Biology subjects

Leshkowitz, D.

Publications and source records attributed to Leshkowitz, D..

7 recordsLinked to original sources

bioRxiv2025

Aberrant inheritance of extrachromosomal DNA amplifications promotes cancer evolution

Gene amplification in the form of extrachromosomal DNA (ecDNA) is a frequent driver in multiple cancer types. As ecDNA lack centromeres, their mitotic segregation does not follow traditional inheritance principles. However, the mechanisms that govern ecDNA fate following mitosis remain unclear. We found that ecDNA undergo numerical and structural optimization under increased selective pressure, with mitotic chromosomal tethering, or detachment, dictating ecDNA fate. When tethered, ecDNA aggregates promote uneven distribution into the newly formed daughter cells, thereby driving inter-cellular numerical heterogeneity and rapid increase of amplification under selective pressure. Mitotically detached ecDNA frequently encapsulate within micronuclei of variable size and content that appear to be highly fragile. Strikingly, ecDNA enclosed in very small micronuclei, which we term nanonuclei, are being actively degraded through autophagy. Together with ongoing structural rearrangements, nanonuclear ecDNA degradation promotes their structural evolution, which facilitates cancer cell adaptation. Our work highlights ecDNA aggregation, micronucleation, and degradation, as pivotal events in directing cancer genome evolution trajectories.

cancer biology↗

bioRxiv2022

Molecular characterization of the intact muscle spindle using a multi-omics approach

The proprioceptive system is essential for the control of coordinated movement, posture and skeletal integrity. The sense of proprioception is produced in the brain using peripheral sensory input from receptors such as the muscle spindle, which detects changes in the length of skeletal muscles. Despite its importance, the molecular composition of the muscle spindle is largely unknown. In this study, we generated comprehensive transcriptomic and proteomic datasets of the entire muscle spindle. We then associated differentially expressed genes with the various tissues composing the spindle using bioinformatic analysis. Immunostaining verified these predictions, thus establishing new markers for the different spindle tissues. Utilizing these markers, we identified the differentiation stages the spindle capsule cells undergo during development. Together, these findings provide comprehensive molecular characterization of the intact spindle as well as new tools to study its development and function in health and disease.

developmental biology↗

bioRxiv2022

Neural plate progenitors give rise to both anterior and posterior pituitary cells

The pituitary is the master neuroendocrine gland, which regulates body homeostasis. It consists of the anterior pituitary/adenohypophysis (AH), which harbors hormones producing cells and the posterior pituitary/neurohypophysis (NH), which relays the direct passage of hormones from the brain to the periphery. It is widely accepted that the AH originates from the oral ectoderm (Rathkes pouch) whereas the neural ectoderm contributes to the NH. Using single cell transcriptomics of the zebrafish pituitary we characterized cyp26b1-positive pituicyte of the NH and prop1-positive adenohypophyseal progenitors. We found that these cell types expressed common markers implying lineage relatedness. Genetic tracing revealed that in contrast to the prevailing dogma, neural plate precursors of both zebrafish (her4.3+) and mouse (Sox1+) contribute to both the neurohypophyseal and adenohypophyseal cells. We further show that pituicytes and prop1+ progenitors reside in close anatomical proximity and pituicyte-derived RA-degrading enzyme Cyp26b1 fine-tunes differentiation of prop1+ progenitors into hormone-producing cells. These results challenge the notion that AH cells are exclusively derived from non-neural ectoderm and demonstrate that a cross-talk between neuro- and adeno-hypophyseal cells fine-tunes the development of pituitary neuroendocrine cells.

developmental biology↗

bioRxiv2021

Pseudo-mutant p53 as a targetable phenotype of DNMT3A-mutated pre-leukemia

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemo-resistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPCs) and lead to chemo-resistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPCs. Intriguingly, a misfolded p53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between a wild-type (WT) and a "pseudomutant" conformations of p53. By combining single cell analyses and p53 conformation-specific monoclonal antibodies we studied preL-HSPCs from primary human DNMT3A AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPCs the pseudomutant conformation is the dominant. HSPCs from non-leukemic samples expressed both conformations to a similar extent. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of p53, specifically eliminated preL-HSPCs that had dysfunctional canonical p53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable p53 dysfunction in human preL-HSPCs carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

cancer biology↗

bioRxiv2020

Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors

The connection between different tissues is vital for the development and function of any organs and systems. In the musculoskeletal system, the attachment of elastic tendons to stiff bones poses a mechanical challenge that is solved by the formation of a transitional tissue, which allows the transfer of muscle forces to the skeleton without tearing. Here, we show that tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, which is regulated by sharing regulatory elements with these cells and by Kruppel-like factors transcription factors (KLF). To uncover the molecular identity of attachment cells, we first applied high-throughput RNA sequencing to murine humeral attachment cells. The results, which were validated by in situ hybridization and single-molecule in situ hybridization, reveal that attachment cells express hundreds of chondrogenic and tenogenic genes. In search for the underlying mechanism allowing these cells to express these genes, we performed ATAC sequencing and found that attachment cells share a significant fraction of accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis further revealed transcriptional enhancer signatures for the majority of these regions. We then examined a subset of these regions using transgenic mouse enhancer reporter. Results verified the shared activity of some of these enhancers, supporting the possibility that the transcriptome of attachment cells is regulated by enhancers with shared activities in tenocytes or chondrocytes. Finally, integrative chromatin and motif analyses, as well as the transcriptome data, indicated that KLFs are regulators of attachment cells. Indeed, blocking the expression of Klf2 and Klf4 in the developing limb mesenchyme led to abnormal differentiation of attachment cells, establishing these factors as key regulators of the fate of these cells. In summary, our findings show how the molecular identity of bi-fated attachment cells enables the formation of the unique transitional tissue that connect tendon to bone. More broadly, we show how mixing the transcriptomes of two cell types through shared enhancers and a dedicated set of transcription factors can lead to the formation of a new cell fate that connects them.

developmental biology↗

bioRxiv2019

Single-cell molecular and cellular architecture of neurohypophyseal cell types in the adult mouse

The neurohypophysis (NH), located at the posterior lobe of the pituitary, is a major neuroendocrine tissue, which mediates osmotic balance, blood pressure, reproduction, and lactation by means of releasing the neurohormones oxytocin and arginine-vasopressin from the brain into the peripheral blood circulation. The major cellular components of the NH are hypothalamic axonal termini, fenestrated endothelia and pituicytes, the resident astroglia. However, despite the physiological importance of the NH, the exact molecular signature defining neurohypophyseal cell types and in particular the pituicytes, remains unclear. Using single cell RNA sequencing, we captured seven distinct cell types in the NH and intermediate lobe (IL) of adult male mouse. We revealed novel pituicyte markers showing higher specificity than previously reported. Single molecule in situ hybridization revealed spatial organization of the major cell types implying intercellular communications. We present a comprehensive molecular and cellular characterization of neurohypophyseal cell-types serving as a valuable resource for further functional research.\n\nSignificance StatementThe neurohypophysis (NH) is a major neuroendocrine interface, which allows the brain to regulate the function of peripheral organs in response to specific physiological demands. Despite its importance, a comprehensive molecular description of cell identities in the NH is still lacking. Utilizing single cell RNA sequencing technology, we identified the transcriptomes of five major neurohypophyseal cell types in the adult male mice and mapped the spatial distribution of selected cell types in situ. We revealed an unexpected cellular heterogeneity of the neurohypophysis and provide novel molecular markers for neurohypophyseal cell types with higher specificity than previously reported.

neuroscience↗

bioRxiv2019

Runx3 prevents spontaneous colitis by directingdifferentiation of anti-inflammatory mononuclear phagocytes

RUNX3 is one of three mammalian Runt-domain transcription factors (TFs) that regulate gene expression in several types of immune cells. Runx3-deficiency in mice is associated with a multitude of defects in the adaptive and innate immunity systems, including the development of early onset colitis. Our study reveals that conditional deletion of Runx3 specifically in mononuclear phagocytes (MNP) (MNPRunx3-/-) but not in T cells, recapitulates the early onset spontaneous colitis seen in Runx3-/- mice.\n\nWe show that Runx3 is expressed in colonic MNP, including resident macrophages (RM) and the dendritic cell cDC2 subsets and its loss results in impaired differentiation/maturation of both cell types. At the transcriptome level, loss of Runx3 in RM and cDC2 was associated with upregulation of pro-inflammatory genes similar to those in the early onset IBD murine model of RMIl10r-/-. The impaired RM maturation in the absence of Runx3 was associated with a marked decrease in expression of anti-inflammatory and TGF{beta}-regulated genes. Similarly, the decreased expression of {beta}-catenin signaling associated genes in Runx3-deficient cDC2 indicates their impaired differentiation/maturation. Analysis of ChIP-seq data suggests that in both MNP cell types a significant fraction of these differentially expressed genes are high confidence Runx3 directly regulated genes. Interestingly, several of these putative Runx3 target genes harbor SNPs associated with IBD susceptibility in humans. Remarkably, the impaired maturation and pro-inflammatory phenotype of MNP lacking Runx3 was associated with a substantial reduction in the prevalence of colonic lamina propria Foxp3+ regulatory T cells and an increase in IFN{gamma}-producing CD4+ T cells, underscoring Runx3 critical role in establishing tolerogenic MNP.\n\nTogether, these data emphasize the dual role of Runx3 in colonic MNP, as a transcriptional repressor of pro-inflammatory genes and an activator of maturation-associated genes including anti-inflammatory genes. Our study highlights the significance of the current MNPRunx3-/- model for understanding of human MNP-associated colitis. It provides new insights into the crucial involvement of Runx3 in intestinal immune tolerance by regulating colonic MNP maturation through TGF{beta}R signaling and anti-inflammatory functions by Il10R signaling, befitting the identification of RUNX3 as a genome-wide associated risk gene for various immune-related diseases in humans including gastrointestinal tract diseases such as celiac and Crohns disease.

genetics↗