Biology subjects

Sasai, N.

Publications and source records attributed to Sasai, N..

8 recordsLinked to original sources

bioRxiv2024

Stepwise Bayesian Machine Learning Uncovers a Novel Gene Regulatory Network Component in Neural Tube Development

Recent advancements in machine learning-based data processing techniques have facilitated the inference of gene regulatory interactions and the identification of key genes from multidimensional gene expression data. In this study, we applied a stepwise Bayesian framework to uncover a novel regulatory component involved in differentiation of specific neural and neuronal cells. We treated naive neural precursor cells with Sonic Hedgehog (Shh) at various concentrations and time points, generating comprehensive whole-genome sequencing data that captured dynamic gene expression profiles during differentiation. The genes were categorized into 224 subsets based on their expression profiles, and the relationships between these subsets were extrapolated. To accurately predict gene regulation among subsets, known networks were used as a core model and subsets to be added were tested stepwise. This approach led to the identification of a novel component involved in neural tube patterning within gene regulatory networks (GRNs), which was experimentally validated. Our study highlights the effectiveness of in silico modeling for extrapolating GRNs during neural development.

systems biology↗

bioRxiv2022

Hedgehog Activity Gradient in Combination with Transcription Network Confers Multiple Hypothalamic Identities

During development, the hypothalamus emerges from the ventral diencephalon of the neural tube and is regionalised into several distinct functional domains. Each domain is characterised by different combinations of transcription factors, the expression of which is regulated by signalling molecules and downstream transcriptional networks. Transcription factors, including Nkx2.1, Nkx2.2, Pax6 and Rx, are expressed in the presumptive hypothalamus and its surrounding regions from an early developmental stage and play critical roles in the development of these areas. However, the regulation of transcription factor expression and the details of the transcriptional network among them have not been fully elucidated. As early hypothalamus development takes place in the ventral region of the forebrain where Sonic Hedgehog (Shh) is expressed, we focused on the relationship between Shh and its downstream transcription factors and investigated the transcriptional regulation along the dorsal-ventral axis. Using a reporter mouse line, in vitro neural differentiation of mouse embryonic stem cells and gene overexpression in chick embryos, we found that Pax6, Nkx2.1 and Nkx2.2 are regulated epistatically by different Shh signal intensities. Nkx2.1 and Nkx2.2 mutually repress each other; however, they induce each other in a non-cell-autonomous manner. Moreover, Rx resides upstream of all these transcription factors and determines the location of the hypothalamic region along the dorsal-ventral and anterior-posterior regulations. Finally, we found that the Shh signal demarcates the diencephalic region from the retinal area. Our findings suggest that Shh signalling, and its downstream transcription network, are required for hypothalamic regionalisation and establishment of diencephalic cell fate.

developmental biology↗

bioRxiv2021

Heterozygous loss of ZBTB38 leads to early embryonic lethality in mice via suppressing Nanog and Sox2

Mammalian DNA methylation is an epigenetic modification which is involved in various biological processes, including gene expression regulation. In mice, methyltransferases are responsible for DNA methylation, which are critical for early embryogenesis. However, the significance of methyl-CpG binding proteins (MBPs) that bind methylated CpG remains largely unknown. We previously demonstrated that ZBTB38/CIBZ-a zinc finger type of MBP-is required for ES cell proliferation by positively regulating Nanog expression. However, the physiological function of ZBTB38 remains unclear. In this study, we generated conditional ZBTB38 knockout mice using Cre-loxP technology. Unexpectedly, our results showed that germline loss of the ZBTB38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. We found that heterozygous loss of ZBTB38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Despite this lethal phenotype, ZBTB38 is dispensable for ES cell establishment and identity. Together, these findings indicate that ZBTB38 is essential for early embryonic development, providing new insights into the roles of MBP in implantation.

developmental biology↗

bioRxiv2020

Light-dependent induction of Edn2 expression and attenuation of retinal pathology by endothelin receptor antagonists in Prominin-1- deficient mice

Retinitis pigmentosa (RP) and macular dystrophy (MD) are prevalent retinal degenerative diseases associated with gradual photoreceptor death. These diseases are often caused by genetic mutations that result in degeneration of the retina postnatally after it has fully developed. The Prominin-1 gene (Prom1) is a causative gene for RP and MD, and Prom1- knockout (KO) mice recapitulate key features of these diseases including light-dependent retinal degeneration and stenosis of retinal blood vessels. The mechanisms underlying progression of such degeneration have remained unknown, however. We here analysed early events associated with retinal degeneration in Prom1-KO mice. We found that photoreceptor cell death and glial cell activation occur between 2 and 3 weeks after birth. High-throughput analysis revealed that expression of the endothelin-2 gene (Edn2) was markedly up-regulated in the Prom1-deficient retina during this period. Expression of Edn2 was also induced by light stimulation in Prom1-KO mice that had been reared in the dark. Finally, treatment with endothelin receptor antagonists attenuated photoreceptor cell death, gliosis, and retinal vessel stenosis in Prom1-KO mice. Our findings suggest that inhibitors of endothelin signalling may delay the progression of RP and MD and therefore warrant further study as potential therapeutic agents for these diseases.

neuroscience↗

bioRxiv2019

Evaluation of the impact of seasonal variations in photoperiod on the hepatic metabolism of medaka (Oryzias latipes)

Organisms living in temperate regions are sensitive to seasonal variations in the environment; they are known to accumulate energy as fat in their livers during the winter when days are shorter, temperatures are lower, and food is scarce. However, the impact of variations in photoperiod alone on hepatic lipid metabolism has not been well-studied. Therefore, in this study, we analyzed lipid metabolism in the liver of medaka, Oryzias latipes, while varying the length of days at constant temperature. Larger amounts of fatty acids accumulated in the liver after 14 days under short-day conditions than under long-day conditions. Metabolome analysis showed no accumulation of the long-chain unsaturated fatty acids required at low temperatures, but showed a significant accumulation of long-chain saturated fatty acids. Short-day conditions induced decreased levels of succinate, fumarate, and malate in the tricarboxylic acid cycle, decreased expression of PPAR, and decreased accumulation of acylcarnitine, which suggested inhibition of lipolysis. In addition, when a high-fat diet was administered to transparent medaka under short-day conditions, larger amounts of fat accumulated and medaka with fatty liver were efficiently produced. Detailed analysis of the relationship between seasonal changes and hepatic steatosis will be important in the future as hepatic diseases become more prevalent in modern society; the findings obtained in our study will be useful for research studies pertaining to the relationship between photoperiod and disorders such as hepatic steatosis and non-alcoholic steatohepatitis.

physiology↗

bioRxiv2019

RNF152 Negatively Regulates mTOR Signalling and Blocks Cell Proliferation in the Floor Plate

The neural tube is composed of a number of neural progenitors and postmitotic neurons distributed in a quantitatively and spatially precise manner. The floor plate, located in the ventral-most region of the neural tube, has a lot of unique characteristics, including a low cell proliferation rate. The mechanisms by which this region-specific proliferation rate is regulated remain elusive.\n\nHere we show that the activity of the mTOR signalling pathway, which regulates the proliferation of the neural progenitor cells, is significantly lower in the floor plate than in other domains of the embryonic neural tube. We identified the forkhead-type transcription factor FoxA2 as a negative regulator of mTOR signalling in the floor plate. We demonstrate that FoxA2 transcriptionally induces the expression of the E3 ubiquitin ligase RNF152, which together with its substrate RagA, regulates cell proliferation via the mTOR pathway. Silencing of RNF152 led to the aberrant upregulation of the mTOR signal and aberrant cell division in the floor plate. Taken together, the present findings suggest that floor plate cell number is controlled by the negative regulation of mTOR signalling through the activity of FoxA2 and its downstream effector RNF152.

developmental biology↗

bioRxiv2019

Membrane Protrusion Formation Mediated by Rho/ROCK Signalling and Modulation of Chloride Flux

Membrane protrusion is an important structural property associated with various cellular functions. The pentaspan membrane protein Prominin-1 (Prom1/CD133) is known to be localised to the protrusions and plays a pivotal role in migration and the determination of cellular morphology; however, the underlying mechanisms have been elusive. Here, we demonstrate that Prom1 is sufficient to trigger membrane protrusion formation. Overexpression of Prom1 in the RPE-1 cells triggers multiple long cholesterol-enriched protrusions, independently from actin and tubulin polymerisation. For this protrusion formation, the five amino acid stretch located at the carboxyl cytosolic region is essential. Moreover, the small GTPase Rho and its effector kinase ROCK are essential for this protrusion formation, and the intersection point of active Rho and Prom1 is where the protrusion formation initiates. Importantly, Prom1 causes the chloride ion efflux induced by calcium ion uptake, and protrusion formation is closely associated with the chloride efflux activity. Altogether, this study has elucidated that Prom1 plays critical roles for the membrane morphology and chloride ion flux.

cell biology↗

bioRxiv2018

GPR17 is an Essential Component of the Negative Feedback Loop of the Sonic Hedgehog Signalling Pathway in Neural Tube Development

Dorsal-ventral pattern formation of the neural tube is regulated by temporal and spatial activities of extracellular signalling molecules. Sonic hedgehog (Shh) assigns ventral neural subtypes via activation of the Gli transcription factors. Shh activity changes dynamically during neural differentiation, but the mechanisms responsible for regulating this dynamicity are not fully understood. Here we show that the P2Y-type G-protein coupled receptor GPR17 is involved in temporal regulation of the Shh signal. GPR17 was expressed in the ventral progenitor regions of the neural tube and acted as a negative regulator of the Shh signal in chick embryos. While the activation of the GPR17-related signal inhibited ventral identity, perturbation of GPR17 expression led to aberrant expansion of ventral neural domains. Notably, perturbation of GPR17 expression partially inhibited the negative feedback of Gli activity. Moreover, GPR17 increased cAMP activity, suggesting that it exerts its function by inhibiting the processing of Gli3 protein. GPR17 also negatively regulated Shh signalling in neural cells differentiated from mouse embryonic stem cells, suggesting that GPR17 function is conserved among different organisms. Our results demonstrate that GPR17 is a novel negative regulator of Shh signalling in a wide range of cellular contexts.\n\nAuthor SummaryDuring neural development, determination of cell fate and the progress of differentiation are regulated by extracellular signal molecules, including Sonic Hedgehog (Shh). Shh forms a gradient within the embryonic organ of the central nervous system, or the neural tube, and a variety of cells are produced corresponding to the concentration. While the signal concentration is critical for cell fate, recent studies have revealed that the intracellular signal intensity does not always correspond to the Shh concentration. Rather, the intracellular signal intensity changes over time. Importantly, the signal intensity peaks and gradually decreases thereafter, and the half-life of the Shh signal contributes to the cell fate determination. However, the mechanisms for this temporal change are not fully understood.\n\nBy using chick embryos and mouse embryonic stem cells as model systems, we demonstrate that the G-protein coupled receptor, GPR17, is an essential regulator for the negative feedback of the Shh signal during neural development. While GPR17 gene expression is induced by the Shh signal, GPR17 perturbs the Shh signalling pathway. This negative function of GPR17 on the Shh signal is conserved among different vertebrate species. The collective data demonstrate that GPR17 is a negative regulator for the Shh signalling pathway in a wide range of the cellular contexts.

developmental biology↗