Tokuichi Iguchi

Smoothened controls cyclinD2 expression and regulates the generation of intermediate progenitors in the developing cortex

Translocation of the Smoothened to the cell membrane is critical for sonic hedgehog activity. However, the biological importance of Smoothened itself has not been fully studied. To address this issue, we disabled Smoothened specifically in the dorsal telencephalon. Birth-date analysis and layer marker expression patterns revealed the slightly impaired development of the superficial layer neurons in the embryos of Emx1-Cre; Smoothened(fl/-) conditional knockout mice. Further analysis of the mutant embryos revealed a decrease in the number of intermediate progenitor cells. In the knockout mice, the expression of cyclin D2, but not cyclin D1 or cyclin E, was reduced in the dorsal telencephalon. In addition, the projections of dopaminergic neurons were affected during development, and the number of activated astrocytes was increased in the neocortex of the mutant mice. Our data suggest that Smoothened signaling, acting through cyclin D2, is critical for the proper development and maturation of the neocortex.

DBZ Regulates Cortical Cell Positioning and Neurite Development by Sustaining the Anterograde Transport of Lis1 and DISC1 through Control of Ndel1 Dual-Phosphorylation

Cell positioning and neuronal network formation are crucial for proper brain function. Disrupted-in-Schizophrenia 1 (DISC1) is anterogradely transported to the neurite tips, together with Lis1, and functions in neurite extension via suppression of GSK3β activity. Then, transported Lis1 is retrogradely transported and functions in cell migration. Here, we show that DISC1-binding zinc finger protein (DBZ), together with DISC1, regulates mouse cortical cell positioning and neurite development in vivo. DBZ hindered Ndel1 phosphorylation at threonine 219 and serine 251. DBZ depletion or expression of a double-phosphorylated mimetic form of Ndel1 impaired the transport of Lis1 and DISC1 to the neurite tips and hampered microtubule elongation. Moreover, application of DISC1 or a GSK3β inhibitor rescued the impairments caused by DBZ insufficiency or double-phosphorylated Ndel1 expression. We concluded that DBZ controls cell positioning and neurite development by interfering with Ndel1 from disproportionate phosphorylation, which is critical for appropriate anterograde transport of the DISC1-complex.

A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.

Background Gene knockdown analyses using the in utero electroporation method have helped reveal functional aspects of genes of interest in cortical development. However, the application of this method to analyses in later stages of brain development or in the adult brain is still difficult because the amount of injected plasmids in a cell decreases along with development due to dilution by cell proliferation and the degradation of the plasmids. Furthermore, it is difficult to exclude the influence of earlier knockdown effects. Methodology/Principal Findings We developed a tightly controlled conditional knockdown system using a newly constructed vector, pT2K-TBI-shRNAmir, based on a Tol2 transposon-mediated gene transfer methodology with the tetracycline-inducible gene expression technique, which allows us to maintain a transgene for a long period of time and induce the knockdown of the gene of interest. We showed that expression of the endogenous amyloid precursor protein (APP) was sharply decreased by our inducible, stably integrated knockdown system in PC12 cells. Moreover, we induced an acute insufficiency of Dab1 with our system and observed that radial migration was impaired in the developing cerebral cortex. Such inhibitory effects on radial migration were not observed without induction, indicating that our system tightly controlled the knockdown, without any expression leakage in vivo. Conclusions/Significance Our system enables us to investigate the brain at any of the later stages of development or in the adult by utilizing a knockdown technique with the aid of the in utero electroporation gene transfer methodology. Furthermore, we can perform knockdown analyses free from the influence of undesired earlier knockdown effects.

Filamin A-interacting protein (FILIP) is a region-specific modulator of myosin 2b and controls spine morphology and NMDA receptor accumulation

Learning and memory depend on morphological and functional changes to neural spines. Non-muscle myosin 2b regulates actin dynamics downstream of long-term potentiation induction. However, the mechanism by which myosin 2b is regulated in the spine has not been fully elucidated. Here, we show that filamin A-interacting protein (FILIP) is involved in the control of neural spine morphology and is limitedly expressed in the brain. FILIP bound near the ATPase domain of non-muscle myosin heavy chain IIb, an essential component of myosin 2b, and modified the function of myosin 2b by interfering with its actin-binding activity. In addition, FILIP altered the subcellular distribution of myosin 2b in spines. Moreover, subunits of the NMDA receptor were differently distributed in FILIP-expressing neurons, and excitation propagation was altered in FILIP-knockout mice. These results indicate that FILIP is a novel, region-specific modulator of myosin 2b.

Developmental downregulation of LIS1 expression limits axonal extension and allows axon pruning

ABSTRACT The robust axonal growth and regenerative capacities of young neurons decrease substantially with age. This developmental downregulation of axonal growth may facilitate axonal pruning and neural circuit formation but limits functional recovery following nerve damage. While external factors influencing axonal growth have been extensively investigated, relatively little is known about the intrinsic molecular changes underlying the age-dependent reduction in regeneration capacity. We report that developmental downregulation of LIS1 is responsible for the decreased axonal extension capacity of mature dorsal root ganglion (DRG) neurons. In contrast, exogenous LIS1 expression or endogenous LIS1 augmentation by calpain inhibition restored axonal extension capacity in mature DRG neurons and facilitated regeneration of the damaged sciatic nerve. The insulator protein CTCF suppressed LIS1 expression in mature DRG neurons, and this reduction resulted in excessive accumulation of phosphoactivated GSK-3β at the axon tip, causing failure of the axonal extension. Conversely, sustained LIS1 expression inhibited developmental axon pruning in the mammillary body. Thus, LIS1 regulation may coordinate the balance between axonal growth and pruning during maturation of neuronal circuits. Summary: Developmental downregulation of LIS1 coordinates the balance between axonal elongation and pruning, which is essential for proper neuronal circuit formation but limits nerve regeneration.

Regulation of dendritic spine morphology by FILIP

ingly, typical ritual behaviors for courtship were normal in Dark-fly, but pairs of Dark-fly male and female copulated quickly even in dark conditions. Therefore, we suggest that multimodal sensory signals regulating courtship behaviors might be stronger in Dark-fly. The composition of Dark-fly’s cuticle hydrocarbons, known as sexual pheromone, was characteristic, and Dark-fly exhibited preference for kin’s chemical signals. On the other hand, courtship song played by Dark-fly male was mostly identical to that of the wild-type male, but Dark-fly seems to be sensitive to a smaller volume of song. Our results suggest that olfactory and auditory signals for courtship behaviors might have evolved under the vision-less environment. To precisely evaluate behaviors excited by sensory signals, we constructed automatic quantitative systems for analyzing fly’s behaviors. We will present the results of analysis of olfactoryand auditory-based behaviors and will discuss Dark-fly’s adaptive behaviors. Research fund: Kyoto University Global COE Program (biodiversity), Leave a nest Co., Leaveanest Grant.

Molecular analysis of amyloid beta precursor protein on the neuronal network formation using in utero electroporation gene transfer combined with inducible gene expression system

p0 progenitor cells and V0 neurons in zebrafish. We generated transgenic lines that express fluorescent proteins in dbx1-expressing cells and/or their progeny. Using these lines, we first examined neurotransmitter properties of V0 neurons. The results indicated that zebrafish V0 neurons consisted of both excitatory and inhibitory neurons, consistent with the situation in V0 neurons in mice. In addition, morphologixal analysis showed excitatory neurons were heterogeneous. Based on the trajectories of axons on the contralateral side of the body, V0 excitatory neurons could be subdivided into three groups: ascending, bifurcating and descending neurons. Next, we examined whether lineages of V0 neurons had any relationship with types of V0 neurons. We performed single-cell labeling of individual p0 progenitors, and followed the fate of their progenies by time-lapse observations. Our results suggested that excitatory and inhibitory neurons were produced from different progenitor cells. Moreover, we found some progenitors that only produced ascending excitatory neurons. These results suggest that there is heterogeneity among p0 progenitor, which may underlie the diversity of V0 neurons.