Elongated Cells Drive Morphogenesis in a Surface-Wrapped Finite-Element Model of Germband Retraction.

During Drosophila embryogenesis, the germband first extends to curl around the posterior end of the embryo and then retracts back; however, retraction is not simply the reversal of extension. At a tissue level, extension is coincident with ventral furrow formation, and at a cellular level, extension occurs via convergent cell neighbor exchanges in the germband, whereas retraction involves only changes in cell shape. To understand how cell shapes, tissue organization, and cellular forces drive germband retraction, we investigate this process using a whole-embryo, surface-wrapped cellular finite-element model. This model represents two key epithelial tissues-amnioserosa and germband-as adjacent sheets of two-dimensional cellular finite elements that are wrapped around an ellipsoidal three-dimensional approximation of an embryo. The model reproduces the detailed kinematics of in vivo retraction by fitting just one free model parameter, the tension along germband cell interfaces; all other cellular forces are constrained to follow ratios inferred from experimental observations. With no additional parameter adjustments, the model also reproduces quantitative assessments of mechanical stress using laser dissection and failures of retraction when amnioserosa cells are removed via mutations or microsurgery. Surprisingly, retraction in the model is robust to changes in cellular force values but is critically dependent on starting from a configuration with highly elongated amnioserosa cells. Their extreme cellular elongation is established during the prior process of germband extension and is then used to drive retraction. The amnioserosa is the one tissue whose cellular morphogenesis is reversed from germband extension to retraction, and this reversal coordinates the forces needed to retract the germband back to its pre-extension position and shape. In this case, cellular force strengths are less important than the carefully established cell shapes that direct them. VIDEO ABSTRACT.

The Attenuation Distribution Across the Long Axis of Breast Cancer Liver Metastases at CT: A Quantitative Biomarker for Predicting Overall Survival.

OBJECTIVE: The objective of our study was to compare attenuation distribution across the long-axis (ADLA) measurements, Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, and Choi criteria for predicting overall survival (OS) in patients with metastatic breast cancer treated with bevacizumab. MATERIALS AND METHODS: We obtained HIPAA-compliant data from a prospective, multisite, phase 3 trial of bevacizumab for the treatment of metastatic breast cancer. For patients with one or more liver metastases measuring 15 mm or larger at baseline, we evaluated up to two target liver lesions using RECIST, Choi criteria, and ADLA measurements, with the latter defined as the SD of the CT attenuation values of each pixel along the tumor long-axis diameter. The optimal percentage change threshold for defining an ADLA response was computed by cross-validation analysis in a Cox model. The log-rank test was applied to evaluate RECIST, Choi criteria, and ADLA for discriminating patients with superior OS. The predictive accuracies of all three techniques were compared using Brier scores and areas under the ROC curve (AUC). All analyses were performed separately using best overall response (BOR) and response at the first follow-up time point (FU1). RESULTS: One hundred sixty-four patients met the inclusion criteria. A 25% decrease in the ADLA measurement from baseline was the optimal ADLA response threshold for BOR and FU1. RECIST, Choi criteria, and ADLA successfully identified patients with superior OS when using BOR (RECIST, p = 0.02; Choi and ADLA, p < 0.001), but only Choi criteria and ADLA measurements were successful when using FU1 (RECIST, p = 0.43; Choi and ADLA, p < 0.001). In a direct comparison, ADLA measurements outperformed both RECIST and Choi criteria using BOR (95% CI for Brier score differences, ADLA-RECIST [-0.58 to -0.08] and ADLA-Choi [-0.55 to -0.06]; 95% CI for AUC differences, ADLA-RECIST [0.16-0.33] and ADLA-Choi [0.17-0.36]) as well as using FU1 (95% CI for Brier score differences, ADLA-RECIST [-0.77 to -0.08] and ADLA-Choi [-0.58 to -0.03]; 95% CI for AUC differences, ADLA-RECIST [0.22-0.39] and ADLA-Choi [0.01-0.22]). CONCLUSION: ADLA measurements may be a useful noninvasive indicator of cancer treatment response. Because ADLA measurements may be extracted relatively easily using existing radiologist workflows, further investigation of the ADLA technique is warranted.

Multiple Mechanisms Drive Calcium Signal Dynamics around Laser-Induced Epithelial Wounds.

Epithelial wound healing is an evolutionarily conserved process that requires coordination across a field of cells. Studies in many organisms have shown that cytosolic calcium levels rise within a field of cells around the wound and spread to neighboring cells, within seconds of wounding. Although calcium is a known potent second messenger and master regulator of wound-healing programs, it is unknown what initiates the rise of cytosolic calcium across the wound field. Here we use laser ablation, a commonly used technique for the precision removal of cells or subcellular components, as a tool to investigate mechanisms of calcium entry upon wounding. Despite its precise ablation capabilities, we find that this technique damages cells outside the primary wound via a laser-induced cavitation bubble, which forms and collapses within microseconds of ablation. This cavitation bubble damages the plasma membranes of cells it contacts, tens of microns away from the wound, allowing direct calcium entry from extracellular fluid into damaged cells. Approximately 45 s after this rapid influx of calcium, we observe a second influx of calcium that spreads to neighboring cells beyond the footprint of cavitation. The occurrence of this second, delayed calcium expansion event is predicted by wound size, indicating that a separate mechanism of calcium entry exists, corresponding to cell loss at the primary wound. Our research demonstrates that the damage profile of laser ablation is more similar to a crush injury than the precision removal of individual cells. The generation of membrane microtears upon ablation is consistent with studies in the field of optoporation, which investigate ablation-induced cellular permeability. We conclude that multiple types of damage, including microtears and cell loss, result in multiple mechanisms of calcium influx around epithelial wounds.

Computational Model of Secondary Palate Fusion and Disruption.

Morphogenetic events are driven by cell-generated physical forces and complex cellular dynamics. To improve our capacity to predict developmental effects from chemical-induced cellular alterations, we built a multicellular agent-based model in CompuCell3D that recapitulates the cellular networks and collective cell behavior underlying growth and fusion of the mammalian secondary palate. The model incorporated multiple signaling pathways (TGFß, BMP, FGF, EGF, and SHH) in a biological framework to recapitulate morphogenetic events from palatal outgrowth through midline fusion. It effectively simulated higher-level phenotypes (e.g., midline contact, medial edge seam (MES) breakdown, mesenchymal confluence, and fusion defects) in response to genetic or environmental perturbations. Perturbation analysis of various control features revealed model functionality with respect to cell signaling systems and feedback loops for growth and fusion, diverse individual cell behaviors and collective cellular behavior leading to physical contact and midline fusion, and quantitative analysis of the TGF/EGF switch that controls MES breakdown-a key event in morphogenetic fusion. The virtual palate model was then executed with theoretical chemical perturbation scenarios to simulate switch behavior leading to a disruption of fusion following chronic (e.g., dioxin) and acute (e.g., retinoic acid) chemical exposures. This computer model adds to similar systems models toward an integrative "virtual embryo" for simulation and quantitative prediction of adverse developmental outcomes following genetic perturbation and/or environmental disruption.

Amnioserosa development and function in Drosophila embryogenesis: Critical mechanical roles for an extraembryonic tissue.

Despite being a short-lived, extraembryonic tissue, the amnioserosa plays critical roles in the major morphogenetic events of Drosophila embryogenesis. These roles involve both cellular mechanics and biochemical signaling. Its best-known role is in dorsal closure-well studied by both developmental biologists and biophysicists-but the amnioserosa is also important during earlier developmental stages. Here, we provide an overview of amnioserosa specification and its role in several key developmental stages: germ band extension, germ band retraction, and dorsal closure. We also compare embryonic development in Drosophila and its relative Megaselia to highlight how the amnioserosa and its roles have evolved. Placed in context, the amnioserosa provides a fascinating example of how signaling, mechanics, and morphogen patterns govern cell-type specification and subsequent morphogenetic changes in cell shape, orientation, and movement. Developmental Dynamics 245:558-568, 2016. © 2016 Wiley Periodicals, Inc.

Pathway to a phenocopy: Heat stress effects in early embryogenesis.

BACKGROUND: Heat shocks applied at the onset of gastrulation in early Drosophila embryos frequently lead to phenocopies of U-shaped mutants-having characteristic failures in the late morphogenetic processes of germband retraction and dorsal closure. The pathway from nonspecific heat stress to phenocopied abnormalities is unknown. RESULTS: Drosophila embryos subjected to 30-min, 38 °C heat shocks at gastrulation appear to recover and restart morphogenesis. Post-heat-shock development appears normal, albeit slower, until a large fraction of embryos develop amnioserosa holes (diameters > 100 µm). These holes are positively correlated with terminal U-shaped phenocopies. They initiate between amnioserosa cells and open over tens of minutes by evading normal wound healing responses. They are not caused by tissue-wide increases in mechanical stress or decreases in cell-cell adhesion, but instead appear to initiate from isolated apoptosis of amnioserosa cells. CONCLUSIONS: The pathway from heat shock to U-shaped phenocopies involves the opening of one or more large holes in the amnioserosa that compromise its structural integrity and lead to failures in morphogenetic processes that rely on amnioserosa-generated tensile forces. The proposed mechanism by which heat shock leads to hole initiation and expansion is heterochonicity, i.e., disruption of morphogenetic coordination between embryonic and extra-embryonic cell types.

Optic nerve sheath fenestration using a Raman-shifted alexandrite laser.

BACKGROUND AND OBJECTIVE: Optic nerve sheath fenestration is an established procedure for relief of potentially damaging overpressure on the optic nerve resulting from idiopathic intracranial hypertension. Prior work showed that a mid-IR free-electron laser could be delivered endoscopically and used to produce an effective fenestration. This study evaluates the efficacy of fenestration using a table-top mid-IR source based on a Raman-shifted alexandrite (RSA) laser. STUDY DESIGN/MATERIALS AND METHODS: Porcine optic nerves were ablated using light from an RSA laser at wavelengths of 6.09, 6.27, and 6.43 µm and pulse energies up to 3 mJ using both free-space and endoscopic beam delivery through 250-µm I.D. hollow-glass waveguides. Waveguide transmission was characterized, ablation thresholds and etch rates were measured, and the efficacy of endoscopic fenestration was evaluated for ex vivo exposures using both optical coherence tomography and histological analysis. RESULTS: Using endoscopic delivery, the RSA laser can effectively fenestrate porcine optic nerves. Performance was optimized at a wavelength of 6.09 µm and delivered pulse energies of 0.5-0.8 mJ (requiring 1.5-2.5 mJ to be incident on the waveguide). Under these conditions, the ablation threshold fluence was 0.8 ± 0.2 J/cm(2) , the ablation rate was 1-4 µm/pulse, and the margins of ablation craters showed little evidence of thermal or mechanical damage. Nonetheless, nominally identical exposures yielded highly variable ablation rates. This led to fenestrations that ranged from too deep to too shallow-either damaging the underlying optic nerve or requiring additional exposure to cut fully through the sheath. Of 48 excised nerves subjected to fenestration at 6.09 µm, 16 ex vivo fenestrations were judged as good, 23 as too deep, and 9 as too shallow. CONCLUSIONS: Mid-IR pulses from the RSA laser, propagated through a flexible hollow waveguide, are capable of cutting through porcine optic nerve sheaths in surgically relevant times with reasonable accuracy and low collateral damage. This can be accomplished at wavelengths of 6.09 or 6.27 µm, with 6.09 µm slightly preferred. The depth of ex vivo fenestrations was difficult to control, but excised nerves lack a sufficient layer of cerebrospinal fluid that would provide an additional margin of safety in actual patients.

Cellular mechanics of germ band retraction in Drosophila.

Germ band retraction involves a dramatic rearrangement of the tissues on the surface of the Drosophila embryo. As germ band retraction commences, one tissue, the germ band, wraps around another, the amnioserosa. Through retraction the two tissues move cohesively as the highly elongated cells of the amnioserosa contract and the germ band moves so it is only on one side of the embryo. To understand the mechanical drivers of this process, we designed a series of laser ablations that suggest a mechanical role for the amnioserosa. First, we find that during mid retraction, segments in the curve of the germ band are under anisotropic tension. The largest tensions are in the direction in which the amnioserosa contracts. Second, ablating one lateral flank of the amnioserosa reduces the observed force anisotropy and leads to retraction failures. The other intact flank of amnioserosa is insufficient to drive retraction, but can support some germ band cell elongation and is thus not a full phenocopy of ush mutants. Another ablation-induced failure in retraction can phenocopy mys mutants, and does so by targeting amnioserosa cells in the same region where the mutant fails to adhere to the germ band. We conclude that the amnioserosa must play a key, but assistive, mechanical role that aids uncurling of the germ band.

A microfluidic-enabled mechanical microcompressor for the immobilization of live single- and multi-cellular specimens.

A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.

Modeling cell elongation during germ band retraction: cell autonomy versus applied anisotropic stress.

The morphogenetic process of germ band retraction in Drosophila embryos involves coordinated movements of two epithelial tissues - germ band and amnioserosa. The germ band shortens along its rostral-caudal or head-to-tail axis, widens along its perpendicular dorsal-ventral axis, and uncurls from an initial 'U' shape. The amnioserosa mechanically assists this process by pulling on the crook of the U-shaped germ band. The amnioserosa may also provide biochemical signals that drive germ band cells to change shape in a mechanically autonomous fashion. Here, we use a finite-element model to investigate how these two contributions reshape the germ band. We do so by modeling the response to laser-induced wounds in each of the germ band's spatially distinct segments (T1-T3, A1-A9) during the middle of retraction when segments T1-A3 form the ventral arm of the 'U', A4-A7 form its crook, and A8-A9 complete the dorsal arm. We explore these responses under a range of externally applied stresses and internal anisotropy of cell edge tensions - akin to a planar cell polarity that can drive elongation of cells in a direction parallel to the minimum edge tension - and identify regions of parameter space (edge-tension anisotropy versus stress anisotropy) that best match previous experiments for each germ band segment. All but three germ band segments are best fit when the applied stress anisotropy and the edge-tension anisotropy work against one another - i.e., when the isolated effects would elongate cells in perpendicular directions. Segments in the crook of the germ band (A4-A7) have cells that elongate in the direction of maximum external stress, i.e., external stress anisotropy is dominant. In most other segments, the dominant factor is internal edge-tension anisotropy. These results are consistent with models in which the amnioserosa pulls on the crook of the germ band to mechanically assist retraction. In addition, they suggest a mechanical cue for edge-tension anisotropy whereby cells do not globally orient their internal elongation axis towards the amnioserosa, but instead orient this axis perpendicular to the local principal stress direction.

Wounding increases nuclear ploidy in wound-proximal epidermal cells of the Drosophila pupal notum.

After injury, tissues must replace cell mass and genome copy number. The mitotic cycle is one mechanism for replacement, but non-mitotic strategies have been observed in quiescent tissues to restore tissue ploidy after wounding. Here we report that nuclei of the mitotically capable Drosophila pupal notum enlarged following nearby laser ablation. Measuring DNA content, we determined that nuclei within 100 µm of a laser-wound increased their ploidy to ~8C, consistent with one extra S-phase. These data indicate non-mitotic repair strategies are not exclusively utilized by quiescent tissues and may be an underexplored wound repair strategy in mitotic tissues.

Live imaging basement membrane assembly under the pupal notum epithelium.

Basement membranes are sheet-like extracellular matrices containing Collagen IV, and they are conserved across the animal kingdom. Basement membranes usually line the basal surfaces of epithelia, where they contribute to structure, maintenance, and signaling. Although adult epithelia contact basement membranes, in early embryos the epithelia contact basement membranes only after basement membranes are assembled in embryogenesis. In Drosophila , the pupal notum epithelium is a useful model for live imaging epithelial cell behaviors, yet it is unclear when the basement membrane assembles in the pupa, as pupae are undergoing metamorphosis, similar to embryogenesis. To characterize the basement membrane in the pupal notum, we used spinning disk fluorescent microscopy to visualize Collagen IV subunit Vkg-GFP and adherens junction protein p120ctnRFP. Bright punctae of Vkg-GFP were observed in the X-Y plane, possibly representing Vkg-containing cells. We found that a thin continuous Vkg-containing basement membrane was evident at 14 h APF, which became more enriched with Vkg-GFP over the next 6 h, indicating the basement membrane is still assembling during that time. Live imaging of the pupal notum during this time could provide insight into formation, assembly, and repair of the basement membranes.

After wounding, a G-protein coupled receptor promotes the restoration of tension in epithelial cells.

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension, we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin about 10 minutes after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

After wounding, a G-protein coupled receptor promotes the restoration of tension in epithelial cells.

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin ∼10 min after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

Apical oscillations in amnioserosa cells: basolateral coupling and mechanical autonomy.

Holographic laser microsurgery is used to isolate single amnioserosa cells in vivo during early dorsal closure. During this stage of Drosophila embryogenesis, amnioserosa cells undergo oscillations in apical surface area. The postisolation behavior of individual cells depends on their preisolation phase in these contraction/expansion cycles: cells that were contracting tend to collapse quickly after isolation; cells that were expanding do not immediately collapse, but instead pause or even continue to expand for ∼40 s. In either case, the postisolation apical collapse can be prevented by prior anesthetization of the embryos with CO2. These results suggest that although the amnioserosa is under tension, its cells are subjected to only small elastic strains. Furthermore, their postisolation apical collapse is not a passive elastic relaxation, and both the contraction and expansion phases of their oscillations are driven by intracellular forces. All of the above require significant changes to existing computational models.

Video force microscopy reveals the mechanics of ventral furrow invagination in Drosophila.

The absence of tools for mapping the forces that drive morphogenetic movements in embryos has impeded our understanding of animal development. Here we describe a unique approach, video force microscopy (VFM), that allows detailed, dynamic force maps to be produced from time-lapse images. The forces at work in an embryo are considered to be decomposed into active and passive elements, where active forces originate from contributions (e.g., actomyosin contraction) that do mechanical work to the system and passive ones (e.g., viscous cytoplasm) that dissipate energy. In the present analysis, the effects of all passive components are considered to be subsumed by an effective cytoplasmic viscosity, and the driving forces are resolved into equivalent forces along the edges of the polygonal boundaries into which the region of interest is divided. Advanced mathematical inverse methods are used to determine these driving forces. When applied to multiphoton sections of wild-type and mutant Drosophila melanogaster embryos, VFM is able to calculate the equivalent driving forces acting along individual cell edges and to do so with subminute temporal resolution. In the wild type, forces along the apical surface of the presumptive mesoderm are found to be large and to vary parabolically with time and angular position, whereas forces along the basal surface of the ectoderm, for example, are found to be smaller and nearly uniform with position. VFM shows that in mutants with reduced junction integrity and myosin II activity, the driving forces are reduced, thus accounting for ventral furrow failure.

Piezo initiates transient production of collagen IV to repair damaged basement membranes.

Basement membranes are sheets of extracellular matrix separating tissue layers and providing mechanical support. Their mechanical properties are determined largely by their most abundant protein, Collagen IV (Col4). Although basement membranes are repaired after damage, little is known about how. To wit, since basement membrane is extracellular it is unknown how damage is detected, and since Col4 is long-lived it is unknown how it is regulated to avoid fibrosis. Using the basement membrane of the adult Drosophila midgut as a model, we show that repair is distinct from maintenance. In healthy conditions, midgut Col4 originates from the fat body, but after damage, a subpopulation of enteroblasts we term "matrix menders" transiently express Col4, and Col4 from these cells is required for repair. Activation of the mechanosensitive channel Piezo is required for matrix menders to upregulate Col4, and the signal to initiate repair is a reduction in basement membrane stiffness. Our data suggests that mechanical sensitivity may be a general property of Col4-producing cells.

A mathematical model of calcium signals around laser-induced epithelial wounds.

Cells around epithelial wounds must first become aware of the wound's presence in order to initiate the wound-healing process. An initial response to an epithelial wound is an increase in cytosolic calcium followed by complex calcium-signaling events. While these calcium signals are driven by both physical and chemical wound responses, cells around the wound will all be equipped with the same cellular components to produce and interact with the calcium signals. Here we have developed a mathematical model in the context of laser ablation of the Drosophila pupal notum that integrates tissue-level damage models with a cellular calcium-signaling toolkit. The model replicates experiments in the contexts of control wounds as well as knockdowns of specific cellular components, but it also provides new insights that are not easily accessible experimentally. The model suggests that cell-cell variability is necessary to produce calcium-signaling events observed in experiments; it quantifies calcium concentrations during wound-induced signaling events, and it shows that intercellular transfer of the molecule IP3 is required to coordinate calcium signals across distal cells around the wound. The mathematical model developed here serves as a framework for quantitative studies in both wound signaling and calcium signaling in the Drosophila system.

Wound-Induced Syncytia Outpace Mononucleate Neighbors during Drosophila Wound Repair.

All organisms have evolved to respond to injury. Cell behaviors like proliferation, migration, and invasion replace missing cells and close wounds. However, the role of other wound-induced cell behaviors is not understood, including the formation of syncytia (multinucleated cells). Wound-induced epithelial syncytia were first reported around puncture wounds in post-mitotic Drosophila epidermal tissues, but have more recently been reported in mitotically competent tissues such as the Drosophila pupal epidermis and zebrafish epicardium. The presence of wound-induced syncytia in mitotically active tissues suggests that syncytia offer adaptive benefits, but it is unknown what those benefits are. Here, we use in vivo live imaging to analyze wound-induced syncytia in mitotically competent Drosophila pupae. We find that almost half the epithelial cells near a wound fuse to form large syncytia. These syncytia use several routes to speed wound repair: they outpace diploid cells to complete wound closure; they reduce cell intercalation during wound closure; and they pool the resources of their component cells to concentrate them toward the wound. In addition to wound healing, these properties of syncytia are likely to contribute to their roles in development and pathology.

Enabling user-guided segmentation and tracking of surface-labeled cells in time-lapse image sets of living tissues.

To study the process of morphogenesis, one often needs to collect and segment time-lapse images of living tissues to accurately track changing cellular morphology. This task typically involves segmenting and tracking tens to hundreds of individual cells over hundreds of image frames, a scale that would certainly benefit from automated routines; however, any automated routine would need to reliably handle a large number of sporadic, and yet typical problems (e.g., illumination inconsistency, photobleaching, rapid cell motions, and drift of focus or of cells moving through the imaging plane). Here, we present a segmentation and cell tracking approach based on the premise that users know their data best-interpreting and using image features that are not accounted for in any a priori algorithm design. We have developed a program, SeedWater Segmenter, that combines a parameter-less and fast automated watershed algorithm with a suite of manual intervention tools that enables users with little to no specialized knowledge of image processing to efficiently segment images with near-perfect accuracy based on simple user interactions.