Biology subjects

Yoshida, J.

Publications and source records attributed to Yoshida, J..

8 recordsLinked to original sources

bioRxiv2023

Membrane molecule bouncer enables follicle fertilization in a viviparous teleost: Poecilia reticulata (guppy)

Fertilization is a fundamental mechanism of sexual reproduction. Generally, oocytes are ovulated from the ovarian follicles and contact the sperm outside the ovarian medulla. Unlike this, follicle fertilization which means the egg contact with sperm in the ovarian medulla without ovulation is known in the viviparous teleost species belonging to the Poeciliidae. In this study, we focused on a viviparous teleost species, Poecilia reticulata (guppy). Our sperm tracking assay indicated that the sperm reached the immature oocytes with a germinal vesicle, and the fertilized immature oocytes were presumed to contribute to littermates. The binding between immature oocytes and sperm is a specific trait in the guppy, which was not observed in Danio rerio (zebrafish) or Oryzias latipes (medaka). The loss- and gain-of-function assays indicated that bouncer plays a critical role in immature oocyte-to-sperm binding. This fertilization trait in immature oocytes may provide certain advantages for females with respect to nutrition or other gestation costs. Our findings shed light on the unique reproductive strategies of guppy and contribute to our understanding of the diverse reproductive mechanisms in vertebrates. Summary statementUnlike general vertebrates, guppys oocyte fertilizes with sperm in the ovarian medulla at the immature stages. The distinctive trait depends on the Ly6/uPAR protein bouncer.

developmental biology↗

bioRxiv2022

Origin, Prospective Identification, and Function of Circulating Endothelial Colony Forming Cells in Mouse and Human

Most circulating endothelial cells are apoptotic, but rare circulating endothelial colony forming cells (C-ECFCs) with proliferative and vasculogenic activity can be cultured; the origin and naive function of these C-ECFCs remains obscure. Herein, detailed lineage tracing reveals murine C-ECFCs emerge in the early postnatal period, display high vasculogenic potential, with enriched frequency of clonal proliferative cells compared to tissue-resident ECFCs, and are not committed to or derived from the bone marrow hematopoietic system but from tissue-resident ECFCs. In human subjects, C-ECFCs are present in the CD34bright cord blood mononuclear subset, possess proliferative potential and in vivo vasculogenic function in a naive or cultured state, and display a single cell transcriptome sharing some umbilical venous endothelial cell features like, higher Protein C Receptor and extracellular matrix gene expression. This study provides an advance for the field by identifying the origin, naive function, and antigens to prospectively isolate C-ECFCs for translational studies.

cell biology↗

bioRxiv2022

Cerebellum Directly Modulates the Substantia Nigra Dopaminergic Activity

Evidence of direct reciprocal connections between the cerebellum and basal ganglia has challenged the long-held notion that these structures function independently. While anatomical studies have suggested the presence of cerebellar projections to the substantia nigra pars compacta (SNc), the nature and function of these connections (Cb-SNc) is unknown. Here we show that the Cb-SNc form monosynaptic glutamatergic synapses with both dopaminergic and non-dopaminergic neurons in the SNc. Optogenetic activation Cb-SNc axons in the SNc rapidly increases SNc activity, elevates striatal dopamine levels, and increases the probability of locomotion. During ongoing behavior, Cb-SNc axons are bilaterally activated prior to ambulation and unilateral lever manipulation. The Cb-SNc axons show prominent activation to water reward, and higher activation for sweet water, suggesting that the pathway also encodes reward value. Thus, the cerebellum directly, rapidly, and effectively modulates basal ganglia dopamine levels and conveys information related to movement initiation, vigor, and possibly reward processing.

neuroscience↗

bioRxiv2022

Disruption of Smarce1, a component of the SWI/SNF chromatin remodeling complex, decreases nucleosome stability in mouse embryonic stem cells and impairs differentiation

The SWI/SNF chromatin remodeling complex consists of more than 10 component proteins that form a large protein complex of > 1 MDa. The catalytic proteins Smarca4 or Smarca2 work in concert with the component proteins to form a chromatin platform suitable for transcriptional regulation. However, the mechanism by which each component protein works synergistically with the catalytic proteins remains largely unknown. Here, we report on the function of Smarce1, a component of the SWI/SNF complex, through the phenotypic analysis of homozygous mutant embryonic stem (ES) cells. Disruption of Smarce1 induced the dissociation of other complex components from the SWI/SNF complex. Histone binding to DNA was loosened in homozygous mutant ES cells, indicating that disruption of Smarce1 decreased nucleosome stability. Sucrose gradient sedimentation analysis suggested an ectopic genomic distribution of the SWI/SNF complex, accounting for the misregulation of chromatin conformations. Unstable nucleosomes remained during ES cell differentiation, impairing the heterochromatin formation that is characteristic of the differentiation process. These results suggest that Smarce1 guides the SWI/SNF complex to the appropriate genomic regions to generate chromatin structures adequate for transcriptional regulation.

molecular biology↗

bioRxiv2022

Hippocampal CA1 represents action and reward events instantly compared to the superficial and deep layers of the lateral entorhinal cortex

The entorhinal cortex (EC) is the main interface between the hippocampus and the neocortex. The EC plays a critical role in learning and memory. We investigated the neuronal representation of behavioral events during operant learning in the hippocampal-entorhinal circuit of head-fixed rats. Both CA1 and lateral entorhinal cortex (LEC) neurons develop task-related activities after learning. Among diverse task-related activities, we compared the transient peak activities that represent action and reward and found a distinct difference in the timing of behavioral event representation between CA1 and LEC. CA1 represents action and reward events in close to real-time, whereas both the superficial and deep layers of the LEC showed delayed representation of those events. Our results suggest that subpopulations exist within which CA1 and LEC neurons process the information in a different order from the anatomically defined hippocampal-entorhinal circuit.

neuroscience↗

bioRxiv2022

Comparison of CRISPR/Cas9-mediated megabase-scale genome deletion methods in mouse embryonic stem cells

The genome contains large functional units ranging in size from hundreds of kilobases to megabases, such as gene clusters, promoter-enhancer loops, and topologically associating domains. To analyze these large functional units, the technique of deleting the entire functional unit is effective. However, deletion of such large regions is less efficient than conventional genome editing, especially in cultured cells, and a method that can ensure success is anticipated. Here, we compared methods to delete the 2.5-Mb Kruppel-associated box zinc finger protein (KRAB-ZFP) gene cluster on chromosome 4 in mouse embryonic stem cells using CRISPR/Cas9. Three methods were used: first, deletion by non-homologous end joining (NHEJ); second, homology-directed repair (HDR) using a single-stranded oligodeoxynucleotide (ssODN) with 70-bp homology arms; and third, HDR employing targeting vectors with a selectable marker and 1-kb homology arms. NHEJ-mediated deletion was achieved in 9% of the transfected cells. The deletion frequency of NHEJ and HDR was found to be comparable when the ssODN was transfected. Deletion frequency was highest when targeting vectors were introduced, with deletions occurring in 31-63% of the drug-resistant clones. Biallelic deletion was observed when targeting vectors were used. This study will serve as a benchmark for the introduction of large deletions into the genome.

genomics↗

bioRxiv2022

Endocytosis-mediated vitellogenin absorption and lipid metabolism in the hindgut-derived pseudoplacenta of the viviparous teleost Xenotoca eiseni

Certain viviparous animals possess mechanisms for mother-to-embryo nutrient transport during gestation. Xenotoca eiseni is one such viviparous teleost species in which the mother supplies proteins and other components to the offspring developing in the ovary. The embryo possesses trophotenia, a hindgut-derived pseudoplacenta to receive the maternal supplement. However, the molecular mechanisms underlying viviparous non-mammalian animals remain elusive. We conducted this study to investigate the mechanism for nutrient absorption and degradation in trophoenia of X. eiseni. The tracer assay indicated that a lipid transfer protein, vitellogenin (Vtg), was absorbed into the epithelial layer cells of trophotaenia. Vtg uptake was significantly suppressed by Pitstop-2, an inhibitor of clathrin-mediated endocytosis. Gene expression analysis indicated that the genes involved in endocytosis-mediated lipolysis and lysosomal cholesterol transport were expressed in trophotaenia. In contrast, plasma membrane transporters expressed in the intestinal tract were not functional in trophotaenia. Our results suggested that endocytosis-mediated lysosomal lipolysis is one of the mechanisms underlying maternal component metabolism. Thus, our study demonstrated how viviparous teleost species have acquired a unique developmental system that is based on the hindgut-derived pseudoplacenta.

zoology↗

bioRxiv2021

Inhibition of N-myristoyltransferase Promotes Naive Pluripotency in Mouse and Human Pluripotent Stem Cells

Naive and primed states are distinct states of pluripotency during early embryonic development that can be captured and converted to each other in vitro. To elucidate the regulatory mechanism of pluripotency, we performed a recessive genetic screen of homozygous mutant mouse embryonic stem cells (mESCs) and found that suppression of N-myristoyltransferase (Nmt) promotes naive pluripotency. Disruption of Nmt1 in mESCs conferred resistance to differentiation. Suppression of Nmt in mouse epiblast stem cells (mEpiSCs) promoted the conversion from the primed to the naive state. This effect was independent of Src, which is a major substrate of Nmt and is known to promote differentiation of mESCs. Suppression of Nmt in naive-state human induced pluripotent stem cells (hiPSCs) increased the expression of the naive-state marker. These results indicate that Nmt is a novel target for the regulation of naive pluripotency conserved between mice and humans.

developmental biology↗