The lectin Dolichos biflorus agglutinin is a sensitive indicator of branching morphogenetic activity in the developing mouse metanephric collecting duct system.

The urine collecting duct system of the metanephric kidney develops by growth and branching morphogenesis of an unbranched progenitor tubule, the ureteric bud. Bud branching is mainly dichotomous and new branches form from existing branch tips, which are also the main sites of cell proliferation in the system. This behaviour, and the fact that some genes (e.g. Wnt11, Sox9) are expressed only in tips, suggests that tip cells are in a specific state of differentiation. In this report, we show that the lectin Dolichos biflorus agglutinin (DBA), hitherto regarded and used as a general marker of developing renal collecting ducts, binds to most of the duct system but does not bind to the very tips of growing branches. The zone avoided by DBA corresponds to the zone that expresses Wnt11, and the zone that shows enhanced cell proliferation. If branching of the ureteric bud of cultured embryonic kidneys is inhibited in organ culture, by blocking the kidney's endogenous glial cell-derived neurothrophic factor (GDNF)-based branch-promoting signals, the DBA-binding zone extends to the very end of the tip but is lost from there when branching is re-activated. Similarly, if excess GDNF is provided to growing kidneys, the DBA-free zone expands. DBA-staining status therefore appears to be a sensitive indicator of the morphogenetic activity of the collecting duct system.

A role for microfilament-based contraction in branching morphogenesis of the ureteric bud.

BACKGROUND: Branching morphogenesis of the ureteric bud/collecting duct epithelium is an important feature of kidney development. Recent work has identified many transcription factors and paracrine signaling molecules that regulate branching, but the physical mechanisms by which these signals act remain largely unknown. The actin cytoskeleton is a common component of mechanisms of morphogenesis. We have therefore studied the expression of, and requirement for actin filaments in the ureteric bud, a branching epithelium of the mammalian kidney. METHODS: Embryonic kidney rudiments were grown in organ culture. Actin expression in kidneys growing normally and those in which branching was inhibited was examined using labeled phalloidin. The morphogenetic effects of inhibiting actin organization and tension using cytochalasin D, butanedione monoxime, and Rho kinase ROCK inhibitors were assessed using immunofluorescence. RESULTS: F-actin is expressed particularly strongly in the apical domains of cells at the tips of branching ureteric bud, but this expression depends on the bud actively growing and branching. Blocking the polymerization of actin using cytochalasin D inhibits ureteric bud branching reversibly, as does blocking myosin function using butadiene monoxime. Inhibiting the activation of ROCK, a known activator of myosin, with the drugs Y27632 or with H1152 inhibits the expression of strong actin bundles in the ureteric bud tips and inhibits ureteric bud branching without inhibiting other aspects of renal development. CONCLUSION: The formation of tension-bearing actin-myosin complexes is essential for branching morphogenesis in the developing kidney.

Pattern and regulation of cell proliferation during murine ureteric bud development.

Branched epithelia determine the anatomy of many mammalian organs; understanding how they develop is therefore an important element of understanding organogenesis as a whole. In recent years, much progress has been made in identifying paracrine factors that regulate branching morphogenesis in many organs, but comparatively little attention has been paid to the mechanisms of morphogenesis that translate these signals into anatomical change. Localized cell proliferation is a potentially powerful mechanism for directing the growth of a developing system to produce a specific final morphology. We have examined the pattern of cell proliferation in the ureteric bud system of the embryonic murine metanephric kidneys developing in culture. We detect a zone of high proliferation at the site of the presumptive ureteric bud even before it emerges from the Wolffian duct and later, as ureteric bud morphogenesis continues, proliferation is localized mainly in the very tips of the branching epithelium. Blocking cell cycling using methotrexate inhibits ureteric bud emergence. The proliferative zone is present at ureteric bud tips only when they are undergoing active morphogenesis; if branching is inhibited either by treatment with natural negative regulators (TGF-beta) or with antagonists of natural positive regulators (GDNF, glycosaminoglycans) then proliferation at the tips falls back to levels characteristic of the stalks behind them. Our results suggest that localized proliferation is an important morphogenetic mechanism in kidney development.

Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation.

Wt1 is a tumour suppressor gene, mutation of which is a cause of Wilms' tumour, a childhood renal nephroblastoma. Wt1 is expressed in a rich pattern during renal development suggesting that it acts at three stages: determination of the kidney area, the differentiation of nephrons and maturation of glomeruli. Wt1-/- mice confirm that Wt1 is essential for the inception of kidney development; cells that ought to form kidneys die by apoptosis instead. Specific human WT1 mutations cause defects of glomerular maturation (Denys-Drash and Frasier syndromes), providing circumstantial evidence for action of Wt1 during glomerular maturation. There is, however, no genetic evidence for a function during nephron differentiation because this stage is never reached in Wt1-/- mice. We have therefore developed a novel technique, based on small interfering RNA (siRNA), to repress the expression of Wt1 and other specific genes at different stages of kidney development in culture. We find that early repression of Wt1 phenocopies the Wt1-/- mouse, but later repression prevents cells differentiating into nephrons and causes them instead to proliferate abnormally, possibly mimicking aspects of Wilms' tumour. In line with established hypotheses about genetic pathways that control kidney development, we find that repressing Pax2 using siRNAs represses Wt1 expression and blocks both bud growth and nephron differentiation, but that repressing Wnt4 blocks nephron differentiation without affecting Wt1 expression. As well as illuminating previously inaccessible aspects of Wt1 biology, our results suggest that siRNA in organ culture will be a powerful method for analyzing other developmental pathways and testing the effects of stage-specific loss of tumour suppressor genes.