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Erk MAP kinase regulates branching morphogenesis in the developing mouse kidney

Centre for Developmental Biology, Department of Biomedical Sciences, University of Edinburgh, George Square, Edinburgh, UK

View larger version (14K): [in a new window]   Fig. 1. A schematic of the classical Erk 1/2 MAP kinase pathway, showing typical connections to and from the pathway, and showing the site of action of the inhibitor, PD98059.

View larger version (17K): [in a new window]   Fig. 2. Blotting using phospho-specific antibodies to activated Erk1/2 MAP kinases shows them to be present in both the ureteric bud (Bud) and the mesenchyme of normal developing kidneys (Mes), the p42 (Erk 2) signal being stronger than that of p44 (Erk1).

View larger version (51K): [in a new window]   Fig. 3. Blocking the Erk MAP kinase pathway using PD98059 inhibits ureteric bud branching. (A) The ureteric buds of E11.5 kidney rudiments cultured for 72 hours in normal medium branch extensively in an organotypic manner. (B,C) Inclusion of PD98059 (15 µM), an inhibitor of Mek-activation of Erk1/2 MAP kinases, greatly decreases the number of branches produced by the ureteric bud although some elongation of existing branches continues. (D) When kidney rudiments are grown for 40 hours in PD98059 (10 µM) and then transferred to normal medium for a further 48 hours, the central, oldest parts of the ureteric bud tree show sparse branching but the outer (newest) parts show a more normal branching density, suggesting that the cells recover from PD98059 treatment. (E) A dose-response curve for PD98059, showing how the number of tips formed by the ureteric bud tree declines with increasing (PD98059); error bars represent the standard error of the mean.

View larger version (82K): [in a new window]   Fig. 4. Effect of PD98059 on ureteric bud cell biology. (A) The pattern of cell proliferation (BrdU incorporation) in normal kidneys; the inset shows both ureteric bud and mesenchyme, and the main image shows only the ureteric bud for clarity (the mesenchyme being masked out electronically). Most BrdU incorporation (green) takes place in the ureteric bud tips. The laminin basement membrane of those tips (red) shows numerous short processes reaching out into the mesenchyme (arrowheads). (B) In kidneys cultures in the presence of 25 µM PD98059, there is less proliferation in the ureteric bud, though it is again localised mainly in the tips. There are also fewer processes and typically, as in this image, the only process points directly forward from the tip (arrowhead). (C,D) Propidium iodide staining of nuclei reveals very little apoptosis in the ureteric buds of either PD98059-treated (C) or control (D) kidneys, although in both cases there is mesenchymal apoptosis away from the immediate vicinity of the ureteric bud (arrowheads). (E,F) Filamentous actin is concentrated strongly at the apices of cells of control kidneys (E) (arrowheads), but much less so in kidneys growing in PD98059 (F). (G,H) Ureteric buds of both PD98059-treated kidneys (G) and controls (H) express Ret. (I,J) Ureteric buds of both PD98059-treated (I) and control (J) kidneys express Ros; many tips of control kidneys show reduced immunoreactivity in the few cells right at the ends of the tips (arrowhead), though PD98059-treated kidneys do not show this effect. Scale bars: 50 µm.

View larger version (50K): [in a new window]   Fig. 5. Evidence from tissue recombinations (A-F) and isolated ureteric buds (G,H) that MAP kinase is required in the ureteric bud itself. (A-C) Lung epithelium undergoes branching morphogenesis in normal medium (A) and also in the presence of 25 µM PD98059 (B), which is sufficient to block ureteric bud morphogenesis in kidneys; even at 75 µM PD98059 (C), some branching continues. Lung mesenchyme is therefore capable of supporting epithelial branching in the presence of PD98059. Ureteric buds recombined with lung mesenchyme and cultured in the presence of GDNF branch organotypically (E), though not as extensively as they do when recombined with kidney mesenchyme (D). In the presence of 25 µM PD98059, however, ureteric buds show markedly reduced branching in lung mesenchyme (F; two ureteric buds are shown in this figure). (G) Isolated ureteric buds cultured in the presence of 50 ng/ml GDNF retain their epithelial organisation, though they round up a little to form almost spherical cysts (this effect has been described previously by Sainio et al. (Sainio et al., 1997)). In the presence of PD98059, however, the ability of GDNF to maintain epithelial organisation of the ureteric bud is lost, and over 24 hours its cells spill out to form a disorganised mass as if no GDNF were present (H).

View larger version (32K): [in a new window]   Fig. 6. The activation of Erk MAP kinases in the ureteric bud is reduced markedly when kidneys are incubated in the presence of function-blocking antibodies to GDNF. (A) The top panel shows a western blot of ureteric buds, isolated after a 7 hour incubation of complete kidneys with and without anti-GDNF, probed with an antibody specific for phosphorylated Erk1/2 (Erk-P). Much less phosphorylation is seen in ureteric buds of kidneys treated with anti-GDNF. The bottom panel shows the same samples probed with an antibody that recognises both phosphorylated and non-phosphorylated Erk, and shows that anti-GDNF treatment does not alter the amounts of Erk protein present in the cells. The blot was prepared after just 7 hours so that net ureteric bud volume would not have changed significantly. (B,C) Micrographs of kidneys cultured in normal medium (B) and anti-GDNF (C) for 48 hours confirm that the anti-GDNF antibody blocks ureteric bud morphogenesis.

View larger version (40K): [in a new window]   Fig. 7. The activation of Erk MAP kinases is greatly reduced when kidney rudiments are grown in the presence of sodium chlorate, an inhibitor of the synthesis of sulphated glycosaminoglycans that is known to inhibit ureteric bud morphogenesis. The top panels show western blots of complete kidney rudiments cultured in the presence of sodium chlorate, or ureteric buds dissected from such kidney rudiments after their incubation, probed with an antibody that recognises only phosphorylated Erk1/2 (Erk-P); chlorate treatment greatly reduces levels of Erk phosphorylation compared with controls (Normal). The right-most lane shows the even more severe reduction in Erk phosphorylation produced by treating kidney rudiments with PD98059. The bottom panels, showing the same samples probed with an antibody that recognises both phosphorylated (Erk-P) and non-phosphorylated Erk, shows that chlorate and PD98059 treatments have little effect on amounts of Erk protein in the tissue.

View larger version (21K): [in a new window]   Fig. 8. The activation of Erk MAP kinases in the ureteric bud is not detectably affected when kidneys are incubated in the presence of 10 ng/ml BMP2. (A) The top panel shows a western blot of ureteric buds, isolated after an 8 hour incubation of complete kidneys with and without BMP2, probed with an antibody specific for phosphorylated Erk1/2 (Erk-P). The bottom panel shows the same samples probed with an antibody that recognises both phosphorylated and non-phosphorylated Erk. (B,C) Micrographs of kidneys cultured in normal medium (C) and 10 ng/ml BMP2 (C) for 30 hours confirm that BMP2 inhibits ureteric bud morphogenesis.

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