Vanessa Barone

All Publications

• Characterization of cellular wound resistance in the giant ciliate Stentor coeruleus. Soft matter Paul, R., Nadkarni, A. V., Sipkova, J., Sato, J. E., Zhang, K. S., Barone, V., Marshall, W. F., Tang, S. K. 2025

  Abstract

  Resistance to mechanical stress is essential for cells to prevent wounding and maintain structural integrity. This capability is especially critical for free-living single-celled organisms, which routinely encounter mechanical stress from their natural habitats. We investigated Stentor coeruleus, a single-celled ciliate known for its remarkable wound repair capacity, as a model for studying mechanical wound resistance. While previous work focused on wound repair in Stentor, the structures that enable it to resist wounding remain poorly understood. We characterized how Stentor resisted mechanical stress during transit through a microfluidic constriction. Using high-speed imaging, we tracked the transit dynamics of the cells and linked them to wounding outcomes. Larger cells experienced longer transit times in the constriction and were more prone to rupture, often failing to recover shape due to membrane rupture and loss of cytoplasm. To elucidate the role of the Stentor cytoskeleton, we performed drug-mediated disruption of KM fibers, which are microtubule bundles in the Stentor cytoskeleton. Drug-treated cells exhibited an increased likelihood of membrane rupture at the constriction, implicating KM fibers in wound resistance. To investigate the resistance of Stentor cells to hydrodynamic stress, we injected the cells at increasing flow rates through the constriction. Interestingly, cells were more resistant to larger hydrodynamic stresses up to a threshold, potentially due to shear-thinning of the cytoplasm. Together, these results suggest that Stentor relies on both cytoskeletal architecture and cytoplasmic rheology to withstand mechanical stress, offering insights into cellular strategies for wound resistance in the absence of rigid extracellular structures.

  View details for DOI 10.1039/d5sm00643k

  View details for PubMedID 41090415

• Advances in culturing of the sea star Patiria miniata. Developmental dynamics : an official publication of the American Association of Anatomists Barone, V., Coronado, L., Ismail, D., Fiaz, S., Lyons, D. C. 2025

  Abstract

  The use of the sea star Patiria miniata as a model system has produced groundbreaking advances in a disparate set of biomedical research fields, including embryology, immunology, regeneration, cell biology, and evolution of development. Nonetheless, the life cycle of P. miniata has not yet been closed in the laboratory, precluding the generation of stable transgenic and mutant lines, which would greatly expand the toolset for experimentation with this model system. Rearing P. miniata in the laboratory has been challenging due to limited knowledge about metamorphosis cues, feeding habits of juveniles, and their relatively long generation time.Here we report protocols to rear P. miniata embryos through sexual maturity in a laboratory setting. We provide detailed staging of early embryonic development at different temperatures, and show that larvae can be raised to competence in as little as 15 days. We find that retinoic acid induces metamorphosis effectively and present methods to rear juveniles on commercially available foods. We show that in a flow-through system, juveniles double in size every 2 months and reach sexual maturity in approximately 2 years.We report the first example of P. miniata raised through sexual maturity in a laboratory setting, paving the way for the generation of stable mutant sea star lines.

  View details for DOI 10.1002/dvdy.70040

  View details for PubMedID 40372060

• Localization and origins of juvenile skeletogenic cells in the sea urchin Lytechinus pictus DEVELOPMENTAL BIOLOGY Tate, H. M., Barone, V., Schrankel, C. S., Hamdoun, A., Lyons, D. C. 2024; 514: 12-27

  Abstract

  The development of the sea urchin larval body plan is well understood from extensive studies of embryonic patterning. However, fewer studies have investigated the late larval stages during which the unique pentaradial adult body plan develops. Previous work on late larval development highlights major tissue changes leading up to metamorphosis, but the location of specific cell types during juvenile development is less understood. Here, we improve on technical limitations by applying highly sensitive hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) to the fast-developing and transparent sea urchin Lytechinus pictus, with a focus on skeletogenic cells. First, we show that HCR-FISH can be used in L. pictus to precisely localize skeletogenic cells in the rudiment. In doing so, we provide a detailed staging scheme for the appearance of skeletogenic cells around the rudiment prior to and during biomineralization and show that many skeletogenic cells unassociated with larval rods localize outside of the rudiment prior to localizing inside. Second, we show that downstream biomineralization genes have similar expression patterns during larval and juvenile skeletogenesis, suggesting some conservation of skeletogenic mechanisms during development between stages. Third, we find co-expression of blastocoelar and skeletogenic cell markers around juvenile skeleton located outside of the rudiment, which is consistent with data showing that cells from the non-skeletogenic mesoderm embryonic lineage contribute to the juvenile skeletogenic cell lineage. This work sets the foundation for subsequent studies of other cell types in the late larva of L. pictus to better understand juvenile body plan development, patterning, and evolution.

  View details for DOI 10.1016/j.ydbio.2024.05.012

  View details for Web of Science ID 001258784800001

  View details for PubMedID 38862087

• Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium. Development (Cambridge, England) Barone, V., Tagua, A., Román, J. Á., Hamdoun, A., Garrido-García, J., Lyons, D. C., Escudero, L. M. 2024

  Abstract

  Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes adopting a shape named "scutoid" that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here we use live-imaging of the sea star embryo coupled with deep learning-based segmentation, to dissect the relative contributions of cell density, tissue compaction, and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing just after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

  View details for DOI 10.1242/dev.202362

  View details for PubMedID 38619327

• Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium. bioRxiv : the preprint server for biology Barone, V., Tagua, A., Andres-San Roman, J. A., Hamdoun, A., Garrido-Garcia, J., Lyons, D. C., Escudero, L. M. 2024

  Abstract

  Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes adopting a shape named "scutoid" that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here we use live-imaging of the sea star embryo coupled with deep learning-based segmentation, to dissect the relative contributions of cell density, tissue compaction, and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing just after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

  View details for DOI 10.1101/2024.02.08.579268

  View details for PubMedID 38370815

• Live imaging of echinoderm embryos to illuminate evo-devo FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY Barone, V., Lyons, D. C. 2022; 10: 1007775

  Abstract

  Echinoderm embryos have been model systems for cell and developmental biology for over 150 years, in good part because of their optical clarity. Discoveries that shaped our understanding of fertilization, cell division and cell differentiation were only possible because of the transparency of sea urchin eggs and embryos, which allowed direct observations of intracellular structures. More recently, live imaging of sea urchin embryos, coupled with fluorescence microscopy, has proven pivotal to uncovering mechanisms of epithelial to mesenchymal transition, cell migration and gastrulation. However, live imaging has mainly been performed on sea urchin embryos, while echinoderms include numerous experimentally tractable species that present interesting variation in key aspects of morphogenesis, including differences in embryo compaction and mechanisms of blastula formation. The study of such variation would allow us not only to understand how tissues are formed in echinoderms, but also to identify which changes in cell shape, cell-matrix and cell-cell contact formation are more likely to result in evolution of new embryonic shapes. Here we argue that adapting live imaging techniques to more echinoderm species will be fundamental to exploit such an evolutionary approach to the study of morphogenesis, as it will allow measuring differences in dynamic cellular behaviors - such as changes in cell shape and cell adhesion - between species. We briefly review existing methods for live imaging of echinoderm embryos and describe in detail how we adapted those methods to allow long-term live imaging of several species, namely the sea urchin Lytechinus pictus and the sea stars Patiria miniata and Patiriella regularis. We outline procedures to successfully label, mount and image early embryos for 10-16 h, from cleavage stages to early blastula. We show that data obtained with these methods allows 3D segmentation and tracking of individual cells over time, the first step to analyze how cell shape and cell contact differ among species. The methods presented here can be easily adopted by most cell and developmental biology laboratories and adapted to successfully image early embryos of additional species, therefore broadening our understanding of the evolution of morphogenesis.

  View details for DOI 10.3389/fcell.2022.1007775

  View details for Web of Science ID 000862351500001

  View details for PubMedID 36187474

  View details for PubMedCentralID PMC9521734

• Lineage tracing shows that cell size asymmetries predict the dorsoventral axis in the sea star embryo BMC BIOLOGY Barone, V., Byrne, M., Lyons, D. C. 2022; 20 (1): 179

  Abstract

  Cell size asymmetries are often linked to cell fate decisions, due to cell volumes and cell fate determinants being unequally partitioned during asymmetric cell divisions. A clear example is found in the sea urchin embryo, where a characteristic and obvious unequal 4th cleavage generates micromeres, which are necessary for mesendoderm cell fate specification. Unlike sea urchin development, sea star development is generally thought to have only equal cleavage. However, subtle cell size asymmetries can be observed in sea star embryos; whether those cell size asymmetries are consistently produced during sea star development and if they are involved in cell fate decisions remains unknown.Using confocal live imaging of early embryos we quantified cell size asymmetries in 16-cell stage embryos of two sea star species, Patiria miniata and Patiriella regularis. Using photoconversion to perform lineage tracing, we find that the position of the smallest cells of P. miniata embryos is biased toward anterior ventral tissues. However, both blastomere dissociation and mechanical removal of one small cell do not prevent dorsoventral (DV) axis formation, suggesting that embryos compensate for the loss of those cells and that asymmetrical partitioning of maternal determinants is not strictly necessary for DV patterning. Finally, we show that manipulating cell size to introduce artificial cell size asymmetries is not sufficient to direct the positioning of the future DV axis in P. miniata embryos.Our results show that although cell size asymmetries are consistently produced during sea star early cleavage and are predictive of the DV axis, they are not necessary to instruct DV axis formation.

  View details for DOI 10.1186/s12915-022-01359-3

  View details for Web of Science ID 000840850300001

  View details for PubMedID 35971116

  View details for PubMedCentralID PMC9380389

• Movement and storage of nematocysts across development in the nudibranch <i>Berghia stephanieae</i> (Valdes, 2005) FRONTIERS IN ZOOLOGY Goodheart, J. A., Barone, V., Lyons, D. C. 2022; 19 (1): 16

  Abstract

  Intracellular sequestration requires specialized cellular and molecular mechanisms allowing a predator to retain and use specific organelles that once belonged to its prey. Little is known about how common cellular mechanisms, like phagocytosis, can be modified to selectively internalize and store foreign structures. One form of defensive sequestration involves animals that sequester stinging organelles (nematocysts) from their cnidarian prey. While it has been hypothesized that nematocysts are identified by specialized phagocytic cells for internalization and storage, little is known about the cellular and developmental mechanisms of this process in any metazoan lineage. This knowledge gap is mainly due to a lack of genetically tractable model systems among predators and their cnidarian prey.Here, we introduce the nudibranch Berghia stephanieae as a model system to investigate the cell, developmental, and physiological features of nematocyst sequestration selectivity. We first show that B. stephanieae, which feeds on Exaiptasia diaphana, selectively sequesters nematocysts over other E. diaphana tissues found in their digestive gland. Using confocal microscopy, we document that nematocyst sequestration begins shortly after feeding and prior to the formation of the appendages (cerata) where the organ responsible for sequestration (the cnidosac) resides in adults. This finding is inconsistent with previous studies that place the formation of the cnidosac after cerata emerge. Our results also show, via live imaging assays, that both nematocysts and dinoflagellates can enter the nascent cnidosac structure. This result indicates that selectivity for nematocysts occurs inside the cnidosac in B. stephanieae, likely in the cnidophage cells themselves.Our work highlights the utility of B. stephanieae for future research, because: (1) this species can be cultured in the laboratory, which provides access to all developmental stages, and (2) the transparency of early juveniles makes imaging techniques (and therefore cell and molecular assays) feasible. Our results pave the way for future studies using live imaging and targeted gene editing to identify the molecular mechanisms involved in nematocyst sequestration. Further studies of nematocyst sequestration in B. stephanieae will also allow us to investigate how common cellular mechanisms like phagocytosis can be modified to selectively internalize and store foreign structures.

  View details for DOI 10.1186/s12983-022-00460-1

  View details for Web of Science ID 000786475900001

  View details for PubMedID 35436919

  View details for PubMedCentralID PMC9016961

• An Effective Feedback Loop between Cell-Cell Contact Duration and Morphogen Signaling Determines Cell Fate DEVELOPMENTAL CELL Barone, V., Lang, M., Krens, S., Pradhan, S. J., Shamipour, S., Sako, K., Sikora, M., Guet, C. C., Heisenberg, C. 2017; 43 (2): 198-+

  Abstract

  Cell-cell contact formation constitutes an essential step in evolution, leading to the differentiation of specialized cell types. However, remarkably little is known about whether and how the interplay between contact formation and fate specification affects development. Here, we identify a positive feedback loop between cell-cell contact duration, morphogen signaling, and mesendoderm cell-fate specification during zebrafish gastrulation. We show that long-lasting cell-cell contacts enhance the competence of prechordal plate (ppl) progenitor cells to respond to Nodal signaling, required for ppl cell-fate specification. We further show that Nodal signaling promotes ppl cell-cell contact duration, generating a positive feedback loop between ppl cell-cell contact duration and cell-fate specification. Finally, by combining mathematical modeling and experimentation, we show that this feedback determines whether anterior axial mesendoderm cells become ppl or, instead, turn into endoderm. Thus, the interdependent activities of cell-cell signaling and contact formation control fate diversification within the developing embryo.

  View details for DOI 10.1016/j.devcel.2017.09.014

  View details for Web of Science ID 000413443700011

  View details for PubMedID 29033362

• Live tracking of moving samples in confocal microscopy for vertically grown roots ELIFE von Wangenheim, D., Hauschild, R., Fendrych, M., Barone, V., Benkova, E., Friml, J. 2017; 6

  Abstract

  Roots navigate through soil integrating environmental signals to orient their growth. The Arabidopsis root is a widely used model for developmental, physiological and cell biological studies. Live imaging greatly aids these efforts, but the horizontal sample position and continuous root tip displacement present significant difficulties. Here, we develop a confocal microscope setup for vertical sample mounting and integrated directional illumination. We present TipTracker - a custom software for automatic tracking of diverse moving objects usable on various microscope setups. Combined, this enables observation of root tips growing along the natural gravity vector over prolonged periods of time, as well as the ability to induce rapid gravity or light stimulation. We also track migrating cells in the developing zebrafish embryo, demonstrating the utility of this system in the acquisition of high-resolution data sets of dynamic samples. We provide detailed descriptions of the tools enabling the easy implementation on other microscopes.

  View details for DOI 10.7554/eLife.26792

  View details for Web of Science ID 000404728300001

  View details for PubMedID 28628006

  View details for PubMedCentralID PMC5498147

• Interstitial fluid osmolarity modulates the action of differential tissue surface tension in progenitor cell segregation during gastrulation DEVELOPMENT Krens, S., Veldhuis, J. H., Barone, V., Capek, D., Maitre, J., Brodland, G., Heisenberg, C. 2017; 144 (10): 1798-1806

  Abstract

  The segregation of different cell types into distinct tissues is a fundamental process in metazoan development. Differences in cell adhesion and cortex tension are commonly thought to drive cell sorting by regulating tissue surface tension (TST). However, the role that differential TST plays in cell segregation within the developing embryo is as yet unclear. Here, we have analyzed the role of differential TST for germ layer progenitor cell segregation during zebrafish gastrulation. Contrary to previous observations that differential TST drives germ layer progenitor cell segregation in vitro, we show that germ layers display indistinguishable TST within the gastrulating embryo, arguing against differential TST driving germ layer progenitor cell segregation in vivo We further show that the osmolarity of the interstitial fluid (IF) is an important factor that influences germ layer TST in vivo, and that lower osmolarity of the IF compared with standard cell culture medium can explain why germ layers display differential TST in culture but not in vivo Finally, we show that directed migration of mesendoderm progenitors is required for germ layer progenitor cell segregation and germ layer formation.

  View details for DOI 10.1242/dev.144964

  View details for Web of Science ID 000402275900007

  View details for PubMedID 28512197

  View details for PubMedCentralID PMC5450835

• Optogenetic Control of Nodal Signaling Reveals a Temporal Pattern of Nodal Signaling Regulating Cell Fate Specification during Gastrulation CELL REPORTS Sako, K., Pradhan, S. J., Barone, V., Ingles-Prieto, A., Mueller, P., Ruprecht, V., Capek, D., Galande, S., Janovjak, H., Heisenberg, C. 2016; 16 (3): 866-877

  Abstract

  During metazoan development, the temporal pattern of morphogen signaling is critical for organizing cell fates in space and time. Yet, tools for temporally controlling morphogen signaling within the embryo are still scarce. Here, we developed a photoactivatable Nodal receptor to determine how the temporal pattern of Nodal signaling affects cell fate specification during zebrafish gastrulation. By using this receptor to manipulate the duration of Nodal signaling in vivo by light, we show that extended Nodal signaling within the organizer promotes prechordal plate specification and suppresses endoderm differentiation. Endoderm differentiation is suppressed by extended Nodal signaling inducing expression of the transcriptional repressor goosecoid (gsc) in prechordal plate progenitors, which in turn restrains Nodal signaling from upregulating the endoderm differentiation gene sox17 within these cells. Thus, optogenetic manipulation of Nodal signaling identifies a critical role of Nodal signaling duration for organizer cell fate specification during gastrulation.

  View details for DOI 10.1016/j.celrep.2016.06.036

  View details for Web of Science ID 000380264200024

  View details for PubMedID 27396324