Kidney Mesenchyme Research Articles

Metastasizing tumors in rats treated with alkylating carcinogens.

Tumors induced in approximately 2000 F344 rats by a number of carcinogenic N-nitroso compounds have been examined for their propensity to metastasize. The objective was to discover relations between the structure of the carcinogen, the tumor induced and the proportion of tumors that formed metastases. Treatments consisted of multiple doses of one of 16 nitrosamines or 19 alkylnitrosoureas, which were administered in drinking water, by gavage or by the intravesicular route. Male and female rats were included. Most of the carcinogens were mutagens in bacteria, but some were not; this had no bearing on the tendency of induced tumors to metastasize, nor did the extent of alkylation of DNA produced in vivo. Fewer malignant tumors appeared in the rats treated with nitrosamines than with alkylnitrosoureas, but more than twice as many of the former metastasized; many were carcinomas or hemangiosarcomas of the liver, of which very few were induced by alkylnitrosoureas. Tumors of the liver, lung, mammary gland and forestomach metastasized most commonly, whereas those of the esophagus, nasal mucosa, Zymbal gland, kidney mesenchyme, thyroid, urinary bladder and mesotheliomas seldom formed metastases; none of the tumors of the brain or intestines metastasized; no differences between males and females were noted. Some rare tumors, osteosarcomas and thymus lymphomas, metastasized frequently. The lungs and lymph nodes were the most common sites for metastases, but less frequently liver, heart, kidney, adrenal gland, omentum, peritoneum, esophagus and pancreas were involved. Higher doses were associated with greater numbers of metastasizing tumors among mutagenic or non-mutagenic carcinogens, as has been reported elsewhere. It appears that directly alkylating alkylnitrosoureas are no more likely (and probably less likely) to induce tumors with metastatic properties than are nitrosamines that require metabolic activation to form reactive proximate carcinogens.

Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4.

The kidney has been widely exploited as a model system for the study of tissue inductions regulating vertebrate organogenesis. Kidney development is initiated by the ingrowth of the Wolfian duct-derived ureteric bud into the presumptive kidney mesenchyme. In response to a signal from the ureter, mesenchymal cells condense, aggregate into pretubular clusters and undergo an epithelial conversion generating a simple tubule. This then undergoes morphogenesis and is transformed into the excretory system of the kidney, the nephron. We report here that the expression of Wnt-4, which encodes a secreted glycoprotein, correlates with, and is required for, kidney tubulogenesis. Mice lacking Wnt-4 activity fail to form pretubular cell aggregates; however, other aspects of mesenchymal and ureteric development are unaffected. Thus, Wnt-4 appears to act as an autoinducer of the mesenchyme to epithelial transition that underlies nephron development.

The regulation of kidney development: new insights from an old model

The embryonic kidney is an excellent model system in which to address many fundamental issues in developmental biology. Inductive interactions are required for proliferation and differentiation of the ureter epithelium and kidney mesenchyme. Recent studies implicate a receptor-type tyrosine kinase as a target of inductive signals in the developing ureter. In the mesenchyme, the early induction response requires at least two transcription factors, WT1 and Pax-2. Through the integrated application of in vitro culture models and gene targeting methods, the molecular mechanisms underlying kidney morphogenesis are becoming clearer.

Pax-2 is required for mesenchyme-to-epithelium conversion during kidney development.

The conversion of mesenchyme to epithelium during the embryonic development of the mammalian kidney requires reciprocal inductive interactions between the ureter and the responding metanephric mesenchyme. The Pax-2 gene is activated in the mesenchyme in response to induction and is subsequently down-regulated in more differentiated cells derived from the mesenchyme. Pax-2 belongs to a family of genes, at least three of which encode morphogenetic regulatory transcription factors. In order to determine the role of Pax-2 during kidney development, we have generated a loss-of-function phenotype using antisense oligonucleotides in mouse kidney organ cultures. These oligonucleotides can specifically inhibit Pax-2 protein accumulation in kidney mesenchyme cells, where the intracellular concentrations are maximal. The kidney organ cultures were stained with uvomurulin and laminin antibodies as markers for epithelium formation. With significantly reduced Pax-2 protein levels, kidney mesenchyme cells fail to aggregate and do not undergo the sequential morphological changes characteristic of epithelial cell formation. The data demonstrate that Pax-2 function is required for the earliest phase of mesenchyme-to-epithelium conversion.

Post-transcriptional regulation of syndecan-1 expression by cAMP in peritoneal macrophages.

Syndecan-1 is a cell surface heparan sulfate proteoglycan that is proposed to serve in cell-cell adhesion, cell-matrix anchorage, and growth factor signaling. Its expression is temporally and spatially regulated during epithelial-mesenchymal interactions in many developing tissues. In some cases, this regulation appears to be achieved at the level of transcription. However, induction of syndecan-1 expression in the embryonic kidney mesenchyme is suggested to occur at the level of mRNA translation (Vainio, S., M. Jalkanen, M. Bernfield, and L. Saxén. 1992. Dev. Biol. 152:221-232). To identify a system in which the regulatory mechanisms controlling syndecan-1 expression can be studied, cells of the monocyte-macrophage lineage, which regulate the expression of many cell surface receptors, were screened for syndecan-1 expression. The syndecan-1 gene is active in blood monocytes as well as resident and thioglycollate-elicited mouse peritoneal macrophages, but expression of the proteoglycan is regulated at two levels. First, elicited macrophages accumulate nine-fold more syndecan-1 mRNA than do resident macrophages or circulating blood monocytes. Another member of the syndecan family of proteoglycans, syndecan-4, shows a distinct pattern of expression, suggesting that this regulation is specific for syndecan-1. Second, utilization of the mRNA for syndecan-1 production encounters a post-transcriptional block in the elicited macrophages that can be overcome by triggering agents such as E-type prostaglandins or dibutyryl cAMP, which raise intracellular cAMP levels. Dibutyryl cAMP does not induce syndecan-1 expression in resident peritoneal macrophages, which lack a pool of stored mRNA. This suggests that this agent promotes the post-transcriptional utilization of stored syndecan-1 mRNA. The induced proteoglycan appears at the cell surface as a integral of 100-kD heparan sulfate-rich isoform of syndecan-1. This suggests that a cAMP-dependent post-transcriptional control mechanism may be present in a variety of tissues when syndecan-1 expression is regulated.

Transient expression of syndecan in mesenchymal cell aggregates of the embryonic kidney

Induction of the embryonic kidney mesenchyme is followed by formation of cell aggregates which subsequently transform into epithelial tubules. Syndecan, which binds various matrix components and growth factors, is a candidate molecule to be involved in this process. We have analyzed the changes in the expression of syndecan during tubule morphogenesis by using in situ hybridization and slot-blot analysis. The expression pattern of syndecan was compared with the distribution of cell proliferation analyzed by immunohistochemistry. Furthermore, the expression of syndecan during formation of the pretubular aggregates was studied in hanging-drop cultures of experimentally induced mesenchymal cells. Syndecan mRNA was expressed in the metanephric mesenchyme prior to induction, was intensely present during formation of the pretubular cell aggregates, but was lost during maturation of the nephron. Slot-blot analyses of the kidney mesenchymes (11-day kidney) cultured in a transfilter situation with a heterotypic inductor tissue that triggers a complete tubulogenic program in the nephric mesenchyme during the first 24 hr suggested the presence of syndecan mRNA in the uninduced mesenchymes with no change during induction. Expression of mRNA was stimulated later (13-day kidney) followed by subsequent decrease. Immunoisolation of sulfate-labeled syndecan, however, revealed a marked stimulation in the induced kidney mesenchyme during the first 24-hr inductive period when the DNA level still remained constant. In hanging-drop cultures where either induced or uninduced mesenchymal cells were dissociated and reaggregated, syndecan was detected only in the induced and aggregating mesenchymal cells. Double-immunostaining demonstrated a close correlation between syndecan expression and cell proliferation analyzed by bromodeoxyuridine incorporation. Thus, it appears that syndecan expression in the mesenchyme is initially induced post-transcriptionally and later during differentiation at the mRNA level. Syndecan may have a dual function during early kidney morphogenesis; it may be involved in cell aggregation through its adhesive properties, and it may contribute to proliferation of the induced mesenchymal cells by binding growth factors.

Monoclonal antibodies against laminin A chain fragment E3 and their effects on binding to cells and proteoglycan and on kidney development

Rat monoclonal antibodies were raised against fragment E3 of the mouse Engelbreth-Holm-Swarm (EHS) tumor laminin and selected according to their exclusive reaction with laminin A chain by immunoblotting and staining pattern in embryonic kidneys by immunofluorescence. Immunochemical studies of nine purified antibodies showed a comparable reaction with unfragmented laminin and fragment E3 but no cross-reaction with several other, unrelated laminin fragments including the major cell-binding fragment E8. Reduction or pepsin digestion of fragment E3 reduced or abolished antibody binding indicating that most of the epitopes involved are conformation dependent and do not include carbohydrates. They are, however, not identical as shown by different reactivities after proteolytic or chemical cleavage of E3. Four of the antibodies were highly active in inhibiting cell adhesion of the teratocarcinoma cell line F9 and the Schwannoma cell line RN22 on fragment E3 (IC 50 ~ 1 μg/ml), while the others were distinctly less active. No inhibition was observed for cell adhesion on unfragmented laminin, consistent with previous findings that this is largely mediated by binding of fragment E8 to α6β1 integrin. A distinct correlation was observed between cell adhesion inhibition and the inhibition of heparansulfate proteoglycan and heparin binding to fragment E3. Since heparin is not very efficient in inhibiting cell adhesion, it indicates that heparin- and cell-binding sites on fragment E3 are in close proximity but not identical. Two of the antibodies also showed partial inhibition of kidney tubule formation in organ culture of embryonic kidney mesenchyme while the other antibodies were inactive. It suggests some but probably minor involvement of the fragment E3 structure of laminin in this developmental process.

Development and growth of mouse embryonic kidney in organ culture and modulation of development by soluble growth factor

Differentiation of the metanephrogenic mesenchyme is triggered by an inductive tissue interaction between an inducer tissue and the mesenchyme. It is generally believed that the epithelial ureter bud acts as an inducer during in vivo development. In response to the inductive stimulus most of the mesenchymal cells convert into epithelial cells, while a small fraction differentiates into stromal cells. In vitro, differentiation of isolated mesenchyme to epithelium can be induced by a variety of embryonic tissues, but nothing is known about the molecular nature of the inducing stimulus. In recent years, large numbers of polypeptide growth factors have been described, which in addition to proliferative effects were shown to exert effects on a variety of biological phenomena such as chemotaxis, inflammation, tissue repair, or induction of embryonic development. We therefore analyzed whether growth factors in the absence of inducer tissue can induce isolated kidney mesenchyme to differentiate into epithelium or interstitium. As expected, both growth and differentiation into epithelium were stimulated by an inducer tissue, the spinal cord. We found that none of the various growth factors tested (including epidermal growth factor, transforming growth factors α and β, insulin-like growth factors I and II, fibroblast growth factor, platelet-derived growth factor, and retinoic acid) could mimick the effect of an inducer tissue, although we tested the factors over a wide concentration range. One of the tested factors, epidermal growth factor (EGF) stimulated the mesenchymal cells to become stromal cells, although it could not stimulate development into epithelium. EGF could stimulate stromal development both when the mesenchyme was cultured in isolation and when the mesenchyme was stimulated by an inducer tissue to become epithelium. The expansion of the stromal compartment in response to EGF treatment occurred at the expense of the epithelial cells, but EGF could not completely suppress the formation of epithelium. These data suggest the presence of EGF receptors in the developing kidney, but since application of soluble EGF leads to abnormal development, soluble EGF cannot be the natural ligand. We suggest that locally produced mitogens with an EGF-like structure may regulate the relative amounts of stroma (interstitium) and epithelium in the developing kidney.

Syndecan, a developmentally regulated cell surface proteoglycan that binds extracellular matrix and growth factors

Cellular behaviour during development is dictated, in part, by the insoluble extracellular matrix and the soluble growth factor peptides, the major molecules responsible for integrating cells into morphologically and functionally defined groups. These extracellular molecules influence cellular behaviour by binding at the cell surface to specific receptors that transduce intracellular signals in various ways not yet fully clear. Syndecan, a cell surface proteoglycan found predominantly on epithelia in mature tissues binds both extracellular matrix components (fibronectin, collagens I, III, V, and thrombospondin) and basic fibroblast growth factor (bFGF). Syndecan consists of chondroitin sulfate and heparan sulphate chains linked to a 31 kilodalton (kDa) integral membrane protein. Syndecan represents a family of integral membrane proteoglycans that differ in extracellular domains, but share cytoplasmic domains. Syndecan behaves as a matrix receptor: it binds selectively to components of the extracellular matrix, associates intracellularly with the actin cytoskeleton when cross-linked at the cell surface, its extracellular domain is shed upon cell rounding and it localizes solely to basolateral surfaces of simple epithelia. Mammary epithelial cells made syndecan-deficient become fibroblastic in morphology and cell behaviour, showing that syndecan maintains epithelial cell morphology. Syndecan changes in quantity, location and structure during development: it appears initially on four-cell embryos (prior to its known matrix ligands), becomes restricted in the pre-implementation embryo to the cells that will form the embryo proper, changes its expression due to epithelial-mesenchymal interactions (for example, induced in kidney mesenchyme by the ureteric bud), and with association of cells with extracellular matrix (for example, during B-cell differentiation), and ultimately, in mature tissues becomes restricted to epithelial tissues. The number and size of its glycosaminoglycan chains vary with changes in cell shape and organization yielding tissue type-specific polymorphic forms of syndecan. Its interactions with the major extracellular effector molecules that influence cell behaviour, its role in maintaining cell shape and its spatial and temporal changes in expression during development indicate that syndecan is involved in morphogenesis.

Transient and locally restricted expression of laminin a chain mRNA by developing epithelial cells during kidney organogenesis

Three polypeptide chains, A, B1, and B2, have been described for mouse laminin, a basement membrane protein. We studied expression of laminin A, B1, and B2 mRNA in the developing mouse kidney. Induction of kidney mesenchyme differentiation in vitro led to an increased expression of B1 and B2 chain mRNA on day 1 of development. In contrast, expression of A chain mRNA increased on day 2, when epithelial cell polarization begins. Laminin A mRNA and polypeptide were expressed only by epithelia during in vivo development as well. Some polarized cell types producing basement membrane (endothelium, some adult epithelia) lacked the A chain mRNA and polypeptide, although thoy did express B chains. Laminin with the 400 kd A chain is therefore a transient form appearing at specific sites of kidney morphogenesis, whereas isoforms with a different A chain or without it have a more widespread distribution.

Levels and patterns of 125I-labeled transferrin binding in mouse embryonic teeth and kidneys at various developmental stages

The iron-transporting serum glycoprotein, transferrin, is necessary for the cell proliferation, morphogenesis, and differentiation of mouse embryonic teeth and kidneys in organ culture. The stimulatory effect of transferrin is mediated by the binding of transferrin to its specific cell-surface receptor and by receptor-mediated endocytosis. Since, in both teeth and kidneys, the requirement for and responsiveness to transferrin depend on the developmental stage of the organ, we studied the binding of transferrin at various stages of tooth and kidney development by incubating tissues with 125I-labeled transferrin. The amount of bound transferrin was determined by measuring the tissue-incorporated radioactivity, and the binding sites were localized by autoradiography. During tooth development in vitro, the requirement for exogenous transferrin is lost as the teeth proceed from the early cap stage to the bell stage. The level of transferrin binding was found to decrease simultaneously, and in bell-stage teeth, the transferrin receptors were concentrated in the areas of most active cell proliferation. In kidneys, the number of transferrin receptors was highest at the stage during which the undifferentiated kidney mesenchyme becomes responsive to transferrin. These receptors were located in both the ureter epithelium and the metanephric mesenchyme, and they dramatically decreased in number with advancing kidney differentiation. The results of the present study indicate that, during the embryonic development of teeth and kidneys, the amount and localization of transferrin binding are correlated with cell proliferation. The number of transferrin receptors is highest during the developmental stages when cell proliferation is most active, and decreases with advancing differentiation. The developmental regulation of transferrin receptors in embryonic teeth and kidneys supports our previous data indicating that transferrin plays an important role in embryonic organogenesis.

Production of a heparin-binding angiogenesis factor by the embryonic kidney.

Embryonic mouse kidneys induce angiogenesis when transplanted on the quail chorioallantoic membrane (Ekblom, P., H. Sariola, M. Karkinen, and L. Saxén, 1982, Cell Differ., 11:35-39). In these experiments all blood vessels were derived from the quail host, suggesting that kidney endothelium is derived from outside blood vessels. We have now analyzed whether kidney angiogenesis is regulated by kidney-derived soluble factors that stimulate the growth of new blood vessels. In the rabbit cornea, 11-d embryonic kidneys induced angiogenesis, whereas uninduced 11-d kidney mesenchymes did not. To characterize and purify this activity from an embryonic organ, we dissected between 600 and 1,000 14-17-d-old embryonic mouse kidneys for each purification experiment. Growth factor activity for capillary endothelial cells was found to bind to heparin-Sepharose and eluted at 0.9-1.1 M sodium chloride. Gel filtration revealed a molecular weight of 16,000-20,000 of this factor. A major 18,000-mol-wt band was seen after gel electrophoresis and silver staining of partially purified growth factor material. The chromatographed factor is mitogenic for endothelial cells but not for smooth muscle cells and stimulates angiogenesis in vivo in the rabbit cornea. Adult kidneys contained two heparin-binding endothelial cell growth factors. The differentiation-dependent production of an angiogenesis factor by the embryonic kidney suggests an important role of angiogenesis in organogenesis.

Control of kidney differentiation by soluble factors secreted by the embryonic liver and the yolk sac

Since transferrin is necessary for the differentiation of the embryonic kidney in organ culture, we have suggested that the component is a growth factor for in vivo development as well. In the present study we demonstrate that transferrin is present in the serum of 11-day-old mouse embryos, at the time when kidney differentiation starts. We have also tested whether various embryonic tissues can replace transferrin as stimulators of the differentiation and proliferation of the metanephric mesenchyme. We used a transfilter model system where nephrogenic mesenchymes are cultured with spinal cord, a known inductor of kidney tubules. The embryonic liver could not replace the spinal cord as an inducer of tubular differentiation. However, when the kidney mesenchymes were cultured together with both the spinal cord and the liver, the mesenchymes proliferated and differentiated also in the absence of exogenous transferrin. In such cocultures the spinal cord had to be in close contact with the mesenchyme while the embryonic liver could be located several cell layers apart. The liver-mediated stimulation of proliferation of the induced mesenchyme could be inhibited by anti-transferrin antibodies. Immunoprecipitation and immunoblotting with these antibodies of the liver-conditioned medium demonstrated that the 11-day mouse liver produces transferrin. Other potential mitogens produced by liver cells, α-fetoprotein, or multiplication stimulating activity, did not in any way stimulate the proliferation of induced mesenchymes. These studies suggest that the mitogen in the liver medium is transferrin. This is supported by data which show that another embryonic transferring producer, the visceral yolk sac, can replace the effect of the liver, whereas a tissue not producing transferrin, the salivary mesenchyme, cannot. In conclusion, an essential function of the inducer is to make the mesenchyme responsive to transferrin. The liver and the yolk sac stimulate early kidney differentiation by producing the soluble factor, transferrin, but they are ineffective as inductors of the transferrin responsiveness.

Role of transferrin in branching morphogenesis, growth and differentiation of the embryonic kidney

Our previous work has suggested that transferrin is an important serum component for differentiation of the kidney. In this study we have analysed more closely the response of cultured mouse embryonic kidney to exogenous transferrin and the dependence of kidney tubule induction on transferrin. Our results show that transferrin causes a dose-dependent increase in cell proliferation in the differentiating kidney mesenchyme, but no stimulation of cell proliferation in the inductor tissue used, the embryonic spinal cord. In cultures of whole kidney rudiments a remarkable increase in the amounts of DNA and protein are caused by transferrin but not by other serum components present in a transferrin-depleted serum. The morphology of the explants was similar when cultured in the presence of human serum and in the transferrin-depleted serum supplemented with transferrin. In transferrin-containing chemically-defined medium the explants flattened and spread out, but the morphology of the kidney tubules was similar as in explants cultured in the presence of serum. Examination of the cultured explants by electron microscopy showed that in all transferrin-containing culture media the mesenchymal cells had differentiated into kidney tubules consisting of epithelial cells lined by a basement membrane. The experiments with the transferrin-depleted serum demonstrate that the main mitogen for kidney development is transferrin, and that other serum factors are mainly required for maintenance of tissue compactness. Our earlier studies have shown that exogenous transferrin is not needed for certain changes preceding overt tubule formation in the kidney mesenchyme, and we suggested that transferrin responsiveness is acquired during the induction of kidney mesenchyme. Our present results do not contradict the postulate, although they demonstrate that the acquisition of the responsiveness is more complicated than previously thought. When the mesenchyme is exposed to inductor tissue for 24 h without transferrin, and then subcultured without the inductor in the presence of transferrin, morphogenesis fails and there is no proliferation of the mesenchyme. The experiment shows that the inductor, the mesenchyme and transferrin must all three be simultaneously present for the acquisition of the transferrin responsiveness. Other experiments show that the induced mesenchyme can be a direct target tissue, since it can proliferate in response to transferrin also in the absence of the inductor. It is evident that the inductor is required for the acquisition of the responsiveness, as suggested.(ABSTRACT TRUNCATED AT 400 WORDS)

Biosynthesis of proteoglycans in organ cultures of developing kidney mesenchyme

The biosynthesis of proteoglycans was studied in organ cultures of differentiating metanephric mesenchymes. When triggered by a contact-mediated inductive interaction, this tissue undergoes transition from a mesenchyme to an epithelium. In the present study, proteoglycans were extracted by guanidinium hydrochloride in the presence of protease inhibitors. We found that, as a response to induction, the differentiating mesenchyme begins to synthesize large size proteoglycans with an apparent molecular weight (MW) of 1 × 10 6 D. The major glycosaminoglycans detected were chondroitin sulfates. Heparan sulfate proteoglycans were also detected, constituting 20% of the proteoglycans. An inhibitor of glucosamine synthesis, 6-diazo-5-oxo-norleucine (DON) was found to inhibit glycosaminoglycan synthesis by approx. 60%, and the size of the proteoglycans was also diminished. Our studies suggest that the transition of the mesenchyme to epithelium is associated with initiation of synthesis of large size proteoglycans.

In vitro isolation and establishment of new embryonal cell lines from rat nephroblastoma.

Two mixed cell lines designated REN-1 and REN-2 were established successfully in continuous in vitro culture from subcutaneously propagated transplant tissue derived from a nephroblastoma which had occurred spontaneously in an Nb hooded rat. In monolayer culture the lines consisted of clumps, islands, and cords of densely crowded, small basophilic cells of epithelioid character, together with mesenchymelike cells occupying the intervening spaces. The proportion of epithelioid cells to mesenchyme could be enhanced by high seeding densities and the intermittent application ofcishydroxyproline to the medium. A third cell form with the appearance of more mature epithelium was observed as a later development in one of the monolayer cultures (REN-1). This larger epithelial cell and a homogeneous mesenchymal population were isolated as cloned cell lines from REN-1 (REN-1-C/2 and REN-1-C/1, respectively), but attempts to clone the dominant basophilic epithelioid cell were not successful. Light and electron microscopy indicated the small, basophilic epithelioid cells to be morphologically consistent with the undifferentiated embryonal blast cells of the parent tumor. They were a distinctive population unlike known malignant cell lines representative of chemically transformed rat kidney mesenchyme and epithelium. The mesenchymelike cells present in the uncloned cell lines, and cloned in REN-1-C/1, were fibroblasts by ultrastructural criteria and therefore distinct from the epithelioid moiety. Ultrastructurally the larger epithelium cloned in REN-1-C/2 displayed the features of differentiated renal epithelium. Subcutaneous transplantation in syngeneic and allogeneic recipients showed the uncloned parent cell lines containing the basophilic epithelioid population to be highly tumorigenic, producing rapidly growing tumors that were unequivocal nephoblastomas. The mesenchymal clone, REN-1-C/1, was also tumorigenic but, consistent with its fibroblastic nature, produced only fibrosarcomas on transplantation. The mature epithelial clone, REN-1-C/2, transplanted as an anaplastic carcinoma with limited tubule formation. Because of their distinctive morphology and growth behavior, and their ability to proliferate into nephroblastomas on transplantation, the dominant population of basophilic epithelioid cells in the parent uncloned cell lines is considered to represent neoplastic kidney cells of undifferentiated embryonal type. The possible host origin of the mesenchymal population from the supporting stroma of the original transplantation tumor is suggested in discussion.

Formation of basement membranes in the embryonic kidney: an immunohistological study.

Specific antibodies to laminin, type IV collagen, basement-membrane proteoglycan, and fibronectin have been used in immunofluorescence microscopy to study the development of basement membranes of the embryonic kidney. Kidney tubules are known to form from the nephrogenic mesenchyme as a result of an inductive tissue interaction. This involves a change in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses fibronectin but no detectable laminin, type IV collagen, or basement-membrane proteoglycan. During the inductive interaction, basement-membrane specific components (laminin, type IV collagen, basement membrane proteoglycan) become detectable in the induced area, whereas fibronectin is lost. While the differentiation to epithelial cells of the kidney requires an inductive interaction, the development of the vasculature seems to involve an ingrowth of cells which throughout development deposits basement-membrane specific components, as well as fibronectin. These cells form the endothelium and possibly also the mesangium of the glomerulus, and contribute to the formation of the glomerular basement membrane. An analysis of differentiation of the kidney mesenchyme in vitro in the absence of circulation supports these conclusions. Because a continuity with vasculature is required for glomerular endothelial cell differentiation, it is possible that these cells are derived from outside vasculature.

Induction of kidney tubules in mouse metanephrogenic mesenchyme by various embryonic mesenchymal tissues

Many, but not all, embryonic mesenchymes were found to be inductively active when combined with kidney mesenchyme. Tissues undergoing ossification are particularly active. There is variation in the time-course of appearance of tubules in kidney mesencyme: 2 days for jaw, 4 days for developing bone, and 5–6 days for salivary mesenchyme or somite.

Inductive interactions in the develoment of the mouse metanephros.

AbstractEmbryonic mouse kidney mesenchymes of various ages were tested for their abilities to initiate and supportureteric bud morphogenesis in vitro.Nephrogenic cord mesenchyme taken from animals before metanephros initiation (10 days) was combined with ureteric buds. The buds showed no morphogenesis. This was true for presumptive metanephric mesenchyme and for mesenchymes (pronephric and mesonephric) already undergoing nephrogenesis. These results indicate basic physiological differences between pronephric, mesonephric and metanephric regions of the cord. When entire nephric systems were cultured alone no metanephros developed. This, and the fact that presumptive metanephric mesenchyme had no influence on ureteric buds, indicates that metanephros initiation (both bud and mesenchyme) results from some activity originating outside the nephrogenic cord.When combined with metanephric mesenchyme at various stages of tubulogenesis (loose, condensed, or tubular), ureteric buds branched only in the presence of the loose mesenchyme. Branching occurred in loose mesenchyme taken from 11 to 15 day kidneys. Apparently once a given group of cells begins tubulogenesis they lose their ability to induce ureteric bud morphogenesis.As tubulogenesis proceeds, the amount of loose mesenchymes per kidney decreases sharply. Ultimate size of the metanephros may be established by depletion of loose mesenchyme resulting in its ureteric bud‐inducing potential falling below some critical level.

Differentiation of Kidney Mesenchyme in an Experimental Model System

Differentiation of Kidney Mesenchyme in an Experimental Model System