Apical abscission alters cell polarity and dismantles the primary cilium during neurogenesis

Abstract

Withdrawal of differentiating cells from proliferative tissue is critical for embryonic development and adult tissue homeostasis; however, the mechanisms that control this cell behavior are poorly understood. Using high-resolution live-cell imaging in chick neural tube, we uncover a form of cell subdivision that abscises apical cell membrane and mediates neuron detachment from the ventricle. This mechanism operates in chick and mouse, is dependent on actin-myosin contraction, and results in loss of apical cell polarity. Apical abscission also dismantles the primary cilium, known to transduce sonic-hedgehog signals, and is required for expression of cell-cycle-exit gene p27/Kip1. We further show that N-cadherin levels, regulated by neuronal-differentiation factor Neurog2, determine cilium disassembly and final abscission. This cell-biological mechanism may mediate such cell transitions in other epithelia in normal and cancerous conditions.

Fig. 1. Apical abscission during neuronal differentiation

(A) Time-lapse sequence of a cell undergoing distinct stages of apical abscission, [Movie S1; these are additional frames of a cell shown in supplementary movie 2 in (6)]. (B-D) Maximum intensity projections of constricting abscission site (white arrowheads) visible in Tuj1⁺ ventricle-contacting cells in chick (B) and mouse embryos (C) and TagRFP-Farn-labelled cell in chick (D); the abscising particle is distal to actin and contains the apical Par-complex, marked by aPKC (3D-reconstructions of B, C and D, in Movies S4, S5, S6 respectively). (E-G) Cells poised to abscise express NeuroM, and Lim1/2 (E) but not HuC/D (F) nor p27 (G) (Magenta arrows). Abscission site (white arrowheads), withdrawing apical cell-process (white arrows), abscised particle (yellow arrows), apical surface (white dashed-line) here and in all figures. Scale bars, 10 μm; enlarged regions, 2 μm.

Fig. 2. Apical abscission depends on actin-myosin activity

(A, B) Time-lapse showing actin localisation (A)(Movie S8) and (B) quantification of Actin-TagRFP intensity during apical abscission (average normalised values for 4 cells, error-bars S.E.M.); (C, D) active myosin (MRLC2^T18DS19D-GFP) (C, green at cell-process tip; Movie S11) localises to abscission site (D) MRLC2^T18DS19D-GFP intensity during apical abscission (average normalised values for 5 cells, error-bars S.E.M). (E-H) Cells exposed to control DMSO undergo abscision (E; Movie S14), but not in the presence of blebbistatin (F; Movie S17) or ML-7 (G; Movie S20). ML-7 abscission inhibition is rescued by expression of, MRLC2^T18DS19D-GFP (H; Movie S23). For definition of apical surfaces, N-Cadherin and aPKC localisation see Figs. S4, S5. In B and D Membrane thinning (black arrow), abscission complete (black arrowhead). Scale bars, 10 μm; enlarged regions, 2 μm.

Fig. 3. Apical abscission dismantles the primary cilium

(A, B) Time-lapse sequences showing centrosome release into the apical cell-process (A; Movie S26), while Arl13b-labelled cilium is retained at the apical membrane (B; Movie S29). (C) Widefield and (C’) structured illumination imaging (white dotted outline)(Movie S32) of TagRFP-Farn labelled apical cell-process and abscised particle containing Arl13b-GFP-labelled cilium. (D, E) Tuj1⁺ cells with ventricle-contacting apical cell-processes exhibit Smo (D; Movie S33) and Gli2 accumulation (E; Movie S34) (empty arrowheads) in their primary cilium (Identified with Arl13b-GFP or Ift88 (Intraflagellar-transport-protein 88) respectively). Scale bars, 10 μm; enlarged regions and C, C’, 2 μm.

Fig. 4. N-Cadherin loss promotes apical abscission

(A) Cells mis-expressing NCad-YFP constrict (white arrowheads) but do not abscise and the centrosome (PACT-tagRFP, magenta arrows) remains at the apical cell-pole (Movie S35). (B) N-Cad-YFP mis-expressing cells do not express p27, (B’) which is normally detected after apical cell-process detachment (cell nuclei; empty arrowheads); (C) Neurog2 mis-expression rescues centrosome release and abscission in NCad-YFP-expressing cells (Movie S38). (D) Neurog2 mis-expression decreases sub-apical NCad-TagRFP levels (magenta arrows), followed by abscission and cell-process withdrawal, note, a second underlying cell has yet to withdraw (Movie S41); Scale bars, 10 μm; enlarged regions, 2 μm.