European Journal of Cell Biology 92 (2013) 280–294

Contents lists available at ScienceDirect

European Journal of Cell Biology journal homepage: www.elsevier.com/locate/ejcb

Essential roles of LEM-domain protein MAN1 during organogenesis in Xenopus laevis and overlapping functions of emerin Michael Reil 1 , Marie-Christine Dabauvalle ∗ Division of Electron Microscopy, Biocenter, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany

a r t i c l e

i n f o

Article history: Received 18 October 2013 Received in revised form 23 October 2013 Accepted 25 October 2013 Keywords: LEM-domain MAN1 Emerin Organogenesis Xenopus laevis Nuclear envelope

a b s t r a c t Mutations in nuclear envelope proteins are linked to an increasing number of human diseases, called envelopathies. Mutations in the inner nuclear membrane protein emerin lead to X-linked Emery–Dreifuss muscular dystrophy, characterized by muscle weakness or wasting. Conversely, mutations in nuclear envelope protein MAN1 are linked to bone and skin disorders. Both proteins share a highly conserved domain, called LEM-domain. LEM proteins are known to interact with Barrier-to-autointegration factor and several transcription factors. Most envelopathies are tissue-specific, but knowledge on the physiological roles of related LEM proteins is still unclear. For this reason, we investigated the roles of MAN1 and emerin during Xenopus laevis organogenesis. Morpholino-mediated knockdown of MAN1 revealed that MAN1 is essential for the formation of eye, skeletal and cardiac muscle tissues. The MAN1 knockdown could be compensated by ectopic expression of emerin, leading to a proper organ development. Further investigations revealed that MAN1 is involved in regulation of genes essential for organ development and tissue homeostasis. Thereby our work supports that LEM proteins might be involved in signalling essential for organ development during early embryogenesis and suggests that loss of MAN1 may cause muscle and retina specific diseases. © 2013 Elsevier GmbH. All rights reserved.

Introduction Envelopathies are human diseases which are caused by mutations in genes encoding for nuclear envelope (NE) proteins. In particular, emerin is encoded by the EMD-gene, which has been identified as the gene mutated in patients suffering from X-linked Emery–Dreifuss muscular dystrophy, EDMD (Bione et al., 1994). This disease is characterized by contractions of main tendons, amyotrophia in the upper limbs and cardiac arrhythmias (Emery, 1989, 2000). An autosomal dominant variant of this disease is caused by point mutations in the LMNA-gene encoding A-type lamins (Bonne et al., 1999; for review see Worman and Bonne, 2007). In human, MAN1, another NE protein is encoded by the LEMD3-gene. Mutations in this gene result in Osteopoikilosis, the Buschke–Ollendorff syndrome and melorheostosis, characterized by increased bone density and skin aberrations (Hellemans et al., 2004). Although these NE proteins are expressed in most differentiated cells, envelopathies are restricted to specific tissues. In order

∗ Corresponding author. Tel.: +49 931 3188055; fax: +49 931 3184252. E-mail addresses: [email protected] (M. Reil), [email protected] (M.-C. Dabauvalle). 1 Tel.: +49 931 3184073. 0171-9335/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ejcb.2013.10.008

to understand this tissue-specific pathology of envelopathies, it is crucial to investigate the functions of NE proteins. Emerin and MAN1 are integral membrane proteins of the NE which is composed of three distinct components: the inner and outer nuclear membranes (INM and ONM), the nuclear pore complexes (NPCs) which are mediating the import and export of macromolecules, and the nuclear lamina (Gruenbaum et al., 2005; Stewart et al., 2007). Facing the cytoplasm, the ONM is continuous with the endoplasmatic reticulum (ER). The INM faces the chromatin and contains specific integral membrane proteins e.g. emerin, MAN1 and the Lamina Associated Polypeptide 2, called LAP2 (Schirmer and Foisner, 2007; Schirmer and Gerace, 2005). The INM is bordered by the so called nuclear lamina. This lamina represents networks of A- and B-type lamins (type-V intermediate filaments) linked to the INM via numerous integral membrane proteins (Dechat et al., 2008). An important and growing group of these lamin binding proteins share a highly conserved domain, called LEM-domain. This domain consists of ∼45 amino acids (Laguri et al., 2001) and was first identified in LAP2, emerin and MAN1 (Lin et al., 2000). Moreover, other LEM proteins were also characterized in Drosophila (Goldberg et al., 1998; Wagner et al., 2004) and Caenorhabditis elegans (Gruenbaum et al., 2002; Lee et al., 2000; Liu et al., 2003). It is well known, that the LEM-domain mediates the interaction with a chromatin-binding protein, called Barrier-to-autointegration factor, BAF (Furukawa,

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

1999). BAF is a small, highly conserved and essential protein with the ability to bind dsDNA, chromatin, histones, lamin binding proteins and various transcription factors (Margalit et al., 2007). Furthermore, LEM proteins are known to bind A- or Btype lamins via discrete domains (for review see Wagner and Krohne, 2007). This implies that lamins are involved in positioning and anchoring LEM proteins to the INM (Brachner et al., 2005; Sullivan et al., 1999; Vaughan et al., 2001). It is also suggested that LEM proteins have multiple roles including gene regulation, chromatin organization, regulation of transcription factor activity at the nuclear periphery and regulation of specific signal pathways (for review see Wagner and Krohne, 2007). Advanced investigations on LEM proteins strengthened their involvement in signalling. For example, emerin is able to bind LIM-domain-only 7 (Lmo7) or ␤-catenin (Holaska et al., 2006; Markiewicz et al., 2006). It is supposed that these interactions regulate the nuclear export of Lmo7 or ␤-catenin (acting as transcription factor), thereby repressing their functions. This inhibition in turn might lead to a misregulated muscle or adipocyte differentiation (Holaska et al., 2006; Tilgner et al., 2009). Furthermore, emerin is able to bind transcription factors like GCL (Holaska et al., 2003) or the BCL-2 related transcription factor, Btf (Haraguchi et al., 2004) and also the splicing factor YT521-B (Wilkinson et al., 2003). This implies a direct contribution of emerin to signalling and gene regulation. Similar to that, MAN1 controls in X. laevis the determination of dorsoventral axis by interactions with Smad-proteins (Osada et al., 2003). This illustrates that MAN1 is involved in modulating transforming growth factor (TGF)-␤ and bone morphogenic protein (BMP)-signalling. It has been further demonstrated that MAN1 has a neuralizing activity that applies to the C-terminal region, featuring a RNA-recognition motif (Birney et al., 1993). In addition to that, the C-terminal region might interact directly with BAF via its SRV-motif (Mansharamani and Wilson, 2005). Like emerin, MAN1 is able to bind transcription factors GCL or Btf (Lin et al., 2005). At the present it is speculated that different LEM proteins might have redundant functions, essential during vertebrate development. For example, LEM proteins NET25 (Lem2) and emerin share partially overlapping functions during myogenesis by regulating ERK-signalling (Huber et al., 2009). Previous studies also indicated that emerin and MAN1 have overlapping functions essential for chromosome segregation and cell division in C. elegans embryos (Liu et al., 2003). In our laboratory we were able to show that emerin is first expressed during Xenopus embryogenesis when MAN1 expression begins to diminish (Gareiss et al., 2005). Furthermore, emerin is able to interact with MAN1 and both proteins share several interactions with transcription factors, lamin proteins and especially BAF (Mansharamani and Wilson, 2005). Thereby it is strongly speculated that MAN1 and emerin have redundant functions in common signal pathways. Actually the functional roles of MAN1 and emerin, in particular during organogenesis are still unclear. For this purpose, we analysed the role of MAN1 in Xenopus laevis development by knockdown experiments. Here, we present evidence that MAN1 is essential for proper development of eyes, skeletal and cardiac muscles. Rescue experiments by coinjection of emerin-mRNA revealed, that emerin is able to compensate MAN1’s functions during organogenesis. These results firstly demonstrate overlapping roles for these two LEM proteins during organ development and tissue homeostasis. Moreover, we conclude that LEM proteins might have common roles in gene regulation and signalling. These findings may generally help to improve the understanding of many human diseases.

281

Materials and methods Biological materials Adult wild type Xenopus laevis (X. laevis) females and males were purchased from the Xenopus Express Farm (Le Bourg, France). Cell lines derived from Xenopus laevis (XLKE-A6) were cultured in DMEM (Gibco® , Life Technologies, Darmstadt, Germany), supplemented with 10% foetal calf serum (Gibco® ) at 27 ◦ C in a 5% CO2 incubator. In vitro fertilization In vitro fertilization of eggs was performed as described (Wolf and Hedrick, 1971). Embryos were cultured at 20 ◦ C in modified ringer solution (MMR, 1:10 diluted; Newport and Kirschner, 1982) on charcoal-agar-plates with 0.5% (w/v) sulfadiazine and staged according to the Normal Table of Xenopus laevis (Nieuwkoop and Faber, 1975). Embryos were documented using a Leica MZ 16 FA stereo microscope (Leica Microsystems, Wetzlar, Germany) combined with a Leica EC3 digital camera and the Leica Application Suite software (Leica LAS EZ v3.0). Video imaging was performed using FRAPSTM as an on-screen capture software (FRAPSTM v3.5.9, Beepa Pty Ltd, VIC, Australia). Beating hearts were recorded at 20 ◦ C (300 images, 30 fps). Cardiac features were measured using Adobe Premiere Pro CS5.5 and Adobe Photoshop CS5 by analysing each frame of recorded videos dependent on related time points. Morpholino design and microinjection Antisense morpholino oligonucleotides were purchased from Gene-Tools (Philomath, Oregon, USA). Morpholino sequences were designed for translational blocking and directed against the translational start site of X. laevis lemd3 (MO-XMAN1: 5 -GCCGCCATTTTGACCACTCGGT-3 ). For control purposes a five-base-mismatch control morpholino (MO-5mm-XMAN1: 5 -GaCGCaATTTTaACCAa TCaGT-3 ; lowercased letters indicate mismatch mutations) as well as a standard control morpholino (MO-CO: 5 -CCTCTTACCTCAGTTACAATTTATA-3 ) were used. Fertilized Xenopus eggs were microinjected as described (Benavente et al., 1985). At the two-cell-stage, one blastomere of the embryo was injected with 50 ng of morpholinos. For microinjection prior to the first cleavage, embryos were injected with 100 ng of morpholinos. Phenotype rescue Rescue experiments were performed by coinjection of morpholinos and Xemerin1-mRNA. The coding sequence of emd1 was cloned into the pCMV-Sport6 vector. The plasmid was linearized by enzymatic digestion and capped Poly(A) mRNA was synthesized using the T7 mMESSAGE mMACHINE Kit and Poly(A)Tailing Kit (both Ambion, Life Technologies, Darmstadt). Phenotype rescue was performed using 40 ng Xemerin1-mRNA and 50 ng MOXMAN1. Antibodies In this study, monoclonal antibodies against Xenopus emerin were used (Gareiss et al., 2005). Polyclonal guinea pig antisera were raised against the recombinant His-tagged C-terminal region of X. laevis MAN1 (amino acids 525–781). For affinity purification of anti-XMAN1 antibodies, we bound the recombinant His-tagged C-terminal region of XMAN1 to CNBractivated SepharoseTM 4B (Amersham, Uppsala, Sweden). Coupling

282

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

of antigens was performed according to the manufacturer’s protocol for CNBr-activated Sepharose. Elution of antibodies was performed using 0.1 M glycine (pH 2.5).

(5 -TCCCTGTACGCTTCT GGTCGTA-3 ) and actin-rev (5 TCTCAAAGTCCAAAGCCACATA-3 ). PCR products were analysed on 1% agarose gels.

Gelelectrophoresis of proteins and immunoblotting

Expression and purification of recombinant proteins

Nuclear envelopes were isolated from cultured cells (A6) as described (Benavente et al., 1985). As a modification, homogenization of cells and nuclei was performed using 10 mM Tris (pH 7.5). Proteins from X. laevis embryos were extracted by homogenization in sodium dodecyl sulphate buffer (1× SDS-buffer, 10 ␮l per embryo) and electrophoretically separated by SDS-PAGE (Thomas and Kornberg, 1975) using 18% polyacrylamide. Immunoblotting was performed as described (Gareiss et al., 2005). The nitrocellulose (NC) was incubated with following primary antibodies, diluted in 5% non-fat dry milk:anti-XMAN1 (1:100), anti-Xemerin (1:10), anti-Fibrillarin (mAb P2G3; 1:1000; Christensen et al., 1986) or anti-alpha-tubulin (Sigma T5168, 1:10,000). Peroxidase-coupled anti-guinea-pig or anti-mouse antibodies were diluted in 10% nonfat dry milk (1:13,000 and 1:5000, respectively; Dianova, Hamburg, Germany).

After bacterial expression of Histidine-tagged XMAN1 and Xemerin1, proteins were extracted followed by purification on NiNTA-agarose beads (Qiagen) according to the Qiagen protocol. The following proteins were purified: C-terminal domain of XMAN1 (CXMAN1, residues 525–781), Xemerin1 lacking the transmembrane domain (Xemerin1TM, residues 1–136).

Whole mount immunostaining For whole mount immunostaining, X. laevis tadpoles were fixed overnight at −20 ◦ C in Dent’s fixative (80% methanol, 20% dimethyl sulfoxide [DMSO]). Bleaching of embryos and labelling was performed as described (Klymkowsky and Hanken, 1991). Following antibodies were used: anti-desmin (Sigma D1033, 1:13), affinity-purified anti-XMAN1 (1:25) and anti-Xemerin (undiluted). Staining of control embryos was performed without incubation of primary antibodies. For detection, the embryos were incubated with appropriate peroxidase-conjugated secondary antibodies (Dianova, 1:3700) and visualized with diaminobenzidine. Electron microscopy and immunogold labelling Ultrastructure of embryos was analysed by electron microscopy using a Zeiss EM-10 or EM-900 following standard procedures (Schoft et al., 2003). Histological sections of embedded embryos were generated using a Leica EM UC7 microtome. Sections were stained with methylene blue AzurII, rinsed with water and air-dried. RNA isolation, cDNA synthesis and RT-PCR analysis Embryos were stored at 4 ◦ C in RNAlater (Qiagen) until further processing. Total RNA of embryos was isolated according to manufacturer’s protocol using the RNeasy Mini Kit (Qiagen). cDNA was synthesized with RevertAid First Strand cDNA Synthesis Kit (Fermentas/Thermo Scientific, Schwerte Germany) using 1 ␮g of total RNA. For RT-PCR analysis, amplification was achieved using Phusion High Fidelity DNA-Polymerase (Fermentas) and the following primer sets: for analysis of eye-specific gene-expression: pax6-fwd (5 GTTTGTCAACGGCCGACCCCT-3 ), pax6-rev (5 -CTGTGCTGGTTGGCC crx-fwd (5 -GAATGGACCTTCTGCACTCAG-3 ), AGGTAC-3 ), crx-rev (5 -GGAACTAG GGGTAACTGCAGT-3 ), otx2-fwd (5 -GCGACCCCCAGGAAACAGAGG-3 ), otx2-rev (5  CAGATGGACACAGGGGCTGTG-3 ); for analysis of the muscle-/heart-specific gene-expression: myoD-fwd (5 AACTGCTCCGATGGCATGATGGATTA-3 ), myoD-rev (5 -AT TGCTGGGAGAAGGGATGGTGATTA-3 ) and myocardin-fwd ACAAGAA-3 ), myocardin-rev (5 (5 -GCCCAAAGCAAATT  GGAAGTCGGTGTTGAAGATAC-3 ). As a control, we analysed expression of actin using following primers: actin-fwd

Results Characterization of Xenopus MAN1 Because no commercial antibody recognizing Xenopus MAN1 (XMAN1) has been available and in order to study the expression of XMAN1, we generated specific antibodies. The polyclonal antibody was raised in guinea pig against the His-tagged C-terminal region of XMAN1 (CXMAN1, residues 525–781). Using our generated XMAN1 antibodies, we were able to identify endogenous XMAN1 by immunoblot as a protein with a Mr of ∼66,000. Further investigations on the intracellular localization of XMAN1 by immunofluorescence on Xenopus A6 cells and oocytes revealed the prominent staining of the NE. Electron microscopy on oocytes showed, that XMAN1 is expectedly localized to the inner nuclear membrane (data not shown). The localization of MAN1 to the inner nuclear membrane has been also demonstrated in human, Drosophila and C. elegans somatic cells (Lee et al., 2000; Lin et al., 2000; Wagner et al., 2006). Interestingly, previous studies reported that LEM-domain protein Xemerin is present in somatic cells but absent in oocytes (Gareiss et al., 2005). For this reason, we were interested in the expression patterns of MAN1 during early Xenopus embryogenesis in comparison to emerin. Xemerin transcripts have been identified in all developmental stages, however transcription levels increased at stage 34 (Gareiss et al., 2005). Since XMAN1 transcription is down-regulated from stage 34 onwards, it has been postulated that XMAN1 translation might also decrease at this stage (Gareiss et al., 2005). Analysing the expression patterns of XMAN1 during Xenopus development by immunoblot revealed that XMAN1 is detectable in all developmental stages with comparable amounts (Fig. 1, upper panel). In contrast, Xemerin was first detected at stage 41 onwards (Fig. 1, second panel) and not at stage 43 as previously observed (Gareiss et al., 2005). Blots were re-probed with fibrillarin antibodies, which served as the loading control. Fibrillarin was detected in each developmental stage with constant levels (Fig. 1, lower panel). Analysis of the expression patterns of XMAN1 and Xemerin by whole mount immunostaining demonstrated that both proteins colocalize within the somites of tadpole stages (data not shown). Knockdown of XMAN1 affects eye, muscle and heart development In order to study the functional role of XMAN1, we manipulated expression levels of XMAN1 in embryos using specific morpholinos (MO-XMAN1). To test the XMAN1 knockdown using antisense morpholinos, we injected embryos at the one-cell-stage using 100 ng of MOXMAN1. Expression levels of XMAN1 during embryogenesis were subsequently analysed by immunoblots (Fig. 2). Embryos injected with MO-XMAN1 showed a significantly decreased expression of XMAN1 at stage 4 and reduced expression levels persisted throughout embryogenesis (Fig. 2, first panel). For comparison, XMAN1 is

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

Fig. 1. Expression patterns of XMAN1 and Xemerin during early Xenopus laevis development. Total protein extracts of 3 defolliculated oocytes (lane 1), embryos of different stages (lanes 2–9) and 5 × 106 nuclear envelopes from A6 nuclei (lane 10, A6 NEs) were separated by SDS-PAGE and immunoblotted with anti-XMAN1. Polypeptides with a molecular mass of ∼66 kDa were detected in all stages including the A6 NEs (upper panel). Immunoblot analysis using monoclonal Xemerin antibodies revealed that Xemerin is first detectable at stage 41 (second panel, Mr 24,000). Xenopus fibrillarin served as the loading control (lower panel). Molecular masses of reference proteins are indicated in kDa. Stg: stage.

283

Fig. 3. Knockdown of XMAN1 leads to a developmental defect of the eye. (A) Stereoscopic microscope images of three days old embryos injected with 50 ng of control morpholinos (MO-CO or MO-5mm-XMAN1, a–c) or MO-XMAN1 (a –c ). The injected side of XMAN1 morphants revealed no or poorly formed eyes (b and c , arrows). Control morphants developed two normal eyes (a and b. arrows). Bars: 1 mm. (B) Quantification of the eye-less phenotype. XMAN1 morphants predominantly displayed a severe (red) or weak (blue) pathological phenotype. The majority of control morphants showed two wild-type eyes (WT, green). n, number of embryos. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

continuously expressed in uninjected control embryos (Fig. 2, third panel). As an internal control, blots were reprobed with antibodies specific for tubulin. Tubulin was detectable in each stage with similar amounts (Fig. 2, second and lower panel). By knockdown of XMAN1, we decided to analyse the development of Xenopus embryos. We therefore performed knockdown experiments by injecting morpholinos into one blastomere of the two-cell-stage. By this way, we were able to use the second (uninjected) side to compare the effects of XMAN1 knockdown to the normal development. Thereby we consequently used 50 ng of MOXMAN1.

Fig. 2. Expression levels of XMAN1 after morpholino-mediated knockdown. Total protein extracts of 5 unfertilized eggs (lane 1), activated and injected eggs (lane 2), embryos of different stages (lanes 3–10) and 5 × 106 nuclear envelopes from A6 nuclei (lane 11) were separated by SDS-PAGE and immunoblotted with antibodies specific for XMAN1. Embryos injected with MO-XMAN1, revealed a significantly decreased expression of XMAN1 at stage 4 (lane 5, upper panel). This decreased expression persisted throughout embryogenesis (lanes 6–10). Note the continuous expression of XMAN1 (Mr 66,000) in uninjected control embryos (third panel). As an internal control, nitrocellulose was reprobed with anti-tubulin. Tubulin expression stayed constant during embryogenesis (second and lower panel). Note reduced amounts of tubulin at lane 7 due to loading problems. Molecular masses of reference proteins are indicated in kDa. Stg: stage.

XMAN1 knockdown leads to a disruption of eye development Knockdown of XMAN1 induced a disrupted eye development within the injected side. This phenotype was first seen at stage 37/38 at the beginning of eye formation and was obvious three days after fertilization (stage 41). The majority of XMAN1 morphants (89%) showed a strong phenotype and displayed no eye (Fig. 3A, b –c , arrows and B). Some embryos (9%) also showed a weak phenotype with barely formed or smaller eyes. Embryos injected with unspecific control morpholinos (MO-CO) as well as with five-basemismatch control morpholinos (MO-5mm-XMAN1) developed two normal eyes (73% and 81%, respectively; Fig. 3A, a–c and B). The occurrence of the weak phenotype “smaller eyes” in 20% and 17%, respectively, of the controls is explained by the sensitivity of the developing embryos to the microinjection procedure. It is important to point out that numbers of controls displaying the strong phenotype were nonsignificant (3%). Hence, the observed weak

284

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

Fig. 4. Morphological analysis of the eye phenotype. (A) Methylene blue stained cephalic transverse sections of control (a) and XMAN1 morphants (b). Histology of XMAN1 morphants revealed aberrant eye structures within the injected side (b, arrow). Bars: 250 ␮m. (B) Retinal structures of WT (a) and aberrant eyes (b) shown by electron microscopy. Photoreceptor cells of aberrant eyes revealed a loss of the membranous outer segment (b, arrow) in combination with disordered membranes (inset in b). XMAN1 morphants also showed a reduced amount and inhomogeneous distribution of pigments within the pigment epithelium, PE (b, arrowhead). C, cone; R, rod. Bars: 5 ␮m and 1 ␮m (insets).

phenotypes in less than 20% of the control morphants appear to represent nonspecific defects due to the experimental manipulation. Although disrupted eye-formation in XMAN1 morphants has been reported in previous studies (Osada et al., 2003), further investigations on the histology are still lacking. In histological sections of XMAN1 morphants, we observed an aberrant eye formation (Fig. 4A, b, arrow) while eyes of the uninjected side appeared unaffected (arrowhead). These embryos also revealed a considerably thickened skin at the injected side (white arrow). Control embryos showed a regular formation of both eyes (Fig. 4A, a). Electron micrographs displaying the retina of XMAN1 morphants revealed severe retinal defects. Firstly, these defects are characterized by a markedly reduced amount and inhomogeneous distribution of pigments in the pigment epithelium (Fig. 4B, b). Pigments were predominantly arranged in one layer and the pigment epithelium showed areas lacking pigments (arrowhead). Secondly, photoreceptor cells of XMAN1 morphants displayed a loss of the outer segments (arrow). These segments are usually harbouring the membrane shelves. Additionally, these membranes appeared more disordered (inset in Fig. 4B, b). Other retinal layers also appeared disorganized in XMAN1 morphants (data not shown). For comparison, the retina of WT eyes showed several layers of regular distributed pigments (Fig. 4B, a, arrowhead) and normal photoreceptor cells with parallel organized membranes (inset in Fig. 4B, a).

Knockdown of XMAN1 causes abnormal muscle formations In addition to malformations of the eye, we observed a disordered body and developmental defects of the somites. A strong phenotype corresponding to a loss of somites and disorganization of muscle structures along the tail was observed in 93% of embryos three days after injection with MO-XMAN1 (Fig. 5A, b –c and B). Only 3% of embryos showed weak phenotypes characterized by linear-shaped somites or a decreased number of somites. Note, that the body of the injected side appeared less pigmented as compared to WT embryos. In contrast, injection of MO-CO and MO-5mmXMAN1 revealed embryos with normal tails showing arrow-shaped somites (Fig. 5A, a–c and B, 100% and 86%, respectively). In order to demonstrate that somites are indeed affected in XMAN1 morphants, we performed whole mount immunostainings using desmin antibodies as a somite marker (Debus et al., 1983). The uninjected side showed the common somite-specific staining for desmin (Fig. 5C, a , arrowhead), but no staining was detectable within the injected side, indicating the absence of proper somite formation (Fig. 5C, b , arrow). In order to specify the morphology of the somites, we first analysed injected embryos by histological sections. XMAN1 morphants displayed no or poorly formed somites in the injected side (Fig. 6A, b, arrow). A thickened or multilayered skin was also observed. In contrast, embryos which were injected with control morpholinos showed fully developed somites in both sides (Fig. 6A, a). Electron microscopy on aberrant somites of XMAN1 morphants confirmed

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

285

a, b with c). In addition, common structures such as the truncus arteriosus or the aortic arches revealed malformations or appeared retarded. Interestingly, ventricle size in XMAN1 morphants was significantly increased. In contrast to WT hearts, ventricle volume during diastole was on average 1.5 fold higher. During systole, ventricle size was even 1.9 fold increased (Fig. 7C). The aberrant ventricles consequently displayed a decreased amplitude (=difference between diastolic and systolic volume). Compared to WT ventricles, amplitude of enlarged ventricles was reduced by approx. 47% (Fig. 7D). As a result, videos of the beating hearts (Movies S1 and S2) revealed, that ventricles appeared permanently filled with blood (also see Fig. 7A, c). Contrary to that, embryos injected with either MO-CO or MO-5mm-XMAN1 developed normal hearts (Fig. 7B, 100% and 94%, respectively; also see Movie S3). Additionally, heart cycles of XMAN1 morphants revealed severe cardiac arrhythmias (Fig. 7E–H). Contractions of the ventricle showed irregular durations and/or remained significantly longer during diastole (Fig. 7E, Morphant 1 and 2, respectively; also see Movies S1 and S2). Quantification of these results showed, that ventricles remained approx. 3.9 fold longer in diastole (Fig. 7F). As a consequence, the entire heart cycle was prolonged (on average 1.7 fold longer; Fig. 7G). Hearts of XMAN1 morphants thus showed a significantly reduced frequency. In contrast to WT hearts, frequency was approx. 40% decreased (Fig. 7H). Comparisons with control embryos showed that WT hearts displayed a periodic alteration between systolic and diastolic phases (Fig. 7E, Control, WT/MO-CO; also see Movie S3). Because our results indicated an impaired development of cardiac muscle, we decided to analyse the ultrastructure of cardiac muscle cells by electron microscopy. Cardiac muscle cells of XMAN1 morphants showed severe cellular disorganisations (Fig. 8B and B ). Muscle filaments were not organized into sarcomeres and appeared to be fractionized. These filament fragments were randomly distributed within the cell (arrowheads). Nuclei of cardiac muscle cells were surrounded by a continuous double membrane but showed numerous lobulations in the nuclear envelope (Fig. 8B). As expected, cardiac muscle cells of WT hearts showed sarcomeres characterized by the common sarcomeric organization and nuclei displaying a normal morphology (Fig. 8A and A ). Altered gene expression by knockdown of XMAN1 Fig. 5. Knockdown of XMAN1 causes aberrant muscle formations. (A) Stereoscopic microscope images of three days old embryos injected with 50 ng of control morpholinos (MO-CO or MO-5mm-XMAN1, a–c) or MO-XMAN1 (a –c ). The injected side of XMAN1 morphants failed to develop somites or displayed an aberrant somite formation (b and c , arrows). Bars: 1 mm. (B) Quantification of the somite phenotypes. The majority of XMAN1 morphants showed a strong (red) or weak (blue) phenotype. Most of the control morphants bilaterally developed normal somites (WT, green). n, number of embryos. (C) Whole mount immunostaining of embryos labelled with antibodies against desmin. WT embryos showed the common staining of somites (a and b, arrowheads). A similar staining within the injected side of XMAN1 morphants was not detected, indicating the absence of proper somite formation (b , arrow). Bars: 1 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

that formed muscle tissues underwent a severe disorganization (Fig. 6B, b). Muscle cells showed, that the amount of sarcomeres was considerably decreased (arrow). Most cells appeared isolated without contacts to neighbouring cells. In comparison, muscle cells of the uninjected side were tightly arranged and shared numerous sarcomeres (Fig. 6B, a). Sagittal sections revealed that muscle cells of the injected side predominantly harboured disordered sarcomeres and several sarcomere fragments (Fig. S1, b). Knockdown of XMAN1 causes cardiac defects Heart development was also affected by knockdown of XMAN1 (Fig. 7). Hearts of 6 days old XMAN1 morphants showed an altered positioning of ventricle and atria (Fig. 7B, 95%; Fig. 7A compare

Since knockdown of XMAN1 showed dramatic disorders during organogenesis, we analysed whether alterations in XMAN1 levels would affect transcription of specific genes. We therefore performed RT-PCR analysis on total RNA isolated from different embryo stages and analysed the expression levels of genes involved in tissue differentiation and organ development (Fig. 9). RT-PCR analysis of genes specific for eye development revealed that in XMAN1 morphants pax6 (paired box transcription factor 6) showed a slightly decreased expression level two days after fertilization (Fig. 9, upper panel, stage 37/38). Analysis of transcription factor crx (cone-rod homeobox) revealed a premature expression during midblastula transition (Fig. 9, second panel, MBT). At the same time, expression of otx2 (orthodenticle homeobox 2), a head- and eye-specific transcription factor, exposed an additional amplificate with a length of ∼580 bp (Fig. 9, third panel). Normally, examined otx2 amplificates appeared at ∼360 bp. Sequencing of this additional amplificate revealed an insertion, flanked by the known 3 and 5 -sequences of otx2, indicating an unspliced variant of this gene. RT-PCR analysis of muscle-specific genes, demonstrated that myocardin (transcription coactivator of the serum response factor) and myoD (myogenic factor 3) revealed significantly decreased expression levels at stage 37/38 (Fig. 9, fourth and fifth panel). For control, we analysed the expression of actin in uninjected control

286

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

Fig. 6. Morphological analysis of somites in XMAN1 morphants. (A) Methylene blue staining of sectioned tails of control morphants (a) and XMAN1 morphants (b). The morpholino-injected side only displayed fragments of muscle structures (b, arrow). Bars: 250 ␮m. (B) Electron micrographs of skeletal muscle cells. Muscle cells of XMAN1 morphants showed a considerably reduced amount of sarcomeres (b, arrow). N, nucleus; S, sarcomeres. Bars: 5 ␮m and 1 ␮m (insets). Also see Fig. S1.

embryos and in XMAN1 morphants (Fig. 9, lower panel). During Xenopus development, actin is continuously expressed at constant expression levels. Xemerin is able to partially rescue the eye, muscle and heart phenotype As previously reported, MAN1 and emerin share overlapping functions (Liu et al., 2003). It has also been described, that emerin plays a role during muscle differentiation (Huber et al., 2009) Thereby it was speculated that emerin might be able to compensate the loss of MAN1. In order to proof this hypothesis, we injected embryos at the two-cell-stage using 50 ng of MO-XMAN1 and 40 ng of Xemerin1-mRNA. The ectopic expression of Xemerin was confirmed by immunoblots (Fig. 10, upper panel). At stage 5, Xemerin was detected and expression persisted during embryogenesis (lanes 3–8). As an internal control, we incubated the nitrocellulose with antibodies specific for tubulin. Tubulin was observed in each stage with comparable amounts (Fig. 10, lower panel). The expected localization of ectopically expressed Xemerin in the nuclear envelope was verified by immunofluorescence on squash preparations of two days old embryos using Xemerinantibodies. A typical prominent staining of the nuclear periphery was observed (data not shown). As the next step, we investigated the phenotype of these injected embryos in relation to eye, somite and heart formation.

Coinjection of Xemerin1-mRNA partially rescues the eye phenotype Whereas knockdown of MAN1 revealed defects during eye formation, the majority (74%, Fig. 11B) of embryos injected with MO-XMAN1 and Xemerin1-mRNA developed eyes at both sides (Fig. 11A, a–c, arrows). As displayed in Fig. 11B, some embryos showed two WT eyes (18%) but most embryos revealed smaller eyes at the injected side (56%). Only a small proportion of embryos retained the eye-less phenotype (26%). Histological sections of rescue morphants displayed a formed eye at the injected side (Fig. 12A, arrow) and the morphological structure appeared normal as seen in WT eyes (arrowhead). Note, that the dermal organization of the injected side appeared restored in contrast to XMAN1 morphants (compare Figs. 12A with 4A, b). Electron microscopy on both eyes revealed the common retinal organization (Fig. 12B, a and b). Xemerin1 is partially able to rescue muscle formation In addition to the rescued eye development, we analysed a formation of muscle structures in our rescue morphants. Contrary to XMAN1 morphants, we observed more embryos (37%) displaying somites within both sides (Fig. 13A, a–c, arrows and B). However most of these embryos (26%) showed a weak phenotype, characterized by linear-shaped and narrowed somites, indicating that somite formation was not completely rescued. Nevertheless, 2/3 of our rescue morphants still failed to form somites.

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

287

Fig. 7. Knockdown of XMAN1 causes cardiac defects. (A) Stereoscopic microscope images of hearts in 6 days old larvae. WT larvae (a) and control morphants injected with 50 ng MO-CO (b) showed the common cardiac morphology. Hearts of XMAN1 morphants displayed larger ventricles combined with an altered morphology (c). Insets represent annotated heart structures. A, atria; TA, truncus arteriosus; V, ventricle. Bars: 500 ␮m and 200 ␮m (insets). (B) Quantification of the heart phenotype. Control embryos predominantly showed WT hearts (green) whereas the majority of XMAN1 morphants exhibited aberrant hearts (red). n, number of embryos. (C-H) Cardiac arrhythmias in XMAN1 morphants analysed by video-based quantification of different heart features. Each column represents the mean dependent on the number of analysed embryos (n). Numbers at the base of certain columns represent related data. Bars indicate s.e.m. Differences were statistically significant as calculated by Student’s t-test relative to WT control embryos: *p > 0.05, **p < 0.05. Also see Movies S1–S3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Whole mount immunostainings on rescue morphants confirmed that somite formation was partially rescued by means of a specific staining for desmin within the injected side (Fig. 13C, b , arrow). In order to verify the morphology of the somites, we analysed histological sections of our rescue embryos. We thereby confirmed that muscle structures were partially formed but still appeared fragmented within the injected side (Fig. 14A, arrow). Nevertheless, electron microscopy on these somites displayed muscle cells undistinguishable from WT cells. (compare Fig. 14B, a and b).

Ectopic expression of Xemerin rescues the heart phenotype Because our results suppose that emerin ensures the formation of skeletal muscle tissues, it is tempting to speculate that emerin is able to compensate the loss of MAN1 during heart development. Following this hypothesis, we analysed the hearts of our rescue morphants (Fig. 15). The majority (82%, Fig. 15B) of these embryos developed normal hearts undistinguishable from WT embryos (compare Figs. 7A a, b with 15A). Since ventricle size was slightly increased in diastole (1.4 fold larger volume), it appeared unaffected during systole. As a consequence to the increased

288

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

Fig. 8. Altered organization of cardiac muscle cells visualized by electron microscopy. (A) Electron micrograph of WT muscle cells. (A ) Higher magnification, showing sarcomeres. (B) XMAN1 morphants display an aberrant organization of cardiac muscle cells. Note that the nuclear envelope displays several lobulations. Sarcomeres appear as fragments (arrowheads). (B ) Higher magnification, showing sarcomere fragments (arrowheads). ID, intercalated disc; N, nucleus; S, sarcomeres. Bars: 5 ␮m (A, B) and 1 ␮m (A , B ).

diastolic volume, ventricles revealed that amplitude was 1.6 fold higher (data not shown). Video analysis of beating hearts revealed normal heart cycles characterized by periodic alterations between systolic and diastolic phases (Fig. 15D, rescue morphant 1 and 2; also see Movies S4 and S5). Since rescue morphants showed on average a slightly prolonged duration during diastole (compare Figs. 7F with 15C), most hearts featured usual diastolic times between 100 and 130 ms (WT hearts: 80–140 ms). Accordingly, the entire heart cycle duration was almost equivalent to that of WT hearts. Following this, frequency of ventricle-contraction was also unchanged in rescue morphants (data not shown). Analysis of the ultrastructure of cardiac muscle cells in our rescue morphants displayed a normal morphology (Fig. S2, A) and well-organized sarcomeres as seen from WT hearts (Fig. S2, B).

Discussion LEM-domain proteins are of special interest, because mutations in related genes lead to tissue-specific diseases, called envelopathies (for review see Worman et al., 2010). LEM proteins are supposed to take part in chromatin organization, maintenance of nuclear architecture and gene regulation (for review see Wagner and Krohne, 2007). Nevertheless, until now a number of questions are still open concerning the physiological roles of LEM proteins in pathology of envelopathies. This report shows that the nuclear envelope protein MAN1 is essential for proper formation of eye- and muscle-tissues during early Xenopus laevis development. Furthermore, we demonstrate that ectopic expression of emerin rescues the loss of MAN1. Our hypothesis is that LEM proteins are key elements during signalling

and chromatin organization enabling accurate gene expression of tissue-specific genes. XMAN1 is essential for organogenesis It has been postulated that XMAN1 has a neuralizing activity and is predominantly expressed in ectoderm and neuroectoderm during tailbud stage (Osada et al., 2003). Our knockdown-experiments strengthen that XMAN1 is essential for proper eye formation. Previous studies, by the group of Osada identified XMAN1 as antagonist of BMP signalling by interacting with Smad proteins. Similar interactions have been shown in Drosophila (Wagner et al., 2010). Inhibition of BMP signalling is known to lead to neural induction during Xenopus development. Thereby the previously reported eyeless phenotypes (Osada et al., 2003) and our results appear to be consistent with an elevated BMP signalling by loss of XMAN1. Interestingly, the injected side of our XMAN1 morphants appeared less pigmented and showed pigmented aggregations posterior to the eye. These observations may indicate a failure in emigration of neural crest cells due to an impaired neuralizing activity of XMAN1. However, by analysing the ultrastructure of the retina, we were further able to show for the first time that XMAN1 is required for photoreceptor differentiation. Knockdown of XMAN1 revealed retinal disorders that were also observed in human retinal degenerative diseases, such as autosomal dominant cone-rod-dystrophy (CORD2) or retinitis pigmentosa (Freund et al., 1997). These diseases are described by degeneration or complete loss of cone and/or rod photoreceptors and disorders within the pigment epithelium (Bird, 1995). Because XMAN1 morphants basically failed to develop eyes, this observed phenotype is probably not due to degeneration as seen in human, but more due to a general defect in photoreceptor development. Nevertheless instead of a complete loss of retinal

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

Fig. 9. RT-PCR analysis on different developmental stages related to the expression of muscle- and heart-specific genes myocardin and myoD, head- and eye-specific genes pax6, crx and otx2. Expression of actin was analysed as a control. Synthesis of cDNA in absence of RT-polymerase served as an internal negative control. Ctrl, uninjected wild-type embryos; +MO, embryos injected at the one-cell-stage using 100 ng of MO-XMAN1; Stg: stage.

Fig. 10. Expression of Xemerin1 in rescue morphants analysed by immunoblot. Total protein extracts of 5 unfertilized eggs (lane 1), activated eggs (lane 2), embryos of different stages (lanes 3–8), 500 ng of recombinant His-tagged Xermerin1 lacking the transmembrane domain (Xemerin1TM-His, lane 9) and 5 × 106 nuclear envelopes from A6 nuclei (lane 10) were separated by SDS-PAGE and transferred to nitrocellulose. After incubation with antibodies specific for Xemerin, protein bands with a Mr of 24,000 were detected at stages 6–41 (upper panel), indicating the synthesis of Xemerin1. As an internal control, nitrocellulose was reprobed with antitubulin. Tubulin was detected in each stage with comparable amounts (lower panel). Molecular masses of reference proteins are indicated in kDa. Stg: stage.

289

Fig. 11. Xemerin is able to partially rescue the eye-less phenotype. (A) Stereoscopic microscope images of rescue morphants three days after injection with MO-XMAN1 and Xemerin1-mRNA. Note, that eye development was rescued at the injected side (b and c, arrows). Bars: 1 mm. (B) Quantification of related eye phenotypes. Rescue morphants successfully developed two wild-type (green) or smaller eyes (blue). Only some embryos failed to develop an eye within the injected side (red). n, number of embryos. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

tissues, some embryos displayed smaller eyes and other retinal layers, e.g. the ganglion cell layer, also appeared disorganized. Because we observed by whole mount immunostaining, that XMAN1 is expressed in somites and XMAN1 transcripts can be found in heart regions during later stages (stages 37–43, data not shown), it is tempting to speculate that XMAN1 has additional roles during muscle formation and differentiation. Accordingly to these data, we were able to show by knockdown-experiments, that XMAN1 is required for proper formation of skeletal as well as cardiac muscle tissues. Other muscle tissues appeared unaffected, indicating that the reported effects are specific for striated muscles. Knockdown of XMAN1 produced embryos showing aberrant heart morphologies in combination with cardiac conduction defects. Similar phenotypes have been reported in humans suffering from dilatative cardiomyopathy, DCM (Taylor et al., 2006). Like our XMAN1 morphants, patients with DCM display ventricular dilatation, cardiac arrhythmias and impaired systolic functions characterized by a decreased ejection fraction. In principle, DCM is caused by mutations in genes encoding for cytoskeletal and sarcomeric proteins. Nevertheless, it has been reported that DCM appears as secondary phenotype of genetic diseases affecting skeletal muscles, such as EDMD or Duchenne muscular dystrophy (Finsterer and Stollberger, 2000). Defects in genes encoding for emerin and lamins thus are able to lead to a DCM accompanied by cardiac conduction defects (Fatkin et al., 1999).

290

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

Fig. 12. Morphological analysis of the rescued eye phenotype. (A) Head sections of rescue morphants were stained with methylene blue. Both eyes generally showed a normal morphology. Bar: 250 ␮m. (B) Electron microscopy of the eye at the injected side (b) revealed a retinal organization undistinguishable from WT eyes (a). C, cone; PE, pigment epithelium; R, rod. Bars: 5 ␮m and 2 ␮m (insets).

Fig. 13. Xemerin1 is partially able to rescue the somite phenotype. (A) Stereoscopic microscope images of three days old embryos injected with MO-XMAN1 and Xemerin1mRNA. Somite formation was partially rescued (b and c, arrows). Bars: 1 mm. (B) Quantification of the related phenotype. More than one third of embryos were partially able to form somites in both sides (green and blue). However, the majority of embryos still missed somites at the injected side (red). n, number of embryos. (C) Whole mount immunostaining for desmin on rescue morphants. The injected side showed a significant but more linear-shaped staining, indicating somites (b , arrow). Bars: 1 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

An involvement of MAN1 during heart development has been implicated in MAN1-null mice revealing failures during heart looping (Ishimura et al., 2008). Furthermore, that study describes a role for MAN1 during formation of left–right asymmetry. More interestingly, genes essential for left–right asymmetry are principally

regulated by signal pathways comprising Smad-proteins (Wright, 2001). Because XMAN1 has been described as antagonist of BMP signalling (Osada et al., 2003), it is tempting to speculate that it represents a key regulator involved in both heart looping and cardiac muscle formation by regulating Smad activity.

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

291

Fig. 14. Morphological analysis of formed somites in rescue morphants. (A) Methylene blue stained transverse section of the tail. The injected side comprised several intermitted muscle structures (arrow). Bar: 250 ␮m. (B) Electron micrographs of skeletal muscle cells. Cells of the injected side (b) showed a normal organization comparable to WT cells (a, uninjected side). N, nucleus; S, sarcomeres. Bars: 10 ␮m and 1 ␮m (insets).

Fig. 15. Xemerin1 is able to rescue the aberrant heart phenotype. (A) Stereoscopic microscope image of a 6 days old rescue morphant showing a normal heart morphology. The inset represents annotated heart structures. A, atria; TA, truncus arteriosus; V, ventricle. Bars: 500 ␮m and 200 ␮m (inset). (B) Quantification of the heart phenotypes. (C) Quantification of different heart phases. Each column represents the mean dependent on the number of analysed embryos (n). Bars indicate s.e.m. Differences were statistically significant as calculated by Student’s t-test relative to WT control embryos (see Fig. 7F): *p > 0.05, **p < 0.05. (D) Video-based analysis of heart cycles depicted as curve chart. Rescue morphants display normal heart cycles and an increased amplitude. Also see Fig. S2, Movies S4 and S5.

Interestingly, loss-of-function mutations in LEMD3 result in Osteopoikilosis and Buschke–Ollendorff syndrome, affecting bone and skin tissues (Hellemans et al., 2004). A reason for misregulated bone formation has been provided by the group of Hellemans, showing that related mutations involve a loss of the XMAN1 Cterminus, responsible for interactions with Smad proteins. Thereby disrupted XMAN1-Smad-interactions might lead to an elevated BMP signalling and increased bone formation. Because of the death of our XMAN1 morphants up to 10 days after fertilization, we were not able to confirm a misregulated bone formation in Xenopus. However, we were able to show that XMAN1 morphants also revealed skin aberrations at the injected side. Until now additional cardiac defects in the mentioned human diseases have not been reported, indicating that roles for MAN1 could be species-specific. Although XMAN1 morphants displayed perturbed formation of skeletal muscle tissues, an involvement of BMP signalling in

skeletal muscle formation has been barely discussed until now. Thereby it will be interesting to analyse an impact of BMP-signalling on skeletal muscle formation. Considering this, analysing the functional roles of other LEM proteins might give insight into signalling pathways during myogenesis. XMAN1 and Xemerin share overlapping functions during organogenesis Functional overlap between LEM proteins has been implicated for different cellular processes in various species (Huber et al., 2009; Liu et al., 2003). Our results strengthen the hypothesis, that roles of emerin are similar to those of XMAN1 during Xenopus organogenesis. By ectopic expression of Xemerin, we were able to rescue the reported phenotypes caused by knockdown of XMAN1. However,

292

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

our observations that some rescue morphants only displayed a partially restored eye or somite formation indicate, that the rescue is stringent but not complete. We presume that injected doses of Xemerin-mRNA appear not sufficient to promote a full rescue. Thereby, using higher doses might result in an increased frequency of wild-type embryos. Regarding muscle differentiation, it has been shown in C2C12 myoblasts that emerin is able to compensate functions of other LEM proteins (Huber et al., 2009). This is in consistence with our results, showing that premature expression of emerin is able to ensure somite formation. More interestingly, we demonstrate that emerin is able to induce proper formation of sarcomeres within both skeletal and cardiac muscle cells. As previously mentioned, roles of XMAN1 during eyedevelopment might arise from regulation of BMP signalling. However, no similar function has been discussed for emerin until now. Because emerin was able to partially rescue the eye-less phenotype, it needs to be clarified whether emerin is able to modulate BMP signalling or interact with Smad proteins. Nevertheless, since our results appear to contradict BMP signalling, it is important to discuss alternative signal pathways for emerin and MAN1. With this intention, BAF appears to be the most evident common factor. Indeed, BAF is known to bind LEM proteins (Furukawa, 1999) and eye-specific transcription factors, like Crx, Otx2 and Pax6 (Wang et al., 2002). It has been also reported that BAF binds to nuclear envelope protein Nemp1 (Mamada et al., 2009). Interestingly, altered expression levels of Nemp1 are also leading to defects in eye development. In fact, our preliminary results obtained by morpholino-mediated knockdown of BAF revealed similar phenotypes as seen in XMAN1 morphants (data not shown). Thereby it is tempting to speculate that XMAN1 acts as regulator at the NE and interactions with BAF might be essential for signalling during organogenesis.

XMAN1 affects gene regulation Because we observed misregulated formations of eyes and different muscle structures, we were interested whether reduced expression levels of XMAN1 affect expression of tissue-specific genes. Transcription of genes during Xenopus development is inactivated until midblastula transition (MBT) but translation of maternal stored mRNA is unaffected (Newport and Kirschner, 1982). Thereby, the observed effects on gene regulation appeared during MBT or later stages. Analysing the expression of eye-specific genes showed that several candidates of the photoreceptor-specific transcription factor network appeared misregulated (Pax6, crx and Otx2). Reduced expression levels of pax6 are consistent with our eye-less phenotype because former studies demonstrated that pax6 is a key regulator of eye development and retina organization (Rungger-Braendle et al., 2010). Interestingly, instead of decreased expression levels, analysis of crx and otx2 revealed an altered gene expression. Whereas crx showed a premature expression at MBT in our XMAN1 morphants, otx2 exhibited at the same time an additional amplificate. We presume that this amplificate represents an unspliced variant of otx2, indicating that loss of XMAN1 might lead to altered splicing. Both genes are known to be essential for eye development. Crx for example is a nuclear transcription factor essential for photoreceptor maturation in rods and cones (Chen et al., 1997). Conversely, Otx2 appears to be critical for general eye development because heterozygous mouse mutants showed an abnormal eye formation (Matsuo et al., 1995). Interestingly, it has been reported in Xenopus, that otx2 acts upstream of eye-specific genes like pax6 in the gene cascade (Danno et al., 2008). Here we report an altered expression of eye-specific genes due to the knockdown of XMAN1. Thereby our

data suggest that XMAN1-mediated regulation of otx2 may affect expression of downstream genes, like pax6. Since previous studies have strengthened the role of myocardin as the major activator of smooth and cardiac muscle reporter genes (Wang et al., 2003), our XMAN1 heart phenotypes appear pursuant to the observed reduced expression levels of myocardin. It has been shown, that myocardin is specific for differentiation of smooth and cardiac muscles but does not activate genes specific for skeletal muscles, such as myoD (Small et al., 2005). Nevertheless, myoD also revealed decreased expression levels in our XMAN1 morphants, thus indicating that XMAN1 regulates expression of myoD in a myocardin-independent manner. Indeed, previous studies reported that downregulation of Lmo7 in C2C12 myoblasts exhibited expression of myf5 or myoD (Dedeic et al., 2011). Interactions between XMAN1 and Lmo7 though have not been described until now. Emerin, however, is able to interact with Lmo7 and inhibits its activity by regulating its nuclear export (Holaska et al., 2006). Following this, ectopic expression of Xemerin in our XMAN1 morphants potentially lead to an inhibition of Lmo7, thereby restoring myoD-expression and leading to the observed rescue of somite formation. As already mentioned, interactions between Xemerin and Smad proteins have not been reported. Interestingly, Xemerin was able to rescue eye formation, maybe in a Smad-independent manner. This hypothesis appears consistent because it has been shown that pax6 expression requires suppression of Wnt/␤-catenin signalling and even elimination of ␤-catenin expression resulted in ectopic lens formation (Smith et al., 2005). Interestingly, Xemerin is capable to bind ␤-catenin and thereby inhibits its activity in the same way like Lmo7 (Markiewicz et al., 2006). We presume that eyeless phenotypes appeared according to a disrupted BMP signalling but rescue of the eye phenotype happened due to a suppressed Wnt/␤-catenin signalling mediated by emerin. Interestingly, preliminary results, obtained from BAF knockdown experiments in our laboratory showed similar phenotypes as seen in our XMAN1 morphants. These XMAN1 morphants showed an altered expression of eye-specific transcription factors, like Otx2, Crx and Pax6. Because BAF is known to bind these transcription factors (Wang et al., 2002), it is most likely that BAF might be essential during signalling downstream to XMAN1. This strongly indicates that phenotype rescue can be alternatively induced by interactions between Xemerin and BAF. In conclusion regulatory interactions between MAN1 and other factors appear complex, our results clearly demonstrate that MAN1 may act as a scaffold protein at the NE regulating upstream signalling of the reported tissue-specific genes. We further report for the first time, that LEM proteins MAN1 and emerin might have fundamental roles during formation of multiple tissues and organs. Because functions of emerin appear to overlap with roles of MAN1, further studies have to investigate whether phenotype rescue happens due to regulation of different pathways or due to interactions of the LEM-domain with common factors like BAF. We postulate that MAN1, like emerin, may form regulatory complexes at the NE either directly with specific transcription factors or indirectly via BAF, enabling correct expression of tissue-specific genes. Interestingly our reported phenotypes could be correlated to the pathology of popular human diseases. Thereby this study demonstrates that Xenopus represent an excellent model organism for studying the impact of nuclear envelope proteins on envelopathies. Acknowledgements We thank Martin Gareiss and Georg Krohne (University of Wuerzburg, Division of Electron Microscopy) for preparation of the XMAN1 antibodies. We are also grateful to Christian Stigloher and Georg Krohne (same institute) as well as Elisabeth

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

Rungger-Braendle for helpful discussion and critical reading of the manuscript. This study received financial support from to the Bavarian Elite Funding Programme. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejcb.2013.10.008. References Benavente, R., Krohne, G., Franke, W.W., 1985. Cell type-specific expression of nuclear lamina proteins during development of Xenopus laevis. Cell 41, 177–190. Bione, S., Maestrini, E., Rivella, S., Mancini, M., Regis, S., Romeo, G., Toniolo, D., 1994. Identification of a novel X-linked gene responsible for Emery–Dreifuss muscular-dystrophy. Nat. Genet. 8, 323–327. Bird, A.C., 1995. Retinal photoreceptor dystrophies LL-Edward-Jackson-Memorial-Lecture. Am. J. Ophthalmol. 119, 543–562. Birney, E., Kumar, S., Krainer, A.R., 1993. Analysis of the RNA-recognition motif and RS and RGG domains – conservation in metazoan pre-messenger-RNA splicing factors. Nucleic Acids Res. 21, 5803–5816. Bonne, G., Di Barletta, M.R., Varnous, S., Becane, H.M., Hammouda, E.H., Merlini, L., Muntoni, F., Greenberg, C.R., Gary, F., Urtizberea, J.A., Duboc, D., Fardeau, M., Toniolo, D., Schwartz, K., 1999. Mutations in the gene encoding lamin A/C cause autosomal dominant Emery–Dreifuss muscular dystrophy. Nat. Genet. 21, 285–288. Brachner, A., Reipert, S., Foisner, R., Gotzmann, J., 2005. LEM2 is a novel MAN1-related inner nuclear membrane protein associated with A-type lamins. J. Cell Sci. 118, 5797–5810. Chen, S.M., Wang, Q.L., Nie, Z.Q., Sun, H., Lennon, G., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Zack, D.J., 1997. Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. Neuron 19, 1017–1030. Christensen, M.E., Moloo, J., Swischuk, J.L., Schelling, M.E., 1986. Characterization of the nucleolar protein, B-36, using monoclonal antibodies. Exp. Cell Res. 166, 77–93. Danno, H., Michiue, T., Hitachi, K., Yukita, A., Ishiura, S., Asashima, M., 2008. Molecular links among the causative genes for ocular malformation, Otx2 and Sox2 coregulate Rax expression. Proc. Natl. Acad. Sci. U. S. A. 105, 5408–5413. Debus, E., Weber, K., Osborn, M., 1983. Monoclonal antibodies to desmin, the muscle-specific intermediate filament proteins. EMBO J. 2, 2305–2312. Dechat, T., Pfleghaar, K., Sengupta, K., Shimi, T., Shumaker, D.K., Solimando, L., Goldman, R.D., 2008. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev. 22, 832–853. Dedeic, Z., Cetera, M., Cohen, T.V., Holaska, J.M., 2011. Emerin inhibits Lmo7 binding to the Pax3 and MyoD promoters and expression of myoblast proliferation genes. J. Cell Sci. 124, 1691–1702. Emery, A.E.H., 1989. Emery–Dreifuss syndrome. J. Med. Genet. 26, 637–641. Emery, A.E.H., 2000. Emery–Dreifuss muscular dystrophy – a 40 year retrospective. Neuromuscul. Disord. 10, 228–232. Fatkin, D., MacRae, C., Sasaki, T., Wolff, M.R., Porcu, M., Frenneaux, M., Atherton, J., Vidaillet, H.J., Spudich, S., De Girolami, U., Seidman, J.G., Seidman, C.E., Muntoni, F., Muehle, G., Johnson, W., McDonough, B., 1999. Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease. N. Engl. J. Med. 341, 1715–1724. Finsterer, J., Stollberger, C., 2000. Cardiac involvement in primary myopathies. Cardiology 94, 1–11. Freund, C.L., GregoryEvans, C.Y., Furukawa, T., Papaioannou, M., Looser, J., Ploder, L., Bellingham, J., Ng, D., Herbrick, J.A.S., Duncan, A., Scherer, S.W., Tsui, L.C., LoutradisAnagnostou, A., Jacobson, S.G., Cepko, C.L., Bhattacharya, S.S., McInnes, R.R., 1997. Cone-rod dystrophy due to mutations in a novel photoreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor. Cell 91, 543–553. Furukawa, K., 1999. LAP2 binding protein 1 (L2BP1/BAF) is a candidate mediator of LAP2-chromatin interaction. J. Cell Sci. 112, 2485–2492. Gareiss, M., Eberhardt, K., Kruger, E., Kandert, S., Bohm, C., Zentgraf, H., Muller, C.R., Dabauvalle, M.C., 2005. Emerin expression in early development of Xenopus laevis. Eur. J. Cell Biol. 84, 295–309. Goldberg, M., Lu, H.H., Stuurman, N., Ashery-Padan, R., Weiss, A.M., Yu, J., Bhattacharyya, D., Fisher, P.A., Gruenbaum, Y., Wolfner, M.F., 1998. Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA. Mol. Cell. Biol. 18, 4315–4323. Gruenbaum, Y., Lee, K.K., Liu, J., Cohen, M., Wilson, K.L., 2002. The expression, lamin-dependent localization and RNAi depletion phenotype for emerin in C. elegans. J. Cell Sci. 115, 923–929. Gruenbaum, Y., Margalit, A., Goldman, R.D., Shumaker, D.K., Wilson, K.L., 2005. The nuclear lamina comes of age. Nat. Rev. Mol. Cell Biol. 6, 21–31. Haraguchi, T., Holaska, J.M., Yamane, M., Koujin, T., Hashiguchi, N., Mori, C., Wilson, K.L., Hiraoka, Y., 2004. Emerin binding to Btf, a death-promoting transcriptional repressor, is disrupted by a missense mutation that causes Emery–Dreifuss muscular dystrophy. Eur. J. Biochem. 271, 1035–1045. Hellemans, J., Preobrazhenska, O., Willaert, A., Debeer, P., Verdonk, P.C.M., Costa, T., Janssens, K., Menten, B., Van Roy, N., Vermeulen, S.J.T., Savarirayan, R.,

293

Van Hul, W., Vanhoenacker, F., Huylebroeck, D., De Paepe, A., Naeyaert, J.M., Vandesompele, J., Speleman, F., Verschueren, K., Coucke, P.J., Mortier, G.R., 2004. Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke–Ollendorff syndrome and melorheostosis. Nat. Genet. 36, 1213–1218. Holaska, J.M., Lee, K.K., Kowalski, A.K., Wilson, K.L., 2003. Transcriptional repressor germ cell-less (GCL) and barrier to autointegration factor (BAF) compete for binding to emerin in vitro. J. Biol. Chem. 278, 6969–6975. Holaska, J.M., Rais-Bahrami, S., Wilson, K.L., 2006. Lmo7 is an emerin-binding protein that regulates the transcription of emerin and many other muscle-relevant genes. Hum. Mol. Genet. 15, 3459–3472. Huber, M.D., Guan, T., Gerace, L., 2009. Overlapping functions of nuclear envelope proteins NET25 (Lem2) and emerin in regulation of extracellular signal-regulated kinase signaling in myoblast differentiation. Mol. Cell. Biol. 29, 5718–5728. Ishimura, A., Chida, S., Osada, S.-I., 2008. Man1, an inner nuclear membrane protein, regulates left–right axis formation by controlling nodal signaling in a node-independent manner. Dev. Dyn. 237, 3565–3576. Klymkowsky, M.W., Hanken, J., 1991. Whole-mount staining of Xenopus and other vertebrates. Methods Cell Biol. 36, 419–441. Laguri, C., Gilquin, B., Wolff, N., Romi-Lebrun, R., Courchay, K., Callebaut, I., Worman, H.J., Zinn-Justin, S., 2001. Structural characterization of the LEM motif common to three human inner nuclear membrane proteins. Structure 9, 503–511. Lee, K.K., Gruenbaum, Y., Spann, P., Liu, J., Wilson, K.L., 2000. C. elegans nuclear envelope proteins emerin, MAN1, lamin, and nucleoporins reveal unique timing of nuclear envelope breakdown during mitosis. Mol. Biol. Cell 11, 3089–3099. Lin, F., Blake, D.L., Callebaut, I., Skerjanc, I.S., Holmer, L., McBurney, M.W., Paulin-Levasseur, M., Worman, H.J., 2000. MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin. J. Biol. Chem. 275, 4840–4847. Lin, F., Morrison, J.M., Wu, W., Worman, H.J., 2005. MAN1, an integral protein of the inner nuclear membrane, binds Smad2 and Smad3 and antagonizes transforming growth factor-beta signaling. Hum. Mol. Genet. 14, 437–445. Liu, J., Lee, K.K., Segura-Totten, M., Neufeld, E., Wilson, K.L., Gruenbaum, Y., 2003. MAN1 and emerin have overlapping function(s) essential for chromosome segregation and cell division in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A. 100, 4598–4603. Mamada, H., Takahashi, N., Taira, M., 2009. Involvement of an inner nuclear membrane protein, Nemp1, in Xenopus neural development through an interaction with the chromatin protein BAF. Dev. Biol. 327, 497–507. Mansharamani, M., Wilson, K.L., 2005. Direct binding of nuclear membrane protein MAN1 to emerin in vitro and two modes of binding to barrier-to-autointegration factor. J. Biol. Chem. 280, 13863–13870. Margalit, A., Neufeld, E., Feinstein, N., Wilson, K.L., Podbilewicz, B., Gruenbaum, Y., 2007. Barrier to autointegration factor blocks premature cell fusion and maintains adult muscle integrity in C. elegans. J. Cell Biol. 178, 661–673. Markiewicz, E., Tilgner, K., Barker, N., van de Wetering, M., Clevers, H., Dorobek, M., Hausmanowa-Petrusewicz, I., Ramaekers, F.C.S., Broers, J.L.V., Blankesteijn, W.M., Salpingidou, G., Wilson, R.G., Ellis, J.A., Hutchison, C.J., 2006. The inner nuclear membrane protein Emerin regulates beta-catenin activity by restricting its accumulation in the nucleus. EMBO J. 25, 3275–3285. Matsuo, I., Kuratani, S., Kimura, C., Takeda, N., Aizawa, S., 1995. Mouse Otx2 functions in the formation and patterning of rostral head. Genes Dev. 9, 2646–2658. Newport, J., Kirschner, M., 1982. A major developmental transition in early Xenopus embryos. 2. Control of the onset of transcription. Cell 30, 687–696. Nieuwkoop, P.D., Faber, J., 1975. Normal Table of Xenopus laevis (Daudin). North-Holland Publ. Comp., Amsterdam. Osada, S.I., Ohmori, S., Taira, M., 2003. XMAN1, an inner nuclear membrane protein, antagonizes BMP signaling by interacting with Smad1 in Xenopus embryos. Development 130, 1783–1794. Rungger-Braendle, E., Ripperger, J.A., Steiner, K., Conti, A., Stieger, A., Soltanieh, S., Rungger, D., 2010. Retinal patterning by Pax6-dependent cell adhesion molecules. Dev. Neurobiol. 70, 764–780. Schirmer, E.C., Foisner, R., 2007. Proteins that associate with lamins: many faces, many functions. Exp. Cell Res. 313, 2167–2179. Schirmer, E.C., Gerace, L., 2005. The nuclear membrane proteome: extending the envelope. Trends Biochem. Sci. 30, 551–558. Schoft, V.K., Beauvais, A.J., Lang, C., Gajewski, A., Prufert, K., Winkler, C., Akimenko, M.A., Paulin-Levasseur, M., Krohne, G., 2003. The lamina-associated polypeptide 2 (LAP2) isoforms beta, gamma and omega of zebrafish: developmental expression and behavior during the cell cycle. J. Cell Sci. 116, 2505–2517. Small, E.M., Warkman, A.S., Wang, D.Z., Sutherland, L.B., Olson, E.N., Krieg, P.A., 2005. Myocardin is sufficient and necessary for cardiac gene expression in Xenopus. Development 132, 987–997. Smith, A.N., Miller, L.A.D., Song, N., Taketo, M.M., Lang, R.A., 2005. The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm. Dev. Biol. 285, 477–489. Stewart, C.L., Roux, K.J., Burke, B., 2007. Blurring the boundary: the nuclear envelope extends its reach. Science 318, 1408–1412. Sullivan, T., Escalante-Alcalde, D., Bhatt, H., Anver, M., Bhat, N., Nagashima, K., Stewart, C.L., Burke, B., 1999. Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J. Cell Biol. 147, 913–919. Taylor, M.R.G., Carniel, E., Mestroni, L., 2006. Cardiomyopathy, familial dilated. Orphanet J. Rare Dis. 1, 27. Thomas, J.O., Kornberg, R.D., 1975. Octamer of histones in chromatin and free in solution. Proc. Natl. Acad. Sci. U. S. A. 72, 2626–2630.

294

M. Reil, M.-C. Dabauvalle / European Journal of Cell Biology 92 (2013) 280–294

Tilgner, K., Wojciechowicz, K., Jahoda, C., Hutchison, C., Markiewicz, E., 2009. Dynamic complexes of A-type lamins and emerin influence adipogenic capacity of the cell via nucleocytoplasmic distribution of beta-catenin. J. Cell Sci. 122, 401–413. Vaughan, A., Alvarez-Reyes, M., Bridger, J.M., Broers, J.L., Ramaekers, F.C., Wehnert, M., Morris, G.E., Whitfield, W.G.F., Hutchison, C.J., 2001. Both emerin and lamin C depend on lamin A for localization at the nuclear envelope. J. Cell Sci. 114, 2577–2590. Wagner, N., Kagermeier, B., Loserth, S., Krohne, G., 2006. The Drosophila melanogaster LEM-domain protein MAN1. Eur. J. Cell Biol. 85 (2), 91–105. Wagner, N., Krohne, G., 2007. LEM-domain proteins: new insights into lamin-interacting proteins. Int. Rev. Cytol.: Survey Cell Biol. 261, 1–46, 261. Wagner, N., Schmitt, J., Krohne, G., 2004. Two novel LEM-domain proteins are splice products of the annotated Drosophila melanogaster gene CG9424 (Bocksbeutel). Eur. J. Cell Biol. 82, 605–616. Wagner, N., Weyhersmueller, A., Blauth, A., Schuhmann, T., Heckmann, M., Krohne, G., Samakovlis, C., 2010. The Drosophila LEM-domain protein MAN1 antagonizes BMP signaling at the neuromuscular junction and the wing crossveins. Dev. Biol. 339, 1–13.

Wang, X.J., Xu, S.Q., Rivolta, C., Li, L.Y., Peng, G.H., Swain, P.K., Sung, C.H., Swaroop, A., Berson, E.L., Dryja, T.P., Chen, S.M., 2002. Barrier to autointegration factor interacts with the cone-rod homeobox and represses its transactivation function. J. Biol. Chem. 277, 43288–43300. Wang, Z.G., Wang, D.Z., Pipes, G.C.T., Olson, E.N., 2003. Myocardin is a master regulator of smooth muscle gene expression. Proc. Natl. Acad. Sci. U. S. A. 100, 7129–7134. Wilkinson, F.L., Holaska, J.M., Zhang, Z.Y., Sharma, A., Manilal, S., Holt, I., Stamm, S., Wilson, K.L., Morris, G.E., 2003. Emerin interacts in vitro with the splicing-associated factor, YT521-B. Eur. J. Biochem. 270, 2459–2466. Wolf, D.P., Hedrick, J.L., 1971. Molecular approach to fertilization. 1. Viability and artificial fertilization of Xenopus laevis gametes. Dev. Biol. 25, 348–359. Worman, H.J., Bonne, G., 2007. “Laminopathies”: a wide spectrum of human diseases. Exp. Cell Res. 313, 2121–2133. Worman, H.J., Ostlund, C., Wang, Y., 2010. Diseases of the nuclear envelope. Cold Spring Harb. Perspect. Biol. 2. Wright, C.V., 2001. Mechanisms of left–right asymmetry: what’s right and what’s left? Dev. Cell 1, 179–186.