Periodic Reporting for period 1 - MiMING (Conserved and divergent mechanisms underlying cytokinesis in early vertebrate and invertebrate embryogenesis)
Periodo di rendicontazione: 2024-05-01 al 2026-04-30
Sintesi del contesto e degli obiettivi generali del progetto
Origin of mechanical force drives membrane ingression underlying meroblastic cytokinesis in early zebrafish embryogenesis
Cell division is a fundamental process incorporating both chromosome segregation and cytokinesis, which divides one parent cell into two daughter cells. The process is not only crucial for growth, repair and reproduction in multicellular organisms, but also constitutes the basis for cell differentiation and tissue formation in early embryonic development. Mainly depending on the distribution and amount of yolk, early embryo division, also termed ‘cleavages’, can be holoblastic (complete seccision of membrane) and meroblastic (incomplete): holoblastic division occurs, for instance, in mammalian embryos, whereas meroblastic division can be observed in fish and most insect early embryos. The fertilized zebrafish egg (zygote) undergoes a series of reductive cell divisions. These cleavages are meroblastic (incomplete) as they only subdivide the blastodisc at the animal pole of zygote, but not at its vegetal portion containing the yolk. Consequently, the initially forming blastomeres are not completely divided by plasma membrane, but remain continuous via the yolky (undivided) portion of the zygote.
Holobastic divisions, similar to the canonical model of cytokinesis in culture cells, is achieved by a contractile cytoskeletal ring, the main force-generating components of which are actin filaments and myosin motors (actomyosin). This contractile actomyosin ring constricts and pinches the cell membrane to form a cytokinetic furrow, which eventually divides the cell into two daughter cells. By contrast, during meroblastic (incomplete) division in early zebrafish embryos, actomyosin forms a contractile band where the meroblastic furrow invaginates at the animal pole of the zygote, rather than a contractile ring found in holoblastic cleavages. Moreover, inhibition of myosin motor function does not block furrow invagination in zebrafish, suggesting that myosin-driven actomyosin contraction might be dispensable for meroblastic furrow ingression in zebrafish zygote. Instead, microtubules have been proposed to constitute the main component that drive furrow invagination. Microtubules filaments not only determine the position of the cleavage plane between the poles of the mitotic spindle, but also transport cellular membrane components to the furrow surface required for furrow ingression.
Meroblastic cleavages also have been observed in Drosophila embryos, where the formed syncytial (multinucleated) blastoderm transforms into a cellular tissue by a process commonly called ‘cellularization’. During cellularization, plasma membrane invaginates between interphase nuclei (meroblastic furrow ingression) and closes at the prospective basal side of the forming cells, two distinct processes that are thought to be mediated by the microtubule (invagination) and actomyosin cytoskeleton (closure), respectively. Specifically, basal closure is mediated by accumulation of Myosin II at the cell cortex leading to basal constriction of plasma membrane. Similar to the situation in zebrafish cleavages, meroblastic furrow ingression appears independent of actomyosin in Drosophila embryos, but relies on an intact microtubule network. These distinct requirements of the microtubule and actomyosin networks during cellularization have been further corroborated by the observation that the cleavage furrow displays different mechanical property at the beginning and end of the cellularization process, indicative for distinct mechanical forces acting on the cleavage furrow during furrow ingression and basal closure.
Meroblastic cleavage in zebrafish embryos requires rapid membrane invagination to partition the blastodisc during the first several cell cycles. Previous work shows that microtubules, F-actin and E-cadherin–based adhesion are essential: mutation of microtubule-associated regulators such as Prc1l disrupts furrow microtubule organization and catenin enrichment, and cdh1 morpholino injection blocks membrane invagination. How cytoskeletal mechanics are integrated with cadherin recruitment to build a stable, invaginating furrow remains unresolved.
Here we combine acute pharmacological perturbations, live imaging and biosensor analyses to define the cytoskeletal and mechanical logic of furrow formation. Microtubule depolymerization with nocodazole, or inhibition of microtubule acetylation with GM90257, disrupts the organization of furrow-associated microtubules and compromises furrow progression, supporting a requirement for organized and stabilized microtubule arrays. In parallel, inhibition of actin polymerization with cytochalasin D prevents completion of membrane invagination, indicating that F-actin is required for sustained furrow ingression.
High-resolution time-lapse imaging reveals highly dynamic protrusive structures distributed along the invaginating membrane. These protrusions are enriched in plasma membrane and F-actin, display leading-edge Rac1 activity, and are abolished by the Rac1 inhibitor EHT1864. We propose that Rac1-dependent protrusions locally increase effective membrane tension and, through tension-coupled membrane flows, promote the propagation and directed migration of E-cadherin toward the furrow. This hypothesis is guided by De Belly et al. (2023), who showed that actin-driven protrusions and contractions can generate rapid long-range membrane tension propagation via membrane flows, and is conceptually consistent with recent work demonstrating that protrusion-associated tension gradients and cortical flows reorganize E-cadherin during contact formation. Together, our results support a model in which microtubule organization, actin‑based protrusive activity, and cadherin‑mediated adhesion are integrated to ensure robust meroblastic cleavage during early zebrafish development.
Cell division is a fundamental process incorporating both chromosome segregation and cytokinesis, which divides one parent cell into two daughter cells. The process is not only crucial for growth, repair and reproduction in multicellular organisms, but also constitutes the basis for cell differentiation and tissue formation in early embryonic development. Mainly depending on the distribution and amount of yolk, early embryo division, also termed ‘cleavages’, can be holoblastic (complete seccision of membrane) and meroblastic (incomplete): holoblastic division occurs, for instance, in mammalian embryos, whereas meroblastic division can be observed in fish and most insect early embryos. The fertilized zebrafish egg (zygote) undergoes a series of reductive cell divisions. These cleavages are meroblastic (incomplete) as they only subdivide the blastodisc at the animal pole of zygote, but not at its vegetal portion containing the yolk. Consequently, the initially forming blastomeres are not completely divided by plasma membrane, but remain continuous via the yolky (undivided) portion of the zygote.
Holobastic divisions, similar to the canonical model of cytokinesis in culture cells, is achieved by a contractile cytoskeletal ring, the main force-generating components of which are actin filaments and myosin motors (actomyosin). This contractile actomyosin ring constricts and pinches the cell membrane to form a cytokinetic furrow, which eventually divides the cell into two daughter cells. By contrast, during meroblastic (incomplete) division in early zebrafish embryos, actomyosin forms a contractile band where the meroblastic furrow invaginates at the animal pole of the zygote, rather than a contractile ring found in holoblastic cleavages. Moreover, inhibition of myosin motor function does not block furrow invagination in zebrafish, suggesting that myosin-driven actomyosin contraction might be dispensable for meroblastic furrow ingression in zebrafish zygote. Instead, microtubules have been proposed to constitute the main component that drive furrow invagination. Microtubules filaments not only determine the position of the cleavage plane between the poles of the mitotic spindle, but also transport cellular membrane components to the furrow surface required for furrow ingression.
Meroblastic cleavages also have been observed in Drosophila embryos, where the formed syncytial (multinucleated) blastoderm transforms into a cellular tissue by a process commonly called ‘cellularization’. During cellularization, plasma membrane invaginates between interphase nuclei (meroblastic furrow ingression) and closes at the prospective basal side of the forming cells, two distinct processes that are thought to be mediated by the microtubule (invagination) and actomyosin cytoskeleton (closure), respectively. Specifically, basal closure is mediated by accumulation of Myosin II at the cell cortex leading to basal constriction of plasma membrane. Similar to the situation in zebrafish cleavages, meroblastic furrow ingression appears independent of actomyosin in Drosophila embryos, but relies on an intact microtubule network. These distinct requirements of the microtubule and actomyosin networks during cellularization have been further corroborated by the observation that the cleavage furrow displays different mechanical property at the beginning and end of the cellularization process, indicative for distinct mechanical forces acting on the cleavage furrow during furrow ingression and basal closure.
Meroblastic cleavage in zebrafish embryos requires rapid membrane invagination to partition the blastodisc during the first several cell cycles. Previous work shows that microtubules, F-actin and E-cadherin–based adhesion are essential: mutation of microtubule-associated regulators such as Prc1l disrupts furrow microtubule organization and catenin enrichment, and cdh1 morpholino injection blocks membrane invagination. How cytoskeletal mechanics are integrated with cadherin recruitment to build a stable, invaginating furrow remains unresolved.
Here we combine acute pharmacological perturbations, live imaging and biosensor analyses to define the cytoskeletal and mechanical logic of furrow formation. Microtubule depolymerization with nocodazole, or inhibition of microtubule acetylation with GM90257, disrupts the organization of furrow-associated microtubules and compromises furrow progression, supporting a requirement for organized and stabilized microtubule arrays. In parallel, inhibition of actin polymerization with cytochalasin D prevents completion of membrane invagination, indicating that F-actin is required for sustained furrow ingression.
High-resolution time-lapse imaging reveals highly dynamic protrusive structures distributed along the invaginating membrane. These protrusions are enriched in plasma membrane and F-actin, display leading-edge Rac1 activity, and are abolished by the Rac1 inhibitor EHT1864. We propose that Rac1-dependent protrusions locally increase effective membrane tension and, through tension-coupled membrane flows, promote the propagation and directed migration of E-cadherin toward the furrow. This hypothesis is guided by De Belly et al. (2023), who showed that actin-driven protrusions and contractions can generate rapid long-range membrane tension propagation via membrane flows, and is conceptually consistent with recent work demonstrating that protrusion-associated tension gradients and cortical flows reorganize E-cadherin during contact formation. Together, our results support a model in which microtubule organization, actin‑based protrusive activity, and cadherin‑mediated adhesion are integrated to ensure robust meroblastic cleavage during early zebrafish development.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
Research Activities and Technical Contributions During the Fellowship Support Period
During my fellowship support period, I focused on two major areas: first, establishing a functional Drosophila work system in a new laboratory environment and conducting quantitative analyses of cytoskeletal functions during cellularization; second, expanding my technical expertise into zebrafish models, where I applied advanced genetic and pharmacological tools to study cell division dynamics. This period significantly broadened my experimental repertoire and deepened my understanding of cytoskeletal regulation during embryonic development.
Part 1: Establishing the Drosophila System and Analyzing Cellularization
A primary achievement was the successful establishment of a fully operational Drosophila melanogaster work system in the new lab. This included setting up embryo collection, staging, fixation, immunostaining, and live-imaging capabilities, as well as maintaining stable fly stocks. Once the system was running, I investigated the roles of the microtubule and F-actin cytoskeletons during the cellularization process in Drosophila embryos—a critical stage of embryogenesis when syncytial blastoderm cells are partitioned into individual cells through furrow invagination.
To quantitatively describe furrow invagination in wild-type embryos, I performed high-resolution time-lapse imaging and developed image analysis pipelines to measure parameters such as furrow depth, progression rate, and morphology over time. This quantitative baseline allowed for rigorous comparison with drug-treated embryos.
I then employed microinjection of specific inhibitors to dissect the contributions of different cytoskeletal components. To disrupt microtubule dynamics, I injected the microtubule polymerization inhibitor Nocodazole. For F-actin and myosin II, I used three complementary inhibitors: SMIFH2 (a formin inhibitor that impairs actin polymerization), Blebbistatin (a non-muscle myosin II ATPase inhibitor), and Y-27632 (a ROCK inhibitor that suppresses myosin II regulatory light chain phosphorylation). By comparing the furrow invagination phenotypes—ranging from incomplete closure to abnormal furrow morphology and altered kinetics—I was able to distinguish the distinct roles of microtubules versus actomyosin contractility during this morphogenetic event. My results revealed that while F-actin/myosin II activity is essential for furrow ingression and constriction, microtubules contribute to furrow stabilization and progression, suggesting a cooperative interaction between the two systems.
Part 2: Expanding to Zebrafish – Genetic Crosses, CRISPR, and Microinjection
In parallel, I sought to broaden my model organism expertise by learning to work with zebrafish (Danio rerio) embryos. I first acquired hands-on training in maintaining zebrafish lines and, importantly, learned how to cross fish lines carrying different genetic backgrounds. This included setting up crosses to generate embryos with specific genotypes, tracking fluorescent markers, and managing large-scale breeding schemes.
Building on these skills, I performed CRISPR/Cas9 genome editing to generate a mutant line for prc1l (protein regulator of cytokinesis 1-like), a gene involved in central spindle assembly and cytokinesis. This involved designing guide RNAs, injecting Cas9 protein and sgRNA into one-cell-stage zebrafish embryos, and screening for mutations using PCR and sequencing. This work positions me to study the role of Prc1l in cytokinesis during zebrafish development.
I also mastered several other key zebrafish embryo techniques. Notably, I performed microinjection of mRNA to express tools for studying Rac1a, a small GTPase that regulates actin dynamics. I injected mRNA encoding a Rac1a biosensor (to monitor real-time Rac1a activity) and constitutively active Rac1a (to hyperactivate downstream signaling). These injections allowed me to manipulate and visualize signaling pathways during early embryogenesis.
To investigate microtubule function in the context of meroblastic division (the incomplete, cleavage-type divisions occurring in the zebrafish blastoderm), I employed a sophisticated optochemical tool: photo-activable combretastatin. This is a caged version of the microtubule-destabilizing drug combretastatin, which can be activated by localized UV illumination. I injected this compound into zebrafish embryos and then selectively uncaged it in specific regions or at precise times during mesoblastic divisions. This spatiotemporally controlled approach allowed me to acutely disrupt microtubules in the forming mesoderm without affecting other cell populations, and then assess the consequences on mitotic spindle orientation, chromosome segregation, and cleavage furrow formation.
Finally, I complemented these perturbations with a systematic pharmacological approach using established zebrafish lines combined with different inhibitors. By treating embryos with inhibitors targeting microtubules (e.g. nocodazole, combretastatin), actin (e.g. latrunculin B), or myosin II (blebbistatin), and then imaging division dynamics in live embryos, I examined how each protein family contributes to meroblastic division. I compared phenotypes across different transgenic lines (e.g. with labelled membranes, microtubules, or histones) to achieve a multi-dimensional view of cell division.
Conclusion and Future Directions
Collectively, the work performed during the fellowship period has achieved two major outcomes. First, I successfully established a complete Drosophila embryology platform in a new laboratory, generating quantitative, inhibitor-based insights into how microtubules and actomyosin cooperate during cellularization. Second, I acquired a broad and transferable skill set in zebrafish research—from genetics (crossing lines and CRISPR mutagenesis) to advanced microinjection (mRNA, biosensors, photo-activable drugs) and live imaging—and applied these tools to dissect the roles of microtubules and other proteins in meroblastic divisions. This dual-model approach has not only advanced my research projects but also prepared me for independent investigations into the cytoskeletal basis of morphogenesis and cell division across species.
During my fellowship support period, I focused on two major areas: first, establishing a functional Drosophila work system in a new laboratory environment and conducting quantitative analyses of cytoskeletal functions during cellularization; second, expanding my technical expertise into zebrafish models, where I applied advanced genetic and pharmacological tools to study cell division dynamics. This period significantly broadened my experimental repertoire and deepened my understanding of cytoskeletal regulation during embryonic development.
Part 1: Establishing the Drosophila System and Analyzing Cellularization
A primary achievement was the successful establishment of a fully operational Drosophila melanogaster work system in the new lab. This included setting up embryo collection, staging, fixation, immunostaining, and live-imaging capabilities, as well as maintaining stable fly stocks. Once the system was running, I investigated the roles of the microtubule and F-actin cytoskeletons during the cellularization process in Drosophila embryos—a critical stage of embryogenesis when syncytial blastoderm cells are partitioned into individual cells through furrow invagination.
To quantitatively describe furrow invagination in wild-type embryos, I performed high-resolution time-lapse imaging and developed image analysis pipelines to measure parameters such as furrow depth, progression rate, and morphology over time. This quantitative baseline allowed for rigorous comparison with drug-treated embryos.
I then employed microinjection of specific inhibitors to dissect the contributions of different cytoskeletal components. To disrupt microtubule dynamics, I injected the microtubule polymerization inhibitor Nocodazole. For F-actin and myosin II, I used three complementary inhibitors: SMIFH2 (a formin inhibitor that impairs actin polymerization), Blebbistatin (a non-muscle myosin II ATPase inhibitor), and Y-27632 (a ROCK inhibitor that suppresses myosin II regulatory light chain phosphorylation). By comparing the furrow invagination phenotypes—ranging from incomplete closure to abnormal furrow morphology and altered kinetics—I was able to distinguish the distinct roles of microtubules versus actomyosin contractility during this morphogenetic event. My results revealed that while F-actin/myosin II activity is essential for furrow ingression and constriction, microtubules contribute to furrow stabilization and progression, suggesting a cooperative interaction between the two systems.
Part 2: Expanding to Zebrafish – Genetic Crosses, CRISPR, and Microinjection
In parallel, I sought to broaden my model organism expertise by learning to work with zebrafish (Danio rerio) embryos. I first acquired hands-on training in maintaining zebrafish lines and, importantly, learned how to cross fish lines carrying different genetic backgrounds. This included setting up crosses to generate embryos with specific genotypes, tracking fluorescent markers, and managing large-scale breeding schemes.
Building on these skills, I performed CRISPR/Cas9 genome editing to generate a mutant line for prc1l (protein regulator of cytokinesis 1-like), a gene involved in central spindle assembly and cytokinesis. This involved designing guide RNAs, injecting Cas9 protein and sgRNA into one-cell-stage zebrafish embryos, and screening for mutations using PCR and sequencing. This work positions me to study the role of Prc1l in cytokinesis during zebrafish development.
I also mastered several other key zebrafish embryo techniques. Notably, I performed microinjection of mRNA to express tools for studying Rac1a, a small GTPase that regulates actin dynamics. I injected mRNA encoding a Rac1a biosensor (to monitor real-time Rac1a activity) and constitutively active Rac1a (to hyperactivate downstream signaling). These injections allowed me to manipulate and visualize signaling pathways during early embryogenesis.
To investigate microtubule function in the context of meroblastic division (the incomplete, cleavage-type divisions occurring in the zebrafish blastoderm), I employed a sophisticated optochemical tool: photo-activable combretastatin. This is a caged version of the microtubule-destabilizing drug combretastatin, which can be activated by localized UV illumination. I injected this compound into zebrafish embryos and then selectively uncaged it in specific regions or at precise times during mesoblastic divisions. This spatiotemporally controlled approach allowed me to acutely disrupt microtubules in the forming mesoderm without affecting other cell populations, and then assess the consequences on mitotic spindle orientation, chromosome segregation, and cleavage furrow formation.
Finally, I complemented these perturbations with a systematic pharmacological approach using established zebrafish lines combined with different inhibitors. By treating embryos with inhibitors targeting microtubules (e.g. nocodazole, combretastatin), actin (e.g. latrunculin B), or myosin II (blebbistatin), and then imaging division dynamics in live embryos, I examined how each protein family contributes to meroblastic division. I compared phenotypes across different transgenic lines (e.g. with labelled membranes, microtubules, or histones) to achieve a multi-dimensional view of cell division.
Conclusion and Future Directions
Collectively, the work performed during the fellowship period has achieved two major outcomes. First, I successfully established a complete Drosophila embryology platform in a new laboratory, generating quantitative, inhibitor-based insights into how microtubules and actomyosin cooperate during cellularization. Second, I acquired a broad and transferable skill set in zebrafish research—from genetics (crossing lines and CRISPR mutagenesis) to advanced microinjection (mRNA, biosensors, photo-activable drugs) and live imaging—and applied these tools to dissect the roles of microtubules and other proteins in meroblastic divisions. This dual-model approach has not only advanced my research projects but also prepared me for independent investigations into the cytoskeletal basis of morphogenesis and cell division across species.
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
In my project, I uncovered a mechanism governing the transition from cortical deformation to adhesive membrane invagination during meroblastic division in zebrafish embryos. I found that Rac1a-induced cortical protrusion is essential for successful meroblastic cleavage. This protrusive activity primes the membrane for subsequent invagination by locally remodeling the actomyosin cytoskeleton. Crucially, my results reveal a novel function for adherens junctions in cell cleavage: beyond their canonical role in cell adhesion, they mediate the mechanical coupling between cortical protrusions and the invaginating membrane furrow. This discovery redefines the role of adherens junctions as dynamic organizers of cleavage furrow architecture during early embryogenesis