Morphogenesis of the <i>C</i>. <i>elegans</i> Intestine Involves Axon Guidance Genes

<div><p>Genetic and molecular studies have provided considerable insight into how various tissue progenitors are specified in early embryogenesis, but much less is known about how those progenitors create three-dimensional tissues and organs. The <i>C</i>. <i>elegans</i> intestine provides a simple system for studying how a single progenitor, the E blastomere, builds an epithelial tube of 20 cells. As the E descendants divide, they form a primordium that transitions between different shapes over time. We used cell contours, traced from confocal optical z-stacks, to build a 3D graphic reconstruction of intestine development. The reconstruction revealed several new aspects of morphogenesis that extend and clarify previous observations. The first 8 E descendants form a plane of four right cells and four left cells; the plane arises through oriented cell divisions and VANG-1/Van Gogh-dependent repositioning of any non-planar cells. LIN-12/Notch signaling affects the left cells in the E8 primordium, and initiates later asymmetry in cell packing. The next few stages involve cell repositioning and intercalation events that shuttle cells to their final positions, like shifting blocks in a Rubik’s cube. Repositioning involves breaking and replacing specific adhesive contacts, and some of these events involve EFN-4/Ephrin, MAB-20/semaphorin-2a, and SAX-3/Robo. Once cells in the primordium align along a common axis and in the correct order, cells at the anterior end rotate clockwise around the axis of the intestine. The anterior rotation appears to align segments of the developing lumen into a continuous structure, and requires the secreted ligand UNC-6/netrin, the receptor UNC-40/DCC, and an interacting protein called MADD-2. Previous studies showed that rotation requires a second round of LIN-12/Notch signaling in cells on the right side of the primordium, and we show that MADD-2-GFP appears to be downregulated in those cells.</p></div

Adherens junction patterns in wild-type and mutant intestines.

<p>The images show adherens junctions (white) in the intestines of embryos with the indicated genotypes; the embryos are about 1–2 hours before hatching. The adherens junctions were stained with mAbMH27; the region shown corresponds to the int1-int5 rings in wild-type animals. In three dimensions, the adherens junction is a polygon surrounding the apical face of a cell (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.g001" target="_blank">Fig 1H</a>). The contiguous adherens junctions of adjacent cells cannot be resolved by light microscopy, and so appear as a single bars of immunostaining. Thus, the 4-cell int1 ring is expected to show four longitudinal bars of staining, and each of the 2-cell int rings is expected to show two bars. The images here are maximum intensity projections to show all of the longitudinal bars simultaneously. Intestinal cells were scored in groups as explained in the legend for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.t004" target="_blank">Table 4</a>, and abnormal cell counts are indicated in red.</p

Intestine anatomy.

<p>(A) Schematic diagram of selected intestinal rings (int rings) and the terminal valve ring in a newly hatched larva. R (right) and L (left) designate the positions of the labeled cells at the E16 stage. The int rings are color coded with different hues; R and L cells within each int ring are colored with different saturations of the same hue. The arrows shown for the body axes indicate the direction of view for all optical stacks. For example, views of optical sections through either the left or right side of the primordium will be displayed with anterior to the left and dorsal top. (B) Diagram of transverse sections through a single embryo at about 515 minutes, showing cell and tissue boundaries. The drawings are tracings from serial-section electron micrographs available at <a href="http://www.wormimage.org/imageList.php?wormName=embryo_RDD" target="_blank">http://www.wormimage.org/imageList.php?wormName=embryo_RDD</a>. Each of the four muscle quadrants consists of a longitudinal row of proximal muscles (with respect to the midline) and a single row of distal muscles. The quadrants also include several muscle processes that are not included in the diagram. (C and D) Images of live, newly hatched larvae expressing transgenic reporters for intestinal cells (green) and for all membranes (red). Panel C is an optical plane through the center of the intestine, showing the closed lumen, and panel D is an optical plane near the surface of the intestine. The inset in panel C shows a single int ring; the membranes around the lumen appear thicker than the lateral membranes because of the numerous microvilli in the brush border. (E and F) Images from 3D reconstructions of the E16 primordium (panel E; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.s005" target="_blank">S1 Video</a>) and of the intestine in a newly hatched larva (panel F; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.s006" target="_blank">S2 Video</a>). The right side of the primordium in panel E is shown as a mirror-right image for comparison with the left side view, and to match the orientation of optical sections presented throughout the paper. (G) High magnification view of the int3 and int4 rings from the reconstruction as in panel F. The 4R cell is removed to show internal cell surfaces, the intestine lumen, and adherens junctions flanking the lumen. Note that the reconstructed apical surface is flattened or closed; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.s004" target="_blank">S4A Fig</a>. (H) Diagram of the intestinal lumen after it opens. (I) Diagram of the basal surfaces of intestinal cells showing possible diagonal contacts between the lateral faces of four cells in adjacent int rings; RaL = Right to anterior Left, LaR = Left to anterior Right. (J) Diagram of the basal surface of a tube of hexagonally packed intestinal cells, cut along the ventral midline and unrolled. Phantom cells (dashed lines) are included to show the complete pattern of diagonal contacts. Panels C and D show strain JJ2360. Bars = 5 microns.</p

Intestinal twist with respect to surrounding tissues.

<p>(A) The top row is a diagram of the rotating cells and surrounding tissues over time; labeling as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.g001" target="_blank">Fig 1A and 1B</a>. The bottom row shows representative images of transverse projections through optical stacks of live embryos; the top and bottom of each stack is indicated (purple lines), and the stacks are rotated to such that the midline is vertical. Additional images and details of the surrounding cells are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.s002" target="_blank">S2 Fig</a>. (B) Image sequence showing the dorsal roof of the primordium over time, imaged at a horizontal plane through the bz1 region (bracket) as defined in panel A. The valve cell v3D invariably rotates counterclockwise under bz1, while R cells typically shift clockwise for variable distances under bz1 (305 minutes). Thereafter 3R, 4R, and later 2R rotate clockwise past bz1, while the int1 and int5 rings shift counterclockwise to center under bz1. (C) Diagram showing the leading poles of R cells (black diamonds) with respect to surrounding, non-intestinal tissues. The data compares wild-type and <i>unc-6</i> mutant embryos, scored between about 361–433 minutes (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.t003" target="_blank">Table 3</a>). Note that all wild-type R cells have moved clockwise (cw) past the dorsal midline, but that <i>unc-6</i> R cells have moved either clockwise or counterclockwise (ccw). (D,E) Transverse and horizontal planes showing, respectively, the clockwise, pre-rotation shift of 2L over ventral neurons (panel D) and behind v3V (panel E). Note that 2R has relatively little contact with the ventral neurons after the shift. (G) <i>lin-12</i> mutant embryo imaged at a ventral horizontal plane through the midline cell v3V (left panel), and at a dorsal horizontal plane through bz1 (right panel). Note that 2L and 2R both contact v3V and ventral neurons, and that the int3 and int4 rings failed to rotate. Panels = A,B, E, F [JJ2360], G [JJ2942]. Bars = 5 microns.</p

Int ring rotation defects in UNC-6/netrin pathway mutants.

<p>(A-D) Examples of defects in the E20 primordium of <i>unc-6</i> mutant embryos; similar defects are observed in <i>unc-40</i> and <i>madd-2</i> mutant embryos (Tables <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.t003" target="_blank">3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.t005" target="_blank">5</a>). (A) Right side of an <i>unc-6</i> primordium showing the lack of L cell rotation; note that 2L, 3L, and 4L each contacts ventral neurons (n). (B-C) Dorsal views of the <i>unc-6</i>, E20 primordium showing no rotation of the int2-4 rings (panel B) and a reversed, counterclockwise rotation of the int2 ring (panel C). (D) Left-dorsal side of the E20 primordium showing partial interdigitation of R and L cells within each of the int3 and int4 rings. (E-I) Transverse views through successive int rings in wild-type or mutant embryos, as indicated. Two <i>unc-6</i> embryos are shown to illustrate directional variation in the partial rotations of the int rings. (J, K) Dorsal surfaces of the <i>unc-6</i> primordium at the indicated times, showing normal RaL asymmetry. (L) Ventral view of an <i>unc-5</i> primordium, showing the normal, pre-rotation, clockwise shift of 2L across ventral neurons and behind v3V. (M) Ventral view of an <i>unc-6</i> primordium showing an abnormal, counterclockwise spreading of 2R over ventral neurons. (N,O) Image sequence and diagram showing the counterclockwise rotation of the int2 ring in a <i>madd-2</i> embryo; panel N shows the ventral surface of the primordium, just above ventral neurons, and panel O shows the dorsal surface of the same primordium. Note the ectopic, ventral RaL contact between 2R and 1L (panel N), and the simultaneous shift of 2R away from the dorsal midline (panel O). Panels = A-D, F, J, K, M [JJ2534], E [JJ2360], G [JJ2530], H,N,O [JJ2528], I [JJ2529]. Bars = 5 microns.</p

Intercalation/closure of the int5 ring.

<p>(A) Transverse view of the reconstructed primordium showing the int5 and int6 cells during int5 intercalation/closure. The nascent apical surface is indicated at 265 and 305 minutes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.ref006" target="_blank">6</a>]. (B) Heat-map representation of GFP fluorescence intensity (red = high) at the leading edge of 5L during intercalation. The increased signal at the leading edge is seen with both of the membrane reporters used in this study, and precedes cytoplasmic flow into the same region (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.s007" target="_blank">S3 Video</a>). We interpret the increased signal as resulting from the transient presence of four membranes (protrusion plus flanking cells), relative to the signal from two membranes at other cell contacts between intestinal cells. (C) Image sequence showing the left side of the E16 primordium during int5 intercalation. The dorsal surface of 3L constricts (purple, inverted double arrow) as 5L begins to shift dorsally. After reaching the roof, 5L expands longitudinally (orange double arrow) as the dorsal surface of 4L constricts (blue, inverted double arrow); see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.s007" target="_blank">S3 Video</a>. (D, E) Image sequences of a wild-type embryo (D) and an <i>efn-4</i> mutant embryo (E) during int5 intercalation, imaged at an optical plane near the roof of the primordium. Note the constriction of 3L and 3R in both embryos. In the <i>efn-4</i> mutant, 3R partially separates from 3L, and detaches entirely from the roof of the primordium; 3R rejoined the roof 64 minutes later. Panels = B,C,D [JJ2360]. E, [JJ2486]. Bars = 5 microns.</p

Summary of intestinal morphogenesis.

<p>(A-C) Key frames from the movie of the reconstructed primordium (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.s005" target="_blank">S1 Video</a>). Major developmental events are summarized for each panel (italics). (A) Left side of the primordium from 169–401 minutes. The dorsal side of the primordium is up and anterior is to the left. (B) The panel at left shows the left side of the primordium at 409 minutes. The right panels show the primordium rotated face on, with successive int rings removed to show rotation of the int2-4 rings. (C) Dorsal views of the primordium at 321 and 393 minutes. (D) Quantitation of R cell rotation in the reconstruction, measured in degrees from the dorsal midline. The circumference of the primordium is about 47 microns, so the rotation of int4 averages about 0.1 μm/min.</p

Intercalation/closure of the int2 ring.

<p>(A) Left lateral view of the E16 primordium during int2 intercalation/closure; note that cells flanking 2L do not appear to undergo dorsal constriction. In this embryo, 2R intercalations lags 2L intercalation, but 2L does not cross the dorsal midline to connect with 2R. (B) Diagram of the two modes of int2 intercalation/closure. Before intercalation, 2L shifts clockwise across ventral neurons and behind the ventral midline valve cell, v3V (red dashed outline). The int1 cells spread ventrally prior to division, across the anterior face of the int2 cells. The int2 cells either intercalate to close the ring and then rotate clockwise (mode 1), or 2L stalls and 2R completes the ring by rotating across the dorsal midline (mode 2). (C) Transverse view of int2 intercalation, mode 1, from the 3D reconstruction. (D) Transverse view of int2 intercalation, mode 2. The dorsal extension of 2L stopped when 1L divided, and the ring was closed by 2R. (E) Left side view of int2 intercalation, mode 2. Here, 2L extends dorsally between 321 and 329 minutes, at which point 1L divides and 2L stalls. Int2 closure occurs only after 2R reaches 2L on the left side. Note that the elapsed time from extension to closure is 58 minutes in this embryo, compared to 24 minutes for mode1 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005950#pgen.1005950.g004" target="_blank">Fig 4C</a>. Panels = A,D,E [JJ2360]. Bars = 5 microns.</p

Intestinal morphogenesis defects in <i>efn-4</i>, <i>mab-20</i>, and <i>sax-3</i> mutant embryos.

<p>(A-B) Intestine primordium in <i>sax-3</i> mutant embryos; these embryos express only the general membrane reporter (red). Panel A shows dorsal and ventral sides of the E20 primordium; note that the second ring consists of three cells (2R, 3R, and 2L). The int3 and int4 rings rotated, but 2R stalled at the RaL contact between 3R and 1LD. Panel B shows a transverse view of an improperly oriented int1 ring. Both R and L cells in the int1 ring contact bz1 in most wild-type embryos (78%, n = 32) and in most <i>sax-3</i> mutant embryos (63%, n = 33). The wild-type R or L cells can cover most or all of bz1 in the remaining embryos, but never extend beyond bz1 (0/32). By contrast, R cells extended past bz1 in 14% of the <i>sax-3</i> mutant embryos. (C) Transverse projections through successive int rings in an <i>efn-4</i> mutant embryo; note that the second int ring contains three cells. (D) Left lateral view of a <i>mab-20</i> embryo showing four cells in the second int ring (3R, 3L, 2L, and 2R). (E) Dorsal view of the primordium in an <i>efn-4</i> mutant embryo. 2R intercalation stalled when 1R divided, as in wild-type embryos. However, 2R failed to resume intercalation and instead retracted. (F) <i>efn-4</i> mutant embryo expressing an <i>end-1</i>::GFP transgene from a non-integrated array. The mosaic expression in 2R shows a persistent lateral protrusion (arrow) between 1RD and 3R, but a failure in int2 intercalation/closure. (G) Early stages of the intestine primordium in a <i>mab-20</i> mutant embryo showing the normal pre-rotation shift of 2L (30/31 embryos) and normal RaL asymmetry (23/25 embryos). (H) EFN-4-GFP expression. The panel at left shows expression in the int2, int5,and int8 cells before int5 intercalation, and the panel at right shows the lack of intestinal expression at later stages. The asterisk indicates EFN-4-GFP accumulation outside the embryo, but within the eggshell. Panels = A,F [JJ2517], B,D [JJ2486], C,G [JJ2583], H [JJ2502]. Bars = 5 microns.</p

Diagonal contacts in the E8 primordium.

<p>Diagonal contacts in the E8 primordium.</p