Biology subjects

Ishizuka, K. J.

Publications and source records attributed to Ishizuka, K. J..

8 recordsLinked to original sources

bioRxiv2026

Anatomical and functional organisation of the cholinergic nervous system in the tunicate Botryllus schlosseri

Acetylcholine (ACh) is an ancient, highly conserved neurotransmitter, yet the functional diversification of cholinergic pathways across early chordates remains incompletely understood. Here, we investigate the spatial and functional organization of the cholinergic system in the colonial tunicate Botryllus schlosseri, a chordate model that undergoes lifelong, cyclical neural regeneration. By integrating HCR-RNA FISH, in vivo pharmacology, extracellular brain electrophysiology, and quantitative reflex assays, we map the core molecular machinery of ACh synthesis (ChAT), vesicular transport (VAChT), AChE, and receptor signaling (CHRNA7, CHRM3). Spatial expression analysis reveals a compartmentalized cholinergic network spanning central ganglia, peripheral sensory cells, and ciliated epithelia. Functionally, nicotinic receptors (nAChRs) mediate rapid mechanosensory burst firing in the brain, evoked siphon reflex contraction, and cilliary arrest, whereas muscarinic receptors (mAChRs), modulate baseline siphon motility and muscle tone. Pharmacological silencing of active siphon motor programs unmasks a slow, vascular-coupled rhythmic motility, while AChE inhibition induces paralysis indicating a critical role for regulated ACh breakdown and non-synaptic transmission. Together, these findings demonstrate that B. schlosseri possesses a sophisticated, dual-effector cholinergic system coordinating both muscular and ciliary networks, providing key insights into the evolutionary diversification of chordate neuromuscular control.

neuroscience↗

bioRxiv2026

Lifelong Invertebrate Chordate Central Nervous System Regeneration is Driven by a Migrating Pool of Conserved Neural Stem Cells Program

Adult central nervous systems (CNS) are generally constrained by limited cellular replacement. How regenerating organisms integrate structural maintenance with continuous, cyclic renewal remains unresolved. Here, we leverage the colonial chordate Botryllus schlosseri which completely resorbs and regenerates its entire CNS de novo weekly. Combining indexed single-cell sorting, Smart-seq3 sequencing, spatial mapping, and temporal deconvolution, our studies suggest that this continuous CNS renewal is mediated by an intrinsic pool of lineage-restricted neural stem cells. Single-cell analysis resolved a conserved SOX2/NOTCH1/NEUROG1 prospective progenitor continuum alongside mature excitability-defined neurons. Brain aging drives a phase-specific desynchronization of this regenerative program, inducing early hyper-activation followed by a sudden collapse. Finally, syngeneic transplantation and in vivo tracking show that neural-complex-derived cells persist after vascular delivery and localize to developing bud and neural-associated territories, with injury-associated accumulation near the neural gland. This work establishes a spatiotemporal framework for lifelong chordate CNS renewal and aging.

cell biology↗

bioRxiv2026

Survival Benefits Outweigh Germline Competition Costs in Kin Chimeras

Chimerism, the coexistence of genetically distinct cells within an organism, is a widespread phenomenon in nature. In the colonial chordate Botryllus schlosseri, fusion between compatible colonies can lead to stem cell competition with a single genotype taking over the gonadal and/or somatic tissue of the chimera. Over five years, we quantified the frequency and consequences of chimeric formation among 809 larvae, assessing the rates of fusion after natural settlement and its long-term impact on colony survival. We found larvae preferentially settle near kin, facilitating high rates of successful fusion. Fusion was concentrated around the first month of life, boosting survival during this period of high mortality. At all timepoints chimerism significantly increased survival relative to naive and rejected colonies and extended overall lifespan. However, fusion triggered intense internal competition: in all cases tested, a single genotype achieved dominance over both germline and somatic tissues within several weeks post-fusion, regardless of initial genotype contribution. We estimate that among kin, the survival benefits of chimerism outweigh the potential costs of germline loss, extending Hamiltons kin selection framework to colonial marine organisms. In this context, chimerism functions as an adaptive strategy that enhances early survival while enabling selection among competing stem cell lineages.

evolutionary biology↗

bioRxiv2025

Germline stem cell isolation, lineage tracing, and aging in a protochordate

Germline stem cells (GSCs), the source of gametes, are the only stem cells capable of passing genes to future generations and are therefore considered units of natural selection. Yet, the factors that influence GSC fitness, and thus govern GSC competition, which exist in both protochordates and mammals, remain poorly understood. We studied how aging affects GSC fitness in the protochordate Botryllus schlosseri, an evolutionary crosspoint between invertebrates and vertebrates. GSCs were isolated and distinguished from developing and mature gametes using flow cytometry and scRNA-Seq, facilitated by a new PacBio genome assembly. Moreover, their function was validated through a novel lineage tracing approach that combines membrane-labeled GSC transplantation with scRNA-Seq. Leveraging our method to isolate them, single-cell transcriptomics showed significant age-related changes between young and old GSCs. Spermatids and sperm, however, showed minimal changes, suggesting that reproductive aging is governed by GSCs rather than by gametes. Reduced expressions of markers like DDX4 and PIWIL1 in aged GSCs mirrored trends in mammalian datasets, pointing to a conserved GSC-driven aging mechanism across chordate evolution. This study provides new techniques that lay the foundation to investigate further drivers of GSC fitness and highlights fertility-related genes as promising targets for therapies to preserve reproductive health.

cell biology↗

bioRxiv2021

Revealing conserved mechanisms of neurodegeneration in a colonial chordate

Loss of the brains functional ability is a common symptom of aging and neurodegenerative diseases1,2. While the genetic and molecular mechanisms underlying human neurodegeneration are studied in-depth3-6, very little is known about the evolutionary origin of these traits and their involvement in loss of nervous system function in aged invertebrate species. Here we study evolutionarily conserved elements of brain degeneration using the colonial chordate model species Botryllus schlosseri. B. schlosseri reproduces both sexually and asexually7, with adult brains regenerating and degenerating multiple times throughout its adult life. Combining microscopy, transcriptomics and behavioral assays, we characterized adult brains from diverse stages and ages. We found that the number of neurons fluctuates each week, reaching a maximum of [~]1000 cells, and thereafter decreasing while the number of immunocytes increases. Comparing the number of neurons in the adult brains of young and old colonies, we found that older brains are smaller and contain fewer cells. Both during weekly degeneration cycles and overall with age, the decrease in neuron number correlates with reduced response to stimuli and with significant changes in the expression of genes with mammalian homologs associated with neural stem cells and neurodegenerative pathways. These results suggest persistent neural stem cell activity across ages and that cellular and molecular mechanisms of neurodegeneration are evolutionary conserved between tunicates and humans.

neuroscience↗

bioRxiv2020

Global Age-Specific Patterns of Cyclic Gene Expression Revealed by Tunicate Transcriptome Atlas

Expression levels of circadian clock genes, which regulate 24-hour rhythms of behavior and physiology, have been shown to change with age. However, a study holistically linking aging and circadian gene expression is missing. Using the colonial chordate Botryllus schlosseri, we combined transcriptome sequencing and stem cell-mediated aging phenomena to test how circadian gene expression changes with age. This revealed that B. schlosseri clock and clock-controlled genes oscillate organism-wide, with daily, age-specific amplitudes and frequencies. These age-related, circadian patterns persist at the tissue level, where dramatic variations in cyclic gene expression of tissue profiles link to morphological and cellular aging phenotypes. Similar cyclical expression differences were found in hundreds of pathways associated with known hallmarks of aging, as well as pathways that were not previously linked to aging. The atlas we developed points to alterations in circadian gene expression as a key regulator of aging. One Sentence SummaryThe Ticking Clock: Systemic changes in circadian gene expression correlates with wide-ranging phenotypes of aging

developmental biology↗

bioRxiv2019

Molecular and Morphological Signatures of Chordate Development: Two Distinct Pathways, One Tunicate

All chordates, including urochordates such as tunicates, develop through embryogenesis. The chordate larvae of colonial tunicates metamorphose to lose all chordate structures such as notochord, neural tube, segmented musculature, and then develop by asexual reproduction [blastogenesis], whereby stem cells form tissues and organs. These two developmental pathways establish the same body axis, morphogenetic patterning and organ formation. It is unknown if this convergent morphology implies convergent cellular and molecular mechanisms, and whether the stem cells that mediate these processes differ. Using the colonial tunicate Botryllus schlosseri, we combined transcriptome sequencing and multiple microscopy techniques to study the molecular and morphological signatures of cells at each developmental stage of embryogenesis and blastogenesis. This revealed that the molecular programs are distinct, but the blastogenic tissue-specific stem cells and embryonic precursor populations share similar molecular profiles. By comparing embryogenesis in other chordates we found shared developmental principles, highlighting transcription factors as key evolutionary conserved elements. This study establishes a platform for advancing the science of stem cell biology and regulation of development and regeneration.

developmental biology↗

bioRxiv2017

Evolutionary Origin of the Mammalian Hematopoietic System Found in a Colonial Chordate

Hematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are hematopoietic stem cells (HSCs), which are multipotent, self-renewing and generate the entire repertoire of blood and immune cells throughout life. Here we studied the hematopoietic system of Botryllus schlosseri, a colonial tunicate that has vasculature, circulating blood cells, and interesting characteristics of stem cell biology and immunity. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other. Using flow-cytometry, whole-transcriptome sequencing of defined cell populations, and diverse functional assays, we identified HSCs, progenitors, immune-effector cells, the HSC niche, and demonstrated that self-recognition inhibits cytotoxic reaction. Our study implies that the HSC and myeloid lineages emerged in a common ancestor of tunicates and vertebrates and suggests that hematopoietic bone marrow and the B. schlosseri endostyle niche evolved from the same origin.

immunology↗