Biology subjects

Shachar, S.

Publications and source records attributed to Shachar, S..

6 recordsLinked to original sources

bioRxiv2026

Branch-specific axon pruning induced by Dpr4/DIP-{Theta} transneuronal interactions

Neuronal remodeling is a conserved, late developmental mechanism to refine neural circuits. Although remodeling typically occurs with remarkable spatiotemporal precision, its underlying molecular mechanisms remain poorly understood. In the Drosophila mushroom body (MB) circuit, {gamma}-Kenyon cells ({gamma}-KCs) undergo stereotyped remodeling during metamorphosis, in which they prune their larval vertical and medial axonal branches and subsequently regrow a medial, adult-specific branch. Our previous transcriptional profiling of developing {gamma}-KCs revealed dynamic expression of Defective proboscis extension response (Dpr) proteins and their binding partners, Dpr-interacting proteins (DIPs), members of the Immunoglobulin (Ig) superfamily. Despite their established roles in neurodevelopment, how Dpr/DIPs function - given their lack of intracellular domains - remains unclear. Here, we show that overexpression of Dpr4 in developing {gamma}-KCs cell-autonomously inhibits axon pruning. Strikingly, this effect is branch-specific: the vertical axonal branch fails to prune, while the medial branch prunes normally. To our knowledge, this represents the first demonstration of branch-specific control of pruning in this system. Moreover, the adult medial branch regrows normally, indicating that pruning and regrowth are independently regulated at the level of individual branches. We demonstrate that this unique branch-specificity arises from trans-neuronal interactions between Dpr4 in {gamma}-KCs and DIP-{theta} in dopaminergic neurons that selectively innervate the vertical larval MB lobe. Furthermore, our findings suggest that this phenotype relies on an Ig2 domain of a Dpr family member, implying the involvement of a third binding partner. Leveraging this robust overexpression phenotype to probe downstream mechanisms, we find that loss of the transmembrane adhesion protein N-Cadherin suppresses the Dpr4-induced pruning defect. Together, our findings highlight the local impact of Dpr/DIP-mediated trans-neuronal interactions on the spatial regulation of remodeling, and provide genetic evidence implicating N-Cadherin as a potential downstream mediator of Dpr/DIP function within a developing neural circuit.

neuroscience↗

bioRxiv2021

Large-scale mapping of positional changes of hypoxia-responsive genes upon activation

Chromosome structure and nuclear organization are important factors in the regulation of gene expression. Transcription of a gene is influenced by local and global chromosome features such as condensation status and histone modifications. The relationship between the position of a gene in the cell nucleus and its activity is less clear. Here, we used high-throughput imaging to perform a large-scale analysis of the spatial location of a set of nearly 100 hypoxia-inducible genes to determine whether their location within the nucleus is correlated with their activity state upon stimulation. Radial distance analysis demonstrated that the majority of HIF- and CREB-inducible hypoxia responsive genes are located in the intermediate region of the nucleus. Radial position of numerous responsive genes changed upon hypoxic treatment. Analysis of the relative distances amongst a subset of HIF target gene groups revealed that some gene pairs also altered their relative location to each other upon hypoxic treatment, suggesting higher order chromatin rearrangements. While these changes in location occurred in response to hypoxic activation of the target genes, they did not correlate with the extent of their activation. These results suggest that induction of the hypoxia-responsive gene expression program is accompanied by spatial alterations of the genome, but that radial and relative gene positions are not directly related to gene activity.

cell biology↗

bioRxiv2020

A Deep Learning Pipeline for Nucleus Segmentation

Deep learning is rapidly becoming the technique of choice for automated segmentation of nuclei in biological image analysis workflows. In order to evaluate the feasibility of training nuclear segmentation models on small, custom annotated image datasets that have been augmented, we have designed a computational pipeline to systematically compare different nuclear segmentation model architectures and model training strategies. Using this approach, we demonstrate that transfer learning and tuning of training parameters, such as the composition, size and pre-processing of the training image dataset, can lead to robust nuclear segmentation models, which match, and often exceed, the performance of existing, off-the-shelf deep learning models pre-trained on large image datasets. We envision a practical scenario where deep learning nuclear segmentation models trained in this way can be shared across a laboratory, facility, or institution, and continuously improved by training them on progressively larger and varied image datasets. Our work provides computational tools and a practical framework for deep learning-based biological image segmentation using small annotated image datasets.

cell biology↗

bioRxiv2018

Effects of Human Sex Chromosome Dosage on Spatial Chromosome Organization

Sex chromosome aneuploidies (SCAs) are common genetic syndromes characterized by the presence of an aberrant number of X and Y chromosomes due to meiotic defects. These conditions impact structure and function of diverse tissues, but the proximal effects of SCA on genome organization are unknown. Here, to determine the consequences of SCAs on global genome organization, we have analyzed multiple architectural features of chromosome organization in a comprehensive set of primary cells from SCA patients with various ratios of X and Y chromosomes by use of imaging-based high-throughput Chromosome Territory Mapping (HiCTMap). We find that X chromosome supernumeracy does not affect the size, volume or nuclear position of the Y chromosome or an autosomal chromosome. In contrast, the active X chromosome undergoes architectural changes as a function of increasing X copy number, as measured by a decrease in size and an increase in circularity, which is indicative of chromatin compaction. With Y chromosome supernumeracy, Y chromosome size is reduced suggesting higher chromatin condensation. The radial positioning of chromosomes is unaffected in SCA karyotypes. Taken together, these observations document changes in genome architecture in response to alterations in sex chromosome numbers and point to trans-effects of dosage compensation on chromosome organization.

cell biology↗

bioRxiv2017

HiCTMap: Detection and Analysis of Chromosome Territory Structure and Position by High-throughput Imaging

The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.

cell biology↗

bioRxiv2017

Causes and consequences of nuclear gene positioning

The eukaryotic genome is organized in a manner that allows folding of the genetic material in the confined space of the cell nucleus, while at the same time enabling its physiological function. A major principle of spatial genome organization is the non-random position of genomic loci relative to other loci and to nuclear bodies. The mechanisms that determine the spatial position of a locus, and how position affects function, are just beginning to be characterized. Initial results suggest multiple, gene-specific mechanisms and the involvement of a wide range of cellular machineries. In this Commentary, we review recent findings from candidate approaches and unbiased screening methods that provide initial insight into the cellular mechanisms of positioning and their functional consequences. We highlight several specific mechanisms including tethering to the nuclear periphery, passage through replication and histone modifications that contribute to gene positioning in yeast, plants and mammals.

molecular biology↗