The expression of RET tyrosine kinase receptor in

The expression of RET tyrosine kinase receptor in UB tip cells is regulated by multiple signaling pathways. For example, WNT-β-catenin activity that first appears in UB and then persists in UB tip cells during kidney development was shown to be required for maintaining UB tip identity from studies using UB-specific β-catenin knockout models (Iglesias et al., 2007; Bridgewater et al., 2008; Marose et al., 2008). Retinoic phorbol myristate acetate (RA) derived from the stromal cells is another signaling pathway that maintains Ret expression in UB tip cells, as the expression of the dominant-negative form of RA receptor was found to inhibit both Ret expression and UB branching (Batourina et al., 2001; Rosselot et al., 2010). Another important signaling pathway for Ret expression and UB branching is fibroblast growth factor (FGF). FGF7 and FGF10 are expressed in stromal and MM cells, and loss of FRS2A/FGFRR2 receptor in UB cells was found to cause a reduction in Ret expression with fewer UB tips (Qiao et al., 1999b; Michos et al., 2010; Bates, 2011). The in vitro UB culture system has been widely used to investigate the regulation of UB branching morphogenesis. These studies have unraveled important roles played by multiple growth factors, including endodermal growth factor, hepatocyte growth factor, epidermal growth factor (EGF)-EGF receptor, FGF, vascular endothelial growth factor A (VEGF-A)-VEGF receptor 2, and transforming growth factor β superfamily members (Perantoni et al., 1991; Santos and Nigam, 1993; Sakurai and Nigam, 1997; Qiao et al., 1999a, 2001; Bush et al., 2004; Woolf et al., 1995; Ishibe et al., 2009; Marlier et al., 2009), as well as soluble factors, such as pleiotrophin (Sakurai et al., 2001) and heregulin (Sakurai et al., 2005), and extracellular matrix, such as type I and IV collagen and Matrigel (Perantoni et al., 1991; Rosines et al., 2007). Serum was also found to be required to promote UB branching morphogenesis in vitro (Takayama et al., 2014). However, the use of culture media containing serum and/or conditioned medium from an MM cell line (BSN-CM) in these studies made it difficult to examine the effect of a specific signaling pathway.
Results
Discussion In this study we showed the feasibility of maintaining UB growth in vitro in an MM- and serum-free culture medium containing defined growth factors. This simplified culture condition allowed us to analyze the specific effect of different signaling pathways. With isolated E11.5 UB, we found that UB cells could not survive in serum-free medium with GDNF alone but required FGF or RA signaling, which worked independently to prevent apoptosis in UB cells and allow them to proliferate. In addition, we found that the maintenance of UB tip cell identity required RET induction by either activation of WNT-β-catenin signaling or RA. Our results therefore demonstrate that there are three critical steps involved in the propagation and maintenance of UB tip cells, i.e., cell survival, proliferation, and RET induction/activation (Figure 7C). The two minimal conditions that we have identified in our MM- and serum-free culture condition are therefore the combination of GDNF, FGF1 with WNT-β-catenin signaling, and the combination of GDNF with RA. However, since RA is known to promote the differentiation of UB cells to collecting duct cells (Takayama et al., 2014) and WNT-β-catenin is known to inhibit such a process (Marose et al., 2008), as we have also found in our present study (Figure 6E), WNT-β-catenin signaling is required to maximize the effect of GDNF and RA in maintaining the proliferation of UB cells. Together with the fact that FGF signaling promotes colony formation from single UB cells, we thus conclude that the combination of GDNF, FGF, RA, and CHIR99021, together with Y-27632, represents the optimum condition to maximize the expansion of dispersed UB cells while maintaining tip cell identity in our serum-free culture condition. Under this condition, FGF1 and FGF7 are equally effective (Figures S1F–S1H). However, we found that this culture condition could not sustain the proliferation of dispersed UB cells after the second round of dissociation from the reconstituted UB structures. Further improvement in our culture system is therefore required to perpetuate the propagation of dispersed UB cells in culture.