DEVELOPMENTAL

BIOLOGY

144,248-261 (1991)

Development and Growth of Mouse Embryonic Kidney in Organ Culture and Modulation of Development by Soluble Growth Factor ANDREAS WELLER,’

LYDIA SOROKIN,~ EVA-MARIA

Friedrich-Mieschw-Lalwratorium

der Max-Planck-Gesellsch& Accepted

December

ILLGEN, AND PETER EKBLOM~ D-7400

Tiibingen, Gerwmny

10, 1990

Differentiation of the metanephrogenic mesenchyme is triggered by an inductive tissue interaction between an inducer tissue and the mesenchyme. It is generally believed that the epithelial ureter bud acts as an inducer during in &no development. In response to the inductive stimulus most of the mesenchymal cells convert into epithelial cells, while a small fraction differentiates into stromal cells. In vitro, differentiation of isolated mesenchyme to epithelium can be induced by a variety of embryonic tissues, but nothing is known about the molecular nature of the inducing stimulus. In recent years, large numbers of polypeptide growth factors have been described, which in addition to proliferative effects were shown to exert effects on a variety of biological phenomena such as chemotaxis, inflammation, tissue repair, or induction of embryonic development. We therefore analyzed whether growth factors in the absence of inducer tissue can induce isolated kidney mesenchyme to differentiate into epithelium or interstitium. As expected, both growth and differentiation into epithelium were stimulated by an inducer tissue, the spinal cord. We found that none of the various growth factors tested (including epidermal growth factor, transforming growth factors (Yand j3, insulin-like growth factors I and II, fibroblast growth factor, platelet-derived growth factor, and retinoie acid) could mimick the effect of an inducer tissue, although we tested the factors over a wide concentration range. One of the tested factors, epidermal growth factor (EGF) stimulated the mesenchymal cells to become stromal cells, although it could not stimulate development into epithelium. EGF could stimulate stromal development both when the mesenchyme was cultured in isolation and when the mesenchyme was stimulated by an inducer tissue to become epithelium. The expansion of the stromal compartment in response to EGF treatment occurred at the expense of the epithelial cells, but EGF could not completely suppress the formation of epithelium. These data suggest the presence of EGF receptors in the developing kidney, but since application of soluble EGF leads to abnormal development, soluble EGF cannot be the natural ligand. We suggest that locally produced mitogens with an EGF-like structure may regulate the relative amounts of stroma (interstitium) and epithelium in the developing kidney. o 1991 Academic press, IX

the formation of epithelial cells expressing markers for different nephron segments occurs within 72 to 96 hr of culture (Ekblom et al, 1981a). Recently, a crucial role for certain extracellular matrix components in the conversion of the kidney mesenthyme to epithelium has been demonstrated. As a consequence of induction, the cells begin to express increased amounts of epithelial matrix proteins which then act as autocrine stimulators of development of epithelial cell polarity (Klein et ab, 1988; Ekblom et aZ., 1990; Sorokin et al, 1990). Very little is known, however, about the molecular mechanisms of the inductive interaction. No inducer substances are known, and the immediate responses to induction are not well understood. One response seems to be an increase in cell proliferation (Vainio et aL, 1965; Saxen et aZ., 1983; Ekblom et aZ., 1983), raising the possibility that local growth factors could act as inducer substances. To date, the only defined factor which has been shown to have a clear effect on proliferation during in vitro kidney development is transferrin, but it does not act during the induction period but rather during the subsequent differentiation stages (Ekblom et ak, 1983). As in many other cell types,

INTRODUCTION

Cell-cell interactions are important driving forces for embryonic development. It is thought that kidney development is driven by inductive interactions between the epithelium of the ureter bud and the kidney mesenthyme. As a consequence of the interactions a fraction of the mesenchymal cells differentiates into epithelium, and the ureter grows and branches into the mesenthyme (Grobstein 1955,1956). In vitro, mesenchyme differentiation to epithelium can be induced by a variety of embryonic tissues other than the ureter. The most used inducer is the spinal cord (Grobstein, 1955; Saxen et al., 1968; Ekblom and Thesleff, 1985). When metanephrogenie mesenchyme is cultured in vitro together with the spinal cord as a heterologous inducer of differentiation, i Present address: Max-Planck-Institute for Immunbiology, Postfach 1169, D-7800 Freiburg, Germany. 2 Present address: Clinical Research Units for Rheumatology, MaxPlanck-Society, Schwabachanlage 10, D-8520 Erlangen, Germany. 3 Present address: Department of Zoophysiology, Uppsala University, Norbyvagen 18 A, S-75122 Uppsala, Sweden. 0012-1606/91 $3.00 Copyright All rights

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

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transferrin acts on proliferation by delivering iron to an intracellular site by receptor-mediated endocytosis (Morgan and Appleton, 1969; Barnes and Sato, 1980), and its effects are probably associated with the increased iron requirements of those kidney cells undergoing division (Landschulz and Ekblom, 1985; Thesleff et al., 1985). Transferrin is not synthesized by the kidney, but by the liver and the yolk sac in the embryo, and it thus operates by an endocrine mechanism (Ekblom and Thesleff, 1985). Since transferrin does not act on the first stage of kidney tubule proliferation, there must be other locally synthesized factors that act by autocrine or paracrine mechanisms as inducer molecules. In addition, there may be serum growth factors other than transferrin which modulate cell proliferation during some stages of kidney development. A large number of polypeptide growth factors have been described. It has been shown that several of them not only stimulate cell proliferation but also can affect other processes such as chemotaxis, activation of inflammatory cells, and tissue repair. Growth factors can apparently act as inducers of development in some parts of the embryo (for reviews see Deuel, 1987; Godsave et al, 1988; Mercola and Stiles, 1988; Sporn and Roberts, 1988; Whitman and Melton, 1989), and it has been proposed that certain growth factors may act as stimulators of kidney tubulogenesis (Bortz et aZ., 1988; Taub et aZ., 1990). Growth factors may be important for epithelial cell regeneration in adult kidneys (Fine and Norman, 1989; Mendley and Toback, 1989), a process recently suggested to be similar to nephrogenesis in the embryo (Bacallao and Fine, 1989). There were thus several reasons to test whether growth factors or other putative morphogens could be involved in the induction of differentiation of the metanephrogenic mesenchyme to epithelium. Over a period of several years, we have tried to stimulate the in vitro growth and morphogenesis of the embryonic mouse kidney by various soluble factors (EGF, PDGF, TGFs, FGFs, IGFs, retinoic acid).4 We have tested whether these factors could act as inducers of proliferation or differentiation of the nephrogenic mesenchyme, or whether they could modulate morphogenesis once the mesenchyme had been induced by a known inducer tissue. We describe these experiments which show that in our hands none of the tested factors acted as inducers of conversion of the mesenchyme to epithelium. However, 4 Abbreviations used: EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; FCS, fetal calf serum; FGF, fibroblast growth factor; IGF, insulin-like growth factor; MEM, minimum essential medium; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; RA, retinoic acid; TGF, transforming growth factor.

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a specific stimulation of mesenchyme differentiation into interstitium was obtained by soluble EGF, both when the mesenchyme was cultured alone and when the kidney mesenchyme was first induced to differentiate into epithelium by inducer tissues. MATERIALS

Embryonic

AND

METHODS

Tissues

Developing embryonic kidneys were microsurgically isolated from National Medical Research Institute (NMRI) X 129 hybrid mouse embryos. The age of the embryos was counted from the day of the appearance of the vaginal plug (Day 0). Culture Media Chemically defined medium consisted of improved Eagle’s MEM (IMEM-ZO) (Richter et ah, 1972; Ekblom et ah, 1981b) custom made by GIBCO (Eggenstein, FRG) supplemented with 50 /*g/ml human transferrin (Collaborative Research, Waltham, MA) and 4 mMglutamine (GIBCO). Serum medium was composed of IMEM-ZO supplemented with 10% fetal calf serum (Biochrom, Berlin, FRG), 50 pg/ml transferrin, and 4 mM glutamine. Growth Factors The growth factors were obtained from the following sources: murine epidermal growth factor (EGF) from Collaborative Research (Waltham, MA) and from Calbiochem (Frankfurt, FRG); transforming growth factor (Y (TGF-(u) from Calbiochem; porcine transforming growth factor p (TGF-P) from R&D (Minneapolis, MN); human insulin-like growth factor-I (IGF-I) from Boehringer (Mannheim, FRG). Human insulin-like growth factor-II (IGF-II) was kindly provided by Dr. Blum and Dr. Ranke (University Children’s Hospital, Tubingen, FRG). Basic fibroblast growth factor (FGF), prepared from bovine brain, was kindly provided by Dr. Risau (Max-Planck-Institute for Developmental Biology, Ttibingen, FRG). Human platelet-derived growth factor (PDGF) was obtained from Paesel (Frankfurt, FRG) and from R&D (Minneapolis, MN), bovine insuline from Collaborative Research, and retinoic acid (RA) (all trans, type XX) from Sigma (Deisenhofen, FRG). The XTC cell line, which produces an inducer molecule important for mesoderm development, (Smith, 1987) was kindly provided by Dr. Smith (Laboratory of Embryogenesis, National Institute for Medical Research, London). The XTC cells were grown at 25°C in 60% Leibovitz L-15 medium and supplemented with 10% FCS. XTC-conditioned medium was obtained by incubating

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confluent cells in serum-free medium for 48 hr (Smith, 1987). This conditioned medium was diluted with an equal volume of IMEM containing 10% FCS and adjusted to 150 mMNaC1 before addition to the organ cultures of uninduced metanephrogenic mesenchyme. Cultures of ll- to l&Day-Old Kidneys and Culture Isolated Metanephrogenic Mesenchyme

of

With isolated mesenchyme two types of experiments were performed. One assessed the ability of the growth factors to induce differentiation or proliferation of the isolated metanephrogenic mesenchyme grown without inducer tissue, and in the other experimental set-up the mesenchyme was exposed to both an inducer tissue and a growth factor. Induction of differentiation of mesenthyme to epithelium in vitro was performed in transfilter cultures with spinal cord as the inducer as previously described (Grobstein, 1956; Saxen et ak, 1968; Ekblom and Thesleff, 1985; Vestweber et aL, 1985). Briefly, kidneys from 11-day-old embryos were dissected in PBS and transferred to PBS containing 0.1 M EDTA. In this buffer the metanephrogenic mesenthymes were microsurgically isolated from the epithelial ureter bud. Three mesenchymes were combined into one explant and placed on a Nuclepore filter, nominal pore size 1.0 pm (Nuclepore, Tubingen, FRG). Pieces of 11-day-old embryonic spinal cord were cemented to the lower side of the filter with a small drop of 1% agar in IMEM-ZO. The tissues were subsequently incubated (Haereus incubator) at 3’7°C and 5% CO2 on a Trowelltype stainless steel grid at the interphase between medium and atmosphere. The incubations were performed either in chemically defined (Ekblom et ah, 198lb) or serum-containing medium, in the presence or absence of various concentrations of growth factors. The ability of the growth factors to alter the course of development induced by the natural inducer, the ureter, was also tested. In these experiments whole kidneys were dissected from 12-day-old embryos and cultured in the presence of serum supplementation (Grobstein, 1955) or in chemically defined medium as described (Ekblom et al, 198la; Thesleff and Ekblom, 1984). Assay

for DNA Content

DNA content was used eration in kidneys which and in metanephrogenic content was assessed H33258 (Riedel de Haen, 100 fluorometer (Hoefer lanta, Heidelberg, FRG) gen (1980).

as a measure of cellular prolifdeveloped in vitro and in viva, mesenchyme cultures. DNA using the dye bisbenzimide Hannover, FRG) and a TKO Scientific Instruments/Ataccording to Labarca and Pai-

VOLUME144,1991

Morphological

Assays

Organ cultures were studied live with a stereomicroscope (Olympus SZH). For histology, the tissues were fixed in Zenker’s solution and mounted in Technovit 7100 embedding plastic (Kulzer, Technical Division, Wehrheim, FRG). Five-micrometer sections were cut and stained with hematoxylin and eosin, and examined using a Zeiss Axiophot microscope (Zeiss, Oberkochen, FRG). RESULTS

Growth of Mouse Embryonic Vitro

Kidneys

in Viva and in

In order to clearly define the effects of growth factors on cellular proliferation, it was valuable to first study the pattern of in vivo growth, and to characterize the patterns of proliferation of whole kidney and metanephrogenic mesenchymes under different culture conditions in vitro. To these ends the DNA content of whole kidneys from 11- to 17-day-old embryos was measured and compared with values obtained from kidneys or mesenchymes cultured in vitro in the absence of added growth factors. Embryonic mouse kidneys grew rapidly during in vivo development. DNA content per kidney increased 2-fold every 24 hr between Days 11 and 17 of embryonic development (Fig. 1). Kidneys grown in vitro on a Nuclepore filter had slightly slower rates of proliferation than kidneys growing in vivo (Fig. 2). The DNA content of kidneys dissected from 12-day-, 13-day-, and 14-day-old embryos increased approximately 1.5-fold every 24 hr during the 72-hr incubation period in medium containing 10% FCS. In contrast, kidneys dissected from llday-old embryos did not show any significant increase in DNA content during the 72-hr incubation period (Fig. 2). In whole kidneys both the ureter and the mesenchyme are present, and the data (Fig. 2) therefore do not distinguish between growth of the ureter and mesenchyme. To study more directly whether induction of differentiation of the mesenchyme to epithelium increases the amount of mesenchymal cells, the transfilter culture was used (Grobstein, 1956). In the culture system, the mesenchyme remains separated from the inducer tissue by the filter and the DNA content of the mesenchyme can be measured. When metanephrogenic mesenchymes were cocultured with embryonic spinal cord in serum medium, the DNA content of the mesenchyme in the explants increased over the first 48 hr of culture to 1.4 pg/explant and plateaued thereafter up to 168 hr of culture (Fig. 3). The onset of this proliferative phase was slightly delayed when incubations were carried out in

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1. Growth of embryonic kidneys in viva Kidneys were dissected from embryos of the indicated ages and assayed for DNA content. Results shown are mean values + standard deviation based on a minimum of six kidneys for each time point. FIG.

chemically defined medium. The increase in DNA content/explant commenced at 24 hr of culture and reached a maximum value of 1.3 pg at 72 hr of culture (Fig. 3). Curiously, explants cultured for 120 hr in chemically defined medium reproducibly had a slightly lower DNA content than explants incubated for 72 or 168 hr (Fig. 3). If mesenchyme was cultured in the absence of spinal cord, the DNA content remained constant for some time, but then gradually declined, both in chemically defined medium and in medium supplemented with fetal calf serum (Fig. 3). Eflects of Growth Factors on Proliferation Mesenchyme in Vitro

0

of

To test whether growth factors could affect cell proliferation of the metanephrogenic mesenchyme to the same extent as an inducer tissue, explants of uninduced mesenchymes were cultured for ‘72 hr in chemically defined medium in the presence of one of the following growth factors: FGF (1, 10, or 100 rig/ml), EGF (1, 10, or 100 rig/ml), TGF-a! (1, 10, or 100 rig/ml), TGF-fl (0.1, 1, or 10 rig/ml), PDGF (5 or 50 rig/ml), IGF-I (1, 10, or 100 rig/ml), IGF-II (1, 10, or 100 rig/ml), insulin (5 pg/ml), or

of in vitro

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FIG. 2. Growth of embryonic kidneys in, vitro. Kidneys were isolated from 11-day to 14-day-old embryos and grown in medium containing 10% FCS. After the indicated times of incubation the explants were assayed for DNA content. Each point represents the mean + standard deviation based on a minimum of six kidneys.

RA (1, 10, or 100 nM). Growth factors were added to the medium at the onset of culture and, after 72 hr, DNA content/explant was measured. At the onset of culture each explant consisting of three uninduced mesenchymes contained a mean of 0.28 pg of DNA. None of the growth factors had a significant effect on the DNA content of uninduced mesenchymes (Fig. 4, bars 2 to 25) compared to the negative control (Fig. 4, bar l), while the presence of inducer tissue resulted in a greater than fourfold increase in DNA content (Fig. 4, bar 26). It is known that during induction kidney mesenchyme acquires responsiveness to the serum mitogen transferrin (Ekblom et ak, 1983). We therefore tested if any of the growth factors could affect the proliferation of kidney rudiments where mesenchymal differentiation into epithelium had been induced. Kidneys from 12-day-old embryos contain the normal inducer and we used 12-day kidneys for these experiments. The kidneys were incubated in chemically defined medium in the presence of FGF (1, 10, or 100 rig/ml), EGF (1,3,5,10, or 100 rig/ml), TGF-a! (1, 10, or 100 rig/ml), TGF-/3 (0.1, 1, 10, or 100

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I 0

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3. Growth of isolated metanephrogenic mesenchyme in vitro. Mesenchyme was microsurgically isolated from kidney rudiments of 11-day-old embryos. For each explant three mesenchymes were combined. The explants were incubated in the presence (closed squares) or absence (open squares) of inducing tissue in either medium containing 10% FCS (solid line) or chemically defined medium (broken line). After the indicated times of incubation the explants were assayed for DNA content. Each point represents the mean f standard deviation based on a minimum of three explants for each time point using a total of over 260 mesenchymes. FIG.

rig/ml), IGF-I (1, 10, or 100 rig/ml), IGF-II (1, 10, or 100 rig/ml), insulin (5 pg/ml), or RA (1, 10, or 100 nM). At the onset of incubation, 12-day kidneys had an average of 0.7 pg DNA (Fig. 5, bar 0). After 72 hr of incubation in the absence of growth factors, the kidneys contained a mean of 3.6 pg of DNA/kidney (Fig. 5, bar 1). This value was not significantly different from the mean DNA content of kidneys incubated in the presence of growth factors (Fig. 5, bars 2 to 26), but there was considerable interassay variability; in some of the experiments the kidneys seemed to be smaller than those of the controls. Thus, although the interassay variability made definite conclusions difficult, it seems that none of the factors could increase the size of the kidneys. Failure to Induce Epithelial Cell Development from Metanephrogenic Mesenchyme with Growth Factors

Even though the tested growth factors did not stimulate proliferation of the metanephrogenic mesenchyme, it was necessary to study whether some small epithelial structures nevertheless had formed in response to treatment by growth factors. The explants were therefore evaluated by stereomicroscopy and by histology. It was evident by direct observation with stereomicroscopy that none of the factors TGF-a (1, 10, or 100 ng/ ml), TGF-0 (1, 10, or 100 rig/ml), FGF (1, 10, or 100 ng/

ml), IGF-I (1, 10, or 100 rig/ml), IGF-II (1, 10, or 100 rig/ml), PDGF (5 or 50 rig/ml), or RA (1, 10, or 100 nM) could mimick the effect of an inducer tissue. The explants cultured with these factors became flat and showed no signs of differentiation after ‘72 hr of incubation and did not in any way differ from mesenchyme cultured alone without growth factors. In mesenchyme induced to differentiate by spinal cord, the abundant formation of epithelium can be easily seen by stereomicroscopy (Fig. 6A). In contrast, explants of mesenthymes incubated in the absence of the inducing tissue with or without growth factors did not differentiate; the tissue flattened (Fig. 6B), and histology revealed that a majority of the cells were necrotic (Fig. 7A). The ability of XTC factor to induce differentiation (Smith, 1987) was also tested. Explants of isolated mesenchymes were cultured in a 50% dilution of XTC cell line-conditioned medium in serum medium. At 72 hr of incubation of the XTC medium the cells appeared necrotic and showed no sign of differentiation (data not shown). EGF Stimulates Kidney Stroma Development

EGF could also not induce epithelial development in the isolated mesenchyme, but the effect of EGF differed from those of the other tested factors. In accordance

WELLER

1

2

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ET AL

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7

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Stimulates

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Embryonic

1213141516171819

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Mesenchyme

2021

2223242526

FIG. 4. Effect of growth factors or inducing tissue on in vitro growth of isolated metanephrogenic mesenchyme. For each explant three mesenchymes were combined. DNA content was measured from explants incubated for ‘72 hr in chemically defined medium in the presence of various concentrations of growth factors (bars 2 to 25). DNA content of explants incubated in the absence of added growth factors (bar 1) and of explants incubated without growth factor but in the presence of inducing tissue for 24 hr (bar 26) are also shown. Results shown are mean values f standard deviation based on a minimum of three explants for each concentration using a total of over 420 mesenchymes. Growth factors tested included: 1, 10, and 100 rig/ml FGF (2-4); 1, 10, and 100 rig/ml EGF (5-7); 1, 10, and 100 rig/ml TGF-CY (8-10); 1,5, and 10 rig/ml TGF-8 (11-13); 5 and 50 rig/ml PDGF (14-15); 1, 10, and 100 rig/ml IGF-I (16-18); 1, 10, and 100 rig/ml IGF-II (19-21); 5 pg/ml insulin (22); 1, 10, and 100 nM retinoic acid (23-25).

with the DNA content data (Fig. 3) morphological observations suggested that EGF did not increase the size of the explants (Fig. 6), but it was evident by stereomicroscopy that mesenchyme treated with EGF was more compact than untreated mesenchyme or mesenchyme treated with the other factors (Figs. 6B, 6C). Histology revealed no epithelial structures in the explants treated with EGF but instead showed well-formed interstitial cells (Fig. 7B). This was in contrast to the untreated explants which contained dead cells (Fig. 7A). For comparison, the epithelial cells that form in response to induction by spinal cord in the absence of EGF are shown (Fig. 7C). It was then tested whether EGF could still exert an effect if the mesenchyme was also induced to become epithelium by inducer tissue. In the mesenthyme simultaneously exposed to spinal cord and EGF, the stromal compartment was clearly larger than in the explants exposed only to the spinal cord. The enlargement of the stromal compartment may have occurred at the expense of the epithelial compartment but a complete suppression of epithelial development did not occur (Fig. 7D). Finally, it.was tested whether EGF treatment led to an increased amount of stroma when epithelial cell development was also induced by the normal inducer. To

this end, 12-day-old embryonic kidneys containing the ureter epithelium were grown in the presence of EGF or other growth factors. In such cultures both the ureter epithelium and the tubular epithelium developed even though EGF was present (Figs. 8 and 9). It seemed that EGF neither enhanced nor inhibited the branching of the ureter epithelium. Because of the many cell lineages present, it is not as easy to study tubular development in these cultures as it is in the cultures of isolated mesenthyme, but it seemed that EGF treatment led to the formation of slightly fewer epithelial tubuli than in control cultures. However, the only clear effect of EGF was an expansion of the mesenchymal tissue surrounding the epithelia (Fig. 8B) compared to control kidney cultures (Fig. 8A). The EGF effect was seen in chemically defined medium (Fig. 8) but the effect was more pronounced when serum supplementation was used. None of the other tested factors seemed to stimulate a similar alteration in morphogenesis. Whereas only a few stromal cells with little extracellular matrix occurred between the epithelial tubuli in the control kidneys (Fig. 9A), the EGF-treated explants contained numerous fibroblast-like cells embedded in a prominent loose extracellular matrix (Fig. 9B), EGF had maximal effects at concentrations of 5 to 10 rig/ml. Higher con-

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FIG. 5. Effect of growth factors on in vitro growth of 12-day-old embryonic kidneys. DNA content was measured in kidneys after a 72-hr incubation in chemically defined medium in the presence of various concentrations of growth factors (bars 2 to 26). DNA content of kidneys before incubation (bar 0) and of kidneys incubated without growth factor (bar 1) are also shown. Results shown are mean values f standard deviation based on a minimum of four kidneys for each concentration using a total of over 440 kidneys. Growth factors tested were: 1, 10, and 100 rig/ml FGF (2-4); 1,3,5,10, and 100 rig/ml EGF (5-9); 1, 10, and 100 rig/ml TGF+ (10-12); 0.1, 1, 10, and 100 rig/ml TGF-P(13-16); 1, 10, and 100 rig/ml IGF-I (17-19); 1, 10, and 100 rig/ml IGF-II (20-22); 5 ag/ml insulin (23); 0.1, 1, and 10 nh4 retinoic acid (24-26).

centrations of EGF were less effective in altering morphogenesis. We conclude that soluble EGF in these organ cultures of metanephric kidney does not primarily affect development of any of the epithelial cell lineages, but instead stimulates development of mesenchyme to stroma. DISCUSSION

In V+UO,a large part of the metanephrogenic mesenthyme differentiates into epithelium, a conversion which can also be induced in vitro. A much used inducer tissue for in vitro development is the spinal cord (Grobstein, 1956). It is shown here that the mesenchyme during the conversion into epithelium in vitro increases its size considerably, approximately threefold. None of the growth factors tested could induce such a growth or a differentiation of mesenchyme to epithelium, although several of the tested substances act as morphogens in other organs. EGF apparently induces epithelial cell development in embryonic lung (Goldin and Opperman, 1980) and it has been suggested that EGF could stimulate epithelial development in the kidney (Taub et cd, 1990) but we found no evidence that EGF stimulates epithelial cells during early stages of mouse kidney development. Instead, EGF was found to slightly increase

the amount of interstitial cells at the expense of epithelial cells. The current data increase the understanding of the segregation of the metanephric mesenchyme into interstitium and epithelium, but also allow us to give some recommendations about organ cultures of the embryonic kidney. Fundamental for the establishment of the points were the initial studies of growth of intact kidneys with both ureter and mesenchyme, and of growth of isolated mesenchymes under various culture conditions. The data show that 11-day kidneys, which contain an unbranched ureter and mesenchyme, do not grow well in vitro in either chemically defined or serum medium. Kidneys from 12-day-old embryos were the earliest stage kidneys which showed both consistent growth and normal morphogenesis in vitro despite some variability in the DNA content. Kidneys from older stage embryos already contain several tubules at advanced stages of differentiation before onset of culture. Thus, even though they have been used for some investigations (Avner et al., 1985,1988) and they grow in vitro, they are not well suited for studies of early stages of kidney development. Even though whole kidneys from 11-day embryos did not grow or differentiate well in vitro, mesenchyme devoid of ureter from kidney rudiments of the same age embryos showed marked growth during the first 2 days

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FIG. 6. Effect of inducing tissue or EGF on in vitro growth of isolated metanephrogenic mesenchyme as seen under the stereomicroscope. Metanephrogenic mesenchyme was microsurgically isolated from kidney rudiments of 11-day-old embryos. For each explant three isolated mesenchymes were combined. (A) The explant was cultured with the heterologous inducer tissue, spinal cord, on the lower side of the filter. The inducer tissue was removed before the photograph was taken. Numerous well-formed tubuli have formed in the explant in response to the inducer tissue. (B) The explant was cultured without inducer tissue. The explant has become very flat. No sign of tubular differentiation is visible. (C) The explant was incubated with 10 rig/ml of EGF in the absence of inducer tissue. The explant does not seem to be bigger than explants cultured without EGF as seen in (B), but it has remained compact. Yet, no differentiation of tubular epithelium can be observed in response to EGF treatment. Note that the explant in A is much bigger than those in B and C. Bar = 0.2 mm.

of culture when cocultured with embryonic spinal cord. DNA content increased more than threefold in both serum medium and chemically defined medium. The ob-

served growth was reproducible both in the chemically defined medium and in the presence of 10% fetal calf serum. In a previous study no net growth of the induced

FIG. 7. Histology of isolated metanephrogenic mesenchymes cultured in the presence or absence of inducer tissue or epidermal growth factor. Spinal cord was used as inducer tissue. Metanephrogenic mesenchyme was microsurgically isolated from kidney rudiments of 11-day-old embryos and cultivated for ‘72hr. In the absence of inducer tissue or EGF no tubule formation was observed and the tissue became necrotic (A). In the absence of inducer tissues and in the presence of EGF (10 rig/ml) well-developed stromal cells were present but no epithelial cells were detected (B). In the presence of inducer tissue most cells were epithelial and very few stromal cells were seen (C), but when the mesenchyme was exposed to both inducer tissue and EGF (10 rig/ml) the number of stromal cells increased at the expense of epithelial cells (D). Bar = 50 pm.

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FIG .8. Histology of 12-day-old embryonic kidneys which had been cultured in chemically defined medium in the absence (A) or presence (.B)of eDide1rmal growth factor. Note that the explants grown in the presence of EGF have slightly more stromal cells than the explant cull lured ut EGF. Bar: 40 pm.

mesenchyme was observed, although the same inducer tissue was used and the medium should have been the same (Ekblom et al., 1983) as the one used here. We attribute this difference to slightly improved organ culture conditions. A more sophisticated incubator was used in the current study, and the culture medium was carefully controlled. The conditions used previously evidently led to loss of cells although differentiation of the surviving cells proceeded normally. Terminal differentiation of the mesenchyme into the three epithelial segments could be demonstrated (Ekblom et al, 1983). It is apparent from the growth curves reported in the

current study that serum substitution is beneficial. The differences between the explants grown in chemically defined medium and in serum are small, and both culture conditions ensure differentiation. If a defined medium is chosen, we advocate the use of the simple medium containing transferrin (Ekblom et aZ., 1981b) rather than more complex media which may contain factors that modulate development. Having established the growth pattern of metanephrogenic mesenchyme, we tested whether growth factors could stimulate proliferation of the uninduced kidney mesenchyme in the absence of inducer tissue. In our

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FIG. 9. High magnification of histology showing the effect of EGF on the mesenchymal compartment of l2-day-old embryonic kidneys cultured in I-MEM medium supplemented with 10% fetal calf serum in the absence (A) or presence (B) of epidermal growth factor. Kidneys were incubated for 72 hr and then processed for histology. The expansion of the mesenchymal stroma in response to 5 rig/ml EGF is clearly visible between the tubular epithelia (B), whereas in the kidneys grown without EGF (A) only a few mesenchymal cells surrounding the numerous tubuli are found. Bar: 20 pm.

hands, none of the various factors tested (EGF, TGF-a, TGF-P, FGF, PDGF, IGF-I, IGF-II, insulin, RA, and XTC-conditioned medium) could mimick the mitogenic effect of the inducer tissue even though a wide range of concentrations was tested. Histological examination confirmed that no differentiation to epithelium had occurred in mesenchyme cultured in the presence of the tested growth factors. The only factor which showed an effect on differentiation was EGF, but it did not stimulate conversion of mesenchyme to epithelium but instead stimulated differentiation into stromal cells. It is noteworthy that this effect went unnoticed in the analy-

sis of DNA content, and only became apparent by morphological assays. The effect with EGF seems to be quite specific because the mesenchyme in the presence of the other tested factors became necrotic during the 3-day culture period when cultured without inducer tissue. The failure to induce development of epithelium from the metanephrogenic mesenchyme by the different morphogens is interesting in light of recent studies from other organs. Growth factors and retinoic acid have been postulated as inducers of embryonic organogenesis for various developing tissues (Strickland and

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Mahdavi, 1978; Tickle et al, 1982; Mercola and Stiles, 1988). On the basis of indirect evidence, it was recently postulated that EGF or TGF-a could be a stimulator of conversion of the kidney mesenchyme to epithelium (Taub et ak, 1990), but in light of our present study this is unlikely. The absence of an inducing effect of FGF, IGFs, and TGF-P on development of kidney tubules is also noteworthy because they are expressed in the kidney (Risau and Ekblom, 1986; Lehnert and Akhurst, 1988; Wilcox and Derynck, 1988; Bortz et al, 1988) and FGF and TGF-P induce mesoderm development to some extent (Slack et ak, 1987; Weeks and Melton, 1987; Kimelman and Kirschner, 1987; Kimelman et al., 1988; Rosa et aZ., 1988). Some of the other factors, such as PDGF and retinoic acid, act on many mesenchymal cell types in other organs (Ross et al., 1986; Gospodarowicz et ab, 1986; Baxter, 1988), and one obvious possibility was that these factors are also active on the metanephrogenie mesenchyme. Yet, our current studies suggest that none of these factors act as inducers of tubular and glomerular epithelial development in the kidney. We can naturally not exclude the possibility that some of the factors would be active in combination with other factors or when applied by different means. Although the majority of the cells of the metanephrogenie mesenchyme convert into epithelium during both in viva and in vitro differentiation, a small fraction differentiate into stromal or interstitial cells (Aufderheide et aZ., 1987). Little is known about factors that stimulate the cells to take the second differentiation pathway into interstitium. In other organs RA can alter the ratio between epithelial and mesenchymal cells during mesenchymal-epithelial interactions (Fell and Mellanby, 1953; Dhouailly and Hardy, 1978; Wedden, 1987; Covant and Hardy, 1990) and it can modify differentiation of the chick limb bud (Maden, 1982; Tickle et al, 1982,1985; Thaller and Eichele, 1987) and the brain (Durston et ah, 1989). PDGF is a mitogen for mesenchymal cell types (Heldin et aZ., 1981) and may be involved in normal gliogenesis in the central nervous system (Richardson et cd., 1988), a process similar to interstitial development in nonneural organs. Finally, some reports have shown that EGF can stimulate embryonic mesenchymal cells (Yoneda and Pratt, 1981; Kaplowitz et aZ., 1982; Partanen et aZ., 1985). Of these putative mesenchymal cell stimulators, only EGF affected the kidney mesenchyme. Soluble EGF was able to slightly increase the proportion of mesenchymal cells becoming stromal (interstitial) cells at the expense of epithelial cells. The effect was seen when differentiation was induced both by the spinal cord and by the ureter, and mesenchyme cultured without inducer tissue also responded. EGF influences development in other organs, but the cells that respond to EGF seem to vary with tissue type (Partanen et ccl.,

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1985; Kim et ak, 1987). For example, EGF has been found to exert effects on the differentiation of tracheal epithelium (Goldin and Opperman, 1980). In the tooth, EGF stimulated proliferation of the dental epithelium while inhibiting proliferation of the dental mesenchyme. In addition EGF stimulated growth of the surrounding, nondental mesenchyme (Partanen et al, 1985). The effects on the mesenchyme surrounding the embryonic tooth and the kidney mesenchyme appear to be similar, and it is thus likely that several embryonic mesenchyma1 cells can be stimulated to become interstitium by molecules that bind to EGF receptors. The manner in which EGF exerted its effect on kidney interstitium is unclear. It may have caused this abnormal morphology by selectively stimulating growth of interstitial cells already committed to the stromal cell lineage, or it could have stimulated differentiation of uncommitted mesenchymal cells to interstitium. Since an effect was seen on isolated mesenchyme it seems clear that neither the presence of ureter nor tubule epithelium is obligatory for responsiveness to EGF. Iodinated EGF has been shown to bind to both the embryonic ureter epithelium and the mesenchyme (Partanen and Thesleff, 1987), and we cannot exclude that the effect of EGF-like molecules on kidney mesenchyme is minimally enhanced by the presence of epithelium. EGF receptors occur on several embryonic tissues (Hortsch et aL, 1983; Adamson and Meek, 1984; Weller et aZ., 1987) including the kidney (Nexs and Kryger-Baggesen, 1989) and EGF can modulate morphogenesis of embryonic organs, as also shown in the present study. Yet, EGF synthesis starts only postnatally (Perheentupa et cd, 1985; Popliker et al., 1987; Fisher et al., 1989; Gattone et al., 1990). It thus seems clear that EGF-receptors are involved in embryonic development, but the natural ligands for the receptors during embryogenesis have not been well defined. Maternally derived EGF or other EGF-like molecules may be present in serum. TGF-a could be an embryonic equivalent to EGF (Twardzik, 1985; Derynck, 1988). Although our failure to see clear effects of TGF-(I! on kidney development does not exclude that TGF-cy is an embryonic growth factor, it suggests that some other EGF-like molecules are the natural ligands binding to embryonic mesenchyme. These ligands are not necessarily soluble molecules but could instead be locally deposited factors. Locally deposited EGF-like mitogens may be present on the cells surface or in the extracellular matrix of embryonic mesenchyme. Several large structural proteins contain EGF-like sequences, and they may act as local growth factors (Engel, 1989). Some developmentally important cell surface proteins contain EGF-like domains (Greenwald, 1985; Wharton et al, 1985; Brachmann et ah, 1989), and many extracellular matrix pro-

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teins implicated in development contain EGF-like repeats (Sasaki et al., 1987; Pearson et ah, 1988; Krusius et al, 1987). Laminin and tenascin isoforms and proteoglycans are deposited into the extracellular matrix of the embryonic kidney mesenchyme (Aufderheide et al., 1987; Platt et aZ., 1987,199O; Klein et ak, 1988; Weller et al., 1991), and it has been shown that laminin and tenascin are mitogens for cells in vitro (Chiquet-Ehrismann et al., 1986; Panayotou et al., 1989). Since these extracellular matrix molecules contain EGF-like repeats they may in part control the relative amounts of epithelial and interstitial cells of the kidney. Local deposition of EGF-like molecules may be better suited for fine-tuning of morphogenesis than soluble EGF, which can act in a less controlled fashion on all cells having EGF receptors. In the mammalian adult kidney, there are very few stromal cells in the cortex (Wohlgast, 1985), and this pattern is established already during embryogenesis. The observed EGF effect on the interstitial compartment is thus apparently not a physiological event, although it could be used to develop models to study interstitial cell development. This developmental pathway is still poorly understood, but there is some evidence that the kidney interstitial cells act as helper cells for the differentiation of the epithelium (Aufderheide et al., 1987; Sariola et al., 1988) and consequently the study of this tissue compartment could be essential for our understanding of kidney development. We thank Dr. Irma Thesleff (University of Helsinki, Finland) for help with some of the initial experiments with epidermal growth factar. This work was supported by grants from the Deutsche Forschungsgemeinschaft (EK 4/l-3) and the Deutsche Krebshilfe/ Mildred-Scheel Stiftung. REFERENCES ADAMSON, E. D. and MEEK, J. (1984). The ontogeny of epidermal growth factor receptors during mouse development. Dev. BioL 103, 62-70. AUFDERHEIDE, E., CHIQUET-EHRISMANN, R., and EKBLOM, P. (1987). Epithelial-mesenchymal interactions in the developing kidney lead to expression of tenascin in the mesenchyme. J. Cell Biol. 105,599608. AVNER, E. D., SWEENEY, W. E., JR., PIESCO, N. P., and ELLIS, D. (1985). Growth factor requirements of organogenesis in serum-free metanephric organ culture. In Vitro Cell. D~IJ. Biol. 21,297-304. AVNER, E. D., PIESCO, N. P., SWEENY, W. E., JR., and ELLIS, D. (1988). Renal epithelial development in organotypic culture. Pediatr. Nephrol.

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