| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
To study cortical neuron patterning, we utilized reaggregation cultures of dissociated retinal cells prepared from chicken embryos. Singly dissociated retinal cells can reaggregate and undergo a certain level of histogenesis, including cell type-specific segregation of cells, but they have never been found to reform into laminated structures. Instead, they form rosetted cell layers within the aggregates. However, it is known that conditioned media (CM) prepared from some cell cultures can induce laminated structures in the reaggregates. We hypothesized that the CM should contain important regulators for layer formation, and attempted to identify them. A series of studies along this line showed that the ciliary margin of the retina had the ability to rearrange rosetted cells into a neuroepithelial structure characteristic of the undifferentiated retinal layer. This activity of the ciliary margin was mimicked by Wnt-2b, which is expressed exclusively in this particular zone of the retina
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. Furthermore, retinal cell aggregates treated with Wnt-2b subsequently developed correctly laminated retinal layers. These observations suggest that the ciliary margin, by producing Wnt-2b, functions as a layer-organizing center in the retina.
We also obtained evidence that a function of Wnt-2b is to maintain retinal stem cells. Retinal stem cells are known to be located in the zone adjacent to the Wnt-2b-positive region. Overexpression of Wnt-2b by use of in ovo electroporation in the central retina inhibited neuronal differentiation, inducing the expression of progenitor cell markers. Blocking of the Wnt downstream signaling pathway by a dominant-negative LEF1 inhibited the proliferation of the cells in the marginal area, which resulted in their premature neuronal differentiation. These results, together with other findings, suggest that Wnt-2b functions to maintain undifferentiated progenitor cells in the ciliary marginal zone, and thus serves as a putative stern cell factor in the retina. Less
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