Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney

Abstract

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...

GDNF/Ret signaling and the development of the kidney

“…Some candidates are localized cell proliferation, changes in cell shape, directed cell movements, differential cell-cell adhesion, and localized remodeling of the extracellular matrix. (68) * How does GDNF signaling interact with other factors and signaling pathways, that either promote (e.g. FGFs) or inhibit (e.g.…”

“…Several cellular and subcellular processes that might underlie ureteric bud branching include localized cell proliferation, directed cell migration, differential adhesion and changes in cell shape. (68,69) While GDNF has been linked to all of these processes in cell culture systems or in the developing nervous system, it remains to be determined which of them contribute to Wolffian duct and ureteric bud branching.…”

“…(66,68,75) Therefore, the UB tip represents a growth center, where most of the new UB cells are generated, and then distributed to the new tips (which form via branching) and to the elongating trunks. Not all the growth occurs at the tips, as UB trunks can elongate without incorporation of cells from the adjacent tips (Fig.…”

Staged in vitro reconstitution and implantation of engineered rat kidney tissue

“…In vitro, these growth factor conditions provided the most robust branch-stimulating conditions (i.e., growth plus most in vivo-like patterning) among the many soluble-factor combinations that we have tested (33)(34)(35)(36)(37). The fact that a combination of BSN-CM, FGF1, and GDNF gave more consistent branching growth than PTN, FGF1, and GDNF suggests that one or more factors within BSN-CM will need to be isolated to achieve ideal minimal conditions.…”

“…Taken together with our previous studies (29,(33)(34)(35)(36), a minimal set of conditions can be defined for UB growth/ branching as pleiotrophin (PTN) plus GDNF (35) in a type IV collagen matrix. The most robust system for both tip and stalk formation, as well as growth, of the in vivo-isolated bud, however, appears to be BSN-CM supplemented with GDNF and FGF1 (36, 37) in a 50% Matrigel solution, which were the culture conditions used for the in vitro-formed UB described below.…”