Skip Navigation


Human Molecular Genetics Advance Access originally published online on May 24, 2006
Human Molecular Genetics 2006 15(13):2138-2145; doi:10.1093/hmg/ddl137
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Material
Right arrow All Versions of this Article:
15/13/2138    most recent
ddl137v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (20)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by de Haro, M.
Right arrow Articles by Botas, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Haro, M.
Right arrow Articles by Botas, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

MBNL1 and CUGBP1 modify expanded CUG-induced toxicity in a Drosophila model of myotonic dystrophy type 1

María de Haro1,3, Ismael Al-Ramahi1,3, Beatrice De Gouyon1, Lubna Ukani1, Alberto Rosa1,{dagger}, Nuno André Faustino2, Tetsuo Ashizawa4, Thomas A. Cooper2 and Juan Botas1,*

1 Department of Molecular and Human Genetics and 2 Departments of Pathology and Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA, 3 Departamento de Biología, Facultad de Ciencias-UAM, Madrid 28049, Spain and 4 Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA

* To whom correspondence should be addressed. Tel: +1 7137985937; fax: +1 713 7985386; Email: jbotas{at}bcm.tmc.edu

Received March 20, 2006; Accepted May 19, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG expansion in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene. It has been hypothesized that the pathogenesis in DM1 is triggered by a toxic gain of function of the expanded DMPK RNA. This expanded RNA is retained in nuclear foci where it sequesters and induces alterations in the levels of RNA-binding proteins (RNA-BP). To model DM1 and study the implication of RNA-BP in CUG-induced toxicity, we have generated a Drosophila DM1 model expressing a non-coding mRNA containing 480 interrupted CUG repeats; i.e. [(CUG)20CUCGA]24. This (iCUG)480 transcript accumulates in nuclear foci and its expression leads to muscle wasting and degeneration in Drosophila. We also report that altering the levels of two RNA-BP known to be involved in DM1 pathogenesis, MBNL1 and CUGBP1, modify the (iCUG)480 degenerative phenotypes. Expanded CUG-induced toxicity in Drosophila is suppressed when MBNL1 expression levels are increased, and enhanced when MBNL1 levels are reduced. In addition, (iCUG)480 also causes a decrease in the levels of soluble MBNL1 that is sequestered in the CUG-containing nuclear foci. In contrast, increasing the levels of CUGBP1 worsens (iCUG)480-induced degeneration even though CUGBP1 distribution is not altered by the expression of the expanded triplet repeat. Our data supports a mechanism for DM1 pathogenesis in which decreased levels of MBNL and increased levels of CUGBP mediate the RNA-induced toxicity observed in DM1. Perhaps more importantly, they also provide proof of the principle that CUG-induced muscle toxicity can be suppressed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Myotonic dystrophy type 1 (DM1) is the most common form of adult onset muscular dystrophy with an incidence of one in 8000 births. Unlike other neuromuscular diseases, DM1 is multisystemic and, in addition to myotonia and muscle degeneration, patients show the presence of cataracts, cardiac conduction defects, insulin resistance, sleeping disorders and testicular atrophy (1).

DM1 is caused by a triple repeat expansion in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene (2–4). While normal individuals have as many as 30 CTG repeats, DM1 patients carry a larger number of repeats ranging from 50 to thousands. The severity of the disease and the age of onset correlates with the number of repeats. Patients with adult onsets show milder symptoms and have less than 100 repeats, juvenile onset DM1 patients carry as many as 500 repeats and congenital cases usually have around a thousand CTG repeats (1).

According to one hypothesis, DM1 is a consequence of DMPK haploinsufficiency (5–7). Another hypothesis postulated that DM1 is a consequence of alterations in the levels of DMPK flanking genes (8,9). However, transgenic mice models generated to test these hypotheses failed to reproduce most of the symptoms of the disease (10–14).

Myotonic dystrophy type 2 (DM2), a disease with similar clinical manifestations, maps to a different locus (15) and is also caused by an untranslated repeat expansion (16). These findings pointed towards an RNA gain-of-function mechanism of pathogenesis for the expanded repeats.

Transgenic mice, expressing expanded CTG repeats in muscle tissue under the control of an actin promoter, display myotonia and some of the histopathological abnormalities of the DM1-affected muscle; i.e. central nuclei and variability in fibre size (17). These results demonstrated that the expanded repeats by themselves could account for at least some of the disease features independent of the DMPK locus.

The expanded transcripts containing CUG repeats form a secondary structure (18,19), accumulate in the nucleus in the form of nuclear foci (20) and sequester RNA-binding proteins (RNA-BP) (18,21,22). Several RNA-BP have been implicated in the disease including muscleblind-like (MBNL) proteins and CUG-binding protein (CUGBP) (21,23). MBNL proteins are homologous to Drosophila muscleblind (Mbl) proteins necessary for photoreceptor and muscle differentiation (24,25). MBNL and CUGBP have been identified as antagonistic splicing regulators of transcripts affected in DM1 such as cardiac troponin T (cTNT), insulin receptor (IR) and muscle-specific chloride channel (ClC-1) (26–30).

MBNL proteins co-localize with the expanded CUG-containing foci (21,22,31–35). A muscleblind knockout mouse model in which only the CUG-binding isoforms were eliminated shows myotonia, cataracts, aberrant splicing of Clcn 1 and abnormal muscle histology (26). These data point towards the depletion of muscleblind as an important mechanism in expanded CUG-induced DM1 pathogenesis.

Two different transgenic mouse lines have been generated that overexpress CUGBP1 either in skeletal muscle alone (36), or in both heart and skeletal muscles (27). These mice show abnormal muscle development and muscular dystrophy and, one of them (27) also shows alternative splicing misregulation of transcripts known to be affected in DM1.

Here, we describe a Drosophila model of DM1 that shows degenerative phenotypes in muscle and eye tissue as well as key histopathological features of the DM1. These features include accumulation of the expanded transcripts in nuclear foci and their co-localization with muscleblind protein. We used this model to further test the hypothesis that DM1 pathogenesis is caused by the depletion of muscleblind. We show that reduced levels of muscleblind aggravate and, more importantly, overexpression suppresses the muscle and eye phenotypes of DM1 flies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Expanded CUG repeats cause eye and muscle degeneration and accumulate in nuclear foci in Drosophila
To test the RNA gain-of-function hypothesis, and to gain insight into the causative mechanisms behind the DM1 pathology, we generated flies expressing a non-coding mRNA containing 480 interrupted CUG repeats [(iCUG)480] made of the sequence: [(CUG)20CUCGA]24. The GAL4/UAS system (37) was used to control the spatial and temporal expression of the (iCUG)480 mRNA.

Expression of (iCUG)480 in the eye causes disorganization and fusion of the ommatidia as well as loss and duplication of inter-ommatidial bristles. Eyes expressing the (iCUG)480 mRNA are also smaller than controls (Fig. 1A–D).


Figure 1371
View larger version (85K):
[in this window]
[in a new window]
  		Figure 1. Expression of (iCUG)480 in Drosophila causes eye and muscle degeneration and accumulation of expanded transcripts in nuclear foci. (A–D) Scanning electron-microscope (SEM) eye images from control flies or flies expressing (iCUG)480. Control eyes show the normal organization of ommatidia and inter-ommatidial bristles (A and C). Expression of (iCUG)480 causes disorganization of the ommatidia and inter-ommatidial bristles, and reduction of the eye size (B and D). (E and F) In situ hybridization in larval muscles using a Cy3-labelled GAC PNA probe of control flies (E) or flies expressing (iCUG)480 (F). Muscles were stained with anti-laminC (blue) to visualize the nuclei. (iCUG)480 repeats accumulate in nuclear foci (red) in muscle cells of (iCUG)480 larvae. (G–L) Transversal resin sections of adult IFM of control flies or flies expressing (iCUG)480 at different ages. At day 20, control flies show normal organization of the IFM (G) and the muscle fibres are well structured. Note the nuclei located in the periphery of muscle fibres, arrows in (J). Ten days after eclosion, animals expressing (iCUG)480 in the IFM show vacuolization (arrowheads) and muscle disorganization (H). Higher magnification shows loss of fibre organization (arrows point nuclei, K). At 20 days of age, muscle degeneration and wasting progresses, the density of myofibrils per muscle decreases and some muscles are absent (arrow, I). The loss of muscle fibre organization and vacuolization (asterisk) is enhanced (arrows point nuclei, L). (M and N) Phalloidin staining of IFM in the adult thorax of control flies or flies expressing the (iCUG)480. At 10 days of age, control flies show normal actin filaments (M). Flies expressing (iCUG)480 at day 10 show vacuolization of the tissue (N). Scale bars: (A and B) 100 µm, (C and D) 10 µm, (E and F) 5 µm, (G–I) 50 µm, (J–L) 10 µm. (M and N) 50 µm. Genotypes are A, C: w; gmr-GAL4/UAS-LacZ; +; B, D: w; gmr-GAL4/UAS-LacZ; UAS-(iCUG)480[M5T]/+ E, G, J, M: w; UAS-LacZ/+; Mhc-GAL4/+; F,H,I,K,L,N: w; UAS-LacZ/+; Mhc-GAL4/UAS-(iCUG)480[M5T].

 
To investigate the effect of (iCUG)480 in somatic muscles, we expressed (iCUG)480 using a muscle-specific myosin heavy chain promoter. A hallmark of DM1 pathology is the accumulation of expanded CUG-containing RNA in nuclear foci. We monitored RNA accumulation using a Cy3 labelled anti-CUG probe. We observe the foci formation in the nuclei of third instar larvae muscles (Fig. 1F). No nuclear foci are detected in muscles from wild-type animals (Fig. 1E) or animals expressing (CUG)20 (data not shown).

We also investigated whether the expression of (iCUG)480 leads to toxicity in the Drosophila muscles. One-day-old adult animals are unable to fly but present no histological evidence of muscle degeneration (data not shown). Ten-day-old (iCUG)480 flies show degeneration and vacuolization of the indirect flight muscles (IFM) (Fig. 1H), and loss of the structure of muscle fibres (compare Fig. 1K with J). In 20-day-old (iCUG)480 flies, the wasting of the IFM is more severe showing a diffuse matrix, the structure of the fibres has been lost and the nuclei appear dispersed (Fig. 1I and L). In contrast, control flies that do not express CUG repeats (Fig. 1G), or expressing (CUG)20 or (CUG)200 (Supplementary Material, Fig. S1) show no muscle phenotype.

Therefore, expression of (iCUG)480 causes IFM dysfunction and progressive muscle degeneration. This degenerative phenotype is also evident by staining dissected Drosophila adult IFM with phalloidin that labels actin in the muscle fibres (Fig. 1M and N).

MBNL1 overexpression suppresses (iCUG)480-induced muscle toxicity
Because MBNL proteins have been implicated in DM1 pathogenesis (21,26,31), we investigated whether altering the levels of muscleblind proteins modulates (iCUG)480-induced toxicity. Reduction of muscleblind levels by means of a heterozygous loss-of-function mutation in muscleblind (mbl) aggravates the (iCUG)480 eye phenotype increasing ommatidial disorganization/fusion and eye size reduction (compare Fig. 2A and C). Eyes from control flies carrying the same mbl mutation but no (iCUG)480 repeats are normal (Supplementary Material, Fig. S2). These results concur with the muscleblind knockout mouse (26) and support the validity of the Drosophila model.


Figure 1372
View larger version (127K):
[in this window]
[in a new window]
  		Figure 2. Modification of (iCUG)480-induced phenotypes by muscleblind. (A–C) SEM eye images from control flies expressing (iCUG)480, (iCUG)480 and MBNL1, or (iCUG)480 and also carrying a heterozygous mutation in the Drosophila muscleblind gene. Flies expressing (iCUG)480 and a control transgene show ommatidial disorganization and fusion, and eye size reduction (A). This phenotype is suppressed by MBNL1 overexpression (B). The (iCUG)480 eye phenotype is enhanced in animals carrying a heterozygous loss-of-function mutation in muscleblind (C). This mutation by itself does not cause an abnormal eye phenotype (see Supplementary Material Fig. S2). (D–I) Transversal semi-thin sections of adult IFM (iCUG)480 control flies, or (iCUG)480 flies also expressing MBNL1. Control 20-day-old (iCUG)480 flies show obvious IFM muscle pathology (D, F and H) that is suppressed by expression of an MBNL1 transgene (E, G and I). Note that the fibres are well organized and the density of myofibril per muscle fibre is higher in the MBNL1-rescued flies (dots in G outline a well-structured muscle fibre) as compared with (iCUG)480 controls where there is vacuolization (asterisk), the structure of the fibres has been lost and the nuclei appear dispersed in the matrix (F). Arrows point to nuclei in F and G. Scale bars: (A–C) 100 µm (insets 10 µm), (D and E) 50 µm, (F–I) 10 µm. Genotypes are A: w; gmr-GAL4/UAS-LacZ; UAS-(iCUG)480[M5T]/+; B: w; gmr-GAL4/UAS-MBNL1[M10M]; UAS-(iCUG)480[M5T]/+; C: w; gmr-GAL4/mblP1514 (4); UAS-(iCUG)480 [M5T]/+(D–I): D, F, H: w; UAS-LacZ/+; Mhc-GAL4/(iCUG)480[M5T]/+; E, G, I: w; UAS-MBNL1[M10M]/+; Mhc-GAL4/UAS-(iCUG)480[M5T].

 
To further test the hypothesis that the (iCUG)480 phenotypes are caused by titration and loss of function of muscleblind protein, we investigated the consequences of increasing its levels. We generated flies to express a human muscleblind transgene (MBNL1) under control of the GAL4/UAS system. As shown in Figure 2B, overexpression of muscleblind dramatically suppresses the (iCUG)480 eye phenotypes (size and organization of the ommatidia).

Next we investigated whether MBNL1 is able to rescue the (iCUG)480 somatic muscle phenotype. We examined the muscles of 20-day-old flies co-expressing (iCUG)480 and human MBNL1. As shown in Figure 2D, F and H, the IFM from (iCUG)480 flies show severe disorganization of the muscle fibres; they also have randomly arranged myofibrils and a diffuse matrix with vacuolization and dispersed nuclei. In contrast, the muscles from (iCUG)480 flies overexpressing MBNL1 at appropriate levels show little or no vacuolization (Fig. 2E and I), have a well-defined myofibril matrix (Fig. 2G) and the IFM are well structured in muscle fibres with peripherally located nuclei (Fig. 2G and I). Note also that the rescued muscle in Figure 2E, G and I is more intensely stained than the control muscle in Figure 2D, F and H because of the higher density of the tissue. The muscle phenotype in (iCUG)480 flies is 100% penetrant, and we also observed rescue in every (iCUG)480 animal also overexpressing MBNL1. In addition, we quantified the rescuing ability of MBNL1 overexpression by counting the number of normal versus abnormal individual IFM. In 20-day-old (iCUG)480 flies, only five out of 35 (14.3%) IFM had normal appearance, whereas in (iCUG)480 flies overexpressing MBNL1 of the same age, 60 out of 67 (89.5%) appeared normal.

Interestingly, high expressing MBNL1 transgenic lines show a muscle phenotype in the absence of (iCUG)480 (data not shown) suggesting that steady state levels of MBNL1 are important.

Because MBNL1 localizes to CUG nuclear foci in DM1 cells (21,22,31), we monitored MBNL1 distribution in the somatic muscle of (iCUG)480 flies. MBNL1 presents a diffuse nuclear distribution in the absence of (iCUG)480 expression (Fig. 3D and E). In contrast, MBNL1 localizes to nuclear foci in muscles expressing (iCUG)480 and MBNL1 simultaneously, and no diffuse MBNL1 is detected in these nuclei (Fig. 3A–C). We also found that the average number of foci observed per nucleus is decreased in (iCUG)480 flies that also overexpress MBNL1 (Fig. 3F).


Figure 1373
View larger version (42K):
[in this window]
[in a new window]
  		Figure 3. Co-localization of MBNL1 protein with the CUG-containing nuclear foci. (A–C) Images of nuclei from larval somatic muscle co-expressing MBNL1 and (iCUG)480 after immunofluorescence and in situ hybridization. MBNL1 protein is detected in muscle nuclei when stained with an antibody against MBNL1 (A, white arrows, green). The (iCUG)480 transcripts are also visualized in the form of nuclear foci after in situ hybridization (B, white arrows, red). Both MBNL1 and (iCUG)480 RNA co-localize when the images are merged (C, white arrows, yellow). Muscles are also stained with anti-CBP antibody to visualize the nuclei (white). (D–E) Images of nuclei from larval somatic muscle expressing MBNL1 but no (iCUG)480 after immunofluorescence. Anti-CBP antibody revealing nuclei outline (D, white), MBNL1 signal shown in green (E). (F) Comparative quantification of nuclei with single versus multiple foci in flies expressing (iCUG)480 alone, or together with MBNL1. Images below the chart illustrate the types of nuclear foci found; no nucleus of any genotype was found without at least one foci. Notice that co-expression of MBNL1 together with (iCUG)480 causes an overall decrease in the number of nuclei with multiple foci when compared with the repeats alone. Error bars represent standard deviation. Data was analyzed using Student's t (P=0.006 in both groups). Total muscle nuclei (~200 per larva) from four to five larvae per genotype were analyzed. Scale bar: (A–F) 5 µm. Genotypes are A–C, F: w; UAS-MBNL1[M10M]/+; Mhc-GAL4/UAS-(iCUG)480[M5T]; D–E: w; UAS-MBNL1[M10M]/+; Mhc-GAL4/+.

 
Increased levels of CUGBP1 enhance expanded CUG toxicity
In cells expressing the CUG-containing expanded mRNA, the levels of CUGBP are increased (23,38). Higher steady state levels of CUGBP1 in DM1 correlate with alternative splicing defects in specific transcripts (39–41). We generated flies expressing human CUGBP1 and we used the DM1 fly model to investigate whether increased levels of CUGBP1 alter the (iCUG)480 eye phenotype. Flies co-expressing CUGBP1 and (iCUG)480 show an aggravated eye phenotype with enhanced ommatidial disorganization in comparison with flies expressing (iCUG)480 alone (Fig. 4A and B). Flies overexpressing CUGBP1 alone show inter-ommatidial bristle loss, but little if any ommatidial disorganization (Fig. 4C); consequently we conclude that GUGBP1 overexpression enhances the (iCUG)480 eye phenotype. Increasing the CUGBP1 levels in the Drosophila somatic muscles leads to a degenerative phenotype (Fig. 4F) similar to the phenotype caused by (iCUG)480 with vacuolization and loss of muscle integrity (Fig. 4I). This phenotype complicates interpreting the effect of CUGBP on (iCUG)480 muscle toxicity. However, co-expressing (iCUG)480 and CUGBP1 leads to an increased wasting of the muscles compared to animals expressing only (iCUG)480 (compare Fig. 4E and H with D and G).


Figure 1374
View larger version (77K):
[in this window]
[in a new window]
  		Figure 4. CUGBP1 enhances the eye and muscle phenotypes caused by (iCUG)480, and does not accumulate in nuclear foci. (A–C) Eye SEM images of flies expressing (iCUG)480, (iCUG)480 and CUGBP1, or CUGBP1 alone. The (iCUG)480 eye phenotype (A), is enhanced by CUGBP1 overexpression (B). Expression of CUGBP1 by itself in the eye shows inter-ommatidial bristle loss, but little if any ommatidial disorganization (C). (D–I) Transversal semi-thin sections of adult IFM expressing (iCUG)480, (iCUG)480 and CUGBP1, or CUGBP1 alone. Ten-day-old flies expressing (iCUG)480 show vacuolization of the tissue (D) and loss of muscle fibre structure (arrows point nuclei, G). Co-expression of (iCUG)480 and CUGBP1 causes a stronger wasting of the muscle in flies of the same age. Notice the absence of some muscle packs (arrow) and the lax distribution of myofibrils in the rest of the tissue (arrowhead) (E). Higher magnification shows a dramatic loss of muscle structure (arrows point nuclei, H). Expression of CUGBP1 alone shows a phenotype similar to the phenotype of (iCUG)480 flies shown in D with vacuolization (F) and disorganization of fibres (arrows point nuclei, I). (J and K) Images of nuclei from larval somatic muscle co-expressing (iCUG)480 and CUGBP1 after immunofluorescence and in situ hybridization. Muscles are stained with TOTO3 to visualize the nuclei (J, blue). CUGBP1 is distributed throughout the nuclei (K, green) and does not accumulate in (iCUG)480 nuclear foci (K, red). (L) Comparative quantification of nuclei with single versus multiple foci in flies expressing (iCUG)480 alone or together with CUGBP1. In nuclei expressing CUGBP1, the average of multiple foci per nucleus is higher than in (iCUG)480 controls. Error bars represent standard deviation. Data was analyzed using Student's t (P=0.006 in both groups). Scale bars: (A–C) 100 µm (insets 10 µm), (D–F) 50 µm, (G–I) 10 µm, (J–L) 5 µm. Genotypes are A: w; gmr-GAL4/UAS-LacZ; UAS-(iCUG)480[M5T]/+; B: w; gmr-GAL4/UAS-CUGBP1[M2A]; UAS-(iCUG)480[M5T]/+; C: w; gmr-GAL4/UAS-CUGBP1[M2A];+ D, G: w; +; Mhc-GAL4/UAS-(iCUG)480[M5T]; E, H: w; UAS-CUGBP1[M2A]/+; Mhc-GAL4/UAS-(iCUG)480[M5T]; F, I: w; UAS-CUGBP1[M2A]/+; Mhc-GAL4/+; J–L: w; UAS-CUGBP1[M2N]/+; Mhc-GAL4/UAS-(iCUG)480[M5T].

 
CUGBP1 does not localize to expanded CUG-containing nuclear foci in DM1 cells (18,31). We monitored the distribution of CUGBP1 in larval muscles co-expressing (iCUG)480 and CUGBP1. Consistent with the existing data, we found that CUGBP1 localizes to the nuclei of muscle cells but is not recruited to the (iCUG)480 nuclear foci (Fig. 4K). Interestingly, overexpression of CUGBP1 increases the average number of (iCUG)480 foci observed per nucleus (Fig. 4L).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Several independent lines of evidence support the hypothesis that DM1 pathogenesis is a consequence of RNA gain-of-function toxicity. A second type of myotonic dystrophy (DM2) is caused by a related but distinct mutation (expansion of CCTG repeats) in a locus unrelated to the DM1 gene (16). In addition, a transgenic mouse model expressing expanded CUG in skeletal muscle displays myotonia and myopathy (17), and a muscleblind knockout also shows many of the pathological features of DM1 (26), supporting the idea that expanded CUG titrates muscleblind proteins.

To further investigate the RNA gain-of-function hypothesis and the mechanisms of expanded CUG-induced pathogenesis, we have generated a transgenic Drosophila DM1 model carrying an interrupted (CTG)480 repeat. Other models of human disease in Drosophila have proved to be a useful tool to investigate the disease mechanisms (42–44). A previously reported DM1 Drosophila model generated with (CTG)162 repeats showed accumulation of the expanded repeats in nuclear foci and co-localization with muscleblind protein, but did not show pathological phenotypes in muscle or other tissues (35). Likewise, flies generated in our laboratory expressing (CUG)200 did not show abnormal phenotypes in the eye (data not shown) or in the muscle (Supplementary Material Fig. S1). Thus, it appears that fewer than 200 CUG repeats are not enough to trigger obvious pathogenic effects in Drosophila.

As reported here, we found that expression of a (iCUG)480 RNA leads to dysfunction and progressive degeneration of somatic muscle. In addition, we find that the expanded transcripts accumulate in nuclear foci in muscle cells. These foci are an important hallmark of DM1 and their presence in fly muscle nuclei supports the validity of the DM1 Drosophila model. We find that overexpression of MBNL1 reduces the percentage of nuclei containing multiple foci (Fig. 3F), whereas, overexpression of GUGBP1 increases this percentage (Fig. 4L). Thus, we find a positive correlation between the number of foci per nuclei and severity of the muscle degenerative phenotype. We also report that expression of (iCUG)480 in the Drosophila eye causes a phenotype that correlates with the muscle phenotype. The eye phenotype provides us with a convenient primary assay for future large-scale genetic screens aimed to identify additional modifiers of (iCUG)480-induced toxicity.

Two families of RNA-BP have been shown to be implicated in DM1 pathogenesis: CELF (CUGBP and ETR-3-like factors) proteins and MBNL proteins (21,31,45). CUGBP and MBNL proteins are antagonistic splicing regulators of two transcripts misregulated in DM1 tissues: cTNT and IR (28,39,40). In DM1, the splicing patterns observed for these transcripts are consistent with increased levels and activity of CUGBP1 (39–41,46) and decreased levels of MBNL proteins (30).

Mouse models expressing CUGBP1 in the heart show disrupted splicing of transcripts (27). Overexpression of CUGBP1 in mice skeletal muscle reproduces some of the muscle histopathology phenotypes observed in DM1 muscle (36). Based on these findings, it could be predicted that increased levels of CUGBP1 would result in an enhancement of the phenotype induced by the expanded RNA. We find that expression of CUGBP1 alone in Drosophila somatic muscles leads to a muscle-wasting phenotype similar to the phenotype observed with expression of (iCUG)480. This observation is consistent with previous reports describing the effect of CUGPB1 expression in mice (27,36). In agreement with data observed in DM1 cells (18,31), the nuclei of Drosophila muscle cells expressing both CUGBP1 and (iCUG)480 show a diffuse pattern of CUGBP1 accumulation but no aggregation or co-localization with the expanded RNA-containing foci. Co-expression of CUGBP1 and (iCUG)480 leads to aggravated muscle wasting and degeneration phenotypes. Because of the phenotype caused by CUGBP1 alone, it is difficult to make a strong conclusion from the enhancement of the (iCUG)480 muscle phenotype; however, these results are consistent with the idea that the increased CUGBP activity in DM1 tissue is relevant for DM1 pathogenesis.

All three mammalian MBNL proteins identified (MBNL1, MBNL2 and MBNL3) have been shown to co-localize with the expanded RNA-containing nuclear foci in vivo (21,22,31). We find that MBNL1 shows a diffuse distribution in the nuclei of Drosophila muscles. However, in the presence of (iCUG)480, MBNL1 accumulates in the nuclear foci and is depleted from the rest of the nucleus.

The phenotype of muscleblind knockout mice strongly supports the hypothesis that sequestration and loss of muscleblind normal function is a critical factor in DM1 pathogenesis. This hypothesis predicts that altering the levels of muscleblind proteins should modulate the phenotypes induced by expanded CUG repeats. We have tested this prediction and found that a heterozygous loss-of-function allele of Drosophila muscleblind enhances the eye phenotype. More importantly, we find that overexpression of human muscleblind is able to dramatically suppress both the somatic muscle and eye (iCUG)480 phenotypes. The degree of suppression is dependent on MBNL1 overexpression levels, as too much MBNL1 also causes muscle pathology. Thus, it appears that the steady state levels of MBNL, like those of CUGBP1, are important for normal muscle physiology. These results support the MBNL sequestration model in DM1 pathogenesis and also constitute proof of the principle that expanded CUG-induced toxicity can be suppressed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Construct generation and transgenic Drosophila lines
The UAS-(iCTG)480 construct was generated by cloning interrupted repeats composed of repeating units of the sequence (CTG)20CTCGA (39) into the pUAST Drosophila expression vector (37). (CTG)20 were also cloned into the pUAST vector to generate transgenic lines. The UAS-MBNL1 and UAS-CUGBP1 constructs were generated by cloning the 41 kDa isoform of MBNL1 with an N-terminal Xpress tag and human CUGBP1, respectively, into the pUAST vector. The UAS-(CTG)200 construct was generated by cloning a 1.4 kb fragment of the DMPK 3' UTR containing 200 CTG repeats into the pUAST-GFP vector. Transgenic Drosophila lines were obtained by injecting these constructs in embryos following standard methods. We generated 13 UAS-(iCTG)480, 13 UAS-(CTG)20, 10 UAS-MBNL1, 7 UAS-CUGBP1 and 13 UAS-(CTG)200 transgenic lines.

The UAS-LacZ, gmr-GAL4 and mblP15144 were obtained from Bloomington Stock Center (Indiana, USA). Mhc-GAL4 was obtained from G. Davis (UCSF).

Scanning electron microscopy of Drosophila eyes
Flies processing for SEM as well as image acquisition were performed following the previously published procedures (47).

Semi-thin sectioning of Drosophila adult thoraxes
Adult thoraxes were dissected out, fixed overnight in glutaraldehyde–paraformaldehyde–cacodylate buffer, washed, fixed for 2 h at 4°C in 1% osmium tetroxide, dehydrated in ethanol, embedded in EPON for transversal semi-thin sections, and then stained with Multiple Stain Solution (Polysciences). Sectioned muscles of different genotypes to be compared with one another were stained simultaneously to ensure the same amount of staining. At least eight flies per genotype were sectioned for each observation.

In situ hybridization and immunofluorescence staining of Drosophila larval muscles
For immunofluorescence staining, larval or adult muscles were dissected and fixed in 4% formaldehyde. Following standard procedures, the tissue was incubated with anti-CUGBP1 3B1 (1:500, Upstate), anti-Xpress tag (1:200, Invitrogen), anti-CBP (1:1000 provided by Dr Mattias Mannervik), anti-LaminC (1:50, Hybridoma Bank), Phalloidin (1:200, Sigma). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories and Molecular Probes. For combined in situ hybridization and immunofluorescence staining after the fixation, the tissue was fixed again with 40% formamide/1xSSC following hybridization with Cy3-labelled (CAG)5 probe (1ng/µl) and washed to continue with the immunofluorescence staining. We studied eight larvae per genotype and 50 nuclei per larvae. For comparative quantification of the nuclear foci, we studied at least four larvae per genotype and an average of 200 nuclei per larvae.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Dr Kwang Choi's Lab at Baylor College of Medicine for assistance with semi-thin sections and Dr Mattias Mannervik for kindly providing anti-dCBP antibody, J. Barrish at the Texas Children's Hospital for help with the SEM and R. Atkinson for advice with the confocal microscopy (supported by MRDDRC and HHMI). We also thank D. Bundman for technical assistance. This work was supported by NIH (NS42179) and seed funding from the Hunter Research Fund to J.B. and funding from the Muscular Dystrophy Association and NIH (AR45653) to T.C.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: Laboratorio de Neurogenética, Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra, Córdoba 5016, Argentina. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

  1. Harper P.S., Brook J.D., Newman E. (2001) Myotonic dystrophy 3rd edn. (Saunders, London).

  2. Fu Y.H., Pizzuti A., Fenwick R.G. Jr, King J., Rajnarayan S., Dunne P.W., Dubel J., Nasser G.A., Ashizawa T., de Jong P., et al. (1992) An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 255:1256–1258.[Abstract/Free Full Text]

  3. Brook J.D., McCurrach M.E., Harley H.G., Buckler A.J., Church D., Aburatani H., Hunter K., Stanton V.P., Thirion J.P., Hudson T., et al. (1992) Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 68:799–808.[CrossRef][Web of Science][Medline]

  4. Mahadevan M., Tsilfidis C., Sabourin L., Shutler G., Amemiya C., Jansen G., Neville C., Narang M., Barcelo J., O'Hoy K., et al. (1992) Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science 255:1253–1255.[Abstract/Free Full Text]

  5. Fu Y.H., Friedman D.L., Richards S., Pearlman J.A., Gibbs R.A., Pizzuti A., Ashizawa T., Perryman M.B., Scarlato G., Fenwick R.G. Jr, et al. (1993) Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy. Science 260:235–238.[Abstract/Free Full Text]

  6. Hofmann-Radvanyi H., Lavedan C., Rabes J.P., Savoy D., Duros C., Johnson K., Junien C. (1993) Myotonic dystrophy: absence of CTG enlarged transcript in congenital forms, and low expression of the normal allele. Hum. Mol. Genet. 2:1263–1266.[Abstract/Free Full Text]

  7. Novelli G., Gennarelli M., Zelano G., Pizzuti A., Fattorini C., Caskey C.T., Dallapiccola B. (1993) Failure in detecting mRNA transcripts from the mutated allele in myotonic dystrophy muscle. Biochem. Mol. Biol. Int. 29:291–297.[Web of Science][Medline]

  8. Wang Y.H., Amirhaeri S., Kang S., Wells R.D., Griffith J.D. (1994) Preferential nucleosome assembly at DNA triplet repeats from the myotonic dystrophy gene. Science 265:669–671.[Abstract/Free Full Text]

  9. Otten A.D. and Tapscott S.J. (1995) Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure. Proc. Natl. Acad. Sci. USA 92:5465–5469.[Abstract/Free Full Text]

  10. Jansen G., Groenen P.J., Bachner D., Jap P.H., Coerwinkel M., Oerlemans F., van den Broek W., Gohlsch B., Pette D., Plomp J.J., et al. (1996) Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice. Nat. Genet. 13:316–324.[CrossRef][Web of Science][Medline]

  11. Reddy S., Smith D.B., Rich M.M., Leferovich J.M., Reilly P., Davis B.M., Tran K., Rayburn H., Bronson R., Cros D., et al. (1996) Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy. Nat. Genet. 13:325–335.[CrossRef][Web of Science][Medline]

  12. Berul C.I., Maguire C.T., Gehrmann J., Reddy S. (2000) Progressive atrioventricular conduction block in a mouse myotonic dystrophy model. J. Interv. Card. Electrophysiol. 4:351–358.[CrossRef][Web of Science][Medline]

  13. Klesert T.R., Cho D.H., Clark J.I., Maylie J., Adelman J., Snider L., Yuen E.C., Soriano P., Tapscott S.J. (2000) Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy. Nat. Genet. 25:105–109.[CrossRef][Web of Science][Medline]

  14. Sarkar P.S., Appukuttan B., Han J., Ito Y., Ai C., Tsai W., Chai Y., Stout J.T., Reddy S. (2000) Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts. Nat. Genet. 25:110–114.[CrossRef][Web of Science][Medline]

  15. Ranum L.P., Rasmussen P.F., Benzow K.A., Koob M.D., Day J.W. (1998) Genetic mapping of a second myotonic dystrophy locus. Nat. Genet. 19:196–198.[CrossRef][Web of Science][Medline]

  16. Liquori C.L., Ricker K., Moseley M.L., Jacobsen J.F., Kress W., Naylor S.L., Day J.W., Ranum L.P. (2001) Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293:864–867.[Abstract/Free Full Text]

  17. Mankodi A., Logigian E., Callahan L., McClain C., White R., Henderson D., Krym M., Thornton C.A. (2000) Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 289:1769–1773.[Abstract/Free Full Text]

  18. Michalowski S., Miller J.W., Urbinati C.R., Paliouras M., Swanson M.S., Griffith J. (1999) Visualization of double-stranded RNAs from the myotonic dystrophy protein kinase gene and interactions with CUG-binding protein. Nucleic Acids Res. 27:3534–3542.[Abstract/Free Full Text]

  19. Napierala M. and Krzyzosiak W.J. (1997) CUG repeats present in myotonin kinase RNA form metastable ‘slippery’ hairpins. J. Biol. Chem. 272:31079–31085.[Abstract/Free Full Text]

  20. Taneja K.L., McCurrach M., Schalling M., Housman D., Singer R.H. (1995) Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. J. Cell. Biol. 128:995–1002.[Abstract/Free Full Text]

  21. Miller J.W., Urbinati C.R., Teng-Umnuay P., Stenberg M.G., Byrne B.J., Thornton C.A., Swanson M.S. (2000) Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J. 19:4439–4448.[CrossRef][Web of Science][Medline]

  22. Fardaei M., Rogers M.T., Thorpe H.M., Larkin K., Hamshere M.G., Harper P.S., Brook J.D. (2002) Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum. Mol. Genet. 11:805–814.[Abstract/Free Full Text]

  23. Timchenko L.T., Miller J.W., Timchenko N.A., DeVore D.R., Datar K.V., Lin L., Roberts R., Caskey C.T., Swanson M.S. (1996) Identification of a (CUG)n triplet repeat RNA-binding protein and its expression in myotonic dystrophy. Nucleic Acids Res. 24:4407–4414.[Abstract/Free Full Text]

  24. Artero R., Prokop A., Paricio N., Begemann G., Pueyo I., Mlodzik M., Perez-Alonso M., Baylies M.K. (1998) The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2. Dev. Biol. 195:131–143.[CrossRef][Web of Science][Medline]

  25. Begemann G., Paricio N., Artero R., Kiss I., Perez-Alonso M., Mlodzik M. (1997) Muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins. Development 124:4321–4331.[Abstract]

  26. Kanadia R.N., Johnstone K.A., Mankodi A., Lungu C., Thornton C.A., Esson D., Timmers A.M., Hauswirth W.W., Swanson M.S. (2003) A muscleblind knockout model for myotonic dystrophy. Science 302:1978–1980.[Abstract/Free Full Text]

  27. Ho T.H., Bundman D., Armstrong D.L., Cooper T.A. (2005) Transgenic mice expressing CUG-BP1 reproduce splicing mis-regulation observed in myotonic dystrophy. Hum. Mol. Genet. 14:1539–1547.[Abstract/Free Full Text]

  28. Ho T.H., Charlet B.N., Poulos M.G., Singh G., Swanson M.S., Cooper T.A. (2004) Muscleblind proteins regulate alternative splicing. EMBO J 23:3103–3112.[CrossRef][Web of Science][Medline]

  29. Ho T.H., Savkur R.S., Poulos M.G., Mancini M.A., Swanson M.S., Cooper T.A. (2005) Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy. J. Cell. Sci. 118:2923–2933.[Abstract/Free Full Text]

  30. Ladd A.N., Stenberg M.G., Swanson M.S., Cooper T.A. (2005) Dynamic balance between activation and repression regulates pre-mRNA alternative splicing during heart development. Dev. Dyn. 233:783–793.[CrossRef][Web of Science][Medline]

  31. Fardaei M., Larkin K., Brook J.D., Hamshere M.G. (2001) In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts. Nucleic Acids Res. 29:2766–2771.[Abstract/Free Full Text]

  32. Mankodi A., Urbinati C.R., Yuan Q.P., Moxley R.T., Sansone V., Krym M., Henderson D., Schalling M., Swanson M.S., Thornton C.A. (2001) Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2. Hum. Mol. Genet. 10:2165–2170.[Abstract/Free Full Text]

  33. Jiang H., Mankodi A., Swanson M.S., Moxley R.T., Thornton C.A. (2004) Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. Hum. Mol. Genet. 13:3079–3088.[Abstract/Free Full Text]

  34. Mankodi A., Lin X., Blaxall B.C., Swanson M.S., Thornton C.A. (2005) Nuclear RNA foci in the heart in myotonic dystrophy. Circ. Res. 97:1152–1155.[Abstract/Free Full Text]

  35. Houseley J.M., Wang Z., Brock G.J., Soloway J., Artero R., Perez-Alonso M., O'Dell K.M., Monckton D.G. (2005) Myotonic dystrophy associated expanded CUG repeat muscleblind positive ribonuclear foci are not toxic to Drosophila. Hum. Mol. Genet. 14:873–883.[Abstract/Free Full Text]

  36. Timchenko N.A., Patel R., Iakova P., Cai Z.J., Quan L., Timchenko L.T. (2004) Overexpression of CUG triplet repeat-binding protein, CUGBP1, in mice inhibits myogenesis. J. Biol. Chem. 279:13129–13139.[Abstract/Free Full Text]

  37. Brand A.H. and Perrimon N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415.[Abstract]

  38. Roberts R., Timchenko N.A., Miller J.W., Reddy S., Caskey C.T., Swanson M.S., Timchenko L.T. (1997) Altered phosphorylation and intracellular distribution of a (CUG)n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice. Proc. Natl. Acad. Sci. USA 94:13221–13226.[Abstract/Free Full Text]

  39. Philips A.V., Timchenko L.T., Cooper T.A. (1998) Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. Science 280:737–741.[Abstract/Free Full Text]

  40. Savkur R.S., Philips A.V., Cooper T.A. (2001) Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat. Genet. 29:40–47.[CrossRef][Web of Science][Medline]

  41. Charlet B.N., Savkur R.S., Singh G., Philips A.V., Grice E.A., Cooper T.A. (2002) Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol. Cell. 10:45–53.[CrossRef][Web of Science][Medline]

  42. Zoghbi H.Y. and Botas J. (2002) Mouse and fly models of neurodegeneration. Trends Genet. 18:463–471.[CrossRef][Web of Science][Medline]

  43. Fortini M.E. and Bonini N.M. (2000) Modeling human neurodegenerative diseases in Drosophila: on a wing and a prayer. Trends Genet. 16:161–167.[CrossRef][Web of Science][Medline]

  44. Shulman J.M., Shulman L.M., Weiner W.J., Feany M.B. (2003) From fruit fly to bedside: translating lessons from Drosophila models of neurodegenerative disease. Curr. Opin. Neurol. 16:443–449.[CrossRef][Web of Science][Medline]

  45. Ladd A.N., Charlet N., Cooper T.A. (2001) The CELF family of RNA-binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol. Cell. Biol. 21:1285–1296.[Abstract/Free Full Text]

  46. Timchenko N.A., Cai Z.J., Welm A.L., Reddy S., Ashizawa T., Timchenko L.T. (2001) RNA CUG repeats sequester CUGBP1 and alter protein levels and activity of CUGBP1. J. Biol. Chem. 276:7820–7826.[Abstract/Free Full Text]

  47. Fernandez-Funez P., Nino-Rosales M.L., de Gouyon B., She W.C., Luchak J.M., Martinez P., Turiegano E., Benito J., Capovilla M., Skinner P.J., et al. (2000) Identification of genes that modify ataxin-1-induced neurodegeneration. Nature 408:101–106.[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. R. O'Rourke and M. S. Swanson
Mechanisms of RNA-mediated Disease
J. Biol. Chem., March 20, 2009; 284(12): 7419 - 7423.
[Abstract] [Full Text] [PDF]

Home page
 DMMHome page
J. A. Hurt and P. A. Silver
mRNA nuclear export and human disease
Dis. Model. Mech., September 1, 2008; 1(2-3): 103 - 108.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
R. J. Osborne and C. A. Thornton
Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats
Nucleic Acids Res., March 27, 2008; 36(4): e24 - e24.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
M. J. Allikian, G. Bhabha, P. Dospoy, A. Heydemann, P. Ryder, J. U. Earley, M. J. Wolf, H. A. Rockman, and E. M. McNally
Reduced life span with heart and muscle dysfunction in Drosophila sarcoglycan mutants
Hum. Mol. Genet., December 1, 2007; 16(23): 2933 - 2943.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
R. J. Osborne and C. A. Thornton
RNA-dominant diseases
Hum. Mol. Genet., October 15, 2006; 15(suppl_2): R162 - R169.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Material
Right arrow All Versions of this Article:
15/13/2138    most recent
ddl137v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (20)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by de Haro, M.
Right arrow Articles by Botas, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Haro, M.
Right arrow Articles by Botas, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?