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1.
JCI Insight ; 7(3)2022 02 08.
Article in English | MEDLINE | ID: mdl-35132963

ABSTRACT

Remodeling of injured sympathetic nerves on the heart after myocardial infarction (MI) contributes to adverse outcomes such as sudden arrhythmic death, yet the underlying structural mechanisms are poorly understood. We sought to examine microstructural changes on the heart after MI and to directly link these changes with electrical dysfunction. We developed a high-resolution pipeline for anatomically precise alignment of electrical maps with structural myofiber and nerve-fiber maps created by customized computer vision algorithms. Using this integrative approach in a mouse model, we identified distinct structure-function correlates to objectively delineate the infarct border zone, a known source of arrhythmias after MI. During tyramine-induced sympathetic nerve activation, we demonstrated regional patterns of altered electrical conduction aligned directly with altered neuroeffector junction distribution, pointing to potential neural substrates for cardiac arrhythmia. This study establishes a synergistic framework for examining structure-function relationships after MI with microscopic precision that has potential to advance understanding of arrhythmogenic mechanisms.


Subject(s)
Body Surface Potential Mapping/methods , Myocardial Infarction/diagnosis , Myocardium/pathology , Sympathetic Nervous System/diagnostic imaging , Action Potentials , Animals , Disease Models, Animal , Male , Mice , Mice, Inbred C57BL , Myocardial Infarction/physiopathology , Sympathetic Nervous System/physiopathology
2.
IEEE Trans Neural Netw Learn Syst ; 32(6): 2521-2534, 2021 Jun.
Article in English | MEDLINE | ID: mdl-32687472

ABSTRACT

Disentangling the sources of visual motion in a dynamic scene during self-movement or ego motion is important for autonomous navigation and tracking. In the dynamic image segments of a video frame containing independently moving objects, optic flow relative to the next frame is the sum of the motion fields generated due to camera and object motion. The traditional ego-motion estimation methods assume the scene to be static, and the recent deep learning-based methods do not separate pixel velocities into object- and ego-motion components. We propose a learning-based approach to predict both ego-motion parameters and object-motion field (OMF) from image sequences using a convolutional autoencoder while being robust to variations due to the unconstrained scene depth. This is achieved by: 1) training with continuous ego-motion constraints that allow solving for ego-motion parameters independently of depth and 2) learning a sparsely activated overcomplete ego-motion field (EMF) basis set, which eliminates the irrelevant components in both static and dynamic segments for the task of ego-motion estimation. In order to learn the EMF basis set, we propose a new differentiable sparsity penalty function that approximates the number of nonzero activations in the bottleneck layer of the autoencoder and enforces sparsity more effectively than L1- and L2-norm-based penalties. Unlike the existing direct ego-motion estimation methods, the predicted global EMF can be used to extract OMF directly by comparing it against the optic flow. Compared with the state-of-the-art baselines, the proposed model performs favorably on pixelwise object- and ego-motion estimation tasks when evaluated on real and synthetic data sets of dynamic scenes.

3.
Neurogastroenterol Motil ; 33(8): e14014, 2021 08.
Article in English | MEDLINE | ID: mdl-33094876

ABSTRACT

BACKGROUND: Intravenous administration of adeno-associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV-PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV-PHP.S with a tunable two-component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). METHODS: A vector system including a tetracycline inducer with a tet-responsive element driving three separate fluorophores was packaged in the AAV-PHP.S capsid. The vectors were injected retro-orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. KEY RESULTS: The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon-axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. CONCLUSIONS & INFERENCES: The multicolor AAV-PHP.S-packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro-circuitry.


Subject(s)
Colon/metabolism , Enteric Nervous System/metabolism , Submucous Plexus/metabolism , Animals , Colon/innervation , Dependovirus , Enteric Nervous System/virology , Female , Gene Transfer Techniques , Green Fluorescent Proteins , Male , Mice , Submucous Plexus/virology
4.
Proc Natl Acad Sci U S A ; 117(45): 28496-28505, 2020 11 10.
Article in English | MEDLINE | ID: mdl-33097671

ABSTRACT

Taxonomic resolution is a major challenge in palynology, largely limiting the ecological and evolutionary interpretations possible with deep-time fossil pollen data. We present an approach for fossil pollen analysis that uses optical superresolution microscopy and machine learning to create a quantitative and higher throughput workflow for producing palynological identifications and hypotheses of biological affinity. We developed three convolutional neural network (CNN) classification models: maximum projection (MPM), multislice (MSM), and fused (FM). We trained the models on the pollen of 16 genera of the legume tribe Amherstieae, and then used these models to constrain the biological classifications of 48 fossil Striatopollis specimens from the Paleocene, Eocene, and Miocene of western Africa and northern South America. All models achieved average accuracies of 83 to 90% in the classification of the extant genera, and the majority of fossil identifications (86%) showed consensus among at least two of the three models. Our fossil identifications support the paleobiogeographic hypothesis that Amherstieae originated in Paleocene Africa and dispersed to South America during the Paleocene-Eocene Thermal Maximum (56 Ma). They also raise the possibility that at least three Amherstieae genera (Crudia, Berlinia, and Anthonotha) may have diverged earlier in the Cenozoic than predicted by molecular phylogenies.


Subject(s)
Fossils , Microscopy/methods , Neural Networks, Computer , Phylogeny , Pollen/classification , Africa , Africa, Western , Machine Learning , Phylogeography , South America
5.
J Neurosci ; 40(3): 585-604, 2020 01 15.
Article in English | MEDLINE | ID: mdl-31767678

ABSTRACT

Study of the neural deficits caused by mismatched binocular vision in early childhood has predominantly focused on circuits in the primary visual cortex (V1). Recent evidence has revealed that neurons in mouse dorsolateral geniculate nucleus (dLGN) can undergo rapid ocular dominance plasticity following monocular deprivation (MD). It remains unclear, however, whether the long-lasting deficits attributed to MD during the critical period originate in the thalamus. Using in vivo two-photon Ca2+ imaging of dLGN afferents in superficial layers of V1 in female and male mice, we demonstrate that 14 d MD during the critical period leads to a chronic loss of binocular dLGN inputs while sparing response strength and spatial acuity. Importantly, MD leads to profoundly mismatched visual tuning properties in remaining binocular dLGN afferents. Furthermore, MD impairs binocular modulation, reducing facilitation of responses of both binocular and monocular dLGN inputs during binocular viewing. As predicted by our findings in thalamic inputs, Ca2+ imaging from V1 neurons revealed spared spatial acuity but impaired binocularity in L4 neurons. V1 L2/3 neurons in contrast displayed deficits in both binocularity and spatial acuity. Our data demonstrate that critical-period MD produces long-lasting disruptions in binocular integration beginning in early binocular circuits in dLGN, whereas spatial acuity deficits first arise from circuits further downstream in V1. Our findings indicate that the development of normal binocular vision and spatial acuity depend upon experience-dependent refinement of distinct stages in the mammalian visual system.SIGNIFICANCE STATEMENT Abnormal binocular vision and reduced acuity are hallmarks of amblyopia, a disorder that affects 2%-5% of the population. It is widely thought that the neural deficits underlying amblyopia begin in the circuits of primary visual cortex. Using in vivo two-photon calcium imaging of thalamocortical axons in mice, we show that depriving one eye of input during a critical period in development chronically impairs binocular integration in thalamic inputs to primary visual cortex. In contrast, visual acuity is spared in thalamic inputs. These findings shed new light on the role for developmental mechanisms in the thalamus in establishing binocular vision and may have critical implications for amblyopia.


Subject(s)
Sensory Deprivation/physiology , Thalamus/growth & development , Thalamus/physiology , Vision, Binocular/physiology , Vision, Monocular/physiology , Vision, Ocular/physiology , Amblyopia/physiopathology , Animals , Brain Mapping , Female , Geniculate Bodies/physiology , Male , Mice , Mice, Inbred C57BL , Photic Stimulation , Space Perception , Visual Acuity/physiology , Visual Cortex/physiology
6.
G3 (Bethesda) ; 9(7): 2171-2182, 2019 07 09.
Article in English | MEDLINE | ID: mdl-31048401

ABSTRACT

Complex spatiotemporal gene expression patterns direct the development of the fertilized egg into an adult animal. Comparisons across species show that, in spite of changes in the underlying regulatory DNA sequence, developmental programs can be maintained across millions of years of evolution. Reciprocally, changes in gene expression can be used to generate morphological novelty. Distinguishing between changes in regulatory DNA that lead to changes in gene expression and those that do not is therefore a central goal of evolutionary developmental biology. Quantitative, spatially-resolved measurements of developmental gene expression patterns play a crucial role in this goal, enabling the detection of subtle phenotypic differences between species and the development of computations models that link the sequence of regulatory DNA to expression patterns. Here we report the generation of two atlases of cellular resolution gene expression measurements for the primary anterior-posterior patterning genes in Drosophila simulans and Drosophila virilis By combining these data sets with existing atlases for three other Drosophila species, we detect subtle differences in the gene expression patterns and dynamics driving the highly conserved axis patterning system and delineate inter-species differences in the embryonic morphology. These data sets will be a resource for future modeling studies of the evolution of developmental gene regulatory networks.


Subject(s)
Body Patterning , Drosophila/embryology , Embryonic Development , Animals , Biomarkers , Body Patterning/genetics , Embryo, Nonmammalian , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Organ Specificity , Species Specificity , Transcriptome
7.
Nat Commun ; 10(1): 1944, 2019 04 26.
Article in English | MEDLINE | ID: mdl-31028266

ABSTRACT

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.


Subject(s)
Heart Rate/physiology , Motor Neurons/physiology , Animals , Cholinergic Neurons/metabolism , Cholinergic Neurons/physiology , Electrophysiology , Female , Male , Mice , Peripheral Nervous System/metabolism , Peripheral Nervous System/physiology , Vagus Nerve/metabolism , Vagus Nerve/physiology
8.
PLoS Genet ; 12(4): e1005985, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27077385

ABSTRACT

Self-renewing organs often experience a decline in function in the course of aging. It is unclear whether chronological age or external factors control this decline, or whether it is driven by stem cell self-renewal-for example, because cycling cells exhaust their replicative capacity and become senescent. Here we assay the relationship between stem cell cycling and senescence in the Caenorhabditis elegans reproductive system, defining this senescence as the progressive decline in "reproductive capacity," i.e. in the number of progeny that can be produced until cessation of reproduction. We show that stem cell cycling diminishes remaining reproductive capacity, at least in part through the DNA damage response. Paradoxically, gonads kept under conditions that preclude reproduction keep cycling and producing cells that undergo apoptosis or are laid as unfertilized gametes, thus squandering reproductive capacity. We show that continued activity is in fact beneficial inasmuch as gonads that are active when reproduction is initiated have more sustained early progeny production. Intriguingly, continued cycling is intermittent-gonads switch between active and dormant states-and in all likelihood stochastic. Other organs face tradeoffs whereby stem cell cycling has the beneficial effect of providing freshly-differentiated cells and the detrimental effect of increasing the likelihood of cancer or senescence; stochastic stem cell cycling may allow for a subset of cells to preserve proliferative potential in old age, which may implement a strategy to deal with uncertainty as to the total amount of proliferation to be undergone over an organism's lifespan.


Subject(s)
Aging/physiology , Caenorhabditis elegans/physiology , Cell Self Renewal/physiology , Cellular Senescence/physiology , DNA Repair/genetics , Animals , Apoptosis/genetics , Caenorhabditis elegans Proteins/genetics , Cellular Senescence/genetics , DNA Damage/genetics , DNA-Binding Proteins/genetics , Female , M Phase Cell Cycle Checkpoints/genetics , Ovary/physiology , Replication Protein A/genetics , Reproduction/physiology , Starvation/physiopathology , Stem Cells , Transcription Factors/genetics
9.
PLoS One ; 11(2): e0148879, 2016.
Article in English | MEDLINE | ID: mdl-26867017

ABSTRACT

Discriminating between black and white spruce (Picea mariana and Picea glauca) is a difficult palynological classification problem that, if solved, would provide valuable data for paleoclimate reconstructions. We developed an open-source visual recognition software (ARLO, Automated Recognition with Layered Optimization) capable of differentiating between these two species at an accuracy on par with human experts. The system applies pattern recognition and machine learning to the analysis of pollen images and discovers general-purpose image features, defined by simple features of lines and grids of pixels taken at different dimensions, size, spacing, and resolution. It adapts to a given problem by searching for the most effective combination of both feature representation and learning strategy. This results in a powerful and flexible framework for image classification. We worked with images acquired using an automated slide scanner. We first applied a hash-based "pollen spotting" model to segment pollen grains from the slide background. We next tested ARLO's ability to reconstruct black to white spruce pollen ratios using artificially constructed slides of known ratios. We then developed a more scalable hash-based method of image analysis that was able to distinguish between the pollen of black and white spruce with an estimated accuracy of 83.61%, comparable to human expert performance. Our results demonstrate the capability of machine learning systems to automate challenging taxonomic classifications in pollen analysis, and our success with simple image representations suggests that our approach is generalizable to many other object recognition problems.


Subject(s)
Image Processing, Computer-Assisted/methods , Picea/physiology , Pollen/classification , Algorithms , Automation , Color , Humans , Machine Learning , Observer Variation , Pattern Recognition, Automated , Pollen/chemistry , Reproducibility of Results , Software
10.
BMC Bioinformatics ; 16: 397, 2015 Nov 25.
Article in English | MEDLINE | ID: mdl-26607933

ABSTRACT

BACKGROUND: Analysis of single cells in their native environment is a powerful method to address key questions in developmental systems biology. Confocal microscopy imaging of intact tissues, followed by automatic image segmentation, provides a means to conduct cytometric studies while at the same time preserving crucial information about the spatial organization of the tissue and morphological features of the cells. This technique is rapidly evolving but is still not in widespread use among research groups that do not specialize in technique development, perhaps in part for lack of tools that automate repetitive tasks while allowing experts to make the best use of their time in injecting their domain-specific knowledge. RESULTS: Here we focus on a well-established stem cell model system, the C. elegans gonad, as well as on two other model systems widely used to study cell fate specification and morphogenesis: the pre-implantation mouse embryo and the developing mouse olfactory epithelium. We report a pipeline that integrates machine-learning-based cell detection, fast human-in-the-loop curation of these detections, and running of active contours seeded from detections to segment cells. The procedure can be bootstrapped by a small number of manual detections, and outperforms alternative pieces of software we benchmarked on C. elegans gonad datasets. Using cell segmentations to quantify fluorescence contents, we report previously-uncharacterized cell behaviors in the model systems we used. We further show how cell morphological features can be used to identify cell cycle phase; this provides a basis for future tools that will streamline cell cycle experiments by minimizing the need for exogenous cell cycle phase labels. CONCLUSIONS: High-throughput 3D segmentation makes it possible to extract rich information from images that are routinely acquired by biologists, and provides insights - in particular with respect to the cell cycle - that would be difficult to derive otherwise.


Subject(s)
Caenorhabditis elegans/growth & development , High-Throughput Screening Assays , Image Processing, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Olfactory Mucosa/cytology , Single-Cell Analysis/methods , Software , Algorithms , Animals , Blastocyst/cytology , Blastocyst/metabolism , Caenorhabditis elegans/metabolism , Cell Cycle/physiology , Cells, Cultured , Computational Biology/methods , Female , Gonads/cytology , Gonads/metabolism , Humans , Male , Mice , Microscopy, Confocal/methods , Olfactory Mucosa/metabolism
11.
Nat Protoc ; 10(11): 1860-1896, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26492141

ABSTRACT

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1-2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.


Subject(s)
Histocytochemistry/methods , Optical Imaging/methods , Pathology/methods , Specimen Handling/methods , Animals , Detergents/isolation & purification , Lipids/isolation & purification , Mice , Rats , Staining and Labeling/methods , Time Factors , Tissue Embedding/methods , Tissue Fixation/methods
12.
Development ; 142(3): 587-96, 2015 Feb 01.
Article in English | MEDLINE | ID: mdl-25605785

ABSTRACT

In developing embryos, gene regulatory networks drive cells towards discrete terminal fates, a process called canalization. We studied the behavior of the anterior-posterior segmentation network in Drosophila melanogaster embryos by depleting a key maternal input, bicoid (bcd), and measuring gene expression patterns of the network at cellular resolution. This method results in a gene expression atlas containing the levels of mRNA or protein expression of 13 core patterning genes over six time points for every cell of the blastoderm embryo. This is the first cellular resolution dataset of a genetically perturbed Drosophila embryo that captures all cells in 3D. We describe the technical developments required to build this atlas and how the method can be employed and extended by others. We also analyze this novel dataset to characterize the degree and timing of cell fate canalization in the segmentation network. We find that in two layers of this gene regulatory network, following depletion of bcd, individual cells rapidly canalize towards normal cell fates. This result supports the hypothesis that the segmentation network directly canalizes cell fate, rather than an alternative hypothesis whereby cells are initially mis-specified and later eliminated by apoptosis. Our gene expression atlas provides a high resolution picture of a classic perturbation and will enable further computational modeling of canalization and gene regulation in this transcriptional network.


Subject(s)
Body Patterning/genetics , Cell Lineage/genetics , Databases, Genetic , Drosophila melanogaster/embryology , Gene Regulatory Networks/genetics , Transcriptome/genetics , Animals , Drosophila Proteins , Homeodomain Proteins , In Situ Hybridization , RNA Interference , Real-Time Polymerase Chain Reaction , Trans-Activators/deficiency
13.
Hum Brain Mapp ; 35(1): 38-52, 2014 Jan.
Article in English | MEDLINE | ID: mdl-22847891

ABSTRACT

Functional brain imaging is a common tool in monitoring the progression of neurodegenerative and neurological disorders. Identifying functional brain imaging derived features that can accurately detect neurological disease is of primary importance to the medical community. Research in computer vision techniques to identify objects in photographs have reported high accuracies in that domain, but their direct applicability to identifying disease in functional imaging is still under investigation in the medical community. In particular, Serre et al. (: In: IEEE Conference on Computer Vision and Pattern Recognition (CVPR-05). pp 994-1000) introduced a biophysically inspired filtering method emulating visual processing in striate cortex which they applied to perform object recognition in photographs. In this work, the model described by Serre et al. [2005] is extended to three-dimensional volumetric images to perform signal detection in functional brain imaging (PET, SPECT). The filter outputs are used to train both neural network and logistic regression classifiers and tested on two distinct datasets: ADNI Alzheimer's disease 2-deoxy-D-glucose (FDG) PET and National Football League players Tc99m HMPAO SPECT. The filtering pipeline is analyzed to identify which steps are most important for classification accuracy. Our results compare favorably with other published classification results and outperform those of a blinded expert human rater, suggesting the utility of this approach.


Subject(s)
Brain Diseases/diagnostic imaging , Brain Mapping/methods , Neural Networks, Computer , Pattern Recognition, Automated/methods , Algorithms , Humans , Positron-Emission Tomography/methods , Tomography, Emission-Computed, Single-Photon/methods
14.
Proc Biol Sci ; 280(1770): 20131905, 2013 Nov 07.
Article in English | MEDLINE | ID: mdl-24048158

ABSTRACT

Taxonomic identification of pollen and spores uses inherently qualitative descriptions of morphology. Consequently, identifications are restricted to categories that can be reliably classified by multiple analysts, resulting in the coarse taxonomic resolution of the pollen and spore record. Grass pollen represents an archetypal example; it is not routinely identified below family level. To address this issue, we developed quantitative morphometric methods to characterize surface ornamentation and classify grass pollen grains. This produces a means of quantifying morphological features that are traditionally described qualitatively. We used scanning electron microscopy to image 240 specimens of pollen from 12 species within the grass family (Poaceae). We classified these species by developing algorithmic features that quantify the size and density of sculptural elements on the pollen surface, and measure the complexity of the ornamentation they form. These features yielded a classification accuracy of 77.5%. In comparison, a texture descriptor based on modelling the statistical distribution of brightness values in image patches yielded a classification accuracy of 85.8%, and seven human subjects achieved accuracies between 68.33 and 81.67%. The algorithmic features we developed directly relate to biologically meaningful features of grass pollen morphology, and could facilitate direct interpretation of unsupervised classification results from fossil material.


Subject(s)
Classification/methods , Poaceae/anatomy & histology , Pollen/anatomy & histology , Fossils , Microscopy, Electron, Scanning , Poaceae/classification , Pollen/classification
15.
IEEE Trans Pattern Anal Mach Intell ; 34(9): 1731-43, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22813957

ABSTRACT

We formulate a layered model for object detection and image segmentation. We describe a generative probabilistic model that composites the output of a bank of object detectors in order to define shape masks and explain the appearance, depth ordering, and labels of all pixels in an image. Notably, our system estimates both class labels and object instance labels. Building on previous benchmark criteria for object detection and image segmentation, we define a novel score that evaluates both class and instance segmentation. We evaluate our system on the PASCAL 2009 and 2010 segmentation challenge data sets and show good test results with state-of-the-art performance in several categories, including segmenting humans.

16.
ACS Chem Neurosci ; 3(6): 433-8, 2012 Jun 20.
Article in English | MEDLINE | ID: mdl-24358503

ABSTRACT

Mitochondria, synaptic vesicles, and other cytoplasmic constituents have to travel long distance along the axons from cell bodies to nerve terminals. Interruption of this axonal transport may contribute to many neurodegenerative diseases including Alzheimer's disease (AD). It has been recently shown that exposure of cultured neurons to ß-amyloid (Aß) resulted in severe impairment of mitochondrial transport. This Letter describes an integrated microfluidic platform that establishes surface patterned and compartmentalized culture of neurons for studying the effect of Aß on mitochondria trafficking in full length of axons. We have successfully quantified the trafficking of fluorescently labeled mitochondria in distal and proximal axons using image processing. Selective treatment of Aß in the somal or axonal compartments resulted in considerable decrease in mitochondria movement in a location dependent manner such that mitochondria trafficking slowed down more significantly proximal to the location of Aß exposure. Furthermore, this result suggests a promising application of microfluidic technology for investigating the dysfunction of axonal transport related to neurodegenerative diseases.


Subject(s)
Axonal Transport/physiology , Cell Compartmentation/physiology , Microfluidic Analytical Techniques/methods , Mitochondrial Membranes/metabolism , Neurons/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Animals , Cells, Cultured , Humans , Image Processing, Computer-Assisted , Kymography/methods , Mitochondrial Membranes/pathology , Neurons/pathology , Neurons/physiology , Rats
17.
PLoS Genet ; 7(10): e1002346, 2011 Oct.
Article in English | MEDLINE | ID: mdl-22046143

ABSTRACT

Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3-4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells.


Subject(s)
Body Patterning/genetics , Drosophila Proteins/metabolism , Drosophila/embryology , Drosophila/genetics , Animals , Biological Evolution , Blastoderm/growth & development , Drosophila Proteins/genetics , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Regulatory Networks/genetics , In Situ Hybridization, Fluorescence , Species Specificity
18.
Adv Mater ; 23(48): 5785-91, 2011 Dec 22.
Article in English | MEDLINE | ID: mdl-22065428

ABSTRACT

A biomimetic substrate for cell-culture is fabricated by plasma treatment of a prestressed thermoplastic shrink film to create tunable multiscaled alignment "wrinkles". Using this substrate, the functional alignment of human embryonic stem cell derived cardiomyocytes is demonstrated.


Subject(s)
Embryonic Stem Cells/cytology , Myocardium/cytology , Myocytes, Cardiac/cytology , Action Potentials , Animals , Biomimetics , Cell Differentiation , Cells, Cultured , Equipment Design , Flow Cytometry/methods , Humans , Materials Testing , Mice , Rats , Stem Cells/cytology , Surface Properties
19.
PLoS One ; 6(11): e26797, 2011.
Article in English | MEDLINE | ID: mdl-22110594

ABSTRACT

The axial bodyplan of Drosophila melanogaster is determined during a process called morphogenesis. Shortly after fertilization, maternal bicoid mRNA is translated into Bicoid (Bcd). This protein establishes a spatially graded morphogen distribution along the anterior-posterior (AP) axis of the embryo. Bcd initiates AP axis determination by triggering expression of gap genes that subsequently regulate each other's expression to form a precisely controlled spatial distribution of gene products. Reaction-diffusion models of gap gene expression on a 1D domain have previously been used to infer complex genetic regulatory network (GRN) interactions by optimizing model parameters with respect to 1D gap gene expression data. Here we construct a finite element reaction-diffusion model with a realistic 3D geometry fit to full 3D gap gene expression data. Though gap gene products exhibit dorsal-ventral asymmetries, we discover that previously inferred gap GRNs yield qualitatively correct AP distributions on the 3D domain only when DV-symmetric initial conditions are employed. Model patterning loses qualitative agreement with experimental data when we incorporate a realistic DV-asymmetric distribution of Bcd. Further, we find that geometry alone is insufficient to account for DV-asymmetries in the final gap gene distribution. Additional GRN optimization confirms that the 3D model remains sensitive to GRN parameter perturbations. Finally, we find that incorporation of 3D data in simulation and optimization does not constrain the search space or improve optimization results.


Subject(s)
Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Genes, Insect/genetics , Models, Genetic , Animals , Drosophila Proteins , Drosophila melanogaster/metabolism , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Trans-Activators/biosynthesis , Trans-Activators/genetics
20.
Tissue Eng Part C Methods ; 17(5): 579-88, 2011 May.
Article in English | MEDLINE | ID: mdl-21235325

ABSTRACT

Nano- and microscale topographical cues play critical roles in the induction and maintenance of various cellular functions, including morphology, adhesion, gene regulation, and communication. Recent studies indicate that structure and function at the heart tissue level is exquisitely sensitive to mechanical cues at the nano-scale as well as at the microscale level. Although fabrication methods exist for generating topographical features for cell culture, current techniques, especially those with nanoscale resolution, are typically complex, prohibitively expensive, and not accessible to most biology laboratories. Here, we present a tunable culture platform comprised of biomimetic wrinkles that simulate the heart's complex anisotropic and multiscale architecture for facile and robust cardiac cell alignment. We demonstrate the cellular and subcellular alignment of both neonatal mouse cardiomyocytes as well as those derived from human embryonic stem cells. By mimicking the fibrillar network of the extracellular matrix, this system enables monitoring of protein localization in real time and therefore the high-resolution study of phenotypic and physiologic responses to in-vivo like topographical cues.


Subject(s)
Biomimetics/methods , Embryonic Stem Cells/cytology , Myocytes, Cardiac/cytology , Tissue Engineering/methods , Animals , Animals, Newborn , Cell Line , Cell Nucleus/metabolism , Embryonic Stem Cells/metabolism , Extracellular Matrix/metabolism , Humans , Mice , Microscopy, Confocal , Myocytes, Cardiac/metabolism
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