Purpose: Both rx1 and pax6 are expressed during the initial formation of the vertebrate eye field, and they are thought to be crucial for maintenance of the retinal stem cells in the ciliary marginal zone. However, both genes continue to be expressed in different layers of the differentiating retina, suggesting that they have additional roles in cell type specification. Because previous work suggested that amacrine cell subtypes are derived from biased progenitors in the eye field, we tested whether altering Rx1 or Pax6 activity during eye field stages affects the production of three neurotransmitter subtypes of amacrine cells.
Methods: Gain-of-function and loss-of-function hormone-inducible constructs of Rx1 and Pax6 were used to alter Rx1 and Pax6 protein or activity levels after the formation of the eye field. The major-retina producing blastomere of the 32-cell stage Xenopus embryo (D1.1.1) was injected with mRNA encoding one of these proteins and mRNA encoding GFP to label the altered lineage. Embryos were treated with synthetic hormone at either early (stage 12) or late (stage 16) eye field stages and they developed to tadpole stages (stage 44/45) when the cells in the central retina have differentiated. Amacrine cell subtypes (dopamine [DA], neuropeptide Y [NPY], gamma aminobutyrate acid [GABA]) were detected by immunofluorescence histology and the numbers of each type of cell produced within the affected lineage were counted. The percent contribution of the D1.1.1 lineage to a particular amacrine subtype after stage 12 or stage 16 hormone treatment were independently compared to those from gfp mRNA-injected control embryos that were similarly treated with hormone.
Results: Increasing Rx1 at early eye field stages promotes NPY amacrine cells and represses GABA and DA amacrine cells, and at late eye field stages significantly represses DA and NPY phenotypes but has a diminished effect on the GABA phenotype. Increasing Pax6 at early eye field stages represses NPY and DA amacrine cells but does not affect the GABA phenotype, whereas in the late eye field it significantly represses only the DA phenotype.
Conclusions: Rx1 and Pax6 differentially modify the ability of eye field precursors to produce different neurotransmitter subtypes of amacrine cells. These effects varied for each of the subtypes investigated, indicating that amacrine cells are not all specified by a single genetic program. Furthermore, some cases were time-dependent, indicating that the downstream effects change as development proceeds.