Branching morphogenesis independent of mesenchymal-epithelial contact in the developing kidney

Abstract

Whether mesenchymal-epithelial interactions leading to branching morphogenesis in developing epithelial tissues such as the kidney require direct cell-cell contact or are due to soluble mediators elaborated by the inducing tissue has been the subject of much debate. Here we demonstrate that ureteric bud (UB) epithelium, from which the kidney collecting system and upper urinary tract are derived, can undergo impressive three-dimensional branching morphogenesis when cultured in the appropriate extracellular matrix context in the absence of direct contact with mesenchymal tissue, indicating that the program for branching morphogenesis is inherent to the UB. Both a soluble factor in BSN cell-conditioned medium (BSN-CM) derived from an immortalized cell line thought to originate in the early metanephric mesenchyme and glial cell line-derived neurotrophic factor (GDNF) were required for early and later events in branching morphogenesis. In the absence of BSN-CM, the isolated UB did not survive; a similar result was obtained in the presence of neutralizing antibodies against glial cell line-derived neurotrophic factor. Preliminary analysis of key activity present in BSN-CM indicates that it is a heat-sensitive, heparin-binding factor with a probable molecular mass greater than 100 kDa. When the in vitro cultured UB was recombined with freshly isolated metanephric mesenchyme, nephric units were induced in the mesenchyme, and the UB branches underwent elongation. Our data suggest that, although UB branching morphogenesis per se does not require direct mesenchymal contact, such contact may play a key role in regulating branch elongation and establishing the pattern of branching. The results also suggest an approach to in vitro engineering of nephron.

Figure 1

A novel system for in vitro branching morphogenesis of the UB. (A) The culture system. UBs free from mesenchyme were microdissected from E-13 rat kidney rudiments and placed in an ECM gel suspension composed of type I collagen and growth factor-reduced Matrigel and cultured in BSN-CM supplemented with 10% FCS and growth factors. Details are given elsewhere in the text. The cultured UB was monitored daily by microscopy. (B) The UB undergoes branching morphogenesis in vitro and develops three-dimensional tubular structures in the absence of mesenchyme. E-13 rat UB was isolated and cultured as described. After culture, UBs were fixed at different time points and processed for DB lectin staining. Three-dimensional reconstructions of confocal images are shown. (a) A freshly isolated UB from an E-13 rat embryonic kidney with a single, branched tubular structure. (b) The same UB shown in (a) cultured for 3 days. The tissue has proliferated and small protrusions have formed. (c) The same UB shown in (a) cultured for 6 days. More protrusions have formed, and the protrusions have started to elongate and branch dichotomously. (d) The same UB shown in (a) cultured for 12 days. The protrusions have undergone further elongation and repeated dichotomous branching to form a structure resembling the developing collecting system of the kidney. The white arrows indicate branch points. At higher power, the structures formed in this in vitro culture system exhibited lumens. Phase microscopic examination and staining for markers revealed no evidence for contamination by other tissue or cells.

Figure 2

BSN-CM and at least one soluble growth factor are required for branching morphogenesis of the isolated UB. (A) The UB cultured in the absence of BSN-CM and growth factors. (B) The UB cultured with the mixture of growth factors (including EGF, IGF, HGF, FGF-2, and GDNF) but no BSN-CM. (C) The UB cultured in the presence of BSN-CM alone. (D) The UB cultured in the presence of both BSN-CM and the mixture of growth factors. All cultures were carried out for about 1 week and then processed for DB lectin staining. Shown is the three-dimensional reconstruction of confocal images. The isolated UB exhibits branching morphogenesis only in the presence of both BSN-CM and the mixture of growth factors.

Figure 3

BSN-CM contains unique soluble factor(s) for branching morphogenesis of the isolated UB. The UBs were cultured in the presence of the key growth factor (GDNF; see Fig. 5) but with different cell-conditioned media. (A) 3T3 fibroblast cell-conditioned medium. (B) Immortalized UB cell-conditioned medium. (C) mIMCD cell-conditioned medium. (D) BSN cell-conditioned medium. After culture, the UBs were fixed and processed for DB lectin staining. Only BSN-CM could promote branching morphogenesis of the isolated UB.

Figure 4

GDNF plus BSN-CM is required for branching morphogenesis. The UBs were cultured in the presence of BSN-CM as in Fig. 2 but with each of single growth factors present in the growth factor mixture. Several examples are shown: with EGF alone (A); with FGF-2 alone (B); with HGF alone (C); and with GDNF alone (D). Only GDNF combined with BSN-CM could promote branching morphogenesis of the isolated UB.

Figure 5

GDNF is required for both early- and late-branching morphogenesis in vitro. (A– C) The antibodies against GDNF are neutralizing antibodies. (A) UB was cultured in the presence of BSN-CM and GDNF without antibodies. (B) Same as A, but normal goat IgG antibody were added. (C) Same as A, but antibodies against GDNF were added. (D– F) GDNF is required for branching morphogenesis. The UBs initially were cultured in the presence of BSN-CM and GDNF, and then the cultures were washed to remove GDNF at different time points; the UBs then were cultured continuously in BSN-CM without GDNF. To ensure neutralization of residual GDNF in the culture, antibodies against GDNF were added after removal and washing of GDNF from the culture medium. (D) The UB was cultured as in A, but GDNF was removed and antibodies against GDNF were added on the first day of culture. (E) Same as D, but the GDNF was removed and antibodies against GDNF were added on the second day of culture. (F) Same as D, but the GDNF was removed and antibodies against GDNF were added on the third day of culture (compare with structures in Fig. 1B). All cultures were carried out until the fifth day and processed with DB lectin staining. Whenever GDNF is depleted, UB growth and branching morphogenesis are aborted, indicating that GDNF is required for both early- and late-branching morphogenesis in vitro.

Figure 6

The cultured three-dimensional tubular structure exhibits markers of UB epithelium and is functionally capable of inducing nephrogenesis when recombined with metanephric mesenchyme in vitro. (A– F) The cultured three-dimensional tubular structure exhibits markers of UB epithelium. The UBs were cultured in the presence of BSN-CM and GDNF and then stained for various markers. (A) Light microscopic-phase photograph of cultured UB. (B) Staining with DB lectin, a ureteric bud-specific lectin that binds to the UB and its derivatives. (C) Staining for vimentin, a mesenchymal marker. (D) Staining for neural cell adhesion molecule, the early marker for mesenchymal-to-epithelial conversion in the kidney. (E) Staining with PNA lectin, a mesenchymally derived renal epithelial cell marker. (F) Staining for cytokeratin, an epithelial marker. (G–I) The cultured three-dimensional tubular structure is capable of inducing nephrogenesis when recombined with metanephric mesenchyme. The isolated UB was first cultured 7–10 days as shown in G. Then, the cultured UB was removed from the ECM gel and recombined with freshly isolated metanephric mesenchyme from E-13 rat kidneys. The recombinant was cultured on a Transwell filter for another 5 days. After culture, the sample was double-stained with DB lectin (FITC) and PNA lectin (tetramethylrhodamine B isothiocyanate) as shown in H and in the enlarged section of H shown in I. Results indicate that the in vitro cultured UB-derived structures are capable of inducing nephrogenesis in vitro.