PHYSIOLOGICAL REVIEWS
Vol. 79, No. 4, October 1999
Printed in U.S.A.
Embryonic Renal Epithelia: Induction, Nephrogenesis,
and Cell Differentiation
MICHAEL F. HORSTER, GERALD S. BRAUN, AND STEPHAN M. HUBER
Physiologisches Institut, Universität München, München, Germany
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I. Introduction to Ontogenesis of the Metanephric Kidney
II. Principles in Nephrogenesis
A. Metanephrogenesis proceeds in defined morphogenic stages
B. Pronephros and mesonephros are embryonic precursors of the metanephros
C. Cell types of the metanephros are derived from different lineages
III. Ureteric Bud and Branching Morphogenesis
A. Induction of the ureteric bud
B. Genes that control the ureteric bud
C. Branching morphogenesis
IV. Metanephric Mesenchyme and Nephrogenic Pathway
A. Uninduced mesenchyme is pluripotent
B. Apoptosis is a regulatory mechanism for mesenchymal stem cells
C. Inductive signaling opens the nephrogenic pathway
D. Postinductive nephron formation
V. Mesenchyme-to-Epithelium Transition and Cell Adhesion
A. MET requires profound changes in gene expression
B. Cell adhesion initiates cellular reorganization in MET
C. Cell adhesion molecules are expressed in cell type-specific patterns
VI. Epithelial Cell Polarization
A. Methods to study embryonic renal epithelia
B. Ionic conductances are expressed before vectorial transport
C. Membrane transporters acquire apicobasal patterns
VII. Growth Factors and Extracellular Matrix
A. Growth factors are signaling molecules in induction and differentiation
B. Growth factor families are expressed in temporospatial patterns
C. ECM and cells interact in epithelial morphogenesis
VIII. Genes That Control Renal Organogenesis
A. Transcriptional regulation
B. Signaling by receptor tyrosine kinases
IX. Genetic Errors in Nephrogenesis
A. Polycystic kidney disease
B. Wilms tumor
C. Renal cell carcinoma
X. Concluding Remarks and Perspectives
Horster, Michael F., Gerald S. Braun, and Stephan M. Huber. Embryonic Renal Epithelia: Induction, Nephrogenesis, and Cell Differentiation. Physiol. Rev. 79: 1157–1191, 1999.—Embryonic metanephroi, differentiating into
the adult kidney, have come to be a generally accepted model system for organogenesis. Nephrogenesis implies a
highly controlled series of morphogenetic and differentiation events that starts with reciprocal inductive interactions
between two different primordial tissues and leads, in one of two mainstream processes, to the formation of
mesenchymal condensations and aggregates. These go through the intricate process of mesenchyme-to-epithelium
transition by which epithelial cell polarization is initiated, and they continue to differentiate into the highly
specialized epithelial cell populations of the nephron. Each step along the developmental metanephrogenic pathway
is initiated and organized by signaling molecules that are locally secreted polypeptides encoded by different gene
families and regulated by transcription factors. Nephrogenesis proceeds from the deep to the outer cortex, and it is
directed by a second, entirely different developmental process, the ductal branching of the ureteric bud-derived
0031-9333/99 $15.00 Copyright © 1999 the American Physiological Society
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collecting tubule. Both systems, the nephrogenic (mesenchymal) and the ductogenic (ureteric), undergo a repeat
series of inductive signaling that serves to organize the architecture and differentiated cell functions in a cascade of
developmental gene programs. The aim of this review is to present a coherent picture of principles and mechanisms
in embryonic renal epithelia.
I. INTRODUCTION TO ONTOGENESIS OF THE
METANEPHRIC KIDNEY
FIG. 1. Histology of early metanephrogenic organization. Section
through human kidney (;20.5 mm embryo) showing structures derived
from Wolffian duct and metanephrogenic blastema in outermost zone of
cortex. Peripheral branch of ureteric tree extends distally into an ampulla (see sect. III). Metanephric blastema has been induced (Fig. 2) to
enter nephrogenic pathway (see sect. IV), and nephron anlage has completed mesenchyme-to-epithelium transition (see sect. V). [Modified
from Horster (112).]
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Most parenchymal epithelial organs follow a fairly
simple scheme of embryonic organogenesis. An epithelial
sheet or tube that is derived from one of the primordia
enters a process of sequential branching (Fig. 1) to generate an arborizing or treelike structure. In the kidney, the
epithelial tube is to become the arborizing nephric ductderived collecting duct system.
The particularly complex situation in renal embryonic development is, however, that it has not one but two
distinct embryological origins. Most of the nephron, i.e.,
the structures beginning with the glomerulus and ending
at the junction of connecting tubule and collecting tubule,
are derived from another primordial tissue, the mesenchymal blastema. These two entirely different tissues interact so that the ureter-derived collecting duct is induced
to branch while the mesenchymal blastema is induced to
enter the critical process of mesenchyme-to-epithelium
conversion or transition (MET). This earliest mesenchyme-derived epithelium follows a structurally well-defined morphogenetic pathway to generate most of the
nephron.
Historically, the basic experimental model for work
in nephrogenesis had been set up by Grobstein (89 –91)
some 45 years ago. These pioneering studies, at the National Institutes of Health, demonstrated in an organ sys-
tem in vitro that 1) kidney rudiments when removed at
embryonic day 11 (E11) (mouse) follow an almost normal
developmental program in culture, 2) the isolated ureteric
bud cannot develop without contact to the metanephric
mesenchyme, and 3) the isolated metanephrogenic mesenchyme can be induced to go through the MET by a
number of tissues, including the embryonic spinal cord
and the ureteric bud. These and later tissue recombination experiments (164, 236, 242, 280) have set the stage for
the application of today’s molecular biology techniques to
nephrogenesis (121, 148, 290).
The two developmental pathways for the two different tissues, the nephrogenic (mesenchymal) and the ductogenic (ureteric), are regulated by transcription factors
and protooncogenes, polypeptide growth factors acting as
signaling molecules, and their receptors. They are modulated by cell adhesion molecule (CAM) complexes and
their associations with the cytoskeleton, by extracellular
matrix (ECM) glycoproteins and ECM receptor molecules
such as the integrin family, and by ECM degrading proteases. Protooncogenes regulate growth particularly in
embryonic organogenesis, and they have the potential to
gain tumorigenesis after gene mutations. Some of the
protooncogenes that encode for receptor tyrosine kinases
are involved in mesenchymal (nephrogenic)-epithelial
(ductogenic) interactions, in which the protooncogene
encoded tyrosine or serine/threonine kinase is the ureteric receptor (see sect. III) for signaling molecules secreted by the other primordial tissue, the metanephrogenic mesenchyme (see sect. IV).
The entire process is governed by changing gene
expression patterns of transcription factors (see sect. VIII)
such as Pax-2, of secreted signaling factors such as
wnt-4, and of protooncogenes such as c-ret. Through the
transfer of techniques from Drosophila to mouse and to
human, some of the genes critical in mammalian development have been identified. These genes are part of complex programs that induce and control sequential morphogenetic and differentiation events, i.e., the stages of
kidney ontogenesis.
To analyze the programs and their downstream effects during embryonic nephrogenesis, a wide spectrum
of techniques is increasingly applied at the individual cell
level. Single determinants of renal morphogenesis and of
epithelial differentiation, uncovered through these techniques, have been presented in a number of excellent
in-depth reviews, specifically on MET (16), conversion of
mesenchyme to epithelium (66), growth factors (93), renal stem cells (100), gene targeting (147), signaling mole-
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cules (201), the transcription factor WT-1 (215), and basement membrane molecules (270).
This review intends to integrate data from these different areas of research. It centers on the progress accomplished over the past 10 years of research in embryonic nephrogenesis, and it intends to provide a
comprehensive view of the many diverse aspects of embryonic renal epithelia. Among these, three processes in
kidney organogenesis are emphasized, namely, 1) acquisition of functional properties in the collecting duct system, 2) MET, and 3) epithelial cell differentiation from an
apolar to an apicobasal polarized phenotype.
II. PRINCIPLES IN NEPHROGENESIS
A. Metanephrogenesis Proceeds in Defined
Morphogenic Stages
The development of the metanephric (permanent)
mammalian kidney begins at gestational week 4 –5 in humans and at E11 in mouse. Organogenesis and its governing principles have been studied mostly in the mouse.
Metanephros formation, i.e., organogenesis of the permanent kidney (242), is initiated by the ureteric bud, which
sprouts out of the posterior end of the Wolffian duct and
invades the metanephrogenic mesenchyme (Fig. 2). The
subsequent interaction between the two primordia induces the ureteric bud to branch dichotomously, thus
initiating the morphogenesis of the collecting duct system
(242). Induced metanephric mesenchyme condenses at
the tips of the ureteric buds (Fig. 1), and mesenchymal
cells form aggregates (Fig. 3), thus beginning the MET.
Each aggregate epithelializes (156) and proceeds in stages
to the vesicle stage, comma stage, and S-stage, from
where each S-shaped body, after fusion with the ureteric
bud-derived collecting duct (Fig. 3F), differentiates into
one of the (2 3 106) nephrons of the human kidneys. The
architectural pattern, therefore, as a result of the sequential ureteric bud arborization, is designed to proceed from
the deep cortex to the periphery in a repeat series of
induction, morphogenesis, and differentiation (Fig. 2).
The epithelial segments of the nephron, unlike the
ureteric bud-derived collecting duct system, are created
from mesenchymal cells by an intricate cascade of events.
The early events (Fig. 2) result in the acquisition of an
essentially epithelial character by the future nephron cells
while these polarized cells form a sphere or vesicle (Fig.
3). The process of modeling the subsequent stages of
comma and S-shape (Fig. 3) is not understood, although
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FIG. 2. Overview of principal events in early nephrogenesis. Ureteric bud, an offspring of Wolffian duct, invades
mesenchymal blastema (left) and initiates reciprocal signaling (middle) between epithelial (ductal) and mesenchymal
(metanephrogenic) cell types. Receptor tyrosine kinases are expressed almost exclusively in ureteric bud cell, whereas
ligands are secreted by adjacent mesenchymal cells. Ligand for c-ros encoded receptor is not yet known. Ligand-receptor
signaling activates successive stages shown in Fig. 3, A—F. Mesenchymal-epithelial interactions, in addition to signaling
growth factors, involve extracellular matrix (ECM), cell adhesion molecules (CAM), transcription factors, and protooncogene-encoded receptor tyrosine kinases (RTK). Mesenchymal blastema expresses stem cells of several cell lineages;
stromogenic and nephrogenic ones are shown. Epithelial S-shaped body connects to ureteric bud-derived collecting duct.
HGF, hepatocyte growth factor; GDNF, glial cell-derived neurotrophic factor. [Modified from Horster et al. (113).]
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Volume 79
plenty of morphoregulatory molecules (see sect. VIIC) and
transcription factors (see sect. VIIIA) are sequentially and
differentially expressed. These stages of morphogenesis
are the onset of nephron differentiation, i.e., epithelial
segments begin to express their specific properties (112).
The mechanisms directing the segmentation of the
nephron have not been identified. Some of the molecules
involved in segmental morphogenesis are characteristically regulated in distinct segments, e.g., some members
of the integrin family are expressed in the late S-stage (a2
distal; a3 proximal), whereas others are upregulated only
in the blastema (a1) or in the vesicle stage (a6) (143).
These stages of nephrogenesis have an ancestry that
begins at the blastula stage, which determines the mesoderm; it follows the induction of the pronephros and the
directed migration of the pronephric duct to proceed
through the stage of the Wolffian duct and to induce the
metanephric mesenchyme, which in turn directs branching of the ureteric tree. Cells of the metanephrogenic
mesenchyme are induced by ureteric bud cells to become
stem cells after rescue from apoptosis (see sect. IVB); they
go on to condense and, guided by regulatory circuits of
gene expression and repression (see sect. VIIIA), to enter
the MET, and to polarize to apicobasal expression patterns (see sect. VI).
B. Pronephros and Mesonephros Are Embryonic
Precursors of the Metanephros
The metanephric kidney is derived from two different early embyronic tissue primordia: the nephric duct
FIG. 3. Morphogenic stages in early metanephrogenesis. Nephrogenic pathway is initiated by inductive signaling between Wolffian ductderived ureteric bud and adjacent mesenchymal blastema (Fig. 2). A:
induced condensing mesenchymal cells adhere and begin cell remodeling to epithelial phenotype. B: globular aggregates close to tip of ureteric
bud express basement membrane proteins and cell adhesion molecules.
C: renal vesicle. Lumen of sphere suggests secreted fluid and solutes. D:
comma-shaped body representing reorganized sphere and (E) S-shaped
body suggest expression of an as yet unknown patterning program. F:
junction of upper domain of mesenchymal blastema-derived S-shape
body with most peripheral branch of Wolffian duct-derived ureteric bud.
Scheme reduces some of morphological features, e.g., not shown is
strictly dichotomous branching of ureteric tree, as visible in Fig. 1.
[Modified from Horster et al. (113).]
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FIG. 4. Embryonic precursors of metanephros. Rudimentary pronephros, transiently functioning mesonephros, and permanent metanephros are sequentially induced and formed, thus recapitulating phylogeny of excretory system. This embryonic continuity also pertains to
some transcription factors and signal molecules. [Modified from Horster
(112).]
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C. Cell Types of the Metanephros Are Derived
From Different Lineages
1. Nephric duct-derived ureteric bud
The embryonic kidney has served for almost 50 years
(89) as a model system to study inductive interactions
between mesenchyme and epithelium. The fact that the
kidney is derived from two distinctly different primordia
that can be grown to develop in culture has aided the
experimental access to signal (growth factor) molecules
and their receptors. The very first step in kidney development is the sprouting of the ureteric bud out off the
Wolffian duct, followed by signaling from the ureteric bud
to the mesenchyme to induce the mesenchyme to become
epithelium.
The ductal system after initial sprouting develops by
sequential dichotomous branching, and the induction of
this repeat process of ureteric bud bifurcation in the
kidney appeared to be through interaction with the nephrogenic mesenchyme (90). The nature of some of the
inductive signals received by the ureteric bud cell from
the mesenchyme has been disclosed, although the cues
for the sequence of events remain elusive.
Branching morphogenesis of the collecting duct system, however, requires not only interactions between the
embedding mesenchyme and the epithelial duct, but also
between basement membrane (ECM) components and
the epithelial cells.
The mechanisms of ductal growth and of ductal
branching (10, 23, 231, 235) as well as differentiation by
expression of plasma membrane transport proteins in the
ureteric bud cell (113, 119) have finally come to be investigated.
2. Mesenchyme-derived stem cell populations
For the metanephric mesenchymal blastema to produce the ;15 epithelial cell types of the metanephric
kidney, it must be induced to undergo a conversion to the
epithelial phenotype and subsequently differentiate into
the highly specialized cell types of the nephron. Hypothetically, this pathway could start from two different points.
One starting point would be a homogeneous mesenchymal population consisting of one multipotent cell type
from which all nephron epithelial cell types are derived.
Alternatively, the primary inductive event is not the conversion to the epithelial phenotype but a commitment of
the mesenchymal cell type to different developmental
pathways, and the secondary inductive event of phenotypic conversion then destines already committed cells to
be recruited for the early nephron (101, 144, 213).
Studies on the temporospatial expression of two
transcription factors, BF-2 (97) and Pax-2 (49, 50), have
shed some light on this situation. It seems now justified to
favor the hypothesis that all peripheral mesenchymal
blastema cell types are induced to become stem cells
through the first signal interactions. This initial step (see
sect. IVA) rescues most of the nephrogenic stem cells now
expressing Pax-2 from apoptosis (6, 145), whereas the
uninduced mesenchymal cells enter programmed cell
death (see sect. IVB).
Induction is a two-step event (6) that had been postulated already from earlier tissue recombination work (243),
where it was found that a short-time (hours) exposure of
uninduced mesenchyme to the ureteric inductor led to the
stem cell phenotype but no further. Nevertheless, this first
step to the stem cell phenotype rescues most of the mesenchyme from apoptosis. The second step, however, very
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and the nephrogenic cord (Fig. 4). The nephric duct becomes the mesonephric duct and continues through the
Wolffian duct stage to the ureteric bud. The nephrogenic
cord, after inductive signaling with the pronephric ductderived cells, develops into the nephroi of mesonephros
and metanephros. The rudimentary pronephros, the transitory mesonephros, and the permanent metanephros
form in sequence during mammalian renal ontogeny (Fig.
4), recapitulating the phylogeny of the excretory system.
Pronephric tubules are formed around E8 (mouse)
from the intermediate mesoderm. In contrast, the primary
nephric duct appears to arise from a distinctly different
cell population within the intermediate mesoderm (198).
The caudal nephric duct (frog) extends along the cranialcaudal axis not by mitotic apposition but through migration of cells from the foremost tip toward their later
caudal organ destination where they reassemble into the
duct (34). Pronephric (zebrafish) and metanephric
(mouse) development, however, are believed to be governed by almost entirely different genetic programs, although the zebrafish Pax-2 and WT-1 homologs appear to
be expressed during pronephros differentiation in a pattern similar to that in mouse metanephros (54).
The nephron of the mesonephros is functioning (173,
266, 298); it is composed of glomerulus and two segments
(proximal/distal) merging in a collecting tubule that empties into the mesonephric or Wolffian duct. Wolffian duct
cells appear to induce mesonephric progenitor cells to
become differentiation competent (100); this interaction,
in the murine mesonephros, occurs around E10.
As the Wolffian duct grows close to a mesenchymal
cell population, it is induced to sprout (E10 —E11) and to
form the ureteric bud, whereupon mesenchymal cells at
the bud tip gather and appear to form a cap (Fig. 4). Next,
the invading bud is directed to branch dichotomously to
form a T-shape (Fig. 1), and this branching mode is maintained sequentially to express the arborizing structure of
the collecting duct in the cortex, which establishes the
general structure of the kidney.
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III. URETERIC BUD AND BRANCHING
MORPHOGENESIS
A. Induction of the Ureteric Bud
Grobstein, in a classic series of experiments (89 –91),
had addressed the question of whether or not mesenchyme and epithelium derived from different embryonic
organs can induce and maintain ductal morphogenesis
and concluded that one common mechanism was not
likely. However, there may be families of morphogenic
molecules, either signaling via the ECM, as transforming
growth factor-b (TGF-b) (230), or mesenchyme derived,
as epimorphin (106), or ligands of protooncogene-encoded receptor tyrosine kinases, such as hepatocyte
grwoth factor (HGF) (234) that participate in a general
pattern governing branching morphogenesis. Several lines
of evidence suggest that branching morphogenesis may
be viewed within an integrated model system in which
cell-matrix receptors (e.g., integrins) and basement membrane components (e.g., laminin-1) on one side and locally secreted signaling molecules [e.g., members of the
bone morphogenetic protein (Bmp) and Wnt families] on
the other organize budding and branching in different
epithelial organs (112, 130, 232). Although specific modes
of interactions between ECM and epithelial cells are expressed during organ morphogenesis, the general pattern
of signal-directed remodeling of the matrix may apply to
branching tubes so diverse as in kidney (232), lung (111),
and salivary glands (130).
B. Genes That Control the Ureteric Bud
1. WT-1
Several genes are presently believed to be regulated
by WT-1. Among those, in addition to Pax-2 (225), is
insulin-like growth factor (IGF)-II and its receptor (53,
296) and TGF-b (47), which are involved in branching
morphogenesis, and platelet-derived growth factor
(PDGF)-A (292). In the mesonephros, WT-1 is clearly
expressed in the mesenchyme, the vesicle, and glomerular structures. In the metanephros, WT-1 is expressed in
the uninduced mesenchyme and increasingly in the induced mesenchyme. Importantly, WT-1 is highly expressed in the proximal limb of the S-shaped body (121a),
specifically in the future podocytes up to the mature
glomerulus (41, 205).
2. c-ret
As a member of the family of growth factor receptors
characterized by an extraordinarily large extracellular
binding domain, c-ret is a protooncogene required for
ureteric bud branching and proliferation. The Ret receptor is first expressed in the Wolffian duct (E8 —E11.5),
and as ureteric arborization proceeds (E13.5—E17.5),
c-ret is expressed only in ureteric bud (Fig. 5) tip cells
(200). It is thus not surprising that in homozygous mutant
mice (RET2k2) the ureteric bud does not outgrow from
the Wolffian duct (247) while the mesenchyme from
RET2k2 mice maintains branching and growth of a wildtype ureter in vitro. Moreover, mesenchymal differentiation appears normal when induced with spinal cord,
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likely differs in molecular nature from the first one (6). Two
hypotheses, at present, are similarly supported by data, although not yet by complete lineage analysis. In one, the
primary inductive interactions between mesenchyme and
bud are believed to determine the distinct and final developmental pathways of both stromal and nephrogenic lineage
(66). In the other, a bivalent stem cell progenitor population
that gathers next to the outermost ureteric bud cells (Fig. 1)
is available throughout nephrogenesis, and it may either
take the nephrogenic (Pax-2) or the stromogenic (BF-2)
pathway (8, 97, 242). It is interesting to note that the endothelial progenitor cell, the angioblast, may derive also from
a bipotential (mesodermal) stem cell precursor (218). The
further fate of the nephrogenic lineage is also determined by
members of the superfamily of signaling peptides, as discussed in section VIIA.
Cell lineage analysis based on classic embryologic
work (92, 100) clearly indicates that the definitive kidney
is derived from two independent tissue compartments of
the intermediate mesoderm, namely, the metanephrogenic mesenchyme and the Wolffian duct. This traditional
view has been broadened by a set of data derived from
embryonic kidney organ culture (213); when uninduced
mesenchyme was isolated and tagged so that cells could
be followed to their final destination, and then cocultured
with isolated ureteric bud, mesenchymal cells were found
to be inserted into the collecting duct, although the majority of collecting duct cells were derived from the ureteric bud.
Organogenesis of the kidney has long become a
model system that represents principles in morphogenesis and cell differentiation. The continuous process of
morphogenesis is guided by cascades of interactions between two different cell populations (Fig. 2). Regulation
involves diverse families of genes and their products,
including protooncogene-encoded receptors (see sect.
VIIIB) and their polypeptide ligands (see sect. VIIA), transcription factors and their target genes (see sect. VIIIA),
and regulating ECM proteins and CAM-mediated signals.
All of these diverse systems interact to initiate and guide
embryonic renal morphogenesis and cell differentiation.
Unraveling the complexity of these interactions, which is
described in the following sections, is a major challenge
for many outstanding laboratories.
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C. Branching Morphogenesis
5. Temporospatial expression of signaling systems in nephrogenic and ureteric bud morphogenic pathways. Receptor tyrosine kinases (Ret, Met, Ros) are encoded by protooncogenes. Growth factor
signaling molecules are expressed and secreted as indicated. Relative
abundance of expression is specified by bold or normal type. Induced
precondensing mesenchyme is shown on top; other stages of metanephric nephrogenesis correspond to those depicted highly schematically in
Fig. 3, A, C, and E. PDGF, platelet-derived growth factor.
FIG.
whereas the ureteric bud from RET2k2 mice did not
interact with wild-type mesenchyme (247). This impressive work indicated that 1) the Ret receptor response to
an inductive signal is independent of the cell type, and 2)
the Ret ligand glial cell-derived neurotrophic factor
(GDNF) activates ureteric cell proliferation and branching very early (Fig. 5), beginning with the first visible
Wolffian duct outgrowth.
3. Limb deformity
Limb deformity (ld) encodes a group of phosphoproteins, the formins, generated by alternative splicing of
mRNA that are all expressed in the kidney. The ld mutation in the mouse results in defects of limb formation and
urogenital development that, interestingly, are expressed
with differing degrees of anomaly (167). The five known
alleles of ld and the formin isoforms are expressed in the
ureteric bud (Fig. 5) and in the mesenchyme. The primary
defect in ld 2/2 mutant is a failure of the ureteric bud to
1. Multiple controls regulate the arborizing
duct system
The embryonic development of several organs, such
as lung, mammary gland, pancreas, tooth, salivary glands,
and kidney, depends decisively on branching morphogenesis. In all of these organs, a small epithelial rudiment is
initially surrounded by mesenchymal cells. In the metanephric kidney (Fig. 2), a sequence of different events is
initiated after reciprocal induction that leads to the formation of dichotomous duct branches. Two processes
result in the arborizing collecting duct system starting
from the first bifurcation of the ureteric bud, namely, the
longitudinal growth of duct epithelia and the branching
process. There is now accumulating evidence that both of
these are regulated separately (40). In fact, a recent model
for renal branching morphogenesis (229 –231) proposes
that a local ratio, at the branching point, of branch-promoting to branch-inhibiting factors might account for the
architecture of the collecting duct system, and it was
demonstrated for the first time that growth (ductogenesis) and branching are regulated by separate mechanism.
Whereas HFG and TGF-a are branching morphogens,
TGF-b is inhibitory to branching but not to ductogenesis.
Nevertheless, HGF/scatter factor (SF) and its c-met receptor are the primary signaling system for branching and for
ductal growth in nephrogenesis. Moreover, this inducing
action on ductal morphogenesis is paralleled by HGF
effects on its mesenchyme-based Met receptor, and anti-
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induce the mesenchyme because outgrowth of the ureteric bud is either arrested or delayed or incomplete
(167). Because formins are believed to operate in intracellular protein-protein interactions (27, 279), they might
be part of a signaling pathway downstream of the ligandreceptor interaction in the ureteric bud tip cell. The epithelial cell at the tip of the invading ureteric bud (Fig. 1)
faces the interspace between the two signaling tissues.
When this cell (Fig. 11) was analyzed by the combined
techniques of electronmicroscopy, electrophysiology, and
molecular biology (119, 121), it was found that it differs
functionally from the cells in deeper parts of the branching ureteric tree, and it undergoes the epithelial polarization and differentiation process in situ. An alternative
hypothesis (100), however, postulates that the bud tip cell
exhibits a mesenchymal phenotype that is capable to
delaminate from the ureteric bud and to be incorporated
into all segments of the mesenchyme-derived nephron.
Ureteric bud cell differentiation, i.e., how this cell acquires the polar organization of apical and basolateral
plasma membrane characteristic for the collecting duct
cell, is discussed in section VI.
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bodies to HGF/SF perturb branching morphogenesis and
the early phase of MET (301).
2. Signaling by GDNF through Ret is
branch promoting
3. Local growth factors interact with the
basement membrane
Extracellular matrix molecules are secreted and localized in complex temporospatial patterns (66, 141), and
they have signaling roles particularly in early nephrogenesis, followed by regulation of basement membrane components and their receptors during epithelial polarization.
When the peptide growth factors IGF-I or IFG-II (220) or
nerve growth factor (NGF) (237) are blocked selectively
by molecular or immunotechniques, mesenchyme and
ureteric bud development is perturbed. On the other
hand, renal morphogenesis is overall normal in mice with
homozygous null mutations of the IGF-II (42) and the
NGF (155) receptors. These apparently contradictory observations are not unusual in polypeptide signaling and
suggest functional redundancy.
Laminin-1, a basement membrane constitutent, acts
during cell differentiation in a dual way, namely, as a
signaling molecule and as a structural component. The
extracellular glycoprotein is a member of a large family
present in basement membranes, and it has several bio-
logically active domains including the proteolytic fragments E3 and E8 (72); importantly, the laminin a1-chain is
expressed at the onset of epithelial polarization (65).
Antisera against the E8 and E3 domains inhibited the MET
in embryonic renal organ culture (141). The role of laminin-1 in early branching morphogenesis was further demonstrated by monoclonal antibodies against the E3 fragment in organ culture of embryonic salivary gland, which
inhibited branching morphogenesis, suggesting a role for
the laminin E3 fragment in structuring basement membranes (130). Dystroglycan is the high-affinity receptor for
laminin-1 and laminin-2, and a-dystroglycan, the receptor
for the E3 fragment, probably links the basement membrane to the cytoskeleton (70), and an antibody against
a-dystroglycan inhibits the embryonic epithelial polarization process (60). In addition to a-dystroglycan, a second
receptor specific for the E8 fragment of laminin-1, the
a6b1-integrin, is expressed on nephrogenic cells during
MET (255). The a6-integrin receptor, therefore, might participate in signal transmission during cell differentiation,
and the a6-integrin subunit associates with the b1-subunit
during the early nephrogenic process (65). The structural
role of the laminins is within the two major complex
networks of laminin and collagen IV that are probably
linked by nidogen that binds to type IV collagen and to a
domain in the laminin g1-chain (174). The crucial role of
nidogen as a link protein has been proven by perturbation
experiments (67), demonstrating that interfering with the
link between nidogen and laminin-1 inhibits ureteric bud
branching morphogenesis. It is of interest in this context
that an unexpected heterogeneity at the histochemical
level (142) was demonstrated for the embryonic collecting duct, and a monoclonal antibody of the IgG-1 subclass
was shown elegantly to react with an epitope at the
basolateral side of the ampullar collecting duct epithelium (259). The roles of antibody and epitope in nephrogenesis remain to be elucidated.
4. Local proteolysis coregulates ductal morphogenesis
Local proteolysis remodels the extracellular matrix
during branching. Matrix proteolysis can be attributed to
secreted matrix metalloproteinases (157) and to plasma
membrane-bound proteases. Matrix metalloproteases and
their regulation thus have a complementary role to the
branch-promoting (157) and branch-inhibiting growth factors. This function implies that inhibitors of proteases
block branching morphogenesis. Proteases can be secreted by epithelial or by adjacent stromal cells. Although
almost all details of the design for collecting duct architecture are unknown, it might be permitted to speculate
that stromally secreted proteases, after binding to epithelial receptors, could guide directed proteolysis along tissue gradients of proteases.
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Ureteric bud cells express high-affinity receptors for
growth factors (see sect. VIIIB) and among them are the
receptor tyrosine kinases Met, Ret, and Ros (Fig. 5). The
polypeptide ligand of Ret, GDNF, is a branch-promoting
growth factor, since GDNF 2/2 mice suffer from delayed
or absent ureteric branching (186, 208, 232). Expression
of GDNF (99) is high in wild-type condensing mesenchymal cells (E11.5) and downregulated after MET (Fig. 5) to
maintain the arborizing morphology. Importantly, expression is maintained high in the outer cortical mesenchymal
cell population, which might enable GDNF to organize the
treelike patterning possibly by radial concentration gradients of GDNF along extracellular matrix components.
Glial cell-derived neurotrophic factor binds to the receptor GDNFR-a (127, 273), and the receptor-ligand complex
binds to Ret (276, 285). Indeed, GDNF and Ret are a
specific ligand-receptor entity, since GDNF when added
to wild-type kidney cultures increased the number of
(aberrant) ureteric branches (285). Ureteric growth and
branching are also dependent on the intact Ret receptor,
which is part of the GDNF signaling system (200, 247).
Among the regulatory genes (Fig. 5) expressed in the
ureteric bud cell, in addition to Pax-2, is ld, and its
mutation inhibits ureteric growth (275). The intracellular
signal systems, however, that link the ligand-dependent
RTK activation to the ureteric bud effector systems for
growth and branching remain undefined.
Volume 79
October 1999
EMBRYONIC RENAL EPITHELIA
IV. METANEPHRIC MESENCHYME
AND NEPHROGENIC PATHWAY
A. Uninduced Mesenchyme is Pluripotent
1. Intermediate mesoderm differentiation is
regulated: Pax-2
The family of Pax genes encodes transcription factors expressed in various embryonic tissues, including the
kidney (50). Two Pax genes are expressed in renal organogenesis, Pax-2 and Pax-8. Pax-2 is central for renal
development, since it appears to specify all epithelial
phenotypes derived from the intermediate mesoderm. It is
first expressed in the nephric duct and in mesonephric
tubules, extending caudally to the ureteric bud (49, 50).
Pax-2 is expressed only in mesenchyme that has been
induced by signaling from the ureteric bud, but not in the
uninduced mesenchyme (207), and Pax-2 becomes the
first indicator of the nephrogenic cell lineage at this early
stage (Fig. 5). Pax-2 continues to be expressed throughout condensation and polarized vesicle formation (Fig.
15), and expression is downregulated in the proximal loop
of the S-shaped body that is the site of the podocyte
precursor cells. This repression of Pax-2 transcription is
related to the expression of WT-1 (215) and its interaction
with the first exon of the Pax-2 gene (225).
2. Pax-2 and WT-1
The regulatory loop between WT-1 and Pax-2 (Fig.
15) may provide clues for further analysis of those renal
diseases in which epithelial cell proliferation continues.
The Pax-2 gene is a member of a family that also includes
genes involved in severe abnormalities as Waardenburg
syndrome (264) and human aniridia (271). Pax-2 is essential for the MET (223), since interference with gene function inhibits mesenchymal condensation and the subsequent steps in nephrogenesis. The complete loss of Pax-2
function, by homologous recombination (272), resulted in
abolished formation of nearly all of the epithelial components derived from the intermediate mesoderm. The central role of Pax-2 was further illustrated in a transgenic
mouse model (52) where the deregulated gene produced
severe abnormalities such as cystic kidney and undifferentiated glomerular epithelia, reminiscent of features in
congenital nephrotic syndrome. In conclusion, the nephrogenic stem cell is characterized by a new pattern of
gene expression, such as Pax-2, both at the level of
transcription and of signaling molecules (Fig. 5).
B. Apoptosis is a Regulatory Mechanism
for Mesenchymal Stem Cells
Apoptosis or programmed cell death is a crucial regulated event in renal morphogenesis, since a large number
of (blastemal) cells are produced and only a few are
guided to follow a developmental program because they
have been rescued from programmed cell death by induction. Cell death occurs by two distinct mechanisms. In
one, necrosis, cellular ATP concentration declines immediately (as in renal ischemia) followed by a sequence of
events, such as cellular swelling, leading to cell lysis. In
the other, apoptosis, the primary event is a regulated
breakdown of DNA into small, 200-bp fragments by the
activation of a calcium-sensitive endonuclease. The cell
constituents condense and ultimately break into fragments that are taken up mostly by macrophages. Mesenchymal cells are destined for apoptosis, as shown in
transfilter culture of rat E13 isolated renal tissue (145,
295), unless they are induced to survive by signaling
interactions with the ureteric bud. Apoptosis is a normal
event in development, and apoptotic cells are frequently
observed next to condensing aggregates and to vesicles
(32, 116). Apoptosis is more prominent in mesenchymal
cells of the nephrogenic outer cortex where cell death
may serve to remove those mesenchymal cells that have
not been chosen for MET. This implies that inductive
signaling may be a two-step event (6) in which rescue
from apoptosis is followed by conversion to the epithelial
phenotype. Although the rescue or survival signal remains
to be disclosed, a hint to a putative pathway may have
come from the observation that LiCl (15 mM), added to
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The classic studies of kidney organogenesis had established (89 –91) that a signal, although its nature and
mechanism could not be identified, must initiate outgrowth and branching of the ureteric bud, and another
signal originating from the ureteric bud must induce MET.
Today, some of the signals essential for interactive early
nephrogenesis have been identified (and many more have
been shown to exist), but the meaning of their temporospatial expression patterns and their precise downstream
roles still remain poorly defined.
The discovery of BF-2 as an embryonic stromal gene
(97) in the mesenchyme, i.e., in the intermediate mesoderm, has rekindled the debate of whether or not two cell
populations are present in the uninduced mesenchyme
whose final destination is either stromal or epithelial. This
view requires that the uninduced mesenchyme had already received a signal, presumably from the early invading outgrowth of the Wolffian duct which is essential for
the continuity of the nephrogenic process, since uninduced mesenchyme goes apoptotic (145). Alternatively,
the decision would be made through molecules of the
inductive interactions within a homogeneous mesenchymal cell type. A case for the latter was made by the
observation (213) that induced ureteric bud cells can
delaminate and incorporate themselves into the mesenchyme to become part of the epithelia-generating cell
population.
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HORSTER, BRAUN, AND HUBER
Volume 79
FIG. 6. Functional complexes in transition
of mesenchymal to epithelial cells (MET). Nephrogenic mesenchymal cells (top) are induced to
enter MET whereby several systems with signaling functions are activated. CAM-mediated signals and ECM-mediated signals interact with
secreted growth factors to express epithelial
phenotype (bottom).
increased mesenchymal apoptosis (148). Also, when cmyc is constitutively expressed in cell lines, the ensuing
apoptosis can be inhibited by coexpression of bcl-2 (17).
Clearly, bcl-2-regulated apoptosis is a basic mechanism in
nephrogenesis, and bcl-2 is an intrinsic negative regulator
of the cell death pathway even beyond the inductive
phase of morphogenesis.
C. Inductive Signaling Opens the Nephrogenic
Pathway
Mesenchymal-to-epithelial transition of the metanephrogenic mesenchyme is the center event in early
nephrogenesis. Mesenchymal cells are nonpolarized,
loosely associated, and apolar cells embedded in ECM
with a fibroblast-like shape and high mobility. Epithelial
cells, in contrast, are asymmetric or polarized, form continuous sheets, express a basement membrane, have a
cuboidal or “cobblestone” shape, and are generally little
mobile. The phenotypic conversion of metanephrogenic
mesenchymal to nephron epithelial cells (Fig. 6) mirrors
changes in the expression of different gene families
(121a), encoding transmembrane receptors, cell adhesion
molecules, growth factors, ECM components, and specific
basement membrane constituents. As discussed, one or
more genes of the Pax family are expressed in each of the
tissues that undergoes MET, and pro-, meso-, and metanephros are all formed through these interactions. The
nature of the inductive signals that were postulated in the
classic embryologic recombination experiments (86, 89)
is now being uncovered. The inductive process is multifactorial and multiphasic. Although much effort has been
invested in studying single factors (e.g., Refs. 37, 39, 102,
206, 295), these single factors appear to address and
express only parts of the developmental program, i.e.,
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the medium of isolated mouse (E11) mesenchyme in the
absence of an inducing signal, was able to rescue these
cells from entering programmed cell death; morphogenesis after the LiCl rescue was initiated up to the expression
of the cell adhesion molecule N-CAM in the comma stage,
but no further (39). In addition, some growth factors, such
as epidermal growth factor (EGF) (295) or basic fibroblast growth factor (FGF) (206) are able to rescue cultured mesenchyme from apoptosis.
The tumor suppressor gene p53 is believed to not
only act as controller in the cell cycle but also in apoptosis (211). However, wild-type p53 expression in commaand S-shaped body does not provide a clue to its function
(245), and a p53 loss-of-function mutation does not dramatically alter organogenesis (48), whereas a gain-offunction mutation induces ureteric alterations and small
kidneys with a reduced number of nephrons and, notably,
accelerated apoptosis (82). Of interest in this context are
the findings that WT-1 binds the p53 protein, thus inhibiting p53-mediated apoptosis (169, 170), and p53 expression can be repressed by Pax-2 (260).
A protooncogene with death repressor activity, bcl-2,
is expressed in nephrogenesis (197, 253). The bcl-2-encoded protein is on the outer mitochondrial membrane, in
the endoplasmic reticulum and the nuclear envelope, and
it is involved probably in an antioxidant pathway (286) to
protect cells from oxidative damage (110). The bcl-2 gene
is highly expressed in condensing mesenchyme and in the
ureteric bud, and it is downregulated, as in most mature
cells, in terminal epithelia and in glomerular cells (154,
253); bcl-2-deficient mice develop hypoplastic polycystic
kidneys (286). In addition, they have abnormal postinductive nephrogenesis either after E13 (193) or weeks after
birth (253, 286), associated with fulminant mesenchymal
apoptosis (194).
WT-1 binds to p53, and WT-1-deficient mice show
October 1999
EMBRYONIC RENAL EPITHELIA
1167
their effects either have not induced tubulogenesis (39,
206, 295), or the inducing factor has activated another
messenger system that in fact induced tubulogenesis (102,
133).
1. Genes in mesenchyme induction
FIG. 7. MET-associated shift in gene expression. Relative abundance
(symbol size) of gene expression or protein production of some CAM
and ECM proteins, and their shift in process of mesenchymal (top left)
to epithelial (top right) cell transition (MET), is shown. Reference
numbers are given in parentheses.
initial phase, however, signaling was discontinued, and
the expression of Lim-1, c-ret, and Pax-2 in ureteric bud,
as well as that of GDNF in the mesenchyme, was greatly
reduced; the ureteric tip did not dilate and branch, Wnt-4
was not expressed, and MET was not initiated. The control experiment showed that wild-type ureteric bud or
spinal cord was capable to induce the mesenchyme of the
mutants, suggesting that Emx2 is required in early ureteric bud cells subsequent to the induction of mesenchymal Pax-2 expression.
Condensed mesenchymal cells, in addition to their
shift from mesenchymal to epithelial surface proteins
(Fig. 7), express a protein encoded by Wnt-4, a member of
a family of genes that encode signaling molecules regulating early embryonic tissues (258). The Wnt-4 protein is
secreted by induced metanephrogenic mesenchyme
(138), and the Wnt-4 gene is expressed in condensates
very soon after induction by tip ureteric bud cells, and
expression persists in the vesicle, comma, and S-stages
(Fig. 5). In mutant Wnt-4 2/2 mice, mesenchymal cells
condense next to the ureteric buds, Pax-2 is normally
expressed (258), but Pax-8 that is normally expressed
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The early stages of kidney organogenesis are governed by four different control genes (Fig. 5). These are
WT-1 (148), Pax-2 (49, 51), Wnt-4 (258), and Bmp7 (55,
165). Their primary roles, as deduced from knockout
experiments, suggest that they are local mediators in
signaling interactions between the ureteric bud and the
nephrogenic blastema.
WT-1 is expressed in the metanephrogenic mesenchyme but not in the ureteric bud cells (4), and the
expression of WT-1 (191) is the earliest sign of commitment in the intermediate mesoderm-derived metanephric
blastema that contains the nephron lineage(s). WT-1 is
essential for mesenchymal competence to later respond
to other inductive signals (148); it directs the genesis of
the first ureteric bud off the Wolffian duct, and it interacts
with Pax-2 in the nephrogenic stem cells at the transcriptional level (Fig. 15).
Another of the control genes in early nephrogenesis
is Bmp-7, which encodes the bone morphogenetic protein-7 (55, 165). Bmp proteins belong to the TGF-b family
of secreted signaling molecules generally involved in morphogenesis (261). Bmp-7 loss-of-function kidneys differentiate up to the comma- and S-shaped stage, but further
epithelial development is defective (56), and the mesenchymal cells undergo rapid apoptosis (165). Consequently, genes of early induction and transition stages are
expressed in Bmp-7-deficient mutants, albeit mostly in
aberrant patterns, such as Pax-2, Wnt-4, WT-1, Ret, and
even Pax-8. Bmp-7 transcripts are seen in the mesonephric kidney, in the metanephric condensates, in comma and
S-stage, and in the collecting duct (166). In line with the
expression pattern of Bmp-7 and the mutant phenotype is
the finding that Bmp-7 induces cultured metanephric
mesenchyme to differentiate, and antibodies or antisense
oligonucleotides inhibit Bmp-7 and tubulogenesis (288).
The homeobox gene lim1 is expressed in the intermediate mesoderm, mesonephric tubules, Wolffian duct,
and induced mesenchymal cells (9, 75). Lim1 2/2 mice
have a complete defect of kidneys and gonads (249) probably due to premetanephric defects, suggesting that lim1
has a primary role in intermediate mesoderm specification. The gene that is expressed in the progeny of the
Wolffian duct appears to have an important role after the
initial signaling events (183). Metanephric mesenchyme
and ureteric bud of Emx2 2/2 mutants correctly expressed transcribed marker molecules of the the first
signaling stage; the ureteric bud invaded the metanephric
mesenchyme and induced Pax-2 expression. After this
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HORSTER, BRAUN, AND HUBER
D. Postinductive Nephron Formation
The inductive process between nephrogenic mesenchyme and ureteric bud ultimately results in aggregates of
induced cells that now express adhesion molecules (Fig.
6). Wnt-4 is expressed in these aggregates, and it is repressed upon completion of the nephron. In Wnt-4 2/2
mice, condensations of induced mesenchymal cells appear, but few only express markers of epithelialization or
Pax-8 (258), indicating that Wnt-4 is an essential regulator of MET, probably by controlling the expression of cell
adhesion molecules (105, 281, 287). Control mechanisms
in the subsequent stage of cell polarization are unknown,
although some putative control genes, such as members
of the Hox family and WT-1, are upregulated during the
transition process. As the mesenchymal-to-epithelial program continues, tenascin is expressed in polarizing mesenchymal cells (5) together with SGP-2 (94).
The MET is completed when cells display the basic
features of an epithelium, e.g., the asymmetry of apicobasal membrane proteins. The condensed and converted
cells form a round body which, by as yet undefined mech-
anisms of fluid secretion, develops a small lumen and
proceeds to the vesicle stage (Fig. 3C). The subsequent
stage, some hours later, is reached by reorganization of
the sphere into a longitudinally curved epithelial body, the
“comma shape” (Fig. 3D). The mechanisms responsible
for this rearrangement and for the intricate pattern formations that follow are unknown. The comma stage develops to the “S-shaped body” by a cleft involution at both
curving ends (Fig. 3E). It is very likely by now that the
S-stage expresses a proximal-distal profile of cell properties (121a) characteristic of the evolving proximal and
distal nephron segments. Nevertheless, in situ hybridization and immunohistochemical work have already demonstrated that some transcription factors are repressed
(e.g., WT-1, Pax-2, N-myc) while others are expressed
(e.g., Pax-8; LFB-1) during this and the subsequent stages
of nephron formation (4, 50, 223, 188, 153, 210). Their
state of molecular differentiation is only now beginning to
be explored (Fig. 12). The most proximal end of the
S-shape enters angiogenesis (218), whereas adjacent tubular parts express a few apical membrane markers (7).
In the most distal part of the S-shape, membrane fusion
ultimately must connect the mesenchyme-derived
nephron segments with those originating from the epithelial ureteric bud (Fig. 3F). Other cells of the distal tail of
the S-body are to express the cell types of the distal
convoluted tubule and of the macula densa. The importance of apoptosis at this stage, regulated by bcl-2 (110,
154), is clearly demonstrated by the fact that bcl-2 2/2
mice, in addition to high apoptotic rates, have polycystic
kidneys (286). Further development into nephron segments is accompanied by basement membrane scaffolding (68) and by signaling of a wide variety of growth
factors (93). Collecting duct growth, in addition, is directed by IGF/R-mediated actions, since antisense oligonucleotides against the receptor inhibit growth of the
collecting duct in organ culture (291). The repression of
Pax-2 at the S-shape stage is a prerequisite for further cell
differentiation; it is as essential here as is the expression
of Pax-2 in previous stages for completion of MET (Fig.
15). Finally, the junction of the mesenchyme-derived segments of the nephron with those derived from the Wolffian duct has not been investigated, and it involves most
likely mechanisms of plasma membrane reorganization
similar to those supposed to take place in branching
morphogenesis (229 –231).
V. MESENCHYME-TO-EPITHELIUM TRANSITION
AND CELL ADHESION
A. MET Requires Profound Changes
in Gene Expression
Conversion of the induced mesenchymal cell to its
epithelial cell phenotype, i.e., MET, requires extensive
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immediately after the condensation stage (Fig. 15) was
not expressed. The failure of nephrogenesis to proceed
beyond the condensation stage suggests that the cascade
of signaling molecules directing the induction process
was interrupted at the Wnt-4 plateau. The Wnt family (77)
indeed has some characteristics of the putative mesenchymal inducer molecule(s), e.g., they are postulated to
associate with secreted matrix or with basement membrane domains. Another Wnt family member, Wnt-1, was
demonstrated to have properties comparable to Wnt-4
(102). In an elegant and important study, Wnt-1-secreting
NIH 3T3 fibroblasts (transfected with Wnt-1 cDNA) were
cocultured with isolated nephrogenic mesenchyme and
shown to induce tubule formation after an initially increased proliferation of induced mesenchymal cells. It is
of interest that Wnt-4 has a temporal expression pattern
very similar to Bmp-7 (166, 288) and that sonic hedgehog
(Shh) genes are coexpressed with Bmp genes (18), but
nothing is known about interactions between these families of morphogens.
To summarize, Wnt-4, in contrast to Wnt-11, which is
expressed exclusively at the very tip of the ureteric bud
(Fig. 5), appears to be essential, possibly as an autoactivator, for the transitional step from the induced mesenchyme to epithelia and most likely in subsequent early
stages of tubulogenesis, but it is not required for the initial
step(s) leading to the induced nephrogenic mesenchymal
cells. This emerging role of Wnt as a regulator of
E-cadherin-mediated cell-cell adhesion (105) denotes a
further very important point along the multistep induction
process.
Volume 79
October 1999
EMBRYONIC RENAL EPITHELIA
B. Cell Adhesion Initiates Cellular Reorganization
in MET
Cell adhesion appears to be regulated through an
interplay of several molecular species in which the cadherin family of adhesion molecules (171), the catenin
family of intracellular adhesive junction proteins, and the
product of the protooncogenes Wnt-1 and src participate
(21, 105). Cadherins and catenins are fundamental molecules in embryonic epitheliogenesis, and changes in cadherin expression patterns designate events in morphogenesis, such as MET. The cadherins E-, N-, and P-, the classic
cadherins, are able to form complexes with specific
catenins, i.e., with a-, b-, and g-catenin (plakoglobin) (21,
199). Cadherins are linked to the actin filament network
by catenins, and cadherin-catenin complexes may interact
with other cytoplasmic or transmembrane proteins (115,
214). Cadherins thus acquire a critical role in early epithelial polarization (152). The a- and b-catenins colocalize
with E-cadherin at the zonula adherens (195), and interactions between the zonula adherens complex, as for
b-catenin/E-cadherin, and the actin filaments may participate in morphogenetic changes such as from the comma
to S-shape. E-cadherin expression increases early at the
contact site between two cells, ;1 h after contact has
been made (176, 177). The next stage is characterized by
interactions between E-cadherin and cytoskeletal proteins that result in the polar distribution of membrane
proteins (176). Truncated N-cadherin lacking the extracellular domain and expressed in Xenopus embryos results in disruption of cell adhesion and abnormal development (137). Furthermore, epithelial cells deprived of
a-catenin are unable to adhere, although they express
b-catenin and E-cadherin; cells that lack cadherins but
express a- and b-catenins can be induced to express an
epithelial adhesive phenotype by the introduction of Ecadherin or N-cadherin, indicating that different cadherins can interact with one catenin subtype (107). Ecadherin is essential early in embryogenesis, since mice
lacking this gene expression are unable to form a trophoectoderm epithelium and die from this preimplantation
defect (217). The final phenotypic step in the polarization
process, some 1.5 days after the first inductive step
(mouse), is the gain of expression of cytokeratin components of the intermediate filament system (Fig. 7) and the
loss of vimentin.
C. Cell Adhesion Molecules Are Expressed in Cell
Type-Specific Patterns
When mesenchymal cells aggregate next to the tip
cells of ureteric buds to form packed condensates, this is
considered the first visible morphological indication of
transformation. Syndecan-1 (14) and N-CAM (196) are
upregulated at the onset of this stage, as is Wnt-4, and
they may therefore be involved in organizing the mesenchymal condensate. N-cell adhesion molecule coexpresses with the low-affinity receptor (p75NGFR) of NGF
(297) and with NGF (237); this fact suggests an early role
for N-CAM and NGF in the predetermined nephrogenic
stem cell (140) even before epithelial polarization is expressed. N-cell adhesion molecule probably is a target
gene for regulation by Pax and Hox gene products (64,
128); it may turn out that these homeobox and paired-box
gene products are an important functional link between
patterning genes and morphoregulatory CAM genes. Acell adhesion molecule has a similar pattern except that
its expression persists in the lower limb of the S-shaped
body during later stages, whereas L-CAM is expressed in
ureteric bud, collecting duct, and the limb of the S-shaped
body that is to become the distal nephron where L-CAM
continues to be expressed in the neonatal kidney (196).
These segment-specific patterns for L-CAM and for ACAM as early as in the S-stage may contribute to the
expression of characteristic nephron segmental properties. In conclusion, upregulation of both syndecan (283)
and N-CAM (140) denotes the onset of epithelial morphogenesis in the mesenchyme. However, it should be mentioned that neither the mechanism of aggregate formation
within the condensations nor the regulation of cell polarization is currently understood. Answers to these open
questions may come from studies on the “reverse” pro-
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alterations in gene expression. Regulatory genes and morphogenic molecules known to participate are considered.
The first step of induction rescues part of the mesenchyme from apoptosis and leaves these mesenchymal
stem cell populations committed for further induction to
enter either the nephrogenic or the stromogenic pathway
(see sect. IIC). Condensing cells transiently express the
surface glycoprotein syndecan (281, 282), and they begin
to express a new set of regulatory genes and of morphogenic molecules, whereas typical mesenchymal markers
are repressed. Mesenchymal vimentin and N-CAM disappear, and epithelial E-cadherin, the a-chain of laminin,
and the a6b1-integrins that act as transmembrane receptors for laminin A (66, 65, 139, 255) appear, to mention
only some of them (Fig. 7). In an attempt to identify
additional surface molecules expressed in the transitional
process, monoclonal antibodies were raised against induced mesenchymal cells (83); their ability to inhibit tubule formation in culture and their expression patterns
should promote further search for the corresponding antigens involved in epitheliogenesis. In conclusion, the
conversion to the epithelial phenotype involves several
levels of cellular, protein, and genetic changes (121a).
Most of the putative interactions between these levels
have not yet been defined.
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VI. EPITHELIAL CELL POLARIZATION
A. Methods to Study Embryonic Renal Epithelia
1. The classic system to study tissue interactions
The embryonic kidney, when taken in organ culture
(mouse E11.5), continues with its tubulogenic developmental program (68, 86, 90, 92, 243, 280). When mesenchymal blastema and ureteric bud are separated before
the inductive interactions, the ureteric bud can be induced to branching morphogenesis only by the renal mesenchyme, whereas the mesenchyme can be induced not
only by the ureteric bud but also by other embryonic
tissues (see sect. II). The fact that the induced metanephrogenic mesenchyme continues its differentiation in vitro
to a certain stage of tubulogenesis provides an indispensable system to study early nephrogenesis because 1) each
embryonic stage can be identified by its characteristic
morphogenetic event, e.g., the comma shape (Fig. 13C), 2)
immunolocalization and in situ hybridization (60) can be
applied directly to the embryonic epithelia to resolve
temporospatial expression patterns (227) of defined molecules, and 3) the in vitro situation provides direct access
for mRNA analysis in single embryonic epithelial cells
(121).
2. Generating knock-out mutations with defective
renal phenotypes
Functions of genes and their successive expression
in the cascade of kidney development are being analyzed
using either transgenic mice or mutants generated by loss
of function/gain of function, i.e., by gene targeting (147).
TABLE
Gene
1. Gene mutations influencing nephrogenesis
Expression Pattern
WT-1
Uninduced mesenchyme
Wnt-4
Mesenchymal condensate
Pax-2
Induced mesenchyme,
Wolffian duct, ureteric
bud
Ureteric bud tip cells
c-ret
lim-1
Mesenchymal
condensates
ld
Mesenchyme, ureteric
bud
Renal Murine Phenotype
No ureteric bud outgrowth,
no induction of
mesenchyme
Arrest of nephrogenesis at
condensation stage, but
initial branching of
ureteric bud
Wolffian duct growth
blocked and
metanephros deleted
Branching morphogenesis
blocked
Absence of metanephros
and precursors and of
Wolffian duct
Ureteric bud outgrowth
blocked, no induction of
mesenchyme, renal
agenesis
Mouse mutants, occurring naturally or engineered genetically,
have contributed much to understanding the roles of developmental
genes. Wild-type expression pattern and phenotype after targeted mutation of a few developmental genes are listed. Abbreviations and references are in text.
Mutation or modification of a gene at its chromosomal
site (“knock-out gene”) can be achieved by gene targeting
in embryonic stem cells in culture; subsequently, these
genetically manipulated cells are reimplantanted into the
mouse, and the mutation can then be investigated in the
whole (embryonic) mouse. In this way, mutations with a
defective kidney phenotype have been created (Table 1),
and their renal phenotype can be compared with the
temporospatial expression pattern of the wild-type gene.
Another approach is to identify gene families and their
role in development by their homology of motifs with
Drosophila genes, since many of the Drosophila melanogaster genes known as developmental control genes are
conserved with evolution, e.g., in mice and humans. This
pertains to the sequence elements of paired box (Pax)
and homeobox (Hox) that are available to search for gene
family members in other species. Ultimately, however, to
understand the developmental (downstream) role of a
gene, it is mandatory to define its function in the whole
embryo under the conditions of constructed misregulation or of absent regulation of the gene under study. By
the loss-of-function (targeted disruption) strategy, WT-1
was shown to prepare the mesenchyme for the inductive
process (148), Pax-2 (272) to control multiple steps in
nephrogenesis downstream of WT-1, Wnt-4 (258) to participate in the transformation from aggregate to epithelial
tubule, Bmp-7 (55, 165) to be involved in stem cell conservation, c-ret (247) to regulate collecting duct branching
and growth, and BF-2 (97) to advance the differentiation
of aggregates.
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cess in which epithelial cells revert to mesenchymal cells,
a phenomenon also seen in carcinogenesis (16).
Syndecan-1 associates with the actin-containing cytoskeleton via its intracellular domain (14), and it is expressed highly in condensing (induced) mesenchymal
cells only, i.e., it appears to be involved in the ligandinduced clustering of the committed mesenchymal cells
(281). The proteoglycan is later reexpressed in bud-derived epithelia, while nephrogenic mesenchymal cells lose
the expression of syndecan-1 with differentiation. Tenascin-C, a mesenchymal ECM glycoprotein, is transiently
expressed close to the condensing (induced) stem cells
and later next to the early epithelia (mouse E13), suggesting that its expression is upregulated by cortical epithelial
signals, whereas in newborn kidney, tenascin C expression declines in the cortex and persists in medullary
mesenchymal cells (5). Epimorphin, a 150-kDa protein, is
expressed with syndecan (130) on the surface of condensing mesenchymal cells, but antibody interference with the
protein had no effect on morphogenesis.
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sense oligonucleotide techniques) with regulatory genes
and analysis of their downstream expression changes.
B. Ionic Conductances Are Expressed Before
Vectorial Transport
3. Cell culture of ureteric bud and of induced
metanephrogenic mesenchyme
A) MONOLAYER CELL CULTURE OF NEPHRIC DUCT-DERIVED EPITHELIA.
The introduction of techniques for primary culture of
single nephron segments (112) opened the in vitro access
not only to most cell types of the nephron (114), but
moreover to the apical plasma membrane (Fig. 8) for ion
channel evaluation (119). In addition, this culture system
(114) was modified to evaluate ion channel expression in
the particular cell population covering the ureteric bud
(Fig. 9) by applying the reverse transcription (RT)-PCR
(Fig. 10) to monolayers and to the single cell (Fig. 11).
These cells at the tip of the ureteric bud express specific
properties, such as ClC-2 mRNA consistent with the notion that this channel is widely expressed in embryonic
cells (see sect. VIC). In a newborn kidney culture system
(259), significantly, a novel antigen, termed PCDAmp1,
was found by immunohistochemical techniques on the
basal aspect of ampullar collecting duct only in embryonic but not in differentiated collecting duct cells derived
from cortical explants of newborn rabbit cortex (259).
This important finding may point to a role of the antigen
in nephrogenesis.
B) MICROCULTURE OF THE METANEPHROGENIC UNIT. The classic embryonic organ culture (90) was modified to culture
a single ureteric bud with the surrounding mesenchyme
(Fig. 12). Morphogenesis of the induced mesenchyme in
vitro continues for several days as shown, e.g., by scanning electron microscopy (unpublished data). This system
holds some promise for the study of cell lineage expression in postinductive stages of nephrogenesis (Fig. 3), for
analysis of membrane transporters (121a) in early nephrogenesis, and in particular for local interference (by anti-
FIG. 9. Scheme of single ureteric bud cell analysis. Ureteric duct
with ureteric buds is microdissected and transferred on collagen matrix
in culture dish. Patch-clamp recording is followed by mRNA harvesting
into patch pipette. mRNA encoding housekeeper and ion channels are
reverse transcribed and amplied by PCR, similar to protocol in Fig. 10.
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FIG. 8. Morphology of ureteric bud cells in culture. Scanning electron microscopy of apical cell side in monolayer (A) suggests that these
cells express a principal cell-like phenotype, as indicated by central
cilium (B).
Ureteric bud cells in culture were analyzed by electrophysiological (patch clamp) and by molecular biological techniques (RT-PCR) during embryonic and early
postnatal development (116 –121).
Embryonic ureteric bud cells (E17) express an apparently constitutively active, outwardly rectifying, whole
cell chloride conductance (120), whereas perinatal and
early postnatal ureteric bud/cortical collecting duct
(CCD) cells acquire the expression of a mature type,
hypotonic swelling-activated chloride conductance between E17 and postnatal day 1 (P1) before the onset of
vectorial transport, i.e., before the fusion of the nephric
duct-derived epithelia with the mesenchyme-derived
nephron (Fig. 13). During this period, the embryonic-type
chloride conductance, interestingly, is downregulated.
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creased by a factor of two in postnatal ureteric bud
(P1—P6) and of five in postnatal CCD (P7—P28), when
compared with the embryonic stage (Fig. 14). This pattern
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FIG. 10. Scheme of quantitative RT-PCR in analysis of embryonic ion
channel expression. Quantitative reverse transcriptase-PCR was applied
to monolayer cultures of ureteric bud cells (Fig. 8) at different stages of
embryonic and postembryonic cell differentiation (Fig. 14).
Vectorial transport systems in nephrogenesis should
be functional at the onset of glomerular filtration in any of
the newly formed nephron generations to prevent loss of
sodium, glucose, amino acids, and water into the urine.
The ontogenic acquisition of one of the transport systems,
therefore, was investigated by comparing the abundance
of mRNA encoding the a-subunit of the epithelial sodium
channel (a-ENaC) in embryonic and perinatal ureteric
bud and in postnatal CCD cultures (117). This expression
of a-ENaC mRNA was compared with the appearance of
low-conductance, sodium-selective channels in the apical
plasma membrane in these ureteric bud and CCD cells
(117). The a-ENaC mRNA could be quantitated in embryonic ureteric bud (E15—E17), and the abundance in-
FIG. 11. Analysis of single ureteric bud cell. Single cells were patchclamped, and cytoplasm for reverse transcriptase (RT)-PCR was harvested to measure ion channel conductance and mRNA expression. A:
light microscopic view of ureteric bud. Inset: entire ureteric tree with
mesenchyme; one bud had been dissected free of mesenchyme to provide access with patch pipette (arrow) to basal cell side. B: acrylamide
gel showing products of ClC-2 chloride channel (bottom) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeper gene (top),
amplified by single cell RT-PCR. Data are from 8 cytoplasmic samples
and 6 controls. C: patch-clamp characterization of single cells at tip (A)
of ureteric bud shows original whole cell traces and derived slowly
activating inward current resembling ClC-2 chloride channel characteristics. [Modified from Huber et al. (121).]
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EMBRYONIC RENAL EPITHELIA
1173
indicates that the principal change in a-ENaC mRNA expression occurs toward the end of morphogenesis. Expression of the functional channel protein, as evaluated
by single-channel recording in apical patches of ureteric
bud and CCD cells, showed the same single-channel characteristics in both stages, albeit at very different densities,
suggesting the same channel type in ureteric bud and
CCD. This was confirmed by the differing response of
whole cell currents to amiloride (117). In conclusion, both
a-ENaC and the functional channel protein are expressed
before glomerular filtrate for vectorial transport reaches
the apical cell membrane.
C. Membrane Transporters Acquire Their
Apicobasal Patterns
1. Ion channels and transporters
Ion channel expression in differentiating epithelia of
the ureteric bud was studied by RT-PCR (Fig. 11) in
cultured ureteric bud cell monolayers (Fig. 14) and by
patch-clamp techniques to measure specific conduc-
tances of whole cell and apical plasma membrane as well
as to identify apical ion channels (Fig. 13). Cortical collecting duct cells in culture express the mature principal
cell phenotype, whereas ureteric bud cells maintain the
embryonic phenotype, i.e., they conserve a nonpolar apicobasal distribution of ion conductances (119). Epithelial
cell ontogeny, from ureteric bud to CCD, is characterized
by a polar differentiation of plasma membrane ionic conductances. This implies the acquisition of amiloride-sensitive sodium channels, low-conductance potassium channels, and 9-pS nonselective cation channels in the apical
plasma membrane (119).
The expression patterns of chloride channel type
mRNA were evaluated during branching morphogenesis
in cultured ureteric bud and CCD epithelia (118). The
temporal pattern of ClC-2 expression suggested a specific
embryonic function of this channel in epithelia undergoing branching morphogenesis, since it is downregulated
after gestation, similar to its regulation in embryonic lung
(192). ClC-2 and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA have embryonic temporal
expression patterns that change in the opposite direction
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FIG. 12. Microculture of metanephrogenic unit. To study MET, a nephrogenic unit as defined by a single ureteric bud
with induced adherent mesenchyme is transferred in collagens. Schematic view illustrates in vitro MET and early
nephrogenesis (Fig. 3, A—F). These processes are documented by electron microscopy and by molecular analysis.
Noninduced mesenchymal cells enter apoptosis, and ureteric bud cells proliferate and migrate to form a monolayer.
[Data from Huber et al. (121a).]
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Volume 79
2. Water channels and glucose transporters
Expression of most known members of the aquaporin (AQP) family was evaluated between E16 and P28 in
FIG. 13. Epithelial cell differentiation in branching morphogenesis:
electrophysiological analysis of ion channel expression. Ureteric bud
cell functional differentiation (E17 to P14) is characterized by patterns
of conductance expression distinct for each channel type. Cell schemes
(bottom) summarize channel types predominantly expressed or acquired
in each of three stages of epithelial cell differentiation. CCD, cortical
collecting duct.
with cell differentiation. While ClC-2 is downregulated, nucleotide-sensitive chloride current (CFTR) is expressed
early, as demonstrated also in human fetal kidney (45), and
it is increasingly expressed during the period of ClC-2 downregulation (Fig. 15). Similar to CFTR, the mRNA of nucleotide-sensitive chloride current (ICln) and of the CFTR
truncated splice variant (TRN-CFTR) are increasingly expressed with embryonic cell differentiation (118, 116).
The developmental acquisition of vectorial transportinvolved apical and basolateral potassium channels of
members of the ATP-dependent Kir subfamily showed
distinctly different patterns for potassium channel types
(21a). ROMK1 and ROMK3 mRNA were not detected in
E15 to P6 developmental stages. ROMK2 mRNA was apparent in postnatal ureteric bud cells and increased at
FIG. 14. Epithelial cell differentiation in branching morphogenesis:
molecular analysis of ion channel expression. Ureteric bud cell functional differentiation (E15 to P14) is characterized by distinct patterns of
ion channel expression. Relative abundance of mRNA expression (bottom) in monolayer cultures (middle) derived from kidney at different
stages of ureteric bud morphogenesis (top) is represented by width of
lines. CFTR, cystic fibrosis transmembrane conductance regulator;
ENaC, epithelial sodium channel.
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P28. The basolateral channel KAB-2 mRNA, similarly, was
not expressed in embryonic cells. Importantly, however,
the expression of Kir6.1 and of SUR was high in embryonic cells and downregulated thereafter (21a). Significantly, later stages of ion channel development in collecting duct cells are characterized by an increase of sodium
and potassium channel density in the apical plasma membrane at constant mature expression pattern (238, 239).
Epithelial cell polarity in renal cells is acquired with cell
differentiation (119). This has been demonstrated not
only for apical membrane ion channel types (Fig. 13), but
also for the isoforms NHE-3 and NHE-1 of the sodium/
hydrogen antiporter in neonatal proximal tubule cells,
where membrane location of these transporters did not
show the mature distribution pattern (11), and for the
Na1-K1-ATPase that is expressed in embryonic ureteric
bud cells in basolateral and in apical plasma membrane
(180), and lastly for an unusual appearance of immunoreactive surface markers (142). In contrast, the temporospatial expression of the Na1-K1-Cl2 symporter (NKCC2)
in embryonic mouse kidney (E14.5) showed the terminal
mature localization in the distal loop of Henle already at
this stage (125).
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1175
VII. GROWTH FACTORS AND EXTRACELLULAR
MATRIX
A. Growth Factors Are Signaling Molecules
in Induction and Differentiation
rat kidney (303), and AQP1 and AQP2 were localized in
human fetal kidney (46). Both AQP1 and AQP2 were
expressed early in fetal kidneys, and AQP2, significantly,
was localized to the apical membrane of fetal CCD principal cells from the onset of expression. As for sodium
and potassium channels in later nephrogenesis (238, 239),
water transport in later (perinatal) stages of CCD ontogeny appears to increase by a density change of water
channel molecules in the plasma membrane (250).
Embryonic sodium-coupled glucose transport is
characterized not only by a lower density of transporter
molecules of the mature-type transporter in the apical
proximal tubule membrane (12). Moreover, detailed analysis of sodium glucose transporter (SGLT)1 and SGLT2
mRNA revealed that expression of both molecular isoforms begins on E18 and E17, respectively, and a change
in size of the SGLT2 mRNA suggested an embryonic splice
variant of the transporter (306).
In conclusion, cell polarization requires a series of
sequential expressions of basement membrane constituents and ECM molecules and the interactions of both with
polypeptide signaling factors (304). It is not clear 1) how
these processes direct epithelial cells to express a polar
distribution of membrane transport proteins and 2) which
developmental programs (54) direct the specific downstream patterns of membrane transporter mRNA expression.
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FIG. 15. Patterns of expression and repression of critical developmental genes. Genes that code for transcription factors and for signaling
molecules interact positively (expression) or negatively (repression) or
behave autoregulatory. Stages of metanephric morphogenesis (Fig. 3)
require profound changes in gene expression, for cell condensation and
adhesion, MET (Fig. 6), epithelial cell apicobasal polarization, nephron
segmental pattern formation, and acquisition of membrane transport
molecules (Fig. 13). Regulation of most expression events and downstream gene targets remains elusive. References to gene expression
patterns are in text. IGF, insulin-like growth factor.
Growth factors, in addition to their mitotic
(growth) action, not only mediate motogenic (migration) and morphogenic inductive signals, but also those
for cell differentiation (polarization), proliferation, and
apoptosis. The roles of the many growth factor families
and their receptors, each of which consists of a multigene family, in regulating developmental programs
through all of its stages appear to be overwhelmingly
complex. The small polypeptides, synthesized and secreted by embryonic renal epithelia, activate in a paracrine/autocrine mode their renal target cells by way of
basolateral plasma membrane recptors (84). They have
been implicated in kidney organogenesis in vitro primarily because of their expression patterns in mice
(93). The best-characterized growth factors pertinent to
renal organogenesis are IGF-I and IGF-II, HGF, TGF-b,
and FGF. Responses of an epithelial renal embryonic
cell can differ widely, from proliferation (123) to apoptosis to cell polarization as a consequence of the
differing intracellular signal pathways activated by the
growth factor ligand-receptor interactions. An additional problem has been posed by the observation that
metanephric uninduced mesenchyme when cultured in
defined medium selectively supplemented with the
growth factors TGF-a, EGF, TGF-b, IGF-I, IGF-II,
PDGF, FGF, or with retinoic acid failed to respond to
any of them with tubulogenesis (295). Interactions are
effective not only between individual soluble growth
factors but also between them and ECM components.
For example, when nephrogenic mesenchyme in culture was incubated with EGF (and pituitary extract) on
different matrices (laminin, fibronectin, collagen type I
or IV), the inductive process was initiated, but morphogenesis failed to be expressed (205). Although none of
the growth factors per se may act as an inducer of
nephrogenesis, the addition of antisense oligonucleotides or antibodies to individual growth factors in vitro
often have revealed inhibitory effects on organogenesis, suggesting that growth factors in nephrogenesis
may act synergistically. Growth factor families shown
to be involved in morphogenesis and differentiation of
the embryonic kidney are, among others, IGF-I/IGF-II,
HGF/SF, TGF-b, TGF-a/EGF, PDGF-A/-B, Bmp, NGF,
and neurotrophin-3. Some of these and their receptor
families (219 –222) have been localized to specific early
structures, and their functional roles are discussed.
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HORSTER, BRAUN, AND HUBER
B. Growth Factor Families Are Expressed
in Temporospatial Patterns
1. IGF-I and IGF-II
2. HGF/SF
Hepatocyte growth factor is produced in mesenchymal cells (209) close to the epithelial ureteric bud target
cells from day 11.5 onward (mouse), i.e., from the time of
induction to the stage of metanephrogenic condensation
(301). Met, its receptor (252), is located on the branching
ureteric cells at the very tip (Fig. 5) where the other
receptor tyrosine kinases, products of c-ros and c-ret, are
also inserted. Signal transduction of HGF to its ureteric
bud receptor may be paracrine in one of the mesenchymal
cell populations and juxracrine in another (301). It is clear
by now that HGF not only stimulates branching morphogenesis, as does TGF-a, but it also is a potent inducer
capable of morphogenic, motogenic (scatter), and mitogenic effects on nephrogenesis (16, 23, 151, 294). The
morphogenic activity of HGF, first described in a cell
culture assay system on a three-dimensional matrix (185),
may also pertain to the MET (234, 277), since Met is
coexpressed with HGF in at least a part of the mesenchy-
mal tubulogenic cell population, where it may act either in
the rescue from apoptosis or else have a part in the
conversion process. When HGF is added to metanephrogenic mesenchymal cells in vitro, epithelial differentiation
is stimulated (134), and anti-HGF serum in vitro inhibited
ureteric bud development as well as nephrogenesis (234),
consistent with the spatial receptor-ligand expression pattern of HGF. These apparently consistent observations,
however, were not confirmed when the HGF gene was
deleted by homologous recombination in mice (246, 278).
The cultured mutant kidneys and the in vivo kidneys did
not reveal any defect until E14, i.e., after tubulogenesis
had begun. In addition, mice with a c-met 2/2 mutation
do not reveal kidney defects (19). The scatter activity of
HGF/SF puts this factor in the context of epithelium-to
mesenchymal transition (EMT), since it dissociates cohesive epithelia that then may transform into spindle-shaped
(mesenchymal-like) cells with increased migration/motility activity (95), suggesting that the “growth factor” induces or mediates those processes that MET and EMT
have in common. Antibody perturbation of HGF in organ
culture affects mesenchyme-to-epithelium conversion, inhibits branching of the ureteric tree, and induces excessive mesenchymal apoptosis (234, 301); however, as mentioned, kidneys of HGF 2/2 mice appear to develop
normally (246, 278), suggesting redundancy of actions
between HGF and other systems such as retinoic acid and
formins might be involved (167, 178).
3. TGF-b
Transforming growth factor-b1, TGF-b2, and TGF-b3
mRNA have distinct expression patterns (31) consistent
with their differential functions in organogenesis (179).
Specifically, TGF-b1 mRNA is expressed in the nephrogenic mesenchyme, whereas later epithelial stages have
low levels of TGF-b2 mRNA and no TGF-b3 expression
(244). In branching morphogenesis of the ureteric bud/
collecting duct system (222, 230), the inhibitory effect of
TGF-b acts in a cross-talk with the activating role of HGF
and TGF-a. Indeed, the early expression pattern of
TGF-b1 and TGF-b3 in embryonic (204) and of TGF-b2 in
neonatal basement membranes (269) suggests that this
growth factor has a primary role in ECM assembly perhaps by differential regulation of collagen constituents.
4. TGF-a/EGF
Transforming growth factor-a is synthesized and secreted in the embryonic kidney (221), where it binds to
the EGF receptor (13), and it may also interact with
laminin (83). The TGF-a peptide and receptors for TGF-a
are present in the fetal human metanephros (85). Branching morphogenesis and subsequent tubulogenesis are inhibited by anti-TGF-a antibodies added to the cultured
embryonic organ (221), suggesting a morphogenetic role
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Early embryonic nephron epithelia express IGF-I,
IGF-II, and IGF-binding proteins (109, 159, 160, 172, 220,
291). The IGF-I receptors are present in nephrogenic mesenchyme and in the ureteric bud cell plasma membranes
(172) where immunoreactivity is particularly high. Antisense oligonucleotides against the IGF-I receptor of embryonic kidney, which mediate most of the actions of
IGF-I and IGF-II, have shown that this signaling system is
probably involved in branching morphogenesis of the collecting duct (291). Expression of IGF-II mRNA appears in
nephrogenic blastema, is upregulated in the condensation
stage (109), and decreases during further morphogenesis.
Both IGF-I and IGF-II produced by the embryonic kidney
act through extracellular IGF-binding proteins, and antiIGF-I or anti-IGF-II antibodies, or anti-IGF-II receptor antibodies, completely inhibit the in vitro development of
the embryonic kidney (220). Both IGF act differentially,
since IGF-II, as shown by in situ hybridization (109), is
localized in human fetal kidney to mesenchymal cells but
not to epithelial cells of the cortex. Moreover, when antisense oligodeoxynucleotides to IGF-I receptor cDNA
were added to mouse organ cultures, growth of induced
condensations was inhibited, and ureteric bud branching
was perturbed (162). Despite this clear evidence from in
vitro studies in which antibodies against IGF-I, IGF-II, and
the IGF-II receptor inhibited ureteric bud branching and
mesenchymal differentiation (220), gene deletion experiments of IGF-I and the IGF receptor indicate that these
genes are not essential for renal morphogenesis, but they
may be important for overall organ size (160).
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EMBRYONIC RENAL EPITHELIA
for TGF-a particularly in early ureteric bud development
(229). Epidermal growth factor/TGF-a also appears to
participate in the rescue of uninduced mesenchymal cells
from apoptosis (32), and in vitro studies have shown that
a mouse kidney cell line cultured on ECM forms aggregates and tubulelike structures when TGF-a (or EGF) is
supplemented (265). Although the role of TGF-a (EGF) in
mesenchymal differentiation remains largely undefined,
its defined impact on branching morphogenesis has assigned this growth factor an important task in kidney
organogenesis.
5. PDGF
6. Bmp
Of the many Bmp genes that are generally involved in
embryonic patterning and mesoderm organization, Bmp-7
is expressed in the kidney (18, 55, 98, 165, 166, 219).
Analysis of the homozygous mutant phenotype has indicated a requirement for Bmp-7 in nephrogenesis (135).
The mouse mutant (56, 165) is characterized by very small
kidneys, by polydactyly of hindlimbs or hind- and forelimbs, and by microphthalmie, indicating that Bmp-7 is
required also for limb and eye development. In the homozygous Bmp-7 mutants, initial branching of the ureteric bud and formation of some early epithelia of the
S-shape stage were observed, but further growth and
differentiation in both cell systems were terminated on
E11.5, followed by massive apoptosis (165). Although the
regulation of Bmp expression is not understood, Bmp
proteins are often expressed in embryonic cells close to
or identical with those expressing hedgehog (18); moreover, Bmp-7 and Bmp-2 RNA colocalize in some organs
(166), suggesting cooperation of both in cell signaling
related to proliferation and apoptosis.
7. NGF and neurotrophin-3
Nerve growth factor receptors have been identified
by in situ hybridiziation (237) in the aggregates close the
ureteric buds, and they are repressed in later stages.
When these metanephric organ cultures were incubated
with antisense NGF receptor oligonucleotide, ureteric
bud branching and tubulogenesis were inibited, consequent to the suppressed nerve growth factor receptor
expression. Although the low-affinity NGF receptor is
expressed in uninduced metanephric mesenchyme (61),
the high-affinity receptor is expressed in stromogenic cortical mesenchymal cells. This interesting, possibly important and nondiffusive signal system (132) awaits further
molecular analysis.
In conclusion, the role of growth factors in nephrogenesis is particularly difficult to evaluate. Embryonic
kidneys in organ culture synthesize and secrete a spectrum of growth factors; when these are added to the
uninduced metanephric mesenchyme, however, growth
and nephrogenesis do not occur, although some of them
increase DNA synthesis. Furthermore, mouse gene
knockout studies of growth factors and their receptors do
not sustain the roles of single growth factors implicated
from the in vitro expression studies. The other problem in
evaluating the role of growth factors arises from the fact
that addition of antisense DNA (or of an antibody) to only
one of the many growth factors produced in the embryonic kidney can entirely block tubulogenesis, although all
other factors are still effective.
C. ECM and Cells Interact in Epithelial
Morphogenesis
Most ECM molecules contain multiple binding domains that are recognized to interact with integrins (72),
their glycoproteins have signal transducing receptors
(240), and they mediate events such as cell adhesion (300)
in embryonic cells. While the composition of ECM depends on cell type and developmental stage (66), interactions between matrix molecules and growth factors are
effective through differing mechanisms. For one, binding
of a growth factor to ECM can alter its local activity (302).
Furthermore, growth factors can alter ECM protein and
receptor production importantly (161), and ECM molecules can modulate (69) the response of cells to growth
factors.
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Platelet-derived growth factor-A and -B are produced, among other cell types of the kidney, in developing
glomerular epithelial cells, in inner medullary collecting
duct cells, and in mesangial cells. The two receptors for
the isoforms of PDGF, the PDGFR-a (73) and PDGFR-b,
are expressed in a variety of renal cells, such as in nephrogenic mesenchyme, interstitial cells, and vascular cells
(2). Platelet-derived growth factor-B is expressed first in
the metanephrogenic mesenchyme, but its later expression is limited to mesangial cells. Capillary vessels of the
embryonic glomerulus are produced very early in the cleft
of the comma-shaped body, but their cell lineage has not
been resolved (218). Subsequent glomerular network organization depends on the expression of PDGF-B and
PDGFR-b, since mutant mice that have no mesangial cells
(158, 254) fail to form an appropriate capillary tuft. Gene
targeting of PDGF-B and of PDGFR-b has further disclosed their critical role for mesangial cells, since PDGF
knock-out embryos completely lack mesangial cells, but
not endothelial cells; consequently, a glomerular capillary
tuft is not formed, implying that the physiological function of mesangial cells (and of PDGF-B/R-b) is the folding
of the glomerular basement membrane and the formation
of capillary branching into loops. Both processes are
likely dependent on the mesangial production of a highly
specialized ECM (1).
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HORSTER, BRAUN, AND HUBER
1. ECM molecules have morphoregulatory functions
2. Laminins transfer ECM signals to the cell
The critical role of laminins is indicated by the fact
that antilaminin antibodies inhibit mesenchymal-epithelial conversion and cell polarization (41, 255). As soon as
the condensation stage has been reached (Fig. 3), laminin
B expression is upregulated, indicating the onset of polarization. WT-1 transcription increases (Fig. 15) and initiates the downregulation of PDGF (76), IGF-II (53), and
the IGF-I receptor (296), all of which are involved in
mesenchymal cell proliferation. Laminin and receptors
for laminin, such as dystroglycan and a6b1-ntegrin, link
ECM and its signals to the cytoskeleton. As the condensation stage proceeds toward polarization (Fig. 6), basement membrane polypeptides (59) such as the B chain of
laminin and collagen IV, the A chain of laminin and its
a6-integrin receptor, and uvomorulin (287) are sequentially expressed. Laminin-1 probably is the principal component during the polarization process. Although laminin-a1 is expressed in embryonic epithelia only,
laminin-b1 and -g1 are localized in both mesenchymal and
epithelial cells. When mesenchymal cells are induced,
laminin-b1 and -g1 mRNA increase immediately, but expression of laminin-a1 remains low until the mRNA expression is upregulated with epithelial cell polarization
(66). In the same stage, expression of E-cadherin and
a6-integrin increase (Fig. 7). Specifically, the E3 and E8
fragments of laminin-1 are importantly involved in the
polarization process. Antibodies against the E8 fragment
of laminin-1 (141), as well as monoclonal antibodies
against the integrin a6-subunit (255), perturb tubulogenesis.
Laminin-1 appears to act via two receptors, the a6b1integrin and dystroglycan (60). The a6-subunit is coexpressed with the laminin-a1 chain during mesenchyme-toepithelium conversion, implying that it may act as
receptor for laminin-1 (60). Importantly, polarization and
further tubulogenesis are blocked at the vesicle stage by
antibodies interfering with the binding of the A-chain
laminin to the a6-integrin (255). Dystroglycan mRNA is
highly expressed in the basal cell membrane of renal
embryonic epithelia, and it appears to be, in addition to
a6b1-integrin, a second important and independent cell
receptor to attach the polarizing embryonic epithelial cell
to the E3 fragment of laminin-1. With nephrogenesis, expression patterns of the many integrin subunits change
differently in different nephron segments (196). Moreover, epithelial maintenance in the mature nephron also
appears to be controlled by cell-matrix interactions as
shown by the fact that basement membrane prevents
apoptosis, suggesting a major role for mature cell-ECM
interactions (74). These studies on the role of dystroglycan (60) have opened a new road of research in epithelial
cell polarization.
3. Integrin expression repertoire changes
with nephrogenesis
The operational scheme common to all integrins
(190) is the interaction with an ECM-derived ligand at its
large extracellular domain (289), and transduction of the
generated signal through a short cytoplasmic domain to
components of the cytoskeleton and other intracellular
signaling systems, as is expected from a conventional
receptor. Synergistic actions between growth factor and
integrin signal pathways (129) may be particularly important in cell adhesion, i.e., in MET. Expression patterns of
integrins first change with induction (190). The uninduced
mesenchyme expresses the a1b1- and the a4b1-integrins.
The a1b1-integrin receptor for fibronectin, interestingly, is
coexpressed with cellular fibronectin in the uninduced
mesenchyme, and they are both lost after induction (143).
As soon as the ureteric bud, which then expresses a2- and
a6-immunoreactivity, invades the mesenchyme, the nowinduced mesenchyme expresses cell-cell contacts, loses
the expression of the a1- and a4b1-integrins, and organizes
into polarized epithelia that now express the a6b1-integrin
receptor for laminin in the cell basal membrane (143).
This expression pattern announces the onset of interactions between the polarizing cell and basement membrane components. With progressing tubulogenesis (Fig.
3), the a3b1-integrin appears expressed in the epithelia of
Bowman’s capsule and in podocytes, whereas a1b1-integrin is expressed in the mesangium and a2b1-intregrin in
endothelial cells. Thus the terminal pattern of integrin
subunit expression is established at the S-stage. In conclusion, these intricate developmental expression patterns of the integrin subunits might represent changing
interactions between basement membrane components
and integrins, and they might mirror events in the cell
polarization process that are poorly understood.
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With induction and the onset of MET, a profound
change to the epithelial phenotype occurs (Fig. 6). Collagen I and II as well as N-CAM expressions are lost,
whereas a multitude of epithelium-specific molecules begin to be expressed (Fig. 7), namely, the basement membrane proteoglycan syndecan-1, the laminin A chain, Ecadherin, a6b1-integrin, and type IV collagen (66).
Laminin B1 and B2 chains, but not the A chain, are
expressed in the precondensation mesenchyme; induced
mesenchymal cells during condensation synthesize high
amounts of A chain for the expression of functionally
active laminin. When laminin or a6b1-integrin functions
are disturbed during this transformation, cell differentiation to the polarized phenotype is inhibited (65, 141, 255).
Thus a large number of ECM components are expressed
by induced mesenchyme, and some of them are known to
be required for epithelial morphogenesis.
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EMBRYONIC RENAL EPITHELIA
VIII. GENES THAT CONTROL RENAL
ORGANOGENESIS
A. Transcriptional Regulation
1. WT-1
where it addresses also the growth factor-receptor pathway of IGF-I/IGF-II/IGFR-I. The finding that the IGF-II
gene is repressed by WT-1 (Fig. 15) denotes a target gene
that is critically important for normal growth and that,
when deregulated, could advance tumor growth. In fact,
IGF-II, the potent fetal mitogen, is overexpressed in most
Wilms tumors, which emphasizes the important physiological downregulation of IGF-II by WT-1 to suppress
mesenchymal proliferation (53, 168).
To summarize, WT-1 is a center transcription factor
in early nephrogenesis (216), where it appears to organize
much if not all of the intricate MET program. Finally,
WT-1 is a striking example of how an alteration in transcription factor function (182) can result in oncogenesis
(see sect. IX).
2. Paired-box genes
Pax-2 is one of the ancient renal genes, since it is
expressed in the pronephros and in the Wolffian duct.
Before renal organogenesis begins, i.e., before E11.5,
Pax-2 is expressed (50) in the intermediate mesoderm
from where the metanephros is derived. Within the
paired-box gene family, Pax-2 and Pax-8 are expressed in
overlapping domains (Fig. 5) in the embryonic mouse
kidney (49 –51, 210). Although Pax-2 proteins have properties of transcription factors such as sequence-specific
DNA-binding and, particularly, transcriptional activation
(51), downstream target genes of Pax-2 are not yet
known. None of these two paired-box genes is expressed
in the uninduced mesenchyme, which renders Pax-2 the
herald of successful inductive interaction. In fact, the very
first stage of cell polarization, i.e., the initial step toward
the epithelial phenotype during mesenchymal-to-epithelial conversion, appears to be a major area of control by
Pax-2 (223). This implies, of course, that the early expression requires induction by the ureteric bud. Also, it is
readily understandable that Pax-2 is not expressed in the
mesenchyme if induction has not occurred, as is the case
in Danforth’s short-tail mice with renal agenesis (207).
Pax-2 expression is downregulated at the beginning of the
S-stage. Persistent expression of Pax-2 in transgenic mice
(52) inhibits terminal differentiation of the nephron and
leads to abnormal glomerular and proximal tubular development, including absent foot processes and microcystic
tubular dilatation. Thus the failure to repress Pax-2 in
tubular and glomerular cells of transgenic mice illustrates
the importance of WT-1 in the downregulation of Pax-2
(Fig. 15) for terminal nephrogenesis. In conclusion, the
Pax-2 gene appears to be a pivotal mediator for signals
transferred from the ureteric bud; other regulating genes,
namely, Wnt-4 (258), Bmp-7 (55, 165), and WT-1 (148),
are fundamentally involved in the Pax-2 stage of nephrogenesis.
Distinctive differences between Pax-2 and Pax-8 ex-
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The Wilms tumor suppressor gene WT-1 is a highly
conserved (24, 136) transcription factor, essential for
nephrogenesis (148, 215), and member of the early growth
response family of the zinc-finger type (15, 26). Although
WT-1 is faintly expressed in uninduced stem cells (Fig.
15), its expression is upregulated after induction (4) in
condensing cells (212), and it continues to be expressed
through the vesicle-, comma-, and S-shaped stages (121a),
whereas high levels in the terminal nephron persist in
podocytes only (Fig. 5). From this location in time and
space, WT-1 is likely to control genes that code for developmental events (33, 168, 224, 225, 292), such as epithelial
cell polarization and expression of differentiated properties. The roles of WT-1 in nephrogenesis were established
from the analysis of WT-1 null mutants (148). In homozygous mice, the ureteric bud does not form and, consequently, the uninduced mesenchyme that is not capable to
enter transition undergoes programmed cell death resulting in renal agenesis, whereas mesonephric duct and
tubule development appear to be largely normal. Thus it
appears by now that one of the major roles of WT-1, apart
from the gene’s roles in other organs (4), is to mediate the
effects of the inductive signals during the MET, i.e., to
render the stem cells competent for induction. WT-1 not
only regulates the expression of several growth factors
and growth factor receptors, but it also targets Pax-2
(225) and itself, the WT-1 (224), and transcriptional repression of both genes is mediated through WT-1 binding
sites in the promoter regions. Therefore, regulation of the
temporospatial expression pattern of Pax-2 (50) can now
be linked to that of WT-1, since a rise of WT-1 protein in
the proximal S-shape concurs with a fall of Pax-2 expression in the same cells (225). Pax-2 expression starts in the
condensing mesenchyme, and Pax-8 expression follows
in the vesicle stage (Fig. 15). It is only when both pairedbox proteins are maximally expressed that WT-1 levels
rise and thereby repress Pax-2 levels (225). This pattern
(Fig. 15), taken together with the high abundance of
Pax-2 in Wilms tumors (51), and with the positive modulation of WT-1 expression through transactivation by
Pax-2 (43) and Pax-8 (44) proteins, indicates important
regulatory interactions (175) between these early expressed and critical genes. A further transcriptional regulation by WT-1 is the repression of bcl-2 and of c-myc
(28, 103), which emphasizes its role in controlling protooncogenes involved in apoptosis (bcl-2) and proliferation (c-myc) of uninduced mesenchyme. Thus WT-1 acts
as a critical regulatory protein in metanephric blastema
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HORSTER, BRAUN, AND HUBER
pression are apparent in nephrogenesis (63, 210). Although Pax-2 is expressed earlier than Pax-8 in the condensed murine mesenchyme, Pax-2 in the S-stage is
downregulated before Pax-8 (Fig. 15). Pax-8 is not expressed in the Wolffian duct and its descendent epithelia
(210), and potential target genes under the control of
Pax-8 have been identified in the thyroid, encoding thyroglobulin and thyroperoxidase, but none has yet been
found in the embryonic kidney. Importantly, Wnt-4 appears to be required for the sequential appearance of
Pax-2 and Pax-8, since mesenchymal cells of Wnt-4 2/2
mutants express Pax-2 after a regular induction, but they
do not proceed to the expression of Pax-8 (258).
BF-2 is first expressed in mesenchymal cells adjacent
to those induced cells that express Pax-2 (97). Thus
Pax-2 and BF-2 are nephrogenic and stromogenic stem
cell markers, respectively. In BF-2 mutants, stromal/interstitial cells persisted throughout embryonic cortical and
medullary morphogenesis (97), whereas normally, stromal cells make up most of the embryonic medulla only
and eventually disappear from there by apoptosis (228).
Some days after the early induction, stromal cells carrying
BF-2 are present in the medulla, and in a peripheral
cortical (stem) cell population (97) that is different from
the other (stem) cell population expressing Pax-2. In the
mesenchymal cell population, BF-2 2/2 kidneys formed
only few but large condensates that expressed Wnt-4 and
c-ret but did not progress to differentiation. Branching
morphogenesis was greatly reduced, and the pattern of
c-ret expression, normally limited to the very tips of the
ureteric buds, now extended throughout the entire cortical system and into the medullary segments of the collecting duct. The important aspect of this latter finding is
that BF-2 in wild-type kidneys is not expressed in the
ureteric bud or the early collecting tubule. The conclusions from these most revealing data are as follows: 1)
BF-2 expression follows the early bifurcating lineage into
nephrogenic (Pax-2) and stromal (BF-2) cells, and 2) the
action of BF-2 as a transcription factor is to regulate the
synthesis and secretion of signaling molecules that guide
morphogenesis and differentiation of both primordial tissues, the nephrogenic and the ductal. Clearly, the highly
organized embryonic expressions of BF-2 and its broad
range of developmental responsibilities will open new
perspectives in nephrogenesis.
4. Homeobox genes
The genes contain a homeobox, a 183-bp sequence
encoding a 61-amino acid sequence that has a DNA-binding motif for transcriptional regulation of sofar nondefined genes. Some of the homologs of Hox genes that exist
in Drosophila are expressed in the meso- and metane-
phros, such as Hoxd-3 (263). Although Hox genes are
believed to be involved primarily in regional patterning
during embryogenesis, their specific roles in kidney organogenesis remain to be discovered, since very few of the
targeted mutations so far have resulted in renal defects
(149), suggesting redundant gene functions in the cluster
or nested expression domains. Hoxb-9 can activate the
promoter for N-CAM (128), a molecule that is expressed,
as mentioned, during the early MET.
5. Hepatocyte nuclear factors
Hepatocyte nuclear factor (HNF)-1a and HNF1-b are
highly expressed in distinctly different temporospatial
patterns in nephrogenesis (153). Although HNF-1a is expressed first in the S-stage only, HNF-1b is already expressed in the mesonephros, in the ureteric bud, in mesenchymal condensations, and in all subsequent stages up
to the mature nephron, suggesting clearly different roles
for the two HNF. The functional role of other HNF families in nephrogenesis (57) is poorly defined, although a
gene homologous to HNF-3, the MFH-1, is highly expressed in uninduced mesenchymal cells (181).
6. The myc gene family
N-myc is generally expressed in epithelia that are
involved in inductive signaling (108), and it regulates, as
demonstrated by homologous recombination in embryonic stem cells, early (precursor) proliferation (29),
branching morphogenesis, and differentiation. The c-myc
transcripts are found in the uninduced mesenchymal cells
and in early differentiating stages, but, importantly, not in
mature epithelia (189), where renal tubular hyperplasia
and cysts occur if the gene is constitutively expressed in
transgenic mice (274). The N-myc protooncogene appears
to have an earlier frame of effects in the embryonic
kidney (188), since as revealed by gene targeting of N-myc
(256), the mesonephros in these transgenic mice had a
lower number and smaller size of tubules and mesenchymal hypoplasia. The wild-type gene is expressed in a
restricted pattern contrasting that of c-myc, since N-myc
begins to be expressed during MET in the condensation
stage, i.e., before Wnt-4 expression (258), and it persists
through the S-stage (189); the mutant embryos die at
E11.5 (256), indicating that not only renal but also generalized effects result from the N-myc loss of function (241).
B. Signaling by Receptor Tyrosine Kinases
Many protooncogenes are assumed or established to
encode receptor proteins that participate in processes
that are activated by growth factor signaling molecules,
and they are expressed during embryonic organogenesis
in distinct temporospatial patterns (248).
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3. BF-2 and the stromal cell lineage
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EMBRYONIC RENAL EPITHELIA
1. c-met
2. c-ret
The c-ret protooncogene encodes Ret, which is developmentally expressed in ureteric bud tip cells (Fig. 5)
but not in the mesenchyme (200). The tip cell expresses a
particular phenotype (121), and it is involved in multiple
signaling loops, since it expresses also the protooncogene
encoded receptor tyrosine kinases c-met and c-ros. The
phenotype of homozygous c-ret mutant mice (247) and
the embryonic expression pattern of c-ret (200) both indicate basic roles for the protooncogene in kidney (126)
and in the intestinal nervous system. In c-ret 2/2 mice,
the ureteric bud does not branch because c-ret directed
signals from the mesenchyme cannot be received; however, mesenchymal cells enter the first stage of nephrogenesis which suggests that, in c-ret 2/2, some early
ureteric bud signaling has occurred.
The kidneys were either dysplastic rudiments characterized by reduced or absent ureteric branching and
extended regions of uninduced mesenchmye, or they
were absent (248). The primary defect of c-ret 2/2 is
intrinsic to ureteric bud, since the metanephrogenic mesenchyme from these mutant embryos developed into tubules when cocultured with embryonic spinal cord (247).
The Ret receptor and its ligand GNDF (58, 276), which is
probably mesenchyme derived, constitute the second system known to signal between the two primordial tissues,
and both share the same signaling pathway (58, 186, 200,
247, 273, 285). Remarkably, GDNF does not bind directly
to the Ret receptor (233), but it binds first to its GDNFR-a,
and this complex interacts with Ret (273). This signaling
pathway induces branching morphogenesis of the ureteric bud, in concert with the HGF-Met signal system. In
conclusion, c-ret is another example of the dual role of
protooncogenes in embryonic development and in cancer.
Although the role of c-ret in embryonic signaling is fairly
obvious from both wild-type localization (200) and renal
and intestinal defects in c-ret recombinant mice (247), the
neoplastic lesions in naturally occurring human mutations are less understood. These are associated with the
familial medullary thyroid carcinoma and the multiple
endocrine neoplasia type 2A (273), and the mutations
appear to alter the extracellular domain of Ret such that
a constitutive increase of the receptor kinase activity may
be considered.
3. c-ros
The murine gene c-ros (267), the homolog of Drosophila sevenless (30, 146), is development-dependently
expressed in intestine, lung, and kidney. The c-ros transcripts Ros were the first protooncogene-encoded receptor tyrosine kinases demonstrated to express a temporospatial pattern in which they are highly expressed in
ureteric bud tip cells in the outer cortex of embryonic
kidney (251) and decline in the neonatal kidney (131). The
ligand for Ros, in contrast to those for Ret and Met, is not
yet known (Fig. 2). Protooncogenes and ECM proteins are
functionally linked. Both c-ros and c-ret antisense oligonucleotides in the culture medium (163) inhibit c-ret expression more than c-ros and, remarkably, the expression
of ECM proteoglycans in the interspace between mesenchymal and ureteric bud cells with resulting dysmorphogenesis (131). In conclusion, ECM glycoproteins modulate morphogenesis, and c-ros transcripts colocalize with
these modulators that may imply that ECM-mediated signaling is part of the early inductive process. Finally, the
signal for activation of Ros could involve direct cell-cell
interactions across the interspace, between the ureteric
bud receptor and the putative mesenchyme-derived ligand molecule (131, 163).
IX. GENETIC ERRORS IN NEPHROGENESIS
Inherited diseases, notwithstanding their deleterious
effects in the patient, provide insight in developmental
events. If renal malformations can be linked to mutations
of specific genes, the role of the latter in normal embryogenesis of an organ can be deduced. In many examples,
these errors in kidney development appear to be caused
by erroneous transcriptional regulation. A few are discussed in their phenotypical context.
A. Polycystic Kidney Disease
Polycystic kidney disease (PKD) is characterized by
intrarenal cysts with an inside single cell layer of epithelial cells. The two major human genetic forms of PKD are
the autosomal dominant polycystic kidney disease (AD-
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The c-met protooncogene product Met is the receptor for HGF, and it is expressed (Fig. 5) in ureteric bud
cells and in early postinduced mesenchyme (3, 20, 252,
294, 301). The mesenchymal expression of c-met transcripts begins in the condensates close to the ureteric bud
tip cells but not in the uninduced mesenchymal cells
(206). The role of this protooncogene and its encoded
receptor tyrosine kinase is unique in that it not only
participates in branching morphogenesis but also in the
induction of epithelial motility (5, 226). Notably, the Met
receptor tyrosine kinase is capable of transducing signals
for the diverse pathways and biologic functions of HGF/
SF, such as proliferation (mitogen), motility (“scatter”),
and branching growth (morphogen); transduction may be
modified by the differential binding characteristics of intracellular signaling molecules to the phosphotyrosine
residues of the activated cytoplasmic receptor domain
(294).
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B. Wilms Tumor
Wilms tumor is the result of faulty MET. It shows as
an embryonic cancer (nephroblastoma) that originates in
early nephrogenesis from mesenchymal stem cells undergoing excessive proliferation, while their normal fate is to
either differentiate into epithelial nephron cells or to enter apoptosis (202, 305). Wilms tumors frequently express
Pax-2 in their epithelial components (62) and contain
cells reminiscent of epithelia, stroma, and blastema. The
genetic errors leading to nephroblastoma are understood
only incompletely (25). The WT-1 protein binds to multiple sites in the fetal promoter of the IGF-II gene and
represses IGF-II transcription (53). In this way, WT-1 can
inhibit IGF-II synthesis, and it could be a negative regulator of renal embryonic mesenchymal growth. Furthermore, the PDGF-A promoter is a target structure for
transcriptional repression by a splice variant of WT-1
(76). Both data suggest that WT-1 acts through the downregulation of growth factor production; WT-1 expression
is upregulated at the onset of condensation (Fig. 15), and
there it suppresses the transcription specifically of IGF-II
and of PDGF-A interrupting normal proliferation of the
mesenchymal cell population. Mutant loss (in some 15%
of all patients with Wilms tumors and in patients with
Denys-Drash syndrome) of this suppressing activity of
WT-1 might be the decisive factor for the massive and
erroneous blastemal growth. In conclusion, WT-1 is a
striking example of the general notion that genes central
to pattern formation in embryonic organogenesis or for
cell lineage differentiation are important also in carcinogenesis. A type of WT-1 mutation is associated with the
Denys-Dash syndrome, a rare congenital disease with renal failure, urogenital dysgenesis, and Wilms tumor. WT-1
mutations in Denys-Drash syndrome patients appear to
have a single nucleotide exchange only (202), which may
have altered the binding characteristics.
C. Renal Cell Carcinoma
Renal cell carcinoma is a frequent adult malignancy
that is assumed to be generated from proximal tubule
epithelium, and a high percentage of primary tumors and
renal cell carcinoma cell lines (73%) expressed Pax-2
(81). Significantly, Pax-2 protein synthesis in the cell lines
could be inhibited by the addition of antisense oligonucleotides directed against Pax-2 mRNA, which resulted in
growth inhibition of the cancer cells, whereas cancer cell
lines that did not express Pax-2 did not change growth
when the same antisense oligonucleotides were used in
culture. These significant data suggest that the reexpression of Pax-2 in mature epithelium may be a predisposition for oncogenesis. In conclusion, developmental control genes regulating cell proliferation and differentiation
in the embryo can be reexpressed in mature cells and in
this unknown way contribute to the initiation and expansion of tumors.
Two human syndromes may be related phenotypically to the human homologs of Pax genes. Waardenburg
syndrome is a mutation of the human homolog of Pax-3,
which is a heritable autosomal dominant combination of
lateral displacement of the inner canthii of the eye, pigment alterations, occasional (one-third) deafness, and
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PKD) and the autosomal recessive polycystic kidney disease (ARPKD). The cyst formation of ADPKD begins in
the embryonic kidney and is maintained by fluid secretion
into the cyst lumen (22, 87), which is mediated by CFTR
(36). The cyst expansion process most likely is slow, since
the disease is detected only in the third decade of life. In
contrast, the recessive or juvenile form of PKD begins
with a rapid embryonic cyst expansion, first in the proximal and next in all of the collecting tubules, leading to an
early failure of the kidney (299) during the first year of
life. In both forms, cell-matrix interactions are altered
(35). The increased expression of the a1-subunit in collecting duct cells and an irregular distribution of a2-, a3-,
and a6-subunits may contribute to the phenotype of the
cystic disease.
Autosomal dominant PKD is caused by the PKD1
gene that encodes the PKD1 460-kDa protein, the polycystin (79, 122, 268, 293), which is a transmembrane protein
(124) mediating interactions between ECM and the cell.
Importantly, a second gene (PKD2) encodes a protein
(PKD2 110 kDa) that possibly interacts with polycystin
(184), and both may be part of a cyst-producing pathway.
Of particular interest is the early embryonic expression of
polycystin (293), which was not seen in uninduced metanephric mesenchyme and weakly expressed in the ureteric bud, in the comma- and S-shaped stages, and in fetal
podocytes, whereas expression in cyst cells appeared to
be prominent. Although polycystin is unequivocally expressed in embryonic renal epithelia (78, 88, 284), its role
in nephrogenesis is not defined. It may be suggested,
assuming normal copies of polycystin to act as ECM
receptors, that this protein could participate in the epithelial polarization process following MET. Several basic
features distinguish ADPKD from ARPKD. In the latter,
the protooncogene c-myc is continued to be expressed,
together with SGP-2, and a link to the cause of ARPKD
may be provided by the fact that transgenic mice that
express c-myc constitutively develop cysts much like
those in ARPKD (274). Another phenotype of PKC results
from a targeted disruption of bcl-2, where the homozygous mutants (194, 286) perinatally showed cystic dilatations in proximal tubule segments and, in addition, cysts
in postnatal collecting duct (187); the gene generating this
cystic phenotype remains to be defined.
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EMBRYONIC RENAL EPITHELIA
mental retardation (264). The AN (aniridia) gene, the
human homolog of Pax-6, is completely or partially deleted in patients with aniridia, a complete or partial absence of the iris and disturbed development of lens, cornea, and retina (104, 271). Aniridia is also expressed in the
WAGR syndrome, which is characterized by several abnormalities such as Wilms tumor (W), aniridia (A), genitourinary tract defects (G), and mental retardation (R)
(203). Lastly, a syndrome that alters early ureteric bud
and mesenchymal development in a dominant mouse mutant, the Danforth’s short tail (Sd), has not been characterized genetically (80).
Nephrogenesis today, through the convergence of
Drosophila, mouse, and human genetics, represents a
model system by which to study the basic regulation of
ductal morphogenesis, inductive mesenchyme-epithelium
tissue interaction, tissue remodeling, cell polarization, expression of epithelial cell transport properties, and the
developmental programs behind. The route presently followed by many groups is to probe the temporospatial
expression pattern of a particular gene and to relate it not
only to a morphogenetic stage but also to simultaneously
expressed patterns of other genes. In this way, patterns of
expression on a time scale, corresponding to a morphogenic scale (Fig. 15), may suggest routes for future experiments. Furthermore, interference with transcriptional or
posttranscriptional expression of single genes has contributed to establish its causal relation to a phenotype.
Nevertheless, although some “upstream” regulations of
gene expression or repression are known, there is little
information about most of the “downstream” gene target
molecules. Although many of the impressive crowd of
registered kidney genes (38) are likely to be involved in
organogenesis, it has been most revealing to work with
only a few of them to establish some activation and
repression patterns as morphogenesis and differentiation
proceed from one stage to the next (Fig. 3). Thus one
important area of progress that emerged in the past decade is the identification of some of the genes that initiate
and direct the stages of nephrogenesis. In this context, the
critical question of signaling molecules, their receptors,
and their signal pathways has only just begun to be investigated, and the discovery of the ligand for the c-rosencoded receptor tyrosine kinase might be one of the next
landmarks in this field. Among the currently most interesting aspects are provided by work on biochemical interactions among gene products, as has been shown impressively for the regulatory circuit between Pax-2 and
WT-1. Although these studies will undoubtedly lead to an
understanding of causal relations in morphogenesis, cell
polarization, and differentiation, they will ultimately serve
to answer the question of how signaling molecules, receptors, and transcription factors contribute to those renal
diseases that are believed to express an aberrant or disrupted developmental program.
Many colleagues in this laboratory have contributed over
the years to the fast-moving field of nephrogenesis, and we
apologize to those authors who could not be quoted.
M. F. Horster thanks Professor Emeritus Klaus Thurau for
his hospitality at the Munich Physiological Institute.
The continuous support from the Deutsche Forschungsgemeinschaft is acknowledged gratefully. This review was written
during the grant period DFG Ho 485/16 –1/16 –2.
Address for reprint requests and other correspondence:
M. F. Horster, Physiologisches Institut, Universität München,
Pettenkoferstrasse 12, D-80336 München, Germany (E-mail:
[email protected]).
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