scholarly journals Reconstruction of cell lineages and behaviors underlying arthropod limb outgrowth with multi-view light-sheet imaging and tracking

2017 ◽  
Author(s):  
Carsten Wolff ◽  
Jean-Yves Tinevez ◽  
Tobias Pietzsch ◽  
Evangelia Stamataki ◽  
Benjamin Harich ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Single Cell ◽  
Limb Development ◽  
Light Sheet ◽  
Limb Bud ◽  
List Type ◽  
Cell Behaviors ◽  
Light Sheet Microscopy ◽  
Organ Morphogenesis ◽  
Parhyale Hawaiensis

SUMMARYDuring development coordinated cell behaviors orchestrate tissue and organ morphogenesis to suit the lifestyle of the organism. We have used here the crustacean Parhyale hawaiensis to study the cellular basis of limb development. Transgenic Parhyale embryos with fluorescently labeled nuclei were imaged at high spatiotemporal resolution with multi-view light-sheet fluorescence microscopy over several days of embryogenesis spanning appendage morphogenesis from early specification up to late differentiation stages. Cell tracking with a new tool called Massive Multi-view Tracker (MaMuT) enabled the reconstruction of the complete cell lineage of an outgrowing thoracic limb with single-cell resolution. In silico clonal analyses suggested that the limb primordium becomes subdivided from an early stage first into anterior-posterior and then into dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb bud formation is associated with the spatial modulation of cell proliferation, while limb elongation is also driven by the preferential orientation of division of epidermal cells along the proximal-distal axis of growth. Cellular reconstructions were predictive of the expression patterns of limb development genes including the Decapentaplegic (Dpp) morphogen.HIGHLIGHTSMulti-view light-sheet microscopy of crustacean embryos from species Parhyale hawaiensis are ideal for cellular-level analysis of organ morphogenesis.Lineages of 3-dimensional organs were reconstructed at single-cell resolution with the Fiji/ImageJ plugin Massive Multi-view Tracker.The Parhyale limb primordium undergoes early lineage restrictions associated with particular cell behaviors and patterns of gene expression.Differential rates of cell proliferation and oriented cell divisions guide appendage proximal-distal outgrowth.

scholarly journals Multi-view light-sheet imaging and tracking with the MaMuT software reveals the cell lineage of a direct developing arthropod limb

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Carsten Wolff ◽  
Jean-Yves Tinevez ◽  
Tobias Pietzsch ◽  
Evangelia Stamataki ◽  
Benjamin Harich ◽  
...  
Keyword(s):  
Limb Development ◽  
Expression Patterns ◽  
Cell Lineage ◽  
Light Sheet ◽  
Limb Bud ◽  
Spatial Modulation ◽  
Spatiotemporal Resolution ◽  
Cell Behaviors ◽  
Light Sheet Microscopy ◽  
Anterior Posterior

During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustacean Parhyale hawaiensis with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT). In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic.

scholarly journals Epithelial morphogenesis in the perinatal mouse uterus

2020 ◽  
Author(s):  
Zer Vue ◽  
Richard R. Behringer
Keyword(s):  
Morphological Changes ◽  
Post Partum ◽  
Fluorescent Microscopy ◽  
Light Sheet ◽  
Uterine Epithelium ◽  
List Type ◽  
Mouse Uterus ◽  
Light Sheet Microscopy ◽  
Uterine Gland ◽  
Uterine Glands

AbstractBackgroundThe uterus is the location where multiple events occur that are required for the start of new life in mammals. The adult uterus contains endometrial or uterine glands that are essential for female fertility. In the mouse, uterine glands are located in the lateral and anti-mesometrial regions of the uterine horn. Previous 3D-imaging of the adult uterus, its glands, and implanting embryos has been performed by multiple groups, using fluorescent microscopy. Adenogenesis, the formation of uterine glands, initiates after birth. Recently, we created a 3D-staging system of mouse uterine gland development at postnatal time points, using light sheet fluorescent microscopy. Here, using a similar approach, we examine the morphological changes in the epithelium of the perinatal mouse uterus.ResultsThe uterine epithelium exhibits mesometrial-antimesometrial (dorsoventral) patterning as early as three days after birth (P3), marked by the presence of the mesometrially-positioned developing uterine rail. Uterine gland buds are present beginning at P4. Novel morphological epithelial structures, including a ventral ridge and uterine segments were identified.ConclusionsThe perinatal mouse uterine luminal epithelium develops mesometrial-antimesometrial (dorsal-ventral) morphologies at 3-4 days post-partum. Between 5-6 days post-partum uterine epithelial folds form, defining alternating left-right segments.Bullet pointsMorphological patterning events in the perinatal uterine epithelium are not well described.Light sheet microscopy was used to generate volumetric reconstructions of the perinatal mouse uterine epithelium.At postnatal day 3 (P3), the uterine epithelium shows the first signs of dorsoventral pattern, with the presence of the forming mesometrially-positioned uterine rail.The first morphological indication of uterine adenogenesis begins at P4.Novel morphological structures were identified from volumetric reconstructions, including the presence of a ventral ridge (another sign of dorsoventral pattern) and uterine segmentation.

scholarly journals 3D projection electrophoresis for single-cell immunoblotting

2019 ◽  
Author(s):  
Samantha M. Grist ◽  
Andoni P. Mourdoukoutas ◽  
Amy E. Herr
Keyword(s):  
Single Cell ◽  
Three Dimensional ◽  
Light Sheet ◽  
Single Cell Protein ◽  
Design Guidelines ◽  
Cell Protein ◽  
Separation Performance ◽  
The Novel ◽  
Light Sheet Microscopy ◽  
Serial Sampling

AbstractWhile immunoassays and mass spectrometry are powerful single-cell protein analysis tools, bottlenecks remain in interfacing and throughput. Here, we introduce highly parallel, synchronous, three-dimensional single-cell immunoblots to detect both cytosolic and nuclear proteins. The novel threedimensional microfluidic device is a photoactive polyacrylamide gel with a high-density microwell array patterned on one face (x-y) for cell isolation and lysis. From each microwell, single-cell lysate is ‘electrophoretically projected’ into the 3rd dimension (z-axis), separated by size, and photo-captured for immunoprobing and three-dimensional interrogation by confocal/light sheet microscopy. Design guidelines for throughput and separation performance are informed by simulation, analyses, and deconvolution postprocessing based on physics of 3D diffusion. Importantly, separations are nearly synchronous, whereas serial analyses can impart hours of delay between the first and last cell. We achieve an electrophoresis throughput of >2.5 cells/s (70X faster than serial sampling) and perform 25 immunoblots/mm2 device area (>10X increase over previous immunoblots). A straightforward device for parallel single-cell immunoblotting, projection electrophoresis promises to advance integration of protein-level profiles into the emerging single-cell atlas of genomic and transcriptomic profiles.

scholarly journals Single-cell mRNA profiling reveals heterogeneous combinatorial expression of Hoxd genes during limb development

2018 ◽  
Author(s):  
Pierre J Fabre ◽  
Marion Leleu ◽  
Benedicte Mascrez ◽  
Quentin Lo Giudice ◽  
John Cobb ◽  
...  
Keyword(s):  
Single Cell ◽  
Limb Development ◽  
Hox Genes ◽  
Global Analysis ◽  
Cell Types ◽  
Cellular Level ◽  
Limb Bud ◽  
Different Parts ◽  
Combinatorial Expression ◽  
Hoxd Gene

A global analysis of gene expression during development reveals specific transcription patterns associated with the emergence of various cell types, tissues and organs. These heterogeneous patterns are instrumental to ensure the proper formation of the different parts of our body, as shown by the phenotypic effects generated by functional genetic approaches. However, variations at the cellular level can be observed within each structure or organ. In the developing mammalian limbs, expression of Hoxd genes is differentially controlled in space and time in cells that will pattern the digits and the arms. Here we analyze single-cell transcriptomes of limb bud cells and show that Hox genes are expressed in specific combinations that match particular cell types. In the presumptive digits, we find that the expression of Hoxd gene is unbalanced, despite their common genomic proximity to known global enhancers, often expressing only a subset of the five genes transcribed in these cells. We also report that combinatorial expression follows a pseudo-time sequence, suggesting that a progression in combinatorial expression may be associated with cellular diversity in developing digits.

scholarly journals Recent Progress in Light Sheet Microscopy for Biological Applications

2018 ◽  
Vol 72 (8) ◽  
pp. 1137-1169 ◽  
Author(s):  
Krishnendu Chatterjee ◽  
Feby Wijaya Pratiwi ◽  
Frances Camille M. Wu ◽  
Peilin Chen ◽  
Bi-Chang Chen
Keyword(s):  
Developmental Biology ◽  
Fluorescence Microscopy ◽  
Single Cell ◽  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
High Signal ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy ◽  
Resolution Imaging

The introduction of light sheet fluorescence microscopy (LSFM) has overcome the challenges in conventional optical microscopy. Among the recent breakthroughs in fluorescence microscopy, LSFM had been proven to provide a high three-dimensional spatial resolution, high signal-to-noise ratio, fast imaging acquisition rate, and minuscule levels of phototoxic and photodamage effects. The aforementioned auspicious properties are crucial in the biomedical and clinical research fields, covering a broad range of applications: from the super-resolution imaging of intracellular dynamics in a single cell to the high spatiotemporal resolution imaging of developmental dynamics in an entirely large organism. In this review, we provided a systematic outline of the historical development of LSFM, detailed discussion on the variants and improvements of LSFM, and delineation on the most recent technological advancements of LSFM and its potential applications in single molecule/particle detection, single-molecule super-resolution imaging, imaging intracellular dynamics of a single cell, multicellular imaging: cell–cell and cell–matrix interactions, plant developmental biology, and brain imaging and developmental biology.

scholarly journals Segmentation of Tissues and Proliferating Cells in Light-Sheet Microscopy Images using Convolutional Neural Networks

2021 ◽  
Author(s):  
Lucas D. Lo Vercio ◽  
Rebecca M. Green ◽  
Samuel Robertson ◽  
Si Han Guo ◽  
Andreas Dauter ◽  
...  
Keyword(s):  
Neural Networks ◽  
Cell Proliferation ◽  
Convolutional Neural Networks ◽  
Light Sheet ◽  
Cell Segmentation ◽  
Segmentation Method ◽  
Tissue Segmentation ◽  
Proliferating Cells ◽  
Light Sheet Microscopy ◽  
Automated Method

AbstractBackground and ObjectiveA variety of genetic mutations are known to affect cell proliferation and apoptosis during organism development, leading to structural birth defects such as facial clefting. Yet, the mechanisms how these alterations influence the development of the face remain unclear. Cell proliferation and its relation to shape variation can be studied in high detail using Light-Sheet Microscopy (LSM) imaging across a range of developmental time points. However, the large number of LSM images captured at cellular resolution precludes manual analysis. Thus, the aim of this work was to develop and evaluate automatic methods to segment tissues and proliferating cells in these images in an accurate and efficient way.MethodsWe developed, trained, and evaluated convolutional neural networks (CNNs) for segmenting tissues, cells, and specifically proliferating cells in LSM datasets. We compared the automatically extracted tissue and cell annotations to corresponding manual segmentations for three specific applications: (i) tissue segmentation (neural ectoderm and mesenchyme) in nuclear-stained LSM images, (ii) cell segmentation in nuclear-stained LSM images, and (iii) segmentation of proliferating cells in Phospho-Histone H3 (PHH3)-stained LSM images.ResultsThe automatic CNN-based tissue segmentation method achieved a macro-average F-score of 0.84 compared to a macro-average F-score of 0.89 comparing corresponding manual segmentations from two observers. The automatic cell segmentation method in nuclear-stained LSM images achieved an F-score of 0.57, while comparing the manual segmentations resulted in an F-score of 0.39. Finally, the automatic segmentation method of proliferating cells in the PHH3-stained LSM datasets achieved an F-score of 0.56 for the automated method, while comparing the manual segmentations resulted in an F-score of 0.45.ConclusionsThe proposed automatic CNN-based framework for tissue and cell segmentation leads to results comparable to the inter-observer agreement, accelerating the LSM image analysis. The trained CNN models can also be applied for shape or morphological analysis of embryos, and more generally in other areas of cell biology.

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