Figure 1.
Top, differential interference contrast micrograph of a C. elegans 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.
Figure 2.
(A) Diagram of the valve with neighboring pharyngeal and intestinal cells; see Figure S1 for full cell names. (B) Left sagittal (S(L)) optical plane through a live embryo expressing reporters for all plasma membranes (pie-1::mCherry::PH(PLC1∂1)) and for intestinal cells (F22B7.9::GFP). The embryo also expresses a pax-1::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 Video S1. 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 int1p cells. The intestinal cell int2D has shifted dorsally and behind the left int1p cell, and is not visible in this transverse view; see Video S1. (E) Reconstruction of the cyst-E20 interface. The int1p 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.
Figure 3.
Dorsal alignment of the valve and intestine.
(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 (int1p cells). (B) Expression of PAR-6 and HMR-1/E-cadherin at the double plate stage (see Figure S2 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 int1p intestinal cells. (D) Transverse plane showing the wedge-shape of the v3D cell body between the left and right int1p 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 int1p 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 die-1(w34) embryo at 350 minutes. (H) v3D in a egl-43(zu471) embryo at 350 minutes; v3D has partially engulfed a cell death. Bars: (A–H) 5 microns.
Figure 4.
Ventral alignment of the valve and intestine.
(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 Figure 2C. 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 int1p cells intercalate ventrally to flank v3V, and remain as the int1p cells divide into the four int1 cells. (B) Ventral valve cells in a die-1(w34) embryo at 360 minutes. Note that the v2L and v3V cells directly contact the germ cell precursors (G). Bars: (A–B) 2.5 microns.
Figure 5.
Intestinal cells influence valve cell polarity.
(A) Terminal stage die-1(w34) mutant embryo. The apical junctions (green, AJM-1) are discontinuous between the pharynx and the intestine, with several nuclei (blue, DAPI) visible in the gap. Compare to the wild-type junctional pattern in panel K. High magnification panel to the right indicates the inferred apicobasal axes of an intestinal cell and a neighboring, presumptive ventral valve cell (asterisk). The valve cell is identified here by its proximity to the ventral mc3 nucleus (#3, magenta; pax-1::HIS-GFP). (B) Developmental sequence of the interface between pharyngeal/valve and intestinal cells; reporters are as in Figure 2B, but the colors are inverted to highlight the intestinal cell bodies (magenta). Note extension from int2V (bold arrow) that reaches the parent of v2L (labeled p) at 275 minutes. After the birth of v2L (asterisk; 299 minutes), int2V spreads between the posterior surface of v2L and a germ cell precursor (labeled G). The association of the intestinal cells with v2L persists as cells remodel and the interface (bracket) condenses. (C) Horizontal plane through the ventral side of the double plate and cyst showing the intercalation of the ventral intestinal cells. (D) Image sequence as in panel C but showing a die-1(w34) mutant embryo. Note that v2L and v2R (asterisks) remain in contact with the germ cell precursors (G). (E) Laminin (red) surrounds the normal cyst and intestinal primordium, except where the posterior cyst contacts intestinal cells (arrowhead); laminin also appears along the midline (M) at this stage. (F) Image as in panel E after ablating the intestinal precursor (dotted outline). Note that laminin extends across the posterior surface of the cyst (arrowhead). (G) Horizontal plane through the ventral side of a wild-type cyst showing the absence of laminin on the posterior surfaces of v2L and v2R (asterisks), where these cells contact intestinal cells (int). (H) Image of a die-1(w34) embryo oriented as in panel G, showing ectopic laminin (arrowhead) across the posterior surfaces of v2L and v2R. (I) Normal pharynx and valve in a newly hatched larva, showing parallel, radial axes of apicobasal polarity of pharyngeal cells and valve cells. (J) Wild-type larva after ablating the intestinal precursor. Note the inferred axis of polarity for the posterior valve cells is oriented to the anterior. (K) Normal apical junctions (AJM-1) and basement membrane (red, UNC-52/Perlecan) in the posterior pharynx and valve. For description of apical junction pattern relative to cell shapes, see [22]. (L) Apical junctions (dlg-1::DLG-1::GFP) in a live, wild-type larva after ablating the intestinal precursor as in panel J. Bars: (A–H) 5 microns; (I–L) 2.5 microns.
Figure 6.
Cyst cells probe their neighbors.
(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, lin-12pm8::HIS-GFP). (B) Time-lapse sequence of the pm8 family at the cyst stage; see also Video S2. The pm8 family members express membrane and nuclear reporters (green, lin-12pm8::mCherry::CAAX; red, lin-12pm8::HIS-GFP). All pharyngeal/valve cells express an additional membrane reporter (red, pha-4::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, lin-12pm8::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 Figure S1C. (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.
Figure 7.
(A) Left sagittal plane showing the pm8 family; cells express nuclear and membrane reporters as in Figure 6B. The right panel shows a time-lapse sequence of pm8 and its sister as pm8 extends ventrally; see also Video S3. (B) Transverse view of pm8 development. This embryo expresses an additional marker (arrowhead) for the midline (red, nmy-2::NMY-2::GFP). (C, D) Surface renderings of the pm8 family as shown for the wild-type embryo in Video S3, and rotated to show cell shapes. (C) die-1(w34) mutant embryo at about 450 minutes. pm8 has extended ventrally, but the pm8 nucleus remains near the midline and pm8 has not completed wrapping. (D) Image sequence of a lag-1(q385) mutant embryo, showing apparently normal pm8 wrapping. (E) pm8 family members expressing a reporter for PAR-6 (red, lin-12pm8::PAR-6::GFP) and for cell bodies (green, lin-12pm8::SAS-5::mCherry). Note that PAR-6 remains apical as pm4L (asterisk) rotates away from other family members (double-headed arrow; see also Figure 6B), and pm8 begins to wrap (arrowhead). (F) Image of the cyst as the presumptive pm8 nucleus begins to travel ventrally in close association with a tract of laminin. The right panels show transverse planes through positions a and b, and show that the laminin tract has a wedge shape centered on the mc3V nucleus. Bars: (A,E,F) 5 microns.
Figure 8.
HMR-1/E-cadherin expression and circumferential intercalations.
(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 Figure 7F. (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 hmr-1(zu389) 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 hmr-1(zu389) 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 [22] for detailed description of junctions at this stage. The apical junctions in the hmr-1 mutants are abnormal, with a large gap in the region of the pm8, v1, and the v2 cells. Similar to the hmr-1 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.
Figure 9.
Notch-related valve defects and target gene expression.
(A) Newly hatched wild-type, lag-1(q385); eff-1(ok1021) and egl-43(zu471) mutant larvae expressing a reporter for pharyngeal muscle nuclei (green, myo-2::HIS-GFP). Open arrows point to nuclei mispositioned posteriorly between intestinal or valve cells. (B) Wild-type cyst oriented as in Figure 7F, showing pax-1::HIS-GFP expression in marginal cell nuclei (#1–3) and in pm8. (C) egl-43(zu471) mutant cyst, staged and oriented as in panel B; note the lack of pax-1::HIS-GFP at the normal position of pm8. (D–G) Notch-regulated target gene expression in larvae (D and E) and in late embryos as listed; see also Figure S4. (H) Late-stage, wild-type embryo as in panels F and G, immunostained to identify pm8 (green, ref-11.8 kb::REF-1::GFP) and showing LAG-1 (red) in the pm8 nucleus. Note that LAG-1 is absent from other embryonic nuclei, although most or all nuclei express LAG-1 in earlier development (data not shown). (I,J) Pharyngeal pumping sequence in a wild-type L1 larva (I) and an inx-20(ok681) (J) mutant larva; see also Video S5. Bars: (A–J) 5 microns.
Figure 10.
EGL-43 expression and egl-43 mutant phenotypes.
(A) Diagram of the EGL-43 protein and egl-43 gene. WRM0622cG11 is a fosmid that rescues the egl-43(zu471) mutant; the site of gfp insertion for the EGL-43::GFP fosmid reporter is indicated. (B) Wild-type, egl-43(zu471) and ceh-51(tm2123) larvae immunostained for the basement membrane component UNC-52/Perlecan (red). The wild-type, but not mutant, basement membrane curves deeply inward, following the posterior surface of the pm8 cell body and nearly separating the pharynx from the valve (see [22]). (C) Wild-type and egl-43 larvae expressing reporters as listed for either pm8 (ref-1153 bp::GFP::CAAX), v1 (egl-43::GFP::CAAX), or v2-v3 (mig-13::MIG-13::GFP). Images are surface renderings as for Video S3, rotated to show posterior views of each cell(s); the midline is indicated by a blue arrow. Note the lack of v2-v3 cells dorsal to the midline in the egl-43 mutant. (D) Images of hatched larvae expressing mig-13::MIG-13::GFP (green) and showing the apparent absence of the v3D valve cell in the egl-43 mutant; the midline is indicated by an open arrowhead. (E,F) Live embryos showing EGL-43-dependent expression of ref-11.8 kb::REF-1::GFP in pm8. Notch-independent expression occurs in other pharyngeal cells, such as the sister cells e2V and mc3V (double-headed arrows); see [36]. (G,H) Live embryos showing the egl-43(+) dependence of a lin-12-derived reporter for the pm8 family (green nuclei, lin-12pm8::HIS-GFP). The embryos are progeny of an egl-43(zu471)/mIn1[dpy-10(e128) mIs14] adult; the mIn1 balancer contains egl-43(+) and a transgene that drives GFP expression in intestinal cells. Thus, only the embryo in G is egl-43(+), and only this embryo expresses the lin-12-derived reporter (see Table 1 for quantification). (I,J) EGL-43 expression in a wild-type embryo (I) and a pha-4(q490) mutant embryo (J, n = 14/14 embryos with similar expression). (K,L) EGL-43::GFP is not expressed in the pharyngeal precursors (blue outline) of most tbx-35 (K) and ceh-51 (L) mutant embryos. In these experiments, the EGL-43::GFP transgene was marked with end-1::mCherry (not shown), and only mCherry-positive embryos were scored. (M) Partial lineage tree showing the MS descendants that express EGL-43::GFP, and showing the fates of these descendants in normal development (see [9]). Bars: (B–D) 5 microns; (E–L) 10 microns.
Table 1.
egl-43 regulates pm8 gene expression.