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