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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01324

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TGFß superfamily signals are required for morphogenesis of the kidney mesenchyme progenitor population

¹ Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
² Department of Pathology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA
³ Department of Pediatrics, Tufts-New England Medical Center, 750 Washington Street, Boston, MA 02111, USA

View larger version (67K): [in a new window]   Fig. 1. Generation and characterization of the Bmp7cre/+ and HoxB7cre kidney deleter strains. (A) Targeting strategy used to generate Bmp7cre/+. (B,C,D) Bmp7cre/+ activates the ROSA26R reporter in Wolffian duct, ureteric bud and the collecting duct system. Mesenchyme excision is scattered through the metanephric blastema at E11.5 (B), and by E12.0 (C), is uniform throughout the mesenchyme condensates surrounding collecting duct tips. A population of cells surrounding the collecting duct trunk is devoid of excision at this stage. At E13.5 (D), excision is limited to the collecting ducts, nephrogenic mesenchyme, renal vesicles, comma- and s-shaped bodies and podocytes. No excision can be seen within stromal cell populations. Bmp7+/lacZ is expressed in nephrogenic mesenchyme (E), the prospective distal end of the comma-shaped body (H), and podocytes and distal tubules of maturing nephrons (I). Proximal tubules and Bowman's capsules do not express Bmp7+/lacZ. Staining in Bmp7cre/+;Rosa26R kidneys is seen throughout the nephron (F,G). HoxB7cre activity marks the Wolffian duct, ureteric bud and collecting ducts at E11.75 (J) and E13.5 (K). The red line in K denotes the border between the collecting duct and nephron. CB, comma-shaped body; CD, collecting duct; dist, distal tubule; Mes, metanephric mesenchyme; neph, nephron; NM, nephrogenic mesenchyme; pod, podocytes; prox, proximal tubule; PS, peripheral stroma; UB, ureteric bud; WD, Wolffian duct; B, BamHI; Na, NaeI; Nc, NcoI; R, EcoRI.

View larger version (162K): [in a new window]   Fig. 2. Localization of active BMP and TGFß signal transduction in the developing kidney. Nuclear immunohistochemical staining for phospho-Smad1 (pSmad1) and phospho-Smad2 (pSmad2) identifies cells actively responding to BMP and TGFß signals, respectively. At E11.5 (A,B), activation is mainly within the metanephric mesenchyme, with responding cells disseminated throughout the mesenchyme. The ureteric bud contains a small number of positive cells. At E12.5 (C,D), pathway activation is predominantly within the peripheral and mature stroma. Few positive cells are seen in nephrogenic mesenchyme and collecting ducts. At E13.5 (E,F), activation is seen in peripheral and mature stroma. Nephrogenic mesenchyme and epithelial structures such as comma-shaped bodies show little staining. Tips and trunks of collecting ducts show activation. (G) RT-PCR for expression of components of the TGFß superfamily pathway in (K) E11.5 kidneys and (E) control E17.5 embryos. CD, collecting duct; CB, comma-shaped body; Mes, metanephric mesenchyme; MS, mature stroma; NM, nephrogenic mesenchyme; TR, collecting duct trunk; UB, ureteric bud.

View larger version (106K): [in a new window]   Fig. 3. Morphology of Bmp7cre/+;Smad4–/CA and HoxB7cre;Smad4–/CA kidneys. (A-C) Sagittal sections of E16.5 kidneys. (A'-C') High magnification images of the cortical zone of each section. Kidneys of HoxB7cre;Smad4–/CA (H7.S4) kidneys (B) are indistinguishable from wild type (A). Bmp7cre/+;Smad4–/CA (B7.S4) kidneys (C) display hydroureter and cyst formation. Nephrogenic mesenchyme is missing from the majority of collecting duct tips, and the cortical cell layer is thickened (bracket in C'). E14.5 Bmp7cre/+;Smad4–/CA kidneys (E) are smaller than wild-type controls (D) and display a prominent thickening of the cortical layer normally composed of nephrogenic mesenchyme and peripheral stroma (arrow). E12.5 Bmp7cre/+;Smad4–/CA kidneys (G) are of comparable size to wild-type controls (F). CD, collecting duct; C, cyst; G, glomerulus; NM, nephrogenic mesenchyme; T, tubules.

View larger version (131K): [in a new window]   Fig. 7. Cell death and proliferation in Bmp7cre/+;Smad4–/CA kidneys. Sections of wild-type (WT; A,C,E) and Bmp7cre/+;Smad4–/CA (Mut; B,D,F) kidneys were examined at E12.5 (A,B), E14.5 (C,D) and E16.5 (E,F) for pyknotic nuclei (marked by arrowheads) as markers of cell death. Wild-type kidneys display only sporadic single dead cells adjacent to nephrogenic mesenchyme. By contrast, mutant kidneys show clusters of cell death within the thickened peripheral cell layer, mainly concentrated to regions distant from collecting duct tips. PCNA immunohistochemistry was used to identify proliferating cells in wild-type (G,I) and mutant (H,J) kidneys at E12.5 (G,H) and E14.5 (I,J). Both wild type and mutant proliferating cells are similarly distributed in collecting ducts and mesenchyme.

View larger version (109K): [in a new window]   Fig. 4. Nephrogenic mesenchyme is disorganized and prematurely depleted in Bmp7cre/+;Smad4–/CA kidneys. Comparison of wild-type (WT; A,C,E,G,I,K,M,O) and Bmp7cre/+;Smad4–/CA (Mut; B,D,F,H,J,L,N,P) kidneys analyzed by section in situ hybridization at E14.5 (A-L) and E16.5 (M-P). (A'-H') High magnification images of cortical regions. Ret (B) and Pax2 (D) expression is maintained in the collecting ducts of mutant kidneys indicating normal morphogenesis of this structure. Pax2 (D) and Wt1 (F,J), markers of nephrogenic mesenchyme, reveal a disorganized distribution of cells in the expanded cortical region of mutant kidneys (red brackets), with occasional clusters of cells surrounding tips of collecting ducts. Raldh2, a marker of peripheral stroma, identifies cells with characteristics of peripheral stroma in the expanded cortical region of mutant kidneys (H,L). Glomerular podocytes are marked by Wt1 in mutant kidneys (F). At E16.5, Wt1 is absent from the cortical region of the mutant kidney (bracket in N), but Raldh2 remains (P), suggesting that the composition of this cell population mirrors that of wild-type peripheral stroma. CD, collecting duct; G, glomerulus; NM, nephrogenic mesenchyme; PS, peripheral stroma.

View larger version (112K): [in a new window]   Fig. 5. Nephrogenic mesenchyme and stroma are incompletely segregated in Bmp7cre/+;Smad4–/CA kidneys. Comparison of wild-type (WT; A,C,E,G,I,K,M,O,Q) and Bmp7cre/+;Smad–/CA (Mut; B,D,F,H,J,L,N,P,R) at E11.5 (A-H), E12.5 (I-P) and E13.0 (Q,R). At E11.5, expression of Wt1 (B) and Pax2 (D) in mutant kidneys is indistinguishable from that of wild type (A,C), indicating that the metanephric blastema is appropriately patterned. Raldh2 (F) reveals a distribution of stromal cells in mutants that is comparable to wild type (E). Expression of Lhx1 (H) at E11.5 indicates that maturing structures such as renal vesicles appear at approximately the same time in mutant kidneys as they do in wild type (G). At E12.5, expression of Wt1 (J) reveals disorganization of the nephrogenic mesenchyme that normally forms around collecting duct tips. Expression of Wt1 is widespread in the wild-type mesenchyme at E12.5 (I), and Wt1-negative peripheral stroma can only be seen in occasional sections. Pax2 expression clearly demarcates nephrogenic mesenchyme and nascent epithelia from stromal cells in the wild type (K). Mutant kidneys show a paucity of nephrogenic mesenchyme and no clear demarcation of stromal cells (L). Raldh2 expression is confined to the stromal cell compartment of the wild-type kidney (M), and is excluded from nephrogenic mesenchyme and collecting ducts. In the mutant (N), Raldh2 expression is spread throughout the majority of the mesenchyme, and is excluded from the limited nephrogenic mesenchyme population and collecting ducts. Lhx1 expression reveals several renal vesicles per section in the wild type (O), but only a limited number in the mutant (P). At E13.0, Gdnf expression is comparable in the wild type (Q) and the mutant (R). CD, collecting duct; G, glomerulus; Mes, metanephric mesenchyme; NM, nephrogenic mesenchyme; PS, peripheral stroma; RV, renal vesicle; UB, ureteric bud.

View larger version (100K): [in a new window]   Fig. 6. Lineage analysis of Bmp7cre/+;Smad–/CA kidneys. The ROSA26R reporter was introduced into the Bmp7cre/+(WT) and Bmp7cre/+;Smad4–/CA (Mut) backgrounds. At E11.5, the ureteric bud and condensing mesenchyme of wild-type (A) and mutant (B) kidneys are labeled by lacZ expression. At E12.0 (C,E), all cells within the nephrogenic mesenchyme of wild-type kidneys are labeled, whereas there is significant mixing of labeled and unlabeled cells in the mutant (D,F). Labeled and unlabeled cells are distributed throughout the mesenchyme of mutant kidneys. At E14.5, nephrogenic mesenchyme is labeled in wild-type kidneys (G), and the stroma surrounding mesenchymal condensates is unlabeled. In mutant kidneys (H), the limited nephrogenic mesenchyme that can be seen is labeled, and there is a disorganized distribution of labeled cells throughout the cortical layer, which is composed of a mixture of labeled and unlabeled cells. At E16.5, nephrogenic mesenchyme is labeled, and unlabeled stromal cells are more obvious in wild-type kidneys (I). In mutant kidneys, a few remaining labeled cells lie within the otherwise unlabeled cortical layer (J). Ectopically placed glomeruli are prominent. CD, collecting duct; G, glomerulus; NM, nephrogenic mesenchyme; PS, peripheral stroma; UB, ureteric bud.

View larger version (96K): [in a new window]   Fig. 8. Bmp7cre/+;Smad4–/CA metanephric mesenchyme responds to induction in vitro. Wild-type (WT) and Bmp7cre/+;Smad4–/CA (Mut) mesenchyme was cultured with and without LiCl, a potent nephrogenic inducer. Untreated mesenchyme rapidly underwent cell death (A,B), whereas treated mesenchyme survived and underwent nephrogenic induction (C,D). Photographs were taken at the same magnification, and the size difference between induced wild-type and mutant mesenchyme is representative. Histological analysis revealed the presence of epithelial condensates within both wild-type (E) and mutant (F) mesenchyme, and the development of tubular structures within the wild type. Basal deposition of laminin surrounding condensates was confirmed by fluorescent immunostaining (G,H). C, condensate; T, tubule.

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