Figure 1. Cell division and elongation challenge the epithelial integrity of the developing C. elegans intestine.

(A, B) A cartoon schematic (A) and corresponding live time course (B) of an embryo expressing an apical microtubule-organizing center (MTOC) marker TBG-1::mCherry (green, orange arrowhead) and a membrane marker intestinal GFP::CAAX (magenta); the anterior and posterior ‘star cells’ divide (blue) and the apical surface elongates (white arrow) as the polarized intestinal primordium develops into an intestine. (C) Top: graph showing intestinal apical length in newly polarized primordia (bean stage, average length = 16.5 ± 2.6 μm, n = 18), ~1.5–2 hr after the start of intestinal elongation (1.5-fold to 1.8-fold, average length = 38.2 ± 2.2 μm, n = 30), and upon hatching in the L1 larval stage (average length = 105.0 ± 11.1 μm, n = 15). Below: corresponding intestinal GFP::CAAX images and approximate age, in minutes post-fertilization (mpf). (D) Cartoon schematic of the anterior star cell divisions illustrating their dorsoventral division in 3D (top) and from a dorsal view (bottom). Dotted line indicates viewing angle of images in (E–H). (E–H) Live imaging of indicated proteins relative to TBG-1/γ-tubulin::mCherry (green) or mCherry::TBA-1/α-tubulin (magenta) and the midline (orange arrowhead) when the right and left cells divided synchronously (gray arrows). Note that in some cases a midline gap (dashed bracket) formed, and in other cases no gap formed (solid bracket). Numbers of embryos with midline gaps in mitotic star cells, assessed by eye: mCherry::TBA-1 (5/5), ZF::GFP::GIP-1 (5/5), TBG-1::mCherry (10/10), mCherry::AIR-1 (10/10), ZYG-9::ZF::GFP (3/3), PTRN-1::GFP (5/5), NOCA-1::ZF::GFP (7/7), VAB-10B::GFP (3/3), HMR-1::GFP (0/8), DLG-1::mNG (1/8), YFP::ACT-5 (0/6), PAR-6::GFP (0/10), GFP::PKC-3 (0/5), and PAR-3::GFP (0/5). All images are maximum intensity Z-projections (0.5–1.5 μm) that capture centrosomes (open white arrowheads) and/or the intestinal midline. Scale bar = 10 μm in (B, C). Scale bar = 5 μm in (E–H).

Figure 1—figure supplement 1. C.elegans intestinal development.

(A) Left: a 3D representation of the intestinal primordium prior to star cell divisions and elongation, with an end-on anterior view showing the apical domains (gold) of intestinal cells facing the central midline, separated from the basolateral domains (blue) by junctions (magenta). One of the 4 star cells (8/9R) and one of the 12 non-star cells (5R) are not in view. Numbers indicate which int ring each cell or its descendants will form (e.g., 3R and 4R will become part of int3 and int4, respectively). Different shades of magenta are used to highlight different cell-cell interactions. Bright magenta indicates junctions between left and right neighbors, which will together build an int ring; dark magenta indicates junctions between anterior and posterior neighbors, which will join adjacent int rings. Right: a 3D representation of the L1 larval intestine. All int rings contain two cells except for the four-celled int1. Apical domains of all cells face the central lumen, and junctions connect left/right pairs of cells within int rings (bright magenta) and also anterior and posterior cells of adjacent int rings (darker magenta), forming a ladder-like pattern. (B) Lateral confocal images of live comma-1.5-fold-stage embryos. Left panels: apical markers tagRFP::PKC-3 (green) and PAR-3::ZF::GFP (magenta). Middle panels: junctional marker DLG-1::mNG (green) with PAR-3::tagRFP (magenta), and the basolateral marker LGL-1::GFP (green) with PAR-3::tagRFP (magenta). Enlarged images of boxed regions shown below. Scale bar = 10 μm for top images, 2 μm for enlarged images. n > 10 embryos per genotype. (C) Enlarged cartoon schematic showing the orientation of anterior star cell divisions. Note that the cleavage furrow divides the apical surface of the star cells longitudinally creating new cell-cell interfaces (yellow lines), related to Figure 1D.

Figure 1—figure supplement 2. Protein localization before and during star cell divisions.

(A) Cartoon schematic of dorsal view of intestinal star cells (blue) and neighboring non-star cells (dark gray) prior to star cell divisions (left panel) and during star cell divisions (right panel). Microtubules (magenta), microtubule-organizing center (MTOC) (green), apical midline (orange arrowhead), and active centrosomes (black arrowheads). (B, C) Dorsolateral confocal images of live bean-stage embryos after pharynx polarization before star cell divisions (left two panels) and during star cell divisions (right two panels). TBG-1::mCherry was co-expressed with PAR-6::GFP, HMR-1::GFP, and DLG-1::mNG, and mCherry::TBA-1 with YFP::ACT-5, to mark the apical MTOC gap during star cell divisions. The approximate position of star cells (blue star) and non-star cells (black star) is indicated. Brackets indicate where a midline gap forms (dashed) or does not form (solid) in dividing star cells, and open arrowheads mark active centrosomes. (D–G) Graphs showing junctional enrichment of DLG-1::mNG (D) and HMR-1::GFP (E) and apical enrichment of TBG-1::mCherry (F) and PAR-6::GFP (G) in star cells (blue) and non-star cells (gray) before and during star cell divisions. Red line indicates level of average cytoplasmic background enrichment. Respective number of embryos scored pre-division and during division: PAR-6: n = 17, n = 10; TBG-1: n = 17, n = 10; HMR-1: n = 9, n = 8; DLG-1: n = 16, n = 10. Statistical analysis: Student’s t-test with Bonferroni correction. Scale bar = 2 μm. *p<0.05, **p<0.01, ***p<0.001.