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First published online 17 September 2008
doi: 10.1242/dev.019760

Development 135, 3333-3343 (2008)
Published by The Company of Biologists 2008
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A functional role for semaphorin 4D/plexin B1 interactions in epithelial branching morphogenesis during organogenesis

Alexander Korostylev1,*, Thomas Worzfeld1,*, Suhua Deng1, Roland H. Friedel2, Jakub M. Swiercz1, Peter Vodrazka1, Viola Maier2, Alexandra Hirschberg1, Yoshiharu Ohoka3, Shinobu Inagaki3, Stefan Offermanns1 and Rohini Kuner1,{dagger}

1 Pharmacology Institute, Im Neuenheimer Feld 366, University of Heidelberg, 69120 Heidelberg, Germany.
2 Institute of Developmental Genetics, Helmholtz Center Munich, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
3 Group of Neurobiology, School of Allied Health Sciences, Osaka University Faculty of Medicine, Yamadaoka 1-7, Suita, Osaka 565-0871, Japan.


Figure 1
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  		Fig. 1. Reciprocal expression of plexin B1 and Sema4d during early stages of branching morphogenesis during kidney development. Boxed areas are magnified in adjacent panels in A-F. Images represent mRNA in situ hybridization (A-E) or lacZ-staining (F,G). (A,B) At E13.5, plexin B1 mRNA is expressed in ureteric tips (arrow in A) whereas Sema4d mRNA is expressed in the condensing mesenchyme (arrowhead in B). (C,D) At E15.5, plexin B1 begins to bt expressed in developing glomeruli (arrow in C) and Sema4d is found in mesenchymal epithelium-derived-S/comma-shaped tubular bodies (asterisk in D). (E) In situ mRNA hybridization for plexin B1 (blue-purple stain) and co-immunostaining with an antibody against WT1 (red) reveal expression of plexin B1 in ureteric tips complementary to WT1-stained condensing mesenchyme at E13.5. (F) Heterozygous reporter mice expressing lacZ via the mouse locus for plexin B1 (Plxnb1lacZ/+) reveal β-galactosidase-stained ureteric tips in the outer cortex of developing kidneys at E17.5 (arrows) in addition to tubular structures in the medulla. Kidneys from wild-type littermates do not show β-galactosidase staining (G). Scale bars: 10 µm in A-G.

 

Figure 2
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  		Fig. 2. mRNA in situ hybridization shows complementary expression of plexin B1 and its ligand Sema4d in epithelial and mesenchymal compartments, respectively, during genesis of several, but not all, organs in mice. (A) Expression of plexin B1 in the developing tooth cusp at embryonic day 13.5 (E13.5) and at day of birth (P0, long arrows), and of Sema4d in the adjacent mesenchyme (short arrows). (B) Expression of plexin B1 in proliferative olfactory epithelium layer (long arrows) and of Sema4d at the border between the olfactory epithelium and mesenchyme (short arrows). Note the reciprocal gradients of plexin B1 and Sema4d expression. (C) Expression of plexin B1 in bronchi (E13.5, long arrow) and branching bronchioles (E15.5, long arrow). Sema4d is expressed on the inner aspect of plexin B1-expressing bronchioles (short arrows), but is not found in the surrounding mesenchyme. In all panels, boxed areas are magnified in insets on the right. Scale bars: 10 µm in A-C.

 

Figure 3
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  		Fig. 3. Development of mouse metanephroi in whole-organ cultures and its modulation by Sema4d. (A-C) Phase-contrast images of a kidney rudiment isolated at E12 (A) and of the same kidney after 24 hours (B) and 48 hours (C) in culture. (D-F) Maximal projections of confocal images of whole cultured metanephroi showing the ureteric tree stained with an anti-calbindin antibody (green, D) and the condensing mesenchyme stained with an anti-WT1 antibody (red, E,F) at E12 (D) and after 24 hours (E) and 48 hours (F) in culture. UT, ureteric tip; CD, collecting duct; MM, metanephrogenic mesenchyme; CB, comma-shape bodies, two examples are indicated by the double arrow. (G) Illustration of the method used for quantifying ureteric branch points (bp) and ureteric tips (arrows, UT) in maximal confocal projections. (H,I) Typical examples of E12 metanephroi cultured for 24 hours in medium supplemented with concentrated medium of mock-transfected HEK293T cells (H) or Sema4d-AP-transfected HEK293T cells (I). The ureteric tree is labelled with an anti-calbindin antibody (green). (J) Western blot analysis demonstrating the efficacy of Sema4d in inducing RhoA activation in a GST-RBD-based assay in HEK293T cells transfected with plexin B1 (PlxnB1) and PDZRhoGEF (PRG). Anti-Myc-positive bands in western blotting serve as loading controls. (K) Sema4d treatment reduces the number of ureteric tips and branch points over mock treatment in sister cultures of metanephroi. (L) Sema4d treatment decreases the size of the developing kidney over mock treatment. Only sister kidneys cultured for 48 hours were taken for analysis. *P<0.001 in comparison with mock, Student's paired t-test. Scale bars: 200 µm in A-I.

 

Figure 4
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  		Fig. 4. Effects of blockade of endogenous Sema4d on metanephroic development. (A) Western blot analysis demonstrating the efficacy of a Sema4d function-blocking antibody (anti-Sema4d), but not preimmune serum (pre.; control), in attenuating Sema4d-induced RhoA activation in a GST-RBD-based assay in HEK293T cells transfected with plexin B1 (plxnB1) and PDZRhoGEF (PRG). Anti-Myc-positive bands in western blotting serve as loading controls. (B,C) Typical examples of the anti-calbindin-immunoreactive ureteric tree (green) in sister metanephroi from E12 embryos cultured in the presence of an anti-Sema4d antibody (C) or preimmune serum (B, control). (D,E) E12 metanephroi cultured with the anti-Sema4d antibody showed significantly more branching (D), but were the same size (E), when compared with metanephroi cultured with preimmune serum (control). *P<0.05, Student's paired t-test. Scale bars: 250 µm.

 

Figure 5
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  		Fig. 5. Pharmacological blockade of receptor tyrosine kinases (RTK) by K252a or by the selective Met inhibitor PHA665752 does not affect Sema4d-induced inhibition of ureteric branching in metanephroi cultured at E12. (A-D) Typical examples of anti-calbindin-stained mouse metanephroi cultured with either Sema4d or mock medium in the presence or absence of the RTK inhibitor, K252a (200 nM). (E-H) Blockade of RTKs by K252a significantly reduced ureteric branching (E,F) and size (G,H) in mock-treated kidneys, but did not affect Sema4d-induced modulation of these parameters in developing kidneys. (I-L) Similar results were obtained when using the selective Met inhibitor PHA665752. *P<0.001 when compared with mock or Sema4d alone; Student's paired t-test.

 

Figure 6
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  		Fig. 6. Sema4d-induced inhibition of ureteric branching morphogenesis requires activation of the Rho-ROCK pathway. (A) Treatment with Sema4d leads to activation of RhoA in mice metanephroi cultured at E14.5. Lysates of kidneys treated with either Sema4d or mock medium for 5 hours were incubated with RBD-coupled sepharose and the amount of RhoA was analysed by western blotting with an anti-RhoA antibody. The amount of G{alpha}13 was comparable in the two samples (internal control). (B-D) Typical examples of anti-calbindin-stained metanephroi cultured with either Sema4d or mock medium in the presence or absence of the ROCK inhibitor Y27632 (1 µM). In the presence of 1 µM Y27632, Sema4d did not attenuate ureteric branching. (E,F) Y27632 (3 µM) led to a hyperplasia and distortion of ureteric structures (arrowheads), to a lesser extent in Sema4d-treated kidneys than in mock-treated kidneys. (G-I) Higher magnification views of phalloidin-stained ureteric tips in Sema4d- or mock-treated kidneys cultured in the presence of 3 µM Y27632. The Y27632-induced distortion of apical wedge cells and actin bundles and the increase in tip lumen occurs to a lesser extent in Sema4d-treated kidneys in comparison with mock-treated kidneys. (J,K) Quantitative summary of the effects of Y27632 at either 1 µM or 3 µM on Sema4d- or mock-treated metanephroi. {dagger}P<0.001 when compared with mock; *P<0.05 when compared with mock or Sema4d alone; ANOVA followed by post-hoc Fisher's test. Scale bars: 200 µm in B-F; 70 µm in G-I.

 

Figure 7
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  		Fig. 7. Early defects in kidney development in mice constitutively lacking plexin B1 expression (Plxnb1-/-). (A) When compared with heterozygous control littermates (Plxnb1+/-), Plxnb1-/- mice show a loss of plexin B1 mRNA, whereas the expression of plexin-B2 mRNA remains unchanged. (B,C) Typical examples of kidneys dissected out of Plxnb1-/- (B) and Plxnb1+/- (C) littermates at E13.5 and stained with an anti-calbindin-28K antibody. (D-G) In comparison with heterozygous littermates, Plxnb1-/- kidneys are larger in size (D,E) and have increased ureteric branching (F,G) at E13.5. (H,I) At E14.5, Plxnb1-/- mice show a larger area of kidneys than heterozygous littermates, but at E15.5 no differences can be detected. (J) The number of developed nephrons is not significantly different between Plxnb1-/- and Plxnb1+/- littermates at E17.5. *P<0.001, Student's t-test. Scale bar: 200 µm.

 

Figure 8
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  		Fig. 8. Effects of Sema4d on metanephroic development are mediated by plexin B1. (A-D) Typical examples of anti-calbindin-stained metanephroi derived from wild-type (wt) or plexin B1-knockout embryos (Plxnb1-/-) cultured with either Sema4d or mock medium. (E-H) The Sema4d-induced inhibition of ureteric branching (E,F) and kidney size (G,H) in wild-type kidneys is lost in kidneys that lack plexin B1.*P<0.05 when compared with mock; Student's paired t-test.

 

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© The Company of Biologists Ltd 2008