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Review
. 2016 May;203(1):35-63.
doi: 10.1534/genetics.116.189357.

The Caenorhabditis Elegans Excretory System: A Model for Tubulogenesis, Cell Fate Specification, and Plasticity

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Free PMC article
Review

The Caenorhabditis Elegans Excretory System: A Model for Tubulogenesis, Cell Fate Specification, and Plasticity

Meera V Sundaram et al. Genetics. .
Free PMC article

Abstract

The excretory system of the nematode Caenorhabditis elegans is a superb model of tubular organogenesis involving a minimum of cells. The system consists of just three unicellular tubes (canal, duct, and pore), a secretory gland, and two associated neurons. Just as in more complex organs, cells of the excretory system must first adopt specific identities and then coordinate diverse processes to form tubes of appropriate topology, shape, connectivity, and physiological function. The unicellular topology of excretory tubes, their varied and sometimes complex shapes, and the dynamic reprogramming of cell identity and remodeling of tube connectivity that occur during larval development are particularly fascinating features of this organ. The physiological roles of the excretory system in osmoregulation and other aspects of the animal's life cycle are only beginning to be explored. The cellular mechanisms and molecular pathways used to build and shape excretory tubes appear similar to those used in both unicellular and multicellular tubes in more complex organs, such as the vertebrate vascular system and kidney, making this simple organ system a useful model for understanding disease processes.

Keywords: Caenorhabditis elegans; WormBook; excretory–secretory system; tubulogenesis.

Figures

Figure 1
Figure 1
Tube topologies and models for unicellular tube formation. (A and B) In multicellular tubes (A) and seamed unicellular tubes (B), adherens junctions (in black) separate and delineate the apical and basal domains. (C and D) In seamless unicellular tubes, junctions appear only at the end(s) of the tube, at the site of connection to another cell. Some multicellular tubes form by a cord-hollowing mechanism (A) involving polarized vesicle trafficking toward the lumen (arrows) (Datta et al. 2011). The excretory pore cell (in blue) (B) forms a seamed tube by wrapping and forming an autocellular junction (AJ). The excretory duct cell (in yellow) (C) initially forms a seamed tube and then converts to a seamless tube via autofusion to remove the AJ (Stone et al. 2009). The canal cell (in red) (D) forms a seamless tube, likely through a cell-hollowing mechanism involving polarized vesicle trafficking toward the lumen. The origin of relevant vesicles is not known. The intercellular junction with the duct may provide a polarizing cue that directs vesicle targeting to this region. For all tubes, both basal and apical sides may secrete extracellular matrices. For all diagrams, lumen is shown in lighter color.
Figure 2
Figure 2
The C. elegans excretory system. (A) Diagram of cells of the C. elegans adult excretory system. (B) DIC with fluorescence photograph of qpIs11[Pvha-1::gfp] adult expressing GFP in the canal cytoplasm. Arrowheads mark anterior and posterior ends of the canals running the length of the organism.
Figure 3
Figure 3
C. elegans excretory system parts list. Schematic representations of the cell types that contribute to the excretory system, their lineal origins (Sulston et al. 1983), and major transcription factor (TF) regulators or markers. Colors represent specific cell types throughout the figures. Anterior is to the left. Junctions (as visualized with AJM-1, DLG-1, or HMR-1/cadherin reporters) are represented with heavy black rings or lines. (A) Canal cell (ventral view). Two ring-shaped junctions connect the canal lumen to the duct and gland. (B) Duct (lateral view). Ring-shaped junctions connect the duct to the canal cell (right), and pore (left). (C and D) G1, G2, or G2p pore (lateral views). An AJ (arrow) seals the tube, and ring-shaped junctions connect the pore to the duct (top) and hypodermis (bottom). (E) Binucleate excretory gland (ventral view). A small ring-shaped secretory–excretory junction (SEJ, arrowhead) connects the gland to the canal cell. (F) CAN neurons (ventral view). *, putative phosphorylation. ^, markers suitable for visualizing the mature cells.
Figure 4
Figure 4
Overview of excretory system development. (A) Excretory system cells are born in disparate locations of the developing embryo and migrate to meet at the ventral midline during ventral enclosure (Sulston et al. 1983). Colors represent cell types as in Figure 3. Dashed line, ventral midline. (B) Sequential Notch and EGFR signaling break symmetry in the ABplpa vs. ABprpa lineages that give rise to the canal, duct, G1 pore, and gland cells (Sulston et al. 1983; Moskowitz and Rothman 1996; Abdus-Saboor et al. 2011). (C) LIN-12/Notch signaling breaks symmetry in the ABplap vs. ABprap lineages to promote the G2 pore vs. W neuroblast fate (Greenwald et al. 1983; Sulston et al. 1983). (D) Near the completion of ventral enclosure, the tube progenitor cells are in contact but have not yet formed junctions or lumen (Abdus-Saboor et al. 2011). Note asymmetric position of the canal cell with respect to the ventral midline. (E) By the 1.5-fold stage, the tubes have junctions consisting of AJM-1, DLG-1, and HMR-1/cadherin (heavy black lines) and a continuous lumen (white) that extends from the canal cell, through the duct and pore, and is open to the extraembryonic environment (Stone et al. 2009). Arrowhead, canal–duct intercellular junction. The gland cells also connect to the canal and duct at this region. Arrows, duct and pore AJs. The duct AJ autofuses at this stage (dashed lines) (Stone et al. 2009). (F) By late embryogenesis, the tubes have undergone morphogenesis to adopt their characteristic shapes. (G) By late in the first larval stage (L1), the G1 pore has delaminated and lost all its prior junctions and lumen, and the G2 cell has replaced it as the pore (Sulston et al. 1983; Parry and Sundaram 2014). Note that in F and G, the canal cell arms are drawn much shorter than actual length, and rotated relative to their actual lateral positions, to show cell shape; in particular the canal arms reach the midbody by late embryogenesis and the rectum by late L1 stage.
Figure 5
Figure 5
Excretory canal cell structure and ultrastructure. (A) TEM of 420 min (comma stage) embryo canal, duct, and pore (tinted as in previous pictures) shows canal lumen developing at the point where canal contacts the duct (TEM courtesy of Shai Shaham, The Rockefeller University). Nuc, cell nuclei; Lu, lumen. (B) TEM of transverse section of an adult anterior canal, shows multitude of canalicular vesicles, some connected to the round central lumen and/or to each other, while others are disconnected. MTs surround the canaliculi, as do ER and mitochondria. The left side of this section is almost entirely gap junction, while the right side is separated from muscle by basal extracellular matrix. The terminal web is stained lightly in this section and surrounds the central lumen (M. Buechner, D. Hall, E. Hedgecock, unpublished results). Note that the membrane of the canalicular vesicles stains more darkly than does the plasma membrane or luminal membrane. (C) DIC micrograph of a section of the posterior canal in N2 adult (M. Buechner and E. Hedgecock, unpublished results). Boundary between lumen and cytoplasm (red guidelines) is highly refractive and shows up well in DIC. Boundary between cytoplasm and hypodermis (blue guidelines) is more difficult to see. (D) High-resolution TEM of canalicular vesicles in adult exc-7(rh252) mutant, in which the cytoplasmic contents appear more dilute. Each canalicular vesicle is surrounded by proteins protruding into the lumen, that appear reminiscent of the lollipop structure of ATPase (M. Buechner, D. Hall, E. Hedgecock, unpublished results).
Figure 6
Figure 6
Canal cell outgrowth. (A) Canal length extends greatly during the L1 stage. Diagrams to scale of animals with left canals shown in red. At hatch, the posterior canals extend only as far as the gonad (light gray). During the next 8 hr the canals extend to the anus (black diagonal line) and have reached full length by the beginning of L2 stage, while the animal has also lengthened considerably. (B) Transverse section of the animal near the canal cell body. The cell body (red) stretches across left and right muscle quadrants beneath the pharynx and extends canals to the hypodermis. Some basal extracellular matrices (basal lamina) are shown, incompletely, in gray. Canal arms must cross the hypodermal basal lamina to extend posteriorward alongside the hypodermis and the CAN neuron (brown). (C) Growth of the tip of a canal during wild-type L1 stage, adapted from fluorescence micrograph (Kolotuev et al. 2013). The basolateral surface of the canals precedes the end of the lumen (apical surface) by several micrometers. In addition, large varicosities of the cytoplasm appear at regular intervals along the canal during growth, while the lumen diameter is less variable. (D) Model for canal tip outgrowth, adapted from Shaye and Greenwald (2015) and Khan et al. (2013) (not to scale). EXC-6/INF2 formin is spread throughout the canal and concentrated at varicosities and especially at the canal tip, where it helps to form a large actin patch to push the membrane forward. EXC-6 is also associated with MTOCs that nucleate MTs growing predominantly toward the cell body. Actin filaments at the tip may extend to connect the basolateral surface to the terminal web of the apical surface, providing guidance for growth of that surface as well. Growth of the apical surface may occur through ERM-1, RAL-1, and exocyst (not shown; see Figure 7)-mediated fusion of canaliculi to the luminal surface, which allows water to pass through aquaporin AQP-8 into the canal lumen; increased osmotic pressure drives luminal expansion.
Figure 7
Figure 7
Model for vesicle trafficking in the canal cell. Speculative model of factors allowing extension and maintenance of the luminal surface. Intermediate filaments, ACT-5/actin, ERM-1, and SMA-1/βH-spectrin surround the luminal surface (Gobel et al. 2004; Praitis et al. 2005; Khan et al. 2013; Kolotuev et al. 2013), where they maintain the smooth diameter of the lumen. ERM-1 interacts with AQP-8 on canaliculi (Khan et al. 2013), and RAL-1 GTPase on canaliculi and the PAR complex on the apical surface attract the exocyst to fuse canaliculi to the apical surface (Armenti et al. 2014), which could allow the activity of vacuolar ATPase to drive water into the lumen via AQP-8 (Figure 6D, Figure 8) (Khan et al. 2013). Various marked endosomes can be viewed moving anteriorward and posteriorward throughout the length of the canals (H. Al-Hashimi and M. Buechner, unpublished results). The canal cytoplasm is rich in MTs (Shaye and Greenwald 2015), which presumably form tracks for this movement. By analogy to its role in vertebrate kidney proximal tubules (Chou et al. 2016), EXC-4/CLIC may mediate vesicle movement in growing canals. In addition, two sets of protein cascades promote trafficking through recycling endosomes: EXC-9/CRIP (Tong and Buechner 2008), EXC-1/IRG (K. Grussendorf, D. Hall, M. Buechner, unpublished results), and EXC-5/FGD (Mattingly and Buechner 2011) activate CDC-42 (Olson et al. 1996; Gao et al. 2001) to stimulate growth of actin filaments and/or transport of endosomes along those filaments. Similarly, CCM-3 and the STRIPAK complex also activate CDC-42 to promote trafficking, possibly toward or from the Golgi (Lant et al. 2015). Presumably, some feature of a weakened or growing luminal cytoskeleton activates EXC-9 and/or CCM-3 to strengthen the cytoskeleton at these points. The recycling endosomes are hypothesized to allow maintenance and/or growth of the lumen, presumably by supplying lipid and/or membrane proteins either directly to the luminal membrane, or indirectly via the canalicular vesicle membrane.
Figure 8
Figure 8
Speculative model of osmoregulatory function of the excretory canals. Excess organismal liquid may flow from the hypodermis into the canals via the many gap junctions between the two tissues and/or may cross the basement membrane from the pseudocoelom to the canals. Canalicular vesicles contain vacuolar ATPase and aquaporin AQP-8 (Khan et al. 2013; Kolotuev et al. 2013). Depending on environmental osmolarity, canaliculi may store water or transport water into the lumen. ATPase pumps protons into the lumen, which may attract a counter-ion into the lumen via EXC-4/CLIC or another associated channel (Berry et al. 2003; Berry and Hobert 2006; Ulmasov et al. 2009). The sodium-proton exchanger (NHX-9) is hypothesized to prevent acidification of the lumen, increasing osmolarity of NaCl sufficient to draw water into the lumen of the channel. Water then flows through the excretory duct and pore to the outside environment. In this model, the duct cuticle and membrane are impermeable to water. Pumps and channels in the duct might retrieve the NaCl to prevent its loss; or pumps and channels in the hypodermis could replenish Na+ and Cl from the environment.
Figure 9
Figure 9
Excretory duct and pore structure and ultrastructure. (A) Early L1 larva expressing apical junction marker AJM-1::GFP (Koppen et al. 2001). The duct and pore are located just anterior and ventral to the posterior bulb of the pharynx (ph). Arrow, pore cell junctions; arrowhead, junction between duct and canal. (B) let-653(cs178) early L1 larva. The spherical dilation near the duct–canal junction is characteristic of mutants with a block in duct or pore lumen continuity (Stone et al. 2009; Mancuso et al. 2012). (C) Duct and G1 pore marked with dct-5promoter::mCherry (cell bodies) and AJM-1::GFP (apical junctions). (D) Duct marked with lin-48promoter::mRFP (cell body) and LET-653::GFP (lumen, line) (H. Gill, J. Cohen and M. Sundaram, unpublished results). Photos in C and D are courtesy of Fabien Soulavie (University of Pennsylvania). (E) TEM of duct lumen in an L4 hermaphrodite. Arrowheads point to membrane stacks (lamellae) that surround the apical membrane. Line indicates lumen, surrounded by darkly staining cuticle, which has detached from the apical membrane in this specimen. TEM image is courtesy of John Sulston (Medical Research Council). (F) TEM of duct and G1 pore in a 420-min (comma stage) embryo. Lines indicate fibrous apical ECM in the lumen. TEM image is courtesy of Shai Shaham (The Rockefeller University).
Figure 10
Figure 10
EGF-Ras-ERK signaling promotes duct vs. G1 pore fate. (A) The biased competition model postulates that asymmetry in canal position results in the left member of the duct/G1 equivalence group receiving more canal-derived LIN-3/EGF signal and therefore becoming the duct cell (Abdus-Saboor et al. 2011). An unknown lateral inhibitory mechanism (Sulston et al. 1983) prevents the cell on the right from receiving or responding to the EGF signal. (B) The EGF-Ras-ERK pathway acts via LIN-1/Ets and EOR-1 to promote duct fate (Howard and Sundaram 2002; Abdus-Saboor et al. 2011). Asterisks indicate potential ERK-dependent phosphorylation (Jacobs et al. 1998; Howell et al. 2010). (C and D) Reduced EGF-Ras-ERK signaling causes a duct-to-G1 pore fate transformation. Constitutive signaling causes a G1 pore-to-duct fate transformation; the two ducts fuse to make a binucleate duct (Yochem et al. 1997; Abdus-Saboor et al. 2011). (E) In mosaics lacking let-60/Ras activity in the ABpl lineage, the duct and G1 pore progenitors switch fates (Yochem et al. 1997). (F) Physical or genetic ablation of the canal cell prevents duct and G1 pore tubulogenesis (Abdus-Saboor et al. 2011).
Figure 11
Figure 11
G2 and W epidermal cells and the G1-to-G2 pore swap. (A–C) Schematic ventral views of apical junctions in the pore region, with G2 and W flanking the G1 pore opening, based on Abdus-Saboor et al. (2011). (A) Lima bean-stage embryo, when the pore tube first forms. (B) Threefold embryo. G2 and W have rectangularly shaped apical junctions. (C) Early L1. G2 and W junctions are ring shaped. (D) At mid-L1, during the G1-to-G2 swap, G1 appears to stretch dorsally and “unzip,” while G2 concomitantly forms an autojunction and “zips up” to form a replacement pore (Parry and Sundaram 2014). An unknown Ras-dependent signal (?) from the duct may promote G1 junction loss (Parry and Sundaram 2014). (E–G) L4 excretory system. (E) In wild type, G2p is the pore. (F) In the absence of G2, as in lin-12(lf) mutants, there is no longer a pore after G1 delaminates, and the duct connects directly to the hypodermis (Abdus-Saboor et al. 2011). (G) In the absence of G1, as in let-60(gf) mutants, the duct connects directly to the hypodermis (see Figure 10D). G2p usually wraps around the base of the duct tube and occasionally inserts to form a true pore (Abdus-Saboor et al. 2011).
Figure 12
Figure 12
The excretory gland and CAN neurons. (A) The A-shaped excretory gland (gl) visualized with B0403.4promoter::GFP (Hunt-Newbury et al. 2007). Image is courtesy of Don Moerman (University of British Columbia). (B) The gland empties into the canal sinus at the secretory junction. Note luminal matrix (gray). Black arrowheads, junctions. TEM image of late threefold embryo is courtesy of Richard Fetter and Cornelia Bargmann (The Rockefeller University). (C) CAN neuron (lateral view) visualized with ceh-23promoter::GFP (Forrester and Garriga 1997).
Figure 13
Figure 13
Adenophorean ES system. Diagram of ES system from Monhystera disjuncta, based on drawings by Bird and Bird (1991) and van de Velde and Coomans (1987).

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