Redirection of renal mesenchyme to stromal and chondrocytic fates in the presence of TGF-β2
Introduction
The permanent kidney (metanephros) is derived from two embryonic precursor tissues, the epithelial ureteric bud (UB) and the metanephric mesenchyme (MM) both of which are derived from intermediate mesoderm. Upon invasion of the UB into the MM reciprocal interactions between these two tissues occur. The MM induces the UB to undergo several generations of branching morphogenesis which gives rise to the collecting ducts, calyces, renal pelvis and ureter. The UB induces a sub-population of MM cells to condense around the ureteric epithelial tips, forming cap mesenchyme. The cap mesenchyme contains nephron progenitors which are capable of self-renewal and also generating all the cell types of the nephron. In addition, the MM contains progenitors giving rise to the renal stroma, smooth muscle cells, and endothelial cells (Clark and Bertram, 1999; Davies and Fisher, 2002; Carroll and McMahon, 2003; Moritz et al., 2008; Kobayashi et al., 2008; Al-Awqati and Oliver, 2002).
In the absence of the UB or key genes expressed in the MM, such as WT1, Eya1, Odd-1 and Six 1 the MM is programmed to undergo apoptosis (Kreidberg et al., 1993; James et al., 2006; Xu et al., 1999, Xu et al., 2003). Several key factors secreted by the UB have been identified in the rat to be capable of maintaining the survival of the MM such as fibroblast growth factor 2 (FGF2), transforming growth factor-α (TGF-α) and ELR+ CXC chemokines (Perantoni et al., 1995; Levashova et al., 2007). TGF-α is also expressed during mesonephric and metanephric development in both rats and humans (Bernardini et al., 1996; Bernardini et al., 2001; Carev et al., 2008). In combination with leukaemia inhibitory factor (LIF) and transforming growth factor-β2 (TGF-β2) these factors cooperate to induce nephrogenesis in isolated rat MM cultures (Plisov et al., 2001; Karavanova et al., 1996; Barasch et al., 1997).
The importance of TGF-β2 in nephron formation has also been demonstrated in the analysis of kidneys from TGF-β2 homozygous (TGF-β2−/−) and heterozygous (TGF-β2+/−) null mutant mice. Although TGF-β2−/− mice present with heart, craniofacial, skeletal, eye, ear, and intestine abnormalities, they also display a range of urogenital abnormalities (Sanford et al., 1997). These include renal agenesis, dilated renal pelvis, dysplastic tubulogenesis, abnormal ureteric branching morphogenesis and reduced nephron number (Sanford et al., 1997; Sims-Lucas et al., 2008). In contrast, TGF-β2+/− mice display an increase in ureteric branching and nephron number demonstrating that the dosage of TGF-β2 plays an important role in kidney development and ultimately regulating nephron number (Sims-Lucas et al., 2008).
To further examine the role of TGF-β2 during nephrogenesis we undertook a gain-of-function approach culturing both rat and mouse metanephroi with exogenous TGF-β2. Previous reports from our laboratory and others (Martinez et al., 2001; Ritvos et al., 1995) have demonstrated that TGF-β2 added to metanephroi results in an expansion of the MM and an inhibition of ureteric branching. This current report extends these findings demonstrating that TGF-β2 is capable of inducing the differentiation of at least two distinct cell types derived from the MM. Immunohistochemistry, molecular analysis and gene expression profiling revealed that TGF-β2 induces subsets of cells within the metanephros to undergo differentiation towards chondrocyte and myofibroblast/smooth muscle cell lineages. TGF-β2 alone was capable of maintaining the survival of isolated mouse MM (iMM) in the absence of serum or any other inductive signal. In turn, TGF-β2 was capable of differentiating the iMM predominantly into chondrocyte-like and myofibroblast/smooth muscle-like cells demonstrating that these cell types are indeed derived from the mesenchyme and not dependent on signals from the ureteric epithelium. Interestingly, the presence of cartilage and muscle in these cultures is reminiscent of cell types present in some Wilms' tumors. These findings demonstrate that the MM contains progenitor cells capable of differentiating away from their renal cell fate in the presence of TGF-β2.
Section snippets
Animals
Time-mated wild-type B6xCBA mice, Hoxb7/GFP (B6x CBA), and BMP4+/lacZ (129/SvEV×Black Swiss) mice were sacrificed at E11.5 and E12.5 by cervical dislocation. Time-mated Sprague-Dawley rats were sacrificed at E14.5 via intraperitoneal injection of sodium pentobarbitone (5 mg/100 g body weight; Abbott Laboratories, Sydney, Australia). Hoxb7/GFP mice were obtained from Dr. Frank Costantini, Columbia University, USA (Srinivas et al., 1999). Mice were housed at Mouseworks, Monash University, Clayton.
Exogenous TGF-β2 inhibits branching morphogenesis and expands populations of metanephric mesenchymal cells
rhTGF-β2 was added to mouse or rat whole metanephric organ culture at a range of concentrations (1–200 ng/ml). At the beginning of the culture period (E12.5 for mice, E14.5 for rats) metanephroi consisted of metanephric mesenchyme surrounding the ureteric bud that had undergone only a few branching events. After 6 days of culture in control media typical growth and differentiation was seen, involving ureteric branching morphogenesis, nephron induction and nephron differentiation (Fig. 1A–C, Fig.
Discussion
In a gain-of-function approach we have demonstrated that TGF-β2 when added to whole rat and mouse metanephric organ culture inhibits both ureteric branching and nephrogenesis and induces the expansion and differentiation of a subset of MM cells towards the chondrocyte and myofibroblast/smooth muscle cell lineages. This phenomenon also occurs in the absence of inductive signals from the ureteric epithelium or serum indicating that TGF-β2 alone and/or TGF-β2 induced signals present in the MM are
Acknowledgements
The authors would like to thank Johnson and Johnson Research for their support and Ian Boundy for his histology expertise. SS-L would like to thank Peter Lucas for all his efforts. ML is an NHMRC Principal Research Fellow.
References (59)
- et al.
Stem cells in the kidney
Kidney Int.
(2002) - et al.
Chondromodulin-I expression in the growth plate of young uremic rats
Kidney Int.
(2004) - et al.
Terminal differentiation of chondrocytes in culture is a spontaneous process and is arrested by transforming growth factor-beta 2 and basic fibroblast growth factor in synergy
Exp. Cell Res.
(1995) - et al.
Overview: the molecular basis of kidney development
- et al.
Murine Wnt-11 and Wnt-12 have temporally and spatially restricted expression patterns during embryonic development
Mech. Dev.
(1995) - et al.
Spatial gene expression in the T-stage mouse metanephros
Gene Expression Patterns
(2006) - et al.
In vitro studies on the roles of transforming growth factor-beta 1 in rat metanephric development
Kidney Int.
(2001) - et al.
Development of human fetal kidney in obstructive uropathy: correlations with ultrasonography and urine biochemistry
Kidney Int.
(1997) - et al.
Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development
Cell Stem Cell
(2008) - et al.
WT-1 is required for early kidney development
Cell
(1993)
CTNNB1 mutations and overexpression of Wnt/beta-catenin target genes in WT1-mutant Wilms' tumors
Am. J. Pathol.
Evidence that bone morphogenetic protein 4 has multiple biological functions during kidney and urinary tract development
Kidney Int.
Expression of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) during fracture healing
Bone
Activin disrupts epithelial branching morphogenesis in developing glandular organs of the mouse
Mech. Dev.
Epithelial–mesenchymal interactions regulate the stage-specific expression of a cell surface proteoglycan, syndecan, in the developing kidney
Dev. Biol.
In vitro chondrogenesis of human bone marrow-derived mesenchymal progenitor cells in monolayer culture: activation by transfection with TGF-beta 2
Tissue Cell
Overexpression of human Dickkopf-1, an antagonist of wingless/WNT signaling, in human hepatoblastomas and Wilms' tumors
Lab. Invest.
Molecular cloning and characterization of CHM1L, a novel membrane molecule similar to chondromodulin-1
Biochem. Biophys. Res. Commun.
Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1
J. Bone Miner. Res.
Development of the renal interstitium
Pediatr. Nephrol.
Immunohistochemical localization of the epidermal growth factor, transforming growth factor alpha, and their receptor in the human mesonephros and metanephros
Dev. Dyn.
TGF-alpha mRNA expression in renal organogenesis: a study in rat and human embryos
Exp. Nephrol.
Ureteric bud cells secrete multiple factors, including bFGF, which rescue renal progenitors from apoptosis
Am. J. Physiol.
Molecular regulation of nephron endowment
Am. J. Physiol.
Expression of intermediate filaments EGF and TGF-alpha in early human kidney development
J. Mol. Histol.
Identifying the molecular phenotype of renal progenitor cells
J. Am. Soc. Nephrol.
Temporal and spatial transcriptional programs in murine kidney development
Physiol. Genomics
Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice
J. Am. Soc. Nephrol.
Genes and proteins in renal development
Exp. Nephrol.
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- 1
These two authors contributed equally to the research described in this article.
- 2
Current address: Rangos Research Center, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA 15201, USA
- 3
Current address: Translational Research Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
- 4
Current address: Department of Anatomy and Cell Biology, University of Melbourne, Melbourne, Victoria, Australia