TISSUE SPECIFIC STEM CELLS

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1 TISSUE SPECIFIC STEM CELLS Brief Report: SRF-Dependent MiR-210 Silences the Sonic Hedgehog Signaling During Cardiopoesis GUOXING ZHENG, a YE TAO, b WEI YU, c ROBERT J. SCHWARTZ c,d a Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas, USA; b Department of Molecular Physiology & Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, USA; c Department of Biology and Biochemistry, The University of Houston, Houston, Texas, USA; d Texas Heart Institute, Houston, Texas, USA Key Words. Developmental biology Differentiation Embryoid bodies Embryonic stem cells Cardiac ABSTRACT Serum response factor (SRF) wields potent gene silencing activity through its regulation over numerous micrornas (mirs). Here, SRF directs embryonic stem cell (ESC) progenitor cell lineage specification in part by silencing genes through mir-210. Viral expression of mir-210 in murine ESCs-derived embryoid bodies (EBs) inhibited cell growth and inhibited the appearance of cardiac progenitor markers Nkx2.5 and Gata4 and terminal differentiated contractile proteins Mlc2v and bmhc. Knockdown of mir-210 expression via antisense RNA activated cardiac progenitor gene activity. mir-210 inhibitory activity was attributed to Disclosure of potential conflicts of interest is found at the end of this article. silencing of the Sonic hedgehog (Shh) signaling pathway, which fosters the cardiac progenitor program. mir-210 directly silenced Shh via targeting of the Shh 3 0 UTR, comparable to the chemical Shh inhibitor, cyclopamine. mir- 210 silencing of Shh/Gli1 signaling also blocked expression of the cell cycle regulators Cyclin D1 and Cyclin D2, and EB cell expansion. Absence of SRF expression in SRF null EBs blocked mir-210 expression, coincident with enhanced Shh, and Gli1 gene activity. Thus, SRF-dependent mir-210 expression may operate as a novel silencer of the Shh signaling pathway. STEM CELLS 2013;31: INTRODUCTION Cardiopoesis provides access into cardiac progenitor cell fate, including regulation of transcription factor networks and extracellular cues. Cardiopoesis is initiated following the hanging drop aggregation of embryonic stem cells (ESCs) into embryoid bodies (EBs), the first step to inhibit stem cell activity and generate cardiac mesoderm [1]. Followed next by the appearance of core cardiac transcription factors led by Nkx2.5, Isl1, Gata4, Mef2C, and Tbx5, which defines these embryonic cells as cardiac progenitors [2]. Then, serum response factor (SRF) the sarcomeric regulatory factor governs the onset of myofibrillogenesis and rhythmic beating in the maturation of terminally differentiated myocytes [3]. SRF is also a powerful silencer and directs the expression of many micrornas (mirs) that influence cardiovascular lineage specification [3,4]. One potential SRF mir target, mir-210, is highly conserved across animal species and may play a critical role in a variety of cardiac diseases [5 8]. We show that mir-210 directed by SRF manages cell lineage specification in cardiovascular development. The regulation of mir-210 on Shh/Gli1 signaling may direct Nkx2.5, Gata4 activity in the appearance of cardiac progenitors, and Cyclin D1 and Cyclin D2 expression in cell proliferation. Our study revealed the novel role of mir-210 in managing early cell fate decisions during murine cardiopoesis. MATERIALS AND METHODS AB2.2 ESCs were cultured on 0.1% gelatin-coated dishes with Dulbecco s modified Eagle s medium supplemented with 15% fetal bovine serum, 2 mm L-glutamine, 1 mm sodium pyruvate, 0.1 mm b-mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.1 mg/ml leukemia inhibitory factor. Mouse AB2.2 ESCs infected with lentivirus carrying mir-210-ires- EGFP cassette were enriched by fluorescence-activated cell sorting (Aria SORP, Becton Dickinson, San Jose, CA, mir-210 antisense RNA (3.75 lm; Life Technologies, Grand Island, NY, was electroporated into ESCs. ESCs at concentration of 400 cells/drop aggregated together to form EB after 3 days hanging drop. The obtained EBs were then plated on 0.1% gelatin-coated dishes and cultured in ESC medium without LIF for 8 days. EBs were analyzed by immunofluorescence. Isolated RNA from EBs was subjected to RT- and qrt-pcr, or hybridized against Affymetrix array 430a2 chips. Independent EB differentiation experiments were repeated at least three times. The mir-210 promoter serum response element (SRE) sites were mutated and cloned upstream of the luciferase reporter gene in the pgl3 vector. The Shh 3 0 UTR and its mir-210 targeting site mutant were also cloned downstream of the luciferase gene. 293FT cells were transfected with Fugene HD Author contributions: R.J.S.: conceived and directed the study, contributed to the acquisition, analysis, interpretation of the data, and wrote the paper; G.Z.: contributed to the acquisition, analysis, interpretation of the data, and wrote the paper, Y.T. and W.Y.: contributed to the acquisition, analysis, and interpretation of the data. Correspondence: Robert J. Schwartz, Ph.D., Department of Biology and Biochemistry, the University of Houston, Houston, Texas 77204, USA. Telephone: ; Fax: ; sjrobert@central.uh.edu or rjschwartz@uh.edu Received December 28, 2012; accepted for publication May 25, 2013; first published online in STEM CELLS EXPRESS July 2, VC AlphaMed Press /2013/$30.00/0 doi: /stem.1464 STEM CELLS 2013;31:

2 2280 mir210 Blocks SHH Signaling Figure 1. Overexpressed mir-210 in murine ESCs repressed EB cell proliferation. (A): Cardiopoesis, is initiated following formation of EBs by hanging drops of AB2.2 ESCs. By day 8 postplating, these cells entered into cardiovascular lineages, which included endothelial, cardiomyocyte and smooth muscle cell types. (B): The strategy to study mir s function during cardiac cell lineage specification. (C): Mouse mir-210 is well conserved across animal species and is located on mouse chromosome 7. (D): Reverse Transcription PCR indicated that mir-210 level in heart varied during mouse development. mir-24 was used as a control to normalize mir-210 expression. (E): AB2.2 ESCs infected with lentivirus carrying mir-210 and a GFP reporter gene were enriched by FACS for GFP. Approximately, 97.4% of our infected ESCs overexpressed mir-210 (F): that was supported by qrt-pcr (76.5-fold, p <.01) *. mir-24 was used for normalization. Student s t test was used to exam the significance of gene expression alterations. (G): Compared to WT EB, the size (38) of mir-210 overexpression EB was much smaller, with the diameter reduction to approximately 1/3. (H): The cell growth curve showed that overexpression of mir-210 significantly reduced cell growth in the EBs. At least three groups of EBs in each time point were counted for the cell number in the curve. (I): The proliferation marker Ki67 was downregulated in mir-210 stable ESC lines (0.25-fold, p <.05). GAPDH was used for normalization. Abbreviations: EB, embryoid body; ES, embryonic stem; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein.

3 Zheng, Tao, Yu et al Figure 2. mir-210 blocked cardiac myocyte lineage specification during embryonic stem cell differentiation. (A): mir-210 downregulated most cardiac gene expression as analyzed in microarray analysis. Microarray analysis shown as a heat map revealed gene expression of day 8 postplated WT EBs versus mir-210 overexpressed EBs. (B): qrt-pcr showed that overexpression of mir-210 reduced cardiomyocyte gene expression (all genes, p <.01). Analysis was performed on total RNA extracted from day 8 postplated WT EBs and day 8 mir-210 overexpressed EBs. In contrast, qrt-pcr showed that knockdown of mir-210 by antisense RNA activated cardiomyocyte gene expressions (all genes, p <.05). GAPDH was used for normalization. Student s t test was used for significance test. (C): Immunostaining of cardiomyocyte sarcomere structure marker-a-actinin (green) was totally lost in mir-210 infected EBs. Scale bar 5 50 lm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; EB, embryoid body. (Roche Applied Science, Indianapolis, IN, applied-science.com/shop/en/us/home) and cultured for 24 hours before b-gal and luciferase activity measurements. Day 8 wild-type EBs were treated with formaldehyde to crosslink proteins to DNA following the EZ-ChIP Chromatin Immunoprecipitation Kit instructions (Millipore, Billerica, MA index.do). Cross-linked DNA fragments were immunoprecipitated with SRF-specific antibody and used to perform RT-PCR with primers flanking each SRE site in mir-210 promoter region. The significance of all results was determined by Student s t test.

4 Figure 3. mir-210 silencing of the Shh signaling pathway blocked EB cell expansion and cardiomyocyte differentiation. (A): Overlapping expression of mir-210, Shh, and Gli1 in WT EBs suggested a potential interrelationship. The expression was normalized by GAPDH and mir- 24. (B): Comparison of WT and mir-210 overexpression EBs by heat maps indicated mir-210 dependent downregulation of Shh/Gli1 signaling, marked by expression like Smo, Shh, Gli1, and Shh/Gli1 targets, like Ccnd2 and Pdfra. (C, E): RT-PCR and qrt-pcr confirmed that overexpression of mir-210 in the day 8 postplated EBs reduced the expression of Shh and its signaling target gene Gli1, which were normalized to GAPDH. Student s t test was used for significance test. (D, F): RT-PCR and qrt-pcr also showed that knockdown of mir-210 via antisense RNA in day 8 postplated EBs activated the expression of Shh and its signaling target, Gli1. Student s t test was used for significance test. (G): Conservation of the mir-210 targeting seed sequence in Shh 3 0 UTR region was defined. (H): mir-210 inhibited the activity of Shh 3 0 UTR luciferase construct, while mutation of the mir-210 targeting sequence prevented inhibition of luciferase activity. b-galactosidase expression plasmid was cotransfected into the cells and used as a control to normalize the luciferase activity. (I): Inhibition of Shh signaling via cyclopamine blocked EB expansion. Embryonic stem cells were treated with cyclopamine or 100% ethanol vehicle just before hanging drop. After plating EBs for 8 days, the diameters of the EBs are measured. Compared with 100% ethanol vehicle-treated EB, the EB size (310) significantly reduced (0.6-fold, n 5 5, p <.05) after treatment with cyclopamine, a potent inhibitor of Shh signaling. (J): Inhibition of Shh signaling blocked cardiac gene expression. qrt-pcr showed that the expression of cardiac genes including Nkx2.5, Gata4, Mlc2v, and bmhc significantly decreased in EBs after cyclopamine treatment. GAPDH was used for normalization. Student s t test was used for significance test. (K): The function of mir- 210 in EBs may be mediated by Gli1 regulation of cyclins and cardiac transcription factors. Vista analysis showed that the cardiac genes like Nkx2.5 and Gata4 and the cell cycle gene cyclins like Cyclin D1 and Cyclin D2 all have Gli1 binding sites in their upstream promoter regions (supporting information Fig. S2). Knockdown of Gli1 expression with shrna in day 8 postplated EBs also downregulated Nkx2.5, Gata4, Cyclin D1, and Cyclin D2 expression by normalized to GAPDH. Abbreviation: EB, embryoid body.

5 Zheng, Tao, Yu et al Figure 4. mir-210 gene activity in embryonic stem cells and mouse embryos is dependent upon SRF. (A): Luciferase reporter assay showed SRF activation on mir-210 promoter transcription activity (18-fold, p <.01). 293T cells were cotransfected with pcgn vector or pcgn-srf and a pgl3-luc reporter containing mir kb promoter region or a series of SRF binding site mutant promoter constructs. b-galactosidase was used as control to normalize the luciferase activity. Student s t test was used for significance test. (B): RT-PCR analysis of the expression of mir-210 and its targets Shh and Gli1 in haploid SRF knockouts (SRF1/2) and homozygous SRF knockouts (SRF2/2) day 8 postplated EBs. mir-210 expression was downregulated while Shh and Gli1 expressions were upregulated in SRF null EBs. (C): RT-PCR analysis of the expression of mir- 210, Shh, and Gli1 in E9.5 embryo hearts of SRF1/LoxP and Nkx2.5-Cre mediated SRF conditional knockout mice. Again, mir-210 expression was downregulated while Shh and Gli1 expressions were upregulated in SRFcko mouse hearts. (D): ChIP analysis of SRF binding to mir-210 upstream SRE. PCR was performed on immunoprecipitated chromatin fragments from WT day 8 postplated EBs. PCR amplified a bp region containing the SRF-binding site of each promoter region. Lane 1: total input DNA (1:100 dilution), Lane 2: ChIP reactions with IgG, Lanes 3: ChIP reactions with anti-srf antibody. (E): Model of SRF-dependent mir-210 attenuates Shh signaling in cardiovascular development. Abbreviations: ChIP, chromatin immunoprecipitation; EB, embryoid body; SRE, serum response element; SRF, serum response factor. RESULTS/DISCUSSION Usually EBs initiate progenitor programs by day 3 and further differentiate into a variety of cardiovascular cell lineages by day 8 (Fig. 1A). Highly conserved Mmu-miR-210 normally appearing in embryonic hearts (Fig. 1B) was virally overexpressed in murine ESCs and during EB formation (Fig. 1C--1I). Compared with wild-type EBs, the size of mir-210 infected EBs was smaller (Fig. 1G), likely caused by inhibiting cell proliferation during ESC differentiation (Fig. 1H, 1I). mir-210-infected EBs failed to beat, indicating a potential block to cardiomyocyte terminal differentiation. Microarray analysis, presented as a heat map (Fig. 2A), revealed the downregulation of many cardiac specified genes after mir-210 overexpression. The downregulated cardiac progenitor cell markers like Nkx2.5, Gata4, Mef2c, Isl1, and contractile proteins such as a-mhc and Mlc2v (Fig. 2B) were independently confirmed (supporting information Fig. S1). Conversely, knockdown of mir-210 activity by antisense RNA significantly stimulated their expression (Fig. 2B). The appearance of sarcomeres was also blocked in mir-210 lentivirus-infected EBs (Fig. 2C). Shh signaling promotes the appearance of cardiomyocytes both in vitro and in vivo [9,10] and plays a role in specification of myocardial progenitor cells [11]. Expression of mir- 210, Shh, and Gli1 overlap during ESC differentiation (Fig. 3A) and we reasoned that mir-210 manages cardiac progenitor cell formation through repression of the Shh signaling. Microarray analysis indicated that the factors in Shh/Gli1 signaling like Smo, Shh, and Gli1 and the Shh/Gli1 targets like Ccnd2 and Pdfra were downregulated after mir-210 overexpression (Fig. 3B), which was further confirmed by RT-PCR and qrt-pcr (Fig. 3C--3F). Knockdown of mir-210 via antisense RNA upregulated the expression of Shh (1.7-fold, p <.01) and Gli1 (1.5-fold, p <.05) (Fig. 3D, 3F). Investigation in human umbilical vein EC revealed the proangiogenic function of mir-210 [12]. mir-210 promoted the capillarylike structures formation of endothelial cell and vascular endothelial growth factor (VEGF)-induced cell migration. We found that mir-210 overexpression activated endothelial gene program marked by the increasing expression of Pecam-1 and Vegfa and decreasing expression of Efna3 (supporting information Fig. S2). Next, analysis of the conserved mir-210 targeting site in the 3 0 UTR region linked to a Luciferase reporter revealed potent inhibition of Shh by mir-210 (0.36-fold, p <.05).

6 2284 mir210 Blocks SHH Signaling Furthermore, three nucleotide substitution mutations (G -> A, A-> C, and A -> T) in the targeting site that corresponded to mir-210 seed sequence eliminated mir-210 s ability to inhibit luciferase activity (0.92-fold, p 5.07) (Fig. 3G, 3H). To test Shh/Gli1 signaling during cardiopoesis, EBs were treated with cyclopamine, an inhibitor of Smo factor in Shh signaling pathway. Compared to the vehicle control, cyclopamine treatments significantly inhibited the expression of cardiac transfactors, such as Nkx2.5 and Gata4, and cardiac cell structural proteins, such as Mlc2v and bmhc, through repression of Gli1 expression (all genes, p <.05) (Fig. 3J). mir- 210 s ability to block cell proliferation and cardiomyocyte progenitor differentiation is attributed to the repression of Sonic Hedgehog (Shh) signaling pathway. Shh plays a role in specification of myocardial progenitor cells [11]. For example, Shh signaling in P19 cells promoted cardiac progenitor markers Nkx2.5 and Gata4 expression [9], which were blocked by Shh inhibition [10]. Also, in zebrafish reducing Shh signaling reduced the number of cardiomyocytes, while increasing Shh signaling produced a surplus of cardiomyocytes [11]. Furthermore, cyclopamine blocked cardiac progenitor cell proliferation in the secondary heart field [13]. Informatics revealed conserved Gli DNA binding sites in promoter regions of Nkx2.5 and Gata4 and the cell cycle regulators Cyclin D1 and Cyclin D2 (supporting information Fig. S3). It is likely that Gli1 regulates Cyclin D1 and D2 gene activity. For example, Cyclopamine also inhibited Cyclin D [14], Gli1 regulates Cyclin D1 expression [15 20], and Gli1 directly targets Cyclin D1 and Cyclin D2 [21,22]. Cyclin Cdk4 binds to Cyclin D1/Cyclin D2 to control cell cycle G0/G1 transition and neonatal cardiomyocyte proliferation [23]. A heat map showed the downregulation of Ccnd1, Cdk4, and Ccnd2 gene activities by mir-210 (supporting information Fig. S3) and quantitative PCR assays (Fig. 3K) supports the idea that Shh/Gli1 signaling may control cell proliferation and cardiac progenitor differentiation. The function of Shh signaling may be mediated by Gli1 s regulation of cardiac key factors Nkx2.5 and Gata4 and cell cycle regulator Cyclin D1, Cyclin D2, and Cdk4. But, cell growth could also play a role for mir-210 in limiting the number of cardiac myocytes. Microarray data of SRFcko mice revealed that mir-210 expression is SRF dependent [3]. SRF significantly activated the luciferase activity of the mir kb promoter (18-fold, p <.01), which was significantly reduced by mutations of SRE1 site (11-fold, p <.05) and SRE3 site (8.4-fold, p <.05; Fig. 4A). Chromatin immunoprecipitation (ChIP) results support mir-210 as a direct downstream target of SRF. Absence of SRF in EBs reduced mir-210 expression and augmented the expression of Shh and Gli1 (Fig. 4B), but blocked sarcomerogenesis and contractility [3,24]. Even in E9.5 SRF cko mouse hearts, mir-210 expression was downregulated and the expression of Shh and Gli1 was upregulated (Fig. 4C). Shh and Gli factors are expressed throughout the mesoderm during gastrulation and may specify early cardiac mesodermal cells that become cardiac progenitors. Mice lacking the Smo gene were embryonic lethal, due to defective cardiac looping which severely downregulated Nkx2.5 expression [25,26]. Later, Smo mutants express normal levels of Nkx2.5, suggesting that there may be both HH-dependent and HHindependent stages in Nkx2.5 regulation. Null Ptc1 mutant mice revealed increased Nkx2-5 in the cardiac crescent in comparison to wild-type mice [26], as expected for the ablation of negative regulator of Shh signaling. We also observed decreased smoothen expression caused by mir-210 expression (Fig. 3B). Therefore, modulation of Shh signaling via Ptc1, Smo, and mir-210 seems to be critical for maintaining wildtype levels of Nkx2-5 expression in the cardiac crescent and ESC-dependent cardiac progenitor cells. ACKNOWLEDGMENTS This work was supported by NIH grants, the Texas Heart Institute with funds from the Cullen Foundation, the Advanced Research Program grant from the Texas Higher Education Coordinating Board, research funds, and Cullen Distinguished Professorship of Biology and Biochemistry from the University of Houston to R.J.S. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest. REFERENCES 1 Nelson TJ, Faustino RS, Chiriac A et al. CXCR41/FLK-11 biomarkers select a cardiopoietic lineage from embryonic stem cells. Stem Cells 2008;26: Islas JF, Liu Y, Weng KC et al. 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