Report

Apical Abscission Alters Cell Polarity and Dismantles the Primary Cilium During Neurogenesis

See allHide authors and affiliations

Science  10 Jan 2014:
Vol. 343, Issue 6167, pp. 200-204
DOI: 10.1126/science.1247521

Developing Neurons Make the Cut

Neurons in the developing central nervous system of vertebrates derive from cells adjacent to the ventricles that then proliferate and differentiate to populate the brain. As one of these cells begins to differentiate, the cell nucleus migrates toward its new residence, away from the ventricle surface, and the cell stretches out. At some point, like any maturing adolescent, the cell has to leave home. Das and Storey (p. 200; see the Perspective by Tozer and Morin) show that instead of letting go and drawing the trailing process up into the migrating cell, the cell cuts off and discards its first roots. The abscission process leaves behind the primary cilium and any signaling systems localized to the cilium.

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.

Newborn neurons detach an apical cell-process from the ventricular surface and then migrate to the lateral neural tube or to form cortical layers within the brain (1, 2). This step is required for the generation of neuronal and tissue architecture (2, 3), and its failure leads to human periventricular heterotopia (4). Down-regulation of N-cadherin is associated with this event (3, 5), as is loss of apical complex proteins (6, 7). The latter may be mediated by down-regulation; protein modification/degradation or relocalization; or loss of apical membrane.

To investigate cell behavior underlying neuron birth, we labeled membranes of individual cells by mosaic transfection of green fluorescent protein– glycosylphosphatidylinositol (pCAGGS-GFP-GPI) into the chick embryonic spinal cord (8). We then monitored neurogenesis in ex vivo embryo slice cultures (1) using wide-field time-lapse microscopy (8). Newborn neurons have a basally located cell body and extend a long, thin cell-process to the apical/ventricular surface. Movies of such cells revealed that shedding of the apical-most cell membrane preceded withdrawal of this cell-process (Fig. 1A). This event, which we name apical abscission, takes ~1 hour (56 min, SD = 18 min, n = 21 cells). It begins with formation of a bulb-like “bouton,” followed by subapical constriction, membrane thinning, and eventual abscission, after which the apical cell-process withdraws (42 abscising cells in 34 embryos; all stages observed in 21 cells) (Fig. 1A, fig. S1, and movies S1 to S3). Abscised particles tracked so far remain at the ventricle.

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 to D) Maximum intensity projections of constricting abscission site (white arrowheads) visible in Tuj1+ ventricle-contacting cells in chick (B) and mouse (C) embryos and TagRFP-Farn–labeled cell in chick (D); the abscising particle is distal to actin and contains the apical Par-complex, marked by aPKC [three-dimensional reconstructions of (B), (C), and (D) in movies S4, S5, and S6, respectively]. (E to 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), and apical surface (white dashed line) here and in all figures. Scale bars, 10 μm; enlarged regions, 2 μm.

Using structured illumination microscopy (8) to generate super-resolution images of abscising cells transfected with membrane-localized Tag–red fluorescent protein–Farnesyl (TagRFP-Farn) revealed a thin membranous connection between apical cell-process and the abscising particle. This confirmed the existence of abscission events in fixed tissue not subject to culture and imaging regimes (n = 5 cells in 3 embryos) (fig. S2 and movie S4). We also observed apical abscission in completely unmanipulated embryos fixed and labeled to reveal the early neuronal marker Tuj1 (class III beta-tubulin). Some Tuj1+ cells with a basally localized nucleus and a ventricle-contacting apical cell-process were found to have a distinct constriction, coincident with subapical actin (n = 31 of 78 cells in 5 embryos) (Fig. 1B and movie S5). To characterize the abscised membrane, we assessed localization of endogenous apical Par-complex protein, atypical protein kinase C (aPKC) (9) in such Tuj1+ cells; aPKC was confined to the abscising particle (n = 31 of 31 cells in 5 embryos) (Fig. 1B and movie S5). This indicates that differentiating neurons experience rapid loss of apical polarity. It is also consistent with the absence of Par-complex proteins from withdrawing cell-processes (6, 7), which, now liberated from apical-junctional complexes, extend transient membrane protrusions (18 cells in 9 embryos) (e.g., see movies S1 and S2). Similar apical constrictions were visible in Tuj1+ ventricle-contacting cells in mouse spinal cord (22 of 40 cells in 4 embryos) (Fig. 1C and movie S6), with aPKC confined to the abscising particle (22 of 22 cells). This demonstrates that apical abscission is conserved across species. In chick, we further characterized cells poised to abscise as indicated by a basally located nucleus and ventricle-contacting apical cell-process revealed by TagRFP-Farn labeling and found a similar localization of actin and aPKC (21 of 21 cells in 6 embryos) (Fig. 1D and movie S7). Many such TagRFP-Farn–labeled cells with this morphology also express low levels of the early neuronal marker, NeuroM (26 of 29 cells in 5 embryos) (Fig. 1, E to G). These NeuroM-positive cells were further found to express the interneuron marker Lim1/2 (23 of 23 cells) but not the later neuronal marker HuC/D (0 of 12 cells) nor the postmitotic cell marker Cdk-inhibitor p27/Kip1 (0 of 11 cells) [(10) and see (7)], identifying these cells as immature neurons that have yet to commit to cell cycle exit (Fig. 1, E to G).

Neuroepithelial cells contain a subapical actin cable that mediates normal cell constriction at the ventricular surface. To investigate whether apical abscission involves actin dynamics, we cotransfected GFP-GPI and Actin-TagRFP vectors into chick neural tube and monitored protein localization. Subapical actin was visible in cells poised to abscise and coincided with the region of constriction before abscission (Fig. 2A and movie S8). As abscission began, Actin-TagRFP signal intensity increased (8), reaching a maximum shortly before abscission completion (Fig. 2B); actin was then depleted from the withdrawing cell-process tip (n = 24 abscising cells in 18 embryos) (Fig. 2A, fig. S3, and movies S8 to S10). This local actin increase raised the possibility that actin-myosin contraction mediates apical abscission.

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

(A and B) Time-lapse sequence showing actin localization (A) (movie S8) and (B) quantification of Actin-TagRFP intensity during apical abscission (average normalized values for four cells; error bars, mean ± SEM). (C and D) Active myosin (MRLC2T18DS19D-GFP) (C, green at cell-process tip; movie S11) localizes to abscission site (D) MRLC2T18DS19D-GFP intensity during apical abscission (average normalized values for five cells error bars, mean ± SEM). (E to H) Cells exposed to control DMSO undergo abscission (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 MRLC2T18DS19D-GFP (H) (movie S23). For definition of apical surfaces, N-cadherin, and aPKC localization, see fig. S6. [(B) and (D)] Membrane thinning, black arrow; abscission complete, black arrowhead. Scale bars, 10 μm; enlarged regions, 2 μm.

We therefore next surveyed myosin localization using a myosin regulatory light chain 2 GFP construct (MRLC2-GFP); this revealed strong subapical localization and diffuse cytosolic distribution in all cells (fig. S4). Because myosin phosphorylation is essential for actin-mediated apical constriction, we next discriminated sites of myosin activity by monitoring MRLC2T18DS19D-GFP, a constitutively active form of MRLC2. To increase the incidence of neuronal differentiation, we cotransfected cells with a plasmid encoding the proneural gene Neurog2 [pCAGGS-Neurog2-IRES-nucGFP (pCIG-Neurog2)], which promotes neuronal differentiation (10). In such cells, also coexpressing TagRFP-Farn to label cell membranes, active MRLC2 localized subapically until shortly after abscission (8) (n = 12 cells in 9 embryos) (Fig. 2, C and D, and movies S11 to S13). Thus, actin is localized and myosin is active in the subapical region of the abscising neuron.

To investigate the requirement for myosin activity, cells were transfected with GFP-GPI and pCIG-Neurog2, and slices were cultured in medium containing blebbistatin (inhibitor of myosin motor function), ML-7 (inhibitor of myosin light chain kinase MLCK, which phosphorylates Myosin II) (see fig. S5), or dimethyl sulfoxide (DMSO) control. Although few cells in control slices failed to abscise and retract their cell-processes within an 8-hour period (n = 4 of 33 cells in 5 embryos) (Fig. 2E and movies S14 to S16), the majority of cells exposed to blebbistatin (n = 33 of 36 cells in 6 embryos) (Fig. 2F and movies S17 to S19; apical surface definition, fig. S6) or ML7 (n = 68 of 83 cells in 15 embryos; Fig. 2G and movies S20 to S22; apical surface definition, fig. S6) remained attached at the ventricle. Furthermore, coexpression of active MRLC2 in the presence of ML-7 rescued its effects, with most cells now abscising within 8 hours (n = 14 of 18 cells in 7 embryos) (Fig. 2H and movies S23 to S25). Misexpression of active MRLC2 alone, however, did not increase neuron numbers, so the potential to increase actin-myosin contraction is by itself insufficient to promote apical abscission (fig. S7). These data indicate that myosin activity is required, but not sufficient, for apical abscission.

Neuroepithelial cells lining the ventricle possess a primary cilium. This projects from the basal body/centrosome located at the apical pole. While this cilium plays a key role in transducing sonic hedgehog (Shh) (and possibly other) signals that maintain neuroepithelial cells in a proliferative state (11), the centrosome is further implicated in positioning axon outgrowth (12). To observe the effect of apical abscission on the primary cilium, we transfected GFP-GPI and pCIG-Neurog2 into the neural tube together with a construct containing a pericentrin-AKAP450 centrosomal targeting (PACT) domain sequence that confers centrosomal localization fused to TagRFP (PACT-TagRFP). As abscission began, the centrosome localized to the withdrawing cell-process (n = 45 cells in 15 embryos) (Fig. 3A and movies S26 to S28). Conversely, the primary cilium, identified with ciliary membrane–associated Arl13b-TagRFP, remained attached to the abscised apical membrane (n = 21 cells in 7 embryos) (Fig. 3B and movies S29 to S31). During apical abscission, the Arl13b-labeled cilium also shortened [two-fold reduction in cilium length; n = 5 abscising cells (8)]. We further used structured illumination microscopy to confirm the presence of Arl13b-GFP in particles abscised from TagRFP-Farn–labeled cells in tissue not subject to culture and live imaging (n = 5 cells) (Fig. 3C and movie S32).

Fig. 3 Apical abscission dismantles the primary cilium.

(A and B) Time-lapse sequences showing centrosome release into the apical cell-process (A) (movie S26), while Arl13b-labeled 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 labeled apical cell-process and abscised particle containing Arl13b-GFP–labeled cilium. (D and 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 and C′, 2 μm.

Active Shh signaling is indicated by accumulation of the Shh transducer Smoothened (Smo) and its key pathway effector Gli2 in the primary cilium (13, 14). Shh signaling is highest in the ventral half of the neural tube, and we therefore assessed localization of endogenous Smo and Gli2 in Tuj1+ cells with ventricle-contacting cell-processes in this region. This revealed many cells with ciliary accumulation of Smo (n = 38 of 43 cells in 4 chick embryos) (Fig. 3D and movie S33) or Gli2 (n = 33 of 35 cells in 3 mouse embryos) (Fig. 3E and movie S34). This localization of proteins suggests that cells poised to abscise are responding to Shh signals and predicts that disjunction of the centrosome and Arl13b-labeled cilium during apical abscission curtails Shh signaling.

Onset of neuronal differentiation is characterized by down-regulation of N-cadherin (3, 5), which forms subapical adherens junctions between neuroepithelial cells (15), and abnormal persistence of N-cadherin inhibits apical cell-process withdrawal (3). Cadherins are connected intracellularly to the contractile actin cable, and this serves to maintain tension at apical junctions and cell-cell adhesion (15). Declining N-cadherin levels within the prospective neuron may therefore trigger apical abscission by loosening cell-cell junctions and connection with the intracellular actin-myosin cable. Because the centrosome is localized in the withdrawing cell-process, it must be released from the cilium before final abscission. To determine how persistent N-cadherin affects apical abscission, we misexpressed N-cadherin-YFP (yellow fluorescent protein) together with GFP-GPI and PACT-TagRFP. Increased N-cadherin blocked cell-process withdrawal, and the centrosome remained at the apical pole (16 cells in 10 embryos) (Fig. 4A and movies S35 to S37). This indicates that N-cadherin down-regulation is required for centrosome release from the apical surface, as well as for final abscission of apical membrane.

Fig. 4 N-cadherin misexpression blocks apical abscission.

(A) Cells misexpressing N-cad-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 misexpressing cells do not express p27 (B′), which is normally detected after apical cell-process detachment (cell nuclei; empty arrowheads). (C) Neurog2 misexpression rescues centrosome release and abscission in N-cad-YFP–expressing cells (movie S38). (D) Neurog2 misexpression decreases subapical N-cad-TagRFP levels (magenta arrows), followed by abscission and cell-process withdrawal. A second underlying cell has yet to withdraw (movie S41). Scale bars, 10 μm; enlarged regions, 2 μm.

One consequence of failure to undergo N-cadherin down-regulation and apical abscission might therefore be maintenance of Shh signaling and therefore inhibition of cell cycle exit. To assess the relationship between cell cycle regulation and apical abscission, we next determined the effect of persistent N-cadherin on expression of p27/Kip1, which normally begins after apical cell-process withdrawal [Figs. 1G and 4B′ and see (7)]. N-cadherin misexpressing cells lacked p27/Kip1 after 24 hours [N-cad-YFP+TagRFP-Farn misexpressing cells 3% p27/Kip1 positive (25 of 689 cells in 4 embryos) (Fig. 4B); control TagRFP-Farn only expressing cells 13% p27/Kip1 positive (76 of 649 cells in 4 embryos) (Fig. 4B′]. These findings therefore place N-cadherin loss and apical abscission, including cilium disassembly, upstream of cell cycle exit as defined by p27/Kip1 expression. Furthermore, driving premature cell cycle exit by p27/Kip1 misexpression did not promote apical abscission or neuronal differentiation (fig. S8), consistent with proneural genes promoting expression of Cdk inhibitors, which then act in concert with other proneural targets to orchestrate neuronal differentiation (16).

N-cadherin down-regulation in prospective neurons is mediated by the transcription factor FoxP2/4, expression of which is promoted by the proneural gene Neurog2 (3, 10). To determine whether Neurog2 misexpression is sufficient to overcome excess N-cadherin, we cotransfected constructs encoding these two genes into the neural tube. Despite excess N-cadherin, cells with excess Neurog2 dismantled their cilium and underwent abscission and cell-process withdrawal (16 cells in 7 embryos) (Fig. 4C and movies S38 to S40). In this context, N-cadherin-TagRFP was localized to the abscission site and then lost before abscission (n = 19 cells in 8 embryos) (Fig. 4D and movies S41 to S43). This indicates that localization and regulation of N-cadherin protein [(as well as transcriptional down-regulation of endogenous N-cadherin (3)] is directed by factors downstream of Neurog2.

These findings uncover a cell biological mechanism, apical abscission, that takes place downstream of N-cadherin loss and involves actin-myosin–dependent cell constriction and dismantling of the primary cilium. This abscission event detaches newborn neurons from the ventricular surface and results in loss of apical-complex–containing cell membrane and therefore apical polarity. By separating centrosome from Arl13b-labeled cilium, apical abscission may curtail active Shh signaling, as indicated by ciliary accumulation of Smo and Gli2 in cells poised to abscise. Consistent with a loss of mitogenic Shh, abscission is also required for expression of cell-cycle exit gene p27/Kip1 (fig. S9). Apical abscission is thus a decisive event in the neuronal differentiation program, which triggers reorganization of the newborn neuron and its withdrawal from the ventricular environment. Abscising the apical membrane and leaving this, at least initially, at the apical surface may also help to maintain tissue integrity. During mitosis, cilia are resorbed or partially internalized (17) rather than shed, and regulated cilium shedding has only been reported in the alga Chlamydomonas (18). Loss of apical complex proteins also characterizes cells undergoing an epithelial to mesenchymal transition, including tumor cell metastasis (19), and some cancers exhibit cilia loss (20). Investigation of apical abscission in normal and also oncogenic epithelia may therefore provide insight into mechanisms that direct critical cell state transitions.

Supplementary Materials

www.sciencemag.org/content/343/6167/200/suppl/DC1

Materials and Methods

Figs. S1 to S9

Movies S1 to S43

References (2130)

References and Notes

  1. Materials and methods are available as supplementary material on Science Online.
  2. Acknowledgments: We thank C. Weijer, A. Muller, and J. Januschke for comments; J. Swedlow for imaging advice; and S. Swift and C. Thomson in the College of Life Sciences Light Microscopy Facility (LMF) for technical support. Structured-illumination microscopy was carried out with assistance of M. Posch (LMF) and L. Ferrand (GE Healthcare) and supported by the Medical Research Council Next Generation Optical Microscopy Award (MR/K015869/1). R.M.D. and K.G.S. are funded by Wellcome Trust program grant 083611/Z/07/Z.
View Abstract

Stay Connected to Science

Navigate This Article