Apical abscission, a novel cell biological mechanism regulating neurogenesis

Abstract

Withdrawal of differentiating neurons from the ventricle of the neural tube is a highly regulated process that is critical for the formation of normal tissue architecture and neural circuitry. We have recently reported a novel cell-biological event that mediates this process and involves local abscission of the apical cell-compartment as cells become neurons. Apical abscission takes place as adherens junctions are lost, depends on actinomyosin contractility, results in loss of apical cell polarity as well as dis-assembly of the primary cilium, and allows cell cycle exit. The molecular mechanisms mediating the series of steps underlying apical abscission are complex and inter-linked and open up many new questions. In this commentary we discuss how these mechanisms may operate and the functions of apical abscission during neurogenesis, in disease, and in other cell biological processes that involve withdrawal of cells from epithelia.

Keywords: :

• neurogenesis
• spinal cord
• adherens junction
• apical polarity
• centrosome
• primary cilium
• cancer
• EMT
• periventricular heterotopia

This article refers to:

Introduction

Becoming a neuron is a complex process. It involves not only exit from the cell cycle but also dramatic change in cell shape and movement. Neurons are generated in the proliferative ventricular layer, located at the lumen of the neural tube and they leave this region to form the mantle layer or cortical layers within the developing brain. A prerequisite for this migration is the detachment of the newborn neuron from the luminal or apical surface. Failure to do so leads to the ectopic accumulation of neurons, which can result in human periventricular heterotopia, a syndrome that includes reduced brain size, epilepsy and dyslexia.¹

Cells about to undergo neuronal differentiation have a very particular morphology: a basally located cell body and a long thin cellular process with an apical endfoot that contributes to the ventricular/apical surface (Fig. 1).² These cells exhibit apico-basal polarity, characterized by localization of the Par complex proteins (Par3, Par6, and aPKC) to the apical-most cell membrane. Such cells also possess an apically positioned primary cilium, which is required to transduce mitogenic Sonic hedgehog (Shh) signals emanating from the floor plate of the neural tube.³ These cells also share sub-apical adherens junctions with neighboring cells that interact with an intracellular contractile actinomyosin cable that transmits forces to the cell cortex. An initial step in the detachment from the ventricle involves breakdown of these inter-cellular connections involving loss of the adherens junction component N-cadherin (Fig. 1).^4-6 Surprisingly, we observed that following N-cadherin loss the prospective neuron undergoes local abscission of its apical membrane, resulting in loss of apical cell polarity. We further demonstrated that this apical abscission event is driven by constriction of the sub-apical actinomyosin cable. During apical abscission, we observed a dis-assembly of the centrosome-primary cilium complex, causing the centrosome to be retained by the withdrawing neuron while the ciliary membrane is left behind in the abscised particle. This suggests that the newborn neuron is now incapable of signaling via Shh; and this, in turn facilitates cell cycle exit. Consistent with this we found that blocking abscission by N-Cadherin mis-expression inhibited onset of the cell cycle exit gene p27.

Figure 1. Steps underlying apical abscission in the neural tube. (A) Schematic of sequence of events taking place as a cell differentiates into a neuron in the early neural tube/spinal cord, beginning with the withdrawal of the apical cell-process from the ventricular surface and subsequent production of a growth cone and primary axon extension. Sonic hedgehog (Shh) signals from the notochord/floor plate promote proliferation and pattern in the spinal cord and their transduction requires an intact primary cilium; (A’) A cell poised to undergo neuronal differentiation, characterized by a basally located cell body and attachment to the apical surface through an elongated apical cell-process. (A”) Schematic of key sub-cellular structures in the apical endfoot of the prospective neuron (white boxed region in A’) (including apical complex containing apical membrane, the primary cilium, cadherin-containing adherens junctions and associated actinomyosin cable) and sequential changes leading to apical abscission following loss of N-cadherin.

Molecular Mechanisms Regulating N-Cadherin Loss

Ectopic maintenance of N-Cadherin also blocked centrosome-cilium dis-assembly as well as abscission, while mis-expression of the proneural factor Neurog2 was able to rescue this by driving depletion of ectopically expressed N-cadherin protein. The molecular mechanisms that deplete N-cadherin from the adherens junction remain unclear, although our observations place Neurog2 upstream of this process. Dynamic instability is a key feature of adherens junctions in vivo and in vitro, and this is mediated by clathrin-dependent endocytic turnover of cadherins. Endocytic turnover is negatively regulated by the cadherin-binding partner p120 catenin^7-9 and the adherens junction-associated protein Numb.¹⁰ Our observations further support that turnover of N-cadherin is a tightly controlled process during neurogenesis and raise the possibility that attenuation of N-cadherin recycling by altered levels of p120 catenin and/or Numb, resulting in less robust cell–cell junctions and increased actinomyosin contractility may regulate progression of apical abscission.

Similarities between Apical Abscission and Cytokinesis

Apical abscission is characterized by a local increase in sub-apical actin density and myosin activity (Fig. 1). This is remarkably similar to cytokinesis where a contractile actinomyosin ring cleaves the cell in to two¹¹ and one possibility is that apical abscission utilizes some cytokinesis machinery. Unlike cytokinesis, however, apical abscission is an asymmetric process, allocating a single centrosome (and nucleus) on one side of the abscission site and the apical and ciliary membrane on the other. While cytokinesis requires de-novo formation of a contractile actinomyosin cable,^12-15 neuroepithelial cells already possess a sub-apical actinomyosin cable that mediates apical constriction.^16-18 The central spindle, consisting of stable antiparallel microtubules,¹⁹ marks the site of the cytokinetic furrow and stimulates actinomyosin contractility during cytokinesis.²⁰ Future studies will determine if formation of a similar microtubule array at the site of apical abscission is required for actinomyosin constriction to proceed. Furthermore, super-resolution structured illumination microscopy revealed a thin membranous connection between the withdrawing apical cell-process and the abscising particle similar to the cytokinetic midbody, suggesting that a final membrane scission event is involved at completion of apical abscission. The molecular machinery that mediates this is likely to be similar to cytokinetic membrane scission, as this is highly evolutionarily conserved.^21,22

Molecular Mechanisms of Primary Cilium Disassembly

During mitosis, the primary cilium is resorbed or partially internalized.^23,24 This is distinct from apical abscission, as here the ciliary membrane is released along with the abscised membrane (Fig. 1). We observed that neurons poised to abscise exhibit accumulation of the hedgehog transducer Smoothened and its pathway effector Gli2 in their primary cilia, which is indicative of cells responding to hedgehog signals;²⁵ apical abscission may therefore effectively curtail such mitogenic signaling, thereby promoting cell cycle exit. The only other example of regulated cilium shedding has been observed during deflagellation in the blue-green alga Chlamydomonas.²⁶ This is mediated by microtubule severing proteins and results in shedding of the whole axonemal structure.²⁷ It will be important to determine if apical abscission also involves severance of the axoneme and whether/how this may be linked to actinomyosin contractility through, for example, the formation of antiparallel microtubule arrays.

Apical Abscission as a Mechanism for Reconfiguring the Newborn Neuron

Apical abscission thus appears to be a pivotal moment in the neuronal differentiation program as it not only mediates acute loss of apical cell polarity, but also transiently releases the cell from the influence of extracellular signals mediated by the primary cilium (Fig. 1). This may provide a critical transition period during which the newborn neuron re-polarizes and re-organizes its cytoskeleton to establish the site of the nascent axon.^6,28 The release of the centrosome from the primary cilium and the discarding of apical complex containing membrane may be necessary for this step, allowing re-deployment of cell polarity machinery and re-positioning of the centrosome, which is thought to influence this process.^29,30 The centrosome will also be required for later signaling through a new cilium, which is regenerated in the differentiating neuron. Indeed, this shedding and replenishing of the ciliary membrane may mediate change in the cell’s response to signals such as Shh, which, although initially mitogenic in the neural tube, is later re-deployed as an axon guidance molecule.³¹ Neuronal primary cilia also play further roles, for example, in the regulation of cortical interneuron migration.³²

Maintenance of Tissue Homeostasis

Abscising the apical membrane and leaving this, at least initially, at the apical surface may allow neighboring cells to adjust to this loss of cell–cell contact and so help to maintain epithelial tissue integrity. Furthermore, Notch and its ligands Dll1 and Jagged1 are enriched at the apical end feet of neural progenitor cells where they mediate lateral inhibition^33-36 and interact with ZO-1, a constituent of the adherens junctions.³⁵ This suggests that interactions at the apical domains of neuroepithelial cells play a key role in regulating neuronal differentiation; when a newborn neuron undergoes apical abscission it may relieve neighboring neural progenitors from Notch signalling, allowing these cells in turn the opportunity to become neurons. It is also possible that persisting discarded apical membrane, enriched for Notch ligands, at least briefly prevents neural progenitors from differentiating into neurons.

Apical Abscission and Human Disease

Human periventricular heterotopia involves failure of neurons to leave the ventricular surface and is anatomically characterized by nodular structures containing these ectopically located neurons. A major cause is mutation in the actin-binding protein Filamin-1 (an X-linked dominant locus), which cross-links actin filaments and creates the meshwork required for actin–myosin interaction.³⁷ Future studies will determine if defective apical abscission, which we show depends on actin–myosin contractility, contributes to this phenotype.

Finally, loss of apical complex proteins and disruption of adherens junctions also characterizes cells undergoing an epithelial to mesenchymal transition (EMT). Indeed, transcription factors (Scratch1/2) belonging to the Snail super-family have been shown to attenuate E-cadherin expression in prospective cortical neurons³⁸ indicating that elements of the EMT machinery also operate during neuronal detachment. However, knockdown of apical complex proteins such as Par3 can increase the probability of tumor cell metastasis even without triggering a full EMT.³⁹ Some cancers also exhibit cilia loss, which depending on the underlying mutation, can augment or attenuate disease progression.^40,41 The mechanism underlying cilia loss in cancer cells is unknown and it will be important to determine whether this and progression to metastasis involve apical abscission events. It is also possible that apical abscission take places in other regions of the embryo undergoing EMT, such as the neural crest and during gastrulation as epiblast cells ingress through the primitive streak and acquire a mesodermal fate.⁴² These important next steps will establish the generality of apical abscission as mechanism for cell state change.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors were supported by a Wellcome Trust Investigator Award WT102817AIA.