Protein-rich fractions inhibitory for isolated ureteric bud (UB) growth were separated from a conditioned medium secreted by cells derived from the metanephric mesenchyme (MM). Elution profiles and immunoblotting indicated the presence of members of the transforming growth factor-beta (TGF-beta) superfamily. Treatment of cultured whole embryonic kidney with BMP2, BMP4, activin, or TGF-beta1 leads to statistically significant differences in the overall size of the kidney, the number of UB branches, the length and angle of the branches, as well as in the thickness of the UB stalks. Thus, the pattern of the ureteric tree is altered. LIF, however, appeared to have only minimal effect on growth and development of the whole embryonic kidney in organ culture. The factors all directly inhibited, in a concentration-dependent fashion, the growth and branching of the isolated UB, albeit to different extents. Antagonists of some of these factors reduced their inhibitory effect. Detailed examination of TGF-beta1-treated UBs revealed only a slight increase in the amount of apoptosis in tips by TUNEL staining, but diminished proliferation throughout by Ki67 staining. These data suggest an important direct modulatory role for BMP2, BMP4, LIF, TGF-beta1, and activin (as well as their antagonists) on growth and branching of the UB, possibly in shaping the growing UB by playing a role in determining the number of branches, as well as where and how the branches occur. In support of this notion, UBs cultured in the presence of fibroblast growth factor 7 (FGF7), which induces the formation of globular structures with little distinction between the stalk and ampullae [Mech. Dev. 109 (2001) 123], and TGF-beta superfamily members lead to the formation of UBs with clear stalks and ampullae. This indicates that positive (i.e., growth and branch promoting) and negative (i.e., growth and branch inhibiting) modulators of UB morphogenesis can cooperate in the formation of slender arborized UB structures similar to those observed in the intact developing kidney or in whole embryonic kidney organ culture. Finally, purification data also indicate the presence of an as yet unidentified soluble non-heparin-binding activity modulating UB growth and branching. The data suggest how contributions of positive and negative growth factors can together (perhaps as local bipolar morphogenetic gradients existing within the mesenchyme) modulate the vectoral arborization pattern of the UB and shape branches as they develop, thereby regulating both nephron number and tubule/duct caliber. We suggest that TGF-beta-like molecules and other non-heparin-binding inhibitory factors can, in the appropriate matrix context, facilitate "braking" of the branching program as the UB shifts from a rapid branching stage (governed by a feed-forward mechanism) to a stage where branching slows down (negative feedback) and eventually stops.

In search of guiding principles involved in the branching of epithelial tubes in the developing kidney, we analyzed branching of the ureteric bud (UB) in whole kidney culture as well as in isolated UB culture independent of mesenchyme but in the presence of mesenchymally derived soluble factors. Microinjection of the UB lumen (both in the isolated UB and in the whole kidney) with fluorescently labeled dextran sulfate demonstrated that branching occurred via smooth tubular epithelial outpouches with a lumen continuous with that of the original structure. Epithelial cells within these outpouches cells were wedge-shaped with actin, myosin-2 and ezrin localized to the luminal side, raising the possibility of a "purse-string" mechanism. Electron microscopy and decoration of heparan sulfates with biotinylated FGF2 revealed that the basolateral surface of the cells remained intact, without the type of cytoplasmic extensions (invadopodia) that are seen in three-dimensional MDCK, mIMCD, and UB cell culture models of branching tubulogenesis. Several growth factor receptors (i.e., FGFR1, FGFR2, c-Ret) and metalloproteases (i.e., MT1-MMP) were localized toward branching UB tips. A large survey of markers revealed the ER chaperone BiP to be highly expressed at UB tips, which, by electron microscopy, are enriched in rough endoplasmic reticulum and Golgi, supporting high activity in the synthesis of transmembrane and secretory proteins at UB tips. After early diffuse proliferation, proliferating and mitotic cells were mostly found within the branching ampullae, whereas apoptotic cells were mostly found in stalks. Gene array experiments, together with protein expression analysis by immunoblotting, revealed a differential spatiotemporal distribution of several proteins associated with epithelial maturation and polarization, including intercellular junctional proteins (e.g., ZO-1, claudin-3, E-cadherin) and the subapical cytoskeletal/microvillar protein ezrin. In addition, Ksp-cadherin was found at UB ampullary cells next to developing outpouches, suggesting a role in epithelial-mesenchymal interactions. These data from the isolated UB culture system support a model where UB branching occurs through outpouching possibly mediated by wedge-shaped cells created through an apical cytoskeletal purse-string mechanism. Additional potential mechanisms include (1) differential localization of growth factor receptors and metalloproteases at tips relative to stalks; (2) creation of a secretory epithelium, in part manifested by increased expression of the ER chaperone BiP, at tips relative to stalks; (3) after initial diffuse proliferation, coexistence of a balance of proliferation vs. apoptosis favoring tip growth with a very different balance in elongating stalks; and (4) differential maturation of the tight and adherens junctions as the structures develop. Because, without mesenchyme, both lateral and bifid branching occurs (including the ureter), the mesenchyme probably restricts lateral branching and provides guidance cues in vivo for di...

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Together with glial-derived neurotrophic factor (GDNF), soluble factors present in a metanephric mesenchyme (MM) cell conditioned medium (BSN-CM) are necessary to induce branching morphogenesis of the isolated ureteric bud (UB) in vitro (Proc. Natl. Acad. Sci. USA 96 (1999) 7330). Several lines of evidence are presented here in support of a modulating role for fibroblast growth factors (FGFs) in this process. RT-PCR revealed the expression of two FGF receptors, FGFR1(IIIc) and FGFR2(IIIb), in isolated embryonic day 13 rat UBs, which by indirect immunofluorescence displayed a uniform distribution. Rat kidney organ culture experiments in the presence of a soluble FGFR2(IIIb) chimera or a neutralizing antibody to FGF7 suggested an important contribution of FGFs other than FGF7 to the branching program. Several FGFs, including FGF1, FGF2, FGF7 and FGF10, in combination with GDNF and BSN-CM were found to affect growth and branching of the isolated UB, albeit with very different effects. FGF1 and FGF7 were at extreme ends of the spectrum, with FGF10 (more FGF1-like) and FGF2 (more FGF7-like) falling in between. FGF1 induced the formation of elongated UB branching stalks with distinct proliferative ampullary tips, whereas FGF7 induced amorphous buds displaying nonselective proliferation with little distinction between stalks and ampullae. Electron microscopic examination demonstrated that FGF1 treatment induced cytoskeletal organization, intercellular junctions and lumens along the stalk portion of the developing tubules, while the ampullary regions contained 'less differentiated' cells with an abundant secretory apparatus. In contrast, FGF7-induced UBs displayed this 'less differentiated' morphology regardless of position on the structure and were virtually indistinguishable from FGF1-induced ampullae. Consistent with this, GeneChip array analysis (employing a novel nanogram-scale assay consisting of two rounds of amplification and in vitro transcription for analyzing small quantities of RNA) revealed that FGF7-induced UBs expressed more markers of cell proliferation than FGF1, which caused the UB to express cytoskeletal proteins, extracellular matrix proteins, and at least one integrin, some of which may be important in UB branch elongation. Thus, while the various FGFs examined all support UB growth, FGF1 and FGF10 appear to be more important for branching and branch elongation, and may thus play a role in determination of nephron number and patterning in the developing kidney. These in vitro data may help to explain results from knockout and transgenic studies and suggest how different FGFs may, together with GDNF and other factor(s) secreted by MM cells, regulate branching morphogenesis of the UB by their relative effects on its growth, branching and branch elongation and differentiation, thereby affecting patterning in the developing kidney.