The twisted pharynx phenotype in C. elegans
© Axäng et al; licensee BioMed Central Ltd. 2007
Received: 15 January 2007
Accepted: 01 June 2007
Published: 01 June 2007
The pharynx of C. elegans is an epithelial tube whose development has been compared to that of the embryonic heart and the kidney and hence serves as an interesting model for organ development. Several C. elegans mutants have been reported to exhibit a twisted pharynx phenotype but no careful studies have been made to directly address this phenomenon. In this study, the twisting mutants dig-1, mig-4, mnm-4 and unc-61 are examined in detail and the nature of the twist is investigated.
We find that the twisting phenotype worsens throughout larval development, that in most mutants the pharynx retains its twist when dissected away from the worm body, and that double mutants between mnm-4 and mutants with thickened pharyngeal domains (pha-2 and sma-1) have less twisting in these regions. We also describe the ultrastructure of pharyngeal tendinous organs that connect the pharyngeal basal lamina to that of the body wall, and show that these are pulled into a spiral orientation by twisted pharynges. Within twisted pharynges, actin filaments also show twisting and are longer than in controls. In a mini screen of adhesionmolecule mutants, we also identified one more twisting pharynx mutant, sax-7.
Defects in pharyngeal cytoskeleton length or its anchor points to the extracellular matrix are proposed as the actual source of the twisting force. The twisted pharynx is a useful and easy-to-score phenotype for genes required in extracellular adhesion or organ attachment, and perhaps forgenes required for cytoskeleton regulation.
The pharynx is a simple muscular epithelial tube responsible for the ingestion and maceration of food in C. elegans. There are 80 cell nuclei from five cell types present in the pharynx: muscle cells, nerve cells, marginal cells, epithelial cells and gland cells [1, 2]. The pharynx is sometimes considered to be evolutionarily related to the heart because: (i) like the heart, the pharynx is a rhythmically contracting muscular pump ; (ii) the muscle cells of the pharynx have autonomous contractile activity reminiscent of cardiac myocytes ; and (iii) ceh-22, the C. elegans homolog to the homeobox gene Nk2x.5 that plays an important role in heart development in vertebrates, participates in pharyngeal development . Parallels between pharyngeal development and mammalian kidney tubulogenesis have also been made based on the similarity of the cellular processes by which these biological tubes form de novo through epithelialization and cell polarity rearrangements .
Several C. elegans mutants have been reported to exhibit a twisted pharynx phenotype. Two of these mutants, dig-1 and mig-4, were isolated in screens for mutants with defects in the shape of the hermaphrodite gonad and likely reflect migration defects by the distal tipcells [7, 8]. The molecular identity of mig-4 is unknown. dig-1 corresponds to the predicted gene K07E12.1a/b, which encodes a large protein of 13 100 amino acids containing several EGF-like domains, immunoglobulin domains, von Willebrand factor type A domains, Fibronectin type III domains, and one Sushi domain (SCR repeat) [9, 10]. DIG-1 likely straddles the basement membrane mediating specific contacts between cells and their environments.
The uncoordinated mutant unc-61 also has a twisted pharynx. unc-61 encodes a septin, that is a GTPase required for cytokinesis and other processes requiring spatial organization of the cell cortex, and which can regulate actin dynamics as well as form filamentous polymers. Interestingly, a mutation in the other C. elegans septin, unc-59, does not cause a twisted pharynx; this is thus one of thefew phenotypes for which unc-59 and unc-61 differ .
Finally, the mnm-4 mutant was isolated in a screen for abnormal morphology of the M2 pharyngeal neurons, and also has a twisted pharynx . The molecular identity of mnm-4 is still unknown.
The aim of the present study was to better understand the twisted pharynx phenotype and its relationship to extracellular matrix components. Our observations suggest that defects at actin anchorage points to the ECM during larval growth cause the progressive twist to develop intrinsic to the organ.
Quantitation of the twisted pharynx phenotype
Degrees of twist within the isthmus in animals of various ages and genotypes.
Twist° in Individuals
Avg + SEM of |Twist|
Samples quantitated from 3D reconstructions
etIs2, Adult (1d)
17, 6, 23, 36
21 ± 6
54, 36, 45
45 ± 5
90, 72, 90, 90
86 ± 5
135, 107, 152, 163
139 ± 12
216, 190, 198, 198, 201
201 ± 4
etIs2;mnm-4, Adult (1d)
180, 198, 198, 198
194 ± 5
etIs2;mnm-4, Adult (6d)
180, 216, 144, 216, 189
189 ± 13
220, 210, 186, 231
212 ± 10
+40, 169, +242, +163
154 ± 42
170, +90, 45, 62
102 ± 32
135, 129, 79, 90
114 ± 15
etEx3;mnm-4 unc-61, Adult
129, 146, 146, 141
141 ± 4
Samples quantitated from photographs
0, 0, 0, 0
229, 264, 210
234 ± 16
220, 169, 244
211 ± 22
79, 71, 71
74 ± 3
+106, +57, +166, 114, 95, 92
99 ± 14
159, 172, 130
154 ± 12
+107, +46, +107, 108, 141
105 ± 15
209, 109, 214
177 ± 34
119, 89, 123, 89, 126, 130, 85
109 ± 8
etIs2;mnm-4, early L2
0, 67, 90, 105
66 ± 23
Samples quantitated from photographs of the same four developing individuals
etIs2;mnm-4, late L2
131, 162, 150, 67
128 ± 21
etIs2;mnm-4, early L3
214, 157, 173, 144
172 ± 15
etIs2;mnm-4, late L3
217, 203, 140, 183
186 ± 17
etIs2;mnm-4, early L4
203, 186, 187, 204
195 ± 5
Properties of the twisted pharynx phenotype
We began by studying the mnm-4 mutant and found that its pharyngeal twist is first visible post-embryonically and increases during development throughout the larval stages, rather than only in sudden jumps during molting, to reach its maximum during the fourth larval stage (Fig. 1; Table 1). Worms heterozygous for the mnm-4 mutation also exhibited a twisted pharynx, although the twist was less pronounced than in age-matched homozygous mutants (Table 1). Furthermore, we noted that mnm-4 always makes a left-handed twist, (i.e. a counter-clockwise turn of the nose end if one imagines looking at the worm along its anterior-posterior axis with the nose at the far end). We then quantitated the pharyngeal twisting and handedness also for the unc-61, dig-1, and mig-4 mutants (Table 1 and Fig. 1) and found that all mnm-4 and unc-61 animals have a left-handed pharyngeal twist, while 25% of dig-1 and mig-4 animals have either a left- or right-handed pharyngeal twist.
The double mutants mnm-4 unc-61 and dig-1;mnm-4 showed intermediate twisting phenotypes compared to the single mutants in the pair, and always twisted to the left (Table 1 and Fig. 1). This result suggests that these three genes may either interact directly or are involved in multiple steps in a single developmental process during larval growth of the pharynx. Also, it is clear that the mnm-4 left-handed twist isdominant over the ambivalence in twisting direction of the dig-1 mutant, which tentatively places mnm-4 downstream of dig-1.
Pharyngeal twist is retained in isolated pharynges, and thickened pharyngeal parts resist twisting
We were interested to test whether the force causing twisting is an intrinsic property of the organ or originates from outside the pharynx. Tothis end, pharynges where dissected away from the rest of the worm, then allowed to relax in an isotonic medium. They were then scored for the presence and extent of twist.
The twisted pharynx is fully functional
Pumping is an unlikely twisting force
mnm-4 6 days old adults showed no increased twisting compared to mnm-4 1 day old adults, indicating that pumping alone is not enough to increase the pharyngeal twist (Table 1). However, pumping rate may influence pharyngeal twisting: eat-3;mnm-4 double mutants have reduced pumping rate and show a slightly reduced twisting compared to age-matched mnm-4 mutants (Table 1 and Fig. 2I–J). However, eat-3;mnm-4 double mutants also showed a 10% reduction in pharyngeal length ("L" in Fig. 1A) compared with mnm-4 mutants, and this may have contributed to the decrease in twist if growth of the pharynx is important. mnm-4 L1 larvae kept in M9 and without foodfor three days showed no increased twist compared to newly hatched L1 larvae (data not shown), suggesting that pharyngeal growth may be required for the twisted phenotype to manifest itself. Note that healthy starved L1's have a pumping rate similar to controls (2.41 ± 0.07 pump/seconds and 2.58 ± 0.05 pump/seconds, respectively; p = 0.08, n = 12), and that starvation stimulates pumping .
The pharyngeal tendons are pulled by the twisted pharynges
Twisted pharynges have a subtle actin cytoskeleton defect
The anterior pharynx is firmly anchored to the lips by intercellular junctions, and more tenuously, via buccal cuticle
More tenuously, the cuticle lining of the pharynx is also continuous with the bodywall cuticle in the region of the arcade cells (buccal cavity). The body cuticle, buccal cuticle, and pharyngeal cuticle are all shed at each molt (Singh and Sulston, 1978), and the regeneration of new cuticle could contribute to twisting. However, as noted above, we see no sudden changes in pharyngeal twist associated with molting (Table 1). Instead, as the animal grows in size, twisting increases gradually during each larval stage, until it reaches its maximal twist by the L4 stage.
Direct contact between the basal laminae of the pharynx and body-wall is very tight in the embryo
Unattached pharynges do not twist in L1 larvae
The unc-61 mutant also occasionally shows pharyngeal attachment defects at the L1 stage. Of 57 unc-61 L1 larvae scored, 4 had normally developed pharynges that were not attached to the buccal cavity (similar to that shown in Fig. 8C), and 2 had a Pun phenotype (Fig. 8D). Neither of these phenotypes was associated with pharyngeal twisting, againsuggesting that it is not attachment to the lips that suppresses twisting at the L1 stage. mnm-4 unc-61 double mutants also showed no increase in the penetration or severity of the unc-61 pharyngeal attachment phenotype: of 51 mnm-4 unc-61 L1 scored, 4 had pharynges unattached to the buccal cavity (Fig. 8C) and 1 had a Pun phenotype.
Mini screen of adhesion mutants
Result of a mini screen of adhesion mutants scored for a pharyngeal twisting phenotype.
The twisted pharynx is a viable phenotype that worsens during post-embryonic development. Although it can be scored by light microscopy, it has sometimes been overlooked in previous mutant screens. We examined several parameters relevant to understanding the twisted pharynx phenotype. Vigorous pharyngeal motions that accompany molting  are not responsible for the twisting force since no sudden increase in twist is produced upon molting (Table 1). This result also suggests that the pharyngeal cuticle is not directly restraining an intrinsic twist. We also found that the attachmentof the pharynx to the bodywall at the lips is not preventing the pharynx from twisting in L1 larvae (Fig. 8), and that the pharyngeal tendons that connect the procorpus to the bodywall appear intact, albeit spirally oriented, in animals with a twisted pharynx (Fig. 4). Twisted pharynges were also functionally indistinguishable from normal pharynges in a bead-uptake assay and pumping rate (Fig. 3). Thus it seems that the twisted pharynx of the studied mutants is functional and properly anchored to the rest of the worm body. Therefore the phenotype likely originates from a relatively minor intrinsic defect within the mutant pharynx. The dissection experiments in which isolated pharynges retained their twisted shape, also show that the twist is an intrinsic property of the pharynges (Fig. 2).
The pharynx features two prominent cytoskeletal filamentous arrays, both of which are oriented in a radial fashion. The marginal epithelial cells have thick bundles of intermediate filaments anchored apically (to cuticle) and basally (to basal lamina) by hemidesmosomes . Since these arrays are not contractile, they likely provide strength and reinforcement to the tissue during pumping, but seem less likely to introduce twisting. Pharyngeal muscles are filled with a space-filling radial array of actomyosin filaments, again anchored to cuticle and basal lamina via specialized actin-based junctions. As the pharynx rhythmically contracts, the actomyosin array foreshortens radially to expand and contract the pharyngeal lumen. This action normally should not introduce any twisting forces, and our observations that the pharynges of 6-day old adult mnm-4 mutants twist no more than that of 1-day old adults, and that mnm-4 L1 larvae kept in M9 for ~72 hours show no twist indicate that pumping, at least if not accompanied by growth, plays a minor role, if any, in the development of the pharyngeal twist.
Twisting occurs very gradually as the animal and pharynx grows. There is no cell replacement during growth, but each existing pharyngeal muscle mustcontinuously add more actomyosin filaments and reorganize their anchorage points to accommodate growth. The time course of twisting and its steady increase matches this continual process of re-shaping the radial array. Thus the most likely cause of the twisting phenotype is a bias in the repositioning of actin binding sites as that radial array grows. The five mutants with twisted pharynges fell into two categories: the unc-61 and mnm-4 mutants always made a left-handed twist with 100% penetrance and the dig-1, mig-4 and sax-7 mutants produced a mixture of right- and left-handed twists and with a 25–30% penetrance. Logically, one might expect these mutations to affect proteinsrequired for actin anchorage, either at the basal lamina, or at the pharyngeal cuticle layer. Chiral ordering of these binding sites as they reassemble with respect to their neighbors may account for the observed tendency to generate a left-handed twist in some mutants. This model of pharyngeal twisting being caused by a chiral bias in mispositioning of cytoskeletal elements is mechanistically similar to the reorientation of collagen fibers in Roller mutants, which also shows an allele-specific handedness .
Actin filaments normally cycle their subunits via rapid "treadmilling" activity . Interference with normal regulation of actin filament length could produce the observed abnormally long filaments (Fig. 5) and also explain a steadily increasing force that could be relieved by gradualtwisting of the whole pharynx to lengthen each sarcomere. However, this mechanism seems less likely to generate twisting in a favored (lefthanded) direction.
dig-1 encodes a large extracellular matrix adhesion molecule and was originally isolated because of abnormal attachment of the gonad primordium [9, 10]. It is likely that its other morphological defects, such as the twisted pharynx, also are caused by impaired adhesion. An interesting observation unrelated to the pharyngeal twist was made when studying the dig-1;mnm-4 double mutant: the gonad arms often completely fail to develop, forming instead a small disorganized clear structure in the mid-body region, completely devoid of germ cell nuclei (unpublished observations). This suggests that mnm-4 and dig-1 have redundant adhesion functions duringgonad development such that the dig-1 gonad adhesion defect is worsened in the double mutant. Interactions between cell adhesion molecules and extracellular matrix are known to be important for gonad development[17, 28–30].
unc-61 is one of two C. elegans septins; unc-59 is the other. Septins are conserved GTP-binding proteins that polymerize to form filamentous structures that act as scaffolds for membrane- and cytoskeleton-binding proteins , a function consistent with our hypothesis that defects in cytoskeleton anchorage points is responsible for the twisted pharynx phenotype. Mutations in the C. elegans septins cause defects in distal tip cell and axon migration, as well as in post-embryonic cytokinesis [11, 26]. In all of these defects single mutants of either septin gave full penetrance, and double mutants did not exhibit worsened phenotype. However, only unc-61 displays the twisted pharynx phenotype. This suggests that their roles in pharyngeal post-embryonic development are independent and distinct from each other. The unc-61;mnm-4 double mutant did not show any increase in twisting of the pharynx nor any additive effect on the gonad phenotype (unpublished observations): it is possible that unc-61 and mnm-4 may function in the same pathway.
The twisted pharynx is a useful and easy-to-score phenotype, allowing one to screen for new genes involved in extracellular adhesion or organ cohesion. Our small pilot screen has already identified one new pharyngeal twisting mutant, sax-7, that encodes a cell adhesion molecule important for the attachment of several types of tissues, including neurons, and that, when mutated, causes pleiotropic phenotypes that include uncoordination and embryonic lethality [27, 32]. That mutants with twisted pharynges are otherwise indistinguishable from wild-type worms (e.g. they feed normally) likely explains why this subtle phenotype has mostly gone unnoticed.
The twisted pharynx phenotype is found in several C. elegans mutants that have defects in adhesion molecules or molecules that regulate attachment of the actin cytoskeleton to the cell cortex.
The twisted pharynx phenotype is not a result of pumping activity nor the consequence of an external twisting force applied to the pharynx: it results from a twisting force intrinsic to the pharynx. That force likely results from the defects in the remodeling of the actin attachment points or actin filament length during pharyngeal growth.
Nematode strains and culturing
General methods for the culture, manipulation, and genetics of C. elegans were as described . The wild-type parent for all strains was C. elegans variety Bristol, strain N2 . All strains were cultured at 20°C (unless otherwise noted) on NGM plates seeded with the E. coli strain OP50 . The following mutations and integrated sequences were used in this study:
LGII: eat-3(ad426), egl-27(n170)
LG III: cdh-4(ok1323), ceh-43(ok479), etIs2 [pRF4 pRIC-19::GFP], ina-1(gm39), mig-4(rh89), dig-1(n1321), pha-1(e2123), rhIs23 [hemicentin::GFP], Y49E10.20(ok1286)
LG IV: dpy-20(e1282), sax-7(ok1244)
LG V: mnm-4(et4), nid-1(cg119), sma-1(ru18), unc-61(e228)
LG X: adm-4(ok265), ham-2(n1323), him-4(e1266), pha-2(ad472), tag-53(gk163), zig-6(ok723)
The integrated transgene jcIs1, which carries an ajm-1::gfp reporter , was also used to visualize adherens junctions and was obtained from the C. elegans Genetics Center.
Generation of strains carrying pRIC-19::GFP
C. elegans transformation was carried out with the use of methodsdescribed by Mello and Fire . Hermaphrodites were injected with a DNA mixture containing 50 μg/m1 plasmid pRF4, which carries the transformation marker rol-6 (su1006dm) , and 50 μg/m1 plasmid pRIC-19::GFP, which expresses the RIC-19 protein fused toGFP from the native ric-19 promoter . Transgenic lines carrying the microinjected DNA on extrachromosomal arrays were established from F2 Rol progeny of injected animals. In some animals, the plasmid pMH86 containing the wild-type dpy-20 gene was used as a transformation marker when injecting dpy-20 mutant hermaphrodites; the resulting extrachromosomal array etEx2 was then crossed away from the dpy-20 background and the transgenic worms identified in later generations by their GFP expression.
Developmental stage study
For each stage of interest, worms of approximately the right sizes were picked from a mixed age plate while observed under a dissecting microscope. Accurate staging was then established by examining the developmental stage of the gonad and vulva using differential interference contrast (DIC) microscopy at 400× and 1 000× magnification using a Zeiss Axioplan 2 microscope. Staged worms were either scored directly or retrieved and mounted for confocal microscopy. For some studies, worms were mounted just before the expected molting, or just after molting had been observed.
DIC and Epifluorescent microscopy
For microscopic analysis, worms were mounted on 2% agarose pads, paralyzed with a small drop of 100 mM levamisole, and covered with a coverslip. These were then examined with a Zeiss Axioplan compound microscope using DIC optics or an FITC filter set to visualize GFP. Digital images were acquired using an attached AxioCam digital camera.
Worms expressing pRIC-19::GFP in their M2 neurons were mounted ondried agarose pads (2% in dH2O), paralyzed with a small drop of 100 mM levamisole and covered with a coverslip. The worms were examined using a Zeiss LSM 510 META system connected to an inverted Zeiss Axiovert 200 microscope. The z-stacks were projected in 360° using 32 or 64 steps and then exported as full resolution images in avi or mov format using the in-microscope software LSM 510 ConfoCor2 Combination, version 3.2. These movies were then used to determine the degree of twisting in the isthmus using video editing software (Sorenson squeeze, trial version). Worms carrying pMH86 was examined using BioRad radiance 2000 setup. The z-stackswas projected in 360° in 36 steps using the in-microscope software Laser Sharp 2000 and thereafter scored using the image processing program ImageJ. "180° twist" means that the distal ends of the M2 neurons would have to be rotated by 180° in order to be parallel with the cell bodies from which they originate.
Dissection of pharynges
Worms to be dissected were held in place on dried agarose pads (2% in H2O) covered with mineral oil. The dissection was done under a Leica stereomicroscope using a tungsten (0.5 mm) dissecting needle. Isolated pharynges were transferred to a drop of M9 on an agarose pad (2% in M9) and allowed to relax by swirling the liquid before applying a coverslip, and examined under the microscope.
Feeding and pumping rate assays
For the feeding assay, L4 larvae of N2, mig-4, dig-1 or mnm-4 genotypes were placed on nematode growth medium (NGM) plates covered with a 500:1 (vol:vol) mixture of OP50 bacteria and Fluoresbrites Multifluorescent microspheres, 0.2 μm (Polyscience, Inc.), essentially as previously described . Briefly, the mixture of bacteria and beads was added to the culture plates 20 h before the worms were allowed to feed for 30 min and then transferred to eppendorf tubes with 1 ml of M9 plus levamisole (1 mM). The tubes were centrifuged at 2000 g for 2 min at room temperature and the supernatant removed, leaving approximately 30 μl in which to resuspend the worms for transfer to a freshly made agarose pad (2% agarose in M9 buffer). The worms were covered with a cover slip and the beads observed by epifluorescence microscopy at 1 000× magnification and counted.
To investigate the transfer rate of bacteria into the intestine, batches of 15–20 worms were placed on NGM plates seeded with OP50 and beads (as above but using 1 000:1 mixtures) and allowed to explore for 2 minutes. The worms were thereafter mounted on agarose pad (2% agarose in M9 buffer) in a drop of 100 mM levamisol, covered with a cover slip and scored for the presence beads in the intestine and the pharynx using epifluorescence microscopy at 400× magnification. Worms that after 2 minutes were outside the bacteria/bead lawn or still remained where they were initially placed were excluded from the analysis.
To determine the pumping rate, eight worms of each genotype were placed onan NGM plate seeded with OP50 and observed using a stereomicroscope for ten consecutive seconds during which the number of pumps was recorded.
Starved L1s: Hermaphrodites were bleached over night on empty NGM plates. Resulting L1 larvae were transferred to 1,5 ml microcentrifuge tubes with M9 and kept at RT for ~72 hours. The tubes where thereafter emptied on unseeded NGM plates and pumping rate was scored in stereomicroscope. Only non-necrotic larvae were scored.
An electrophysiology setup was modified essentially as described in a published protocol to record electropharyngeograms . To encourage pumping, the worms were kept in Dent's saline containing 1 μM serotonin . Several dozen pharyngeal pumps were recorded from at least ten worms for each studied genotype.
Specimens were immersion fixed using buffered aldehydes and then osmium tetroxide as described previously . Three or four animals were aligned within agar blocks then embedded in plastic resin and sectioned together. Thin cross sections were collected on slot grids, post-stained with uranyl acetate and lead citrate, then examined with a Philips CM10 electron microscope. For embryo studies, a laserhole protocol was instead used to permit fixative to pass the eggshell .
For close examination of basal lamina, wild type animals were fixed by high pressure freezing and freeze substitution  before embedment into resin. Thin sections were again post-stained and examined in a Philips CM10 electron microscope. Additional wild type animals were immersion fixed and exposed to microwave energy using a Pelco Biowave oven to help aldehydefixatives and osmium fixatives to cross the cuticle efficiently. Fixed animals were then mounted in agar blocks and embedded in plastic resin following standard protocols for thin sectioning.
We thank to Peter Carlsson and Magnus Holm for comments on an earlier version of this manuscript, and Zeynep Altun for helpful discussions on terminology. We thank the Swegene Centre for Cellular Imaging at Gothenburg University for the use of imaging equipment and for the continuous support from the staff, Marika Hellqvist-Greberg for some solutions and phalloidin, and Bruce Vogel for the rhIs23 strain. Many thanks also to Eric Hanse for assistance with the electrophysiology experiments. We thank the C. elegans Genetics Center (funded by the NIH Center for Research Resources) and particularly Theresa Stiernagle for providing many of the strains used in this study. We also thank Carolyn Norris, Ken Nguyen and Frank Macaluso for help with some electron microscopy studies. The original TEM images of the wild type adult head (Fig. 3C) were donated to the archives of the Hall laboratory by Ed Hedgecock from studies originally conducted at the MRC in Cambridge. This work was supported by the Swedish funding agencies Vetenskaprådet, Cancerfonden and Carl Trygger Stiftelse, and by NIH RR 12596 (Center for C. elegans Anatomy, to DHH).
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