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Results

Reconstruction
The organization of the C. elegans secretory-excretory system conforms generally to that found in other rhabditidae (Chitwood and Chitwood, 1950) and consists of four cells: (1) The large, H-shaped excretory cell extends bilateral canals anteriorly and posteriorly nearly the entire length of the worm. The cell body contains an excretory sinus, a system of small channels which joins the lumena of the four excretory canals with the origin of the excretory duct. (2) The duct cell surrounds the cuticle-lined excretory duct from the origin of the duct to the pore cell boundary. A lamellar system, formed by multiple invaginations of the duct cell plasma membrane, greatly increases the surface area of the cell at the duct lining. (3) The pore cell encloses the terminal one-third of the duct and underlies the excretory pore at the ventral surface of the animal. (4) A binucleate, A-shaped gland cell extends bilaterally symmetrical processes from cell bodies just behind the terminal bulb of the pharynx anteriorly to the nerve ring where the processes join and apparently receive synaptic input. The gland cell processes are also joined across the anterior edge of the excretory cell body, where the gland cell, duct cell, and excretory cell are all joined at a tight junction. We call this point of intercellular transfer the secretory-excretory junction.

The reconstruction of secretory-excretory anatomy from transverse serial-section micrographs of a second-stage (L2) larva is shown in Figs. 1-4. These results were confirmed in a second specimen. The L2 stage precedes the molt at which dauer larvae may be formed and provides an appropriate point of reference for anatomical comparisons with dauer larvae. In Figs. 1-4, both lateral and ventral projections have been divided into two drawings for clarity. Figures 1 and 2 show ventral and lateral views of the gland and excretory cells together with an outline of the excretory duct. Figures 3 and 4 show the duct and pore cells. The figures show a portion of the worm beginning at the circumpharyngeal nerve ring approximately 65 micrometer behind the tip of the nose and ending just behind the pharyngeal-intestinal valve. This represents a length of about 30 micrometer or 8% of the total body length of a second-stage larva. We have also examined these cells in dauer larvae, L4 larvae recovered from the dauer stage, and in adults. Some aspects of subcellular organization become more elaborate with increasing maturity, although the secretory-excretory system is presumably fully functional at the time of hatching, or very soon thereafter.

FIGS. 1-4. Reconstruction of C. elegans secretory-excretory anatomy from transverse serial-section electron micrographs of a second-stage (L2) larva. The area shown encompasses 30 micrometer beginning approximately 65 micrometer from the tip of the nose. Lateral and ventral projections have been divided into two drawings each for clarity. Outlines of the pharynx and posterior portion of the nerve ring (thin dashed lines) have been included for reference. X 1900.
FIG. 1. Ventral view of the gland cell (G), the excretory cell (E), and the excretory duct (labeled in Fig. 2). Excretory canals (EC), excretory cell nucleus (EN).
FIG. 2. Lateral view of the cells shown in Fig. 1. Excretory pore (EP), excretory duct (ED), excretory cell nucleus (EN), excretory canal (EC), gland cell (G), gland cell nucleus (GN).
FIG. 3. Ventral view of the duct cell (D) and pore cell (P). Excretory duct (ED), excretory pore (EP).
FIG. 4. Lateral view of the region shown in Fig. 3. Pore cell (P), pore cell nucleus (PN), duct cell (D), duct cell nucleus (DN).

Many features of secretory-excretory morphology are visible in Nomarski interference-contrast microscopy. Figures 5 and 6 show two different focal planes of the region around the terminal bulb of the pharynx in an L4 larva. These light micrographs show the excretory cell nucleus, pore cell nucleus, excretory duct and excretory pore along the ventral midline (Fig. 5), and the duct cell nucleus and one of the gland cell nuclei just above the focal plane of the excretory cell (Fig. 6). The granular cytoplasm of the gland cell and the highly refractile secretory-excretory junction are also visible. A large nucleolus is visible in each nucleus.

Excretory Cell
The excretory cell is an exceptionally large cell, the nucleus of which is located on the ventral side of the terminal pharyngeal bulb (Fig. 5). The cell body forms a bridge which connects the subventral, bilateral excretory canals (Fig. 1). This is in contrast to some nematodes, such as Ascaris, in which the bilateral canals are connected only by a small duct (Chitwood and Chitwood, 1950). Much of the cell body is occupied by the large nucleus (Fig. 7).

The excretory canals are fluid-filled channels surrounded by the cytoplasm of the excretory cell processes. These processes, which extend both anteriorly and posteriorly from the excretory cell body, form gap junctions with the adjacent hypodermis (Fig. 9). The plasma membrane surrounding the canal does not form junctions with other tissues such as adjacent neurons or muscle cells, but the canal membranes are exposed to the pseudocoelom. Within the canals are canaliculi (small dead-end channels) which usually appear as small circles in the transverse-section micrographs (Fig. 9). The canaliculi, which are contiguous with the lumen of the canals, increase surface area. Mitochondria are a prominent feature of the canal cytoplasm. At the cell body, the canals fuse to form a sinus just anterior to the excretory cell nucleus (Fig. 8). This excretory sinus joins with the excretory duct at its origin (Figs. 8, 15), where the excretory cell, gland cell, and duct cell form a complex, branched tight junction (Figs. 16, 17) morphologically similar to the zonula occludens in vertebrate epithelial tissue. In addition to forming gap junctions with the hypodermis via the lateral canals, the excretory cell also forms gap junctions with the duct cell and the pore cell (Fig. 16).

FIGS. 5 AND 6. Nomarski micrographs taken at two focal planes through the secretory-excretory anatomy of a fourth-stage (L4) larva. X 1940.
FIG. 5. Excretory duct (ED), excretory pore (EP), pore cell nucleus (PN), excretory cell nucleus (EN), secretory-excretory junction (SEJ).
FIG. 6. Focal plane slightly above that of the excretory cell nucleus (EN). Duct cell nucleus (DN), one of the gland cell nuclei (GN), terminal pharyngeal bulb (TB), intestine (I).
FIG. 7. Excretory cell. The cell body is characterized by a prominent nucleus (EN) and sinuses (ES) which join the excretory canal (EC) lumena in more anterior sections. Gland cell process (G), neurons (N), terminal pharyngeal bulb (TB). L2. X 13 300.
FIG. 8. Detail of excretory cell sinus (ES) and duct cell (D). The sinus is open to the excretory duct at the secretory-excretory junction (SEJ). Although this L2 had been starved for 3 days prior to fixation (see text), excretory cell morphology was not appreciably affected, whereas body wall muscle filaments (mf) show considerable disorganization. Duct cell nucleus (DN), excretory duct (ED), gland cell process (G), mitochondria (m), secretory granules (sg). X 21 300.

Duct Cell
The duct cell is adjacent to the pharynx at the antero-ventral side of the terminal bulb. Its nucleus, containing a prominent nucleolus (Fig. 10), is located just anterior and lateral (either left or right) to the excretory cell body (Fig. 6). The cell cytoplasm is rich in mitochondria (Fig. 8). The duct cell completely surrounds the excretory duct from its origin to the boundary of the pore cell. The excretory duct is a channel lined with a collagenous cuticular wall and the plasma membrane of the duct cell (Fig. 10). The duct wall is continuous with the body-wall cuticle at the excretory pore. The duct forms a loop through the cell so that, in the L2, a distance of only 2 micrometer is traversed by a 9-micrometer segment of the duct (Fig. 2). The looped path taken by the duct is such that a single transverse section may transect the duct as many as four times.

The cytoplasm around the excretory duct is bordered by stacks of parallel lamellar membranes (Fig. 10). The sheetlike membrane stacks are invaginations of the plasma membrane surrounding the duct. Small vesicles are occasionally associated with these membranes. The looped path of the duct through the cell, combined with extensive lamellar folding of the adjacent cell membrane dramatically increases the surface area of the cell's interface with the duct. The lamellar profiles become more elaborate in specimens of greater maturity, so that they eventually become a dominant feature of the cell in an older adult (not shown). The duct cell of an L4 larva (Fig. 10) exhibits an intermediate degree of lamellar proliferation.

Observations of live nematodes with Nomarski interference-contrast microscopy reveal that the excretory duct of the dauer larva pulsates. Periodic swelling of the duct, beginning at its origin, is followed by a release of fluid through the excretory pore. We have not detected any musclelike structures within the duct cell, even at high magnification of glutaraldehyde-fixed specimens, so duct pulsation may be nonmuscular.

Pore Cell
A pore cell has been described in other nematode species (Narang, 1972; Dick and Wright, 1974), but C. elegans provides an example of an excretory duct contained by two cells, a duct cell and a pore cell. The structural involvement of the pore cell in C. elegans was beyond the resolution of previous histological studies (Mounier, 1981). The point at which the excretory duct is transferred from the duct cell to the pore cell is shown in Figs. 11-13. The duct cell forms a tight junction with the pore cell at the point where the duct is transferred. The pore cell surrounds the duct by wrapping around it and forming a tight junction with itself (Fig. 13). This intracellular tight junction follows along the duct to the excretory pore. In this manner the pore cell encloses the terminal one-third of the duct (a distance of about 5 micrometer in an L2 larva) and joins the duct with the external cuticle at the excretory pore (Fig. 16).

The pore cell divides the nerve bundle which connects the circumpharyngeal nerve ring with the retrovesicular ganglion. It underlies the body-wall cuticle in the immediate region around the excretory pore (Figs. 16, 17). In the dauer larva, a stage resistant to treatment with detergents and other chemical agents (Cassada and Russell, 1975), the excretory pore remains open as it is in other stages. The cuticular lining of the dauer pore lacks the radially striated layer (Fig. 20), which is characteristic of the dauer body-wall cuticle (Popham and Webster, 1978; Cox et al, 1981).

FIG. 9. Transverse section through the left posterior excretory canal (EC) in an L2. Canaliculi (c), small dead-end channels, surround and are contiguous with the canal lumen, center. Gap junctions (arrow heads) form between the excretory canal and adjacent hypodermis (H). Mitochondrion (m). X 20 500.
FIG. 10. Duct cell (D) in an L4 which developed from a dauer larva. Cytoplasm surrounding the cuticle-lined excretory duct (ED) is characterized by stacks of parallel membranes (pm) which are continuous with the plasma membrane surrounding the duct (arrow). Small vesicles (v) are occasionally associated with the membrane stacks. Secretory-excretory junction (SEJ). X 12 500.
FIGS. 11-13. Excretory duct transition between the duct cell and pore cell of an L2. X 34 400.
FIG. 11. Excretory duct (ED) entirely within the duct cell (D). Pore cell (P). Duct cell-pore cell junction (arrowhead).
FIG. 12. Three sections posterior to that shown in Fig. 11. The excretory duct is just inside the pore cell (P). The duct cell membrane (single arrowhead) and pore cell membrane (double arrowhead) are apposed, but no longer within the tight junction. Obliquely sectioned membranes have been marked with dots to emphasize boundaries between portions of the duct (D) and pore cells.
FIG. 13. Two sections posterior to that shown in Fig. 12. The pore cell (P) has surrounded the excretory duct and formed a tight junction (arrow) with itself. The small duct cell process (D) terminates within the next four sections.
FIG. 14. Excretory gland cell body morphology. The cytoplasm is characterized by an extensive network of rough endoplasmic reticulum (er) which takes on a sinuslike appearance due to the dilated cisternae. Small clusters of electron-dense secretory granules (sg) are observed, frequently in close proximity to Golgi complexes (gc). Ribosomes and mitochondria (m) are abundant. The area shown is located just anterior to the gland nuclei. Hypodermis (H), terminal pharyngeal bulb (TB), pharyngeal-intestinal valve (PIV). L2. X 1 5 300.

FIGS. 15-18. Excretory gland morphology at selected regions anterior to the cell bodies. Transverse-section micrographs of L2s proceed from posterior to anterior.
FIG. 15. At the secretory-excretory junction, part of the gland cell secretory membrane (sm) is exposed to the origin of the excretory duct (ED). Filaments protruding from the excretory cell sinus (ES) can be seen in the duct. The two gland cell processes (G) are also visible. X 23 100.
FIG. 16. The gland processes fuse, forming a cytoplasmic bridge rich in secretory granules (sg). This portion of the excretory gland is bound to the duct cell (D) and excretory cell (E) by a complex tight junction (tj). Gap junctions join the excretory cell to the duct cell (double arrowheads) and pore cell (single arrowhead). Pore cell (P), excretory pore (EP), Golgi complex (gc), secretory membrane (sm). X 17 800.
FIG. 17. The cytoplasmic bridge region reveals that the secretory membrane (sm) can be oriented such that the channels are cut transversely rather than longitudinally. In this orientation, the tight junction (tj) encircles the secretory membrane. X 17 000.
FIG. 18. A single excretory gland process (G) is formed by the fusion of the two anterior gland processes (see Fig. 1) just before entry into the base of the circumpharyngeal nerve ring. The gland cell presumably receives synaptic input from one or more neurons (N). One possible junction appears as a dense plaque (arrow). X 22 100.
The pore cell cytoplasm completely lacks the lamellar sheets found in the duct cell. The cytoplasm, however, contains numerous mitochondria, rough endoplasmic reticulum, and Golgi complexes (Fig. 16), suggesting the cell may secrete the cuticle around the pore. The pore cell forms an extensive gap junction with the excretory cell (Figs. 16, 17).

Excretory Gland Cell
The excretory gland cell is a binucleate, A-shaped cell whose bilateral, subventral nuclei are located just posterior to the pharyngeal-intestinal valve (Figs. 1, 2, 6). An extensive network of endoplasmic reticulum (ER) is the most prominent feature of the cell body cytoplasm (Fig. 14). The dilated cisternae of the rough ER give the cell a sinuslike appearance. The cytoplasm is densely populated with ribosomes and contains numerous mitochondria. Small clusters of electron-dense granules are frequently found in close proximity to Golgi complexes. These membrane-bound granules are presumably transported anteriorly and are concentrated where the bilateral processes fuse, forming a granule-filled bridge across the anterior edge of the excretory cell body (Figs. 15-17). Several classes of secretory granule can be distinguished on the basis of electron density in osmium-fixed specimens. A uniquely specialized portion of the gland cell membrane, the "secretory membrane" (Figs. 15-17), is connected to the origin of the excretory duct. We have not observed granules within the duct or in the excretory sinus.

Anterior to the granule-filled bridge of the gland cell, the bilateral processes separate again but are fused a second time just prior to entry into the base of the circumpharyngeal nerve ring (Fig. 18). Presumably, the gland cell receives synaptic input from one or more of the many neurons surrounding the anterior tip of the cell. One possible synaptic junction can be seen as a dense plaque in Fig. 18.

The gland cell can be stained with the histochemical reagent paraldehyde-fuchsin (PAF). The portion of the cell which stains most intensely is the region where secretory granules are most concentrated (Fig. 23). Although PAF-staining is sometimes considered to be specific for neurosecretory cells, we cannot resolve any PAF-positive neurons in C. elegans. The only tissues found to stain positively are the excretory gland cell, the lining of the pharynx, the body-wall cuticle and, rarely, the pharyngeal gland (g1 and g2) cell bodies (Figs. 23, 24).

Secretory-Excretory Junction
The secretory-excretory junction, centered at the anterior edge of the excretory cell body, is the point at which the gland and the excretory cell converge with the duct cell at the origin of the excretory duct. A tight junction binds the three cells together (Figs. 16, 17). The junction, surrounded by a diffuse band of dense cytoplasm, is pentilaminar, and 90-110 Angstrom wide in four animals from which measurements were taken (high-magnification micrograph not shown). The two adjacent membranes appear to be fused or very closely apposed.

At the extreme posterior origin of the duct, the excretory sinus opens directly into the open channel. This area contains an accumulation of filaments of varying lengths and orientations which protrude from the sinus into the duct (Figs. 8, 15). Directly anterior and adjacent to this point, the gland cell is connected to the excretory duct via the secretory membrane (Figs. 15, 20). Thus, any glandular secretions entering the duct conceivably may also enter the excretory sinus (Fig. 20). The gland-duct junction is between the postero-dorsal side of the gland cell "bridge" and the ventral edge of the excretory duct. The secretory membrane is encircled by a tight junction (Fig. 17). Its orientation with respect to the axis of the worm is quite variable, so that it was sectioned longitudinally in Fig. 16, but transversely in Fig. 17. A similar comparison between such orientations can be made between Figs. 19 and 20. Figures 16, 17, and 19-22 all show portions of the secretory membrane and the adjacent tight junction in slightly different orientations. In some orientations, the secretory membrane appears to be structured into an assembly of "secretory channels." The membrane-enclosed channels are about 40-50 nm in diameter (Fig. 19), and are lined with amorphous, dense material.

Physiological and Developmental Variations in Excretory Gland Morphology
In L2 and post-dauer L4 larvae, and in adults, numerous secretory granules are concentrated in the gland cell near the secretory-excretory junction. Similar granules are associated with Golgi complexes in the cell bodies. The gland cell enlarges in proportion to the size of the growing animal, and adults contain many more granules than do young larvae. In two L2s and in two post-dauer L4 larvae examined, the majority of the secretory granules are extremely osmiophilic, whereas in two adults examined, a greater proportion of the granules are of intermediate and low electron density (not shown).

Dauer larvae totally lack secretory granules. Whether the dauer stage is induced by starvation (Fig. 19) or by exogenously added pheromone in the presence of abundant food (Fig. 20), the gland cell cytoplasm appears to contain only a loose membranous network. In the gland cell bodies, the nuclei, mitochondria, and ribosomes are visible but are narrowly confined within a fibrous cytoplasmic network as if the cisternae of the endoplasmic reticulum swelled to occupy all available extraorganellar volume. Near the secretory-excretory junction, the intracellular network may be ER, or the membranous residue of depleted secretory vesicles, or a combination of both. Because the absence of secretory granules was observed in two starvation-induced and two pheromone-induced (nonstarved) dauer larvae, we conclude that the change in gland morphology is correlated with the developmental state itself, rather than the nutritional condition of the animal prior to dauer larva formation. We also examined the gland in an L2 larva that had been starved by incubation in M9 buffer for 3 days, the approximate age of the dauer larvae studied. Although a reduced number of secretory granules was observed in the starved L2 (Figs. 8, 21), the cellular morphology remained similar to that of well-fed worms (Figs. 15-17). Thus, starvation alone does not account for the type of vacuolation unique to the dauer larva gland.

FIGS. 19-22. Variation in excretory gland morphology correlated with the physiological state and/or developmental stage. X 15 000.
FIG. 19. The excretory gland of a starvation-induced dauer larva (see text) totally lacks the secretory granules normally observed in an L2 (see Figs. 15-17). This portion of the gland contains only a membranous network of cytoplasm in addition to the secretory membrane (sm). The morphology of the secretory membrane does not differ from that of the L2.
FIG. 20. Gland cell morphology of a dauer larva induced in the presence of food (bacteria) by exogenous application of a Caenorhabditis-specific pheromone (see text). Gland morphology is like that observed in the starvation-induced dauer larva (Fig. 19). This section also shows the excretory cell sinus (ES) and the gland secretory membrane (sm) simultaneously open to the duct and to each other. Excretory pore (EP), pore cell (P).
FIG. 21. Excretory gland of an L2 starved for 3 days prior to fixation. Secretory granules are present, though in reduced number, and the cytoplasmic morphology is similar to that observed in well-fed L2s (see Figs. 16, 17).
FIG. 22. Examination of the first post-dauer developmental stage (L4) reveals a return to predauer (L2) gland morphology.
FIGS. 23 AND 24. Light micrographs of paraldehyde-fuchsin (PAF) stained nematodes. Animals were grown asynchronously on plates seeded with bacteria and stained as described under Materials and Methods. X 1075.
FIG. 23. PAF-positive gland cell (G) of a nonstarved adult. The body-wall cuticle and pharyngeal lining also stain.
FIG. 24. An L4 starved for 36 hr lacks PAF staining in gland cell. Terminal pharyngeal bulb (TB), pharyngeal lining (PL).

The post-dauer fate of the gland cell also was examined (Fig. 22). Starvation-induced dauer larvae were placed in food and allowed to molt and develop into L4s. Examination of two specimens revealed that a subcellular morphology rich in secretory granules similar to those in the L2 stage was regained.

In contrast to dauer-specific elimination of secretory granules, staining of the gland cell with PAF seems to be highly dependent on nutritional state as well as developmental state. The gland cells of all growing developmental stages are strongly PAF-positive (Fig. 23). Body-wall cuticle and the pharyngeal lining also are stained. Although pharyngeal and cuticular staining are exhibited by dauer larvae, the excretory gland does not stain (not shown). Furthermore excretory gland staining is eliminated in any larval stage that has been starved for as little as 6 hr prior to fixation. A PAF-stained L4 larva, starved for 36 hr, is shown in Fig. 24. Again, pharyngeal and cuticular staining are not affected under these conditions. The loss of PAF staining is readily reversible. In one experiment, staining was restored to more than 90% (109 of 117 animals observed) of the L2 larvae that were fed E. coli for 3.5 hr after 24 hr of starvation. The ability of the gland to be stained with PAF may be correlated with a high density of secretory granules or with a higher level of gland cell activity than may be typical of either dauer larvae or starved animals per se.

Adapted by Yusuf KARABEY for WORMATLAS, 2003