Toshiyuki Ohtsuka

Continuous neurogenesis in the adult forebrain is required for innate olfactory responses

Although the functional significance of adult neurogenesis in hippocampal-dependent learning and memory has been well documented, the role of such neurogenesis in olfactory activity is rather obscure. To understand the significance of adult neurogenesis in olfactory functions, we genetically ablated newly born neurons by using tamoxifen-treated Nestin-CreERT2;neuron-specific enolase-diphtheria toxin fragment A (NSE-DTA) mice. In these mice, tamoxifen-inducible Cre recombinase allows the NSE (Eno2) gene to drive DTA expression in differentiating neurons, leading to the efficient ablation of newly born neurons in the forebrain. These mutant mice were capable of discriminating odors as competently as control mice. Strikingly, although control and mutant mice frequently showed freezing behaviors to a fox scent, a predator odor, mutant mice approached this odor when they were conditioned to associate the odor with a reward, whereas control mice did not approach the odor. Furthermore, although mutant males and females showed normal social recognition behaviors to other mice of a different sex, mutant males displayed deficits in male–male aggression and male sexual behaviors toward females, whereas mutant females displayed deficits in fertility and nurturing, indicating that sex-specific activities, which are known to depend on olfaction, are impaired. These results suggest that continuous neurogenesis is required for predator avoidance and sex-specific responses that are olfaction dependent and innately programmed.

Accelerating the Tempo of the Segmentation Clock by Reducing the Number of Introns in the Hes7 Gene

Periodic somite segmentation is controlled by the cyclic gene Hes7, whose oscillatory expression depends upon negative feedback with a delayed timing. The mechanism that regulates the pace of segmentation remains to be determined, but mathematical modeling has predicted that negative feedback with shorter delays would give rise to dampened but more rapid oscillations. Here, we show that reducing the number of introns within the Hes7 gene shortens the delay and results in a more rapid tempo of both Hes7 oscillation and somite segmentation, increasing the number of somites and vertebrae in the cervical and upper thoracic region. These results suggest that the number of introns is important for the appropriate tempo of oscillatory expression and that Hes7 is a key regulator of the pace of the segmentation clock.

Dynamic expression of notch signaling genes in neural stem/progenitor cells.

In neural stem/progenitor cells, expression of the Notch effector Hes1, a transcriptional repressor, oscillates with a period of 2–3u2009h by negative feedback, and Hes1 oscillations induce the oscillatory expression of the proneural gene Neurogenin2 (Ngn2) and the Notch ligand gene Delta-like1 (Dll1). Dll1 oscillation leads to the mutual activation of Notch signaling between neighboring cells, thereby maintaining a group of cells in the undifferentiated state. Not all cells express Hes1 in an oscillatory manner: cells in boundary regions such as the isthmus express Hes1 in a sustained manner, and these cells are rather dormant with regard to proliferation and differentiation. Thus, Hes1 allows cell proliferation and differentiation when its expression oscillates but induces dormancy when its expression is sustained. After Hes1 expression is repressed, Ngn2 is expressed in a sustained manner, promoting neuronal differentiation. Thus, Ngn2 leads to the maintenance of neural stem/progenitor cells by inducing Dll1 oscillation when its expression oscillates but to neuronal differentiation when its expression is sustained. These results indicate that the different dynamics of Hes1 and Ngn2 lead to different outcomes.

The role of Hes genes in intestinal development, homeostasis and tumor formation.

Notch signaling regulates intestinal development, homeostasis and tumorigenesis, but its precise downstream mechanism remains largely unknown. Here we found that inactivation of the Notch effectors Hes1, Hes3 and Hes5, but not Hes1 alone, led to reduced cell proliferation, increased secretory cell formation and altered intestinal structures in adult mice. However, in Apc mutation-induced intestinal tumors, inactivation of Hes1 alone was sufficient for reducing tumor cell proliferation and inducing differentiation of tumor cells into all types of intestinal epithelial cells, but without affecting the homeostasis of normal crypts owing to genetic redundancy. These results indicated that Hes genes cooperatively regulate intestinal development and homeostasis and raised the possibility that Hes1 is a promising target to induce the differentiation of tumor cells.

MicroRNA9 regulates neural stem cell differentiation by controlling Hes1 expression dynamics in the developing brain.

Earlier studies show that Hes1 expression is oscillatory in neural stem cells but sustained and high in the roof plate and the floor plate, and that such different dynamics of Hes1 expression (oscillatory versus sustained) regulate different proliferation and differentiation characteristics of these cells (active in neural stem cells but rather dormant in roof/floor plate cells). The mechanism of how different dynamics of Hes1 expression is controlled remains to be determined. Here, we found that the seed sequence of microRNA‐9 (miR‐9) is complementary to the 3′‐UTR sequence of Hes1 mRNA. MiR‐9 is highly expressed in the ventricular zone of the developing brain, which contains neural stem cells, but it is not expressed in the roof plate or the floor plate. Over‐expression of miR‐9 negatively regulates the Hes1 protein expression by interacting with the 3′‐UTR of Hes1 mRNA, thereby inducing cell cycle exit and neuronal differentiation. Conversely, knockdown of miR‐9 inhibits neuronal differentiation. Furthermore, knockdown of miR‐9 inhibits the oscillatory expression of Hes1 mRNA in neural stem cells. These results indicate that miR‐9 regulates the proliferation and differentiation of neural stem cells by controlling the dynamics of Hes1 expression in the developing brain.

Genetic visualization of notch signaling in mammalian neurogenesis

Notch signaling plays crucial roles in fate determination and the differentiation of neural stem cells in embryonic and adult brains. It is now clear that the notch pathway is under more complex and dynamic regulation than previously thought. To understand the functional details of notch signaling more precisely, it is important to reveal when, where, and how notch signaling is dynamically communicated between cells, for which the visualization of notch signaling is essential. In this review, we introduce recent technical advances in the visualization of notch signaling during neural development and in the adult brain, and we discuss the physiological significance of dynamic regulation of notch signaling.

Gene expression profiling of neural stem cells and identification of regulators of neural differentiation during cortical development.

During mammalian brain development, neural stem cells transform from neuroepithelial cells to radial glial cells and finally remain as astrocyte‐like cells in the postnatal and adult brain. Neuroepithelial cells divide symmetrically and expand the neural stem cell pool; after the onset of neurogenesis, radial glial cells sequentially produce deep layer neurons and then superficial layer neurons by asymmetric, self‐renewing divisions during cortical development. Thereafter, gliogenesis supersedes neurogenesis, while a subset of neural stem cells retain their stemness and lurk in the postnatal and adult brain. Thus, neural stem cells undergo alterations in morphology and the capacity to proliferate or give rise to various types of neural cells in a temporally regulated manner. To shed light on the temporal alterations of embryonic neural stem cells, we sorted the green fluorescent protein‐positive cells from the dorsolateral telencephalon (neocortical region) of pHes1‐d2EGFP transgenic mouse embryos at different developmental stages and performed gene expression profiling. Among dozens of transcription factors differentially expressed by cells in the ventricular zone during the course of development, several of them exhibited the activity to inhibit neuronal differentiation when overexpressed. Furthermore, knockdown of Tcf3 or Klf15 led to accelerated neuronal differentiation of neural stem cells in the developing cortex, and neurospheres originated from Klf15 knockdown cells mostly lacked neurogenic activities and only retained gliogenic activities. These results suggest that Tcf3 and Klf15 play critical roles in the maintenance of neural stem cells at early and late embryonic stages, respectively. STEM CELLS 2011;29:1817–1828

Hes1 and Hes5 regulate vascular remodeling and arterial specification of endothelial cells in brain vascular development.

The vascular system is the first organ to form in the developing mammalian embryo. The Notch signaling pathway is an evolutionarily conserved signaling mechanism essential for proper embryonic development in almost all vertebrate organs. The analysis of targeted mouse mutants has demonstrated essential roles of the Notch signaling pathway in embryonic vascular development. However, Notch signaling-deficient mice have so far not been examined in detail in the head region. The bHLH genes Hes1 and Hes5 are essential effectors for Notch signaling, which regulate the maintenance of progenitor cells and the timing of their differentiation in various tissues and organs. Here, we report that endothelial-specific Hes1 and Hes5 mutant embryos exhibited defective vascular remodeling in the brain. In addition, arterial identity of endothelial cells was partially lost in the brain of these mutant mice. These data suggest that Hes1 and Hes5 regulate vascular remodeling and arterial fate specification of endothelial cells in the development of the brain. Hes1 and Hes5 represent critical transducers of Notch signals in brain vascular development.

The identification of transcriptional targets of Ascl1 in oligodendrocyte development

The basic helix‐loop‐helix (bHLH) transcription factor Ascl1 plays crucial roles in both oligodendrocyte development and neuronal development; however, the molecular target of Ascl1 in oligodendrocyte progenitor cells (OPCs) remains elusive. To identify the downstream targets of Ascl1 in OPCs, we performed gene expression microarray analysis and identified Hes5 as a putative downstream target of Ascl1. In vivo analysis revealed that Ascl1 and Hes5 were coexpressed in early developmental oligodendrocytes in both the telencephalon and the ventral spinal cord. We also found that Hes5 expression was reduced in the OPCs of Ascl1 mutant mice. Furthermore, we demonstrated that Ascl1 directly binds to an E‐box region within the Hes5 promoter and regulates Hes5 expression at the transcriptional level. Taken together, these in vivo and in vitro data suggest that Ascl1 induces Hes5 expression in a cell‐autonomous manner. Considering the previously known function of Hes5 as a repressor of Ascl1, our data indicate that Hes5 is involved in the negative feedback regulation of Ascl1.

Essential roles of the histone methyltransferase ESET in the epigenetic control of neural progenitor cells during development

In the developing brain, neural progenitor cells switch differentiation competency by changing gene expression profiles that are governed partly by epigenetic control, such as histone modification, although the precise mechanism is unknown. Here we found that ESET (Setdb1), a histone H3 Lys9 (H3K9) methyltransferase, is highly expressed at early stages of mouse brain development but downregulated over time, and that ablation of ESET leads to decreased H3K9 trimethylation and the misregulation of genes, resulting in severe brain defects and early lethality. In the mutant brain, endogenous retrotransposons were derepressed and non-neural gene expression was activated. Furthermore, early neurogenesis was severely impaired, whereas astrocyte formation was enhanced. We conclude that there is an epigenetic role of ESET in the temporal and tissue-specific gene expression that results in proper control of brain development.