Pharyngeal cyst development in wild-type and pha-4 mutant C. elegans embryos. (A,B) Longitudinal optical sections through the centers of wild-type (A) and pha-4(q490) (B) embryos at ∼420 minutes in development. Embryos express a nuclear reporter (pha-4::HIS-GFP, green) in all pharyngeal precursor cells (PPCs) and in intestinal cells (int). Because the PPC reporter does not encode the PHA-4 protein, it does not rescue the pha-4 mutation and ‘PPCs’ in the mutant embryos do not differentiate as pharyngeal cells. Arrows in A indicate the periphery of the pharyngeal cyst and the red arrowhead indicates the developing apical lumen. (C) Partial cell lineage diagram indicating the origins of early cells described in this study. Body wall muscles (BWMs) are a non-pharyngeal type of muscle. (D) Optical sectioning planes used in this study superimposed on a depiction of an embryo midway through development. The internal position of the PPCs is indicated (green). D, dorsal; V, ventral; A, anterior; P, posterior. Scale bar: 5 μm.

PPCs organize into a bilaterally symmetric double plate. (A,B) Confocal fluorescence image sequence of single live C. elegans embryos expressing a PPC nuclear reporter (pha-4::HIS-GFP) and a membrane reporter (memb-mCherry). Images are transverse optical sections oriented as in ; images were collected ∼15 μm from the anterior pole of the embryo. (A) PPCs are ingressing from the left and right ventral (bottom) surface of the embryo, then moving dorsally (top). By 290 minutes, the double plate is 6-8 cells deep. (B) Examples of left-right PPC divisions in the double plate. Cells are indicated before (single arrow) and after (double arrow) division. The dashed line indicates the midplane of the double plate. (C) Representation of the ventral surface of a 24-cell embryo (∼100 minutes), and a transverse section through an embryo at the double plate stage (∼290 minutes). Nuclei with bold outlines are PPCs and are color-coded as in ; red nuclei are BWM precursors. (D) Transverse optical section of a live ∼250 minute embryo expressing the same PPC and membrane reporters as in A and B, but with an additional reporter for BWMs (hlh-1::HIS-mCherry). (E) Longitudinal optical section through a terminal stage mex-1(RNAi) embryo (anterior at top). Note the single large cluster of PPCs (green); the fainter, larger nuclei toward the bottom are intestinal nuclei (int). (F,G) Ventral surface views of live wild-type and wrm-1(ts) embryos raised at the restrictive temperature until MS cell division, then imaged at the permissive temperature. Images show before (top) and after (bottom) the fifth cell division of the MS-derived PPCs. Cells are color-coded as in , with white lines indicating sister cells; cells that have ingressed are shown with lighter coloring. Note the irregular left-right positioning of cells in the wrm-1(ts) embryo. (H,I) Live wrm-1(ts) embryos raised at the permissive temperature (H) or temperature shifted (I) as in G; reporters and optical sectioning as in A. Note the failure of PPCs to form a double plate after shifting from the restrictive temperature (n=15/15 embryos). Scale bars: 5 μm in A,D,E,H,I; 2.5 μm in B,F,G.

PHA-4 shapes and maintains the double plate. (A) Transverse optical sections through a live wild-type C. elegans embryo showing the PPC (pha-4::HIS-GFP) transition from double plate to cyst. Images are from supplementary material Movie 1; the dashed line represents the midplane. (B) pha-4(q490) embryo showing PPCs inappropriately crossing the midplane (arrow) and the failure to form a cyst. Images are from supplementary material Movie 2. (C-D″) Longitudinal optical sections through the anterior of a fixed wild-type embryo and a pha-4(q490) mutant embryo immunostained as follows: PPC nuclei [anti-GFP (pha-4::HIS-GFP)], BWM nuclei (hlh-1::HIS-mCherry), laminin (mAbGJ2) and total nuclei (DAPI). Laminin outlines the body in both embryos (blue arrowhead). Laminin localizes to the periphery of the developing pharynx in the wild-type embryo (yellow arrow) and separates PPCs from the surrounding BWMs (see inset of bracketed region in C″). Note that laminin is present in an irregular pattern throughout the pha-4 mutant PPCs (D′) and does not separate PPCs from BWMs (see inset of bracketed region in D″). The insets in C and D show merged images of PPC nuclei (green) and total nuclei (blue). Note that PPCs do not intermingle with non-PPCs in wild-type embryos, but mix in pha-4 mutants. Time is in minutes. Scale bars: 5 μm.

Cyst formation by apical constriction of double plate PPCs. (A-D′) Transverse optical sections of live C. elegans embryos showing the transition from double plate to cyst. Arrows at the first time point indicate the left and right margins of the double plate. (A) Image sequence from supplementary material Movie 3 showing all cell membranes (memb-mCherry). (B) Image sequence showing cell shape changes for a single PPC marked by zuEx254, which is expressed in only a few PPCs. (C) F-actin expression (pha-4::GFP::dMoesin-ABD) in the PPCs. Note the enrichment along the midplane/apical surface. (D) Non-muscle myosin expression (nmy-2::NMY-2::GFP). (D′) High magnification of the periphery of the double plate (arrow with bracket in D). Note that non-muscle myosin is initially enriched at the periphery of PPCs, but disappears from the periphery as it concentrates apically. (E) Average membrane length (μm) of midplane and peripheral PPC surfaces. Error bars indicate 95% confidence intervals. (F) The left panel is a longitudinal view (anterior at top) of the double plate PPCs (DAPI, blue) at ∼310 minutes showing the positions of centrosomes (IFA, red) relative to the midplane (dashed line). Note the number of midplane-facing centrosomes. The right panel quantifies centrosome positions over time (n>55 centrosomes per time point). (G) Quantification of midplane enrichment of PAR-6 (pie-1::mCherry::PAR-6) and NMY-2 (nmy-2::NMY-2::GFP) in single embryos expressing both reporters (n=4 embryos). See also supplementary material Movie 4. Error bars indicate 95% confidence intervals. Scale bars: 5 μm in A,C,D; 2.5 μm in B,F; 1 μm in D′.

Onset of apical and basal polarity in double plate PPCs. (A-C) Transverse optical sections of live C. elegans embryos showing the transition from double plate to cyst. Arrows indicate the left and right margins of the double plate. Embryos express the following reporters: (A) PAR-6 (pie-1::mCherry::PAR-6), (B) laminin β (lam-1::LAM-1::GFP) and (C) β-integrin (pat-3::PAT-3::GFP). See supplementary material Movie 6 for onset of lam-1::LAM-1::GFP expression. (D,E) Longitudinal optical sections through the center of the double plate (brackets) and intestine (int, arrowheads). The embryos were immunostained for PAR-3 and either laminin α (mAbGJ1) (D) or β-integrin (MH25) (E). The embryos were selected for the earliest detectable asymmetry of PAR-3 in the PPCs. Note that at this stage both laminin α and β-integrin are enriched in puncta at the periphery of the double plate; at later stages, laminin α and β-integrin uniformly coat the basal PPC surfaces (see ; supplementary material Fig. S4). LAM-3 is not enriched at the periphery of the intestinal cells (arrowheads). (F) Kymograph analysis (top) from supplementary material Movie 6 of an embryo co-expressing pie-1::mCherry::PAR-6 and lam-1::LAM-1::GFP showing the width of the double plate and surrounding BWMs. Beneath are shown fluorescence intensity line scans across the kymographs at 35 and 60 minutes. The dashed line indicates the midplane. Scale bars: 5 μm.

PPC polarity in mex-1 embryos. (A) High-magnification view of superficial PPCs in a mex-1(RNAi) C. elegans embryo. The embryo was immunostained for laminin α (mAbGJ1), PPCs [anti-GFP (pha-4::PHA-4::GFP)] and a lumenal marker (mAbPL1). Note that laminin accumulates at the external, contact-free periphery of the superficial PPCs (arrowheads) and that lumenal markers differentiate one cell diameter inward from the periphery, similar to the wild-type cyst. (B) Images from supplementary material Movie 5 of superficial PPCs in a live mex-1(RNAi) embryo expressing reporters for PAR-6 (pie-1::mCherry::PAR-6) and PPC nuclei (pha-4::PHA-4::GFP). Note the accumulation of PAR-6 at the center of a cyst-like structure. (C) Superficial PPCs in a mex-1(RNAi); lam-1(ok3139) embryo immunostained for PAR-3 and PPC nuclei [anti-GFP (pha-4::PHA-4::GFP)]. Note that in the absence of laminin, PAR-3 accumulates with inverted polarity on the contact-free surface. Time is in minutes. Scale bar: 2.5 μm.

Laminin is required to specify the single pharyngeal lumen. (A,B) Brightfield images of late stage wild-type and lam-1(ok3139) C. elegans embryos. Arrowheads indicate the cuticle-lined pharyngeal lumen. Note the multiple lumens in the lam-1(–) mutant. (C,D) Immunostained wild-type and lam-1(–) embryos at 470 minutes showing PPC nuclei [anti-GFP (pha-4::PHA-4::GFP)] and a marker for the pharyngeal lumen (mAbPL1). Insets are reconstructed transverse views through the pharynx. Note that both of the apparent lumens in the lam-1(–) embryo are surrounded by PPCs. (E,F) Brightfield and fluorescent images of the intestine in live wild-type and lam-1(–) embryos at ∼280 minutes, showing localization of PAR-6 (par-6::PAR-6::GFP) to a single lumen (arrow). Although lam-1(–) embryos showed some mispositioning of intestinal cells, each of 28 embryos examined localized PAR-3 or PAR-6 only to the apical surfaces of intestinal cells. (G-J) Brightfield images of newly hatched larvae showing a single internal pharyngeal lumen in null mutants for LAM-3/laminin α (G), EPI-1/laminin α (H), UNC-52/perlecan (I) and EMB-9/type IV collagen (J). n>50 animals examined for each genotype. The pharynx is outlined (blue dashed line). (K-M) Newly hatched homozygous lam-1(–) mutant larvae that are mosaic for LAM-1 expression [zuEx288: lam-1(+) transgene marked by sur-5::SUR-5::GFP]. Note the formation of a single pharyngeal lumen (arrowhead) when zuEx288 is present only in the intestine (L) or in hypodermis and BWMs (M). Scale bars: 5 μm.

Laminin cues PPC polarity. (A,B) Longitudinal optical sections through the double plate PPCs of immunostained wild-type and lam-1(ok3139) C. elegans embryos. Anterior is top. Images show PPC nuclei [anti-GFP (pha-4::PHA-4::GFP)], total nuclei (DAPI) and PAR-3 as maximum intensity projections through the double plate; arrows indicate the midplane. Note that PAR-3 localizes to midplane surfaces in the wild-type double plate, but predominantly to peripheral surfaces in the lam-1(–) embryo [n>50 for wild type; n=18/18 for lam-1(–)]. (C,D) Optical sections as in A and B of live embryos expressing a PAR-6 reporter (par-6::PAR-6::GFP). Note that PAR-6 localization begins, and persists, at the peripheral surfaces of the double plate in the lam-1(–) embryos (n=16/16). See supplementary material Movies 7 and 8 for cell membranes of PPCs. (E,F) High-magnification images of part of a longitudinal optical section through the double plate of live wild-type (E) and lam-1(–) (F) embryos showing PAR-6 (par-6::PAR-6::GFP) and plasma membranes (memb-mCherry). Line scans through the double plate are shown beneath each image. Note that ectopic PAR-6 is localized within the PPCs in the lam-1(–) embryo. (G-G″) Images from supplementary material Movie 9 showing transverse optical sections of a live lam-1(–) embryo expressing reporters for non-muscle myosin (nmy-2::NMY-2::GFP) and plasma membranes (memb-mCherry). Images in G″ are high-magnification views of PPCs at the periphery of the double plate (arrowhead and bracketed region in G and G′). The diagrams show tracings of individual color-coded PPCs within the double plate (light gray region), taken from movie frames at left. Time is in minutes. Scale bars: 2.5 μm in A-F,G″; 5 μm in G.

Model of cyst morphogenesis in C. elegans. Summary of the formation of the pharyngeal cyst (transverse views). Left (yellow) and right (light blue) PPCs ingress from the ventral (bottom) surface of the embryo and aggregate to form the bilaterally symmetric double plate. Laminin accumulates on the exposed peripheral surface of the double plate in wild-type embryos, but is absent in lam-1(–) embryos. Laminin at the peripheral surface precedes, and is required for, localization of proteins in the PAR-3 complex to the opposite, midplane surface. In lam-1 mutants, PAR proteins accumulate ectopically on the peripheral surface. Cell surface constriction occurs where PAR proteins localize, leading to midplane constriction in wild-type embryos and the formation of a single-lumen cyst. In lam-1 mutants, peripheral constriction and shifts in PPC position internalize the PAR-containing peripheral surfaces, leading to a multi-lumen cyst.