Article |
Address correspondence to Donald G. Moerman, Dept. of Zoology, University of British Columbia, 6270 University Blvd., Vancouver, BC, Canada V6T 1Z4. Tel.: (604) 822-3365. Fax: (604) 822-2416. E-mail: moerman{at}zoology.ubc.ca
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Abstract |
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Key Words: actin cytoskeleton; spectrin; morphogenesis; epithelia; muscle
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Introduction |
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The spectrin-based membrane cytoskeleton was first characterized in erythrocytes and has subsequently been studied in great detail. In the erythrocyte, spectrin forms a submembrane cytoskeletal network that is important for maintaining the structural integrity of the membrane and maintaining cell shape (Lux and Palek, 1995). However, the role of the spectrin-based membrane cytoskeleton in nonerythrocytes has remained elusive. Studies in Drosophila and Caenorhabditis elegans are providing new information on the function of the spectrin cytoskeleton in nonerythrocytes. In Drosophila and C. elegans, three spectrin genes have been identified. One gene encodes an spectrin, and the other two genes encode ß spectrins. The
and one of the ß spectrins are highly similar to the nonerythroid spectrins (Dubreuil et al., 1987; Moorthy et al., 2000). However, the other ß spectrin, ßHeavy (ßH)* spectrin, is unusual in that it encodes a very large spectrin molecule
400 kD (Dubreuil et al., 1990; Thomas and Kiehart, 1994; McKeown et al., 1998) compared with the nonerythroid ß spectrin, which is
220 kD (Dubreuil et al., 1987). In Drosophila,
and ß spectrin are widely expressed in most tissues (Pesacreta et al., 1989). Conversely, the ßH isoform is restricted to certain tissues, such as the epithelia (Pesacreta et al., 1989; Thomas and Kiehart, 1994). Similarly, C. elegans ß spectrin is found in most tissues (Moorthy et al., 2000), whereas the C. elegans ßH spectrin is restricted to the epidermis (hypodermis), pharynx, and intestine (McKeown et al., 1998). In Drosophila epithelial tissues, two spectrin isoforms associate with different membrane domains: the (
ß)2 isoform localizes to the lateral and basal membrane, and the (
ßH)2 isoform localizes to the apical membrane (Dubreuil et al., 1997).
Examination of Drosophila spectrin mutants reveals several developmental defects, which implicate spectrin in the processes of cell growth, differentiation, and specification (de Cuevas et al., 1996; Lee et al., 1997a; Thomas et al., 1998). Examination of ß spectrin mutant animals in Drosophila and C. elegans implicate this molecule in body wall muscle function, axon pathfinding, synapse function, and the maintenance of protein localization at the membrane (Dubreuil et al., 2000; Hammarlund et al., 2000; Moorthy et al., 2000; Featherstone et al., 2001). ßH mutants have defects in cell morphological events including embryonic elongation of C. elegans embryos (McKeown et al., 1998) and epithelial morphogenesis in Drosophila oocyte development (Zarnescu and Thomas, 1999).
We have identified a mutation in the C. elegans spectrin gene, spc-1. C. elegans
spectrin localizes to cell membranes in all tissues examined during embryogenesis. Phenotypic characterization of the spc-1 mutant embryos reveals a defect in embryonic elongation and defects in body wall muscle differentiation. spc-1 mutants have a slow rate of elongation and fail to elongate beyond twofold in length. This defect in elongation results from the failure of the apical actin cytoskeleton within the hypodermis to organize properly. Also, the body wall muscle displays an abnormal arrangement of myofilaments, and the body wall muscle quadrants are twofold wider than normal muscle quadrants. This wider partitioning is mirrored by the underlying basement membrane and in the hypodermal body wall muscle attachment structures. These results indicate that the spectrin-based membrane cytoskeleton is required for the normal development of these two tissues in C. elegans.
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Results |
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To determine if ß spectrin is required for the localization of spectrin, the localization of
spectrin was examined in ß spectrin and ßH spectrinnull mutants, unc-70(s1639) and sma-1(ru18), respectively (McKeown et al., 1998; Hammarlund et al., 2000). In ß spectrin mutants,
spectrin immunofluorescence is not observed in any tissues except for bright immunofluorescence in the apical membrane region of the intestine and hypodermis (Fig. 2 B and Fig. 3 A). Faint immunofluorescence is also observed in the nerve ring (unpublished data). Interestingly, this indicates that ß spectrin is required for the localization of
spectrin in all tissues except the apical membrane in the hypodermis and intestine. Conversely, ßH spectrin was not required for the localization of
spectrin. In all tissues,
spectrin localized normally including the apical membrane region of the hypodermis and intestine (Fig. 3 B). To complement this analysis,
spectrin mutant embryos (see below) were stained with ß spectrin antisera (Moorthy et al. 2000). In
spectrin mutants, ß spectrin localized to the membrane; however, the staining was weak in all tissues except for the nervous system (Fig. 3 D). Surprisingly, this indicates that
spectrin is required for the stability but not the localization of ß spectrin at the membrane.
Loss of spectrin leads to a failure to complete embryonic elongation and lethality
In a mutagenesis screen designed to identify mutations in genes involved in body wall muscle development, an ethylnethanesulfanate-induced mutation in the C. elegans spectrin gene was isolated. This mutation, spc-1(ra409), was genetically mapped to the left arm of the X chromosome between two cloned genes, unc-2 and unc-20. Several approaches were used to determine that the spc-1(ra409) mutation is in the
spectrin gene. First, the spc-1(ra409) mutant phenotype was rescued by transformation rescue with a cosmid containing the full-length
spectrin gene (M01F12). Second, since this cosmid contained other genes, the spc-1(ra409) phenotype was rescued with a subclone of M01F12 containing only the
spectrin gene. Third, injection of double-stranded RNA (dsRNA) specific to the
spectrin coding region was found to phenocopy spc-1(ra409) mutants. Fourth, a transposon-induced allele of spc-1(ra417::Tc1) (Fig. 1 A) fails to complement spc-1(ra409). Finally, by sequencing the entire
spectrin gene in spc-1(ra409) homozygotes the molecular lesion was identified, which introduces a premature stop at amino acid position 2111 (Fig. 1 A). To determine if spc-1(ra409) is a null mutation, it was crossed into a strain carrying a deficiency that deletes the spc-1 locus (syDf1), and the heterozygous progeny were examined. spc-1(ra409)/syDf1 progeny exhibited the same phenotype (described below) as spc-1(ra409) homozygotes, suggesting that spc-1(ra409) is a null allele of the spc-1 locus.
To determine the role of spectrin in embryogenesis, the phenotype of homozygous spc-1(ra409) mutant animals was analyzed using light microscopy. spc-1(ra409) mutant embryos undergo embryogenesis without any obvious defects until the embryo begins to elongate. Elongation converts the embryo from a ball of cells (lima bean stage) into the elongated threefold vermiform shape. Embryonic elongation is composed of two phases: (a) extension from lima bean to twofold, which is thought to occur by cell changes in the hypodermis, and (b) progressing from two to threefold, which is thought to be driven by body wall muscle contraction (Chin-Sang and Chisholm, 2000). spc-1(ra409) mutant embryos undergo elongation at a slower rate than wild-type animals during the first phase of elongation and fail to elongate beyond twofold in length (Fig. 4). In wild-type animals, it takes 60 min to extend to twofold. Conversely, in spc-1 mutants it takes 180 min to extend to twofold. The resultant twofold embryos hatch but die soon after as small L1 larvae. Within the egg, spc-1(ra409) animals initiate body wall muscle contraction normally; however, their movement is not as vigorous as wild-type animals. Additionally, pharyngeal contraction is infrequent and irregular in spc-1(ra409) mutants. The pharynx in these mutant animals is much shorter than normal (Fig. 4, arrows). After hatching, spc-1(ra409) animals display muscle contraction, but their movement is uncoordinated.
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Elongation is required for correct morphogenesis of the body wall muscle
During the analysis of the hypodermal cytoskeleton, abnormalities in the distribution of the thin filaments were observed in the body wall muscle of spectrin mutant animals. This defect is not only associated with the thin filaments, since similar defects are seen when the thick filaments of
spectrin mutants are examined by immunofluorescence microscopy with a monoclonal antibody specific to body wall muscle myosin. Specifically, the muscle quadrants of these mutants appear twofold wider than normal (Fig. 6, B and E). In addition, the myofilaments are abnormally oriented to the longitudinal axis. Normally, the sarcomeres are arranged at an oblique angle (6°) to the longitudinal axis of the nematode (Fig. 6 A) (Waterston, 1988). In
spectrin mutants, this oblique striation is exaggerated to an almost 20° angle. This deflection of the sarcomeres is particularly prevalent in the anterior muscle cells (Fig. 6 B). Additionally, there is a large gap between the myofilament lattice in neighboring muscle cells within the same muscle quadrant that is not observed in muscle quadrants of wild-type animals (Fig. 5 B and Fig. 6 B). In wild-type animals, the myofilament lattice in neighboring cells is closely associated (Fig. 5 A and Fig. 6 A). In
spectrin mutant animals, the myofilament lattice is not tightly associated, and there is a 12 micron gap between the myofilament lattice in neighboring cells (Fig. 5 B and Fig. 6 B). This defect is not the result of the failure of the muscle cells to polarize, since the myofilaments are normally located against the basal membrane (unpublished data).
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spectrin, ßH spectrin, let-502, and mlc-4 mutants have the same body wall muscle phenotype, and they have a slower rate of elongation than normal (Fig. 4 D) (McKeown et al., 1998). To examine whether a slower embryonic elongation up to twofold influences muscle cell shape, muscle cell changes during elongation were monitored. Wild-type and mutant embryos at various stages of elongation were examined with ß spectrin and myosin immunofluorescence microscopy to visualize muscle cell shape changes. The cell shape changes that convert round body wall muscle cells into thin spindle-shaped cells were defective in the slow elongation mutants (Fig. 7). In these mutants, the muscle cells are square and have not lengthened (Fig. 7, B and C), but this process occurs normally in egl-19 embryos even though these mutants only elongate to twofold (Fig. 7 D). Although egl-19 mutants arrest elongation at twofold, these mutants elongate up to twofold at the same rate as wild-type embryos. These data indicate that the process of elongation from lima bean to twofold, not just the failure to elongate beyond, is critical for the normal development of the body wall muscle.
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Discussion |
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ßH spectrin has been reported to localize to the apical region of epithelial tissues in both C. elegans and Drosophila (Thomas, 2001) and therefore may be required for the apical localization of spectrin. However, examination of
spectrin localization in ßH spectrin (sma-1) mutants reveals a normal distribution of
spectrin, indicating that ßH spectrin is not required for
spectrin localization in C. elegans. This suggests that the mechanisms that localize
spectrin to the apical verses the basolateral membrane are distinct.
The localization pattern of the different spectrin isoforms and the examination of and ß spectrin localization in the reciprocal mutants suggests that the spectrin cytoskeleton is organized into two populations: an (
ß)2 spectrin that localizes to all tissues except the apical membrane in epithelia and an (
ßH)2 spectrin that localizes to the epithelial apical membrane. This model is further corroborated by the analysis of the double mutants (this study; Moorthy et al., 2000). Double mutants constructed with null alleles of ß and ßH spectrin resulted in a phenotype identical to that of the single loss of function allele of
spectrin.
The (ßH)2 spectrin cytoskeleton is essential for embryonic morphogenesis
Time-lapse video recording of spectrin (spc-1) mutants during embryogenesis indicates that these mutants have a slow rate of elongation resulting in L1 larvae that are half the size of wild-type L1 larvae. Circumferential contraction of the apically localized actin cytoskeleton in the hypodermis is the driving force behind the process of elongation during morphogenesis (Priess and Hirsh, 1986; Costa et al., 1997). Examination of this cytoskeleton in
spectrin mutants reveals that the actin filaments are discontinuous and disorganized. The same defect is observed in ßH spectrin mutants but is not observed in ß spectrin mutant animals. Also, ßH spectrin mutants have defects in elongation, whereas ß spectrin mutants do not. These data indicate that the (
ßH)2 spectrin cytoskeleton, and not (
ß)2, is essential for the proper organization and function of the apical actin cytoskeleton in the hypodermis and is a key component involved in epithelial morphogenesis. The importance of the spectrin cytoskeleton in epithelial morphogenic events has been documented recently in Drosophila. Zarnescu and Thomas (1999) have shown that ßH spectrin is required for normal epithelial morphogenesis in Drosophila follicle cells during oocyte development. Additionally, spectrin has been implicated in morphogenic events that occur during mouse neural tube formation (Sadler et al., 1986) and during gastrulation in sea urchin embryos (Wessel and Chen, 1993). These observations together with our data suggest that the spectrin cytoskeleton may function globally in coordinating the cytoskeleton within epithelial tissues during morphogenic events.
The process of elongation occurs in two steps. First, the hypodermal apical actin cytoskeleton is required to extend the embryo to twofold, and second, body wall muscle contraction induces hypodermal cell changes that extend the embryo to threefold (Chin-Sang and Chisholm, 2000). Unlike Pat mutants, the elongation mutants described here (spc-1, sma-1, let-502, and mlc-4) are capable of muscle contraction and have a slow rate of elongation from lima bean to twofold. Pat mutants elongate to twofold normally, but they fail to elongate beyond twofold because their lack of muscle contraction fails to induce changes in the hypodermis. spc-1, sma-1, let-502, and mlc-4 have defects in the hypodermal actin cytoskeleton and in extending to twofold. However, these mutants are capable of body wall muscle contraction. Presumably, the induction of hypodermal changes by body wall muscle contraction fails or is inefficient in these mutants because the completion of this induction event requires an intact hypodermal actin cytoskeleton, which is absent in these mutants.
Hypodermal-mediated contraction determines the shape of body wall muscle cells
Two lines of evidence indicate that the expression and function of spectrin in the hypodermis, as opposed to spectrin expression within body wall muscle, is critical for determining the shape of the developing muscle cells. First, unlike
spectrin mutants, the ß spectrin mutant, unc-70, does not have any defects in embryonic elongation or body wall muscle development. In ß spectrin mutants,
spectrin fails to localize to the muscle cell membrane. These observations imply that spectrin expression within muscle is not important for determining muscle cell shape. Since unc-70 mutants do not impede elongation, it appears that the presence of spectrin within the hypodermis is sufficient for embryonic elongation to occur. A second line of evidence implicating the hypodermis in influencing muscle cell shape comes from the study of genes regulating the apical actin cytoskeleton. The genes we examined that influence the apical cytoskeleton with the exception of spc-1, including let-502, sma-1, and mlc-4, are expressed in the hypodermis but not in muscle (Wissmann et al., 1997; McKeown et al., 1998; Shelton et al., 1999). Mutants in all three genes elongate to twofold more slowly than wild-type embryos, and all three have broader muscles than wild-type embryos.
Embryonic elongation leads to a change in shape of the body wall muscle in wild-type embryos. A slower rate of elongation in the phase leading to a twofold embryo leads to a muscle cell with greater surface area in contact with the underlying hypodermis. Why a slower elongation rate leads to this final muscle cell shape is not clear. However, the consequences, a twofold increase in basement membrane in contact with the basal face of the muscle and a wider hemidesmosomal complex within the hypodermis, are quite dramatic. This is perhaps the most striking phenotype of these mutants and is what first drew our attention to the spc-1 mutants.
During C. elegans embryogenesis, the myofilaments of muscle and the hypodermal hemidesmosomes assemble concurrently and have the same spatial distribution (Hresko et al., 1994). How the complementary patterning of hemidesmosomes and myofilament attachment sites is achieved is an intriguing problem in development. Does the pattern within muscle determine the pattern within the hypodermis, or is it the other way around? The hypodermis could direct the formation of the body wall muscle via the hemidesmosomal-like components that anchor the body wall muscle to the cuticle. Specifically, the hypodermal cytoskeletal rearrangements that are required for elongation could also be required to restrict hemidesmosomal placement. If these rearrangements are perturbed, this could lead to a wider distribution of the hemidesmosomes and result in wider muscle quadrants. Conversely, it is possible that the body wall muscle cells determine where the basement membrane and hypodermal hemidesmosomal-like structures are assembled. Laser ablation of body wall muscle precursors can lead to a gap in a muscle quadrant. Within this gap, no perlecan accumulates, and within the hypodermis adjacent to the gap, no hemidesmosomes accumulate (Moerman et al., 1996; Hresko et al., 1999). One interpretation of these observations is that muscle is essential for organizing hemidesmosomal complex assembly within the hypodermis. If muscle is acting as an inducer, wider muscle cells may therefore lead to a wider hemidesmosomal complex within the hypodermis.
Our data cannot distinguish between these two models. What our observations do underscore is that during embryogenesis there is a dynamic interaction between muscle and hypodermis to ensure that force transmission from muscle is distributed evenly across the apposing face of the hypodermis. We have shown that spectrin has a critical role in this interaction. Future studies using the power of C. elegans and the spectrin mutants may enable us to describe the function of the spectrin cytoskeleton during development in still greater detail.
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Materials and methods |
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spc-1(ra409) was isolated in a mutant screen designed to identify mutations in genes required for muscle development (KRN and DGM [unpublished data]). The spc-1(ra409) mutation was mapped to the X chromosome. To further localize spc-1, three factor mapping was conducted. unc-2 + lon-2/+ spc-1 + and dpy-3 +unc-20/ + spc-1 + animals were generated and allowed to self. 12/48 Unc-2 nonLon-2 and 6/51 Unc-20 nonDpy-3 recombinants were mutant for spc-1, placing spc-1 between unc-2 and unc-20. Additionally, spc-1(ra409) mutants failed to complement the deficiency syDf1.
Transgenic strains were generated as described by Mello and Fire (1995). N2 animals were injected with two different cocktails of DNA. The first cocktail consisted of 80 µg/ml of pRF4 (dominant rol-6 mutation; rol-6[su1006dm]) and 10 µg/ml of M01F12. The second cocktail consisted of 80 µg/ml of pRF4 and 20 µg/ml of DM#214 (subclone of M01F12 containing only the spectrin ORF).
Molecular biology
PCR was conducted as described by Barstead et al. (1991). DNA sequencing reactions were performed directly on PCR amplified genomic DNA and cDNA clones by using the BRL dsDNA Cycle Sequencing System as described by Rogalski et al. (1993) or by the Nucleic Acid/Protein Service unit at the University of British Columbia. Sequence alignments and comparisons were performed using BLAST (National Center for Biotechnology Information server [Altschul et al., 1997]) and ClustalW (Mac Vector). Exon/intron boundaries were determined by sequencing cDNAs yk205f3, yk44a4, (gifts from Y. Kohara, National Institute of Genetics, Mishima, Japan), cm9a10, DM#455, and PCR-amplified fragments from a cDNA library (a gift from R. Barstead, Oklahoma Medical Research Center, Oklahoma City, OK).
General molecular biological techniques were performed as described in Sambrook et al. (1989). The bacterial strains DH5 (BRL) and XL1-Blue (Stratagene) were used for subcloning and fusion protein expression. To generate the full-length
spectrin clone, a 4.7-kb Avr II SpeI fragment from the cosmid M01F12 (a gift from A. Coulson, Sanger Center, Hinxton, England) was subcloned into SpeI-digested pBluescript (Stratagene) to produce DM#213. Subsequently, a 9.1-kb SpeI fragment from the cosmid M01F12 was subcloned into the SpeI site of DM#213, producing a clone (DM#214) that contains the full-length C. elegans
spectrin gene including 2-kb upstream and 1-kb downstream.
To generate a fusion protein encoding part of the C. elegans spectrin gene, the complete third spectrin repeat plus part of the second and fourth spectrin repeat (amino acids 208449) encoded by exon 4 was cloned into pGEX 4T-1 to produce DM#215. Specifically, PCR was performed to amplify a 957 base pair region of the fourth exon from genomic DNA. Subsequently, the clone DM#215 was produced by digesting this PCR product with ApoI, and the fragment was subcloned into the EcoR1 site of the pGEX 4T-1 vector (Amersham Pharmacia Biotech).
The production of dsRNA and RNAi were conducted as described in Norman and Moerman (2000). The template used for RNA production was the EST clone yk205f3 and DM#455.
Antibody production
The GST spectrin fusion protein was produced and purified by the procedure described previously (Smith and Johnson, 1988). To generate polyclonal antisera, two New Zealand white rabbits were injected subcutaneously with purified fusion protein emulsified in Freund's complete adjuvant (
0.5 mg protein/rabbit). Rabbits were boosted by intramuscular injections at 46-wk intervals with fusion protein emulsified in Freund's incomplete adjuvant (
0.3 mg protein/rabbit), and blood samples were taken 1012 d after injection. Immune response was monitored by Western blotting of purified fusion proteins and worm extracts and by immunofluorescence staining of nematodes. Antibodies were affinity purified according to Miller and Shakes (1995). Antibodies were further purified with a bacterial-GST acetone powder that removed all GST reactivity.
Immunofluorescence and microscopy
Embryo fixation, antibody staining, and microscopy were performed as described in Norman and Moerman (2000). Fluorescence microscopy was conducted on a ZEISS Axiovert equipped with the Radiance 2000 confocal system (Bio-Rad Laboratories, Inc). A minimum of 10 animals was examined for each genotype. Animals were staged according to pharyngeal development. The antibodies used in this study were a mouse monoclonal antibody to myosin heavy chain A (DM5.6 [Miller et al., 1983]), a mouse monoclonal antibody to nematode intermediate filaments (MH4 [Francis and Waterston, 1991]), a rabbit polyclonal to nematode perlecan (GM1 [Moerman et al., 1996]), a rabbit polyclonal to nematode spectrin (AS1), and a rabbit polyclonal antibody to nematode ß spectrin (Moorthy et al., 2000). DM5.6, GM1, and the AS1 were diluted 1:50; MH4 was diluted 1:100; the ß spectrin antibody was diluted 1:500. The secondary antibodies used were FITC-labeled donkey antirabbit IgG F(ab')2 and Texas redlabeled donkey antimouse IgG F(ab')2 (Jackson ImmunoResearch Laboratories) and were diluted 1:200. For control, spc-1(RNAi) embryos, using yk205f3 and DM#455 for dsRNA production, were labeled with AS1 to test for specificity. Additionally, AS1 antiserum was incubated over night at 4°C with the spectrinGST fusion protein that was used to generate the AS1 antiserum and stain wild-type embryos. No immunofluorescence signal was detected in these experiments. Embryos for FITC-phalloidin staining were prepared as described by Costa et al. (1997). Differential interference contrast images were collected on a ZEISS Axiophot microscope equipped with differential interference contrast optics. The images were collected using a Dage-MTI CCD-100 digital camera and Scion image software. Adobe Photoshop 4.0® was used to present images. Time-lapse video recording was performed as described by McKeown et al. (1998) except embryos were imaged every 5 s.
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Footnotes |
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Acknowledgments |
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This work was supported by grants from the Canadian Institute for Health Research and the Heart and Stroke Foundation of Canada.
Submitted: 13 November 2001
Revised: 13 March 2002
Accepted: 21 March 2002
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References |
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