Biomedical subjects

Kyoji Ohyama

Publications and source records attributed to Kyoji Ohyama.

7 recordsLinked to original sources

PubMed2006

A robust system for RNA interference in the chicken using a modified microRNA operon.

RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.

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PubMed2006

Regional morphogenesis in the hypothalamus: a BMP-Tbx2 pathway coordinates fate and proliferation through Shh downregulation.

A central challenge in embryonic development is to understand how growth and pattern are coordinated to direct emerging new territories during morphogenesis. Here, we report on a signaling cascade that links cell proliferation and fate, promoting formation of a distinct progenitor domain within the developing chick hypothalamus. We show that the downregulation of Shh in floor plate-like cells in the forebrain governs their progression to a distinctive, proliferating hypothalamic progenitor domain. Shh downregulation occurs via a local BMP-Tbx2 pathway, Tbx2 acting to repress Shh expression. We show in vivo and in vitro that forced maintenance of Shh in hypothalamic progenitors prevents their normal morphogenesis, leading to maintenance of the Shh receptor, ptc, and preventing progression to an Emx2(+)-proliferative progenitor state. Our data identify a molecular pathway for the downregulation of Shh via a BMP-Tbx2 pathway and provide a mechanism for expansion of a discrete progenitor domain within the developing forebrain.

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PubMed2005

Directed differentiation of neural cells to hypothalamic dopaminergic neurons.

Hypothalamic neurons play a key role in homeostasis, yet little is known about their differentiation. Here, we demonstrate that Shh and Bmp7 from the adjacent prechordal mesoderm govern hypothalamic neural fate, their sequential action controlling hypothalamic dopaminergic neuron generation in a Six3-dependent manner. Our data suggest a temporal distinction in the requirement for the two signals. Shh acts early to specify dopaminergic neurotransmitter phenotype. Subsequently, Bmp7 acts on cells that are ventralised by Shh, establishing aspects of hypothalamic regional identity in late-differentiating/postmitotic cells. The concerted actions of Shh and Bmp7 can direct mouse embryonic stem cell-derived neural progenitor cells to a hypothalamic dopaminergic fate ex vivo.

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PubMed2004

Receptor-like protein tyrosine phosphatase zeta/RPTP beta is expressed on tangentially aligned neurons in early mouse neocortex.

Protein tyrosine phosphatase zeta (PTPzeta)/RPTPbeta is a chondroitin sulfate proteoglycan predominantly expressed in the brain. In this study, we examined immunohistochemical localisation of PTPzeta in the mouse telencephalon from embryonic day 9.5 (E9.5) to E15.5. During E10.5-E12.5, immunoreactivities for PTPzeta are specifically observed on the tangentially aligned neurons at the preplate (PP) of the neocortex, as well as on the neurons at the mantle layer (ML) of the ganglionic eminences (GEs). Likewise, neurons immunoreactive for CR50, a marker for Cajal-Retzius neurons, are aligned from the ML of the ganglionic eminences to the PP of the neocortex and co-express PTPzeta. During E13.5-E15.5, PTPzeta-positive neurons are present at the subplate (SP) as well as at the marginal zone (MZ) of the neocortex. These results indicate that PTPzeta is a useful marker for early-generated neocortical neurons in mice: Cajal-Retzius neurons as well as the subplate neurons.

Aging↗

PubMed2004

Neural cell adhesion molecule L1 is required for fasciculation and routing of thalamocortical fibres and corticothalamic fibres.

To examine the role of neural cell adhesion molecule L1 in thalamocortical projections, we analysed L1 deficient (L1-/y) mice. Immunohistochemistry of pleiotrophin/HB-GAM, a marker for thalamocortical axons and axonal tracing experiments showed that thalamocortical axons were abnormally and highly fasciculated when they pass through the developing internal capsule. Within the cortex, however, their course was more diffuse. The corticofugal fibres immunoreactive for TAG-1 were also more strongly fasciculated and their number was decreased in L1-/y mice. Furthermore, no TAG-1-positive corticofugal axons reached the dorsal thalamus. These data suggest that L1 plays an important role in the fasciculation and routing of axons connecting between the thalamus and the cortex.

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PubMed2002

Coordinate expression of beta1 integrins and their regulator, TGF beta2 at the floor plate of the medulla oblongata is correlated with the crossing of the fibers of olivocerebellar projection in mice.

During embryonic day 11 (E11) to E16, contact-dependent interacting molecules beta1 integrins and their putative regulator TGF beta2 are coordinately expressed at the floor plate in the caudal part of mouse myelencephalon. Their expression disappears at E18. Consistent with the peak of their expression (E13-E16), olivocerebellar fibers primarily cross the floor plate. These data indicate that spatiotemporal expression of beta1 integrins and TGF beta2 is correlated with the crossing of olivocerebellar fibers.

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PubMedDate not supplied

Mutual dependence of murine fetal cutaneous regeneration and peripheral nerve regeneration.

Mammalian fetal cutaneous wounds made at certain developmental stages show complete regeneration. It is reported that wound healing in both adult and fetal skin is disrupted by denervation. Furthermore, fetal cutaneous regeneration has unique aspects such as epidermal wrinkle texture regeneration and dermal regeneration that depend on developmental stage. Therefore, we have examined the relationship of fetal cutaneous regeneration with denervation. We made cutaneous wounds on fetal mice at various developmental time points including embryonic days (E)13, E15, and E17, and compared the regenerating patterns of peripheral nerves in the skin. We found that when the fetuses are wounded at an early stage of development, peripheral nerves regenerate quicker than at later stages of development when peripheral nerve regeneration is delayed. Next, we denervated the intercostal nerves and made wounds at the denervated sites on E13 and E15. We found that epidermal wrinkling and dermal regeneration were disrupted by denervation. These findings indicate that components of fetal cutaneous regeneration and peripheral nerve regeneration are mutually dependent.

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