Mami Matsuo-Takasaki

Self-Organized Formation of Polarized Cortical Tissues from ESCs and Its Active Manipulation by Extrinsic Signals

Here, we demonstrate self-organized formation of apico-basally polarized cortical tissues from ESCs using an efficient three-dimensional aggregation culture (SFEBq culture). The generated cortical neurons are functional, transplantable, and capable of forming proper long-range connections in vivo and in vitro. The regional identity of the generated pallial tissues can be selectively controlled (into olfactory bulb, rostral and caudal cortices, hem, and choroid plexus) by secreted patterning factors such as Fgf, Wnt, and BMP. In addition, the in vivo-mimicking birth order of distinct cortical neurons permits the selective generation of particular layer-specific neurons by timed induction of cell-cycle exit. Importantly, cortical tissues generated from mouse and human ESCs form a self-organized structure that includes four distinct zones (ventricular, early and late cortical-plate, and Cajal-Retzius cell zones) along the apico-basal direction. Thus, spatial and temporal aspects of early corticogenesis are recapitulated and can be manipulated in this ESC culture.

Dorsalization of the Neural Tube by Xenopus Tiarin, a Novel Patterning Factor Secreted by the Flanking Nonneural Head Ectoderm

We have isolated a novel secreted dorsalizing factor of the neural tube, Xenopus Tiarin, which belongs to the olfactomedin-related family. Tiarin expression starts at the late gastrula stage in the nonneural ectoderm adjacent to the anterior neural plate. Overexpression of Tiarin in the embryo causes expansion of dorsal neural markers and suppression of ventral markers. In the eye-forming field, Tiarin overexpression induces the retinal markers and represses optic stalk markers. Tiarin directly dorsalizes neural tissues in the absence of mesodermal tissues and antagonizes the ventralizing activity of Sonic hedghog (Shh). Unlike BMP4, another dorsalizing factor, Tiarin does not display antineuralizing activity on the ectoderm or mesoderm-ventralizing activity. These findings show that Tiarin is a novel patterning signal candidate acting in the specification of the dorsal neural tube.

Cloning and expression of a novel zinc finger gene, Fez, transcribed in the forebrain of Xenopus and mouse embryos

We have identified and cloned a novel zinc finger gene, Fez (forebrain embryonic zinc-finger), as a potential downstream determinant of anterior neural plate formation in Xenopus. Fez was isolated as one of several neural-specific genes that was induced by the neuralizing factor, noggin (Smith and Harland, 1992. Cell 70, 829-840), in uncommitted ectoderm. Fez has an open reading frame comprising 466 amino acids, and contains six C(2)H(2) type zinc finger domains, which are highly conserved among Drosophila, zebrafish, mouse, and human. In Xenopus, the expression of Fez begins at stage 12 in the rostral end of the neural plate, and by stage 45, it is localized to several telencephalic regions, including the olfactory bulbs, nervus terminalis, and ventricular zone. The mouse homologue of Fez is similarly expressed in the mouse forebrain by embryonic day 11.

An essential role of Xenopus Foxi1a for ventral specification of the cephalic ectoderm during gastrulation

During gastrulation in Xenopus, the head ectoderm is subdivided into the central nervous system (CNS) anlage (neural plate) and the non-CNS ectoderm (i.e. epidermis, placodes and neural crest). The winged-helix transcription factor Xfoxi1a is one of the earliest markers for the preplacodal region at the mid-neurula stage. Interestingly, before the establishment of the preplacodal region, Xfoxi1a expression is detected in the entire cephalic non-neural ectoderm at the mid- and late gastrula stages. The present study focuses on the role of Xfoxi1a particularly at the gastrula stages. The early Xfoxi1a expression in the anteroventral ectoderm is dependent on Bmp signals and suppressed by Wnt signals. Inhibition of Xfoxi1a activities by injection of antisense oligonucleotides leads to suppression of non-CNS ectodermal markers (e.g. keratin) and expansion of the anterior expression domain of the CNS marker Sox2. Conversely, misexpression of Xfoxi1a suppresses Sox2 and induces keratin in the anterior neural plate. In the animal cap, Xfoxi1a overexpression antagonizes the neuralizing activity of Chordin (Chd). Studies using an inducible Xfoxi1a construct (GR-Xfoxi1a) show that the ventralizing function of Xfoxi1a is confined to the gastrula stage. Thus, Xfoxi1a is an essential regulator of ventral specification of the early head ectoderm during gastrulation.

Generation of Induced Pluripotent Stem Cells from Human Nasal Epithelial Cells Using a Sendai Virus Vector

The generation of induced pluripotent stem cells (iPSCs) by introducing reprogramming factors into somatic cells is a promising method for stem cell therapy in regenerative medicine. Therefore, it is desirable to develop a minimally invasive simple method to create iPSCs. In this study, we generated human nasal epithelial cells (HNECs)-derived iPSCs by gene transduction with Sendai virus (SeV) vectors. HNECs can be obtained from subjects in a noninvasive manner, without anesthesia or biopsy. In addition, SeV carries no risk of altering the host genome, which provides an additional level of safety during generation of human iPSCs. The multiplicity of SeV infection ranged from 3 to 4, and the reprogramming efficiency of HNECs was 0.08–0.10%. iPSCs derived from HNECs had global gene expression profiles and epigenetic states consistent with those of human embryonic stem cells. The ease with which HNECs can be obtained, together with their robust reprogramming characteristics, will provide opportunities to investigate disease pathogenesis and molecular mechanisms in vitro, using cells with particular genotypes.

Anterior neural development requires Del1, a matrix-associated protein that attenuates canonical Wnt signaling via the Ror2 pathway

During early embryogenesis, the neural plate is specified along the anterior-posterior (AP) axis by the action of graded patterning signals. In particular, the attenuation of canonical Wnt signals plays a central role in the determination of the anterior brain region. Here, we show that the extracellular matrix (ECM) protein Del1, expressed in the anterior neural plate, is essential for forebrain development in the Xenopus embryo. Overexpression of Del1 expands the forebrain domain and promotes the formation of head structures, such as the eye, in a Chordin-induced secondary axis. Conversely, the inhibition of Del1 function by a morpholino oligonucleotide (MO) represses forebrain development. Del1 also augments the expression of forebrain markers in neuralized animal cap cells, whereas Del1-MO suppresses them. We previously reported that Del1 interferes with BMP signaling in the dorsal-ventral patterning of the gastrula marginal zone. By contrast, we demonstrate here that Del1 function in AP neural patterning is mediated mainly by the inhibition of canonical Wnt signaling. Wnt-induced posteriorization of the neural plate is counteracted by Del1, and the Del1-MO phenotype (posteriorization) is reversed by Dkk1. Topflash reporter assays show that Del1 suppresses luciferase activities induced by Wnt1 and β-catenin. This inhibitory effect of Del1 on canonical Wnt signaling, but not on BMP signaling, requires the Ror2 pathway, which is implicated in non-canonical Wnt signaling. These findings indicate that the ECM protein Del1 promotes forebrain development by creating a local environment that attenuates the cellular response to posteriorizing Wnt signals via a unique pathway.

XTsh3 is an essential enhancing factor of canonical Wnt signaling in Xenopus axial determination.

In Xenopus, an asymmetric distribution of Wnt activity that follows cortical rotation in the fertilized egg leads to the dorsal–ventral (DV) axis establishment. However, how a clear DV polarity develops from the initial difference in Wnt activity still remains elusive. We report here that the Teashirt‐class Zn‐finger factor XTsh3 plays an essential role in dorsal determination by enhancing canonical Wnt signaling. Knockdown of the XTsh3 function causes ventralization in the Xenopus embryo. Both in vivo and in vitro studies show that XTsh3 substantially enhances Wnt signaling activity in a β‐catenin‐dependent manner. XTsh3 cooperatively promotes the formation of a secondary axis on the ventral side when combined with weak Wnt activity, whereas XTsh3 alone has little axis‐inducing ability. Furthermore, Wnt1 requires XTsh3 for its dorsalizing activity in vivo. Immunostaining and protein analyses indicate that XTsh3 is a nuclear protein that physically associates with β‐catenin and efficiently increases the level of β‐catenin in the nucleus. We discuss the role of XTsh3 as an essential amplifying factor of canonical Wnt signaling in embryonic dorsal determination.

The POU domain gene, XlPOU 2 is an essential downstream determinant of neural induction.

The POU domain gene, XlPOU 2, acts as a transcriptional activator during mid-gastrulation in Xenopus. Overexpression or misexpression of VP16-POU-GR, a fusion protein consisting of the strong activator domain of VP16 and the POU domain of XlPOU 2, results in ectopic expression of the neural-specific genes, nrp-1, en-2, and beta-tubulin. In contrast, overexpressing a dominant-inhibitory form of XlPOU 2 inhibits the chordin-induced neuralization of uncommitted ectoderm, and results in a loss of nrp-1 and en-2 expression in embryos. Furthermore, in uncommitted ectoderm, XlPOU 2 regulates the developmental neural program that includes a number of pre-pattern genes and at least one proneural gene, X-ngnr-1, thus playing a key role during neural determination.

Dual Functions of Hypoxia-Inducible Factor 1 Alpha for the Commitment of Mouse Embryonic Stem Cells Toward a Neural Lineage

Embryonic stem (ES) cells are useful for elucidating the molecular mechanisms of cell fate decision in the early development of mammals. It has been shown that aggregate culture of ES cells efficiently induces neuroectoderm differentiation. However, the molecular mechanism that leads to selective neural differentiation in aggregate culture is not fully understood. Here, we demonstrate that the oxygen-sensitive hypoxia-inducible transcription factor, Hif-1α, is an essential regulator for neural commitment of ES cells. We found that a hypoxic environment is spontaneously established in differentiating ES cell aggregates within 3 days, and that this time window coincides with Hif-1α activation. In ES cells in adherent culture under hypoxic conditions, Hif-1α activation was correlated with significantly greater expression of neural progenitor-specific gene Sox1 compared with ES cells in adherent culture under normoxic conditions. In contrast, Hif-1α-depleted ES cell aggregates showed severe reduction in Sox1 expression and maintained high expression of undifferentiated ES cell marker genes and epiblast marker gene Fgf5 on day 4. Notably, chromatin immune precipitation assay and luciferase assay showed that Hif-1α might directly activate Sox1 expression. Of additional importance is our finding that attenuation of Hif-1α resulted in an increase of BMP4, a potent inhibitor of neural differentiation, and led to a high level of phosphorylated Smad1. Thus, our results indicate that Hif-1α acts as a positive regulator of neural commitment by promoting the transition of ES cell differentiation from the epiblast into the neuroectoderm state via direct activation of Sox1 expression and suppressing endogenous BMP signaling.

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. Author Summary During 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. By 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.