Pattern and regulation of cell proliferation during murine ureteric bud development

Authors



Dr L. Michael and Dr J. A. Davies, Genes and Development Group, Anatomy Building, University of Edinburgh College of Medicine, Teviot Place, Edinburgh EH8 9AG, UK. E: Lydia.Michael@ed.ac.uk, Jamie.Davies@ed.ac.uk

Abstract

Branched epithelia determine the anatomy of many mammalian organs; understanding how they develop is therefore an important element of understanding organogenesis as a whole. In recent years, much progress has been made in identifying paracrine factors that regulate branching morphogenesis in many organs, but comparatively little attention has been paid to the mechanisms of morphogenesis that translate these signals into anatomical change. Localized cell proliferation is a potentially powerful mechanism for directing the growth of a developing system to produce a specific final morphology. We have examined the pattern of cell proliferation in the ureteric bud system of the embryonic murine metanephric kidneys developing in culture. We detect a zone of high proliferation at the site of the presumptive ureteric bud even before it emerges from the Wolffian duct and later, as ureteric bud morphogenesis continues, proliferation is localized mainly in the very tips of the branching epithelium. Blocking cell cycling using methotrexate inhibits ureteric bud emergence. The proliferative zone is present at ureteric bud tips only when they are undergoing active morphogenesis; if branching is inhibited either by treatment with natural negative regulators (TGF-β) or with antagonists of natural positive regulators (GDNF, glycosaminoglycans) then proliferation at the tips falls back to levels characteristic of the stalks behind them. Our results suggest that localized proliferation is an important morphogenetic mechanism in kidney development.

Introduction

Branched tubular epithelial structures are found in most animals and function to transport gases and liquids in the body. Many essential organs such as the lung, mammary gland, pancreas and kidney are composed of ramifying networks of epithelial tubes that develop through branching morphogenesis (Davies & Fisher, 2002). The branched epithelia of these organs arise from initially unbranched buds, which grow, elongate and bifurcate in a highly reproducible way to form a characteristic three-dimensional, tree-like structure (Hogan, 1999). Growth and bifurcation of the epithelial bud is co-ordinated by positive and negative reciprocal feedback between signalling centres in the mesenchyme and epithelium.

The developing mouse metanephric kidney is an established system for studying organogenesis because of its ability to develop well in culture (Saxen, 1987). Of its two epithelial components, the collecting duct system develops by typical branching morphogenesis from an initially unbranched epithelium, the ureteric bud (Davies & Bard, 1998). The nephrons, by contrast, develop by a mesenchymal to epithelial transition (Davies, 1996) and connect to the collecting ducts later. Intensive work on the kidney has identified a large number of extracellular regulators of branching such as members of the GDNF family (Moore et al. 1996; Sainio et al. 1997; Milbrandt et al. 1998; Davies et al. 1999), TGF-β family (Ritvos et al. 1995), BMP-2 (Piscione et al. 1997), HGF (Woolf et al. 1995) and others (reviewed in Davies & Davey, 1999; Davies, 2001). By contrast, little is known about the basic morphogenetic processes within the ureteric bud/collecting duct cells that translate these signals into anatomical change.

One potentially powerful mechanism of epithelial morphogenesis is cell proliferation. Proliferation in general can in principle explain the bulk increase in size of the collecting duct system, but accurately localized cell proliferation could do more than that and could in principle drive extension of the epithelium in specific directions. Differential rates of cell division are able to produce characteristic shape changes during organ formation. In teeth, for example, areas of non-dividing epithelial cells (enamel knot) are surrounded by areas of strongly proliferative cells, the disparity of proliferation rates forcing dental epithelia to fold (Jernvall & Thesleff, 2000).

Studies on branching epithelia derived from other germ layers support the idea that proliferation is tightly localized during epithelial branching. In the developing lung and exocrine pancreas, both derived from endoderm, there is evidence that implicates localized intense cell proliferation in bud formation (Goldin et al. 1984). In the ectodermally derived mammary gland, the terminal end buds are highly mitotic during branching morphogenesis (Daniel et al. 1984; Humphreys et al. 1996). The association between branching and localized proliferation is not universal, however. In Drosophila, the tracheal system forms without cell proliferation and new branches are formed by migration, elongation and rearrangement of tracheal cells (Samakovlis et al. 1996). The cells of the Drosophila Malpighian tubules, the insect analogue of the vertebrate excretory system, undergo a tightly regulated programme of cell divisions to reach a finite number of cells. Only then do the tubules undergo most of their morphogenesis, by cell growth rather than division (Skaer, 1989, 2003). In both of those systems, cell enlargement and rearrangement are adequate mechanisms for morphogenesis, and proliferation is not therefore a prerequisite for organogenesis.

Even if cell proliferation is involved in growth and branching of the ureteric bud, a mesodermally derived structure, it may not be restricted to the part that is obviously moving forwards, as epithelia can elongate by means of convergent extension cell rearrangements so that cells throughout their lengths contribute to the advancement of the ends. Examples of morphogenetic changes by means of convergent extension occur during gastrulation and shaping and patterning of the neural ectoderm in Xenopus laevis (Keller & Danilchik, 1988; Shih & Keller, 1992; Elul et al. 1997), and during the primary invagination in the forming archenteron of the sea urchin, in which localized cell proliferation is not involved (Burke et al. 1991).

We have therefore performed a careful study on the location of cell proliferation in ureteric buds at different developmental stages in kidneys growing in culture, from the very first signs of ureteric bud emergence to the development of a highly branched tree. We have also used known regulators of ureteric bud branching to study the correlation between proliferation and branching activity. We find that proliferation is localized mainly to the very tips of the epithelium. Furthermore, a zone of tightly localized proliferation precedes the first emergence of the ureteric bud from the nephric duct, timing suggesting that proliferation may be a cause rather than a consequence of ureteric bud protrusion. Furthermore, this pattern of proliferation is seen only when branching is actively taking place and disappears when branching is inhibited by a variety of experimental interventions.

Materials and methods

Organ cultures

Whole organs

Metanephric kidney rudiments were isolated from embryos of MF1 mice at E11.5 (morning of discovery of vaginal plug was taken to be E0.5) and were cultured on 5-µm isopore membrane filters (Millipore; Sigma) placed on Trowell screens in culture medium as previously described (Davies, 1994). The culture medium used was Eagle's minimal essential medium with Earle's salts (Sigma) with non-essential amino acids, supplemented with 10% newborn calf serum (Labtech) and 5% antibiotics (penicillin/streptomycin; Sigma). Kidneys were pooled and assigned randomly to control or treatment groups. For studies on ureteric bud emergence, nephrogenic ridges (nephric ducts with their surrounding mesenchyme) were isolated by microdissection from E10.5 embryos, which are too young to have formed a ureteric bud. They were cultured for a time period according to the experiment as described above. All cultures were incubated in a humidified atmosphere at 5% CO2 at 37 °C.

Cell proliferation assay

BrdU incorporation and experimental treatments

Cell proliferation was measured using 5-bromo-2′-deoxy-uridine (BrdU; Sigma) which was introduced to the media at a final concentration of 100 µm for either a time course of: 0–16, 16–32, 32–48 and 48–64 h or for 4 or 24 h prior to fixation. In the case of the Wolffian ducts, BrdU was present for the total culture period.

The experimental treatments were as follows. Whole kidney rudiments were cultured for 48 h in the presence of 30 mm sodium chlorate (AnaLaR, BDH), an inhibitor of sulphated glycosaminoglycan synthesis, which blocks ureteric bud branching in culture (Davies et al. 1995). Exogenous GDNF (Sigma) was added to the cultures at a final concentration of 100 ng mL−1 for 24 h and 48 h. For local application of growth factor, Biorad Affigel Blue beads were washed in serum-free medium for 30 min at 37 °C, and were then soaked in 1% bovine serum albumin (BSA) in PBS (control) or 50 µg mL−1 GDNF (experimental) for a further 30 min in 37 °C. The beads were then placed on one side of the T-stage E11.5 kidney rudiments for 48 h. Kidney rudiments were also cultured in 10 µg mL−1 function-blocking anti-GDNF (R & D systems) for 48 h. TGF-β1 (Sigma) was added to the complete medium at a final concentration of 1 nm for 24 or 72 h. Wolffian ducts were cultured for a time course of 4, 6, 8, 12 and 24 h in order to establish the approximate time that the ureteric bud evaginates from the duct in culture. Then Wolffian ducts were cultured for 8 h in the presence of 100 µm BrdU. DNA synthesis was blocked by 500 nm methotrexate (Sigma) for 24 h (Davies et al. 1995).

Immunohistochemistry

The procedure followed was that of Davies et al. (1995) with some modifications. Cultured rudiments were fixed in −20 °C methanol for 10 min at room temperature (RT). After fixation, kidneys (or Wolffian ducts) were washed in PBS for 30 min then incubated in 4% paraformaldehyde (PFA; BDH) for 30 min at RT (we found that this improved tissue integrity by the end of the BrdU protocol). They were then washed in PBS, incubated in 0.5 mg mL−1 trypsin (Sigma) for 7 min at 37 °C, refixed in 4% PFA for 1.5 h at RT and washed in PBS. To denature the DNA for BrdU detection, rudiments were incubated in 95% formamide, 5% 0.15 m trisodium citrate (BDH) for 1 h at 70 °C. They were washed in PBS for 30 min (RT) and incubated at 4 °C overnight in the presence of 1 : 200 anti-calbindin D28k (Chemicon) or 1 : 100 anti-laminin (Sigma) and 1 : 50 anti-BrdU (mAb BU-33, Sigma) in PBS. Samples were then washed for 30 min in PBS and incubated in secondary antibodies overnight at 4 °C. The secondary antibodies used were: 1 : 100 FITC or TRITC-conjugated anti-rabbit IgG (Sigma) and 1 : 100 TRITC or FITC-conjugated anti-mouse IgG (Sigma) in PBS. After a last PBS wash, samples were mounted on slides and viewed on a confocal laser scanning microscope.

Confocal microscopy

The samples were examined on the TCS NT Leica confocal laser scanning microscope (Leica Microsystems, Heidelburg, Germany). For dual-labelled samples, both FITC and TRITC emissions were acquired simultaneously, once appropriate checks had been carried out for any cross-talk between the channels. Serial 3-µm optical sections of each kidney were acquired.

Quantification of cell proliferation

Quantification of BrdU-positive nuclei was performed on stacks of three middle optical sections (each of 3 µm thickness), which were selected from points in the original set of confocal sections, at which the relevant tip was its widest.

The software packages used for the analysis were Adobe Photoshop (version 5.0) and Scion Image (version beta 4.0.2) For measuring cell proliferation along the branching ureteric bud epithelium or along the Wolffian ducts, the quantification method described in Fisher et al. (2001) was used. Using Adobe Photoshop, successive lines of 100 µm were drawn across the ureteric bud tubules starting from the terminal tip edge towards the stalk. These lines divided the tubule into 100-µm-long segments. The BrdU-positive nuclei, in each segment, were then counted. Data are expressed as mean ± SEM throughout. In the case of Wolffian ducts, the lines started from the caudal tip of the duct and continued towards the mesonephros. The tissue area that the lines defined was measured (µm2) and the number of BrdU-positive nuclei per tissue area was counted. Data are expressed as mean BrdU-positive nuclei per 1000 µm2 of epithelium ± SEM.

For measuring cell proliferation at the ureteric bud tips under the experimental treatments, optical sections stacks were imported to Scion Image, calibrated to the appropriate scale (500 µm2) as all the original optical sections were scanned at ×20 objective, and were converted into greyscale images. Using the free-hand tool to draw round the tip, the tip area was measured (µm2). A user-defined adjustment of the fluorescence background threshold was applied. The threshold value was taken as three times higher than the mean background value plus the standard deviation of the selected area. Anything above this value was considered as a BrdU-positive nucleus. (In practice, this coincided well with a qualitative ‘eyeballing’ assessment.) The BrdU-positive nuclei inside the selected area were counted.

Some of the experimental treatments affected the morphology of the epithelium. For measuring cell proliferation at the ureteric bud tips, a specific length was drawn across a well-defined control ampullary tip from the outer edge towards the stalk and was used in all the tips (control and experimental) of each experiment to be analysed. The choice of the applied length was made upon measuring an average sized and well-structured control tip. This selection was made anew for each experiment (but in practice varied little between experiments).

Results

Localized cell proliferation is confined to the tips of ureteric bud branching epithelium

The hypothesis that localized cell proliferation is a feature of the ureteric bud tip during branching morphogenesis of the developing epithelium was tested by culturing mouse kidney rudiments in the presence of BrdU, a thymidine analogue that is incorporated into cell DNA during the S phase of the cell cycle. E11.5 metanephroi were cultured (Fig. 1) and cell proliferation was measured for the culture period of 16–32 h (Fig. 1B), BrdU being present for the last 24 h of the culture period (or for the total culture period, whichever was lesser). Qualitatively, it was obvious that more proliferating nuclei were localized at the tips of the ureteric bud than in the stalks distal to them (Fig. 1). It is conventional for BrdU incorporation to be expressed quantitatively as the ratio of labelled to total nuclei in a given area. In our system, however, the thickness of the whole-mounts made counting of the total number of nuclei (labelled with ToPro and similar dyes) unreliable even with a confocal microscope, but physical sectioning ran an unacceptable risk of obliquely sectioned curving tubules being mistaken for tips. We therefore chose to express proliferation as the number of labelled nuclei per unit area of ureteric bud (see Materials and methods for details). The mean number of BrdU-positive nuclei at the tip (the proximal 100 µm) was 12.1 ± 1.60; in the subsequent 100 µm, the proximal zone of the stalk, it had reduced to only 3.47 ± 0.67 and in the next 100 µm of stalk it was 3.50 ± 1.05 (Fig. 1E). These results suggest that proliferation is concentrated at the ureteric bud tips. A similar concentration of BrdU labelling in the tips was seen when BrdU was present only for the last 4 h of culture, although the absolute numbers of labelled nuclei were of course lower (Fig. 1F).

Figure 1.

Pattern of cell proliferation in the ureteric bud epithelium during a time course of (A) 0–16 h, (B) 16–32 h, (C) 32–48 h and (D) 48–64 h in culture. The arrows show examples of BrdU-positive nuclei, which are mainly confined to the branching tips regardless of whether a 24-h (E) or 4-h (f) BrdU pulse is used. Immunofluorescence double staining: TRITC–anti-BrdU, FITC–anti-laminin. Scale bar = 100 µm.

Localized cell proliferation is the first sign of ureteric bud outgrowth from the Wolffian duct

Because cell proliferation is localized at the ureteric bud tips, it is possible that localized proliferation precedes or even drives the formation of the first tip, the ureteric bud itself. In order to test this hypothesis, BrdU was incorporated into Wolffian ducts at the stage prior to and around the moment of emergence of the ureteric bud.

The caudal regions of intermediate mesoderm, including Wolffian ducts, were isolated from E10 nephrogenic ridges and cultured for 4, 6, 8, 12 and 24 h (Fig. 2); the ureteric bud was found to emerge at around 8 h of culture (Figs 2C and 3A). At each of these stages, BrdU incorporation was quantified along consecutive 100-µm intervals along the Wolffian duct from the caudal end up towards the mesonephros (Fig. 3) and was expressed as the average number of BrdU-positive nuclei per 1000 µm2 measured along these lengths. At the first and most caudal part of the duct, the average number of proliferating cells was quite low (2.44 ± 1.29); it then increased along the three consecutive 100-µm lengths to reach a maximum of 9.91 ± 1.59 positive nuclei/1000 µm2 and then dropped gradually (Fig. 3C). These results support the hypothesis that at the presumptive metanephric area there is an area of increased localized cell proliferation where the ureteric bud will evaginate. Outside the presumptive metanephric area, along the nephric duct towards the position of the mesonephros, the average number of proliferating epithelial cells showed no specific pattern (Fig. 3B).

Figure 2.

Confocal images of the caudal areas of developing Wolffian ducts. (A) Wolffian ducts cultured for 4 h, (B) 6 h, (C) 8 h when the caudal part of the duct starts to enlarge, (D) 12 h when the ureteric bud has emerged and (E) 24 h when the ureteric bud has already branched twice (yellow arrows). Immunofluorescence: FITC–anti-calbindin D28k (A–D), FITC–anti-laminin (E). Scale bars = 100 µm.

Figure 3.

(A) Two immediately adjacent confocal sections of Wolffian duct cultured for 8 h, showing presumptive site of ureteric bud emergence. Arrows show the BrdU-positive nuclei at the metanephric area. Quantification of BrdU incorporation along the nephric duct in the area craniad to the presumptive metanephric area shows random variation (B), whereas at the area where ureteric bud is forming there is a sustained increase in cell proliferation (C). The yellow arrow shows the site of the mesonephros, and the inset figures show the manner in which the duct was divided for measurement. Treatment of cultures with the DNA synthesis inhibitor, methotrexate, significantly inhibits ureteric bud emergence (D). Immunofluorescence double staining: FITC–anti-BrdU, TRITC–anti-calbindin D28k. Scale bar = 100 µm.

To investigate whether proliferation is necessary for ureteric bud outgrowth or is simply a ‘downstream’ effect of that outgrowth, E10 nephrogenic ridges were treated with methotrexate, an inhibitor of DNA synthesis and therefore of cell cycling. Although all control cultures produced a ureteric bud over the following 24 h, significantly fewer methotrexate-treated cultures did so (Fig. 3D; P = 0.03). We assume that the variability between treated cultures reflected variability in their precise stage of embryonic development and the numbers of cycling cells that had already passed S phase.

Localized cell proliferation correlates with branching activity

In order to test whether the higher proliferation rate observed at the ureteric bud tips is present only while branching morphogenesis is taking place, branching was arrested by a variety of methods and the pattern of BrdU incorporation was compared with that in normally growing controls.

First, E11.5 kidney rudiments were cultured in 30 mm sodium chlorate, which inhibits the synthesis of sulphated glycosaminoglycans and blocks ureteric bud development (Davies et al. 1995, 1999; Kispert et al. 1996; Milbrandt et al. 1998). Branching in the chlorate-treated kidneys was inhibited by this treatment but the remaining tips were significantly larger than control tips. Qualitatively, proliferation at the ureteric bud tips was reduced in the kidneys cultured in chlorate (Fig. 4A,B). Quantitatively, BrdU incorporation in the ureteric bud tips of chlorate-treated kidneys was only 0.58 ± 0.16/1000 µm2, compared with 1.28 ± 0.16/1000 µm2 in controls (Fig. 4E). The difference is statistically significant (P = 0.008). The use of a function blocking antibody to GDNF causes a reduction in ureteric bud branching (Towers et al. 1998; Davies et al. 1999). The addition of 10 µg mL−1 anti-GDNF in culture for 48 h resulted in significantly less branching (Fig. 4C,D) (P = 0.01) and loss of the typically swollen character of the ampullary tip. Quantification of BrdU incorporation revealed a significant reduction in cell proliferation at the tips when GDNF signalling is blocked (Fig. 4F; P = 0.03) without any effect at the proliferation in the stalks (P = 0.89).

Figure 4.

Proliferation in ureteric buds treated with sodium chlorate (A,B) and function-blocking antibody to GDNF (C,D). Controls: (A) and (C), respectively. Quantification of BrdU incorporation shows reduction in cell proliferation at the tips of the ureteric epithelia when branching is blocked with 30 mm sodium chlorate (E) or with 10 µg mL−1 anti-GDNF without affecting the proliferation in the stalks (F). Immunofluorescence: double staining TRITC–anti-BrdU, FITC–anti-laminin (A,B); FITC–anti-BrdU, TRITC–anti-laminin (C,D). Scale bars = 100 µm.

TGF-β, an endogenous growth factor, has previously been reported to exert an inhibitory effect on branching morphogenesis of the ureteric bud (Ritvos et al. 1995; Sakurai & Nigam, 1997; Clark et al. 2001). We found that, by 72 h of culture in 1 nm TGF-β (Fig. 5A,B), kidneys showed a significant reduction in branching morphogenesis, which was confirmed by counting the numbers of tips (control average number of tips: 15.71 ± 1.02; TGF-β: 8.17 ± 0.40, P = 0.00014) (Fig. 5E). The morphology of the ureteric buds fell into a range of phenotypes, which included short branches emerging from the stalks. In order to examine whether 1 nm TGF-β affects cell proliferation at the bud tips during the time that branching was taking place and the foundations were being laid for the final difference in morphology, BrdU was added to kidneys for 24 h (Fig. 5C,D). After this short time, the TGF-β-treated kidneys showed less branching, although this was not yet quite statistically significant because not even the controls had had much time to branch significantly (Fig. 5E), but the average of proliferating tip cells was significantly lower (P = 1.59 × 10−5) than in controls (Fig. 5F). By 72 h, the cumulative differences in development between experimental and control cultures had produced statistically significant differences in the extent of branching (Fig. 5E).

Figure 5.

Confocal sections of the effect of TGF-β on ureteric bud branching morphogenesis (A,B) and cell proliferation in culture (C,D). Arrows in (C) and (D) show representative BrdU-positive nuclei at the tips of the epithelium. Quantification of branching (expressed as the number of tips) shows a significant reduction after 72 h in TGF-β but not after 24 h (E). Measuring cell proliferation at the tips of the ureteric buds shows a significant reduction even after 24 h (F). Immunofluorescence: TRITC–anti-laminin (A,B), double staining TRITC–anti-BrdU, FITC–anti-calbindin D28k (C,D). Scale bars = 100 µm.

Together, these results support the hypothesis that local proliferation is a feature of active tips.

The effect of exogenous GDNF on cell proliferation at the ureteric bud tips

GDNF is an important paracrine factor involved in metanephric development. It is produced by the mesenchyme and binds and signals to the ureteric bud tips (Sainio et al. 1997) and exogenous GDNF drives ureteric bud overgrowth and can produce ectopic ureteric buds. In order to test whether GDNF-induced abnormal morphogenesis is associated with abnormal proliferation, we tested the effect of exogenous GDNF on cell proliferation at the ureteric bud tips. When applied to bulk culture medium, 100 ng mL−1 GDNF did not result in a significant increase in the number of tips, but rather in an abnormal morphology of the epithelium with distorted branching pattern. Most obviously, the existing tips became very large and distended while new tips emerged from the stalk region of the ureter and, where it was present, from the Wolffian duct (Fig. 6B). The expansion of the tips could be seen quantitatively as well as qualitatively (Table 1).

Figure 6.

Confocal sections of kidneys in culture after application of GDNF either generally (B) or locally (D). (A) A control kidney in culture and (C) a control bead (steeped in 1% BSA). Quantification of the BrdU incorporation at the tips of the epithelium when GDNF is applied generally (E) or locally on a bead (F). White dotted arrows show the BrdU-positive nuclei at the tips of the epithelium (A–D), yellow arrows show the ectopic tips (B). Immunofluorescence: double staining FITC–anti-BrdU (A–D), TRITC–anti-calbindin D28k (A,B), TRITC–anti-laminin (C,D). Scale bars = 100 µm.

Table 1.  The effect of GDNF application on the ureteric bud tip size
TreatmentAverage tip size (µm2)
Control4.8 × 103
GDNF (bulk medium)2.6 × 104
Control bead5.3 × 103
GDNF bead2.6 × 104

Quantification of the BrdU incorporation showed that the average number of BrdU-positive nuclei per 1000 µm2 of tip epithelium was not significantly different in control and GDNF tips (P = 0.38), whereas the cell proliferation at the ectopic tips was significantly lower than at the control tips (P = 0.02) (Fig. 6E). This result suggests that exogenous GDNF does not exert a detectable mitogenic effect on ureteric bud epithelial tips in culture.

To supply cultured kidneys with a local source of GDNF (Sainio et al. 1997), an agarose bead soaked in 50 µg mL−1 GDNF was placed adjacent to one of the tips of T-shaped stage E11.5 ureteric buds. The effect was similar to that seen when the growth factor was added to bulk medium (Fig. 6C,D). The tips were enlarged compared with controls, and ectopic buds emerged from the stalks. Quantification of BrdU incorporation revealed that, under these circumstances, GDNF did cause an increase in the number of proliferating cells at the ureteric bud tips (Fig. 6F). The average number of BrdU-labelled nuclei per 1000 µm2 of epithelium for the control beads was 2.76 ± 0.44 and for the GDNF beads 5.69 ± 1.43 (P = 0.05).

A summary of these results is shown in Table 2.

Table 2.  Summary of effects of culture conditions on proliferation within the ureteric bud tips
TreatmentEffect on proliferation
ChlorateReduced
Anti-GDNFReduced
GDNF (bulk medium)No significant difference
GDNF (local source)Increased (locally)
TGFβReduced

Discussion

During branching morphogenesis, the emergence of new buds is an active process, which is often associated with high cell proliferation; an example of this is also found in the branching prostate (Sugimura et al. 1986). In the present study we found that locally high levels of proliferation are a feature of the tips, but not the stalks, of actively branching ureteric buds. Furthermore, we found that a zone of intense proliferation appears at the presumptive site of ureteric bud emergence from the Wolffian duct even before any overt morphogenesis of the bud has taken place. This suggests that localized cell proliferation might be an important morphogenetic mechanism that underlies the emergence of the bud and, by extension, the emergence of all future buds/tips during branching morphogenesis. Local proliferation is present only when morphogenesis is actively taking place, and disappears when kidneys are treated either with anti-branching growth factors (TGF-β) or with agents that block the action or synthesis of branch-promoting molecules (anti-GDNF, chlorate ions).

The GDNF family of molecules have long been known to be key regulators of ureteric tip development; GDNF itself is necessary for the formation of a normal ureteric bud tree (which fails in gdnf–/– animals) and in culture all members of the GDNF family tested so far – GDNF, neurturin and persephin – can promote the formation of new ureteric bud tips (Pichel et al. 1996; Sainio et al. 1997; Milbrandt et al. 1998; Davies et al. 1999). Although GDNF is known to promote ureteric bud branching in culture (Towers et al. 1998), it is not clear from published evidence whether it acts directly as a mitogen in this system. The molecule has the ability to act as a mitogen for other cell types, for example neural crest (Chalazonitis et al. 1998; Heuckeroth et al. 1998; Taraviras et al. 1999; Worley et al. 2000) and the sertoli cells of the testis (Hu et al. 1999). Pepicelli et al. (1997) cultured kidneys in the presence of added GDNF and, as well as reporting distortions of the tips similar to those that we have reported here, suggested that GDNF increased proliferation. In experiments that set out specifically to test the mitogenic effect of GDNF on ureteric bud cells, but which used an unusual culture system consisting of buds isolated from their surrounding mesenchyme and culture in hanging drops, exogenous GDNF failed to increase cell numbers and instead affected cell adhesion (Sainio et al. 1997). This result was clear for that culture system, but the cells in it may not have responded normally in the absence of other mesenchyme-derived signals with which they would normally be provided. In our experiments, addition of exogenous GDNF to the complete culture medium had no significant effect on proliferation of tips in the main ureteric bud tree.

Application of a point source of GDNF did result in increased proliferation in tips immediately next to that point source, a result that appears at variance with the results of adding GDNF to the bulk medium. There are two possible explanations for this. The first is that, because the bead was steeped in a high concentration of GDNF (50 µg mL−1), the local concentration in the immediate vicinity of the bead is significantly higher than that which could be applied to the bulk medium, and this massive concentration is enough to drive proliferation. The other possible explanation is that the cells respond mitogenically to a gradient of GDNF rather than to an absolute value (only application by the bead would have created a significant gradient). In the intact kidney the main source of GDNF is the population of ‘uninduced’ mesenchyme cells, not yet contacted by the ureteric bud and lying cortically. Ret is expressed only by the bud itself, and the co-receptor, GFRα1, by all cells. By invading the mesenchyme and inducing it to differentiate, the ureteric bud switches off the production of GDNF by that mesenchyme. Therefore, one would expect there to be a gradient of GDNF in the normal developing kidney, with the highest concentrations in the cortical regions that have not yet been invaded by the bud. If this gradient were sensed by the bud and used to direct its proliferation, the direction of advance of the tubule tips would be set automatically towards areas of mesenchyme not yet invaded.

The GDNF family of growth factors, which are positive regulators of ureteric bud morphogenesis, are themselves members of the TGF-β superfamily. Other members of this superfamily, such as the bone morphogenetic proteins and TGF-β itself, inhibit branching in a variety of systems (see Davies & Fisher, 2002, for review). Our results show that addition of TGF-β results in a reduction of branching after 72 h in culture, consistent with the findings of Clark et al. (2001) in which TGF-β1 added to cultured rat metanephroi inhibits overall metanephric and ureteric bud growth, leading to reduced nephron endowment, and of Ritvos et al. (1995), in which TGF-β resulted in spindly and underbranched ureteric bud systems. We find that TGF-β treatment causes a significant reduction in cell proliferation at the tips of the ureteric bud epithelium. This is similar to studies in other developing branching organs such as the lung, mammary gland and pancreas. In the lung organ culture, TGF-β1 reduces branching and overall lung size (Serra et al. 1994; Bragg et al. 2001) and a reduction in [3H]thymidine- or BrdU-labelling of nuclei in the epithelium (Serra et al. 1994). Local administration of TGF-β to mammary gland cultures results in inhibition of normal branching morphogenesis so that ducts of the treated glands do not elongate and instead resemble growth-quiescent thin-walled ducts with blunt-tipped terminal ends (Silberstein & Daniel, 1987). Again, DNA synthesis at the tips of the ducts (‘end buds’) is reduced to levels similar to that in growth-quiescent ducts (Silberstein & Daniel, 1987; Daniel et al. 1989). In the cultured embryonic pancreas addition of TGF-β results in a reduction in epithelial cell proliferation (Sanvito et al. 1994).

The reduced rates of proliferation seen in ureteric bud tips of kidneys treated with TGF-β may account directly for the short branches that are observed in these kidneys. Indeed, in some cell types, TGF-β is known to inhibit cell-cycle progression by causing the retention of the retinoblastoma gene product (Rb) in the underphosphorylated, growth suppressive state (reviewed in Laiho et al. 1990; Massague, 1990). The explanation for the short buds may, however, be indirect; for example, TGF-β may stimulate the deposition of extracellular matrix components or tissue inhibitor of metalloproteinases (TIMP) around the developing ureteric bud tips, as it does in other systems (reviewed by Massague, 1990; Daniel & Robinson, 1992), and consequently limits their development.

To summarize, in the present study we have shown that the pattern of cell proliferation in the mesodermally derived ureteric bud/collecting duct system is similar to that seen in the branching epithelia of other ectodermally and endodermally derived mammalian organs, in that proliferation is concentrated mainly in the ampullary tips. Furthermore, we have shown that localized cell proliferation is a specialization of the actively growing ureteric bud tips and is absent when branching is blocked. This pattern suggests that local proliferation is a key morphogenetic mechanism of renal development; understanding how the many signals that converge on the ureteric bud are integrated to control cell proliferation is therefore an important subject for future research.

Acknowledgements

We gratefully acknowledge the help and good humour of Linda Wilson, who provided us with advice on confocal microscopy, Peter Bush who advised us on image analysis, and John West for helpful discussions. We thank the Wellcome and the Leverhulme Trusts for their support.

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