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-β (TGF-β) superfamily. Treatment of cultured whole embryonic kidney with BMP2, BMP4, activin, or TGF-β1 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-β1-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-β1, 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-β 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-β-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.
Bibliographical noteFunding Information:
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK57286 and RO1-DK49517 (to S.K.N) and an American Heart Association Scientist Development Award 9730096N (to K.T.B). D.L. Steer is a recipient of a National Research Service Award (1F32DK09974). H. Sakurai is a recipient of an American Heart Association Scientist Development Award (0330182N). K.T. Bush is also supported by a Normon S. Coplon Extramural Grant from Satellite Healthcare, Inc. The care and use of animals described in this investigation conform to the procedures of the laboratory's Animal Protocol approved by the Animal Subjects Program and the IACUC of the University of California, San Diego.
- Bone morphogenetic proteins
- Branching morphogenesis
- Fibroblast growth factors
- Kidney development
- Transforming growth factor-β
- Ureteric bud