We also measured TAM check details receptor and ligand mRNAs in the retina and eye using quantitative

RT-PCR with mRNA prepared from multiple isolated tissues: RPE, choroid, eye cup (tissue remaining after removal of neural retina and RPE), ciliary body (CB), and neural retina (minus RPE; see Experimental Procedures) (Figure 6). Although the apical microvilli of RPE cells express both Mer and Tyro3 (Prasad et al., 2006), Mertk−/− single gene mutants yield a strong PR degeneration phenotype ( Figures 2 and 3), whereas Tyro3−/− mutants have a normally configured ONL ( Prasad et al., 2006). To assess the relative expression level of the two TAM receptor genes in these cells, we measured the relative expression of Mertk and Tyro3

mRNAs in isolated RPE. As before ( Prasad et al., 2006), we used a collagenase/hyaluronidase dissociation protocol to cleanly peel off the RPE layer from adult 129/C57Bl/6 retinae, and prepared mRNA from this purified RPE layer. Quantitative RT-PCR revealed that RPE cells express slightly more than four times as much Mertk mRNA as Tyro3 mRNA, and no detectable Axl mRNA ( Figure 6A). In these same qRT-PCR experiments, we measured Gas6 and Pros1 mRNA levels. Gas6 ( Figure 6B) and Pros1 ( Figure 6C) mRNAs were detected in all ocular tissues, with measured Gas6 mRNA levels being approximately an order of magnitude higher than those for Pros1

mRNA throughout the eye. We observed a modest (∼2.5-fold) upregulation of Pros1 mRNA expression in the CB of Gas6−/− mutants, with no substantial signaling pathway change in this mRNA in other Gas6−/− ocular tissues ( Figure 6C). Similarly, there was no substantial change in Gas6 mRNA levels measured in the retina of either Pros1fl/-/Nes-Cre or Pros1fl/-/Trp1-Cre mice ( Figure 6B). The complete degeneration phenotype evident in the central retina of the Pros1fl/fl/Nes-Cre/Gas6−/− mice suggests that blood-derived Protein S is unlikely to be a major reservoir of ligand for the Mer receptor of the RPE. The TAM RTKs play key roles in immune regulation, the phagocytosis of apoptotic cells (ACs) and membranes, the facilitation of viral infection, and the progression of cancer (Lemke and Burstyn-Cohen, 2010; Lemke and STK38 Rothlin, 2008; Meertens et al., 2012; Morizono et al., 2011; Verma et al., 2011). However, the relative importance and contribution of the ligands that activate these receptors has yet to be assessed genetically in any setting in vivo. Although there have been previous biochemical studies that document Protein S and/or Gas6 binding to or activation of Tyro3, Axl, or Mer (Lemke and Rothlin, 2008), most of these in vitro studies were performed with cultured cells or membranes in which endogenous TAM receptor and ligand expression were unknown.