the kidney || formation and development of nephrons

I. Introduction

II. Morphogenesis

III. Induction

IV. Intrinsic Factors That Control the Induction Response

IV. Factors That Drive Mesenchyme-to-Epithelial Conversion

VI. Summary

References

I. Introduction

The development of renal tubules in the adult kidney, ormetanephros, begins with the reciprical, inductive inter-actions between the ureteric bud epithelium and the meta-nephric mesenchyme. The classical model predicts that signalsemanating from the ureteric bud epithelial promote conversionof the metanephric mesenchyme to tubular epithelium, where-as signals from the mesenchyme promote growth and branch-ing morphogenesis of the ureteric bud. As growth and invasionof the ureteric bud have been discussed in previous chapters,this chapter focuses on the metanephric mesenchyme, itsresponses to inductive signals, and the factors required forthe conversion of the mesenchyme to tubular epithelium.

Prior to induction, the metanephric mesenchyme lineageis already fixed within the environment of the intermediatemesoderm. Several key factors are now known to mark thisas the renal anlagen and are required for epithelial specifi-

cation. The signals that induce the mesenchyme to becomeepithelium are also being elucidated, although many ques-tions still remain. Finally, the chapter discusses factors thatpromote growth, remodeling, and cell type specification ofthe nephron segments.

The conversion of metanephric mesenchyme to tubularepithelium has been studied intensively since the 1960s.The morphology of the developing tubules, from uninducedmesenchyme to fully differentiated epithelium, is describedin detail in a classic monograph by Saxén (1987). With respectto the parts of the nephron, the prevailing view held that theglomerular, the proximal tubular, and the distal tubularepithelia were derived from the metanephric mesenchyme,whereas the branching ureteric bud generated the collectingducts and tubules. However, precise cell lineage mapping hasrevealed more plasticity between the two renal primordial celltypes than previously appreciated (Herzlinger et al., 1992;Qiao et al., 1995). Epithelial cells from the ureteric bud areable to delaminate and contribute to more proximal parts ofthe nephron. Thus, if we consider the proximal distal axisalong the tubular epithelium of the nephron, from glomerulusto collecting duct, it becomes more difficult to draw a clearboundary between ureteric bud-derived and metanephricmesenchyme-derived tubular epithelium. Nevertheless, it isclear that much of the tubular epithelium is generatedfrom the induced mesenchyme through a well-characterizedsequence of events.

This chapter introduces genetic and biochemical mech-anisms that are implicated in the development of the renalepithelia. As with any scientific endeavor, there is some con-troversy and conflicting data regarding potential mechanisms

14Formation and Development

of NephronsEun Ah Cho and Gregory R. Dressler

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that regulate early nephron formation. This chapter attemptsto relate recent insights, gained primarily from the pheno-typic analysis of mutant mice, with more classical in vitroand biochemical data regarding induction and differentia-tion. To put these factors into a morphological context, abrief description of the process of mesenchyme-to-epithelialconversion follows.

II. Morphogenesis

In the mouse and rat, the mammalian species studied mostextensively, the ureteric bud makes contact with the meta-nephric mesenchyme at approximately embryonic days 11(E11) and E13, respectively. As outlined in Fig. 14.1, theinitial mesenchymal response to ureteric-bud-derived induc-tive signals is condensation of mesenchymal cells, three tofour cell layers thick, around the tip of the ureteric bud. Asthe bud undergoes dichotomous branching, this condensedmesenchyme forms an aggregate that remains associated, atone side, with the ureteric bud epithelium. While the uretericbud proliferates outward, along the radial axis of the kidney,the aggregate cells begin to show signs of epithelial polarity.Expression of new cadherin genes is induced and a laminin-containing basement membrane is laid down. The polarizedaggregate is known as the renal vesicle. This vesicle developstwo clefts, resulting in a characteristic S-shaped structure,whereas the part of the renal vesicle abutting the uretericbud epithelium fuses to form a continuous epithelial lumen.At this stage, the S-shaped body is already compartmental-ized into distinct groups of cells, based on the expression ofa variety of marker genes. The cleft furthest from the uretericbud becomes the glomerular epithelium, consisting of thevisceral epithelium or podocyte layer and the parietal epithe-lium of Bowmans capsule. Endothelial cells invade thisdistal cleft–distal with respect to the ureteric bud, althoughit generates the proximal part of the nephron–and begin to form the glomerular tuft. The junction between themesenchymal-derived epithelium and the ureteric bud epi-thelium is not entirely clear, as cell lineage tracing reveals adegree of cell mixing between these two precursor cellpopulations. Presumably, that part of the S-shaped body thatconnects to the ureteric bud will generate the distal andcollecting tubules. Proximal tubule precursors are locatedbetween the two ends of the S-shaped structure.

As this rudimentary nephron continues to proliferate, theproximal tubules form a highly convoluted mass of epitheliumthat then leads straight into the medullary zone to generatethe descending and ascending limbs of Henle’s loop. Theascending limb of Henle’s loop returns to establish contactwith the juxtaglomerular cells of the same nephron at themacula densa, a system designed to provide feedback andcontrol the release of intrarenal renin. Epithelial cells alongthe nephron are highly specialized and express many unique

markers, depending on their position. Little is known aboutthe terminal differentiation of epithelial subtypes in thenephron. However, the proper localization of transmembranewater and ion channels and ion pumps is essential for main-taining electrolyte and water homeostasis and regulatingblood pressure.

Much of the early morphogenetic events can be mimickedin vitro using organ culture methods. The kidney rudimentis dissected out of an E11 or E13 mouse or rat embryo, justafter induction when the ureteric bud has invaded the mesen-chyme and formed an ampullae-shaped structure. After 2days, the cultured kidneys exhibit extensive ureteric budbranching and mesenchymal condensations and epithelialderivatives (Fig. 14.2). Expression of early marker genes isvery similar to the in vivo pattern of gene expression, withsome notable differences. In vitro-cultured kidneys undergoonly an initial burst of cell proliferation, do not develop therenal vasculature, and do not exhibit the characteristicascending and descending limbs of Henle’s loop. Despitethese limitations, the early events of mesenchyme-to-epithelial conversion have been modeled quite successfullyin organ culture up to about the S-shaped body stage.

III. Induction

A. Nature of Inductive Signals

In the developing kidney, epithelial and mesenchymalcell–cell interactions are necessary for the survival anddifferentiation of both cell types. The fundamental tenetsgoverning these interactions were first proposed by Grobstein(1956), who utilized primarily in vitro organ culture methodsto formulate his conclusions (see Fig. 14.3). He was able tomicrodissect the ureteric bud from the metanephric mesen-chyme at a time when the mesenchyme had not shown anymeasurable response to the bud (E11.0) and culture the twotissues separately and in combination. In these experiments,neither the bud epithelium nor the metanephric mesenchymewere able to proliferate or differentiate in vitro because of thelack of inductive signals emanating from the other tissue.Thus, in the model of reciprocal inductive signaling, themesenchyme provided essential signals to promote growthand branching of the ureteric bud, whereas the bud epithe-lium provided signals to promote aggregation and differentia-tion of mesenchyme into the epithelium of the developingnephron. Furthermore, the metanephric mesenchyme couldbe induced by tissues other than the ureteric bud. Among theheterologous tissues tried, the dorsal embryonic neural tubeproved a powerful inducer of kidney mesenchyme that waseasy to isolate and grow (Lombard and Grobstein, 1969;Unsworth and Grobstein, 1970). However, the metanephricmesenchyme would always form characteristic renal epithe-lium, no matter what inducing tissue was used, suggesting

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14 Formation and Development of Nephrons 197

Figure 14.1 Morphogenesis of the nephron. Sequential stages of the development of a single nephron are outlined schematically. At the inductionphase, mesenchymal cells aggregate at the tips of the ureteric bud. Not all mesenchyme becomes aggregates, as some cells remain along the periphery aspotential mesenchymal stem cells and others become interstitial mesenchyme. The aggregate becomes polarized into a renal vesicle, which remains closelyassociated with the ureteric bud. Meanwhile, a new aggregate forms at the growing tip of the bud as the next generation of nephron is induced. At the S-shaped body stage, the polarized vesicle elongates and forms two visible clefts, the most distal of which becomes infiltrated with endothelial precursor cells.The glomerulus is beginning to take shape and much of the epithelia now express specific markers for proximal or distal tubules. Proliferation of tubularepithelium generates a highly convoluted proximal and distal part, whereas the tubules of Henle’s loop now extend toward the medullary zone along the radialaxis of the kidney. The glomerular tuft takes shape as endothelial cells, and the visceral epithelia, or podocytes, come into contact to form the filtration unit.

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that the inductive signals derived from the ureteric bud wereprimarily permissive rather than instructive, a distinction firstproposed by Saxén (1987). The metanephric mesenchyme wasalready fated to become renal epithelium, needing only asignal to initiate and maintain the process of differentiation.Heterologous inducers could not reprogram the mesenchymeto generate other epithelial cell types. Morphologically, themetanephric mesenchyme is patterned with respect to thesurrounding intermediate mesoderm and appears as a distinctaggregate of cells adjacent to the nephric duct. A numberof specific genes, such as WT1, GDNF, and Pax2, arealready expressed prior to induction and mark the meta-nephric anlagen.

Properties of the inductive signals emanating from the ure-teric bud and spinal cord were characterized in detail usingthe transfilter organ culture system (Saxén, 1987). The indu-cers were not small, freely diffusible molecules, as a mini-

mum pore size was necessary for signaling to occur acrossthe filter. Indeed, induction was coincident with cellularprocesses from the inducing tissue permeating the filters.Thus, the idea of cell contact, being a necessary event forinduction, was established. Early attempts to use conditionedmedia from spinal cord or ureteric bud also failed. However,more recent experiments using conditioned media from ratpituitary (Perantoni et al., 1991) or from ureteric bud cell lines(Barasch et al., 1999; Karavanova et al., 1996), in conjunc-tion with survival factors such as fibroblast growth factor(FGF) and transforming growth factor α (TGF-α), suggestthat inducing activity could be found in a soluble form.

As much of the data indicated a requirement for cellcontact, the question still remained how the inductive signalwas able to penetrate the mesenchyme such that cells notimmediately around the bud could also be induced. Saxénand colleagues put two hypotheses to the test in an elegantseries of experiments. The signal may penetrate several celllayers deep into the mesenchyme by attachment to cellularprocesses or extracellular matrix that extends into the mesen-chymal aggregate. Alternatively, mesenchyme cells imme-diately abutting the ureteric bud, once induced, could passon the inductive signals to neighboring uninduced cells. Ifthe signal was passed from one induced mesenchyme cell toanother, then isolated mesenchyme, which had been exposedto an inducer for 24 h, could now be utilized as a source ofinductive signals for naive mesenchyme. However, Saxénand Saksela (1971) demonstrated that only the mesenchymeoriginally exposed to the inducer was able to form tubules,whereas the uninduced mesenchyme remained as such. Thus,it appears that each mesenchymal cell requires contact withthe primary source of the inducing signal. This was consis-tent with the observation that the cellular process emanatingfrom the inducing tissues could be observed extending threeto four cell layers deep into the mesenchyme.

B. Wnt Genes as Candidate Inducers

Many of the properties associated with the inductive sig-nals emanating from the ureteric bud are common to the wntfamily of secreted signaling peptides. The first clue that Wntgenes may be important signaling molecules for kidneydevelopment, specifically for induction of the metanephricmesenchyme, came from phenotypic analysis of mouse mu-tants for the wnt4 gene (Stark et al., 1994). The Wnt genefamily encodes secreted signaling peptides that are associatedwith the extracellular matrix and have a variety of develop-mental functions (Nusse and Varmus, 1992; Wodarz andNusse, 1998). The prototype Wnt protein is Drosophila wing-less, which controls segment polarity, patterning of the wingdisc, and epithelial planer cell polarity. Wnt4 is expressed inthe early mesenchymal aggregates (Fig. 14.4), just after induc-tion by the ureteric bud and prior to formation of the polari-zed renal vesicle. Homozygous mice for a wnt4 null alleleexhibit severe renal agenesis due to a developmental arrest


Figure 14.2 in vitro tubulogenesis. Whole-mount antibody stainingagainst proteins expressed in the early kidney rudiment. (A) After 2 days inculture, multiple branches of the ureteric bud (UB) are evident and Pax2-positive cells of the condensed mesenchyme (CM) are closely associatedwith the bud tips. Some Pax2-positive cells have undergone polarizationinto renal vesicles and comma-shaped bodies (CB). (B) Expression of thecell adhesion molecules E-cadherin and cadherin-6 mark ureteric bud epi-thelia and mesenchymal aggregates. The comma-shaped bodies are alreadycompartmentalized with respect to cadherin expression. (C) Staining of the basement membranes with antilaminin clearly marks the S-shapedstructures (S) that are derived from the mesenchyme. Epithelium derivedfrom the ureteric bud is stained with an antipan cytokeratin antibody. Notethe junction of the mesenchymal-derived epithelium and the ureteric bud-derived epithelium (arrow). (D) By 4 days in culture, glomeruli-likestructures are evident with anti-WT1 antibodies (arrows). Levels of WT1are particularly high in the podocytes and their precursors.

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Figure 14.3 The transfilter induction assay. E11 or E13 mouse or rat kidneys are microdissected from the embryo, generally with fine tungsten needlesor 30-gauge syringe needles. The tissues are cultured on a polycarbonate filter, with a defined pore size, and are suspended above the media on a solidsupport. For the mesenchyme induction assay, the inducer is fixed to the bottom of the filter and the mesenchyme is placed on top. The inducing signalpermeates the pores of the filter and stimulates the conversion of mesenchyme to epithelium over a period of 24–48 h.

Figure 14.4 Expression of Wnt genes in the metanephric kidney. Thesecreted components of the Wnt signaling pathway are expressed in a tem-poral and spatial manner during induction and mesenchymal aggregation.At E11.5, Wnt4 is in the aggregates, whereas Wnt11 is in the ureteric bud,especially the tips. By E12, Wnt11 becomes restricted to the ureteric budsand Wnt4 continues to be in the aggregates and renal vesicles. The secretedfrizzled-related proteins, potential inhibitors of Wnt signaling, are nowobserved in the comma-shaped structures (sFRP2) and in the uninducedmesenchyme (sFRP1).

show no evidence of a mesenchyme-derived epithelium. ThePax2 gene is detectable, albeit at low levels, in these wnt4mutant aggregates, but there is no expression of the Pax8 gene,which normally is activated in more developed aggregates.While it appears that wnt4 is not the primary ureteric bud-derived inductive signal, based on its expression pattern andon the early aggregation of the wnt4 mutant mesenchyme,wnt4 is a secondary signal for the induced mesenchyme toundergo polarization. Alternatively, given the relationshipbetween the stromal lineage and the epithelial lineage in themesenchyme (see Section V), wnt4 may act as a paracrinesignal from the mesenchymal aggregates that stimulatesstromal cells to secrete survival factors for both inducedmesenchyme and ureteric bud epithelium. In the absence ofthis paracrine signaling, developmental arrest occurs.

Evidence that Wnt signals can function as the primaryinducers of mesenchyme-to-epithelial conversion is alsocompelling. By expressing wnt1 in NIHT3 cells, Herzlingeret al. (1994) could confer the ability of 3T3 cells to inducemetanephric mesenchyme in the transfilter assay, indicatingthat either Wnt1 can itself induce the mesenchyme or thatwnt1 activates a factor in 3T3 cells capable of inducing mesen-chyme. Similar experiments further demonstrated that manyWnt proteins are capable of inducing tubulogenesis in themetanephric mesenchyme. Indeed, the different Wnt proteins

shortly after the ureteric bud invades the mesenchyme. Thebud is able to branch a few times, and there is evidence ofearly aggregation of the mesenchyme. However, wnt4 mutants

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examined by Kispert et al. (1998) fall into two basic cate-gories: those able to induce mesenchyme include wnt 1, 3a,4, 7a, and 7b and those unable to induce the mesenchyme,including wnt5a and wnt11. Thus, the ability of the spinalchord to induce the mesenchyme can be explained by the pre-valence and diversity of wnt genes expressed there. Unfor-tunately, the only wnt gene expressed in the ureteric bud tips,wnt11 (Kispert et al., 1996), proved incapable of inducingtubules in the transfilter assay.

The canonical wnt signaling pathway is activated whenthe wnt protein binds to the frizzled family of transmem-brane receptors (Figure 14.5; Wodarz and Nusse, 1998). Thisactivates the cytoplasmic protein disheveled, which can inhibitthe kinase, GSK3. The β-catenin protein is a target of GSK3phosphorylation and, once phosphorylated, is shuttled to theubiquitin degradation pathway via the β-catenin-bindingproteins axin and APC. Inhibition of GSK3 results in the accu-mulation and nuclear translocation of β-catenin where it caninteract with the TCF/LEF family of DNA-binding proteinsand activate transcription. In the absence of nuclear β-catenin,

the TCF/LEF factors cooperate with the Groucho family ofproteins to repress transcription (Eastman and Grosschedl,1999). Strikingly, lithium is a potent inhibitor of GSK3 (Kleinand Melton, 1996) and can simulate many of the inducing pro-perties of wnt signals. Upon incubation of metanephric mesen-chyme with lithium ions, aggregation and early evidence ofepithelialization are observed (Davies and Garrod, 1995).However, the lithium-treated mesenchyme cannot progressto a fully polarized epithelium, perhaps because its effects aretoo broad. During normal kidney devlopment, wnt signalingmay be modulated by local inhibitors, such as the secretedfrizzled-related proteins (sFRP)(Leyns et al., 1997), whichare highly expressed in the early mesenchymal derivatives(Leimeister et al., 1998; Lescher et al., 1998). Because lithiumacts directly on GSK3, its effects cannot be regulated nega-tively by sFRPs, which inhibit signaling by competing withthe transmembrane frizzleds for wnt binding. In addition toPax-8, the secreted frizzled-related protein-2 (sFRP-2) wasnot detected in wnt4 homozygous mutant embryonic kidneys(Lescher et al., 1998). However, culturing wnt-4 –/– met-anephric mesenchyme in the presence of wnt-4 inducedexpression of both molecules. Thus, Wnt-4 may activate itsown potential inhibitor, perhaps to negate the effects of wntsignaling in more developed epithelial cells.

Wnt signaling can activate other pathways, independentof β-catenin. The specification of epithelial planar cell polarityin the fly is mediated partly through wnt activation of the c-jun N-terminal kinase (JNK; Mlodzik, 1999). Activation ofJNK requires disheveled and appears to work through theGTPase RhoA. The disheveled protein has at least threedifferent protein–protein interaction domains: the amino-terminal DIX domain stabilizes β-catenin, the carboxy-terminal DEP domain activates JNK, and between theseterminal domains lies a PDZ domain whose function is lessclear (Axelrod et al., 1998; Boutros et al., 1998; Yanagawaet al., 1995). Thus, it appears that disheveled is a branchpoint that can differentially activate alternative biochemicalpathways in a wnt-dependent manner. However, how disheve-led is activated by different frizzled proteins remains to bedetermined and is fundamental to understanding WNT signal-ing specificity in different developing tissues.

Early transfilter experiments suggested that mesenchymal-inducing signals were not freely diffusible. Although secre-ted, wnt proteins are rarely detected in the media of expres-sing cells. Instead, they appear to be associated with theextra-cellular matrix through interactions with heparin-sulfated glycoproteins. In the fly, Dally encodes a sulfatedproteoglycan that is required for wingless signaling andwhose overexpression can mimic a wingless gain-of-functionphenotype in the absence of extra wingless protein (Lin andPerrimon, 1999; Tsuda et al., 1999). In the kidney, chloratetreatment inhibits Wnt-inducing activity and reduces the accu-mulation of wnt11 mRNA at the ureteric bud tips (Kispertet al., 1996). In addition, mouse mutants carrying a null allele


Figure 14.5 The simple model of Wnt signaling. Wnt proteinsinteract with the transmembrane receptor frizzled (Frz) and the LDLreceptor-related protein (LRP) to activate the disheveled (Dvl) protein.Disheveled can inhibit the kinase GSK3, which is also inhibited by lithium.Disheveled can also activate the c-jun N-terminal kinase (JNK) pathway.GSK3 phosphorylates β-catenin such that β-catenin binds the proteins axinand APC and is targeted for degradation. Upon inhibition of GSK3, β-catenin levels rise and can trasnlocate to the nucleus where it interacts withthe TCF/Lef family of DNA-binding proteins. The TCF/Lef family bindsDNA but suppresses transcription in the absence of β-catenin, also throughinteractions with the groucho family of repressors. Nuclear β-cateninprovides an activation domain in trans such that the complex can nowactivate target gene expression.

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for the gene encoding heparan sulfate 2-sulfotransferaseexhibit renal agenesis (Bullock et al., 1998), reminiscent of awnt4 phenotype. Thus, wnt signaling requires association withglycoproteins found in the extracellular matrix, consistent withthe observation of contact and process extension into themesenchyme as a prerequisite for induction.

Clearly there is a definitive role for wnt signaling in theearly mesenchyme-to-epithelial transition. Although wntsignals can mimic the inductive signals emanating from theureteric bud and the spinal chord, it has not been shown con-clusively that a specific wnt signal is the primary inducer ofthe metanephric mesenchyme. Furthermore, the receptors forindividual wnt proteins, of which at least six are expressedin the kidney, have not been described in sufficient detailso that individual ligands can be assigned for each frizzledfamily member.

C. Induction of Tubules by Soluble Factors

Since the initial observations by Grobstein, many growthfactors have been tested for inducing activity (Weller et al.,1991). Although early transfilter experiments were not consis-tent with the possibility of soluble, diffusible factors, severalreports have demonstrated mesenchyme induction using acocktail of growth factors and extracts. Rat pituitary extractsin combination with epidermal growth factor and extra-cellular matrix could stimulate epithelial conversion of ratmetanephric mesenchyme (Perantoni et al., 1991). Morerecently, basic fibroblast growth factor and transforminggrowth factor-α, together with conditioned media from aureteric bud cell line, were used as an inducer of rat meta-nephric mesenchyme (Barasch et al., 1996). The active ingre-dient in the conditioned medium of ureteric bud cell lineswas determined to be leukocyte inhibitory factor (LIF), which,together with a defined concentration of bFGF and TGF-α,had inducing activity (Barasch et al., 1999). While these dataare thought provoking and cause one to reexamine some ofthe early transfilter experiments, a number of caveats makethe interpretation difficult. The experiments done with rattissue, and at least in one case, were not reproducible with thecorresponding mouse mesenchyme (Perantoni et al., 1991).Given the conservation of genetic pathways between organi-sms as disparate as humans and flies, this is rather unex-pected and points to an inherent problem. Furthermore, mousemutants do not support the role of any of these factors inkidney induction. There is no appreciable renal phenotype inbFGF mutant mice (Dono et al., 1998). Knockout mice carry-ing a null allele of the LIF receptor gp130 show only a slightreduction in kidney size, with no real effect on induction orepithelial differentiation (Yoshida et al., 1996). In the classicin vitro induction experiment, mesenchyme is micro-dissected away from the ureteric bud, yet it has already beenin contact and may have received a primary signal. In culture,24 h of contact was required before a full response was seen

in the mesenchyme (Saxén and Lehtonen, 1978), as judgedby the formation of tubules. If the appropriate mixture ofgrowth and survival factors is present, perhaps this need forextended contact is reduced. Thus, at the time of dissectionand explant, cells that have been exposed to the primaryinducer can now survive and generate tubules in sufficientnumber. These issues need to be examined before the role ofLIF and FGFs during primary induction can be establisheddefinitively. Regardless of their potential as primary inducers,FGF and LIF may provide important survival or secondarysignals for the mesenchyme and its derivatives.

IV. Intrinsic Factors That Control theInduction Response

Prior to induction by the ureteric bud, the metanephricmesenchyme is an aggregate of distinct cells, expressing aunique combination of genes. Some of these early genes arenecessary for the survival of the mesenchyme and for itsability to respond to the inductive signals emanating from thebud. However, little is known about the positional cues thatpattern the posterior intermediate mesoderm. In frog and chickembryos, paraxial mesoderm is required for formation of thepro- and mesonephric fields, as marked by the expression ofPax2 and lim1, whereas lateral plate mesoderm or notochordis not required (Seufert et al., 1999; Mauch et al., 2000).What signals may preprogram the mesenchyme such that itsdevelopmental fate is restricted remain to be identified.However, it can be said with some assurance that the processof tubule development begins before induction of the mesen-chyme, as genetic mechanisms have fixed, at least in part,the renal epithelial lineages.

A. Pax2 Gene

An essential gene for early urogenital development istranscription factor Pax2. Members of the Pax gene familywere first identified in Drosophila as genes regulatingsegmentation and neural cell identity. In the mouse, Paxgenes are required for the development of the eye, the verte-bral column, certain derivatives of the neural crest, B lympho-cytes, the thyroid, and various neural structures (Dahl et al.,1997; Noll, 1993; Stuart et al., 1993). In both mice andhumans, Pax genes are haploinsufficient, such that hetero-zygous individuals already exhibit phenotypes in organsystems regulated by the respective gene. The phenotype ofheterozygous individuals is variable and less severe thanthat found among homozygotes. In at least one case, additionalcopies of a wild-type Pax6 gene also disturb normal eyedevelopment (Schedl et al., 1996). Thus, data indicate a strictrequirement for Pax gene dosage during normal development.

In the developing kidney, Pax2 expression is first detect-ed in the intermediate mesoderm, prior to formation of the


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nephric duct, beginning at approximately E8.5. Mousemutants lacking Pax2 fail to develop mesonephric tubules,the metanephros, and the sex-specific epithelial componentsderived from this region (Torres et al., 1995). Despite its earlyexpression pattern, Pax2 is not required for initiation andextension of the nephric duct epithelium. Within the area ofthe mesonephros, Pax2 mutants show no evidence of tubuleformation, indicating that the periductal mesenchyme is unableto generate epithelium or respond to signals derived from thenephric duct. Metanephric mesenchyme isolated from Pax2mutants is also unable to respond to inductive signals fromheterologous-inducing tissues in the organ culture assay. Thusit appears that Pax2 is necessary for specifying the region ofintermediate mesoderm destined to undergo mesenchyme-to-epithelium conversion. However, the Pax2 mutant nephricduct does not respond to GDNF and shows no evidence ofureteric bud outgrowth, despite expression of at least somemarkers of normal ductal epithelium, suggesting also thatPax2 in the duct epithelium is required for maintenance orresponsiveness (Brophy et al., 2001).

Pax2-expressing cells in the metanephric mesenchymeare closely associated with the ureteric bud. This observationled to the proposition that Pax2 expression was activated byinductive signals emanating from the bud (Dressler et al.,1990; Dressler and Douglass, 1992). Further support for thedependence of Pax2 expression on inductive signals camefrom studies with the Danforth’s short tail (Sd) mouse, inwhich Pax2 expression was detected in the nephric duct andureteric bud but not in the mesenchyme, presumably due tolack of ureteric bud invasion (Phelps and Dressler, 1993).However, the primary defect in the Sd mouse is posteriordegeneration of the notocord. Thus, gene expression in theintermediate mesoderm could be affected by loss of notocord-derived patterning signals. Until recently, it has proveddifficult to separate Pax2 expression in the mesenchyme fromureteric bud invasion, as it is not easy to define the mesen-chyme morphologically prior to E11. However, we haveobserved Pax2 expression in the metanephric mesenchymeof E11.5 RET homozygous mutants, which have no uretericbud but essentially wild-type mesenchyme. Thus, Pax2expression in the metanephric mesenchyme predates uretericbud invasion and marks the posterior intermediate meso-derm as the renal anlagen (Brophy et al., 2001).

B. WT1 Gene

One potential target of Pax2 activation is the Wilms’tumor suppressor gene, WT1. Wilms’ tumor is an embryonickidney neoplasia that consists of undifferentiated mesen-chymal cells, poorly organized epithelium, and surroundingstromal cells. Because the neoplastic cells of the tumor areable to differentiate into a wide variety of cell types, the genesresponsible for Wilms’ tumor were thought to be importantregulators of early kidney development (van Heyningen and

Hastie, 1992; Chapter 22). Expression of WT1 is regulatedspatially and temporally in a variety of tissues and is furthercomplicated by the presence of at least four isoforms,generated by alter-native splicing. In the developing kidney,WT1 can be found in the uninduced metanephric mesenchymeand in differen-tiating epithelium after induction (Armstronget al., 1992; Pritchard-Jones et al., 1990). Initial expressionlevels are low in the mesenchyme. Strikingly, WT1 becomesupregulated at the S-shaped body stage in the precursor cellsof the glome-rular epithelium, the podocytes. High WT1 levelspersists in the podocytes of the glomerulus into adulthood.

How WT1 impacts kidney development is still under in-tense investigation. However, some conclusions can be drawnfrom the phenotypes of both mouse and human mutations.The homozygous null WT1 mouse has complete renal age-nesis (Kreidberg et al., 1993) because the ureteric bud failsto grow out of the nephric duct and the metanephric mesen-chyme undergoes apoptosis. The arrest of ureteric bud growthappears to be noncell autonomous and is most probably dueto lack of signaling by the WT1 mutant mesenchyme. How-ever, the mesenchyme is unable to respond to inductive signalseven if a wild-type inducer is utilized in the organ cultureassay. Taken together, it appears that WT1 is required earlyin the mesenchyme to promote cell survival, such that cellscan respond to inductive signals and express ureteric budgrowth promoting factors.

What might the WT1 gene be doing at later stages ofkidney development? Increased expression levels in podocyteprecursor cells correlate with repression of the Pax2 gene(Ryan et al., 1995). WT1 binds to the promoters of a varietyof genes, including Pax2, IGF2, and the IGF2 receptor, bydirect contact of the zinc finger DNA-binding domain to aconserved GC-rich nanomer. Much data indicate that theamino-terminal region of WT1 acts as a transcription repres-sion domain (Madden et al., 1991), consistent with theincreased levels of growth factor expression observed inWilms’ tumors. However, in other cases, WT1 is able to func-tion as an activator. Furthermore, specific isoforms of WT1proteins have been colocalized to the mRNA splicing machi-nery, suggesting a posttranscriptional role (Davies et al.,1998). Thus, many questions still remain to be addressed.The expression levels of WT1 are regulated precisely duringkidney development and may indicate different functionsdepending on the threshold level of protein present. How-ever, it is not even clear if all WT1 isoforms are expressedequally in different cell types. This raises the possibility thata shift in the ratio of WT1 isoforms may specify repression,activation, or posttranscriptional regulatory functions.

C. Eya-1

The mammalian homologues of the Drosophila eyes absentgene constitute a small gene family, of which at least onemember is an essential regulator of early kidney development.


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In humans, mutations in the Eya1 gene are associated withBranchio-Oto-Renal syndrome, a complex multifacetedphenotype (Abdelhak et al., 1997). In mice homozygous foran Eya1 mutation, kidney development is arrested at E11because ureteric bud growth is inhibited and the mesen-chyme remains uninduced (Xu et al., 1999).

Eya1 expression during kidney development is first detectedin both the mesonephric and the metanephric mesenchymeand appears to regulate the expression of GDNF. In Eya1mutants, Pax2 and WT1 expression appears normal, but Six2and GDNF expression are lost in the mesenchyme. The lossof GDNF expression most probably underlies the failure ofureteric bud growth. However, it is not clear if themesenchyme is competent to respond to inductive signals ifa wild-type inducer were to be used in vitro. Eya proteinsshare a conserved domain but do not have DNA-bindingactivity. Eya proteins appear to function through direct inter-actions with the Six family of homeodomain proteins. MouseSix genes are homologues of the Drosophila sina oculishomeobox gene and encode DNA-binding proteins that alsointeract directly with the Eya family of nuclear factors. Eyaproteins, in turn, can also bind to the Dachshund family ofproteins. This cooperative interaction between Six and Eyaproteins is necessary for nuclear translocation and tran-scriptional activation of Six target genes (Ohto et al., 1999).In Drosophila eye development (Pignoni et al., 1997) andduring muscle cell specification in the chick embryo(Heanue et al., 1999), the regulatory network involving Pax,Eya, Six, and dachshund genes is well conserved. Based onconservation of expression patterns and mutant phenotypes,a similar regulatory network has been proposed in the dev-eloping mouse kidney (Xu et al., 1999), with Pax2 being up-stream of Six2 and Eya1. A dachshund family homologuehas not yet been described in the kidney, but there is noreason to believe it will not be found.

V. Factors That Drive Mesenchyme-to-Epithelial Conversion

The response of the metanephric mesenchyme to induc-tive signals has been under intense scrutiny for many years(Ekblom, 1989). The aggregation and polarization of themesenchyme have been utilized as a readout for the presenceof inductive signals and for the characterization of the cellularchanges leading to a fully polarized epithelium. Presently, theability to follow the cellular changes in response to induc-tion has been advanced greatly by the identification of manynew gene products that are activated in the induced cell andthat may drive the process of epithelialization.

The mesenchyme undergoes extensive remodeling afterinduction. Proliferation and differentiation are tightly linkedwithin the newly formed tubules. The activation of genesspecific for epithelia and the repression of mesenchymal

specific genes must be regulated by transcription factors thatrespond, at least initially, to the inductive interactions. Thedriving forces underlying cell aggregation and polarizationwould include the activation of new cellular adhesion mole-cules and extracellular matrix components.

A. Aggregation and Cell Adhesion

Among the most intensely studied cell adhesion mole-cules, cadherins are transmembrane proteins that promotehomophillic adhesion between cells. Adhesion is mediatedby an extracellular cadherin repeat whose structural con-formation changes with increasing calcium concentration(Gumbiner, 1996; Koch et al., 1999). Cadherins are classi-fied into type I and type II subfamilies based on the sequenceof the extracellular domain and functional properties of theintracellular domain. Type I cadherins, which include E-,N-, P-, and R-cadherin, contain five extracellular cadherinrepeats, the first of which mediates specific homophilic inter-actions. This repeat promotes interactions between cadherinsof the same type on opposing cells and quite possibly withinthe same cell (Shapiro et al., 1995). The intracellular domainsof type I cadherins bind to the catenins, a family of cyto-plasmic proteins linking cadherins to the actin cytoskeleton(Kemler and Ozawa, 1989; Ozawa et al., 1989). Within thesame cell, association with the catenins is also important forclustering cadherins at the cell surface (Katz et al., 1998;Yap et al., 1997, 1998). The adherents junction, a special-ized site of cell–cell contact that binds cells into solid tissue,forms as cadherins cluster at points of contact between twocells expressing like cadherins (Gumbiner, 1996). In epithelia,disruption of the adherents junction has a profound affect onthe maintenance of other contacts and ultimately to a loss ofepithelial polarity and integrity. Thus, adhesive forces canremodel developing tissues and compartmentalize the tubulesinto distinct units. The type II subfamily of cadherins,including cadherin-6, cadherin-11, and F-cadherin, are closelyrelated to the classical cadherins in both structure and function(Cho et al., 1998; Hoffmann and Balling, 1995; Kimura etal., 1995). Type II cadherins can also mediate cell adhesionand bind to the actin-based cytoskeleton via catenins (Inoueet al., 1997; Nakagawa and Takeichi, 1995).

During the development of renal tubules, cadherin expres-sion is dynamic and complex (Fig. 14.6). Prior to induction,the metanephric mesenchyme expresses cadherin-11, whereasthe ureteric bud expresses E-cadherin (Cho et al., 1998).Upon aggregation of the metanephric mesenchyme, in res-ponse to inducing signals from the ureteric bud, the expressionof cadherin-11 is downregulated and the expression of R-cadherin and cadherin-6 is upregulated (Cho et al., 1998;Rosenberg et al., 1997). The onset of R-cadherin andcadherin-6 expression is coincident with formation of therenal vesicle and the deposition of a basement membrane.Thus, formation of the first polarized epithelium derived


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from the mesenchyme correlates with profound changes incadherin expression. As the renal vesicle proliferates to formthe S-shaped body, R-cadherin is primarily restricted to theepithelial structures furthest from the ureteric bud, E-cadherinis expressed in the bud and most distal tubules, and cadherin-6is located between E-cadherin and R-cadherin expressiondomains (Cho et al., 1998; Rosenberg et al., 1997). Theexpression of cadherin-6 marks the proximal tubule precursorcells, including the ascending and descending limbs of Henle’sloop at later developmental stages. Within the glomerulus,R-cadherin becomes restricted to the parietal epithelium ofBowman’s capsule and the most proximal tubule cells, where-as P-cadherin becomes evident in the podocytes of the visceralepithelium in the glomerular tuft. As proximal tubules mature,E-cadherin becomes more prominent and cadherin-6 expres-sion is suppressed. These dynamic changes in the expressionof type II and type I cadherins correlate with proliferation,cell polarity, and tissue remodeling, strongly indicating that

cadherins mediate aspects of the differential cell adhesionrequired for nephrogenesis.

Within the kidney, cadherin function has been addressedin vitro and in vivo. Cell adhesion can be neutralized in vitrousing Fab fragments of antibodies against the cadherin extra-cellular domains. In kidney organ cultures, anti-cadherin-6Fabs inhibit mesenchymal cell adhesion and the subsequentformation of comma and S-shaped structures, without affect-ing ureteric bud branching (Cho et al., 1998). Similar experi-ments using neutralizing antibodies to E-cadherin have noapparent effect on the conversion of mesenchyme to epithe-lium (Vestweber et al., 1985). Cadherin-6 mutant mice havealso been described, yet their phenotype is not nearly asstriking as the in vitro experiments might suggest (Mah etal., 2000). The mesenchyme of cadherin-6 mutants appearsdelayed in its ability to form a fully polarized epithelium.This delay may compromise the ability of the mesenchymal-derived epithelium to fuse with the ureteric bud-derived


Figure 14.6 A model of cadherin gene switching during nephrogenesis. This schematic model presents a composite of published expression patterns(Cho et al., 1998; Goto et al., 1998; Mah et al., 2000; Rosenberg et al., 1997; Tassin et al., 1994). The model tries to reconcile any discrepancies in theliterature as fairly as possible. The uninduced mesenchyme expresses cadherin-11, which is widely distributed in other embryonic mesenchymal tissue.Upon aggregation of the induced mesenchyme, R-cadherin and cadherin-6 are activated. E-Cadherin is prevalent in the ureteric bud epithelium and can alsobe seen in part of the aggregates close to the bud tips. By the S-shaped body stage, R- and P-cadherin can be detected in the podocyte precursor cells, R-cadherin in much of the proximal part of the developing nephron, cadherin-6 in the proximal part of the nephron, and E-cadherin in the more distal parts ofthe developing nephron. In the maturing nephron, P- and R-cadherin are mostly in the glomerular tuft, the podocytes, whereas cadherin-6 becomes morerestricted to the proliferating epithelia, such as the loop of Henle. E-cadherin is widely distributed both in more mature proximal and distal tubules and allcollecting ducts.

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epithelium and results in a high frequency of dead-endingnephrons. Despite some similarites between in vitro antibody-mediated disruption experiments and knockout cadherin-6mutants, it is clear that the organ culture assay is much moresensitive to perturbations. in vitro development proceeds ata slower rate and lacks certain cell types, such as the vascu-lature. Alternatively, the antibody inhibition experimentsmight inhibit adhesion mediated not only by cadherin-6, butalso by potential heterodimeric cadherins. The recent dis-covery that at least some cadherins can form heterodimerswithin the same cell and function as heterodimeric adhesionmolecules between cells raises the possibility that antibodiesmay inhibit the function of more than just one type ofcadherin protein (Shan et al., 2000). In any event, in vitroinhibition studies must be evaluated with caution before anyassignment of protein function can be defined.

B. Cell–Matrix Interactions

Changes in cell adhesion occur not only at the junctionsbetween cells, but also at the interface between cells and theextracellular matrix (ECM). The integrin family of trans-membrane proteins regulates specific interactions amongcells and various extracellular substrates. Integrins are hetero-dimeric receptors that consist of an α and β subunit, suchthat a single β subunit is able to heterodimerize with multipleα subunits (Giancotti and Ruoslahti, 1999). The specific com-bination of the α and the β subunits determines specificityof binding to the ECM, whereas the β subunit can interactwith cytoplasmic signal transduction molecules. In the dev-eloping renal tubules, at least four different α subunits, incombination with the β1 subunit, are found in different celltypes at different times. The α8β1 integrin is expressed inthe undinduced mesenchyme and is upregulated to high levelsin the induced mesenchyme surrounding the ureteric budtips. Homozygous mice carrying a mutant allele for the α8subunit show severe renal agenesis, with a varying degree ofpenetrance (Muller et al., 1997). Most newborn α8 homo-zygous mutants had bilateral renal agenesis and no urethraspresent. However, some homozygous mutant animals had asingle kidney rudiment and about 25% had a single, appa-rently normal, kidney. At E11 in 100% of the α8 mutants,the ureter had not invaded the mesenchyme and not under-gone branching morphogenesis. Thus it would appear thatthe α8β1 integrin in the mesenchyme provides a permissiveenvironment for optimal ureteric bud invasion. This permis-sive environment may be due to interactions of the α8β1 inte-grin with the ECM of the growing ureteric bud. Alterna-tively, the α8β1 integrin may affect the expression of budguidance or survival factors, such as GDNF, through anintegrin-linked signaling role in the mesenchyme.

Epithelial cells of the renal tubules are polarized (Chapter16). The apical side faces the lumen of the tubules, whereasthe basal side contacts the basement membrane that com-

pletely sur-rounds the outside of individual tubules. Thebasement membrane is made up of collagens, laminins,entactin, and sulfated proteoglycans. During renal tubuledevelopment, composition of the basement membranesshifts both spatially and temporally such that specificisoforms of both collagen and laminin are found in differentparts of the developing nephron (Miner, 1999). Theseisoforms of the matrix compo-nents appear to have uniquefunctions, as genetic mutations of specific isoforms canhave dramatic effects on both early and late development.

The assembly of the epithelial basement membrane canbe detected as early as the renal vesicle stage. The hetero-trimeric protein laminin consists of a single α, β, and γ chainand is a major structural determinant of the basementmembrane. The initial isoform detected in the renal vesicleis laminin-1, which encompasses an α1, β1, and γ1 chain. Inthe induced mesenchyme, expression of the laminin α1chain may be a rate-limiting step in formation of the earlybasement membrane, as expression of the laminin β1 and γ1chains can already be detected in the uninduced mesen-chyme in a filamentous pattern. In kidney organ cultures,antibodies against the α1 chain of laminin-1 inhibit the con-version of mesenchyme to epithelium, presumably by dis-turbing the formation of the epithelial basement membraneor integrin–matrix interactions (Klein et al., 1988).

As tubules develop to the S-shaped body stage, the glo-merular epithelial precursors begin to express laminin-10(α5β1γ1). In the mature glomerular basement membrane, anessential element of the filtration barrier, laminin-10 is re-placed by laminin-11 (α5β2γ1) (Miner et al., 1997). Thefunction of these seemingly homologous isoforms has beenexamined in mouse knockout models by mutating individuallaminin chains. Homozygous mice carrying a laminin β2null allele develop severe proteinuria (Noakes et al., 1995).In the β2 null mouse, podocyte cells of the glomerulus areabnormal and fused, yet the basement membrane appearsnormal at the ultratructural level. The laminin-β1 is found inthe mature glomerular basement membrane, but cannot fullycompensate for the lack of the laminin-β2 chain. Thus, theshift in laminin isoform expression has a profound effect onthe visceral epithelium of the glomerulus. Perhaps thematuring podocytes receive signals, via integrins, from theglomerular-specific laminin chains. Alternatively, as inte-grin expression in the podocytes shifts, a compensatory shiftin laminin chain expression is necessary to maintain tightcell–matrix adhesion.

Gene targeting of the laminin α5 chain also reveals anessential function during development of the glomerular epi-thelium. Although some mice homozygous for the lama5allele show bilateral or unilateral renal agenesis, most of theaffected mutants show some degree of nephrogenesis with agross defect in the glomerulus (Miner and Li, 2000). Thelaminin α5 chain is expressed in the basement membrane ofthe S-shaped body when endothelial cells are first observed


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invading the prospective glomerular cleft. This is coincidentwith a shift from laminin-1 to laminin-10. At this time, upre-gulation of the WT1 gene marks the podocyte precursorcells, which, form a crescent-shaped layer along the cleftand subsequently outline the developing Bowmans capsule.Rather than this single layer of podocyte precursors, lama5mutants exhibit multiple layers that ultimately displace theinvading endothelial cells from the glomerular cleft.

Another major structural component of the renal base-ment membrane is collagen IV. There are six distinct chainsof collagen IV, α1–α6, which form a triple helical rod-likestructure. Collagen IV isoforms are expressed differentiallydepending on the type of basement membrane and the deve-lopmental stage (Miner and Sanes, 1994). α1 and α2 chainsare expressed in the basement membrane of the renal vesi-cle, the comma-shaped bodies, and the S-shaped bodies,whereas α1 and α5 are detected in the developing glome-rulus at the capillary loop stage. However, the mature glo-merular basement membrane contains mostly α3, α4, andα5. Although little is known about the function of distinctcollagen IV chains in developing tubules, the glomerular base-ment membrane has been the focus of much attention due tothe effect of collagen IV mutations on the filtration barrier.

In humans, mutations in any of the α3, α4, or α5 chains cancause Alport syndrome (Barker et al., 1990; Hudson et al.,1993), which is characterized by decreased glomerular filtra-tion, increased mesangial matrix deposition, glomerularsclerosis, and tubule atrophy. The persistence of collagen IV α1 and α2 chains in the Alport syndrome kidneysappears to increase the susceptibility of the glomerular base-ment membrane to damage.

C. Epithelium–Stroma Interactions

The classic view of tubulogenesis focused on the role ofinductive signaling from the ureteric bud and epithelialconversion of the mesenchyme. However, not all of the meta-nephric mesenchyme is fated to become epithelium. Certain-ly by E11.5 in the mouse there are epithelial and stromalprecursor cells and there is limited evidence of endothelialprecursors. The effects of nonepithelial cell types on the pro-cess of tubulogenesis are profound. Both survival and proli-feration signals are now known to emanate from the stromalpopulation within the developing kidney that act in a para-crine fashion on the developing tubules (for summary, seeFig. 14.7). The genetic factors that designate the stromal and


Figure 14.7 A model of cellular interactions among different lineage precursors. Inductive signals from the ureter bud act on either a single renal stemcell or on multiple progenitor cells whose fate may be restricted prior to induction. The epithelial lineage is marked by Pax2 expression, whereas the stromallineage expresses BF-2. Endothelial cell precursors, which express the VEGF receptor Flk1, have been observed as early as E11.5 and may represent a thirdlineage. BMP7 is expressed in both the ureteric bud and the condensed mesenchyme and promotes survival of BF-2 positive stroma, and potentially alsouninduced mesenchyme. Stromal cells feedback to the epithelial lineage by providing as yet unidentified survival factors. Retinoids also act on the stromallineage and, indirectly, on the ureteric bud cells by activating a branching stimulant, which increases the expression of RET. FGFs, whose source is notclear, promote survival of the stromal lineage at the expense of the epithelial lineage. Wnt4 is expressed in the mesenchymal aggregates and may providean autocrine signal for epithelial polarization or a paracrine signal to stromal cells. More differentiated structures, such as the S-shaped body, expresspotential inhibitors of Wnt4, perhaps to localize the effects of Wnt4 signaling to less differentiated cell types. VEGF becomes expressed in the presumptiveglomerular epithelium and functions to attract endothelial cells into the glomerular tuft.

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epithelial cell populations are now being addressed using acombination of genetic and tissue culture models.

Within the induced mesenchyme, the transcription factorBF-2 is restricted to those cells not undergoing epithelialconversion after induction (Hatini et al., 1996). BF-2expres-sion is localized along the periphery of the kidneyand in the interstitial mesenchyme, or stroma. Indeed, thereis little over-lap between the expression domains of BF-2and Pax2, which marks the mesenchymal aggregates andearly epithelial derivatives. Whether mesenchyme cells mayalready be partitioned into a BF-2-positive stromal precursorand a Pax2-positive epithelial precursor prior to inductionremains to be deter-mined. However, the mouse knockoutfor BF-2 reveals an essential role for the stroma in pro-moting tubulogenesis (Hatini et al., 1996). In homozygousBF-2 null mice, ureteric bud growth and initial branchingare unaffected, as is the for-mation of the first mesenchymalaggregates. However, within 2 days after induction, mesen-chymal aggregates fail to dif-ferentiate into comma and S-shaped bodies at a rate similar to wild type. This results inreduced branching of the ureteric bud and coincidentally fewernew mesenchymal aggregates forming. Early mesenchymalaggregates are able to form epi-thelium and express theappropriate markers, such as Pax2, wnt4, and WT1. How-ever, in the absence of BF-2-expressing stromal cells, growthof both ureteric bud epithelium and mesenchymal aggre-gates is slowed or inhibited. BF-2 knockout studies revealan essential role for stromal cells in supporting the survivaland proliferation of epithelial precursor cells. As epithelialcells become induced to form aggregates, a self-renewingpopulation of epithelial precursor cells must be maintainedalong the periphery of the developing kidney such that newaggregates can form around the tips of the next ureteric bud end point. Factors secreted from the stroma mayprovide the cues for this self-renewing population of epi-thelial precursors, in the absence of which the mesenchymeis quickly exhausted of all cells fated to become epithelia.

What might some of these stromal-derived factors be?Evidence points to both fibroblast growth factors and bonemorphogenetic proteins (BMPs) as influential players in thedecision to go stromal or epithelial. Among the early candi-dates for the inductive signal, BMP7 is expressed in theureteric bud epithelium and in the induced mesenchyme(Vukicevic et al., 1996). However, BMP7 mutants do notshow a fundamental defect in early mesenchyme induction.Rather, they are developmentally arrested after the inductionphase, do exhibit ureteric bud branching morphogenesis, andexpress early markers of induced mesenchyme (Dudley etal., 1995; Luo et al., 1995). The ability of BMP7 to inducemesenchyme was not confirmed in more recent studies(Dudley et al., 1999). Using the in vitro organ culture model,Dudley et al., (1999) demonstrated the effect of BMP7 onmesenchymal cell survival. Thus, uninduced mesenchyme,which dies very quickly in culture, can survive for severaldays and still respond to inductive signals. In combination

with FGF2, the survival of the mesenchyme and the abilityto respond to induction are enhanced greatly by BMP7. Infact, both factors together appear to increase the proportionof cells expressing BF-2, the stromal population, at theexpense of the Pax2 expressing epithelial cells. These in vitroexperiments point to a delicate balance between stromal andepithelial progenitor cells, the proportion of which must bewell regu-lated by both autocrine and paracrine factors, suchthat neither cell population is exhausted prematurely.

Other factors within the stroma that may affect epithelialcell proliferation and tubulogenesis include retinoids and theirreceptors. That vitamin A deficiency leads to congenital renaldefects, including a reduction in the number of nephrons, iswell documented (Lelievre-Pegorier et al., 1998). The stromalcell population expresses both retinoic acid receptors (RAR)α and β2. A striking renal phenotype has been reported inmice that are homozygous null for both receptors(Mendelsohn et al., 1999). A general developmental arrest isevident that can be explained in large part due to the inhi-bition of ureteric bud branching. Consistent with this view,down-regulation of the RET receptor is evident in theureteric bud epithelial of RAR α/β2 null animals. Reducedexpression of BF-2 is also observed, indicating that proli-feration of the stromal population may be limited. Using exo-genous retinoids in the organ culture model, activation ofRET and a stimulation of ureteric bud branching morpho-genesis is observed (Vilar et al., 1996). Thus the effects ofretinoids appear to be mediated by the stromal population.The stromal cells must provide signals that maintain RETexpression and provide an environment for branching of theureteric bud. Failure to do so results in a general develop-mental arrest and an inhibition of tubulogenesis.

VI. Summary

This chapter outlined the current state of knowledge withrespect to the genetic and biochemical basis of tubule develop-ment in the nephron. Intrinsic factors, such as Pax2,WT1,and Eya1, specify the metanephric mesenchyme from sur-rounding intermediate mesoderm. Expression of these genesmost likely depends on positional cues from somitic meso-derm and perhaps surface ectoderm. While Pax2 and WT1are required to enable the mesenchyme to respond to induc-tive signals, they may also be necessary for the proliferationand differentiation of tubular epithelium after inductionoccurs. The expression of both genes is highly dynamicduring the course of nephron development. Subsequentchapters discuss the relevance of Pax2 and WT1 to humanrenal disease, further underscoring the importance ofactivation and repression of Pax2 and WT1.

The inductive signals emanating from the ureteric budhave not been defined conclusively. However, evidence thatthe secreted Wnt proteins can induce the metanephric mesen-chyme is compelling. In contrast, the trio of secreted


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signaling molecules bFGF, TGF-α, and LIF has also beenimplicated as a mesenchymal inducer, although genetic evi-dence supporting their in vivo role is lacking. These issuesneed to be clarified. In theory, a mouse carrying a mutationin the ureteric bud-derived inducer should exhibit growth andbranching of the ureteric bud without any evidence of pri-mary induction in the mesenchyme. Unfortunately, such agenetic mutant has not been identified to date, leaving only thein vitro assays as biochemical evidence of inductive capacity.

The response of the mesenchyme to inductive signals hasbeen characterized over many years. However, there are manycritical elements missing. While activation of cell adhesionmolecules must underlie the aggregation and perhaps thepolarization of the early epithelia, how the inductive signalsare translated into morphological changes remains obscure.As the nephron develops, compartmentalization occurs alongthe axis of the tubular epithelia. This is evident by segment-specific gene expression of cadherins, integrins, laminins,and other markers. How are changes in integrins and cadherinsregulated so precisely along the developing nephron? Whyis there a need for a shift in extracellular matrix collagen andlaminin isoforms? What are the factors that determine special-ized epithelial cell types along the axis of the nephron? Thesequestions remain unanswered and will surely be addressedin the coming decade as a more complete understanding ofnephrogenesis is at hand.

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