Structural Components of the Nonstriated Contractile Apparatuses in the Caenorhabditis elegans Gonadal Myoepithelial Sheath and Their Essential Roles for Ovulation

Abstract

Ovulation in the nematode Caenorhabditis elegans is regulated by complex signal transduction pathways and cell–cell interactions. Myoepithelial sheath cells of the proximal ovary are smooth muscle-like cells that provide contractile forces to push a mature oocyte into the spermatheca for fertilization. Although several genes that regulate sheath contraction have been characterized, basic components of the contractile apparatuses of the myoepithelial sheath have not been extensively studied. We identified major structural proteins of the contractile apparatuses of the myoepithelial sheath and characterized their nonstriated arrangement. Of interest, integrin and perlecan were found only at the dense bodies, whereas they localized to both dense bodies and M-lines in the striated body wall muscle. RNA interference of most of the myofibrillar components impaired ovulation in a soma-specific manner. Our results provide basic information that helps understanding the mechanism of sheath contraction during ovulation and establishing a new model to study morphogenesis of nonstriated muscle.

Introduction

Ovulation in the nematode Caenorhabditis elegans is a highly coordinated process involving communications among sperm, oocytes, and the somatic gonad. A C. elegans hermaphrodite has a pair of U-shaped gonadal arms (Fig. 1). The germline and oocytes are surrounded by 10 sheath cells (pairs 1–5), and ovulation requires a signal from sperm that induces ovarian contraction and oocyte maturation, which are then followed by dilation of the spermatheca and fertilization (Ward and Carrel, 1979; McCarter et al., 1997, 1999; Hubbard and Greenstein, 2000; Yamamoto et al., 2006). The proximal sheath cells (pairs 3–5; Fig. 1) are smooth muscle-like cells with distinct thin and thick filaments and designated as the myoepithelial sheath (Hirsh et al., 1976; Strome, 1986; Ardizzi and Epstein, 1987; Hall et al., 1999). Contraction of the myoepithelial sheath is periodically enhanced upon maturation of the most proximal oocytes (McCarter et al., 1997, 1999). Sheath contraction is negatively regulated by ceh-18 encoding a POU-class homeoprotein (Rose et al., 1997; Miller et al., 2003) and gap junctions that are formed between the myoepithelial sheath and maturing oocytes (Whitten and Miller, 2006). On the other hand, it is enhanced by major sperm protein (Miller et al., 2001; Kosinski et al., 2005) by means of the VAB-1 Eph receptor (Miller et al., 2003). In addition, NMR-1 NMDA receptor and UNC-43 Ca²⁺-calmodulin–dependent kinase II negatively regulate sperm-dependent sheath contraction (Corrigan et al., 2005), whereas ITR-1 inositol triphosphate receptor (Yin et al., 2004), VAV-1 Rho/Rac guanine nucleotide exchange factor (GEF), and RHO-1 small GTPase (Norman et al., 2005) enhance contraction. Probably, precise regulation of sheath contraction by a complex mechanism is required for tight coupling of contraction with oocyte maturation and spermathecal dilation. However, functional interactions among these signaling molecules are still poorly understood.

Fig. 1.

Schematic representation of the Caenorhabditis elegans hermaphroditic gonad. A hermaphrodite has two gonadal arms. The distal tip cells are located at the most distal part of the gonad. Five pairs of the sheath cells surround the germline and oocytes and are designated as pairs 1 to 5 (not all cells are shown). Pairs 3–5 are the myoepithelial sheath that has distinct thin and thick filaments and are responsible for pushing a mature oocyte into the spermatheca during ovulation. After fertilization, embryos begin development in the uterus. Note that sheath cells are shown only in the anterior gonadal arm and that the posterior gonadal arm also has the same set of sheath cells.

One of the critical signals for sheath contraction is increase in the intracellular Ca²⁺ level (Yin et al., 2004; Xu et al., 2005). Troponin and tropomyosin function as actin-linked Ca²⁺ sensors in the thin filaments of the myoepithelial sheath cells and are required for ovulation (Myers et al., 1996; Ono and Ono, 2004). Thus, these nonstriated muscle cells are atypical, as troponin is generally expressed in striated muscle. Instead, vertebrate smooth muscle uses a myosin-linked system to activate contraction upon elevation of Ca²⁺ (Marston, 1995). However, potential regulators of myosin activity including VAV-1 Rho/Rac GEF, RHO-1, LET-502 Rho-kinase, and MEL-11 myosin phosphatase regulatory subunit are also required for ovulation (Wissmann et al., 1999; Norman et al., 2005). A biochemical study on purified actin and myosin from C. elegans suggests that there are both actin- and myosin-linked regulatory systems for actomyosin interaction (Harris et al., 1977). In addition, recent studies in vertebrate smooth muscle have shown that regulators of actin polymerization are important for contraction (Tang and Tan, 2003; Tang et al., 2005). Therefore, actomyosin contractility in the myoepithelial sheath might also be regulated by multiple mechanisms.

The myoepithelial sheath cells express MYO-3 and UNC-54 myosin heavy chains (Ardizzi and Epstein, 1987), which are also expressed in striated body wall muscle (Miller et al., 1983). Ovulation is perturbed by disturbing functions of several structural proteins, including MUP-2 troponin T (Myers et al., 1996), PAT-10 troponin C (Ono and Ono, 2004), LEV-11 tropomyosin (Ono and Ono, 2004), PAT-3 β-integrin (Lee et al., 2001), talin (Cram et al., 2003), PAT-4 integrin-linked kinase (Xu et al., 2006), and UNC-112 Mig-2 (Xu et al., 2006). However, overall organization and molecular components of the contractile apparatuses of the myoepithelial sheath are not clearly understood. In this study, we identified and localized major components of the contractile apparatuses in these nonstriated muscle cells and showed that many of these proteins are required for proper ovulation.

Results

Expression Patterns of Cytoskeletal Proteins in the C. elegans Somatic Gonad

To identify and localize the components of the contractile apparatuses of the gonadal myoepithelial sheath, antibodies against well-characterized cytoskeletal proteins were used for immunofluorescent staining of the somatic gonad. We detected expression of major cytoskeletal proteins in the myoepithelial sheath, and, interestingly, some of them were also expressed in other parts of the somatic gonad (Fig. 2). As described previously by Strome (1986), visualization of actin filaments by tetramethylrhodamine-phalloidin revealed a nonstriated meshwork of the filaments in the sheath cells and filamentous staining in other gonadal cells including germline (Fig. 2H,Q). Immunostaining of actin by mouse monoclonal (our unpublished data) or rabbit polyclonal anti-actin antibodies (Fig. 2B,E,K,N) yielded indistinguishable patterns of actin filaments in the sheath cells. Therefore, we used either tetramethylrhodamine-phalloidin or anti-actin antibody to label actin filaments, depending on the optimal fixation method. Many antibodies were optimal with fixation with cold methanol, which was not compatible with staining with tetramethylrhodamine-phalloidin.

Fig. 2.

Localization of structural proteins in the somatic gonad. A,B,D,E,G,H,J,K,M,N,P,Q: Dissected hermaphroditic gonads were doubly stained for MYO-3 myosin heavy chain (A), UNC-15 paramyosin (D), UNC-89 (G), UNC-52 perlecan (J), DEB-1 vinculin (M), or PAT-3 β-integrin (P), and actin (B,E,H,K,N,Q). C,F,I,L,O,R: Merged images of actin (red), other proteins (green), and DNA (blue), are shown. Positions of the spermatheca (Sp), the myoepithelial sheath (MS), and the distal tip (DT) are indicated. Scale bar = 100 μm.

As reported previously (Ardizzi and Epstein, 1987), MYO-3 myosin heavy chain A (myoA; Fig. 2A–C) and UNC-15 paramyosin (Fig. 2D–F) were specifically expressed in the myoepithelial sheath. We previously demonstrated that PAT-10 troponin C and Ce-kettin, a large immunoglobulin-like repeat protein, were also specific in the myoepithelial sheath (Ono and Ono, 2004; Ono et al., 2006). We found that the monoclonal antibody MH42 against UNC-89, a large obscurin-like thick-filament-associated protein (Benian et al., 1996), specifically stained the proximal myoepithelial sheath (Fig. 2G–I). Although MH42 weakly stained the germline, RNAi of unc-89 specifically removed signals in the myoepithelial sheath but not in the germline (data not shown), suggesting that the germline staining was nonspecific. In contrast, components of the adhesion structures, UNC-52 perlecan, DEB-1 vinculin, and PAT-3 β-integrin, were present in all somatic gonadal cells from the distal tip to the spermatheca (Fig. 2J–R). In addition, we previously reported that Ce-tropomyosin was expressed in the proximal myoepithelial sheath and the spermatheca (Ono and Ono, 2004). All of these structural proteins, except for actin, were not significantly detected in the germline as observed at different focal planes (data not shown). As summarized in Figure 3 and Table 1, all these cytoskeletal proteins are the cytoskeletal components of the smooth muscle-like proximal myoepithelial sheath, and some proteins are limited in these cells, suggesting their specific roles in the regulation of sheath contraction. However, some are present in other somatic gonadal cells, suggesting that they are involved in multiple aspects of the gonadal activities.

Fig. 3.

Schematic representation of the distribution patterns of structural proteins in the somatic gonad. Results of this study and previous studies (Ardizzi and Epstein, 1987; Ono and Ono, 2004; Ono et al., 2006) are summarized.

TABLE 1. Fixation and Staining Conditions for Fluorescence Microscopy^a.

Figure	Panel	Protein	Fixationb	Primary antibodies/probe	Secondary antibodiesc	Primary antibodies	Zenon-IgG2ad
2 & 4	A-C	MYO-3	A	5-6	A488-GAM	NA	NA
		Actin		AAN01	Cy3-DAR
	D-F	UNC-15	A	5-23	A488-GAM	NA	NA
		Actin		AAN01	Cy3-DAR
	G-I	UNC-52	B	MH2	A488-GAM	NA	NA
		Actin		TRITC-phalloidin	-
	J-L	UNC-89	A	MH42	A488-GAM	NA	NA
		Actin		AAN01	Cy3-DAR
	M-O	DEB-1	C	MH24	A488-GAM	NA	NA
		Actin		AAN01	Cy3-DAR
	P-R	PAT-3	B	MH25	A488-GAM	NA	NA
		Actin		TRITC-phalloidin	-
5	A-C	UNC-15	A	5-23	Cy3-DAM	-	-
		MYO-3		-	-	5-14	A488
	D-F	UNC-89	A	MH42	Cy3-DAM	-	-
		MYO-3		-	-	5-14	A488
6	A-D,	Actin	A	AAN01	Cy3-DAR	-	-
	Q-T	PAT-3		-	-	MH25	A488
		MYO-3		5-6	A647-GAM	-	-
	E-H	Actin	A	AAN01	Cy3-DAR	-	-
		PAT-3		-	-	MH25	A488
		UNC-89		MH42	A647-GAM	-	-
	I-L	Actin	A	AAN01	Cy3-DAR	-	-
		PAT-3		-	-	MH25	A488
		DEB-1		MH24	A647-GAM	-	-
	M-P	Actin	A	AAN01	Cy3-DAR	-	-
		PAT-3		-	-	MH25	A488
		UNC-52		MH2	A647-GAM	-	-

^aNA, not applicable

^bFixation methods: A, methanol (5 min, –20 °C); B, 4% paraformaldehyde in cytoskeleton buffer (138 mM KCl, 3 mM MgCl₂, 2 mM EGTA, and 10 mM MES-KOH, pH 6.1) containing 0.32 M sucrose (20 min at room temperature) followed by phosphate-buffered saline containing 0.5% Triton X-100 and 30 mM glycine (20 min at room temperature); and C, methanol (5 min, –20 °C) followed by acetone (5 min, –20 °C).

^cA488-GAM, Alexa488-labeled goat anti-mouse IgG; A647-GAM, Alexa647-labeled goat anti-mouse IgG; Cy3-DAM, Cy3-labeled donkey anti-mouse IgG; Cy3-DAR, Cy3-labeled donkey anti-rabbit IgG.

^dZenon Alexa Fluor mouse IgG2a labeling reagents (Invitrogen).

Myofibrillar Components of the Gonadal Myoepithelial Sheath

We focused on the proximal myoepithelial sheath and examined localization of the cytoskeletal proteins at a higher magnification. Thin and thick filaments were arranged in a nonstriated manner, and major components of the adhesion structures were only associated with the thin filaments but not with the thick filaments.

Thin filaments

As described previously by Strome (1986), actin filaments are arranged in a nonstriated meshwork in the sheath cells (Fig. 4B,E,H,K,N,Q). The filaments were apparently continuous, and breaks at the thick filaments and dense bodies were not observed (Fig. 4B,E,H,K,N,Q) as clearly as in the striated body wall muscle (Waterston, 1988; Moerman and Fire, 1997). This finding suggests that the thin filaments in the myoepithelial sheath may have variable length and that the filament ends may not be tightly aligned. We have previously shown that tropomyosin (CeTM) and PAT-10 troponin C localize to the thin filaments in the myoepithelial sheath (Ono and Ono, 2004). Ce-kettin (MH44 antigen) also localizes to the thin filaments in the myoepithelial sheath, but it is limited to a narrow region near the dense bodies (Ono et al., 2005, 2006).

Fig. 4.

Localization of structural proteins in the myoepithelial sheath cells. A,B,D,E,G,H,J,K,M,N,P,Q: Dissected hermaphroditic gonads were doubly stained for MYO-3 myosin heavy chain (A), UNC-15 paramyosin (D), UNC-89 (G), UNC-52 perlecan (J), DEB-1 vinculin (M), or PAT-3 β-integrin (P), and actin (B,E,H,K,N,Q), and the myoepithelial sheath was observed at a high magnification. C,F,I,L,O,R: Merged images of actin (red) and other proteins (green) are shown. Scale bar = 10 μm.

Thick filaments

Previous studies by others have shown that the myoepithelial sheath cells express MYO-3 (myoA; Fig. 4A–C) and UNC-54 (myoB) myosin heavy chains (Ardizzi and Epstein, 1987) that are also expressed in the striated body wall muscle and that UNC-54 localizes to the outer regions, whereas MYO-3 is limited to the central part of the thick filaments (Rose et al., 1997) as observed in the thick filaments in the striated body wall muscle (Miller et al., 1983). UNC-15 paramyosin was also expressed in the myoepithelial sheath (Ardizzi and Epstein, 1987; Fig. 4D–F). Staining with the monoclonal anti–UNC-15 antibody (5-23) often resulted in punctate patterns (Fig. 4D). Double staining of UNC-15 paramyosin and MYO-3 showed that most of the UNC-15-spots (Fig. 5A, and red spots in C, arrowheads) are associated with the surface, not in the core, of the MYO-3 filaments (Fig. 5B, and green filaments in C, arrowheads). UNC-15 paramyosin is one of the core components of the nematode thick filaments (Harris and Epstein, 1977; Epstein et al., 1985, 1988). However, a previous biochemical study has demonstrated the existence of a population of paramyosin that can be readily dissociated from the thick filaments by a high-salt buffer (Deitiker and Epstein, 1993). Therefore, these spots of UNC-15 may represent the epitope of a subset of UNC-15 that is exposed on the surface of the thick filaments.

Fig. 5.

Localization of UNC-15 paramyosin, UNC-89, and MYO-3 myosin heavy chain on the thick filaments. A,B,D,E: The proximal myoepithelial sheath was doubly stained for UNC-15 paramyosin (A) or UNC-89 (D), and MYO-3 myosin heavy chain (B,E). C,F: Merged images are shown (MYO-3 in green and others in red). Arrowheads in C show typical MYO-3 filaments with punctate UNC-15 staining. Arrows in F indicate representative MYO-3 filaments with concentration of UNC-89 in the center. Scale bar = 10 μm.

UNC-89, a component of the M-lines in the striated muscles (Benian et al., 1996), was expressed in the myoepithelial sheath and accumulated into spots (Fig. 4G–I). Double staining of UNC-89 and MYO-3 showed that these proteins colocalize on the thick filaments (Fig. 5D–F), whereas UNC-89 was often highly accumulated at the center of the MYO-3 filaments (Fig. 5F, arrows). Because these central structures of the thick filaments are not organized into M-lines as in striated muscle, we designate these UNC-89–containing structures as the M-spots.

Adhesion structures

In the body wall muscle, myofibrils are attached to the plasma membrane at the dense bodies and the M-lines (Waterston, 1988; Moerman and Fire, 1997). The ultrastructural analysis of the myoepithelial sheath identified the “hemi-adherens junctions,” which are dense body-like structures (Hall et al., 1999). However, the molecular composition of the adhesion structures in the myoepithelial sheath has not been characterized. We found UNC-52 perlecan (Fig. 4J–L), DEB-1 vinculin (Fig. 4M–O), and PAT-3 β-integrin (Fig. 4P–R; Francis and Waterston, 1985) were expressed in the myoepithelial sheath and localized to dot-like structures. Surprisingly, α-actinin, a major component of the dense bodies in the body wall muscle, was not detected by two different monoclonal antibodies MH35 and MH40 (Francis and Waterston, 1985; Table 2, data not shown). Double staining for actin and UNC-52 perlecan, DEB-1 vinculin, or PAT-3 β-integrin showed that these proteins were associated with the actin filaments (Fig. 4J–R), suggesting strongly that these dot-like structures are dense bodies and link the thin filaments to the plasma membrane. However, the spots of PAT-3 β-integrin did not colocalize with MYO-3 myosin heavy chain that is located in the center of the thick filaments (Fig. 6A–D, see absence of overlap between green spots [PAT-3 β-integrin] [arrows] and blue filaments [MYO-3] [arrowheads] in D). This result was further confirmed by triple staining for actin, PAT-3 β-integrin, and UNC-89 (Fig. 6E–H) in which PAT-3 β-integrin (Fig. 6H, arrows) and UNC-89 (Fig. 6H, arrowheads), a marker of the M-spots, do not colocalize (Fig. 6H). These results strongly suggest that PAT-3 β-integrin does not link the M-spots and the thick filaments to the plasma membrane. These were unexpected results, as PAT-3 β-integrin localizes to the M-lines in the body wall muscle (Francis and Waterston, 1985, 1991). This is not likely to be an artifact of the fixation/staining conditions because, under the same fixation/staining conditions, the monoclonal antibody against PAT-3 β-integrin (MH25) stained only the dense bodies in the myoepithelial sheath (Fig. 6A–D). However, it stained both the dense bodies and the M-lines in the body wall muscle (Fig. 6Q–T).

TABLE 2. Components of the Contractile Apparatuses in the Gonadal Myoepithelial Sheath and Body Wall Muscle.

Gene	Protein	Antibody	MSa location	Reference	BWMb location	Reference
act-?c	Actin	C4 or AAN01	Thin filaments	(Strome, 1986)	Thin filaments	(Waterston et al., 1984)
myo-3	Myosin heavy chain A	5-6	Thick filaments (core)	(Ardizzi and Epstein, 1987)	Thick filaments (core)	(Miller et al., 1983)
unc-54	Myosin heavy chain B	5-8	Thick filaments (outer region)	(Ardizzi and Epstein, 1987)	Thick filaments (outer region)	(Miller et al., 1983)
unc-15	Paramyosin	5-23	Thick filaments (core)	(Ardizzi and Epstein, 1987)	Thick filaments (core)	(Epstein et al., 1985)
unc-89	UNC-89	MH42	M-spots	This study	M-lines	(Benian et al., 1996)
lev-11	Tropomyosin	Anti-CeTM	Thin filaments	(Ono and Ono, 2004)	Thin filaments	(Ono and Ono, 2002)
pat-10	Troponin C	Anti-PAT-10	Thin filaments	(Ono and Ono, 2004)	Thin filaments	(Terami et al., 1999)
ketn-1	Kettin	MH44	Thin filaments (near dense bodies)	(Ono et al., 2006)	Thin filaments (near dense bodies)	(Francis and Waterston, 1985)
pat-3	β-integrin	MH25	Dense bodies	This study	Dense bodies and M-lines	(Francis and Waterston, 1985)
deb-1	Vinculin	MH24	Dense bodies	This study	Dense bodies	(Francis and Waterston, 1985)
unc-52	Perlecan	MH2	Dense bodies	This study	Dense bodies and M-lines	(Francis and Waterston, 1991)
atn-1	β-actinin	MH35 or MH40	Absent	This study	Dense bodies	(Francis and Waterston, 1985)
unc-60B	ADF/cofilin	Anti-UNC-60B	Absent	This study	Thin filaments	(Ono et al., 1999)
unc-78	AIP1	Anti-UNC-78	Absent	This study	Thin filaments	(Mohri and Ono, 2003)
ajm-1	AJM-1	MH27	Absent	This study	Absent (adherens junction in the hypodermis)	(Hresko et al., 1994)
let-805	Myotactin	MH1	Absent	This study	Absent (hemidesmosomes in the hypodermis	(Hresko et al., 1999)

^aMS, myoepithelial sheath.

^bBWM, body wall muscle.

^cCaenorhabditis elegans has five actin genes. It is not known which actin gene is expressed in the myoepithelial sheath.

Fig. 6.

Accumulation of PAT-3 β-integrin and other cell attachment proteins to the dense bodies but not the M-spots. A–C,E–G,I–K,M–O: The proximal myoepithelial sheath was triply stained for actin (A,E,I,M), PAT-3 β-integrin (B,F,J,N), and MYO-3 myosin heavy chain (C), UNC-89 (G), DEB-1 vinculin (K), or UNC-52 perlecan (O). D,H,L,P: Merged images of actin (red), PAT-3 β-integrin (green), and other proteins (blue) are shown. In the merged images, arrows indicate representative spots of PAT-3 β-integrin where segregation from MYO-3 (arrowheads in D) or UNC-89 (arrowheads in H) or colocalization with DEB-1 vinculin (L) or UNC-52 perlecan (P) is clearly seen. Q–T: The body wall muscle was triply stained for actin (Q), PAT-3 β-integrin (R), and MYO-3 myosin heavy chain (S) under the same conditions for A–D, and a merged image actin (red), PAT-3 β-integrin (green), and MYO-3 (blue) is shown in T. Scale bars = 10 μm.

Moreover, other components of the dense bodies, DEB-1 vinculin (Fig. 6I–L, arrows in L) and UNC-52 perlecan (Fig. 6M–P, arrows in P), showed nearly identical localization patterns with PAT-3 β-integrin at the dense bodies. Vinculin is a component of the dense bodies but not M-lines in the body wall muscle (Francis and Waterston, 1985; Barstead and Waterston, 1989), while UNC-52 localizes to both dense bodies and M-lines in the body wall muscle (Francis and Waterston, 1991; Rogalski et al., 1993). These observations suggest that the contractile apparatuses of the myoepithelial sheath are attached to the plasma membrane only at the dense bodies or that the thick filaments are attached to the membrane by an unknown mechanism. Thus, the gonadal myoepithelial sheath and the body wall muscle share common cell attachment proteins, but they are organized in different manners.

Other components

We also examined distribution of AJM-1, a component of adherens junctions (Koppen et al., 2001), myotactin, a component of hemidesmosomes (Hresko et al., 1999), UNC-78 actin-interacting protein 1 (Ono, 2001), and UNC-60B actin depolymerizing factor/ cofilin (Ono et al., 1999, 2003). However, these proteins were detected only in the spermatheca but not in the myoepithelial sheath (our unpublished data).

Roles of the Myofibrillar Components in Ovulation

To determine the roles of the structural components of the myoepithelial sheath in ovulation, they were knocked down by RNA interference (RNAi) and the resultant phenotypes in ovulation were examined. When ovulation fails, a mature oocyte remains in the proximal ovary, repeats DNA synthesis without cell division, and becomes endomitotic (Iwasaki et al., 1996; McCarter et al., 1997, 1999). This phenotype is designated as Emo (endomytotic oocytes in the proximal ovary) and is characterized by intense staining by 4′6-diamidino-2-phenylindole, dihydrochloride (DAPI) of overly amplified nuclear DNA in endomitotic oocytes (Fig. 7C, arrows; Iwasaki et al., 1996). The ovulation process is also regulated by signals from sperm and oocytes. Therefore, to distinguish the effects of RNAi in the somatic gonads from those in the germ cells, we compared the RNAi phenotypes in wild-type and rrf-1 backgrounds. The rrf-1 mutants are defective with RNAi in the somatic cells but not in the germ cells (Sijen et al., 2001). Thus, if function of an RNAi target is important in the somatic gonad (either sheath contraction [McCarter et al., 1997] or spermathecal dilation [Clandinin et al., 1998; Aono et al., 2004]), ovulation will be defective in wild-type but normal in the rrf-1 background.

Fig. 7.

Effects of RNA interference (RNAi) knockdown of the structural proteins on ovulation. A–P: Wild-type (A,C,E,G,I,K,M,O) or an rrf-1 strain (B,D,F,H,J,L,N,P) was treated with RNAi for control (vector with no insert, A,B), myo-3 (C,D), unc-15 (E,F), unc-52 (G,H), unc-89 (I,J), deb-1 (K,L), pat-3 (M,N), or ketn-1 (O,P). Worms were fixed and stained with tetramethylrhodamine-phalloidin and 4′6-diamidino-2-phenylindole, dihydrochloride (DAPI) to visualize actin and DNA, respectively, and merged images are shown in red (actin) and blue (DNA). Positions of the spermatheca are indicated by asterisks. Defective ovulation was characterized by the presence of endomitotic oocytes (arrows in C) that have intense DNA staining (Emo phenotype). Scale bar = 50 μm.

Previous studies have shown that unc-54 myosin heavy chain (Rose et al., 1997), lev-11 tropomyosin (Ono and Ono, 2004), pat-10 troponin C (Ono and Ono, 2004), and mup-2 troponin T (Myers et al., 1996) are required for ovulation. Here, we found that the Emo phenotype (22–51%; n = 100) was induced by RNAi treatments in wild-type background for myo-3 myosin heavy chain, unc-15 paramyosin, pat-3 β-integrin, deb-1 vinculin, or unc-52 perlecan (Fig. 7C,E,G,K; Table 3). However, RNAi of unc-89 or ketn-1 Ce-kettin did not significantly increase the percentages of the Emo phenotype (Fig. 7I,O; Table 3). Only background levels of the Emo phenotype (0–5%) were detected in the rrf-1 background after RNAi of myo-3, unc-15, unc-52, or deb-1 (Table 3), suggesting strongly that these genes function in the somatic cells. pat-3(RNAi) caused 99% P0 sterility with the Emo phenotype (n = 100) in wild-type, but it is significantly reduced in rrf-1 (Fig. 6M,N; Table 3). A slightly higher rate of the Emo phenotype (10%) by pat-3(RNAi) in rrf-1 than the background level may indicate a possible role of pat-3 in the germline or the gut, which is a known source of yolk proteins (Kimble and Sharrock, 1983; Grant and Hirsh, 1999). RNAi of unc-89 did not cause the Emo phenotype. Instead, the unc-89(RNAi) animals, in wild-type but not rrf-1 background, were defective in egg laying and accumulated embryos in the uterus (our unpublished data), suggesting that UNC-89 is functionally important in the vulval and/or uterine muscle. Together, these results indicate that the structural components of the contractile apparatuses in the myoepithelial sheath, except for UNC-89 and Ce-kettin, are essential for ovulation. However, we cannot rule out the possibilities that UNC-89 and Ce-kettin are also essential for ovulation, because the gene products might not have been knocked down by the RNAi treatments sufficiently to cause phenotypes.

TABLE 3. Effects of RNAi Treatments on Ovulation.

RNAi treatment (generation observed)	% Emo phenotype (n = 100)
	Wild-type	rrf-1
Control (F1)	1	3
myo-3 (F1)	51	3
unc-15(F1)	22	5
unc-52 (F1)	30	1
unc-89 (F1)	0	3
deb-1 (F1)	30	0
ketn-1 (F1)	0	3
Control (P0)	0	0
pat-3 (P0)	99	10

Discussion

In this study, we characterized organization and function of nonstriated contractile apparatuses in the C. elegans gonadal myoepithelial sheath. Based on our results, we propose a model for the organization of the contractile apparatuses in the myoepithelial sheath (Fig. 8A). The thin filaments are anchored to the plasma membrane at the dense bodies where integrin is accumulated. The dense bodies lack α-actinin. Therefore, vinculin, talin, or other actin-binding proteins may connect actin with integrin. The thick filaments have M-spots but do not have integrin-based attachments with the plasma membrane. An alternative model (Fig. 8B) is that cytoplasmic actin-anchoring structures, possibly containing α-actinin–like molecules, links the thin filaments to the integrin-based structure and that the thick filaments are attached to the membrane through non–integrin-based structures. Whether the thick filaments in the myoepithelial sheath are physically connected with the plasma membrane is unknown. Previous ultrastructural studies on the somatic gonad did not reveal such connections (Hirsh et al., 1976; Strome, 1986; Hall et al., 1999).

Fig. 8.

Models of the organization of structural components of the contractile apparatuses in the myoepithelial sheath. A: Model 1 shows that the thin filaments are attached to the plasma membrane at the integrin-based structures, while the thick filaments lack a physical link with the membrane. B: Model 2 shows that the thin filaments are connected to the integrin-based structures through unknown anchoring components, whereas the thick filaments are attached to the membrane with unknown attachment structures. C: An established model of the structure of the contractile apparatuses of the body wall muscle (Waterston, 1988; Moerman and Fire, 1997). Arrows of the thin filaments are pointing toward the pointed ends of the actin filaments.

Myofibrils in the myoepithelial sheath and the body wall muscle are not only different in their organization but also in their size. The myoepithelial sheath cells are ∼1 μm thick and the thin and thick filaments are loosely arranged in a meshwork pattern (Hall et al., 1999). In contrast, the body wall muscle cells are 2- to 3-μm thick and the myofibrils are densely packed within 1–1.5 μm from the plasma membrane to transmit contractile forces to the hypodermis and the cuticles (Fig. 8C; Waterston et al., 1980; Francis and Waterston, 1985). Therefore, the body wall muscle may require very rigid attachment structures for both thin and thick filaments. These muscles are also different in their manners of contraction. The myoepithelial sheath needs to be flexible in changing their cell shape to support transport of oocytes. Thus, nonstriated filamentous organization and lack of integrin-based links between the thick filaments and the membrane might be beneficial for such a function. On the other hand, the body wall muscle always contracts in the longitudinal direction, and tight arrangement of sarcomeres in a uniform direction will be very efficient to coordinate contractile forces.

pat-3 is the single gene for β-integrin in C. elegans and is essential for assembling the adhesion structures in body wall muscle (Francis and Waterston, 1985; Williams and Waterston, 1994; Gettner et al., 1995). PAT-3 β-integrin is expressed in a variety of muscle and nonmuscle cells and also involved in cell migration, gonadal morphogenesis, and axon guidance (Lee et al., 2001, 2005; Poinat et al., 2002; Xu et al., 2006). Localization of PAT-3 β-integrin to the dense bodies and the M-lines in body wall muscle and cell attachment structures in several other cell types has been reported previously (Francis and Waterston, 1985; Gettner et al., 1995). However, this study is the first to describe intracellular localization of PAT-3 β-integrin in the myoepithelial sheath, supporting previous observations that PAT-3 β-integrin is required for ovulation (Lee et al., 2001; Xu et al., 2005, 2006). Limited localization of PAT-3 β-integrin to the dense bodies in the myoepithelial sheath is a remarkable difference from its localization to both dense bodies and M-lines in the body wall muscle. Integrin plays critical roles in mediating extracellular signals to trigger several intracellular signaling events, including reorganization of the actin cytoskeleton (Brakebusch and Fassler, 2003; Wozniak et al., 2004; Lo, 2006). Thus, the difference in the integrin localization between striated and nonstriated muscles suggests that integrin has different functions in these muscles. One possible role for integrin might be to regulate the morphogenetic processes of nonstriated or striated myofibrils by sending signals to different cytoskeletal regulators. Currently, little is known about the mechanism of morphogenesis of the myoepithelial sheath, and further studies may reveal how integrin might be involved in this process.

RNA interference of the major contractile proteins in the myoepithelial sheath caused defective ovulation. However, RNAi of myo-3, unc-15, unc-52, or deb-1 resulted in 22–51% sterility, which is weaker than nearly 100% sterility caused by RNAi of pat-3 (this study), pat-10 troponin C, or lev-11 tropomyosin (Ono and Ono, 2004). This finding is likely due to incomplete knockdown of these genes by the RNAi treatments, as strong loss-of-function or null mutation of deb-1, myo-3, or unc-52 results in embryonic lethality (Waterston, 1989; Barstead and Waterston, 1991; Rogalski et al., 1995). Nonetheless, these weak RNAi phenotypes allowed us to characterize the ovulation defects without having severe embryonic defects. We also did not observe severe disorganization of the actin filament structures in the myoepithelial sheath even after the RNAi treatments, suggesting that a mild decrease in the amounts of these contractile proteins might be sufficient to impair sheath contraction but insufficient to cause severe morphogenetic defects.

Our study also suggests that the C. elegans myoepithelial sheath might be a good model to study structure and function of smooth muscle, myoepithelial cells, and myofibroblasts, which have nonstriated contractile apparatuses. To date, a genetic model for cytoskeletal assembly in such nonstriated contractile cells has not been developed. Smooth muscles are present in both vertebrates and invertebrates and play a variety of functions (Somlyo and Somlyo, 1994). Myoepithelial cells are often found in the outer layer of exocrine glands and have characteristics of both smooth muscle and epithelial cells (Lazard et al., 1993; Foschini et al., 2000; Furuse et al., 2005). Myofibroblasts are present in many organs and express several sarcomeric proteins (Mayer and Leinwand, 1997; Walker et al., 2001; Rice and Leinwand, 2003). Regulatory mechanisms for contraction is also various. Vertebrate smooth muscles are regulated by a myosin-linked system (Marston, 1995), whereas ascidian smooth body wall muscle (Endo and Obinata, 1981) and C. elegans myoepithelial sheath (Ono and Ono, 2004) are regulated by an actin-linked troponin-tropomyosin system. However, nonstriated organization of the contractile apparatuses is the common feature of all smooth muscles, myoepithelial cells, and myofibroblasts, and little is known about the mechanism by which nonstriated myofibrils are assembled during development. Thus, C. elegans might be a suitable model to investigate this fundamental mechanism. We preliminarily found that known regulators of actin dynamics in the body wall muscle, including unc-60B actin depolymerizing factor/cofilin (Ono et al., 1999, 2003) and unc-78 actin-interacting protein 1 (Ono, 2001), are not required for assembly of nonstriated myofibrils in the myoepithelial sheath (our unpublished data). Although we previously reported that unc-60B might be involved in ovulation antagonistically to lev-11 tropomyosin (Ono and Ono, 2004), we have recently shown that the observed genetic interaction between unc-60B and lev-11 was due to a background mutation that affected the efficiency of RNAi of lev-11 but not due to a direct effect of the unc-60B mutation (Yu and Ono, 2006). These data suggest that nonstriated and striated muscles in C. elegans use different sets of genes to regulate actin organization.

Experimental Procedures

Nematode Strains

Wild-type C. elegans strain N2 and an RNAi-defective strain NL2098 rrf-1(pk1417) (Sijen et al., 2001) were obtained from the Caenorhabditis Genetics Center (Minneapolis, MN). Nematodes were grown under standard conditions at 20°C (Brenner, 1974).

Antibodies and Fluorescent Probes

Anti-actin mouse monoclonal antibody (C4) was purchased from MP Biomedicals. Anti-actin rabbit polyclonal antibody (AAN01) was purchased from Cytoskeleton, Inc. Anti-myoA (5-6; Miller et al., 1983) and anti-paramyosin (5-23; Ardizzi and Epstein, 1987) mouse monoclonal antibodies were provided by Dr. Henry Epstein (University of Texas Medical Branch, Galveston, TX). Anti–PAT-3 β-integrin (MH25; Francis and Waterston, 1985), anti–DEB-1 vinculin (MH24; Francis and Waterston, 1985), anti–UNC-52 perlecan (MH2; Francis and Waterston, 1991) monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Anti–UNC-89 monoclonal antibody (MH42) (Benian et al., 1996) was provided by Drs. Pamela Hoppe (Western Michigan University, Kalamazoo, MI) and Robert Waterston (University of Washington, Seattle, WA). Tetramethylrhodamine-phalloidin and DAPI were purchased from Sigma-Aldrich. All Alexa dye-conjugated secondary antibodies and Zenon Mouse IgG2a Labeling Reagents were purchased from Invitrogen/Molecular Probes. Cy3-conjugated donkey anti-rabbit IgG and anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories.

Fluorescence Microscopy

The gonads were dissected by cutting adult hermaphrodites at the level of pharynx (Fig. 1)–on poly-lysine–coated slides as described previously (Rose et al., 1997). These samples were fixed by an optimal method and stained with antibodies as listed in Table 1. When the host animals of the primary antibodies were different, they were mixed and reacted with the samples simultaneously, and followed by treatments with appropriate fluorescently labeled secondary antibodies (Table 1). We also performed differential labeling of two mouse monoclonal antibodies and one rabbit antibody by using secondary antibodies that distinguish IgG isotypes and host species as described previously (Ono et al., 2006). Briefly, the samples were first treated with a mixture of mouse IgG1 antibody and rabbit antibody, and then with secondary antibodies (nonspecific for IgG isotypes). They were washed with phosphate buffered saline (PBS) and blocked with 0.1 mg/ml mouse IgG (Rockland Immunochemicals) in 1% bovine serum albumin in PBS for 10 min, and reacted with a mouse IgG2a antibody. Then, it was visualized by Zenon Alexa Fluor Mouse IgG2a Labeling Reagents (Invitrogen/Molecular Probes) (Table 1).

Samples were viewed by epifluorescence using a Nikon Eclipse TE2000 inverted microscope with a ×40 or ×60 CFI Plan Fluor objective. Images were captured by a SPOT RT Monochrome CCD camera (Diagnostic Instruments) and processed by the IPLab imaging software (Scanalytics, Inc.) and Adobe Photoshop 6.0.

RNA Interference Experiments

Namatodes were treated with RNAi by feeding Escherichia coli–expressing double-stranded RNA (Timmons and Fire, 1998; Timmons et al., 2001). Except for pat-3(RNAi), L4 larvae were treated with RNAi, and phenotypes were characterized in their F1 generation as described previously (Ono and Ono, 2002). pat-3(RNAi) caused strong P0 sterility. Therefore, L1 larvae were treated with pat-3(RNAi), and phenotypes were characterized when they became adults. RNAi clones were obtained from the C. elegans RNAi library from Geneservice (Cambridge, United Kingdom), and the clone numbers are as follows: myo-3, V-8003; unc-15, I-3N01; unc-52, II-9A20; unc-89, I-1B22; pat-3, III-1P02; and ketn-1, V-2F01. Because an RNAi clone for deb-1 was not available from the library, we made a construct by cloning a 1.0-kb genomic fragment of the deb-1 gene into an RNAi vector plasmid L4440 (provided by Dr. Andrew Fire, Stanford University) and transformed E. coli HT115(DE3) with the recombinant plasmid. Control experiments were performed with E. coli HT115(DE3) that was transformed with L4440 with no insert. Staining of RNAi-treated worms with tetramethylrhodamine-phalloidin and DAPI was performed as described previously (Ono, 2001).