The columns show wild-type (left) and lin-12 glp-1 mutant (right) animals either after hatching at 14 hours (A–F), or at 7 hours in embryogenesis (G–J). Transgenic reporters are as listed; white lines indicate perimeter of the pharynx (C,D) or epithelial cyst (I,J). Nuclei labeled “n” in panels C and D are neurons outside the pharynx that express ceh-24::GFP. Bars = 5 µm (A–F), 10 µm (G–J). Embryos are approximately 50 µm in length.

(A) Diagram comparing (1) a box-like cell with a simple, flat apical surface, (2) a topologically equivalent C-shaped cell, and (3) a topologically distinct toroid with a cylindrical apical surface; apical junctions are shown as bold lines. (B–B”) Apical junctions at the pharynx/valve boundary in a 7.5 hour wild-type embryo stained for the apical junction marker AJM-1 (panel B) and for eff-1::EFF-1::GFP (panel B’). In this longitudinal view, the intercellular junctions at the ends of pm8 and vpi1 appear as vertical lines (panel B). The inset in (B) shows the same region in an eff-1 mutant with an autocellular junction linking the intercellular junctions in vpi1, but not pm8. (C–C”) Same region and stage as in panel B, showing aff-1::GFP expression in pm8 and an autocellular junction in pm8 in an aff-1 mutant (inset). (DD”) lag-1 embryo with binucleate cell expressing eff-1::EFF-1::GFP. (E,E’) lag-1 embryo showing a mononucleate cell expressing eff-1::EFF-1::GFP.(F,F’) different focal plane of the same embryo in panel (E) showing a second eff-1::EFF-1::GFP-expressing cell. Note that both cells have a ventral-directed process but neither cell extends completely through the cyst. Bars= 2.5 µm (B–F).

(A) Diagram of some of the cells in the terminal bulb of the pharynx and in the valve. The three group 3 marginal cells are shown in purple, and names of the principle cells mentioned in the text are indicated. (B) Diagram of cell positions in the cyst (top) and pharynx/valve (bottom). Note the reorientation of the basal lamina-associated, basal surfaces of pm8 and vpi1. (C) pm8 in an adult and newly hatched larva (inset) visualized by ref-1::GFP-PM; white lines indicate perimeter of terminal bulb. (D) Optical longitudinal section through the middle of an embryo showing the epithelial cyst (bracket). (E–E”) High magnification of region corresponding to double-headed arrow in panel D after immunostaining for apical junctions (AJM-1) and LIN-12/Notch to visualize pm8 (see also Figure 3D). Note that pm8 contacts, but does not cross, the midline (arrow). Polygonal shapes are the apical surfaces of various cells in the cylindrical array around the midline. Bars =2 µm (C), 10 µm (D), 2.5 µm (E–E”).

(A–A’) Ventral view of an embryo before formation of the cyst showing lag-2 expression in MSaapa. (B) ref-1::GFP expression in a cyst-stage, wild-type embryo after killing MSaapa; embryo shown is the same stage and orientation as in Figure 2I. ref-1::GFP expression in pm8 was observed after killing the following cells: MS (0/4 embryos), MSaa (0/4- MS and MSaa are precursors of pm8), MSap (6/6), MSaap (0/4), MSaapa plus MSaapp (0/3), MSaapp (5/5), MSaapa (4/4 when the ablated cell entered the body cavity, 0/3 when it remained outside). (C–C”) Dorsal view of a 6 hour embryo immunostained for GFP (lag-2::GFP) and LIN-12/Notch; white lines indicate boundary of epithelial cyst. The approximate position of vpi1 is indicated based on light microscopy of living embryos at this stage and orientation. (D–D”) Lateral view of embryo at about the same stage as panel C; ref-1::GFP is expressed in the Notch-independent cells e2V and mc3V, but not in pm8. (E–E”) Embryo approximately 20 minutes later than in panel D showing ref-1::GFP in pm8. Note lamella from pm8 (arrow in panel E) extending ventrally across the midline of the cyst (dashed line).

(A) Optical longitudinal section through the middle of a 7 hour embryo stained for laminin. Laminin is present in the basal lamina surrounding the cyst (bracket). A transverse tract of laminin is evident in the posterior, ventral half of the cyst (arrow indicates the cyst midline). (B) lin-12 glp-1 embryo at 7.5 hours. (C–F) Sequence of successively older wild-type embryos showing the appearance (D) and disappearance (E) of the laminin tract, followed by the deposition of laminin on the posterior surface of pm8 after morphogenesis (F). (G–I) Single embryos immunostained for LAM-3 and GFP (ref-1::GFP-PM) before (G), during (H), and near the completion (I) of pm8 migration. Arrows indicate the midline of the cyst. The fixation required for LAM-3 staining compromises GFP-PM localization in pm8 (compare with Figures 4A–D). (J) lam-3; epi-1 double mutant at about 9 hours; pm8 has failed to migrate to the ventral side. This embryo has a shape similar to younger, 7.5 hour wild-type embryos because of defects in body morphogenesis, but has the well-formed tail spike (arrow) characteristic of wild-type 9 hour embryos. (K) ina-1 mutant larva showing pm8 extension into the valve/anterior intestine; similar defects occur in 74% of the hatched animals (n =46). Bars= 10µm (A,B) and 5 µm (G–I).

(A–D) ref-1::GFP-PM expression in pm8 and the group 3 marginal cell mc3V in successively older embryos. Images are optical sections through the midline of the cyst; a complete image series corresponding to panel D is shown in Movie S4. (E–E”) vpi1 expressing eff-1::EFF-1::GFP and stained with phalloidin to visualize F-actin at the midline (arrow); note relative absence of actin in pm8 (asterisk indicates position of pm8 nucleus). Panel E”’ is a diagram of vpi1 with the approximate position of the pm8 cell body included for reference (see Fig S3D). (F) Electron micrograph and diagram of a cross section through pm8 in an embryo near hatching. The three marginal cell fingers (numbered 1–3) are evident in the Y-shaped lumenal channel (white) of pm8. (G) Apical junctions in a wild-type, third stage (L3) larva; cells in the pharynx and valve do not divide during larval development, but increase in size and allow better visualization of apical junctions. Apical surfaces of cell like pm7 resemble broad triangles, while the group 3 marginal cells (numbered 1–3) have long, thin apical surfaces (see also Movie S1). Note how fingers from the marginal cells extend through the apical surface of pm8. The region indicated by the double-headed arrow is diagrammed to show a superposition of the pm8 and vpi1 cell bodies on their apical surfaces; apical junctions are drawn in black. (H, I) Same region as in panel F in wild-type (H) and lin-12 glp-1 mutant (I) embryos near hatching. Bars = 1 µm (F), 5 µm (F–H).