January 26, 2007;
Changes in Rx1 and Pax6 activity at eye field stages differentially alter the production of amacrine neurotransmitter subtypes in Xenopus.
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
activity during eye
field stages affects the production of three neurotransmitter subtypes of amacrine cells. Gain-of-function and loss-of-function hormone-inducible constructs of Rx1
were used to alter Rx1
protein or activity levels after the formation of the eye
field. The major-retina
of the 32-cell stage
(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
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. 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. Rx1
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.
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Figure 2. Labeling of amacrine cell subtypes. Sections of retina were labeled with antibodies to distinguish amacrine subtypes (red cells); those descended from the injected blastomere express GFP (green cells). Top row: Large numbers of amacrine cells express GABA (left panel). Large arrowheads indicate two GABA amacrine cells descended from D1.1.1 blastomere (green in middle panel and double-labeled in merged right panel). Small arrow indicates a GABA amacrine cell that is not GFP-labeled. Middle row: Dopamine (DA) amacrine cells are less abundant (left panel). Large arrowhead indicates a DA amacrine cell descended from D1.1.1 blastomere (green in middle panel and double-labeled in merged right panel). Small arrows indicate two DA amacrine cells that are not GFP-labeled. Bottom row: NPY amacrine cells also are less abundant (left panel). Large arrowhead indicates a NPY amacrine cell descended from D1.1.1 blastomere (green in middle panel and double-labeled in merged right panel). Small arrows indicate two NPY amacrine cells that are not GFP-labeled. Each image was collected with 40x oil lens, zoom set at 1.9, in a 1024x1024 pixel field, and pixel size equal to 0.12 μm.
Figure 3. Altered Rx1 levels/activity affect all three amacrine cell subtypes. rx1 gain-of-function (dark blue bars) and loss-of function (rx1-EnR; light blue bars) were induced at early (St. 12) or late (St. 16) eye field stages. The percent contribution that the injected blastomere lineage made to the total number of the subtype was determined for: (A) GABA, (B) DA and (C) NPY amacrine cells. Bars indicate SEM. A single asterisk (*) indicates a significant difference (p<0.05) compared to gfp mRNA-injected control embryos that also were treated with dexamethasone (green bars). A double asterisk (**) over a stage 16 bar indicates a significant difference (p<0.05) compared to stage 12 induction data. All samples passed the equal variance test. rx1 gain-of-function at both eye field stages caused a significant reduction of GABA and DA amacrine cells, but the GABA reduction was significantly less at stage 16. In contrast, NPY amacrine cells were reduced only at the late stage. rx1 loss-of-function increased GABA cell production and reduced DA amacrine cell production equivalently at both eye field stages; it significantly decreased NPY amacrine cells only at stage 12.
Figure 4. Altered Pax6 levels/activity differentially affect amacrine cell subtypes. pax6 gain-of-function (dark yellow bars) and loss-of-function (dnpax6, light yellow bars) were induced at early (St. 12) and late (St. 16) eye field stages, and analyzed as in Figure 3. pax6 gain-of-function at either eye field stage significantly reduced the D1.1.1 contribution to DA amacrine cells, whereas it only affected NPY cells at stage 12. pax6 loss-of-function at both stages significantly increased GABA cells and significantly decreased DA cells. NPY amacrine cells were not significantly affected by pax6 loss-of-function at either stage.
Figure 5. The developmental programs that produce amacrine cell subtypes are differentially affected by Rx1 and Pax6 in a time-dependent manner. The effects on the number of amacrine cells produced after induction of the different Rx1 and Pax6 constructs at the two eye field stages are summarized. Retinal stem cells (RSC) in the stage 12 eye field are repressed by Rx1 from producing GABA and DA amacrine cells, but Rx1 is required for NPY amacrine cells. In contrast, Pax6 represses the production of DA and NPY amacrine cells. Loss-of-function of either Rx1 (by Rx1EnR) or Pax6 (by dnPax6) is required for GABA amacrine cell production. In the stage 16 eye field, which is likely comprised of both RSC and retinal progenitor cells (RPC) , Rx1 continues to repress DA amacrine cells, but the effect on GABA amacrine cells is much reduced. In addition, Rx1 now represses NPY amacrine cells. Pax6 continues to repress DA cells, but the effect on NPY cells is no longer detectable. As at stage 12, loss-of-function of either Rx1 (by Rx1EnR) or Pax6 (by dnPax6) is required for GABA amacrine cell production. These data indicate that Rx1 and Pax6 differentially affect the production of the different amacrine subtypes over time. Because both genes are considered to be transcriptional activators [28,43], their inhibitory effects on amacrine subtypes are likely to be transcriptionally indirect.
Requirement of multiple basic helix-loop-helix genes for retinal neuronal subtype specification.