Departments of Pediatrics and Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0693, USA
*Author for correspondence (e-mail: snigam{at}ucsd.edu)
Accepted June 10, 2001
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SUMMARY |
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Key words: Ureteric bud, Kidney development, Mesenchymal epithelial interaction, Pleiotrophin, Rat, GDNF
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INTRODUCTION |
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Soluble factors that have been hypothesized to function in such a morphogenetic capacity include hepatocyte growth factor (HGF) and epidermal growth factor (EGF) receptor ligands, which have been shown to induce branching tubular structures in epithelial cells cultured in collagen gels (Barros et al., 1995; Cantley et al., 1994; Montesano et al., 1991; Sakurai et al., 1997b). However, these studies have been carried out largely on adult cell lines. In a cell culture model that employs UB cells, an epithelial cell line derived from embryonic day 11.5 (E11.5) mouse UB, neither HGF, EGF receptor ligands, or many other factors tested (alone or in combination), were able to induce UB cells to form branching tubular structures with lumens (Sakurai et al., 1997a). However, UB cells undergo branching tubulogenesis in the presence of a conditioned medium elaborated by a cell line derived from the MM also isolated from an E11.5 mouse (BSN cells; Sakurai et al., 1997a). This data suggests that other, yet to be identified, soluble factors present in BSN-CM are important for UB cell morphogenesis. These potentially novel factors that are presumably secreted by the MM may be as (or more) important for the development of the collecting system as those currently receiving attention.
This MM-derived cell conditioned medium (BSN-CM), when supplemented with glial cell-derived neurotrophic factor (GDNF), also induces the isolated rat UB (in the absence of MM) to undergo dichotomous branching reminiscent of that seen in the developing kidney (Qiao et al., 1999a). This indicates that the MM-derived cell line, presumably reflecting the MM itself, secretes soluble factors capable of inducing branching morphogenesis of the UB. This isolated UB culture system can serve as a powerful assay system since it directly assesses the effect of soluble factors on UB morphogenesis.
We have isolated a UB branching morphogenetic activity from the BSN-CM and identified it as an 18 kDa heparin binding protein, pleiotrophin. Pleiotrophin was originally discovered as a fibroblast proliferative factor (Milner et al., 1989) and a neurite outgrowth-promoting factor (Rauvala, 1989). Outside the nervous system, pleiotrophin is generally detected in embryonic organs in which mesenchymal-epithelial interactions are thought to play an important role, such as salivary glands, lung, pancreas, and kidney (Mitsiadis et al., 1995; Vanderwinden et al., 1992). Although pleiotrophin has been shown to be mitogenic for certain epithelial cells (Li et al., 1990; Sato et al., 1999), there has been no compelling evidence for a key role for pleiotrophin during epithelial organogenesis. Here, we have shown that purified pleiotrophin induces impressive branching morphogenesis of the isolated UB (in the presence of GDNF) as well as tubule formation in a UB cell line in vitro. We suggest that pleiotrophin is a key metanephric mesenchymally derived factor that plays a critical role in branching morphogenesis of the UB during kidney development.
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MATERIALS AND METHODS |
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Cell culture and conditioned medium
BSN cells were grown to confluency in DMEM/F12 supplemented with 10% fetal calf serum (FCS). The growth medium was removed and the cells were then incubated in serum-free DMEM/F12 for 3-4 days followed by collection of the conditioned medium (Qiao et al., 1999a). Swiss 3T3 cells (ATCC) were grown to confluency in DMEM with 10% FCS. Once the cells were confluent, the growth medium was replaced with DMEM supplemented with 2% FCS and the cells were cultured for an additional 3-4 days. The conditioned medium was collected and used for the experiments. UB cells were cultured in DMEM supplemented with 10% FCS at 32°C in an atmosphere of 5% CO2 and 100% humidity.
Isolated ureteric bud and whole embryonic kidney culture
Timed pregnant female Sprague-Dawley rats at day 13 of gestation (day 0 being the day of appearance of the vaginal plug) were sacrificed and the uteri were removed. The embryos were dissected free of surrounding tissues and the kidneys were isolated. For the culture of the whole kidney rudiment, 2-3 kidneys were applied directly to the top of a polyester Transwell filter (0.4 µm pore size; Corning-Costar). The Transwells were then placed within individual wells of a 24-well tissue culture dish containing 400 µl DMEM/F12 supplemented with 10% FCS with or without purified pleiotrophin. Following 7 days in culture, the kidneys were fixed in 2% paraformaldehyde and double-stained with fluorescein-conjugated Dolichos biflorus lectin, which binds specifically to UB-derived structures (Laitinen et al., 1987), and rhodamine-conjugated peanut agglutinin, a lectin which binds to structures derived from the MM (Laitinen et al., 1987), as described previously (Qiao et al., 1999a). Fluorescent staining was detected using a laser-scanning confocal microscope (Zeiss).
In the case of culture of the isolated UB, the isolated kidneys were trypsinized for 15 minutes at 37°C in L-15 medium containing 2 µg/ml trypsin (Sigma). Trypsin digestion was arrested by the addition of 10% FCS and the kidneys were removed to fresh L-15 where the UBs were isolated from surrounding MM by mechanical dissection. Isolated UBs were suspended within an extracellular matrix gel [1:1 mixture of growth factor reduced Matrigel (BD) and Type 1 collagen (BD)] applied to the top of a polyester Transwell filter (0.4 µm pore size; Corning-Costar). The Transwells were placed within individual wells of a 24-well tissue culture dish containing 400 µl of either whole BSN-CM, purified fractions of BSN-CM, or DMEM/F12 which were supplemented with human recombinant fibroblast growth factor 1 (FGF1; 250 ng/ml; R&D Systems), rat recombinant GDNF (125 ng/ml; R&D Systems) and 10% FCS and cultured as previously described (Qiao et al., 1999a). Phase-contrast photomicrographs of the developing UB were taken using a RT-Slider Spot digital camera (Diagnostic Instruments Inc.) attached to a Nikon Eclipse TE300 inverted microscope.
Three-dimensional UB cell culture
Confluent monolayers of UB cells were removed from tissue culture dishes by light trypinization and the cells (20,000 cells/ml) were suspended in an extracellular matrix gel composed of 80% Type 1 collagen and 20% growth factor-reduced Matrigel (Sakurai et al., 1997a). 100 µl of the UB cell-containing gel was then aliquoted into individual wells of a 96-well tissue culture plate. After gelation, 100 µl of growth medium (DMEM/F12 with or without purified pleiotrophin) supplemented with 1% FCS was applied to each well and the cultures were incubated at 32°C in 5% CO2 and 100% humidity. Following 4 days in culture, the percentage of cells and/or colonies processes per colony was counted as an indicator of the tubulogenic activity. Phase-contrast photomicrographs were taken as described above.
Purification of morphogenetic factor
1.5-2 l of BSN-CM collected as described above was filtered to remove extraneous cellular debris using a 0.22 µm polyethersulphone membrane filter (Corning). The BSN-CM was then concentrated approx. 40-fold using a Vivaflow 200 concentrator with a 5 kDa molecular mass cutoff (Sartorius). After adjusting the salt concentration to 0.4 M NaCl, the concentrated BSN-CM was then subjected to sequential liquid column chromatography using an AKTA purifier (Amersham-Pharmacia). Initial fractionation was performed using a heparin sepharose chromatography column (HiTrap heparin, 5 ml; Amersham-Pharmacia). The flow-through fraction was collected and individual 5 ml fractions of the heparin-bound proteins were eluted using increasing concentrations of NaCl (0.4 M-2.0 M) buffered to pH 7.2 with 50 mM Hepes. Aliquots of each fraction were subjected to buffer exchange by dia-filtration using an Ultrafree 500 spin column (Millipore) according to the manufacturers instructions and then tested for morphogenetic activity using the isolated UB culture system.
An active fraction corresponding to the 1.21.4 M NaCl eluate was identified based on its ability to induce branching morphogenesis of the isolated UB. After adjusting this fraction to 1.7 M ammonium sulfate (pH 7.2) it was subjected to further fractionation using a Resource phenyl sepharose hydrophobic interaction column (1 ml; Amersham-Pharmacia). The flow-through was collected and 1 ml fractions of bound proteins were eluted with decreasing concentrations of ammonium sulfate (1.7 M-0 M). After buffer exchange, the individual fractions were again tested for their ability to induce UB branching morphogenesis.
The morphogenetically active fractions from the hydrophobic interaction column were diluted 10-fold with 50 mM Hepes and applied to a Resource S cation exchange column (1 ml; Amersham-Pharmacia). The flow-through was collected and individual 1 ml fractions of bound proteins were eluted using increasing NaCl concentrations (0 M-2.0 M) and assayed for their ability to induce branching morphogenesis.
The active fractions from the Resource S cation exchange column were subjected to further fractionation using a Superdex 200 gel filtration column (Amersham-Pharmacia). Individual 1 ml fractions were collected and assayed for morphogenetic activity. In addition, the active fractions from the Resource S cation exchange column were subjected to SDS-PAGE and the proteins were visualized using Coomassie Blue (Colloidal Coomassie; Invitrogen) staining. Individual protein bands were cut out of the gels and submitted for microsequencing. Sequence analysis of the protein bands was performed at the Harvard Microchemistry Facility by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry (µLC/MS/MS) on a Finnigan LCQ DECA quadrupole ion trap mass spectrometer.
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RESULTS |
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UBs isolated from E13 rat embryos, when suspended in an extracellular matrix gel and cultured in the presence of BSN-CM (with GDNF), grew to form impressive multiply branching tubular structures comparable to those seen in in vivo kidney development (though the growth was non-directional) (Fig. 1B). As previously shown (Qiao et al., 1999a), in the absence of BSN-CM, however, the UBs failed to develop (Fig. 1C). Thus, BSN-CM apparently contains an additional soluble factor(s) necessary for epithelial cell branching morphogenesis. Using this isolated UB culture model as an assay, we attempted to purify the key morphogenetic factor present in the BSN-CM.
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The pattern of pleiotrophin induced UB morphogenesis depends upon its concentration
During the course of purification, we observed differences in the morphology of the branching UB, depending upon the amount of pleiotrophin present in the fraction (detected by immunoblotting). This was examined more carefully using the purified protein in which the pleiotrophin concentration was determined by immunoblotting using recombinant human pleiotrophin as a standard. High concentration (5 µg/ml) pleiotrophin resulted in robust proliferation with less elongation, while lower concentrations of pleiotrophin (156 ng/ml-2.5 µg/ml) induced dichotomous branching and elongation of the stalk (Fig. 5A), similar to that seen with whole BSN-CM.
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Pleiotrophin also induces branching morphogenesis of UB cells in three-dimensional culture
As discussed previously, it has been shown that E11.5 mouse UB-derived cells (UB cells) develop into branching tubular structures with lumens in the presence of BSN-CM. DNA array, PCR analysis and immunostaining have confirmed the epithelial and UB-like characteristics of these cells (Barasch et al., 1996; Pavlova et al., 1999; Sakurai et al., 1997a). Using this model for UB branching morphogenesis, pleiotrophin was also capable of inducing the formation of branching structures of UB cells at concentrations of 200 ng/ml and above. As in the isolated UB culture model, the extent of UB branching morphogenesis was found to be concentration dependent, with higher concentrations resulting in more extensive growth and branching (Fig. 6A). Morphologically, the structures were similar to those induced by whole BSN-CM (Fig. 6B), although there was a higher fraction of spiny cysts.
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DISCUSSION |
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The identification of specific MM-derived soluble factors mediating UB branching morphogenesis remains a central question in this field. HGF has been shown to induce the formation of branching tubular structures with lumens in three-dimensional cultures of epithelial cell lines derived from adult kidneys (i.e., MDCK and mIMCD cells; Barros et al., 1995; Cantley et al., 1994; Montesano et al., 1991; Santos et al., 1993). However, incubation of three-dimensional cultures of an embryonic cell line derived from the UB (UB cells) with HGF had only a slight morphogenetic effect, and the formation of branching tubular structures with lumens was not observed (Sakurai et al., 1997a). Furthermore, HGF, alone or in the presence of GDNF, does not induce branching morphogenesis of the isolated UB (unlike that seen with the MM cell conditioned medium) (Qiao et al., 1999a). These findings suggest that HGF is not an essential factor for early branching morphogenesis of the embryonic UB, though it may play a facilitory role. This notion is supported by the fact that genetic deletion of hgf or its receptor (c-met) apparently has little, if any, effect on kidney development (Bladt et al., 1995; Schmidt et al., 1995).
Another group of soluble factors implicated in branching morphogenesis of epithelial cells are the family of EGF receptor ligands. EGF receptor ligands are capable of inducing the formation of branching tubular structures with lumens in three-dimensional cultures of mIMCD cells, a kidney cell line derived from adult collecting duct cells (Barros et al., 1995; Sakurai et al., 1997b). However, as is the case with HGF, EGF receptor ligands are not capable of inducing the formation of branching tubular structures in three-dimensional cultures of the embryonically derived UB cells (Sakurai et al., 1997a), nor are they capable of inducing branching morphogenesis of the isolated UB (Qiao et al., 1999a). Deletion of the EGF receptor gene results in cystic dilation of collecting ducts in mice of certain genetic backgrounds, perhaps suggesting a role in final maturation of these structures (Threadgill et al., 1995). However, as with HGF, most experimental evidence indicates that the EGF receptor ligands are not essential for early steps in UB branching morphogenesis.
In fact, among many growth factors hypothesized to play a role in kidney development, no single factor, or combination of factors, has been shown to induce UB cells to form branching tubular structures. Only the conditioned medium elaborated by the MM-derived cell line, BSN-CM, consistently induced UB cells in three-dimensional culture to form branching tubular structures with clearly distinguishable lumens (Sakurai et al., 1997a). Likewise, in the isolated UB culture system (in the presence of GDNF), no growth factor, alone or in combination, could induce the extensive branching morphogenesis observed when the isolated UB was cultured with BSN-CM (Qiao et al., 1999a).
An essential role for GDNF in UB development is supported by a number of studies, including gene knockouts. For example, null mutations of gdnf, its receptor, c-ret, or its co-receptor, gfr, result in abnormal kidney development, although variable phenotypes have been reported in the gdnf and c-ret knockout mice (Enomoto et al., 1998; Moore et al., 1996; Schuchardt et al., 1996). Moreover, although the proliferative effect of GDNF on UB cells has been debated (Pepicelli et al., 1997; Sainio et al., 1997), it has been shown to initiate UB growth (Sainio et al., 1997), and it is required for branching morphogenesis of the isolated UB (Qiao et al., 1999a). Nevertheless, GDNF is not sufficient to induce branching morphogenesis of either the isolated UB (Qiao et al., 1999a) or cultured UB cells (Sakurai et al., 1997a), again consistent with the view that there are additional factors in BSN-CM that are critical to the branching morphogenesis of the UB.
Studies in the developing mammalian lung and Drosophila trachea indicate that members of the FGF family function in branching morphogenesis of epithelial tissues (Hogan, 1999; Metzger and Krasnow, 1999; Zelzer and Shilo, 2000). Furthermore, null mutations of either fgf7 or fgf10 have also been reported to affect kidney development (Ohuchi et al., 2000; Qiao et al., 1999b), although in both cases the kidneys appear to be mildly affected. For example, in fgf7-null kidneys, there is a 30% reduction in the number of nephrons, and the kidneys appear to function normally (Qiao et al., 1999b). Moreover, since FGF7 is detected in the developing kidney only after several iterations of UB branching have already occurred, it is likely that other factors are necessary for the early steps of the branching program. In the case of FGF10, the defect appears similar, although the phenotype has yet to be investigated in detail since the embryos die at birth due to severe lung defects (Ohuchi et al., 2000). Nevertheless, by potentiating the effect of an essential branching morphogen produced by the MM, certain FGFs may play a facilitory role in early UB branching morphogenesis (see below).
In this study, serial liquid column chromatographic fractionation of BSN-CM lead to the isolation of an active morphogenetic fraction that contained an apparent single protein (capable of inducing branching morphogenesis comparable to whole BSN-CM). This protein was identified as pleiotrophin (Fig. 2). Immunoblot analysis of BSN-CM (Fig. 7A) as well as in situ hybridization data of developing kidney (Vanderwinden et al., 1992), clearly demonstrated that the embryonic MM is a source of pleiotrophin. In addition to its ability to induce branching morphogenesis in the isolated UB, pleiotrophin also induced a UB cell line to form branching tubular structures with lumens, and is thus the only soluble factor so far identified with this capability (Fig. 6). Based on these in vitro studies with the isolated UB as well as the UB cell line, we propose that pleiotrophin could act as a UB morphogenetic factor produced by the MM.
Pleiotrophin and another heparin binding growth factor, midkine, make up a distinct growth factor family. These proteins share about 50% sequence homology (Rauvala, 1989), both are expressed widely during organogenesis (Mitsiadis et al., 1995), and are highly conserved among species (Kurtz et al., 1995). Both have been implicated in neurite outgrowth (Li et al., 1990; Rauvala et al., 1994), a phenomenon that has some similarity to branching morphogenesis (particularly as it occurs in cultured cells), and they exhibit a spatiotemporal expression pattern in other developing organ systems that suggests a role in mesenchymal-epithelial interactions (Mitsiadis et al., 1995). However, other than the finding that pleiotrophin enhances bone formation (Imai et al., 1998) and limb cartilage differentiation (Dreyfus et al., 1998), little is known about the role of pleiotrophin in organogenesis. It will be important to confirm an in vivo role for pleiotrophin in branching morphogenesis during epithelial organogenesis. To our knowledge, a pleiotrophin gene knockout has not been reported. However, an in vivo study, which utilized dominant-negative mutant chimera mice, did suggest a role for pleiotrophin in spermatogenesis, although other organs including brain, kidney and bone appear normal in these mice (Zhang et al., 1999). There has also been some question about the mitogenic activity of pleiotrophin (Hampton et al., 1992; Souttou et al., 1997; Szabat and Rauvala, 1996), which seems to be affected by the source and purification method (reviewed by Zhang and Deuel, 1999). Nevertheless, in our studies, pleiotrophin freshly purified to apparent homogeneity from either BSN cells or 3T3 cells induced impressive growth and branching morphogenesis of the isolated UB.
A wide range of concentrations of pleiotrophin has been reported to exhibit biological activity (up to 50 µg/ml) in various systems (Imai et al., 1998; Li et al., 1990; Rauvala et al., 1994; Souttou et al., 1997). Pleiotrophin binds to the extracellular matrix, which may explain why concentrations of 200-600 ng/ml were required for morphogenetic activity in the systems employed in our study (Figs 5A and 6). The UB cells and isolated UB were cultured within basement membrane Matrigel, which could conceivably bind a large fraction of the pleiotrophin. It seems improbable, though not inconceivable, that another protein could have co-purified with pleiotrophin through 4 very different chromatography steps and not been detected by silver staining and microsequencing. If such a protein were there, it would have to possess morphogenetic activity in the subnanogram to nanogram range and have physical properties (i.e. size, charge, hydrophobicity) very similar to pleiotrophin.
To date, several glycoproteins, including brain-specific proteoglycans, the receptor type tyrosine phosphatase beta (Maeda and Noda, 1998; Meng et al., 2000) and syndecan 3 (Raulo et al., 1994) have been postulated to function as receptors for pleiotrophin. The UB has been shown to express syndecan 1 (Vainio et al., 1989), and while pleiotrophin is capable of binding to the syndecan 1 (Mitsiadis et al., 1995), it remains to be determined whether syndecan 1 mediates pleiotrophin binding and signal transduction during UB branching morphogenesis. Whether proteoglycans serve as co-receptors for pleiotrophin, as is the case for FGF signaling (Schlessinger et al., 1995), or whether they directly transduce the pleiotrophin signal is presently unclear.
The possible involvement of proteoglycans in pleiotrophin-mediated branching morphogenesis of the UB is particularly interesting in light of several studies demonstrating the importance of proteoglycans in kidney development (Bullock et al., 1998; Davies et al., 1995; Kispert et al., 1996). In these studies of whole kidney, chemical or genetic depletion of sulfated proteoglycans inhibits UB branching morphogenesis, and this is accompanied by decreased GDNF expression, and loss of c-Ret at the UB tips (Bullock et al., 1998; Kispert et al., 1996). Even at early time points, when c-Ret expression is still preserved, addition of exogenous GDNF alone does not completely restore UB branching morphogenesis (Sainio et al., 1997), suggesting that other molecules are required in this process. One possibility is that depletion of sulfated proteoglycans also affects pleiotrophin-mediated signaling or binding.
Together, our results suggest that pleiotrophin functions as a MM-derived morphogen acting upon the UB. Moreover, the results support the idea that UB branching morphogenesis is likely to be regulated by more than a single factor. At least two soluble factors, GDNF and pleiotrophin are necessary for the morphogenetic changes. GDNF may initiate the UB outgrowth (Sainio et al., 1997), and pleiotrophin may induce proliferation and/or facilitate branching (Figs 5 and 8). Whether pleiotrophin acts primarily through control of epithelial proliferation, survival, or elongation/branching requires further study. In vivo loss-of-function studies could potentially clarify the role of pleiotrophin in UB branching morphogenesis. In addition, a FGF family member may play a facilitory role, since FGF1 potentiates the effects of purified pleiotrophin on UB branching morphogenesis, though by itself (with GDNF present) exerts little if any morphogenetic activity (K. T. B., J. Qiao, D. L. Steer, R. O. Stuart, H. S. and S. K. N., unpublished). There may also be a similar set of inhibitory factors, which may include members of the transforming factor-beta family (Sakurai and Nigam, 1997). As previously argued (Nigam, 1995), gradients of positive and negative factors, most of which are matrix-bound, may exist in the mesenchyme and stroma. By regulating proliferation, apoptosis and the expression of morphogenetic molecules at branch tips, branch points and stalks, the global and local balance of these stimulatory and inhibitory factors could be a crucial determinant of branching patterns during collecting system development. In addition, it is likely that sulfated proteoglycans must also be present either to maintain expression of these soluble factors or to secure their binding sites. At later stages, other soluble factors such as HGF and/or EGF receptor ligands may play supplementary roles, either during branching (particularly in the later stages) or shaping/maturation of tubular structures.
Lastly, it should be noted that the concentration-dependent morphogenetic changes induced by pleiotrophin in the UB (Fig. 5A), raise the possibility that pleiotrophin represents a classical "morphogen", in the sense of activin in early Xenopus development (Green and Smith, 1990). Such a molecule might be expected to produce different phenotypic changes in the responding tissue depending upon its concentration. In this regard, the basement membrane of the developing UB, to which pleiotrophin is localized, could potentially act as a "reservoir". Release of pleiotrophin from the basement membrane at the UB tips, perhaps through digestion by matrix degrading proteases, could produce a local concentration gradient, resulting in increased growth and proliferation of tips, while lower amounts of pleiotrophin along the length of the stalk would appear to induce elongation of the forming tubule. Such a concentration gradient of pleiotrophin could provide a basis for modulating the shape and directionality of the developing UB.
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ACKNOWLEDGMENTS |
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