Metanephric mesenchyme gives rise to both the epithelial cells of the nephron and the stromal cells of the mature kidney. The function of the stroma. in kidney morphogenesis is poorly understood. We have generated mice with a null mutation in the Winged Helix (WH) transcription factor BF-2 to examine its function during development. BF-2 expression within the developing kidney is restricted to the stromal cell lineage. Homozygotes die within the first 24 hr after birth with abnormal kidneys. Mutant kidneys are small, fused longitudinally, and rotated 90 degrees ventrally. Histological examination reveals a smaller collecting system, numerous large condensations of mesenchyme, and a decrease in the number of nephrons. Using molecular markers we show that induction and condensation of the nephrogenic mesenchyme occurs normally in mutant. The disruption of BF-2 reduces the rate of differentiation of the condensed mesenchyme into tubular epithelium, as well as the rate of growth and branching of the ureter and collecting system. Our findings demonstrate that BF-2 and stromal cells have essential functions during kidney morphogenesis. Furthermore, they suggest that BF-2 controls the production, by the stroma, of signals or factors that are required for the normal transition of induced mesenchyme into tubular epithelium and full growth and branching of the collecting system.

During kidney morphogenesis, the formation of nephrons begins when mesenchymal nephron progenitor cells aggregate and transform into epithelial vesicles that elongate and assume an S-shape. Cells in different regions of the S-shaped body subsequently differentiate into the morphologically and functionally distinct segments of the mature nephron. Here, we have used an allelic series of mutations to determine the role of the secreted signaling molecule FGF8 in nephrogenesis. In the absence of FGF8 signaling, nephron formation is initiated, but the nascent nephrons do not express Wnt4or Lim1, and nephrogenesis does not progress to the S-shaped body stage. Furthermore, the nephron progenitor cells that reside in the peripheral zone, the outermost region of the developing kidney, are progressively lost. When FGF8 signaling is severely reduced rather than eliminated, mesenchymal cells differentiate into S-shaped bodies. However, the cells within these structures that normally differentiate into the tubular segments of the mature nephron undergo apoptosis, resulting in the formation of kidneys with severely truncated nephrons consisting of renal corpuscles connected to collecting ducts by an abnormally short tubular segment. Thus, unlike other FGF family members, which regulate growth and branching morphogenesis of the collecting duct system, Fgf8 encodes a factor essential for gene regulation and cell survival at distinct steps in nephrogenesis.

WT1 encodes a zinc finger transcription factor implicated in kidney differentiation and tumorigenesis. In reporter assays, WT1 represses transcription from GC- and TC-rich promoters, but its physiological targets remain uncertain. We used hybridization to high-density oligonucleotide arrays to search for native genes whose expression is altered following inducible expression of WT1. The major target of WT1 was amphiregulin, a member of the epidermal growth factor family. The WT1(-KTS) isoform binds directly to the amphiregulin promoter, resulting in potent transcriptional activation. The in vivo expression profile of amphiregulin during fetal kidney development mirrors the highly specific pattern of WT1 itself, and recombinant Amphiregulin stimulates epithelial branching in organ cultures of embryonic mouse kidney. These observations suggest a model for WT1 as a transcriptional regulator during kidney differentiation.

In addition to the traditional renin-angiotensin system, a great deal of evidence favors the existence of numerous independent tissue-specific renin-angiotensin systems. We report that mast cells are an additional source of renin and constitute a unique extrarenal renin-angiotensin system. We use renin-specific antibodies to demonstrate that cardiac mast cells contain renin. Extending this observation to the human mast cell line HMC-1, we show that these mast cells also express renin. The HMC-1 renin RT-PCR product is 100% homologous to Homo sapiens renin. HMC-1 cells also contain renin protein, as demonstrated both by immunoblot and immunocytochemical analyses. Renin released from HMC-1 cells is active; furthermore, HMC-1 cells are able to synthesize renin. It is known that, in the heart, mast cells are found in the interstitium in close proximity to nerves and myocytes, which both express angiotensin II receptors. Inasmuch as myocardial interstitium contains angiotensinogen and angiotensinconverting enzyme, and because we were able to detect renin only in mast cells, we postulate that the release of renin from cardiac mast cells is the pivotal event triggering local formation of angiotensin II. Because of the ubiquity of mast cells, our results represent a unique paradigm for understanding local renin-angiotensin systems, not just in the heart, but in all tissues. Our findings provide a rationale for targeting mast cells in conjunction with renin-angiotensin system inhibitors in the management of angiotensin II-related dysfunctions.T raditionally the renin-angiotensin system (RAS) has been viewed as a circulating axis, whereby renin is released into the circulation from the kidneys in response to decreased renal perfusion pressure, decreased delivery of NaCl at the macula densa, and͞or increased renal sympathetic nerve activity (1). The rate-limiting step in the formation of angiotensin II (ANG II) is the proteolytic action of renin, which cleaves angiotensinogen (Aogen) to the intermediate angiotensin I (ANG I). ANG I is then converted to ANG II by angiotensin-converting enzyme (ACE) at the endothelial surface (2, 3).In addition to this conventional pathway, many tissues, including heart and brain, are thought to be capable of local ANG II production via tissue-specific RAS (4, 5). Although Aogen, ANG I, and ACE, have been demonstrated in various organs, the presence of renin of extrarenal origin has been more difficult to prove and remains controversial. In this investigation, experiments were designed to determine whether renin could be detected in native tissue other than kidney. Because ANG II plays such a crucial role in cardiovascular disease, we focused our efforts on heart tissue, which we screened for renin by using an established polyclonal anti-renin Ab made to recombinant human renin (6). We found that cardiac mast cells were immunopositive for renin. In addition, we used a cultured mast cell line to extrapolate our observations from fixed heart slices to living cells.Our findings indicate that ma...

Mammalian kidney morphogenesis begins when the ureteric bud (UB) induces surrounding metanephric mesenchyme to differentiate into nephrons, the functional units of the mature organ. Although several genes required for this process have been identified, the mechanisms that control final nephron number and the localization of distinct tubular segments to cortical and medullary zones of the kidney remain poorly understood. This finding is due, in part, to the lack of quantitative studies describing the acquisition of mature renal structure. We have analyzed the following parameters of the developing murine kidney throughout embryogenesis: nephron and UB tip number, distance between UB branch points and total kidney, and cortical and medullary volume. Results of this morphometric analysis reveal previously unrecognized changes in the pattern of UB growth and rate of nephrogenesis. In addition, this morphometric index provides a much-needed reference for accurately describing renal patterning defects exhibited by genetically altered mice. Developmental Dynamics 231:601-608, 2004.

Abstract. During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin-D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis.Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelia requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation.

A dodecapeptide corresponding to the carboxyl terminus of the lac carrier of Escherichia coli was synthesized, coupled to thyroglobulin, and the conjugate was used to generate site-directed polyclonal antibodies. The antibodies react with the carboxyl-terminal peptide and with the lac carrier protein, while monoclonal antibody 4B1 reacts with intact lac carrier protein, but not with the carboxyl-terminal peptide. Antibody 4B1 binds preferentially to right-side-out membrane vesicles relative to inside-out vesicles, confirming the presence of the 4B1 epitope on the periplasmic surface of the membrane. Alternatively, anti-carboxyl-terminal antibody binds preferentially to inside-out vesicles, demonstrating that the carboxyl terminus of the lac carrier protein is on the cytoplasmic surface. Surprisingly, both antibodies bind to proteoliposomes reconstituted with purified lac carrier protein, and quantitative binding assays indicate that the epitopes are equally accessible. When proteoliposomes containing purified lac carrier protein are digested with carboxypeptidases A and B, binding of anti-carboxyl-terminal antibodies decreases by >80%, while binding of antibody 4B1 and various transport activities remain essentially unchanged. It is suggested that during reconstitution, the lac carrier protein undergoes intramolecular dislocation of the carboxyl terminus with no significant effect on its catalytic activity.The lac carrier protein (i.e., lac permease) in Escherichia coli is an intrinsic membrane protein, the product of the lacY gene, that catalyzes the coupled translocation of 3-galactosides with hydrogen ion in a symport reaction (cf. ref. 1 for a recent review). As such, this protein is representative of a large class of substrate-specific polypeptides that couple downhill movement of a cation to uphill transport of solute in response to a transmembrane electrochemical ion gradient. lac permease has been purified to homogeneity in a functional state, and proteoliposomes reconstituted with a single polypeptide species catalyze all of the transport activities observed in intact cells and right-side-out (RSO) membrane vesicles with full efficiency (1-7). In addition, other similarities between native and purified lac carrier protein have been documented (6, 7), and it is evident from the findings as a whole that /3galactoside transport in E. coli requires a single gene product, that of the lacY gene.The permease is a 46.5-kDa polypeptide containing 417 amino acid residues of known sequence (8). Based on circular dichroic measurements indicating that the protein has an exceptionally high helical content and based on analysis of the sequential hydropathic character of the protein, a secondary structure model has been proposed (9). The model suggests that the protein consists of 12 or 13 hydrophobic ahelical segments that traverse the membrane in a zigzag fashion connected by more hydrophilic loops. Accordingly, the model makes explicit predictions regarding those portions of the molecule that should be acces...

Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17 to 27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the