Abstract
Purpose: :
The mechanism of horizontal cell differentiation has been largely unknown. We examined the function of Prox1, which was previously shown to be essential for horizontal cell differentiation, in retinal development by a gain-of-function analysis in mouse retinal explant cultures. However, Prox1 was not sufficient for the complete differentiation of horizontal cells. This result suggests that a network of transcription factors is required for the terminal differentiation of horizontal cells. Therefore, we searched transcription factors, which may be involved in horizontal cell differentiation.
Methods: :
We searched public databases of retinal in-situ hybridization expression patterns at different developmental stages, and selected candidate genes by virtue of their expression in horizontal cells. We performed gain-of-function analysis by retrovirus mediated gene transfer into mouse retinal explant cultures. Loss of function analysis was done by using knockout mice.
Results: :
We first examined the role of Prox1, Pax6, and Lim1 in horizontal cell differentiation by examining the effect of overexpression of these genes in E17 mouse retinal explant cultures. Expressing each gene singly or in combination did not achieve terminal differentiation of horizontal cells, indicating a requirement for additional gene(s). Among candidate genes, we focused on Sall3 as it was expressed in horizontal cells. The role of Sall3 in vertebrate retina has not been reported. The gain of function analysis in mouse retinal explant showed that the Sall3-expressed cells preferentially localized in the outer plexiform layer (OPL) as compared with control cells and did not express the horizontal cell marker calbindin. We then examined the retinal in Sall3 deficient (Sall3-KO) mice. Since Sall3-KO died immediate after birth, we made retinal explant cultures from the P0 retina. After 10 days in culture, a lack of the horizontal cell markers, calbindin and NF160, was observed, suggesting that Sall3 is essential for horizontal cell differentiation. When we examined the retina at E17, calbindin expression was observed in cells in the appropriate position and number; however, NF160 signals were weaker than control mice. These results indicate that horizontal cells are appropriately specification in the absence of Sall3, but that they fail to terminally differentiate.
Conclusions: :
These studies revealed that Sall3 is not required for the specification of horizontal cells, but regulates the terminal differentiation of horizontal cells.
Keywords: horizontal cells • transcription factors • transgenics/knock-outs