Journal of Cell Science, Supplement 17, 61-64 (1993)Printed in Great Britain © The Company of Biologists Limited 1993

61

Phenotypic conversions in renal development

D. Herzlinger1, R. Abramson2 and D. Cohen1department of Physiology and Biophysics, and 2Division of Urology, Department of Surgery, Cornell University Medical College, New York, NY 10021, USA

SUMMARY

The transporting epithelia of the kidney are derived from an embryonic rudiment containing two distinct cell populations: ureteric bud epithelia and mesenchymal cells of the metanephric blastema. The ureteric bud is a caudal outgrowth of the Wolffian Duct and gives rise to the renal collecting system by branching morpho­genesis. The metanephric blastema gives rise to diverse cells of the nephron after receiving an inductive stimu­lus. It has been proposed that mesenchymal progenitors of the metanephric blastema derive directly from inter­mediate mesoderm, although this hypothesis has never been tested directly. Utilizing direct lineage analysis techniques we demonstrate, in an organ culture system, that mesenchymal nephron progenitors are immediate descendants of ureteric bud epithelia. Ureteric bud epithelia can give rise to mesenchymal nephron prog-

enitors that populate the metanephric blastema by undergoing an epithelial-to-mesenchymal transition fol­lowed by delamination. If this process occurs in vivo, renal morphogenesis can be characterized by two phenotypic conversions: an epithelial-to-mesenchymal transition leading to the generation of mesenchymal- nephron progenitors, followed by a mesenchymal- to-epithelial transition leading to the generation of diverse nephron epithelial cell types. We have immor­talized an embryonic renal mesenchymal cell line and demonstrate that the clonal cell line, RSTEM-1, under­goes phenotypic conversions in vitro, providing a suit­able model to study the regulation of the epithelial phenotype.

Key words: epithelia, mesenchyme, kidney, development

INTRODUCTION

Epithelial cells can be characterized by several criteria, including a polarized cell surface, the presence of junctional complexes, and the expression of epithelial-specific proteins such as cytokeratin and uvomorulin. Much insight into the cellular and genetic regulation of the epithelial phenotype has been gained by utilizing epithelial and fibroblastic cell culture systems, where the epithelial phenotype can be experimentally manipulated (see review by Rodriguez- Boulan and Nelson, 1989). However, a model system to elucidate the in vivo biological signals that regulate the epithelial phenotype has been lacking.

Epithelial-to-mesenchymal, and the opposite, mesenchy- mal-to-epithelial conversions occur throughout embryonic development, mediating the formation of diverse organ sys­tems (Gilbert, 1991). The developing kidney can be utilized as a model system to study the biogenesis of epithelial cell surface polarity because the polarized epithelia of the nephron are derived from unpolarized mesenchymal prog­enitors of the metanephric blastema (Grobstein, 1956; Saxen, 1987; Ekblom, 1981). Utilizing recently developed lineage tracing techniques we have re-examined renal differentiation, and show that mesenchymal progenitors of the metanephric blastema are direct descendants of the ureteric bud.

DEVELOPMENT OF THE URETERIC BUD

Serial sections of rat kidney rudiments at the first identifi­able stage of metanephric kidney formation were examined. The rudiment at this stage of development (gestation day 12.5) consists of the epithelial ureteric bud surrounded by the mesenchymal cells of the metanephric blastema. Sec­tions of such rudiments were assayed for the binding of the lectin Dolichos Bifloris (DB), specific for the ureteric bud, and antibodies directed against epithelial-specific interme­diate filament proteins (cytokeratin) and vimentin, the only intermediate filament protein expressed by mesenchymal cells (Franke, 1982). Such studies demonstrate that the majority of cells comprising the ureteric bud are DB+ epithelia (Table 1). However, cells of the ureteric bud at its terminal, branching tips exhibit a mesenchymal phenotype identical to cells of the metanephric blastema (Table 1). In addition, utilizing antibodies directed against collagen type IV, we observed basement membrane discontinuities at the terminal tips of the ureteric bud. These results suggested that ureteric bud epithelia may undergo an epithelial-to- mesenchymal transition and delaminte into the metanephric blastema.

To test this hypothesis, ureteric buds were isolated from gestation day 12.5 kidney rudiments. The purity of such preparations was assessed by electron microscopy. Isolated

Table 1. Im m unostaining of ureteric bud cellsUreteric bud Metanephric

Ureteric bud terminal tip blastema

Dolichos Bifloris (DB) +Cytokeratin + - -Vimentin +/— + +

Gestation day 12.5 rat kidney rudiments were processed for cryosectioning as described by Herzlinger et al. (1982). Serial frozen sections (4 |im) were incubated with FITC-labeled Dolichos Bifloris (DB), an antibody directed against all cytokeratin isoforms, and antibodies directed against vimentin. The binding of such antibodies was visualized by FITC-labeled second antibody incubations. Most cells of the ureteric bud stained with the lectin, Dolichos Bifloris, and expressed the epithelial- specific cytoskeletal protein, cytokeratin. Many of such cells co-expressed vimentin with cytokeratin. Cells at the terminal tips of the ureteric bud but within the confines of its basement membrane were DB-negative and expressed only vimentin, exhibiting an identical phenotype to cells of the metanephric blastema.

62 D. Herzlinger, R. Abramson and D. Cohen

buds were labeled with Dil (Honig and Hume, 1989) and the fate of such tagged ureteric bud epithelia and their immediate progeny followed after culture in the presence of unlabeled metanephric blastema to facilitate renal differ­entiation (Fig. 1). Conversely, control cultures containing labeled metanephric blastema and unlabeled ureteric buds were established to determine the extent of Dil transfer by cell-cell contact. In such control cultures, no Dil-labeled ureteric bud epithelia were observed, demonstrating that the label can only be transferred in a heritable manner. Results from cultures established with Dil-labeled ureteric bud demonstrate that clusters of Dil-labeled cells integrate into the surrounding unlabeled metanephric blastema (Fig. 1). Such cell clusters were clearly separated from the branch­ing portion of the ureteric bud. Thus, the ureteric bud under­goes delamination as well as branching morphogenesis during renal development. The phenotype of Dil-labeled

Table 2. Im m unostaining of developing ureteric bud epithelia

PhenotypeDays of culture(Dil+cells) Ureteric bud Mesenchyme Renal epithelia

1 + — —

Around tip of ub +3 + + -5 + + +

Frozen sections of organ cultures established with Dil-labeled ureteric buds were prepared after 1, 3 and 5 days of culture (Herzlinger et al., 1982). Dil labeling was colocalized with the binding of FITC labeled ureteric-bud-specific lectin, Dolichos Bifloris, to identify ureteric bud. Dil labeling was also co-localized with the binding of antibodies directed against the following terminal differentiation antigens: glomerulus (the clearance atrial naturetic peptide receptor); proximal tubule epithelia (aminopeptidase N and maltase, provided by A. Quaroni), and distal tubule epithelia (Tamms-Horsfall protein). By 1 day of culture, the majority of Dil-labeled cells were defined as ureteric bud. Some Dil- labeled cells exhibited a mesenchymal morphology, and were clearly outside the confines of the ureteric bud basement membane. By 3 days of culture, a large proportion of Dil-labeled cells exhibited a mesenchymal morphology and were clearly intermixing with unlabeled metanephric mesenchyme. By 5 days of culture, Dil labeled-cells could be seen to express terminal differentiation antigens.

ureteric bud epithelia and their immediate progeny was fol­lowed over five days (Fig. 2, Table 2). Results of such analyses demonstrate that over time Dil-labeled cells formed metanephric blastema and ultimately differentiated into nephron epithelia.

The fate of ureteric bud epithelia was also assessed by retroviral-mediated gene transfer (Jaenisch and Soriano, 1986; Sanes, et al., 1986; Price et al., 1987; Herzlinger et al., 1992). Importantly, retroviral-mediated gene transfer is not dependent on the purity of ureteric bud preparations, since the progenies of a single viral-infected cell are exam-

Fig. 1. Dil-labeled ureteric bud. (A) Ureteric buds were isolated from gestation 12.5-13 day rat kidney rudiments and labeled with Dil (Honig and Hume, 1989). Representative example of isolated bud after Dil-labeling before culture with unlabeled metanephric blastema. (B) Localization of Dil-labeled cells from labeled buds after three days of co-culture with unlabeled metanephric blastema. Dil-labeled cells have left the branching portion of the ureteric bud (out of focus, bright center) and have integrated into the unlabeled metanephric blastema.

Phenotypic conversions in renal development 63

Fig. 2. Frozen section of Dil-labeled ureteric bud after three days of culture with unlabeled metanephric blastema assayed for Dolichos Bifloris binding. Frozen sections were prepared and processed for the binding of FITC-labeled ureteric bud-specific lectin, Dolichos Bifloris, as described. Dil-labeled cells were observed in the ureteric bud (yellow, due to Dil- and FITC-labeling) as well as in the metanephric blastema (red only). By three days of culture, the ureteric bud contains many Dil-negative cells.

Fig. 3. Clonally derived retroviral tagged colonies present in organ cultures established with BAG- infected ureteric buds. Ureteric buds were isolated from gestation 12.5 day rat kidney rudiments and incubated with a limiting dilution of the BAG retrovirus as described by Herzlinger et al. (1992). Cultures containing single, (3-gal- expressing colonies were processed for Dolichos Bifloris histochemistry utilizing peroxidase-labeled DB.(A) Micrograph of a colony containing (3-gal- labeled cells restricted to the ureteric bud.(B) Colony exhibiting (3-gal-labeled ureteric bud cells as well as (3-gal-labeled cells in more proximal nephron segments ((3-gal- positive, DB-negative).

64 D. Herzlinger, R. Abramson and D. CohenTable 3. Differentiation of RSTEM-1 cells into diverse

renal epithelial cell typesPhenotype of RSTEM-1

RenalDays after trypsinization Ureteric bud Mesenchyme epithelia

3 +14 +

RSTEM-1 cell cultures were trypsinized and new cultures initiated at a density of 3x l05 cells/cm2 on polycarbonate filters. Cultures were assayed at the given times and conditions for DB binding, the expression of cytoskeletal proteins vimentin and cytokeratin, and terminal differentiation antigens as described as Table 2. Ureteric bud epithelia were characterized as DB-positive and cytokeratin-positive. Mesenchyme was characterized by the exclusive expression of vimentin and the expression of the p75 nerve growth factor receptor. Renal epithelia were characterized by the expression of cytokeratin and terminal differentiation antigens.

ined. Isolated ureteric buds were infected with decreasing concentrations of the BAG retrovirus and cultured with uninfected metanephric blastema for seven days prior to fix­ation and visualization of P-galactosidase (p-gal) activity and Dolichos Bifloris binding. The dilution of retrovirus resulting in single p-gal-expressing colonies per sample was determined. Such samples, containing single clonally dervied colonies were serially sectioned and the phenotype of cells contained in clonal colonies determined. Impor­tantly, DB is specific for the ureteric bud in these cultures. All clonal colonies examined exhibited DB+ cells, demon­strating a ureteric bud derivation. Whole-mount analysis of such colonies demostrate that many clonal colonies con­tained |3-gal-labeled cells in tubules classified as ureteric bud (DB+), as well as more proximal segments of the nephron that were DB negative (Fig. 3). Serial sections of the latter colonies demonstrate that [3-gal-positi ve cells were present on segments of forming nephrons, including the primitive glomeruli. These data corroborate the results obtained with Dil-labeling, that is, that the ureteric bud is a multipotent renal stem cell, that forms the metanephric blastema.

To examine a phenotypic conversion mediating nephro- genesis, we established a clonal, immortalized, renal mes­enchymal cell line. Gestation 12.5 day rat kidney rudiments were infected with the ts58 retrovirus, which encodes for temperature-sensitive SV40 large T antigen and neomycin resistance (Frederikson et al., 1988). Such immortalized cell populations were selected by growth in the neomycin ana­logue, G418, and clonal cell lines prepared by three rounds of limiting dilution cloning.

One clonal cell line, designated RSTEM-1, fulfills the criteria expected of a multipotent renal progenitor cell. At the non-permissive temperature, large T antigen expression is turned off in RSTEM-1. RSTEM-1 cells integrate into the metanephric blastema of gestation day 12.5 rat kidney rudiments and form tubules after extended growth in co­culture. Additionally, after extended growth as monolayers without passage, on conventional tissue culture substrates, RSTEM-1 changes from a homogeneous population of mes­

enchymal cells to a population of epithelial cells that ulti­mately express terminal differentiation antigens character­istic of glomerular, proximal and distal tubule epithelia (Table 3) (Ekblom et al., 1981). These results demonstrate that RSTEM-1 has the potential to undergo mesenchymal to epithelial transitions leading to terminal differentiation in culture.

CONCLUSIONS

In conclusion, we demonstrate that nephrogeneis may be a process that can be divided into two phenotypic conver­sions. First, the ureteric bud delaminates, forming the mes­enchymal nephron progenitors of the metanephric blastema and second, this blastema differentiates into the diverse epithelia of the neprhon. The renal stem cell line (RSTEM- 1) undergoes this latter transition in culture.

We thank C. Cepko and R. McKay for providing the retro­viruses utilized for this study, and A. Quaroni for providing anti­bodies to the proximal tubule.

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