Daichi Kawaguchi

Non-cell-autonomous action of STAT3 in maintenance of neural precursor cells in the mouse neocortex.

The transcription factor STAT3 promotes astrocytic differentiation of neural precursor cells (NPCs) during postnatal development of the mouse neocortex, but little has been known of the possible role of STAT3 in the embryonic neocortex. We now show that STAT3 is expressed in NPCs of the mouse embryonic neocortex and that the JAK-STAT3 signaling pathway plays an essential role in the maintenance of NPCs by fibroblast growth factor 2. Conditional deletion of the STAT3 gene in NPCs reduced their capacity to form neurospheres in vitro, as well as promoted neuronal differentiation both in vitro and in vivo. Furthermore, STAT3 was found to maintain NPCs in the undifferentiated state in a non-cell-autonomous manner. STAT3-dependent expression of the Notch ligand Delta-like1 (DLL1) appears to account for the non-cell-autonomous effect of STAT3 on NPC maintenance, as knockdown of DLL1 by RNA interference or inhibition of Notch activation with a γ-secretase inhibitor abrogated the enhancement of neurosphere formation by STAT3. Our results reveal a previously unrecognized mechanism of interaction between the JAK-STAT3 and DLL1-Notch signaling pathways, as well as a pivotal role for this interaction in maintenance of NPCs during early neocortical development.

Mechanisms that regulate the number of neurons during mouse neocortical development

Cortical development progresses through an early phase of progenitor expansion, a middle phase of neurogenesis, and a final phase of gliogenesis. During the middle phase, the neurogenic phase, the neocortical primordium balances the production of neurons against the maintenance of neural precursor cells (NPCs). The final number of neurons is determined by the duration of the neurogenic phase, the rate of NPC division, and the mode of NPC division, that is, whether a division gives rise to two NPCs, one NPC and one cell committed to the neuronal lineage, or two committed cells. We discuss here recent advances in understanding these key aspects that are fundamental for normal brain development.

Selection of differentiating cells by different levels of delta-like 1 among neural precursor cells in the developing mouse telencephalon

During the neurogenic phase of mammalian brain development, only a subpopulation of neural precursor cells (NPCs) differentiates into neurons. The mechanisms underlying this selection remain unclear. Here we provide evidence that the Notch-Delta pathway plays an important role in this selection in the developing mouse telencephalon. We found that the expression patterns of the Notch ligand delta-like 1 (Dll1) and of the active form of Notch1 were mutually exclusive and segregated into distinct NPC subpopulations in the ventricular zone of the telencephalon. When Dll1 was overexpressed in a small, but not a large, proportion of NPCs, these cells underwent neuronal differentiation in vitro and in vivo. This Dll1-induced neuronal differentiation did not occur when cells were plated at lower densities in an in vitro culture. Importantly, conditional deletion of the Dll1 gene in a small proportion of NPCs reduced neurogenesis in vivo, whereas deletion in a large proportion promoted premature neurogenesis. These results support the notion that different levels of Dll1 expression determine the fate of NPCs through cell-cell interactions, most likely through the Notch-Delta lateral inhibitory signaling pathway, thus contributing to the selection of differentiating cells.

In Vivo Expression of a Light-Activatable Potassium Channel Using Unnatural Amino Acids

Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been used to manipulate neuronal activity, a method for optical control of neuronal proteins using unnatural amino acids (Uaa) in vivo is lacking. Here, we describe the genetic incorporation of a photoreactive Uaa into the pore of an inwardly rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel nonconducting, and, on brief light illumination, was released to permit outward K(+) current. Expression of this photoinducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expanded the genetic code of mammals to express PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. These principles could be generally expanded to other proteins expressed in the brain to enable optical regulation.

Dll1 maintains quiescence of adult neural stem cells and segregates asymmetrically during mitosis

Stem cells often divide asymmetrically to produce one stem cell and one differentiating cell, thus maintaining the stem cell pool. Although neural stem cells (NSCs) in the adult mouse subventricular zone have been suggested to divide asymmetrically, intrinsic cell fate determinants for asymmetric NSC division are largely unknown. Stem cell niches are important for stem cell maintenance, but the niche for the maintenance of adult quiescent NSCs has remained obscure. Here we show that the Notch ligand Delta-like 1 (Dll1) is required to maintain quiescent NSCs in the adult mouse subventricular zone. Dll1 protein is induced in activated NSCs and segregates to one daughter cell during mitosis. Dll1-expressing cells reside in close proximity to quiescent NSCs, suggesting a feedback signal for NSC maintenance by their sister cells and progeny. Our data suggest a model in which NSCs produce their own niche cells for their maintenance through asymmetric Dll1 inheritance at mitosis.

Vulnerability to cavitation differs between current‐year and older xylem: non‐destructive observation with a compact magnetic resonance imaging system of two deciduous diffuse‐porous species

Development of xylem embolism during water stress in two diffuse-porous hardwoods, Katsura (Cercidiphyllum japonicum) and Japanese white birch (Betula platyphylla var. japonica), was observed non-destructively under a compact magnetic resonance imaging (MRI) system in addition to conventional quantitation of hydraulic vulnerability to cavitation from excised stem segments. Distribution of white and dark areas in MR images corresponded well to the distribution of water-filled/embolized vessels observed by cryo-scanning electron microscopy in both species. Water-filled vessels were observed in MR images as white areas in Katsura and as white dots in Japanese white birch, respectively, and embolisms could be detected as a change to dark areas. The increase in the relative embolized area (REA: %) in the cross-sectional area of total xylem during water stress, which was estimated from the binarized MR images, was consistent with the hydraulic vulnerability curves of these species. From the non-destructive MRI observations, cavitation induced by water stress was shown to develop earlier in 1- or 2-year-old xylem than in the current-year xylem in both species; that is, the vulnerability to cavitation differs between vessels in the current-year xylem and those in older annual rings.

Area-Specific Regulation of Quiescent Neural Stem Cells by Notch3 in the Adult Mouse Subependymal Zone

In the adult mammalian brain, neural stem cells (NSCs) generate new neurons throughout the mammals lifetime. The balance between quiescence and active cell division among NSCs is crucial in producing appropriate numbers of neurons while maintaining the stem cell pool for a long period. The Notch signaling pathway plays a central role in both maintaining quiescent NSCs (qNSCs) and promoting cell division of active NSCs (aNSCs), although no one knows how this pathway regulates these apparently opposite functions. Notch1 has been shown to promote proliferation of aNSCs without affecting qNSCs in the adult mouse subependymal zone (SEZ). In this study, we found that Notch3 is expressed to a higher extent in qNSCs than in aNSCs while Notch1 is preferentially expressed in aNSCs and transit-amplifying progenitors in the adult mouse SEZ. Furthermore, Notch3 is selectively expressed in the lateral and ventral walls of the SEZ. Knockdown of Notch3 in the lateral wall of the adult SEZ increased the division of NSCs. Moreover, deletion of the Notch3 gene resulted in significant reduction of qNSCs specifically in the lateral and ventral walls, compared with the medial and dorsal walls, of the lateral ventricles. Notch3 deletion also reduced the number of qNSCs activated after antimitotic cytosine β-D-arabinofuranoside (Ara-C) treatment. Importantly, Notch3 deletion preferentially reduced specific subtypes of newborn neurons in the olfactory bulb derived from the lateral walls of the SEZ. These results indicate that Notch isoforms differentially control the quiescent and proliferative steps of adult SEZ NSCs in a domain-specific manner. SIGNIFICANCE STATEMENT In the adult mammalian brain, the subependymal zone (SEZ) of the lateral ventricles is the largest neurogenic niche, where neural stem cells (NSCs) generate neurons. In this study, we found that Notch3 plays an important role in the maintenance of quiescent NSCs (qNSCs), while Notch1 has been reported to act as a regulator of actively cycling NSCs. Furthermore, we found that Notch3 is specifically expressed in qNSCs located in the lateral and ventral walls of the lateral ventricles and regulates neuronal production of NSCs in a region-specific manner. Our results indicate that Notch3, by maintaining the quiescence of a subpopulation of NSCs, confers a region-specific heterogeneity among NSCs in the adult SEZ.

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K(+) current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Layer-specific morphological and molecular differences in neocortical astrocytes and their dependence on neuronal layers

Non-pial neocortical astrocytes have historically been thought to comprise largely a nondiverse population of protoplasmic astrocytes. Here we show that astrocytes of the mouse somatosensory cortex manifest layer-specific morphological and molecular differences. Two- and three-dimensional observations revealed that astrocytes in the different layers possess distinct morphologies as reflected by differences in cell orientation, territorial volume, and arborization. The extent of ensheathment of synaptic clefts by astrocytes in layer II/III was greater than that by those in layer VI. Moreover, differences in gene expression were observed between upper-layer and deep-layer astrocytes. Importantly, layer-specific differences in astrocyte properties were abrogated in reeler and Dab1 conditional knockout mice, in which neuronal layers are disturbed, suggesting that neuronal layers are a prerequisite for the observed morphological and molecular differences of neocortical astrocytes. This study thus demonstrates the existence of layer-specific interactions between neurons and astrocytes, which may underlie their layer-specific functions.Several studies have suggested that astrocytes in the neocortex are more diverse than previously thought. Here, the authors describe layer-specific differences in morphology and molecular characteristics of astrocytes that depend on the neurons within those layers.

Genetically Encoding Unnatural Amino Acids in Neurons In Vitro and in the Embryonic Mouse Brain for Optical Control of Neuronal Proteins

Deciphering neuronal networks governing specific brain functions is a longstanding mission in neuroscience, yet global manipulation of protein functions pharmacologically or genetically lacks sufficient specificity to reveal a neuronal proteins function in a particular neuron or a circuitry. Photostimulation presents a great venue for researchers to control neuronal proteins with high temporal and spatial resolution. Recently, an approach to optically control the function of a neuronal protein directly in neurons has been demonstrated using genetically encoded light-sensitive Unnatural amino acids (Uaas). Here, we describe procedures for genetically incorporating Uaas into target neuronal proteins in neurons in vitro and in embryonic mouse brain. As an example, a photocaged Uaa was incorporated into an inwardly rectifying potassium channel Kir2.1 to render Kir2.1 photo-activatable. This method has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. Uaas with other properties can be similarly incorporated into neuronal proteins in neurons for various applications.