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Yukihiro Baba, Sumiko Watanabe; Induction Of Horizontal Cell Differentiation In Mouse Embryonic Retinal Cells By Combinatorial Expression Of Foxn4, Prox1 And Sall3. Invest. Ophthalmol. Vis. Sci. 2012;53(14):432.
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Several transcription factors such as Foxn4, Ptf1a, and Prox1 have been shown to be essential for horizontal cell fate determination. Subsequently, LIM class homeodomain transcription factor Lim1 was found to regulate horizontal cell migration toward the outerneuroblastic layer. However, none of these factors were sufficient to induce horizontal cells from retinal progenitor cells in mouse. In addition, we found that the spalt-like zinc finger protein family Sall3 was essential for maturation of horizontal cells, but again it could not induce horizontal cell fate by itself. Therefore, it is suggested that combinational function of these transcription factors may be required for horizontal cell differentiation. To verify this hypothesis, gain-of-function analysis was performed with transcription factors in combination to induce horizontal cell differentiation.
Condition of electroporation was carefully adjusted to achieve multiple gene introductions to retinal progenitor cells. Various combinations of pCAG plasmids encoding Foxn4, Ptf1a, Prox1, Lim1, and Sall3 were introduced into mouse retina at embryonic day 17 by electroporation. After 11 days of explant culture, the explants were harvested and frozen-sectioned. Retinal sub-types of gene transfected cells were examined by immunohistochemical analysis using cell type specific markers. In addition, morphology of transfected cells was observed after monolayer culture.
Single overexpression of any candidate genes failed to enhance the expression of horizontal cell markers, in agreement with earlier studies. On the other hand, combination of Foxn4, Prox1, and Sall3 induced the expression of horizontal cell markers, NF160, NFL, and Calbindin-D 28k, whereas expression of other cell type of markers was inhibited. In addition, transfected cells localized at the appropriate position of mature horizontal cells. Furthermore, transfected cells in the monolayer culture showed horizontal cell-like morphology, such as long neurites.
Induction of horizontal cell differentiation was achieved by combinational expression of Foxn4, Prox1, and Sall3, suggesting that collaborative function of these genes is essential for horizontal cell differentiation.
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