Methodology to examine kidney development

Whole metanephric organ culture, developed by Clifford Grobstein in the 1950s (Grobstein 1956) has been the most important in vitro model system to date for studying kidney development. With this technique, whole rat or mouse metanephroi are cultured in defined media or in the presence of serum from approximately 13.5 or 11.5 dpc, respectively, when the ureteric tree contains just a few branches and the kidney contains no mature nephrons. The metanephroi are cultured at an air-media interface on Transwell filters for up to approximately 6 days, by which time they typically contain at least 50 glomeruli at different stages of maturation, and at least this number of ureteric branches. Developing glomeruli can be visualized and quantified by whole-mount immunostaining the cultures with WT-1, which is a marker of developing podocytes. The development of the ureteric tree can be visualized by immunostaining with calbindin-D_28K-or cytokeratin (Fig. 7). Alternatively, metanephroi from Hoxb7/GFP transgenic mice in which the entire ureteric epithelium expresses green fluorescent protein (GFP) can be cultured to monitor the development of the ureteric epithelium (see Sect. 5.5 below).