Biology subjects

Atsuta, Y.

Publications and source records attributed to Atsuta, Y..

11 recordsLinked to original sources

bioRxiv2025

Bleb-based extravasation: conserved morphodynamics, divergent calcium control

Extravasation, the exit of cells from blood vessels, is essential in both development and disease, from germ cell migration to cancer metastasis. We show that all extravasating cells examined, avian primordial germ cells and diverse human cancer lines, form Ca{superscript 2}-dependent membrane blebs during transendothelial migration. Yet the source of Ca{superscript 2} diverges by lineage. PGCs and HT-1080 fibrosarcoma cells rely on store-operated Ca{superscript 2} entry (SOCE), whereas epithelial cancer cells such as PC-3 and MDA-MB-231 use IPR-mediated Ca{superscript 2} release from the endoplasmic reticulum. These findings establish bleb-based extravasation as a conserved morphodynamic strategy powered by distinct regulatory modules. Evolutionarily, they map onto an ancestral ER-release pathway and a metazoan-derived SOCE pathway. Conceptually, cancer blebbing emerges as a composite strategy that reactivates both ancient survival programs and developmental toolkits to maximize invasive success. This unified framework highlights Ca{superscript 2} supply as a critical bottleneck of vascular escape, offering new angles for targeting metastatic dissemination.

cell biology↗

bioRxiv2025

The heterochronic activation of TGF-β signaling drives the diversity of the avian sterna

Adaptive evolution has driven the diversification of vertebrate skeletal morphology, enabling a wide array of locomotor modes and lifestyles. In volant birds (carinates), the sternum develops a ventral keel that provides an attachment site for massive pectoral muscles essential for powered flight. In contrast, flightless ratites have lost both flight capability and the keel, resulting in a striking morphological contrast between their sterna. Here, we interrogated the cellular and molecular basis underlying this morphological divergence by using chicken embryos as a carinate model and emu embryos as a ratite model. Through a series of analyses including spatiotemporal transcriptomics and a spheroid culture system, we found that TGF-{beta} signaling, which promotes proliferation of ventral sternal chondroprogenitors, is activated in both species until the stage when the left and right sternal progenitors meet; however, in chicken this activation persists beyond this stage, driving ventral extension of the keel primordium, whereas in emu it shuts off early, culminating in the absence of a protruding keel. Together, our findings suggest that skeletal morphological changes associated with behavioral transitions can arise from heterochrony in developmental signaling, thereby deepening our understanding of the evolutionary logic shaping skeletal diversification.

developmental biology↗

bioRxiv2025

Optimization of culture and transfection methods for primary snake cells

Snakes serve as important models for understanding how changes in genes and genome sequences drive vertebrate morphological evolution. However, the lack of established primary culture methods and gene delivery techniques for snake cells has hindered functional analyses of evolutionarily modified genes and genomic elements. Here, we optimized primary culture conditions and screened for efficient transfection methods using corn snake embryonic fibroblasts. Our culture optimization experiments revealed that TeSR medium, designed for stem cells, with fetal bovine serum supplementation, and incubation at 28 provided a suitable condition for primary snake fibroblasts. Transcriptome analysis further demonstrated that under this optimized condition, genes associated with cytoskeletal organization, extracellular matrix components, and sterol biosynthetic process were upregulated, likely promoting snake cell proliferation. Additionally, screening of various gene transfection methods identified three efficient approaches, including electroporation. These findings enhance the utility of snake cells and pave the way for functional analyses of genes and genomic elements using snake cell-based systems.

developmental biology↗

bioRxiv2024

The yolk sac vasculature in early avian embryo provides a novel model for the analysis of cancer extravasation

Hematogenous metastasis, a key trait of cancer cells, involves a complex sequence of cell migration steps, including intravasation, circulation, arrest in capillary vessels, and extravasation. Among these steps, extravasation is challenging to image in amniotes like humans and mice due to its unpredictable timing and location, limiting our understanding of cellular and molecular mechanisms through imaging. Establishing a new cancer carrier model with high-resolution imaging capabilities in amniotes is crucial. In this study, we investigated the yolk sac vasculature (YSV) of early avian embryos (chickens and quail) as a new model for studying extravasation, offering excellent imaging capabilities. We examined the YSV structure and attempted fluorescent labeling to enhance visibility. We then injected mCherry-labeled HT-1080 cells into YSV and observed their behavior, revealing distinct morphologies and extravasation dynamics. Our findings suggest that the YSV model holds promise as a novel cancer carrier model for elucidating cellular and molecular mechanisms through imaging-based approaches.

cancer biology↗

bioRxiv2024

The universal labeling and sorting of avian primordial germ cell utilizing Lycopersicon Esculentum lectin

Avian species serve as vital resources in human society, with the preservation and utilization of these species heavily reliant on primordial germ cells (PGCs). However, methods for effectively isolating these rare cells alive from embryos remain elusive in avian species beyond chickens. Even within chicken species, existing techniques have shown limited efficiency. In our study, we present a rapid, simple, and cost-effective method for labeling and sorting circulating-stage PGCs across various avian species, including Carinatae and Ratitae, utilizing Lycopersicon Esculentum lectin. Significantly, this method demonstrates high sorting efficiency by identifying a wide range of PGC subtypes while preserving the proliferative potential of chicken PGCs. This approach is anticipated to make a significant contribution to the conservation, research, and agricultural industries related to birds worldwide.

cell biology↗

bioRxiv2023

The SOCE system is critical for membrane bleb formation to drive avian primordial germ cell migration

Amoeboid cell migration is driven by the specialized cell protrusion, membrane bleb. A recent in vitro analysis of bleb formation using a cancer cell line showed that store-operated calcium entry (SOCE)-mediated local elevation of Ca2+ concentration triggers bleb formation, but it remains unknown how commonly this system is utilized in the bleb formation and bleb-driven cell migration in vivo. We demonstrate with avian primordial germ cell (PGC) model that that chick PGCs use SOCE, triggered by stem cell factor (SCF), to induce bleb formation and that it is essential for in vivo migration including trans-endothelial and mesenchymal cell migration. Our discovery also provides insight into the correlation between cancer metastasis/invasion and SOCE-mediated bleb formation.

developmental biology↗

bioRxiv2022

Differential modularity of the mammalian Engrailed 1 enhancer network directs eccrine sweat gland development

Enhancers are context-specific regulators of expression that drive biological complexity and variation through the redeployment of conserved genes. An example of this is the enhancer-mediated control of the transcription of Engrailed 1 (EN1), a pleiotropic gene whose expression is required for the specification of eccrine sweat glands in mammals. We have previously identified an enhancer, ECE18 that has been highly and repeatedly derived on the human lineage to potentiate ectodermal EN1 and induce our species uniquely high eccrine gland density. Intriguingly, ECE18 quantitative activity is negligible outside of primates and ECE18 is not required for En1 regulation and eccrine gland specification in mice, raising the possibility that distinct enhancers have evolved to modulate the same trait. Here we report the identification of the ECE20 enhancer and show it has conserved functionality in mouse and human developing skin ectoderm. Unlike ECE18, knock-out of ECE20 in mice reduces ectodermal En1 and eccrine gland number. Notably, we find ECE20, but not ECE18, is also required for En1 expression in the embryonic mouse brain, demonstrating that ECE20 is a pleiotropic En1 enhancer. Finally, that ECE18 deletion does not potentiate the eccrine phenotype of ECE20 knockout mice supports the secondary incorporation of ECE18 into the regulation of this trait in primates. Our findings reveal that the mammalian En1 regulatory machinery diversified to incorporate both shared and lineage-restricted enhancers to regulate the same phenotype, and also have implications for understanding the forces that shape the robustness and evolvability of developmental traits.

evolutionary biology↗

bioRxiv2022

Prime editing in chicken fibroblasts and primordial germ cells

CRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and primordial germ cells (PGCs). By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chick PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.

developmental biology↗

bioRxiv2021

Direct Reprogramming of Non-limb Fibroblasts toCells with Properties of Limb Progenitors

The early limb bud consists of mesenchymal progenitors (limb progenitors) derived from the lateral plate mesoderm (LPM) that produce most of the tissues of the mature limb bud. The LPM also gives rise to the mesodermal components of the trunk, flank and neck. However, the mesenchymal cells generated at these other axial levels cannot produce the variety of cell types found in the limb bud, nor can they be directed to form a patterned appendage-like structure, even when placed in the context of the signals responsible for organizing the limb bud. Here, by taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28) normally expressed in the early limb bud, that are capable of imparting limb progenitor-like properties to non-limb fibroblasts. Cells reprogrammed by these factors show similar gene expression profiles, and can differentiate into similar cell types, as endogenous limb progenitors. The further addition of Lin41 potentiates proliferation of the reprogrammed cells while suppressing differentiation. These results suggest that these same four key factors may play pivotal roles in the specification of endogenous limb progenitors.

developmental biology↗

bioRxiv2020

In ovo electroporation of chick limb bud ectoderm

Deciphering how ectodermal tissues form, and how they maintain their integrity, is crucial for understanding epidermal development and pathogenesis. However, lack of simple and rapid gene manipulation techniques limits genetic studies to elucidate mechanisms underlying these events. Here we describe have an easy method for electroporation of chick embryo limb bud ectoderm, enabling gene manipulation during ectoderm development and wound healing. Taking advantage of a small parafilm well that constrains DNA plasmids locally and the fact that the limb ectoderm arises from a defined site, we target the limb ectoderm forming region by in ovo electroporation. This approach results in efficient transgenesis of the limb ectodermal cells. Further, using a previously described Msx2 promoter, gene manipulation can be specifically targeted to the apical ectodermal ridge (AER), a signaling center regulating limb development. Using the electroporation technique to deliver a fluorescent marker into the embryonic limb ectoderm, we show its utility in performing time-lapse imaging during wound healing. This analysis revealed previously unrecognized dynamic remodeling of the actin cytoskeleton and lamellipodia formation at the edges of the wound. We find that the lamellipodia formation requires activity of Rac1 GTPase, suggesting its necessity for wound closure. Our method is simple and cheap, and permits high throughput tests for gene function during limb ectodermal development and wound healing.

developmental biology↗

bioRxiv2020

An Engrailed1 enhancer underlies human thermoregulatory evolution

Humans rely on sweating to cool off and have the highest eccrine sweat gland density among mammals. We investigated whether altered regulation of the Engrailed 1 (EN1) gene, the levels of which are critical for patterning eccrine glands during development, could underlie the evolution of this defining human trait. First, we identify five EN1 candidate enhancers (ECEs) using comparative genomics and validation of enhancer activity in mouse skin. The human ortholog of one ECE, hECE18, contains multiple derived substitutions that together dramatically increase the activity of this enhancer in keratinocytes. Targeted repression of hECE18 reduces EN1 expression in human keratinocytes, indicating hECE18 upregulates EN1 in this context. Finally, we find that hECE18 increases ectodermal En1 in a humanized knock-in mouse to increase eccrine gland number. Our study uncovers a genetic basis for the evolution of one of the most singular human adaptations and implicates the recurrent mutation of a single enhancer as a novel mechanism for evolutionary change.

evolutionary biology↗