Image analysis of neural stem cell division patterns in the zebrafish brain.

Proliferating stem cells in the adult body are the source of constant regeneration. In the brain, neural stem cells (NSCs) divide to maintain the stem cell population and generate neural progenitor cells that eventually replenish mature neurons and glial cells. How much spatial coordination of NSC division and differentiation is present in a functional brain is an open question. To quantify the patterns of stem cell divisions, one has to (i) identify the pool of NSCs that have the ability to divide, (ii) determine NSCs that divide within a given time window, and (iii) analyze the degree of spatial coordination. Here, we present a bioimage informatics pipeline that automatically identifies GFP expressing NSCs in three-dimensional image stacks of zebrafish brain from whole-mount preparations. We exploit the fact that NSCs in the zebrafish hemispheres are located on a two-dimensional surface and identify between 1,500 and 2,500 NSCs in six brain hemispheres. We then determine the position of dividing NSCs in the hemisphere by EdU incorporation into cells undergoing S-phase and calculate all pairwise NSC distances with three alternative metrics. Finally, we fit a probabilistic model to the observed spatial patterns that accounts for the non-homogeneous distribution of NSCs. We find a weak positive coordination between dividing NSCs irrespective of the metric and conclude that neither strong inhibitory nor strong attractive signals drive NSC divisions in the adult zebrafish brain. (c) 2017 International Society for Advancement of Cytometr

NG2-glia transiently overcome their homeostatic network and contribute to wound closure after brain injury.

In the adult brain, NG2-glia represent a cell population that responds to injury. To further investigate if, how and why NG2-glia are recruited to the injury site, we analyzed in detail the long-term reaction of NG2-glia after a lesion by time-lapse two-photon in vivo microscopy. Live imaging over several weeks of GFP-labeled NG2-glia in the stab wounded cerebral cortex revealed their fast and heterogeneous reaction, including proliferation, migration, polarization, hypertrophy, or a mixed response, while a small subset of cells remained unresponsive. At the peak of the reaction, 2-4 days after the injury, NG2-glia accumulated around and within the lesion core, overcoming the homeostatic control of their density, which normalized back to physiological conditions only 4 weeks after the insult. Genetic ablation of proliferating NG2-glia demonstrated that this accumulation contributed beneficially to wound closure. Thus, NG2-glia show a fast response to traumatic brain injury (TBI) and participate in tissue repair

A single-cell micro-trench platform for automatic monitoring of cell division and apoptosis after chemotherapeutic drug administration

Abstract Cells vary in their dynamic response to external stimuli, due to stochastic fluctuations and non-uniform progression through the cell cycle. Hence, single-cell studies are required to reveal the range of heterogeneity in their responses to defined perturbations, which provides detailed insight into signaling processes. Here, we present a time-lapse study using arrays of micro-trenches to monitor the timing of cell division and apoptosis in non-adherent cells at the single-cell level. By employing automated cell tracking and division detection, we precisely determine cell cycle duration and sister-cell correlations for hundreds of individual cells in parallel. As a model application we study the response of leukemia cells to the chemostatic drug vincristine as a function of cell cycle phase. The time-to-death after drug addition is found to depend both on drug concentration and cell cycle phase. The resulting timing and dose-response distributions were reproduced in control experiments using synchronized cell populations. Interestingly, in non-synchronized cells, the time-to-death intervals for sister cells appear to be correlated. Our study demonstrates the practical benefits of micro-trench arrays as a platform for high-throughput, single-cell time-lapse studies on cell cycle dependence, correlations and cell fate decisions in general

Twist1 induces distinct cell states depending on TGFBR1-activation.

Basic helix-loop-helix transcription factor Twist1 is a master regulator of Epithelial-Mesenchymal Transition (EMT), a cellular program implicated in different stages of development as well as metastatic dissemination of carcinomas. Here, we show that Twist1 requires TGF-beta type-I receptor (TGFBR1)-activation to bind an enhancer region of downstream effector ZEB1, thereby inducing ZEB1 transcription and EMT. When TGFBR1-phosphorylation is inhibited, Twist1 generates a distinct cell state characterized by collective invasion, simultaneous proliferation and expression of endothelial markers. By contrast, TGFBR1-activation directs Twist1 to induce stable mesenchymal transdifferentiation through EMT, thereby generating cells that display single-cell invasion, but lose their proliferative capacity. In conclusion, preventing Twist1-induced EMT by inhibiting TGFβ-signaling does not generally block acquisition of invasion, but switches mode from single-cell/non-proliferative to collective/proliferative. Together, these data reveal that transient Twist1-activation induces distinct cell states depending on signaling context and caution against the use of TGFβ-inhibitors as a therapeutic strategy to target invasiveness

Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation

Astrocytes are thought to have important roles after brain injury, but their behavior has largely been inferred from postmortem analysis. To examine the mechanisms that recruit astrocytes to sites of injury, we used in vivo two-photon laser-scanning microscopy to follow the response of GFP-labeled astrocytes in the adult mouse cerebral cortex over several weeks after acute injury. Live imaging revealed a marked heterogeneity in the reaction of individual astrocytes, with one subset retaining their initial morphology, another directing their processes toward the lesion, and a distinct subset located at juxtavascular sites proliferating. Although no astrocytes actively migrated toward the injury site, selective proliferation of juxtavascular astrocytes was observed after the introduction of a lesion and was still the case, even though the extent was reduced, after astrocyte-specific deletion of the RhoGTPase Cdc42. Thus, astrocyte recruitment after injury relies solely on proliferation in a specific niche