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RESEARCH ARTICLE
Generation of the podocyte and tubular components of an
amniote kidney: timing of specification and a role for Wnt
signaling
ABSTRACT
Kidneys remove unwanted substances from the body and regulate
the internal body environment. These functions are carried out by
specialized cells (podocytes) that act as a filtration barrier between
the internal milieu and the outside world, and by a series of tubules
and ducts that process the filtrate and convey it to the outside. In the
kidneys of amniote vertebrates, the filtration (podocyte) and tubular
functions are tightly integrated into functional units called nephrons.
The specification of the podocyte and tubular components of amniote
nephrons is currently not well understood. The present study
investigates podocyte and tubule differentiation in the avian
mesonephric kidney, and presents several findings that refine our
understanding of the initial events of nephron formation. First, well
before the first morphological or molecular signs of nephron
formation, mesonephric mesenchyme can be separated on the basis
of morphology and the expression of the transcription factor Pod1 into
dorsal and ventral components, which can independently differentiate
in culture along tubule and podocyte pathways, respectively. Second,
canonical Wnt signals, which are found in the nephric duct adjacent
to the dorsal mesonephric mesenchyme and later in portions of the
differentiating nephron, strongly inhibit podocyte but not tubule
differentiation, suggesting that Wnt signaling plays an important role
in the segmentation of the mesonephric mesenchyme into tubular
and glomerular segments. The results are discussed in terms of their
broader implications for models of nephron segmentation.
KEY WORDS: Wnt signaling, Chick embryo, Kidney, Mesonephros,
Podocyte, Renal vesicle
INTRODUCTION
Vertebrate kidneys are composed of functional units called nephrons
(Saxen, 1987; Dressler, 2006). Each nephron has several distinct
functional and morphological compartments. At the proximal end of
each nephron is a glomerulus, which filters the blood. The
glomerulus contains vascular capillaries and specialized epithelial
cells, the podocytes. The combination of fenestrated slits between
the capillary endothelial cells, the basement membrane between
endothelial cells and podocytes, and specialized foot processes of
the podocytes together form the glomerular filtration barrier. Small
molecules can pass through the barrier into the interior of the
nephron, whereas larger molecules and cells remain in the vascular
1
Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine,
Technion-Israel Institute of Technology, Haifa 31096, Israel. 2Department of
Physiology and Biophysics, Weill Medical College of Cornell University, New York,
NY 10065, USA.
*Author for correspondence ([email protected])
Received 1 April 2013; Accepted 27 August 2013
system. After passing the glomerular filtration barrier, the filtrate
enters the tubule, where it is processed by absorption and secretion
carried out by tubular epithelial cells. Finally, the processed filtrate,
now called urine, enters the collecting system, a system of ducts that
drains the urine to the outside.
Because of the crucial role that kidneys play in regulating the
internal body environment, it is not surprising that the structure of
kidney tissues varies greatly between different species, as well as at
different stages of the life cycle of the same organism (Fraser, 1950).
Classically, three types of vertebrate kidneys are discussed: the
pronephros, which serves as the embryonic kidney in anamniotes
and is a transient, largely non-functional structure in amniotes; the
mesonephros, which is the adult kidney is anamniotes and the
functional embryonic/fetal kidney in amniotes; and the metanephros,
which is the functional amniote adult kidney and which is not
formed in anamniotes. Each of these kidneys comprises collections
of nephrons, which can vary in number from a single nephron in the
zebrafish pronephros to ~1 million nephrons in the human
metanephros (Vize et al., 2003b).
Understanding how nephrons form is crucial for developing
approaches to repair or regenerate damaged kidney tissue. The most
common model system for studying vertebrate nephron formation
has been the rodent metanephros. Metanephric nephrons form as the
consequence of mutual inductive interactions between the
metanephric mesenchyme and an epithelial tube called the ureteric
bud (UB) (Yu et al., 2004; Dressler, 2009; Costantini and Kopan,
2010). According to current models, as a result of initiating signals
from the UB, the mesenchyme undergoes a sequence of
morphological changes that leads to nephron formation. First, the
mesenchyme undergoes localized condensation to form pretubular
aggregates (PTAs), which then undergo a mesenchymal-to-epithelial
transition (MET) to form renal vesicles (RVs). The RVs
subsequently undergo elongation and differentiation, with the
proximal end interacting with local endothelial cells to form the
glomerulus, the middle part forming the tubule, and the distal end
connecting to the branching UB, which will form the collecting
system.
Knowledge of the signaling pathways that regulate specification
of nephrogenic mesenchyme into glomerular and tubular fates is still
incomplete. Wnt signaling is required for nephron formation and is
thought to provide an initial inductive signal for PTA and RV
formation (Herzlinger et al., 1994; Stark et al., 1994; Carroll et al.,
2005; Park et al., 2007), and one report has linked Wnt4 expression
to induction of glomerular fates (Naylor and Jones, 2009). Notch
signaling has been found to be required for generation of glomerular
and proximal tubular nephron components in several species
(McLaughlin et al., 2000; McCright et al., 2001; Cheng et al., 2003;
Taelman et al., 2006; Cheng et al., 2007; Wingert et al., 2007;
Naylor and Jones, 2009), but the stage(s) at which Notch signaling
4565
Development
Mor Grinstein1, Ronit Yelin1, Doris Herzlinger2 and Thomas M. Schultheiss1,*
acts during this process is not yet clear. Retinoic acid has also been
found to promote glomerulus formation at the expense of tubules in
zebrafish embryos (Wingert et al., 2007).
In most current models of metanephric nephron formation,
podocytes as well as tubular epithelium are thought to be derived
from a uniform pool of undifferentiated cells that constitutes the
PTA and that is subsequently patterned to form the different nephron
segments (Costantini and Kopan, 2010). However, genetic fatemapping studies in the mouse metanephros (Boyle et al., 2008;
Kobayashi et al., 2008) have not yet yielded a clear picture of when
the podocyte and tubular lineages diverge. Interestingly, there are
numerous examples in both vertebrates and invertebrates of kidney
tissues in which the glomerular and the tubular functions are not
tightly integrated (Ruppert, 1994). In the Xenopus pronephros (Vize
et al., 1997), the kidneys of oligochaete worms (Ruppert, 1994), and
insect nephrocytes (Weavers et al., 2009), filtration of blood or body
fluids takes place in one compartment, and the processing of the
filtrate and its excretion to the outside takes place in a separate
compartment. The two compartments are typically separated by the
internal body cavity or coelom (Ruppert, 1994). This raises the
question of whether distinct developmental pathways of the
glomerular and tubular components of the nephron are a general
property of nephrons, a feature that may have become obscured in
the highly compact and integrated metanephric nephron.
The development of the avian mesonephros may be able to shed
light on the specification of filtration and tubular components of
integrated nephrons. Avian mesonephric nephrons are similar in
morphology to their metanephric counterparts and pass through
similar stages in their development, including PTA and RV stages
(Hamilton, 1952) (see also Results, below). However, the avian
mesonephros is much simpler in structure than the metanephros,
being a linear organ with only a few nephrons per body segment,
and thus easier to manipulate and analyze. The current study
investigated the initial stages of podocyte and tubule specification
in the avian mesonephros. It was found that, well before the
appearance of PTAs or RVs, tubule and podocytes precursors can be
separated from each other and can differentiate independently.
Evidence is also presented that canonical Wnt signaling plays a role
in distinguishing between these two alternative mesonephric cell
fates by repressing differentiation of podocytes.
RESULTS
Initial stages of nephron formation are similar in the avian
mesonephros and mammalian metanephros
Qualitative analysis of early avian mesonephros development was
performed in order to evaluate how closely nephron formation in the
mesonephros resembles that in the more widely described
mammalian mesonephros. The chick mesonephros develops in the
intermediate mesoderm (IM) at axial levels between somites 15 and
30 (Hamilton, 1952). At the onset of mesonephric nephron
formation, the future mesonephros consists of the nephric duct
(which constitutes the future drainage system of the mesonephros)
and the nephrogenic cord, a strip of mesoderm that lies ventral to
the nephric duct and dorsal to the aorta (Fig. 1F). As mesonephros
differentiation proceeds, the region undergoes a dorsal-to-lateral
rotation, so that eventually the nephric duct lies lateral to the
differentiating mesonephric nephrons (Fig. 1I).
Because chick development proceeds from anterior to posterior,
examination of a single embryo during the period of mesonephros
formation allows one to observe nephrons at various stages of
maturation, with the less mature nephrons located in more posterior
segments and more mature nephrons being found more anteriorly.
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Development (2013) doi:10.1242/dev.097063
Fig. 1. Development of the chick mesonephros. (A-C) Plastic sections
from a single stage 18 embryo, from posterior (A) to anterior (C) axial levels,
illustrating stages in the differentiation of mesonephric nephrons, including
pretubular aggregate (ag), renal vesicle (rv) and S-shaped body (sb). (D,E) In
situ hybridization at stage 16 for Pax2 (D) and Wnt4 (E), showing Pax2
expression in nephric duct and nephrogenic cord (D) and Wnt4 expression in
the condensing pretubular aggregate (E). (G,H) In situ hybridization at stage
25 for Lhx1 (G) and Nphs2 (H) showing Lhx1 expression in the nephric duct
and tubules (G) and Nphs2 expression in the podocytes of the glomerulus
(H). (F,I) Diagrams of sections through the mesonephric regions of stage 15
and 18 embryos for orientation purposes. Note that in F, the undifferentiated
nephrogenic cord is located between the nephric duct and the aorta. In I, the
embryo has begun to fold, resulting in the nephric duct being located laterally,
but the relationships between the nephric duct, the IM and the aorta are
preserved. Within the differentiating IM in I, the tubules are located laterally,
closer to the duct, and the glomeruli are located medially, closer to the aorta.
ag, pretubular aggregate; ao, aorta; c, coelom; g, glomerulus; nc,
nephrogenic cord; nd, nephric duct; no, notochord; nt, neural tube; rv, renal
vesicle; s, somite; sb, s-shaped body; t, tubule; vcp, posterior cardinal vein.
Figure 1A-C shows plastic sections through the region of the
mesonephros at axial levels from posterior to anterior in a single
Hamburger-Hamilton (HH) (Hamburger and Hamilton, 1951) stage 18
embryo, and illustrates the morphological changes that occur during
nephron formation. Initially, mesenchyme adjacent to the nephric duct
begins to condense to form PTAs (Fig. 1A, ag) and subsequently
undergoes epithelialization to form RVs (Fig. 1B, rv). In more anterior
sections, ‘S-shaped bodies’ can be found, in which an emerging tubule
can be seen with a distal end connecting to the nephric duct and a
proximal end beginning to show signs of glomerular differentiation
(Fig. 1C, sb). These stages are morphologically similar to the stages
that have been well described during nephron formation in the mouse
and rat metanephros (Iino et al., 2001).
We also examined a set of molecular markers that have been shown
to play key roles during mouse metanephric nephron formation. As in
the mouse metanephros (Dressler et al., 1990), Pax2 is expressed in
both the nephric duct and in the undifferentiated mesonephric
mesenchyme (Fig. 1D) and Wnt4 (Stark et al., 1994) is expressed in
newly condensing mesonephric pretubular aggregates (Fig. 1E).
Development
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2013) doi:10.1242/dev.097063
Shortly thereafter, Lhx1 (Lim1) (Fujii et al., 1994; Shawlot and
Behringer, 1995) begins to be expressed as the aggregates undergo
MET to form renal vesicles (data not shown). At later stages, markers
of specific nephron segments can be found, including Lhx1 in the
nephric duct and tubules (Fig. 1G) and Nphs2 (podocin) (Boute et al.,
2000), a component of the slit diaphragm that is expressed exclusively
in podocytes (Fig. 1H).
Thus, with respect to expression of markers, as well as
morphological appearance, nephron formation in the avian
mesonephros appears to be very similar to that of the mouse
metanephros and can therefore potentially serve as a useful model
for studying formation of the type of integrated nephron seen in the
mammalian metanephros.
Distinct dorsal and ventral zones within the mesonephric IM
Close inspection of plastic sections through the mesonephric IM
before the onset of PTA formation revealed that the IM at this stage
is not homogeneous, but appears to consist of two morphologically
distinct zones: a dorsal zone adjacent to the nephric duct, which
typically appears to be more compact, and a looser ventral zone
adjacent to the dorsal aorta (Fig. 2A,A′). The dorsal and ventral IM
regions appear to be continuations of the somatopleuric and
sphlanchnopleuric layers of the lateral plate, respectively, with the
difference that the dorsal and ventral layers of the IM are not
separated by a coelomic space as they are in the lateral plate.
In a survey of genes known to be expressed in the developing
mammalian kidney, the transcription factor gene Pod1 (also called
Tcf21 and capsulin) (Hidai et al., 1998; Lu et al., 1998; Quaggin et
al., 1999) was found to be expressed in the mesonephric region
(Fig. 2C), and in sections was found to be expressed in the ventral
IM and the adjacent splanchnic (ventral) portion of the lateral plate,
but not in the dorsal IM or the somatopleuric lateral plate (Fig. 2B).
Upon appearance of differentiated nephrons, Pod1 was expressed
specifically in glomerular podocytes (Fig. 2D).
Dorsal and ventral IM zones generate distinct segments of
the nephron in culture
The existence of two distinct zones in the early mesonephric IM,
and the observation that the ventral IM zone specifically expresses
a marker (Pod1) that is later expressed in podocytes but not tubular
parts of the nephron, raised the question of whether the ventral and
dorsal zones of the IM might give rise to separate parts of the
nephron. In order to begin to address this question, we developed an
in vitro system for studying mesonephric nephron differentiation.
Slices ~150 microns in thickness (~1.5 somites in thickness) were
cut through the undifferentiated mesonephric region at the axial
level of the most newly formed somite and cultured on a filter at the
air-medium interface. A micrograph of a representative slice at the
beginning of the culture period in shown in Fig. 3A. Importantly, the
mesonephric mesenchyme at the start of the culture period does not
express Wnt4, the earliest known molecular marker of PTA initiation
(Kispert et al., 1998; Carroll et al., 2005). The slices were taken
from the axial level of the most newly formed somite, where the
nephrogenic cord is Wnt4 negative (Fig. 2E). Wnt4 expression is
initiated in vivo approximately 6 hours later, after four more somites
have formed (Fig. 2F). The nephrogenic cord at the axial level at
which the slices were taken is also negative for the tubular marker
Lhx1, which is expressed at this stage only in the nephric duct
(Fig. 2G), and is also negative for the podocyte marker Nphs2,
which is not expressed in the embryo at all at this stage (data not
shown).
When whole slices were cultured for two days, robust nephron
differentiation occurred. By bright-field microscopy, multiple
tubular structures were detected (Fig. 3B). By immunofluorescence,
abundant differentiation was seen of Lhx1-expressing nephric duct
and tubule cells and Nphs2-expressing podocytes (Fig. 3C; 100% of
40 cultures expressed both Lhx1 and Nphs2). The podocytes were
typically arranged in clusters resembling glomeruli and located at
the periphery of the explants, immediately adjacent to the Lhx1expressing tubules, suggesting that integrated nephrons had formed
in culture.
In order to determine the developmental potential of the dorsal and
ventral zones of the IM, the embryo slices were dissected into ventral
and dorsal halves (Fig. 3A) and cultured separately. Dissection was
aided by the fact that the more compact dorsal and looser ventral
regions of the nephrogenic cord could be visually distinguished under
the dissecting microscope. After two days in culture, dorsal cultures
exhibited essentially normal Lhx1-expressing tubule formation, but
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Development
Fig. 2. Distinct dorsal and ventral zones within the undifferentiated mesonephric IM. (A,A′) Plastic sections through region of newly formed somites of a
stage 15 embryo, illustrating distinct dorsal (d) and ventral (v) morphological zones within the undifferentiated IM (arrowheads in A indicate the plane of
separation between the two zones, which are more clearly seen in A⬘). (B-D) Expression of Pod1 by in situ hybridization at stage 15 (B,C) and stage 25 (D).
Line in C indicates approximate level of section shown in B. Note that Pod1 is expressed in the ventral portion of IM and the sphlanchnopleuric lateral plate but
not in the dorsal IM or somatopleuric lateral plate (B). At stage 25 (D), Pod1 is expressed specifically in the glomeruli of mesonephric nephrons.
(E-G) Expression of Wnt4 (E,F) and Lhx1 (G) at a similar stage and axial level to B. Note in E that Wnt4 is absent at the axial level indicated in F, and is only
activated in more mature, anterior axial levels (arrow in F). Lhx1 is expressed in the nephric duct, with no expression in the nephrogenic cord (G). ao, aorta; c,
coelom; g, glomerulus; lp, lateral plate; nc, nephrogenic cord; nd, nephric duct; no, notochord; nt, neural tube; s, somite; t, tubule.
RESEARCH ARTICLE
Development (2013) doi:10.1242/dev.097063
expressed Nphs2) but did not express the marker podocalyxin (data
not shown). As Nphs2 is such a specific marker for podocytes, we
hypothesize that the cyst-like structures represent an early or
abnormal form of podocyte differentiation.
Podocytes originate from the ventral IM
The experiments shown in Fig. 3A-G suggest that the tissues of the
ventral explants are a source of podocytes. However, the ventral
explants contain several distinct tissues, including ventral somites,
IM, and lateral plate splanchnopleuric mesoderm, all of which could
potentially be a source of podocytes. In order to narrow down the
source of the podocytes, the lipophilic dye SP-DiO was used to label
individual tissues at the onset of the culture period, and the fate of
the labeled cells was determined after the formation of nephrons.
Only injection into the IM led to labeling of cells that ended up in
the vicinity of Nphs2-expressing podocytes (Fig. 3H,J) (three out of
four experiments). By contrast, labeled cells from the lateral plate
mesoderm (Fig. 3I,K) or somites (data not shown) gave rise to cells
that were located far from the podocytes (seven out of seven
experiments). These results, together with the results of the culture
experiments shown in Fig. 3A-G, suggest that podocytes originate
from the ventral IM.
Podocyte differentiation in the absence of duct or tubules:
experimental blockage of nephric duct migration
showed a marked reduction in Nphs2 expression, with a majority of
cultures having a complete loss of Nphs2 (Fig. 3D,E). Of 25 dorsal
cultures, 100% expressed Lhx1 whereas only 16% expressed Nphs2.
The minority of cultures expressing some Nphs2 might be attributable
to inaccuracy of dissection along the dorsal-ventral boundary. These
results indicate that podocyte formation does not take place if ventral
tissue is not present.
When the ventral region is cultured alone, Lhx1-expressing cells
were never observed (0% of 20 cultures), indicating an absence of
tubule formation. Ventral cultures typically contained one or more
hollow, single-cell-thick epithelial cyst-like structure that expressed
the podocyte marker Nphs2 (Fig. 3F,G; 80% of 20 cultures
4568
Podocyte differentiation in the absence of tubules in vivo:
external glomeruli
In addition to the typical nephrons of the mesonephros and
metanephros, avian embryos have long been known to form
structures in the pronephric region called external glomeruli
(Hamilton, 1952; Hiruma and Nakamura, 2003; Vize et al., 2003a).
In these structures, the glomerulus is not integrated into a nephron
but instead bulges into the coelomic space. Often, a tubular opening
is located nearby. Thus, these external glomeruli resemble the
pronephric tubules of some anamniotes, in that there is a separation
of glomerular and tubular functions.
Development
Fig. 3. Developmental fate of dorsal and ventral IM regions.
(A-G) Independent developmental pathways for dorsal and ventral regions.
(A) Slice of a stage 15 embryo illustrating dorsal and ventral zones cultured
in subsequent panels. (B,C) Bright-field (B) and fluorescent (C) images of
dorsal and ventral regions cultured together. C is stained with antibodies to
Nephs2 (red) to mark podocytes and Lhx1 (green) to mark duct and tubules.
Note differentiation of both glomerular podocytes (g) and tubules (t).
(D,E) Bright-field (D) and fluorescent (E) images of dorsal region cultured
alone. Note differentiation of tubules but not podocytes. (F,G) Bright-field (F)
and fluorescent (G) images of ventral region cultured alone. Note formation
of a cyst-like structure (asterisk) expressing Nephs2 and no expression of
Lhx1. (G′) Higher magnification of cyst-like structure from ventral culture with
confocal imaging. Note the epithelial nature of the cysts. (H-K) Fate mapping
of lateral plate and IM in slice cultures. SP-DiO was injected into the ventral
intermediate mesoderm (H) or the lateral plate coelomic lining (I). After
culturing for 48 hours and confocal imaging, labeled cells can be found in the
vicinity of glomerular podocytes (g) in IM cultures (J) but not lateral plate
cultures (K). ao, aorta; im, intermediate mesoderm; lp, lateral plate; nd,
nephric duct; s, somite; t, tubule.
The avian nephric duct originates in the pronephric region, adjacent
to somites 8-10. Subsequently, the duct extends posteriorly into the
mesonephric and metanephric regions, where it induces nephron
formation (Obara-Ishihara et al., 1999; Schultheiss et al., 2003; Attia
et al., 2012). Classical experiments have demonstrated that if posterior
extension of the nephric duct is prevented, mesonephric tubules will
not form (Gruenwald, 1937; Waddington, 1938; Soueid-Baumgarten
et al., 2013). We utilized this knowledge in order to investigate
whether podocytes can differentiate in the absence of both the nephric
duct and tubules. A foil barrier was placed in the migration path of the
nephric duct so that the duct was prevented from reaching the
mesonephric zone (Fig. 4A). The opposite side of the embryo did not
receive a barrier and served as a control. Eggs were incubated for a
further 48 hours and then examined for expression of tubule and
podocyte markers. Examination of Lhx1 expression found that tubule
formation was completely blocked on the side with the barrier (data
not shown). However, even in the complete absence of a nephric duct
or tubules, Nphs2-expressing cells could be identified (Fig. 4B) (five
out of five embryos). These Nphs2-expressing cells were often found
within large cystic structures, which could possibly represent a greatly
enlarged Bowman’s space, although this is uncertain. The number of
Nphs2-expressing cells generated on the operated side was typically
smaller than on the control side. These results indicate that presence
of a nephric duct or kidney tubule is not required for formation of at
least some podocyte-like cells in the mesonephric region.
RESEARCH ARTICLE
The formation of external glomeruli in avians has not been
examined at the molecular level. We reasoned that the study of the
formation of a natural structure that exhibits a separation of
glomerulus and tubule components of the nephron could yield
insights into the relationship between these components during
nephron differentiation. Chick embryos were examined at stage 18
in the pronephric region for expression of Nphs2, the podocyte
marker, and Lhx1, which marks both tubules and the nephric duct.
Serial sections of multiple embryos revealed many cases in which
well-developed glomeruli bulging into the coelom were found in the
absence of nearby tubular structures (Fig. 4C,D; the Lhx1expressing structure in Fig. 4D is the nephric duct). Other examples
were found of Nephs2-expressing cells embedded within
mesenchyme (not bulging into the coelom), also in the absence of
adjacent tubules (Fig. 4E). Thus, it appears that formation of these
podocytes does not depend on a process that simultaneously
generates tubules. It should be emphasized that the Nphs2expressing cells in the duct block experiment shown in Fig. 4A,B
are not external glomeruli, because external glomeruli develop in the
pronephric region, whereas the duct-blocked embryos were analyzed
in the mesonephric region.
Canonical Wnt signaling selectively inhibits podocyte
differentiation
It has been well established that Wnt signaling components,
particularly Wnt9b and Wnt4, are required for nephron formation
in both the metanephros and mesonephros (Stark et al., 1994;
Kispert et al., 1998; Carroll et al., 2005). As Wnt9b is expressed
in the nephric duct (Carroll et al., 2005), which is located on the
dorsal side of the mesonephric mesenchyme (Fig. 2), we explored
whether Wnt signals might be involved in patterning the
mesonephros into tubular and glomerular domains. Wnt9b is
thought to work via the canonical Wnt signaling pathway (Carroll
et al., 2005; Park et al., 2007). Mesonephric slices were taken from
embryos before the initiation of PTA formation, as in Fig. 3, and
cultured in the presence or absence of 6-bromoindirubin-3′-oxime
(BIO), which inhibits the enzyme Gsk3 and therefore mimics
activation of the canonical Wnt signaling pathway (Meijer et al.,
2003; Sato et al., 2004). Addition of BIO to the culture medium
resulted in loss of the podocyte marker Nphs2 in a dose-dependent
manner (Fig. 5A-D), without significantly affecting expression of
the duct/tubule marker Lhx1 (Fig. 5E-H). At higher doses of BIO,
expression of Nphs2 was completely repressed (Fig. 5C,D). The
podocyte-repressing effects of BIO were similar to those obtained
by inhibition of Notch signaling (Fig. 5M,N), which has been
found to repress glomerulus formation (Cheng et al., 2003; Cheng
and Kopan, 2005). In complementary experiments, explants were
treated with IWR-1-endo, an inhibitor of Axin degradation and
thus an inhibitor of the canonical Wnt signaling pathway (Chen et
al., 2009). In preliminary experiments (data not shown), an IWR1-endo concentration of 20-50 μM was found to be capable of
activating cardiac gene expression in cultures of posterior gastrulastage chick mesoderm, a known effect of Wnt inhibition (Marvin
et al., 2001). Consistent with the results of BIO treatment, IWR1-endo-treated mesonephric explants exhibited stronger expression
of Nphs2, whereas expression of the duct/tubule marker Lhx1 was
modestly reduced (Fig. 5I-L).
DISCUSSION
In developing nephrons of the mammalian metanephros, distinct
glomerular and tubular regions can first be detected at the S-shaped
body stage, using both morphological and molecular criteria
(Dressler, 2006; Quaggin and Kreidberg, 2008; Georgas et al.,
2009). The history of the glomerular and tubular nephron
components prior to this stage is not clear. Current models of
nephron segmentation generally hold that the pretubular aggregate
and the renal vesicle are composed of equivalent uncommitted
precursor cells that subsequently, under the influence of poorly
defined regionalizing signals, differentiate into glomerular and
tubular cell types (reviewed by Costantini and Kopan, 2010). The
current work modifies and supplements this model in several ways.
First, the studies reported here provide several strands of evidence
indicating that the developmental pathways of the podocyte and
tubular components of the nephron are distinct earlier than
previously thought. Well before the formation of the first PTA or RV,
the IM can be separated into dorsal and ventral regions, with the
dorsal region generating tubules but no podocytes, and the ventral
region exhibiting podocyte but not tubular gene expression (Fig. 3).
In addition, blocking the formation migration of the nephric duct
and the subsequent formation of tubules does not completely prevent
the appearance of Nphs2-expressing podocyte-like cells in vivo
(Fig. 4). The current experiments do not establish when the
podocyte and tubular lineages become specified. It is possible that
the lineages are already specified at the time when dorsal and ventral
parts of the IM were separated in the experiments shown in Fig. 3AG. The expression of Pod1 in the ventral but not the dorsal IM
(Fig. 2B), as well as the morphological differences between the two
IM regions (Fig. 2A,A′), supports the idea that the podocyte and
tubular lineages may already be somewhat distinct at this stage.
However, it is also possible that one or both of the IM regions
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Development
Fig. 4. Development of podocytes independent of duct or tubules in
vivo. (A) Diagram illustrating location of the barrier placed to block migration
of the nephric duct into the mesonephric region. (B) Section of a ductblocked embryo analyzed for expression of Nphs2 (red) and stained with
DAPI (blue), showing the presence of Nphs2-expressing structures (g*)
within an expanded cavity on the blocked (left) side of the embryo. Note the
presence of extensive tubule formation on the control side but not the
blocked side. (C-E) Sections of unmanipulated stage 18 embryos in the
pronephric region stained by in situ hybridization for Nephs2 (C) or
immunofluorescence to Nphs2 (red) and Lhx1 (green) (D,E). C and D
illustrate classic external glomeruli with podocytes projecting into the
coelomic space. In D, no Lhx1-expressing tubules are seen near the external
glomerulus (the Lhx1-exressing structure is the nephric duct). E shows an
example of an internal glomerulus that is not associated with a nearby tubule.
ao, aorta; c, coelom; g, glomerulus; nd, nephric duct; no, notochord.
Development (2013) doi:10.1242/dev.097063
RESEARCH ARTICLE
Development (2013) doi:10.1242/dev.097063
continues to receive essential signals during the culture period from
other tissues included in the dorsal and ventral explants. It does
appear to be the case, however, that neither tubule nor podocyte
lineages require the presence of the other lineage in order to undergo
initial differentiation.
Second, the finding that canonical Wnt signaling is a strong
repressor of podocyte formation suggests that Wnt signaling is likely
to play a role in nephron segmentation. Wnt9b is expressed in the
nephric duct and is required and sufficient for induction of nephrons
(Carroll et al., 2005; Park et al., 2007). The current results suggest
that Wnt9b (or other sources of canonical Wnt signaling) not only
initiates PTA formation, but also is involved in patterning the
nephron, with regions that receive high levels of Wnt signaling
becoming tubules and regions that receive lower levels of signaling
becoming podocytes. In support of this model, Wnt4, which is also
required for nephron formation (Stark et al., 1994), is expressed in
the PTA, RV and S-shaped bodies of the mouse metanephros, with
a bias towards the future dorsal side of the nephron and away from
the proximal end where the podocytes are located (Stark et al., 1994;
Kispert et al., 1998) [see also GUDMAP Embryo #8209 (McMahon
et al., 2008)]. Opposing results have been obtained in the Xenopus
pronephros, in which overexpression of Wnt4 leads to an expansion
of glomerular gene expression (Naylor and Jones, 2009). The
difference between the chick and the Xenopus results could be due
to timing (in Xenopus, Wnt4 RNA was introduced into the eight-cell
4570
embryo), or to differences between pronephric and mesonephric
patterning. A working model for initial events in nephron
segmentation in the mesonephros is presented in Fig. 6A. In the
model, Wnt signaling from the nephric duct induces formation of
tubules and represses formation of podocytes. Podocyte formation
may be a ‘default’ differentiation pathway that occurs in the absence
of Wnt signaling, or may require active inductive signaling, perhaps
from the aorta or other tissue adjacent to the ventral IM. Notch and
retinoic acid signaling have also been found to be involved in
regulation of podocyte formation (McLaughlin et al., 2000;
McCright et al., 2001; Cheng et al., 2003; Taelman et al., 2006;
Cheng et al., 2007; Wingert et al., 2007; Naylor and Jones, 2009).
An important aim in future work will be to understand the
relationship between Wnt, Notch and retinoic acid signaling in
regulating segmentation of the nephron.
A third issue raised by these studies is the question of the
relationship of the PTA and the RV to the fully developed nephron
(Fig. 6B,C). Because the current studies find that podocyte and
tubule specification begin prior to PTA and RV formation, they
disfavor a model in which both podocytes and tubules derive from
a common unsegmented precursor structure, such as the PTA or RV,
which then subsequently differentiates into podocyte and tubular
lineages under local signals. It is possible, however, that the PTA
and/or RV are hybrid structures that are built from two separate
sources of already-patterned podocyte and tubule precursors
Development
Fig. 5. Regulation of podocyte formation by manipulation of Wnt signaling. (A-N) Whole-slice explants were taken from the axial level of the most newly
formed somite of stage 15-16 chick embryos and cultured in control medium (A,E,I,K,M), or in medium containing the canonical Wnt pathway activator BIO
(B-D,F-H), the Wnt pathway inhibitor IWR-1-endo (J,L) or the Notch inhibitor Compound E (N) at the indicated concentrations. After 48 hours, cultures were
analyzed by whole-mount in situ hybridization for Nphs2 (A-D,I,J,M,N) or Lhx1 (E-H,K,L). BIO produced a dose-dependent inhibition of Nphs2 expression but
not of Lhx1 expression, while IWR-endo-1 produced an increase in Nphs2 expression and a modest decrease in Lhx1 expression (Lhx1 is also expressed in a
subset of neurons in the neural tube). Compound E also inhibited Nphs2 expression. IWR produced an increase in the podocyte marker Nphs2 expression and
a mild decrease in the duct/tubule marker Lhx1. n, neural tube; p, podocytes; t, tubules.
RESEARCH ARTICLE
Development (2013) doi:10.1242/dev.097063
(Fig. 6B, b1). Alternatively, the PTA and RV may be precursors to
only the tubule component of the nephron, with podocyte precursors
joining at some point prior to the formation of the S-shaped body
(Fig. 6B, b2). Distinguishing between these models will require
single cell-resolution fate mapping of the mesonephric IM, the PTA
and the RV, experiments that are currently underway in our
laboratory.
Although isolated ventral IM can initiate the expression of the
podocyte-specific gene Nphs2, it does not exhibit full podocytetype differentiation. The podocin-expressing cyst-like structures in
ventral-isolated explants bear a resemblance to the early epithelial
stage of podocyte differentiation (Quaggin and Kreidberg, 2008),
suggesting that these cysts may represent an arrested stage of
podocyte differentiation. The ability of this same ventral IM to
form well-differentiated podocytes when cultured together with the
tubule-forming dorsal IM (Fig. 3B,C) suggests that podocyte
precursors require interaction with a component of the dorsal IM
in order to fully differentiate. An important future goal will be to
understand the factors that promote differentiation and maturation
of the podocyte precursors as well as the integration of other
glomerular components, including mesangial cells and
endothelium.
The focus of the current experiments was the chick mesonephros,
and it is important to consider how these results apply to the
formation of the mammalian mesonephros, which has received
much more attention. We have shown here (Fig. 1) that
nephrogenesis in the chick mesonephros resembles closely the
formation of nephrons in the mouse metanephros, both in terms of
morphological stages of nephron formation and expression of
molecular markers. The finding in the current work that podocyte
and tubule develop along distinct developmental pathways from
very early stages in kidney formation must now be investigated in
the mammalian metanephros. The metanephros undergoes
significantly more expansion than the mesonephros, and thus aspects
of the establishment of podocyte and tubule precursors are likely to
be different in the two cases. There is currently no evidence for a
prepattern of proximal and distal components of metanephric
tubules; however, it must be said that there is also no definitive
evidence against the possibility. A genetic lineage-tracing study
reported that individual Six2-expressing cells can give rise to both
podocyte and tubular nephron components (Kobayashi et al., 2008),
but because Six2 is expressed very early in the whole mesonephric
mesenchyme (Oliver et al., 1995), this does not rule out the
subsequent segregation of progeny of the original clone into separate
podocyte and tubule precursor pools. One potentially interesting
point to investigate will be the expression of Pod1, which is
expressed specifically in the chick ventral mesonephric IM (Fig. 2).
In the mouse metanephros, Pod1 (also known as Tcf21) is expressed
in a subset of metanephric mesenchyme precursor cells and is later
expressed in podocytes and stroma, but not tubular epithelia
(Quaggin et al., 1998; Quaggin et al., 1999). Genetic lineage studies
using mouse Pod1-cre lines have been somewhat conflicting
regarding the fate map of Pod1-expressing cells, probably owing to
differences in the timing and efficiency of cre induction and reporter
activation (Acharya et al., 2011; Maezawa et al., 2012). A thorough
re-examination of Pod1-cre mice will help to determine whether
Pod1 marks a pre-podocyte and/or pre-stroma lineage in the mouse
metanephros, and whether there is any connection between the
podocyte and stromal lineages.
Although the mesonephric and metanephric nephrons of amniotes
have glomeruli directly connected to tubules, this is not generally
the case in kidneys throughout the animal kingdom (Goodrich,
1895; Goodrich, 1930; Ruppert, 1994). In many cases, the
podocyte/blood filtration function is spatially separated from the
tubule/processing and excretion functions. Typically, these two
components are separated by an expanse of the body cavity, or
coelom, with podocytes filtering body waste into the coelom, and
tubules and nephric duct processing and draining the filtrate from
the coelom to the outside. In Xenopus, evidence exists for the
separability of glomerular and tubular developmental programs
(Urban et al., 2006). In organisms requiring a high kidney
throughput, the podocyte and tubules have been brought closer
together, eventually reducing the coelomic space to the Bowman’s
capsule that directly links the glomerulus to the entrance to the
tubule, as seen in amniote vertebrates. The current results indicate
that, despite this closer and closer apposition of the glomerular and
tubular nephron components, a distinction between the initial
developmental pathways of the podocyte and the tubule appears to
be conserved in amniote integrated nephrons. Thus, the existence of
separate developmental pathways of the filtration and tubular
components of the nephric system might be a fundamental feature
of animal excretory systems.
4571
Development
Fig. 6. Models of intermediate mesoderm regionalization and nephron patterning. (A) Summary of data from the current study. Wnt signaling from the
nephric duct prior to the initiation of PTA formation promotes dorsalization of the mesonephric mesenchyme and specification of tubules, and inhibits
specification of podocytes. There may be also podocyte-promoting signals from ventral tissues such as the aorta, although this is currently not clear.
(B,C) Alternative models for the contribution of the renal vesicle to the S-shaped body. In b1, the RV is a hybrid structure comprising dorsal tubule precursors
and ventral podocyte precursors. In b2, the RV is a precursor structure to the tubule, and the pre-podocytes reside in the mesenchyme ventral to the RV.
Regardless of which model from part B is correct, the S-shaped body (C) comprises contributions from both the ventral and the dorsal mesenchyme
compartments. ao, aorta; d, nephric duct; po, podocytes; rv, renal vesicle; tu, tubule.
RESEARCH ARTICLE
Chick embryos
Fertile White Leghorn chick eggs were incubated at 38.5°C in a humidified
incubator and staged according to Hamburger and Hamilton (Hamburger
and Hamilton, 1951).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was preformed as previously described
(Wilkinson and Nieto, 1993; Schultheiss et al., 1995; Attia et al., 2012).
Anti-sense probe to chick Pod1 was generated from a plasmid generously
provided by S. Dietrich (von Scheven et al., 2006). Following signal
development, embryos were embedded in sucrose/gelatin, cryosectioned at
20-μm intervals, and examined using a Zeiss Axioimager microscope with
differential interference contrast (DIC) optics.
Section in situ hybridization
Embryos were fixed overnight at 4°C in 4% paraformaldehyde, dehydrated
through a series of graded ethanols, and embedded in paraffin. Sections of
10-μm thickness were collected, and section in situ hybridization with
alkaline phosphatase detection was performed as previously described
(Murtaugh et al., 1999; Nissim et al., 2007). Digoxigenin-labeled probes
were generated for Pax-2 (Burrill et al., 1997), Lhx1 (Tsuchida et al., 1994),
Wnt4 (GenBank Locus BU418913, MRC Geneservice Clone
ChEST934g18), Pod1 (von Scheven et al., 2006) and podocin (Nphs2)
(GenBank Locus BU377446, ARK Genomics Clone ChEST790p13).
Nphs2 antibody
A rabbit polyclonal antibody to the N-terminus of chick Nphs2 was
generated to the peptide RKRESPATQRKQEKRERSKEASK (SigmaAldrich). Antiserum was affinity purified against the target peptide using an
Aminolink column (Pierce) according to the manufacturer’s instructions.
Immunofluorescence
For immunofluorescence on sections, embryos were fixed in 4%
paraformaldehyde for 1 hour at room temperature. Fixed embryos were
embedded in 7.5% gelatin and 15% sucrose in PBS, and cryosectioned at
10-μm intervals. After permeabilization in 0.25% Triton X-100 in PBS for
15 minutes and blocking (1% bovine serum albumin, 1% goat serum, 1%
horse serum and 0.02% Tween 20 in PBS) for 15 minutes, sections were
incubated with the primary antibodies rabbit anti-Nphs2 (1:4000) and mouse
anti-Lhx1 (1:10) (Developmental Studies Hybridoma Bank, Clone 4F2).
Alexa Fluor 488-, Cy3- and DyLight 649-conjugated secondary antibodies
(Jackson ImmunoResearch) were used at 1:250 in blocking solution. DAPI
at 1 μg/ml was used to visualize nuclei. Images were collected on a Zeiss
Axioimager microscope with a Qimaging ExiBlue digital camera and Image
Pro plus imaging software.
For immunofluorescence of explant cultures, explants were fixed in 4%
paraformaldehyde for 24 hours, washed three times in PBS and permeabilized
in 0.5% Triton X-100 and 0.1% saponin in PBS (TSP) for at least 24 hours.
Explants were blocked in TSP with 1% sheep serum for several hours and then
incubated overnight at 4°C with the primary antibodies rabbit anti-Nphs2
(1:4000) and mouse anti-Lhx1 (1:10) (Developmental Studies Hybridoma
Bank, Clone 4F2). The next day, the explants were washed in TSP (three × 15
minutes), and incubated in secondary antibodies Alexa Fluor 488-, Cy3- and
DyLight 649-conjugated secondary antibodies (Jackson ImmunoResearch) at
1:250 in blocking solution.
Plastic sections
After overnight fixation in 3% glutaraldehyde, embryos were embedded in
epon resin and 1-μm-thick sections were cut using a diamond knife. Slides
were stained with Methylene Blue and coverslipped using methacrylate
mounting medium.
Explant culture and microdissection
Embryos were pinned to a silicone dish dorsal side up using insect pins and
slices ~1.5 somites in thickness (150 μm) were cut with a microscalpel
(Feather) at the axial level of the most newly formed somites. In some cases,
4572
slices were further microdissected using a microscalpel (see Results).
Explants were incubated in drops of chilled growth medium (DMEM with
4.5 g/ml glucose, 10% fetal bovine serum, 2% chick extract, 2 mM
glutamine and 50 μg/ml PenStrep) until all explants were collected, and
were then grown for 48 hours at the air-medium interface on Millicell cell
culture inserts (Millipore). In some cases, the canonical Wnt pathway
activator 6-bromoindirubin-3′-oxime (BIO) (Calbiochem), the canonical
Wnt pathway inhibitor IWR-1-endo (Calbiochem), or the γ-secretase
inhibitor Compound E (Calbiochem) were added to the medium at the
indicated concentrations.
DiO fate mapping
SP-DiO (Molecular Probes) was diluted in 0.3 M sucrose to a final
concentration of 0.1 μg/μl. To mark the intermediate mesoderm, DiO was
injected into the ventral IM in slice explant cultures using a pulled glass
capillary (1.0 mm outer diameter; Drummond) with a 20-μm diameter tip
attached to a Picospritzer III (Parker-Hannifin), with settings of pressure=10
psi; duration=20 mseconds. For coelom injections, DiO was injected into
the embryonic coelom in ovo, and subsequently slices were prepared and
cultured as described above.
Nephric duct barriers
A thin aluminium foil sheet was placed between two pieces of parafilm and
small rectangles were cut (about 1.5 mm × 3 mm) using a scalpel knife. A
small crosswise cut was made on one side of stage 9-11 embryos in ovo, at
the axial level of about two somites posterior to the last somite using a thin
tungsten needle (0.005 inch diameter). The aluminium barrier, isolated from
the parafilm, was gently pushed to position using a thicker tungsten needle
(0.01 inch diameter). Embryos were cultured for 48 hours in ovo in a 38.5°C
humidified incubator.
Acknowledgements
The authors would like to thank A. McMahon and T. Jessell for providing probes for
in situ hybridization.
Competing interests
The authors declare no competing financial interests.
Author contributions
M.G., R.Y. and T.M.S. conducted the experiments. All the authors were involved in
designing and analyzing the experiments and writing the paper.
Funding
This work was supported by grants from the March of Dimes [FY12-343 to T.M.S.];
the Israel Science Foundation [1328/08 and 1573/12 to T.M.S.]; the Rappaport
Family Foundation [T.M.S.]; the Charles Krown Research Fund [T.M.S.]; and the
National institutes of Health (NIH) [DK45218 to D.H.]. D.H. was the recipient of a
Visiting Professor Fellowship from the Lady Davis Fellowship Trust. Deposited in
PMC for release after 12 months.
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