LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages

Mutations in the human LIS1 gene cause the smooth brain disease classical lissencephaly. To understand the underlying mechanisms, we conducted in situ live cell imaging analysis of LIS1 function throughout the entire radial migration pathway. In utero electroporation of LIS1 small interference RNA and short hairpin dominant negative LIS1 and dynactin cDNAs caused a dramatic accumulation of multipolar progenitor cells within the subventricular zone of embryonic rat brains. This effect resulted from a complete failure in progression from the multipolar to the migratory bipolar state, as revealed by time-lapse analysis of brain slices. Surprisingly, interkinetic nuclear oscillations in the radial glial progenitors were also abolished, as were cell divisions at the ventricular surface. Those few bipolar cells that reached the intermediate zone also exhibited a complete block in somal translocation, although, remarkably, process extension persisted. Finally, axonal growth also ceased. These results identify multiple distinct and novel roles for LIS1 in nucleokinesis and process dynamics and suggest that nuclear position controls neural progenitor cell division.     

Timing is crucial for consequences of migratory connectivity

Migratory connectivity can have important consequences for individuals, populations and communities. We argue that most consequences not only depend on which sites are used but importantly also on when these are used and suggest that the timing of migration is characterised by synchrony, phenology, and consistency. We illustrate the importance of these aspects of timing for shaping the consequences of migratory connectivity on individual fitness, population dynamics, gene flow and community dynamics using examples from throughout the animal kingdom. Exemplarily for one specific process that is shaped by migratory connectivity and the timing of migration – the transmission of parasites and the dynamics of diseases – we underpin our arguments with a dynamic epidemiological network model of a migratory population. Here, we quantitatively demonstrate that variations in migration phenology and synchrony yield disease dynamics that significantly differ from a time‐neglecting case. Extending the original definition of migratory connectivity into a spatio‐temporal concept can importantly contribute to understanding the links migratory animals make across the globe and the consequences these may have both for the dynamics of their populations and the communities they visit throughout their journeys. Synthesis Migratory connectivity quantifies the links migrant animals make across the globe and these can have manifold consequences – from individual fitness, population dynamics, gene flow to transmission of pathogens and parasites. We show through the use of empirical examples and a conceptual model that these consequences not only depend on which sites are used but importantly also on when these are used. Therefore, we specify three dimensions of migration timing – phenology, synchrony and consistency, which describe the timing of migration 1) relative to development of key resources; 2) relative to the migration of other individuals; and 3) relative to previous migration events. Each of these dimensions can alter the consequences, but typically through different mechanisms.     

Quantifying Neurite Growth Mediated by Interactions among Secretory Vesicles, Microtubules, and Actin Networks

Neurite growth is a fundamental process of neuronal development, which requires both membrane expansions by exocytosis and cytoskeletal dynamics. However, the specific contribution of these processes has not been yet assessed quantitatively. To study and quantify the growth process, we construct a biophysical model in which we relate the overall neurite outgrowth rate to the vesicle dynamics. By considering the complex motion of vesicles in the cell soma, we demonstrate from biophysical consideration that the main step of finding the neurite initiation site relies mainly on a two-dimensional diffusion/sequestration/fusion at the cell surface and we obtain a novel formula for the flux of vesicles at the neurite base. In the absence of microtubules, we show that a nascent neurite initiated by vesicular delivery can only reach a small length. By adding the microtubule dynamics to the secretory pathway and using stochastic analysis and simulations, we study the complex dynamics of neurite growth. Within this model, depending on the coupling parameter between the microtubules and the neurite, we find different regimes of growth, which describe dendritic and axonal growth. To validate one aspect of our model, we demonstrate that the experimental flux of TI-VAMP but not Synaptobrevin 2 vesicles contributes to the neurite growth. We conclude that although vesicles can be generated randomly in the cell body, the search for the neurite position using the microtubule network and diffusion is quite fast. Furthermore, when the TI-VAMP vesicular flow is large enough, the interactions between the microtubule bundle and the neurite control the growth process. In addition, all of these processes intimately cooperate to mediate the various modes of neurite growth: the model predicts three different growing modes including, in addition to the stable axonal growth and the stochastic dendritic growth, a fast oscillatory regime. Finally our study demonstrates that cytoskeletal dynamics is necessary to generate long protrusion, while vesicular delivery alone can only generate small neurite.     

A Novel Microfluidic Device-Based Neurite Outgrowth Inhibition Assay Reveals the Neurite Outgrowth-Promoting Activity of Tropomyosin Tpm3.1 in Hippocampal Neurons

Overcoming neurite inhibition is integral for restoring neuronal connectivity after CNS injury. Actin dynamics are critical for neurite growth cone formation and extension. The tropomyosin family of proteins is a regarded as master regulator of actin dynamics. This study investigates tropomyosin isoform 3.1 (Tpm3.1) as a potential candidate for overcoming an inhibitory substrate, as it is known to influence neurite branching and outgrowth. We designed a microfluidic device that enables neurons to be grown adjacent to an inhibitory substrate, Nogo-66. Results show that neurons, overexpressing hTpm3.1, have an increased propensity to overcome Nogo-66 inhibition. We propose Tpm3.1 as a potential target for promoting neurite growth in an inhibitory environment in the central nervous system.     

Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth.     

Neuronal migration and the role of reelin during early development of the cerebral cortex

During development, neurons migrate to the cortex radially from periventricular germinative zones as well as tangentially from ganglionic eminences. The vast majority of cortical neurons settle radially in the cortical plate. Neuronal migration requires an exquisite regulation of leading edge extension, nuclear translocation (nucleokinesis), and retraction of trailing processes. During the past few years, several genes and proteins have been identified that are implicated in neuronal migration. Many have been characterized by reference to known mechanisms of neuronal and non-neuronal cell migration in culture; however, probably the most interesting have been identified by gene inactivation or modification in mice and by positional cloning of brain malformation genes in humans and mice. Although it is impossible to provide a fully integrated view, some patterns clearly emerge and are the subject of this article. Specific emphasis is placed on three aspects: first, the role of the actin treadmill, with cyclic formation of filopodial and lamellipodial extensions, in relation to surface events that occur at the leading edge of radially migrating neurons; second, the regulation of microtubule dynamics, which seems to play a key role in nucleokinesis; and third, the mechanisms by which the extracellular protein Reelin regulates neuronal positioning at the end of migration.     

Multi-cellular logistics of collective cell migration

During development, the formation of biological networks (such as organs and neuronal networks) is controlled by multicellular transportation phenomena based on cell migration. In multi-cellular systems, cellular locomotion is restricted by physical interactions with other cells in a crowded space, similar to passengers pushing others out of their way on a packed train. The motion of individual cells is intrinsically stochastic and may be viewed as a type of random walk. However, this walk takes place in a noisy environment because the cell interacts with its randomly moving neighbors. Despite this randomness and complexity, development is highly orchestrated and precisely regulated, following genetic (and even epigenetic) blueprints. Although individual cell migration has long been studied, the manner in which stochasticity affects multi-cellular transportation within the precisely controlled process of development remains largely unknown. To explore the general principles underlying multicellular migration, we focus on the migration of neural crest cells, which migrate collectively and form streams. We introduce a mechanical model of multi-cellular migration. Simulations based on the model show that the migration mode depends on the relative strengths of the noise from migratory and non-migratory cells. Strong noise from migratory cells and weak noise from surrounding cells causes "collective migration," whereas strong noise from non-migratory cells causes "dispersive migration." Moreover, our theoretical analyses reveal that migratory cells attract each other over long distances, even without direct mechanical contacts. This effective interaction depends on the stochasticity of the migratory and non-migratory cells. On the basis of these findings, we propose that stochastic behavior at the single-cell level works effectively and precisely to achieve collective migration in multi-cellular systems.     

Distinct roles of Rac1/Cdc42 and Rho/Rock for axon outgrowth and nucleokinesis of precerebellar neurons toward netrin 1

During embryonic development, tangentially migrating precerebellar neurons emit a leading process and then translocate their nuclei inside it (nucleokinesis). Netrin 1 (also known as netrin-1) acts as a chemoattractant factor for neurophilic migration of precerebellar neurons (PCN) both in vivo and in vitro. In the present work, we analyzed Rho GTPases that could direct axon outgrowth and/or nuclear migration. We show that the expression pattern of Rho GTPases in developing PCN is consistent with their involvement in the migration of PCN from the rhombic lips. We report that pharmacological inhibition of Rho enhances axon outgrowth of PCN and prevents nuclei migration toward a netrin 1 source, whereas inhibition of Rac and Cdc42 sub-families impair neurite outgrowth of PCN without affecting migration. We show, through pharmacological inhibition, that Rho signaling directs neurophilic migration through Rock activation. Altogether, our results indicate that Rho/Rock acts on signaling pathways favoring nuclear translocation during tangential migration of PCN. Thus, axon extension and nuclear migration of PCN in response to netrin 1 are not strictly dependent processes because: (1) distinct small GTPases are involved; (2) axon extension can occur when migration is blocked; and (3) migration can occur when axon outgrowth is impaired.     

Effect of thymosin beta15 on the branching of developing neurons

The thymosin betas (Tbetas) are polypeptide regulators of actin dynamics that are critical for the growth and branching of neurites in developing neurons. We found that mRNAs for Tbeta4, Tbeta10, and Tbeta15 were highly expressed in the developing rat brain during neuritogenesis, supporting a role for the Tbetas in this process. Overexpression of the Tbetas increased the number of neurite branches per neuron in cultured hippocampal and cerebral cortex neurons, and Tbeta15 had the greatest effect. Actin binding activity appears to be essential for the branch-promoting activity of Tbetas because two mutants of Tbeta15 lacking monomeric actin binding activity failed to stimulate branch formation. We also found that transfection of siRNA against Tbeta15 reduced branching. Taken together, these data suggest that the three Tbetas, and especially Tbeta15, stimulate neurite branching during brain development.     

Drebrin Regulates Neuroblast Migration in the Postnatal Mammalian Brain

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis.     

A two-phase growth strategy in cultured neuronal networks as reflected by the distribution of neurite branching angles

Neurite outgrowth and branching patterns are instrumental in dictating the wiring diagram of developing neuronal networks. We study the self-organization of single cultured neurons into complex networks focusing on factors governing the branching of a neurite into its daughter branches. Neurite branching angles of insect ganglion neurons in vitro were comparatively measured in two neuronal categories: neurons in dense cultures that bifurcated under the presence of extrinsic (cellular environment) cues versus neurons in practical isolation that developed their neurites following predominantly intrinsic cues. Our experimental results were complemented by theoretical modeling and computer simulations. A preferred regime of branching angles was found in isolated neurons. A model based on biophysical constraints predicted a preferred bifurcation angle that was consistent with this range shown by our real neurons. In order to examine the origin of the preferred regime of angles we constructed simulations of neurite outgrowth in a developing network and compared the simulated developing neurons with our experimental results. We tested cost functions for neuronal growth that would be optimized at a specific regime of angles. Our results suggest two phases in the process of neuronal development. In the first, reflected by our isolated neurons, neurons are tuned to make first contact with a target cell as soon as possible, to minimize the time of growth. After contact is made, that is, after neuronal interconnections are formed, a second branching strategy is adopted, favoring higher efficiency in neurite length and volume. The two-phase development theory is discussed in relation to previous results.     

Neurite consolidation is an active process requiring constant repression of protrusive activity

During development, neurons extend projections that pathfind to reach their appropriate targets. These projections are composed of two distinct domains: a highly dynamic growth cone and a stable neurite shaft, which is considered to be consolidated. Although the regulation of these domains is critical to the appropriate formation of neural networks, the molecular mechanisms that regulate neurite shape remain poorly understood. Here, we show that calpain protease activity localizes to the neurite shaft, where it is essential for the repression of protrusive activity by limiting cortactin levels and inhibiting actin polymerization. Correspondingly, inhibition of calpain by branching factors induces the formation of new growth cones along the neurite shaft through cAMP elevation. These findings demonstrate that neurite consolidation is an active process requiring constant repression of protrusive activity. We also show that sprouting is, at least in part, accomplished by turning off the mechanism of consolidation.     

A two‐phase growth strategy in cultured neuronal networks as reflected by the distribution of neurite branching angles

Neurite outgrowth and branching patterns are instrumental in dictating the wiring diagram of developing neuronal networks. We study the self‐organization of single cultured neurons into complex networks focusing on factors governing the branching of a neurite into its daughter branches. Neurite branching angles of insect ganglion neurons in vitro were comparatively measured in two neuronal categories: neurons in dense cultures that bifurcated under the presence of extrinsic (cellular environment) cues versus neurons in practical isolation that developed their neurites following predominantly intrinsic cues. Our experimental results were complemented by theoretical modeling and computer simulations. A preferred regime of branching angles was found in isolated neurons. A model based on biophysical constraints predicted a preferred bifurcation angle that was consistent with this range shown by our real neurons. In order to examine the origin of the preferred regime of angles we constructed simulations of neurite outgrowth in a developing network and compared the simulated developing neurons with our experimental results. We tested cost functions for neuronal growth that would be optimized at a specific regime of angles. Our results suggest two phases in the process of neuronal development. In the first, reflected by our isolated neurons, neurons are tuned to make first contact with a target cell as soon as possible, to minimize the time of growth. After contact is made, that is, after neuronal interconnections are formed, a second branching strategy is adopted, favoring higher efficiency in neurite length and volume. The two‐phase development theory is discussed in relation to previous results. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Dynamic maximum entropy provides accurate approximation of structured population dynamics

Realistic models of biological processes typically involve interacting components on multiple scales, driven by changing environment and inherent stochasticity. Such models are often analytically and numerically intractable. We revisit a dynamic maximum entropy method that combines a static maximum entropy with a quasi-stationary approximation. This allows us to reduce stochastic non-equilibrium dynamics expressed by the Fokker-Planck equation to a simpler low-dimensional deterministic dynamics, without the need to track microscopic details. Although the method has been previously applied to a few (rather complicated) applications in population genetics, our main goal here is to explain and to better understand how the method works. We demonstrate the usefulness of the method for two widely studied stochastic problems, highlighting its accuracy in capturing important macroscopic quantities even in rapidly changing non-stationary conditions. For the Ornstein-Uhlenbeck process, the method recovers the exact dynamics whilst for a stochastic island model with migration from other habitats, the approximation retains high macroscopic accuracy under a wide range of scenarios in a dynamic environment.     

A Stochastic Tick-Borne Disease Model: Exploring the Probability of Pathogen Persistence

We formulate and analyse a stochastic epidemic model for the transmission dynamics of a tick-borne disease in a single population using a continuous-time Markov chain approach. The stochastic model is based on an existing deterministic metapopulation tick-borne disease model. We compare the disease dynamics of the deterministic and stochastic models in order to determine the effect of randomness in tick-borne disease dynamics. The probability of disease extinction and that of a major outbreak are computed and approximated using the multitype Galton–Watson branching process and numerical simulations, respectively. Analytical and numerical results show some significant differences in model predictions between the stochastic and deterministic models. In particular, we find that a disease outbreak is more likely if the disease is introduced by infected deer as opposed to infected ticks. These insights demonstrate the importance of host movement in the expansion of tick-borne diseases into new geographic areas.     

miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling

The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR-124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS-specific microRNA miR-124 and connect the regulation of RhoG expression by miR-124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR-124 to control axonal and dendritic branching.     

INFLUENCE OF VIRAL REPLICATION MECHANISMS ON WITHIN‐HOST EVOLUTIONARY DYNAMICS

Viruses replicate their genomes using a variety of mechanisms, leading to different distributions of mutations among their progeny. Yet, models of viral evolution often only consider the mean mutation rate. To investigate when and how replication mechanisms impact viral evolution, we analyze the early dynamics of within‐host infection for two idealized cases: when all offspring virions from an infected cell carry the same genotype, mutated or not; and when mutations occur independently across offspring virions. Other replication life histories fall between these extremes. Using branching process models, we study the probability that viral infection becomes established when mutations are lethal, and in the more general case of two strains of different fitness. For a given mean mutation rate, we show that a lineage of viruses with correlated mutations is less likely to survive than with independent mutations, but when it survives, the viral population grows faster. While this holds true for all parameter regimes, replication life history has a quantitatively significant influence on viral dynamics when stochastic effects are important and when mutations are crucial for survival—conditions typical of evolutionary escape situations.     

Neuronal calcium sensor-1 modulation of optimal calcium level for neurite outgrowth

Neurite extension and branching are affected by activity-dependent modulation of intracellular Ca2+, such that an optimal window of [Ca2+] is required for outgrowth. Our understanding of the molecular mechanisms regulating this optimal [Ca2+]i remains unclear. Taking advantage of the large growth cone size of cultured primary neurons from pond snail Lymnaea stagnalis combined with dsRNA knockdown, we show that neuronal calcium sensor-1 (NCS-1) regulates neurite extension and branching, and activity-dependent Ca2+ signals in growth cones. An NCS-1 C-terminal peptide enhances only neurite branching and moderately reduces the Ca2+ signal in growth cones compared with dsRNA knockdown. Our findings suggest that at least two separate structural domains in NCS-1 independently regulate Ca2+ influx and neurite outgrowth, with the C-terminus specifically affecting branching. We describe a model in which NCS-1 regulates cytosolic Ca2+ around the optimal window level to differentially control neurite extension and branching.     

Proneural transcription factors regulate different steps of cortical neuron migration through Rnd-mediated inhibition of RhoA signaling

Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program.