Fig 2: Inhibition of Cer1-induced ureteric bud branching by Bmp4 and acquired sensitivity to Gdnf signalling.A-G) The kidneys were prepared at E11 and cultured for 48 hrs. A) The ureteric bud in a cultured embryonic kidney branched a few times, as revealed by Troma-1 immunostaining (in orange). The mesenchyme was induced to undergo nephrogenesis, as shown by the expression of an early tubule differentiation marker Pax2, as identified with an antibody (in green). B) A Cer1 gain of function enhanced ureteric bud branching to a moderate extent and also created some ectopic ureteric side branches (arrows). C) A double ureteric bud formed occasionally in the cultured embryonic kidney in response to the Cer1 gain of function (in orange, arrow). D) Supplementation of Gdnf in culture induced some Wolffian duct-derived epithelial bud formation in the wild-type embryonic kidneys (arrows), while this was more pronounced in a kidney overexpressing Cer1 (E, arrows, arrowheads), with elevated Pax2 expression. (F) Culture of a normal embryonic kidney in the presence of Bmp4 (in green). G) An embryonic kidney expressing Cer1 cultured in the presence of Bmp4 for 48 hrs. H) Numbers of ureteric bud tips calculated from several cultured kidneys, as depicted in A-G. I) Note that supplementation of the culture medium with Bmp4 in reduces Gdnf and Wnt11 gene expression in normal kidneys as compared with controls analysed by qPCR. Cer1, transgenic kidney overexpressing the Cer1 gene; Wt, wild-type. Scale bar: 100 µm.

Fig 3: BIAcore sensograms indicate that Cer1 binds to Bmp2 and Bmp4 but not to Gdnf.(A) The purified recombinant mCer1 protein used for the BIAcore binding studies, as identified by SDS-PAGE with molecular size markers. B) BIAcore sensorgrams of BMP2, BMP4, or Gdnf to Cer1, which was immobilized on a chip. The BIAcore sensograms indicate that BMP2 and BMP4 bind to mCer1 while Gdnf does not. The dissociation constants (KD) of BMP2 and BMP4 for mCer1 were 3.38±1.29×10−9 and 2.34 ±0.16×10−8, respectively. The measurements were performed in triplicate. The sensograms represent mean values.

Fig 4: Cer1 gain of function leads to reduced Bmp4 expression but induced Gdnf and Wnt11 expression.A, B) Localization of Gdnf gene expression (arrows) in the mesenchyme of a wild-type (A) and Cer1+ embryonic kidney (B). The dotted lines in (A) and (B) depict the border of the ureteric bud. C, D) Ret expression reveals that the ureteric bud is advanced in development at E11.5 in the case of Cer1+ as compared with a stage-matched control (arrows in C and D). E-F) Wnt11 expression reveals that the Cer1 gain of function has promoted ureteric bud development at E12.5 as compared with the degree of tip development in the control. G-J) Analysis of changes in Gdnf, Wnt11, Bmp4 and Gremlin (Grem) expression brought about by qPCR point to trend for average reductions of 24% in Gdnf (G), 14% in Wnt11 (H), 21% in Bmp4 (I) and 15% in Grem (J) relative to the controls upon Cer1 gain of function. Cer1, transgenic kidney overexpressing the Cer1 gene; Wt, wild-type. Scale bar, 50 µm.

Fig 5: Changes in Smad reporter and Wnt4 expression in response to Bmp4 and in Bmp4 and Wnt in response to Cer1.A) mK4 cells were transfected with the Bmp reporter BRE2-luc and cultured in the presence of various amounts of Bmp4 protein. Bmp4 stimulated the activity of the reporter in a dose-dependent manner up to a plateau reached at 33 ng/ml of Bmp4. B) The mode of Bmp4-mediated regulation of Wnt4 gene expression was dependent on the Bmp4 dose. C) CHO cells were transfected with the Bmp reporter BRE2-luc and various amounts of Cer1 cDNA and the cells were cultured in the presence of Bmp4 protein. Cer1 inhibited the activity of the BMP4 protein-induced Bmp reporter in a dose-dependent manner. D, E) mK4 or CHO cells were transfected with a plasmid that encoded Bmp4 (10 ng/well), Cer1 and the BRE2-luc reporter construct in the amounts depicted in the figure. Cer1 inhibited the activity of the BMP4-induced Bmp reporter in dose-dependent manner. F) mK4 cells were transfected with plasmids that encode Bmp4 (10ng/well) or Noggin and BRE2-luc. Noggin is a robust inhibitor of Bmp4-mediated reporter activity (F). G) CHO cells were transfected with Wnt11 (25 ng/well), Cer1 (100 ng/well) and the SuperTopFlash reporter plasmids. While Wnt3a activates the canonical Wnt signalling pathway reporter, the presence of Wnt11 activity inhibits it. Expression of mCer1 does not alter the degree of Wnt11-mediated inhibition of the Wnt pathway reporter activity. It is significant that Cer1 is sufficient to achieve moderate inhibition of the Wnt3a-mediated induction of the canonical Wnt signalling pathway reporter Top Flash. Luciferase activity in the control transfections for the reporter assays was set to 100.