Cell Lineages and Stem Cells in the Embryonic Kidney

Chapter 73

Cell Lineages and Stem Cells in the Embryonic Kidney Gregory R. Dressler Department of Pathology, University of Michigan, Ann Arbor, MI, USA

Chapter Outline The Anatomy of Kidney Development Genes That Control Early Kidney Development Genes That Determine the Nephrogenic Field Genes That Function at the Time of Metanephric Induction The Establishment of Additional Cell Lineages

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The goal of developmental biology is to understand the genetic and biochemical mechanisms that determine cell growth and differentiation and the three-dimensional patterning of a complex organism. This knowledge can reframe the pathogenesis of human diseases such as cancer within a developmental context and can also be applied to create new therapies for the regeneration of damaged tissues. Given the emphasis on stem cells throughout this book, it would seem prudent to discuss the origin of the embryonic kidney with respect to pluripotent renal stem cells and how they may differentiate into the multiplicity of cell types found in an adult kidney. Unfortunately, no such renal embryonic stem (ES) cells have been conclusively identified. Instead, the kidney develops from a region of the embryo, over a relatively long window of time, while undergoing a sequential anterior to posterior transition that reflects in part the evolutionary history of the organ. The question then arises at what stage are cells fated to become kidney cells and when are they restricted in their developmental potential? To address these issues, it is necessary to review the early morphogenesis of the urogenital system. We can then appreciate which genes and molecular markers contribute to early renal development and what cell lineages arise from the region of the embryo devoted to creating the urogenital system.

Epithelia Versus Stroma Cells of the Glomerular Tuft What Constitutes a Renal Stem Cell? Acknowledgments References

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THE ANATOMY OF KIDNEY DEVELOPMENT For a detailed description of early renal development and nephron formation, I refer the reader to several reviews (Kuure et al., 2000; Dressler, 2002). A brief summary of renal patterning and the origin of the renal progenitor cells must begin with gastrulation. In vertebrates, the process of gastrulation converts a single pluripotent sheet of embryonic tissue, the epiblast or embryonic ectoderm, into the three primary germ layers: the endoderm, the mesoderm, and the ectoderm (Figure 73.1A). In mammals, gastrulation is marked by a furrow called the primitive streak, which extends from the posterior pole of the epiblast. Extension of the primitive streak occurs by proliferation and migration of more lateral epiblast cells to the furrow, followed by invagination through the furrow and migration back laterally under the epiblast sheet. At the most anterior end of the primitive streak is the node or organizer, called Hensen’s node in the chick, and the functional equivalent to the blastopore lip or Speeman’s organizer in the amphibian embryo. The node is a signaling center that expresses a potent combination of secreted factors for establishing the body axes and lefteright asymmetry. The node is positioned at the anterior pole of the primitive streak at

Handbook of Stem Cells, Two-Volume Set. DOI: http://dx.doi.org/10.1016/B978-0-12-385942-6.00073-1 Copyright Ó 2013 Elsevier Inc. All rights reserved.

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(A)

E7.0 Paraxial mesoderm

Embryonic ectoderm

Primitive streak

Anterior

Dorsal

Node Embryonic endoderm

Posterior Primitive streak

(B)

Ventral

Lateral mesoderm

E8.5 Head folds

Neural crest

Neural tube

Somites Somite Intermediate Notochord mesoderm Lateral plate mesoderm Primitive streak regression

FIGURE 73.1 The origin of the intermediate mesoderm. (A) At gastrulation, cells of the epiblast, or embryonic ectoderm, migrate through the primitive streak. In the mouse, the single sheet of embryonic ectoderm lines a cup-shaped egg cylinder. The left panel is a planar representation of a mouse epiblast looking down into the cup from above. The streak begins at the posterior and moves toward the anterior. Fate mapping studies indicate that lateral plate mesoderm originates from the posterior epiblast, whereas axial mesoderm is derived from more anterior epiblast cells. The intermediate mesoderm most likely originates from cells between these two regions. At the mid-streak stage, the expression of lim1 can already be detected before gastrulation in cells of the posterior epiblast. The schematic on the right is a cross section through the primitive streak, showing the formation of mesoderm as cells of the epiblast migrate toward the streak then invaginate and reverse direction underneath the epiblast sheet. (B) At the time of primitive streak regression, the notochord is formed along the ventral midline. Paraxial mesoderm begins to form segments, or somites, in an anterior to posterior direction. The lateral plate mesoderm, consists of two sheets called the somatopleure (dorsal) and the splanchnopleure (ventral). The region between the somite and the lateral plate is the intermediate mesoderm, where the first renal epithelial tube will form.

approximately the midpoint of the epiblast. The more anterior epiblast generates much of the head and central nervous system and does not undergo gastrulation in the same manner. Once the streak reaches the node, it begins to regress back to the posterior pole. During this process of primitive streak regression, the notochord is formed along the midline of the embryo and just ventral to the neural plate. The notochord is a second critical signaling center for dorsal ventral patterning of both neural plate and paraxial mesoderm. The axial mesoderm refers to the most medial mesodermal cells, which in response to regression of the streak become segmented into somites, blocks of cells surrounded by a simple epithelium. At the stage of the first somite formation, going medial to lateral, the notochord marks the midline, the somites abut the notochord on either

Adult and Fetal Stem Cells

side, and the unsegmented mesoderm is termed intermediate near the somite and lateral plate more distally (Figure 73.1B). It is this region of intermediate mesoderm within which the kidney will form that is the primary focus of this chapter. The earliest morphologic indication of unique derivatives arising from the intermediate mesoderm is the formation of the pronephric duct, or primary nephric duct. This single-cell-thick epithelial tube runs bilaterally beginning at around the 12th somite in birds and mammals. The nephric duct extends caudally until it reaches the cloaca. As it grows, it induces a linear array of epithelial tubules, which extend medioventrally and are thought to derive from periductal mesenchyme (Figure 73.2). The tubules are referred to as pronephric or mesonephric, depending on their position and degree of development, and represent an evolutionarily more primitive excretory system that forms transiently in mammals until it is replaced by the adult or metanephric kidney. Along the nephric duct, there is a graded evolution of renal tubule development, with the most anterior, or pronephric tubules, being very rudimentary, and the mesonephric tubules becoming well developed with glomeruli and convoluted proximal tubule-like structures. In contrast, the pronephros of the zebrafish larvae is a fully developed, functional filtration unit with a single midline glomerulus (Drummond et al., 1998). Amphibian embryos such as Xenopus laevis have bilateral pronephric glomeruli and tubules that are functional until replaced by a mesonephric kidney in the tadpole (Vize et al., 1997). In fact, it is not altogether obvious in mammals where to draw the

FIGURE 73.2 Expression of Pax2 at the time of metanephric induction. One side of the intermediate mesoderm-derived nephric chord was microdissected from an E11.5 mouse embryo and stained with antiPax2 antibodies. The micrograph shows Pax2 in the nephric duct (nd) and the ureteric bud (ub) branching from the posterior duct. Pax2 is in mesonephric tubules (mt), even those more posterior that are not connected to the nephric duct. At the posterior end, Pax2 is in the metanephric mesenchyme cells (mm), surrounding the ureteric bud, that has not yet begun to form epithelia.

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distinction between pronephric tubules and mesonephric tubules. Mature mesonephric tubules are characterized by a vascularized glomerulus at the proximal end of the tubule that empties into the nephric duct; the most anterior and posterior mesonephric tubules are more rudimentary, and the most posterior tubules are not connected to the duct at all. The adult kidney, or metanephros, is formed at the caudal end of the nephric duct when an outgrowth, called the ureteric bud or metanephric diverticulum, extends into the surrounding metanephric mesenchyme. Outgrowth or budding of the epithelia requires signals emanating from the mesenchyme. Genetic and biochemical studies indicate that outgrowth of the ureteric bud is mediated by the transmembrane tyrosine kinase RET, which is expressed in the nephric duct, and the secreted neurotrophin glial derived neurotrophic factor (GDNF), which is expressed in the metanephric mesenchyme. Once the ureteric bud has invaded the metanephric mesenchyme, inductive signals

(A)

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emanating from the bud initiate the conversion of the metanephric mesenchyme to epithelium (Figure 73.3). The induced, condensing mesenchymal cells aggregate around the tips of the bud and will form a primitive polarized epithelium, the renal vesicle. Through a series of cleft formations, the renal vesicle forms first a comma then an Sshaped body, whose most distal end remains in contact with the ureteric bud epithelium and fuses to form a continuous epithelial tubule. This S-shaped tubule begins to express genes specific for glomerular podocyte cells at its most proximal end, markers for more distal tubules near the fusion with the ureteric bud epithelia, and proximal tubules markers in between. Endothelial cells begin to infiltrate the most proximal cleft of the S-shaped body as the vasculature of the glomerular tuft takes shape. At this stage, the glomerular epithelium consists of a visceral and parietal component, with the visceral cells becoming podocytes and the parietal cells the epithelia surrounding the urinary space. The capillary tuft consists of capillary endothelial

(B)

Mesenchyme

Ureteric buds

Ureteric bud Stromal cells

Renal vesicle

Condensed mesenchyme

(C)

Collecting duct Distal tubule Proximal tubule Endothelial cells

Glomerulus

(D)

Proximal tubule Collecting duct Distal tubule

Efferent arteriole Afferent arteriole Podocytes Perietal glomerular epithelium

Thick ascending limb , Henle s loop

FIGURE 73.3 The sequential conversion of metanephric mesenchyme to renal epithelia. A schematic of the condensation and polarization of the metanephric mesenchyme at the tips of the ureteric bud epithelia is shown. (A) Epithelial precursors aggregate at the tips, whereas stromal cells remain peripheral. (B) The initial aggregates form a primitive sphere, the renal vesicle, as branching ureteric bud epithelia cells extend outward to induce new aggregates. Stromal cells begin to migrate into the interstitium. (C) At the S-shaped body stage, the mesenchymal derived structure is fused to the ureteric bud epithelial, which will make the collecting ducts and tubules. The proximal cleft of the S-shaped body is invaded by endothelial cells. Expression of glomerular, proximal tubule, and more distal tubule specific markers can be seen at this stage. (D) The architecture of the nephron is elaborated. Podocyte cells of the visceral glomerular epithelium contact the capillary tuft and the glomerular basement membrane is laid down. The proximal tubules become more convoluted and grow into the medullary zone to form the descending and ascending limbs of Henle’s loop. The distal tubules and collecting ducts begin to express markers for more differentiated, specialized epithelia.

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cells and a specialized type of smooth muscle cell, termed the mesangial cell, whose origin remains unclear. As these renal vesicles are generating much of the epithelia of the nephron, the ureteric bud epithelia continues to undergo branching morphogenesis in response to signals derived from the mesenchyme. Branching follows a stereotypical pattern and results in new mesenchymal aggregates induced at the tips of the branches, as new nephrons are sequentially induced. This repeated branching and induction results in the formation of nephrons along the radial axis of the kidney, with the oldest nephrons being more medullary and the younger nephrons located toward the periphery. However, not all cells of the mesenchyme become induced and convert to epithelia, some cells remain mesenchymal and migrate to the interstitium. These interstitial mesenchymal cells, or stromal cells, are essential for providing signals that maintain branching morphogenesis of the ureteric bud and survival of the mesenchyme. From a stem cell perspective, defining the population of cells that generate the kidney depends in part on which stage one considers. At the time of metanephric mesenchyme induction, there are at least two primary cell types: the mesenchyme and the ureteric bud epithelia. Although these cells are phenotypically distinguishable, they do express some common markers and share a common region of origin. As development progresses, it was thought that most of the epithelium of the nephron was derived from the metanephric mesenchyme, whereas the branching ureteric bud epithelium generates the collecting ducts and the most distal tubules. This view has been challenged by cell lineage tracing methods in vitro, which indicate some plasticity at the tips of the ureteric bud epithelium such that the two populations may intermingle (Qiao et al., 1995). Thus, at the time of induction, epithelial cells can convert to mesenchyme, just as the mesenchymal aggregates can convert to epithelia. Regardless of how the mesenchyme is induced, the cells are predetermined to make renal epithelia. Thus, their potential as renal stem cells has begun to be explored. To understand the origin of the metanephric mesenchyme, we begin with the patterning of the intermediate mesoderm.

GENES THAT CONTROL EARLY KIDNEY DEVELOPMENT Genetic studies in the mouse have provided substantial new insights into the regulatory mechanisms underlying renal development. Although there are many genes that can affect the growth and patterning of the kidney, of particular importance with respect to potential renal stem cells are the genes that control formation of the nephric duct epithelia

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and proliferation and differentiation of the metanephric mesenchyme (Table 73.1).

Genes That Determine the Nephrogenic Field From a perspective of stem cells and potential therapy, the adult or metanephric kidney should be a primary focus. However, the early events controlling the specification of the renal cell lineages may be common among the pronephric and mesonephric regions. Indeed, many of the same genes expressed in the pronephric and mesonephric tubules are instrumental in early metanephric development. Furthermore, the earliest events that underlie regional specification have been studied in more amenable organisms, including fish and amphibians, in which pronephric development is less transient and of functional significance. Although formation of the nephric duct is the earliest morphologic evidence of renal development, the expression of intermediate mesodermal-specific markers precedes nephric duct formation temporally and marks the intermediate mesoderm along much of the anterioreposterior (AeP) body axis. The earliest markers specific of intermediate mesoderm are two transcription factors of the Pax family (Figure 73.4): Pax2 and Pax8, which appear to function redundantly in nephric duct formation and extension (Bouchard et al., 2002). The homeobox gene lim1 is also expressed in the intermediate mesoderm but is initially expressed in the lateral plate mesoderm before becoming more restricted (Tsang et al., 2000). Genetics implicates all three genes in some aspects of regionalization along the intermediate mesoderm. In the mouse, Pax2 mutants begin nephric duct formation and extension but lack mesonephric tubules and the metanephros (Torres et al., 1995). Pax2/Pax8 double mutants have no evidence of nephric duct formation and do not express the lim1 gene. Null mutants in lim1 also lack the nephric duct and show reduced ability to differentiate into intermediate mesoderm-specific derivatives (Tsang et al., 2000). The reduced expression of Pax2 in lim1 mutants could explain the inability of these cells to differentiate into urogenital epithelia. Although in Pax2/Pax8 double null embryos (Bouchard et al., 2002), the lack of lim1 expression may also be an integral part of the phenotype. Because lim1 expression precedes Pax2 and Pax8 and is spread over a wider area in the pregastrulation and postgastrulation embryo and is Pax independent, it seems likely that maintenance and restriction of lim1 expression within the intermediate mesoderm requires activation of the Pax2/8 genes at the five- to eight-somite stage. Are Pax2/8 sufficient then to specify the renal progenitors? This question was addressed in the chick embryo by Bouchard et al. (2002) using replication competent retroviruses expressing a Pax2b cDNA. Ectopic nephric ducts were generated within the

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TABLE 73.1 Genes that Regulate Early Kidney Cell Lineages Gene

Expression

Mutant Phenotype

Reference

lim1

Lateral plate

No nephric duct, no kidneys

Tsang et al., 2000

Inter. mesoderm

No mesonephric tubules

Torres et al., 1995

Nephric duct

No metanephros

Brophy et al., 2001

Pax2/Pax8

Inter. mesoderm

No nephric duct, no kidneys

Bouchard et al., 2002

WT1

Inter. mesoderm

Fewer mesonephric tubules

Kreidberg et al., 1993

Mesenchyme

Apoptosis of mesenchyme

Eya1

Met. mesenchyme

No induction of mesenchyme

Xu et al., 1999

wnt4

Mesenchymal aggregates

No polarization of aggregates

Stark et al., 1994

bmp7

Ureter bud and met.

Developmental arrest postinduction

Dudley et al., 1995

Mesenchyme

Some branching, few nephrons

Met. mesenchyme

Developmental arrest, few nephrons

Interstitial stroma

Limited branching

pod-1

Stroma and podocytes

Poorly differentiated podocytes

Quaggin et al., 1999

pdgf(r)

S-shaped body

No vascularization of glomerular tuft

Soriano, 1994

Nephric duct Pax2

bf-2

Hatini et al., 1996

inter., intermediate; met., metanephric.

general area of the intermediate mesoderm upon retrovirally driven Pax2b expression. These ectopic nephric ducts were not obtained with either lim1 or Pax8 alone. Strikingly, the ectopic nephric ducts paralleled the endogenous ducts and

were not found in more paraxial or lateral plate mesoderm. This would suggest that Pax2’s ability to induce duct formation does require some regional competence, perhaps only in the lim1-expressing domain.

Head fold

Optic placode Ismuth

3–4 Somites

Intermediate mesoderm 8 Somites

FIGURE 73.4 The activation of Pax2 expression in the intermediate mesoderm. One of the earliest markers for the nephrogenic region Pax2 expression is activated around the four- to five-somite stage in the cells between the axial and lateral plate mesoderm. The embryos shown carry a Pax2 promoter driving the LacZ gene and expression is visualized by staining for beta-galactosidase activity. By the eight-somite stage, Pax2 marks the growing intermediate mesoderm even before the nephric duct is formed. Pax2 is also expressed in parts of the nervous system, especially the mid-brainehindbrain junction, known as the rhombencephalic ismuth, and in the optic placode and cup.

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If Pax2/8 and lim1 restriction in the intermediate mesoderm are the earliest events that distinguish the nephrogenic zone from surrounding paraxial and lateral plate mesoderm, the question then remains as to how these genes are activated. In the axial mesoderm, signals derived from the ventral notochord pattern the somites along the dorsaleventral axis. Similar notochord derived signals could also pattern mesoderm along the mediolateral axis. However, this does not appear to be the case. In the chick embryo, the notochord is dispensable for activation of the Pax2 gene in the intermediate mesoderm. Instead, signals derived from the somites, or paraxial mesoderm, are required for activation of Pax2 (Mauch et al., 2000). It remains to be determined what these somite derived signals are, although it is worth noting that in the amphibian embryo a combination of retinoic acid and activin-A is able to activate early markers of the pronephric field including lim1 (Chan et al., 2000; Osafune et al., 2002). In addition to somite-derived signals, additional signals may be required for the formation of epithelia within the predisposed intermediate mesoderm. Activation of Pax2/8 is found more anterior to and precedes formation of the nephric duct. Thus, not all Pax2 positive cells will make the primary nephric duct. In the chick embryo, the overlying ectoderm is necessary for formation of the nephric duct, which initiates adjacent to somites 10e12, significantly more posterior to the initial Pax2 positive domain (ObaraIshihara et al., 1999). Removal of overlying ectodermal tissues, which express the secreted signaling molecule bone morphogenetic protein-4 (BMP4), blocks nephric duct formation and extension. This block can be overcome by recombinant BMP4 protein, suggesting that this is indeed the essential ectodermally derived factor. If Pax2/8 mark the entire nephric region, there must be additional factors that specify the position of elements along the AeP axis in the intermediate mesoderm. Such patterning genes could determine whether a mesonephric or metanephric kidney is formed within the Pax2 positive domain. Among the known AeP patterning genes are members of the HOX gene family. Indeed, mice that have deleted all genes of the Hox11 paralogous group have no metanephric kidneys (Wellik et al., 2002), although it is not clear whether this is truly a shift in AeP patterning or a lack of induction. AeP patterning of the intermediate mesoderm may also depend on the FoxC family of transcription factors. Foxc1 and Foxc2 have similar expression domains in the presomitic and intermediate mesoderm, as early as E8.5 (Kume et al., 2000). As nephric duct extension progresses, Foxc1 is expressed in a dorsoventral gradient, with the highest levels near the neural tube and lower levels in the BMP4 positive ventrolateral regions. In Foxc1 homozygous null mutants the anterior boundary of the metanephric mesenchyme, as marked by GDNF expression, extends rostrally (Kume et al., 2000). This results in

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a broader ureteric bud forming along the AeP axis and eventual duplication of ureters. Similar defects are observed in compound heterozygotes of Foxc1 and Foxc2, indicating some redundancy and gene dosage effects. Thus, Foxc1 and Foxc2 may set the anterior boundary of the metanephric mesenchyme, at the time of ureteric bud outgrowth, by suppressing genes at the transcriptional level.

Genes That Function at the Time of Metanephric Induction Pax2 and Pax8 are coexpressed in the nephric duct, but the Pax2 expression domain is broader and encompasses the mesonephric tubules and the metanephric mesenchyme. Thus, Pax8 mutants have no obvious renal phenotype, Pax2 mutants have a nephric duct but no mesonephric tubules or metanephros, and Pax2/8 double mutants lack the nephric duct completely. Thus, either Pax2 or Pax8 is enough for duct formation, but Pax2 is clearly essential for conversion of the metanephric mesenchyme into epithelia. The Pax2 phenotype is complex. Despite the presence of a nephric duct, there is no evidence of ureteric bud outgrowth. Ureteric bud outgrowth is controlled primarily by the receptor type tyrosine kinase RET, which is expressed on the nephric duct epithelia, the secreted signaling protein GDNF, which is expressed in the metanephric mesenchyme, and the GPi linked protein GFRa1, which is expressed in both tissues. Pax2 mutants have no ureteric buds because they do not express GDNF in the mesenchyme and fail to maintain high levels of RET expression in the nephric duct (Brophy et al., 2001). Despite the lack of bud, the metanephric mesenchyme is morphologically distinguishable in Pax2 mutants. Although lacking GDNF, it does express other markers of the mesenchyme, such as Six2 (Torres et al., 1995). In vitro recombination experiments using Pax2 mutant mesenchyme, surgically isolated from E11 mouse embryos, and heterologous inducing tissues indicate that Pax2 mutants are unable to respond to inductive signals (Brophy et al., 2001). Thus, Pax2 is necessary for specifying the region of intermediate mesoderm destined to undergo mesenchymeto-epithelium conversion. In humans, the necessity of Pax2 function is further underscored because the loss of a single Pax2 allele is associated with renal-coloboma syndrome, which is characterized by hypoplastic kidneys with vesicoureteral reflux (Sanyanusin et al., 1995). A second essential gene for conversion of the metanephric mesenchyme to epithelia is Eya1, a vertebrate homologue of the Drosophila eyes absent gene. In humans, mutations in the Eya1 gene are associated with branchiooto-renal syndrome, a complex multifaceted phenotype (Abdelhak et al., 1997). In mice homozygous for an Eya1 mutation, kidney development is arrested at E11 because

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ureteric bud growth is inhibited and the mesenchyme remains uninduced, although Pax2 and WT1 expression appears normal (Xu et al., 1999). However, two other markers of the metanephric mesenchyme, Six2 and GDNF expression, are lost in the Eya1 mutants. The loss of GDNF expression most probably underlies the failure of ureteric bud growth. However, it is not clear if the mesenchyme is competent to respond to inductive signals if a wild-type inducer were to be used in vitro. The eyes absent gene family is part of a conserved network that underlies cell specification in several other developing tissues. Eya proteins share a conserved domain but lack DNA binding activity. The Eya proteins interact directly with the Six family of DNA-binding proteins. Mammalian Six genes are homologues of the Drosophila sina oculis homeobox gene. This cooperative interaction between Six and Eya proteins is necessary for nuclear translocation and transcriptional activation of Six target genes (Ohto et al., 1999). The Wilms tumor suppressor gene, WT1, is another early marker of the metanephric mesenchyme and is essential for its survival. Wilms tumor is an embryonic kidney neoplasia that consists of undifferentiated mesenchymal cells, poorly organized epithelium, and surrounding stromal cells. Expression of WT1 is regulated spatially and temporally in a variety of tissues and is further complicated by the presence of at least four isoforms, generated by alternative splicing. In the developing kidney, WT1 can be found in the uninduced metanephric mesenchyme and in differentiating epithelium after induction (Pritchard-Jones et al., 1990; Armstrong et al., 1992). Early expression of WT1 may be mediated by Pax2 (Dehbi et al., 1996). Initial expression levels are low in the metanephric mesenchyme, but become up-regulated at the S-shaped body stage in the precursor cells of the glomerular epithelium, the podocytes. High WT1 levels persist in the adult podocytes. In the mouse, WT1 null mutants have complete renal agenesis (Kreidberg et al., 1993), because the metanephric mesenchyme undergoes apoptosis and the ureteric bud fails to grow out of the nephric duct. The arrest of ureteric bud growth is most probably due to lack of signaling by the WT1 mutant mesenchyme. As in Pax2 mutants, the mesenchyme is unable to respond to inductive signals even if a heterologous inducer is used in vitro. Thus, it appears that WT1 is required early in the mesenchyme to promote cell survival, enabling cells to respond to inductive signals and express ureteric bud growth promoting factors.

THE ESTABLISHMENT OF ADDITIONAL CELL LINEAGES At the time of ureteric bud invasion, there appear to be at least two cell lineages established: the metanephric mesenchyme and the ureteric bud epithelia. As branching

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morphogenesis and induction of the mesenchyme progresses, additional cell lineages are evident. The early E11.5 mouse metanephros contains precursors for almost all cell types, including endothelial, stromal, epithelial, and mesangial cells. However, it is far from clear if these cell types share a common precursor or if the metanephric mesenchyme is a mixed population of precursors. The latter point may well be true for the endothelial lineage. Although transplantation studies with lineage markers indicate that the vasculature can be derived from E11.5 metanephric kidneys, the Flk1 positive endothelial precursors have been observed closely associated with the ureteric bud epithelium, shortly after invasion, and are probably not derived from the metanephric mesenchyme. The lineages most likely to share a common origin within the metanephric mesenchyme are the stromal and epithelial lineages. The maintenance of these two lineages is essential for renal development, because the ratio of stroma to epithelia is a critical factor for the renewal of mesenchyme and the continued induction of new nephrons.

Epithelia Versus Stroma What are the early events in the induced mesenchyme that separates the stromal lineage from the epithelial lineage? In response to inductive signals, Pax2 positive cells aggregate at the tips of the ureteric bud. Activation of the Wnt4 gene in these early aggregates appears critical to promote polarization. Wnt genes encode a family of secreted peptides that are known to function in the development of many tissues. Mice homozygous for a Wnt4 mutation exhibit renal agenesis resulting from growth arrest shortly after branching of the ureteric bud (Stark et al., 1994). Although some mesenchymal aggregation has occurred, there is no evidence of cell differentiation into a polarized epithelial vesicle. Expression of Pax2 is maintained but reduced. Thus, Wnt4 may be a secondary inductive signal in the mesenchyme that propagates or maintains the primary induction response in the epithelial lineage. The transcription factor FoxD1/BF-2 is expressed in uninduced mesenchyme and becomes restricted to those cells not undergoing epithelial conversion after induction (Hatini et al., 1996). FoxD1 expression is found along the periphery of the kidney and in the interstitial mesenchyme, or stroma. After induction, there is little overlap between FoxD1 and the Pax2 expression domain, prominent in the condensing pretubular aggregates. Clear lineage analysis is still lacking, although the expression patterns are consistent with the interpretation that mesenchyme cells may already be partitioned into a FoxD1 positive stromal precursor and a Pax2 positive epithelial precursor before or shortly after induction. Mouse mutants in FoxD1 exhibit severe developmental defects in the kidney that point to an essential role for FoxD1 in maintaining growth and structure (Hatini

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et al., 1996). Early ureteric bud growth and branching are unaffected, as is the formation of the first mesenchymal aggregates. However, at later stages (E13e14) these mesenchymal aggregates fail to differentiate into commaand S-shaped bodies at a rate similar to wild-type. Branching of the ureteric bud is greatly reduced at this stage, resulting in fewer new mesenchymal aggregates forming. The fate of the initial aggregates is not fixed, because some are able to form epithelium and most all express the appropriate early markers, such as Pax2, wnt4, and WT1. Nevertheless, it appears that the FoxD1 expressing stromal lineage is necessary to maintain growth of both ureteric bud epithelium and mesenchymal aggregates. Perhaps factors secreted from the stroma provide survival or proliferation cues for the epithelial precursors, in the absence of which the non-self-renewing population of mesenchyme is exhausted. Some survival factors that act on the mesenchyme have already been identified. The secreted transforming growth factor beta (TGFb) family member BMP7 and the fibroblast growth factor-2 (FGF2) in combination dramatically promote survival of uninduced metanephric mesenchyme in vitro (Dudley et al., 1999). FGF2 is necessary to maintain the ability of the mesenchyme to respond to inductive signals in vitro. BMP7 alone inhibits apoptosis but is not sufficient to enable mesenchyme to undergo tubulogenesis at some later time. After induction, exogenously added FGF2 and BMP7 reduce the proportion of mesenchyme that undergoes tubulogenesis while increasing the population of FoxD1 positive stromal cells (Dudley et al., 1999). At least after induction occurs, there is a delicate balance between a self-renewing population of stromal and epithelial progenitor cells, the proportion of which must be well regulated by both autocrine and paracrine factors. Whether this lineage decision has already been made in the uninduced mesenchyme remains to be determined. The role of stroma in regulating renal development is further underscored by studies with retinoic acid receptors. It is well documented that vitamin A deficiency results in severe renal defects (Lelievre-Pegorier et al., 1998). In organ culture, retinoic acid stimulates expression of RET to dramatically increase the number of ureteric bud branch points, increasing the number of nephrons (Vilar et al., 1996). However, it is the stromal cell population that express the retinoic acid receptors (RARs), specifically RARa and RARb2. Genetic studies with RARa and RARb2 homozygous mutant mice indicate no significant renal defects when either gene is deleted. However, double homozygotes mutant for both RARa and RARb2 exhibit severe growth retardation in the kidney (Mendelsohn et al., 1999). These defects are primarily due to decreased expression of the RET protein in the ureteric bud epithelia and limited branching morphogenesis. Surprisingly, overexpression of RET with a HoxB7/RET transgene can

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completely rescue the double RAR mutants (Batourina et al., 2001). These studies suggest that stromal cells provide paracrine signals for maintaining RET expression in the ureteric bud epithelia and that retinoids are required for stromal proliferation. Reduced expression of stromal cell marker FoxD1, particularly in the interstitium of RAR double mutants, supports this hypothesis.

Cells of the Glomerular Tuft The unique structure of the glomerulus is intricately linked to its ability to retain large macromolecules within the circulating bloodstream while allowing for rapid diffusion of ions and small molecules into the urinary space. The glomerulus consists of four major cell types: the endothelial cells of the microvasculature, the mesangial cells, the podocyte cells of the visceral epithelium, and the parietal epithelium. The development of the glomerular architecture and the origin of the individual cell types are just beginning to be understood. The podocyte is a highly specialized epithelial cell whose function is integral to maintaining the filtration barrier in the glomerulus. The glomerular basement membrane separates the endothelial cells of the capillary tufts from the urinary space. The outside of the glomerular basement membrane, which faces the urinary space, is covered with podocyte cells and their interdigitated foot processes. At the basement membrane, these interdigitations meet to form a highly specialized cellecell junction, called the slit diaphragm. The slit diaphragm has a specific pore size to enable small molecules to cross the filtration barrier into the urinary space, while retaining larger proteins in the blood stream. The podocytes are derived from condensing metanephric mesenchyme and can be visualized with specific markers at the S-shaped body stage. Although there are a number of genes expressed in the podocytes, there are only a few factors known to regulate podocyte differentiation. These include the WT1 gene, which is required early for metanephric mesenchyme survival but whose levels increase in podocyte precursors at the S-shaped body stage. In the mouse, complete WT1 null animals lack kidneys, but reduced gene dosage and expression of WT1 results in specific podocyte defects (Guo et al., 2002). Thus, the high levels of WT1 expression in podocytes appear to be required and make these precursor cells more sensitive to gene dosage. The basic helix-loop-helix protein Pod1 is expressed in epithelial precursor cells and in more mature interstitial mesenchyme. At later developmental stages, Pod1 is restricted to the podocytes. In mice homozygous for a Pod1 null allele, podocyte development appears arrested (Quaggin et al., 1999). Normal podocytes flatten and wrap their foot processes around the glomerular basement membrane. Pod1 mutant podocytes remain more columnar and fail to

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Cell Lineages and Stem Cells in the Embryonic Kidney

fully develop foot processes. Because Pod1 is expressed in epithelial precursors and in the interstitium, it is unclear whether these podocyte effects are due to a general developmental arrest because of the stromal environment or a cell autonomous defect within the Pod1 mutant podocyte precursor cells. Within the glomerular tuft, the origin of the endothelium and the mesangium is less clear. At the S-shaped body stage, the glomerular cleft forms at the most proximal part of the S-shaped body, furthest from the ureteric bud epithelium. Vascularization of the developing kidney is first evident within this developing tuft. The origin of these invading endothelial cells has been studied in some detail. Under normal growth conditions, kidneys excised at the time of induction and cultured in vitro do not exhibit signs of vascularization, leading to the presumption that endothelial cells migrate to the kidney some time after induction. However, hypoxygenation or treatment with vascular endothelial growth factor (VEGF) promotes survival or differentiation of endothelial precursors in these same cultures, suggesting that endothelial precursors are already present and require growth differentiation stimuli (Tufro-McReddie et al., 1997; Tufro et al., 1999). In vivo transplantation experiments using lacZ expressing donors or hosts also demonstrate that the E11.5 kidney rudiment has the potential to generate endothelial cells, although recruitment of endothelium is also observed from exogenous tissue depending on the environment (Hyink et al., 1996; Robert et al., 1996, 1998). The data are consistent with the idea that cells within the E11.5 kidney have the ability to differentiate along the endothelial lineage. Are these endothelial cells generated from the metanephric mesenchyme? Using a lacZ knockin allele for the endothelial specific receptor Flk-1, Robert et al. (1998) showed presumptive angioblasts are dispersed along the periphery of the E12 kidney mesenchyme, with some positive cells invading the mesenchyme along the aspect of the growing ureteric bud. At later stages, Flk-1 positive angioblasts were localized to the nephrogenic zone, the developing glomerular cleft of the S-shaped bodies, and the more mature capillary loops, whereas VEGF localizes to the parietal and visceral glomerular epithelium. Injection of neutralizing VEGF antibodies into newborn mice, at a time when nephrogenesis is still ongoing, disturbs vascular growth and glomerular architecture (Kitamoto et al., 1997). The data suggest that endothelial cells originate independently of the metanephric mesenchyme and invade the growing kidney from the periphery and along the ureteric bud. The mesangial cells are located between the capillary loops of the glomerular tuft and have been referred to as specialized pericytes. The pericytes are found within the capillary basement membranes and have contractile abilities, much like a smooth muscle cell. Whether the mesangial cell is derived from the endothelial or epithelial lineage

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remains unclear. However, genetic and chimeric analyses in the mouse have revealed a clear role for the platelet-derived growth factor receptor (PDGFR) and its ligand plateletderived growth factor (PDGF). In mice deficient for either PDGF or PDGFR (Leveen et al., 1994; Soriano, 1994), a complete absence of mesangial cells results in glomerular defects, including the lack of microvasculature in the tuft. PDGF is expressed in the developing endothelial cells of the glomerular tuft, whereas the receptor is found in the presumptive mesangial cell precursors. Using ES cell chimeras of Pdgfr e/e and þ/þ genotypes, only the wildtype cells could contribute to the mesangial lineage (Lindahl et al., 1998). This cell autonomous effect indicates that signaling from the developing vasculature promotes proliferation and/or migration of the mesangial precursor cells. Also, expression of PDGFR and smooth muscle actin supports a model in which mesangial cells are derived from smooth muscle of the afferent and efferent arterioles during glomerular maturation (Lindahl et al., 1998).

WHAT CONSTITUTES A RENAL STEM CELL? The issue of renal stem cells is beginning to draw more attention as new information regarding development and lineage specification is unraveled (Al-Awqati and Oliver, 2002). A prospective renal stem cell should be selfrenewing and able to generate all of the cell types in the kidney. Whether such a cell exists remains to be demonstrated in vivo, although the in vitro data of Oliveret al. (2002) appear promising. In simplest terms, all of the cells in the kidney could be generated by a single stem cell population as outlined in Figure 73.5. Despite the potential, there are several issues outstanding. Even at the earliest stage of kidney development, there are already two identifiable cell types. The presence of early endothelial precursors at the time of induction would make a third distinct cell type. If the three earliest cell types can indeed be derived from the metanephric mesenchyme, a single stem cell may indeed exist and continue to proliferate as development progresses. At present, the data suggest that stroma and epithelia may share a common origin, whereas endothelial cells and their potential smooth muscle derivatives constitute a second lineage. However, even if there are three separate lineages already demarcated within the metanephric mesenchyme, the most relevant with respect to the repair of renal tissue is the epithelial lineage. Thus, if we consider the possibility of an epithelial stem cell, the following points would be among the criteria for selection: (1) the cells would most likely be a derivative of the intermediate mesoderm, (2) the cells would express a combination of markers specific for the metanephric mesenchyme, and (3) the cells should be able to contribute

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VOLUME | 2

Metanephric mesenchymal stem cell Single Stem Cell Epithelial precursor

Angioblast Stroma

Multiple Stem Cells

Simple epithelia Endothelial cell Mesangial cell Interstitial stroma Podocyte

Distal and Proximal collecting tubule tubule

FIGURE 73.5 The major cell lineages of the kidney. Whether the kidney arises from a single renal stem cell or from multiple independent lineages remains to be determined. However, the basic differentiation scheme is becoming clearer. The cell lineage relationships are outlined schematically, with dotted lines reflecting ambiguity in terms of direct lineages. The metanephric mesenchyme contains angioblasts and stromal and epithelial precursors. Whether angioblasts arise from mesenchymal cells or are a separate lineage that surrounds the mesenchyme is not entirely clear. Similarly, the origin of the mesangial cell is not well defined. Stromal and epithelial cells may share a common precursor, the metanephric mesenchyme, but segregate at the time of induction. The epithelial cell precursors, found in the aggregates at the ureteric bud tips, generate almost all of the epithelial cell types in the nephron.

to all epithelial components of the nephron, in vitro and in vivo. Unlike ES cells, it seems improbable that cells from the intermediate mesoderm can be cultured indefinitely without additional transformation or immortalization taking place. Embryonic fibroblasts can be cultured from the mouse, but, in almost every case, a limited number of cell divisions occurs. The problem is apparent even in vivo, because the E11 metanephric mesenchyme is essentially quiescent and does not proliferate in the absence of induction. However, growth conditions that mimic induction might be able to allow for the mesenchyme cells to proliferate while suppressing their differentiation into epithelium. Alternatively, it may be possible to differentiate ES cells into intermediate mesodermal cells using combinations of growth and patterning factors, as has been done for motor neuron differentiation. If metanephric mesenchymal cells were able to proliferate in vitro, the markers they might express should include the following: Pax2, lim1, WT1, GDNF, Six2, and FoxD1. Expression of these markers would indicate a mesenchymal cell that had not decided between the

Adult and Fetal Stem Cells

stromal and epithelial lineage. If cells express Pax2, WT1, and Wnt4, but not FoxD1, they could be epithelial stem cells. Such epithelial stem cells when injected into, or recombined with, an in vitro cultured metanephric kidney should be able to make all of the epithelial cells along the proximaledistal axis of the nephron. Such epithelial stem cells could prove significant in regenerating damaged tubules in acute and chronic renal injury. At present, the complexity of the kidney still impedes progress in the area of tissue and cell-based therapies. Not only must the right cells be made but they must be able to organize into a specialized three-dimensional tubular structure capable of fulfilling all of the physiologic demands put on the nephrons. Developmental biology can provide a framework for understanding how these cells arise and what factors promote their differentiation and growth. Although we may not be able to make a kidney from scratch, it seems within the realm of possibility to provide the injured adult kidney with cells or factors to facilitate its own regeneration. Given the high incidence and severity of acute and chronic renal insufficiency, such therapies would be most welcome indeed.

ACKNOWLEDGMENTS I thank the members of my laboratory for valuable discussion regarding this topic, particularly Pat Brophy, Yi Cai, and Sanj Patel. G.R.D. is supported by NIH grants DK54740 and DK39255 and a grant from the Polycystic Kidney Research Foundation.

REFERENCES Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samson, D., Vincent, C., et al., 1997. A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family. Nat. Genet. 15, 157e164. Al-Awqati, Q., Oliver, J.A., 2002. Stem cells in the kidney. Kidney Int. 61, 387e395. Armstrong, J.F., Pritchard-Jones, K., Bickmore, W.A., Hastie, N.D., Bard, J.B.L., 1992. The expression of the Wilms’ tumor gene, WT1, in the developing mammalian embryo. Mech. Dev. 40, 85e97. Batourina, E., Gim, S., Bello, N., Shy, M., Clagett-Dame, M., Srinivas, S., et al., 2001. Vitamin A controls epithelial/mesenchymal interactions through Ret expression. Nat. Genet. 27, 74e78. Bouchard, M., Souabni, A., Mandler, M., Neubuser, A., Busslinger, M., 2002. Nephric lineage specification by Pax2 and Pax8. Genes Dev. 16, 2958e2970. Brophy, P.D., Ostrom, L., Lang, K.M., Dressler, G.R., 2001. Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene. Development 128, 4747e4756. Chan, T.C., Takahashi, S., Asashima, M., 2000. A role for Xlim-1 in pronephros development in Xenopus laevis. Dev. Biol. 228, 256e269. Dehbi, M., Ghahremani, M., Lechner, M., Dressler, G., Pelletier, J., 1996. The paired-box transcription factor, PAX2, positively modulates

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expression of the Wilms’ tumor suppressor gene (WT1) Oncogene 13, 447e453. Dressler, G.R., 2002. Development of the excretory system. In: Rossant, Ja.T.P.T (Ed.), Mouse Development: Patterning, Morphogenesis, and Organogenesis. Academic Press, San Diego, CA, pp. 395e420. Drummond, I.A., Majumdar, A., Hentschel, H., Elger, M., SolnicaKrezel, L., Schier, A.F., et al., 1998. Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. Development 125, 4655e4667. Dudley, A.T., Godin, R.E., Robertson, E.J., 1999. Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme. Genes Dev. 13, 1601e1613. Dudley, A.T., Lyons, K.M., Robertson, E.J., 1995. A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev. 9, 2795e2807. Guo, J.K., Menke, A.L., Gubler, M.C., Clarke, A.R., Harrison, D., Hammes, A., Hastie, N.D., Schedl, A., 2002. WT1 is a key regulator of podocyte function: reduced expression levels cause crescentic glomerulonephritis and mesangial sclerosis. Hum. Mol. Genet. 11, 651e659. Hatini, V., Huh, S.O., Herzlinger, D., Soares, V.C., Lai, E., 1996. Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2. Genes Dev. 10, 1467e1478. Hyink, D.P., Tucker, D.C., St John, P.L., Leardkamolkarn, V., Accavitti, M.A., Abrass, C.K., et al., 1996. Endogenous origin of glomerular endothelial and mesangial cells in grafts of embryonic kidneys. Am. J. Physiol. 270, F886eF899. Kitamoto, Y., Tokunaga, H., Tomita, K., 1997. Vascular endothelial growth factor is an essential molecule for mouse kidney development: Glomerulogenesis and nephrogenesis. J. Clin. Invest. 99, 2351e2357. Kreidberg, J.A., Sariola, H., Loring, J.M., Maeda, M., Pelletier, J., Housman, D., et al., 1993. WT1 is required for early kidney development. Cell 74, 679e691. Kume, T., Deng, K., Hogan, B.L., 2000. Murine forkhead/ winged helix genes Foxc1 (Mf1) and Foxc2 (Mfh1) are required for the early organogenesis of the kidney and urinary tract. Development 127, 1387e1395. Kuure, S., Vuolteenaho, R., Vainio, S., 2000. Kidney morphogenesis: cellular and molecular regulation. Mech. Dev. 92, 31e45. Lelievre-Pegorier, M., Vilar, J., Ferrier, M.L., Moreau, E., Freund, N., Gilbert, T., et al., 1998. Mild vitamin A deficiency leads to inborn nephron deficit in the rat. Kidney Int. 54, 1455e1462. Leveen, P., Pekny, M., Gebre-Medhin, S., Swolin, B., Larsson, E., Betsholtz, C., 1994. Mice deficient for PDGFD B show renal, cardiovascular, and hematological abnormalities. Genes Dev. 8, 1876e1887. Lindahl, P., Hellstrom, M., Kalen, M., Karlsson, L., Pekny, M., Pekna, M., et al., 1998. Paracrine PDGF-B/PDGF-Rbeta signaling controls mesangial cell development in kidney glomeruli. Development 125, 3313e3322. Mauch, T.J., Yang, G., Wright, M., Smith, D., Schoenwolf, G.C., 2000. Signals from trunk paraxial mesoderm induce pronephros formation in chick intermediate mesoderm. Dev. Biol. 220, 62e75. Mendelsohn, C., Batourina, E., Fung, S., Gilbert, T., Dodd, J., 1999. Stromal cells mediate retinoid-dependent functions essential for renal development. Development 126, 1139e1148. Obara-Ishihara, T., Kuhlman, J., Niswander, L., Herzlinger, D., 1999. The surface ectoderm is essential for nephric duct formation in intermediate mesoderm. Development 126, 1103e1108.

899

Ohto, H., Kamada, S., Tago, K., Tominaga, S.I., Ozaki, H., Sato, S., et al., 1999. Cooperation of six and eya in activation of their target genes through nuclear translocation of Eya. Mol. Cell Biol. 19, 6815e6824. Oliver, J.A., Barasch, J., Yang, J., Herzlinger, D., Al-Awqati, Q., 2002. Metanephric mesenchyme contains embryonic renal stem cells. Am. J. Physiol. Renal Physiol. 283, F799eF809. Osafune, K., Nishinakamura, R., Komazaki, S., Asashima, M., 2002. In vitro induction of the pronephric duct in Xenopus explants. Dev. Growth Differ. 44, 161e167. Pritchard-Jones, K., Fleming, S., Davidson, D., Bickmore, W., Porteous, D., Gosden, C., et al., 1990. The candidate Wilms’ tumor gene is involved in genitourinary development. Nature 346, 194e197. Qiao, J., Cohen, D., Herzlinger, D., 1995. The metanephric blastema differentiates into collecting system and nephron epithelia in vitro. Development 121, 3207e3214. Quaggin, S.E., Schwartz, L., Cui, S., Igarashi, P., Deimling, J., Post, M., et al., 1999. The basic-helix-loop-helix protein pod1 is critically important for kidney and lung organogenesis. Development 126, 5771e5783. Robert, B., St. John, P.L., Abrahamson, D.R., 1998. Direct visualization of renal vascular morphogenesis in Flk1 heterozygous mutant mice. Am. J. Physiol. 275, F164eF172. Robert, B., St. John, P.L., Hyink, D.P., Abrahamson, D.R., 1996. Evidence that embryonic kidney cells expressing flk-1 are intrinsic, vasculogenic angioblasts. Am. J. Physiol. 271, F744eF753. Sanyanusin, P., Schimmenti, L.A., McNoe, L.A., Ward, T.A., Pierpont, M.E.M., Sullivan, M.J., et al., 1995. Mutation of the Pax2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux. Nat. Genet. 9, 358e364. Soriano, P., 1994. Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev. 8, 1888e1896. Stark, K., Vainio, S., Vassileva, G., McMahon, A.P., 1994. Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature 372, 679e683. Torres, M., Gomez-Pardo, E., Dressler, G.R., Gruss, P., 1995. Pax-2 controls multiple steps of urogenital development. Development 121, 4057e4065. Tsang, T.E., Shawlot, W., Kinder, S.J., Kobayashi, A., Kwan, K.M., Schughart, K., et al., 2000. Lim1 activity is required for intermediate mesoderm differentiation in the mouse embryo. Dev. Biol. 223, 77e90. Tufro, A., Norwood, V.F., Carey, R.M., Gomez, R.A., 1999. Vascular endothelial growth factor induces nephrogenesis and vasculogenesis. J. Am. Soc. Nephrol. 10, 2125e2134. Tufro-McReddie, A., Norwood, V.F., Aylor, K.W., Botkin, S.J., Carey, R.M., Gomez, R.A., 1997. Oxygen regulates vascular endothelial growth factor-mediated vasculogenesis and tubulogenesis. Dev. Biol. 183, 139e149. Vilar, J., Gilbert, T., Moreau, E., Merlet-Benichou, C., 1996. Metanephros organogenesis is highly stimulated by vitamin A derivatives in organ culture. Kidney Int. 49, 1478e1487. Vize, P.D., Seufert, D.W., Carroll, T.J., Wallingford, J.B., 1997. Model systems for the study of kidney development:use of the pronephros in the analysis of organ induction and patterning. Dev. Biol. 188, 189e204. Wellik, D.M., Hawkes, P.J., Capecchi, M.R., 2002. Hox11 paralogous genes are essential for metanephric kidney induction. Genes Dev. 16, 1423e1432. Xu, P.X., Adams, J., Peters, H., Brown, M.C., Heaney, S., Maas, R., 1999. Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nat. Genet. 23, 113e117.