The role of neurogenesis in olfaction-dependent behaviors.

Newly born neurons continuously migrate into the main and accessory olfactory bulbs and modulate the output of projection neurons. Despite some contradictory results, it is becoming clear that these newly born neurons play an important role in the response to some odorant cues. In this minireview, we discuss the recent findings surrounding the functional significance of adult neurogenesis in olfaction-dependent behaviors

Real-time imaging of bHLH transcription factors reveals their dynamic control in the multipotency and fate choice of neural stem cells

The basic-helix-loop-helix (bHLH) transcription factors Ascl1/Mash1, Hes1, and Olig2 regulate the fate choice of neurons, astrocytes, and oligodendrocytes, respectively; however, these factors are coexpressed in self-renewing multipotent neural stem cells (NSCs) even before cell fate determination. This fact raises the possibility that these fate determination factors are differentially expressed between self-renewing and differentiating NSCs with unique expression dynamics. Real-time imaging analysis utilizing fluorescent proteins is a powerful strategy for monitoring expression dynamics. Fusion with fluorescent reporters makes it possible to analyze the dynamic behavior of specific proteins in living cells. However, it is technically challenging to conduct long-term imaging of proteins, particularly those with low expression levels, because a high-sensitivity and low-noise imaging system is required, and very often bleaching of fluorescent proteins and cell toxicity by prolonged laser exposure are problematic. Furthermore, to analyze the functional roles of the dynamic expression of cellular proteins, it is essential to image reporter fusion proteins that are expressed at comparable levels to their endogenous expression. In this review, we introduce our recent reports about the dynamic control of bHLH transcription factors in multipotency and fate choice of NSCs, focusing on real-time imaging of fluorescent reporters fused with bHLH transcription factors. Our imaging results indicate that bHLH transcription factors are expressed in an oscillatory manner by NSCs, and that one of them becomes dominant during fate choice. We propose that the multipotent state of NSCs correlates with the oscillatory expression of several bHLH transcription factors, whereas the differentiated state correlates with the sustained expression of a single bHLH transcription factor

Genetic Methods to Identify and Manipulate Newly Born Neurons in the Adult Brain

Although mammalian neurogenesis is mostly completed by the perinatal period, new neurons are continuously generated in the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. Since the discovery of adult neurogenesis, many extensive studies have been performed on various aspects of adult neurogenesis, including proliferation and fate-specification of adult neural stem cells, and the migration, maturation and synaptic integration of newly born neurons. Furthermore, recent research has shed light on the intensive contribution of adult neurogenesis to olfactory-related and hippocampus-mediated brain functions. The field of adult neurogenesis progressed tremendously thanks to technical advances that facilitate the identification and selective manipulation of newly born neurons among billions of pre-existing neurons in the adult central nervous system. In this review, we introduce recent advances in the methodologies for visualizing newly generated neurons and manipulating neurogenesis in the adult brain. Particularly, the application of site-specific recombinases and Tet inducible system in combination with transgenic or gene targeting strategy is discussed in further detail

可動性超分子キラリティーの識別:有機触媒を用いたアシル化による分子変換

京都大学0048新制・課程博士博士(薬科学)甲第19660号薬科博第48号新制||薬科||6(附属図書館)32696京都大学大学院薬学研究科薬科学専攻(主査)教授 川端 猛夫, 教授 高須 清誠, 教授 竹本 佳司学位規則第4条第1項該当Doctor of Pharmaceutical SciencesKyoto UniversityDFA

Cooperative functions of Hes/Hey genes in auditory hair cell and supporting cell development.

Notch-mediated lateral inhibition has been reported to regulate auditory hair cell and supporting cell development from common precursors. While the Notch effector genes Hes1, Hes5 and Hey1 are expressed in the developing cochlea, inactivation of either of them causes only mild abnormality, suggesting their functional redundancy. To explore the roles of Hes/Hey genes in cochlear development, we examined compound heterozygous or homozygous mutant mice that lacked Hes1, Hes5 and Hey1 alleles. We found that a reduction in Hes/Hey gene dosage led to graded increase of hair cell formation. However, if at least one allele of Hes1, Hes5 or Hey1 was intact, excessive hair cells were accompanied by overproduction of supporting cells, suggesting that the hair cell increase does not occur at the expense of supporting cells, and that each Hes/Hey gene functions to induce supporting cells. By contrast, when all alleles of Hes1, Hes5 and Hey1 were inactivated, the number of hair cells increased more drastically, whereas that of supporting cells was unchanged compared with control, suggesting that supporting cell formation was balanced by their overproduction and fate conversion into hair cells. The increase of the cell numbers seemed to occur after the prosensory domain formation in the mutants because the proliferation state and the size of the prosensory domain were not affected. Thus, Hes1, Hes5 and Hey1 cooperatively inhibit hair cell formation, and one allele of Hes1, Hes5 or Hey1 is sufficient for supporting cell production probably by lateral inhibition in the sensory epithelium. Strikingly, Hes/Hey mutations lead to disorganized cell alignment and polarity and to hearing loss despite hair cell overproduction. These results suggest that Hes/Hey gene dosage is essential not only for generation of appropriate numbers of hair cells and supporting cells by controlling cell proliferation and lateral inhibition but also for the hearing ability by regulating the cell alignment and polarity

Optimization of Light-Inducible Gal4/UAS Gene Expression System in Mammalian Cells

遺伝子発現を光で制御できる新技術を開発 --発生・幹細胞研究への貢献に期待--. 京都大学プレスリリース. 2020-08-28.Light-inducible gene expression systems represent powerful methods for studying the functional roles of dynamic gene expression. Here, we developed an optimized light-inducible Gal4/UAS gene expression system for mammalian cells. We designed photoactivatable (PA)-Gal4 transcriptional activators based on the concept of split transcription factors, in which light-dependent interactions between Cry2-CIB1 PA-protein interaction modules can reconstitute a split Gal4 DNA-binding domain and p65 transcription activation domain. We developed a set of PA-Gal4 transcriptional activators (PA-Gal4cc), which differ in terms of induced gene expression levels following pulsed or prolonged light exposure, and which have different activation/deactivation kinetics. These systems offer optogenetic tools for the precise manipulation of gene expression at fine spatiotemporal resolution in mammalian cells

Three-dimensional live imaging of Atoh1 reveals the dynamics of hair cell induction and organization in the developing cochlea

During cochlear development, hair cells (HCs) and supporting cells differentiate in the prosensory domain to form the organ of Corti, but how one row of inner HCs (IHCs) and three rows of outer HCs (OHCs) are organized is not well understood. Here, we investigated the process of HC induction by monitoring Atoh1 expression in cochlear explants of Atoh1-EGFP knock-in mouse embryos and showed that only the cells that express Atoh1 over a certain threshold are selected for HC fate determination. HC induction initially occurs at the medial edge of the prosensory domain to form IHCs and subsequently at the lateral edge to form OHCs, while Hedgehog signaling maintains a space between IHCs and OHCs, leading to formation of the tunnel of Corti. These results reveal dynamic Atoh1 expression in HC fate control and suggest that multi-directional signals regulate OHC induction, thereby organizing the prototype of the organ of Corti