Cell Interactions and Patterned Intercalations Shape and Link Epithelial Tubes in <i>C. elegans</i>

<div><p>Many animal organs are composed largely or entirely of polarized epithelial tubes, and the formation of complex organ systems, such as the digestive or vascular systems, requires that separate tubes link with a common polarity. The <i>Caenorhabditis elegans</i> digestive tract consists primarily of three interconnected tubes—the pharynx, valve, and intestine—and provides a simple model for understanding the cellular and molecular mechanisms used to form and connect epithelial tubes. Here, we use live imaging and 3D reconstructions of developing cells to examine tube formation. The three tubes develop from a pharynx/valve primordium and a separate intestine primordium. Cells in the pharynx/valve primordium polarize and become wedge-shaped, transforming the primordium into a cylindrical cyst centered on the future lumenal axis. For continuity of the digestive tract, valve cells must have the same, radial axis of apicobasal polarity as adjacent intestinal cells. We show that intestinal cells contribute to valve cell polarity by restricting the distribution of a polarizing cue, laminin. After developing apicobasal polarity, many pharyngeal and valve cells appear to explore their neighborhoods through lateral, actin-rich lamellipodia. For a subset of cells, these lamellipodia precede more extensive intercalations that create the valve. Formation of the valve tube begins when two valve cells become embedded at the left-right boundary of the intestinal primordium. Other valve cells organize symmetrically around these two cells, and wrap partially or completely around the orthogonal, lumenal axis, thus extruding a small valve tube from the larger cyst. We show that the transcription factors DIE-1 and EGL-43/EVI1 regulate cell intercalations and cell fates during valve formation, and that the Notch pathway is required to establish the proper boundary between the pharyngeal and valve tubes.</p></div

Pharynx/valve morphogenesis.

<p>Top, differential interference contrast micrograph of a <i>C. elegans</i> larva, false colored to highlight the pharynx, valve and intestine. Bottom, diagram illustrating major stages in the formation of the cyst, and subsequent events as described in this report on the morphogenesis of the valve tube from the cyst; the diagram shows transverse views of all stages. Future pharyngeal and valve cells aggregate into an intermediate structure called the double plate. Laminin (bold black) at the periphery, or basal surface, of the double plate cues the opposite localization of apical proteins (blue), and apical constriction transforms the double plate into a cyst. Two future valve cells (red, called v3D and v3V) dock at the left-right boundary of the intestinal primordium (yellow). Docking begins slightly before, and continues during, apical constriction. The v3 valve cells remain at the left-right boundary until the two intestinal cells divide and form the final, four-cell terminus of the intestine. Valve cells and other cells in the cyst appear to explore their neighbors through actin-rich lamellipodia, and in many cases reposition their cell bodies. pm8 (green) and valve cells wrap partially or completely around the midline, thereby extruding the valve tube from the cyst.</p

Cyst cells probe their neighbors.

<p>(A) Embryo at the double plate stage showing LIN-12/Notch immunostaining (green) and simultaneous expression of a nuclear-localized transcriptional reporter for the pm8 family (red, <i>lin-12</i>^pm8::HIS-GFP). (B) Time-lapse sequence of the pm8 family at the cyst stage; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003772#pgen.1003772.s008" target="_blank">Video S2</a>. The pm8 family members express membrane and nuclear reporters (green, <i>lin-12</i>^pm8::mCherry::CAAX; red, <i>lin-12</i>^pm8::HIS-GFP). All pharyngeal/valve cells express an additional membrane reporter (red, <i>pha-4</i>::GFP::CAAX). Sequence shows several dynamic lamellipodia, including one from pm4L (arrow) that appears to lead it through a row of marginal cells (1 = mc1DL, 2 = mc2DL, 3 = mc3DL). (C) pm8 family members expressing the above membrane reporter (green) plus a reporter for filamentous actin (red, <i>lin-12</i>^pm8::GFP::dMoeABD). Note concentration of actin at tip of pm4 lamellipodium (arrow) and other lamellipodia. (D) Horizontal plane through the dorsal roof of the cyst, showing lamellipodia extending from pm8 and v1 and covering the anterior face of v3D; cells in this panel are identified in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003772#pgen.1003772.s001" target="_blank">Figure S1C</a>. (E) Image sequence similar to panel D, but showing HMR-1/E-cadherin expression (green, HMR-1::GFP). (F) Cartoon summarizing circumferential intercalations in the cyst. (G) Diagrams of the posterior cyst shown flayed along the dorsal margin and flattened. Superimposed on each diagram is the intercalation path of the cell outlined in bold. Bars: (A) 10 microns, (B–E) 2.5 microns.</p

Dorsal alignment of the valve and intestine.

<p>(A) Horizontal optical plane near the center of an embryo shortly after the birth of v3D; the top panel shows the E16 primordium and the bottom panel is the same plane showing all cell membranes. Note that v3D is deeply embedded between the left and right anterior intestinal cells (int1^p cells). (B) Expression of PAR-6 and HMR-1/E-cadherin at the double plate stage (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003772#pgen.1003772.s002" target="_blank">Figure S2</a> for developmental sequence). Closed arrowheads indicate contacts between intestinal cells and v3D, and open arrowheads indicate contacts between intestinal cells and other cyst cells. (C) High magnification through a plane as in panel A, but taken at the late double plate stage. A shallow cleft (arrow) occupied by a process from v3D remains at the left-right boundary between the int1^p intestinal cells. (D) Transverse plane showing the wedge-shape of the v3D cell body between the left and right int1^p cells. (E) Transverse plane just anterior to the plane shown in panel D, showing the cuboidal shapes of other double plate cells. (F) Time-lapse sequence to 350 minutes showing the intercalation of v3D dorsally across the int1^p cells. The arrow indicates a lamellipodium that leads v3D intercalation. The bold black line indicates the dorsal margin of the double plate. (G) v3D in a <i>die-1(w34)</i> embryo at 350 minutes. (H) v3D in a <i>egl-43(zu471)</i> embryo at 350 minutes; v3D has partially engulfed a cell death. Bars: (A–H) 5 microns.</p

HMR-1/E-cadherin expression and circumferential intercalations.

<p>(A) Low magnification of an immunostained embryo showing HMR-1::GFP (green) and the apical junction protein AJM-1 (red). Note the high level of HMR-1 in the region of the developing valve compared to the level in most other embryonic cells. The right panel is a high magnification of the valve region showing HMR-1 enrichment at the boundary between v1 and v3D, and around the v2R cell body. (B) Time-lapse images of a live embryo showing HMR-1::GFP on the left side of the cyst as pm8 moves ventrally; the pm8 nucleus is indicated by an asterisk. Note that HMR-1 associated with the ventral lamellipodium from pm8 (arrow) appears to split as the pm8 nucleus moves toward and into the lamellipodium. (C) Lower, sagittal focal plane of the same embryo as in panel B at 430 minutes. The pm8 nucleus has moved further ventral, and is now adjacent to mc3V (the mc3V nucleus is labeled 3). The line of HMR-1 between pm8 and mc3V is in the same position as the laminin tract shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003772#pgen-1003772-g007" target="_blank">Figure 7F</a>. (D) Right sagittal focal plane at 430 minutes of the same embryo shown in panels B and C; the HMR-1::GFP signal has been removed to show cell shapes. v2R has intercalated dorsally, with its bulk cell body past the dorsal-ventral boundary (open arrowhead) between the int1 intestinal cells. (E) Time-lapse of a <i>hmr-1(zu389)</i> mutant embryo. The v2R nucleus and most of the cell body remain below the dorsal-ventral int1 boundary through 496 minutes, although the v2R lamellipodium (closed arrowhead) has extended dorsally. (F,G) Apical junctions (red, AJM-1) in a wild-type embryo (F) and a <i>hmr-1(zu389)</i> mutant embryo (G), taken at about 440 minutes. For cell identification, the panels to the right in each figure show an image of cell bodies and/or nuclei (green) taken 1.5 microns to the left of the midline and superimposed on the apical junction image. At the stage shown, pm8 and v1 have wrapped around the midline, but not undergone autofusion into donuts; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003772#pgen.1003772-Rasmussen2" target="_blank">[22]</a> for detailed description of junctions at this stage. The apical junctions in the <i>hmr-1</i> mutants are abnormal, with a large gap in the region of the pm8, v1, and the v2 cells. Similar to the <i>hmr-1</i> embryo in panel E, the v2R cell has failed to move dorsally, although its lamellipodium (arrowhead) has intercalated between v1 and v3D. Bars: (A) 10 microns; (B–G) 2.5 microns.</p

Ventral alignment of the valve and intestine.

<p>(A) Time-lapse sequence to 360 minutes showing a horizontal plane through the ventral side of the cyst; all four valve cells in the ventral cluster are visible, and colored as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003772#pgen-1003772-g002" target="_blank">Figure 2C</a>. Note that v3V does not track the left-right boundary between the int2 cells as that boundary (white arrow) shifts clockwise (down in panel). Processes from the dorsal int1^p cells intercalate ventrally to flank v3V, and remain as the int1^p cells divide into the four int1 cells. (B) Ventral valve cells in a <i>die-1(w34)</i> embryo at 360 minutes. Note that the v2L and v3V cells directly contact the germ cell precursors (G). Bars: (A–B) 2.5 microns.</p

Valve anatomy and origins.

<p>(A) Diagram of the valve with neighboring pharyngeal and intestinal cells; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003772#pgen.1003772.s001" target="_blank">Figure S1</a> for full cell names. (B) Left sagittal (S^(L)) optical plane through a live embryo expressing reporters for all plasma membranes (<i>pie-1</i>::mCherry::PH(PLC1∂1)) and for intestinal cells (F22B7.9::GFP). The embryo also expresses a <i>pax-1</i>::HIS-GFP reporter used for spatial reference; this reporter is expressed in the nuclei of all three groups of marginal cells (numbered 1,2, or 3 throughout this report), pm8 and v1. The solid white line indicates the margins of the double plate. The germ cell precursors (G) and a cell death (x) are visible in this plane. (C) Images from 3D reconstruction of the cell interface between the double plate and E16 intestinal primordia; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003772#pgen.1003772.s007" target="_blank">Video S1</a>. Transverse views show all pharyngeal or valve cells that contact intestinal cells, and then selected subsets as labeled. Cells that undergo an additional division are labeled according to their daughters; for example, M5/I6 is the parent of the M5 and I6 neurons, and g2L/x is the parent of the left g2 gland cell and a cell death (x). (D) Reconstruction of the cyst-E16 interface. The pharyngeal and valve cells have acquired wedge shapes through apical constriction. The midline (M) is indicated here and elsewhere by a dashed arrow pointing to the anterior. Note that v3D has shifted dorsally, and begun to center on the left-right boundary between the int1^p cells. The intestinal cell int2D has shifted dorsally and behind the left int1^p cell, and is not visible in this transverse view; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003772#pgen.1003772.s007" target="_blank">Video S1</a>. (E) Reconstruction of the cyst-E20 interface. The int1^p cells have divided to form the final int1 ring, and both v3D and v3V are centered on the left-right boundaries of the intestinal cells. Bars: (B) 5 microns.</p

Capsids accumulate in the mid-pachytene gonad core as adults age at 15°C.

<p>(A–D) Longitudinal, optical sections through gonads showing increased accumulation of capsids (green) with adult age, as indicated. The gonad in panel B is immunostained for HIM-4/hemicentin (red) to visualize the apical membrane (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat-1002591-g001" target="_blank">Figure 1B</a>). Note that capsids localize predominately in the core, outside the ring channel and away from nuclei (blue, DAPI). (C) Capsids grouped in wavy lines and tangles of lines in the mid-pachytene region of a day 3 adult. (D) Low magnification of the mid-pachytene region. Note variation in capsid abundance between the two sides of the gonad core (double-headed arrow), and that the wavy lines of capsids disappear as germ cells move proximally into late pachytene (bracketed region). (E–G) The late-pachytene/diplotene region, stained and imaged as for panel B; two of the somatic sheath cells that surround the gonad are visible in this image. Germ cells from regions F and G are shown at high magnification, oriented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat-1002591-g001" target="_blank">Figure 1B</a>. An optical rotation of a similar region of the gonad is shown in Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591.s005" target="_blank">Video S1</a>. (H) Optical section through germ nuclei in the late-pachytene/diplotene region showing capsids (green) and P granules (red, αPGL-1). Note that capsids localize close to the nuclear envelope, but most are not directly on, or within, P granules. Electron micrographs of capsids within P granules are shown in Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591.s001" target="_blank">Figure S1H</a>. Scale bars: A–C, F–H (5 µm), D–E (10 µm).</p

<i>Cer1</i> GAG particles in <i>C. elegans</i> wild strains.

a<p>Data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002591#ppat.1002591-Palopoli1" target="_blank">[31]</a>.</p

<i>Cer1</i> capsids localize on microtubules.

<p>(A–F) Electron microscopy of gonads from 15°C adults; regions of the gonad are as indicated at the top of the panels. Capsids localize predominantly to the core in the mid-pachytene region, but concentrate near nuclei in late-pachytene germ cells (C–E) and in oogonia (F). Note that many capsids localize with microtubules (arrowheads) both in the core (panel A) and in germ cells (panel C). P granules are visible in panels C (arrow) and in panel D; arrowheads in panel D indicate examples of nuclear pores. (E) Cluster of capsids near the nucleus of an early oogonium. (F) A “crescent” of capsids by an oogonium nucleus; there are at least 64 capsids and numerous microtubules visible at higher magnifications of this single thin section (data not shown). Scale bars: A–F (0.2 µm).</p