Fig. 4. Shh and Gli3 interact to control cell proliferation and expression of cell cycle regulatory proteins. (A-D) In situ analysis of BrdU incorporation at E14.5. Pregnant mice were injected with BrdU and sacrificed 4 hours later. BrdU incorporation was detected by an in situ BrdU incorporation assay. Red-stained nuclei are BrdU positive. Tissues were counterstained with Hematoxylin (blue). In wild-type (A), Gli3^–/– (C) and Shh^–/–;Gli3^–/– (D) mice, BrdU incorporation was observed in metanephric mesenchyme cells, mesenchyme-derived epithelial structures and ureteric bud tips. In Shh^–/– mice (B), BrdU incorporation was markedly decreased. Scale bar: 100 µm. (E) Quantitative analysis of BrdU incorporation at E14.5. BrdU incorporation is expressed as a fraction of the total number of cells in the ureteric bud (white bars) and its branches or in the metanephric mesenchyme (black bars). Shh deficiency decreased BrdU incorporation in both ureteric bud and mesenchyme-derived epithelial structures significantly compared with wild type. Removal of Gli3 in Shh^–/– mice rescued BrdU incorporation to levels not significantly different than those observed in wild type. Removal of Gli3 increased BrdU incorporation in ureteric bud derived cells to a level greater than that observed in wild type. (F) Western analysis of E14.5 kidney tissue lysates. Expression of cyclin D1 and MYCN was markedly decreased in Shh^–/– compared with wild type and was rescued to wild-type levels in Shh^–/–;Gli3^–/– mice. By contrast, expression of cyclin D2 and MYC was not affected by Shh deficiency.