Glial Cell Line-Derived Neurotrophic Factor Stimulates Ureteric Bud Outgrowth and Enhances Survival of Ureteric Bud Cells in vitro

Abstract

Development of epithelial organs requires co-ordinated interactions between epithelial and mesenchymal tissues. Studies using null mutant mice have indicated that the ret receptor and its ligand, glial cell line-derived neurotrophic factor (GDNF), are crucial for initiation of development of the metanephric kidney. However, the role of this signalling system in other branching organs has not been analysed. Here we describe detailed expression studies of ret, GDNF, and a co-receptor for GDNF (GDNFRα) in the developing mouse metanephros, lung, and submandibular salivary gland. Also, we examined the role of this signalling system in the development of these organs in vitro. In situ hybridisation revealed differences in the spatial distribution of the three transcripts in the different organs. At the initiation of metanephric development, late on embryonic day 10 (E10), ret and GDNFRα were detected in the Wolffian duct (including the presumptive ureteric bud) whilst the presumptive metanephric mesenchyme expressed GDNFRα and GDNF. Later in development, all three transcripts were restricted to the nephrogenic zone. In contrast, expression in the lung was not detectable by in situ hybridisation until after initiation of development, at E13.5. At this time ret was expressed throughout the epithelium; GDNF was detected throughout the mesenchyme, and GDNFRα was present in the proximal epithelium and mesenchyme only. Ret and GDNF were not detected in the epithelium or mesenchyme of the developing salivary gland, however, GDNFRα was expressed in the mesenchyme at E13.5 and E16.5. Functional studies demonstrated that in organ culture, GDNF significantly increased branching morphogenesis of the E11.5 metanephros, and induced the formation of ectopic ureteric buds from the base of the bud and from the Wolffian duct. The development of lung and salivary primordia were not affected under similar growth conditions. In a novel ureteric bud primary culture system, GDNF significantly increased cell numbers at 24 and 48 h. In cells cultured on laminin this increase was due to increased survival and proliferation, whereas in cells cultured on fibronectin, only survival was enhanced. Our data suggest that GDNF stimulates outgrowth of the ureteric bud, in part, by enhancing cell survival and possibly by increasing proliferation.

GDNF/Ret signaling and the development of the kidney

“…Why is Ret signaling specifically required for tip formation? One potential explanation is that it is important for cell survival in the UB tips, (63) but this was not supported by the data, as dying Ret À/À cells were not observed in the tips. Alternatively, Ret mutant cells might have a migration defect and fail to migrate into the tips in response to GDNF.…”

“…(10) Addition of GDNF to kidney cultures can increase the number of UB tips, which suggested a stimulation of branching. (42,62) Conversely, GDNF antibodies (42,63) or a Ret-Ig fusion protein (64) inhibit growth of the kidney in vitro. Finally, GDNF is continuously required (but is not sufficient) for the branching of isolated UBs grown in Matrigel with conditioned medium from a mesenchymal cell line.…”

OFD1, the Gene Mutated in Oral-Facial-Digital Syndrome Type 1, Is Expressed in the Metanephros and in Human Embryonic Renal Mesenchymal Cells

“…Cells from all cultures appeared uniformly "mesenchymal" in shape, with irregular, elongated outlines; although uncloned, this phenotype was maintained for more than 20 passages. The mesenchymal phenotype of the cells was further examined by assessing the expression of proteins characteristic of this lineage in vivo (Figure 5A), including the WT1 (21) and PAX2 transcription factors (21), HGF (22) and GDNF (23). All lines expressed GDNF, as assessed by Western blot, with a prominent band at 18 kD ( Figure 5A).…”

Glial Cell–Derived Neurotrophic Factor–Independent Ureteric Bud Outgrowth from the Wolffian Duct

“…Gdnf knockout mice are known to have renal agenesis, 2-4 and mice lacking GDNF receptor genes such as Gfra1 5 and Ret 6,7 also exhibit similar phenotypic abnormalities. In vitro studies have also demonstrated that GDNF signaling through GFR␣1 and Ret is a central signaling system involved in both the UB outgrowth from the WD 8,9 and branching morphogenesis of the UB. 10 Nevertheless, a substantial percentage of knockout animals lacking Gdnf (27% 2 ) or its receptors (40 to 45% of Ret Ϫ/Ϫ 7 ) have seemingly normal UB formation, suggesting that the GDNF-dependent budding pathway of the WD may be bypassed by activation of other signaling pathways.…”