PHA-1 is required for pharyngeal cell reorientation and the stable attachment of arcade cells to pharyngeal epithelial cells

The C. elegans pharynx (foregut) is an organ derived from 95 primordial cells that differentiate into seven distinct cell types (arcade, epithelial, muscle, gland, marginal, valve, and neural) (Albertson and Thomson 1976; Mango 2007, 2009). A principal function of the pharynx is to ingest food, to mechanically process it, and to deposit it into the intestine. Studies of pharyngeal morphogenesis by Portereiko and Mango (2001) defined three temporally separated steps (Figure 1, A and B). In the first step, termed reorientation, anterior pharyngeal epithelial cells are converted from a radial configuration into two parallel rows through changes in cell shape, location, and apicobasal polarity. In the second step, termed epithelialization, reorientated pharyngeal cells attach to the neighboring arcade cells, which are anterior to the pharynx, to form a contiguous epithelial tube connecting the nascent buccal cavity (mouth) to the intestine. In the third step, termed contraction, localized forces pull the arcade and pharyngeal epithelial cells closer together, and the pharynx shifts en masse slightly toward the anterior. This initiates the conversion of the primordial pharynx from a ball of cells into an elongated bilobed tube connecting the mouth to the intestine (Figures 1, B and C). Notably, the first step, reorientation, is essential for all subsequent steps of morphogenesis to occur.

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Figure 1

Pharyngeal morphogenesis in wild-type and pha-1 mutants. (A) Schematic diagram of early stages of pharyngeal morphogenesis (after Portereiko and Mango 2001) showing the initial state, reorientation, and epithelialization/contraction (left to right). Arcade cells are indicated in light shading, pharyngeal cells in dark shading. Thick solid lines indicate the locations of basement membranes. (B) Overview of pharyngeal morphogenesis during embryogenesis in wild type and pha-1 mutants. Note differences between the phenotypes of pha-1(0) and pha-1(ts) mutants. Also note that pha-1(0) mutants fail to progress beyond the twofold stage of embryogenesis. Dashed lines indicate pharyngeal boundaries. (C) Representative images of P_pha-4::membrane–GFP reporter expression in wild-type, pha-1(ts) at 25°, and pha-1(0) embryos. Anterior is to the left, dorsal is up. Corresponding DIC (left) and fluorescence images (right) are paired for each embryo. Bars in wild-type DIC and fluorescence images, 10 µm for DIC and fluorescence images, respectively. Solid arrows indicate the anterior border of the developing pharynx. Note that both wild type (50/50) and pha-1(ts) mutants at 25° (47/48) show extension by the 1.5-fold stage. In contrast, the large majority of pha-1(0) embryos do not undergo any obvious extension (38/44). Furthermore, in pha-1(0) mutants that did undergo pharyngeal extension (lower right paired panels), the width of the pharynx was often narrower along the dorsal–ventral axis than in wild type.

Previous phenotypic analyses of pha-1 employed what are now known to be partial-LOF alleles (e.g., pha-1(e2123) (referred to as pha-1(ts)), which may differ from the phenotypes displayed by null mutants (Schnabel and Schnabel 1990; Granato et al. 1994a; Fay et al. 2004; Mani and Fay 2009). We therefore examined pharyngeal morphogenesis using strains homozygous for the pha-1(tm3671) (pha-1(0)) null deletion, which contain a 203-bp deletion that removes the N-terminal portion of the second pha-1 exon and creates a premature stop codon (Fay et al. 2012). Cell-shape changes during pharyngeal morphogenesis were visualized using a pha-4-driven plasma membrane–GFP reporter that is expressed exclusively within cells of the pharyngeal primordium, intestine, and arcade cells (Portereiko and Mango 2001; Fay et al. 2003).

Consistent with our previous observations (Fay et al. 2004), a large majority of pha-1(ts) mutants (47/48) incubated at the nonpermissive temperature of 25° were capable of completing reorientation, initiating attachment to the arcade cells, and establishing a transient connection to the nascent buccal cavity (Figure 1, B and C). In contrast, the large majority of pha-1(0) embryos (38/44) did not undergo reorientation and failed to integrate with the arcade cells (Figure 1, B and C). Furthermore, in pha-1(0) mutants that did undergo reorientation, the developing anterior pharynx was often abnormally constricted along the dorsal–ventral axis (Figure 1C). A role for PHA-1 in reorientation is also consistent with reorientation defects observed in lin-35; pha-1(fd1) and lin-35; ubc-18 double mutants (Fay et al. 2003, 2004). We also note that equivalent levels of expression of the P_pha-4::membrane–GFP reporter in wild type, pha-1(ts) and pha-1(0) mutants indicates that a reduction or loss of PHA-1 activity does not affect the specification of the pharyngeal primordium (Figure 1).

The highly penetrant pharynx unattached (Pun) phenotype of pha-1(ts) mutants indicates that PHA-1 is also required for the maintenance of pharyngeal attachment to the buccal capsule at later stages of development. Furthermore, on the basis of the arcade cell marker, bath-15::GFP (Shaw et al. 2010), we found that the buccal capsule in pha-1(ts) mutants is typically severed or discontinuous, with the break occurring at the junction of the arcade and pharyngeal epithelial cells (Supporting Information, Figure S1). In more subtle cases, a morphologically abnormal X-shaped (Chi) structure was detected at the junction of the arcade and pharyngeal cells (Figure S1), which may interfere with the ability of these animals to feed, thereby leading to larval arrest. Taken together, our findings demonstrate that PHA-1 is critical for both the initiation and maintenance of pharyngeal epithelial cell attachment to the arcade cells.

Several epithelial apical junction markers are misregulated in pha-1 mutants

To further characterize pharyngeal defects in pha-1(0) mutants, we made use of a fluorescence reporter for AJM-1, an apical junction protein that is broadly required for morphogenesis and the integrity of epithelia in C. elegans (Koppen et al. 2001; McMahon et al. 2001; Lynch and Hardin 2009). For these assays, AJM-1::GFP served as a marker of epithelialization for both the pharynx and arcade cells (Portereiko and Mango 2001). Consistent with previous reports, AJM-1::GFP is first detected in wild-type embryos beginning at the lima-bean stage (∼350 min postfertilization) in the central region of the primordial pharynx (Figure 2). Expression of AJM-1::GFP is also observed in the region corresponding to the arcade cells beginning at around the comma stage (∼400 min), coincident with the onset of reorientation. Expression of AJM-1::GFP increases throughout the 1.5- and 2.0-fold stages of embryogenesis in both the pharynx and arcade regions of wild-type embryos (∼430–450 min; Figure 2).

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Figure 2

AJM-1::GFP expression in pha-1 mutants. Representative images of AJM-1::GFP (kuIs46) in wild-type, pha-1(ts) at 25°, and pha-1(0) embryos at the indicated stages. Anterior is to the left, dorsal is up. Location of the pharynx (p) is indicated with a solid white line, and arcade cells (a) are indicated with a dashed white line (sensory depression, s). Arrowheads indicate AJM-1 puncta in the developing epidermis. Bar, 5 µm. Graph shows quantification of AJM-1::GFP expression patterns for each genotype and stage. The number of embryos examined for each stage and genotype ranged from 21 to 32.

Although pha-1(ts) mutants at 25° displayed a similar pattern of AJM-1::GFP to that of wild-type embryos, expression was typically weaker and less uniform than expression in wild type at corresponding stages (Figure 2). In contrast, AJM-1::GFP expression was dramatically reduced in pha-1(0) mutants with little or no detectable expression in the region of the primordial pharynx through the 2.0-fold stage (Figure 2). In addition, pha-1(0) mutants that failed to undergo reorientation never expressed AJM-1::GFP in the region of the arcade cells, consistent with the model that epithelialization of the arcade cells strictly follows integration with the anterior pharynx (Portereiko and Mango 2001). Although some 2.0-fold pha-1(0) embryos did not express AJM-1::GFP, expression was always observed in late-stage, terminally arrested pha-1(0) embryos, although levels were strongly reduced as compared with wild type (Figure S2). Similar results were also observed using an independent array of AJM-1::GFP integrated on a different chromosome (data not shown). Finally, AJM-1::GFP expression defects in pha-1(0) mutants were fully rescued by an extrachromosomal array containing wild-type copies of pha-1 or by inhibition of sup-35 by RNAi (Figure S2). These findings indicate that PHA-1 plays an important role in the timing and strength of AJM-1 expression and more generally in the process of pharyngeal epithelialization. They also demonstrated that suppression and rescue of pha-1(0) defects correlates with the normal expression of AJM-1.

In our studies examining the expression of AJM-1::GFP in the embryonic pharynx, we noticed that this marker was expressed at much lower levels in the developing embryonic epidermis (formerly termed the hypodermis) of pha-1(0) null mutants as compared with wild-type or pha-1(ts) embryos (Figure 2 and Figure 3B). To see if our observation was specific to AJM-1 or reflected a more generalized defect in the epithelia of pha-1(0) mutants, we examined expression patterns of several markers associated with adherens junctions and integrity in 1.5-fold pha-1(0) embryos. Two well-characterized junctional complexes in C. elegans include a classical cadherin–catenin complex (HMR-1/cadherin, JAC-1/p120-catenin, HMP-1/α-catenin and HMP-2/β-catenin) and the more basal AJM-1 and DLG-1/discs large complex (Labouesse 2006; Lynch and Hardin 2009). DLG-1, a binding partner of AJM-1, is essential for proper localization of AJM-1 (Koppen et al. 2001), whereas SAX-7 has been proposed to function as the transmembrane component of the complex (Chen et al. 2001). Loss of pha-1 resulted in a strong reduction in AJM-1::GFP in the pharynx and intestine (4.6-fold), and lateral epithelial cells (5.4-fold; Figure 3B). In addition, a modest increase in DLG-1::dsRed expression (1.5- to 1.7-fold) was observed in both the epidermis and gastrointestinal tract of pha-1(0) mutants, whereas no obvious changes were detected with SAX-7::GFP (Figure 3B). In the case of the cadherin–catenin complex, expression of HMR-1::GFP was reduced ∼1.9-fold in both the pharynx and epidermis of pha-1(0) mutants, whereas JAC-1 expression was enhanced 1.4- to 1.6-fold in the pharynx and epidermis (Figure 3C). In contrast, HMP-1::GFP showed no change in pha-1(0) mutants (Figure 3C). Our results indicate that components of two major junctional complexes are misregulated in epithelial cells of pha-1(0) mutants, consistent with the role of PHA-1 in regulating epidermal morphogenesis.

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Figure 3

Loss of PHA-1 affects normal expression of epithelial junction markers. (A) Schematic representation of the proposed organization of epithelial junctions in C. elegans. Arrows show the direction of expression changes in the pha-1(0) background. (B and C) Quantification of fluorescence intensities of members of the DLG-1/AJM-1 complex (B) and cadherin–catenin complex (C) in wild-type and pha-1(0) mutant backgrounds. Also shown are representative fluorescence images of 1.5-fold-stage embryos. Fluorescence is expressed relative to wild type, which was arbitrarily set to 1.0. Error bars indicate 95% confidence intervals. Statistical analysis was performed using Student’s t-test; **P < 0.001.

PHA-1 is not required autonomously for the differentiation or function of most pharyngeal cells

To better understand the role of PHA-1 in pharyngeal development and to more broadly identify tissues in which PHA-1 performs important functions, we carried out a mosaic analysis using the pha-1(0) allele strain. These strains have a mitotically unstable extrachromosomal array containing wild-type pha-1 [pha-1(+)] rescuing sequences along with a broadly expressed visible marker, SUR-5::GFP (Yochem et al. 1998). These strains produce animals containing a mixture of wild-type and pha-1(0) mutant cells that can be distinguishable by the presence or absence of nuclear GFP. The pattern of array inheritance was used to determine the cell lineages in which the array was retained or lost during development. On the basis of the phenotype of individual worms and the inheritance profile of the array, it was possible to infer tissue-specific requirements for pha-1 and to correlate losses within particular lineages with specific phenotypes. A schematic summary of the mosaic analysis is shown in Figure 4A.

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Figure 4

pha-1(0) mosaic analysis. (A) Early embryonic lineages are indicated along with major cell types produced by each lineage. Solid lines indicate lineages where pha-1 plays an essential function. Numbers indicate viable L4 larvae or adults that were isolated with losses at the indicated cell divisions. (B–K) DIC, showing B, D, F, H, and J and corresponding fluorescence C, E, G, I, and K images of pha-1(0) mosaic animals carrying the fdEx182 extrachromosomal array, which expresses both wild type pha-1 and the SUR-5::GFP marker. Anterior is to the left. (B and C) L4-stage larva in which pha-1 is absent from the MS lineage. Note the normal appearance of the posterior pharyngeal bulb and isthmus (dashed outline) despite the absence of pha-1 activity. (D and E) Adult ABa-mosaic animal with a normal pharynx in which pha-1 is present only in the left ventral tripartite epidermal section (pm2, e1, e2; indicated by arrows in E) derived from ABal. (F and G) P1-minus arrested L1 larva. Note the presence of a normal-appearing pharynx; solid arrows indicate the pharyngeal lumen in F. Also note normal body morphology. (H and I) Embryo (1.5-fold stage) that is missing pha-1 in all cells derived from ABa. Dashed line indicates the boundary of the pharynx. Note the absence of any anterior pharyngeal extension. (J and K) Arrested L1 larvae in which pha-1 is missing within the E lineage. Solid arrows in J indicate pharyngeal lumen; bracket in J, posterior bulb of pharynx (MS-plus); dashed line encircles the intestine (GFP-minus). (L) A clear (Clr) fluid-filled adult containing multiple vacuoles (v) that was missing pha-1 in the excretory cell. The solid and open dashed lines denote the outlines of the intestine and gonad, respectively. Bar in B, 10 µm (for B, C, F–K) and bar in D, 10 µm (for D, E, and L).

Intriguingly, in view of previous reports suggesting a role for PHA-1 in pharyngeal cell specification and differentiation, our data strongly indicate that PHA-1 was not required autonomously for the specification, differentiation, or function of most pharyngeal cells. Evidence includes our isolation of three adult animals in which pha-1(+) was missing entirely from the MS lineage (i.e., MS-minus), as well as three animals in which pha-1(+) was missing in one of the daughters of MS (MSa/p). These MS-mosaic animals developed at normal rates and had pharynxes with wild-type morphology (Figure 4, B and C). Given that about half of the cells comprising the pharynx are derived from MS (Sulston et al. 1983), it appears that PHA-1 is dispensable for the normal development and function of at least half of the cells of this organ. Furthermore, although we failed to isolate adults in which pha-1 was missing entirely from ABa or its daughters ABal and ABar, we did identify adults that had suffered multiple losses within the ABa sublineages that give rise to the pharynx. This included an adult in which only the left ventral tripartite epidermal section (pm2, e1, and e2, which are derived from ABal and ABar) retained the array within the ABa-derived pharyngeal lineage (Figure 4, D and E). Taken together, these findings suggest that PHA-1 does not function autonomously to promote the differentiation, morphogenesis, or function of the large majority of pharyngeal cells.

To complement the above analysis, we also analyzed embryos and arrested L1 larvae that were mosaic for pha-1. We identified seven P1-minus mosaics, which uniformly arrested as L1 larvae but contained pharynxes with relatively normal morphology (Figure 4, F and G). We also observed one L1 in which pha-1 was missing within both the MS and ABar lineages. Although arrested, this animal had a pharynx with relatively normal morphology (data not shown). Given that the anteriormost pharyngeal epidermal cells, e1D, e1VL, and e1VR, are all derived from ABar, this would suggest that pha-1 is not required in the e1 lineage for normal morphogenesis. (For pharyngeal anatomy, D signifies dorsal, whereas VL VR, and DL indicate ventral left, ventral right and dorsal left respectively; ant and post indicate anterior and posterior.) It also suggests that arcade cells derived from ABal (post-VL, post-DL, ant-DL, and ant-V) could be a focus for pha-1. Additional arrested L1s with little or no detectable expression in ABa-derived pharyngeal lineages were also observed, although the patterns of array inheritance within these animals were often quite complex. Nevertheless, such animals typically contained elongated pharynxes with relatively normal morphologies, suggesting that pha-1 is not required within most ABa-derived pharyngeal cells for pharyngeal differentiation or morphogenesis. We also identified one ABa-minus ABp-plus embryo in which pharyngeal cell reorientation had failed (Figure 4, H and I). This finding suggested that although pha-1 may not be autonomously required within most pharyngeal cells, there is nevertheless a requirement for pha-1 within the ABa lineage for pharyngeal morphogenesis to occur. Taken together, these findings suggest a requirement for PHA-1 within a subset of arcade cells, but because one or more pharyngeal epithelial cells is always close to one or more arcade cells in the ABa part of the cell lineage (Sulston et al. 1983), the possibility has not been excluded that PHA-1 acts within a subset of the ABa-derived pharyngeal cells (e.g., e2) or that PHA-1 may be required in some combination of arcade and anterior pharyngeal cells. In addition, it is possible that PHA-1 also acts in other cells that are close to the pharyngeal epithelium and arcade cells in the cell lineage, such as the marginal cells.

PHA-1 performs essential functions in tissues outside of the pharynx

Interestingly, our mosaic analysis with the null allele also revealed several previously unknown tissue requirements for PHA-1. Most notably, we failed to observe viable larvae or adults in which pha-1 was missing entirely from the intestinal lineage (n > 10,000). Rather, our data indicate that the absence of pha-1 within the intestine leads to L1 larval arrest. Consistent with this, we identified 19 arrested L1 larvae in which the pha-1-rescuing array was lost in either the P1 (5), EMS (7), or E (7) lineages (Figure 4, J and K). These animals failed to display gross morphological defects of their intestines, and their intestinal cells appeared to contain microvilli (data not shown). Nevertheless, it is possible that these intestines contained subtle structural defects that we did not detect. It is also possible that these larvae arrested for reasons unrelated to intestinal defects, although their gross morphologies, including the pharynx, appeared normal (Figure 4, F and J). Moreover, the complete absence of viable intestine-minus mosaics strongly indicates that PHA-1 is required in this tissue. Somewhat surprisingly, we identified several adults that were missing pha-1 in the majority of intestinal cells but were nevertheless viable. This included one sick, slow-growing adult that contained only four PHA-1-positive posterior gut nuclei (out of a total of 20 binucleate intestinal cells in the adult). Taken together, our data suggest that pha-1 is autonomously required for some aspect of intestinal cell function but that this activity is not strictly required in all intestinal cells for growth and development to proceed.

We also observed a small percentage of mosaic animals (∼1%) that became clear, vacuolated, and filled with fluid (the Clr phenotype), a defect associated with abnormal osmotic regulation (Nelson and Riddle 1984). The Clr phenotype did not become evident until ∼2–3 days into adulthood, at which time clear fluid-filled animals containing multiple vacuoles could be detected (Figure 4L). Osmotic regulation in C. elegans is controlled by four cells that comprise the excretory system, a primitive analog of the renal system of vertebrates (Nelson et al. 1983). We examined the excretory system for the presence of the pha-1 array in Clr adults. Notably, 21 of 24 Clr adults lacked detectable GFP expression in the excretory cell (one of four cells within the excretory system). In contrast, array expression in the excretory cell was detected in 25 of 25 adults that did not show the Clr phenotype. Furthermore, in the three Clr adults that showed GFP expression in the excretory cell, expression was not detected in the anatomically and lineally connected excretory duct cell. Taken together, our observations suggested a role for PHA-1 in the excretory system, in particular the excretory cell. We note, however, that correlating GFP expression with specific excretory cells using DIC optics was difficult, and the poor cellular morphology of the Clr adults further compounded this problem. Moreover, given the relatively late onset of the Clr phenotype, the role of PHA-1 in the excretory system may be subtle.

Although, as noted above, MS-minus animals grew normally to become viable adults, abnormal-looking proximal gonads were often observed, especially in older adult hermaphrodites. Specifically, PHA-1 appears to promote normal spermatogenesis, ovulation, or both. This is notable because MS gives rise to both Z1 (through MSp) and Z4 (through MSa), which produce the large majority of anterior and posterior somatic gonad cells, respectively (Figure 4A). Gonadal defects correlated with the reduction or absence of PHA-1 expression within individual gonad arms; MSa-minus/MSp-plus (Z1⁺/Z4⁻) mosaics showed defects specifically in the posterior gonad, whereas MSa-plus/MSp-minus (Z1⁻/Z4⁺) mosaics showed defects in the anterior gonad (Figure 5, A–D). Gonad arms deficient for pha-1 produced endomitotic oocytes (Figure 5, C and D), which arise under conditions where oocytes fail to undergo timely fertilization (McCarter et al. 1997, 1999). We further identified 12 animals that were missing pha-1 within sublineages of MS that give rise to the sheath and spermathecal cells of the somatic gonad (SS lineage). Although the patterns of array inheritance in these animals were often complex, we observed a clear correlation between the absence of pha-1 expression within somatic gonad arms and the incidence of fertilization defects (Figure 5, E–J). This led to a reduction in the number of sperm and to the abnormal persistence of residual bodies, which are anucleate byproducts of spermatogenesis (L’Hernault 2006). In addition, MS-minus animals often contained abnormal embryos, oocytes, or oocyte fragments within the uterus (Figure 5K). Consistent with these data, MS-mosaic animals had reduced fecundity. Whereas nonmosaic controls had an average brood size of 264 (SD = 15.1; n = 3), animals that contained one positive and one negative gonad arm had an average brood size of 144 (SD = 34.4, n = 4). Nevertheless the complete absence of PHA-1 in the MS lineage did not preclude the production of progeny as we observed a single MS-minus animal with a brood size of 201. Collectively, these findings indicate an autonomous role for PHA-1 in the somatic gonad. In contrast, we found no essential role for PHA-1 within the developing germline (Figure 4). F₁ progeny from germline-minus adults were, as expected, embryonic lethal, and displayed a phenotype identical to that of pha-1(0) animals that failed to inherit the pha-1(+) array in the parental strain (data not shown).

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Figure 5

Gonadal defects in pha-1 mosaic adults. (A–D) DIC and corresponding Hoechst staining of proximal gonad arms in an animal that is Z4-plus and Z1-minus for PHA-1. Z1 and Z4 give rise to the large majority of anterior and posterior somatic gonad cells, respectively. (A and B) Posterior gonad arm (pha-1-plus) with normal-appearing chromosomes (arrows) within the proximalmost oocyte (dashed line). (C and D) Anterior gonad arm (pha-1-minus) containing endomitotic oocytes. Arrowheads indicate swollen polyploid nuclei. Although not shown, more distal oocytes in the anterior gonad had normal nuclear morphologies. (E–H) DIC and corresponding GFP-fluorescence images from an adult with partial transmission of the pha-1-rescuing array within the MS lineage. o, oocytes; e, embryos. The proximal portion of each gonad is to the left. (E and F) Anterior gonad arm that contains normal-appearing sperm (arrows in E) within a pha-1-plus spermatheca (dashed line). (G and H) Posterior gonad arm that appears devoid of sperm within a pha-1-minus spermatheca (dashed line). (I and J) DIC and corresponding GFP-fluorescence image of the anterior proximal gonad region in an MS-minus pha-1 mosaic adult. The proximal region of the gonad arm is to the left. Note the presence of spermatocyte residual bodies (arrows in I) in the region of the spermatheca. (K). DIC image of a uterus within an MS-minus pha-1 mosaic showing abnormal oocytes or oocyte fragments that have been ovulated. Bar in A, 10 µm (for A–D); bar in D, 10 µm (for D–J); bar in K, 10 µm.

Additional phenotypes observed in pha-1 mosaic animals included irregularly shaped (Lpy) and short and fat (Dpy) larvae and adults. The Lpy/Dpy phenotypes are consistent with previously described roles for PHA-1 during embryonic morphogenesis (Schnabel and Schnabel 1990; Granato et al. 1994a; Fay et al. 2004). Though difficult to study using classical mosaic analysis, Lpy/Dpy animals could be expected to arise when the array is absent within particular regions of the epidermis or possibly when the copy number of the array within the hypodermal syncytium is strongly reduced. Notably, loss of the array within either the ABa or ABp lineages, which are the major contributors to the epidermis, was never detected in hatched larvae (Figure 4). In contrast, loss within the C lineage, which contributes to the epidermis but to a lesser extent than ABa or ABp, was detected in morphologically normal viable adults (Figure 4). Finally, we occasionally observed adult hermaphrodites that retained abnormal numbers of eggs (Egl; data not shown). Although bloated, these Egl animals had vulvae that appeared normal and were able to lay eggs. Therefore, PHA-1 may play a minor role in some aspect of vulval development or in the neurons or muscles that are required for rhythmic egg laying.