Rho kinase acts at separate steps in ureteric bud and metanephric mesenchyme morphogenesis during kidney development.

In this study, five different in vitro assays, which together recapitulate much of kidney development, were used to examine the role of the Rho-associated protein serine/threonine kinase (ROCK) in events central to ureteric bud (UB) and metanephric mesenchyme (MM) morphogenensis, in isolation and together. ROCK activity was found to be critical for (1) cell proliferation, growth, and development of the whole embryonic kidney in organ culture, (2) tip and stalk formation in cultures of isolated UBs, and (3) migration of MM cells (in a novel MM migration assay) during their condensation at UB tips (in a UB/MM recombination assay). Together, the data indicate selective involvement of Rho/ROCK in distinct morphogenetic processes necessary for kidney development and that the coordination of these events by Rho/ROCK provides a potential mechanism to regulate overall branching patterns, nephron formation, and thus, kidney architecture.

Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney.

In search of guiding principles involved in the branching of epithelial tubes in the developing kidney, we analyzed branching of the ureteric bud (UB) in whole kidney culture as well as in isolated UB culture independent of mesenchyme but in the presence of mesenchymally derived soluble factors. Microinjection of the UB lumen (both in the isolated UB and in the whole kidney) with fluorescently labeled dextran sulfate demonstrated that branching occurred via smooth tubular epithelial outpouches with a lumen continuous with that of the original structure. Epithelial cells within these outpouches cells were wedge-shaped with actin, myosin-2 and ezrin localized to the luminal side, raising the possibility of a "purse-string" mechanism. Electron microscopy and decoration of heparan sulfates with biotinylated FGF2 revealed that the basolateral surface of the cells remained intact, without the type of cytoplasmic extensions (invadopodia) that are seen in three-dimensional MDCK, mIMCD, and UB cell culture models of branching tubulogenesis. Several growth factor receptors (i.e., FGFR1, FGFR2, c-Ret) and metalloproteases (i.e., MT1-MMP) were localized toward branching UB tips. A large survey of markers revealed the ER chaperone BiP to be highly expressed at UB tips, which, by electron microscopy, are enriched in rough endoplasmic reticulum and Golgi, supporting high activity in the synthesis of transmembrane and secretory proteins at UB tips. After early diffuse proliferation, proliferating and mitotic cells were mostly found within the branching ampullae, whereas apoptotic cells were mostly found in stalks. Gene array experiments, together with protein expression analysis by immunoblotting, revealed a differential spatiotemporal distribution of several proteins associated with epithelial maturation and polarization, including intercellular junctional proteins (e.g., ZO-1, claudin-3, E-cadherin) and the subapical cytoskeletal/microvillar protein ezrin. In addition, Ksp-cadherin was found at UB ampullary cells next to developing outpouches, suggesting a role in epithelial-mesenchymal interactions. These data from the isolated UB culture system support a model where UB branching occurs through outpouching possibly mediated by wedge-shaped cells created through an apical cytoskeletal purse-string mechanism. Additional potential mechanisms include (1) differential localization of growth factor receptors and metalloproteases at tips relative to stalks; (2) creation of a secretory epithelium, in part manifested by increased expression of the ER chaperone BiP, at tips relative to stalks; (3) after initial diffuse proliferation, coexistence of a balance of proliferation vs. apoptosis favoring tip growth with a very different balance in elongating stalks; and (4) differential maturation of the tight and adherens junctions as the structures develop. Because, without mesenchyme, both lateral and bifid branching occurs (including the ureter), the mesenchyme probably restricts lateral branching and provides guidance cues in vivo for directional branching and elongation as well as functioning to modulate tubular caliber and induce differentiation. Selective cadherin, claudin, and microvillar protein expression as the UB matures likely enables the formation of a tight, polarized differentiated epithelium. Although, in vivo, metanephric mesenchyme development occurs simultaneously with UB branching, these studies shed light on how (mesenchymally derived) soluble factors alone regulate spatial and temporal expression of morphogenetic molecules and processes (proliferation, apoptosis, etc.) postulated to be essential to the UB branching program as it forms an arborized structure with a continuous lumen.

Regulation of ureteric bud branching morphogenesis by sulfated proteoglycans in the developing kidney.

Glycosaminoglycans in the form of heparan sulfate proteoglycans (HSPG) and chondroitin sulfate proteoglycans (CSPG) are required for normal kidney organogenesis. The specific roles of HSPGs and CSPGs on ureteric bud (UB) branching morphogenesis are unclear, and past reports have obtained differing results. Here we employ in vitro systems, including isolated UB culture, to clarify the roles of HSPGs and CSPGs on this process. Microarray analysis revealed that many proteoglycan core proteins change during kidney development (syndecan-1,2,4, glypican-1,2,3, versican, decorin, biglycan). Moreover, syndecan-1, syndecan-4, glypican-3, and versican are differentially expressed during isolated UB culture, while decorin is dynamically regulated in cultured isolated metanephric mesenchyme (MM). Biochemical analysis indicated that while both heparan sulfate (HS) and chondroitin sulfate (CS) are present, CS accounts for approximately 75% of the glycosaminoglycans (GAG) in the embryonic kidney. Selective perturbation of HS in whole kidney rudiments and in the isolated UB resulted in a significant reduction in the number of UB branch tips, while CS perturbation has much less impressive effects on branching morphogenesis. Disruption of endogenous HS sulfation with chlorate resulted in diminished FGF2 binding and proliferation, which markedly altered kidney area but did not have a statistically significant effect on patterning of the ureteric tree. Furthermore, perturbation of GAGs did not have a detectable effect on FGFR2 expression or epithelial marker localization, suggesting the expression of these molecules is largely independent of HS function. Taken together, the data suggests that nonselective perturbation of HSPG function results in a general proliferation defect; selective perturbation of specific core proteins and/or GAG microstructure may result in branching pattern defects. Despite CS being the major GAG synthesized in the whole developing kidney, it appears to play a lesser role in UB branching; however, CS is likely to be integral to other developmental processes during nephrogenesis, possibly involving the MM. A model is presented of how, together with growth factors, heterogeneity of proteoglycan core proteins and glycosaminoglycan sulfation act as a switching mechanism to regulate different stages of the branching process. In this model, specific growth factor-HSPG combinations play key roles in the transitioning between stages and their maintenance.

In silico dissection of cell-type-associated patterns of gene expression in prostate cancer.

Prostate tumors are complex entities composed of malignant cells mixed and interacting with nonmalignant cells. However, molecular analyses by standard gene expression profiling are limited because spatial information and nontumor cell types are lost in sample preparation. We scored 88 prostate specimens for relative content of tumor, benign hyperplastic epithelium, stroma, and dilated cystic glands. The proportions of these cell types were then linked in silico to gene expression levels determined by microarray analysis, revealing unique cell-specific profiles. Gene expression differences for malignant and nonmalignant epithelial cells (tumor versus benign hyperplastic epithelium) could be identified without being confounded by contributions from stroma that dominate many samples or sacrificing possible paracrine influences. Cell-specific expression of selected genes was validated by immunohistochemistry and quantitative PCR. The results provide patterns of gene expression for these three lineages with relevance to pathogenetic, diagnostic, and therapeutic considerations.

Rival penalized competitive learning (RPCL): a topology-determining algorithm for analyzing gene expression data.

DNA arrays have become the immediate choice in the analysis of large-scale expression measurements. Understanding the expression pattern of genes provide functional information on newly identified genes by computational approaches. Gene expression pattern is an indicator of the state of the cell, and abnormal cellular states can be inferred by comparing expression profiles. Since co-regulated genes, and genes involved in a particular pathway, tend to show similar expression patterns, clustering expression patterns has become the natural method of choice to differentiate groups. However, most methods based on cluster analysis suffer from the usual problems (i) dead units, and (ii) the problem of determining the correct number of clusters (k) needed to classify the data. Selecting the k has been an open problem of pattern recognition and statistics for decades. Since clustering reveals similar patterns present in the data, fixing this number strongly influences the quality of the result. While there is no theoretical solution to this problem, the number of clusters can be decided by a heuristic clustering algorithm called rival penalized competitive learning (RPCL). We present a novel implementation of RPCL that transforms the correct number of clusters problem to the tractable problem of clustering based on the degree of similarity. This is biologically significant since our implementation clusters functionally co-regulated genes and genes that present similar patterns of expression. This new approach reveals potential genes that are co-involved in a biological process. This implementation of the RPCL algorithm is useful in differentiating groups involved in concerted functional regulation and helps to progressively home into patterns, which are closely similar.

Changes in gene expression patterns in the ureteric bud and metanephric mesenchyme in models of kidney development.

BACKGROUND: In a recent study, the pattern of gene expression during development of the rat kidney was analyzed using high-density DNA array technology (Stuart RO, Bush KT, Nigam SK, Proc Natl Acad Sci USA 98:5649-5654, 2001). This approach, while shedding light on global patterns of gene expression in the developing kidney, does not provide insight into the contributions of genes that might be part of the morphogenetic program of the ureteric bud (UB) and metanephric mesenchyme (MM), the two tissues that interact closely during nephron formation. METHODS: We have now used high-density DNA arrays together with a double in vitro transcription (dIVT) approach to examine gene expression patterns in in vitro models for morphogenesis of the rat UB (isolated UB culture) and MM (coculture with embryonic spinal cord) and compared this data with patterns of gene expression in the whole embryonic kidney at different stages of development. RESULTS: The results indicate that different sets of genes are expressed in the UB and MM as morphogenesis occurs. The dIVT data from the in vitro UB and MM culture models was clustered hierarchically with single IVT data from the whole embryonic kidney obtained at different stages of development, and the global patterns of gene expression were remarkably compatible, supporting the validity of the approach. The potential roles of genes whose expression was associated with the individual tissues were examined, and several pathways were identified that could have roles in kidney development. For example, hepatocyte nuclear factor-6 (HNF-6), a transcription factor potentially upstream in a pathway leading to the expression of KSP-cadherin was highly expressed in the UB. Embigin, a cell adhesion molecule important in cell/extracellular matrix (ECM) interactions, was also found in the UB and may serve as a Dolichos biflorus binding protein in the kidney. ADAM10, a disintegrin-metalloprotease involved in Delta-Notch signaling and perhaps Slit-Robo signaling, was also highly expressed in late UB. Celsr-3, a protein, which along with members of the Wnt-frizzled transduction cascade, might be involved in the polarization of the forming nephron, was found to be highly expressed in differentiating MM. DDR2, a member of the discoidin domain receptor family, which is thought to function in the activation of matrix metalloproteinase-2 (MMP-2), was also found to be highly expressed in differentiating MM. It is also interesting to note that almost 10% of the highly expressed genes in both tissues were associated with neuronal growth and/or differentiation. CONCLUSION: The data presented in this study point to the power of combining in vitro models of kidney development with high-density DNA arrays to identify the genes involved in the morphogenetic process. Clear differences were found between patterns of genes expressed by the UB and MM at different stages of morphogenesis, and many of these were associated with neuronal growth and/or differentiation. Together, the high-density microarray data not only begin to suggest how separate genetic programs in the UB and MM orchestrate the formation of the whole kidney, but also suggest the involvement of heretofore largely unexplored developmental pathways (involving HNF-6, ADAM-10, Celsr-3, DDR2, and other genes) in nephrogenesis.

Debt91, a putative zinc finger protein differentially expressed during epithelial morphogenesis.

In a differential screen for genes that might be important in the regulation of epithelial morphogenesis, we identified a novel gene, Debt91 (differentially expressed in branching tubulogenesis), which is up-regulated in an in vitro model of renal tubulogenesis and branching. Debt91 appears to encode a 381 amino acid molecule with high Ser and Thr composition and is highly conserved at its N-terminus across species. Sequence analysis suggests that it is a coiled-coil nuclear phosphoprotein with zinc finger motifs at the N-terminal conserved region, which is rich in cysteine and histidine. Debt91 is located on mouse chromosome 6 at a region that has conserved synteny with human chromosome 2p11.2, and appears to express two transcripts in several mouse cell lines and adult tissues. On whole murine embryo blots Debt91 expresses primarily its small transcript and is differentially regulated during development. Analysis of expression in in vitro cell culture models suggests that Debt91 is an immediate early gene up-regulated during growth factor-induced branching tubulogenesis.

Maternal hypercholesterolemia during pregnancy promotes early atherogenesis in LDL receptor-deficient mice and alters aortic gene expression determined by microarray.

BACKGROUND: Maternal hypercholesterolemia during pregnancy is associated with markedly enhanced fatty streak formation in human fetal aortas and accelerated progression of atherosclerosis in normocholesterolemic children. METHODS AND RESULTS: To establish the causal role of maternal hypercholesterolemia in a genetically homogeneous murine model and to test the hypothesis that pathogenic events during fetal development result in persistent changes in arterial gene expression, female LDL receptor-deficient (LDLR(-/-)) mice were fed regular chow or high-fat diets supplemented with 0.075% or 1.25% cholesterol during pregnancy. Lesion sizes were determined in the aortic origin of their chow-fed offspring at 3 months. Maternal hypercholesterolemia more than doubled lesion sizes in male offspring (P<0.0001 for the 0.0075% cholesterol group). Microarray analysis of the expression of 11 000 murine genes in the nonatherosclerotic descending aorta by Affymetrix gene chips suggested that 139 genes were significantly regulated in offspring of hypercholesterolemic mothers. A subset of 12 of the upregulated transcripts was subjected to secondary analysis by semiquantitative PCR of pooled RNA and 4 genes were found to be upregulated >1.7-fold. Quantitative PCR for one of these genes using RNA from individual mice yielded similar results. Comparative immunostaining for several of the above genes also indicated increased protein content in offspring of hypercholesterolemic mothers. CONCLUSIONS: These findings establish an atherogenic effect of maternal hypercholesterolemia in genetically uniform mice and indicate that changes in aortic gene expression persist long after fetal exposure to hypercholesterolemia. In addition to elucidating pathogenic mechanisms initiated during fetal development, this approach may identify genes in morphologically normal arteries that influence the susceptibility to classical risk factors of atherosclerosis.