© 2013. Published by The Company of Biologists Ltd | Development (2013) 140, 4565-4573 doi:10.1242/dev.097063


Generation of the podocyte and tubular components of an amniote kidney: timing of specification and a role for Wnt signaling Mor Grinstein1, Ronit Yelin1, Doris Herzlinger2 and Thomas M. Schultheiss1,*

ABSTRACT system. After passing the glomerular filtration barrier, the filtrate Kidneys remove unwanted substances from the body and regulate enters the tubule, where it is processed by absorption and secretion the internal body environment. These functions are carried out by carried out by tubular epithelial cells. Finally, the processed filtrate, specialized cells (podocytes) that act as a filtration barrier between now called urine, enters the collecting system, a system of ducts that the internal milieu and the outside world, and by a series of tubules drains the urine to the outside. and ducts that process the filtrate and convey it to the outside. In the Because of the crucial role that kidneys play in regulating the kidneys of amniote vertebrates, the filtration (podocyte) and tubular internal body environment, it is not surprising that the structure of functions are tightly integrated into functional units called nephrons. kidney tissues varies greatly between different species, as well as at The specification of the podocyte and tubular components of amniote different stages of the life cycle of the same organism (Fraser, 1950). nephrons is currently not well understood. The present study Classically, three types of vertebrate kidneys are discussed: the investigates podocyte and tubule differentiation in the avian , which serves as the embryonic kidney in anamniotes mesonephric kidney, and presents several findings that refine our and is a transient, largely non-functional structure in amniotes; the understanding of the initial events of nephron formation. First, well , which is the adult kidney is anamniotes and the before the first morphological or molecular signs of nephron functional embryonic/fetal kidney in amniotes; and the metanephros, formation, mesonephric mesenchyme can be separated on the basis which is the functional amniote adult kidney and which is not of morphology and the expression of the transcription factor Pod1 into formed in anamniotes. Each of these kidneys comprises collections dorsal and ventral components, which can independently differentiate of nephrons, which can vary in number from a single nephron in the in culture along tubule and podocyte pathways, respectively. Second, zebrafish pronephros to ~1 million nephrons in the human canonical Wnt signals, which are found in the nephric duct adjacent metanephros (Vize et al., 2003b). to the dorsal mesonephric mesenchyme and later in portions of the Understanding how nephrons form is crucial for developing differentiating nephron, strongly inhibit podocyte but not tubule approaches to repair or regenerate damaged kidney tissue. The most differentiation, suggesting that Wnt signaling plays an important role common model system for studying vertebrate nephron formation in the segmentation of the mesonephric mesenchyme into tubular has been the rodent metanephros. Metanephric nephrons form as the and glomerular segments. The results are discussed in terms of their consequence of mutual inductive interactions between the broader implications for models of nephron segmentation. metanephric mesenchyme and an epithelial tube called the (UB) (Yu et al., 2004; Dressler, 2009; Costantini and Kopan, KEY WORDS: Wnt signaling, Chick embryo, Kidney, Mesonephros, 2010). According to current models, as a result of initiating signals Podocyte, Renal vesicle from the UB, the mesenchyme undergoes a sequence of morphological changes that leads to nephron formation. First, the INTRODUCTION mesenchyme undergoes localized condensation to form pretubular Vertebrate kidneys are composed of functional units called nephrons aggregates (PTAs), which then undergo a mesenchymal-to-epithelial (Saxen, 1987; Dressler, 2006). Each nephron has several distinct transition (MET) to form renal vesicles (RVs). The RVs functional and morphological compartments. At the proximal end of subsequently undergo elongation and differentiation, with the each nephron is a glomerulus, which filters the blood. The proximal end interacting with local endothelial cells to form the glomerulus contains vascular capillaries and specialized epithelial glomerulus, the middle part forming the tubule, and the distal end cells, the podocytes. The combination of fenestrated slits between connecting to the branching UB, which will form the collecting the capillary endothelial cells, the basement membrane between system. endothelial cells and podocytes, and specialized foot processes of Knowledge of the signaling pathways that regulate specification the podocytes together form the glomerular filtration barrier. Small of nephrogenic mesenchyme into glomerular and tubular fates is still molecules can pass through the barrier into the interior of the incomplete. Wnt signaling is required for nephron formation and is nephron, whereas larger molecules and cells remain in the vascular 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 1Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, to induction of glomerular fates (Naylor and Jones, 2009). Notch Technion-Israel Institute of Technology, Haifa 31096, Israel. 2Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, signaling has been found to be required for generation of glomerular NY 10065, USA. and proximal tubular nephron components in several species (McLaughlin et al., 2000; McCright et al., 2001; Cheng et al., 2003; *Author for correspondence ([email protected]) Taelman et al., 2006; Cheng et al., 2007; Wingert et al., 2007;

Received 1 April 2013; Accepted 27 August 2013 Naylor and Jones, 2009), but the stage(s) at which Notch signaling Development

4565 RESEARCH ARTICLE Development (2013) doi:10.1242/dev.097063 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 fate- mapping 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 Fig. 1. Development of the chick mesonephros. (A-C) Plastic sections light on the specification of filtration and tubular components of from a single stage 18 embryo, from posterior (A) to anterior (C) axial levels, integrated nephrons. Avian mesonephric nephrons are similar in illustrating stages in the differentiation of mesonephric nephrons, including morphology to their metanephric counterparts and pass through pretubular aggregate (ag), renal vesicle (rv) and S-shaped body (sb). (D,E) In similar stages in their development, including PTA and RV stages 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 (Hamilton, 1952) (see also Results, below). However, the avian the condensing pretubular aggregate (E). (G,H) In situ hybridization at stage mesonephros is much simpler in structure than the metanephros, 25 for Lhx1 (G) and Nphs2 (H) showing Lhx1 expression in the nephric duct being a linear organ with only a few nephrons per body segment, and tubules (G) and Nphs2 expression in the podocytes of the glomerulus and thus easier to manipulate and analyze. The current study (H). (F,I) Diagrams of sections through the mesonephric regions of stage 15 investigated the initial stages of podocyte and tubule specification and 18 embryos for orientation purposes. Note that in F, the undifferentiated in the avian mesonephros. It was found that, well before the 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, appearance of PTAs or RVs, tubule and podocytes precursors can be but the relationships between the nephric duct, the IM and the aorta are separated from each other and can differentiate independently. preserved. Within the differentiating IM in I, the tubules are located laterally, Evidence is also presented that canonical Wnt signaling plays a role closer to the duct, and the glomeruli are located medially, closer to the aorta. in distinguishing between these two alternative mesonephric cell ag, pretubular aggregate; ao, aorta; c, coelom; g, glomerulus; nc, fates by repressing differentiation of podocytes. 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. RESULTS Initial stages of nephron formation are similar in the avian mesonephros and mammalian metanephros Figure 1A-C shows plastic sections through the region of the Qualitative analysis of early avian mesonephros development was mesonephros at axial levels from posterior to anterior in a single performed in order to evaluate how closely nephron formation in the Hamburger-Hamilton (HH) (Hamburger and Hamilton, 1951) stage 18 mesonephros resembles that in the more widely described embryo, and illustrates the morphological changes that occur during mammalian mesonephros. The chick mesonephros develops in the nephron formation. Initially, mesenchyme adjacent to the nephric duct (IM) at axial levels between somites 15 and begins to condense to form PTAs (Fig. 1A, ag) and subsequently 30 (Hamilton, 1952). At the onset of mesonephric nephron undergoes epithelialization to form RVs (Fig. 1B, rv). In more anterior formation, the future mesonephros consists of the nephric duct sections, ‘S-shaped bodies’ can be found, in which an emerging tubule (which constitutes the future drainage system of the mesonephros) can be seen with a distal end connecting to the nephric duct and a and the nephrogenic cord, a strip of mesoderm that lies ventral to proximal end beginning to show signs of glomerular differentiation the nephric duct and dorsal to the aorta (Fig. 1F). As mesonephros (Fig. 1C, sb). These stages are morphologically similar to the stages differentiation proceeds, the region undergoes a dorsal-to-lateral that have been well described during nephron formation in the mouse rotation, so that eventually the nephric duct lies lateral to the and rat metanephros (Iino et al., 2001). differentiating mesonephric nephrons (Fig. 1I). We also examined a set of molecular markers that have been shown Because chick development proceeds from anterior to posterior, to play key roles during mouse metanephric nephron formation. As in examination of a single embryo during the period of mesonephros the mouse metanephros (Dressler et al., 1990), Pax2 is expressed in formation allows one to observe nephrons at various stages of both the nephric duct and in the undifferentiated mesonephric maturation, with the less mature nephrons located in more posterior mesenchyme (Fig. 1D) and Wnt4 (Stark et al., 1994) is expressed in

segments and more mature nephrons being found more anteriorly. newly condensing mesonephric pretubular aggregates (Fig. 1E). Development

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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 AЈ). (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.

Shortly thereafter, Lhx1 (Lim1) (Fujii et al., 1994; Shawlot and a marker (Pod1) that is later expressed in podocytes but not tubular Behringer, 1995) begins to be expressed as the aggregates undergo parts of the nephron, raised the question of whether the ventral and MET to form renal vesicles (data not shown). At later stages, markers dorsal zones of the IM might give rise to separate parts of the of specific nephron segments can be found, including Lhx1 in the nephron. In order to begin to address this question, we developed an nephric duct and tubules (Fig. 1G) and Nphs2 (podocin) (Boute et al., in vitro system for studying mesonephric nephron differentiation. 2000), a component of the slit diaphragm that is expressed exclusively Slices ~150 microns in thickness (~1.5 somites in thickness) were in podocytes (Fig. 1H). cut through the undifferentiated mesonephric region at the axial Thus, with respect to expression of markers, as well as level of the most newly formed somite and cultured on a filter at the morphological appearance, nephron formation in the avian air-medium interface. A micrograph of a representative slice at the mesonephros appears to be very similar to that of the mouse beginning of the culture period in shown in Fig. 3A. Importantly, the metanephros and can therefore potentially serve as a useful model mesonephric mesenchyme at the start of the culture period does not for studying formation of the type of integrated nephron seen in the express Wnt4, the earliest known molecular marker of PTA initiation mammalian metanephros. (Kispert et al., 1998; Carroll et al., 2005). The slices were taken from the axial level of the most newly formed somite, where the Distinct dorsal and ventral zones within the mesonephric IM nephrogenic cord is Wnt4 negative (Fig. 2E). Wnt4 expression is Close inspection of plastic sections through the mesonephric IM initiated in vivo approximately 6 hours later, after four more somites before the onset of PTA formation revealed that the IM at this stage have formed (Fig. 2F). The nephrogenic cord at the axial level at is not homogeneous, but appears to consist of two morphologically which the slices were taken is also negative for the tubular marker distinct zones: a dorsal zone adjacent to the nephric duct, which Lhx1, which is expressed at this stage only in the nephric duct typically appears to be more compact, and a looser ventral zone (Fig. 2G), and is also negative for the podocyte marker Nphs2, adjacent to the dorsal aorta (Fig. 2A,A′). The dorsal and ventral IM which is not expressed in the embryo at all at this stage (data not regions appear to be continuations of the somatopleuric and shown). sphlanchnopleuric layers of the lateral plate, respectively, with the When whole slices were cultured for two days, robust nephron difference that the dorsal and ventral layers of the IM are not differentiation occurred. By bright-field microscopy, multiple separated by a coelomic space as they are in the lateral plate. tubular structures were detected (Fig. 3B). By immunofluorescence, In a survey of genes known to be expressed in the developing abundant differentiation was seen of Lhx1-expressing nephric duct mammalian kidney, the transcription factor gene Pod1 (also called and tubule cells and Nphs2-expressing podocytes (Fig. 3C; 100% of Tcf21 and capsulin) (Hidai et al., 1998; Lu et al., 1998; Quaggin et 40 cultures expressed both Lhx1 and Nphs2). The podocytes were al., 1999) was found to be expressed in the mesonephric region typically arranged in clusters resembling glomeruli and located at (Fig. 2C), and in sections was found to be expressed in the ventral the periphery of the explants, immediately adjacent to the Lhx1- IM and the adjacent splanchnic (ventral) portion of the lateral plate, expressing tubules, suggesting that integrated nephrons had formed but not in the dorsal IM or the somatopleuric lateral plate (Fig. 2B). in culture. Upon appearance of differentiated nephrons, Pod1 was expressed In order to determine the developmental potential of the dorsal and specifically in glomerular podocytes (Fig. 2D). ventral zones of the IM, the embryo slices were dissected into ventral and dorsal halves (Fig. 3A) and cultured separately. Dissection was Dorsal and ventral IM zones generate distinct segments of aided by the fact that the more compact dorsal and looser ventral the nephron in culture regions of the nephrogenic cord could be visually distinguished under The existence of two distinct zones in the early mesonephric IM, the dissecting microscope. After two days in culture, dorsal cultures

and the observation that the ventral IM zone specifically expresses exhibited essentially normal Lhx1-expressing tubule formation, but Development

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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 (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 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, will not form (Gruenwald, 1937; Waddington, 1938; Soueid-Baumgarten Fig. 3. Developmental fate of dorsal and ventral IM regions. et al., 2013). We utilized this knowledge in order to investigate (A-G) Independent developmental pathways for dorsal and ventral regions. whether podocytes can differentiate in the absence of both the nephric (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 duct and tubules. A foil barrier was placed in the migration path of the dorsal and ventral regions cultured together. C is stained with antibodies to nephric duct so that the duct was prevented from reaching the Nephs2 (red) to mark podocytes and Lhx1 (green) to mark duct and tubules. mesonephric zone (Fig. 4A). The opposite side of the embryo did not Note differentiation of both glomerular podocytes (g) and tubules (t). receive a barrier and served as a control. Eggs were incubated for a (D,E) Bright-field (D) and fluorescent (E) images of dorsal region cultured further 48 hours and then examined for expression of tubule and alone. Note differentiation of tubules but not podocytes. (F,G) Bright-field (F) podocyte markers. Examination of Lhx1 expression found that tubule and fluorescent (G) images of ventral region cultured alone. Note formation of a cyst-like structure (asterisk) expressing Nephs2 and no expression of formation was completely blocked on the side with the barrier (data Lhx1. (G′) Higher magnification of cyst-like structure from ventral culture with not shown). However, even in the complete absence of a nephric duct confocal imaging. Note the epithelial nature of the cysts. (H-K) Fate mapping or tubules, Nphs2-expressing cells could be identified (Fig. 4B) (five of lateral plate and IM in slice cultures. SP-DiO was injected into the ventral out of five embryos). These Nphs2-expressing cells were often found intermediate mesoderm (H) or the lateral plate coelomic lining (I). After within large cystic structures, which could possibly represent a greatly culturing for 48 hours and confocal imaging, labeled cells can be found in the enlarged Bowman’s space, although this is uncertain. The number of vicinity of glomerular podocytes (g) in IM cultures (J) but not lateral plate cultures (K). ao, aorta; im, intermediate mesoderm; lp, lateral plate; nd, Nphs2-expressing cells generated on the operated side was typically nephric duct; s, somite; t, tubule. 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. showed a marked reduction in Nphs2 expression, with a majority of cultures having a complete loss of Nphs2 (Fig. 3D,E). Of 25 dorsal Podocyte differentiation in the absence of tubules in vivo: cultures, 100% expressed Lhx1 whereas only 16% expressed Nphs2. external glomeruli The minority of cultures expressing some Nphs2 might be attributable In addition to the typical nephrons of the mesonephros and to inaccuracy of dissection along the dorsal-ventral boundary. These metanephros, avian embryos have long been known to form results indicate that podocyte formation does not take place if ventral structures in the pronephric region called external glomeruli tissue is not present. (Hamilton, 1952; Hiruma and Nakamura, 2003; Vize et al., 2003a). When the ventral region is cultured alone, Lhx1-expressing cells In these structures, the glomerulus is not integrated into a nephron were never observed (0% of 20 cultures), indicating an absence of but instead bulges into the coelomic space. Often, a tubular opening tubule formation. Ventral cultures typically contained one or more is located nearby. Thus, these external glomeruli resemble the hollow, single-cell-thick epithelial cyst-like structure that expressed pronephric tubules of some anamniotes, in that there is a separation

the podocyte marker Nphs2 (Fig. 3F,G; 80% of 20 cultures of glomerular and tubular functions. Development

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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 Fig. 4. Development of podocytes independent of duct or tubules in treated with IWR-1-endo, an inhibitor of Axin degradation and vivo. (A) Diagram illustrating location of the barrier placed to block migration thus an inhibitor of the canonical Wnt signaling pathway (Chen et of the nephric duct into the mesonephric region. (B) Section of a duct- al., 2009). In preliminary experiments (data not shown), an IWR- blocked embryo analyzed for expression of Nphs2 (red) and stained with 1-endo concentration of 20-50 μM was found to be capable of DAPI (blue), showing the presence of Nphs2-expressing structures (g*) within an expanded cavity on the blocked (left) side of the embryo. Note the activating cardiac gene expression in cultures of posterior gastrula- presence of extensive tubule formation on the control side but not the stage chick mesoderm, a known effect of Wnt inhibition (Marvin blocked side. (C-E) Sections of unmanipulated stage 18 embryos in the et al., 2001). Consistent with the results of BIO treatment, IWR- pronephric region stained by in situ hybridization for Nephs2 (C) or 1-endo-treated mesonephric explants exhibited stronger expression immunofluorescence to Nphs2 (red) and Lhx1 (green) (D,E). C and D of Nphs2, whereas expression of the duct/tubule marker Lhx1 was illustrate classic external glomeruli with podocytes projecting into the modestly reduced (Fig. 5I-L). 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. DISCUSSION ao, aorta; c, coelom; g, glomerulus; nd, nephric duct; no, notochord. 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 The formation of external glomeruli in avians has not been (Dressler, 2006; Quaggin and Kreidberg, 2008; Georgas et al., examined at the molecular level. We reasoned that the study of the 2009). The history of the glomerular and tubular nephron formation of a natural structure that exhibits a separation of components prior to this stage is not clear. Current models of glomerulus and tubule components of the nephron could yield nephron segmentation generally hold that the pretubular aggregate insights into the relationship between these components during and the renal vesicle are composed of equivalent uncommitted nephron differentiation. Chick embryos were examined at stage 18 precursor cells that subsequently, under the influence of poorly in the pronephric region for expression of Nphs2, the podocyte defined regionalizing signals, differentiate into glomerular and marker, and Lhx1, which marks both tubules and the nephric duct. tubular cell types (reviewed by Costantini and Kopan, 2010). The Serial sections of multiple embryos revealed many cases in which current work modifies and supplements this model in several ways. well-developed glomeruli bulging into the coelom were found in the First, the studies reported here provide several strands of evidence absence of nearby tubular structures (Fig. 4C,D; the Lhx1- indicating that the developmental pathways of the podocyte and expressing structure in Fig. 4D is the nephric duct). Other examples tubular components of the nephron are distinct earlier than were found of Nephs2-expressing cells embedded within previously thought. Well before the formation of the first PTA or RV, mesenchyme (not bulging into the coelom), also in the absence of the IM can be separated into dorsal and ventral regions, with the adjacent tubules (Fig. 4E). Thus, it appears that formation of these dorsal region generating tubules but no podocytes, and the ventral podocytes does not depend on a process that simultaneously region exhibiting podocyte but not tubular gene expression (Fig. 3). generates tubules. It should be emphasized that the Nphs2- In addition, blocking the formation migration of the nephric duct expressing cells in the duct block experiment shown in Fig. 4A,B and the subsequent formation of tubules does not completely prevent are not external glomeruli, because external glomeruli develop in the the appearance of Nphs2-expressing podocyte-like cells in vivo pronephric region, whereas the duct-blocked embryos were analyzed (Fig. 4). The current experiments do not establish when the in the mesonephric region. podocyte and tubular lineages become specified. It is possible that the lineages are already specified at the time when dorsal and ventral Canonical Wnt signaling selectively inhibits podocyte parts of the IM were separated in the experiments shown in Fig. 3A- differentiation G. The expression of Pod1 in the ventral but not the dorsal IM It has been well established that Wnt signaling components, (Fig. 2B), as well as the morphological differences between the two particularly Wnt9b and Wnt4, are required for nephron formation IM regions (Fig. 2A,A′), supports the idea that the podocyte and in both the metanephros and mesonephros (Stark et al., 1994; tubular lineages may already be somewhat distinct at this stage.

Kispert et al., 1998; Carroll et al., 2005). As Wnt9b is expressed However, it is also possible that one or both of the IM regions Development

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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. continues to receive essential signals during the culture period from embryo), or to differences between pronephric and mesonephric other tissues included in the dorsal and ventral explants. It does patterning. A working model for initial events in nephron appear to be the case, however, that neither tubule nor podocyte segmentation in the mesonephros is presented in Fig. 6A. In the lineages require the presence of the other lineage in order to undergo model, Wnt signaling from the nephric duct induces formation of initial differentiation. tubules and represses formation of podocytes. Podocyte formation Second, the finding that canonical Wnt signaling is a strong may be a ‘default’ differentiation pathway that occurs in the absence repressor of podocyte formation suggests that Wnt signaling is likely of Wnt signaling, or may require active inductive signaling, perhaps to play a role in nephron segmentation. Wnt9b is expressed in the from the aorta or other tissue adjacent to the ventral IM. Notch and nephric duct and is required and sufficient for induction of nephrons retinoic acid signaling have also been found to be involved in (Carroll et al., 2005; Park et al., 2007). The current results suggest regulation of podocyte formation (McLaughlin et al., 2000; that Wnt9b (or other sources of canonical Wnt signaling) not only McCright et al., 2001; Cheng et al., 2003; Taelman et al., 2006; initiates PTA formation, but also is involved in patterning the Cheng et al., 2007; Wingert et al., 2007; Naylor and Jones, 2009). nephron, with regions that receive high levels of Wnt signaling An important aim in future work will be to understand the becoming tubules and regions that receive lower levels of signaling relationship between Wnt, Notch and retinoic acid signaling in becoming podocytes. In support of this model, Wnt4, which is also regulating segmentation of the nephron. required for nephron formation (Stark et al., 1994), is expressed in A third issue raised by these studies is the question of the the PTA, RV and S-shaped bodies of the mouse metanephros, with relationship of the PTA and the RV to the fully developed nephron a bias towards the future dorsal side of the nephron and away from (Fig. 6B,C). Because the current studies find that podocyte and the proximal end where the podocytes are located (Stark et al., 1994; tubule specification begin prior to PTA and RV formation, they Kispert et al., 1998) [see also GUDMAP Embryo #8209 (McMahon disfavor a model in which both podocytes and tubules derive from et al., 2008)]. Opposing results have been obtained in the Xenopus a common unsegmented precursor structure, such as the PTA or RV, pronephros, in which overexpression of Wnt4 leads to an expansion which then subsequently differentiates into podocyte and tubular of glomerular gene expression (Naylor and Jones, 2009). The lineages under local signals. It is possible, however, that the PTA difference between the chick and the Xenopus results could be due and/or RV are hybrid structures that are built from two separate

to timing (in Xenopus, Wnt4 RNA was introduced into the eight-cell sources of already-patterned podocyte and tubule precursors Development

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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.

(Fig. 6B, b1). Alternatively, the PTA and RV may be precursors to but because Six2 is expressed very early in the whole mesonephric only the tubule component of the nephron, with podocyte precursors mesenchyme (Oliver et al., 1995), this does not rule out the joining at some point prior to the formation of the S-shaped body subsequent segregation of progeny of the original clone into separate (Fig. 6B, b2). Distinguishing between these models will require podocyte and tubule precursor pools. One potentially interesting single cell-resolution fate mapping of the mesonephric IM, the PTA point to investigate will be the expression of Pod1, which is and the RV, experiments that are currently underway in our expressed specifically in the chick ventral mesonephric IM (Fig. 2). laboratory. In the mouse metanephros, Pod1 (also known as Tcf21) is expressed Although isolated ventral IM can initiate the expression of the in a subset of metanephric mesenchyme precursor cells and is later podocyte-specific gene Nphs2, it does not exhibit full podocyte- expressed in podocytes and stroma, but not tubular epithelia type differentiation. The podocin-expressing cyst-like structures in (Quaggin et al., 1998; Quaggin et al., 1999). Genetic lineage studies ventral-isolated explants bear a resemblance to the early epithelial using mouse Pod1-cre lines have been somewhat conflicting stage of podocyte differentiation (Quaggin and Kreidberg, 2008), regarding the fate map of Pod1-expressing cells, probably owing to suggesting that these cysts may represent an arrested stage of differences in the timing and efficiency of cre induction and reporter podocyte differentiation. The ability of this same ventral IM to activation (Acharya et al., 2011; Maezawa et al., 2012). A thorough form well-differentiated podocytes when cultured together with the re-examination of Pod1-cre mice will help to determine whether tubule-forming dorsal IM (Fig. 3B,C) suggests that podocyte Pod1 marks a pre-podocyte and/or pre-stroma lineage in the mouse precursors require interaction with a component of the dorsal IM metanephros, and whether there is any connection between the in order to fully differentiate. An important future goal will be to podocyte and stromal lineages. understand the factors that promote differentiation and maturation Although the mesonephric and metanephric nephrons of amniotes of the podocyte precursors as well as the integration of other have glomeruli directly connected to tubules, this is not generally glomerular components, including mesangial cells and the case in kidneys throughout the animal kingdom (Goodrich, endothelium. 1895; Goodrich, 1930; Ruppert, 1994). In many cases, the The focus of the current experiments was the chick mesonephros, podocyte/blood filtration function is spatially separated from the and it is important to consider how these results apply to the tubule/processing and excretion functions. Typically, these two formation of the mammalian mesonephros, which has received components are separated by an expanse of the body cavity, or much more attention. We have shown here (Fig. 1) that coelom, with podocytes filtering body waste into the coelom, and nephrogenesis in the chick mesonephros resembles closely the tubules and nephric duct processing and draining the filtrate from formation of nephrons in the mouse metanephros, both in terms of the coelom to the outside. In Xenopus, evidence exists for the morphological stages of nephron formation and expression of separability of glomerular and tubular developmental programs molecular markers. The finding in the current work that podocyte (Urban et al., 2006). In organisms requiring a high kidney and tubule develop along distinct developmental pathways from throughput, the podocyte and tubules have been brought closer very early stages in kidney formation must now be investigated in together, eventually reducing the coelomic space to the Bowman’s the mammalian metanephros. The metanephros undergoes capsule that directly links the glomerulus to the entrance to the significantly more expansion than the mesonephros, and thus aspects tubule, as seen in amniote vertebrates. The current results indicate of the establishment of podocyte and tubule precursors are likely to that, despite this closer and closer apposition of the glomerular and be different in the two cases. There is currently no evidence for a tubular nephron components, a distinction between the initial prepattern of proximal and distal components of metanephric developmental pathways of the podocyte and the tubule appears to tubules; however, it must be said that there is also no definitive be conserved in amniote integrated nephrons. Thus, the existence of evidence against the possibility. A genetic lineage-tracing study separate developmental pathways of the filtration and tubular reported that individual Six2-expressing cells can give rise to both components of the nephric system might be a fundamental feature

podocyte and tubular nephron components (Kobayashi et al., 2008), of animal excretory systems. Development

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MATERIALS AND METHODS slices were further microdissected using a microscalpel (see Results). Chick embryos Explants were incubated in drops of chilled growth medium (DMEM with Fertile White Leghorn chick eggs were incubated at 38.5°C in a humidified 4.5 g/ml glucose, 10% fetal bovine serum, 2% chick extract, 2 mM incubator and staged according to Hamburger and Hamilton (Hamburger glutamine and 50 μg/ml PenStrep) until all explants were collected, and and Hamilton, 1951). were then grown for 48 hours at the air-medium interface on Millicell cell culture inserts (Millipore). In some cases, the canonical Wnt pathway Whole-mount in situ hybridization activator 6-bromoindirubin-3′-oxime (BIO) (Calbiochem), the canonical Whole-mount in situ hybridization was preformed as previously described Wnt pathway inhibitor IWR-1-endo (Calbiochem), or the γ-secretase (Wilkinson and Nieto, 1993; Schultheiss et al., 1995; Attia et al., 2012). inhibitor Compound E (Calbiochem) were added to the medium at the Anti-sense probe to chick Pod1 was generated from a plasmid generously indicated concentrations. provided by S. Dietrich (von Scheven et al., 2006). Following signal development, embryos were embedded in sucrose/gelatin, cryosectioned at DiO fate mapping 20-μm intervals, and examined using a Zeiss Axioimager microscope with SP-DiO (Molecular Probes) was diluted in 0.3 M sucrose to a final differential interference contrast (DIC) optics. 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 Section in situ hybridization capillary (1.0 mm outer diameter; Drummond) with a 20-μm diameter tip Embryos were fixed overnight at 4°C in 4% paraformaldehyde, dehydrated attached to a Picospritzer III (Parker-Hannifin), with settings of pressure=10 through a series of graded ethanols, and embedded in paraffin. Sections of psi; duration=20 mseconds. For coelom injections, DiO was injected into 10-μm thickness were collected, and section in situ hybridization with the embryonic coelom in ovo, and subsequently slices were prepared and alkaline phosphatase detection was performed as previously described cultured as described above. (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), Nephric duct barriers Wnt4 (GenBank Locus BU418913, MRC Geneservice Clone A thin aluminium foil sheet was placed between two pieces of parafilm and ChEST934g18), Pod1 (von Scheven et al., 2006) and podocin (Nphs2) small rectangles were cut (about 1.5 mm × 3 mm) using a scalpel knife. A (GenBank Locus BU377446, ARK Genomics Clone ChEST790p13). 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 Nphs2 antibody tungsten needle (0.005 inch diameter). The aluminium barrier, isolated from A rabbit polyclonal antibody to the N-terminus of chick Nphs2 was the parafilm, was gently pushed to position using a thicker tungsten needle generated to the peptide RKRESPATQRKQEKRERSKEASK (Sigma- (0.01 inch diameter). Embryos were cultured for 48 hours in ovo in a 38.5°C Aldrich). Antiserum was affinity purified against the target peptide using an humidified incubator. Aminolink column (Pierce) according to the manufacturer’s instructions. Acknowledgements The authors would like to thank A. McMahon and T. Jessell for providing probes for Immunofluorescence in situ hybridization. For immunofluorescence on sections, embryos were fixed in 4% paraformaldehyde for 1 hour at room temperature. Fixed embryos were Competing interests embedded in 7.5% gelatin and 15% sucrose in PBS, and cryosectioned at The authors declare no competing financial interests. 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% Author contributions horse serum and 0.02% Tween 20 in PBS) for 15 minutes, sections were M.G., R.Y. and T.M.S. conducted the experiments. All the authors were involved in incubated with the primary antibodies rabbit anti-Nphs2 (1:4000) and mouse designing and analyzing the experiments and writing the paper. anti-Lhx1 (1:10) (Developmental Studies Hybridoma Bank, Clone 4F2). Funding Alexa Fluor 488-, Cy3- and DyLight 649-conjugated secondary antibodies This work was supported by grants from the March of Dimes [FY12-343 to T.M.S.]; (Jackson ImmunoResearch) were used at 1:250 in blocking solution. DAPI the Israel Science Foundation [1328/08 and 1573/12 to T.M.S.]; the Rappaport at 1 μg/ml was used to visualize nuclei. Images were collected on a Zeiss Family Foundation [T.M.S.]; the Charles Krown Research Fund [T.M.S.]; and the Axioimager microscope with a Qimaging ExiBlue digital camera and Image National institutes of Health (NIH) [DK45218 to D.H.]. D.H. was the recipient of a Pro plus imaging software. Visiting Professor Fellowship from the Lady Davis Fellowship Trust. Deposited in For immunofluorescence of explant cultures, explants were fixed in 4% PMC for release after 12 months. 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. References Acharya, A., Baek, S. T., Banfi, S., Eskiocak, B. and Tallquist, M. D. (2011). Efficient Explants were blocked in TSP with 1% sheep serum for several hours and then inducible Cre-mediated recombination in Tcf21 cell lineages in the heart and kidney. incubated overnight at 4°C with the primary antibodies rabbit anti-Nphs2 Genesis 49, 870-877. (1:4000) and mouse anti-Lhx1 (1:10) (Developmental Studies Hybridoma Attia, L., Yelin, R. and Schultheiss, T. M. (2012). Analysis of nephric duct Bank, Clone 4F2). The next day, the explants were washed in TSP (three × 15 specification in the avian embryo. Development 139, 4143-4151. 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