SCIENCE IN RENAL MEDICINE www.jasn.org

How Does the Ureteric Bud Branch?

Sanjay K. Nigam*†‡ and Mita M. Shah†

Departments of *Pediatrics, †Medicine, and ‡Cellular and Molecular Medicine, University of California, San Diego, San Diego, California

ABSTRACT Many genes that modulate kidney development have been identified; however, (Figure 1A). A comprehensive discus- the molecular interactions that direct arborization of the ureteric bud (UB) remain sion regarding the many factors that have incompletely understood. This article discusses how “systems” approaches may been discovered to modulate UB branch- shed light on the structure of the gene network during UB branching morphogen- ing and mesenchymal-to-epithelial tran- esis and the mechanisms involved in the formation of a branched collecting system sition is beyond the scope of this article from a straight epithelial tube in the context of a stage model. In vitro and genetic and has been the subject of excellent re- studies suggest that the stages seem to be governed by a conserved network of views.1–3 Here we discuss the general genes that establish a “tip-stalk generator”; these genes sustain iterative UB principles that lead to the formation of a branching tubulogenesis through minimal alterations in the network architecture as branched ureteric tree; such principles a budding system shifts to one that autocatalytically branches through budding. may apply to other branching epithelia as The differential expression of stage-specific positive and inhibitory factors in the well. mesenchyme, likely presented in the context of heparan sulfate proteoglycans, and effector molecules in the epithelium seems to regulate advancement between stages; similar principles may apply to other branching epithelia such as the lung, CREATION OF TIPS AND STALKS salivary gland, pancreas, mammary gland, and prostate. Active mesenchymal in- teractions with the UB seem to govern vectorial arborization and tapering of the In vitro and genetic approaches have collecting system and its terminal differentiation. Cessation of branching correlates helped to identify many promoters and in- with induction of mesenchyme as well as local extracellular matrix changes. Per- hibitors of UB branching (reviewed in1,2), turbations of these mechanisms and/or single-nucleotide polymorphisms in genes but the fundamental mechanism of how a regulating UB branching may predispose to a variety of renal diseases (e.g., straight epithelial tube gives rise to a hypertension and chronic kidney disease) by altering nephron number. Decentral- branched tree remains obscure. Before the ization of the gene–protein interaction network may explain the relative paucity of advent of systems specifically designed to branching phenotypes in mutant mice and in human disease. study the UB, branching morphogenesis of the UB was described through the analysis J Am Soc Nephrol 20: 1465–1469, 2009. doi: 10.1681/ASN.2008020132 of branching in renal cell lines such as Ma- din-Darby canine kidney (MDCK) and UB cells4–6; however, recent data suggest Ureteric bud (UB) branching morpho- The renal architecture primarily that UB branching proceeds through a genesis is fundamental to establishing arises through the growth and morpho- fundamentally different mechanism of the architecture of the kidney and is a key genesis of two progenitor tissues, the UB outpouching of wedge-shaped cells that determinant of nephron number. This and metanephric mesenchyme (MM). are created through an apical cytoskeletal process is important not only for normal Through a process of mutual induction “purse-string” mechanism (Figure 2A).7 renal function but also from the stand- between these tissues, the UB is formed point of disease. Although it is clear that through an outpouching of the Wolffian kidney malformations such as renal duct (WD) and undergoes a number of Published online ahead of print. Publication date agenesis and dysplasia are caused by de- iterative dichotomous branching events available at www.jasn.org. fective morphogenesis of the UB, emerg- to form the urinary collecting system Correspondence: Dr. Sanjay K. Nigam, University of California, San Diego, 9500 Gilman Drive, La Jolla, ing data suggest that the predisposition while the MM is induced to undergo a CA 92093-0693. Phone: 858-822-3482; Fax: 858- to several common diseases such as hy- mesenchymal-to-epithelial transforma- 822-3483; E-mail: [email protected] pertension and chronic kidney disease tion to form the nephron, from the epi- Copyright ᮊ 2009 by the American Society of have similar developmental origins. thelial glomerulus to the distal tubule Nephrology

J Am Soc Nephrol 20: 1465–1469, 2009 ISSN : 1046-6673/2007-1465 1465 SCIENCE IN RENAL MEDICINE www.jasn.org

Figure 1. (A) The kidney originates from two mesenchymally derived components: The Wolffian duct (WD) and the metanephric mesenchyme (MM). The initiating step in kidney development is outpouching of the uteric bud (UB) from the WD, an event directed by inductive signals emanating from the MM. After formation of the UB, reciprocal induction between the UB and thee MM leads to multiple iterations of branching and elongation of the UB to form the collecting system, while the mesenchyme is induced to condense and epithelialize around the branched tips and undergo a mesenchymal-to-epithelial transformation. These mesenchymal aggregates then proceed through several morphologic stages, including comma- and S-shaped bodies, forming metanephric tubules that eventually mature into the nephron (proximal and distal tubules, as well as the epithelial glomerulus). In vitro analyses, combined with global patterns of gene expression during kidney development, suggest that UB branching can be conceptualized in terms of developmental stages: (1) Outgrowth of the UB from the WD; (2) early branching in which the UB undergoes rapid, iterative branching; (3) later UB branching characterized by deceleration of branching with accompanied differentiation of the metanephric mesenchyme; and (4) cessation of branching and completion of mesenchymal differentiation. These stages can be recapitulated in in vitro modules that can be reconstituted into engineered kidney tissue. The modest changes in gene expression that occur between the formation of the UB and multiple rounds of branching suggest that a “budding” and “branching” network exists. Thus, iterative branching can occur through minimal alterations in the expression of a small set of genes (represented by the multicolored circles) such that an autocatalytic network, or “tip-stalk generator,” is established. Such alterations may occur through the capture of nodes and/or the “tightness” of links between nodes (represented by the varying thickness of the connections between the multicolored circles). (B) General schematic of the principles that are hypothesized to contribute to vectorial branching and the formation of UB tips and stalks. Patterning of the ureteric tree via vectorial branching and tubule spacing likely occurs through the formation of morphogen gradients. Bud-promoting factors (represented as orange circles) differentially binding to heparan sulfate proteoglycans (HSPG) located on the cell surface UB cells may be an important mechanism for gradient formation (GF) along the ureteric epithelium. New UB branches are created through a budding-out process via the formation of wedge-shaped cells through an apical actin cytoskeletal “purse-string” mechanism (yellow). The “branching through budding” model implies that cells destined to become new UB tips have differential localization of growth factor receptors, extracellular matrix (ECM) components, matrix-degrading enzymes, and possibly differences in local basement membrane composition, such as various molecular weight hyaluronic acid chains at UB tips versus stalk regions.

The “branching through budding” model KIDNEY DEVELOPMENT IS A of the metanephric mesenchyme; and (4) implies that the remodeling of the contig- STAGED PROCESS termination of branching and completion uous epithelial tube occurs through the of mesenchymal differentiation (Figure creation of a secretory epithelium via dif- In addition to the creation of tips and 1A).11,13,14 These stages are separable into ferential localization of growth factor re- stalks, branching morphogenesis under- in vitro modules that have been used to re- ceptors and matrix-degrading enzymes to pins the basic architecture of the kidney. constitute “engineered” kidney tissue that UB tips relative to stalks that initiate new Global patterns of gene expression during is capable of early vascularization and rudi- branch formation, a concept supported by kidney organogenesis, together with in mentary tubular function.15 Each stage is cell lineage studies,8 and microarray analy- vitro and genetic data and morphologic typified by various sets of heparin-binding sis of differential gene expression in UB tip analyses, suggest that branching morpho- growth factors, receptor tyrosine kinases, versus stalk cells.9 Thus, this challenges the genesis of the UB is an iterative yet simul- signaling pathways involving intracellular traditional notion that the epithelial tube is taneously vectorial process that can be kinases, and effectors that mediate cell composed of homogeneous cells and sug- broadly conceptualized in terms of devel- adhesion and basement membrane re- gests that microenvironments within the opmental stages: (1) Outgrowth of the UB modeling (reviewed in2,16–18). Although UB, possibly in the form of gradients, are from the WD; (2) rapid, iterative branch- disruption of certain pathways produces key to vectorial branching morphogenesis ing of the UB; (3) deceleration of UB catastrophic effects (i.e., renal agenesis), and the generation of new tips.10–12 branching accompanied by differentiation numerous instances in which mutation

1466 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1465–1469, 2009 www.jasn.org SCIENCE IN RENAL MEDICINE of key molecules has minimal apparent (e.g., FGFs, pleiotrophin, heregulin) and signaling by morphogen gradients (Fig- phenotypic consequences exist, suggest- inhibitory (e.g., TGF-␤1, bone morpho- ure 1B).33 In this context, heparan sulfate ing that each of these stages is character- genic protein 4, activin) molecules have proteoglycan diversity has been pro- ized by a distinct network structure of been shown to modulate the extent of posed to be a key (although largely unex- gene and protein interactions that confer ureteric branching, but, in general, posi- plored) driving mechanism of stage-spe- varying resilience to mutation.19,24,25 tive feedback mechanisms seem to pre- cific regulation of UB morphogenesis.34 dominate.14 The iterative, or “feed-forward,” na- UB OUTGROWTH ture of the branching tree suggests that LATE UB BRANCHING the expression of a particular set of pro- The initiating step in metanephric devel- teins becomes stable and self-maintain- As organogenesis comes to completion, opment is emergence of the UB from the ing such that an autocatalytic network, branching slows down, presumably ow- WD; failure of this critical step leads to re- or “tip-stalk generator,” is established ing to negative feedback. In the kidney nal agenesis, whereas incorrect positioning (Figure 1A)25; therefore, only a minimal and other branching systems, it seems of the UB leads to a variety of urinary tract alteration in the expression of a small set that members of the TGF-␤ superfam- anomalies ranging from mega-ureter to of genes may be required for the impres- ily are the primary molecules involved vesicoureteral reflux. A multitude of posi- sive morphologic changes that occur be- in branching inhibition, but there is tive and negative regulatory factors, con- tween the stages of UB branching. Such a mounting evidence that negative feed- verging on glial cell line–derived neurotro- “consensus set” of conserved signaling back signals may also arise from mesen- phic factor (GDNF) signaling, play key molecules can also be found among a chyme cell surface molecules.5,14 This is roles in this stage (reviewed in16). Genetic number of organs, and it is the relative anatomically suggested in the kidney as deletion of GDNF or its receptor, Ret, most “weight” of certain pathways (along with fusion between a lateral ureteric branch, often results in renal agenesis, although ru- those guiding final differentiation) that and metanephric tubule effectively re- dimentary kidneys form in up to 50% of may be organ specific.26 Thus, it may be moves the ureteric branch from further these mice,20–22 suggesting that GDNF-de- the structure of the branching network divisions; however, signals to slow branch- pendent budding of the WD may be by- through the capture of nodes and the ing may be present even earlier during passed through activation of other signal- “tightness” of the links between nodes MM-derived tubule formation, where it ing pathways. This concept has been that determines epithelial cell fate.25 has been noted that branch-inhibitory fac- validated through in vitro studies in which Such self-organization has been pro- tors are expressed in comma and S-shaped the combined effect of stimulatory (fibro- posed in gene regulatory networks in or- bodies and thus may regulate the extent blast growth factor 7 [FGF7]) and blockade igin of life scenarios.27 and pattern of branching (reviewed in26). of inhibitory (activin) molecules is able to The question remains, then, how is Mutations in the pathways that regu- induce bud formation in isolated WDs.23 the complex patterning of the nephron late early and late UB branching usually The existence of such a bypass pathway achieved? To explain patterning during do not result in significant branching de- may be sufficient to explain the relative in- embryogenesis, the concept of gradient fects.11 The phenotypes that are manifest frequency of renal agenesis despite the morphogens was proposed more than a are generally quantitative leading to a de- seeming dependence of UB outgrowth on century ago.28 Secreted proteins of the crease in nephron number. There is evi- GDNF signaling; therefore, it may be that a WNT; hedgehog; and members of the dence that low nephron number in hu- “second hit” is necessary to manifest the EGF, FGF, and TGF-␤ families, all of mans predisposes to hypertension and phenotype. which have been found to be important chronic kidney disease35,36; although it is in kidney morphogenesis, have been rec- unlikely that essential hypertension is ognized as candidate substances to pro- due to a loss-of-function mutation in a EARLY UB BRANCHING vide positional information (reviewed single gene, it is conceivable that variant in12,29,30). That TGF-␤ may be an arbiter alleles, identified by single-nucleotide The next conceptualized stage of collect- of tubule spacing has been explored in polymorphisms, that interact differently ing system development, rapid iterative other branching systems in which TGF-␤ with modifiers, suppressors, and en- UB branching, depends on reciprocal ep- repulses epithelial cell processes to space hancers are sufficient to cause subtle ithelial–mesenchymal interactions. A out the branches of the epithelial changes in nephron number that eventu- common theme emerging from the tree.12,31,32 It is interesting to note that ally lead to disease.11 study of diverse branching systems is the most of the secreted proteins involved in existence of both positive and negative UB branching are heparin binding. feedback loops in the complex interplay Heparan sulfate proteoglycans are BRANCHING CESSATION among epithelium and mesenchyme.24 known to modulate cell surface localiza- Like UB outgrowth (and subsequent tion of ligands and thus are ideal candi- The events that signal the termination of stages), a combination of stimulatory dates as positive and negative regulators of branching remain enigmatic. Miscues in

J Am Soc Nephrol 20: 1465–1469, 2009 How Does the Ureteric Bud Branch? 1467 SCIENCE IN RENAL MEDICINE www.jasn.org the cessation of kidney development can the signaling network, resulting in a soluble growth factors. Proc Natl Acad Sci lead to a variety of renal disorders rang- branching module. It seems likely that USA94: 6279–6284, 1997 7. Meyer TN, Schwesinger C, Bush KT, Stuart ing from reduced nephron number to this branching module is conserved RO, Rose DW, Shah MM, Vaughn DA, Steer cystic kidney disease, which can be con- among multiple branching organs,26 and DL, Nigam SK: Spatiotemporal regulation of sidered a disorder of tubule mainte- although it seems plausible to hypothe- morphogenetic molecules during in vitro nance.11 In vitro studies suggest soluble size a unifying theory of epithelial branching of the isolated ureteric bud: To- factors produced by the differentiating branching through the establishment of ward a model of branching through budding in the developing kidney. Dev Biol 275: 44– MM modulate the expression of specific an autocatalytic network, there are un- 67, 2004 subsets of matrix proteases that can doubtedly pathways that are stage and 8. Shakya R, Watanabe T, Costantini F: The modify the extracellular matrix and in- organ specific. It will be through elucida- role of GDNF/Ret signaling in ureteric bud fluence branch termination.12 For exam- tion of those pathways that we will un- cell fate and branching morphogenesis. Dev ple, release of endostatin at UB tips mod- derstand what makes a kidney a kidney Cell 8: 65–74, 2005 9. Schmidt-Ott KM, Yang J, Chen X, Wang H, ulates UB branching and specific sizes and why perturbation of specific path- Paragas N, Mori K, Li JY, Lu B, Costantini F, and concentrations of the basement ways results in renal disease. Schiffer M, Bottinger E, Barasch J: Novel membrane component hyaluronic acid regulators of kidney development from the seems to independently regulate UB tips of the ureteric bud. J Am Soc Nephrol branching and promote tubular matura- 16: 1993–2002, 2005 10. Stuart RO, Barros EJ, Ribeiro E, Nigam SK: 37,38 ACKNOWLEDGMENTS tion. Thus, extracellular matrix com- Epithelial tubulogenesis through branching ponents may act as a potential switch for S.K.N. is supported by National Institute of morphogenesis: Relevance to collecting sys- ending branching morphogenesis, as well tem development. J Am Soc Nephrol 6: Diabetes and Digestive and Kidney Diseases as initiating nephron differentiation. 1151–1159, 1995 grants RO1-DK57286 and RO1-DK65831. Stop/maturation signals also seem to 11. Shah MM, Sampogna RV, Sakurai H, Bush M.M.S. is supported by a Research Career KT, Nigam SK: Branching morphogenesis be correlated with the differentiation of Award from the National Institute of Diabe- and kidney disease. Development 131: the MM.39 UB-derived soluble factors tes and Digestive and Kidney Diseases (K08- 1449–1462, 2004 that promote mesenchymal epithelial- 12. Nigam SK: Determinants of branching tubu- DK069324). ization, such as leukemia inhibitory fac- logenesis. 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