Methods for measuring the properties of individual cells within their native 3D environment will enable a deeper understanding of embryonic development, tissue regeneration, and tumorigenesis. However, current methods for segmenting nuclei in 3D tissues are not designed for situations in which nuclei are densely packed, nonspherical, or heterogeneous in shape, size, or texture, all of which are true of many embryonic and adult tissue types as well as in many cases for cells differentiating in culture. Here, we overcome this bottleneck by devising a novel method based on labelling the nuclear envelope (NE) and automatically distinguishing individual nuclei using a tree-structured ridge-tracing method followed by shape ranking according to a trained classifier. The method is fast and makes it possible to process images that are larger than the computer’s memory. We consistently obtain accurate segmentation rates of >90%, even for challenging images such as mid-gestation embryos or 3D cultures. We provide a 3D editor and inspector for the manual curation of the segmentation results as well as a program to assess the accuracy of the segmentation. We have also generated a live reporter of the NE that can be used to track live cells in 3 dimensions over time. We use this to monitor the history of cell interactions and occurrences of neighbour exchange within cultures of pluripotent cells during differentiation. We provide these tools in an open-access user-friendly format.

Cell-cell interactions govern differentiation and cell competition in pluripotent cells during early development, but the investigation of such processes is hindered by a lack of efficient analysis tools. Here we introduce SyNPL: clonal pluripotent stem cell lines which employ optimised Synthetic Notch (SynNotch) technology to report cell-cell interactions between engineered “sender” and “receiver” cells in cultured pluripotent cells and chimaeric mouse embryos. A modular design makes it straightforward to adapt the system for programming differentiation decisions non-cell-autonomously in receiver cells in response to direct contact with sender cells. We demonstrate the utility of this system by enforcing neuronal differentiation at the boundary between two cell populations. In summary, we provide a new adaptation of SynNotch technology which could be used to identify cell interactions and to profile changes in gene or protein expression that result from direct cell-cell contact with defined cell populations in culture and in early embryos, and which can be customised to generate synthetic patterning of cell fate decisions.

Methods for measuring the properties of individual cells within their native 3D environment will enable a deeper understanding of embryonic development, tissue regeneration, and tumorigenesis. However current methods for segmenting nuclei in 3D tissues are not designed for situations where nuclei are densely packed, non-spherical, heterogeneous in shape, size, or texture, all of which are true of many embryonic and adult tissue types as well as in many cases for cells differentiating in culture.Here we overcome this bottleneck by devising a novel method based on labelling the nuclear envelope (NE) and automatically distinguishing individual nuclei using a tree structured ridge tracing method followed by shape ranking according to a trained classifier. The method is fast and makes it possible to process images that are larger than the computer's memory. We consistently obtain accurate segmentation rates of >90% even for challenging images such as mid-gestation embryos or 3D cultures. We provide a 3D editor and inspector for the manual curation of the segmentation results as well as a program to assess the accuracy of the segmentation.We have also generated a live reporter of the NE that can be used to track live cells in three dimensions over time. We use this to monitor the history of cell interactions and occurrences of neighbour exchange within cultures of pluripotent cells during differentiation.We provide these tools in an open-access user-friendly format. 2 35 40 45 50 2012; Carpenter et al., 2013; Meijering et al., 2016).Here, we report a new approach to overcome these bottlenecks in quantitative image analysis of individual cells in 3D. Rather than relying on staining for nuclear content (for example DAPI or Hoescht staining), we instead detect the nuclear envelope (NE). This makes it easier to identify individual nuclei that are in close contact with each other and does not suffer from segmentation problems associated with textured nuclear staining, unusually shaped nuclei, or cell debris. Furthermore, the NE of individual nuclei are easily discernable by eye in crowded tissues, and so manual correction of any mis-segmented nuclei becomes easier than is the case for DAPI stained nuclei. We provide a user-friendly 3D-4D editing tool to rapidly correct any

Cell-cell interactions govern differentiation and cell competition in pluripotent cells during early development, but the investigation of such processes is hindered by a lack of efficient analysis tools. Here we introduce SyNPL: clonal pluripotent stem cell lines which employ optimised Synthetic Notch (SynNotch) technology to report cell-cell interactions between engineered sender and receiver cells in cultured pluripotent cells and chimaeric mouse embryos. A modular design makes it straightforward to adapt the system for programming differentiation decisions non-cell-autonomously in receiver cells in response to direct contact with sender cells. We demonstrate the utility of this system by enforcing neuronal differentiation at the boundary between two cell populations. In summary, we provide a new tool which could be used to identify cell interactions and to profile changes in gene or protein expression that result from direct cell-cell contact with defined cell populations in culture and in early embryos, and which can be adapted to generate synthetic patterning of cell fate decisions.

It is a common misconception to view the "cyto"-skeleton as just the filament systems in the "cyto"-plasm. In fact, the cytoskeleton extends into the nucleus where the complex network connects to chromatin, and it also connects through the plasma membrane to the cytoskeleton of adjacent cells and to the "exo"-skeleton of the extracellular matrix. This review will focus principally on the subcomplex of the cytoskeleton associated with the nucleus, often referred to as the nucleoskeleton, but in the context of its extensive interconnectivity with the rest of the nucleus and with cytoplasmic filament systems all the way to the exoskeleton. The nucleoskeleton, made principally of type-V intermediate filament lamins, connects across the double membrane system of the nuclear envelope to likely all three primary cytoplasmic filament systems. It provides structural stability to the nucleus, and also incredible flexibility. In both its core structural aspect and through specificity gained by tissue-specific partner proteins, it contributes to genome organization and regulation as well as to signal transduction, both through chemical signaling cascades and likely through mechanotransduction. Defects in the nucleoskeleton have far-ranging effects due to its interactions with cytoplasmic filament systems, from mispositioning of nuclei to disruption of cell polarity and both decreased and increased cell migration depending on the defect. Accordingly, it is not surprising that many nucleoskeletal components are linked to a wide range of human diseases from specific types of cancer to muscular dystrophies, neuropathies, dermopathies, and premature aging syndromes.