| Abstract No.: | C-A3012 |
| Country: | Canada |
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| Title: | IDENTIFYING THE DOWNSTREAM TARGETS OF THE PUTATIVE RETINAL TRANSCRIPTION FACTOR PRDM8 |
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| Authors/Affiliations: | 2 Katie Allen*; 2 Cynthia Jung; 1 Martin Klein; 1 David Birch; 2 Roderick McInnes;
1 Retina Foundation of the Southwest, Dallas, TX, USA; 2 The Hospital for Sick Children/University of Toronto, ON, Canada;
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| Content: | Objectives: PRDM8 (PR-domain containing 8) is a vertebrate conserved putative transcription factor identified in a screen for novel retinal developmental regulators. At least five other PR-domain family members are known to be required for the regulation of cell fate and maintenance in other tissues, suggesting that Prdm8 plays a similar role in the retina. During retinal development, Prdm8 expression is low in retinal progenitors, but expression is pronounced in differentiating retinal neurons. To begin to understand the role of Prdm8 in retinal biology, we made a mouse loss of function mutant (Prdm8GFP/GFP). Prdm8 knockout mice have a near complete absence of rod bipolar interneurons in the adult retina. Our current goal is to identify downstream targets of Prdm8, and to define its role in retinal development and maintenance.
Materials and Methods: Prdm8 expression in wildtype mice was characterized by Northern blot and RNA in situ hybridization. The Prdm8 ORF was disrupted by insertion of a nuclear-localized eGFP reporter (Prdm8GFP/GFP) to generate a loss of function allele. Immunohistochemistry and electrotetinograms were carried out to determine the effect of the loss of Prdm8 in the mutant retina. To identify potential transcriptional targets of Prdm8, microarray analysis was used to identify differentially expressed genes in Prdm8GFP/GFP vs. wildtype retinas at postnatal day 6, when Prdm8GFP/GFP retinas still appear morphologically normal.
Results: In the adult mouse, the Prdm8 transcript is expressed predominantly in the nervous system, most notably in the retina, hippocampus and cerebellum. Early in life (P6), following bipolar cell genesis, Prdm8GFP/GFP retinas appear morphologically normal and retinal expression of the pan-bipolar marker Chx10 is comparable to wildtype. These findings indicate that Prdm8 is not required for bipolar cell specification. In the mature retina, however, the Prdm8 knockout retinas have near complete loss of rod (PKCα+) bipolar cells and a significant reduction of Type 2 OFF (recoverin, bHLHb5+) cone bipolar cells. Electroretinograms showed that Prdm8 mutants exhibit significant b-wave deficits, consistant with a loss of rod pathway signalling. The microarray analysis revealed 18 differentially expressed genes at a false discovery rate of 15%. A number of these genes are also transcription factors expressed elsewhere in the nervous system, including Sox6, Lhx3 and Lhx4. The microarray results are being confirmed using real-time PCR, in situ hybridization and immunohistochemistry.
Conclusions: We conclude that 1) Prdm8 is required for the maintenance of rod bipolar cells and Type 2 OFF cone bipolar interneurons in the mammalian retina, and 2) Prdm8 is likely to regulate essential molecules of the late rod bipolar differentiation. These studies will contribute to our knowledge of normal retinal neuronal development and maintenance.
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