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First published online September 5, 2008
doi: 10.1242/10.1242/dev.022442

Development 135, 3301-3310 (2008)
Published by The Company of Biologists 2008
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Teashirt 3 is necessary for ureteral smooth muscle differentiation downstream of SHH and BMP4

Xavier Caubit1,*, Claire M. Lye2,*, Elise Martin1,*, Nathalie Coré1, David A. Long2, Christine Vola1, Dagan Jenkins3, Alistair N. Garratt4, Helen Skaer5, Adrian S. Woolf2,{dagger} and Laurent Fasano1,{dagger}

1 Institut de Biologie du Développement de Marseille-Luminy (IBDML), UMR6216, CNRS, Université de la Méditerranée, F-13288 Marseille cedex 09, France.
2 Nephro-Urology Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.
3 Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
4 Max-Delbrueck-Center for Molecular Medicine, Robert-Roessle-Strasse 10, 13125 Berlin, Germany.
5 Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.


Figure 1
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  		Fig. 1. Expression of TSHZ3 in the developing ureter and metanephros. (A-C) Immunostaining for TSHZ3 (green) and PAX2 (red) that visualises the UB and metanephric mesenchyme (mm) at E11.5 (A), E12.5 (B) and E12.75 (C). (D-H) Immunostaining for TSHZ3 (green) and nuclei (DAPI, blue) at E15.5 in metanephros (D), ureter (E) and bladder (F) and at E18.5 in ureter (G) and the bladder (H). Broken line in H separates the submucosal layer and the detrusor. d, detrusor SM; e, epithelium; mm, metanephric mesenchyme; mms, metanephric medullary stroma; pUB, proximal ureteric bud; sl, submucosal layer; UBs, ureteric bud stalk.

 

Figure 2
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  		Fig. 2. Targeted disruption of Tshz3. (A) Gene targeting strategy. The mouse Tshz3 locus consists of two exons and one intron, and spans more than 75 kb; the first and second exons encode, respectively, 13 and 1068 amino acids. The in-frame insertion of the lacZ-coding sequence (hatched box) and the neomycin expression cassette (pTK-neo; black box) into exon 2 (ex2; light dotted box) resulted in complete deletion of the zinc-finger motifs and created an XbaI digest size difference between wild-type and targeted loci. Restriction sites are abbreviated as follows: B, BamHI; E, EcoRI; S, SalI, N, NotI, X, XbaI. (B) Southern blot analysis of DNA from wild-type (+/+) and targeted (+/-) ES cell clones using XbaI-digested genomic DNA and a 3' external probe (3' probe; white box in A). (C) Appropriate recombination 5' to the locus was confirmed by PCR with primers shown in A (short arrows). (D) PCR-based genotyping of wild-type (+/+), heterozygous (+/-) and homozygous null (-/-) embryos. (E) Immunostaining of TSHZ3 (left panel) on sections from E15.5 wild-type (+/+) and Tshz3lacZ/lacZ mutant (-/-) ureter; TSHZ3 is not detected in mutant ureter. Co-immunostaining of TSHZ3 and β-gal (right panel) on a section from E14.5 Tshz3lacZ/+ heterozygote ureter.

 

Figure 3
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  		Fig. 3. Tshz3lacZ/lacZ mice develop congenital hydronephrosis. (A,B) Whole E18.5 urinary tracts. In comparison with wild-type littermates (A), Tshz3lacZ/lacZ kidneys had bilateral hydronephroses (B). (C) Higher magnification view of proximal hydroureter shown in B. (D-K) Haematoxylin and Eosin-stained transverse sections through E17.5 embryos (D,E) and E18.0 ureters (F-K). Wild-type proximal (F,G) and distal (H) ureter. Tshz3lacZ/lacZ proximal (I,J) and distal (K) ureter. The mutant ureter shown in I has collapsed post mortem, which is why it does not look dilated. (L-O) Sections are stained with Masson's trichrome and Haematoxylin which results in a red colour for muscles, blue for nuclei and a blue/green for collagens (epithelia also appear red). E16.0 wild-type (L) and mutant (N) ureters with mesenchymal cells surrounding proximal ureter ephithelia indicated by arrowheads. (M,O) High-power magnification images of longitudinal sections from wild-type (L) and Tshz3lacZ/lacZ (N) E16.0 proximal ureters demonstrate aggregating mesenchyme in wild type and early disorganisation of mesenchymal cells in mutants. (P,Q) E16.5 autopsies in which India ink was injected into the right renal pelvis of a normal (P) and a null mutant (Q) mouse: in both, ink flowed into the bladder (arrowhead). (R) Inset shows dilated proximal ureter filled with ink. bl, bladder; k, kidney; s, stromal layer; u, ureter; ue, ureteric epithelium; um, ureteral mesenchyme; ut, uterus.

 

Figure 4
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  		Fig. 4. Tshz3 is required for harmonised ureteric peristalisis. (A,B) Cultured ureter of wild-type (A) and Tshz3lacZ/lacZ (B) embryos. (C,D) Graphs illustrating peristaltic contractions of cultured ureters of wild-type (C) and Tshz3lacZ/lacZ (D) ureters. Measurements of luminal diameters were made at four different positions from their proximal to distal ends from a 20-second digital recording of contracting ureters (see Movies in the supplementary material). Data are presented as the percentage of the relaxed diameter. In the trace from the wild-type ureter (C), the contraction (downward motion from baseline) initiates about one-fifth of the way down (red line) the ureter; this is rapidly followed by both contraction of the more proximal segment (blue line) and also by distal propagation (pink and yellow lines). Null mutant trace (D) shows absent proximal segment contraction together with a variable delay of distal relaxation (pink line). Representative traces are shown for at least five samples from each genotype.

 

Figure 5
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  		Fig. 5. Characterisation of Tshz3-positive mesenchymal cells in the developing ureter. (A-F) Immunostaining for SMAA (green) and β-gal (red) in tissue section of E15.5 (B), E18.5 (D) and adult (F) Tshz3lacZ/+ ureters. Corresponding DAPI staining are shown (A,C,E). (G) Expression of Tshz3 in E18.5 ureter from TshzlacZ/+ mice, revealed by X-Gal staining (blue) and immunohistochemistry for RALDH2 (brown): inset shows RALDH2/X-Gal double-positive cells.

 

Figure 6
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  		Fig. 6. Ureteric SMC differentiation in wild-type and Tshz3-null mutants. (A-L) Transverse sections of E15.5 proximal ureters. In A-F, all nuclei were stained with DAPI (blue) and immunohistochemistry (green positive signals) is shown for SMMHC (A,D), SM22 (B,E) and SMAA (C,F). (G,J) Sections from wild-type (G) or null mutant (J) immunostained for SMAA (green), MYOCD (red) and DAPI (blue); arrow in J indicates MYOCD- and SMAA-positive cells in artery. In situ hybridisation for Myocd (H,K) and Smaa (I,L) (positive purple signal): arrowheads in I and L indicate the Smaa-positive signal in arteries. (M-P) Longitudinal (M,O) and transverse (N,P) sections were counterstained with Haematoxylin (blue) and immunostained for SMAA (brown). In Tshz3 mutant ureter (O) at E17.5, no SMAA was detected in the proximal ureter, in contrast to wild-type ureter (M). Distally, the immunostaining for SMAA was reduced in mutant (P) versus wild-type (N) ureters. (Q-T) E18.5 proximal ureter sections from wild-type (Q,R) or null mutant (S,T) stained with DAPI (blue) and immunostained for RALDH2 (Q,S, red) or UPK (R,T, red). Asterisks indicate the lumen of the ureter; arrowheads in Q and S indicate RALDH2-positive cells.

 

Figure 7
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  		Fig. 7. Molecular characterisation of Tshz3lacZ/lacZ ureters. (A-F) In situ hybridisation on transverse sections of E15.5 proximal ureters of wild type (A,C,E) and Tshz3-null mutant (B,D,F). Patterns of expression (red dots) were similar in both genotypes, with Shh expressed in urothelia (A,B), and Ptch1 (C,D) and Bmp4 (E,F) expressed in peri-urothelial cells. Silver grains were pseudo-coloured in Adobe Photoshop. (G,H) E14.5 proximal ureter sections from wild type (G) or null mutant (H) immunostained for pSMAD 1/5/8 (red) and DAPI (blue).

 

Figure 8
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  		Fig. 8. Tshz3 induction by BMP4. (A) Whole-mount images of E12.5 explants (each comprising a metanephros and attached ureter) cultured for 4 days in absence (left and middle panels) or in presence (right panel) of recombinant BMP4 (100 ng/ml). Ureters were immunoprobed with antibodies directed against SMAA (green) and E-cadherin (red; E-Cad) to detect SMC and epithelia, respectively. In each frame, the proximal ureter is outlined by a box; note the paucity of SMAA expression in this region in the mutants, whether or not exposed to exogenous BMP4. (B) Effects of BMP4 on gene expression in E13.5 wild-type explants cultured for 3 days in absence or presence of exogenous BMP4 (100 ng/ml). After factoring for Hprt, real-time Q-PCR revealed significant increases for Id3, Tshz3 and Myocd. Statistical analyses were performed by one-tailed paired t-tests. Expression data are represented as mean±s.e.m. with n=5 in each group.

 

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© The Company of Biologists Ltd 2008