Biology subjects

Eisenhoffer, G. T.

Publications and source records attributed to Eisenhoffer, G. T..

7 recordsLinked to original sources

bioRxiv2023

Assessing mechanical agency during apical apoptotic cell extrusion

Epithelial tissues maintain homeostasis through the continual addition and removal of cells. Homeostasis is necessary for epithelia to maintain barrier function and prevent the accumulation of defective cells. Unfit, excess, and dying cells can be removed from epithelia by the process of extrusion. Controlled cell death and extrusion in the epithelium of the larval zebrafish tail fin coincides with oscillation of cell area, both in the extruding cell and its neighbors. Both cell-autonomous and non-autonomous factors have been proposed to contribute to extrusion but have been challenging to test by experimental approaches. Here we develop a dynamic cell-based biophysical model that recapitulates the process of oscillatory cell extrusion to test and compare the relative contributions of these factors. Our model incorporates the mechanical properties of individual epithelial cells in a two-dimensional simulation as repelling active particles. The area of cells destined to extrude oscillates with varying durations or amplitudes, decreasing their mechanical contribution to the epithelium and surrendering their space to surrounding cells. Quantitative variations in cell shape and size during extrusion are visualized by a hybrid weighted Voronoi tessellation technique that renders individual cell mechanical properties directly into an epithelial sheet. To explore the role of autonomous and non-autonomous mechanics, we vary the biophysical properties and behaviors of extruding cells and neighbors such as the period and amplitude of repulsive forces, cell density, and tissue viscosity. Our data suggest that cell autonomous processes are major contributors to the dynamics of extrusion, with the mechanical microenvironment providing a less pronounced contribution. Our computational model based on in vivo data serves as a tool to provide insights into the cellular dynamics and localized changes in mechanics that promote elimination of unwanted cells from epithelia during homeostatic tissue maintenance.

biophysics↗

bioRxiv2023

Macrophage Migration Inhibitory Factor on Apoptotic Extracellular Vesicles Regulates Compensatory Proliferation

Apoptotic cells can signal to neighboring cells to stimulate proliferation and compensate for cell loss to maintain tissue homeostasis. While apoptotic cell-derived extracellular vesicles (AEVs) can transmit instructional cues to mediate communication with neighboring cells, the molecular mechanisms that induce cell division are not well understood. Here we show that macrophage migration inhibitory factor (MIF)-containing AEVs regulate compensatory proliferation via ERK signaling in epithelial stem cells of larval zebrafish. Time-lapse imaging showed efferocytosis of AEVs from dying epithelial stem cells by healthy neighboring stem cells. Proteomic and ultrastructure analysis of purified AEVs identified MIF localization on the AEV surface. Pharmacological inhibition or genetic mutation of MIF, or its cognate receptor CD74, decreased levels of phosphorylated ERK and compensatory proliferation in the neighboring epithelial stem cells. Disruption of MIF activity also caused decreased numbers of macrophages patrolling near AEVs, while depletion of the macrophage lineage resulted in a reduced proliferative response by the epithelial stem cells. We propose that AEVs carrying MIF directly stimulate epithelial stem cell repopulation and guide macrophages to cell non-autonomously induce localized proliferation to sustain overall cell numbers during tissue maintenance.

cell biology↗

bioRxiv2022

Disruption of fos causes craniofacial anomalies in developing zebrafish

Craniofacial development is a complex and tightly regulated process and disruptions can lead to structural birth defects, the most common being nonsyndromic cleft lip and palate (NSCLP). Previously, we identified FOS as a candidate regulator of NSCLP through family-based association studies, yet its specific contributions to oral and palatal formation are poorly understood. This study investigated the role of fos during zebrafish craniofacial development through genetic disruption and knockdown approaches. Fos was expressed in the periderm, olfactory epithelium and other cell populations in the head. Genetic perturbation of fos produced an abnormal craniofacial phenotype with a hypoplastic oral cavity that showed significant changes in midface dimensions by quantitative facial morphometric analysis. Loss and knockdown of fos caused increased cell apoptosis in the head, followed by a significant reduction in cranial neural crest cells (CNCCs) populating the upper and lower jaws. These changes resulted in abnormalities of cartilage, bone and pharyngeal teeth formation. Periderm cells surrounding the oral cavity showed altered morphology and a subset of cells in the upper and lower lip showed disrupted Wnt/{beta}-catenin activation, consistent with modified inductive interactions between mesenchymal and epithelial cells. Taken together, these findings demonstrate that perturbation of fos has detrimental effects on oral epithelial and CNCC-derived tissues suggesting that it plays a critical role in zebrafish craniofacial development and a potential role in NSCLP. Summary statementPerturbation of fos, a candidate gene associated with nonsyndromic cleft lip and palate in humans, causes a distinctive orofacial phenotype in zebrafish as a result of abnormal development of craniofacial tissues. Disruption of fos in the oral epithelial, cranial neural crest and Wnt responsive cell populations around the oral cavity causes anomalies that suggest a potential role in the etiology of NSCLP.

developmental biology↗

bioRxiv2022

zFACE: Facial Analytics from a Coordinate Extrapolation System for Developing Zebrafish

Facial development requires a complex and coordinated series of cellular events, that when perturbed, can lead to structural birth defects. A standardized quantitative approach to quickly assess morphological changes could address how genetic or environmental inputs lead to differences in facial development. Here we report on a method to rapidly analyze craniofacial development in zebrafish embryos that combines a simple staining and mounting paradigm with Facial Analytics based on a Coordinate Extrapolation system, termed zFACE. Confocal imaging of frontal/rostral mounted embryos generates high-resolution images to capture facial structures and morphometric data is quantified based on a coordinate system that assesses 26 anatomical landmarks present at defined times in development. The semi-automated analysis can be applied to embryos at different stages of development and quantitative morphometric data can detect subtle phenotypic variation. Shape analysis can also be performed with the coordinate data to inform on global changes in facial morphology. We applied this new approach to show that loss of smarca4a in developing zebrafish leads to craniofacial anomalies, microcephaly and alterations in brain morphology. These changes are characteristic of humans with Coffin-Siris syndrome (CSS), a rare genetic disorder associated with mutations in SMARCA4 that is defined by anomalies in head size, intellectual disabilities and craniofacial abnormalities. We observed that smarca4a is expressed in craniofacial tissues and our multivariate analysis facilitated the classification of smarca4a mutants based on changes in specific phenotypic characteristics. Together, our approach provides a way to rapidly and quantitatively assess the impact of genetic alterations on craniofacial development in zebrafish.

developmental biology↗

bioRxiv2019

CreLite: An Optogenetically Controlled Cre/loxP System Using Red Light

Precise manipulation of gene expression with temporal and spatial control is essential for functional studies and the determination of cell lineage relationships in complex biological systems. The Cre-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre or CreER (tamoxifen-inducible). Here we present CreLite, an optogenetically-controlled Cre system using red light in developing zebrafish embryos. Cre activity is disabled by splitting Cre and fusing the inactive halves with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. In addition, PhyB-PIF6 binding requires phycocyanobilin (PCB), providing an additional layer of control. Upon exposure to red light (660 nm) illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of PCB to restore Cre activity. Red-light exposure of transgenic zebrafish embryos harboring a Cre-dependent multi-color fluorescent protein reporter (ubi:zebrabow) injected with CreLite mRNAs and PCB, resulted in Cre activity as measured by the generation of multi-spectral cell labeling in various tissues, including heart, skeletal muscle and epithelium. We show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different stages of development. CreLite provides a novel optogenetic tool for precise temporal and spatial control of gene expression in zebrafish embryos that may also be useful in cell culture, ex vivo organ culture, and other animal models for developmental biology studies.

molecular biology↗

bioRxiv2018

FishNET: An automated relational database for zebrafish colony management.

The zebrafish Danio rerio is a powerful model system to study the genetics of development and disease. However, maintenance of zebrafish husbandry records is both time intensive and laborious, and a standardized way to manage and track the large amount of unique lines in a given laboratory or centralized facility has not been embraced by the field. Here we present FishNet, an intuitive, open source, relational database for managing data and information related to zebrafish husbandry and maintenance. By creating a \"virtual facility\", FishNET enables users to remotely inspect the rooms, racks, tanks and lines within a given facility. Importantly, FishNET scales from one laboratory, to an entire facility with several laboratories, to multiple facilities, generating a cohesive laboratory and community-based platform. Automated data entry eliminates confusion regarding line nomenclature and streamlines maintenance of individual lines, while flexible query forms allow researchers to retrieve database records based on user-defined criteria. FishNet also links associated embryonic and adult biological samples with data, such as genotyping results or confocal images, to enable robust and efficient colony management and storage of laboratory information. A shared calendar function with email notifications and automated reminders for line turnover, automated tank counts and census reports promote communication with both end-users and administrators. The expected benefits of FishNET are improved vivaria efficiency, increased quality control for experimental numbers, and flexible data reporting and retrieval. FishNets easy, intuitive record management and open source, end user-modifiable architecture provides an efficient solution to real-time zebrafish colony management for users throughout a facility and institution, and in some cases across entire research hubs.

genetics↗

bioRxiv2018

Cellular Crowding Influences Extrusion and Proliferation to Facilitate Epithelial Tissue Repair

Epithelial wound healing requires a complex orchestration of cellular rearrangements and movements to restore tissue architecture and function after injury. While it is well-known that mechanical forces can affect tissue morphogenesis and patterning, how the biophysical cues generated after injury influence cellular behaviors during tissue repair is not well understood. Using time-lapsed confocal imaging of epithelial tissues in living zebrafish larvae, we provide evidence that localized increases in cellular crowding during wound closure promote the extrusion of non-apoptotic cells via mechanically regulated stretch-activated ion channels (SACs). Directed cell migration toward the injury site promoted the rapid changes in cell number and generated shifts in tension at cellular interfaces over long spatial distances. Perturbation of SAC activity resulted in failed extrusion and increased proliferation in crowded areas of the tissue. Together, we conclude that localized cell number plays a key role in dictating cellular behaviors that facilitate wound closure and tissue repair.

cell biology↗