions occurs between the ureteric bud and the nephrogenic mesenchyme. A population of mesenchymal cells distinct from nephrogenic precursors and termed stromal cells, have been relatively understudied. Yet existing knowledge indicates that stromal cells are critical regulators in the normal and diseased kidney. This commentary reviews current knowledge regarding the origin and functional roles of the stromal cell population during kidney development. Gaps in our current understanding of renal stromal cells and future directions needed to advance this expanding field of study are highlighted. Developmental Dynamics 243:853–863, 2014. © 2014 Wiley Periodicals, Inc.     

Transplantation of embryonic spleen tissue reveals a role for adult non‐lymphoid cells in initiating lymphoid tissue organization

In this report we describe a transplantation system where embryonic spleens are grafted into adult hosts. This model can be used to analyze the cellular and molecular requirements for the development and organization of splenic microenvironments. Whole embryonic day 15 (ED15) spleens, grafted under the kidney capsule of adult mice, were colonized by host‐derived lymphocytes and DC and developed normal splenic architecture. Grafts were also able to form germinal centers in response to T‐dependent antigen. Using this system we demonstrated that adult host‐derived lymphotoxin (LT) α was sufficient for the development of ED15 LTα^?/? grafts. Grafting of ED15 LTα^?/? spleens into RAG^?/? hosts followed by transfer of LT α^?/? splenocytes revealed no requirement for lymphocyte‐derived LT α in the induction of CCL21 or the development of T‐zone stroma. These data suggest that interactions between adult lymphoid‐tissue inducer‐like cells and embryonic stromal cells initiated T‐zone development. Furthermore, adult lymphoid tissue inducer‐like cells were shown to develop from bone marrow‐derived progenitors. The model described here demonstrates a method of transferring whole splenic microenvironments and dissecting the stromal and hematopoietic signals involved in spleen development and organization.     

Transforming growth factor beta1 exerts an autocrine regulatory effect on human endometrial stromal cell apoptosis,involving the FasL and Bcl-2 apoptotic pathways

Transforming growth factor beta1 (TGFbeta1) is expressed in human endometrium. It regulates epithelial cell proliferation and apoptosis. The aim of the present work was to examine the role of TGFbeta1 on human endometrial stromal cell apoptosis. Primary cultures of isolated stromal cells were obtained from biopsies of late secretory phase endometrium. We have found the following: (i) TGFbeta1 induced apoptosis of stromal cells in a time- and dose-dependent manner; (ii) blockade of TGFbeta1's autocrine/paracrine effect by TGFbeta1-neutralizing antibodies diminished the basal rate of stromal cell apoptosis; (iii) semi-quantitative Western blot analysis showed that TGFbeta1 caused a rapid but transient elevation of the pro-apoptotic FasL protein, without affecting the levels of Fas receptor; (iv) TGFbeta1 increased the levels of the anti-apoptotic Bcl-2 and Bcl-xL proteins, while having no significant effects on the pro-apoptotic proteins Bax and Bak, suggesting the activation of a transient survival mechanism activated in stromal cells as a parallel rescue response to the apoptosis-inducing FasL protein. In conclusion, our data provide evidence that TGFbeta1 exerts an autocrine pro-apoptotic effect on human endometrial stroma, via the FasL/Fas system.     

PDGFR-alpha signaling is critical for tooth cusp and palate morphogenesis.

Platelet-derived growth factor receptor alpha (PDGFR-alpha) and PDGF ligands are key regulators for embryonic development. Although Pdgfralpha is spatially expressed in the cranial neural crest (CNC)-derived odontogenic mesenchyme, mice deficient for Pdgfralpha are embryonic lethal, making it impossible to investigate the functional significance of PDGF signaling in regulating the fate of CNC cells during tooth morphogenesis. Taking advantage of the kidney capsule assay, we investigated the biological function of PDGF signaling in regulating tooth morphogenesis. Pdgfralpha and Pdgfa are specifically and consistently expressed in the CNC-derived odontogenic mesenchyme and the dental epithelium, respectively, throughout all stages of tooth development, suggesting a paracrine function of PDGF signaling in regulating tooth morphogenesis. Highly concentrated expression patterns of Pdgfralpha and Pdgfa are associated with the developing dental cusp, suggesting possible functional importance of PDGF signaling in regulating cusp formation. Loss of the Pdgfralpha gene does not affect proper odontoblasts proliferation and differentiation in the CNC-derived odontogenic mesenchyme but perturbs the formation of extracellular matrix and the organization of odontoblast cells at the forming cusp area, resulting in dental cusp growth defect. Pdgfralpha-/- mice have complete cleft palate. We show that the cleft palate in Pdgfralpha mutant mice results from an extracellular matrix defect within the CNC-derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Collectively, our data suggests that PDGFRalpha and PDGFA are critical regulators for the continued epithelial-mesenchymal interaction during tooth and palate morphogenesis. Disruption of PDGFRalpha signaling disturbs the growth of dental cusp and interferes with the critical extension of palatal shelf during craniofacial development.     

Heterogeneous expression of nestin in myofibroblasts of various human tissues

In this study we explored the distribution of nestin‐positive cells in extraneural human tissues with special reference to stromal myofibroblasts. Tissue microarrays were constructed from various tissues with normal histology and tissues with fibrosing disorders. Sections were immunostained for nestin, alpha‐smooth muscle actin (alpha‐SMA), desmin, vimentin, CD34, and other stromal markers. Nestin was expressed in the myoepithelium of the breast, podocytes of the renal glomerulus, and endothelial cells of most organs. Nestin was also expressed in the stroma of several organs, including the intestine, uterine cervix, and endometrium. Nestin‐positive fibroblast‐like cells appeared in the stroma of the kidney, pancreas, lung, and skin in fibrosing conditions. With the notable exception of endometrial stromal cells, most of these nestin‐positive stromal cells were alpha‐SMA‐positive. Interestingly, we observed a concomitant appearance of nestin‐ and CD34‐positive myofibroblasts under fibrosing conditions. Further investigation showed that nestin was expressed by stromal fibroblasts in cervical squamous cell carcinoma, but not in lung adenocarcinoma, pointing to heterogeneity of cancer stroma. In conclusion, nestin was expressed in variable proportions of stromal myofibroblasts in human tissues. The differential expression of nestin may indicate phenotypic and functional heterogeneity. Nestin‐positive myofibroblast may represent a relatively immature subpopulation of cells with multipotentiality.     

Cystathionine γ-lyase expression during avian embryogenesis

Cystathionine γ-lyase (CSE) is a key enzyme in the trans-sulphuration pathway for the biosynthesis of cysteine from methionine and catalyses the hydrolysis of cystathionine into cysteine. It has been reported to be expressed in mammalian liver and kidney but so far no comprehensive developmental expression analysis of CSE has been available. We cloned a 600 bp fragment of chick CSE cDNA and analysed its expression pattern during avian embryonic development until embryonic day 13. We found CSE expression in various developing organs including the notochord, eye, neural tube, limb bud mesenchyme and sclerotomal compartment of the somites. Notably, prominent expression was found in renal epithelia throughout kidney development, i.e. in the tubular structures of pronephros, mesonephros and metanephros. Our data introduce CSE as a novel marker gene to study avian kidney development.     

Induction of ureter branching as a response to Wnt-2b signaling during early kidney organogenesis.

Epithelial-mesenchymal tissue interactions play a central role in vertebrate organogenesis, but the molecular mediators and mechanisms of these morphogenetic interactions are still not well characterized. We report here on the expression pattern of Wnt-2b during mouse organogenesis and on tests of its function in epithelial- mesenchymal interactions during kidney development. Wnt-2b is expressed in numerous developing organs in the mouse embryo, including the kidney, lung, salivary gland, gut, pancreas, adrenal gland, and genital tubercle. Additional sites of expression include the branchial arches and craniofacial placodes such as the eye and ear. The data suggest that the expression of Wnt-2b is associated with organs regulated by epithelial-mesenchymal interactions. It is typically localized in the capsular epithelium or peripheral mesenchymal cells of organ rudiments, e.g., the perinephric mesenchymal cells in the region of the presumptive renal stroma in the developing kidney at E11.5. Functional studies of the kidney demonstrate that cells expressing Wnt-2b are not capable of inducing tubule formation but instead stimulate ureter development. Incubation of isolated ureteric buds on such cells supports bud growth and branching. In addition, recombination of Wnt-2b-pretreated ureteric bud tissue with isolated nephrogenic mesenchyme results in a recovery of organogenesis and the expression of epithelial genes within the reconstituted organ explant. Lithium, a known activator of Wnt signaling (Hedgepeth et al. [1997] Dev Biol 185:82-91), is also sufficient to promote ureter branching in the reconstituted kidney in a comparable manner to Wnt-2b signaling, whereas Wnt-4, which induces tubules, neither supports the growth of a ureteric bud nor leads to reconstitution of the ureteric bud with the kidney mesenchyme. We conclude that Wnt-2b may act in the mouse kidney as an early mesenchymal signal controlling morphogenesis of epithelial tissue, and that the Wnt pathway may regulate ureter branching directly. In addition, Wnt signals in the kidney differ qualitatively and are specific to either the epithelial ureteric bud or the kidney mesenchyme.