The BMP-7 Smad1/5/8 Pathway Promotes Kidney Repair After Obstruction Induced Renal Injury

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1 The BMP-7 Smad1/5/8 Pathway Promotes Kidney Repair After Obstruction Induced Renal Injury Scott R. Manson, Robert A. Niederhoff, Keith A. Hruska and Paul F. Austin* From the Division of Pediatric Urology, Department of Surgery, and Division of Pediatric Nephrology, Departments of Medicine and Pediatrics (KAH), Washington University, St. Louis Children s Hospital, St. Louis, Missouri Purpose: Urinary tract obstruction causes hydroureteronephrosis and requires surgical intervention to prevent permanent renal injury. While many studies have focused on the development of renal injury, we examined the molecular mechanisms that promote renal recovery after correcting obstruction. Materials and Methods: A reversible murine model of ureteral obstruction was used to examine the bone morphogenic protein-7 and transforming growth factor- signaling pathways during renal recovery after obstruction induced injury. Analysis was done using standard molecular techniques, including reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, immunoblotting and co-immunoprecipitation. Results: After correcting obstruction the up-regulation of bone morphogenic protein-7 inhibited the transforming growth factor- dependent profibrotic pathways that are central to renal injury pathogenesis. The inhibitory effects of bone morphogenic protein-7 were mediated in part by the activation of its downstream target proteins, SMA and MAD related proteins 1, 5 and 8, which suppress the activity of transforming growth factor- dependent Smad proteins and in turn inhibit the expression of transforming growth factor- dependent genes. Activation of the bone morphogenic protein-7-smad related protein 1/5/8 pathway during renal recovery promoted renal architecture restoration and fibrosis resolution in the kidney after correcting obstruction. Conclusions: Together these findings show that the bone morphogenic protein-7 Smad1/5/8 pathway promotes kidney repair after obstruction induced injury. Accordingly the pathway represents an important therapeutic target to stimulate this innate repair mechanisms of the kidney during treatment for obstruction induced renal injury. Abbreviations and Acronyms BMP-7 bone morphogenic protein-7 GAPDH glyceraldehyde-3- phosphate dehydrogenase PBS phosphate buffered saline PCR polymerase chain reaction SMAD SMA and MAD related protein TGF- transforming growth factor- UUO unilateral ureteral obstruction Study received institutional animal care and use committee approval. Supported by the National Institutes of Health and Midwest Stone Institute. * Correspondence: Washington University, 4990 Children s Pl., Suite 1120, Campus Box 8242, St. Louis, Missouri (telephone: ; FAX: ; austinp@ wustl.edu). Key Words: kidney, ureteral obstruction, fibrosis, Smad proteins, mice OBSTRUCTIVE uropathy is a leading cause of renal injury, chronic renal insufficiency and renal failure. 1 3 While current surgical approaches often alleviate urinary tract obstructions, the possibility of irreversible renal injury remains even after treatment. 2,3 Thus, the clinical management of obstructive uropathy would benefit from the development of therapeutic approaches that promote renal recovery after injury. While the development of renal injury is well understood, 2,3 renal recovery after injury is less clearly understood. Although it lacks true regenerative ability, 4 the mature kidney has innate ability to restore its structure and function after injury. However, this reparative ability is decreased after severe renal injury. 5,6 Understanding the processes that promote renal recovery and their molecular regulation may /11/ /0 Vol. 185, , June 2011 THE JOURNAL OF UROLOGY Printed in U.S.A by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC. DOI: /j.juro

2 2524 BMP-7 SMAD1/5/8 PATHWAY PROMOTES KIDNEY REPAIR AFTER INJURY enable the development of therapeutic approaches to stimulate the innate repair mechanisms of the kidney during treatment for obstruction induced injury. At the molecular level activation of the TGF- pathway has a central role in the pathogenesis of renal injury by promoting apoptosis, epithelial-mesenchymal transformation, matrix protein synthesis and other profibrotic events that lead to the disruption of renal structure and function. 7 Accordingly neutralizing TGF- inhibits the progression of obstruction induced renal injury Another member of the TGF- superfamily, BMP-7, has the distinguishing property of inhibiting TGF- dependent biological functions. 11 TGF- and BMP-7 signal through receptor complexes that phosphorylate Smad transcription factors TGF- and BMP-7 have different functions, in part since they promote the activating phosphorylation of distinct Smad proteins. TGF- activates Smad2/3 while BMP-7 activates Smad1/5/8. These phosphorylated Smad proteins then bind Smad4 protein and regulate the transcription of target genes in a pathway specific manner While the signaling pathways downstream of TGF- and BMP-7 have been well defined, the mechanisms by which BMP-7 counters TGF- in the kidney have not yet been clearly defined. Treatment with exogenous BMP-7 inhibits the development of renal injury We explored the possibility that BMP-7 may inhibit the pathogenesis of renal injury, in part by promoting kidney repair. While several studies show that exogenous BMP-7 reverses the progression of chronic renal injury, 17,18 chronic injury models have not effectively differentiated between the effects of BMP-7 on the development and on the repair of renal injury since in these models the injurious stimuli is continuously present. To begin to determine the role of the BMP-7 pathway in renal recovery after injury several important questions remain to be answered. 1) Is the BMP-7 pathway regulated during renal recovery? 2) By what mechanisms does BMP-7 counter TGF- in the injured kidney and are these counter regulatory mechanisms subject to regulation during renal recovery? 3) Is pharmacological manipulation of the BMP-7 pathway an effective therapeutic approach to stimulate the innate repair mechanisms of the kidney during treatment for renal injury? We began to address these questions to better understand the molecular mechanisms that contribute to renal recovery after obstruction induced injury. MATERIALS AND METHODS Reversible UUO Eight to 10-week-old C57BL/6J mice were obstructed by placing a vascular clamp on the proximal ureter and, when indicated, obstruction was reversed by subsequently removing the clamp. 6 Mice were treated with PBS or 300 g/kg purified BMP-7 (R & D Systems ), as indicated. Histology/Immunostaining Kidneys were fixed in HistoChoice. Slides containing 5 m tissue slices were stained using Masson s trichrome reagent (Richard Allen Scientific, Kalamazoo, Michigan) according to product specifications. Slides were prepared as described, subjected to antigen retrieval by boiling in 10 mm citrate buffer (ph 6.0) and stained using rabbit anti-collagen IV/procollagen IV (Millipore ) and fluorescein isothiocyanate conjugated anti-rabbit antibodies (Sigma ). Three photographs per sample were uniformly taken of the region bounded by the renal capsule and the corticomedullary junction adjacent to the papillae at 200 magnification. Interstitial/tubular volume were visually quantified by overlaying a grid on slide photographs and counting the grid points in the interstitial/tubular regions. 19 Total Kidney Collagen Content Quantification Kidneys were hydrolyzed, resuspended in citrateacetate buffer (ph 6.0) and oxidized with 0.35% chloramine T. Hydroxyproline was quantitated by measuring the conversion of 7% dimethyl-aminobenzaldehyde to a colorimetric product by hydrolyzates using spectrophotometry (558 nm) and comparison to hydroxyproline standards. Collagen content was determined using the approximation that collagen contains 14% hydroxyproline. 20 Reverse Transcriptase-PCR Kidneys were pulverized in liquid nitrogen and homogenized in TRIzol. RNA was isolated according to product specifications. Semiquantitative reverse transcriptase PCR was done using SuperScript for RT-PCR for 24 to 32 cycles with primers for -smooth muscle actin (5=-ctgagcgtggctattccttc-3= and 5=-gggggccaccctataataaa-3=), collagen 1(I) (5=-actggtacatcagcccgaac-3= and 5=-ggtggagggagtttacacga-3=) or GAPDH (5=-actccactcacggcaaattc-3= and 5=-ccttccacaatgccaaagtt-3=). Enzyme-Linked Immunosorbent Assay Kidneys were pulverized in liquid nitrogen and homogenized in 20 mm tris-hcl (ph 7.5), 2 M NaCl, 0.1% Tween and 1 mm ethylenediaminetetraacetic acid. Protein levels were determined using a BMP-7 enzyme-linked immunosorbent assay kit (R & D Systems) according to product specifications. Immunoblotting Kidneys were pulverized in liquid nitrogen and lysed in 83.3 mm tris, 150 mm NaCl, 4% sodium dodecyl sulfate and 100 mm dithiothreitol supplemented with Complete Protease/PhosSTOP inhibitors. Immunoblotting was performed using rabbit anti-phospho-smad 2/3 (Cell Signaling Technology ), rabbit anti-phospho-smad 1/5/8, rabbit anti-smad4 (Santa Cruz Biotechnology, Santa Cruz, California) or mouse anti-gapdh (Chemicon ) and horseradish peroxidase-goat anti-rabbit or horseradish peroxidase-goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania).

3 BMP-7 SMAD1/5/8 PATHWAY PROMOTES KIDNEY REPAIR AFTER INJURY 2525 TRICHROME TYPE IV COLLAGEN to Protein G beads (Dynal ). Immunoblotting was performed as described. SHAM Statistical Analysis Data are shown as the mean SD. Statistical significance was analyzed by NOVA with the Bonferroni correction. 2D UUO 2D UUO/ 3D REC 2D UUO/ 10D REC Figure 1. Repair after UUO injury. Views of kidney in 3 mice each with sham operation (SHAM), 2 days (D) of UUO, 2 days of UUO followed by reversal and 3 days of recovery (REC) or2 days of UUO followed by reversal and 10 days of recovery. Masson s trichrome stain, reduced from 100. Type IV collagen immunofluorescence, reduced from 200. Co-Immunoprecipitation Kidneys were pulverized in liquid nitrogen and lysed in 50 mm tris-hcl, 150 mm NaCl, 1 mm ethylenediaminetetraacetic acid, 1% Triton, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate supplemented with Complete Protease/PhosSTOP phosphatase inhibitors. Lysates were immunoprecipitated with rabbit anti-smad4 conjugated RESULTS Reversible UUO as Renal Recovery Model To better understand the mechanisms that contribute to renal recovery from obstructive uropathy we studied a model of acute renal injury in response to UUO. 6 In this model the subsequent reversal of obstruction mimics the surgical correction of obstructive uropathy in patients and serves as a model of kidney recovery from acute, obstruction induced injury. Obstructive uropathy potentially results in decreased renal function through the disruption of renal architecture and the promotion of renal fibrosis. 2,3 These deleterious changes were effectively reproduced in our model since UUO causes renal fibrosis (fig. 1), as characterized by interstitial expansion, loss of tubular volume and collagen accumulation (p 0.01, 0.01 and 0.005, respectively, fig. 2). When using the UUO model to examine renal recovery, we found that mice that undergo 2 days of UUO show renal fibrosis but after obstruction reversal and a recovery period much of the renal damage subsides (fig. 1). Obstruction reversal promotes decreased interstitial volume, increased tubular volume and decreased collagen (p 0.005, 0.01 and 0.005, respectively, fig. 2). These findings show that fibrosis resolution and renal architecture restoration are processes that contribute to renal recovery. We next examined the Figure 2. Repair after UUO injury. Kidney findings in 3 mice each underwent sham operation (SHAM), 2 days (D) of UUO, 2 days of UUO followed by reversal and 3 days of recovery (REC) or 2 days of UUO followed by reversal and 10 days of recovery. A, interstitial volume. Single asterisk indicates p Triple asterisks indicate p B, tubular volume. Double asterisks indicate p C, kidney collagen content.

4 2526 BMP-7 SMAD1/5/8 PATHWAY PROMOTES KIDNEY REPAIR AFTER INJURY Figure 3. BMP-7 Smad1/5/8 pathway was activated during renal recovery (REC) after UUO induced renal injury. Three mice each underwent sham operation (SHAM), 2 days (D) of UUO, 2 days of UUO followed by reversal and 3 days of recovery or 2 days of UUO followed by reversal and 10 days of recovery. A, BMP-7 enzyme-linked immunosorbent assay. Triple asterisks indicate p B, Western blot for phospho-smad 1/5/8 and GAPDH as control. Results represent 3 or more independent experiments. molecular regulation of these innate repair mechanisms. Activation of BMP-7 Inhibits Profibrotic Pathways We hypothesized that BMP-7 has a role in renal recovery after obstruction induced injury. When examining this possibility, we found that while BMP-7 was down-regulated after UUO, BMP-7 was restored after UUO reversal (p 0.005, fig. 3, A). Similarly we found that BMP-7 up-regulation was associated with restoration of the activity of its target proteins, Smad1/5/8 (fig. 3, B). Together these findings show that the BMP-7 Smad1/5/8 pathway is activated during the repair of obstruction induced injury. Although the BMP-7 pathway is activated during renal recovery, the possibility remained that the activation of the BMP-7 pathway is merely a consequence of renal recovery itself. To determine the functional importance of BMP-7 we examined the molecular consequences of BMP-7 pathway activation in the injured kidney. Treatment with exogenous BMP-7 inhibited the transcription of several TGF- dependent genes that are central to the pathogenesis of renal injury and are normally increased in the injured kidney, including collagen 1(I), a gene that encodes a protein that is a significant contributor to fibrosis (p 0.05) 21 and -smooth muscle actin, a gene that encodes a protein that contributes to the differentiation of profibrotic myofibroblasts (p 0.005, fig. 4). 22 We next determined the mechanisms by which BMP-7 inhibits the activity of TGF- dependent pathways. Exogenous BMP-7 promoted the formation of BMP-7 regulated Smad1/5/8-Smad4 transcription factor complexes and decreased the obstruction induced formation of TGF- regulated Figure 4. BMP-7 suppressed TGF- dependent profibrotic pathways in injured kidneys. Three mice each underwent sham operation (SHAM) or 2 days (D) of UUO and were treated with PBS as control or 300 g/kg BMP-7 daily. Reverse transcriptase-pcr was done for TGF- dependent target genes. Expression was normalized as ratio to GAPDH and values were compared to those in sham operated mice. A, type I collagen. Single asterisk indicates p B, -smooth muscle actin. Triple asterisks indicate p

5 BMP-7 SMAD1/5/8 PATHWAY PROMOTES KIDNEY REPAIR AFTER INJURY 2527 Figure 5. BMP-7 Smad1/5/8 pathway suppressed formation of TGF- dependent Smad4 transcription factor complexes. Three mice each underwent sham operation (SHAM) or 2 days (D) of UUO and were treated with PBS as control or 300 g/kg BMP-7 daily. A, immunoprecipitation (IP) for anti-smad4, followed by Western blot for phospho-smad 2/3 or 1/5/8, or Smad4 as control. B, Western blot for phospho-smad 2/3 and 1/5/8 proteins, and GAPDH as control. Results represent 3 or more independent experiments. Smad2/3-Smad4 transcription factor complexes in the injured kidney despite continued increased levels of activated/phosphorylated Smad2/3 proteins (fig. 5) and TGF- (data not shown). Together these findings show that activation of the BMP-7 Smad 1/5/8 pathway during renal recovery inhibits the activity of TGF- dependent Smad proteins in the injured kidney. Activation of BMP-7 Promotes Structural Repair of Kidney Given that the BMP-7 up-regulation during renal recovery suppressed TGF- dependent pathways that are central to the pathogenesis of renal injury, we hypothesized that these molecular mechanisms contribute to the repair of renal injury. Accordingly we examined the effects of augmenting BMP-7 activity after correcting obstruction. While mice with 2 days of UUO followed by correction of obstruction and a 3-day recovery period showed a 31.2% decrease in renal collagen during the recovery period, those treated with exogenous BMP-7 during the recovery period showed a 53.1% decrease in renal collagen (p 0.05, fig. 6, A). Similarly BMP-7 treatment during recovery promoted the restoration of renal architecture by stimulating a 59.7% decrease in interstitial volume compared to a 34.9% decrease in untreated mice and a 63.0% decrease in tubular volume loss compared to a 34.5% decrease in untreated mice (each p 0.01, fig. 6, B and C). Together these findings reveal that activation of the BMP-7 path- Figure 6. BMP-7 promoted kidney repair after UUO induced injury. Three mice each underwent sham operation (SHAM), 2 days (D) of UUO or 2 days of UUO followed by reversal and 3 days of recovery (REC). Mice were treated with PBS as control or 300 g/kg BMP-7 daily during obstruction, recovery or obstruction and recovery. A, kidney collagen content. Single asterisk indicates p Double asterisks indicate p B, interstitial volume. Triple asterisks indicate p C, tubular volume.

6 2528 BMP-7 SMAD1/5/8 PATHWAY PROMOTES KIDNEY REPAIR AFTER INJURY way during renal recovery stimulates the restoration of renal architecture and the resolution of fibrosis in the kidney. Notably the renal protective effects of BMP-7 treatment during recovery were only minimally improved by treatment with BMP-7 during obstruction and recovery (fig. 6). Accordingly these findings show that the renal protective effects of BMP-7 during the repair of established renal injury may exceed even those of BMP-7 during the development of renal injury. Figure 7. Model of BMP-7 mediated suppression of TGF- dependent profibrotic pathways during repair of renal injury. During development of UUO induced renal injury activation of TGF- -Smad2/3 pathway unchecked by BMP-7-Smad1/5/8 pathway promoted transcription of TGF- dependent gene products, and renal architecture disruption, renal fibrosis and renal insufficiency. During renal recovery after injury BMP-7 was up-regulated and Smad1/5/8 pathway was activated, resulting in redistribution of Smad4 transcription factor complexes, suppression of activated TGF- dependent Smad2/3 proteins, resolution of fibrotic lesions in kidney and restoration of renal architecture.

7 BMP-7 SMAD1/5/8 PATHWAY PROMOTES KIDNEY REPAIR AFTER INJURY 2529 DISCUSSION An important long-term goal in the treatment of obstructive uropathies is the development of therapeutic approaches to stimulate the regression of fibrosis, the repair of structural damage and ultimately the recovery of renal function after obstruction induced injury. 1 3 However, little progress has been made toward achieving this goal, in part since the processes that promote renal recovery after injury are poorly understood. 5 In our reversible UUO model the BMP-7 Smad1/ 5/8 pathway was activated during kidney repair after acute, reversible obstruction induced injury. Activation of the BMP-7 pathway promoted the resolution of fibrosis and the restoration of renal architecture during renal recovery. While BMP-7 was previously reported to prevent the development of renal injury, our studies reveal that BMP-7 has renal protective effects that extend beyond preventing renal injury per se since BMP-7 also stimulated the repair of established renal injury after the correction of obstruction. In our model the renal protective effects of treatment with exogenous BMP-7 during recovery were only minimally improved by treatment with exogenous BMP-7 during obstruction and recovery. Given the absent synergistic effect, these findings suggest that the repair promoting functions of BMP-7 may have had a significant role in previous studies of the renal protective effects of BMP-7 during the development of obstruction induced renal injury At the molecular level the restoration of BMP-7 levels after obstruction correction is associated with the activation of its downstream target proteins, the Smad1/5/8 proteins. The activation of BMP-7 dependent Smad proteins opposes TGF- dependent pathways in the injured kidney by inhibiting the formation of TGF- dependent Smad2/3-Smad4 transcription factor complexes and the transcription of TGF- dependent genes (fig. 7). Given that TGF- dependent pathways are required for the pathogenesis of renal injury, 7 10 these findings suggest that the activation of BMP-7 dependent Smad proteins has an important role in renal recovery after injury and in the renal protective effects of BMP-7. Nonetheless, the precise molecular mechanisms downstream of BMP-7 that promote renal recovery remain to be elucidated in future studies. It is likely that at least some repair promoting functions of BMP-7 may be attributable to its ability to counter TGF-. For example, BMP-7 may oppose TGF- induced matrix protein synthesis, protease inhibition, and myofibroblast recruitment and differentiation. 7,23 Other repair promoting functions of BMP-7 may be attributable to functions independent of TGF-, such as its ability to act as a renal morphogen. 14 BMP-7 is indispensable for kidney development during embryogenesis 14,23 and in turn for tissue repair and the replacement of damaged cells through regeneration involved processes analogous to embryogenesis. 24 Along with identifying the downstream molecular mechanisms that contribute to BMP-7 stimulated kidney repair, important future goals are to determine why these pathways are impaired in cases of prolonged obstruction that leads to permanent renal injury and also develop therapeutic approaches to reactivate these innate repair mechanisms in the kidney during treatment for obstructive uropathy. CONCLUSIONS As we work toward a better understanding of renal recovery after injury, reversible models of acute renal injury will serve as invaluable tools to elucidate the innate repair mechanisms of the kidney. We report that activation of the BMP-7-Smad1/5/8 pathway promotes the resolution of fibrosis and the restoration of renal architecture during renal recovery after the reversal of obstruction. Together these findings show that the BMP-7 Smad1/5/8 pathway has an important role in the repair of renal injury that results from obstructive uropathy. Accordingly the BMP-7 pathway represents a potential target of adjuvant therapy to stimulate the innate repair mechanisms of the kidney during treatment for obstructive uropathy. REFERENCES 1. Roth KS, Koo HP, Spottswood SE et al: Obstructive uropathy: an important cause of chronic renal failure in children. Clin Pediatr (Phila) 2002; 41: Chevalier RL: Pathogenesis of renal injury in obstructive uropathy. Curr Opin Pediatr 2006; 18: Chevalier RL: Obstructive nephropathy: towards biomarker discovery and gene therapy. Nat Clin Pract Nephrol 2006; 2: Hostetter TH: Progression of renal disease and renal hypertrophy. Annu Rev Physiol 1995; 57: Little MH: Regrow or repair: potential regenerative therapies for the kidney. J Am Soc Nephrol 2006; 17: Cochrane AL, Kett MM, Samuel CS et al: Renal structural and functional repair in a mouse model of reversal of ureteral obstruction. J Am Soc Nephrol 2005; 16: Bottinger EP: TGF-beta in renal injury and disease. Semin Nephrol 2007; 27: Gagliardini E and Benigni A: Role of anti-tgfbeta antibodies in the treatment of renal injury. Cytokine Growth Factor Rev 2006; 17: Isaka Y, Tsujie M, Ando Y et al: Transforming growth factor-beta 1 antisense oligodeoxynucle-

8 2530 BMP-7 SMAD1/5/8 PATHWAY PROMOTES KIDNEY REPAIR AFTER INJURY otides block interstitial fibrosis in unilateral ureteral obstruction. Kidney Int 2000; 58: Miyajima A, Chen J, Lawrence C et al: Antibody to transforming growth factor-beta ameliorates tubular apoptosis in unilateral ureteral obstruction. Kidney Int 2000; 58: Miyazono K, Kusanagi K and Inoue H: Divergence and convergence of TGF-beta/BMP signaling. J Cell Physiol 2001; 187: Massague J: How cells read TGF-beta signals. Nat Rev Mol Cell Biol 2000; 1: Derynck R and Zhang YE: Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 2003; 425: Patel SR and Dressler GR: BMP7 signaling in renal development and disease. Trends Mol Med 2005; 11: Zeisberg M, Bottiglio C, Kumar N et al: Bone morphogenic protein-7 inhibits progression of chronic renal fibrosis associated with two genetic mouse models. Am J Physiol Renal Physiol 2003; 285: F Hruska KA, Guo G, Wozniak M et al: Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction. Am J Physiol Renal Physiol 2000; 279: F Zeisberg M, Hanai J, Sugimoto H et al: BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med 2003; 9: Zeisberg M, Shah AA and Kalluri R: Bone morphogenic protein-7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney. J Biol Chem 2005; 280: Morrissey J, Hruska K, Guo G et al: Bone morphogenetic protein-7 improves renal fibrosis and accelerates the return of renal function. J Am Soc Nephrol, suppl., 2002; 13: S Samuel CS: Determination of collagen content, concentration, and sub-types in kidney tissue. Methods Mol Biol 2009; 466: Ritzenthaler JD, Goldstein RH, Fine A et al: Regulation of the alpha 1(I) collagen promoter via a transforming growth factor-beta activation element. J Biol Chem 1993; 268: Hautmann MB, Madsen CS and Owens GK: A transforming growth factor beta (TGFbeta) control element drives TGFbeta-induced stimulation of smooth muscle alpha-actin gene expression in concert with two CArG elements. J Biol Chem 1997; 272: Hogan BL: Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev 1996; 10: Martin P and Parkhurst SM: Parallels between tissue repair and embryo morphogenesis. Development 2004; 131: 3021.