Biology subjects

Fernandez-Gonzalez, R.

Publications and source records attributed to Fernandez-Gonzalez, R..

17 recordsLinked to original sources

bioRxiv2026

Cell intrinsic dynamics guide neuroblast ingression independent of tissue fluidity

Morphogenesis involves the coordination of multiple cellular processes that occur simultaneously within developing tissues. During early Drosophila embryogenesis, neuroblast (NB) ingression occurs concurrently with germ band extension (GBE), yet whether these processes interact mechanistically remains unclear. Here, we combine mathematical modelling with quantitative live imaging to investigate whether tissue-level mechanics during GBE influence NB ingression dynamics. Mathematical modelling predicted that reducing tissue fluidity through impaired cellular rearrangements should slow NB ingression by increasing mechanical resistance. Experimental analysis of mutants in which cell intercalation and GBE are disrupted revealed a dramatic reduction in tissue fluidity. However, NB ingression rates remained largely unaffected when tissue fluidity decreased. Incorporating cell-intrinsic myosin anisotropy and endocytosis-contractility coupling into our mathematical model rescued the rate of neuroblast ingression in solid-like tissues. Thus, our findings suggest that cell-intrinsic mechanisms, rather than tissue-level fluidity, maintain ingression kinetics. More broadly, these results illustrate how developmental systems can achieve robustness by insulating critical cellular events from tissue-level mechanical variability. Statement of SignificanceMorphogenesis requires coordination of cellular processes in tissues undergoing mechanical transitions. While recent work highlights the importance of tissue fluidity in morphogenesis, whether tissue-level mechanical changes influence concurrent cellular events remains unclear. Combining mathematical modelling with quantitative live imaging, we tested whether tissue fluidization during Drosophila germ-band extension regulates neuroblast ingression. Shape-based vertex models failed to predict tissue mechanical states when myosin was disrupted. Instead, incorporating cellular rearrangement delays recapitulated phenotypes independent of cell shape. Strikingly, we showed that neuroblast ingression remains robust despite tissue solidification in vivo. Biophysical modelling suggests that coupled cell contractility and endocytosis maintains robust ingression despite tissue solidification. Our work shows that developmental programs can be mechanically insulated from tissue-scale changes, thus enabling developmental robustness.

developmental biology↗

bioRxiv2026

Rab4 and Rab11 GTPases cooperate to reinforce adherens junctions at the leading edge to promote rapid embryonic wound healing

Collective cell movements drive the formation and repair of tissues and contribute to the spread of metastatic disease. Cells must remodel their cell-cell adhesions and actomyosin cytoskeleton to enable migration, but the mechanisms that drive these molecular rearrangements are unclear. We used wound healing in the Drosophila embryonic epidermis to investigate these mechanisms. Upon wounding, a supracellular cable composed of actin and myosin assembles around the wound. In parallel, adherens junction proteins, including E-cadherin, are depleted from the wound edge via endocytosis and accumulate at former tricellular junctions around the wound (wTCJs) through unknown mechanisms. We found that the small GTPases Rab4 and Rab11, implicated in endosomal trafficking, are necessary for rapid wound repair. Manipulations of Rab4 and Rab11 activity reduced wound closure rates but did not have effects on tissue mechanics or myosin polarization that could explain the defect. Instead, Rab4 and Rab11 redistributed E-cadherin to accumulate at wTCJs in a process necessary for collective cell movement. Together, our results show that adherens junction reinforcement via endosomal recycling is a key step of coordinated cell migration that controls the rate of wound healing independent of cytoskeletal remodeling. Summary StatementDuring embryonic wound repair, Rab4 and Rab11 GTPases transport E-cadherin to reinforce specialized cell adhesions at the wound edge, which are essential to drive rapid collective cell migration.

cell biology↗

bioRxiv2026

Quantitative image analysis reveals no sexual dimorphism in the cellular dynamics of Drosophila heart tube formation

Congenital heart defects affect females and males differently. Several congenital heart defects arise during the formation of the heart tube, suggesting that heart tube morphogenesis may differ between females and males. We investigated if the fruit fly Drosophila melanogaster displays sexual dimorphisms in the cellular mechanisms of heart tube formation. Quantitative microscopy revealed no differences between females and males in the migration of cardiac progenitors to form the heart tube. Our results suggest that Drosophila do not display sexual dimorphisms in early cardiac development, and support the omission of sex as an experimental variable when investigating Drosophila heart tube morphogenesis.

developmental biology↗

bioRxiv2025

Cell divisions both challenge and refine tissue boundaries in the Drosophila embryo

Tissue boundaries pattern embryos, suppress tumours, and provide directional cues. Tissue boundaries are associated with supracellular cables formed by actin and the molecular motor non-muscle myosin II. Actomyosin cables generate tension that prevents cell mixing. Whether other cellular behaviours contribute to the formation of linear interfaces between cell populations remains unclear. In the Drosophila embryo, an actomyosin-based boundary separates the ectoderm from the mesectoderm, a group of neuronal and glial progenitors. Mathematical modelling predicted that cell divisions in the ectoderm challenge the mesectoderm-ectoderm (ME) boundary. Consistent with this, suppressing ectoderm cell divisions in vivo prevented cell mixing across the ME boundary when actomyosin-based tension was lost. Our mathematical model also predicted that cell divisions sharpen the ME boundary by reducing tension and increasing cell motility in the ectoderm. We found that inhibiting ectoderm divisions in vivo reduced boundary linearity. Using laser ablation and cell tracking, we demonstrated that cell divisions reduced junctional tension and increased cell movement in the ectoderm. Together, our results reveal that cell divisions facilitate cellular rearrangements to increase fluidity in a novel mechanism for boundary refinement.

developmental biology↗

bioRxiv2025

Inferring active and passive mechanical drivers of epithelial convergent extension

What can we learn about the mechanical processes that shape tissues by simply watching? Several schemes suggest that static cell morphology or junctional connectivity can reveal where chains of cells transmit force or where force asymmetries drive cellular rearrangements. We hypothesize that dynamic cell shape changes from time lapse sequences can be used to distinguish specific mechanisms of tissue morphogenesis. Convergent extension (CE) is a crucial developmental motif wherein a planar tissue narrows in one direction and lengthens in the other. It is tempting to assume that forces driving CE reside within cells of the deforming tissue, as CE may reflect a variety of active processes or passive responses to forces generated by adjacent tissues. In this work, we first construct a simple model of epithelial cells capable of passive CE in response to external forces. We adapt this framework to simulate CE from active anisotropic processes in three different modes: crawling, contraction, and capture. We develop an image analysis pipeline for analysis of morphogenetic changes in both live cells and simulated cells using a panel of mechanical and statistical approaches. Our results allow us to identify how each simulated mechanism uniquely contributes to tissue morphology and provide insight into how force transmission is coordinated. We construct a MEchanism Index (MEI) to quantify how similar live cells are to simulated passive and active cells undergoing CE. Applying these analyses to live cell data of Xenopus neural CE reveals features of both passive motion and active forces. Furthermore, we find spatial variation across the neural plate. We compare the inferred mechanisms in the frog midline to tissues undergoing CE in both the mouse and fly. We find that distinct active modes may have different prevalences depending on the model system. Our modeling framework allows us to gain insight from tissue timelapse images and assess the relative contribution of specific cellular mechanisms to observed tissue phenotypes. This approach can be used to guide further experimental inquiry into how mechanics influences the shaping of tissues and organs during development.

biophysics↗

bioRxiv2024

Cofilin promotes actin turnover and flexibility to drive coordinated cell movements in vivo

Embryos display a striking ability to repair wounds rapidly, with no inflammation or scarring. Embryonic wound healing is driven by the collective movement of the cells adjacent to the wound. The cells at the wound edge polarize actin and the molecular motor non-muscle myosin II, forming a supracellular cable around the wound that generates force and coordinates cell movements to close the lesion. Actin network contraction has been associated with the disassembly of the actin filaments that form the network. We found that the actin-severing protein Cofilin and its co-factor Aip1 accumulated at the edge of epidermal wounds in Drosophila embryos. Reducing Cofilin activity or levels slowed down wound closure, indicating that Cofilin is necessary for rapid wound healing. Using quantitative microscopy, we showed that Cofilin controls F-actin turnover at the wound edge, but not F-actin polarity or contractile force generation. Combining genetic and pharmacological manipulations, we found that F-actin turnover at the wound edge must be tightly regulated for wounds to close rapidly. Computational modelling suggested that Cofilin may contribute to rapid wound repair by maintaining a flexible actin network around the wound. Consistent with this model, fluorescence fluctuation analysis revealed that F-actin networks at the wound edge were significantly more rigid when we reduced Cofilin activity. Together, our results indicate that Cofilin promotes F-actin turnover at the wound margin to maintain a flexible actin network and facilitate rapid contraction and wound healing.

cell biology↗

bioRxiv2024

ReSCU-Nets: recurrent U-Nets for segmentation of multidimensional microscopy data

Segmenting multi-dimensional microscopy data requires high accuracy across many images (e.g. timepoints or Z slices) and is thus a labour-intensive part of biological image processing pipelines. We present ReSCU-Nets, recurrent convolutional neural networks that use the segmentation results from the previous frame as a prompt to segment the current frame. We demonstrate that ReSCU-Nets outperform state-of-the-art image segmentation models in different segmentation tasks on time-lapse microscopy sequences.

bioinformatics↗

bioRxiv2024

Oviductin sets the species-specificity of the mammalian zona pellucida

The zona pellucida (ZP) is vital for species-specific fertilization as this barrier mediates sperm-oocyte binding. Here, we determined whether sperm from distant mammalian orders (Carnivora, Primates, and Rodentia) could penetrate bovine oocytes by examining the role of bovine oviductal fluid and species-specific oviductal glycoprotein (OVGP1 or oviductin) from bovine, murine, or human sources in modulating the species-specificity of bovine and murine oocytes. Sperm from all the species were found to penetrate intact bovine ovarian oocytes to form hybrid embryos. However, contact with oviductal fluid or bovine, murine, or human OVGP1, conferred the ZP species-specificity, allowing only the penetration of the corresponding sperm regardless of the ZPs origin. Glycolytic and microstructural analyses revealed that OVGP1 covers the pores present in the ZP and that OVGP1 glycosylation determines sperm specificity. This suggests specific fertilization capacity is acquired in the oviduct through the ZPs incorporation of specific oviductin. TeaserThe oocyte zona pellucida needs to interact with an oviduct protein called OVGP1 to ensure that only sperm from the same species can fertilize the egg.

developmental biology↗

bioRxiv2023

mTOR inhibits autophagy to facilitate cell swelling and rapid wound repair

Embryonic wounds repair rapidly, with no inflammation or scarring. Embryonic wound healing is driven by collective cell movements facilitated by the swelling of the cells adjacent to the wound. The mechanistic target of rapamycin complex 1 (mTORC1) is often associated with cell growth. We found that disrupting mTORC1 signalling prevented cell swelling and slowed down wound repair. Catabolic processes, such as autophagy, can inhibit cell growth. Using five-dimensional time-lapse microscopy, as well as pharmacological and genetic manipulations, we demonstrated that the number of autophagosomes decreased during wound repair, suggesting that autophagy must be tightly regulated for rapid wound healing. Quantitative image analysis showed that mTOR inhibition increased autophagy, and that activating autophagy prevented cell swelling and slowed down embryonic wound closure. Finally, reducing autophagy in embryos in which mTORC1 signalling was disrupted rescued rapid wound repair. Together, our results show that mTORC1 activation upon wounding negatively regulates autophagy, allowing cells to increase their volumes to facilitate rapid wound healing.

cell biology↗

bioRxiv2023

Cortical tension regulates Hippo signaling via Par-1-mediated Kibra degradation

The Hippo pathway is an evolutionarily conserved regulator of tissue growth. Multiple Hippo signaling components are regulated via proteolytic degradation. However, how these degradation mechanisms are themselves modulated remains unexplored. Kibra is a key upstream pathway activator that promotes its own ubiquitin-mediated degradation upon assembling a Hippo signaling complex. Here, we demonstrate that Hippo complex-dependent Kibra degradation is modulated by cortical tension. Using classical genetic, osmotic, and pharmacological manipulations of myosin activity and cortical tension, we show that increasing cortical tension leads to Kibra degradation, whereas decreasing cortical tension increases Kibra abundance. Our study also implicates Par-1 in regulating Kib abundance downstream of cortical tension. We demonstrate that Par-1 promotes ubiquitin-mediated Kib degradation in a Hippo complex-dependent manner and is required for tension-induced Kib degradation. Collectively, our results reveal a previously unknown molecular mechanism by which cortical tension affects Hippo signaling and provide novel insights into the role of mechanical forces in growth control.

cell biology↗

bioRxiv2023

Integrin-based adhesions promote cell-cell junction remodelling and cytoskeletal rearrangements to drive embryonic wound healing

Embryos have a remarkable ability to repair wounds rapidly, with no inflammation or scarring. Embryonic wound healing is driven by the collective movement of the surrounding cells to seal the lesion. During embryonic wound closure, the cells adjacent to the wound polarize the cytoskeletal protein actin and the molecular motor non-muscle myosin II, which accumulate at the wound edge forming a supracellular cable around the wound. Adherens junction proteins including E-cadherin are internalized from the interface with the lesion and localize to former tricellular junctions at the wound margin, in a process necessary for cytoskeletal polarity. Using quantitative live microscopy, we found that the cells adjacent to wounds in the Drosophila epidermis also polarized Talin, a core component of cell-extracellular matrix (ECM) adhesions that links integrins to the actin cytoskeleton. Integrin knock-down and inhibition of integrin binding delayed wound closure and were associated with a reduction in actin levels around the wound. Additionally, disrupting integrins caused a defect in E-cadherin reinforcement at tricellular junctions along the wound edge, suggesting crosstalk between integrin-based and cadherin-based adhesions. Together, our results show that cell-ECM adhesion contributes to embryonic wound repair and reveal an interplay between cell-cell and cell-ECM adhesion in the collective cell movements that drive rapid wound healing.

cell biology↗

bioRxiv2022

Rap1 coordinates cell-cell adhesion and cytoskeletal reorganization to drive collective cell migration in vivo

Collective cell movements contribute to tissue development and repair, and spread metastatic disease. In epithelia, cohesive cell movements require reorganization of adherens junctions and the actomyosin cytoskeleton. However, the mechanisms that coordinate cell-cell adhesion and cytoskeletal remodelling during collective cell migration in vivo are unclear. We investigated the mechanisms of collective cell migration during wound healing in the Drosophila embryonic epidermis. Upon wounding, the cells adjacent to the wound internalize cell-cell adhesion molecules and polarize actin and the motor protein myosin II to form a supracellular cable around the wound that coordinates cell movements. The cable anchors at former tricellular junctions (TCJs) along the wound edge, and TCJs are reinforced during wound closure. We found that the small GTPase Rap1 was both necessary and sufficient for rapid wound repair. Rap1 promoted actomyosin polarization to the wound edge and E-cadherin accumulation at TCJs. Using embryos expressing a mutant form of the Rap1 effector Canoe/Afadin that cannot bind Rap1, we found that Rap1 signals through Canoe for adherens junction remodelling, but not for actomyosin cable assembly. Rap1 was necessary and sufficient for RhoA/Rho1 activation at the wound edge. Consistent with this, the RhoGEF Ephexin localized to the wound edge in a Rap1-dependent manner, and Ephexin was necessary for myosin polarization and rapid wound repair, but not for E-cadherin redistribution. Together, our data show that Rap1 coordinates the molecular rearrangements that drive embryonic wound healing and independently drives actomyosin cable assembly through Ephexin-Rho1, and E-cadherin redistribution through Canoe, thus enabling rapid collective cell migration in vivo.

cell biology↗

bioRxiv2022

Myosin waves and a mechanical asymmetry guide the oscillatory migration of Drosophila cardiac progenitors

Heart development begins with the formation of a tube, as cardiac progenitors migrate from opposite sides of the embryo and meet medially. Defective cardiac progenitor movements cause congenital heart defects. However, the mechanisms of cell migration during early heart development remain poorly understood. We investigated the mechanisms of movement of the Drosophila cardiac progenitors, the cardioblasts. Using quantitative time-lapse microscopy, we found that cardioblasts did not advance monotonically. Instead, cardioblasts took periodic forward and backward steps as they migrated. The forward steps were greater in both amplitude and duration, resulting in net forward movement of the cells. The molecular motor non-muscle myosin II displayed an alternating pattern of localization between the leading and trailing ends of migrating cardioblasts, forming oscillatory waves that traversed the cells. The alternating pattern of myosin polarity was associated with the alternative contraction and relaxation of the leading and trailing edges of the cell. Mathematical modelling predicted that forward migration requires the presence of a stiff boundary at the trailing edge of the cardioblasts. Consistent with this, we found a supracellular actin cable at the trailing edge of the cardioblasts. When we reduced the tension sustained by the actin cable, the amplitude of the backward steps of cardioblasts increased, thus reducing the net forward speed of migration. Together, our results indicate that periodic cell shape changes coupled with a polarized actin cable produce asymmetrical forces that guide cardioblast migration.

developmental biology↗

bioRxiv2022

Crumbs complex-directed apical membrane dynamics in epithelial cell ingression

Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cell lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. Here, we show that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. Our findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer.

developmental biology↗

bioRxiv2021

The recycling endosome protein Rab25 coordinates collective cell movements in the zebrafish surface epithelium

In emerging epithelial tissues, cells undergo dramatic rearrangements to promote tissue shape changes. Dividing cells remain interconnected via transient cytokinetic bridges. Bridges are cleaved during abscission and currently, the consequences of disrupting abscission in developing epithelia are not well understood. We show that the Rab GTPase, Rab25, localizes near cytokinetic midbodies and likely coordinates abscission through endomembrane trafficking in the epithelium of the zebrafish gastrula during epiboly. In maternal-zygotic Rab25a and Rab25b mutant embryos, morphogenic activity tears open persistent apical cytokinetic bridges that failed to undergo timely abscission. Cytokinesis defects result in anisotropic cell morphologies that are associated with a reduction of contractile actomyosin networks. This slows cell rearrangements and alters the viscoelastic responses of the tissue, all of which likely contribute to delayed epiboly. We present a model in which Rab25 trafficking coordinates cytokinetic bridge abscission and cortical actin density, impacting local cell shape changes and tissue-scale forces.

cell biology↗

bioRxiv2021

Functional plasticity of polarity proteins controls epithelial tissue architecture

The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1- PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1 and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.

cell biology↗

bioRxiv2019

The Crk adapter protein is essential for Drosophila embryogenesis, where it regulates multiple actin-dependent morphogenic events

AbstractSmall SH2/SH3 adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The Crk family has tissue-specific roles in phagocytosis, cell migration and neuronal development, and mediates oncogenic signaling in pathways like that of Abelson kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crks full role in embryogenesis. We circumvented these limitations with shRNA and CRISPR technology to assess Crks function in Drosophila morphogenesis. We found Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates levels of the Arp2/3 complex, its regulator SCAR, and F-actin in actin caps and pseudocleavage furrows. Crk loss leads to loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics.

cell biology↗